Medicine, Faculty of
Medical Genetics, Department of
DSpace
UBCV
Petkau, Terri Lynn
Doctor of Philosophy - PhD
University of British Columbia
Frontotemporal lobar degeneration (FTLD) is a devastating, late-onset neurodegenerative disorder that causes profound behavioral abnormalities, language impairment, and alterations in personality in affected patients. As many as 40% of cases have a family history, and of these approximately 25% are due to loss-of-function mutations in the progranulin (GRN) gene. Progranulin-mediated FTLD, inherited in an autosomal dominant manner, is caused by haploinsufficiency. The purpose of this thesis was to characterize a novel mouse model of progranulin deficiency in order to study the role of progranulin in the brain and to understand how progranulin deficiency leads to neurodegeneration. The expression pattern of progranulin in the murine brain was examined using a lacZ reporter transgenic mouse line and immunohistochemistry. Progranulin is expressed at varying levels in all regions of the brain examined. Expression is strongest in the thalamus and hippocampus, moderate in the cortex, and low in the striatum and cerebellum. Double immunofluorescent labeling identified progranulin expression in mature neurons and microglia, the resident immune cells of the brain. Subsequently, a mouse model of progranulin deficiency was generated and characterized for abnormalities in behavior and neuropathology. The tests and outcome measures used were designed to reflect the behavioral and neuropathological changes observed in FTLD patients carrying GRN mutations. Heterozygous loss of progranulin produced no measurable alterations in behavior or pathology. Homozygous loss of progranulin in mice caused subtle behavioral abnormalities, intense microgliosis and astrogliosis throughout the brain, and exaggerated deposition of the aging pigment lipofuscin. Strain-dependent differences in the penetrance and expressivity of these phenotypes was observed, indicating that progranulin-dependent neurodegenerative phenotypes are modified by genetic background. In conclusion, progranulin-deficient mice develop neuropathological phenotypes that indicate increased neuronal stress and a decline in overall brain health that progresses with age. Although dramatic behavioral changes and overt neuronal loss similar to what is seen FTLD patients is not apparent in progranulin-deficient mice, the presence of robust, age-dependent neurodegenerative abnormalities makes progranulin knockout mice a useful model for certain aspects of progranulin-dependent FTLD.
THE ROLE OF PROGRANULIN IN THE BRAIN by Terri Lynn Petkau B.Sc., The University of Calgary, 2003 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate and Postdoctoral Studies (Genetics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2013 ? Terri Lynn Petkau, 2013 ii Abstract Frontotemporal lobar degeneration (FTLD) is a devastating, late-onset neurodegenerative disorder that causes profound behavioral abnormalities, language impairment, and alterations in personality in affected patients. As many as 40% of cases have a family history, and of these approximately 25% are due to loss-of-function mutations in the progranulin (GRN) gene. Progranulin-mediated FTLD, inherited in an autosomal dominant manner, is caused by haploinsufficiency. The purpose of this thesis was to characterize a novel mouse model of progranulin deficiency in order to study the role of progranulin in the brain and to understand how progranulin deficiency leads to neurodegeneration. The expression pattern of progranulin in the murine brain was examined using a lacZ reporter transgenic mouse line and immunohistochemistry. Progranulin is expressed at varying levels in all regions of the brain examined. Expression is strongest in the thalamus and hippocampus, moderate in the cortex, and low in the striatum and cerebellum. Double immunofluorescent labeling identified progranulin expression in mature neurons and microglia, the resident immune cells of the brain. Subsequently, a mouse model of progranulin deficiency was generated and characterized for abnormalities in behavior and neuropathology. The tests and outcome iii measures used were designed to reflect the behavioral and neuropathological changes observed in FTLD patients carrying GRN mutations. Heterozygous loss of progranulin produced no measurable alterations in behavior or pathology. Homozygous loss of progranulin in mice caused subtle behavioral abnormalities, intense microgliosis and astrogliosis throughout the brain, and exaggerated deposition of the aging pigment lipofuscin. Strain-dependent differences in the penetrance and expressivity of these phenotypes was observed, indicating that progranulin-dependent neurodegenerative phenotypes are modified by genetic background. In conclusion, progranulin-deficient mice develop neuropathological phenotypes that indicate increased neuronal stress and a decline in overall brain health that progresses with age. Although dramatic behavioral changes and overt neuronal loss similar to what is seen FTLD patients is not apparent in progranulin-deficient mice, the presence of robust, age-dependent neurodegenerative abnormalities makes progranulin knockout mice a useful model for certain aspects of progranulin-dependent FTLD. iv Preface All of the work presented henceforth was conducted at the Center for Molecular Medicine and Therapeutics, part of the Child and Family Research Institute at the University of British Columbia, Children?s and Women?s Hospital campus. All projects and associated methods were approved by the University of British Columbia?s Animal Care Committee (Certificate # A09-0673). Chapter 2: Figures 2-1 and 2-3 as well some of the text describing the methodology for generation of progranulin-targeted mice have been published in Petkau TL, Neal SJ, Milnerwood A, Mew A, Hill AM, Orban P, Gregg J, Lu G, Feldman HH, Mackenzie IR, Raymond LA, Leavitt BR. Synaptic dysfunction in progranulin-deficient mice. Neurobiol Dis. 2012 Feb;45(2):711-22 (Reproduced with permission, license no. 3205010630134). Figure 2-4 and some of the text describing the validation of the lacZ reporter mice and antibody specificity have been published in Petkau TL, Neal SJ, Orban PC, MacDonald JL, Hill AM, Lu G, Feldman HH, Mackenzie IR, Leavitt BR. Progranulin expression in the developing and adult murine brain. J Comp Neurol. 2010 Oct 1;518(19):3931-47 (Reproduced with permission, license no. 3204971135557). The text describing the generation of constitutive Grn-null mice was published as Petkau TL, Zhu S, Lu G, Fernando S, Cynader M, Leavitt BR. Sensitivity to neurotoxic stress is not increased in progranulin-deficient mice. Neurobiol Aging. 2013 Nov;34(11):2548-50 (Reproduced v with permission, license no. 3217620497041). S.J. Neal was responsible for all aspects of construct design and assembly in the production of progranulin-targeted mice. I performed all experiments for the validation of progranulin deficiency in targeted mice and for the expression of the lacZ reporter gene. Chapter 3: A version of Chapter 3 has been published as Petkau TL, Neal SJ, Orban PC, MacDonald JL, Hill AM, Lu G, Feldman HH, Mackenzie IR, Leavitt BR. Progranulin expression in the developing and adult murine brain. J Comp Neurol. 2010 Oct 1;518(19):3931-47 (Reproduced with permission, license no. 3204971135557). I contributed to experimental design and was responsible for all major areas of data collection and analysis, and all of the manuscript composition. S.J. Neal created the constructs for the generation of progranulin-targeted mice and assisted with antibody validation. P.C. Orban provided the backbone vector for generation of the targeting construct. J.L. MacDonald assisted with antibody validation and Western blots. A.M. Hill acted as a second, blinded investigator semi-quantitative expression analysis. G. Lu provided performed intra-striatal quinolinic acid injections. H.H. Feldman and I.R. Mackenzie critically reviewed the manuscript. B.R. Leavitt aided in the design of experiments and provided critical review of the manuscript. vi Chapter 4: A version of Chapter 4 has been published as Petkau TL, Neal SJ, Milnerwood A, Mew A, Hill AM, Orban P, Gregg J, Lu G, Feldman HH, Mackenzie IR, Raymond LA, Leavitt BR. Synaptic dysfunction in progranulin-deficient mice. Neurobiol Dis. 2012 Feb;45(2):711-22 (Reproduced with permission, license no. 3205010630134). I contributed to experimental design and was responsible for all major areas of data collection and analysis, and the majority of manuscript composition. S.J. Neal designed the targeting construct used to make the conditional progranulin-knockout mice used for this work. A. Milnerwood performed the electrophysiology experiments and wrote the relevant section of the manuscript. A. Mew traced cells for the analysis of neuronal morphology. A.M. Hill and J. Gregg performed behavioral analysis on heterozygous progranulin-knockout mice. P. Orban provided the backbone vector for generation of the targeting vector. G. Lu provided technical assistance in perfusing mice for collection. H.H. Feldman, I.R. Mackenzie, and L.A. Raymond critically reviewed the manuscript. B.R. Leavitt aided in the design of experiments and provided critical review of the manuscript. Chapter 5: Chapter 5 contains unpublished data that is part of a manuscript in preparation. I contributed to experimental design and was responsible for all major areas of data collection and analysis, and manuscript composition. K. Lilly performed a portion of the behavioral testing. G.Lu perfused the mice that were collected for vii neuropathology. C. Chen assisted with cryosectioning brains for neuropathology. A.M. Hill performed stereological analysis of brain volume and neuronal counts. viii Table of Contents Abstract ...........................................................................................................................ii ?Preface ...........................................................................................................................iv ?Table of Contents ........................................................................................................viii ?List of Tables ...............................................................................................................xiv ?List of Figures...............................................................................................................xv ?Acknowledgements ..................................................................................................xviii ?Dedication ....................................................................................................................xix ?Glossary of Terms........................................................................................................xx ?1 ? Introduction...............................................................................................................1 ?1.1 ? Frontotemporal lobar degeneration............................................................................. 1 ?1.1.1 ? Clinical presentations................................................................................................ 2 ?1.1.2 ? Neuroimaging features.............................................................................................. 2 ?1.1.3 ? Neuropathology......................................................................................................... 3 ?1.1.4 ? Genetics of FTLD...................................................................................................... 5 ?1.1.5 ? FTLD due to GRN mutations..................................................................................... 9 ?1.1.6 ? GRN genetic variability in other neurodegenerative disorders................................ 11 ?1.1.7 ? Genetic variation contributing to incomplete penetrance in GRN mutation carriers 16 ?1.1.8 ? The clinical and neuropathological phenotype of FTLD due to GRN mutations ..... 19 ? ix 1.1.9 ? Conclusions............................................................................................................. 23 ?1.2 ? Progranulin................................................................................................................... 23 ?1.2.1 ? Progranulin structure and function .......................................................................... 24 ?1.2.2 ? Regulation of progranulin expression...................................................................... 29 ?1.2.3 ? Peripheral functions of progranulin ......................................................................... 33 ?1.2.4 ? Progranulin in the immune system.......................................................................... 35 ?1.2.5 ? Progranulin in cancer biology.................................................................................. 40 ?1.2.6 ? Progranulin and its interacting partners .................................................................. 42 ?1.2.7 ? Use of mouse models in brain diseases ................................................................. 44 ?1.2.8 ? Mouse models of FTLD........................................................................................... 46 ?1.2.9 ? Mouse models of progranulin deficiency................................................................. 49 ?1.3 ? Objectives..................................................................................................................... 50 ?2 ? Generation of Progranulin-deficient Mice ............................................................52 ?2.1 ? Introduction .................................................................................................................. 52 ?2.2 ? Materials and methods................................................................................................ 56 ?2.2.1 ? Progranulin targeting vector design ........................................................................ 56 ?2.2.2 ? Generation of Grn-targeted mice using the pFleo gene-trap vector........................ 61 ?2.2.3 ? Generation of ?floxed? Grn-targeted mice ................................................................ 61 ?2.2.4 ? Generation of constitutive Grn-null mice ................................................................. 62 ?2.2.5 ? Real-time quantitative PCR..................................................................................... 63 ?2.2.6 ? Tissue preparation .................................................................................................. 64 ?2.2.7 ? Immunohistochemistry ............................................................................................ 64 ?2.2.8 ? Competition assay................................................................................................... 65 ? x 2.2.9 ? Western blot analysis.............................................................................................. 66 ?2.2.10 ? Progranulin ELISA................................................................................................. 67 ?2.3 ? Results .......................................................................................................................... 67 ?2.3.1 ? Characterization of GrnKO mice carrying the pFleo allele ...................................... 67 ?2.3.2 ? Confirmation of lacZ expression and antibody specificity ....................................... 70 ?2.3.3 ? Characterization of constitutive Grn+/- and Grn-/- mice............................................. 74 ?2.4 ? Discussion.................................................................................................................... 76 ?3 ? Progranulin expression in the brain .....................................................................78 ?3.1 ? Introduction .................................................................................................................. 78 ?3.2 ? Materials and methods................................................................................................ 82 ?3.2.1 ? Generation of knock-in reporter Grn-targeted mice ................................................ 82 ?3.2.2 ? Preparation of tissue sections................................................................................. 82 ?3.2.3 ? Immunohistochemistry ............................................................................................ 82 ?3.2.4 ? Antibody characterization........................................................................................ 83 ?3.2.5 ? X-gal staining to detect ?-galactosidase activity ..................................................... 87 ?3.2.6 ? Semiquantitative evaluation of regional expression ................................................ 87 ?3.2.7 ? Intra-striatal injection of quinolinic acid ................................................................... 89 ?3.2.8 ? Image collection ...................................................................................................... 89 ?3.2.9 ? Quantification of immunofluorescent intensity......................................................... 90 ?3.3 ? Results .......................................................................................................................... 90 ?3.3.1 ? Progranulin is expressed in microglia but not in neuronal progenitors in the developing CNS. ................................................................................................................. 91 ?3.3.2 ? Progranulin is expressed in multiple regions of the adult brain............................... 99 ? xi 3.3.3 ? Progranulin is expressed in neurons and microglia but not astrocytes. ................ 105 ?3.3.4 ? Progranulin expression is up-regulated in activated microglia. ............................. 107 ?3.3.5 ? Progranulin expression in neurons is marginally down-regulated after physical injury?.............................................................................................................................. 111 ?3.4 ? Discussion.................................................................................................................. 111 ?3.5 ? Additional discussion from subsequent literature................................................. 118 ?4 ? Synaptic dysfunction in progranulin-deficient mice .........................................122 ?4.1 ? Introduction ................................................................................................................ 122 ?4.2 ? Materials and methods.............................................................................................. 124 ?4.2.1 ? Mice ...................................................................................................................... 124 ?4.2.2 ? Immunohistochemistry .......................................................................................... 124 ?4.2.3 ? Behavioral tests .................................................................................................... 125 ?4.2.4 ? Electrophysiology.................................................................................................. 128 ?4.2.5 ? Neuronal morphology............................................................................................ 130 ?4.2.6 ? Image analysis ...................................................................................................... 131 ?4.2.7 ? Statistical analysis................................................................................................. 131 ?4.3 ? Results ........................................................................................................................ 132 ?4.3.1 ? Generation of progranulin knockout mice ............................................................. 132 ?4.3.2 ? Behavior at 8 months of age in GrnKO mice......................................................... 132 ?4.3.3 ? Electrophysiology.................................................................................................. 138 ?4.3.4 ? Neuronal morphology............................................................................................ 144 ?4.3.5 ? Neuropathology..................................................................................................... 151 ?4.4 ? Discussion.................................................................................................................. 154 ? xii 4.5 ? Additional discussion from subsequent literature................................................. 161 ?5 ? Genetic background modifies neuropathology in mice deficient for the FTLD-associated protein progranulin.................................................................................167 ?5.1 ? Introduction ................................................................................................................ 167 ?5.2 ? Materials and methods.............................................................................................. 171 ?5.2.1 ? Mice ...................................................................................................................... 171 ?5.2.2 ? Behavioral testing ................................................................................................. 171 ?5.2.3 ? Immunohistochemistry .......................................................................................... 174 ?5.2.4 ? Analysis of regional brain volumes ....................................................................... 175 ?5.2.5 ? Stereological estimation of total cell number......................................................... 176 ?5.2.6 ? Image analysis ...................................................................................................... 176 ?5.2.7 ? Analysis of neuronal morphology.......................................................................... 177 ?5.2.8 ? Statistical analysis................................................................................................. 177 ?5.3 ? Results ........................................................................................................................ 177 ?5.3.1 ? Longitudinal analysis of survival and behavior...................................................... 177 ?5.3.2 ? Lipofuscinosis in progranulin-knockout mice is penetrant on both background strains and most prominent in the thalamus ..................................................................... 183 ?5.3.3 ? Astrogliosis and microgliosis are exaggerated in progranulin-knockout mice....... 188 ?5.3.4 ? Neuroinflammation in progranulin knockout mice is exaggerated on the B6 background ....................................................................................................................... 192 ?5.3.5 ? Neuronal loss in Grn-/- mice is present on the B6 background only ...................... 195 ?5.3.6 ? Neuronal morphology in the hippocampus is significantly altered in Grn-/- mice on the 129 background only................................................................................................... 200 ? xiii 5.4 ? Discussion.................................................................................................................. 204 ?6 ? Discussion.............................................................................................................214 ?6.1 ? Progranulin-targeted mice as a model of FTLD...................................................... 214 ?6.2 ? Progranulin-targeted mice as a tool to study progranulin biology....................... 216 ?6.3 ? Future directions ....................................................................................................... 220 ?6.4 ? Conclusion ................................................................................................................. 