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Generation and characterization of embryonic stem cell lines derived from the YAC128 mouse model of.. Thiele, Jenny 2010

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GENERATION AND CHARACTERIZATION OF EMBRYONIC STEM CELL LINES DERIVED FROM THE YAC128 MOUSE MODEL OF HUNTINGTON DISEASE  by Jenny Thiele  A THESIS SUBMITTED IN PARTIAL FULLFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate Studies (Medical Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2010 © Jenny Thiele, 2010 i  Abstract Huntington Disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the Huntingtin gene. Patients typically present  in  mid-life  with  progressive  motor  dysfunction,  cognitive  deficits,  and  neuropsychiatric abnormalities. Recently, researchers have provided evidence that HD is associated with significant pathology in peripheral tissues as well. At the current time no effective treatment has been proven to alter or cure progression of HD which leads to complete loss of independence and eventual death an average of 20 years after disease onset. The ability to model Huntington disease in animals has enabled studies which have provided new insights into the mechanisms of HD pathogenesis. However, the development of simple cell culture-based systems will be useful to accelerate our research efforts into the basic underlying pathogenic pathways of HD and will allow dissection of cellular interactions and the identification of novel targets for intervention that offer the greatest hope of a cure. The YAC mouse model of HD expresses full-length human Huntingtin with either 18 polyglutamines (YAC18) or 128 polyglutamines (YAC128), and develops age-dependent cognitive deficits, motor dysfunction, and selective striatal neurodegeneration similar to that seen in human HD patients. I have generated novel embryonic stem (ES) cell lines from wild-type, YAC18 and YAC128 mice on two genetic backgrounds. These cell lines have been cultured under defined conditions over long periods of time, and express characteristic markers of pluripotency, such as alkaline phosphatase, Oct-4 and Nanog. Neurons and macrophages derived from these novel cell lines using established in vitro protocols have been characterized via immunocytochemistry and challenged in functional assays. To confirm results attained from functional assays in our ES-derived macrophages, I examined primary macrophages and microglia cultures derived from the YAC mice and determined the functional response of these cells to endotoxin stimulation. Primary cell cultures isolated from YAC128 mice produced significantly more IL-6 than wild-type cultures. In comparison, with the same endotoxin stimulation, YAC18 primary macrophages and microglia responded with similar levels of IL-6 release as cultures of wild-type cells, suggesting that the over-activity in the YAC128 cytokine response is caused by the mutant Huntingtin transgene.  ii  Table of contents Abstract.................................................................................................................................. ii Table of contents ................................................................................................................. iii List of tables .......................................................................................................................... v List of figures ....................................................................................................................... vi List of abbreviations .......................................................................................................... viii Acknowledgements ............................................................................................................. ix Dedication.............................................................................................................................. x Co-authorship statement..................................................................................................... xi 1. Introduction ....................................................................................................................... 1 1.1 Huntington Disease....................................................................................................... 1 1.1.1 History .................................................................................................................... 1 1.1.2 Clinical findings ...................................................................................................... 3 1.1.3 Genetics ................................................................................................................. 4 1.1.4 Neuropathology ...................................................................................................... 6 1.1.5 Peripheral alterations ............................................................................................. 7 1.1.6 Pathogenesis.......................................................................................................... 8 1.1.7 Models.................................................................................................................. 10 1.2 Hypothesis and objectives .......................................................................................... 13 1.3 References.................................................................................................................. 14 2. Generation and characterization of embryonic stem cell lines from YAC18 and YAC128 transgenic mice................................................................................................ 22 2.1 Introduction ................................................................................................................. 22 2.2 Materials and methods................................................................................................ 25 2.2.1 Generation of novel ES cell lines ......................................................................... 25 2.2.2 Characterization ................................................................................................... 27 2.3 Results ........................................................................................................................ 32 2.3.1 Generation of 17 novel ES cell lines .................................................................... 32 2.3.2 Characterization of novel ES cell lines using stem cell markers .......................... 33 2.3.3 Serial culturing...................................................................................................... 36 2.3.4 ES cell differentiation into neurons....................................................................... 38 2.3.5 ES cell differentiation into macrophages .............................................................. 41 2.4 Discussion................................................................................................................... 48 2.5 References.................................................................................................................. 53  iii  3. Assessment of macrophages and microglia from YAC128 mice in a model of acute inflammation ................................................................................................................... 57 3.1 Introduction ................................................................................................................. 57 3.2 Materials and methods................................................................................................ 61 3.2.1 Ex vivo studies ..................................................................................................... 61 3.2.2 In vivo study ......................................................................................................... 64 3.3 Results ........................................................................................................................ 65 3.3.1 Ex vivo studies ..................................................................................................... 65 3.3.2 In vivo study ......................................................................................................... 72 3.4 Discussion................................................................................................................... 75 3.5 References.................................................................................................................. 78 4. Conclusion....................................................................................................................... 81 4.1 Summary..................................................................................................................... 81 4.2 Advantages and disadvantages of the chosen models ............................................... 84 4.3 Future directions and significance............................................................................... 87 4.4 References.................................................................................................................. 90 Appendix.............................................................................................................................. 92  iv  List of tables Table 1: List of primer sequences used to identify wild-type and YAC ES cell lines............. 27 Table 2: List of primary antibodies used for ICC. . ................................................................ 28 Table 3: List of primer sequences used to evaluate cDNA derived from ES cell lines.......... 28 Table 4: Overview of all established novel ES cell lines. . .................................................... 48 Table 5: List of primary antibodies used for ICC and IHC..................................................... 62 Table 6: Numbers of animals used to isolate primary microglia in each ex vivo CSE experiment. . ........................................................................................................... 64  v  List of figures Figure 1: Workflow of the generation of novel ES cell lines. ................................................. 26 Figure 2: Isolation of novel mouse ES cells from YAC mice. ................................................ 32 Figure 3: Novel ES cell lines stained for alkaline phosphatase (AP). ................................... 34 Figure 4: Nuclear expression of Oct4 in novel ES cell lines.................................................. 35 Figure 5: Novel ES cell lines expressed known ES cell markers shown by RT-PCR. . ........ 35 Figure 6: Novel ES cell lines were able to maintain the characteristic morphology and high levels of Oct4 expression after 50 passages. ...................................................... 37 Figure 7: After 50 passages, the novel ES cell lines did not stain for F4/80 and only occasional cells were found to stain weakly for Nestin after 50 passages. .......... 