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Contribution of Piezo1 mechanosensitive channels to astrocyte calcium signaling Ko, Rebecca Wai Yin 2018

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CONTRIBUTION OF PIEZO1 MECHANOSENSITIVE CHANNELS TO ASTROCYTE CALCIUM SIGNALING by  Rebecca Wai Yin Ko  B.Sc., The University of British Columbia, 2009  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  June 2018  © Rebecca Wai Yin Ko, 2018 ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled:   Contribution of Piezo1 mechanosensitive channels to astrocyte calcium signaling                      i  submitted by  Rebecca Wai Yin Ko                  in partial fulfillment of the requirements for the degree of  Doctor of Philosophy                                        I                                                                                           in                    Neuroscience                                                                                                           i  Examining Committee: Brian MacVicar, Neuroscience                                                                                                         I Supervisor  Terrance Snutch, Neuroscience                                                                                                        I  Supervisory Committee Member  Eric Accili, Neuroscience                                                                                                                 I University Examiner  Vanessa Auld, Neuroscience                                                                                                            I University Examiner   Additional Supervisory Committee Members:  Ann Marie Craig, Neuroscience                                                                                                       I  Supervisory Committee Member  Yu Tian Wang, Neuroscience                                                                                                           I Supervisory Committee Member   iii  Abstract  Astrocyte calcium (Ca2+) signaling is involved in the regulation of physiological processes such as synaptic activity and vascular tone in the brain.  Recent developments in tools to monitor Ca2+ signals have revealed a novel type of spontaneous Ca2+ transient that is localized to microdomains in the fine astrocytic processes.  However, the molecular mechanisms underlying these signals have not been fully characterized.  Based on data from rodent brain transcriptomic and proteomic studies, we identified Piezo1, a mechanosensitive cation channel, as a potential candidate for mediating these Ca2+ transients.  In Chapter 2, we performed two-photon imaging of the membrane-tethered, genetically encoded Ca2+ indicator Lck-GCaMP5 in cultured astrocytes and developed an algorithm for extracting and analyzing the microdomain Ca2+ signals.  Using a combination of pharmacological and siRNA approaches, we showed that Piezo1 channels contribute to these spontaneous Ca2+ transients.  We also conducted preliminary imaging experiments in brain slice astrocytes and found that spontaneous Ca2+ signals in the endfoot compartments were sensitive to pharmacological modulators of Piezo1.  In Chapter 3, we performed immunostaining using strategies that were optimized to target subcellular locations where Piezo1 expression had previously been reported in other cell types.  Our results indicated that Piezo1 is localized to subcellular compartments relevant to mechanosensation; Piezo1 immunoreactivity was localized to discrete clusters on the plasma membrane and associated with focal adhesion and actin stress fibers in cultured astrocytes, and Piezo1 expression was observed within the endfoot processes of iv  brain slice astrocytes.  Lastly, in Chapter 4, we showed that an osmotic stress model of astrocyte swelling could activate Piezo1-mediated Ca2+ microdomain signals in cultured astrocytes.  Taken together, the data provide evidence that Piezo1 contributes to spontaneous Ca2+ microdomain signals in astrocytes in both cell culture and acute brain slices, and suggest that astrocyte Ca2+ signaling may play a role in integrating mechanical stimuli to regulate brain function in physiological and pathological processes involving changes in mechanical force.   v  Lay Summary  Astrocytes are brain cells that display intracellular calcium signals, and these signals have been shown to regulate physiological processes such as neuronal activity and cerebral blood flow.  Recent advances in imaging tools revealed a novel type of spontaneous calcium signals within astrocytes, but the underlying mechanisms have not been fully characterized.  Here we present evidence that Piezo1, a channel sensitive to mechanical forces, is involved in mediating these signals.   We performed imaging experiments and showed that the signals can be modulated by strategies that activate or block Piezo1.  We conducted staining experiments and demonstrated that Piezo1 is located in astrocyte regions associated with force sensing.  Furthermore, we found that cell swelling could activate Piezo1 in astrocytes.  Taken together, the data suggest that these calcium signals may represent astrocyte integration of mechanical stimuli, raising interesting possibilities for the role of astrocytes in physiological and pathological processes involving changes in mechanical force. vi  Preface  The experiments in this dissertation were jointly designed with Dr. Brian MacVicar.  I conducted all the experiments and data analyses, with the following exceptions:  In Chapter 2, Alexa Nelson assisted with preparation of the Lck-GCaMP5 plasmid.  Dr. Leigh Wicki-Stordeur conducted the immunoblotting experiments.  Dr. John Tyson from Dr. Terry Snutch’s lab designed the luciferase and Piezo1 siRNA sequences, and performed the qPCR analyses.  Joslyn Quick from Dr. Pieter Cullis’ lab prepared the lipid nanoparticle plasmid DNA and siRNA encapsulations.  Jeffrey Ledue contributed greatly to the Matlab algorithms used for data analysis.  Dr. Xiling Zhou from Dr. Ann Marie Craig’s lab provided the astrocyte cultures.  In Chapter 3, Dr. Esperanza Garcia cultured the Neuro2A cells and assisted with designing the immunostaining experiments.  Karen Jones performed immunoblotting and Neuro2A cell staining experiments.  Dr. John Tyson conducted the qPCR analyses.  All collaborators were from Dr. Terry Snutch’s lab.  All animal procedures performed for this dissertation were approved by the University of British Columbia Animal Care Committee under certificates A15-0209 and A15-0086.  vii  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Abbreviations ...................................................................................................................xv Acknowledgements .................................................................................................................. xviii Chapter 1: Introduction ................................................................................................................1 1.1 Astrocyte Ca2+ signaling ................................................................................................. 1 1.1.1 Evolution of approaches to study astrocyte Ca2+ signaling ........................................ 2 1.1.2 Spontaneous Ca2+ microdomain signals in astrocytes ................................................ 6 1.2 Astrocytes as mechanosensitive cells ........................................................................... 10 1.2.1 Astrocytes detect mechanical stimuli to regulate cell physiology ............................ 10 1.2.2 Membrane proteins linked to mechanosensitivity in astrocytes ............................... 12 1.3 Piezo1: a mechanosensitive cation channel .................................................................. 16 1.3.1 Piezo1 channel topology and the structural basis of function................................... 18 1.3.2 Properties of the Piezo1 channel ............................................................................... 20 1.3.3 Modulation of Piezo1 channel activity by second messengers ................................. 23 1.3.4 Piezo1 pharmacology ................................................................................................ 24 1.3.5 Piezo1 expression and physiological functions ........................................................ 26 viii  1.3.6 Pathologies associated with Piezo1 dysfunction....................................................... 27 1.4 Rationale and hypotheses .............................................................................................. 29 1.4.1 Objective 1: To determine the contribution of Piezo1 to spontaneous Ca2+ transients in astrocytes .......................................................................................................................... 30 1.4.2 Objective 2: To characterize the subcellular localization of Piezo1 in astrocytes .... 30 1.4.3 Objective 3: To examine the activation of Piezo1 in hypotonicity-induced astrocyte swelling ................................................................................................................................. 31 Chapter 2: Piezo1 mediates astrocyte Ca2+ microdomain signals ...........................................32 2.1 Overview ....................................................................................................................... 32 2.2 Methods......................................................................................................................... 34 2.2.1 Chemicals and reagents............................................................................................. 34 2.2.2 Astrocyte cultures ..................................................................................................... 35 2.2.3 siRNA sequences for Piezo1 knockdown ................................................................. 36 2.2.4 Lck-GCaMP5 plasmid preparation ........................................................................... 36 2.2.5 Preparation of siRNA- and plasmid DNA-containing lipid nanoparticles ............... 37 2.2.6 Astrocyte transfection with LNPs ............................................................................. 38 2.2.7 Evaluation of transfection viability and efficiency ................................................... 38 2.2.8 Evaluation of Piezo1 siRNA effect on mRNA levels ............................................... 39 2.2.9 Evaluation of Piezo1 siRNA effect on protein levels ............................................... 39 2.2.10 Culture imaging .................................................................................................... 40 2.2.11 Transgenic GLAST-GCaMP5 mice and tamoxifen treatment.............................. 41 2.2.12 Hippocampal slice preparation ............................................................................. 41 2.2.13 Slice imaging ........................................................................................................ 42 ix  2.2.14 Image processing and data analysis ...................................................................... 42 2.2.15 Statistics ................................................................................................................ 43 2.3 Results ........................................................................................................................... 44 2.3.1 LNP delivery of plasmid DNA and siRNA in cultured astrocytes ........................... 44 2.3.2 Development of a novel method to analyze astrocyte Ca2+ signals .......................... 47 2.3.3 Piezo1 activation selectively increases the frequency of microdomain Ca2+ signals 49 2.3.4 Yoda1-induced Ca2+ microdomain signals are sensitive to pharmacological and genetic Piezo1 inhibition....................................................................................................... 50 2.3.5 Yoda1-induced Ca2+ microdomain signals depend on extracellular Ca2+ entry ....... 54 2.3.6 Spontaneous Ca2+ microdomain signals are sensitive to Piezo1 antagonists ........... 55 2.3.7 Preliminary imaging of brain slice astrocyte Ca2+ signals show sensitivity to pharmacological Piezo1 modulations ................................................................................... 57 2.4 Discussion ..................................................................................................................... 59 Chapter 3: Subcellular localization of Piezo1 in astrocytes .....................................................65 3.1 Overview ....................................................................................................................... 65 3.2 Methods......................................................................................................................... 66 3.2.1 Cell culture ................................................................................................................ 66 3.2.2 Astrocyte transfection and treatment ........................................................................ 67 3.2.3 qPCR for Neuro2A cells ........................................................................................... 68 3.2.4 Immunoblotting......................................................................................................... 68 3.2.5 Immunocytochemistry .............................................................................................. 69 3.2.6 Immunohistochemistry ............................................................................................. 71 3.2.7 Confocal image acquisition and analysis .................................................................. 72 x  3.3 Results ........................................................................................................................... 73 3.3.1 Antibody testing in Neuro2A cells that endogenously express Piezo1 .................... 73 3.3.2 Piezo1 immunoreactivity clusters in discrete domains in the astrocyte plasma membrane .............................................................................................................................. 74 3.3.3 Piezo1 immunoreactivity is present in actin filaments and focal adhesions ............. 79 3.3.4 Forskolin-induced disorganization of the cytoskeleton alters Piezo1 distribution ... 82 3.3.5 Piezo1 immunoreactivity colocalizes with astrocyte markers in the mouse brain ... 84 3.4 Discussion ..................................................................................................................... 86 Chapter 4: Role of Piezo1 in astrocyte swelling ........................................................................93 4.1 Overview ....................................................................................................................... 93 4.2 Methods......................................................................................................................... 96 4.2.1 Cell culture and transfection ..................................................................................... 96 4.2.2 Culture imaging ........................................................................................................ 96 4.2.3 Data analysis and statistics........................................................................................ 97 4.3 Results ........................................................................................................................... 97 4.3.1 Piezo1 mediates Ca2+ microdomain signals in astrocytes under hypotonic stress .... 97 4.3.2 Forskolin-induced reorganization of the cytoskeleton does not alter the pharmacological profile of Piezo1 or affect Piezo1-mediated Ca2+ transients to hypotonic challenge ............................................................................................................................... 99 4.3.3 Piezo1 mediates hypotonicity-induced swelling in astrocytes ................................ 103 4.4 Discussion ................................................................................................................... 104 Chapter 5: Conclusion ...............................................................................................................109 5.1 Summary of research findings .................................................................................... 109 xi  5.2 Research significance.................................................................................................. 109 5.3 Future directions ......................................................................................................... 113 5.3.1 Transgenic mice for investigating Piezo1-mediated Ca2+ signaling in vivo ........... 114 5.3.2 Role of Piezo1 in regulation of cerebral vasculature tone ...................................... 115 5.3.3 Role of Piezo1 in astrocyte differentiation ............................................................. 116 5.3.4 Role of Piezo1 in transmitter release ...................................................................... 117 5.3.5 Piezo1 and pathology .............................................................................................. 118 References ...................................................................................................................................120  xii  List of Tables  Table 2-1.  LNP properties. ........................................................................................................... 38  xiii  List of Figures  Figure 1-1.  Genetically-encoded Ca2+ indicators (GECIs) reveal distal astrocyte processes that are not detected by bulk-loaded Ca2+ indicator dyes. ..................................................................... 5 Figure 1-2.  Structure of mouse Piezo1. ....................................................................................... 18 Figure 2-1.  Ca2+ signal analysis algorithm flowchart. ................................................................. 43 Figure 2-2.  Lipid nanoparticle (LNP) delivery of Lck-GCaMP5 plasmid DNA and Piezo1 siRNA is non-toxic and effective in cultured astrocytes. ............................................................. 46 Figure 2-3.  The Ca2+ signal analysis algorithm isolates Ca2+ microdomain events by masking Ca2+ waves. ................................................................................................................................... 48 Figure 2-4.  The Piezo1 agonist, Yoda1, selectively increases the number of Ca2+ microdomain signals. .......................................................................................................................................... 50 Figure 2-5.  Yoda1-induced increases in Ca2+ microdomain signals are partially inhibited by Piezo1 antagonists GsMTx4 and Gd3+. ......................................................................................... 52 Figure 2-6.  Yoda1-induced increases in Ca2+ microdomain signals are inhibited by Piezo1 siRNA. .......................................................................................................................................... 53 Figure 2-7.  Yoda1-induced increases in Ca2+ microdomain signals depend on extracellular Ca2+ entry. ............................................................................................................................................. 55 Figure 2-8.  Spontaneous Ca2+ microdomain signals are inhibited by Piezo1 antagonists GsMTx4 and Gd3+. ....................................................................................................................................... 57 Figure 2-9.  The frequency of astrocyte endfeet Ca2+ transients are sensitive to pharmacological modulators of Piezo1. ................................................................................................................... 58 xiv  Figure 3-1.  Flowchart of immunocytochemistry protocols for optimization of Piezo1 staining in different subcellular regions. ........................................................................................................ 70 Figure 3-2.  Schematic illustrating the epitopes targeted by the Alomone and Proteintech Piezo1 antibodies. ..................................................................................................................................... 71 Figure 3-3.  Neuro2A cells show distinct clusters of Piezo1 immunoreactivity. ......................... 74 Figure 3-4.  Astrocytes exhibit different patterns of Piezo1 immunoreactivity at the plasma membrane. ..................................................................................................................................... 75 Figure 3-5.  Piezo1 immunoreactivity is distributed in clusters located on astrocyte plasma membranes. ................................................................................................................................... 77 Figure 3-6.  Piezo1 siRNA reduces the number of Piezo1 clusters in astrocytes. ........................ 79 Figure 3-7.  Piezo1 immunoreactivity is found in actin filaments and focal adhesions of the cytoskeleton. ................................................................................................................................. 81 Figure 3-8.  Forskolin induces astrocyte stellation and Piezo1 redistribution. ............................. 84 Figure 3-9.  Piezo1 immunoreactivity co-localizes with astrocyte markers in brain slices. ......... 86 Figure 4-1.  Hypotonic solution induces an increase in astrocyte microdomain Ca2+ signals that is inhibited by Piezo1 siRNA treatment. .......................................................................................... 99 Figure 4-2.  Forskolin treatment does not affect the pharmacological profile of Piezo1. .......... 101 Figure 4-3.  Hypotonic solution induces an increase in forskolin-treated astrocyte microdomain Ca2+ signals that is inhibited by Piezo1 siRNA treatment. ......................................................... 102 Figure 4-4.  Piezo1 siRNA treatment inhibits hypotonicity-induced swelling in forskolin-treated astrocytes..................................................................................................................................... 104   xv  List of Abbreviations  AQP4    Aquaporin-4 ASIC    Acid-sensing ion channel ATP    Adenosine triphosphate BAPTA   1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid Ca2+    Calcium cAMP    Cyclic adenosine monophosphate CED    C-terminal extracellular loop domain CHO-K1   Chinese hamster ovary K1 cell line CNS    Central nervous system CTD    C-terminal domain DEG/ENaC   Degenerin/epithelial Na+ channel DiIC18 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine                             perchlorate DLin-KC2-DMA  2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane DLin-MC3-DMA  heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate DSPC    1,2-distearoyl-sn-glycero-3-phosphocholine ECM    Extracellular matrix ECS    Extracellular space EGTA    Ethylene glycol tetraacetic acid ER    Endoplasmic reticulum GABA    Gamma-aminobutyric acid xvi  Gd3+    Gadolinium GECI    Genetically-encoded Ca2+ indicator GFAP    Glial fibrillary acidic protein GFP    Green fluorescent protein GPCR    G-protein-coupled receptor GsMTx4   Grammostola spatulata mechanotoxin 4 HEK293   Human embryonic kidney 293 cell line HPLC    High-performance liquid chromatography IP3    Inositol-1,4,5-trisphosphate IP3R2    IP3 type 2 receptor     K2P    Two-pore domain K+ channel La3+    Lanthanum LNP    Lipid nanoparticle mPTP    Mitochondrial permeability transition pore mRNA    Messenger RNA NMDAR   N-methyl-D-aspartate receptor PBS    Phosphate buffered saline PEG-DMG   Polyethylene glycol (2K)-dimyristolglycerol PI(4,5)P2   Phosphatidylinositol-4,5-bisphosphate qPCR    Quantitative polymerase chain reaction ROI    Region of interest ROS    Reactive oxygen species RVD    Regulatory volume decrease xvii  SERCA   Sarco/endoplasmic reticulum Ca2+ ATPase siRNA    Small interfering ribonucleic acid SOPC    1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine STOML3   Stomatin-like protein 3 TRP    Transient receptor potential VRAC    Volume-regulated anion channel xviii  Acknowledgements  I would like to thank my supervisor, Dr. Brian MacVicar, for his mentorship and guidance.  His scientific insight and passion for research have been truly inspiring.  Thank you for providing me with opportunities to attend courses, participate in collaborations, and present my work in both national and international settings, which have greatly benefitted my growth as a scientist.  I would also like to thank my committee members Dr. Ann Marie Craig, Dr. Terry Snutch, and Dr. Yu Tian Wang for sharing their expertise and providing invaluable feedback for my projects throughout the years.   My PhD training was financially supported by the University of British Columbia Four-Year Fellowship and the Canadian Institutes of Health Research Master’s and Doctoral Research Awards.  To all past and present members of the MacVicar lab, I am fortunate to have crossed paths with you.  Thank you for the scientific collaborations, troubleshooting advice, and moral support, especially during the inevitable failures that are a part of scientific research.   Lastly, thank you to my family and friends for your unconditional support and encouragement.  This thesis would not be possible without you.   1  Chapter 1: Introduction  1.1 Astrocyte Ca2+ signaling Astrocytes are a type of glial cell found throughout the vertebrate central nervous system, accounting for approximately 20 – 40% of the cells in the brain (Herculano-Houzel, 2014).  They were first described by Rudolf Virchow in 1846 as a connective substance, or ‘glue’, that acts as a scaffold to hold neurons together (García-Marín et al., 2007).  Later developments in staining methods allowed the star-like morphology of these cells to be visualized, leading to the introduction of the term ‘astrocyte’ (García-Marín et al., 2007).  However, researchers lacked techniques to study dynamic processes in living astrocytes; in contrast to neuronal activity, which could be probed with electrophysiology, astrocytes were electrically unexcitable.  Therefore, it was assumed that astrocytes were uniformly passive cells that merely provided structural and trophic support for neurons (Navarrete and Araque, 2014).   