225 ?Bibliography ...............................................................................................................228 ? xiv List of Tables Table 3-1 Antibodies used for Immunohistochemistry ...................................................84 ?Table 3-2 Semi-quantitative analysis of progranulin expression in the adult murine brain......................................................................................................................................100 ?Table 5-1 Summary of behavior testing schedule for both B6 and 129 cohorts. .........173 ?Table 5-2 Summary of strain-dependent neuropathological phenotypes in Grn-/- mice.......................................................................................................................................205 ?Table 6-1 Cre-expressing transgenic mice ..................................................................223 ? xv List of Figures Figure 1-1 A summary of the subtypes of FTLD and their respective underlying pathologies and genetics..................................................................................................7 ?Figure 1-2 Organization and conservation of the progranulin gene...............................25 ?Figure 1-3 The structure of the human progranulin gene and protein. ..........................27 ?Figure 2-1 Generation of the gene-trap ?pFleo? Grn-targeting vector.............................59 ?Figure 2-2 Schematic of the targeted alleles that can be generated from the pFleo construct. ........................................................................................................................60 ?Figure 2-3 Characterization of progranulin-targeted mice. ............................................69 ?Figure 2-4 Specificity of progranulin antibody and X-gal staining..................................73 ?Figure 3-1 Progranulin is expressed at low levels in the developing brain. ...................94 ?Figure 3-2 Progranulin is not expressed in neuronal progenitors in the embryonic brain.........................................................................................................................................96 ?Figure 3-3 Progranulin is expressed in amoeboid microglia in the developing CNS. ....98 ?Figure 3-4 Progranulin is expressed in multiple regions in the adult brain. .................102 ?Figure 3-5 Immunoblot analysis of progranulin expression. ........................................104 ?Figure 3-6 Progranulin is expressed in neurons and microglia but not in astrocytes. .106 ?Figure 3-7 Progranulin expression is up-regulated in quinolinic acid-injected striatum.......................................................................................................................................108 ? xvi Figure 3-8 Progranulin expression is up-regulated in individual microglia in quinolinic acid-injected striatum....................................................................................................110 ?Figure 4-1 Progranulin-deficient mice show sex-specific behavioral abnormalities at 8 months of age...............................................................................................................137 ?Figure 4-2 Reduced hippocampal synaptic connectivity and impaired LTP in GrnKO male mice. ....................................................................................................................143 ?Figure 4-3 Progranulin-deficient CA1 pyramidal neurons display decreased dendritic length in the apical dendritic arbor in vivo. ...................................................................148 ?Figure 4-4 Progranulin-deficient CA1 hippocampal neurons display decreased spine density on the apical dendrite.......................................................................................150 ?Figure 4-5 Progranulin-deficient mice exhibit neuropathological changes at 18 months of age............................................................................................................................153 ?Figure 5-1 Longitudinal analysis of behavior did not reveal any significant or progressive differences between Grn+/+ and Grn-/- mice. .............................................181 ?Figure 5-2 Lipofuscinosis is exaggerated in Grn-/- mice on both the B6 and 129 backgrounds. ................................................................................................................185 ?Figure 5-3 Lipofuscinosis in Grn-/- mice is exaggerated in brain regions with high progranulin expression in Grn+/+ mice. .........................................................................187 ?Figure 5-4 GFAP immunoreactivity is significantly increased in all brain regions of Grn-/- mice compared to Grn+/+ mice on both backgrounds. ..................................................189 ? xvii Figure 5-5 Increased Iba1 immunoreactivity in Grn-/- mice is apparent in most brain regions, but is more pronounced on the B6 background..............................................191 ?Figure 5-6 The number of activated microglia in the striatum is significantly increased in Grn-/- mice only on the B6 background. ........................................................................194 ?Figure 5-7 Regional brain volumes at 18 months of age are similar between Grn+/+ and Grn-/- mice except for reduced hippocampal volume in Grn-/- mice on the B6 background.......................................................................................................................................197 ?Figure 5-8 Neuronal loss is apparent in the hippocampus and thalamus of Grn-/- mice on the B6 but not on the 129 background. ...................................................................199 ?Figure 5-9 Age-dependent alterations in neuronal morphology and spine density in the CA1 subfield of the hippocampus are present in Grn-/- mice on the 129 but not the B6 background...................................................................................................................202 ? xviii Acknowledgements First of all, I would like to thank my supervisor, Dr. Blair R. Leavitt, for his guidance throughout the course of this work and for teaching me to interpret results rather than just report them. I would also like to thank the remainder of my supervisory committee ? Pamela Hoodless, Daniel Goldowitz, and Ian Mackenzie ? for their guidance along the way. Second, I would like to thank my colleagues in the Leavitt Lab. I would like to thank Austin Hill, Ge Lu. Colum Connolly, and Chen Chen for their technical and theoretical contributions to my research. I would like to think Angela Gurney for her help in all things non-science related. I would especially like to thank Scott Neal and Jessica Macdonald for being amazing mentors during my first few years in the lab. Finally, I would like to thank my family for the incredible support that they have given me as I completed my thesis work. I would like to thank my mom Randolyn and my dad Wesley for showing me what hard work, determination, and strength look like. I would like to thank my husband, Fernando, for instilling in me his philosophy that the impossible is always possible. I would like to thank my children, Nikko and Tiago, for showing me what the meaning of life and of my work really is. xix Dedication To my mother, my greatest inspiration xx Glossary of Terms AD (Alzheimer?s disease) A progressive neurodegenerative condition and currently the most common cause of dementia. ALS (amyotrophic lateral sclerosis) A progressive neuromuscular disorder that causes degeneration of upper and lower motor neurons. Clinical and neuropathological features may overlap between ALS and FTLD. Astrogliosis An abnormal increase in the number and activation state of astrocytes in the brain in response to neuronal injury, stress, or death. ?-galactosidase The enzyme encoded by the lacZ gene. When provided with the organic substrate X-gal, the enzyme catalyzes a hydrolysis reaction, creating a blue precipitate that can be detected using light microscopy. FTD (Frontotemporal dementia) A collective term used to describe a diverse set of clinical syndromes associated with degeneration of the frontal and temporal lobes of the brain. FTLD (Frontotemporal lobar degeneration) A collective term that refers to the pathological processes underlying the clinical syndromes of FTD. Haploinsufficiency A disease mechanism brought about by the loss of a single functional copy of a gene in a diploid organism. If the single remaining gene copy is not xxi sufficient to maintain wild-type function of the resultant protein, that gene is referred to as haploinsufficient. Inbred mouse strain A strain of mice where every individual is genetically identical and homozygous at all loci. lacZ gene A bacterial gene which encodes for the ?-galactosidase gene. It is commonly employed in the generation of transgenic mice as a reporter gene. Lipofuscin An autofluorescent pigment that accumulates with age in post-mitotic cells. It is composed of lipids, sugars and metals, and is often associated with mitochondrial or lysosomal dysfunction. Microgliosis An abnormal increase in the number and activation state of microglia in the brain which can occur in response to neuronal injury, stress, or death, or to an immune challenge to the brain. Neurite Any projection from the cell soma of a neuron. The term neurite is frequently employed when the exact identity of a projection (dendrite or axon) cannot be determined. Neuropathology The study of brain tissue in a disease or neurological condition. Neuropathological examination is required for post-mortem diagnosis of neurodegenerative diseases. xxii Pleiotropy The condition where a single gene affects multiple, seemingly unrelated phenotypic traits. Spine A small membranous protrusion from a dendrite that receives excitatory synaptic input, typically from an axon. TDP-43 The protein encoded by the TARDBP gene, called trans-activating response element with an approximate molecular weight of 43kDa. It is the major ubiquitinated protein in many cases of FTLD, Alzheimer?s disease, and ALS. 1 1 Introduction 1.1 Frontotemporal lobar degeneration Frontotemporal lobar degeneration (FTLD) collectively describes a group of neurodegenerative disorders that cause relatively selective atrophy of the frontal and temporal lobes of the brain. Clinically, pathologically, and genetically, FTLD is highly heterogeneous. The spectrum of clinical disorders, referred to as frontotemporal dementia (FTD), is characterized by progressive changes in behavior, personality, executive function, and language. FTLD represents a significant cause of early-onset dementia with 75-80% of cases presenting between 45-64 years of age (Sieben et al., 2012). The prevalence of FTLD in the 45-64 year age group has been estimated to be the same as that for Alzheimer?s disease, about 15 per 100 000 (Ratnavalli et al., 2002), though it has been reported to be somewhat lower (4 per 100 000) in other populations (Rosso et al., 2003). No Food and Drug Administration approved medications that alter the course of disease are available for FTLD patients; treatments for select symptoms are available, including trazodone for behavior symptoms, galantamine for aphasia, and selective serotonin uptake inhibitors as antipsychotics (Anneser et al., 2007; Kertesz, 2009; Vossel and Miller, 2008). 2 1.1.1 Clinical presentations FTD can be divided into three subtypes based on the predominant early clinical features: behavioral variant, characterized by progressive decline in behavior and executive function and predominant frontal lobe atrophy; semantic dementia, characterized by loss of knowledge of semantic language and corresponding asymmetric, predominantly left hemisphere atrophy of the anterior temporal lobe; and progressive non-fluent aphasia, characterized by deficits in expressive or motor speech and primarily left hemisphere atrophy in the peri-sylvian area. In addition, FTD may overlap clinically, pathologically, or genetically with motor neuron disease/amyotrophic lateral sclerosis (FTD-MND) and the parkinsonian syndromes progressive supranuclear palsy and corticobasal syndrome (reviewed in Seltman and Matthews, 2012). 1.1.2 Neuroimaging features FTD can be distinguished from Alzheimer?s disease and other dementias using volumetric magnetic resonance imaging (MRI) (Davatzikos et al., 2008) and hypometabolism on 18-F fluorodeoxyglucose positron emission tomography, both of which show salient abnormalities in the frontal and temporal lobes of the neocortex in FTD patients (O'Brien, 2007). In longitudinal studies, FTD patients show more rapid rates of atrophy than patients with Alzheimer?s disease (Krueger et al., 2010). To date, neuroimaging data does not unequivocally distinguish between the clinical subtypes of FTD. Nevertheless, a considerable amount of effort has been invested in analyzing the 3 neuroimaging characteristics of the three main subtypes of FTD (reviewed in Whitwell and Josephs, 2012). Behavioral variant FTD is characterized by progressive atrophy in the frontal cortex, in particular the medial frontal cortex (Brambati et al., 2007). Diffusion tensor imaging has demonstrated widespread white matter atrophy, and resting-state functional MRI shows a pronounced decrease in connectivity in the salience network involving the anterior cingulated and frontoinsular regions in behavioral variant FTD cases (Borroni et al., 2012). Semantic dementia is associated with a consistent pattern of atrophy and hypometabolism in the left anterior temporal lobe, as well as corresponding degeneration in white matter tracts that project to that area. Progressive non-fluent aphasia, conversely, is characterized by primarily frontal lobe atrophy and hypometabolism, particularly in the posterior frontal premotor cortex. 1.1.3 Neuropathology Pathological confirmation of FTLD is assigned to clinically diagnosed cases after neuropathological examination. In most cases of FTLD, prominent atrophy in the frontal and temporal lobes of the brain is present. Involvement of the parietal lobe is common, while the occipital lobe is almost always spared. Advanced disease causes more widespread atrophy and a variable degree of pathology is common in the striatum, hippocampus, globus pallidus, substantia nigra, and cerebellum (Whitwell and Josephs, 2012). 4 Immunohistochemical analysis of post-mortem brain tissue can be used to classify FTLD cases into several subtypes based on the main component(s) of pathological protein aggregates that accumulate in affected brain regions. Tau pathology refers to cases where deposits of hyper-phosphorylated tau protein are present primarily in neurons and astrocytes (Pickering-Brown et al., 2002). Most tau-negative cases have neuronal cytoplasmic inclusions that are immunoreactive for ubiquitin, previously referred to as FTLD-U. FTLD-U is further broken down into subtypes depending on the major ubiquitinated protein present. Trans-activating DNA binding protein with a molecular weight of 43kDa (TDP-43) is the major ubiquitinated protein in ubiquitin-positive cases of FTLD (Cairns et al., 2007), Pathologic TDP-43 is truncated and hyper-phosphorylated (Neumann et al., 2009a). TDP-43-immunoreactive inclusions are common in neurons and can be cytoplasmic, nuclear, or dendritic. White matter TDP-43 pathology, primarily in oligodendrocytes, is present in many cases of FTLD (Neumann et al., 2007). The distribution and relative abundance of these inclusions have led to another layer of classification, namely four types of TDP-43 pathologies referred to as Types A-D (Mackenzie et al., 2011). Fused in sarcoma (FUS) and other protein family members (known as FET proteins) are the major ubiquitinated proteins in cases of FTLD-U that are tau and TDP-43-negative, including the majority of sporadic cases that present with this pathology (Neumann et al., 2009b). There remain a small number of patients with FTLD-U pathology where the major ubiquitinated protein(s) are still 5 unidentified. This pathology is currently referred to as FTLD-ubiquitin proteasome system, or FTLD-UPS. 1.1.4 Genetics of FTLD Similar to Alzheimer?s disease and ALS, the majority of cases of FTLD are sporadic. Approximately 30-50% of cases of FTLD have a positive family history (Rohrer et al., 2009b; Seelaar et al., 2011). A clear pattern of autosomal dominant inheritance is present in 10-27% of cases and varies by pathological subtype. Just under a quarter of familial cases of FTLD can be explained by mutations in the MAPT gene, encoding the microtubule-associated protein tau (Gass et al., 2006; Pickering-Brown et al., 2008). Mutations in the progranulin (GRN) gene are slightly more common than MAPT mutations (Le Ber et al., 2007), accounting for about 23% of familial FTLD (Gass et al., 2006; Pickering-Brown et al., 2008). A hexanucleotide repeat expansion in C9ORF72 is one of the most common mutations that cause of FTLD (DeJesus-Hernandez et al., 2011; Majounie et al., 2012), and accounts for a large proportion of cases of FTD-MND linked to chromosome 9. (DeJesus-Hernandez et al., 2011; Renton et al., 2011). More rare mutations in valosin-containing protein (VCP), charged multivesicular body protein 2B (CHMP2B), TDP-43 (TARDBP), and fused-in-sarcoma (FUS) account for less than 5% of familial FTLD (epidemiology and genetics of FTLD have been recently reviewed in (Seelaar et al., 2011; Seltman and Matthews, 2012; Sieben et al., 2012). 6 A summary of the different neuropathological subtypes of FTLD and their respective underlying protein substrates and genetics is provided in Figure 1-1. The figure includes the recent discovery of transportin 1 (TRN1) as part of the ubiquitinated inclusions present in cases of FTLD-FUS (Neumann et al., 2012). 7 Figure 1-1 A summary of the subtypes of FTLD and their respective underlying pathologies and genetics. *TARDBP, UBQLN2, and FUS are common causes of familial ALS and only rarely cause FTLD. #TMEM106B is a genetic risk factor for FTLD that works indirectly by affecting progranulin levels. Box size does not reflect the relative frequency of the different pathologies or genetic mutations. 8 It is important to note that considerable overlap exists between FTLD and other neurodegenerative disorders. Protein aggregates and intra-neuronal inclusions that are immunoreactive for TDP-43 are present in many cases of Alzheimer?s disease (Amador-Ortiz et al., 2007; Uryu et al., 2008) and amyotrophic lateral sclerosis (ALS) (Arai et al., 2006). Mutations in TARDBP, although rare, are causative of a small proportion of cases of ALS as well as FTLD (Kabashi et al., 2008; Sreedharan et al., 2008). Mice expressing pathological TDP-43 mutations develop motor symptoms and motor neuron degeneration similar to ALS patients (Tsao et al., 2012). FUS mutations are also a cause of familial ALS (Kwiatkowski et al., 2009; Vance et al., 2009) and FUS pathology is common in both sporadic and familial forms of ALS (Deng et al., 2010). Our current understanding of the clinical, neuropathological, and molecular basis of FTLD and ALS suggests that these two pathological entities are part of a continuum of related disorders. No clear genetic modifiers for FTLD have been defined to date. The heterogeneity present within the spectrum of FTLD disorders increases the sample size required for informative genome-wide association studies. To address this, many studies have attempted to homogenize their sample population by looking at patients with a defined pathology or defined genetic cause, and with some success. Variants in TMEM106B, which codes for a transmembrane protein, were identified as risk-modifying factors for FTLD in patients with TDP-43 immunoreactive inclusions (Van Deerlin et al., 2010). 9 Subsequent work has linked TMEM106B and progranulin, which is discussed in section 1.1.7. 1.1.5 FTLD due to GRN mutations Null mutations in GRN as a cause of familial FTLD were first reported in 2006 (Baker et al., 2006; Cruts et al., 2006). The discovery of GRN mutations was confounded by the close proximity of GRN to MAPT on chromosome 17q21, mutations in which were discovered in 1998 as a cause of FTLD linked to chromosome 17 (Hutton et al., 1998). GRN mutations occur throughout the gene, but all pathological mutations reported to date are confirmed or predicted to be loss-of-function alleles (Yu et al., 2010). The mechanism of function loss is diverse and includes: mutations introducing a premature stop codon leading to transcript degradation by nonsense-mediated decay (Baker et al., 2006; Bronner et al., 2007; Masellis et al., 2006); missense mutations or deletions in splice donor sites, leading to nuclear degradation of the transcript (Skoglund et al., 2011); missense mutations in the translation start site preventing translation of the protein (Cruts et al., 2006); missense mutations predicted to affect protein folding and/or processing (van der Zee et al., 2007); 5? regulatory region variations predicted to affect transcriptional activity (van der Zee et al., 2007), missense mutations in exon 1 causing a non-function signal peptide that allows for translation but prevents secretion of the protein (Mukherjee et al., 2008; Shankaran et al., 2008); missense mutations in key cysteine residues, which produce normal levels of mRNA and protein but to have 10 impaired function and alterations in cleavage products (Wang et al., 2010); and finally, complete or near-complete locus deletion (Gijselinck et al., 2008; Rovelet-Lecrux et al., 2008). No alterations in gene dosage have been discovered to date (Skoglund et al., 2009b). An approximately 50% reduction in mRNA level (Baker et al., 2006; Chiang et al., 2008; Mukherjee et al., 2008) and 33% reduction in protein level is reported in heterozygous GRN mutation carriers (Baker et al., 2006). A single report of two siblings with homozygous null mutations in GRN described the patients as presenting with late-onset neuronal ceroid lipofuscinosis (Smith et al., 2012). The neuronal ceroid lipofuscinoses (NCL) are a group of lysosomal storage disorders that typically present in childhood and manifest with mental and motor decline, epilepsy, vision loss, and premature death (Kousi et al., 2012). This finding is intriguing and may represent a distinct clinical and neuropathological phenotype for homozygous null GRN mutation carriers. Alternatively, the clinical phenotype of FTLD patients is highly heterogeneous, and it remains possible that neuropathological examination will reveal FTLD pathology in these patients, in which case they would represent early-onset FTLD with a highly atypical clinical presentation. The frequency of GRN mutations has been estimated at 6.9% (30 of 434) of all FTLD-spectrum cases, 21.4% (9 of (28 of 175) of FTLD-spectrum cases with a family history 11 of a similar neurodegenerative disease, and 56.2% (9 of 16) of cases of FTLD-U with a family history in a large, multi-center study in the United States of America (Yu et al., 2010). Similar or slightly lower estimates have been reported in other studies (Borroni et al., 2008b; Guerreiro et al., 2008; Pickering-Brown et al., 2008). 1.1.6 GRN genetic variability in other neurodegenerative disorders Confirmed pathogenic mutations in GRN in cases where the clinical diagnosis is verified by neuropathological examination have thus far been identified primarily in FTLD. A confirmed GRN mutation in the first pathologically confirmed case of ALS was recently reported (Cannon et al., 2013). However, GRN mutation screening in two different cohorts of ALS patients did not reveal any pathogenic mutations, indicating that GRN mutations are not a common cause of ALS (Del Bo et al., 2011; Schymick et al., 2007). Both predicted and confirmed pathogenic mutations in GRN have been identified in cohorts of clinically diagnosed Alzheimer?s disease patients (Brouwers et al., 2008; Wojtas et al., 2012) and patients with Parkinson?s disease (Brouwers et al., 2007), though lack of pathological confirmation precludes unequivocal disease diagnosis. At least one case report has described a patient with a confirmed GRN mutation that displayed both TDP-43-immunoreactive inclusions and Alzheimer?s disease pathology (Perry et al., 2013). On the contrary, Yu et al. (2010) did not identify any pathogenic mutations in GRN in 111 non-FTLD case-controls where TDP-43 pathology was present, and other studies in Parkinson?s disease patients have not detected any GRN 12 mutations (Nuytemans et al., 2008). Thus, GRN mutations primarily cause FTLD-spectrum disorders despite clinical evaluations that are highly heterogeneous and may overlap with other neurodegenerative diseases. In one report describing a large kindred with a unique GRN mutation, in which 10 individual were affected, the most common initial diagnosis was Alzheimer?s disease, though neuropathological characteristics in these patients were similar to other GRN mutation carriers (Kelley et al., 2010). This suggests that GRN mutations do not cause Alzheimer?s disease but instead that the spectrum of clinical presentations suggestive of GRN mutations should be extended to include an Alzheimer?s disease-like phenotype. Though loss-of-function GRN mutations do not appear to be causative in neurodegenerative disorders other than FTLD, there is evidence to suggest that common GRN variability can act as a risk factor for neurodegenerative phenotypes. This seems likely, given that GRN mutations cause FTLD through a haploinsufficiency mechanism. An association study in sporadic FTLD patients identified a tagging single nucleotide polymorphism (SNP) in the GRN gene for which the risk genotype was significantly associated with disease (Galimberti et al., 2010). It is predicted that the associated variants are in regulatory regions and affect progranulin levels. Subsequent analysis of the progranulin promoter revealed increased methylation in the sporadic FTLD patients, with a concurrent but non-significant decrease in progranulin mRNA level (Galimberti et al., 2012a). 13 Expression of GRN, and many other genes, is regulated post-transcriptionally by micro-RNAs. Common genetic variability in a micro-RNA binding site in the 3?-UTR of GRN (rs5848) can affect progranulin levels through translational regulation and contribute to risk for FTLD-TDP regardless of mutation status (Rademakers et al., 2008). It is unclear why this finding has not been replicated in other cohorts of FTLD patients (Rollinson et al., 2011). Nevertheless, the functional consequence of homozygosity for the risk allele for rs5848 appears to be reduced circulating progranulin levels (Hsiung et al., 2011; Kamalainen et al., 2013). Furthermore, the risk allele is over-represented in cases of hippocampal sclerosis (Dickson et al., 2010; Pao et al., 2011). Taken together, the data suggest that GRN variants that result in decreased in progranulin expression may be risk factors both for FTLD and for other dementias. Candidate gene association studies assessing GRN gene variability have been performed for a number of neurodegenerative disorders. Common GRN variants are associated with earlier age of onset and shorter disease duration for ALS patients in some populations (Sleegers et al., 2008). There is evidence that GRN is a disease-modifying gene in Alzheimer?s disease (Fenoglio et al., 2009; Lee et al., 2011). An association study in multiple sclerosis looking at GRN genetic variants suggested that GRN haplotypes may contribute to risk for multiple sclerosis, though only in males 14 (Fenoglio et al., 2010). A small association study in a German cohort showed that variants of the GRN gene are associated with decreased risk for bipolar disorder and schizophrenia (Galimberti et al., 2012b). A link between schizophrenia and GRN mutations has also been reported in other instances (Momeni et al., 2010) and thus may warrant further investigation. Overall, variability in GRN has been associated with multiple neurological and neurodegenerative phenotypes, suggesting that progranulin plays an important role in overall neuronal health and/or brain function. The fact that the effect of GRN genetic variability as a disease modifier is often small and not always reproducible suggests that complex interactions likely exist between progranulin and other genetic and environmental factors. Uniquely in FTLD due to GRN mutations, cerebrospinal fluid and plasma progranulin levels are non-overlapping between normal individuals and mutation carriers, providing a valuable clinical tool for predicting GRN mutations (Antonell et al., 2012; Carecchio et al., 2009; Finch et al., 2009; Ghidoni et al., 2008; Ghidoni et al., 2012; Lopez de Munain et al., 2008; Pietroboni et al., 2011; Rossi et al., 2011; Schofield et al., 2010). Missense mutations that partially reduce progranulin levels are also detectable by circulating progranulin levels (Sleegers et al., 2009). GRN mutations in apparently sporadic cases of FTLD have been discovered using peripheral screening (Le Ber et al., 2007), demonstrating the potential application of more systematic screening in sporadic FTLD. 15 The potential for predicting genetic variation in GRN by a simple enzyme-linked immunosorbent assay, as opposed to expensive, labor-intensive sequencing, is both potentially valuable and problematic. Lower than average levels of circulating progranulin have been observed in a cohort of patients with Parkinson?s disease (Mateo et al., 2013) and in children with autism (Al-Ayadhi and Mostafa, 2011). These findings, if replicated and validated, could mean that progranulin plays an important role in multiple neurological disorders. On the other hand, the ease of screening for progranulin levels in peripheral blood, although useful in specific clinical settings, raises the risk of its overuse and over-interpretation until more is known about progranulin biology and variation within the general population. In a large cohort of patients with chronic kidney disease, serum progranulin levels were shown to increase with increasing disease stage, indicating that renal failure leads to progranulin accumulation in the blood (Richter et al., 2013). A genome-wide association study identified a SNP near the neurotrophin receptor sortilin (SORT1) that showed significant association with progranulin plasma levels in a healthy control population (Carrasquillo et al., 2010). Further examination showed that this SNP was indeed associated with an increase in SORT1 mRNA, but only in the liver. The result is that increased sortilin expression in the liver leads to decreased extracellular progranulin, resulting in reduced plasma progranulin levels. This reduction in plasma is entirely independent of progranulin levels, and SORT1 expression, in the brain. Thus, caution is warranted when interpreting 16 progranulin plasma levels for patients, especially in patients with disorders other than FTLD where causative GRN mutations have not yet been described. Given the pleiotropic expression and actions of progranulin in the periphery (further discussed in Sections 1.2.3 - 1.2.5), peripheral screening of progranulin levels, although useful, should be approached with caution and rigorously validated to avoid over-interpretation or mis-interpretation of the results. 