37 Figure 8: ES-derived neuronal cultures stained similar to primary neurons.......................... 38 Figure 9: ES-derived neurons and astrocytes. ..................................................................... 40 Figure 10: Homogeneous ES-derived cultures stain for the macrophages markers F4/80 and CD11b. . ...................................................................................................... 41 Figure 11: Culture time influenced the cell size of alveolar macrophages. ........................... 42 Figure 12: Upon LPS stimulation ES-derived macrophages released TNFα into the culture media.................................................................................................................. 43 Figure 13: Rolipram treatment reduced TNFα release. . ...................................................... 44 Figure 14: Rolipram dose-response of ES-derived macrophages. ....................................... 44 Figure 15: ES-derived macrophages internalize 2 um latex beads by forming actin cups like primary macrophages. ....................................................................................... 45 Figure 16: Novel ES-derived macrophages. ......................................................................... 46 Figure 17: Upon CSE stimulation novel ES-derived macrophages released TNFα into the culture media. ..................................................................................................... 47 Figure 18: Upon CSE stimulation novel ES-derived macrophages released IL-6 into the culture media. ..................................................................................................... 47 Figure 19: Primary cells isolated from the lung expressed glycoproteins characteristic for macrophages...................................................................................................... 65 Figure 20: Primary macrophages isolated from the lungs of wild-type FVB/N mice were able to engulf latex particles....................................................................................... 65 Figure 21: HD monocytes, macrophages, and microglia were overactive when stimulated. 66  vi  Figure 22: Mixed glia cultures appeared to be multi-layered consistent of microglia and astrocytes at different activation levels............................................................... 67 Figure 23: Primary glial cultures expressed common markers of microglia and astrocytes, but not neurons................................................................................................... 68 Figure 24: Microglia isolated from neonatal mice released IL-6 when stimulated with CSE. 69 Figure 25: YAC128 primary microglia were over-active when stimulated with CSE. . .......... 70 Figure 26: YAC128 microglia exhibited elevated IL-6 levels in response to 24 hours of CSE exposure when compared to wild-type and YAC18 microglia. . ......................... 71 Figure 27: YAC128 microglia release higher levels of cytokines than YAC18 after 20 hours of CSE exposure. . ................................................................................................. 72 Figure 28: Injections of CSE into the striatum of FVB/N mice caused up-regulation of activated microglia. ............................................................................................ 73 Figure 29: Striatal injections of CSE caused a 4-fold increase of IBA-1 signal in YAC128 mice compared to wild-type mice. . .................................................................... 74 Figure 30: Novel ES cell lines but not MEFs or whole brain samples express known ES cell markers shown by RT-PCR. .............................................................................. 92 Figure 31: A representative karyotype of a ES cell line after prolonged culture. .................. 92  vii  List of abbreviations AP  alkaline phosphatase  CSE  control standard endotoxin  DAPI  4',6-diamidino-2-phenylindole  DMEM  Dulbecco's modified Eagle medium  DIC  days in culture  dpc  days post coitus  EB  embryoid body  ES  embryonic stem  FBS  fetal bovine serum  HD  Huntington disease  HTT  human huntingtin gene  Htt or Hdh  mouse huntingtin gene  ICC  immunocytochemistry  ICM  inner cell mass  IL-6  interleukin-6  IMDM  Iscove’s modified Dulbecco’s medium  INF-γ  Interferon-γ  KSR  knockout serum replacement  LPS  lipopolysaccharide  MEF  mouse embryonic fibroblast  MSN  medium spiny neuron  NEAA  non-essential amino acids  NMDA  N-methyl-D-aspartic acid  NGS  normal goat serum  Oct4  Octamer 4  PBS  phosphate-buffered saline  PBS-T  phosphate-buffered saline with triton-X100  RT  room temperature  RT-PCR  reverse transcription polymerase chain reaction  TNFα  tumor necrosis factor-α  TUNEL  terminal deoxynucleotidyl transferase dUTP nick end labelling  ULC  ultra low attachment cluster  YAC  yeast artificial chromosomes  viii  Acknowledgements First and foremost, I would like to thank to Dr. Blair Leavitt, my supervisor and mentor. I am grateful for all his support, encouragement, and trust over the years. His guidance has enabled me to grow with my projects while still giving me the room to work in my own way. I could have not wished for a better supervisor. I would also like to show my sincere gratitude to the members of my supervisory committee, Drs. Simpson and Rossi, for their advice and constructive criticism on my thesis projects. I am indebted to many of my colleagues at the CMMT who supported me over the course of my graduate studies. My thanks go to all members of the Leavitt lab for their help, advice and friendship. Gelareh Mazarei, Laura Wagner, Rebecca De Souza, Colúm Connolly, Terri Sosa, Angela Gruney, Austin Hill, Ge Lu and Scott Neal thank you for endless talks, late night hours of hard work, continuous proof-reading, speed-pipetting competitions, Japanese fun and last-minute ordering. I also would like to thank Kathy Banks and Russell Bonaguro from the Simpson lab, without their advice I would have had no ES cell lines. Further, I would like to show my gratitude to Cheryl Bishop, the Graduate Secretary at the Department of Medical Genetics, for her help and encouragement. Finally, I would like to thank all my friends and family, in particular my husband Veikko Thiele. His courage, self-discipline and strength inspire and mesmerize me every day. I am very grateful to have him.  ix  Dedication                                                                                                                                                              gÉ Åç uxÄÉäxw ytÅ|Äç       x  Co-authorship statement In September 2007, I started my graduate studies as a Master of Science candidate in the Department of Medical Genetics at the University of British Columbia. Since then, approximately 85 % of my time has been dedicated to planning and conducting my experiments, and corresponding with collaborators. I was fully responsible for performing the majority of experimental procedures, including troubleshooting at all stages. Procedures which had to be performed in the transgenic core facility at the Centre for Molecular Medicine and Therapeutics, such as setting up mouse matings, collecting blastocysts, and mouse striatal injections, were carried out by a team of Research Technicians including: Jenny Gregg, Kathy Banks, Rewa Grewal, and Ge Lu. During my graduate work, Colúm Connolly and Yuanyuan Gao helped performing tissue culture, ICC, and ELISA experiments at various times. Ge Lu performed the animal surgeries to isolate primary macrophages. Austin Hill carried out all the genotyping experiments to identify wild-type, YAC18, and YAC128 status of the mice used and the generated cell lines. Gelareh Mazarei quantified the IBA-1 signal in brain sections of PBS or CSE injected mice. This thesis also incorporates the results of a joint research project published in: Björkqvist M, Wild EJ, Thiele J et al, A novel pathogenic pathway of immune activation detectable before clinical onset in Huntington's disease, The Journal of Experimental Medicine 205(8): 1869-1877 (2008). The collaboration is covered in Chapter 3 (Figure 21) of my thesis. I have obtained written permission from Dr. Maria Björkqvist and Dr. Edward Wild to include the above stated material in my thesis. I was fully in charge of the preparation of this thesis and the prospective manuscripts resulting from this project. Finally, the entire project was at all times under the supervision of Dr. Blair Leavitt.  xi  1. Introduction 1.1 Huntington Disease Huntington disease (HD) is an inherited neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat in the HTT gene that encodes for a polyglutamine expansion in the huntingtin protein. Affected individuals typically present motor dysfunctions, cognitive deficits, and neuropsychiatric abnormalities. However, recent studies have demonstrated that HD is also associated with malfunctions in peripheral tissues. In Caucasians, HD has a prevalence of 4-8 affected per 100,000 individuals. Much lower rates have been reported for most populations of Asian (Japan only 0.5 affected individuals per 100,000) and African ancestry (reviewed by Harper, 1992). Currently, there is no effective treatment available that alters the course of this devastating disease and progressive disability and loss of function eventually leading to death about 20 years after onset.  1.1.1 History George Huntington’s classic description of adult-onset hereditary chorea, in the paper ‘On chorea’, ultimately led to the currently used designation of the disease. Interestingly however, there is much evidence in the literature that he was not the first one to describe the characteristic disease features and inheritance patterns. Epidemic dancing mania or choreomania (Greek: khoreia = 'dance' and mania = 'madness’) was first mentioned in Europe as early as 1374. Documentations affirm hundreds of people, in towns along the River Rhine in Germany and later in north-eastern France and the Netherlands, danced through the streets, screaming of visions and hallucinations (reviewed by Waller, 2009). Numerous hypotheses were proposed such as demonic possession, heretical dancing cults, epilepsy and ergot poisoning. However, more recent evidence from the fields of psychology, history and anthropology suggests that the medieval European dancing plagues were in fact a mass psychogenic illness (Waller 2009).  