Despite the lack of sophisticated technology, early researchers studied static images of astrocyte morphology and made important observations revealing the diversity of these cells, as well as predictions about their relevance in brain function.  For example, Andriezen (1893) found that astrocytes could be classified based on different process morphologies and distribution in the brain.  Furthermore, by refining staining methods, Ramón y Cajal was able to observe the highly complex organization of fine astrocyte processes; based on their close association with synapses and blood vessels, he proposed that astrocytes may release substances that affect neuronal activity and mediate vasoconstriction or dilation (García-Marín et al., 2007; Navarrete and Araque, 2014).  Advances in imaging and genetic tools have since demonstrated that astrocytes 2  are molecularly and functionally heterogeneous (Khakh and Sofroniew, 2015; Haim and Rowitch, 2017), and provided evidence supporting the role of astrocytes in regulation of synaptic transmission and blood flow (Araque and Navarrete, 2010; Attwell et al., 2010).  Calcium (Ca2+) is a ubiquitous second messenger that regulates multiple cellular functions.  A key development in the field of astrocyte biology was the introduction of fluorescence microscopy and Ca2+-sensitive fluorescent dyes, which allowed researchers to monitor changes in cytosolic Ca2+ in response to synaptic activity and sensory stimuli (Verkhratsky et al., 2012; Volterra et al., 2014).  These observations suggested that astrocytes exhibit a form of excitability based upon variations in intracellular Ca2+ concentrations rather than through action potential propagation, and indicated that astrocytes may also be involved in information processing in the brain (Volterra et al., 2014).  The role of astrocytes is now established in a number of processes including cellular homeostasis, synapse formation and pruning, synaptic modulation, and neurovascular coupling (Shigetomi et al., 2016).  New insights in the field of astrocyte research are intimately linked to the improving tools for monitoring Ca2+ signals, and recent developments in imaging techniques, such as genetically encoded Ca2+ indicators (GECIs), have revealed further complexities in astrocyte Ca2+ signaling and the functions they govern.    1.1.1 Evolution of approaches to study astrocyte Ca2+ signaling The discovery of Ca2+ signaling as the mode of astrocyte excitability changed the perception of astrocytes from passive support cell to potential partner in brain information processing.  This important advance was possible due to tools such as bulk-loaded Ca2+ indicator dyes that preferentially loaded astrocytes in brain slices, allowing astrocyte Ca2+ signals to be monitored 3  (Porter and McCarthy, 1995), and caged Ca2+ compounds that when activated selectively elevate Ca2+ levels in astrocyte somata.  By using these strategies, pioneering studies in the early 1990s demonstrated that astrocytes in culture and brain slices respond to exogenous or synaptically released glutamate with oscillatory increases in somatic Ca2+ concentrations and propagating cytoplasmic Ca2+ waves (Cornell-Bell et al., 1990; Dani et al., 1992; Porter and McCarthy, 1996).  In addition to glutamate, other neurotransmitters that act on Gq-linked G protein-coupled receptors, including norepinephrine, acetylcholine, and ATP, can also evoke astrocyte Ca2+ signals through inositol-1,4,5-trisphosphate (IP3) – dependent release from endoplasmic reticulum stores, suggesting that astrocytes can respond to a broad range of neuronal activity (Perea et al., 2009).  The use of techniques to selectively monitor and manipulate astrocyte cytosolic Ca2+ levels led to findings implicating astrocyte Ca2+ signaling in physiological processes such as modulation of synaptic strength and cerebral blood flow (Bazargani and Attwell, 2016).  The physiological relevance of astrocyte Ca2+ signaling has been challenged in subsequent studies examining the mechanisms of Ca2+ elevation in astrocytes, and the functional consequences of inhibiting such elevations.  For instance, Sun et al. (2013) showed that the Gq-linked metabotropic glutamate receptor, mGluR5, is both developmentally downregulated and that mGluR5 agonists failed to increase intracellular Ca2+ levels in adult mouse astrocytes.  The authors therefore speculated that astrocytes may no longer respond to synaptic glutamate release beyond the juvenile developmental stage in rodents.  Furthermore, genetic deletion of the IP3 type 2 receptor (IP3R2), which is known to be highly expressed in astrocytes, abolished astrocyte Ca2+ elevations to Gq-linked GPCR agonists, but the transgenic mice still exhibited excitatory 4  synaptic activity and neurovascular coupling, leading to the proposal that astrocyte intracellular Ca2+ changes may be functionally irrelevant (Petravicz et al., 2008; Nizar et al., 2013; Bonder and McCarthy, 2014).    A critical insight in addressing these discrepancies was the realization that bulk-loaded Ca2+ indicators were restricted to the astrocyte soma and proximal processes, leading to severe undersampling of ~90% of the astrocyte volume that is comprised of fine processes (Reeves et al., 2011).  Recent developments utilizing GECI proteins or dialysis of patch-loaded Ca2+ dyes allowing for Ca2+ fluctuations to be monitored in the fine processes (Fig. 1-1) have revealed a previously unappreciated type of astrocyte Ca2+ signaling differing from soma signals in their spatial and temporal characteristics (Shigetomi et al., 2013a; Rungta et al., 2016).  For example, Srinivasan et al. (2015) showed that while soma Ca2+ signals were greatly reduced in IP3R2 knockout mice, a substantial proportion of microdomain transients persisted in the fine processes.  Removal of extracellular Ca2+ did not affect soma Ca2+ transients, but reduced the frequency of fine process signals.  Furthermore, Gq-linked GCPR stimulation evoked Ca2+ elevations in the processes but not in the soma.  Taken together, these data indicate that astrocytes exhibit a diverse range of Ca2+ signals that vary in their subcellular compartmentalization, induction mechanism, and Ca2+ source (Khakh and Sofroniew, 2015).  5   Figure 1-1.  Genetically-encoded Ca2+ indicators (GECIs) reveal distal astrocyte processes that are not detected by bulk-loaded Ca2+ indicator dyes. Two-photon microscopy was used to acquire maximum intensity projection images of hippocampal brain slices from A) C57BL/6 mice that were bulk-loaded with Rhod-2 AM and B) GLAST-GCaMP5 mice that expressed the fluorescent cell marker tdTomato (red) and the GECI GCaMP5G (green) under the preferential astrocyte promoter GLAST.  Astrocyte proximal processes (solid arrowheads) were visible by both bulk-loading and GECI expression, while the fine distal processes (empty arrowheads) were only detected by the GECI.  Scale bars = 15 µm.  The physiological relevance of astrocytes has been re-examined in the context of these newly described Ca2+ signals.  For instance, synaptic activity-dependent microdomain transients were measured in hippocampal astrocyte processes by dialyzing the Ca2+ indicator Fluo-4 into individual cells, and with addition of the Ca2+ chelator BAPTA resulted in decreased release probability and increased synaptic failure rate at neighbouring synapses (Di Castro et al., 2011; Panatier et al., 2011).  Moreover, in mice with astrocytic expression of the GECI GCaMP3, physiological stimulation of olfactory sensory neurons was demonstrated to trigger Ca2+ signals in astrocyte processes that preceded the onset of functional hyperemia (Otsu et al., 2015).  These studies provide evidence for the role of astrocytes in modulating synaptic events and neurovascular coupling, and further demonstrate the importance of considering the contributions of different types of Ca2+ signals to astrocyte physiology. 6  1.1.2 Spontaneous Ca2+ microdomain signals in astrocytes The use of GECIs and patch-loaded Ca2+ indicator dyes revealed a diverse range of astrocyte Ca2+ signals in different subcellular compartments; up to seven distinct kinds of signals have been described (Khakh and Sofroniew, 2015).  Previous studies have shown that neuronal activity-associated astrocyte Ca2+ signals can be observed with GECIs or bulk- and patch-loaded Ca2+ dyes (Perea et al., 2009; Khakh and Sofroniew, 2015).  Intriguingly, the ability to monitor Ca2+ in the distal astrocyte processes with the new approaches has revealed a spontaneous Ca2+ transient that is localized to the fine processes (Shigetomi et al., 2010).  However, the mechanisms underlying these spontaneous Ca2+ signals are incompletely characterized.        Spontaneous Ca2+ transients were first described by Nett et al. (2002) in juvenile mouse brain slices wherein individual astrocytes were dialyzed with the Ca2+ dye Oregon Green BAPTA-1.  The authors imaged both proximal and distal processes, and found that astrocyte Ca2+ fluctuations persisted in the presence of the Na+ channel blocker tetrodotoxin and the vesicular H+-ATPase inhibitor bafilomycin A1, suggesting that the signals are independent of neuronal action potentials and spontaneous vesicular glutamate release.  The Ca2+ transients in astrocyte processes occurred at a higher frequency compared to soma signals.  Furthermore, the process signals were localized as microdomains, and the signals occurred asynchronously as if each microdomain was functionally independent.  Similar observations have since been described in astrocytes from culture, brain slices, and in vivo, using both dye dialysis and GECI-based approaches (Wang et al., 2006; Di Castro et al., 2011; Panatier et al., 2011; Shigetomi et al., 2012, 2013a; Rungta et al., 2016).  7  The mechanisms mediating the spontaneous Ca2+ transients are under investigation with three components thus far proposed as the Ca2+ source: release from the endoplasmic reticulum, release from mitochondria, and influx across the plasma membrane.  Pharmacological and genetic tools have indicated that multiple pathways are likely to be involved.  For example, the microdomain signals are inhibited by cyclopiazonic acid and thapsigargin, agents that deplete intracellular endoplasmic reticulum Ca2+ stores (Nett et al., 2002; Agarwal et al., 2017).  However, the observation that these Ca2+ signals are reduced, but not abolished, in IP3R2 KO mice, suggests contributions from other sources.  Agarwal et al. (2017) suggested that the spontaneous signals may be mediated by mitochondria, as signal frequency and amplitude were decreased by blockade of the mitochondrial permeability transition pore with cyclosporine A and rotenone.  Another possibility is Ca2+ entry through plasma membrane channels or transporters, since the spontaneous transients are blocked by removal of Ca2+ from the extracellular solution (Shigetomi et al., 2012; Rungta et al., 2016).   Shigetomi et al. (2012) has proposed the transient receptor potential channel TRPA1 as a candidate for mediating this transmembrane Ca2+ influx, based on the observations that treatment with TRPA1 antagonists or siRNA reduced the microdomain signals in cultured astrocytes, while TRPA1 agonists or overexpression in a heterologous system increased the transients.  However, subsequent experiments in hippocampal brain slices showed that spontaneous Ca2+ transients persisted in the presence of TRPA1 blockers, as well as in TRPA1 knockout mice (Shigetomi et al., 2013b; Rungta et al., 2016; Agarwal et al., 2017).  Inhibition of other plasma membrane proteins implicated in Ca2+ flux in astrocytes, such as voltage-gated Ca2+ channels, Na+-Ca2+ exchangers, ryanodine receptors, and Ca2+ release-activated channels have also failed to block the Ca2+ transients (Rungta et al., 2016; 8  Agarwal et al., 2017).  Therefore, the exact Ca2+ entry pathways contributing to the spontaneous Ca2+ microdomain signals remain to be determined.  Several studies have examined the physiological significance of these ubiquitous spontaneous Ca2+ signals.  Experiments conducted by Agarwal et al. (2017) demonstrated that microdomain signaling is enhanced by elevated reactive oxygen species (ROS) levels through Ca2+ efflux from mitochondria stores.  Moreover, since ROS are produced during the process of oxidative phosphorylation (Mailloux and Harper, 2012), the authors speculated that the spontaneous signals may reflect the metabolic state of the cell.  Interestingly, increased spontaneous Ca2+ transients have also been observed in astrocytes from mouse models of Alzheimer’s disease (Kuchibhotla et al., 2009; Delekate et al., 2014).  Together with the observation that Ca2+ can increase ATP production by acting on enzymes involved in glycolysis and oxidative phosphorylation (Ververken et al., 1982; McCormack et al., 1990), it is possible that Ca2+ signals facilitate coupling of ATP synthesis to disease states wherein energy metabolism is likely compromised.   Spontaneous Ca2+ signals have also been implicated in the control of basal Ca2+ levels in astrocytes, which may be important in regulation of synaptic activity and resting vessel tone.  For instance, as measured by ratiometric Fura-2 imaging, Shigetomi et al. (2012) demonstrated that pharmacological inhibition of TRPA1-mediated Ca2+ transients was associated with a reduction in basal Ca2+ concentrations from 120 nM to 50 nM.  This result is supported by the observations from Rungta et al. (2016) that removal of extracellular Ca2+ abolished Ca2+ transients in Fluo-4-dialyzed astrocytes, and resulted in a ~40% decrease in baseline fluorescence.  Reduction of 9  astrocyte intracellular free Ca2+ concentration by TRPA1 block or by dialysis of the Ca2+ chelator BAPTA was found to reduce the amplitude of interneuron mini inhibitory postsynaptic currents.  The authors demonstrated that decreased basal Ca2+ was associated with decreased expression of the GABA transporter GAT-3 in astrocytes, leading to increased extracellular levels of the neurotransmitter GABA and subsequent GABAA receptor desensitization (Shigetomi et al., 2012).  Pharmacological inhibition or genetic deletion of TRPA1 also suppressed LTP in hippocampal CA1 pyramidal neurons, which was rescued by exogenous application of the NMDA receptor (NMDAR) coagonist D-serine (Shigetomi et al., 2013b).  Similar results were obtained when astrocyte intracellular free Ca2+ was reduced to 50 – 80 nM with the chelator EGTA (Henneberger et al., 2010), suggesting that a certain level of basal Ca2+ must be maintained for constitutive D-serine release and NMDAR-dependent plasticity.  Finally, Rosenegger et al. (2015) showed that introducing BAPTA into astrocytes with a patch pipette resulted in vasoconstriction of adjacent arterioles.  This effect was occluded by cyclooxygenase inhibition, indicating that astrocytes may constitutively release prostaglandins in a Ca2+-dependent manner to maintain constant vasodilation.  Taken together, these examples demonstrate that spontaneous Ca2+ transients in astrocytes may play important roles in the modulation of metabolism, synaptic activity, and vascular tone in the brain.  In conclusion, continual improvements in the techniques employed to monitor Ca2+ levels have demonstrated that astrocytes exhibit a complex range of Ca2+ signals within distinct subcellular compartments, with different contributions from intracellular and transmembrane Ca2+ sources.  This nuanced view of astrocyte Ca2+ signaling is critical for understanding the role of astrocytes in brain function.      10  1.2 Astrocytes as mechanosensitive cells Astrocytes exhibit fluctuations in intracellular Ca2+ concentration thought to represent a form of cellular excitability (Parpura and Verkhratsky, 2012).  While neuronal activity-associated astrocyte Ca2+ signaling is well-established, other studies have indicated that astrocytes are also sensitive to mechanical stimulation.  Early studies demonstrated that membrane deformation or substrate stretch increased intracellular Ca2+ levels in cultured astrocytes, raising the possibility that they can transduce mechanical stress into chemical signals (Charles et al., 1991; Ostrow et al., 2000).  Mounting evidence suggests that astrocytes express mechanosensitive channels and can respond to a variety of mechanical signals including blood flow, matrix stiffness, and osmotic stress (Kirischuk, 2009).  1.2.1 Astrocytes detect mechanical stimuli to regulate cell physiology Cells are continuously exposed to mechanical forces that are integrated by the plasma membrane, cytoskeleton, extracellular matrix (ECM), cell adhesion proteins, and ion channels to influence cell functioning (Tyler, 2012).  In particular, astrocytes are structurally well-suited to detecting a diverse range of mechanical stimuli.  For example, astrocyte membranes form specialized processes known as endfeet that ensheath the blood vessels in the brain (Gordon et al., 2011).  Benfenati et al. (2007) described enriched expression of the mechanosensitive cation channel TRPV4 in these endfeet compartments, raising the possibility that astrocyte endfeet may be involved in detecting hemodynamic forces such as blood flow.  The hypothesis was supported by experiments in cannulated brain slice parenchymal arterioles demonstrating that increases in blood flow stimulated astrocyte Ca2+ signals and subsequent vasoconstriction; Ca2+ elevation and vessel response were both attenuated by patch-dialysis of the Ca2+ chelator BAPTA into 11  astrocytes, or by pharmacological inhibition or genetic deletion of TRPV4 (Kim et al., 2015).  Hence, astrocytic TRPV4 was suggested to play a significant role in sensing blood flow and regulating parenchymal arteriole tone.  Astrocyte functions have also been intimately linked to the extracellular environment.  Astrocytes secrete a number of ECM proteins, including hevin, tenascin-C, and fibronectin which provide a microenvironment promoting proper synaptogenesis, synaptic plasticity, and blood-brain barrier integrity (Faissner et al., 2010; Benarroch, 2015).  Interestingly, astrocyte morphology and function are also regulated by interactions with the ECM.  Astrocytes express integrins, a family of transmembrane cell-adhesion molecules that act as receptors for various ECM proteins (Milner et al., 1999; Tanigami et al., 2012).  The cytoplasmic domains of integrin proteins are associated with signaling complexes and cytoskeletal components such as actin stress fibers (Geiger et al., 2009; Campbell and Humphries, 2011).  Thus, astrocytes can sense physical properties of their environment through the forces generated from integrin-mediated adhesions to the ECM, which are then transduced intracellularly to modulate cellular response (Schwartz, 2010).  For instance, the molecular composition and mechanical stiffness of the cell substrate affect proliferation, reactive oxygen species production, glutamate uptake, and migration in cultured astrocytes (Johnson et al., 2015; Wilson et al., 2016).  Since the protein composition of the ECM and tissue stiffness vary with age and disease state (Lau et al., 2013; Arani et al., 2015; Murphy et al., 2016), the ability of astrocytes to discriminate between different extracellular environments may play an important role in regulating both physiological and pathological astrocyte functions.    12  Astrocytes have also been implicated in the maintenance of volume and ion homeostasis in the brain (Simard and Nedergaard, 2004).  Electron microscopy studies revealed that aquaporin-4 (AQP4), the predominant water channel in the brain (Nagelhus and Ottersen, 2013), is selectively localized to astrocytes, with the strongest staining observed in brain regions associated with detection and regulation of osmolarity (Oliet and Bourque, 1993; Nielsen et al., 1997).  AQP4 expression is enriched in astrocyte endfeet lining blood vessels and the pial surface, and these endfeet compartments have been proposed to form a paravascular compartment as part of a ‘glymphatic’ system that promotes interstitial fluid and osmolyte clearance from the brain (Thrane et al., 2014).  The selective expression of AQP4 in astrocytes may underlie the cell’s sensitivity to osmotic changes.  Wasterlain and Torack (1968) examined rat brain cell morphology two hours following intraperitoneal injections of water; this model of water intoxication induced astrocyte swelling while neurons and oligodendrocytes were unaffected.  In addition to AQP4 channels, astrocytes also express a range of channels and transporters involved in uptake of ions and neurotransmitters from the extracellular space (ECS), which is followed by obligatory water entry to maintain the osmotic gradient across the membrane (Simard and Nedergaard, 2004; Vargova and Sykova, 2014).  Therefore, astrocyte swelling is observed in response to both physiological and pathological perturbations to ECS homeostasis (Kimelberg et al., 1993; Florence et al., 2012).  Astrocyte swelling can activate mechanosensitive membrane channels, which will be discussed in Section 1.2.2.  1.2.2 Membrane proteins linked to mechanosensitivity in astrocytes Mechanosensitive channels in the CNS can be activated by plasma membrane perturbations such as osmotic stress (Tyler, 2012).  Initial studies by Kimelberg and O’Connor (1988)  13  demonstrated that perfusion with hypoosmotic solution induced cell swelling and reversible membrane depolarization in cultured astrocytes, and subsequent ion-replacement studies indicated that both cation- and anion-permeable channels may contribute to the hypotonic-induced membrane potential changes (Kimelberg et al., 1990; Pasantes-Morales et al., 1994).  Furthermore, the mechanosensitive channel blocker GsMTX4 inhibited swelling-activated currents in astrocytes (Suchyna et al., 2000).  Together, the data support that astrocytes express channels that can be activated by hypoosmotic stimuli-induced swelling.  While their molecular identities have not been fully characterized, putative astrocyte mechanosensitive channels are described below.  The transient receptor potential (TRP) family is a group of non-selective cation channels that responds to a diverse range of stimuli, including temperature, pressure, and inflammatory agents (Moran et al., 2004).  Six types of TRP channels have been identified in mammals: the classical TRPs (TRPCs), the vanilloid receptor TRPs (TRPVs), the melastatin TRPs (TRPMs), the mucolipins (TRPMLs), the polycystins (TRPPs), and the ankyrin transmembrane protein 1 (TRPA1) (Moran et al., 2004).  Amongst the TRPs, the best candidate for sensing osmotic stimuli in astrocytes is the TRPV4 channel (Liedtke and Kim, 2005; Plant, 2014).  The earliest evidence of TRPV4 as an osmosensor came from Ca2+ imaging and electrophysiological studies.  In HEK293 or CHO-K1 cells transfected with TRPV4, perfusion of hypoosmotic solution induced an increase in intracellular Ca2+ and activated cationic currents, which were both inhibited by non-selective mechanosensitive channel blockers such as ruthenium red and La3+ (Liedtke et al., 2000; Strotmann et al., 2000).  TRPV4 expression has been identified using immunofluorescence approaches in both cultured and brain slice rat cortical astrocytes 14  (Benfenati et al., 2007).  Electron microscopy studies suggest that TRPV4 is enriched at astrocyte endfeet bordering blood vessels and the pial surface (Benfenati et al., 2007).  Astrocyte TRPV4 channels are functional, as application of a TRPV4 agonist or hypotonicity-induced swelling caused intracellular Ca2+ elevations in cultured astrocytes, and these signals were inhibited by ruthenium red (Benfenati et al., 2007).  Interestingly, TRPV4 has been shown to be involved in a homeostatic mechanism termed regulatory volume decrease (RVD), where cell volume recovers following hypotonicity-induced swelling.  Overexpression of recombinant TRPV4 conferred RVD to a cell line that could not reduce its volume following hypoosmotic stress (Becker et al., 2005), while RVD was impaired in astrocytes with siRNA-mediated TRPV4 knockdown (Benfenati et al., 2011).  Therefore, TRPV4 may contribute to both detection of volume change and subsequent volume recovery in response to osmotic stimuli in astrocytes.  Mechanosensitive K+-selective channels have also been found expressed in astrocytes, although they remain to be fully characterized at the molecular level (Bowman et al., 1992; Kirischuk, 2009).  These channels belong to the two-pore domain K+ (K2P) channel family, a group of structurally similar K+ channels that are formed by dimerization (Feliciangeli et al., 2015).  Of the fifteen K2P channels identified in mammals, only the TREK-1, TREK-2, and TRAAK channels are mechanosensitive (Ryoo and Park, 2016).  Gnatenco et al. (2002) found that cultured astrocytes contained TREK-2 mRNA, and functional expression was confirmed with single-channel recordings.  Of note, TREK-2 activity was increased in astrocytes exposed to hypotonic solution (Gnatenco et al., 2002), consistent with previous studies suggesting that TREK channels are activated by membrane stretch (Patel et al., 1998).  Although TREK-1 15  expression has also been reported in astrocytes (Zhou et al., 2009), its functions related to mechanotransduction have not been described.  Previous studies have also recognized that osmotic swelling activates Cl- currents in astrocytes (Lascola and Kraig, 1996; Crépel et al., 1998).  The currents were proposed to be mediated by volume-regulated anion channels (VRACs) that, in addition to being permeable to Cl-, can also flux organic osmolytes such as glutamate, aspartate, and taurine (Kimelberg et al., 2006).  Recent work has identified the LRRC8 family of transmembrane proteins as the molecular constituents of VRACs (Qiu et al., 2014; Voss et al., 2014), an important advance that has allowed for development of genetic tools to specifically target these channels.  LRRC8 mRNA has since been identified in cultured astrocytes, and siRNA-mediated LRRC8 knockdown was found to inhibit hypoosmotic solution-induced amino acid efflux, suggesting that astrocytes express functional LRRC8 proteins (Hyzinski-García et al., 2014; Schober et al., 2017).  However, VRACs have been demonstrated to be activated by reduction of intracellular ionic strength in cells maintained at constant volume (Pedersen et al., 2015).  Hence, while VRACs may respond to hypotonic stimuli, the channels may not be intrinsically mechanosensitive.  