1.1.7 Genetic variation contributing to incomplete penetrance in GRN mutation carriers The penetrance of GRN mutations is incomplete. It is estimated to be approximately 50% by 60 years of age and increases to 90% by 70 years of age (Gass et al., 2006; Rademakers et al., 2007). The contribution of environmental factors to FTLD etiology remains largely unexplored; genetic variation affecting progranulin expression has, however, become of a topic of considerable interest. Genetic variability, both on the wild-type GRN allele and in other genes, is hypothesized to contribute to disease pathogenesis because of the striking variability in the clinical and neuropathological characteristics of FTLD patients with GRN mutations. Analysis of 30 families, all of which carry the common Arg493X nonsense mutation in GRN, found at least four different clinical presentations and a 25-year range in age of onset in affected patients (Rademakers et al., 2007). Another compelling piece of evidence that genetic background plays a significant role in disease pathogenesis in GRN mutation carriers 17 comes from a pair of affected monozygotic twins, who are reported to have strikingly similar clinical and neuroimaging features (McDade et al., 2012). There is an emerging list of genetic modifiers and risk factors associated with FTLD in GRN mutation carriers. GRN mutation carriers that are also homozygous for the minor allele of TMEM106B, which codes for a trans-membrane protein that associates with progranulin in endo-lysosomes, have a reduced disease penetrance (Finch et al., 2011). Increased expression of TMEM106B increases intracellular progranulin (Chen-Plotkin et al., 2012), presumably sequestering it from secretion and/or increasing the rate of its degradation. The tagging SNP for TMEM106B is associated with age at onset in GRN mutation carriers, with carriers of the risk allele having an age at onset an average of 13 years earlier than non-carriers (Cruchaga et al., 2011). A replication study did not find an association with age of onset, but did determine that homozygous carriers of the protective TMEM106B allele reduced the risk of developing FTLD by 50% (van der Zee et al., 2011). It is also associated with lower plasma progranulin levels both in patients and in healthy elderly individuals. A reasonable hypothesis as to the source of genetic variation that causes variable phenotypes in GRN mutation carriers is that risk factors for other neurodegenerative disorders contribute to disease pathogenesis. Aside from a causative role in FTLD with 18 tau pathology, the MAPT gene has haplotypes associated with multiple neurodegenerative phenotypes including Alzheimer?s disease and Parkinson?s disease (Caffrey and Wade-Martins, 2007). The haplotype associated with the APOE4 isoform of the apolipoprotein E gene is a well-known risk factor for Alzheimer?s disease (Cedazo-Minguez and Cowburn, 2001). No association with MAPT haplotypes and age-of-onset has been detected in GRN mutation carriers, but the presence of one APOE4 allele increased age-of-onset in two different patient series (Beck et al., 2008; Gass et al., 2006). The modifying effect of APOE4 may be small, as it showed no association with age-of-onset in a third patient series, but was instead associated with early memory deficits (Rademakers et al., 2007). Polymorphisms in the prion protein 129 codon were significantly associated with age-of-onset in a series of patients carrying a unique splicing mutation in GRN (Moreno et al., 2011). Genetic variability in the prion protein gene has been associated with multiple neurological phenotypes, but the authors noted that the biological consequences of this potential relationship remain entirely speculative. It is difficult to assess the relative contribution of many environmental factors to disease pathogenesis due to the lack of accurate medical records. There is a paucity of data exploring potential medical and environmental risk factors for FTLD. Traumatic brain 19 injury is one of the few medically documented variables that may contribute to neurodegeneration. A recent study in a veteran population found an increased prevalence of traumatic brain injury in patients that were diagnosed with FTLD (Kalkonde et al., 2012). A connection between FTLD, progranulin, and traumatic brain injury had been previously put forward (Jawaid et al., 2009) and is now being explored in mouse models of progranulin deficiency (Tanaka et al., 2013a; Tanaka et al., 2013b). 1.1.8 The clinical and neuropathological phenotype of FTLD due to GRN mutations FTLD due to GRN mutations is highly heterogeneous in terms of age of onset, clinical presentation, and neuroimaging profiles. (Benussi et al., 2008; Coppola et al., 2012; Gabryelewicz et al., 2010; Kelley et al., 2009; Le Ber et al., 2008; Mackenzie and Rademakers, 2007; Moreno et al., 2009; Pietroboni et al., 2011; Skoglund et al., 2009a). In a consanguineous family harboring a single GRN mutation, the age of onset in nine affected patients ranged over five decades (Bruni et al., 2007). A similar degree of heterogeneity in families carrying the same GRN mutation has been noted by others (Larner, 2012; Leverenz et al., 2007; Marcon et al., 2011; Rossi et al., 2011). Nevertheless, several studies have examined clinical and/or neuroimaging data for signature phenotypes that predict an underlying mutation in GRN. An international, 20 multi-center study comparing FTLD cases with TDP-43 pathology found that cases with GRN mutations had a significantly earlier age-of-onset and younger age-at-death compared to non-GRN cases (Chen-Plotkin et al., 2011). A later age-of-onset, but shortened disease progression, was found in a cohort from the United Kingdom (Beck et al., 2008). GRN mutation carriers may present with any of the defined clinical syndromes of FTLD, including corticobasal syndrome (Benussi et al., 2009; Lopez de Munain et al., 2008; Masellis et al., 2006; Rohrer et al., 2009a), primary progressive aphasia (Cerami et al., 2013; Mesulam et al., 2007), and FTLD with parkinsonism (Di Fabio et al., 2010). Early apraxia, visual hallucinations, and parkinsonism are common in GRN mutation carriers (Bruni et al., 2007; Josephs et al., 2007; Le Ber et al., 2008; Le Ber et al., 2007; Mackenzie and Rademakers, 2007; Rohrer et al., 2008b; Taipa et al., 2012), though concomitant motor neuron disease is rare (Josephs et al., 2007). A primary diagnosis of progressive non-fluent aphasia is significantly more common in GRN mutations carriers compared to MAPT mutation carriers in some patient series (Pickering-Brown et al., 2008). Language impairments are more severe in GRN mutation carriers than in other FTLD patients in some patient series (Bruni et al., 2007; Josephs et al., 2007) but not in others (Van Deerlin et al., 2007). Disease is often rapidly progressing and frequently results in a secondary diagnosis within two years of the primary diagnosis for GRN mutation carriers (Moreno et al., 2009). Despite clear efforts to determine specific 21 clinical phenotypes that are associated with FTLD due to GRN mutations, no clear phenotypic signature has yet emerged. Neuroimaging data is more robust in terms of distinguishing GRN mutation carriers from other mutation carriers among FTLD patients. The most defining neuroimaging feature of GRN mutation carriers is asymmetric brain atrophy (Beck et al., 2008; Bozzali et al., 2013; Ghetti et al., 2008; Rohrer et al., 2011; Rohrer et al., 2010; Taipa et al., 2012; Whitwell and Josephs, 2012). An underlying GRN mutation in a case of apparently sporadic FTLD was discovered based on asymmetric atrophy in diagnostic reports (Passov et al., 2011). Asymmetric atrophy also distinguishes FTLD with parkinsonism due to GRN mutations from cases where the underlying genetic mutation is in MAPT (Ghetti et al., 2008). Asymmetric damage to white matter may be an additional feature of FTLD associated with GRN mutations (Bozzali et al., 2013). More severe cortical atrophy than is present in non-carriers and greater involvement of the parietal lobe is also often apparent in GRN mutation carriers (Beck et al., 2008; Moreno et al., 2009; Rohrer et al., 2008a; Whitwell et al., 2007; Whitwell et al., 2009). When compared to MAPT mutation carriers, GRN mutation carriers have a higher degree of whole brain atrophy (Whitwell et al., 2011). Functional imaging suggests that GRN mutation carriers have impaired connectivity of the parietotemporal regions, but that compensation in other areas may occur (Premi et al., 2013). 22 Ubiquitinated, neuronal inclusions are a defining feature for several subtypes of FTLD. In GRN mutation carriers, these inclusions are immunoreactive for TDP-43, may be intranuclear, and consistently found in the frontal cortex as well as in the striatum (Davion et al., 2007; Josephs et al., 2007; Kelley et al., 2009; Mackenzie, 2007a; Mackenzie et al., 2006b). The pattern of FTLD-TDP found in GRN mutation carriers is characteristically subtype A according to the classification system of Mackenzie et al. (2011). Progranulin itself is not a component of the neuronal inclusions in FTLD (Baker et al., 2006). Predominant dystrophic neurites in the superficial layers of the cortex (Josephs et al., 2007) and short, delicate, dystrophic TDP-43-positive neurites in the CA1 region of the hippocampus (Hatanpaa et al., 2008) are also common. The spatial pattern of TDP-43 pathology in GRN mutation carriers suggests that neuronal cytoplasmic and intranuclear inclusions affect distinct clusters of neurons in cortico-cortical and cortico-hippocampal circuits (Armstrong and Cairns, 2011). Asymmetrical distribution of TDP-43, which is predominantly in the left hemisphere, may distinguish between GRN mutations and TDP-43 pathology in Alzheimer?s disease (Gliebus et al., 2010). Evidence is beginning to emerge that GRN mutation carriers may show early signs of brain atrophy prior to the onset of clinical symptoms. Diffusion tensor imaging of pre- 23 symptomatic GRN mutation carriers versus non-carrier controls has suggested that early loss of white matter integrity occurs in asymptomatic carriers (Borroni et al., 2008a). Another recent imaging study identified early cortical thinning, particularly in the temporal lobe, which increased with age in asymptomatic GRN mutation carriers (Moreno et al., 2013). Thus, there are potential neuroimaging markers of disease onset that could assist with timing early therapeutic interventions for GRN mutation carriers. 1.1.9 Conclusions FTLD refers to a spectrum of disorders that involve diverse clinical presentations, neuroimaging characteristics, and neuropathological features. Clinical presentation predicts neither a clear neuroimaging signature nor a defined, invariable underlying pathology. Emerging evidence appears to support a tighter correlation between genetic cause and neuropathology than between any other two factors. FTLD due to GRN mutations is particularly heterogeneous in terms of clinical features, but asymmetric brain atrophy and a distinct pattern of TDP-43 pathology appear to be the best predictors of GRN mutations. 1.2 Progranulin Progranulin is a secreted growth factor expressed in multiple tissues and cell types throughout the body. It has pleiotropic actions, including major roles in cancer and 24 inflammation. Its role in the brain is largely unknown but has become a topic of intense research since the discovery of its role in FTLD in 2006. 1.2.1 Progranulin structure and function The human progranulin gene (GRN) is located on chromosome 17q21 and encodes a 593 amino acid protein. It is a single-copy gene with 13 exons and 12 introns (Baba et al., 1993b). The orthologous murine gene (Grn) is located on chromosome 11 (Bucan et al., 1996) and encodes a 589 amino acid protein sharing a similar predicted structure and high sequence identity at both the DNA (76%) and protein (79%) levels with human GRN and progranulin respectively. Homologous genes to human GRN have been reported in pigs (Yang et al., 2006), zebrafish (Cadieux et al., 2005), tilapia (Chen et al., 2007), teleost fish (Belcourt et al., 1995; Uesaka et al., 1995), leeches (Hong and Kang, 1999), tunicates (Zhao et al., 2013a), and a multitude of other invertebrates (Hanington et al., 2008; Larade and Storey, 2008; Nara et al., 2004; Smout et al., 2009). The sweet potato has, at the very least, a granulin-domain-containing cysteine protease (Chen et al., 2006), suggesting incredible antiquity of the granulin domain. A comparison of homology of the progranulin gene in mammals is shown in Figure 1-2. 25 Figure 1-2 Organization and conservation of the progranulin gene. (A) Scaled schematics of the human and mouse progranulin (GRN and Grn respectively) loci on chromosomes 17 and 11, respectively. (B) Conservation plot from the UCSC genome browser (http://genome.ucsc.edu; accessed September, 2007) aligned to the human GRN gene. Conservation of the progranulin locus among vertebrates and pairwise conservation with certain species is shown. (C) Conserved granulin motifs are found within the translated human and mouse progranulin coding sequences (https://www.ncbi.nlm.nih.gov/blast). 26 Full-length progranulin is a 68kDa protein that consists of seven and a half repeats of a unique 12-cysteine motif and an N-terminal 17 amino acid signal peptide for secretion (Zhou et al., 1993). A schematic representation of both the human gene and protein structure is shown in Figure 1-3. When secreted, progranulin has an apparent molecular weight of 88kDa due to being highly glycosylated (Zhou et al., 1993). Granulin peptides are approximately 6kDa proteins that consist of about 20% cysteine residues, some of which form disulphide bridges essential for biological activity (Shoyab et al., 1990). The granulin motif forms a parallel stack of beta-hairpins held together by six disulphide bridges (Hrabal et al., 1996) resulting in a conformationally stable tertiary structure (Tolkatchev et al., 2000). The specific arrangement of the cysteine residues in granulin domains appears to be sufficient to produce the beta-hairpin structure, given that a similar tertiary structure is present in granulin-like domains found in plant cysteine-proteases, which share very little sequence homology with mammalian granulins (Tolkatchev et al., 2001). 27 Figure 1-3 The structure of the human progranulin gene and protein. A) The human GRN gene has 12 coding exons and is located on chromosome 17q21.32. B) The progranulin protein is composed of seven and a half repeats of the granulin domain joined by short linking sequences. Note that each coding exon codes for either the N-terminal or C-terminal portion of a granulin domain. The granulin domains are lettered based on the order in which they were discovered. C) The conserved granulin consensus sequence. *Location of the human mutation 388_391delCAGT modeled in the progranulin-deficient mice generated in this thesis (see Section 2.2.1) Adapted from http://endolab.mcgill.ca/research/images/grn_gene_str_ps.jpg 28 Analysis of the individual human granulin peptides has shown that granulins A, C, and F form the stable beta-hairpin stack, but the other granulins are poorly structured (Tolkatchev et al., 2008). Functionally, GrnA is inhibitory of growth in ovarian cancer cells, GrnF is stimulatory, and the poorly structured granulins have little or no activity (Tolkatchev et al., 2008). Granulin-like repeats are proposed to be an important structural feature in protein-protein interactions. Granulins A and B, also known as epithelins 1 and 2 respectively, have opposing effects on cell growth in rodent keratinocytes and fibroblasts (Plowman et al., 1992). These two granulins share just 47% sequence homology, but the spacing of their unique 12-cysteine motif is conserved (Plowman et al., 1992). Secreted, full-length progranulin can be cleaved into its shorter granulin constituents by serine-threonine proteases, including matrix metalloproteinase 14 (Butler et al., 2008), the metalloproteinase ADAMTS-7 (Bai et al., 2009), proteinase 3, and neutrophil elastase (Kessenbrock et al., 2008). Secretory leukocyte protease inhibitor binds progranulin and prevents its cleavage by neutrophil elastase (Zhu et al., 2002). This interaction is may be important for the proliferation and cell survival effects of both progranulin and secretory leukocyte protease inhibitor in ovarian cancer cells (Simpkins et al., 2008). Secreted progranulin forms homodimers (Nguyen et al., 2013), which may be an additional biological mechanism that protects the full-length protein from 29 cleavage. Protecting progranulin from extracellular cleavage is, therefore, a potential therapeutic strategy that warrants further investigation. Characterization of a potential receptor for granulins in both breast cancer carcinoma cells (Culouscou et al., 1993) and a mink lung epithelial cell line (Xia and Serrero, 1998) indicates the presence of high- and low-affinity binding sites on a unique receptor shared by both peptides. The presence of this putative receptor with similar characteristics in multiple cell lines suggests that the progranulin signaling pathway is widely distributed in cells of distinct embryonic origins. 1.2.2 Regulation of progranulin expression Early characterization of the mouse progranulin gene (Grn) predicted several transcription factor binding sites, including the SP-1 consensus site, the MTF binding site, and the IL-1/IL-6 responsive element (Baba et al., 1993b). The human gene has two transcriptional start sites as well as many predicted transcription factor binding sites and multiple palindromic sequences and direct or inverted repeats, which are indicative of enhancer and/or repressor activity (Bhandari et al., 1996). Over the past several years, an exponential increase in the number of publicly available expression databases, advancements in sequencing technology, and increased computing capacity has contributed to a massive expansion in our ability to use computational biology better 30 understand gene regulation. A more extensive examination and re-evaluation of both the mouse and human progranulin genes would likely contribute significantly to our understanding of how the gene is regulated. Progranulin regulation has been studied to a limited extent in different cell types. In cells of the myeloid lineage, GRN mRNA levels are regulated by all-trans retinoic acid, which acts in a cell-specific manner and can both affect mRNA turnover and/or act in a manner dependent on NF?B signaling (Ong et al., 2006). The histone deacetylase inhibitor suberoylanilide hydroxamic acid up-regulates progranulin transcription in multiple cell lines and in progranulin-deficient human lymphoblasts (Cenik et al., 2011). In fact, at least one other histone deacetylase inhibitor has been shown to up-regulate progranulin expression (Fass et al., 2013), suggesting a potential point of therapeutic intervention for progranulin deficiency. Several lines of evidence suggest that progranulin expression is induced by steroids. In breast cancer and endometrial cancer cell lines, progranulin expression is induced by estrogens (Lu and Serrero, 1999) and co-localizes with estrogen receptors (Jones et al., 2006). Autocrine progranulin signaling mediates the mitogenic effects of estrogen via stimulation of cyclin D1 (Lu and Serrero, 2001). Estrogen can also stimulate progranulin expression in vivo. Suzuki et al. (2001) demonstrated that administration of an estrogen 31 compound to either pregnant dams or neonatal rats increased progranulin expression in the hypothalamus 8-15 days later (Suzuki et al., 2001). They further characterized progranulin as an androgen-inducible gene highly expressed in the hypothalamus during the critical neonatal period when sexual dimorphism is established in the rodent brain (Suzuki and Nishiahara, 2002). This work was furthered by Lee et al. (2006), who showed a similar increase in hypothalamic progranulin expression after feeding rats a diet enriched in various phytoestrogens and a subsequent perturbation of sexual behavior (Lee et al., 2006). Progranulin is up-regulated in the hippocampus one month after estrogen treatment in adult rats, co-incident with increased cell proliferation in the dentate gyrus (Chiba et al., 2007). Voluntary running, used as a method of increasing neurogenesis in the hippocampus, increases expression of progranulin in CA1 and CA3 pyramidal neurons as well as in the interneurons of the dentate gyrus (Asakura et al., 2011). Progranulin is a stress-response factor in many cell types. Progranulin expression is up-regulated in fibroblasts, which do not express progranulin in basal conditions, in response to hypoxia and acidosis (Guerra et al., 2007). Progranulin expression increases in many mouse tissues with increasing age (Swindell, 2009), which is co-incident with multiple stress response factors. However, expression of many inflammatory markers also increases with age, and progranulin may fit equally well into this category. 32 Regulation of progranulin expression is also controlled post-transcriptionally. Using a straightforward, candidate gene approach, Jiao et al. (2010) identified microRNA-29b as a regulator of progranulin expression through a 3?-UTR binding site in both the mouse and the human gene (Jiao et al., 2010). At the level of translation, intracellular and secreted levels of progranulin can be increased by alkalizing agents (Capell et al.). Progranulin also binds to chaperone proteins of the endoplasmic reticulum, presumably prior to secretion, providing another potential point of therapeutic intervention for increasing or decreasing its release from the cell (Almeida et al., 2011). The signaling pathways activated downstream of progranulin that have thus far been identified are typical of extra-cellular growth factor signaling. Activation of the mitogen-activated protein kinase (MAPK) and phosphotidylinositol 3-kinase (PI3K) pathways are commonly seen in response to progranulin over-expression in cancer cells (Bose et al., 2011; Cuevas-Antonio et al., 2010; Wang et al., 2003). Activation of the extracellular signal regulated kinase (ERK) 1/2 pathway may also result from progranulin-mediated signaling (Monami et al., 2006). All three of these pathways are inter-related and part of a complex signaling cascade that regulates cell proliferation, differentiation, and cell death. During chondrogenesis, progranulin signals through the Akt and PI3K pathways to stimulate NF?B nuclear translocation, one of the targets of which is cyclin D1 (Wu et 33 al., 2011). Blocking progranulin expression by antisense oligonucleotides caused a decrease in cyclin D1 and CDK4 expression in ovarian cancer cell lines, suggesting a possible link between these cell cycle modulators and progranulin expression (Liu et al., 2007). Progranulin can substitute for insulin-like growth factor 1 (IGF-1) signaling in regulation of muscle growth through the PI3K/Akt/mTor pathway (Hu et al., 2012). Progranulin expression itself is stimulated in human neuroblastoma cell lines after hypoxic stress through the MAPK and MEK signaling pathways (Piscopo et al., 2010). In cholangiocarcinoma cells, signaling through the ERK1 pathway increased progranulin expression, which in turn leads to activation of the Akt pathway (Frampton et al., 2012). Blocking the protein kinase C pathway lowers progranulin protein levels but not mRNA (Diaz-Cueto et al., 2012). Thus, signaling cascades typically involved in cell proliferation, cell differentiation, and cell death both activate progranulin expression and are activated by progranulin expression. Further delineation of progranulin signaling in different cell types is required to understand all of the mechanisms that likely exist to regulate this positive feedback loop. 1.2.3 Peripheral functions of progranulin Progranulin is expressed in multiple cell types originating from all three germ layers (Bhandari et al., 1993; Daniel et al., 2000) and has pleiotropic functions. Progranulin 34 was identified independently at least four times in different cell types and in relation to different biological processes, speaking to its diverse roles. The biological roles of progranulin can be grouped into three broad categories: progranulin as a growth factor in development and cancer; progranulin as a regulator of inflammation; and progranulin in the brain in health and disease. A growth factor highly expressed during mammalian spermatogenesis, progranulin was also named acrogranin when it was purified from guinea pig testes (Baba et al., 1993a). A putative role for progranulin in sperm function in humans has subsequently been identified (Thomas et al., 2013). Progranulin promotes blastocyst hatching, adhesion, and outgrowth in vitro and is highly expressed in mouse (Diaz-Cueto et al., 2000; Qin et al., 2005) and mink blastocysts (Desmarais et al., 2008) and in select cell types in human placenta (Stubert et al., 2011). It has a putative role in preeclampsia and fetal growth restriction (Stubert et al., 2012). Developmentally, progranulin is involved in tooth formation (Cao et al., 2010), liver morphogenesis (Li et al., 2010), myogenesis (Li et al., 2013; Wang et al., 2012a), and cartilage formation and function (Feng et al., 2010; Wu et al., 2011). Peptides termed ?granulins? were first reported as a novel class of leukocyte peptide with potential cytokine-like properties, isolated from both rat and human leukocytes and bone 35 marrow (Bateman et al., 1990). High expression of the full-length precursor, progranulin was demonstrated in multiple cell types of the myeloid lineage (Bhandari et al., 1992), thus defining a role for progranulin in immunity (see Section 1.2.4). Short peptides with opposing activities on proliferation in epithelial cells were purified from rat kidney and termed epithelins (Shoyab et al., 1990). It was recognized immediately that granulins and epithelins referred to the same peptides. Subsequently, an 88kDa secreted and glycosylated protein with mitogenic properties derived from PC cells, a highly tumorigenic, insulin-independent teratoma cell line, was termed PC cell derived growth factor and shown to be the likely precursor for granulins and/or epithelins (Zhou et al., 1993). The growth modulating effects of progranulin and its derived granulin peptides have subsequently been extensively studied with respect to cancer (see Section 1.2.5). 