1  In the 16th century Paracelsus suggested ‘Chorea Sancti Viti’ – the rapid, jerking physical movements of medieval pilgrims who journeyed to the shrine of Saint Vitus, patron of dancers, young people and dogs – to be an organic medical condition (Aubert 2005). Thomas Sydenham, an English physician, used the very same term to describe childhood chorea in 1686, which was later recognized as a late manifestation of rheumatic fever by Charcot (Koutouvidis 1995, Aubert 2005). By the 1900s several doctors, including Charles Oscar Waters, Charles Gorman and Johan Christian Lund, later studying a secluded population in Setesdalen (Norway), described the hereditary nature of chorea before George Huntington published his findings in 1872 (Harper PS 2002, Harper B 2005), but these reports were not well known. It is believed however, that Huntington was the first clinician to conclude that once an individual did not develop the disease during a normal life span it would not be transmitted to subsequent generations, instead of skipping a generation before re-appearing (Wexler 2006). During the following century, the neuropathology of HD was well described, the worldwide distribution of the disorder was characterized and its juvenile form was documented, but advances in our understanding of HD were limited. Until in March 1993 ‘The Huntington's Disease Collaborative Research Group’ determined the genetic cause of the disease as CAG trinucleotide repeat expansion in a gene called IT15 (interesting transcript 15), later known as the Huntingtin or HTT gene. Shortly after this discovery, researchers at UBC lead by Dr. Michael Hayden described the inverse correlation between the triplet repeat expansion size and the age of onset that accounts for the non-mendelian genetics of the disorder, specifically the phenomenon of anticipation (Andrew 1993, Duyao 1993, Snell 1993). Since then various animal models have been developed, which I will discuss briefly later in this chapter. Current research is focussing on identifying the wild-type function of the affected protein huntingtin, as well as molecular mechanisms by which mutant polyglutamine expanded huntingtin causes the disease, and in identifying treatment options which alter HD pathogenesis. Furthermore, several extensive longitudinal studies have been undertaken to identify biomarkers of disease progression.  2  1.1.2 Clinical findings Before the illness manifests itself, affected individuals are healthy showing minimal detectable functional impairment or obvious clinical abnormalities. The first symptoms can appear at any time between the ages of 1 and 80 years, with a mean age of onset around 40 years (Andrew 1993). The healthy phase and the pre-manifest (or pre-diagnostic) phase are occasionally defined as the presymptomatic stage, however during the pre-manifest phase many patients already exhibit subtle changes to personality, cognition, and motor control of which they are often unaware (Paulsen 2008, Tabrizi 2009). Family members may describe the affected person as irritable, disinhibited or restless, multitasking becomes difficult and events of forgetfulness and anxiety may occur more frequently (Walker 2007). Large ongoing studies of pre-manifest and early manifest HD subjects, such as Predict-HD and TRACK-HD, have identified neurobiological and clinical alterations that are quantifiable and significant decades before more severe motor symptoms like chorea and motor impersistence are noticeable (Paulsen 2008, Tabrizi 2009). However, right now the formal clinical diagnosis of HD is based on the presence of specific motor symptoms (an extrapyramidal movement disorder) in the setting of a known family history of HD and is verified through genetic testing for the disease allele. Chorea is slowly progressive and mainly observed during the early stages of HD, but often peaks in the mid-stages and then becomes replaced by a clinical picture dominated by bradykinesia, dystonia and rigidity as the disease progresses (Leavitt 2000). In comparison, motor impersistence, the inability to maintain a constant voluntary muscle contraction arises independently of chorea and correlates with disease progression. Quantifiable abnormalities in fine motor skills such as tongue force and finger tapping rhythm and rate may be useful in the future for establishing an early diagnosis of HD (Paulsen 2008, Tabrizi 2009), whereas changes in gait, balance and posture are often observed later (Busse 2009, Grimbergen 2008, Rao 2009). Cognitive dysfunction can greatly vary in different individuals affected with HD. However it often affects executive functions such as organising skills and problem solving, but also visuo-spatial abilities, emotional processing (Sprengelmeyer 1996) and smell identification (Tabrizi 2009). These changes can appear early in pre-manifest subjects before motor symptoms are noticeable and once they occur they do tend to worsen as the disease progresses (Montoya 2006, Paulsen 2008, Tabrizi 2009).  3  Psychiatric and behavioural symptoms can occur at any point in the illness, independent of disease state, and are very common and vary from depression, low selfesteem and guilt to aggression, anxiety, obsession, compulsions, hallucinations and suicide (Huntington Study Group 1996). A small percentage (5-10 %) of HD subjects develops symptoms before the age of 20. These juvenile cases are often present with rigidity, bradykinesia, ataxia as well as seizures occasionally without the typical chorea of adult-onset HD (Andrew 1993, Nance 2001, Ruocco 2006). They are sometimes detected through declining motor milestones or school performance. As the disease progresses, motor and cognitive deficits generally become severe, and patients typically die about 20 years after being diagnosed, often from cardiac failure, complications of falls, inanition, dysphagia, or aspiration (Lanska 1988, Sørensen 1992). HD research has advanced over the last decades, however survival rates in areas where medical support is largely unavailable, like Venezuela, are similar to those of populations with wide access to treatments (Walker 2007). As of now, no effective intervention has been identified to treat or cure HD progression. Tetrabenezine or neuroleptics can be used to treat severe chorea, but cause bradykinesia, rigidity, and depression or sedation in high doses. Counselling, medication, and psychiatric treatment are applied to deal with behavioural symptoms and affective disorders (Walker 2007).  1.1.3 Genetics HD is inherited in a dominant fashion and caused by CAG repeat expansion in the HTT (alternative HD, IT15) gene (The Huntington's Disease Collaborative Research Group 1993, NCBI Entrez Gene). In humans the HTT locus is located on chromosome 4p16.3 consisting of 180 Kb within 67 exons (as listed in NCBI Entrez Gene). Two alternatively polyadenylated transcripts were identified, a 13.7 kb large form was expressed mainly in fetal and adult brain, and a 10.3 kb form was present more widely (Lin 1993). Putative orthologs were discovered in many species, including chimpanzee, dog, mouse, rat and chicken (as listed in NCBI HomoloGene). The coding sequence of the mouse homologue (Htt, Hdh), located on chromosome 5, is 86 % identical to the human at the DNA level and 91% identical at the protein level (Barnes 1994). Different however is the CAG repeat size, encoding for only seven consecutive glutamines in wild-type mice (Barnes 1994).  4  In human, the size of the trinucleotide repeat sequence is polymorphic ranging from 10-35 repeats in the unaffected population (Andrew 1993, Duyao 1993, Snell 1993). The illness has been reported as incompletely penetrant in individuals with 36-40 CAG repeats, but appears fully penetrant in cases of 41 or more CAG repeats (McNeil 1997, Quarrell 2007). Furthermore, the number of CAG repeats inversely correlates with the age of onset, so that longer CAG repeats are associated with earlier disease manifestation. It is thought that 50 to 70 % of the variation in age of onset is caused by CAG repeat size and the remainder represented by modifying genes and environment (Andrew 1993, Myers 2004). HD patients with large CAG repeats of ≥60 generally have a juvenile onset (Andresen 2007), among these cases the relationship between repeat size and onset age is clear (Andrew 1993). This correlation appears to be much weaker for individuals with ≤55 repeats where the repeat size is not entirely predictive of onset age (Andrew 1993). Monozygotic twins often have a very similar age of onset; however their clinical features may vary significantly (Friedman 2005, Gómez-Esteban 2007). Patients carrying homozygous mutant alleles show no significant differences in age of onset when compared to heterozygotes, but may have more severe disease progression (Squitieri 2003). Similar to other trinucleotide repeat expansion disorders, CAG repeats greater than 28 appear to be instable upon replication, in the case of HD most leading to expansion (Andrew 1993). Greater expansion of the trinucleotide repeat and higher frequency of juvenile-onset cases have been observed with paternal transmission (Andrew 1993, Ranen 1995). The CAG repeat size instability often accounts for the observed effect of anticipation, in which the age of onset appears earlier in consecutive generations (Andrew 1993, Ranen 1995). Moreover, somatic instability of the CAG repeat size was found to be greatest in brain regions most affected in the CNS and relatively stable in the cerebellum. This instability has been suggested to contribute to the selective vulnerability of specific brain regions in HD patients (Telenius 1994). Predictive genetic and prenatal testing is available in many countries. Strict regulations are in place to ensure confidentiality, counselling before and after testing as well outlining sources of support, however less than 5% of individuals at risk for HD choose to actually pursue genetic verification (Walker 2007, Bombard 2008).  