In summary, astrocytes express a variety of channels activated by osmotic stress, and the examples described above may represent just a subset.  Future experiments examining the concerted activation of these channels in response to hypotonic stimuli are required to determine whether the channels are functionally redundant, or if they act in a complementary fashion to encode different aspects of the mechanical force.  16  1.3 Piezo1: a mechanosensitive cation channel Mechanosensitive cation channels mediate the transduction of mechanical forces into chemical or electrical signals that allow cells to sense and adapt to changes in their environment (Gillespie and Walker, 2001).  Studies in Caenorhabditis elegans and Drosophila melanogaster mutants initially implicated mechanosensitive channels in sensory modalities such as touch and hearing, and identified three classes of channel proteins as putative mechanosensory channels: the degenerin/epithelial Na+ channel (DEG/ENaC) family, the transient receptor potential (TRP) channels, and the two-pore-domain K+ (K2P) channels (reviewed in Chalfie, 2009).  In mammals, mechanical stimuli such as touch, pressure, and tendon stretch are converted to electrical signals by mechanosensory transducers on specialized somatosensory neurons that can lead to depolarization and generation of action potentials that propagate to the CNS (Ernstrom and Chalfie, 2002; Delmas et al., 2011).  However, given the inaccessibility of the small sensory nerve endings and the sparseness of the mechanosensing molecules, the molecular entities responsible for mammalian mechanotransduction have been difficult to study (Chalfie, 2009; Delmas et al., 2011).   Early experiments measuring the speed of sensory transduction in the hair cells of the bullfrog sacculus found that an electrical response was produced within 40 µs, suggesting that the mechanism was too rapid to involve chemical signaling; rather, the electrical response was proposed to arise from direct activation of an ion channel (Corey and Hudspeth, 1979).  Researchers therefore focused on channel proteins that may be directly gated by mechanical stimuli in their search for the molecules underlying mechanotransduction.  However, investigations of homologous proteins identified by the invertebrate mutant screens yielded 17  mixed results, as deletions of the genes in mouse models were rarely associated with a clear loss of phenotype (Alloui et al., 2006; Bautista et al., 2006).  For example, while DEG/ENaC channels mediate touch sensitivity in C. elegans (Chalfie and Sulston, 1981), loss of the homologous ASIC2 in knockout mice did not produce touch defects, suggesting that the invertebrate channels may not be functionally conserved in mammals (Drew et al., 2004).  Therefore, the molecular identities of mechanosensitive channels in mammalian cells remain incompletely characterized.  In 2010, Coste et al. identified a novel class of mammalian mechanosensitive channels by screening siRNA sequences against candidate channels for efficacy in inhibiting endogenous mechanically activated ionic currents in the Neuro2A mouse neuroblastoma cell line.  The authors found that knockdown of the Fam38A gene reduced pressure- and stretch-induced currents in the Neuro2A cells, and renamed the gene Piezo1, from the Greek word píesi meaning ‘pressure’.  A second related gene, Piezo2, was later cloned from dorsal root ganglia neurons.  While the Piezo proteins are conserved amongst animal, plant, and other eukaryotic species, they lack homology to other channels.  Piezo1 or Piezo2 overexpression in multiple mammalian cell lines demonstrated large mechanically activated cation currents that were sensitive to ruthenium red and gadolinium, two non-specific blockers of stretch-activated channels.  Taken together, the data suggest that the proteins are involved in generating currents in response to mechanical force.  The role of Piezo proteins in mechanotransduction has since been demonstrated in a number of organ systems including the bladder, the lung, and the vasculature (Miyamoto et al., 2014; Ranade et al., 2014; Nonomura et al., 2016).    18  1.3.1 Piezo1 channel topology and the structural basis of function Mammalian Piezo1 is a large transmembrane protein of over 2500 amino acids (Coste et al., 2010).  Piezo1 was first proposed to assemble as an oligomeric protein based on evidence from photobleaching experiments of mouse Piezo1 – GFP fusion proteins expressed in Xenopus oocytes, and size analysis of glutathione S-transferase-tagged Piezo1 proteins resolved by gel electrophoresis (Coste et al., 2012; Ge et al., 2015).  The model was later refined by cryo-electron microscopy that allowed the three-dimensional structure of Piezo1 to be determined at a resolution of 4.8 Å.  Mouse Piezo1 was found to be a homotrimer, with three propeller-like wings consisting of transmembrane helices and extracellular blade domains, supported by intracellular beam and anchor domains that surround an ion-conducting pore (Fig. 1-2) (Ge et al., 2015).    Figure 1-2.  Structure of mouse Piezo1. A) Extracellular, B) side, and C) intracellular views of the cyro-electron microscopy structures of Piezo1.  D) Schematic diagram of Piezo1 showing the major structural features of the channel, with the putative mechanotransducing and pore modules illustrated in blue and red, respectively.  CED, C-terminal extracellular loop domain; CTD, C-terminal domain.  Adapted from Geng et al., 2017.  Piezo1 topology was further characterized with biochemical mapping of Flag- or Myc-tagged amino acids to intracellular and extracellular domains, leading to a prediction of 14 – 18 19  transmembrane helices for each wing domain (Coste et al., 2015; Ge et al., 2015).  Subsequent studies suggested that the C-terminal extracellular loop domain (CED) and the intracellular C-terminal domain (CTD) form the central pore region of Piezo1, as replacing this region of the mouse protein with the analogous sequence from Drosophila resulted in conductance properties that resembled the characteristics of the fly Piezo (Zhao et al., 2016).  Site-directed mutagenesis studies also indicated that this pore region determines the conduction properties of Piezo1, including unitary conductance, ion selectivity, and sensitivity to pore blockers (Coste et al., 2015; Zhao et al., 2016).    In order to elucidate the structural components of Piezo1 that mediate mechanotransduction, Zhao et al. (2016) replaced the Piezo1 pore region with the mechano-insensitive ASIC1 channel, resulting in a chimeric protein that can respond to both mechanical force and pH.  This indicated that the non-pore-containing region of Piezo1 can confer mechanosensitivity to the ion-fluxing pore (Zhao et al., 2016).  The authors proposed that the extracellular blades and transmembrane wing domains may sense mechanical stimuli, which are then transmitted through the beam and interfacing anchor domains to cause conformational changes of the pore module (Zhao et al., 2016).  Hence, the structure of Piezo1 suggests that the channel contains mechanotransduction components and raises the possibility that Piezo1 may be inherently mechanosensitive (Wang and Xiao, 2017).  Together, these topological and structural studies of Piezo1 provide insight into the mechanotransduction mechanism of the channel, and form a foundation for designing pharmacological agents to modify Piezo1 activity.  20  1.3.2 Properties of the Piezo1 channel  Piezo1 channels, like all mechanosensitive channels, mediate the transduction of physical forces into conformational changes associated with channel opening.  Two models have been proposed to explain the mechanism of molecular force transduction: the “force through lipid” model wherein bilayer tension directly acts on the channel, and the “force through filaments” model where mechanical stimuli are transduced through physical tethers to the cytoskeleton or the extracellular matrix (ECM) (Ranade et al., 2015).  While some mechanosensitive channels have been shown to be exclusively gated by one mechanism, most membrane channels are also associated with scaffolding proteins that are linked to cytoskeletal and ECM proteins (Zhang et al., 2000; Cox et al., 2017), suggesting that both models may contribute to channel activation.    To address whether Piezo1 can be activated by membrane tension alone, Piezo1-transfected HEK293 cells were treated with hypoosmotic solution to create membrane blebs, protrusions of the bilayer that are uncoupled to the underlying cytoskeleton.  Mechanically stimulated Piezo1 currents could be recorded from patched blebs, and Piezo1 activity persisted in cells that had been pretreated with drugs to disrupt microtubule assembly and actin depolymerization (Cox et al., 2016).  In addition, spontaneous Piezo1 activity was observed in channels reconstituted in asymmetric bilayers (Syeda et al., 2015).  The data suggest that Piezo1 may be inherently mechanosensitive in the absence of other cellular components, as predicted by its cryo-electron microscopy structure (Zhao et al., 2016), and support that mechanical force can be transmitted directly from the membrane to the channel.  21  Mechanical stresses might affect cell shape and produce distortion of the cell surface and several studies have examined the involvement of the cytoskeleton and associated scaffolding proteins in the transduction mechanism responsible for the activation of Piezo1 in response to mechanical forces propagated from the ECM.  For example, mechanically-stimulated Piezo1 whole-cell currents were inhibited by actin disruption in a heterologous expression system (Gottlieb et al., 2012).  The scaffold protein stomatin-like protein 3 (STOML3), a molecule necessary for mechanosensitive ion channel function in mouse sensory neurons, was found to sensitize Piezo1 channels in Neuro2A cells, possibly via cholesterol binding and modulation of plasma membrane stiffness (Wetzel et al., 2007; Poole et al., 2014; Qi et al., 2015).  Gaub and Müller (2017) found that plating Piezo1-transfected HEK293 cells onto ECM proteins sensitized Ca2+ signals induced by membrane deformation compared to using a glass substrate.  Furthermore, Pathak et al. (2014) described spontaneous Piezo1-mediated Ca2+ signals in neural stem cells that were modified by substrate stiffness, and disruption of the traction force used by the cells to sense ECM rigidity was associated with loss of Piezo1 activity.  Taken together, the data suggest that force from both membrane tension and physical tethers can affect Piezo1 activity, and that the contribution from each may depend on the cell type and/or mechanical stimulus experienced by the cell (Nourse and Pathak, 2017).    Piezo1-mediated cationic currents decay rapidly after activation by a mechanical stimulus (Coste et al., 2010).  Previous studies have shown that Piezo1 inactivation regulates the ability of the channel to act as a frequency filter of repetitive mechanical stimuli (Lewis et al., 2017).  In contrast to activation, Piezo1 inactivation is independent of membrane tension and actin cytoskeleton modulation (Gottlieb et al., 2012; Wu et al., 2017).  Rather, channel inactivation 22  was observed to be voltage-dependent, with faster inactivation occurring at more hyperpolarized membrane potentials (Bae et al., 2013b).  Therefore, inactivation-dependent modulation of Piezo1 may be particularly important in excitable cells such as neurons and cardiomyocytes.  Mutations in the C-terminal extracellular loop domain (CED) and intracellular C-terminal domain (CTD) were associated with slowing of inactivation (Bae et al., 2013b).  The involvement of the CED in regulating Piezo1 inactivation kinetics was further observed in chimeric proteins where the CEDs of Piezo1 and Piezo2 were exchanged (Wu et al., 2017).  In addition, mutation of a single lysine residue within the inner pore helix abolished the voltage-dependency of Piezo1 inactivation (Wu et al., 2017).  The data suggest that the CED and inner pore module may act in concert to mediate Piezo1 inactivation.    Electrophysiological recordings from Piezo1-overexpressing HEK293 cells revealed mechanically activated inward currents that were blocked when cations in the extracellular solution were substituted by non-permeant N-methyl-D-glucamine, suggesting that Piezo1 is a non-selective cation channel (Coste et al., 2010).  Piezo1 was found to be permeable to both monovalent and divalent cations, with the highest permeability for K+ (Gnanasambandam et al., 2015).  Site-directed mutagenesis studies have indicated that the ion selectivity and single-channel conductance of Piezo1 is determined by specific glutamate residues in the C-terminal region forming the pore module (Coste et al., 2015; Zhao et al., 2016), where the negatively charged amino acids may act as a selectivity filter to allow for preferential flux of cations over anions through the Piezo1 channel.  23  1.3.3 Modulation of Piezo1 channel activity by second messengers While mechanosensitive channels are primarily gated by mechanical stimuli, their functions can be modulated by intracellular second messengers such as phospholipid-derived signaling molecules and protein kinases.  For instance, the mechanosensitive K+ channel TREK-1 has been shown to be inhibited by protein kinase A- and protein kinase C-mediated phosphorylation downstream of G-protein-coupled receptor (GPCR) activity (Noël et al., 2011).  Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), the most abundant inositol phospholipid in the plasma membrane (Fruman et al., 1998), has also been demonstrated to regulate TREK-1 activity (Chemin et al., 2005).  These data raise the possibility that Piezo1 activity may also be similarly sensitive to modulation by second messenger pathways.  Borbiro et al. (2015) published one of the first reports documenting the effects of PI(4,5)P2 on Piezo1 activity.  Using a heterologous expression system coexpressing the heat-sensitive TRPV1 channel with Piezo1, the researchers found that Piezo1-mediated mechanosensitive currents were attenuated by TRPV1 activation with capsaicin.  This inhibition was dependent on TRPV1-mediated Ca2+ influx, activation of the phospholipase C δ (PLCδ) isoform, and subsequent decrease in PI(4,5)P2 levels.  Depletion of PI(4,5)P2 with a chemically inducible phosphatase system decreased Piezo1 currents, while intracellular application of excess PI(4,5)P2 rescued the capsaicin-induced inhibition of Piezo1 currents.  The data suggest a functional link between TRPV1 and Piezo1, and that PI(4,5)P2 is required for Piezo1 activity.  A recent study suggested that Piezo1 activity can also be modified by GPCR signaling pathways (Lawrence et al., 2017).  Piezo1 was found to promote Ca2+ accumulation and cell death of 24  human chondrocytes, and that was blocked by either stimulation of endogenous Gαs-linked receptors or pharmacological elevation of cAMP production.  In addition, phospholipase A2 inhibition was observed to reduce Piezo1-mediated cell death, leading to the speculation that a yet-unidentified lipid pathway may contribute to Piezo1 activation.  However, it is currently unclear whether these putative lipid modifiers of Piezo1 activity interact directly or indirectly with the channels (Borbiro and Rohacs, 2017).  The factors regulating the relevance of these signaling pathways in different Piezo1-expressing cells also remain to be determined.  1.3.4 Piezo1 pharmacology To establish Piezo1 as a mechanosensitive channel, initial studies tested the sensitivity of Piezo1-mediated currents to the non-specific mechanosensitive ion channel blockers, gadolinium (Gd3+) and ruthenium red (Coste et al., 2010).  Gd3+ has been proposed to bind to negatively charged headgroups of membrane phospholipids, affecting membrane curvature and force distribution within the bilayer, with direct consequences for channel gating (Ermakov et al., 2010).  This membrane-based mechanism of channel inhibition supports previous observations that the lipid environment can modulate Piezo1 activity (see section 1.3.3).  In contrast, ruthenium red likely acts as a pore blocker, since mutations of specific glutamate residues within the Piezo1 pore module resulted in loss of ruthenium red blockade (Coste et al., 2012; Zhao et al., 2016).  This pharmacological approach has been used to demonstrate the role of Piezo1 in mediating functions such as ATP release from red blood cells and stretch-induced proliferation of epithelial cells (Cinar et al., 2015; Gudipaty et al., 2017).  However, both inhibitors block a variety of other targets, such as voltage-gated Ca2+ and Na+ channels (Biagi and Enyeart, 1990; Elinder and 25  Arhem, 1994; Malécot et al., 1998), and therefore cannot be used as the sole strategy to rule out contributions from other channels.    The first specific blocker for mechanosensitive channels was isolated from the venom of the tarantula Grammostola spatulata, which had previously been reported to block mechanically activated currents in Xenopus oocyte and chick cardiac cells (Niggel et al., 1996).  By separating venom components using reverse phase HPLC and testing each fraction against stretch-activated channels in astrocytes, Suchyna et al. (2000) identified a 34-amino acid peptide Grammostola spatulata mechanotoxin 4 (GsMTx4) that inhibited mechanically activated currents without affecting voltage-sensitive currents.  GsMTx4 was found to inhibit mechanically induced currents in Piezo1-transfected HEK293 cells, with a rightward shift in the activation curve, suggesting the toxin acts as a gating modifier (Bae et al., 2011).  Interestingly, both D and L enantiomers of GsMTx4 were effective, indicating that the toxin does not bind to a specific complementary binding site on Piezo1 (Bae et al., 2011).  Instead, GsMTx4 may mediate Piezo1 inhibition by modulating the lipids surrounding the channel to decrease the efficiency of force transduction between the membrane bilayer and the channel (Gnanasambandam et al., 2017).  While GsMTx4 is an important tool for studying the physiological and pathological roles of mechanosensitive channels, the toxin does not exclusively block Piezo1 (Bowman et al., 2007) and to date, no specific Piezo1 pharmacological antagonists have been identified.    Recently, a Piezo1 activator, Yoda1, was identified by screening synthetic compounds for their ability to induce Ca2+ entry in Piezo1-transfected HEK293 cells (Syeda et al., 2015). Electrophysiological recordings demonstrated that Yoda1 could modify Piezo1-mediated 26  mechanically activated currents by slowing their inactivation and shifting the activation curve to the left, indicating sensitization of Piezo1 to mechanical stimulation (Syeda et al., 2015).  To study the mechanism of Yoda1 action, its effects on Piezo1 activity were examined on channels reconstituted in an artificial cell membrane.  Yoda1 was found to induce single-channel currents with increased open time (Syeda et al., 2015).  Of note, Yoda1 did not affect Piezo2 activity.  Since Yoda1 can specifically activate Piezo1 in the absence of other cellular components, Syeda et al. (2015) proposed that Yoda1 may interact directly with Piezo1.  The development of Yoda1 suggests that Piezo1 is not exclusively gated by mechanical forces (Syeda et al., 2015).      1.3.5 Piezo1 expression and physiological functions Piezo1 is widely expressed in various mechanically sensitive cells.  In the mouse, Piezo1 mRNA was identified in tissues such as the lung, bladder, kidney, skin, and colon (Coste et al., 2010), with subsequent studies characterizing the channel’s functional role in different organs.  For instance, Piezo1 has been shown to mediate Ca2+ influx in bladder urothelial cells upon mechanical stretch, leading to downstream ATP release that may act as signal for bladder contractions (Miyamoto et al., 2014).  Piezo1 is also expressed by chondrocytes in cartilage, where it mediates compression-evoked Ca2+ transients with possible consequences for cytoskeleton remodeling and energy homeostasis (Lee et al., 2014).  In addition, the function of the channel in vivo has been demonstrated in the vascular system, where Piezo1 in endothelial cells can detect shear stress to mediate proper cell alignment; Piezo1 knockdown with siRNA was associated with abnormal endothelial cell alignment, while genetic deletion led to vascular abnormalities and embryonic lethality (Ranade et al., 2014).  27  Previous studies have also raised the possibility for Piezo1-mediated mechanosensing in the CNS.  For example, cultured rat hippocampal neurons associate with astrocytes in a manner dependent on the roughness of the cell substrate, an association lost with GsMTx4 treatment, suggesting that Piezo1 may mediate neuronal sensing of substrate topology (Blumenthal et al., 2014).  In neural stem cells, Piezo1-mediated Ca2+ influx was demonstrated to be affected by substrate stiffness, with downstream consequences for differentiation along either a neuronal or astrocytic lineage (Pathak et al., 2014).  Furthermore, Xenopus retinal ganglion cells were proposed to use Piezo1 as a detector for sensing brain tissue stiffness and promoting axon growth toward softer tissues, as Piezo1 knockdown or GsMTx4 treatment led to disrupted axonal pathfinding (Koser et al., 2016).  Piezo1 expression in astrocytes is supported by transcriptomic and proteomic studies.  Piezo1 mRNA was found in cortical astrocytes from both P7 and P80 mice (Zhang et al., 2014; Srinivasan et al., 2016), while the protein was detected in primary astrocyte cultures as well as in adult mouse brains (Sharma et al., 2015).  An early report by Satoh et al. (2006) suggested that Piezo1 may be upregulated in reactive astrocytes associated with amyloid-beta plaques in Alzheimer’s disease.  However, the physiological functions of Piezo1 in astrocytes has not been characterized.    1.3.6 Pathologies associated with Piezo1 dysfunction Consistent with the role of Piezo1 in mechanosensing, mutations in human Piezo1 have been linked to multiple hereditary disorders (Alper, 2017).  While most mutations remain to be fully characterized, Piezo1 loss-of-function mutations are associated with congenital lymphatic 28  dysplasia, wherein abnormal function of the lymphatic system results in symptoms such as generalized edema, seizures, and microencephaly (Fotiou et al., 2015; Lukacs et al., 2015).  HEK293 cells overexpressing the G2029R Piezo1 loss-of-function mutant displayed impaired Yoda1-induced Ca2+ responses, minimal mechanically activated currents, and reduced Piezo1 surface expression (Lukacs et al., 2015).  The mutation may also have hematological effects, as some patients exhibited red blood cell swelling and mild hemolysis (Fotiou et al., 2015).       In contrast, Piezo1 gain-of-function mutations have been linked to a type of hemolytic anemia known as autosomal dominant hereditary xerocytosis, or dehydrated stomatocytosis (Zarychanski et al., 2012; Andolfo et al., 2013; Alper, 2017).  In HEK293 cells overexpressing Piezo1 with two gain-of-function mutations, pressure- or osmotic stress-induced cationic currents exhibited delayed inactivation (Albuisson et al., 2013).  Mechanistically, this results in increased Ca2+ influx into red blood cells, leading to K+ channel activation and K+ efflux, followed by osmotically driven dehydration (Cahalan et al., 2015).  The clinical phenotypes of both gain-of-function and loss-of-function Piezo1 mutants suggest that a balanced level of mechanical sensitivity is likely required to maintain normal cell health.    Since Piezo1 was first identified as a mechanosensitive cation channel, the understanding of force transduction in mammalian cells has advanced rapidly.  These developments were aided by structure-function studies and pharmacological tools, and it is now well-established that Piezo1 is involved in many physiological responses in a wide variety of cell types.  The importance of Piezo1 function is further supported by examples of Piezo1 mutations that lead to pathologies associated with impaired mechanical sensing. 29  1.4 Rationale and hypotheses Over the last few decades of research, technological advances in the field of astrocyte Ca2+ signaling have allowed Ca2+ levels to be monitored in fine astrocyte arborizations, revealing a new and diverse repertoire of Ca2+ signals and refining our understanding of the physiological roles they mediate.  These new approaches have revealed an intriguing spontaneous Ca2+ signal localized to microdomains in the fine astrocytic processes.  Recent studies have provided evidence for the role of these signals in regulating astrocyte basal Ca2+ levels, with implications for synaptic activity and vessel tone.  However, the molecular mechanisms underlying the spontaneous Ca2+ transients have not been fully characterized.  While the signals are dependent on transmembrane Ca2+ influx, they are resistant to pharmacological block or genetic deletion of a range of Ca2+-permeable candidate channels.  Therefore, determining the molecular identity of the channel or transporter mediating these signals would be an important contribution to the overall understanding of the role of astrocytes in the physiology and pathology of the CNS.  We initially searched for novel Ca2+ fluxing proteins in recently published rodent brain transcriptomic and proteomic databases, and found that both Piezo1 mRNA and protein were expressed in astrocytes.  Piezo1 is a non-selective cationic channel that is a potential candidate for mediating Ca2+ entry across the plasma membrane.  Importantly, astrocytes are known to be mechanosensitive cells that respond to physical forces such as extracellular matrix stiffness and hypotonicity-induced swelling, mechanical stimuli previously shown to activate Piezo1 in other cell types.  We therefore decided to investigate the role of Piezo1 in astrocytes, from the perspective of a Ca2+ signal mediator as well as a force transducer.  We hypothesize: 1) that spontaneous Ca2+ transients in astrocytes are mediated by Piezo1 channel activity, 2) that 30  Piezo1 is expressed in astrocyte subcellular compartments relevant to mechanotransduction, and 3) that Piezo1 can be activated by hypoosmotic stress-induced astrocyte swelling.    1.4.1 Objective 1: To determine the contribution of Piezo1 to spontaneous Ca2+ transients in astrocytes Astrocytes exhibit spontaneous Ca2+ microdomain signals that are abolished in the absence of extracellular Ca2+, suggesting the involvement of a yet-undefined Ca2+ entry pathway.  