1.2.4 Progranulin in the immune system Progranulin is highly expressed in cells of the myeloid lineage, including macrophages, microglia, and monocyte-derived dendritic cells (Ong et al., 2006). Its function in immune cells appears to be two-fold. In flounder, two shorter granulin transcripts have been identified as opposed to one longer precursor. One of these granulins is constitutively expressed in many tissues and the other is activated in response to an 36 inflammatory stimulus (Nam et al., 2009). The goldfish granulin homologue regulates cell proliferation in undifferentiated macrophages but its potency as a mitogen decreases with increasing myeloid differentiation state (Hanington et al., 2006). Progranulin expression in carp head kidney cells is induced only after immunostimulation (Sakai et al., 2005). Taken together, the evidence from lower organisms points to a dual role for progranulin in the immune system as both a mitogenic factor and an activation response element. In many inflammatory diseases, progranulin expression is up-regulated. Chronic inflammation is associated with central obesity and diabetes, and progranulin is significantly increased in serum in patients with Type II diabetes (Qu et al., 2013; Youn et al., 2009). Circulating progranulin levels and mRNA expression in peripheral blood mononuclear cells also shows a positive correlation with body mass index and visceral fat (Hossein-Nezhad et al., 2012). Plasma progranulin levels are elevated in patients with systemic lupus erythematosus (Tanaka et al., 2012) and can be lowered by glucocorticoid treatment (Qiu et al., 2013). Interestingly, both its high expression in untreated patients and its lowered expression after glucocorticoid treatment correlate with expression of several pro-inflammatory cytokines. Regulation of progranulin expression in a mouse model of lupus nephritis mediates disease severity, at least partially by altering macrophage phenotype via MAPK-dependent signaling pathways (Chen et al., 2013b). Patients with systemic lupus erythematosus and rheumatoid 37 arthritis have neutralizing anti-progranulin antibodies present in serum that are not present in the majority of healthy controls, implicating progranulin as a target in autoimmune diseases (Thurner et al., 2013). A screen for differentially expressed genes in synovial tissue from rheumatoid arthritis patients identified an up-regulation of progranulin (Justen et al., 2000). Follow-up work showed that progranulin is over-expressed in patients with both osteoarthritis and rheumatoid arthritis, and that progranulin is a specific inhibitor of the degradation of cartilage oligomeric matrix protein (Guo et al., 2010b). Progranulin is an independent marker of liver fibrosis in non-alcoholic fatty liver disease (Yilmaz et al., 2011) and atherosclerosis in patients without metabolic syndrome (Yoo et al., 2013). There are several instances where the role of full-length progranulin appears to differ from, and in fact counteract, the role of the proteolytically processed granulin peptides. Progranulin has been identified as a modulator of the wound response (Zhu et al., 2002), acting as a growth factor that promotes cell division, migration, and capillary-like tubule formation in dermal fibroblasts and epithelial cells (He et al., 2003). In this context, short granulin peptides prevented epithelial cell proliferation and increased interleukin-8 secretion, which could be considered pro-inflammatory actions, while full-length progranulin blocked neutrophil activation. An endogenous protease inhibitor 38 released by leukocytes at the wound site binds to progranulin and prevents its cleavage into smaller granulin peptides by neutrophil elastase, thus preserving the full-length protein and all of its anti-inflammatory properties (Zhu et al., 2002). Progranulin expression and regulation has also been implicated in atherosclerosis, where its primary role is thought to be to regulate the inflammatory response (Kojima et al., 2009). In this system again, progranulin and granulins have opposing actions, where full-length progranulin decreases, while short granulin peptides increase, the secretion of pro-inflammatory cytokines. Okura et al. (2010) were able to demonstrate that progranulin forms a complex with the anti-inflammatory molecule high-density lipoprotein derived from human monocyte-derived macrophages (Okura et al., 2010). The authors suggest that this binding interaction may prevent the conversion of progranulin to granulins and thus exert a protective, anti-inflammatory effect similar to the action of secretory leukocyte protease inhibitor during wound healing. In several different models of inflammatory disease in mice, progranulin deficiency exacerbates the disease phenotype. Progranulin deficiency increases the susceptibility to and disease severity of induced inflammatory arthritis in mice, which is rescued by progranulin over-expression (Tang et al., 2011). Acute lung injury in mice, induced by the administration of the inflammatory agent lipopolysaccharide, can be rescued by 39 progranulin administration (Guo et al., 2012). Induced dermatitis in mice is exaggerated on a progranulin-deficient background (Zhao et al., 2013b). Progranulin deficiency in mice prevents the induction of insulin resistance by a high fat diet by blocking increases in the pro-inflammatory cytokine intereleukin-6 (Matsubara et al., 2012). Conversely insulin resistance could be induced by chronic progranulin administration. Progranulin also plays a role in hypothalamic glucose-sensing and appetite regulation in rodents (Kim et al., 2011). The mechanism(s) through which progranulin works to mediate inflammation are still being explored. Toll-like receptors are critical for both innate and adaptive immune responses. Toll-like receptor 9 is activated by CpG oligonucleotides typically derived from bacteria. Progranulin acts as a co-factor for the delivery of CpG oligonucleotides to toll-like receptor 9, a process that is impaired in progranulin knockout macrophages (Park et al., 2011a). Tumor necrosis factor receptors 1 and 2 were identified as important progranulin receptors that mediate its anti-inflammatory effects in induced arthritis in mice (Tang et al., 2011). The binding of progranulin to tumor necrosis factor receptors has been disputed (Chen et al., 2013a), but tumor necrosis factor receptor 2 does appear to mediate progranulin-dependent reduction in inflammatory markers in acute lung injury (Guo et al., 2012). 40 1.2.5 Progranulin in cancer biology Progranulin is over-expressed in multiple tumor types and different human cancers, including breast cancer (Serrero and Ioffe, 2003), ovarian cancer (Cuevas-Antonio et al., 2010), hepatocellular carcinoma (Cheung et al., 2004), renal cell carcinoma (Donald et al., 2001), multiple myeloma (Wang et al., 2003), gastric cancer (Line et al., 2002), glioblastoma (Liau et al., 2000; Markert et al., 2001), intracranial meningioma (Kim et al., 2010), prostate cancer (Pan et al., 2004), uterine leiomyosarcoma (Matsumura et al., 2006), laryngeal squamous cell carcinoma (Kong et al., 2007), esophageal squamous cell carcinoma (Chen et al., 2008), and multiple types of skin cancer (Huang et al., 2012). It has been identified as an important regulator of tumorigenicity in multiple cancer cell lines (He and Bateman, 1999; Jones et al., 2003; Lu and Serrero, 2000; Tangkeangsirisin and Serrero, 2004). Many studies have correlated high progranulin expression with increased tumor invasiveness (Elkabets et al., 2011; Monami et al., 2006; Swamydas et al., 2011; Tangkeangsirisin and Serrero, 2004) and angiogenesis (Li et al., 2011) and in fact one study suggested that progranulin may play a direct role in the malignant conversion of ovarian epithelial cells (Miyanishi et al., 2007). Progranulin is a potential prognostic marker of ovarian carcinoma and glioblastoma, its over-expression being correlated with poor prognosis (Davidson et al., 2004; Wang et al., 2012b). Progranulin is also a prognostic biomarker for recurrence in some breast cancers (Asaka et al., 2006; Koo et al., 2012; Serrero et al., 2012) and is potentially an independent prognostic biomarker for ovarian epithelial cancers (Han et al., 2011). 41 Progranulin may even be useful as a serum biomarker for breast cancer (Tkaczuk et al., 2011). In colorectal cancer cells (Bose et al., 2011), hepatocellular carcinoma (Cheung et al., 2011), multiple myeloma (Wang et al., 2006), breast cancer cell lines (Abrhale et al., 2011), and ovarian cell lines (Pizarro et al., 2007), progranulin confers or contributes to the development of chemoresistance. Growth-regulated cells, such as mouse embryonic fibroblasts, typically require multiple growth factors for growth. Progranulin expression alone is sufficient to allow for growth in insulin-like growth factor receptor-negative cells, through activation of the MAPK and PI3K pathways (Xu et al., 1998; Zanocco-Marani et al., 1999). Studies in breast cancer and hepatocellular carcinoma (Cheung et al., 2006) have noted the progranulin expression in positively correlated with expression of the oncogene p53. Given its involvement in a broad spectrum of different cancers, progranulin is an attractive therapeutic target. Neutralizing antibodies and/or antisense oligonucleotide transfection to decrease progranulin have shown to increase apoptosis, decrease proliferation rate, and decrease the invasive index in ovarian cancer cell lines (Kamrava et al., 2005; Liu et al., 2007) and prostate cancer cell lines (Monami et al., 2009). The use of RNA interference against progranulin is being investigated in hepatocellular carcinoma (Park et al., 2011b). A study using human liver cancer cells in nude mice and anti-progranulin monoclonal antibodies showed efficacy of anti-progranulin treatment without any measurable detrimental effects on normal liver cells (Ho et al., 2008). 42 Interestingly, the single progranulin over-expressing mouse reported in the literature has a hair growth phenotype (Kato et al., 2009). 1.2.6 Progranulin and its interacting partners Protein-protein interactions are an important aspect of functional biology. The yeast-two-hybrid screening system has been employed multiple times in an attempt to identify interacting partners of progranulin. As a secreted protein, progranulin was identified by yeast-two-hybrid screening as an interacting protein for human immunodeficiency virus (HIV) Tat proteins through its cysteine residues. Tat proteins are secreted from HIV-infected cells and serve as mitogenic factors. Progranulin may be a co-factor for Tat proteins in this role (Trinh et al., 1999). Progranulin is also reported to interact with cyclin T1 and modulate transcription (Hoque et al., 2003). A follow-up study provided evidence that progranulin can repress transcription from many different cellular promoters when it is prevented from exiting the cell (Hoque et al., 2010). The yeast-two-hybrid system identified an interaction between progranulin and Type III hexokinase, a member of a family of kinases involved in glucose metabolism (Sui and Wilson, 2000). The potential functional consequence of this interaction is unclear. Progranulin was identified in a yeast-two-hybrid screen to interact with cartilage oligomeric matrix protein, an interaction that is required for cell proliferation in chondrocytes (Xu et al., 2007). 43 Subsequent work demonstrated a key role for progranulin in chondrocyte differentiation (Feng et al., 2010). In whole cell lysates from liver cancer cell lines, tropomyosin 3, an actin-binding protein expressed in multiple cell types, was identified as a major interacting protein for progranulin (Lam et al., 2012). The yeast-two-hybrid system was used to identify progranulin as an interacting partner with midkine, a heparin-binding growth factor important in liver tumorigenesis (Yan et al., 2012). Progranulin is also reported to interact with perlecan, a basement membrane/cell surface protein involved in angiogenesis (Gonzalez et al., 2003). The diverse set of putative progranulin-interacting proteins brings into question two key points. First, although there are instances where secreted proteins have an intracellular action, most commonly through redirection to an intracellular compartment from the endoplasmic reticulum (Arnoys and Wang, 2007), cautious interpretation is required when examining protein-protein interactions in systems employing non-physiological methods. The use of tagged or mutated proteins, over-expression of these proteins, and cell-free systems that take proteins out of their cellular context all allow for otherwise improbable interactions to occur. Second, the perhaps unlikely interacting partners for progranulin that have been identified suggest that progranulin is prone to non-specific interactions and/or that the yeast-two-hybrid screening system is prone to false positive detections. The tertiary structure of progranulin, as already discussed, is 44 conformationally very stable and predicted to have a high potential for protein-protein interactions. 1.2.7 Use of mouse models in brain diseases The use of animals to model human disease is a widespread and well-accepted scientific practice, despite some limitations. For many disorders, including but not limited to several types of cancer, heart disease, hypertension, metabolic and hormonal disorders, diabetes, obesity, osteoporosis, glaucoma, skin pigmentation diseases, blindness, deafness, neurodegenerative disorders (such as Huntington's or Alzheimer's disease), psychiatric disturbances (including anxiety and depression), and birth defects (such as cleft palate and anencephaly), disease-modeling in mice provides a useful setting in which to study complex biological interactions and test novel therapeutic interventions in vivo in a mammalian system. Indeed, mice as a model organism have been central to the vast majority of the major biologic and medical breakthroughs of this century. Practically every current-day protocol for the prevention, control, and cure of disease is based on laboratory animal research. The availability of inbred mouse strains, which provide a defined genetic background, combined with technologies that allow for targeted genetic manipulations has further increased the value of mice as a model system. The classic genetic approach of comparing wild-type organisms to those with a specific genetic deletion or alteration in a mammalian system is a useful 45 approach for improving our understanding of the biological functions of genes related to human diseases. The human brain is unique for several reasons, all of which make the study of human brain diseases in mice challenging. Some of our defining features as human are higher consciousness, complex language, and executive function, all of which can be attributed to an expansion of the cerebral cortex, particularly the frontal lobes. The human brain is composed of numerous folds, known as gyri, that allow approximately 1000 times more neurons to fit inside the skull than would fit in a smooth, unfolded structure. As such, the complexity of the human brain is immense and unequaled by most other mammals. Nevertheless, in order to study brain function using intrusive techniques, an animal model is an absolute requirement. Many human neurodegenerative diseases have a corresponding mouse model that recapitulates at least some of the core features of the disease. In some cases, genetic manipulation of an endogenous mouse gene can reproduce a neurological phenotype, as is the case with conditional presenilin knockout mice as a model of Alzheimer?s disease (Saura et al., 2004). In others, introduction of a mutant human transgene can replicate key elements of the human disease phenotype(s), as is the case with mouse models of Huntington?s disease. Sometimes multiple genetic manipulations have been 46 introduced in an attempt to reproduce as many features of the human disease as possible in the mouse, an approach exemplified by triple transgenic mouse model of Alzheimer?s disease (Oddo et al., 2003). In short, though rarely exact replicas of the human disorder, mouse models of human brain disorders are capable of recapitulating neurological disease in an experimental mammalian system. They are therefore useful and important for understanding disease pathogenesis, for exploring the molecular mechanisms of disease, and for developing and testing novel therapeutic strategies. 1.2.8 Mouse models of FTLD A recent synopsis of mouse models of FTLD described transgenic, knockin, or knockout mice for all of the five known FTLD-related disease genes (Roberson, 2013). Mutations in MAPT were the first to be discovered in FTLD, and so not surprisingly a relatively large number of transgenic mice expressing human tau mutations have been developed (Gotz and Ittner, 2008). Mice transgenic for the P301L tau mutation develop age-dependent accumulation of neurofibrillary tangles and hyper-phosphorylated tau (Deters et al., 2008), characteristic of FTLD patients with MAPT mutations. Another line of tau transgenic mice, known as K3 mice, display Parkinsonism and memory deficits due to selectively impaired anterograde axonal transport (Ittner et al., 2008). In the JNPL3 mouse model, Wnt-activation through glycogen synthase kinase-3-? is activated early and causes hyper-phosphorylation of tau (Wiedau-Pazos et al., 2009). The rTg4510 47 mouse model, which displays severe cortical and hippocampal neuron loss, has been used to test sirtuin 2 inhibitors as a potential therapeutic (Spires-Jones et al., 2012). A more sophisticated model that uses a regulatable human MAPT gene carrying a mutation that is prone to aggregation has been used to show that tau-mediated deficits in the hippocampus are reversible and that the expression human mutant tau appears to have a gain-of-function toxicity (Sydow et al., 2011). Overall, there is considerable evidence that there is a soluble form of phospho-tau oligomers that are toxic to cells, synaptic dysfunction is an early and important event related to mutant tau expression but perhaps independent of tau aggregation, and at least some of the behavioral and neuropathological changes associated with mutant tau expression can be reversed. The defined role of tau in microtubule stabilization and its pathological phosphorylation state have allowed for the development and testing of directed therapeutic approaches, including kinase inhibitors and microtubule stabilizers. The causative mutations in VCP are largely focused in an N-terminal substrate recognition domain. The mouse models of VCP mutations, all of which have included mutations in this N-terminal domain, exhibit age-dependent phenotypes that reflect FTLD patients, including neuronal dysfunction, gliosis, and abnormal accumulation of TDP-43 (Badadani et al., 2010; Custer et al., 2010; Muller et al., 2007). 48 CHMP2B mutations are a rare cause of FTLD and the four pathogenic mutations described to date all affect the C-terminal region of the protein. A transgenic mouse expressing a human mutant form of CHMP2B has reduced survival and develops ubiquitinated neuronal inclusions similar to humans (Ghazi-Noori et al., 2012) in addition to axonal deficits. Missense mutations in TARDBP primarily cause ALS and are only a very rare cause of FTLD, despite the relative abundance of TDP-43-positive inclusions in the brains of FTLD patients. Numerous transgenic mouse models expressing either a wild-type or mutant form of human TDP-43 have been generated (Tsao et al., 2012). In general, these models display overt motor neuron degeneration and severely reduced survival, which is in line with a primary role for TARDBP mutations in ALS. Because over-expression of TDP-43, whether wild-type or mutant, causes a severe phenotype, and because mice null for TDP-43 are embryonic lethal, no evaluation of FTLD-related phenotypes in TDP-43 mouse models has yet been reported. One distinct advantage to the mouse models of FTLD described so far is that they express the mutant form of the human protein and do not rely on alterations of the endogenous mouse gene to produce a phenotype. This is possible, of course, because 49 the pathological mutations in MAPT, VCP, CHMP2B, and TARDBP are gain-of-function mutations. This is an advantage for mouse models for two reasons. First, providing that a genomic DNA construct is used, expressing the human gene means that the mutation is expressed in its endogenous genetic context, guaranteeing that any potential regulatory or enhancer regions in the gene that may affect expression or have functional consequences on the mutation are included. Second, should a relevant phenotype be observed, the efficacy of preclinical testing of therapeutics can be evaluated in a system that is at least partially human-specific. This is particularly important for treatments aimed at specifically targeting the mutant allele or mutant protein. The main disadvantage of this system is that non-physiological over-expression of the mutant protein is often required, which can lead to toxicity and off-target phenotypes. 1.2.9 Mouse models of progranulin deficiency When GRN mutations were first identified as a common genetic cause of FTLD in 2006, there was no published literature addressing the role of progranulin in the brain. Likewise, there were no published reports of progranulin knockout mice in the literature. Subsequent to the discovery GRN mutations in FTLD, several groups developed mouse models that targeted the murine progranulin gene, reports of which became available throughout the course of this work. Several aspects of this project were guided and/or re-directed as new information became available with respect to progranulin function in the brain and progranulin-deficient mouse models, which will be discussed in the 50 following chapters when relevant to my work. For Chapters 3 and 4, which are based on published manuscripts, a section entitled ?Additional discussion from subsequent literature? has been added to place my work in the context of the field as it stands today, while leaving the ?Discussion? section for discussion of the literature as it was best interpreted at the time of publication. 1.3 Objectives The primary objectives of this thesis were 1) to characterize a mouse model of progranulin deficiency with respect to behavioral and neuropathological phenotypes in order to evaluate the utility of progranulin knockout mice as a model of FTLD and 2) to improve our understanding of the role of progranulin in the brain. We generated a progranulin-deficient mouse model using a conditional targeting strategy. The targeting strategy used allows for regulation of progranulin expression in different cell types or at different developmental stages. Although this strategy is more complex than the production of a straightforward constitutive null allele, it allows for greater flexibility in future studies and avoids the potential downfall of embryonic lethality. 51 The conditionally targeted progranulin-null mice that we generated also carry the lacZ reporter gene. Using reporter gene expression combined with standard immunohistochemistry, we evaluated expression of progranulin in the brain throughout development and in the adult. We then characterized progranulin knockout mice for multiple behavioral and neuropathological phenotypes. We began with a cross-sectional study in a mixed background cohort in which we performed a battery of behavioral tests, followed by an analysis of electrophysiology and neuronal morphology in the hippocampus. Several neuropathological endpoints were also assessed. Based on these findings, we subsequently generated two cohorts of progranulin knockout mice and their wild-type counterparts on two different inbred background strains in which we repeated our previous analyses. The use of multiple background strains is a means of evaluating the penetrance and expressivity of progranulin-dependent phenotypes and is an important step towards identifying genetic modifiers of disease. 