5  1.1.4 Neuropathology Although HTT is expressed throughout the body, the main site affected by dysfunction and cell death is the brain, and specifically the corpus striatum - which in humans is comprised of the caudate nucleus and the putamen. Many HD features, such as motor dysfunction and neuropsychiatric behaviour are thought to be directly caused by the dysfunction or death of neurons within the striatal network (DeLong 1986, Calabresi 1997) and the cortex (reviewed by Mattson, 2008). Vonsattel et al established a rating scale (grade 0-4) to classify the severity of caudate atrophy in post-mortem brain tissue. Grade 0 represents the least affected grade of pathology, with no gross neuropathological abnormalities, but up to 30-40% loss of neurons in the caudate nucleus when examined microscopically. As the disease progresses pathological changes become more widespread. In grade 1 brain samples approximately 50% of neurons are lost in the caudate nucleus which increases to 95% in grade 4 (Vonsattel 1985). The effects of the mutation, although selective, are not limited to the striatum. Using advanced Magnetic Resonance Imaging (MRI) technology combined with volumetric analysis researchers identified significant reductions in whole-brain volume, cortical thinning as well as regional grey and white matter differences even in presymptomatic HD individuals up to 10 years before the predicted age of onset (Tabrizi 2009). Changes are detected early in the caudate nucleus and putamen, but increasingly spread through the brain over the course of HD. Affected areas other than the striatum include the substantia nigra, cortical layers 3, 5, and 6, parts of the hippocampus, cerebellum, hypothalamus, and the thalamus (reviewed by Walker, 2007). Interestingly, the neuropathology appears to be more widespread and accelerated in juvenile cases (Myers 1988). At late stages of HD up to 40 % of brain weight is lost, attributed to atrophy. Ventricles are drastically enlarged. The medium spiny projection neurons (MSN), which make up about 95 % of striatal neurons are particularly vulnerable to degeneration in HD, while interneurons remain intact even as the disease progresses (reviewed by Mitchell et al, 1999). Among the MSN population, neurons of the caudate nucleus appear to be affected earlier than those of the putamen. MSNs exert inhibitory effects to the basal ganglia by using the transmitter γaminobutyric acid (GABA) and co-transmitters such as enkephalin or substance P (Reiner 1988). Evidence suggests that enkephalin positive MSNs are more sensitive than neurons that contain substance P/ dynorphin (Sapp 1995). The loss of the inhibitory input of these  6  cells may account for the choreic movements typically observed in early HD patients (Joel 2001). Another pathologic feature of HD is the presence of cytoplasmic and nuclear inclusions containing misfolded wild-type and mutant huntingtin protein, which were first identified in transgenic mouse models of HD (Davies 1997, Slow 2003) and later in human postmortem brain tissue (DiFiglia 1997). It is unclear whether these aggregates are toxic, protective or an epiphenomenone of the disease mechanism (reviewed by Gil et al, 2008). It has been argued that the clinical manifestations of HD and other brain disorders cannot be explained exclusively by neuronal loss. Especially early subtle symptoms occurring before the more obvious manifestation of motor symptoms maybe attributed to complex changes in neuronal function, tissue repair, and circuitry reorganization (reviewed by Paulsen, 2009). In addition to neurons being affected in HD, astrocytes and reactive microglia have been reported to be increased at various stages of the disease (Vonsattel 1985, Sapp 2001) predominately in the striatum and cortex. It was first believed that this may be a consequence of the ongoing neuronal death; however there is a growing body of evidence that implicates a role for activated glia cells in HD pathogenesis. I will talk about this hypothesis in more detail in chapter 3 of my thesis.  1.1.5 Peripheral alterations Historically, HD research has focussed on neuropathology since core symptoms are thought to be directly derived from brain dysfunction. However, in mammals huntingtin is expressed ubiquitously throughout the body with the highest expression levels in the brain and testes. Although its wild-type function remains unclear, evidence suggests that huntingtin is functionally important for many tissues. Recent studies in mice and humans identified testicular abnormalities, skeletal-muscle wasting, cardiac failure, weight loss and widespread changes of the innate immune system to be associated with mutant huntingtin expression (reviewed in Van der Burg, 2009). For example, reduced testosterone levels and numbers of spermatids as well as abnormal morphology of seminiferous tubule were observed in human HD subjects and two mouse models (Papalexi 2005, Van Raamsdonk 2007c). Although, some of these alterations may be a consequence of hypothalamic dysfunction, not all are thought to be secondary to neurodegeneration. In vitro cultures of human monocytes and mouse macrophages, expressing the mutant protein, are also overactive in comparison to wild-type cultures when stimulated with endotoxin (Björkqvist 2008).  7  Furthermore,  83  residue-long  polyglutamine  repeats  conditionally  expressed  in  cardiomyocytes of otherwise wild-type mice reduced cardiac function leading to dilation and subsequently death, whereas control mice had normal cardiac function, morphology, and life span (Pattison 2008). Together, these results provide evidence that dysfunction can occur isolated in peripheral tissues independent of neuronal pathology and HD could therefore be recognized as systemic disorder rather than brain disorder alone.  1.1.6 Pathogenesis It has been hypothesized that the CAG repeat expansion in the huntingtin gene leads to a toxic gain-of-function and not to haploinsufficiency, a mechanism by which an autosomal-dominant mutation causes deficient amounts of a product to be generated and hence disrupts cell function. Supporting this are observations from hemizygous null Htt+/mice and humans with terminal deletion of the HTT gene as occurs in Wolf-Hirschhorn syndrome none of which present with the HD phenotype (Ambrose 1994, Duyao 1995). Furthermore, it has been demonstrated that homozygous knock-out mice (Htt-/-) are embryonic lethal (Duyao 1995). A loss-of-function mutation would infer that individuals with the CAG repeat expansion on both HTT alleles are not viable as well; however homozygous humans have been reported to develop HD in a similar manner as heterozygous HTT cases (Squitieri 2003). Interestingly, similar genetic disorders caused by polyglutamine expansion, such as various types of spinocerebellar ataxia (reviewed by Paulson 2009) and dentatorubropallidoluysian atrophy (reviewed by Takeda et al 1996; Yamada 2004) also exhibit selective neurodegeneration, neuronal inclusions as well as the inverse correlation of age of onset to CAG repeat size. So far no evidence suggests that any of these diseases are caused by haploinsufficiency and the leading theory is that mutant huntingtin causes HD by a toxic gain-of-function mechanism. In addition to a toxic gain of function, mutant huntingtin might also have a dominant negative effect, by which it impairs the wild-type function as a component of disease pathogenesis. The biochemical mechanisms by which a gain-of-function mutation could cause HD are unclear. The HTT gene encodes a large protein comprised of 3,144 amino acid residues with the polymorphic polyglutamine repeat at the N-terminus (The Huntington's Disease Collaborative Research Group 1993). Within the protein a number of different protein cleavage sites have been identified and it has been demonstrated that proteolysis by caspase-3 and calpain is increased at the mutant protein (Goldberg 1996, Kim 2001). It is  8  believed that the proteolysis of mutant huntingtin generates toxic N-terminal fragments which may diffuse to the nucleus and aggregate more often (Martindale 1998, Wellington 2002, Gafni 2004). Some evidence suggests that the truncated and aggregated fragments maintain active binding sites and recruit other proteins into their matrix, affecting nuclear and cytoplasmic proteins that regulate transcription, apoptosis, mitochondrial function, tumour suppression, vesicular and neurotransmitter release, axonal transport (Steffan 2000, Li H 2000, Li SH 2000, Panov 2002, Benchoua 2006). Interestingly, researchers demonstrated that YAC128 mice resistant to cleavage by caspase-6, but not caspase-3, did not exhibit striatal pathology up to 10 months of age and were resistant to quinolinic acid (QA) induced excitotoxicity, a acute model of striatal neurodegeneration (Graham 2006). The cytoplasmic and nuclear inclusions, containing wild-type and mutant huntingtin together with other proteins, were initially thought to be toxic species that mediated cellular dysfunction, as they are found predominately in the striatum and the cerebral cortex (Davies 1997, DiFiglia 1997). Researchers suggested that their formation could account for both the delayed disease onset as well as the inverse correlation of age at onset with CAG repeat length. However these aggregates are not limited to the brain, but are found in tissues that are relatively spared in HD, such as muscle (Orth 2003, Ciammola 2006). Striatal interneurons, which are often spared throughout the disease, have also been shown to exhibit high numbers of inclusions (Kuemmerle 1999). Furthermore, cultured striatal neurons transfected with mutant huntingtin are more prone to cell death if the formation of nuclear inclusions is impaired (Saudou 1998). Some evidence suggests that these large inclusions of aggregated misfolded protein may even play a protective function in cells by sequestering soluble mutant species, because their formation in a cell culture model correlated with improved neuronal survival (Arrasate 2004). YAC128 short-stop mice express a truncated version of the human HTT gene together with two endogenous copies of mouse wild-type Htt. These mice show large numbers of widespread nuclear inclusions, but develop no HD-like phenotype (Slow 2005). However, it is also possible that inclusions are an epiphenomenone and are unrelated to the disease mechanism (Gil 2008). Two models have been suggested on how these inclusions may form. The polar zipper model assumes that the tertiary conformation of the mutant protein is impaired through the CAG repeat expansion and subsequently leads to binding other polyglutamine-containing proteins to form insoluble sheets using hydrogen bonds (Perutz 1994). The second model considers transglutaminases, enzymes that cross-link glutamine residues, which have  9  been shown to increase their activity with increasing CAG repeat length (Kahlem 1998, Karpuj 1999).  