I will monitor Ca2+ microdomain signals in cultured astrocytes using the membrane-tethered GECI, Lck-GCaMP5, and assess the effects of pharmacological Piezo1 agonists and antagonists and siRNA-mediated Piezo1 knockdown to examine the role of Piezo1 in mediating the spontaneous Ca2+ signals.  1.4.2 Objective 2: To characterize the subcellular localization of Piezo1 in astrocytes Piezo1 expression has been identified in various subcellular compartments in different cell types; some compartments, such as focal adhesions, are associated with mechanotransduction, in contrast with other structures such as the endoplasmic reticulum.  I will identify the subcellular distribution of Piezo1 in cultured astrocytes using antibody labeling and fluorescent-tagged protein approaches, and compare the staining patterns to those of the organelle-specific markers.   31  1.4.3 Objective 3: To examine the activation of Piezo1 in hypotonicity-induced astrocyte swelling Previous studies found that Piezo1 responds to a range of mechanical forces, including extracellular matrix rigidity, shear stress, membrane stretch, and osmotic stress.  Astrocytes are involved in maintaining CNS volume homeostasis and exhibit swelling in response to both physiological and pathological stimuli.  I will utilize hypoosmotic solution as a tool to induce cell swelling and perform Ca2+ imaging simultaneously to test the possibility that a mechanical force relevant to astrocytes is capable of activating Piezo1 channels.            32  Chapter 2: Piezo1 mediates astrocyte Ca2+ microdomain signals  2.1  Overview Pioneering studies in astrocyte Ca2+ signaling have established the functional importance of these signals in the brain (Shigetomi et al., 2016).  By combining techniques such as bulk-loaded Ca2+ indicator dyes and caged Ca2+ compounds, researchers could selectively monitor and manipulate Ca2+ levels in the astrocyte soma (Mulligan and MacVicar, 2004).  This has led to findings such as the ability of various neurotransmitters to evoke Ca2+ release from astrocyte intracellular stores (Dani et al., 1992; Duffy and MacVicar, 1995; Porter and McCarthy, 1996; Pasti et al., 1997), resulting in gliotransmitter and arachidonic acid metabolite release from astrocytes (Parpura et al., 1994; Bezzi and Volterra, 2001) that in turn acts on neurons and vascular smooth muscle to regulate synaptic plasticity and blood flow (reviewed in Volterra et al., 2014; Bazargani and Attwell, 2016).  However, subsequent studies demonstrated that IP3 type 2 receptor (IP3R2) knockout mice had normal synaptic activity and neurovascular coupling, despite impaired astrocyte soma Ca2+ responses (Petravicz et al., 2008; Nizar et al., 2013; Bonder and McCarthy, 2014).  Therefore, the physiological relevance of astrocyte Ca2+ signals remained controversial.  Recent developments in Ca2+ imaging techniques have revealed both spontaneous and neurotransmitter-evoked microdomain Ca2+ signals within astrocyte fine processes (Shigetomi et al., 2010; Rungta et al., 2016; Agarwal et al., 2017).  These Ca2+ transients, detected by patch-loaded dialysis of Ca2+ dyes or genetically encoded Ca2+ indicators (GECIs), show different spatial and temporal characteristics compared to the somatic signals.  Importantly, experiments 33  with IP3R2 knockout mice indicated that a substantial proportion of microdomain transients persisted in the fine processes while Ca2+ signals in the astrocyte soma were largely abolished (Srinivasan et al., 2015), suggesting that Ca2+ signals in different subcellular compartments may have different underlying mechanisms and functions.  These Ca2+ microdomain signals have since been linked to regulation of neuronal and vascular function via modulation of basal astrocyte Ca2+ levels and gliotransmitter release or uptake (Di Castro et al., 2011; Panatier et al., 2011; Shigetomi et al., 2012; Otsu et al., 2015; Rosenegger et al., 2015).  However, the mechanisms mediating Ca2+ transients remain incompletely described.  Previous studies have shown that the spontaneous microdomain Ca2+ signals depend on extracellular Ca2+ entry (Shigetomi et al., 2012; Rungta et al., 2016).  One possible mediator of these Ca2+ transients is the TRPA1 channel, as indicated by the potency of a TRPA1 antagonist in blocking the signals in cultured astrocytes (Shigetomi et al., 2012).  However, subsequent experiments found that a proportion of the signals persisted in brain slices from TRPA1 knockout mice (Shigetomi et al., 2013b; Rungta et al., 2016), indicating that other mediators for Ca2+ sparks exist.  Given that no treatment thus far has completely abolished the signals, the data suggest that an unidentified transmembrane channel or transporter may also be involved.   Thus, elucidating the molecular pathway mediating these spontaneous Ca2+ microdomain signals will contribute to the overall understanding of astrocyte physiology.   Piezo1 is a mechanosensitive cation channel that was first cloned from the Neuro2A neuroblastoma cell line using an siRNA approach (Coste et al., 2010).  Piezo1 channels have since been identified in the brain as mechanosensors for detecting extracellular matrix 34  topography and rigidity, with consequences for neural stem cell differentiation and retinal ganglion cell axon growth (Pathak et al., 2014; Koser et al., 2016).  Intriguingly, transcriptome and proteome analyses have also suggested that Piezo1 is expressed in astrocytes (Zhang et al., 2014; Sharma et al., 2015; Srinivasan et al., 2016), although little is known about the contribution of the channels to astrocyte physiology.    In this chapter, we present evidence for the role of Piezo1 in mediating astrocyte Ca2+ microdomain transients.  Two-photon imaging was performed in cultured astrocytes transfected with the Ca2+ indicator Lck-GCaMP5.  We developed an analysis algorithm that allowed microdomain signals to be quantified independently of other types of Ca2+ signals, and assessed the effect of both pharmacological and genetic manipulations of Piezo1.  Finally, we repeated pharmacological experiments in brain slices to extend the findings to astrocytes in situ.   2.2 Methods  2.2.1 Chemicals and reagents Stock solutions (1000x) of BAPTA-AM (Invitrogen), HC067047 (Tocris), thapsigargin (Tocris), and Yoda1 (Tocris) were dissolved in dimethyl sulfoxide, while EGTA (Sigma) and gadolinium (III) chloride (Tocris) were prepared in water.  GsMTx4 (Smartox) was dissolved in water as a 50x stock solution.  All chemicals were diluted in aCSF immediately prior to use.   35  2.2.2 Astrocyte cultures Rat astrocytes were prepared using a protocol adapted from Kaech and Banker (2006).  Brains were harvested from E19 Sprague Dawley rat pups, minced with micro-scissors, and incubated at 37°C for 15 minutes in balanced salt solution (Gibco) with 0.25% trypsin (Gibco) and 0.1% DNAse (Roche Applied Science).  The dissociated tissue was filtered through 75 µm nylon mesh, centrifuged, and resuspended in glia media (MEM (ThermoFisher Scientific) supplemented with 14% fetal bovine serum (FBS; ThermoFisher Scientific), 0.6% glucose (Sigma), penicillin (100 units/mL, ThermoFisher Scientific) and streptomycin (0.1 mg/mL, ThermoFisher Scientific)).  Cells were plated at 2-3 x 106 cells per 75 cm2 flask and grown for 7 – 9 days until confluent.  Cells were then detached with 0.25% trypsin, centrifuged, resuspended with glia media, and aliquoted into cryogenic vials for storage in liquid nitrogen.  For experiments, astrocytes were thawed, resuspended in glial medium, and plated onto 35 mm glass bottom petri dishes (poly-D-lysine-coated, 10 mm glass diameter; MatTek) at a density of 4 x 103 cells / dish.  Cells were grown for 3 to 5 days in a 37°C incubator with 5% CO2 in glia media.  Mouse astrocytes were prepared from heterozygous Piezo1-tdTomato mice expressing Piezo1 fused to the fluorescent marker tdTomato (Ranade et al., 2014; Jax stock no. 029214).  Cortices were harvested between P0 – P10 and triturated.  Cells were plated in DMEM/F12 (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) and grown for 2 – 3 weeks.  The mixed glial culture was then shaken for 4 -5 hours at 120 rpm to dislodge microglial cells, resulting in an enriched astrocyte culture.  Astrocytes were passaged 1 – 3 times and maintained in a 37°C incubator with 5% CO2 in glia media.  36  2.2.3 siRNA sequences for Piezo1 knockdown An siRNA approach was used for Piezo1 knockdown.  All siRNA oligonucleotides were ordered from Integrated DNA Technologies.  Uppercase letters represent unmodified bases, lowercase letters represent 2’-O-methyl-modified bases, and dTsdT represents two phosphorothioate-linked deoxythymidine bases.  These modifications increase siRNA resistance to nuclease degradation (Deleavey and Damha, 2012).   Luciferase siRNA was described by Addepalli et al. (2010):  Sense strand, 5’-cuuAcGcuGAGuAcuucGAdTsdT-3’  Antisense strand, 5’-UCGAAGuACUcAGCGuAAGdTsdT-3’  Piezo1 siRNA: Sense strand, 5’-GAgGUUUAgAuGaUUGUACUUCuUggu-3’   Antisense strand, 5’-caAgAaGUACAaUcAuCuAAACCuC-3’  2.2.4 Lck-GCaMP5 plasmid preparation Ca2+ signals were monitored with the membrane-targeted GECI Lck-GCaMP5.  The pN1 Lck-GCaMP5 plasmid was a gift from Baljit Khakh (Addgene plasmid #34924; Akerboom et al., 2012).  The plasmid was expanded in E. coli (DH5alpha strain) grown on agar plates (1.5% agar (InvivoGen) with kanamycin (50 µg/mL; Alfa Aesar)) overnight at 37 °C, and colonies were selected and grown at 37°C in LB broth (Sigma) for 8 hours to create a starter culture.  The starter culture was used to inoculate a flask of LB broth and grown overnight at 37 °C.  The broth was then centrifuged, and plasmid DNA was isolated from the pelleted bacteria by midi prep (Qiagen) according to the manufacturer’s instructions.    37  2.2.5 Preparation of siRNA- and plasmid DNA-containing lipid nanoparticles Lipid nanoparticles (LNPs), the ionizable cationic lipid system used to package and deliver siRNA and plasmid DNA to astrocytes, were synthesized as previously described (Rungta et al., 2013; Kulkarni et al., 2017).  For siRNA encapsulation, LNPs were composed of heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA; Biofine International), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids), cholesterol (Sigma), (R)-2,3-bis(octadecyloxy)propyl-1-(methoxy polyethylene glycol 2000) carbamate (PEG-DMG; synthesized as previously described in Akinc et al., 2008), and the lipophilic carbocyanine dye 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiIC18; Invitrogen) at a molar ratio of 50/10/37.5/1.5/1.  Plasmid DNA was encapsulated in LNPs consisting of 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA; Biofine International), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC; Avanti Polar Lipids), cholesterol, and PEG-DMG at a molar ratio of 50/10/39/1.    Lipids were solubilized in ethanol and mixed with siRNA or plasmid DNA dissolved in sodium acetate buffer (25 mM, pH 4.0) at a volumetric ratio of 1:3 using a microfluidic micromixer (Precision NanoSystems) or T-junction mixer.  Ethanol was removed from the resulting nanoparticles and replaced with phosphate-buffered saline (PBS, pH 7.4; Sigma) by dialysis.  LNPs were concentrated using Amicon Ultra centrifugal filter units (Millipore).  Particle sizes and polydispersity index (PDI) were quantified by dynamic light scattering (Malvern Zetasizer NanoZS).  Total lipid concentration of LNPs was determined by measuring cholesterol content using the Cholesterol E assay (Wako Chemicals).  Nucleic acid concentration was measured by absorbance at 260 nm, and encapsulation efficiency was measured using the Quanti-IT 38  Ribogreen RNA and Picogreen dsDNA assays (Life Technologies) for siRNA and plasmid DNA, respectively.  LNP properties are summarized in Table 2-1. Table 2-1.  LNP properties. Nucleic acid Size (nm) PDI % encapsulated Lck-GCaMP5 plasmid 72.67 ± 3.34 0.073 ± 0.020 94.92 ± 2.84 Piezo1 siRNA 43.53 ± 3.85 0.072 ± 0.053 95.16 ± 1.84 Luciferase siRNA 40.78 ± 3.28 0.088 ± 0.031 95.25 ± 3.45  2.2.6 Astrocyte transfection with LNPs Astrocyte cultures were treated with LNP-siRNA complexes (0.125 µg/mL) for five days prior to experiments.  Lck-GCaMP5 plasmids were either packaged in LNPs or transfected using Effectene (Qiagen) in accordance with the manufacturer’s instructions.  Astrocytes were exposed to the plasmid (500 ng/mL) for two hours, and experiments were conducted after 48 hours.  The treatments were combined by first treating astrocytes with LNP-siRNA for three days, followed by a two-hour incubation with LNP-Lck-GCaMP5, and experiments were performed after 48 hours.  2.2.7 Evaluation of transfection viability and efficiency Fluorescence microscopy images were acquired using the EVOS FL Cell Imaging System (Invitrogen) to evaluate the optimal conditions for Lck-GCaMP5 plasmid transfection efficiency and cell viability.  Lck-GCaMP5 plasmid transfection efficiency was determined by the number of cells expressing green fluorescence.  Cells were then fixed with 4% paraformaldehyde (Ted Pella) at room temperature for ten minutes and stained with DAPI (Sigma) for visualization of 39  cell nuclei.  Green fluorescence and DAPI images were taken with a 20x/0.40 NA and 4x/0.13 NA objective lens, respectively.  Five fields from each coverslip were sampled.    2.2.8 Evaluation of Piezo1 siRNA effect on mRNA levels To measure the effect of Piezo1 siRNA on mRNA levels, quantitative polymerase chain reaction (qPCR) was performed on primary astrocyte cultures treated with 0.125 µg/mL LNP-siRNA complexes (luciferase or Piezo1) for five days.  Cells were treated with TRIzol (Invitrogen), and total RNA was purified with the MagMAX-96 Microarray Total RNA Isolation Kit (Life Technologies) followed by cDNA synthesis using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).  Gene-specific qPCR was performed with the KAPA Probe Fast Universal qPCR Kit (KAPA Biosystems) using a CFX384 Real-Time PCR System (Bio-Rad).  TaqMan probes were used to amplify PIEZO1 (rPiezo1-1 Rn.PT.58.12896870; rPiezo1-2 Rn.PT.58.46120084), with GAPDH (rGAPDH Rn.PT.58.35727291) as the internal control.    2.2.9 Evaluation of Piezo1 siRNA effect on protein levels Immunoblot analysis was conducted to assess the effect of Piezo1 siRNA knockdown on protein levels.  Primary astrocyte cultures were treated with 0.125 µg/mL LNP-siRNA complexes (luciferase or Piezo1) for five days. Cells were collected and homogenized on ice in RIPA buffer (Tris Buffered Saline with 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with cOmplete ULTRA protease inhibitor cocktail (Sigma Aldrich), phosSTOP phosphatase inhibitors (Roche), and 1 mM EDTA. Cell homogenates were passed three times through a 27-gauge syringe, incubated on ice for 30 minutes, then centrifuged at 4°C for 20 min at 12,000 rpm 40  to remove debris. Protein concentration was determined using a DC Protein Assay (Bio-Rad), and equal protein amounts were incubated at room temperature for two hours in Laemmli Sample buffer under reducing conditions (5% β-mercaptoethanol) before loading onto gels. Gels were transferred to 0.2 µm pore-size polyvinyldene fluoride (PVDF) membrane using Trans-Blot Turbo semi-dry transfer system (Bio-Rad). Blocking and antibody incubations took place in blocking buffer (5% skim milk in TBS with 0.1% Tween 20). Primary antibodies used were: 1/3000 Piezo1 (Alomone); 1/500 tdTomato (Biorbyt); 1/4000 Actin (Santa Cruz Biotechnology); 1/1000 GAPDH (Santa Cruz Biotechnology). Secondary antibodies used were: 1/2000 horseradish peroxidase (HRP)-conjugated donkey anti-rabbit; 1/2000 HRP-conjugated donkey anti-goat (both from Jackson Immunoresearch). Blots were imaged using a C-DiGit western blot scanner (LI-COR).    2.2.10 Culture imaging Experiments were performed in static baths (35 mm petri dishes) at room temperature with HEPES-aCSF containing (in mM): NaCl, 140.4; HEPES, 10.8; KCl, 2.5; glucose, 10; MgCl2, 2.0; CaCl2, 2.0.  HEPES buffer was used in place of NaHCO3 and NaH2PO4 was omitted to prevent precipitation with gadolinium.  Cells were imaged using a two-photon laser scanning microscope (LSM 7 MP; Zeiss) with a 20x-W/1.0 numerical aperture objective lens coupled to a Chameleon Ultra2 laser (Coherent).  Images were acquired as time series of 256 x 256 pixels using 8-line averaging with a frequency of 0.41 Hz. Lck-GCaMP5 was excited at 920 nm, and the emission was detected by a photomultiplier tube after passing through a 500-550 nm filter.  41  2.2.11 Transgenic GLAST-GCaMP5 mice and tamoxifen treatment Slc1a3(GLAST)-cre/ERT (Jax stock no. 012586) and PC-G5-tdT (Jax stock no. 024477) mice were bred according to protocols approved by the University of British Columbia Animal Care Committee.  The resulting GLAST-GCaMP5 mice expressed the Ca2+ indicator GCaMP5 and the red fluorescent protein tdTomato in astrocytes upon tamoxifen administration.  Mice were housed under standard conditions (12-hour light/dark cycle), and food and water were provided ad libitum.  Tamoxifen (Sigma) was dissolved in peanut oil (Sigma; 20 mg/mL), and mice (postnatal day 30) were given three doses (200 mg/kg intraperitoneally) at 48-hour intervals.  Experiments were performed at least three weeks after the last tamoxifen injection.  2.2.12 Hippocampal slice preparation Mice (postnatal day 50 to 60) were decapitated without anesthesia according to protocols approved by the University of British Columbia Animal Care Committee.  Brains were rapidly extracted and submerged in ice-cold slicing solution saturated with carbogen (95% O2 / 5% CO2).  The slicing solution contained (in mM): N-methyl-D- glucamine, 120; KCl, 2.5; NaH2PO4, 1.2; NaHCO3, 25; CaCl2, 1.0; MgCl2, 7.0; sodium pyruvate, 2.4; sodium ascorbate, 1.3; glucose, 20.  Transverse hippocampal slices, 300 µm thick, were cut using a Leica VT 1200S vibratome and recovered in artificial cerebrospinal fluid (aCSF) saturated with carbogen at room temperature for 1 hour.  aCSF contained (in mM): NaCl, 126; NaHCO3, 26; KCl, 2.5; glucose, 10; NaH2PO4, 1.25; MgCl2, 2.0; CaCl2, 2.0.    42  2.2.13 Slice imaging Experiments were performed at room temperature with continuous perfusion (2.5 mL/min) of HEPES-aCSF saturated with oxygen.  Hippocampal slices were imaged using a two-photon laser scanning microscope as described in section 2.2.10.  Cells were imaged in the CA1 stratum radiatum at depths between 70 – 90 µm.  Frame images for time-lapse analysis were collected at 256 x 160 pixels using 8-line averaging with a frequency of 0.26 Hz.  GCaMP5 and tdTomato were excited at 920 nm, and signals were detected with photomultiplier tubes after passing through 500-550 nm and 600-660 nm emission filters, respectively.      2.2.14 Image processing and data analysis Time-lapse images of astrocyte culture Ca2+ signals were processed using ImageJ (NIH).  Stacks were aligned to remove drift in the x and y axes, and cell borders were delimited by hand.  Images were median filtered to remove noise, and thresholding was applied to identify the signals from the background.  Signals were then analyzed with the 3D components function in MATLAB (MathWorks), which recognizes signals continuous in the x, y, and t axes.  Large spreading signals, such as Ca2+ waves, that exceeded the empirically-determined size threshold of 1000 pixels were assigned a value of zero in a binary mask, and the mask was applied to the aligned images to generate a stack containing only microdomain Ca2+ transients.  Microdomain regions of interest (ROIs; 6 x 6 pixels) were then detected based on maximum fluorescence intensity, and the intensity profile of each ROI was baseline-corrected using the rolling ball filter.  Microdomain events were defined as signals larger than 10x the standard deviation of the ROI baseline intensity, and parameters of frequency, amplitude, and duration were calculated and binned over five-minute intervals.  The analysis algorithm is summarized in Fig. 2-1. 43   Figure 2-1.  Ca2+ signal analysis algorithm flowchart.    2.2.15 Statistics Statistical analyses were conducted with GraphPad Prism (version 5.0).  Data were analyzed using the Shapiro-Wilk test to assess if they were from a normal distribution.  Unpaired t-tests were used to compare the significance of difference in means between two groups.  For multiple group comparisons, one-way ANOVA analysis was used, followed by Bonferroni’s post-test.  44  When comparing different treatments conducted on the same cell, a paired t-test or repeated-measures ANOVA analysis followed by Bonferroni’s post hoc test was performed.  The n values represent the number of cells imaged.  Data are reported as mean ± 95% confidence interval, and p < 0.05 was considered statistically significant.  2.3 Results  2.3.1 LNP delivery of plasmid DNA and siRNA in cultured astrocytes To study the role of Piezo1 in mediating astrocyte Ca2+ signals, a safe and efficient strategy was necessary for transfecting the Lck-GCaMP5 Ca2+ indicator into astrocytes.  The LNP technology, a system that packages molecules within inverted micelles of ionizable cationic lipids for delivery into cells (Leung et al., 2012), was chosen as our lab had previously demonstrated its effectiveness in delivering nucleic acids for both in vitro and in vivo applications in the brain (Rungta et al., 2013, 2015).  To determine whether LNPs are a feasible approach for transfecting astrocytes with Lck-GCaMP5 plasmid DNA, we compared its cytotoxicity and transfection efficiency with the commercially available transfection reagent Effectene, which had previously been used to transfect cultured astrocytes with the Lck-GCaMP3 plasmid (Shigetomi et al., 2012).  Cytotoxicity, assessed by the number of adherent cells following transfection, was quantified by counting the number of DAPI-positive cell nuclei per visual field, while transfection efficiency was determined by quantifying the number of astrocytes with green fluorescence from Lck-GCaMP5 expression (Fig. 2-2A).  We found that LNP-packaged Lck-GCaMP5 plasmid DNA transfection had no effect on cell viability (approximately 1000 cells counted per coverslip; untreated: 100 ± 76%; LNP: 106 ± 31%; Effectene: 130 ± 70%; p > 0.05) 45  (Fig. 2-2B), with similar transfection efficiency achieved by both LNP and Effectene (approximately 125 cells counted per coverslip; untreated: 0%; LNP: 10.6 ± 9.1%; Effectene: 9.2 ± 10.3 %; p < 0.05 compared to untreated) (Fig. 2-2C).    Previous studies in our lab have shown that LNP-siRNA complexes can safely and effectively knock down target genes in neurons (Rungta et al., 2013).  Here we validate the use of LNP-packaged Piezo1 siRNA in astrocytes by performing qPCR and immunoblotting to measure mRNA and protein levels respectively.  Using two probes targeted to different locations of the Piezo1 sequence, we found that five-day treatment of astrocytes in cell culture with LNP-packaged Piezo1 siRNA significantly reduced Piezo1 mRNA levels relative to the luciferase siRNA-treated control (Probe 1, luciferase: 100.0 ± 4.2%; Probe 1, Piezo1: 18.2 ± 1.2%; Probe 2, luciferase: 100 ± 5.6%; Probe 2, Piezo1: 15.6 ± 0.5%; p < 0.001 compared to luciferase treatment) (Fig. 2-2D).  Similarly, LNP-Piezo1 siRNA treatment knocked down Piezo1 protein expression in cultured wild-type rat astrocytes relative to the untreated and luciferase siRNA controls, and this knockdown was observed in the Piezo1-tdTomato mouse astrocytes as detected by both the Piezo1 and tdTomato antibodies (Fig. 2-2E).  The single and double band patterns have been demonstrated in immunoblots of other rodent tissue samples, although it remains to be determined whether the molecular weight differences are due to post-translational modifications or splice variants (Andolfo et al., 2013; Ranade et al., 2014).  Taken together, the data suggest that LNPs are a safe and effective method for transfecting plasmid DNA and siRNA into astrocyte cultures.   46   Figure 2-2.  Lipid nanoparticle (LNP) delivery of Lck-GCaMP5 plasmid DNA and Piezo1 siRNA is non-toxic and effective in cultured astrocytes. A) Representative images of DAPI-stained nuclei (top row) and GCaMP5 fluorescence (bottom row) from astrocytes two days post-transfection with 500 ng Lck-GCaMP5 plasmid DNA using LNP or Effectene compared to untreated cells.  Scale bar = 200 µm.  Quantification of B) the percentage of adherent astrocytes relative to untreated cells and C) the percentage of cells expressing green fluorescence when transfected with LNPs or Effectene.  n = 3 independent experiments.  D) qPCR analysis of Piezo1 mRNA levels in astrocytes following five-day treatment with LNP-packaged Piezo1 siRNA (0.125 µg / mL) relative to luciferase controls, as measured using two probes.  n = 3 coverslips.  E) Immunoblot analysis of Piezo1 protein expression in LNP-Piezo1 (Pz1) siRNA-treated wild-type (WT) rat or Piezo1-tdTomato (PztdT) mouse cultured astrocytes relative to untreated and luciferase (Luc) siRNA-treated controls.  Data represent mean ± 95% CI; * p < 0.05 compared to untreated group by one-way ANOVA with Bonferroni’s multiple comparison post hoc test and *** p < 0.001 compared to luciferase siRNA-treated group by one-tailed unpaired t-test.  47  2.3.2 Development of a novel method to analyze astrocyte Ca2+ signals Astrocytes exhibit various types of Ca2+ signals with distinct spatial and temporal characteristics that reflect different mechanisms of Ca2+ release or entry (Srinivasan et al., 2015).   In cultured astrocytes transfected with the Lck-GCaMP5 construct, we observed at least two distinct patterns of spontaneous Ca2+ signals- Ca2+ waves with long duration that appeared to expand and contract from their point of origin, and Ca2+ transients that were spatially restricted in microdomains (Fig. 2-3A).  Since Ca2+ waves and transients overlap in the xy-plane, we developed an algorithm to distinguish and independently analyze these signals.    A key feature of the algorithm involves using the MATLAB 3D connected components function to identify Ca2+ signals that are continuous in the x, y, and t axes, allowing the signals to be classified based on total area (Fig. 2-3B).  The masking threshold was set at 1000 pixels (1186 µm2) empirically to separate Ca2+ microdomain events from the Ca2+ waves (Fig. 2-3C).  The algorithm then identifies microdomain regions of interest (ROIs) based on fluorescence intensity (Fig. 2-3D), and generates intensity-time course traces (Fig. 2-3E) and raster plots (Fig. 2-3F) of the Ca2+ signals detected for each ROI.  