52 2 Generation of Progranulin-deficient Mice 2.1 Introduction A knockout mouse model is a powerful tool for understanding the wild-type function of a gene of interest. Phenotypes that arise in the knockout mouse provide important clues regarding the function of the missing protein. Phenotyping a mutant mouse is neither trivial nor straightforward and depends largely on what tests are chosen, what order the tests are given in, the age of the mice at testing, and multiple other factors. The most practical examination for a knockout phenotype is one that is directed and specific, which can be done, for example, when the gene of interest is implicated in a human disease. We generated a knockout mouse model based on the fact that loss-of-function progranulin mutations cause FTLD with the express purpose of evaluating it for behavioral and neuropathological phenotypes. Unlike mutations in MAPT, TARDBP, CHMP2B, and VCP, GRN mutations as a cause of FTLD are unique in that they are loss-of-function mutations. This means that a traditional knockout strategy is the most appropriate for the generation of a mouse model of FTLD caused by GRN mutations. The most straightforward approach to producing a null allele is to remove all of the coding exons of the gene. When first discovered, it was proposed that non-sense mediated decay was responsible for 53 degrading the majority of aberrant GRN transcripts, causing subsequent loss of the protein. Instead of opting for a total locus deletion, we instead decided to create an allele that we predicted would be degraded by non-sense mediated decay. We chose to model a human GRN mutation from one of the families used in the original identification of GRN mutations from the FTLD research group at the University of British Columbia, known as family UBC-4. This family carries a 4 bp deletion at position 388, in exon 4. The deletion causes a frameshift that introduces a premature stop codon in exon 5. Our strategy for targeting murine progranulin closely mimics this mutation. The involvement of progranulin in blastocyst hatching and embryo outgrowth in the mouse (Qin et al., 2005), combined with the fact that all of the initially reported FTLD patients carrying GRN mutations were heterozygous carriers, led us to hypothesize that constitutive progranulin loss may be embryonic lethal. We thus chose a conditional strategy for targeting the progranulin locus. The Cre-lox system is a versatile method of genomic manipulation that is widely employed in the development of conditional transgenic mouse models. The system requires that the gene of interest, or a portion thereof, be flanked by specific 34 base pair sequences, known as loxP sites. The loxP sites do not typically interfere with gene expression, such that the ?floxed? allele expresses similar to the wild-type allele. The Cre 54 recombinase enzyme, derived from P1 bacteriophage, recognizes the loxP sites and initiates a recombination event that circularizes the DNA in between the two sites, leaving a single loxP site present in the original sequence and excising a circular DNA ring including the second loxP site with the intervening sequence. The loss of the excised sequence is sufficient to interrupt gene expression, producing a null allele. The conditional aspect of the Cre-lox technology results from the introduction of the Cre recombinase enzyme, which is also done through transgenic technology as neither Cre recombinase nor loxP sites are endogenous to the mammalian genome. The introduction of Cre expression can be under the control of a ubiquitous promoter, a cell type-specific or tissue-specific promoter, or a combination of any of these with an inducible element. This allows for incredible flexibility for the control of recombination in time and space in an in vivo setting. To avoid embryonic lethality, a ?floxed? allele can be recombined after development is complete through, for example, the introduction of Cre recombinase under the control of a drug-inducible promoter. Alternatively, for genes such as progranulin that are expressed in multiple tissues and organs, a cell type-specific promoter driving Cre expression could produce recombination in the tissue of interest without affecting expression in areas critical for development. 55 A similar but separate recombination system is the FLP1/FRT system, derived from the yeast Saccharomyces cerevisiae. It works by the same principle as the Cre-lox system, whereby a genomic sequence flanked by FRT sites can be excised through the introduction of the FLP1 recombinase enzyme. The versatility and efficiency of these two systems have made them very popular and widely used, such that a large selection of FLP1- and Cre-expressing transgenic mice is available commercially. The development and use of mice expressing reporter genes is another aspect of transgenic technology that has gained in popularity as the relative ease of producing transgenic mice has increased. A reporter gene is any gene that produces a protein product that can be easily visualized in the tissue of interest. When expression of a reporter gene is under the control of a specific promoter, reporter gene expression serves as a surrogate for expression of the endogenous gene. Because detection of the reporter gene product is straightforward and specific, this system avoids the technical difficulties of detecting gene expression using methods such as in situ hybridization. In the case of progranulin, a secreted protein, it is also of interest to visualize both the cells that produce progranulin, via the expressed reporter gene, as well as the location of the mature protein, which can be detected using standard immunohistochemistry. We chose to target the murine progranulin gene using a conditional construct that carries the lacZ reporter gene. The lacZ gene encodes the ?-galactosidase enzyme, the activity of which is rapidly visualized as a blue precipitate after the addition of the substrate X-gal. 56 Thus, our targeting strategy for producing progranulin knockout mice includes 1) an excisable gene-trap allele that expresses lacZ under the control of the endogenous progranulin promoter and simultaneously disrupts progranulin expression; a ?floxed? allele that can be conditionally recombined to disrupt progranulin expression; and 3) the introduction of a premature stop codon after removal of the targeted exons, which creates a null allele through nonsense-mediated decay and mimics a known human mutation. 2.2 Materials and methods 2.2.1 Progranulin targeting vector design Using the mouse genomic sequence available from the Mouse Genome Assembly (March 2006) polymerase chain reaction (PCR) primers were designed to amplify the progranulin (Grn) locus in three pieces from mEMS128 embryonic stem (ES) cell (strain of origin: 129S1/SvImJ) DNA [5? long arm of homology (7,080 bp from ?4.4 kb to intron 2), an internal fragment to be flanked by loxP sites (?floxed?; 538 bp including exons 3 and 4) and a 3? short arm of homology (2,613 bp from intron 4 to intron 12]. The mEMS128 ES cells have been successfully used to generate conditional gene targeted mice in the past (Devon et al., 2006) and it was important to use isogenic DNA to maximize the probability of homologous recombination in the transfected ES cells. The 57 PCR products were inserted into the pFlrt1 vector. This is a second-generation conditional targeting vector that features both Cre and FLP1 recombinase sites and has successfully been used to generate gene-targeted mice in our lab. The conditional targeting vector was then modified by sub-cloning a lacZ/neomycin fusion protein (?geo) coding sequence from the gene-trap vector pIFS into the pFlrt1 construct where it replaces the neomycin resistance cassette (referred to as ?pFleo? allele). The complete ?geo cassette comprises a consensus splice acceptor, and an internal ribosome entry site (IRES) preceding the ?geo coding sequence and a polyadenylation signal following the stop codon, such that it is spliced downstream of Grn exons 1-4 and expressed as would be native Grn. The IRES serves to uncouple the translation of the reporter from that of the Grn sequence. The ?geo cassette is situated between FRT sites, allowing its removal via the introduction of FLP1 recombinase. We elected to ?flox? exons 3 and 4, such that they may be excised by transient expression of Cre recombinase. The removal of these exons generates a frameshift mutation in exon 5, inducing a premature stop codon in exon 6 (p.Asp47IlefsX70). It is expected that, as observed in human patients, this nonsense allele will be subject to nonsense-mediated decay since the stop codon is located prior to 3?-most exon and it is located more than 55 nucleotides from the end of the transcript (Maquat, 2005). 58 However, even if a mutant protein is made, it will consist solely of 46 in-frame amino acids including the signal peptide and a short loop domain and will not contain any granulin motifs. Schematics how the targeting construct was made (Figure 2-1) and the Grn-targeted alleles that can be derived from the original construct (Figure 2-2) are provided. 59 Figure 2-1 Generation of the gene-trap ?pFleo? Grn-targeting vector. (A) The endogenous murine Grn locus. (B) The final targeting vector was generated using a two-step process. First, three segments of the murine progranulin gene were cloned into the pFlrt1 vector. Restriction sites are shown in green text and the location of the loxP and FRT sites are shown by pink and green arrows, respectively. The neomycin resistance cassette (NeoR) is flanked by the phosphoglycerate kinase (PGK) promoter and poly-adenylation (pA) sequences and is inserted antisense to the host gene. Second, the NeoR cassette was replaced with the ??geo? cassette from the pIFS vector. The ?geo cassette consists of a splice acceptor (sa) sequence, an internal ribosome entry site (IRES), a neomycin phosphotransferase/beta-galactosidase fusion protein (?geo) coding sequence and a pA signal. (C) The final ?pFleo? gene-trap targeting allele. 60 Figure 2-2 Schematic of the targeted alleles that can be generated from the pFleo construct. (A) The murine Grn locus on chromosome 11, showing 12 coding exons. Exon 1 (not shown) is a non-coding exon in the mouse. (B) The ?pFleo? gene-trap allele, comprised of a ?geo fusion protein coding sequence coupled to a splice acceptor site (sa) and an internal ribosomal entry site (IRES) and followed by a poly-adenylation (pA) sequence. The entire ?geo sequence is situated between FRT sites so that it can be removed with the introduction of FLP1 recombinase. Exons 3 and 4 are flanked by loxP sites. The red bars indicate the location of the 5?, neomycin, and 3? probes used for Southern blotting. (C) After FLP1-mediated recombination, a ?floxed? allele is produced. (D) Excision of the ?floxed? exons by Cre recombinase introduces a frameshift in exon 5 (*) followed by a premature stop codon in exon 6. The resulting allele is a null Grn allele. 61 2.2.2 Generation of Grn-targeted mice using the pFleo gene-trap vector The complete pFleo targeting vector was assembled and verified by DNA sequencing. The integrity of the recombinase sites in the vector was confirmed by transforming it into E. coli strains that constitutively express Cre or FLP1 recombinase (294Cre and 294FLP, respectively) (Buchholz et al., 1996). The validated construct was then electroporated into mEMS128 ES cells and G418 antibiotic selection was carried out to select for neomycin-resistant cells. G418 resistant clones were screened by PCR to establish proper targeting of the 3? end followed by confirmation of the 5? targeting by PCR. Clones positive in the PCR screen were confirmed by Southern blot and then micro-injected into C57BL/6 blastocysts to produce chimeric mice. Potential founding chimeras, identified by coat color, were genotyped by PCR to confirm the presence of the targeting construct and then crossed to C57BL/6 mice to test for germline transmission. Three independent founders were identified and maintained by backcrossing to the C57BL/6J background strain. 2.2.3 Generation of ?floxed? Grn-targeted mice Heterozygous mice carrying the pFleo Grn-targeted allele were crossed to mice carrying a modified FLP1 recombinase gene under the control of the human ?-actin promoter (Jackson Laboratories, strain name: B6.Cg-Tg(ACTFLP1e)9205Dym/J; Jacks #005703). 62 These mice express FLP recombinase in a wide selection of tissue from early in development and useful as a deleter strain (Rodriguez et al., 2000). Offspring in which recombination had occurred were identified by PCR using DNA extracted from a tail tip sample as the template. A ?floxed? allele can be distinguished from a wild-type allele by a small difference in size due to the presence of the loxP sites and residual FRT site on the ?floxed? allele. 2.2.4 Generation of constitutive Grn-null mice A heterozygous FLP-recombined animal was subsequently crossed to one carrying the Cre recombinase gene under the control of the human ?-actin promoter (generous gift from E. Simpson, originally from Jackson Laboratories, strain name: FVB/N-Tg(ACTB-cre)2Mrt/J, Jax #003376; backcrossed for 5 generations to C57BL/6 before being crossed to mice carrying the FLP-recombined allele). Once again, successful Cre recombination was detected by PCR on tail tip DNA. FLP- and Cre-recombined mice (denoted Grn+/-) were backcrossed to both C57BL/6 and 129/SvImJ mice for 5 generations to produce constitutive progranulin knockout mice incipient congenic on two inbred backgrounds. 63 2.2.5 Real-time quantitative PCR Cortical tissue from 2 Grn+/+, 7 Grn+/fleo, and 3 Grnfleo/fleo mice at 3 months of age was collected and immediately frozen at ?80?C. Striatal tissue from 3 Grn+/+, 3 Grn+/-, and 3 Grn-/- mice at 3 months of age was collected in a similar manner. Homogenization of each sample was performed using a Fastprep Homogenizer (ThermoScientific). Total RNA from homogenized tissues was isolated using the PureLink RNA mini kit (Invitrogen). Reverse transcription was carried out using the Superscript VILO kit (Invitrogen) according to the manufacturer's instructions. Quantitative analyses of mRNA expression were performed using PowerSYBR?green mix according to the manufacturer's instructions (Applied Biosystems). Amplification of cDNA was performed using the StepOne Plus Real-Time PCR System (Applied Biosystems). The wild-type (WT) Grn allele was amplified using primers that spanned the junction between exons 4 and 5 (forward primer, 5?-CTGTAGTGCAGATGGGAAATCCTGCT-3?; reverse primer, 5?-GTGGCAGAGTCAGGACATTCAAACT-3?); the Grn-targeted pFleo allele was amplified using the same forward primer and a reverse primer within the splice acceptor site of the targeting construct (reverse primer, 5?-AAAGACCGCGAAGAGTTTGTCCTC-3?). For constitutive Grn-null where the full construct had been recombined by FLP and Cre-mediated recombination, a forward primer in exon 2 (5?-CGGAACACAGTGTCCAGATG-3?) and a reverse primer in exon 3 (5?-GTGTCTGGGACTTCCAGCTG-3?) were used. Quantification of mRNA levels was calculated using the standard curve method, with amplification of target mRNA and control genes in separate wells. Standard curves were 64 created using 10-fold serial dilutions of whole brain cDNA from heterozygous-targeted animals. Each sample was run in duplicate. The relative amount of mRNA in each well was calculated as the ratio between the target mRNA and the endogenous level of the HPRT transcripts. 2.2.6 Tissue preparation Mice were anaesthetized by intraperitoneal injection of 0.5mg/g avertin (2,2,2 tribromoethanol) and terminally perfused through the ascending aorta with ice-cold 1X phosphate-buffered saline (PBS) followed by ice-cold 4% paraformaldehyde. The brains were then removed and post-fixed in 4% paraformaldehyde overnight. Post fixation, the tissue was equilibrated in 10% then 30% sucrose in PBS overnight at 4oC, after which it was embedded in Optimal Cutting Temperature compound (Sakura), frozen in liquid nitrogen, and stored at -80 oC. Twelve micron sections of embedded tissue were cut on a cryostat and thaw-mounted onto glass slides (Fisherbrand Superfrost Plus). 2.2.7 Immunohistochemistry Slides were warmed to 55oC for 20min on a slide warmer and post-fixed in 4% paraformaldehyde for 10min. Antigen retrieval was performed by microwaving the sections on high power for 10min in 0.01M citric acid pH 6.0, after which the sections were permeabilized in 0.1% Triton X-100 in PBS, blocked with 4% normal donkey 65 serum, and incubated at 4?C for 12-20hr in primary antibody. Immunofluorescent detection was achieved by 1hr incubation at room temperature with secondary antibodies appropriate to the primary antibodies used. Secondary antibodies were donkey anti-sheep or donkey anti-rabbit IgG antibodies complexed to either Alexa 488 or Alexa 594 dyes. All sections were then incubated for 5min in 4',6-diamidino-2-phenylindole (DAPI; 1:10 000; Sigma) at room temperature. Sections were coverslipped using Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and stored at 4oC. For colorometric detection, endogenous peroxidase activity was quenched by a 10min wash in 0.5% H2O2 in PBS in PBS to tissue permeabilization. After overnight primary antibody incubation in 2% normal serum, a 30min incubation at room temperature with a biotinylated secondary antibody (1:200) was followed by an amplification step with an avidin?biotin?HRP complex kit and the peroxidase substrate kit Vector DAB (all from Vector Laboratories), used according to the manufacturer?s instructions. Sections were dehydrated through a series of alcohols and xylene, and mounted with DEPEX (Electron Microscopy Sciences). For all immunohistochemical staining, we evaluated a minimum of three individual animals and stained multiple sections per animal; images presented are representative. 2.2.8 Competition assay The progranulin antibody (R&D Systems, Minneapolis; see Table 3-1) was incubated with a 10-fold by mass excess of recombinant full-length mouse progranulin protein 66 (R&D Systems) in a solution of 2% normal donkey serum in 1X PBS for three hours at room temperature with gentle shaking. Immunohistochemical staining was performed as described in Section 2.2.7. 2.2.9 Western blot analysis To prepare protein homogenate for Western blot analysis, mice were lightly anaesthetized and then immediately sacrificed by cervical dislocation or decapitation. Brains were removed and micro-dissected on ice, and the individual samples were snap frozen in dry ice and isopentane and stored at -80 oC. Protein was extracted from frozen tissue by homogenization for 30s in homogenization buffer [50mM Tris-HCl pH 8.0, 1% TritonX-100, 150mM NaCl, 100?g/ml phenylmethane sulphonyl fluoride (PMSF), 1% protease inhibitor cocktail (Sigma), 1% phosphatase inhibitor cocktail (Sigma), 200?g/ml sodium vanadate (Sigma), and 0.02mM Z-VAD-FMK (R&D Systems)] followed by centrifugation at 20800g for 5min and collection of the supernatant. Approximately 100?g of total protein homogenate was mixed with 5X sample loading buffer [0.3125M Tris-HCl pH6.8, 50% glycerol, 10.25% SDS, 0.078g/mL DTT, 0.025mg/ml bromophenol blue] and denatured in a 70oC water bath for 20min. Proteins were run on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. Western blotting was conducted using ?-Grn antibody (R&D Systems, 1:1000) followed by detection using enhanced chemiluminescence (Supersignal West Femto Maximum Sensitivity substrate, Pierce) and exposure to film (Kodak BioMax MR film, Sigma). 67 2.2.10 Progranulin ELISA The quantity of Grn in whole brain homogenate was determined by an enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Mouse progranulin ELISA; Adipogen, Korea). 75?g of total protein was loaded per well. All samples were run in duplicate. The ELISA was conducted according to the manufacturer?s instructions. Data represent the average of 2-6 samples per condition, and all conditions that were compared directly were run on the same plate. 2.3 Results 2.3.1 Characterization of GrnKO mice carrying the pFleo allele Progranulin knockout mice were generated using a gene-trap vector that disrupts expression of endogenous Grn and instead expresses a lacZ/neomycin fusion protein under the control of the endogenous Grn promoter. The production of ES cell clones that had correctly undergone homologous recombination with the targeting vector was confirmed by Southern blotting (Figure 2-3, A). Blastocyst injection of correctly targeted ES cell clones resulted in the establishment of three lines of Grn-targeted mice. Heterozygous and homozygous pups carrying the pFleo gene-trap allele (Grn+/fleo and Grnfleo/fleo mice respectively) were identified by PCR. Using one set of primers that spans the junction between exons 4 and 5 and a second set that span from exon 4 to the 68 5?end of the gene-trap construct, we confirmed expression of the WT and targeted alleles by real-time quantitative PCR using cDNA derived from cortical tissue from Grn+/+, Grn+/fleo, and Grnfleo/fleo mice. The level of expression of the pFleo allele is lower than that of the wild-type allele, probably due to decreased efficiency of either transcription or splicing of the larger transcript (Figure 2-3, B). As expected, a polyclonal anti-progranulin antibody (R&D Systems) demonstrated strong immunoreactivity in the cortex and hippocampus of wild-type brain sections but no immunoreactivity in sections from a Grnfleo/fleo mouse (Figure 2-3 C). Western blot analysis of whole brain homogenate with a polyclonal anti-Grn antibody confirmed the absence of progranulin in our gene-trap knockout mice (Figure 2-3, D). For the sake of clarity, Grnfleo/fleo mice are referred to as GrnKO to ensure that the reader is aware that these mice are null for progranulin despite the presence of the lacZ reporter gene, while constitutive progranulin-null mice are labeled Grn-/- throughout this thesis. For consistency, mice homozygous for the wild-type allele are termed WT or Grn+/+ as the respective littermates of GrnKO or Grn-/- mice. 69 Figure 2-3 Characterization of Grn-targeted mice. (A) Southern blot was used to confirm correct targeting of the Grn locus with the pFleo construct. Genomic DNA was digested using a combination of PshAI and AseI, separated on an agarose gel and blotted to a nylon membrane. The membrane was probed using a 5? external probe, which detects a size difference between the WT and the targeted alleles. The blot was stripped (lower panel) and re-probed with a neomycin phosphotransferase probe, which detects only the targeted allele. pFleo_vector served as a negative control in the upper panel and a positive control in the lower panel. (B) Relative mRNA expression in Grn+/+, Grn+/fleo, and Grnfleo/fleo mice. (C) Immunoreactivity for Grn in WT and GrnKO sections. (D) Immunoblot for Grn in Grn+/+, Grn+/fleo, and Grnfleo/fleo mice. Ponceau S (lower panel) is shown to indicate similar protein loading in all lanes. 70 2.3.2 Confirmation of lacZ expression and antibody specificity We evaluated a commercially available antibody raised against the mouse progranulin protein for specificity to progranulin by immunohistochemistry. We performed a dilution series on both post-natal day 7 (P7) and three-month old adult brain sections with the anti-progranulin antibody and chose the lowest concentration that gave clear and specific staining. A comparison of the staining pattern both using citric acid antigen retrieval and omitting it indicated that the staining pattern is identical but more robust and clear using the antigen retrieval step. All further immunohistochemical staining was therefore performed using antigen retrieval. The specificity of the anti-progranulin antibody was demonstrated by reciprocally staining sections of WT and GrnKO cortex from P7 animals. As expected, progranulin immunoreactivity was strong in WT cortex (dark brown color, Figure 2-4, A) but completely absent from GrnKO tissue (Figure 2-4, B). Staining with the anti-progranulin antibody was also abolished by pre-incubation with a 10-fold excess of recombinant mouse progranulin protein (Figure 2-4, compare C and D) and absent in GrnKO cerebellum (Figure 2-4, E). Mice hemizygous for the targeted pFleo allele express one copy of the lacZ reporter gene as well as the remaining wild-type progranulin allele. Mice homozygous for the 71 targeted allele express two copies of the lacZ reporter gene but are null for Grn (GrnKO). Expression of the lacZ transgene, which can be visualized by assaying for the activity of its gene product ?-galactosidase, using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside (X-gal) provides a read-out of the regional and cellular pattern of Grn promoter activation. ?-galactosidase activity is commonly considered a more sensitive assay than immunohistochemistry or in situ hybridization (Parent et al., 2004; Shintani et al., 1998). We evaluated the specificity of X-gal staining in Grn-targeted mice. WT sections from P7 cortex did not stain with X-gal (Figure 2-4, F) whereas GrnKO sections stained intensely (blue color, Figure 2-4, G). We then compared the X-gal staining pattern in hemizygous mice to the immunohistochemical expression pattern observed with the anti-progranulin antibody. Mice hemizygous for Grn appeared phenotypically normal and showed no overt alterations in development or behavior. Sequential staining of P7 cerebellum from a hemizygous Grn-targeted mouse for X-gal and anti-progranulin immunoreactivity labeled an identical set of cells (Figure 2-4, H-J). This provides evidence that the pattern of expression of the lacZ transgene faithfully replicates the endogenous Grn expression pattern, and conversely, further confirms the specificity of the anti-progranulin antibody for mouse progranulin. 72 73 Figure 2-4 Specificity of progranulin antibody and X-gal staining. (A) Immunohistochemistry in WT P7 cortex, showing strong progranulin immunoreactivity with the anti-progranulin antibody. (B) GrnKO P7 cortex stained with the anti-progranulin antibody, showing no immunoreactivity. (C) Immunoreactivity in WT P7 cerebellum using the anti-progranulin antibody. (D) Competition assay using recombinant mouse Grn protein, where progranulin staining is completely abolished. (E) GrnKO cerebellum, also showing no progranulin immunoreactivity. Note that microglia, stained with Iba1, are still present in GrnKO tissue. (F) X-gal staining in WT P7 cortex, showing no staining. (G) X-gal staining in GrnKO P7 cortex, showing abundant staining (blue). (H) X-gal staining for ?