1.1.7 Models Despite significant advances in our basic understanding of HD pathogenesis, there are still no effective therapies available that slow or reverse disease progression. Identifying the causal pathways and how they interrelate will assist in the development of rational pharmacotherapy in HD. Human blood, cerebrospinal fluid (CSF) and biopsy samples as well as post-mortem tissue from HD patients and corresponding control cases are used to study the pathogenesis of the disorder; however they are very valuable and often difficult to obtain and store. Furthermore, confounding biases like age, gender, ancestry, but more important diet, access to medical care, health and social status are often present and need to be controlled for. Therefore, in vitro and in vivo animal models within a controlled environment are attractive. They provide material for histopathological, biological and biochemical studies, as well as platforms for high-throughput drug screening and therapeutic compound trials. Most important, these models allow genetic modification of the HD phenotype by manipulation of various genes of interest. Unfortunately however, HD has not been reported to occur naturally in any species other than man, leaving the question of how applicable animal models can be to the human condition. Ideally, a model system should be simple but genetically and phenotypically faithful to the human condition, reflecting characteristics of the most affected cell types. In the beginning, to study the disease phenotype researchers often used acute toxin-induced models of cell death. These models were based on the vulnerability of striatal neurons to excitotoxic compounds and compounds disrupting the mitochondrial machinery. Quinolinic acid and Kanic acid have been shown to induce cell death through binding to N-methyl-Daspartic acid (NMDA) and non-NMDA receptors (Coyle 1976, Stone 1987, Norman 1991), respectively expressed on striatal neurons, whereas 3-nitropropionic acid and malonic acid result in neuronal death via mitochondrial dysfunction (Alston 1977, Beal 1993a,b, Greene 1993). Although, these non-genetic models produce some neuropathologic phenotypes that are similar to what is seen in HD, these are very acute models of synchronous neuronal death and do not permit the study of the pre-manifest state and/or age-related disease progression. With the identification of the genetic cause of HD in 1993 however, scientists have developed a variety of different transgenic, knock-in and recently virus-mediated models of  10  HD. Since then, a range of huntingtin fragment and full-length models have been established in Caenorhabditis elegans (Parker 2001), Drosophila melanogaster (Lee 2004), mice (Mangiarini 1996, Slow 2003), rat (Kántor 2006), monkeys (Yang 2008), and sheep (unpublished, data was presented at the Society for Neuroscience conference in 2009). To study the function of wild-type and mutant huntingtin mammalian cells have been transfected transiently or stably to express fragments or even the full-length huntingtin protein (Li 1998, Lunkes 1998, Martindale 1998). In addition primary cultures of neurons (Zeron 2002), fibroblasts (Archer 1983), lymphocytes (McGovern 1982) and monocytes from human and mouse models (Björkqvist 2008) are being used to examine the impact of toxins and mutant huntingtin on cell processes. More recently, embryonic and neural stem cell models were also established, which I will review in more detail in chapter 2 of my thesis. Neurodegeneration and nuclear localization of huntingtin were observed in a Drosophila model of HD, which expresses N-terminal fragments of human huntingtin in photoreceptor neurons in the eye (Jackson 1998). Furthermore, researchers showed evidence for the correlation between the age of onset and severity of neuronal degeneration with CAG repeat length (Jackson 1998). Similar to these results, studies of the transgenic C. elegans model expressing human huntingtin fragments, reported that tracts of 150 residues led to progressive degeneration of sensory neurons and formation of protein aggregates (Faber 2002). Despite the fact that the short life span of invertebrates enables fast and inexpensive possibilities to screen compounds, it is uncertain how applicable these models are for human neurologic disease. A more complex phenotype can be observed in mammalian models of HD. So far mice have been the species of choice for generating HD models, mainly because of the Nobelprize winning discovery which enables the modification or deletion of specific genes in mice using engineered embryonic stem cells (Bradley 1984, Thomas 1987). Transgenic mouse models of HD can be categorized into i) fragment models, ii) full-length models and iii) knock-in models. Interestingly, it appears that the more genetically true the model is, the more subtle the phenotype (Ferrante 2009). Huntingtin fragment models including R6/2 transgenic mice, R6/1 transgenic mice, and N171-82Q transgenic HD mice that were generated by pronuclear injection of N-terminal fragments of human mutant huntingtin in mouse blastocysts (Mangiarini 1996). The R6/2 mouse line, the first and still most widely employed transgenic mouse model of HD, expresses exon 1 of the human protein with approximately 144–150 CAG repeats, R6/1  11  transgenic mice express a similar huntingtin fragment at a lower level of expression (Mangiarini 1996). N171-82Q mice have an N-terminal fragment of exon 1 and 2 of the human huntingtin gene with 82 CAG repeats under the mouse prion promoter which restricts expression to neurons (Schilling 1999). These mice show early non-specific neurodegeneration, intra-nuclear inclusions, hypoactivity, weight reduction, behavioural abnormalities and a shortened lifespan (Mangiarini 1996, Schilling 1999, Stack 2005). However, the phenotype is most pronounced in the R6/2 model. Due to the large size of the HTT genomic region (210 Kb) yeast artificial chromosomes (YACs; Hodgson 1996 and 1999, Slow 2003) and bacterial artificial chromosomes (BACs; Gray 2008) were used to generate the full-length transgenic mouse models. YAC mouse models like the YAC18 and the YAC128 mice contain the normal intronic 5’ genomic regulatory elements and express the entire human huntingtin protein with 18 to 128 CAG repeats respectively. In comparison to wild-type and YAC18 mice, YAC128 mice show progressive motor deficits, cognitive impairment, selective neuronal loss and atrophy similar to what is seen in human HD (reviewed in Van Raamsdonk 2007b). Huntingtin inclusions in the striatum and cortex are detected after 12 to 18 months, depending on the genetic mouse strain background (Van Raamsdonk 2007a). Interestingly, BAC and YAC mice also show a significant gain in body weight, likely due to the over-expression of functional human huntingtin on the background of mouse huntingtin (Van Raamsdonk 2006, Gray 2008). Potential pitfalls associated with these transgenic mouse models are the random insertion of the transgenes into the genome of each mouse line, and these mice often contain multiple copies of the mutant human gene which they express together with two copies of endogenous murine huntingtin. Therefore, knock-in mice, created through introduction of an expanded CAG repeat into exon one of the mouse huntingtin homologue, could be considered to be a more accurate genetic model of HD. However, these mice show often a very mild HD-like phenotype. An alternative view is that the CAG repeat expansion only causes disease in the context of the human gene and a knock-in into the mouse gene may lack the required genomic background for features of HD pathogenesis. A number of lines, homozygous or heterozygous for the mutation have been generated, such as Hdh/Q72–80, HdhQ111, CAG140, and CAG150 (reviewed by Menalled 2005). However, they exhibit a mild but measurable behavioral and neuropathological phenotype with normal life span in comparison to fragment models, making them a useful confirmatory model (reviewed by Menalled 2005).  12  Lentiviral delivery of mutant huntingtin was employed in rat to generate animals with 44, 66, and 82 repeat fragments (de Almeida 2002). These rats exhibit formation of ubiquitinated huntingtin inclusions one week after injection increasing in number by four weeks. Furthermore, specific GABAergic MSN degeneration, decreased DARPP-32 staining and cell death were reported to occur over the following six months (de Almeida 2002). In 2003, von Hörsten et al developed transgenic rats expressing a truncated Huntingtin cDNA fragment with 51 CAG repeats under the native rat huntingtin promoter. Using this model, the researchers identified adult-onset neurological phenotypes in transgenic animals similar to human patients (anxiety, cognitive impairments, motor dysfunction) as well as neuronal nuclear inclusions, striatal shrinkage and a reduced brain glucose metabolism (von Hörsten 2003). Both toxin (Hantraye 1990) and genetic (Yang 2008) primate HD models have been developed. Huntingtin aggregates were present in the brains of recently developed transgenic monkeys; these monkeys had decreased survival, but striatal neuron degeneration was not observed (Yang 2008). Researchers continue to study these monkeys as it may be years before they exhibit a full HD-like phenotype. Recent reports have also suggested that transgenic HD sheep have been generated (unpublished, data was presented at the Society for Neuroscience conference in 2009).  1.2 Hypothesis and objectives For my first thesis project I hypothesize that embryonic stem (ES) cells isolated from wild-type, YAC18 and YAC128 mice can be used to develop an in vitro model of HD that will be useful for compound screening. Therefore, the research objectives include the generation and characterisation of these novel ES cell lines, as well as the assessment of ES-derived neurons and macrophages in functional assays. For the second project of my thesis I hypothesise that inflammatory changes take place in the YAC128 model of HD similar to the human condition. These alterations are quantifiable in microglia of the CNS and macrophages from the periphery. Accordingly, the research objectives for this project are: 1). the successful establishment of primary microglia and macrophage cultures from mouse models of HD. 2). 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Generation and characterization of embryonic stem cell lines from YAC18 and YAC128 transgenic mice1 2.1 Introduction Despite significant advances in our basic understanding of the biology of wild-type and mutant huntingtin and their role in Huntington disease (HD), there are still no effective therapies that alter the progression of this devastating disorder. Additional insights into the molecular mechanisms of HD pathogenesis and the development of cellular assays that will facilitate the screening of potential therapeutic approaches are required. Many rodent models are genetically faithful to the human condition and exhibit HD-like phenotypes; however they are often more expensive and time-consuming than in vitro assays. Therefore, I hypothesized that development of a simple yet genetically accurate cell-based model system would provide a platform to study HD pathogenesis and test possible interventions. For this project, I decided to generate novel embryonic stem (ES) cell lines from a wellcharacterized HD mouse model, the YAC128 mice. The model was developed in 2003 through stable integration of a yeast artificial chromosome (YAC) enclosing the entire human huntingtin gene (HTT) together with 25 kb of upstream sequence and 120 kb of downstream sequence to ensure the presence of all required endogenous regulatory regions (Slow 2003). The YAC128 mouse line (line 53) expresses high levels of full-length human protein with 128 polyglutamine repeats and develops an age-dependent phenotype similar to that seen in HD patients, including cognitive deficits, motor dysfunction, and selective neurodegeneration (Slow 2003, Van Raamsdonk 2005). It has been demonstrated that 12 month old YAC128 mice are similar in phenotype to early-manifest human HD patients (reviewed by Van Raamsdonk et al, 2007b). YAC18 transgenic mice (line 212) express transgenic human wild-type huntingtin with 18 polyglutamines and do not exhibit a disease phenotype similar to the YAC128 mice; however significant gain in body weight was reported relative to their wild-type littermates (Van Raamsdonk 2006). YAC18 differ from YAC128 mice in the length