Parameters such as event frequency, intensity, and duration can then be quantified to assess the effect of different interventions on the microdomain transients selectively.   48   Figure 2-3.  The Ca2+ signal analysis algorithm isolates Ca2+ microdomain events by masking Ca2+ waves.   A) The original image shows the maximum intensity projection of the spontaneous Ca2+ signals recorded from an Lck-GCaMP5-transfected astrocyte over 20 minutes.  Ca2+ microdomains (solid arrowheads) and waves (empty arrowheads) are highlighted.  Scale bar = 100 µm.  B) MATLAB’s 3D connected components analysis identifies pixels that are connected in the x, y, and t axes as belonging to the same Ca2+ signal.  The colour coding represents signals that were distinguished by the algorithm as discrete events separated in time and space.  C) Ca2+ waves were filtered out in the masked image, revealing microdomain signals (solid arrowheads) that were initially occluded.  The blacked-out region in the middle of the cell represents static fluorescent puncta that were manually traced and removed from analysis.  D) Microdomain regions of interest (ROIs) can then be automatically detected based on fluorescence intensity, as marked by the red squares in ROI selection.  Ca2+ signal events for the 10 ROIs are depicted as E) intensity (dF/F; calculated as change in fluorescence divided by baseline fluorescence) versus time plots and F) raster plots where yellow and blue bars (example highlighted by arrows in ROI 10) respectively represent the beginning and end of each event.  49  2.3.3 Piezo1 activation selectively increases the frequency of microdomain Ca2+ signals To determine if Piezo1 is involved in mediating Ca2+ signals in astrocytes, we analyzed the effect of the Piezo1 agonist, Yoda1, on Ca2+ waves and microdomain transients in LNP-Lck-GCaMP5-transfected cells.  Yoda1 was applied at 1 µM, corresponding to the concentration found to induce single-channel currents in Piezo1 channels reconstituted in artificial bilayers (Syeda et al., 2015).  The maximum intensity projection images from the 3D connected components analysis show that discrete Ca2+ wave and microdomain signals occurred both spontaneously at baseline and following Yoda1 treatment (Fig. 2-4A).  Yoda1-mediated Piezo1 activation did not affect astrocyte Ca2+ waves as monitored by frequency (baseline: 1.4 ± 1.2 events/10 min; Yoda1: 1.7 ± 1.1 events/10 min; p > 0.05) (Fig. 2-4B), maximum area of spread in the xy-plane (baseline: 2100 ± 2200 µm2; Yoda1: 1000 ± 520 µm2; p > 0.05)(Fig. 2-4C), and duration (baseline: 98 ± 65 s; Yoda1: 110 ± 76 s; p > 0.05)(Fig. 2-4D).  In contrast, Yoda1 increased the frequency of microdomain signals (baseline: 0.98 ± 0.77 events/ROI/5 min; Yoda1: 3.3 ± 1.2 events/ROI/5 min; p < 0.01)(Fig. 2-4E) without altering their amplitude (baseline: 1.5 ± 0.6 a.u.; Yoda1: 1.5 ± 0.3 a.u.; p > 0.05)(Fig. 2-4F) or duration (baseline: 6.7 ± 4.0 s; Yoda1: 7.1 ± 2.1 s; p > 0.05)(Fig. 2-4G).    Our results suggest that Piezo1 activation in astrocytes causes localized increases in Ca2+ concentration proximal to the plasma membrane, as detected by the membrane-targeted Lck-GCaMP5 indicator.  Furthermore, the selective effect of Yoda1 supports the interpretation that Ca2+ waves and microdomain transients are physiologically distinct, and validates the importance of analyzing these signals separately.  We focused on the Ca2+ signal frequency in subsequent experiments, as this was the only parameter that was affected by Yoda1 application. 50   Figure 2-4.  The Piezo1 agonist, Yoda1, selectively increases the number of Ca2+ microdomain signals. A) The 3D connected components analysis shows the Ca2+ waves (top row) and microdomain events (bottom row) that occurred during baseline and following Yoda1 (1 µM) application.  Images represent 10-minute projections.  Scale bar = 50 µm.  Quantification of the effect of Yoda 1 on B) the frequency, C) the maximum area of spread in the xy-plane, and D) the duration of Ca2+ waves.  Quantification of the effect of Yoda on E) the frequency, F) maximum amplitude (measured in arbitrary units (a.u.) normalized to baseline), and G) duration of Ca2+ microdomain events.  n = 5-6 cells from individual coverslips.  Data represent mean ± 95% CI; ** p < 0.01 compared to baseline by one-tailed paired t-test.  2.3.4 Yoda1-induced Ca2+ microdomain signals are sensitive to pharmacological and genetic Piezo1 inhibition To further test that the Yoda1-induced microdomain transients were mediated through Piezo1 activation, we tested the sensitivity of the Ca2+ signals to different Piezo1 antagonists.  Lck-GCaMP5-transfected astrocytes were treated with Yoda1 followed by the blocker (Fig. 2-5A), and the number of events was normalized to time-matched controls where only Yoda1 was 51  applied.  We first tested GsMTx4, a toxin purified from the spider Grammostola spatulata that blocked mechanosensitive currents in Piezo1-transfected HEK cells (Bae et al., 2011).  As shown in Fig. 2-5B, Yoda1-evoked microdomain Ca2+ transients were partially inhibited by GsMTx4 (20 µM) (Yoda1: 100 ± 29%; Yoda1 + GsMTx4: 54 ± 19%; p < 0.05).  Application of Gd3+ (100 µM), a non-specific blocker of stretch-activated channels (Hamill and McBride, 1996), also partially inhibited the signals (Yoda1 + Gd3+: 37 ± 45%; p < 0.05).  As a negative control, we tested HC067047 (10 µM), a selective inhibitor of the Ca2+-permeable mechanosensitive TRPV4 channel expressed in astrocytes (Benfenati et al., 2007; Dunn et al., 2013) and found it had no effect on the frequency of Ca2+ signals (Yoda1 + HC067047: 77 ± 20%; p > 0.05).         52   Figure 2-5.  Yoda1-induced increases in Ca2+ microdomain signals are partially inhibited by Piezo1 antagonists GsMTx4 and Gd3+. A) Images represent masked 10-minute maximum intensity projections of astrocytes transfected with Lck-GCaMP5 during baseline, following Yoda1 (1 µM) application, and after subsequent addition of either GsMTx4 (20 µM), Gd3+ (100 µM) or HC067047 (10 µM).  Scale bars = 10 µm.  B) Quantification of the effects of the antagonists on Yoda1-induced Ca2+ microdomain transients.  n = 6 – 22 cells from individual coverslips.  Data represent mean ± 95% CI; * p < 0.05 compared to Yoda1 by one-way ANOVA with Bonferroni’s multiple comparison post hoc test.  Since pharmacological blockers can have non-specific effects, we further studied the involvement of Piezo1 in Yoda1-induced microdomain signals using an siRNA approach to knock down Piezo1 expression.  Astrocyte cultures were treated with LNP-packaged siRNA (0.125 µg/mL) for five days based on the mRNA and protein knockdown data (Fig. 2-2D and E).  Yoda1-induced Ca2+ microdomain transients were significantly reduced in Piezo1 siRNA-treated astrocytes relative to the control luciferase siRNA condition (luciferase siRNA baseline: 0.3 ± 0.3 events/ROI/5 min; luciferase siRNA Yoda1: 5.0 ± 1.5 events/ROI/5 min; p < 0.001; 53  Piezo1 siRNA baseline: 0.2 ± 0.1 events/ROI/5 min; Piezo1 siRNA Yoda1: 1.8 ± 1.0 events/ROI/5 min; p < 0.05)(Fig. 2-6A and B).  Our data support that Yoda1 evokes microdomain Ca2+ signals in astrocytes through Piezo1 activation.   Figure 2-6.  Yoda1-induced increases in Ca2+ microdomain signals are inhibited by Piezo1 siRNA. A) Images represent masked 10-minute maximum intensity projections of Lck-GCaMP5-transfected astrocytes after five days of luciferase or Piezo1 siRNA treatment, during baseline and following Yoda1 (1 µM) application.  Scale bars = 25 µm.  B) Quantification of the effect of Piezo1 siRNA treatment on Yoda1-induced Ca2+ microdomain transients relative to the control luciferase siRNA condition.  n = 11 – 17 cells from individual coverslips.  Data represent mean ± 95% CI; * p < 0.05, ** p < 0.01 and *** p < 0.001 by one-way ANOVA with Bonferroni’s multiple comparison post hoc test.  C) Quantification of the number of ROIs mediating spontaneous Ca2+ microdomain transients during baseline in astrocytes treated with luciferase or Piezo1 siRNA.  n = 11 – 17 cells from individual coverslips.  Data represent mean ± 95% CI; ** p < 0.01 by one-tailed unpaired t-test.  We also attempted to quantify the effect of Piezo1 knockdown on basal Ca2+ microdomain signals, which was difficult due to the low number of events at baseline.  A trend towards reduction of spontaneous Ca2+ transients was observed with Piezo1 siRNA treatment, although it did not reach significance (Luciferase siRNA (n = 11): 4.0 ± 2.8 events/5 minutes; Piezo1 siRNA 54  (n = 17): 2.9 ± 1.5 events/5 minutes; p > 0.05).  However, the number of ROIs that mediated the spontaneous microdomain Ca2+ signals was significantly decreased by Piezo1 siRNA treatment (Luciferase siRNA: 4.4 ± 1.8 ROIs; Piezo1 siRNA: 2.2 ± 1.0 ROIs; p < 0.01)(Fig. 2-6C).  The data suggest that Piezo1 siRNA knockdown reduces the number of basally active Ca2+ microdomains in astrocytes.  2.3.5 Yoda1-induced Ca2+ microdomain signals depend on extracellular Ca2+ entry To elucidate the Ca2+ source underlying Yoda1-induced microdomain transients, we first tested the effect of chelating intracellular Ca2+ with BAPTA-AM (1 µM).  As shown in Fig. 2-7A, Ca2+ signals were completely inhibited by BAPTA-AM, consistent with the intracellular localization of the Lck-GCaMP5 indicator (Yoda1: 3.5 ± 1.5 events/ROI/5 min; BAPTA-AM: 0 events/ROI/5 min; p < 0.05).  Yoda1-evoked transients were also blocked in the absence of extracellular Ca2+ (aCSF containing 0 mM Ca2+ and 2 mM EGTA)(0 Ca2+/EGTA: 0 events/ROI/5 min; p < 0.01 compared to Yoda1 alone).  In contrast, depleting intracellular Ca2+ stores by blocking SERCA with thapsigargin (1 µM) had no effect (thapsigargin: 5.2 ± 1.8 events/ROI/5min; p > 0.05 compared to Yoda1 alone).  The results from sections 2.3.4 and 2.3.5 are summarized in the schematics in Fig. 2-7B, which illustrate Yoda1-induced Ca2+ microdomain signals in astrocytes occur via Ca2+ influx through the Piezo1 channel.   55   Figure 2-7.  Yoda1-induced increases in Ca2+ microdomain signals depend on extracellular Ca2+ entry. A) Quantification of the effects of intracellular Ca2+ chelation with BAPTA-AM (1 µM), removing Ca2+ from the extracellular solution (0 mM Ca2+ with 2 mM EGTA), and blocking the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) with thapsigargin (1 µM) on Yoda1-induced Ca2+ microdomain transients.  n = 4 – 13 cells from individual coverslips.  Data represent mean ± 95% CI; * p < 0.05 and ** p < 0.01 compared to Yoda1 by one-way ANOVA with Bonferroni’s multiple comparison post-test.  B) Schematics summarizing the pharmacological and genetic approaches that blocked Yoda1-induced microdomain Ca2+ signals or had no effect.  2.3.6 Spontaneous Ca2+ microdomain signals are sensitive to Piezo1 antagonists In our previous experiments, Yoda1 was used as a tool to study the effect of Piezo1 activation on astrocyte Ca2+ transients.  We now focus on the mechanism underlying spontaneous Ca2+ microdomain signals exhibited by Lck-GCaMP5-transfected astrocytes (Fig. 2-4).  These spontaneous Ca2+ transients have been shown to affect astrocyte basal Ca2+ levels, which in turn have been implicated in functions such as control of blood vessel tone and regulation of synaptic 56  activity via neurotransmitter uptake (Shigetomi et al., 2012; Rosenegger et al., 2015).  We tested whether spontaneous Ca2+ signals have a Piezo1-mediated component using the same pharmacological tools as in section 2.3.4.  The Piezo1 antagonists GsMTx4 (20 µM) and Gd3+ (100 µM) decreased the basal Ca2+ signals (Baseline: 0.8 ± 0.2 events/ROI/5 min; GsMTx4: 0.5 ± 0.3 events/ROI/5 min; p < 0.01; Baseline: 1.2 ± 0.9 events/ROI/5 min; Gd3+: 0.2 ± 0.2 events/ROI/5 min; p < 0.05)(Fig. 2-8A – C), while HC067047 (10 µM) had no effect (Baseline: 1.4 ± 1.2 events/ROI/5 min; HC067047: 0.9 ± 0.5 events/ROI/5 min; p > 0.05)(Fig. 2-8A and D).  Together with the observation that Piezo1 siRNA reduces the number of basally active Ca2+ microdomains (Fig. 2-6C), the data suggest that Piezo1 may contribute to astrocyte spontaneous Ca2+ transients.      57   Figure 2-8.  Spontaneous Ca2+ microdomain signals are inhibited by Piezo1 antagonists GsMTx4 and Gd3+. A) Images represent masked 10-minute maximum intensity projections of astrocytes transfected with Lck-GCaMP5 during baseline, and following application of either GsMTx4 (20 µM), Gd3+ (100 µM) or HC067047 (10 µM).  Scale bars = 50 µm.  Quantification of the effects of B) GsMTx4, C) Gd3+, and D) HC067047 on spontaneous Ca2+ microdomain transients.  n = 4 – 14 cells from individual coverslips.  Data represent mean ± 95% CI; * p < 0.05 and ** p < 0.01 compared to baseline by one-tailed paired t-test.  2.3.7 Preliminary imaging of brain slice astrocyte Ca2+ signals show sensitivity to pharmacological Piezo1 modulations Astrocyte fine processes exhibit spontaneous Ca2+ transients, but the underlying mechanism has not been fully defined.  To determine whether Piezo1 may be involved in mediating these signals in the brain, we performed Ca2+ imaging using hippocampal slices from GLAST-GCaMP5 mice with astrocyte-specific expression of the GCaMP5 Ca2+ indicator.  This mouse model was selected to keep the GECI consistent with the Lck-GCaMP5 indicator used for the cultured astrocyte experiments.  In preliminary experiments, Yoda1 (25 µM) was observed to increase the 58  frequency of Ca2+ signals in the astrocyte endfeet (Fig. 2-9A), while Gd3+ (100 µM) application inhibited the transients (Fig. 2-9B).  These results are consistent with the pharmacological profile of the Ca2+ signals observed in the Lck-GCaMP5-transfected astrocyte cultures, suggesting that Piezo1 contributes to spontaneous Ca2+ transients in the intact brain.  Figure 2-9.  The frequency of astrocyte endfeet Ca2+ transients are sensitive to pharmacological modulators of Piezo1. A) Two-photon imaging of brain slices from mice with inducible astrocyte-specific expression of GCaMP5 showing the effect of Yoda1 (25 µM) and B) Gd3+ (100 µM) on the frequency of Ca2+ transients in the endfeet, as highlighted with the dashed circles.  The representative baseline, Yoda1, and Gd3+ images correspond to the black, green, and grey arrowheads, respectively, on the intensity-time traces to the right.  Scale bar = 10 µm.  59  2.4 Discussion In these studies, we demonstrated that Piezo1 channels contribute to microdomain Ca2+ signals in cultured astrocytes, suggesting that Piezo1 may be physiologically important in regulating astrocyte basal Ca2+ level and function.  The Piezo1 agonist Yoda1 increased the number of microdomain Ca2+ transients, which were completely abolished by removal of extracellular Ca2+ but unaffected by depletion of intracellular Ca2+ stores with thapsigargin.  In addition, both Yoda1-evoked and spontaneous signals were inhibited by the Piezo1 antagonists GsMTx4 and Gd3+.  The pharmacological data were further supported by the sensitivity of Yoda1-induced Ca2+ signals to Piezo1 siRNA treatment.  Furthermore, we performed preliminary imaging experiments using brain slices from GLAST-GCaMP5 mice, and our observations suggest that astrocyte endfeet Ca2+ transients may also involve a Piezo1-mediated component.    Numerous chemical, physical, and viral strategies have been developed and refined to maximize transfection efficiency while preserving cell viability (Gresch and Altrogge, 2012).  Differentiated mammalian cells in primary culture are often difficult to transfect, with transfection efficiency rates of ~3% previously reported for liposome-mediated transfection of primary astrocytes (Wu et al., 2000).  In our experiments, we utilized LNP technology for delivery of both plasmid DNA and siRNA into cultured astrocytes due to its advantages in ease of preparation, low immunogenicity, and adaptability for in vivo gene delivery and knockdown (Nayerossadat et al., 2012; Rungta et al., 2013, 2015; Zatsepin et al., 2016).  In preliminary control experiments, we found LNPs were well-tolerated and equally effective as the non-liposomal transfection reagent Effectene in delivering plasmid DNA to astrocytes (Shigetomi et al., 2012), with transfection efficiency rate ~10%.  We also verified that LNP-packaged Piezo1 60  siRNA was effective in silencing mRNA and reducing protein expression in astrocytes, as we have previously shown for neurons (Rungta et al., 2013).  While multiple approaches are available for gene knockdown in the brain (Heidenreich and Zhang, 2016), an siRNA approach was selected for its simplicity in generating sequences against specific targets, ease of delivery into cells, and relatively low cost.  Furthermore, effective Piezo1 knockdown by siRNA has been demonstrated in various cell types (Coste et al., 2010; McHugh et al., 2010; Lee et al., 2014; Pathak et al., 2014).    Studies on Piezo1 activity as a cationic channel have relied on patch clamp electrophysiological techniques.  However, these methods inherently apply a mechanical force to the cell, resulting in alterations of membrane tension or cytoskeletal structure that may affect the properties of mechanosensitive channels (Nourse and Pathak, 2017).  In contrast, several groups have used Ca2+ imaging as a non-invasive technique to measure Piezo1 function.  For instance, Piezo1-mediated intracellular Ca2+ elevations have been recorded in Piezo1-transfected HEK293 cells and chondrocytes in response to mechanical deformation of the plasma membrane (Lee et al., 2014; Gaub and Müller, 2017).  Intriguingly, Pathak et al. (2014) observed Piezo1-dependent spontaneous Ca2+ microdomain signals in neural stem cells that were loaded with Fluo-4 and imaged with total internal reflection fluorescence microscopy (TIRFM), which allowed the authors to selectively measure signals at the plasma membrane.    Alternatively, we used the membrane-tethered GECI, Lck-GCaMP5, to monitor near-membrane Ca2+ microdomain signals in cultured astrocytes.  While patch-dialysis of organic dyes is a viable strategy for recording microdomain Ca2+ signals in fine astrocyte processes, cultured astrocytes 61  have a polygonal morphology that lacks the highly branched processes characteristic of in vivo astrocytes.  The Lck domain consists of tandem palmitoylation and myristoylation domains from the N-terminal of a Src tyrosine kinase, which targets the GCaMP5 to the plasma membrane (Shigetomi et al., 2010).  Using such Lck-linked constructs, Shigetomi et al. (2010) previously demonstrated that both global and localized Ca2+ signals could be detected in cultured astrocytes.  Furthermore, while GECIs are Ca2+ buffers that may alter endogenous Ca2+ homeostasis, no studies to date have been published on GECI-induced alterations to astrocyte physiology (Shigetomi et al., 2016).  Importantly, the microdomain Ca2+ signals reported here share a number of characteristics with the transients recorded from the fine processes of brain slice and in vivo astrocytes, including dependence on extracellular Ca2+ and resistance to treatments that inhibit Ca2+ release from intracellular stores (Shigetomi et al., 2010; Srinivasan et al., 2015; Rungta et al., 2016).    Initial studies of astrocyte Ca2+ signaling relied on measuring large, robust responses in the soma (Volterra et al., 2014).  However, the discovery of microdomain Ca2+ transients, with different spatial and temporal characteristics, presented a new challenge for analysis.  Early work on developing algorithms to detect and quantify Ca2+ transients was conducted in muscle cells, where Ca2+ release from intracellular stores appeared as ‘sparks’ in confocal line scan images (Cheng et al., 1999).  Researchers recognized the need for an automatic analysis program to quantify many events, and to prevent detection bias against events with low signal-to-noise ratio (Cheng et al., 1999; Picht et al., 2007).  In the astrocyte Ca2+ imaging field, algorithms such as automatic segmentation, GECIquant, and CaSCaDe have been developed to analyze 62  microdomain signals (Di Castro et al., 2011; Srinivasan et al., 2015; Agarwal et al., 2017; Bindocci et al., 2017).    Here we developed a semi-automatic method to quantify astrocyte Ca2+ microdomain signals.  The analysis algorithm shares several similarities, such as image alignment to eliminate x-y axis drift, median filtering to remove noise, separation of microdomain and wave signals based on area, and thresholding to identify microdomains events.  One key feature is our use of the 3D connected components function in MATLAB, which allows discrete Ca2+ signals to be identified across time.  Hence, Ca2+ waves that expanded and contracted from their point of origin could be masked effectively, revealing microdomain signals that occupied the overlapping space in the xy-plane.  Intracellular Ca2+ wave events and localized microdomain transients were analyzed separately due to differences in their spatial and temporal characteristics, as well as their Ca2+ source.  Previous studies have found that intrinsic somatic Ca2+ waves have a greater dependence on IP3R-mediated release from endoplasmic reticulum (ER) stores, and the extent of their spread may be determined by the spatial distribution of IP3Rs in the ER (Khakh and McCarthy, 2015; Srinivasan et al., 2015).  In contrast, microdomain Ca2+ signals involve a transmembrane Ca2+ entry pathway rather than an IP3R-mediated mechanism (Shigetomi et al., 2012; Rungta et al., 2016), consistent with the observation that fine astrocyte processes have little ER content (Verkhratsky and Nedergaard, 2014).  Using the Piezo1 agonist Yoda1, we were able to selectively increase the frequency of microdomain transients, underscoring the need to consider different types of Ca2+ signals separately.  63  Analysis of cultured astrocytes showed that spontaneous microdomain Ca2+ events occurred with a frequency of 0.20 ± 0.06 events/ROI/min (mean ± SEM), comparable to the 0.5 ± 0.1 and ~0.4 events/ROI/min reported by other groups from imaging astrocytes in brain slices (Srinivasan et al., 2015; Agarwal et al., 2017).  Furthermore, we observed that microdomain transients exhibited slow kinetics with an average duration of 6.7 ± 1.5 s (mean ± SEM), which is consistent with the previously reported duration of 8.5 ± 1.7 s (Agarwal et al., 2017).  The convergence of the parameters across different models, Ca2+ indicators, and analysis algorithms suggests our methods for imaging and quantifying Ca2+ microdomain signals are reliable.  The data suggest a role for Piezo1 in mediating spontaneous Ca2+ microdomain transients, in agreement with previous studies of reconstituted Piezo1 channels that identified Piezo1-mediated currents in the absence of applied pressure (Syeda et al., 2015).  Yoda1 induced an increase in the frequency of Ca2+ signals, consistent with reports that Yoda1 lowers the threshold for Piezo1 activation (Syeda et al., 2015).  Yoda1 has also been shown to slow Piezo1 inactivation kinetics (Syeda et al., 2015); however, we did not observe any changes in the duration of the Ca2+ transients, possibly because our scanning rate of 2.4 s/frame was too slow to capture any differences.  Our finding that Yoda1-evoked microdomain Ca2+ signals are dependent on Ca2+ entry recapitulates other studies demonstrating that such localized Ca2+ transients in astrocytes are mediated by a transmembrane Ca2+ pathway rather than by release from intracellular stores (Srinivasan et al., 2015; Rungta et al., 2016), and suggests that subcellular compartmentalization of Ca2+ signaling is preserved in cultured astrocytes.  64  While our work suggests that Piezo1 is involved in mediating astrocyte Ca2+ microdomain signals, it is interesting to note that only ~40% of the spontaneous signals were blocked by the Piezo1 antagonist GsMTx4, while ~80% block was attained with the nonspecific mechanosensitive channel blocker Gd3+.  Furthermore, siRNA-mediated Piezo1 knockdown reduced the number of Ca2+ microdomains but did not completely abolish the signals.  This suggests that in addition to Piezo1, other Ca2+-fluxing pathways, such as TRPA1 (Shigetomi et al., 2012), are likely to contribute to the Ca2+ transients.  Another candidate is Ca2+-induced Ca2+ release through the mitochondrial permeability transition pore (mPTP); Agarwal et al. (2017) found that mPTP block was associated with a 70% reduction in the frequency of Ca2+ transients.  These transporters or channels could act in concert or contribute in different proportions under different physiological conditions to mediate the spontaneous Ca2+ transients.   65  Chapter 3: Subcellular localization of Piezo1 in astrocytes  3.1  Overview As reported in the previous Chapter, pharmacological and genetic manipulation of Piezo1 in astrocytes affects the frequency of microdomain Ca2+ transients, suggesting that astrocytes express functional Piezo1 channels.  This finding is supported by recent transcriptomic and proteomic studies of both cultured and acutely isolated rodent astrocytes demonstrating Piezo1 mRNA and protein expression (Zhang et al., 2014; Sharma et al., 2015; Srinivasan et al., 2016).  While our data point to the presence of Piezo1 at the plasma membrane to mediate transmembrane Ca2+ influx, the channel may also be expressed in other cellular domains that are relevant for mechanotransduction.    Li et al. (2014) observed green fluorescent protein-tagged Piezo1 (Piezo1-GFP) localized to cytoskeleton-associated focal adhesion zones in cultured human umbilical vein endothelial cells, where the channels may sense shear stress.  The authors further suggested that this association with the cytoskeleton may be related to the role of Piezo1 in mediating Ca2+ entry to activate calpain-dependent reorganization of the cytoskeleton for proper endothelial cell alignment.  Furthermore, McHugh et al. (2010) reported colocalization of GFP-tagged Piezo1 or Piezo1 immunoreactivity with the endoplasmic reticulum (ER) marker SERCA2 in Chinese hamster ovary and HeLa cells, and suggested Piezo1 involvement in recruiting R-Ras GTPase to the ER, followed by Ca2+ release from ER stores and subsequent activation of integrin to mediate cell adhesion.  Taken together, the studies reveal that while cell type-specific Piezo1 expression pattern in different organelles can mediate a diverse range of functions, there appears to be a 66  common association with structural locations related to mechanosensing and mechanotransduction.  