-galactosidase activity in the cerebellum of a P7 hemizygous transgenic mouse, showing clearly positive cells (blue). (I) Sequential immunofluorescent staining with the anti-progranulin antibody in the same section as in (H). (J) Merge of (H) and (I), demonstrating that the antibody labels an identical set of cells to those expressing the lacZ transgene. Images with X-gal staining are counterstained with Neutral Red, except for (H) in which no counterstain was used. Immunofluorescent images are counterstained with DAPI (blue). Scale bar = 25 ?m. 74 2.3.3 Characterization of constitutive Grn+/- and Grn-/- mice After crossing a heterozygous Grn+/fleo mouse to a deleter FLP-expressing line, the ?geo fusion protein coding sequence was excised from the targeted allele, leaving a ?floxed? Grn allele. A subsequent cross between a heterozygous ?floxed? mouse and a deleter Cre-expressing line produced a constitutive null Grn allele. An intercross of two Grn+/- mice produced pups of all three genotypes: Grn+/+, Grn+/-, and Grn-/-. We confirmed that progranulin levels were reduced in Grn+/- mice and absent in Grn-/- mice at the mRNA level (Figure 2-5, A) using quantitative PCR and at the protein level using ELISA (Figure 2-5, B). 75 Figure 2-5 Reduced progranulin levels in constitutive Grn-null mice. (A) Relative progranulin mRNA levels in striatal tissue from Grn+/+, Grn+/-, and Grn-/- mice. (B) Progranulin protein level in whole brain homogenate in Grn+/+, Grn+/-, and Grn-/- mice using a commercially available ELISA kit. N = 3 animals per genotype in (A); for (B), N = 7 Grn+/+, 4 Grn+/-, and 2 Grn-/- mice. 76 2.4 Discussion Despite the involvement of progranulin in blastocyst hatching and embryogenesis, progranulin knockout mice are fertile and viable. Functional redundancy is common in growth factor families, and the critical process of development occurs in a milieu of growth peptides, all of which appear to act to promote cell proliferation and metabolic events (Schultz and Heyner, 1993). Based on the viability of progranulin knockout mice, one must conclude that progranulin is non-essential for development. One risk associated with the targeting strategy employed here, where all coding exons were not deleted, is that it is possible that alternative splicing events that exclude the targeted exons occur in some situations. Using both ELISA and Western blot, we detected no progranulin protein in whole brain lysates from Grn-/- mice, and approximately 50% wild-type levels in Grn+/- mice. The antibody used for Western blotting is a polyclonal antibody raised against the full-length recombinant mouse progranulin protein. This is reasonable evidence to indicate that no progranulin protein is produced in our mice, though a more extensive selection of epitope-specific antibodies would be useful to perform a more exhaustive search for partial progranulin fragments. 77 In conclusion, we have generated and characterized a novel mouse model of progranulin deficiency. Our targeting strategy for producing progranulin knockout mice resulted in two lines of Grn-null mice: 1) mice carrying a gene-trap allele that expresses lacZ under the control of the endogenous progranulin promoter and simultaneously disrupts progranulin expression and 2) mice carrying a constitutive Grn-null allele which is produced via a frameshift mutation that subsequently leads to nonsense-mediated decay of the transcript and mimics a known human mutation. 78 3 Progranulin expression in the brain 3.1 Introduction A detailed analysis of the expression pattern of a protein of interest is a key first step in understanding its biological role in a given tissue or organ. Considerable information about a protein?s function can be inferred from its spatial and temporal expression pattern at the regional and cellular level. To maximize the information obtained from an analysis of gene expression in the brain, it is important to consider the brain?s structural make-up at both of these levels. The brain is organized into multiple different regions, each with a particular function or set of functions and all interconnected into a complex neuronal network. In many neurodegenerative diseases, neuronal loss starts in a highly selective region-specific manner, suggesting that it may be possible to define a unique pattern of gene expression for different brain regions. There are several cases where the gene responsible for a hereditary neurodegenerative disorder is not restricted in expression to vulnerable brain regions. Genes such as huntingtin, presenilin 1, MAPT, superoxide dismutase 1, TARDBP, and FUS are all ubiquitously expressed proteins that, when mutated, are responsible for selective neuronal degeneration in Huntington?s disease, Alzheimer?s disease, FTLD, and ALS. Even so, regional variability in gene expression 79 may provide clues as to which neural circuits and related higher level behaviors are affected by the gene of interest. The brain is a complex tissue made of two main cell types, neurons and glia. Neurons are post-mitotic cells that vary widely in size, shape, and connectivity but maintain their primary function of transmission of information via electrical signal propagation. Glia, although often referred to collectively as support cells, are composed of several distinct cell types. Astrocytes are the most abundant glial cells in the central nervous system and act in multiple ways to maintain homeostasis during neuronal signal transduction. Microglia are the immune cells of the brain, thought to be derived from peripheral monocytes, and capable of mounting an inflammatory response to local insult or injury. Other types of glia, including oligodendrocytes and ependymal cells, have additional important supporting functions but are less involved in aging and neurodegenerative processes and thus not the focus of this work. As a first step in understanding the role of progranulin in the brain and how its loss may lead to neurodegeneration, its expression pattern must be established. Progranulin expression in the human brain has been examined to a limited extent. Post-mortem brain tissue both from FTLD and Alzheimer?s disease patients as well as from unaffected control individuals is immunoreactive for progranulin in a subset of cortical 80 neurons and in activated microglia (Baker et al., 2006). The neuronal cytoplasmic and intranuclear inclusions characteristic of FTLD are not immunoreactive for progranulin (Baker et al., 2006). Instead, these inclusions are immunoreactive for TAR DNA binding protein 43 (TDP-43), a DNA/RNA binding protein with multiple functions and widespread expression. The expression of progranulin mRNA has been described in healthy adult rodent brains (Daniel et al., 2000). In situ hybridization results indicated that the level of mRNA is high in neurons within the superficial lamina of the cerebral cortex, in Purkinje cells of the cerebellum, and in pyramidal neurons and granule cells of the hippocampus. In the mouse embryo at E14.5, progranulin transcript expression is detected during development of the forebrain, olfactory lobes, retinal ganglion cells, and spinal cord (Daniel et al., 2003). In vitro results using recombinant human progranulin protein and one of its proteolytic fragments suggest that progranulin can promote neuronal survival and neuritic outgrowth and that it may therefore be involved in the development and maintenance of mature neurons and of neuronal connectivity (Van Damme et al., 2008). Progranulin does not, however, appear to be a mitotic factor for neuronal precursors because it is lacking in areas of proliferation during the process of neuronal maturation within the neopallial cortex (Daniel et al., 2003). 81 Thus, a basic description of Grn expression in the murine brain, assessed by in situ hybridization, has been put forth. In situ hybridization as a means of detecting mRNA expression is a useful tool, but it has, as does every technique, its limitations and disadvantages. It requires a sequence-specific probe, good permeabilization of the tissue being examined, and proper preservation of the mRNA of interest. In addition, it is not amenable to double-labeling with cell-type specific markers, so the exact identity of expressing cells cannot be unequivocally determined in many cases. To simplify the process of detecting expression from the progranulin promoter, we developed a knock-in mouse model in which the lacZ reporter gene is expressed under the control of the endogenous murine progranulin promoter. The expression of lacZ can be detected by a simple histochemical staining procedure that detects activity of the lacZ gene product, ?-galactosidase. This reporter system allows for high sensitivity and single-cell resolution in examining expression from the Grn promoter without the technical difficulties that accompany in situ hybridization. Here we report the results of a detailed immunohistochemical analysis of progranulin expression in WT mice in the developing and mature murine nervous system. In addition, we examine Grn gene expression with transgenic lacZ reporter Grn-targeted mice. We find that progranulin is expressed in both neurons and microglia within the CNS, but that its expression pattern in each cell type differs during development, in the adult, and in response to excitotoxic injury induced by quinolinic acid injection. 82 3.2 Materials and methods 3.2.1 Generation of knock-in reporter Grn-targeted mice Generation of Grn-targeted mice is described in detail in Chapter 2. Two-to three heterozygous pFleo mice at each experimental age were used to evaluate ?-galactosidase expression in the brain. 3.2.2 Preparation of tissue sections Tissue sections were prepared as described in Section 2.2.6 from 2-3 wild-type adult and early post-natal mice of the C57BL/6 and 129/SvImJ inbred strains. Embryonic specimens were harvested from pregnant females perfused as described for adult mice. For P0 pups, mice were decapitated without perfusion and the whole head was processed as described above. 3.2.3 Immunohistochemistry Immunohistochemical staining was performed as described in Section 2.2.7. Secondary antibodies used were donkey anti-goat, donkey anti-sheep, donkey anti-rabbit, or donkey anti-mouse IgG antibodies complexed to either Alexa 488 or Alexa 594 dyes (Molecular Probes, Invitrogen; 1:1000). 83 3.2.4 Antibody characterization Please see Table 3-1for a list of all antibodies used. 84 Table 3-1 Antibodies used for immunohistochemistry Use Antibody Antigen Description Manufacturer Dilution Grn antibody ?-progranulin Full length recombinant mouse progranulin Sheep polyclonal R&D Systems 1:250 Cell stage markers ?-PCNA Proliferating cell nuclear antigen Mouse monoclonal 424 Chemicon 1:1000 ?-nestin Nestin, concentrate; radial glia Rat-401 Developmental Studies Hybridoma Bank 1:200 ?-dcx Doublecortin, human Rabbit polyclonal Cell Signaling Technology 1:1000 Cell type markers ?-NeuN Neuronal specific nuclear protein, neurons Mouse monoclonal A60 Chemicon 1:500 ?-GFAP Glial fibrillary acidic protein, astrocytes Cy3 direct conjugate Sigma 1:500 ?-Iba1 Calcium adapter binding protein 1, microglia Rabbit polyclonal Wako 1:1000 85 Full characterization of the sheep polyclonal anti-Grn antibody (R&D Systems, Minneapolis) is described in this study (see Section 2.3.2). Mouse monoclonal anti-PCNA clone 19A2 (Chemicon, Temecula, CA) stains a single 36-kD band in Western blot analysis (Ogata et al., 1987). Detailed analysis of staining with this antibody in the adult dentate gyrus indicates that it is specific for dividing cells (Mandyam et al., 2004). The nestin antiserum (Developmental Studies Hybridoma Bank, University of Iowa) recognizes a doublet with an apparent molecular weight of 200kDa on homogenized Sprague-Dawley E15 rat spinal cord and by immunohistochemistry stains cells with a morphology that matches that of radial glia (progenitor) cells (Hockfield and McKay, 1985). The staining pattern seen in the current study is identical to previously published results (Johe et al., 1996; Lendahl et al., 1990; Nakamura et al., 2000; Sakakibara et al., 2001). The rabbit polyclonal anti-DCX (Cell Signaling Technology, MA) recognizes one or two bands at 45 kDa by immunoblot on mouse and rat brain homogenates 86 (manufacturer?s technical information). Specific immunoreactivity in the mouse CNS was absent when tissue was incubated with the DCX preimmune sera (Gleeson et al., 1999). The monoclonal mouse anti-NeuN antibody (Chemicon, Temecula, CA) recognizes the neuron-specific protein NeuN in all vertebrates tested (see manufacturer's technical data sheet for list of species; (Mullen et al., 1992)). Using mouse brain homogenate, this antibody recognizes four bands on Western blot (Unal-Cevik et al., 2004) and is thought to reflect multiple phosphorylation states of NeuN (Lind et al., 2005). The glial fibrillary acidic protein (GFAP) antiserum (Sigma-Aldrich, St. Louis) recognizes a protein of 50 kDa in porcine spinal cord homogenate (Debus et al., 1983) The staining pattern seen in the current study was identical to that previously published for mouse brain sections (Sakakibara and Okano, 1997). The rabbit anti-Iba-1 polyclonal antibody recognizes a single 17-kD band by Western blot analysis of homogenized primary microglia cultures and labels microglia and macrophages but not neurons or astrocytes by immunohistochemistry in both primary mixed brain cell cultures and in fixed rat brain sections (Ito et al., 1998). 87 3.2.5 X-gal staining to detect ?-galactosidase activity ?-galactosidase activity was assayed using the chromogenic substrate X-gal. Mice were perfused with 0.2% glutaraldehyde and 2% paraformaldehyde and the brains were removed and processed as for immunohistochemistry. Warmed mounted sections were post-fixed in 0.2% glutaraldehyde and 2% paraformaldehyde for 10min and washed 3 x 10min in lacZ wash buffer (2mM magnesium chloride, 0.01% sodium deoxycholate, 0.02% Nonidet P-40 in 0.1M phosphate buffer). Sections were stained overnight in lacZ staining solution (1 mg/mL X-gal (Gibco), 5mM potassium ferricyanide, 5mM potassium ferrocyanide in 1X lacZ wash buffer). After washing 3 x 10min in 1X lacZ wash buffer, sections were dehydrated in a graded series of alcohol and coverslipped with DEPEX (Electron Microscopy Sciences). Counterstaining, when required, was performed after the last wash step and prior to coverslipping. Sections were washed briefly in distilled H20 and then immersed in 0.5% Neutral Red solution for 60s, followed by brief rinses in distilled H20 until an appropriate amount of counterstain remained. 3.2.6 Semiquantitative evaluation of regional expression Two sagittal sections per animal from 3 individual 3-month-old C57BL/6 animals of mixed sex were processed for immunofluorescent detection of progranulin using the R&D Systems anti-Grn antibody (Table 3-1) and counterstained with DAPI nuclear stain as described above. Intact sections corresponding to approximately Bregma lateral 88 1.525 mm and lateral -0.20 mm were chosen from each animal to cover all brain regions evaluated. Using a Zeiss Axioplan 2 imaging microscope (Zeiss, Germany), brain regions were identified under the DAPI fluorescent filter using a mouse brain atlas to verify landmarks. For the visual evaluation of relative expression levels, two independent observers evaluated each brain region for fluorescence intensity based on the following scale: - for no expression present, + for weak expression, ++ for moderate expression, and +++ for strong expression. Results from three animals and from each observer were averaged and rounded to the nearest whole value. Integrated optical density measurements of immunofluorescent signal intensity in each brain region were collected for a separate set of 3 individual 3 month old C57BL/6 animals of mixed sex to provide an objective validation of the observed relative expression levels. Microphotographs were acquired using a Zeiss Axioplan 2 imaging microscope (Zeiss, Germany). A fixed exposure time was established based on the most immunofluorescent image in a given run. Focusing was achieved using the DAPI filter to avoid photobleaching the immunostaining of interest. A measurement for fluorescence intensity was obtained using MetaMorph 6.2 analysis software (Meridian Instrument Co., Kent, WA). Threshold intensity was set based on the dimmest image in a given run. The area of interest was then contoured and the integrated optical density (IOD) was measured. The data herein represent the average value of the IOD/total contoured area for three measurements (one per animal) in arbitrary units. The presence or absence of ?-galactosidase activity, indicated as either + or ? respectively, in each of the same 89 brain regions was evaluated in three hemizygous Grn-targeted mice by two observers and the results were averaged as described above. 3.2.7 Intra-striatal injection of quinolinic acid Unilateral intra-striatal injection of 8 nmol quinolinic acid (QA) was performed in four C57BL/6 x 129/SvImJ 3-month-old wild-type mice according to previously described methods (Zeron et al., 2002). Seven days post-injection mice were collected as described above for tissue sections. For immunohistochemical analysis, 25?m coronal sections covering the entire striatum were cut on a cryostat and collected into PBS in a 24-well plate. Sections were then hand-mounted onto slides and processed for immunohistochemistry as described above. 3.2.8 Image collection All images were captured using a Zeiss Axioplan 2 imaging fluorescent microscope (Zeiss, Germany) and MetaMorph 6.2 analysis software (Meridian Instrument Co., Kent, WA). The images were compiled using Photoshop 7.0 software (Adobe Systems, San Jose, CA) and were adjusted for sharpness, contrast, and brightness. 90 3.2.9 Quantification of immunofluorescent intensity Quantification of the immunofluorescent signal intensity in immunohistochemically-labeled fixed brain sections was performed as described above for the semi-quantitative evaluation of regional progranulin expression. For low power images of the striatum, equivalent sections for each animal were chosen and the entire striatum was captured in one picture per hemisphere. For individual cell analysis, five different fields of view within the lesioned area (injected hemisphere) or within the striatum (uninjected hemisphere) were photographed at 3 different focal planes. Cells were analyzed only if their nucleoli were in sharp focus. The data herein represent the fold change in expression calculated from an average of the IOD/total area for four animals (striatal measurement) or approximately 40 microglial cells from a total of four different animals (individual cell analysis). All experiments were carried out in accordance with protocols approved by the UBC Committee on Animal Care and the Canadian Council on Animal Care. 3.3 Results For all immunohistochemical and X-gal staining, we evaluated a minimum of three individual animals of mixed sex from two different mouse strains at each time point and stained multiple sections per animal. For the adult time point, mice 3 to 4 months of age were used. As no strain differences were detected, results from both strains were 91 pooled. At all time points examined, variability in regional expression patterns and staining intensity among animals was negligible. Images presented herein are representative. 3.3.1 Progranulin is expressed in microglia but not in neuronal progenitors in the developing CNS. Grn expression was examined in the developing mouse brain at time points throughout embryogenesis and into early post-natal life, from embryonic day 13.5 (E13.5) to P7. Both X-gal staining in hemizygous Grn-targeted mice and immunohistochemistry in WT mice suggest that there is restricted and very low progranulin expression in the embryonic brain (Figure 3-1, A). ?-galactosidase activity in transgenic animals at E15.5 was strong in various peripheral tissues, indicating that the lacZ transgene is expressed and that its product is detectable at this age; however, lacZ expression was limited to a small population of cells scattered throughout the developing brain (Figure 3-1, B). Most of these lacZ-expressing cells were associated with blood vessels (arrows, Figure 3-1, B) and epithelia (arrowheads; Figure 3-1, B). ?-galactosidase activity in neurons increased gradually with age; it was rare at E15.5 (Figure 3-1, C), became initially apparent at P0 (Figure 3-1, D), further increased by P7 (Figure 3-1, E), and peaked in the adult (Figure 3-1, F). 92 Immunohistochemical detection identified minimal progranulin expression throughout the brain of WT E13.5 mice, which gradually increased throughout late embryogenesis (E15.5 ? E18.5; Figure 3-1, G) and into early postnatal development (P0-P7; Figure 3-1, H and I) and appeared to peak in the adult (Figure 3-1, J). This pattern of expression correlated precisely with the detection of ?-galactosidase activity in hemizygous Grn-targeted tissue. 93 94 Figure 3-1 Progranulin is expressed at low levels in the developing brain. (A) A low power image of an E15.5 hemizygous Grn-targeted embryo expressing the lacZ transgene and stained for ?-galactosidase activity. Note that while several regions have strong ?-galactosidase activity (blue color), including the epidermis and the meninges of the pia mater of the brain, the brain itself shows very low ?-galactosidase activity. (B) Higher magnification image of the boxed area (E15.5 dorsal cortical plate (midline)) in (A). Scattered cells are positive for ?-galactosidase activity, but most are associated with blood vessels (arrows) or epithelia (arrowheads). (C-J) X-gal staining in hemizygous Grn-targeted transgenic mice (C-F) and immunohistochemical staining for progranulin expression (G-J) at different time points. (C, G) The developing neocortex at E15.5, showing a few scattered progranulin-positive cells (arrowheads) in the IZ. (D,H) The neocortex at P0, showing increased progranulin expression in cortical neurons. Note that X-gal staining in hemizygous animals at this age labeled numerous cells but that the intensity of the staining was weak in any given cell. (E,I) P7 cortex, demonstrating stronger progranulin expression than at earlier ages. (D,J) Adult cortex, showing the strongest progranulin expression. Images stained with X-gal are counterstained with Neutral Red except in A and B, where no counterstain was used. Abbreviations: cortex (ctx), VZ (ventricular zone), IZ (intraventricular zone). Scale bar = 300 ?m in (B), 25 ?m in (C-J). 95 To further characterize progranulin expression during neurogenesis, we examined neuroblast proliferation and differentiation in the neocortex. Neocortical development follows a series of stages whereby neurons born from proliferating progenitor cells in the ventricular zone (VZ) become post-mitotic and migrate along radial glia through the intermediate zone (IZ) to the cortical plate (CP), forming the layers of the cortex in an inside-out manner (Stitt et al., 1991). Beginning around E15.5, cells progressing through all of these stages can be identified (Figure 3-2). Double-immunofluorescence staining indicated that progranulin is not present in proliferating cells (immunoreactive for PCNA; Figure 3-2, B) nor in nestin-positive neuronal precursors in the VZ (Figure 3-2, C). Immature, migrating neurons in the IZ immunoreactive for doublecortin (dcx) are similarly devoid of progranulin immunoreactivity (Figure 3-2, D). A number of post-mitotic neurons expressing the NeuN antigen can be seen in layer VI of the piriform cortex (arrowheads, Figure 3-2, E) but not even these cells are immunoreactive for progranulin (Figure 3-2, E). However, starting at about P0, progranulin staining in post-mitotic neurons becomes stronger and more distinct (Figure 3-2, F), and appears to increase gradually into early adulthood. 96 Figure 3-2 Progranulin is not expressed in neuronal progenitors in the embryonic brain. (A) A low power image of an E15.5 sagittal brain section immunofluorescently labeled for progranulin in red and PCNA in green. The white box indicates the approximate area where higher power photographs were taken in (B-E). (B) Double labeling for progranulin and PCNA, indicating a lack of expression of progranulin in proliferating cells in the VZ. (C) Double labeling for progranulin and nestin, showing that progranulin is not expressed in immature neuronal precursors. (D) Double labeling for progranulin and doublecortin, demonstrating that progranulin is not expressed in post-mitotic, migrating immature neurons. (E) Double labeling for progranulin and NeuN. The lack of co-labeling indicates that progranulin expression in mature neurons is turned on later than NeuN expression. (F) Double labeling for progranulin and NeuN at P0, now showing progranulin expression in NeuN-positive mature neurons. All images are counterstained with DAPI nuclear stain (blue). Abbreviations: lateral ventricle (lv), cortical plate (cp), ventricular zone (VZ), intraventricular zone (IZ). Scale bar = 25 ?m. 97 Although most neurons in the embryonic brain stained only very weakly for progranulin, several scattered and brightly staining cells were observed throughout the developing brain (Figure 3-3, A). Interestingly, this staining co-localized with staining for the Iba1 antigen (Figure 3-3, B-C), indicating that these cells are likely microglia. Microglia originate as myeloid cells in the spleen, which differentiate into monocytes and infiltrate the CNS during late embryogenesis (Chan et al., 2007). During development, microglia have amoeboid cell bodies which project thin processes and contain numerous lysosomes and hydrolytic enzymes (Kaur et al., 2007). Progranulin-positive microglia were detected throughout the brain at all embryonic time points examined, including at E15.5 (Figure 3-3, D; arrowheads), in the white matter tracts of the developing cerebellum from P0 to P14 (P3 and P7 are shown in Figure 3-3, E and F), and in the corpus callosum of post-natal brains from P0 to P14 (P7 is shown in Figure 3-3, G). In the adult, these amoeboid microglial cells disappear from white matter tracts (Figure 3-3, H-I). 