of the polyglutamine tract, but not in transgenic huntingtin expression levels. In addition to the  1  A version of this chapter will be submitted for publication. Thiele J, Connolly C, Crackower MA, and Leavitt (2010) Generation and characterization of embryonic stem cell lines from YAC18 and YAC128 transgenic mice.  22  human protein, two alleles of endogenous mouse huntingtin homolog on chromosome 5 remain intact in both mouse lines. Generating wild-type, YAC18 and YAC128 ES cell lines will allow us to utilize the unique characteristics of ES cells in the development of a novel in vitro HD model system. Since their first isolation in 1981 by Evans/ Kaufman and Martin independently, mouse ES cells have been studied extensively in vitro and in vivo. ES are derived during early embryogenesis from an embryo at the blastocyst-stage (Evans 1981, Martin 1981). In mice, 3.5 days post coitus (dpc) - before implantation - the dividing cells of the embryo form a hollow structure, containing the inner cell mass (ICM; embryoblast) within the blastocyst cavity (blastocoel) completely encased by trophoblast cells and the zona pellucida. The ICM will form the three germ layers, endoderm, mesoderm and ectoderm, and hence all tissues of the embryonic body. Therefore, ES cells which are isolated from the ICM remain pluripotent and have the capacity to self-renew indefinitely. Pluripotency describes the ability of ES cells to differentiate into any somatic cell of the embryonic body. At a molecular level, pluripotency seems to be established and maintained in vivo and in vitro predominately by the expression of three nuclear regulators Octamer 3/4 (Oct3/4 alternatively Oct4; Nichols 1998, Niwa 2000), SRY (sex determining region Y)-box gene 2 (Sox2; Avilion 2003) and Nanog (Chambers 2003, Mitsui 2003). These transcription factors, essential for the pluripotent state, are typically expressed during early embryogenesis and are linked in a recursive self-reinforcing circuit (reviewed by Chambers et al 2004 and Niwa 2007). Silva and Smith portrayed the pluripotent state as a transcription factor battlefield in which the trinity suppresses the functional expression and activity of lineage specification factors (Silva 2008). The forced expression of Oct4, Sox2, c-Myc and Klf4 via transfection was shown to re-program mouse and human somatic cells (Takahashi 2006, Takahashi 2007). However, recent evidence suggests that Oct4 together with Sox2 also regulate the expression of fibroblast growth factor 4 (FGF4) which interacts with extracellular signal-regulated kinase 1/2 (Erk1/2 alternatively Mapk3/1) and therefore poise ES cells for unspecific lineage commitment (Kunath 2007). Consequently, FGF4/Erk signalling appears to make ES cells responsive for further differentiation (Kunath 2007, Silva 2008). Furthermore, it has been demonstrated that ES cell cultures are not homogenous as expression levels of key factors can vary from cell to cell, resulting in different developmental potential and self-renewal ability (Silva 2008). Epigenetic mechanisms have also been suggested to play a role in maintaining pluripotency by providing or denying access to the target genes, however they seem to contribute to a  23  lesser degree. Pluripotency represents a valuable tool, as the process of differentiation as well as the derived cell type can be studied extensively using ES cells in vitro. Stem cells have a unique characteristic that distinguishes them from other mammalian cell types: they can divide symmetrically as well as asymmetrically (reviewed by Morrison et al, 2006), producing one daughter cell that maintains the stem cell identity (self-renewal) and a second daughter cell that develops into more specialized cells (differentiation). Under defined conditions, cultured ES cells have been shown to proliferate over long periods of time without being immortalized through the induction of oncogenes or loss of tumor suppressor genes, and are still able to contribute to form chimaeric mice (Bradley 1984, Pease 1990). Extracellular matrix components as well as the signal transducer and activator of transcription 3 (STAT3; Niwa 1998) and phosphatidylinositol 3-kinase (PI3K; Paling 2004) have been shown to promote self-renewal, while the mitogen-activated protein kinases (MAPK) family members ERK1 and ERK2 negatively regulate it (Burdon 1999, Kunath 2007). Oct4 (Niwa 2000) and the leukemia inhibitory factor (LIF; Smith 1988, Williams 1988) can influence these pathways. To support the undifferentiated ES cell state mouse ES cells are often cultured on a mouse embryonic fibroblast (MEF) layer with LIF as substrate in the culture media. Transplantation of ES cells into an animal causes teratoma formation (Evans 1981, Martin 1981); however injection of ES cells into the blastocyst results in chimera formation (Bradley 1984). Today, genetically engineered ES cells with targeted mutations generated in vitro (i.e. by homologous recombination) are widely used to evaluate the roles of specific genes by generating genetically modified mice (Thomas 1987, Thompson 1989). Furthermore, ES cells hold potential for regenerative medicine as a source of specific types of ES cell-derived donor cells (reviewed by Murry et al, 2008). ES cells were initially generated from 129 mouse strains and ES cells derived from these mice continue to be widely used. However the genetic background of 129 mice is often not ideal for all studies, recent technical advances have lead to the development of more efficient ES cell generation protocols and lead to production of ES cells from other common mouse inbred strains such as C57BL/6 (reviewed by Glaser et al, 2005).  24  2.2 Materials and methods 2.2.1 Generation of novel ES cell lines Animals The YAC128 (line 53) mouse line expresses high levels of full-length human huntingtin with 128 polyglutamine repeats and is a well-established model of HD. In comparison, YAC18 mice (line 212) express transgenic human wild-type huntingtin with only 18 polyglutamine repeats and do not exhibit a Huntington disease phenotype. Wild-type females from a pure 129S1/SvIm1 (JAX stock #002448; here referred to as 129) strain (super-ovulated or crowded) were mated to YAC128 transgenic males (single-housed) on a pure 129S1/SvIm1 to generate WT128-129 and YAC128-129 ES cell lines. 129xFVB F1hybrid ES cell lines have been derived from crosses between wild-type females (129) and YAC18 or YAC128 transgenic males on the FVB/N strain background. All mice were maintained in a pathogen-free environment at the transgenic core facility at the Centre for Molecular Medicine and Therapeutics (CMMT).  ES cell generation ES cell generation was based on a protocol developed by Cheng et al (2004). All novel ES cells lines described here have been derived using the following steps. 3 days post coitum mouse blastocysts were harvested in phosphate-buffered saline (PBS; Invitrogen) by flushing the uterine horn of the pregnant female following standard operating procedures. Any remaining blood and tissue was carefully washed off before transferring each embryo into a separate 96 well containing a confluent layer of mouse embryonic fibroblasts (MEF) in serum replacement (KSR) media (Knockout™ D-MEM [Invitrogen], 16 % Knockout™ Serum Replacement [Invitrogen], 2 mM L-glutamine [Invitrogen], 0.1 mM MEM non-essential amino acids solution [NEAA; Invitrogen], 1000 U/ ml ESGRO® [Millipore]). The blastocysts were left undisturbed for eight to nine days at 37 ºC and 5 % CO2. On day 8/9 each wells were trypsinized for 4 minutes at 37 ºC and 5 % CO2 dissociating the outgrown inner cell mass (ICM). After quenching the trypsin (Invitrogen) with soya bean trypsin inhibitor (Invitrogen) the cells were transferred into a 24 well containing a fresh MEF feeder layer and cultured for 4 more days in KSR-media at 37 ºC and 5 % CO2. Starting day 12/13 the cells were adjusted to ES-media (Dulbecco's modified Eagle medium [DMEM; Invitrogen], 16 % fetal bovine serum [FBS; PAA], 2 mM L-glutamine, 0.1 mM  25  NEAA, 1000 U/ ml ESGRO®, 0.01 % β-mercaptoethanol [Sigma-Aldrich]). The KSR culture media was replaced gradually over the following 4 days to 25, 50, 75 and then 100 % ESmedia. Wells containing ES cell-like colonies were then expanded according to standard tissue culture procedures. Early passages were frozen and sufficient amounts of cells were prepared for genotyping and characterization.  Culture of MEF feeder layer MEFs were derived from E12.5-14.5 embryos as described by Meissner et al (2009). A confluent layer of mitotically inactivated fibroblasts was cultured overnight on gelatincoated 96-well plates (BD Falcon) in regular MEF-media (DMEM, 10 % FBS, 2 mM Lglutamine, 0.1 mM NEAA). The culture media was changed from MEF-media to KSRmedia 5 hours before plating the embryos or to ES-media right before plating ES cells.  Figure 1: Workflow of the generation of novel ES cell lines.  Mouse blastocyts were isolated about 3 days post coitus (dpc) and seeded into individual 96 wells containing mouse embryonic fibroblasts (MEF) in serum-free media. After 8 to 9 days in culture each outgrown clone was transferred individually into a 24 well containing MEFs in serum-free media. On day 12/13 all cells were adjusted to regular FBS-containing ES-media over the period of 4 days and expanded afterwards.  26  Genotyping Each cell line was genotyped using GoTaq flexi (Promega).  Designation  Sequence  Right YAC arm (Forward)  CTT-GAG-ATC-GGG-CGT-TCG-ACT-CGC  Right YAC arm (Reverse)  CCG-CAC-CTG-TGG-CGC-CGG-TGA-TGC  Left YAC arm (Forward)  CCT-GCT-CGC-TTC-GCT-ACT-TGG-AGC  Left YAC arm (Reverse)  GTC-TTG-CGC-CTT-AAA-CCA-ACT-TGG  IL-2 (Forward)  GAG-CAG-AGT-GTT-CAT-GTT-CCC-AGT-T  IL-2 (Reverse)  TCC-TCT-AGG-CCA-CAG-AAT-TGA-AAG-A  Table 1: List of primer sequences used to identify wild-type and YAC ES cell lines.  2.2.2 Characterization Serial culture and alkaline phosphatase (AP) staining All novel ES cell lines were sub-cultured a minimum of 25 times or higher while their morphology was microscopically monitored. After about 25 passages, the AP activity of all established cell lines was examined. The AP staining was performed using the AP Detection Kit (Millipore).  Immunocytochemistry (ICC) Cells were plated onto gelatin or poly-D-lysine/ laminin treated glass coverslips in a 24well plate. Culture media was aspirated and cells were washed once with PBS and fixed with 3 % paraformaldehyde for 10 minutes at room temperature (RT). Cells were then washed and permeabilized three times 5 minutes with PBS-T (T = 0.3 % Triton X-100), followed by a block step with 2 % normal goat serum (NGS) in PBS-T for 30 minutes, both at RT. Primary antibodies were diluted in PBS-T with 2 % NGS, added and left overnight at 4 °C. A list of all primary antibodies used is provided below (Table 2). About 15 hours later cells were washed three times 5 minutes with PBS before fluorescent-labelled secondary antibodies (diluted in PBS) were added. All following steps were performed at RT with minimal light exposure. After 1 hour antibody incubation cells were washed twice for 3 minutes with PBS and then stained with 200 nM 4',6-diamidino-2-phenylindole (DAPI) solution for 5 minutes. Cells were washed once more for 5 minutes with PBS. The coverslips were transferred out of the 24-well plate and air-dried. Each coverslip was  27  dipped into xylene before being mounted with DePeX MOUNTING MEDIUM (Electron Microscopy Sciences) onto glass slides.  