Of note, astrocytes have been shown to express mechanosensitive channels (Bowman et al., 1992), and the cells exhibit changes in morphology and function in response to stimuli such as extracellular matrix stiffness (Summers et al., 2010; Wilson et al., 2016).  Therefore, it is interesting to study the subcellular localization of Piezo1 as it can provide information about the protein’s interaction partners and physiological roles in astrocytes (Stadler et al., 2013).       In this chapter, we provide evidence for Piezo1 association with the plasma membrane and cytoskeletal components in astrocytes.  Subcellular localization of Piezo1 was determined by optimized immunocytochemistry protocols, expression of exogenous fluorescent protein – tagged Piezo1, and verified by siRNA-mediated Piezo1 knockdown.  To further investigate the link between Piezo1 and the cytoskeleton, astrocytes were treated with forskolin to induce cytoskeleton reorganization followed by immunostaining.  Finally, we performed preliminary immunohistochemistry experiments in fixed brain slices to assess Piezo1 localization in astrocytes in situ.  3.2 Methods  3.2.1 Cell culture Astrocyte cultures were prepared as described in section 2.2.2.  For staining experiments, P1 astrocytes were thawed, resuspended in glial medium, and plated onto sterile 15 mm No. 1.5 glass coverslips (MatTek) at a density of ~9 x 103 cells per coverslip.  For immunoblotting 67  experiments, cells were seeded at a density of 4.5 x 104 cells per T25 flask.  Cells were grown for 3 to 7 days in a 37°C incubator with 5% CO2 in glia media.  Neuro2A cells (ATCC CCL-131, P185 at arrival) were cultured in Eagle’s Minimum Essential Medium (SAFC Biosciences) supplemented with 10% FBS and 0.5 mM sodium pyruvate.  Cells were seeded at a density of 5 x 104 cells/mL in T25 flasks for immunoblotting and qPCR experiments, and onto sterile 15 mm No. 1.5 glass coverslips for immunostaining experiments.  Cells were grown for 24 hours in a 37°C incubator with 5% CO2 prior to experiments.  3.2.2 Astrocyte transfection and treatment For Piezo1 knockdown experiments, astrocyte cultures were treated with LNP-siRNA complexes as described in section 2.2.6.  The Piezo1-1591-GFP plasmid was a gift from Dr. Philip Gottlieb (SUNY, Buffalo), corresponding to a fluorescence-tagged Piezo1 protein with GFP inserted at amino acid position 1591 (Cox et al., 2016).  The plasmid was expanded in E. coli (HST08 strain) with colonies selected by kanamycin, and plasmid DNA was isolated by midi prep (Qiagen) according to manufacturer’s instructions.  Cultures were transfected with the plasmid (0.125 µg/mL) using Effectene (Qiagen) according to the manufacturer’s instructions, and cells were used for staining experiments after 48 hours post-transfection.  In experiments with forskolin treatment, cultures were incubated with serum-free media for 1 hour followed by 1-hour incubation with forskolin (1 µM, diluted from 1 mM stock in DMSO; Sigma) prior to imaging. 68  3.2.3 qPCR for Neuro2A cells Quantitative polymerase chain reaction (qPCR) was performed on Neuro2A cells as described in Section 2.2.8.  Probes from Integrated DNA Technologies were used to amplify PIEZO1 (mPiezo1-1 Mm.PT.58.11048868; mPiezo1-2 Mm.PT.58.10591550), PIEZO2 (mPiezo2-1 Mm.PT.58.6238757; mPiezo2-2 Mm.PT.58.30174298), and tyrosine hydroxylase (mTh Mm.PT.58.33106186), with GAPDH (mGAPDH Mm.PT.39a.1) as the internal control.  3.2.4 Immunoblotting Astrocyte and Neuro2A cultures were collected using 1X Laemmli buffer with cOmplete protease inhibitor cocktail (Roche).  Samples were heated for 10 minutes at 65°C, homogenized with an insulin syringe, and heated for another 5 minutes at 65°C.  Wild-type C57BL/6 mice (P30) were anesthetized with isoflurane and decapitated according to protocols approved by the University of British Columbia Animal Care Committee.  Lung tissue was flash-frozen in liquid nitrogen and homogenized with 2X Laemmli buffer supplemented with protease inhibitor cocktail.  Samples were heated for 10 minutes at 65°C, homogenized with a 23G syringe followed by an insulin syringe, and heated for another 5 minutes at 65 °C.  All samples were then centrifuged for 10 minutes at 10,000 rpm at 4°C to remove debris.  Protein concentration was determined with the BCA protein assay (Pierce) measured at 660 nm with a microplate reader.  Samples were resolved on 3-8% Tris-acetate gradient gels (Invitrogen), and transferred overnight to HyBond membranes (GE Life Sciences).  Membranes were then equilibrated in TBS-Tween (TBST) for 10 minutes and blocked with TBST + 2% milk for one hour at room temperature.  Primary antibodies used were: 1/1000 Piezo1 (Proteintech); 1/20,000 vinculin (Sigma).  Secondary antibodies used were: 1/5000 horseradish peroxidase (HRP)-conjugated 69  goat anti-rabbit; 1/20,000 HRP-conjugated goat anti-mouse (both from Pierce).  Blots were incubated using SuperSignal West Pico Chemiluminescent Substrate (Pierce), and developed on film.  3.2.5 Immunocytochemistry Based on the previously reported distribution of Piezo1 in different cell types, we used different staining protocols that have been optimized to target those subcellular compartments to study Piezo1 localization in astrocytes, as depicted in Fig. 3-1.  For example, strong detergents such as Triton-X allow antibodies to access intracellular cytoskeletal epitopes, but may also extract membrane proteins along with lipids, suggesting that a single staining protocol is insufficient for achieving optimal staining for all cellular structures (Jamur and Oliver, 2010; Bocksteins et al., 2012; Maity et al., 2013).  We therefore modified the duration of permeabilization and compared the staining pattern of Piezo1 with organelle-specific markers to assess Piezo1 localization to the corresponding subcellular compartments.  To confirm the Piezo1 staining pattern, we compared experiments performed using two commercial antibodies raised against different epitopes of the rat Piezo1 protein (Fig. 3-2).  Cells were fixed for 10 minutes with 4% paraformaldehyde (PFA; Ted Pella), followed by two 5-minute washes with phosphate buffered saline (PBS; NaCl 110 mM, K2HPO4 16 mM, KH2PO4 3 mM, pH 7.4).  In the ‘with permeabilization’ conditions, cells were treated with either 0.1% Triton X-100 (Thermo Scientific) or 0.01% saponin (Sigma), followed by two PBS washes.  Cells were then blocked with 4% normal goat serum (Abcam) for 30 minutes, and incubated overnight at 4°C with primary antibodies diluted in 0.1% bovine serum albumin (BSA; Sigma).  70  Samples were washed twice with PBS and incubated with secondary antibodies diluted in 0.1% BSA for 1 hour, followed by two PBS washes.  Cells were incubated with Hoechst dye for 10 minutes to label the nuclei.  After washing, coverslips were mounted using Vectashield mounting media (Vector Labs) and sealed with nail polish.  As negative controls, Piezo1 antibody was omitted or replaced with rabbit IgG (Santa Cruz Biotechnology).  Primary antibodies and probes used were: 1/300 Piezo1 (Alomone and Proteintech); 1/200 vinculin (Sigma); 1/400 calnexin (Abcam); 1/2000 GFP (Abcam); 1/200 phalloidin-633; 1/200 wheat germ agglutinin-555; 1/1000 Hoechst 33342 (all from Invitrogen).  Secondary antibodies used were: 1/200 goat anti-rabbit Alexa Fluor 488 and 546; 1/200 goat anti-chicken Alexa Fluor 488; 1/200 goat anti-mouse Alexa Fluor 546 (all from Invitrogen).  Figure 3-1.  Flowchart of immunocytochemistry protocols for optimization of Piezo1 staining in different subcellular regions. 71   Figure 3-2.  Schematic illustrating the epitopes targeted by the Alomone and Proteintech Piezo1 antibodies. The Piezo1 topology is adapted from Cox et al. (2016) and Zhao et al. (2016).  According to information from the commercially supplied technical sheets, the Alomone and Proteintech antibodies were raised against epitopes corresponding to amino acid sequences 1844 – 1888 and 2199 – 2437, respectively that we mapped to the rat Piezo1 protein sequence (NCBI reference sequence NP_001070668.2).  3.2.6 Immunohistochemistry Wild-type C57BL/6 mice (P30) were anesthetized with halothane and given an intraperitoneal injection of urethane (0.1 mL of 30% urethane per 10 g body weight).  Mice were then transcardially perfused with 0.1 M PBS (Oxoid) followed by 4% PFA (FD Neuro Technologies).  Brains were extracted and immersed in post-fix (10% sucrose in 4% PFA) at 4°C overnight, followed by cryoprotection in 30% sucrose in 0.1 M PBS overnight. Using a cryostat (Leica), 40 µm coronal sections were collected and stored in cryoprotectant (30% sucrose, 30% ethylene glycol, and 1% polyvinyl-pyrrolidone in 0.1 M PBS) at -20°C.    Free-floating sections were rinsed in PBS and blocked in 10% normal donkey serum (Jackson Immunoresearch Laboratories) and 0.4% Triton-X (Sigma) for one hour, followed by incubation with primary antibodies diluted in buffer (2.5% donkey serum + 0.04% Triton-X) overnight at 4°C.  Sections were then rinsed in PBS and incubated with secondary antibodies for 1.5 hours at 72  room temperature.  Sections were rinsed with PBS, mounted onto glass slides, and coverslipped using FluorSave Reagent (Calbiochem).  Primary antibodies used were: 1/100 Piezo1 (Alomone and Proteintech); 1/250 GFAP (Invitrogen); 1/250 Aquaporin 4 (Santa Cruz Biotechnology).  Secondary antibodies used were: 1/500 donkey anti-rabbit Alexa Fluor 488; 1/500 donkey anti-rat Alexa Fluor 546; 1/500 donkey anti-goat Alexa Fluor 546 (all from Invitrogen).  3.2.7 Confocal image acquisition and analysis Cells and tissue sections were imaged using a TCS SP8 confocal laser scanning microscope (Leica) with a 25x-W/0.95 NA and a 63x-O/1.4 NA objective lens.  Images were acquired as 512 x 512-pixel frames using 4-line averaging.  For siRNA-treated coverslips, 12 random fields were acquired per coverslip.  We observed that non-permeabilized astrocytes displayed a heterogeneous staining pattern, and decided to classify the cells into different categories based on the characteristics of their Piezo1 immunoreactivity, as described in Fig. 3-4.  Quantitative analysis was focused on astrocytes exhibiting Type 1 staining pattern (cells with multiple discrete Piezo1-positive clusters) as these cells had the clearest signal-to-noise ratio, and the staining pattern resembled the Yoda1-activated Ca2+ microdomain signals imaged in Chapter 2.  Statistics were performed as described in section 2.2.15, where unpaired t-tests were used to compare the significance of difference in means between two groups.  Data are reported as mean ± 95% confidence interval, and p < 0.05 was considered statistically significant.  73  3.3 Results  3.3.1 Antibody testing in Neuro2A cells that endogenously express Piezo1 We initially decided to test the Piezo1 staining procedure and antibody using Neuro2A mouse neuroblastoma cells as a positive control, since Piezo1 was initially cloned from this cell line (Coste et al., 2010).  Neuro2A cells contained mRNA for Piezo1, but not the homolog Piezo2, as quantified by qPCR with two sets of probes against independent regions of each gene (Fig. 3-3A).  In addition, tyrosine hydroxylase was probed as a phenotypic marker for Neuro2A cells, which can be induced to differentiate into dopaminergic-like neurons (Tremblay et al., 2010).  Although a variety of commercially available antibodies against Piezo1 exist, the Proteintech antibody was selected as it had been shown to stain bladder urothelial cells (Miyamoto et al., 2014), a tissue known to express Piezo1 mRNA (Coste et al., 2010).  Immunoblot analysis demonstrated that the antibody recognized the Piezo1 protein with a single band from cultured astrocyte lysate (Fig. 3-3B), albeit with a higher molecular weight, compared to the Neuro2A cell lysate and mouse lung tissue positive controls (Coste et al., 2010).  Interestingly, this predominant band has a higher molecular weight than the previously observed band pattern at ~280 kDa (Fig. 2-2E), and may represent Piezo1 dimers that have been described by other groups (Coste et al., 2012).  Immunocytochemistry performed in the absence of membrane permeabilization revealed that Piezo1 immunoreactivity is located in clusters at the surface of Neuro2A cells (Fig. 3-3C).  The ability of the Proteintech antibody to detect endogenous Piezo1 protein by immunoblotting and staining in Neuro2A cells suggests that it may also be used for Piezo1 immunostaining in astrocytes. 74   Figure 3-3.  Neuro2A cells show distinct clusters of Piezo1 immunoreactivity. A) mRNA levels of Piezo1 and Piezo2 in Neuro2A cells from three different samples (Neuro2A-P186, -P187, and -P188) as determined by qPCR using two sets of independent probes for each sequence (mouse Piezo1: mPiezo1-1 and mPiezo1-2; mouse Piezo2: mPiezo2-1 and mPiezo2-2).  Mouse tyrosine hydroxylase (mTh) expression was used as a phenotypic marker for Neuro2A cells.  Data were normalized to Gapdh.  n = 3 independent experiments.  B) Immunoblot analysis of Piezo1 protein expression in cultured astrocyte lysate, with Neuro2A and mouse lung samples as positive controls, and vinculin as the loading control.  C) Confocal microscopy image of Piezo1 immunoreactivity (highlighted by white arrowheads) in Neuro2A cells.  Phalloidin and Hoechst staining label actin filaments and nuclei, respectively.  Scale bar = 10 µm.  3.3.2 Piezo1 immunoreactivity clusters in discrete domains in the astrocyte plasma membrane Previous literature showed Piezo1 expression in the plasma membrane of HEK293 cells (Cox et al., 2016).  We therefore optimized our immunocytochemistry protocol for astrocyte cell surface staining by eliminating permeabilization with Triton-X.  We observed that astrocytes exhibited 75  different patterns of Piezo1 staining, which we categorized according to the presence and characteristics of the immunopositive ‘plaque’-like clusters (Fig. 3-4).      Figure 3-4.  Astrocytes exhibit different patterns of Piezo1 immunoreactivity at the plasma membrane. Representative confocal images of cultured astrocytes stained in the absence of cell permeabilization for Piezo1 (green), actin filaments (blue), and nuclei (teal).  Scale bar = 50 µm.    We repeated the Piezo1 staining with the addition of wheat germ agglutinin to label the glycolipids and glycoproteins on the plasma membrane, while phalloidin and Hoechst stain were used to stain actin filaments and cell nuclei, respectively.  Approximately 10% of the astrocytes exhibited multiple round, discrete clusters typical of the Type 1 staining pattern, and this was 76  reminiscent of both spontaneous and Yoda1-induced Ca2+ microdomains observed in Chapter 2 (Fig. 3-5A).  Similarly, astrocytes transfected with Piezo1-GFP showed a clustered immunofluorescence pattern that co-localized with Piezo1 antibody staining (Fig. 3-5B).  This aggregate pattern was not observed in negative controls where the Piezo1 primary antibody had been omitted (Fig. 3-5C) or replaced with an isotype control (Fig. 3-5D).  The clustered staining pattern of Piezo1 was replicated using an alternate Piezo1 antibody from Alomone Labs that was raised against a different immunogen (Fig. 3-5E).    77   Figure 3-5.  Piezo1 immunoreactivity is distributed in clusters located on astrocyte plasma membranes. A) Single plane confocal images showing the pattern of Piezo1 staining with the Proteintech antibody (green) in cultured astrocytes in the absence of permeabilization.  Wheat germ agglutinin (WGA; red) was used to detect the 78  glycolipids and glycoproteins on the cell plasma membrane, and phalloidin (blue) was used as an actin filament marker.  B) Confocal images of astrocytes transfected with 0.25 µg Piezo1-GFP DNA and stained with anti-GFP (green) and anti-Piezo1 antibody (red).  Co-localization between the GFP-tagged Piezo1 and the Piezo1 antibody signal is highlighted with white arrowheads.  Negative controls where Piezo1 primary antibody was C) omitted, or D) replaced with the IgG isotype control; phalloidin and Hoechst staining was imaged in the same fields to show the actin filaments and nuclei, respectively. E) Confocal images as in part A), with Piezo1 being detected by an antibody from Alomone Labs.  Scale bar = 50 µm.  We further examined Piezo1 expression in the astrocyte membrane by conducting immunostaining following protein knockdown with Piezo1 siRNA.  We focused on astrocytes showing Type 1 staining based on the high signal-to-noise ratio of the Piezo1 clusters that allowed them to be quantified accurately, and because the staining pattern resembles the Ca2+ microdomains imaged in Chapter 2.  Piezo1 siRNA reduced the number of Piezo1 clusters in these astrocytes (luciferase siRNA: 55 cells analyzed from 4 coverslips, 14 ± 2.8 clusters/cell; Piezo1 siRNA: 52 cells analyzed from 4 coverslips, 9.8 ± 2.9 clusters/cell; p < 0.01)(Fig. 3-6A and B).      79   Figure 3-6.  Piezo1 siRNA reduces the number of Piezo1 clusters in astrocytes. A) Confocal images of Type 1 astrocytes treated with luciferase or Piezo1 siRNA (0.125 µg /mL) for five days and stained without permeabilization.  B) Quantification of the effect of Piezo1 siRNA on the number of Piezo1-positive clusters per Type 1 cell.  Scale bars = 50 µm.  n = 4 coverslips.  Data represent mean ± 95% CI; ** p < 0.01 compared to luciferase siRNA treatment group by one-tailed unpaired t-test.  3.3.3 Piezo1 immunoreactivity is present in actin filaments and focal adhesions Since Piezo1 has previously been shown to localize to intracellular compartments such as the endoplasmic reticulum (ER) and focal adhesions (McHugh et al., 2010; Li et al., 2014), we adapted our staining protocol for those targets by varying the duration of Triton-X permeabilization.  When astrocytes were permeabilized for 45 minutes for optimal cytoskeleton staining (Maity et al., 2013), Piezo1 immunoreactivity appeared to overlap with vinculin and phalloidin fluorescence, markers for focal adhesions and actin filaments, respectively (Fig. 3-7A).  To demonstrate that the staining is independent of the permeabilization agent, we replaced Triton-X with saponin and obtained similar staining patterns (Fig. 3-7E).  In astrocytes transfected with Piezo1-GFP, we also observed fluorescent protein signals overlapping with Piezo1 antibody staining in focal adhesion-like compartments and actin filaments (Fig. 3-7B).  As in section 3.3.2, we controlled for our staining procedure by omitting the primary antibody 80  (Fig. 3-7C) or replacing it with rabbit IgG as the species isotype control (Fig. 3-7D); a faint nuclear signal was detected in the isotype control, but neither controls showed staining in cytoskeleton components.  We repeated the experiments using the Alomone Piezo1 antibody and achieved the same observation (data not shown).  Interestingly, Piezo1-GFP-transfected astrocytes also exhibited a perinuclear fluorescent signal resembling ER staining (Fig. 3-7B).  However, ectopic localization of fluorescently tagged proteins is a known issue, as overexpressed proteins can be trapped in the endomembrane system (Stadler et al., 2013).  We therefore examined whether Piezo1 is expressed in the ER by permeabilizing cells with Triton-X for 5 minutes and compared the staining pattern to the ER marker calnexin.  Since the antibodies are from the same host species, cells were independently stained with Piezo1 or calnexin and the staining pattern was analyzed on separate coverslips.  Piezo1 immunoreactivity was more filamentous and peripheral, indicative of focal adhesions and actin filaments, which is distinct from the perinuclear pattern of calnexin staining (Fig. 3-7F).  The difference between the staining patterns of Piezo1 and calnexin suggest that ER localization of Piezo1-GFP might be an artifact.  81   Figure 3-7.  Piezo1 immunoreactivity is found in actin filaments and focal adhesions of the cytoskeleton. A) Single plane confocal images showing the pattern of Piezo1 immunoreactivity with the Proteintech antibody (green) in cultured astrocytes following 45 minutes of Triton-X permeabilization.  Focal adhesions and actin stress fibers were respectively detected by vinculin (red) and phalloidin (blue).  The regions highlighted by the solid and 82  dashed rectangles are shown at higher magnification in the right-most panels, respectively representing the merged images between Piezo1 and vinculin (top image) and between Piezo1 and phalloidin (bottom image).  Scale bars = 10 µm.  B) Confocal images of astrocytes transfected with 0.25 µg Piezo1-GFP DNA and stained with anti-GFP antibody (green), anti-Piezo1 antibody (red), and phalloidin (blue).  The regions highlighted by the solid and dashed rectangles are shown at higher magnification in the right-most panels, respectively representing the merged images between Piezo1-GFP and Piezo1 immunoreactivity with the antibody (top image) and between Piezo1-GFP and phalloidin (bottom image).  Scale bars = 10 µm.  Negative controls where Piezo1 primary antibody was C) omitted, or D) replaced with the IgG isotype control; phalloidin and Hoechst staining was imaged in the same fields to show the actin filaments and nuclei, respectively.  E) Confocal images as in part A), with permeabilization using saponin instead of Triton-X.  Piezo1 staining associated with focal adhesion structures (solid arrowheads) and actin stress fibers (empty arrowheads) are highlighted.  F) Confocal images showing Piezo1 and calnexin immunoreactivity following 5 minutes of Triton-X permeabilization.  Scale bars = 50 µm.  3.3.4 Forskolin-induced disorganization of the cytoskeleton alters Piezo1 distribution Piezo1 is a mechanically activated channel, and our immunostaining data and the literature suggest that it may be associated with cytoskeletal components that are involved in mechanosensing.  To further probe the interactions between Piezo1 and the cytoskeleton, we assessed the effect of altering the cytoskeletal architecture with forskolin on the Piezo1 staining pattern.  Forskolin increases cAMP levels in astrocytes, leading to cell stellation characterized by membrane retraction and loss of focal adhesions and actin stress fibers (Perez et al., 2005; Rodnight and Gottfried, 2013; Vardjan et al., 2014).  In this study, we tested different concentrations of forskolin treatment and observed that 1-hour incubation with 1 µM forskolin resulted in a typical stellation response.  These morphological changes are shown in Fig. 3-8A, in cells that were permeabilized with Triton-X for 45 minutes.  Forskolin-induced stellation (astrocyte highlighted by the dotted line in the ‘Piezo1’ panel) induced complete loss of Piezo1 83  immunoreactivity in focal adhesion and actin stress fiber-like compartments.  This was accompanied by stress fiber depolymerization, as seen by the loss of parallel structures detected by phalloidin in the stellate astrocyte, although stress fibers remained present in the surrounding non-stellate cells (marked by solid arrowheads).  Stellation also induced loss of focal adhesions in the periphery of the astrocyte cell body, in contrast to the distribution of vinculin immunoreactivity displayed by non-stellate cells (marked by empty arrowheads, Fig. 3-8B).  In the absence of permeabilization, forskolin treatment resulted in redistribution of Piezo1 immunoreactivity to the membranes of astrocyte processes (highlighted by empty arrowheads); phalloidin staining was also performed to show fragmentation of the actin stress fibers into globular clumps (highlighted by solid arrowheads) (Fig. 3-8C).  Taken together, our data indicate that forskolin treatment alters intracellular and membrane Piezo1 redistribution, suggesting an association between Piezo1 and the cytoskeleton.  84   Figure 3-8.  Forskolin induces astrocyte stellation and Piezo1 redistribution. A) Single plane confocal images showing the effect of forskolin (1 µM for 1 hour) on Piezo1 immunoreactivity in cultured astrocytes that were permeabilized with Triton-X for 45 minutes.  A stellate astrocyte is highlighted by the dotted lines in the ‘Piezo1’ panel.  Phalloidin-positive actin filaments in neighbouring non-stellate cells are highlighted by solid arrowheads.  B) Vinculin-positive focal adhesions in non-stellate cells are highlighted by empty arrowheads.  C) Staining was repeated in the absence of permeabilization to study the effect of forskolin on Piezo1 distribution at the astrocyte surface.  Empty arrowheads indicate Piezo1 clusters redistributed to the processes, while white arrowheads highlight the depolymerization of phalloidin-positive actin stress fibers into globular clumps.  Scale bar = 50 µm.  3.3.5 Piezo1 immunoreactivity colocalizes with astrocyte markers in the mouse brain Our results show that Piezo1 is localized to the plasma membrane and in domains associated with cytoskeletal proteins in cultured astrocytes.  Next, we performed immunohistochemistry 85  with fixed mouse coronal brain sections to determine whether Piezo1 is also present in astrocytes in situ.  Piezo1 immunoreactivity was observed to colocalize with the astrocyte markers glial fibrillary acidic protein (GFAP) and aquaporin4 (Aqp4) in layer I of the cortex and in the CA1 region of the hippocampus (Fig. 3-9).  In addition to astrocyte cell bodies, a diffuse ‘spongiform’ staining pattern was observed surrounding the large processes marked by GFAP, characteristic of the morphology of fine processes present in mature astrocytes (Bushong et al., 2004).  Interestingly, Piezo1 immunoreactivity was also detected in the astrocyte endfeet, specialized processes that contact the cerebral vasculature (highlighted by arrowheads).  No staining was observed in negative controls where the Piezo1 primary antibody had been omitted (data not shown).  Together the results indicate astrocytic expression of Piezo1 in the brain. 86   Figure 3-9.  Piezo1 immunoreactivity co-localizes with astrocyte markers in brain slices. Confocal images of layer I cortical and CA1 hippocampal regions from coronal brain sections of transcardially perfused mice showing Piezo1 immunoreactivity (green) and the astrocyte markers, glial fibrillary acidic protein (GFAP) and aquaporin4 (Aqp4) (red).  Astrocyte endfeet adjacent to blood vessels are highlighted by white arrowheads.  Scale bar = 10 µm.  3.4 Discussion In this Chapter, we describe experimental evidence that Piezo1 localized to the plasma membrane and cytoskeleton components in cultured astrocytes using both antibody- and fluorescent protein-based approaches.  Piezo1 immunoreactivity appeared as clusters on the cell 87  surface that resembled the Ca2+ microdomains imaged in Chapter 2, and these clusters were reduced by Piezo1 siRNA treatment.  