98 Figure 3-3 Progranulin is expressed in amoeboid microglia in the developing CNS. (A) Progranulin-positive cells in the white matter tracts of the cerebellum at P7. (B) Iba1 immunoreactivity in the same section. (C) Merge, indicating clear co-localization of Grn and Iba1. (D) Progranulin immunohistochemistry at E15.5, showing brightly staining, scattered progranulin-positive microglia in the area of the developing cerebellum. (E) P3 cerebellum showing progranulin-positive microglia migrating through the white matter into the cerebellum. (F) P7 cerebellum, showing progranulin-positive microglia still present in the developing post-natal cerebellum. (G) Progranulin-positive microglia in the corpus callosum at P7. (H) Adult corpus callosum, showing a nearly complete lack of progranulin-positive microglia, though surrounding cortical neurons are clearly stained. (I) Adult cerebellum, showing that progranulin-positive microglia are largely absent. All images are counterstained with DAPI nuclear stain (blue). Abbreviations: fourth ventricle (IV ven), cerebellum (cer), corpus callosum (cc), hippocampus (hip), Purkinje cell layer (PCL). Scale bar = 50?m. 99 3.3.2 Progranulin is expressed in multiple regions of the adult brain. The expression pattern of progranulin was examined by immunohistochemistry in adult wild-type brain sections and by X-gal staining in hemizygous transgenic Grn-targeted lacZ knock-in mice. A semi-quantitative evaluation of progranulin immunoreactivity in different brain regions is given in Table 3-2. Prominent immunoreactivity was seen in the neocortex and hippocampus (Figure 3-4). In the neocortex, immunoreactive cells were present in all cell layers, though staining appeared more intense in the large pyramidal neurons of layers V/VI (Figure 3-4, A). This pattern was closely mirrored by X-gal staining (Figure 3-4, B). In the pyramidal cells of the hippocampus, progranulin immunoreactivity was strongest in the CA3 region and much less pronounced in the CA1 region, both by immunohistochemistry and X-gal staining (Figure 3-4, C-D). Progranulin expression was moderate in the thalamus, hypothalamus, amygdala, and midbrain, and weak but detectable in the striatum (Figure 3-4, E-F) and brain stem. The large Purkinje neurons were the only progranulin-positive cells in the cerebellum (Figure 3-4, G); no staining was apparent in the granular or molecular layers. Interestingly, X-gal staining was absent in the cerebellum, including in Purkinje neurons (Figure 3-4, H). 100 Table 3-2 Semi-quantitative analysis of progranulin expression in the adult murine brain. Brain Region Relative Grn Expression Visual evaluation IOD/area ?-Gal Activity Prelimbic +++ 0.62 + Somatomotor +++ 0.60 + Anterior cingulate +++ 0.37 + Cortex Visual +++ 0.23 + Striatum + 0.01 + Lateral Septal Complex + 0.01 + CA1 ++ 0.56 + CA3 +++ 1.00 + Dentate gyrus + 0.33 + Hippocampal Formation Subiculum ++ 0.12 + Thalamus +++ 0.91 + Hypothalamus +++ 0.64 + Reticular nucleus +++ 0.70 + Superior colliculus ++ 0.20 + Midbrain Substantia nigra ++ 0.20 + Cerebellum Granular cell layer - 0.00 - Purkinje cell layer ++ 0.27 - Molecular cell layer - 0.00 - Medulla + 0.01 + Brainstem Pons + 0.03 + 101 102 Figure 3-4 Progranulin is expressed in multiple regions in the adult brain. (A) Immunohistochemical staining with anti-progranulin antibody and (B) X-gal staining in lacZ-expressing hemizygous transgenic animals showing neuronal staining throughout the layers of the cortex, with the most intense staining in the large pyramidal neurons of layer V. (C) Progranulin immunohistochemistry and (D) X-gal staining in the hippocampus showing strong expression in the CA3 region but weak expression in the CA1 region. (E) Progranulin immunoreactivity and (F) lacZ expression in the striatum. (G) Progranulin immunohistochemistry in the cerebellum showing staining only in Purkinje neurons. (H) ?-galactosidase staining in the cerebellum. Note that the Purkinje neurons (arrows) are negative for ?-galactosidase activity. All images with X-gal staining are counterstained with Neutral Red. Abbreviations: Purkinje cell layer (PCL). Scale bar = 50 ?m. 103 We examined progranulin expression in micro-dissected brain regions by Western immunoblot (Figure 3-5). We detected a band of approximately 80kDa in all regions tested (Figure 3-5, A) which was absent from GrnKO tissue (Figure 3-5, B). Despite varied and repeated efforts, we did not obtain consistently strong bands suitable for quantification. A qualitative assessment of band intensity supported the conclusions of our immunohistochemical staining (see Table 3-2). 104 Figure 3-5 Immunoblot analysis of progranulin expression. (A) Western blot analysis of micro-dissected brain tissue homogenates from a WT adult mouse using the ?-Grn antibody. (B) Western blot analysis of whole brain homogenate from a litter of P0 pups born to two hemizygous Grn-targeted transgenic parents. The first lane is homogenate from WT C57BL/6 whole brain, used as a positive control. The genotype for each of the six pups (geno) as determined by polymerase chain reaction is shown below each lane. 105 3.3.3 Progranulin is expressed in neurons and microglia but not astrocytes. To evaluate cell-type specific expression of progranulin in the adult brain, we performed immunofluorescent double-labeling with specific markers for neurons (NeuN), astrocytes (GFAP), and microglia (Iba1). Cells positive for the mature neuronal marker NeuN were double-labeled with the progranulin antibody throughout the brain (Figure 3-6, A-C). Although the subcellular localization of NeuN (nuclear) and progranulin (cytoplasmic) differ, the two antigens are clearly present in the same cells (arrowheads, Figure 3-6, C). Nearly all neurons in expressing regions (see Table 3-2), such as the neocortex and hippocampus, are progranulin-positive. Double-labeling is also apparent between progranulin and the microglia marker Iba1 (Figure 3-6, D-F). Progranulin immunoreactivity in microglia appears to be cytoplasmic and the relative intensity of the staining is greater than in neurons. No progranulin immunoreactivity is observed in cells detected with the astrocyte marker GFAP (Figure 3-6, G-I). 106 Figure 3-6 Progranulin is expressed in neurons and microglia but not in astrocytes. (A-C) NeuN and Grn expression in the adult neocortex, demonstrating co-expression in mature neurons. Note that while NeuN staining is mainly nuclear and progranulin staining is cytoplasmic, the same set of cells clearly express both antigens (arrowheads). (D-F) Iba1 and Grn expression in the cortex, showing co-expression in resting microglia. (G-I) GFAP expression in the corpus callosum and progranulin expression in the nearby cortex, showing a lack of co-expression. All images are counterstained with DAPI nuclear stain (blue). Scale bar = 50 ?m. 107 3.3.4 Progranulin expression is up-regulated in activated microglia. Microglial cells are resident brain macrophages. They exist scattered ubiquitously throughout the grey matter in a ramified, resting state in healthy brain tissue and upon injury or insult, transform into activated immune cells capable of cytokine secretion and phagocytosis (Kaur et al., 2007). Given that progranulin is expressed in resting microglia, we hypothesized that its expression may be altered by microglial activation. To address this question, we performed intra-striatal injections of the N-methyl-D-aspartic acid (NMDA) receptor agonist quinolinic acid (QA). QA injection causes excitotoxic neuronal cell loss (Leavitt et al., 2006) and intense microglial activation in the lesioned area, as shown by Iba1 immunohistochemistry (Figure 3-7, compare A and B). Progranulin expression is also extensively up-regulated in the lesioned area (Figure 3-7, compare C and D). The strongest progranulin immunoreactivity overlaps with Iba1, indicating that microglia and not surrounding neurons are upregulating progranulin expression following excitotoxic brain injury (Figure 3-7, compare E and F). Quantification of immunoreactivity in the QA-injected hemisphere versus the uninjected contralateral hemisphere indicates that Iba1 expression increases approximately 6-fold after QA injection, as expected (Ito et al., 2001), and that progranulin expression is approximately 500-fold higher than its resting level (Figure 3-7, G). 108 Figure 3-7 Progranulin expression is up-regulated in quinolinic acid-injected striatum. Images were captured at the same exposure for each antigen. (A) Low power representative microphotograph of Iba1 immunoreactivity showing baseline expression in the uninjected striatum. (B) Baseline progranulin expression in uninjected striatum. Note that progranulin immunoreactivity is undetectable due to the low exposure required for the QA-injected hemisphere. (C) Merge of baseline expression (D) Iba1 immunoreactivity in the QA-injected hemisphere, showing clear up-regulation. (E) Progranulin immunoreactivity in the QA-injected striatum, showing strong up-regulation. (F) The merged picture of Iba1 and progranulin immunoreactivity after QA injection, indicating that progranulin upregulation occurs largely in microglia. (G) Quantification of the staining intensity in uninjected versus QA-injected striatum for Iba1 and progranulin. Graphs represent the fold change in IOD/total area averaged from 4 animals. Scale bar = 200 ?m. 109 The immune response of microglia involves both the up-regulation of specific inflammatory factors in individual cells and the recruitment of additional microglia and peripheral macrophages to the site of injury. Since our measurement of progranulin immunoreactivity in the entire QA-injected hemisphere of the striatum does not distinguish between these two phenomena, we performed the same measurement using high power microphotographs of individual cells. Visual inspection of representative microphotographs taken at a fixed exposure indicates that the expression of both Iba1 and progranulin is clearly up-regulated in individual activated microglia cells (Figure 3-8, A-F). Quantification of the increased immunoreactivity shows that Iba1 expression in individual microglia cells is up-regulated approximately 3-fold over resting cells, while progranulin expression is over 60-fold higher (Figure 3-8, G). Overall, our results indicate that progranulin expression is highly up-regulated in activated microglia cells in response to excitotoxic injury in the striatum. 110 Figure 3-8 Progranulin expression is up-regulated in individual microglia in quinolinic acid-injected striatum. Images were captured at the same exposure for each antigen. (A) Baseline expression of Iba1 in an individual microglia cell in the uninjected striatum. (B) Baseline expression of progranulin in an individual cell in the uninjected striatum. (C) Merge of baseline expression. (D) Iba1 expression in the QA-injected striatum, indicating clear upregulation of Iba1 in individual microglial cells. (E) Progranulin expression in an individual activated microglia cell, indicating that progranulin is also clearly upregulated after microglia activation. (F) Merged picture of Iba1 and progranulin expression. (G) Quantification of the staining intensity in uninjected versus QA-injected striatum for Iba1 and progranulin. Graph represents the fold change in IOD/total area averaged for approximately 40 cells counted from a total of 4 animals. Scale bar = 10 ?m. 111 3.3.5 Progranulin expression in neurons is marginally down-regulated after physical injury. We also investigated progranulin expression in neurons in areas injured by QA injection. A quantitative evaluation in the lesioned area in the striatum was not possible due to the low baseline expression of progranulin in striatal neurons and the intense up-regulation of progranulin expression in surrounding microglia. We performed a qualitative assessment of progranulin expression in cortical neurons in the region of the cortex injured by insertion of the needle used for stereotaxic QA injection. Examination of progranulin immunoreactivity by two independent observers suggests that progranulin expression was marginally down-regulated in cortical neurons present near the needle track (data not shown). 3.4 Discussion Using the multiple approaches of immunohistochemistry in WT mice and X-gal staining in hemizygous Grn-targeted lacZ-expressing knock-in transgenic mice, we have shown that progranulin is expressed in both neurons and microglia in the developing and adult CNS. Progranulin expression in microglia is highest in the embryonic brain and in activated microglia, while its expression in neurons is restricted to fully mature cells and is most prominent in the adult. 112 The generation of transgenic mice expressing the lacZ reporter gene under the control of endogenous Grn regulatory elements provides a powerful tool for evaluating the pattern of Grn expression and for monitoring gene regulation under different conditions. Because the transgene disrupts expression of Grn from the targeted allele, the caveat exists that hemizygous targeted mice expressing only one copy of Grn may have an altered Grn expression pattern when compared to WT mice. We acknowledge this caveat; however, several lines of evidence suggest that mice heterozygous for Grn likely exhibit a near-normal developmental expression pattern of Grn. First, our own hemizygous Grn-targeted animals have no overt phenotype with respect to development, behavior, or neuropathology to the extent that we have evaluated these phenotypes thus far. Second, in the first published report on progranulin-null mice, Kayasuga et al. (2007) reported that while subtle behavioral abnormalities were observed in progranulin-null males, these differences were not apparent in heterozygous littermates (Kayasuga et al., 2007). Finally, our comparison of progranulin immunoreactivity in WT mice and lacZ expression in hemizygous Grn-targeted mice indicates that lacZ expression very closely mimics progranulin expression in the brain. Thus, our Grn-targeted mice expressing the lacZ reporter gene represent a novel and useful tool for the study of Grn expression and Grn function in the murine brain. Expression of progranulin in the CNS is low in early development and increases with age. Progranulin is not detected in neuronal precursor cells, nor is it present in migrating 113 immature neurons. This suggests that progranulin is involved in the function and/or maintenance of mature neurons, which is consistent with a role in late-onset neurodegeneration. In vitro results suggest that progranulin can promote neurite outgrowth and neuronal survival (Van Damme et al., 2008), and Van Damme et al. suggest that it may therefore be involved in neurodevelopment. Since our results indicate that progranulin expression is confined to mature neurons, it is more likely that these capabilities are used for fine-tuning neuronal connections and maintaining mature cell populations in the adult brain. Progranulin is also a potent mitogen in epithelial cells (He and Bateman, 1999), but its lack of expression in proliferating neuronal cells in the developing brain suggests that it has a distinct role in the CNS. We did not see strong progranulin immunoreactivity nor lacZ reporter expression in the cortical plate at E15.5, which is in contrast to previously reported results for Grn mRNA expression in the mouse neopallial cortex at E14.5 (Daniel et al., 2003). The previous study used in situ hybridization to examine Grn mRNA expression, a technique that does not allow for identification of specific cell types. Given that angiogenesis occurs throughout fetal development (Kurosaka and Kashina, 2008) and that progranulin expression has been reported in developing fetal microvasculature (Daniel et al., 2003), it is possible that progranulin expression in the cortical plate region represents non-neuronal cells involved in angiogenesis and/or monocytes and microglia invading the central nervous system through the vasculature. 114 In the embryonic murine CNS, progranulin expression was most conspicuous in amoeboid microglia. The exact origin of microglia in the brain is an issue still under debate, but it is most likely that microglia are derived from the myeloid cell lineage and that they arise in the periphery, colonizing the CNS during late embryogenesis (Chan et al., 2007). Amoeboid microglia spread through the CNS along tangential and radial migration pathways (Marin-Teva et al., 2004), and in this state express some markers typical of activation, such as OX-42 (Ling et al., 1990). It is therefore possible that the expression of progranulin in these cells is a passive reflection of the semi-activated state of non-stationary microglia. On the other hand, it is also possible that progranulin plays a specific role in the ability of microglia to migrate. Given that one of the core pathological features of FTLD is prominent gliosis (Baker et al., 2006), it is unlikely that the heterozygous loss of GRN, as occurs in patients, causes a major impairment to microglia migration. It remains to be seen whether progranulin-null mice show deficiencies in the migratory ability of microglia. Whether or not microglia play a functional role in the developing brain is not yet clear, but there is evidence to suggest that these CNS macrophages are active in clearing cellular debris produced by neurons undergoing developmental apoptotic cell death (Marin-Teva et al., 2004), in pruning inappropriate or superfluous axons (Marin-Teva et 115 al., 2004) and in promoting axonal migration and growth (Polazzi and Contestabile, 2002). It will be important be to assess whether or not progranulin is required for or directly involved in these functions in the future. Consistent with the pathology of FTLD patients carrying GRN mutations (Mackenzie, 2007b) and previously published in situ hybridization results (Daniel et al., 2000), high progranulin expression is observed in the adult neocortex and hippocampus. The elevated expression in areas involved in complex behavior and learning is also consistent with the clinical presentation of FTLD patients. The greatest pathology in FTLD patients with GRN mutations occurs in the upper layers of the neocortex, in the hippocampal dentate and CA1 regions, and in the caudate nucleus (Mackenzie, 2007a). In this study the highest progranulin expression in mice was observed in layers V/VI of the neocortex, in the pyramidal cells of the CA3 region in the hippocampal formation, and in the thalamus. These results suggest that it may not be the cells that produce a high level of progranulin that are most vulnerable in FTLD, but instead the afferent and/or efferent neuronal populations associated with them. The difficulty that we encountered in detecting progranulin by Western blot, in addition to the widespread but relatively low progranulin expression in other brain regions, suggests that the overall level of progranulin in the healthy brain is likely to be very low. That the regions in the mouse brain with a low abundance of progranulin coincides with the regions of greatest 116 pathology in FTLD brains may offer a possible explanation as to why even partial loss of progranulin in FTLD patients is pathologic. The only immunoreactivity detected in the cerebellum was in the large Purkinje neurons, which were not stained by X-gal in hemizygous transgenic mice. This discrepancy could indicate that progranulin is synthesized elsewhere and transported into Purkinje neuron cell bodies, or alternatively that the lacZ reporter construct used here is not correctly expressed in Purkinje cells. It is possible that Purkinje neurons express an alternative splice variant of Grn that excludes the lacZ reporter construct, located in exon 5. This has been known to occur in other situations where transgene expression shows mild discrepancies from in situ hybridization (Delaloy et al., 2006). Considering that previously reported in situ hybridization results indicate that Grn mRNA is present at high levels in Purkinje neurons (Daniel et al., 2000), alternative splicing seems the most plausible explanation for the lack of X-gal staining in Grn-targeted lacZ-expressing mice. Chronic neuroinflammation is implicated in the pathogenesis of a variety of neurodegenerative disorders, including AD (Eikelenboom et al., 2006), Huntington?s Disease (Bjorkqvist et al., 2008), and FTLD caused by mutations in the MAPT gene (Yoshiyama et al., 2007). Activated microglia in pathologic FTLD and AD patient brains show marked progranulin immunoreactivity (Baker et al., 2006). Accordingly, while we 117 detected a baseline level of progranulin expression in resting microglia in the adult murine brain, we observed an approximately 500-fold increase in progranulin immunoreactivity in QA-lesioned striatum (Figure 3-7) and a greater than 60-fold up-regulation of progranulin in individual microglia activated by the injection of QA (Figure 3-8). The apparent discrepancy in both progranulin and Iba1 immunoreactive intensity between individual cells and total lesion area may be due to the invasion of additional microglia from surrounding areas, the proliferation of existing microglia in the lesion area, the recruitment of additional blood macrophages to the site of injury, or most likely, from a combination of all of these events. In addition, progranulin is a secreted protein that can be cleaved extracellularly into biologically active granulin peptides (Bateman and Bennett, 1998; Plowman et al., 1992). It is possible that the immense up-regulation in progranulin immunoreactivity seen at the regional level represents both intracellular full-length progranulin and extracellular granulin peptides. Full-length progranulin inhibits the respiratory burst in cultured neutrophils (Zhu et al., 2002) and mediates the wound response in the periphery (He et al., 2003). It therefore seems likely that progranulin takes part in the process of neuroinflammation, which will be an important area of future investigation. In conclusion, our results show that progranulin is differentially expressed in neurons and microglia within the CNS throughout development and into adulthood. Expression in neurons increases as they mature whereas expression in microglia subsides as they 118 attain their quiescent state. This suggests that progranulin plays distinct roles in neurons and microglia in contributing to overall neuronal health and function. Understanding its various roles in the specific cell types of the CNS will be essential for determining how the loss of progranulin leads to neurodegeneration and for identifying potential therapeutic targets for the treatment of FTLD. 3.5 Additional discussion from subsequent literature It has been suggested that, in addition to neurons and microglia, progranulin may be expressed at low levels in astrocytes and ependymal cells (Ahmed et al., 2010). Although possible that progranulin or granulin peptides are taken up by cells other than those that express Grn, immunohistochemistry, as used by Ahmed and colleagues (2010) is not an appropriate technique to definitively define the cell types that produce a secreted protein. Using our lacZ reporter mice, no expression from the Grn promoter was detected in cells with the morphology or distribution of astrocytes or ependymal cells. As already noted, some caveats, such as the possibility of alternative splice variants, do exist with our reporter mice. Whether progranulin is actually expressed in cells other than neurons and microglia has therefore not conclusively been determined. Increased expression of progranulin in immune cells after injury is a robust finding that has now been replicated in several different systems. Progranulin expression is up- 119 regulated after spinal cord injury in microglia and macrophages for up to 6 months post-injury in rats (Byrnes et al., 2011; Naphade et al., 2010). Using sciatic axotomies as an in vivo model of neuronal injury in mice, Moisse et al. (2009) used immunohistochemistry to demonstrate that microglia up-regulate progranulin expression in response to neuronal injury. Intense progranulin immunoreactivity in activated microglia is a common feature found in both patients and mouse models of several neurodegenerative diseases. Increased glial expression of progranulin is present in pathologically affected areas of the spinal cord and brain of ALS patients (Irwin et al., 2009) and in mouse models of ALS (Philips et al., 2010). Increased progranulin, attributed to expression from microglia and macrophages, is detectable in CSF from late-stage ALS mouse models (Philips et al., 2010). In fact, up-regulation of progranulin expression was detected by microarray in a screen for differentially expressed genes in ALS patients even before GRN mutations were identified in FTLD patients (Malaspina et al., 2001). Progranulin expression in the brains of patients with Lewy body dementia co-localizes and correlates with beta-amyloid plaques (Revuelta et al., 2008). Progranulin immunoreactivity is increased in microglia in both human Alzheimer?s disease patients and in several Alzheimer?s disease mouse models (Gliebus et al., 2009; Pereson et al., 2009). Progranulin expression is also up-regulated, primarily in activated microglia, in the brains and in cerebrospinal fluid in patients with multiple sclerosis (Vercellino et al., 2011). 120 Given that GRN mutations that cause FTLD are loss-of-function mutations, one would predict that progranulin expression in mutation carriers would be reduced compared to normal controls. A comprehensive analysis of mRNA levels in patients versus controls showed that regions of little or no pathology in GRN-mutation carriers, such as the cerebellum and occipital cortex, had significantly lower GRN mRNA levels, as expected (Chen-Plotkin et al., 2010). Interestingly, in areas of substantial pathology, GRN mRNA levels were actually increased in patients, the increase being correlated with an increase both in the number and in the activation state of microglia. The extensive evidence for increased progranulin expression in activated microglia after all forms of acute and chronic injury, and particularly even in patients with loss-of-function GRN mutations, indicates that progranulin plays a role in neuroinflammatory processes. It also suggests that the causative role of decreased progranulin levels in FTLD patients is likely neuronal. Whether or not neurons also regulate the level of progranulin in response to injury is less clear. What has been observed in Alzheimer?s disease mouse models indicates an increase in progranulin immunoreactivity in neurons and neurites (Pereson et al., 2009). In acute spinal cord injury, it has been suggested that neuronal progranulin decreases in response to injury (Moisse et al., 2009). The differences in these two observations could be the result of numerous factors, including 121 but not limited to different organisms, different neuronal subpopulations, and/or different forms of neuronal injury. As a technical note, it can be difficult to assess the relative contribution of different cell types to the observed staining when progranulin immunoreactivity in activated microglia constitutes the vast majority of all of the staining present and is considerably more intense than what is seen in neurons. 