Designation  Clone and/or catalogue # (company)  Oct4  # ab19857 (Abcam)  F4/80  BM8, # MF48000 (Caltag Labs- Invitrogen)  Nestin  Rat-401 (Developmental Studies Hybridoma Bank)  neuronal class β-III-tubilin  TUJ1, # 01409 (Stemcell Technology)  GFAP  # AB5040 (Chemicon - Millipore)  NeuN  A60, # MAB377 (Chemicon - Millipore)  CD11b  M1/70.15 (Caltag Labs- Invitrogen)  Table 2: List of primary antibodies used for ICC.  RNA isolation from novel ES cell lines and reverse transcription polymerase chain reaction (RT-PCR) To avoid contamination with other cell types, colonies with ES cell-like morphology were cultured a minimum of five passages on gelatin coated plates without MEF feeder layer before RNA isolation was performed. The RNeasy Plus Mini Kit (QIAGEN) was utilized to isolate and purify total RNA from cultured cells. 30-100 ng purified RNA from each cell line was then examined using the OneStep RT-PCR Kit (QIAGEN). Primers were designed using Primer3 provided at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cg iT.  Designation  Sequence  Pou5f1_Forward  ATC-ACT-CAC-ATC-GCC-AAT-CA  Pou5f1_Reverse  AAG-GTG-TCC-CTG-TAG-CCT-CA  Nanog_Forward  TGC-GGA-CTG-TGT-TCT-CTC-AG  Nanog_Reverse  GTG-CTG-AGC-CCT-TCT-GAA-TC  Gapdh_Forward (oEMS2574)  CTC-GTC-TCA-TAG-ACA-AGA-TGG-TGA-AG  Gapdh_Reverse (oEMS2575)  AGA-CTC-CAC-GAC-ATA-CTC-AGC-ACC  Table 3: List of primer sequences used to evaluate cDNA derived from ES cell lines.  28  An aliquot of the Gapdh primer pair was obtained from Charles de Leeuw (Simpson Laboratory, CMMT) and has been described previously by Barberi et al (2003).  ES cell-derived neurons: 4-/4+ protocol This protocol has been described by Bain et al (1995) and defined by Li et al (1998). Briefly, ES cells were sub-cultured as normal and seeded to achieve approximately 80 % confluency the next day. The cells were trypsinized the following day and the trypsin quenched with ES-mediaW (DMEM, 16 % FBS, 2 mM L-glutamine, 0.1 mM NEAA). Cells were pelleted, re-suspended in 10 ml ES-mediaW and plated onto a 10 cm bacterial-grade plastic petri-dish (Fisher Scientific) and culture as normal. About 48 hours later embryoid bodies (EB) formed. The EBs were transferred to a 15 ml tube (BD Falcon) using a 10 ml plastic pipette. The EBs were allowed to settle to the bottom of the tube (about 5 minutes), media was replaced by fresh ES-mediaW and cells were seeded into a new petri-dish. On the 4th and 6th day of differentiation the media was changed as described earlier. This time ES-mediaW was supplemented with 1x10-6 M all-trans retinoic acid (Sigma) in dimethyl sulfoxide. On day 8 of differentiation EBs were collected, washed twice with PBS and trypsinized for 10 minutes to dissociate the cells. After quenching, the trypsinized cells were transferred through a cell strainer (BD Falcon), pelleted and re-suspended in 1 ml 1:1 media (one part neurobasal medium [NBM; Invitrogen] with B27 [Invitrogen] and one part DMEM/F12 [Invitrogen] with N2 [modified by Meng Li and Austin Smith (Institute Stem Cell Research, Edinburgh); 25 ug/ ml insulin, 100 ug/ ml apo-transferrin, 20 ng/ ml progesterone, 16 ug/ ml putrescine, 30 nM sodium selenite, 75 ug/ ml bovine serum albumin (fraction V)], supplemented with 10 ng basic-FGF/ ml [Peprotech] and 2 mM L-glutamine). After single cells were counted they were diluted and seeded 8x104 cells per well (24-well plate) onto poly-D-lysine/ laminin treated coverslips. About 24 hours later culture media was replaced with fresh 1:1 media. On the 11th day of differentiation the media was changed to 4:1 media [four parts NBM and one part DMEM/F12, 2 mM L-glutamine] to induce terminal differentiation, this process needed approximately one week. The culture medium was changed every 48 hours. On day 21 differentiation ICC (as described above) and NMDA toxicity assays were performed to evaluate the differentiated cells.  29  N-methyl-D-aspartic acid (NMDA) toxicity assay and TUNEL staining At day 21 of the neuronal differentiation NMDA stimulation was performed using a protocol modified from Metzler et al (2007). Briefly, ES derived neuronal cultures were cultured on coverslips in 24 well plates. The media of each well was collected and cells were washed twice with assay buffer (AB; 140 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 10 mM HEPES, 25 mM glucose, pH 7.35) containing 1 mM MgCl2. Cells were then stimulated for 5 minutes with 200 uM NMDA and 50 uM glycine in AB without MgCl2 at 37 ºC and 5 % CO2. Afterwards cells were washed twice with AB containing MgCl2 and conditioned media was added back. Cultures were left undisturbed for 20 hours at 37 ºC and 5 % CO2. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining was performed as follows. Cultures were washed twice with PBS and permeabilized in PBS-T for 2 minutes on ice. After three washes in PBS cells were stained using the ‘In Situ Cell Death Detection Kit, TMR red’ (Roche Applied Science) according to the manufactory’s instructions. Positive controls were treated with DNaseI prior to TUNEL staining. Cells were subsequently counter-stained with TUJ1 and DAPI using the ICC protocol described above.  ES cell-derived macrophages The protocol used was modified from Lindmark et al (2004). In brief, ES cells were sub-cultured as previously described and seeded to achieve approximately 80% confluency the next day. On the following day, the ES-media was changed to IMDM-1 (Iscove’s modified Dulbecco’s medium [IMDM; Invitrogen], 10 % FBS, 0.1 mM NEAA, 0.01 % β-mercaptoethanol). After 3 hours, the cells were passaged as normal (5 minutes at 37 ºC and 5 % CO2), trypsinization was inhibited with IMDM-1 and cells were pelleted, resuspended in 0.5 - 1.5 ml IMDM-1 and counted. 5x104 cells per well of a ultra low attachment cluster 6-well plate (ULC-plate; Corning) were seeded in 3 ml MethoCult™ GF M3434 (Stemcell Technologies) and cultured at 37 ºC and 5 % CO2. By day 11 of differentiation, EBs had formed. Each well was transferred into a 15 ml tube and washed with IMDM-1. Then EBs were seeded in IMDM-2 (IMDM-1, 20 ng/ ml mouse interleukin-3, 20 ng/ ml mouse macrophages-colony stimulation factor, 10 ug/ ml human insulin, 20 ng/ ml mouse interleukin-1β) into ULC-plates and cultured at 37 ºC and 5 % CO2. The media was changed every second day. After about 5 to 10 days macrophages-like cells had attached to the surface, and were subsequently trypsinized, counted and re-seeded to be used in ICC (described above) or CSE assay.  30  Control standard endotoxin (CSE) assay On day 21 of the differentiation procedure, ES-derived macrophages from four wildtype and three YAC128 ES cell lines, all derived from mice of pure 129 strain background, were counted and seeded at 4x104 cells per ml onto BD Primaria™ 96-well microplates (BD Biosciences) and incubated in IMDM-2 media. After 24 hours the media was replaced by 1% IMDM-1 medium (IMDM, 1% FBS, 0.1 mM NEAA, 0.01 % β-mercaptoethanol). Functional studies were performed after an additional 24 hours. Media containing interferon-γ (IFN-γ; R&D Systems, final concentration 10 ng/ ml) with or without control standard endotoxin (CSE; Associates of Cape Cod, final concentration 100 ng/ ml) was added to the cells and incubated for 24 hours. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) concentrations were measured in supernatants from one well of INF-γ condition and two to three independent wells of CSE condition from each cell line, using a commercial mouse IL-6 and TNFα ELISA kit (eBioscience). The cells were lysed and total protein amounts were determined using the ‘Micro BCA Protein Assay Kit’ (Thermo Scientific).  Phagocytosis assay Latex beads (Sigma-Aldrich) were rinsed according to manufacturer’s instructions and opsonised with 1 mg/ ml human immunoglobulin G (IgG; Sigma) for 1 hour at 37 °C and 5 % CO2 The beads were then washed three times to remove excess IgG and re-suspended in PBS. ES-derived macrophages were seeded onto coverslips (24-well plate) one day prior and were pre-cooled for 15 minutes to 4 °C and then incubated 30 minutes with IgGlabelled beads to allow binding. Approximately 200 beads per cell were applied. The cold media was replaced by preheated (37 °C) media. After 1 hour incubation at 37 °C and 5 % CO2 cells were washed three times with ice-cold PBS and stained with Alexa Fluor®488 goat anti-human IgG (Invitrogen) for 5 minutes on ice. Cells were fixed then cells using 2 % paraformaldehyde in PBS for 10 minutes at RT, followed by 3 washes with PBS. The cells were then permeabilized in PBS-T for 3 minutes at RT, washed again three times with PBS and subsequently stained with, 2.5 Units/ ml PBS Alexa Fluor®594 phalloidin (Invitrogen) for 15 minutes at RT. Cells were washed once more for 5 minutes with PBS. The coverslips were transferred out of the 24-well plate and air-dried. Each coverslip was dipped into xylene before being mounted it with DePeX MOUNTING MEDIUM (Electron Microscopy Sciences) onto glass slides.  31  2.3 Results 2.3.1 Generation of 17 novel ES cell lines We collected 20 morulae and 40 blastocysts from timed matings of 129 females and YAC128-transgenic 129 males. All of these embryos initially attached to the MEF feeder layer, but only 26 embryos continued to expand (Figure 2b) 24 to 48 hours later. Outgrowths of the inner cell mass (ICM) were dissociated after 8 to 9 days in culture (DIC) and re-seeded onto fresh MEF feeder layers. At this stage, cells of the ICM as well as other cell types, likely trophoblast cells were visible. ES cell-like colonies were formed by 12 to 13 DIC, which grew larger as the KSR culture media was gradually replaced to 100 % ES media. Of 26 clones only 6 clones established 10 stable cell lines, 4 wild-type and 6 transgenic. The undifferentiated cells grew in sphere-like, compact, shiny colonies with smooth outlines atop the MEF layer which appeared darker as they were very tightly adherent to the plastic surface as shown in Figure 2d-e.  