Forskolin-induced disorganization of focal adhesions and actin filaments was associated with loss of intracellular Piezo1 staining and redistribution of the surface membrane Piezo1 clusters to the processes of the stellate astrocytes, suggesting that the cytoskeleton components may be involved in organizing Piezo1 domains.  Finally, immunohistochemistry using fixed brain slices revealed Piezo1 immunoreactivity in cortical and hippocampal astrocytes, indicating that Piezo1 may also be expressed in astrocytes in situ.    Protein localization is commonly visualized by imaging of either immunofluorescence or fluorescent-tagged proteins.  Immunofluorescence relies on an antibody-antigen interaction to label endogenous proteins of interest in fixed tissues or cells, but artifacts may arise from antibody recognition of off-target proteins, or from the protocols used for fixation and permeabilization (Schnell et al., 2012; Stadler et al., 2013).  In contrast, fluorescent-tagged proteins allow protein distribution to be visualized in live or fixed cells without the use of antibodies.  However, protein trafficking may be altered by overexpression of these proteins above endogenous protein levels, or by the fluorescent tag itself (Stadler et al., 2013).  As each technique exhibits distinct advantages and disadvantages, we combined both approaches to strengthen our findings.  Several commercially available and in-house antibodies have been used to examine Piezo1 distribution in different cell types (Coste et al., 2010; McHugh et al., 2010; Blumenthal et al., 2014; Miyamoto et al., 2014; Gudipaty et al., 2017).  To our knowledge, our immunostaining using the Proteintech antibody is the first demonstration of surface Piezo1 immunofluorescence 88  in Neuro2A cells, where Piezo1 is functionally expressed (Coste et al., 2010).  The antibody also detected Piezo1 protein in astrocytes with both immunoblotting and immunostaining approaches, consistent with previous transcriptome and proteome studies (Zhang et al., 2014; Sharma et al., 2015; Srinivasan et al., 2016).    Our finding that Piezo1 is distributed as clusters on astrocyte plasma membranes is supported by some examples from other cell types in the literature.  For instance, Coste et al. (2010) showed overlap between Piezo1 staining and the control surface marker TRPA1 in HEK293 cells transfected with Piezo1.  Other groups observed that fluorescent protein-tagged Piezo1 formed clustered ‘domains’ at the surface of transfected HEK293 cells (Cox et al., 2016; Gnanasambandam et al., 2017).  The staining pattern observed is consistent with the organization of discrete spatial domains described using electrophysiological techniques that suggest groups of Piezo1 channels change kinetics simultaneously (Bae et al., 2013a).  However, in contrast to the irregularly-shaped clusters observed by Cox et al. (2016), astrocyte Piezo1 appeared to aggregate as round ‘plaque’-like domains as detected by antibody and fluorescent protein approaches.  These Piezo1-immunopositive clusters bear a striking resemblance to the Ca2+ microdomains observed in Chapter 2, suggesting that the near-membrane Ca2+ signals detected by Lck-GCaMP5 may accurately represent the spatial orientation of Piezo1 channels mediating Ca2+ entry at the astrocyte plasma membrane.    We observed a variety of different Piezo1 staining patterns at the plasma membrane of cultured astrocytes.  Heterogeneity of Piezo1 expression has also been described by Gudipaty et al. (2017) in canine kidney epithelial cells, where Piezo1 distribution and function depended on density-89  induced mechanical forces experienced by the cells.  Preliminary observations in our lab indicate that increasing astrocyte culture density is associated with increased Piezo1 expression as quantified by immunoblotting.  However, we did not quantify cell density in the immunostaining experiments, as the cells detach during the fixation procedure, and it remains to be determined if Piezo1 localization in astrocytes is also density-dependent.  Previous studies have demonstrated that astrocyte density relies on factors including brain region, physiological development and ageing, and pathological proliferation as seen in reactive gliosis in response to CNS injury such as stroke or traumatic brain injury (Sofroniew and Vinters, 2010; Olude et al., 2015).  Hence it would be worthwhile to examine how astrocyte Piezo1 subcellular distribution and function are affected by different physiological and pathological states.  The range of staining patterns observed in astrocytes may also be related to heterogeneity in astrocyte functional state.  For instance, the greatest number of discrete Piezo1 clusters was observed in large astrocytes, suggesting that Piezo1 expression may also be dependent on progression through the cell cycle, metabolic state, or interactions with the extracellular matrix and signaling pathways that regulate cell morphology and growth (Lloyd, 2013; Levy et al., 2014).    Our experiments indicated that Piezo1 siRNA reduced the number of Piezo1-immunopositive domains at the astrocyte plasma membrane, correlating with the result that the number of basal Ca2+ microdomains are also sensitive to siRNA knockdown (Fig. 2-6C).  However, we did not observe an absolute absence of immunolabeled Piezo1 protein, supported by the Ca2+ imaging experiments showing that Yoda1 could still evoke an increase in Ca2+ microdomain signals in Piezo1 siRNA-treated astrocytes (Fig. 2-6B).  Previous studies have suggested that the degree of siRNA-mediated protein knockdown depends on factors such as the relation between siRNA 90  stability, mRNA transcription rate and half-life, and Piezo1 turnover rate (Stadler et al., 2013).  In addition, Thul et al. (2017) recently demonstrated that cells can exhibit variations in protein expression levels and spatial distribution depending on their cell cycle stage.  Thus, the efficiency of siRNA-mediated knockdown may be improved by treating astrocytes with more frequent, lower concentration applications of siRNA.  Piezo1 immunoreactivity in astrocytes was also found in the same cellular domains as focal adhesions and actin stress fibers, and this expression pattern was lost in cells treated with forskolin to induce cytoskeleton disorganization, supporting Piezo1 distribution in these cytoskeletal compartments.  This is an interesting observation given the importance of these structures in the molecular mechanism for sensing extracellular matrix (ECM) rigidity.  Previous studies have indicated that ventral stress fibers are composed of actin filaments and myosin II motor proteins that are anchored at both ends by large protein complexes known as focal adhesions.  These stress fibers exert traction forces that continually pull at focal adhesion complexes.  In addition, focal adhesions contain transmembrane adhesion molecules of the integrin family that act as receptors for various ECM proteins.  Therefore, focal adhesions mediate cell detection of ECM stiffness by transmitting traction forces generated by the stress fibers to the substrate (Geiger et al., 2009; Kobayashi and Sokabe, 2010; Tojkander et al., 2012).    The first demonstration that mechanosensitive channels may form a complex with focal adhesions and actin stress fibers to transduce information about substrate stiffness into Ca2+ signals was performed by Hayakawa et al. (2008) in cultured human umbilical vein endothelial cells.  By using optical tweezers to stretch single stress fibers and performing simultaneous Ca2+ 91  imaging, the authors found that mechanical stimulation of the actin cytoskeleton can generate Ca2+ microdomains located near focal adhesions.  These microdomain signals were inhibited by Gd3+ or by removal of Ca2+ from the extracellular solution, suggesting the involvement of putative mechanosensitive channels.  Subsequent experiments by Pathak et al. (2014) found that neural stem cells also exhibited microdomain Ca2+ transients that were sensitive to cytoskeletal mechanosensing; for example, the Ca2+ signals increased in amplitude and frequency with increasing substrate stiffness, while the signals were abolished by inhibition of myosin II-mediated traction forces by blebbistatin.  Strikingly, the Ca2+ transients were also blocked by GsMTx4 and Piezo1 siRNA treatment, indicating that Piezo1 may be involved in sensing mechanical properties of the substrate.  Our findings are consistent with these studies, raising the possibility that Piezo1 may associate with focal adhesions and stress fibers in astrocytes to function as a mechanosensing unit that can also respond to different cell substrates.  Fluorescence-tagged Piezo1 has been found to localize to the ER by other researchers (Satoh et al., 2006; McHugh et al., 2010).  While we also observed a perinuclear fluorescent signal resembling ER labeling in astrocytes that were transfected with Piezo1-GFP, subsequent immunocytochemistry experiments showed that the Piezo1 labeling distribution differed from the ER marker calnexin, suggesting that under our experimental conditions, the astrocyte ER signal may represent ectopic Piezo1-GFP overexpression or a mislocalization artifact from sequence differences between the fluorescent and native Piezo1 proteins (Nourse and Pathak, 2017).    92  To extend our findings to astrocytes in situ, Piezo1 colocalization with astrocyte markers was assessed in the cortex and hippocampus of fixed brain slices.  Interestingly, strong Piezo1 immunoreactivity was observed in the astrocyte endfeet compartment, where Ca2+ transients were found to be sensitive to pharmacological manipulation of Piezo1 (section 2.3.7).  The Ca2+ imaging and immunostaining experiments suggest that Piezo1 expression may be concentrated at the endfeet of astrocytes in the brain.  It is attractive to speculate that Piezo1 may be well-positioned to sense mechanical forces exerted by dilating or constricting blood vessels, modulating astrocyte Ca2+ microdomain activity that in turn can stimulate release of vasoactive compounds to regulate brain blood flow.   93  Chapter 4: Role of Piezo1 in astrocyte swelling  4.1  Overview Previous studies have shown that Piezo1 channels are involved in a variety of physiological processes related to sensation and transduction of mechanical forces.  For instance, Piezo1 has been implicated in detecting shear stress and realigning vascular endothelial cells to the direction of blood flow by mediating focal adhesion and actin stress fiber reorganization, a process that is critical to normal vascular development (Li et al., 2014; Ranade et al., 2014).  Piezo1 has also been suggested to sense plasma membrane tension to regulate cell volume, as shown in red blood cells where pharmacological activation and genetic knockdown of the channel were associated with cell shrinkage and swelling, respectively (Faucherre et al., 2014; Cahalan et al., 2015).  The data presented in Chapter 3 indicate that Piezo1 is expressed in astrocyte plasma membrane and is associated with cytoskeletal components such as cell adhesion structures, suggesting a potential relevance for mechano-regulated responses that are functionally important for astrocyte biology (section 1.2).  We therefore examined the possibility of activating Piezo1-mediated Ca2+ signals in response to osmotic swelling as a model of mechanical stimulation.  We studied astrocyte Piezo1 activation using osmotic swelling because astrocytes are involved in ion and volume homeostasis in the brain, and cell swelling has been observed under both physiological and pathological conditions (Kimelberg et al., 1993; Florence et al., 2012; Xiang et al., 2016).  Previous studies have used hypotonicity-induced swelling as a strategy to characterize the molecular components of mechanosensitive channels in astrocytes by measuring their activation as swelling-activated currents or increases in intracellular Ca2+ (Suchyna et al., 94  2000; Benfenati et al., 2007).  Furthermore, Piezo1 channels reconstituted into lipid bilayers have been activated by osmotic stress (Syeda et al., 2016).  Hence we speculated that hypotonicity-induced astrocyte swelling and subsequent plasma membrane stretch would activate Piezo1 channels, leading to an increase in Ca2+ microdomain transients.  One common strategy to induce astrocyte swelling is the application of hypotonic solution, where reduction in extracellular osmolarity triggers water movement into cells.  In these experiments, we used hyponatremia, defined as serum Na+ concentration below 135 mM (Schober and Mongin, 2015), to induce swelling, as this model has been well-characterized in previous studies examining the consequences of astrocyte swelling.  For instance, hyponatremia treatment has been shown to activate stretch-sensitive TRPV4 channels, induce release of osmolytes such as glutathione and taurine, and alter glutamine synthesis in astrocytes (Clark et al., 1996; Benfenati et al., 2011; Hyzinski-García et al., 2011; Schober and Mongin, 2015).  Interestingly, hyponatremia is of clinical relevance because it is associated with neuropathologies involving reactive astrogliosis such as traumatic brain injury, subarachnoid hemorrhage, and brain tumors (Kimelberg et al., 1993; Giuliani and Peri, 2014).  Since the data from Chapter 3 indicated that Piezo1 is also localized to components of the astrocyte cytoskeleton, we wondered whether this association is also involved in mediating Piezo1 responses to astrocyte swelling.  Experimental evidence indicates that the sensitivity of Piezo1 to mechanical forces is influenced by interactions with the cytoskeleton and extracellular matrix (ECM) (Bavi et al., 2017).  However, these interactions are complex; on one hand, the cytoskeleton may act as a physical barrier to shield the channels from mechanical stress, but on 95  the other hand, cytoskeletal proteins may also activate Piezo1 by transducing mechanical forces via direct physical coupling with the channel (Bavi et al., 2017; Nourse and Pathak, 2017).  In support of the idea that the cytoskeleton is mechanoprotective, Cox et al. (2016) patched Piezo1 channels in membrane blebs that lacked cytoskeleton, and showed that the pressure threshold for Piezo1 activation was lowered.  This result was consistent with the finding that deletion of filamin A, a scaffold protein linking actin filaments to membrane proteins, increased pressure-induced Piezo1 activation in smooth muscle cells (Retailleau et al., 2015).  In contrast, other studies have demonstrated mechanosensitive channel activation in endothelial cells in response to direct actin cytoskeleton stimulation (Hayakawa et al., 2008), and mechanically-activated Piezo1 currents were reduced by actin cytoskeleton disruption with cytochalasin D treatment (Gottlieb et al., 2012).  Therefore, it is likely that factors such as cell type and mode of mechanical stimulus act in concert to determine the contribution of the cytoskeleton to mechanosensing.  In this chapter, we aimed to test the hypothesis that hypotonicity-induced astrocyte swelling can activate Piezo1 by combining Ca2+ imaging in Lck-GCaMP5-transfected astrocytes with Piezo1 siRNA treatment in response to hyponatremia.  We also used forskolin-induced cytoskeleton reorganization as a tool to explore the relevance of Piezo1 – cytoskeleton interactions in hypoosmotic stress-mediated cell swelling and channel activation.    96  4.2 Methods  4.2.1 Cell culture and transfection Astrocyte cultures were prepared as described in section 2.2.2.  Cells were plated at a density of ~9 x 103 cells / mL onto sterile 15 mm No. 1.5 glass coverslips (MatTek) that were coated with collagen IV (50 µg/mL; Corning).  Previous studies have found that cells in vitro respond best when grown in substrates that mimic their physiological extracellular matrix (ECM) (Kleinman et al., 1987).  Our data suggest that astrocyte endfeet processes may be a possible site of concentrated Piezo1 expression in the brain (see Sections 2.3.7 and 3.3.5), and imaging studies have shown that astrocyte endfeet are closely associated with the basal lamina of endothelial cells surrounding cerebral capillaries (Kacem et al., 1998).  Collagen IV, an abundant ECM protein of the basement membrane, has been proposed to be a major component of the matrix at the endfeet – endothelial cell interface (Benarroch, 2015).  Therefore, astrocytes were plated on collagen IV-coated coverslips to allow osmotic stress-induced Piezo1 activation to be studied in a condition that better mimics the physiological ECM environment.  Cells were grown for 3 to 5 days in a 37°C incubator with 5% CO2 in glia media.  Cultures were transfected with LNP-siRNA and LNP-plasmid complexes as described in section 2.2.6.  In experiments with forskolin treatment, cultures were incubated with serum-free media for 1 hour followed by 1-hour incubation with forskolin (1 µM; Sigma) prior to imaging.  4.2.2 Culture imaging Cells were imaged as described in section 2.2.10.  Transmitted light images were collected using infrared differential interference contrast optics.  Experiments were performed at room 97  temperature with continuous perfusion (2.5 mL/min) of HEPES-aCSF (300 mOsm).  Hypotonic solution (150 mOsm) contained (in mM): NaCl, 59.9; HEPES, 10.8; KCl, 2.5; glucose, 10; MgCl2, 2.0; CaCl2, 2.0.    4.2.3 Data analysis and statistics Astrocyte Ca2+ microdomain signals were isolated and analyzed as described in section 2.2.14.  In contrast, Ca2+ transients in forskolin-treated cells were localized to segments of astrocyte processes rather than presenting as circular ROIs.  Discrete Ca2+ events that had connected pixels in the x, y, and t axes were therefore identified by the 3D Connected Components function in Matlab to allow the total number of events to be quantified per unit time.  Hyponatremia-induced astrocyte swelling was analyzed as the percent increase in cross-sectional area of the soma relative to the baseline in isotonic solution.  Statistics were performed as described in section 2.2.15, where unpaired t-tests were used to compare the significance of difference in means between two groups, while paired t-tests were used to compare different treatments (i.e. isotonic versus hypotonic) conducted on the same cell.  Data are reported as mean ± 95% confidence interval, and p < 0.05 was considered statistically significant.  4.3 Results  4.3.1 Piezo1 mediates Ca2+ microdomain signals in astrocytes under hypotonic stress Astrocytes have been shown to respond to osmotic stress by elevating cytosolic Ca2+ as measured by bulk-loaded dyes such as Fluo-4 AM and Fura-2 AM (Hua et al., 2010; Mola et al., 2016).  To determine whether Ca2+ microdomain transients are also affected by hypotonic stimulus, we 98  performed Ca2+ imaging in Lck-GCaMP5-transfected astrocytes that were exposed to hyponatremia for 10 minutes.  We found that perfusion of hypotonic solution increased the frequency of Ca2+ microdomain signals by three-fold (baseline: 0.71 ± 0.27 events/ROI/5 min; hypotonic: 2.17 ± 0.86 events/ROI/5 min; p < 0.01)(Fig. 4-1A and B).  Furthermore, these evoked Ca2+ signals were inhibited by Piezo1 siRNA treatment compared to the luciferase control (luciferase baseline: 0.5 ± 0.5 events/ROI/5 min; luciferase hypotonic: 1.2 ± 0.8 events/ROI/5 min; p < 0.01; Piezo1 baseline: 0.7 ± 0.4 events/ROI/5 min; Piezo1 hypotonic: 0.8 ± 0.4 events/ROI/5 min; p > 0.05)(Fig. 4-1A, C and D).  Together, the data indicate that hyponatremia-induced Ca2+ microdomain transients are mediated by Piezo1.        99   Figure 4-1.  Hypotonic solution induces an increase in astrocyte microdomain Ca2+ signals that is inhibited by Piezo1 siRNA treatment. A) Images represent five-minute maximum intensity projections of microdomain Ca2+ signals in Lck-GCaMP5-transfected astrocyte cultures before and after hypotonic challenge.  Astrocytes were either untreated (control) or exposed to luciferase or Piezo1 siRNA (0.125 µg/mL) for five days prior to imaging. Scale bars = 25 µm.  B) Quantification of the effect of hypotonic solution on the number of microdomain Ca2+ signals in B) untreated astrocytes (n = 11 cells), C) cells treated with luciferase siRNA (n = 6 cells), and D) cells treated with Piezo1 siRNA (n = 7 cells).  ** p < 0.01 compared to baseline in isotonic solution by one-tailed paired t-test; data represent mean ± 95% CI.  4.3.2 Forskolin-induced reorganization of the cytoskeleton does not alter the pharmacological profile of Piezo1 or affect Piezo1-mediated Ca2+ transients to hypotonic challenge To examine whether cytoskeleton-dependent Piezo1 activation may underlie the Ca2+ transients induced by hypotonic stimulation, we used forskolin as a tool to induce cytoskeleton 100  reorganization.  Previous immunostaining results indicated that forskolin treatment induces remodeling of actin stress fibers and loss of focal adhesions in astrocytes, as well as Piezo1 redistribution at the plasma membrane (section 3.3.4).  Here we observed that forskolin-treated astrocytes exhibited spontaneous Ca2+ transients as detected by Lck-GCaMP5, but these transients were localized along astrocyte processes instead of showing the circular microdomain phenotype observed in control cells.  Therefore, discrete Ca2+ events were quantified by 3D connected components (Fig. 4-2A) rather than identifying microdomains based on fluorescence intensity of 6 x 6-pixel ROIs (Fig. 2-3D).  The frequency of Ca2+ transients was increased by application of the Piezo1 agonist Yoda1 and blocked by subsequent addition of Gd3+ (baseline: 43 ± 66 events/5 min; Yoda1: 203 ± 78 events/5 min; Yoda1 + Gd3+: 63 ± 61 events/5 min; p < 0.01), suggesting that the channels are functional, independent of cytoskeleton organization (Fig. 4-2B and C).     101   Figure 4-2.  Forskolin treatment does not affect the pharmacological profile of Piezo1. A) Astrocyte cultures were treated with forskolin (1 µM) for one hour before imaging.  The left panel shows the maximum intensity projection of a 20-minute recording of spontaneous Ca2+ signals as detected by Lck-GCaMP5 fluorescence.  The signals were analyzed by 3D connected components as shown in the right panel to account for the change in ROI morphology.  Each colour represents a discrete Ca2+ transient signal, defined by pixels connected in the x, y, and t axes.  B) Images represent 10-minute maximum intensity projections of the microdomain Ca2+ signals in Lck-GCaMP5-transfected astrocyte cultures before and after treatment with Yoda1 (1 µM) and Gd3+ (100 µM).  C) Quantification of the effect of Yoda1 and Gd3+ on the frequency of astrocyte Ca2+ signals.  Scale bars = 50 µm.  n = 5 cells from independent coverslips.  ** p < 0.01 by one-way ANOVA with Bonferroni’s multiple comparison post hoc test.  Next, Lck-GCaMP5-transfected astrocytes were treated with forskolin and imaged during a 10-minute application of hypotonic solution.  Hypotonic stimulation increased the frequency of Ca2+ transients (baseline: 86 ± 56 events/5 min; hypotonic: 97 ± 58 events/5 min; p < 0.05)(Fig. 4-3A and B), and these signals were sensitive to Piezo1 siRNA treatment compared to the luciferase 102  control (luciferase baseline: 78 ± 61 events/5 min; luciferase hypotonic: 131 ± 63 events/5 min; p < 0.05; Piezo1 baseline: 79 ± 27 events/5 min; Piezo1 hypotonic: 87 ± 21 events/5 min; p > 0.05)(Fig. 4-3A, C and D).  Taken together, the data indicate that Ca2+ transients evoked by hypotonic challenge are mediated by Piezo1, and may be independent of the interaction between the channel and the cytoskeleton.  Figure 4-3.  Hypotonic solution induces an increase in forskolin-treated astrocyte microdomain Ca2+ signals that is inhibited by Piezo1 siRNA treatment. A) Images represent five-minute maximum intensity projections of the microdomain Ca2+ signals in Lck-GCaMP5-transfected astrocyte cultures before and after hypotonic challenge.  Signals are depicted as the output of the 3D connected components analysis, where different colours represent discrete events.  Astrocytes were either untreated (control) or exposed to luciferase or Piezo1 siRNA (0.125 µg/mL) for five days, then treated with forskolin (1 µM) for one hour prior to imaging.  Scale bars = 25 µm.  Quantification of the effect of hypotonic solution on the number of microdomain Ca2+ signals in B) untreated astrocytes (n = 9 cells), C) cells treated with luciferase siRNA (n = 5 cells), and D) cells treated with Piezo1 siRNA (n = 8 cells).  * p < 0.05 compared to baseline in isotonic solution by one-tailed paired t-test; data represent mean ± 95% CI. 103  4.3.3 Piezo1 mediates hypotonicity-induced swelling in astrocytes Our observation that hypotonic challenge can evoke astrocyte Ca2+ transients is intriguing, as previous studies have suggested that these signals may act as a trigger for the homeostatic mechanism known as regulatory volume decrease (RVD) (O’Connor and Kimelberg, 1993; Hua et al., 2010).  In RVD, ion channels or transporters are activated to decrease intracellular osmolarity in response to external hypotonicity, allowing cells to revert to resting volume to prevent cell lysis and death (Lang et al., 1998).  Therefore, to assess whether Piezo1-mediated Ca2+ transients are involved in regulation of cell volume in response to hypotonic challenge, the cross-sectional areas of astrocyte soma were measured during baseline in isotonic solution and following 10 minutes of perfusion with hyponatremia solution.  Since control cells did not appear to swell in the xy-plane, changes in cross-sectional area could not be measured.  Imaging was therefore performed in astrocytes that had been treated with forskolin (Fig. 4-4A).   Piezo1 siRNA treatment inhibited hyponatremia-mediated astrocyte swelling relative to the luciferase control (luciferase siRNA: 121 ± 16%; Piezo siRNA: 102 ± 9%; p < 0.05), indicating that Piezo1 may be required for astrocyte swelling in response to hypotonic stimulus (Fig. 4-4B). 104   Figure 4-4.  Piezo1 siRNA treatment inhibits hypotonicity-induced swelling in forskolin-treated astrocytes. A) Transmitted light images of astrocyte cultures treated with luciferase or Piezo1 siRNA (0.125 µg/mL; red puncta) for five days prior to imaging.  Luciferase siRNA-treated cells exhibit swelling in the soma and processes (highlighted by yellow arrowheads) in response to the hypotonic challenge, while the effect is inhibited by Piezo1 siRNA treatment, as quantified in B).  