122 4 Synaptic dysfunction in progranulin-deficient mice 4.1 Introduction Progranulin is expressed at varying levels in neurons throughout the brain and in microglia (see Chapter 3). Functionally, the role of progranulin in the brain is not well understood. In different in vitro paradigms, progranulin deficiency enhances neuron vulnerability to excitotoxic and oxidative stress (Guo et al., 2010a; Yin et al.), alters TAR DNA binding protein 43 (TDP-43) localization and solubility (Guo et al., 2010a; Kleinberger et al., 2010), reduces neuronal survival and neurite outgrowth (Gao et al., 2010; Ryan et al., 2009; Van Damme et al., 2008), and alters the stress response (Piscopo et al., 2010). Progranulin-deficient macrophages and microglia are cytotoxic to neurons in culture (Yin et al., 2010a). Progranulin is a sex-steroid inducible gene that may be involved in the masculinization of the rat brain (Suzuki et al., 2000; Suzuki and Nishiahara, 2002; Suzuki et al., 2001; Suzuki et al., 1998). Progranulin-deficient mice display alterations in male sexual behavior, increased male aggression, and increased anxiety in young adult male mice (Kayasuga et al., 2007). Subsequent work showed an increase in the volume and number of cells in progranulin knockout mice in the locus coeruleus, a sexually dimorphic brain region involved in regulating anxiety and anxiety-like behaviors (Chiba et al., 2009). 123 The first characterization of progranulin-deficient mice with respect to neurobehavioral phenotypes relevant to FTLD described increased depression- and disinhibition-like behavior, as well as deficits in social recognition and impaired spatial learning in aged progranulin-deficient mice (Yin et al., 2010b). Progranulin knockout mice display progressive neuropathological changes, including the accumulation of ubiquitin immunoreactivity and lipofuscinosis as well as increased microgliosis and astrocytosis (Ahmed et al., 2010; Yin et al., 2010a; Yin et al., 2010b). Although Yin et al. (2010) reported the appearance of mis-localized TDP-43 in aged progranulin knockout mice, similar to what is seen in FTLD patients, Ahmed et al. (2010) found no abnormal TDP-43 localization in mice up to 23 months of age. Heterozygous progranulin-deficient mice are phenotypically similar to wild-type mice for all behavioral and neuropathological endpoints that have been evaluated (Ahmed et al., 2010; Kayasuga et al., 2007). Despite reasonable evidence that progranulin deficiency in mice causes behavioral abnormalities and progressive neuropathological changes, the mechanism(s) through which progranulin exerts its effects in vivo is not known. We report the first evidence of reduced basal synaptic efficacy, impaired synaptic plasticity, and abnormal neuronal morphology in 10-12 month old progranulin knockout male mice, and suggest that 124 strategies aimed at increasing or maintaining synaptic connectivity may prove beneficial in the treatment of FTLD. 4.2 Materials and methods 4.2.1 Mice The generation of progranulin knockout mice harboring the ?pFleo? gene-trap allele was described in Chapter 2. Two heterozygous Grn+/fleo animals were bred to produce experimental cohorts of Grnfleo/fleo mice and their Grn+/+ littermates as controls. Analysis of behavior and neuronal morphology in Grnfleo/fleo mice (GrnKO) at 8-10 months of age were done after two backcrosses (N=2); electrophysiology and neuropathology were assessed in a mixed cohort of early generation (N=2) and incipient congenic (N=5) mice. All animal work was approved by the UBC Animal Care Committee and the Canadian Council on Animal Care. 4.2.2 Immunohistochemistry Immunohistochemistry was performed as previously described (Mackenzie et al., 2006a, Petkau et al., 2010) on 5?m paraffin-embedded brain sections from 3 WT and 3 GrnKO mice at 8 and 18 months of age. 125 4.2.3 Behavioral tests Prior to initiating any behavior tests, mice were allowed to acclimatize to the testing room for 10 minutes. The equipment for each test was cleaned with ethanol in between animals, and females were tested before males. All video analysis was done using EthoVision XT 7.0 software (Noldus, VA). A modified SHIRPA examination specific for behavior and cognition phenotyping was performed according to the protocol from the EMPReSS database (http://empress.har.mrc.ac.uk). Briefly, mice were evaluated in a viewing jar for body position, tremor, palpebral closure, coat appearance, lacrimation, and defecation; in an open field arena for transfer arousal, locomotor activity, gait, tail elevation, startle response, and touch escape; and under manual restraint for positional passivity, trunk curl, limb grasping, pinna and corneal reflexes, limb grasping, contact righting, biting, and vocalization. Immediately following the SHIRPA evaluation, grip strength was tested using an automated digital grip strength meter (Columbus Instruments, OH). Mice were placed on the wire grid and allowed to grab on with all four feet for approximately 5 sec before being gently pulled off by the tail. The maximum forced produced was recorded. Each mouse was given five consecutive trials, and the highest and lowest scores were disregarded so that the score for each mouse is the average of the three middle scores. The open field test consisted of one 10-minute trial per day over three consecutive days in a 50cm x 50cm x 20cm arena. The center zone was defined as a square covering 16% of the total arena area (20cm x 20cm central square). Spontaneous motor activity was measured using an automated open-field 126 system (San Diego Instruments, San Diego, CA). The activity of mice in the open field was assessed in the dark during the light cycle and was measured automatically as the number of photobeam breaks during each trial. Activity was recorded for the duration of a 30 min period. For the resident-intruder test of social interactions, a group-housed male C57BL/6 intruder mouse of similar age and weight was introduced into the test animal?s home cage and social interactions were recorded by an overhead camera for 10 minutes. In the event of an aggressive encounter, the trial was stopped immediately. Trials that ran the complete 10 minutes were manually scored (from video) for latency to first interaction, number of interactions, total time spent interacting, anogenital sniffing, and climbing behavior. Motor learning and coordination were assessed on an accelerating rotarod. Mice were placed on an accelerating rotarod (Ugo Basile, Italy) for three consecutive days with three trials per day spaced two hours apart. For each trial the rotarod was accelerated from 5 to 40 RPM over 300 sec and the latency to fall was recorded. For novel object recognition (NOR), mice were tested 24 hours after the final open field trial, which was used for habituation to the testing arena. Mice were allowed to explore two identical objects placed in opposite corners of the arena for 5 min. Any animal that did not explore each object at least once was removed from the analysis. Approximately 5 hours later, mice were returned to the arena in which one of the objects had been replaced with a novel object, and again allowed to explore the objects for 5 minutes. Total time spent exploring each object was defined as the amount of time that the nose point of the animal was detected in the zone around each object, a square 127 approximately 5cm x 5cm. The object zones were defined such that the animal moving around the arena near the wall was not detected in the object zone. For the Morris water maze (MWM), a round pool of approximately 100cm diameter was filled with water (20-21oC) to a depth of approximately 50cm and painted with non-toxic white tempera paint. Visible cues of geometric shapes were placed around the pool at a level visible to the mice. On Day 1, a habituation trial was performed by placing the mouse in a random but pre-determined release point in the pool and allowing it to explore for 60 sec. On days 2-4, training trials were performed in blocks of 4 trials per day performed at approximately the same time each day. For the training trials, each mouse was assigned a random platform location in one of four possible positions in the pool (NW, NE, SE, or SW), which was kept consistent throughout training. The platform (10cm diameter) was submerged approximately 1cm under the water. On each training day, the mouse was released from each of four possible release locations (N, S, E, W) in a random order and allowed to search for the hidden platform for a maximum of 60 sec. The latency to reach the platform was recorded for each trial. If a mouse failed to find the platform in 60 sec, a time of 61 seconds was recorded and the mouse gently guided to the platform by the experimenter. After each trial, the mouse remained on the platform for 10-15 sec before being returned to its home cage. Consecutive trials on each day were spaced approximately 30 min apart. On Day 5, a probe trial was performed where the hidden platform was removed from the pool. Each mouse was released from a semi-randomized released point. The mouse was allowed to search for 128 the platform for 60 sec, after which it was returned to its home cage. For the analysis, the pool was divided into four quadrants and the time spent in each quadrant was recorded. The forced swim test was conducted as previously described (Pouladi et al., 2009) except that video was scored using Ethovision XT 7.0 software (Noldus, VA). Passive avoidance was evaluated as previously described (Wong et al.) 4.2.4 Electrophysiology Male mice were used for both electrophysiological experiments and assessment of neuronal morphology. Females were intentionally avoided due to known effects of estrous on synaptic plasticity (Foy) and spine density in the hippocampus (Gould et al., 1990). Mice were killed by decapitation and the brain removed and immersed in oxygenated (95% O2, 5% CO2), chilled (~8oC) artificial cerebrospinal fluid (ACSF, containing mM: 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 5 MgCl2, 2 CaCl2, 25 glucose, pH 7.3?7.4, 300?310 mOsmol l?1). Sagittal slices (400?m) were cut on a vibratome (Leica VT1000), the hippocampal formation was cut at the CA3/2 border, and slices were placed in ACSF at 37oC (45 min), then room temperature (>1h). Slices were transferred to a submerged recording chamber on a DIC-IR microscope (Axioskop, Zeiss) continuously perfused (3-5 ml/min) with oxygenated ACSF (as for dissection, excepting 1mM MgCl2) at 28-30oC. 129 Hippocampal CA1 field excitatory post-synaptic potentials (fEPSPs) were evoked by constant current (40?s) applied to CA3 Schaffer-collateral commissural projections via glass microelectrodes placed in the stratum radiatum ~100?m subicular of the CA2/3 cut. fEPSPs were recorded via an extracellular glass microelectrode placed in the stratum radiatum of CA1 (300-400?m subicular to stimulating electrode) and acquired with a multiclamp700A and pClamp10 (Axon Instruments, USA). High-frequency tetani consisted of single 100 Hz trains of 100 shocks delivered twice with a five-minute interval. Long-term potentiation (LTP) was defined as a stable increase (>25%) in fEPSP slope at 45 minutes post-conditioning similarly to that in Milner et al. (2004). Depotentiation of fEPSPs by low frequency stimulation (LFS) consisted of 900 shocks at 1 Hz. For assessing probability, successful induction was defined as a stable reduction (>10%) of the fEPSP slope 30 minutes post-conditioning. Analysis of synaptic responses was set to measure the fEPSP initial linear slope at a fixed latency (Clampfit10). Stimulus intensity was set to produce 50-70% of the maximal fEPSP slope and evoked at 0.033 Hz for at least 15 min, to ensure a stable baseline. Data are expressed as percent change in fEPSP slope ? S.E.M. LTP and depotentiation values were calculated as the change in mean fEPSP slope between the 5-min period prior to conditioning and 40-45 minute (LTP) or 25-30 minute (depotentiation) period post conditioning. 130 4.2.5 Neuronal morphology Mice were decapitated, brains were removed and immediately placed in amber jars filled with Golgi-Cox fixative solution (1% KCr2O7, 1% HgCl2 0.75% K2CrO4) and stored for 12 days at room temperature. This solution was changed one time after 48 hours. The brains were then rinsed in distilled water for 5 min then immersed in 70% and 96% ethanol overnight followed by 100% ethanol for 8 hours and finally 1:1 ethanol:diethyl ether overnight. Brains were then embedded in 12% celloidin and cut at 100?m on a vibratome. Every other section was selected for staining to encompass the entire hippocampus. The staining procedure consisted of a 5 min rinse in ddH2O followed by 30 min in 16% ammonia, 2 min rinse in ddH2O, 7 min in 1% sodium thiosulphate, 10 min rinse in ddH2O and dehydration in a graded series of ethanol. Sections were then placed in xylenes for 30 seconds before being mounted on slides and coverslipped with Cytoseal (Electron Microscopy Sciences, PA). Pyramidal neurons of the dorsal CA1 region were selected for study from both hemispheres of four brain sections corresponding to approximately Bregma -1.34mm to -2.80mm based on the atlas of Paxinos and Watson (1986). Five neurons per animal from 4 WT and 4 GrnKO males were traced using a camera lucida attached to the microscope at a magnification of 400? (DMLS, Zeiss Microscope). The dendritic trees were studied using the method of Sholl (Gibb and Kolb, 1998; Sholl, 1953). The density of dendritic spines was estimated by selecting clearly visible dendritic segments with an appropriate branch order (order 2-6 for apical dendrites; order 1-6 for basal dendrites) with a length ? 10?m at 1000x 131 magnification and tracing visible spines using Neurolucida 9.0 software (MBF Bioscience, VT). No attempt was made to correct for spines hidden beneath or above the dendritic segment so the spine density values are likely to underestimate the actual density of the dendritic spines. 4.2.6 Image analysis Integrated optical density (IOD) measurements of signal intensity were acquired as previously described Section 3.2.9 using 2-3 images of the hippocampus (dentate gyrus and CA3 region) in each hemisphere acquired at a fixed exposure for 3 WT and 3 GrnKO mice at 18 months of age. 4.2.7 Statistical analysis Statistical analyses were performed by one-way ANOVA and Student?s t-test where stated (Prism 6, Graphpad Software Inc.). All behavior tests were analyzed by two-way ANOVA using sex and genotype as factors and Bonforroni post-hoc analysis. When indicated, a repeated measures ANOVA was performed individually for each sex using genotype and time as factors. 132 4.3 Results 4.3.1 Generation of progranulin knockout mice GrnKO mice were viable and fertile and intercrosses of two heterozygous parents resulted in a normal Mendelian distribution of genotypes [Grn+/+, n=81 (28.1%); Grn+/fleo, n=140 (48.6%); Grnfleo/fleo, n=67 (23.3%)]. At 3 months of age, GrnKO mice were indistinguishable from their WT littermates in size, appearance, and home cage behavior. 4.3.2 Behavior at 8 months of age in GrnKO mice A battery of behavioral tests was performed in our GrnKO mice at 8 months of age. Body weight measurement, a modified neurological SHIRPA screen, and grip strength measurement revealed no differences between WT and GrnKO mice (data not shown). To assess locomotor activity and anxiety-like behavior, GrnKO mice and their WT littermates were assessed in an open field (Figure 4-1, A). Activity levels were similar between WT and GrnKO mice with the exception of the first day of testing, where GrnKO males were hypoactive when compared to WT males (Bonferroni posttest p<0.05). WT males have been reported to display less anxiety-like behavior than WT females given by the amount of time spent in the center of an open field (Kayasuga et al., 2007; Voikar et al., 2001), and we observed this sexually dimorphic behavior in this study (Figure 4-1, B; 2-way ANOVA sex: F(1,40) = 4.62, p<0.05). GrnKO mice, however, 133 did not mimic their WT counterparts in this behavior (2-way ANOVA sex: F(1,36) = 3.65, p>0.05) and in fact showed trends towards a reversal of sex roles with respect to center crossing. GrnKO males crossed the center of the open field less than WT males (Day 1 Bonferroni posttest p<0.05), while GrnKO females tended to cross the center zone more often than WT females. To confirm that the deficits seen in GrnKO males reflected an anxiety-related phenotype and not a deficit in motor ability, spontaneous motor activity was assessed in a subset of animals from the behavior cohort. WT and GrnKO mice showed similar activity levels in a novel cage over a 30-minute trial conducted in the dark (WT female 408 ? 99 beam breaks; WT male 615 ? 108 beam breaks; GrnKO female 483 ? 70 beam breaks; GrnKO male 579 ? 46 beam breaks; 2-way ANOVA genotype: F(1,23) = 0.095, p>0.05). We examined social interactions in our GrnKO male mice using a resident-intruder paradigm. WT males showed a modest attack rate in this study, attacking the intruder mouse in 25% of cases (3 of 12 mice). However, GrnKO males were never observed to attack an intruder male (0 of 10 mice). In addition, GrnKO males displayed a longer latency to the first interaction with the intruder mouse (Figure 4-1, C) and tended to display passive and/or disinterested behavior in two other measures (climbing and/or grooming intruder [WT: 4.3 ? 3.9 sec; GrnKO: 31.6 ? 17.9 sec], anogenital sniffing [WT: 17.0 ? 8.5 sec; GrnKO: 9.6 ? 4.4 sec]). 134 Motor coordination and motor learning were evaluated using the rotarod task. GrnKO males took longer to learn the rotarod task the WT males, as indicated by shorter latencies to fall over 9 trials (Figure 4-1, D; repeated measures ANOVA genotype: F1,20 = 6.20, p<0.05). No differences were observed between WT and GrnKO females (data not shown). In the test of novel object recognition, preference for a novel object, indicated by ?60% of total exploratory time spent with the novel object, was clearly demonstrated by WT males, but absent in GrnKO males (Figure 4-1, E), despite an equal amount of total exploratory time in both groups GrnKO females also showed a clear preference for the novel object, but surprisingly, WT female mice did not. Although this may call into question the robustness of this assay, total exploratory time was reduced in WT females (Figure 4-1, F), which provides an equally likely explanation for the poor performance of WT female mice. Spatial memory and learning were assessed using the Morris water maze. Swim speed assessed in a habituation trial on the first day of testing revealed no differences between WT and GrnKO mice (2-way ANOVA genotype: F1,38 = 0.323, p=0.57). Both WT and GrnKO mice learned the location of the hidden platform over the course of 12 training trials with similar average latencies to find the hidden platform on all three days (Figure 4-1, G). A probe trial conducted 24 hours after completion of the training trials revealed no differences in spatial memory between WT and GrnKO mice, with both groups crossing the platform location an equal number of times (Figure 4-1, 135 H). WT and GrnKO mice performed similarly in a passive avoidance paradigm and the forced swim test of depression (data not shown). 136 137 Figure 4-1 Progranulin-deficient mice show sex-specific behavioral abnormalities at 8 months of age. (A) Distance traveled in an open field arena over a 10-minute trial on 3 consecutive days. (B) Frequency of center crosses in an open field arena over a 10-minute trial on 3 consecutive days. (C) Latency to first interaction between a resident mouse (WT or GrnKO) and a WT male intruder mouse. (D) Latency to fall from an accelerating rotarod in WT and GrnKO male mice (2-way ANOVA, genotype *p<0.05, F1,8=6.2). Nine trials were conducted over 3 consecutive days. (E) Preference for a novel object expressed as [time spent with novel object / (time with novel object + time with familiar object)*100]. Dashed line indicates cut-off for novel object preference. (F) Total time spent exploring either the familiar or novel object in a 5-minute period (. (G) Latency to find the hidden platform during training trials in the Morris water maze. Data shown are the average of four trials per day ? SEM. (H) Number of platform crosses in the probe trial of the Morris water maze. WT female: n=10; WT male: n=12; GrnKO female: n=10; GrnKO male: n=10. Error bars indicate mean ? SEM. 138 We evaluated heterozygous GrnKO mice at 18 months of age in the open field apparatus and in the rotarod test of motor coordination. We observed no significant differences between heterozygous GrnKO mice and their WT littermates in total distance moved, time spent in the center of the open field arena, or rotarod performance (data not shown). Overall, our behavioral analysis indicates that GrnKO mice show moderate sex-specific abnormalities in anxiety-related behavior, social interactions, motor coordination, and novel object recognition at 8 months of age. 4.3.3 Electrophysiology We investigated hippocampal field excitatory post-synaptic potentials (fEPSPs) in GrnKO male mice at 10-12 months of age (WT, 14 slices prepared from 8 mice; GrnKO, 10 slices prepared from 6 mice). Females were intentionally avoided due to known effects of estrous on synaptic plasticity (Foy, 2010) and spine density in the hippocampus (Gould et al., 1990). Hippocampal neurons from both WT and GrnKO male mice exhibited grossly normal responses to increasing stimulation intensities (Figure 4-2, A-B, input output curve), although fEPSPs in GrnKO mice were generally much smaller (Figure 4-2, B.i). This was not apparent in the mean fEPSP slope data (Figure 4-2, B.ii) due to one GrnKO slice exhibiting extremely large responses (and afferent volleys). Interpretation of absolute values generated by increasing electrical 139 stimulation is confounded by the variability inherent in stimulating electrode placement and afferent fiber recruitment. However, hippocampal fEPSPs allow analysis of the afferent volley (AV), the amplitude of which is determined by the number of axon fibers recruited by stimulation. When normalized to the AV amplitude, the fEPSP slope / AV ratio (Plath et al., 2006) reveals that postsynaptic responsiveness is significantly reduced in GrnKO slices (Figure 4-2, B.iii). For example, at 50-70% maximal output (350?A), GrnKO response ratios were ~50% of those in WT slices (WT = 0.98 ? 0.15, GrnKO = 0.49 ? 0.12, p<0.05 by t-test). Reduced fEPSP slope in response to a given afferent volley in GrnKO slices could be a result of reduced release probability of presynaptic glutamate. When given in short succession (<300 ms), the second of two hippocampal fEPSPs evoked by paired-pulse stimulation is enhanced. This short-term plasticity is traditionally held to be due to the recruitment of lower probability of release (Pr) synapses in the second response, due to the facilitatory effect of residual presynaptic calcium from the first unsuccessful release signal. Thus, paired-pulse facilitation (PPF) is seen as a measure of presynaptic Pr (Sudhof, 2004; Zucker, 1973). Analysis of PPF revealed no significant differences between slices from WT and KO mice (Figure 4-2, C), suggesting that Pr is either equivalent between genotypes, or altered in a manner independent of residual presynaptic calcium. 140 In order to study hippocampal synaptic plasticity, we employed a bi-directional plasticity paradigm (Milner et al., 2004) which first induces LTP and later depotentiates fEPSPs, by application of high- and low-frequency stimulation, respectively (HFS, LFS; Figure 4-2, D-F). In WT slices, two brief bursts of tetanic HFS, separated by 5 minutes of basal frequency stimulation, produced typical post-tetanic potentiation (PTP), which partially decayed to a robust and stable potentiated state (LTP; Figure 4-2, D-E.). In slices from GrnKO mice PTP was also apparent and, while reduced, the difference between genotypes was not significant (PTP1 and PTP2; Figure 4-2, E). At 45 minutes post-HFS conditioning, the resultant mean LTP in GrnKO hippocampi was less than half of that observed in WT slices (Figure 4-2, D-E; WT = 58.8 ? 8.5%, GrnKO = 25.8 ? 14.1%, p<0.05 by t-test). In WT slices, the probability of successful LTP induction was 86% (12 of 14 slices), whereas LTP was only induced in 40% of KO slices (4 of 10 slices). Furthermore, 30% of GrnKO slices exhibited HFS-induced depression, a phenomenon that was not observed in WT hippocampi. The data demonstrate that hippocampal LTP is not entirely absent in GrnKO mice and can be induced in a subset of slices; however, in these cases LTP is dysfunctional when compared to WT littermates. Slices from WT mice exhibited modest, but reliable, depotentiation (Figure 4-2, F). The probability of successful LFS-induced depotentiation (>10% reduction) was 57% with all but one of the remaining slices exhibiting smaller decreases in fEPSP slope (mean depotentiation WT = -11.0 ? 2.3%). In GrnKO slices, the response to LFS was less 141 clear; similarly to WT slices the probability of LFS-induced depotentiation (>10% reduction) was 50% (mean depotentiation GrnKO = -17.6 ? 8.6%); however, unlike WT slices this subset in GrnKO slices included all those which exhibited significant reductions, and the degree of depotentiation was >20% in 40% of all slices. In contrast, the maximum depotentiation achieved in WT slices was 20%. These qualitative differences in response to LFS between genotypes are reflected in the highly significant differences in the variances between the data sets, but no significant difference in the mean level of depotentiation (Figure 4-2, F). Analysis of only those slices in which successful LFS-induced reductions in fEPSP slope did occur demonstrated that the mean depression was significantly greater in GrnKO slices (WT = -15.8 ? 1.6%, GrnKO = -38.1 ? 12.2%, p<0.02 by t-test). Perhaps surprisingly, there was no clear correlation between the degree (or failure) of LTP and the magnitude (or failure) of depotentiation in GrnKO slices. The data suggest that LFS is capable of inducing depotentiation in slices from GrnKO mice and that, when they do occur, LFS-induced reductions in GrnKO hippocampi are much greater in magnitude than those observed in slices from WT mice.
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Genetics
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The role of progranulin in the brain
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