Scale bar = 50 µm.  n = 9 – 10 cells from independent coverslips.  * p < 0.05 compared to luciferase siRNA treatment group by one-tailed unpaired t-test; data represent mean ± 95% CI.  4.4 Discussion In this Chapter, we demonstrated that Piezo1 channels contribute to the hypotonicity-induced increase in astrocyte Ca2+ microdomain signals.  Our results suggest that activation of Piezo1 by osmotic stress was independent of interactions between the channel and the focal adhesions and actin stress fibers, as the increase in Ca2+ transients was preserved in the presence of stress fiber disorganization with forskolin treatment.  In addition, Piezo1 may be required for astrocyte swelling in response to hypotonic stimulus. 105  Our data indicate that in vitro hyponatremia treatment increases the frequency of microdomain Ca2+ signals in astrocytes via a Piezo1-dependent mechanism.  This result is consistent with earlier studies demonstrating that osmotic stress-induced cytosolic Ca2+ elevations, as detected by bulk-loaded Ca2+ indicators in astrocytes, is mediated by a Ca2+ influx pathway that is sensitive to the nonspecific mechanosensitive channel antagonists Gd3+ and ruthenium red (Liu et al., 2006; Benfenati et al., 2011; Galizia et al., 2012; Mola et al., 2016).  Interestingly, Benfenati et al. (2011) showed that the intracellular Ca2+ increases can also be inhibited by TRPV4 siRNA treatment, suggesting that TRPV4 is involved in mediating Ca2+ influx following hypotonic challenge.  While our previous data suggest that TRPV4 does not mediate spontaneous Ca2+ transients (Section 2.3.6), it is possible that TRPV4, or other mechanosensitive channels, are activated in addition to Piezo1 by astrocyte swelling.  To examine the contribution of the interaction between Piezo1 and the cytoskeleton to channel activation by hypotonic stress, we studied the effect of cytoskeleton disorganization with forskolin pre-treatment on the hypotonicity-induced Ca2+ microdomain signals.  Despite Piezo1 redistribution to the processes of stellate astrocytes, its pharmacology and role in mediating Ca2+ signals in response to hypotonic stimulus were unaltered by forskolin, suggesting that Piezo1 basal function and activation by osmotic swelling can occur independently of interactions with focal adhesion proteins or actin stress fibers.  This result is supported by previous studies showing Piezo1 activation by osmotic gradient when the channel was reconstituted in cytoskeleton-free lipid bilayers (Syeda et al., 2016).  However, our data do not rule out cytoskeleton involvement in astrocyte Piezo1 activation by other types of mechanical stimuli in vivo.   106  What is the functional significance of these hypotonicity-induced Ca2+ microdomain signals?  Previous studies have observed that astrocytes, when exposed to hypotonic solution, exhibit an initial swelling phase followed by a return to original cell volume in a homeostatic process termed regulatory volume decrease (RVD).  Several groups have proposed that RVD may be triggered by stretch-induced activation of mechanosensitive ion channels that then permit Ca2+ entry, either through the channels directly or by depolarizing the cell to activate voltage-gated Ca2+ channels.  Subsequent opening of Ca2+-activated channels and volume-regulated anion channels leads to net efflux of K+ and Cl- to regulate osmolyte balance across the membrane, allowing cells to revert to resting volume (Lang et al., 1993, 1998; O’Connor and Kimelberg, 1993).  Consistent with this idea, removal of extracellular Ca2+ has been shown to abolish hypotonicity-induced Ca2+ signals and RVD in astrocytes (O’Connor and Kimelberg, 1993; Hua et al., 2010).  However, other studies have indicated that RVD can occur independently of Ca2+ influx (Morales-Mulia et al., 1998; Mola et al., 2016).  Therefore, the role of Ca2+ in mediating RVD is still incompletely characterized.  We reasoned that if hypotonicity-induced Ca2+ microdomain transients were involved in mediating RVD, then inhibition of the Ca2+ signals with Piezo1 siRNA treatment may be associated with impaired volume homeostasis, leading to exacerbated cell swelling.  Surprisingly, our results indicated that swelling was inhibited by Piezo1 knockdown.  While Piezo1 activity may be required for astrocyte swelling, we did not observe any appreciable increases in cell volume following application of the Piezo1 agonist Yoda1 (Section 4.3.2), suggesting that Piezo1-mediated Ca2+ signaling alone does not induce cell swelling.  It is possible that Piezo1 may act synergistically with other molecules or cellular pathways as a 107  permissive signal for swelling.  Another possible explanation is that Piezo1 siRNA treatment may have altered aquaporin-4 expression, thereby inhibiting water influx and astrocyte swelling in response to hypoosmotic stress.  One limitation of our experiments is that the soma cross-sectional area may not sensitive enough to reflect true changes in cell volume, as swelling in the z-axis would not be measurable.  Other groups have loaded astrocytes with fluorescent dyes and quantified cell swelling as a function of dye intensity decrease (Liu et al., 2006; Benfenati et al., 2011; Mola et al., 2016).  However, the relationship between dye intensity and cell volume is only linear for a specific range, and dye efflux may occur due to active transport or leak when the cell is swollen (Kimelberg et al., 1993; Liu et al., 2006).  An alternate strategy relies on impedance-based methods to monitor cell volume in real time, where the electrical resistance of the cells is inversely related to cell volume (O’Connor et al., 1993; Hua et al., 2010).  These techniques may provide a more accurate measure of cell volume, particularly for flat polygonal astrocytes that do not appreciably swell in the xy-plane.  Another complexity of measuring RVD is the heterogeneity of the phenomenon.  For instance, reports of the time it took for RVD to return astrocytes to baseline volume varied from ~1 minute to ~30 minutes (Hua et al., 2010; Mola et al., 2016).  Furthermore, RVD dependence on Ca2+ influx is controversial, as some groups still observed RVD in the absence of external Ca2+ (Mola et al., 2016).  While the parameters underlying these differences is unclear, presentation of RVD may be affected by cell culture, experimental temperature, perfusion speed, magnitude of hypotonic challenge, and technique used to monitor cell volume.  Therefore, future studies can 108  be conducted to optimize the experimental parameters to examine the contribution of Piezo1 to RVD.  Intriguingly, previous studies have found that RVD in keratinocytes and CHO cells is dependent on interactions between TRPV4 and the actin cytoskeleton (Becker et al., 2009).  This suggests that RVD may be impaired in forskolin-treated astrocytes used in our experiments.  It would be interesting to study the contribution of Piezo1 to hypotonic-evoked RVD with astrocytes in vivo, where putative interactions between the cytoskeleton and the ECM are preserved.                    109  Chapter 5: Conclusion  5.1 Summary of research findings In this thesis, I provide evidence that the activation of mechanosensitive cation channel Piezo1 contributes to spontaneous microdomain Ca2+ signals in rodent astrocytes in both cell culture and acute brain slices.  The Piezo1 agonist Yoda1 increased the frequency of microdomain Ca2+ transients, while these were inhibited by the mechanosensitive channel antagonists GsMTx4 and Gd3+.  The pharmacological data were supported by the reduction of Yoda1-induced Ca2+ signals after siRNA-mediated Piezo1 knockdown.  Immunostaining indicated that Piezo1 expression is localized to subcellular compartments relevant to mechanosensation.  Piezo1 immunoreactivity was distributed as distinct plasma membrane clusters and associated with focal adhesion and stress fiber compartments in cultured astrocytes.  Piezo1 channels appeared to be concentrated within the endfeet processes of brain slice astrocytes.  Furthermore, Piezo1-mediated Ca2+ microdomain signals were activated by an osmotic stress model of astrocyte swelling.  Taken together, the data demonstrate that cultured and brain slice astrocytes express functional Piezo1 channels, suggesting that astrocyte Ca2+ signaling may play a role in integrating mechanical stimuli to regulate physiological processes in the brain.  5.2 Research significance Recent advances in techniques used to study astrocyte Ca2+ signaling have revealed a previously unappreciated type of spontaneous Ca2+ signal localized to the microdomains of fine astrocytic processes.  Recent work from our lab and others has demonstrated that these signals depend on transmembrane Ca2+ influx (Shigetomi et al., 2012; Rungta et al., 2016), but the underlying 110  molecular mechanisms have not been fully characterized.  In this dissertation, I combined Ca2+ imaging with siRNA-mediated gene silencing and pharmacological approaches in an astrocyte culture model, and identified the Piezo1 channel as a novel Ca2+ entry pathway that contributes to the Ca2+ microdomain signals.  Previous studies have shown that spontaneous Ca2+ transients modulate basal astrocyte Ca2+ levels, with implications for regulation of synaptic activity and arteriole tone (Shigetomi et al., 2012, 2013b; Rosenegger et al., 2015).  As Piezo1 is a mechanosensitive channel, this finding raises the intriguing possibility that astrocytes may serve as a transducer of mechanical stimuli to affect neuronal and vascular function in the brain.   Our discovery of functional Piezo1 channels in astrocytes extends the repertoire of mechanically activated channels expressed in these cells.  Piezo1 appears to share several characteristics with other astrocyte mechanosensitive channels; for instance, TRPV4 is also distributed in astrocyte endfeet, and mediates Ca2+ signals in response to osmotic stress (Benfenati et al., 2007, 2011).  However, one caveat to interpreting these previous studies is their use of non-selective pharmacological blockers such as Gd3+ and ruthenium red to assign function to a specific channel (Benfenati et al., 2007).  These tools block both TRPV4 and Piezo1 as well as other channels, and therefore cannot distinguish between the contributions of Piezo1 and TRPV4.  In our experiments, we showed that astrocyte Ca2+ microdomain transients in cultured astrocytes are sensitive to GsMTx4, but not to the selective TRPV4 antagonist HC067047, suggesting that different mechanosensitive channels may have distinct functions in the same cell.  This idea is supported by studies comparing different modes of mechanical stimulation in chondrocytes, which also express both TRPV4 and Piezo1; deflection of cell-substrate contact points activated both Piezo1- and TRPV4-mediated currents, while membrane stretch only activated Piezo1 111  (Servin-Vences et al., 2017).  Furthermore, Miyamoto et al. (2014) demonstrated that Piezo1 was more sensitive than TRPV4 in detecting stretch stimulation of urothelial cells as measured by intracellular Ca2+ elevations.  Therefore, it is likely that Piezo1 acts in a complementary manner with other mechanosensitive channels to refine astrocyte sensitivity to different types and magnitudes of mechanical force.  In this thesis, we presented a novel method for quantitative analysis of microdomain Ca2+ signals.  We observed that Lck-GCaMP5-transfected cultured astrocytes exhibited two types of spontaneous Ca2+ signals: transient, spatially restricted Ca2+ microdomain signals, and long-lasting Ca2+ waves that expanded and contracted from their point of origin.  Since these signals have different spatiotemporal properties and Ca2+ sources, we developed an algorithm to analyze them separately.  A key feature in our analysis algorithm is the use of the 3D connected components function in Matlab, which identified discrete Ca2+ signals and allowed them to be ranked according to total area.  Thus Ca2+ waves could be selectively masked using a size threshold while preserving the microdomain signals.  In addition, up to 35 distinct microdomains were observed in each cell, requiring the development of a strategy to objectively identify and analyze the Ca2+ signals from these regions.  An algorithm was written to automatically detect these regions of interest based on fluorescence intensity, define Ca2+ transients, and calculate their frequency, amplitude and duration.  Interestingly, our independently developed algorithm shares a number of similar features with recently published methods for quantifying microdomain signals (Srinivasan et al., 2015; Agarwal et al., 2017), and the convergence of the results further supports the reliability of our analysis approach.  However, our algorithm has not yet been tested on astrocytes in vivo, and it remains to be determined whether adaptations such as 112  masking the cell soma or large proximal processes are required to optimize the analysis for in vivo applications.    The immunostaining data provide the first evidence of Piezo1 distribution to subcellular localizations relevant to mechanosensing in cultured astrocytes.  We observed that Piezo1 immunoreactivity formed discrete clusters at the plasma membrane, where the channels are well-situated to detect membrane stretch.  These clusters bore a striking resemblance to the spontaneous and Yoda1-activated Ca2+ microdomain signals, and were sensitive to siRNA-mediated Piezo1 knockdown.  In addition, the data presented in Chapter 4 indicated that the hypoosmotic stress model of cell swelling was associated with increased frequency of Ca2+ microdomain transients, and that was inhibited by Piezo1 siRNA treatment.  Taken together, the data suggest that Piezo1 channels expressed in the astrocyte plasma membrane may function as a membrane stretch detector, where spontaneous Ca2+ signals may reflect basal Piezo1 activation by resting membrane tension and with changes in Ca2+ signaling frequency encoding information such as the rate, onset, or magnitude of cell swelling.    In cultured astrocytes, Piezo1 immunoreactivity was identified in subcellular compartments associated with focal adhesions and actin stress fibers.  We examined the relevance of this putative channel – cytoskeleton interaction to hypotonic stress-induced Piezo1 activation by treating the cells with forskolin.  Hypotonic stimulation of forskolin-treated astrocytes still increased microdomain Ca2+ signal frequency.  However, we did not observe differences in Piezo1-mediated Ca2+ microdomain signaling changes in response to hypotonic stimulation in forskolin-treated versus untreated cells, despite striking changes in astrocyte morphology, 113  redistribution of plasma membrane Piezo1-positive clusters to the processes, and loss of Piezo1 immunoreactivity distributed to focal adhesion and actin stress fiber structures.  As such, our data indicate that at least in cultured astrocytes, the interactions with focal adhesions and actin stress fibers are not a major contributor to Piezo1 activation by hypotonic stress, and suggest that Piezo1 may mediate different functions depending on its specific subcellular location.    Piezo1 function may also depend on the developmental or proliferative state of the cell.  This interesting observation was recently described in Madin Darby canine kidney epithelial cells by Gudipaty et al. (2017), where Piezo1 was shown to maintain epithelial cell number homeostasis by matching the rate of cell division to the rate of cell death.  Piezo1 was demonstrated to be involved in sensing crowding in regions of high cell density and stretching in regions of low cell density to stimulate cell extrusion and cell division, respectively (Gudipaty et al., 2017).  Therefore, Piezo1 may also mediate different functions at different timepoints of development, or following damage where astrocytes become reactive and proliferate.  These observations will guide further experiments on Piezo1 function under various physiological and pathological states in vivo.  Preliminary experiments described in Section 5.3 indicate that these are important considerations, as Piezo1 expression in astrocytes is upregulated during early postnatal development and after stroke damage.  5.3 Future directions The identification of Piezo1 as a mammalian mechanosensitive channel in 2010 has led to rapid progress in the field of mammalian mechanotransduction.  Increasingly higher resolution structures of Piezo1 provided a better understanding of the structural basis underlying channel 114  function (Guo and MacKinnon, 2017; Saotome et al., 2017; Zhao et al., 2018), and studies have begun to characterize protein partners that interact with Piezo1 to regulate channel activation (Zhang et al., 2017).  Recent studies have demonstrated novel roles for Piezo1 in mediating mechanosensation in different organ systems (Liang et al., 2017; Rode et al., 2017), and exciting possibilities exist for further research on the contributions of Piezo1 to physiological and pathological conditions.    5.3.1 Transgenic mice for investigating Piezo1-mediated Ca2+ signaling in vivo In this dissertation, we utilized an astrocyte culture model to test pharmacological and genetic tools for modulating Piezo1 activity, and to develop an analysis algorithm for quantifying their effects, culminating in the finding that Piezo1 contributes to spontaneous Ca2+ microdomain signals in astrocytes in vitro.  A next step to understanding the functional role of Piezo1 in astrocyte physiology is to determine whether Piezo1 also contributes to astrocyte microdomain Ca2+ signals in vivo.  Preliminary observations from our lab indicate that endfoot Ca2+ signaling is enhanced by the Piezo1 activator Yoda1 in brain slice astrocytes.  Furthermore, preliminary data from GCaMP7 mouse brain slices, where the GECI is expressed in both neurons and astrocytes, have shown spontaneous Ca2+ signals that are inhibited by Gd3+ in astrocyte compartments identified by SR101 staining.  Recently, Srinivasan et al. (2016) generated a transgenic mouse line in which the Lck-GCaMP6f GECI was expressed under the astrocyte-specific Aldh1l1 promoter, resulting in a mouse model where spontaneous Ca2+ microdomain transients could be monitored in fine astrocyte processes throughout the brain.  This mouse model is likely well-suited for examining whether these spontaneous signals are also sensitive to pharmacological treatments or siRNA-mediated knockdown targeting Piezo1 in vivo.   115  Previous studies have demonstrated that constitutive Piezo1 knockouts are embryonically lethal (Ranade et al., 2014).  Therefore, another important tool to study the contribution of Piezo1 to astrocyte function is the generation of transgenic mouse lines wherein conditional Piezo1 knockout can be achieved in astrocytes.  For example, the Aldh1l1 promoter line contains a Cre/ERT2 insertion at its start codon (Srinivasan et al., 2016) and this Aldh1l1-Cre/ERT2 mouse line could be crossed with mice developed by the Patapoutian lab in which Piezo1 is flanked by loxP sites, resulting in a mouse model where astrocyte-specific deletion of Piezo1 could induced by tamoxifen administration.  Thus, this model could be used to investigate the functional role of astrocyte Piezo1 at different developmental stages, as well as during different pathological states.  5.3.2 Role of Piezo1 in regulation of cerebral vasculature tone Our preliminary brain slice Ca2+ imaging and immunostaining suggest that astrocyte Piezo1 expression may be concentrated at endfeet processes that surround the cerebral vasculature.  As we consider the physiological role of Piezo1-mediated mechanotransduction in the context of the brain, it is tempting to speculate that Piezo1 may be involved in sensing mechanical forces exerted by dilating or constricting blood vessels, resulting in changes to astrocyte Ca2+ microdomain activity that can stimulate release of vasoactive compounds to regulate brain blood flow.  This idea is supported by studies demonstrating that disruption of astrocyte Ca2+ signals with BAPTA is associated with alterations in prostaglandin release and arteriole tone (Rosenegger et al., 2015).  The role of Piezo1 in detecting vascular changes can be studied by measuring the effect of agents, such as the α2 adrenoceptor agonist clonidine, that induce vasoconstriction by acting on smooth muscle cells.  The direct stimulation of vascular constriction by this method should stretch the astrocyte endfeet, and thereby would be predicted 116  to cause Piezo1-dependent endfoot Ca2+ transients in transgenic mice expressing Lck-GCaMP6f driven by the Aldh1l1 promoter.  These vasomotion-induced responses in astrocyte endfeet can then be compared to the responses of astrocytes where Piezo1 is pharmacologically blocked or genetically deleted.  5.3.3 Role of Piezo1 in astrocyte differentiation As described in Chapter 3, our observations of Piezo1 localization to focal adhesions and actin stress fibers in cultured astrocytes suggest a role for the mechanosensitive channel in detecting extracellular matrix (ECM) rigidity, and accumulating evidence indicates that this link to matrix mechanics may have important consequences for cell differentiation.  For instance, Pathak et al. (2014) showed that neural stem cells exhibit Piezo1-mediated spontaneous Ca2+ microdomain signals dependent on substrate stiffness, and pharmacological inhibition or siRNA-induced knockdown of Piezo1 resulted in increased differentiation into astrocytes and decreased neuron formation.  In addition, Sugimoto et al. (2017) demonstrated that the hydrostatic pressure of extracellular fluid induced differentiation of mesenchymal stem cells to osteoblasts, and this process required Piezo1-mediated regulation of bone morphogenetic protein 2 (BMP2) expression.  Interestingly, BMP2 upregulation has also been shown to promote differentiation of oligodendroglial-astroglial progenitor cells into astrocytes during development (Mabie et al., 1997).  Our lab has recently investigated Piezo1 expression in the brain using a transgenic mouse line where Piezo1 is fused to the fluorescent reporter tdTomato (Ranade et al., 2014), and the preliminary immunoblotting data indicate that Piezo1 expression levels are age-dependent.  Notably, Piezo1 expression was highest during early development (postnatal day 2), with levels in the cortex and hippocampus declining to the lower levels found in the adult by postnatal day 117  14 – 21; importantly, this early period of high Piezo1 expression correlates with an active stage of gliogenesis (Bushong et al., 2004; Freeman, 2010).  It is therefore plausible that Piezo1-mediated spontaneous Ca2+ transients may be relaying information about the mechanical properties of the ECM to guide astrocyte differentiation.  This hypothesis can be tested in the conditional astrocyte Piezo1 knockout mice, where tamoxifen injections can be administered at different timepoints throughout development to assess the contribution of Piezo1 to astrocyte differentiation.  Astrocytes from adult Aldh1l1-Lck-GCaMP6f mice can also be cultured on substrates of varying stiffness, followed by Ca2+ imaging in combination with Piezo1 pharmacology, to examine whether Piezo1 is involved in sensing ECM rigidity in mature astrocytes.  5.3.4 Role of Piezo1 in transmitter release Previous studies have indicated that activation of mechanosensitive channels can lead to downstream efflux of various transmitters.  For example, stretch stimulation of cultured astrocytes has been demonstrated to induce Ca2+ influx and endothelin-1 release, both of which were inhibited by the mechanosensitive channel inhibitor GsMTx4 (Ostrow et al., 2000).  Similarly, Piezo1-dependent Ca2+ elevation and ATP efflux have been identified in red blood cells (Cinar et al., 2015).  Preliminary studies from our lab suggest that treatment with the Piezo1 agonist Yoda1 is associated with ATP release from cultured astrocytes, but its mechanism of efflux and functional significance are currently unknown.  ATP has been shown to contribute to various types of astrocyte Ca2+ signaling in vivo, such as the spontaneous Ca2+ waves in Bergmann glia of the cerebellum (Nimmerjahn et al., 2009), raising the possibility that Piezo1 activity may also be involved in mediating different Ca2+ signals in distinct brain regions.  118  However, the physiological stimuli that evoke Piezo1-mediated ATP release from in vivo astrocytes remain to be determined.    5.3.5 Piezo1 and pathology Piezo1 was initially observed in astrocytes associated with β-amyloid plaques (Satoh et al., 2006).  Although its function in Alzheimer’s disease pathology has not been characterized, Piezo1 expression in astrocytes has since been found in other pathological conditions.  For example, Choi et al. (2015) performed quantitative PCR (qPCR) on astrocytes isolated from the optic nerve head and observed an increase in Piezo1 mRNA three days following optic nerve crush injury.  Furthermore, using a photothrombotic stroke model, our lab has found elevated levels of Piezo1 mRNA and protein in the glial scar astrocytes lining the borders of the infarct tissue, as quantified by qPCR, immunoblotting, and immunohistochemistry seven days post-stroke.  Given the increase in Piezo1 expression in various pathological states, it is possible that Piezo1 is upregulated in reactive astrocytes as a general response to CNS injury.    Reactive gliosis encompasses a broad spectrum of cellular responses that ranges from reversible gene expression changes to formation of glial scars and permanent tissue reorganization (Sofroniew and Vinters, 2010).  While glial scars are considered to be an adaptive response for creating a barrier around wounded brain tissue, it is unclear how reactive astrocytes determine the boundaries of the scar (Burda et al., 2016).  Interestingly, previous studies have identified Piezo1 as an activator of integrin receptors; these molecules are involved in cell – extracellular matrix adhesion, and have also been implicated in cell migration (McHugh et al., 2010; Huttenlocher and Horwitz, 2011).   Indeed, siRNA-mediated Piezo1 knockdown has been 119  associated with altered migration of cancer cells (McHugh et al., 2012; Yang et al., 2014).  Furthermore, Piezo1 has been shown to regulate retinal ganglion cell axonal pathfinding by sensing brain stiffness and promoting axonal growth toward softer tissue (Koser et al., 2016).  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