Figure 2: Isolation of novel mouse ES cells from YAC mice. Blastocysts were plated onto MEF feeder layers (a). Within 1 to 2 days of culture, the embryo hatched as the zona pellucida (ZP) separated from the blastocyst (b). By day 8 to 9 of culture, the outgrowth of the inner cell mass (ICM) was well established (c). First ES cell-like colonies appeared after dissociation and re-seeding, and grew bigger as the media was changed to serum-containing media (d). With further sub-culture, the number of novel ES cell colonies increased and their morphology appeared to be similar to colonies of control cell lines (e). bar = 100 um  There were only a small number of embryos collected from each mating of 129 females with FVB/N transgenic males. All together, 15 blastocysts and 8 morulae were collected from 129 x 212 (YAC18) matings, from those 4 cell lines (2 wild-type and 2 YAC18) were established. For the 129 x 53 (YAC128) matings a total of 25 embryos were collected of which 10 embryos successfully generated ES cell lines (3 wild-type and 7 YAC128).  32  The presence of the YAC18 or YAC128 transgene in transgenic cell lines was confirmed via PCR, a 230 bp band represented the left arm of the YAC construct and 170 bp band corresponded to the right arm of the YAC construct. In wild-type cell lines a 400 bp interleukin-2 amplicon, only detected in the multiplex reaction in the absence of the YAC transgene, was present. After all ES cell lines were genotyped, cells for each genotype at low passage number were cryo-preserved.  2.3.2 Characterization of novel ES cell lines using stem cell markers The expression of previously described stem cell markers was examined in our newly generated cell lines to assess whether these cells were indeed ES cells. Alkaline phosphatase (AP) activity was detected in undifferentiated colonies of 17 of the 24 initial cell lines isolated. 2 wild-type and 5 YAC128 cell lines on the 129 x FVB/N F1 hybrid background did not exhibit significant AP activity using this assay. No staining was observed in mitotically inactivated MEF cultures (Figure 3). Wild-type (Figure 3a-c) and YAC128 (Figure 3e-g) ES cells from both strain backgrounds exhibited similar staining patterns when compared to the control ES cell line mEMS159 (Figure 3d). The color intensity was not completely uniform, suggesting the specific populations in these cultures were not homogeneous; however all colonies exhibited some degree of AP activity. The proportion of differentiated cells, lacking AP staining was slightly increased when cell lines were cultured on gelatin coated culture dishes; making this a sub-optimal condition in comparison to cells cultured on feeder layers. It is worth noting that cell lines from YAC18129/FVB mice (Figure 3b,f) appeared to generate more differentiated cells with fewer cells expressing clear AP activity, even at a much lower passage number. Furthermore, immunocytochemistry (ICC) for Oct4, which is involved in the maintenance of the pluripotent state of ES cells (Nichols 1998, Niwa 2000), was performed for all of the cell lines generated. As shown in Figure 4, Oct4 was highly expressed in the nuclei of undifferentiated cells of all novel cell lines. The intensity of the Oct4 signal in cells appeared to be equal in undifferentiated cells from each cell line. As observed earlier the YAC18 cell lines exhibited the same pattern, with frequent differentiated cells and fewer Oct4 positive cells. It was also noted that MEFs showed very weak nuclear staining, but it was punctuated and clearly different in expression pattern compared to that of ES cell lines.  33  Figure 3: Novel ES cell lines stained for alkaline phosphatase (AP). The wild-type cell lines 21.1wt128-129 (a), 5wt18-129/FVB (b) and 8wt128-129/FVB (c.) cultured on MEF feeder layers as well as the transgenic cell lines 12Y128-129 (e), 2Y18129/FVB (f) and 2Y128-129/FVB (g) express the cell surface enzyme AP (red) similar to the control ES cell line mEMS159 (d) showing evidence of their undifferentiated status. In comparison to the pluripotent ES cell lines the MEF feeder layer (h) does not stain for AP. bar = 100 um  The 7 cell lines that had been observed to lack AP activity also did not exibit any Oct4 expression, confirming their non-ES cell status. These cell lines showed similar AP and Oct4 staining to MEF feeder layers and were excluded from further experiments. To verify the ICC results RT-PCR (reverse transcription – polymerase chain reaction) was performed to prove expression of Pou5f1 (Oct4) and Nanog, a homeodomain protein that has been identified as a critical factor underling pluripotency (Chambers 2003, Mitsui 2003). The novel cell lines but not MEF feeder layer or whole mouse brain (for later data see Appendix Figure 30) showed high levels of these stem cell markers (Figure 5). The noRNA input as well as the no-RT controls did not amplify any product. Glyceraldehyde-3phosphate dehydrogenase (Gapdh) was amplified from all RNA samples as control of their quality.  34  Figure 4: Nuclear expression of Oct4 in novel ES cell lines. The wild-type cell lines 15.2wt128-129 (a), 5wt18-129/FVB (b) and 8wt128-129/FVB (c.) cultured on MEF feeder layers as well as the transgenic cell lines 12Y128-129 (e), 7Y18129/FVB (f) and 5Y128-129/FVB (g) stained for Oct4 (green) similar to the control ES cell line mEMS159 (d) independently of the presence of the human HTT transgene. In comparison to the pluripotent ES cell lines, the MEF feeder layer (h) stained only weakly for Oct4. Nuclei were counterstained with DAPI (blue). bar = 100 um  Figure 5: Novel ES cell lines expressed known ES cell markers shown by RT-PCR. Panel I shows the expression of the Pou5f1 gene (Oct4 protein, 136 bp amplicon) in ES cell lines (lanes 1,3-8). In comparison, Pou5f1 is not expressed in MEF feeder layers (lane 2). Panel II shows the expression of the Nanog gene (222 bp product). Nanog is not expressed in MEF feeder layers (lane 2); the band seen was most likely due to contamination (see Appendix Figure 30). Gapdh (305 bp product) is expressed in all samples as shown in panel III. In all cases the ‘no-RNA input’ (lane 9) and ‘no-RT’ control were free of contamination. Samples were loaded as followed: (1) mEMS159 (2) MEF feeder layers (3) 15.2wt-129 (4) 7Y128-129 (5) 5wt18-129/FVB (6) 7Y18-129/FVB (7) 8wt128-129/FVB (8) 5Y128-129/FVB (9) no-RNA input. (L) MassRulerTM DNA ladder, Low Range  35  In addition, I analyzed our cell lines for the expression of F4/80 and Nestin, both markers for more differentiated cells - macrophages and neural stem cells respectively, via ICC. Mouse F4/80, a transmembrane protein and member of the EGF-TM7 family, is expressed on a wide range of mature tissue macrophages (Haidl 1996, Schaller 2002). The newly derived ES cell lines did not exhibit any F4/80 staining when compared to isolated alveolar macrophages or ES-derived macrophages. However, depending of the method of sub-culturing, some cell lines showed small numbers of cells expressing Nestin. Nestin, an intermediate filament protein, is expressed by proliferating cells of the CNS, PNS and in myogenic and other tissues at early stages of embryonic development (Michalczyk 2005). Nestin expression is downregulated after terminal differentiation, but can be re-induced in the adult during pathological situations, like the formation of the glial scar after CNS injury (Michalczyk 2005). I observed Nestin-expressing cells among large cell clusters, especially when cells were cultured on gelatin coated dishes. Colonies with typical ES cell morphology under the bright-field microscope often did not stain for Nestin. ES cell morphology was therefore strictly monitored before and during serial culturing and differentiation experiments.  2.3.3 Serial culturing The self-renewal capacity of all remaining novel ES cell lines was tested by serial culture up to 25 passages. All cell lines were able to maintain the compact, shiny and sphere-like morphology characteristic of established ES cell lines. It is worth noting that the ES cell lines on the 129 background were easier to maintain than the cell lines on the F1 hybrid background. In total, three wild-type and three YAC128 cell lines on the 129 background were chosen and together with the control ES cell line mEMS159 sub-cultured for more then 50 passages. As shown in Figure 6, these cell lines were able to maintain ES morphology, and continued expressing high levels of Oct4 similar to earlier ES cell cultures independent of the YAC transgene. An average doubling-time of about 15 hours for these cell lines was stable throughout this experiment. Moreover, these serial cultured cells did not express F4/80, providing evidence that these cells had not differentiated into macrophages-like cells during the extensive period in culture (Figure 7, panel I). When cultured on MEF feeder layers, most of the ES cell lines did not express Nestin (Figure 7, panel II), but again some cells in the  36  culture differentiated quickly when maintained under sub-optimal conditions such as when placed on gelatin-coated culture dishes.  Figure 6: Novel ES cell lines were able to maintain the characteristic morphology and high levels of Oct4 expression after 50 passages. Shown in panel I (bright-field) and panel II (Oct4 ICC; Oct4 green and blue DAPI counterstaining) are the wild-type cell line 15.2wt128-129 (a), the control ES cell line mEMS159 (b) and the transgenic cell line 12Y128-129 cultured on MEF feeder layer (d). bar = 100 um  Figure 7: After 50 passages, the novel ES cell lines did not stain for F4/80 and only occasional cells were found to stain weakly for Nestin. Shown in panel I (F4/80 red and blue DAPI counter-staining) and panel II (Nestin red and blue DAPI counter-staining) are the wild-type cell line 15.2wt128-129 (a) and the transgenic cell line 12Y128-129 (c) culture on MEF feeder layer (d) in comparison to primary alveolar macrophages (b, panel I) or mEMS159-derived neurons (b, panel II). White arrow heads indicate ES cell colonies negative for Nestin, green arrow heads indicate colonies with weak Nestin staining detectable. bar = 100 um 37  2.3.4 ES cell differentiation into neurons ES cell have been shown to develop in vitro into many cell types derived from the three germlayers (Bain 1995, Moritoh 2003, Lindmark 2004). Therefore, the ability of our novel cell lines to differentiate into embryoid bodies (EB) and TUJ1+ neurons was examined. First, following the protocol by Bain et al (1995) and Li et al (1998), the control ES cell line mEMS159 was differentiated into EBs, Nestin+ neuronal precursors and TUJ1+ neurons. ES-derived neuronal cultures stained for Nestin, a marker that identifies the most primitive neuroepithelium, while primary neuronal cultures only contained a small population of Nestin+ cells (Figure 8a,d). Furthermore, 15 DIC old ES-derived neurons and 9 to 11 DIC old primary neuronal cultures stained for TUJ1, a marker for neuron specific class III β-tubulin (Figure 8b,e).  Figure 8: ES-derived neuronal cultures stained similar to primary neurons. After 9 to 11 days in vitro only a small population of primary cells (a) stained positive for Nestin (green), whereas almost all ES-derived cells (d) are positive for this marker at 15 DIC. TUJ1 (green) staining was detected in both, primary neurons (b) as well as ESderived neurons (e). GFAP+ astrocytes (red) were almost absent in primary neuronal cultures (c), but appeared in clusters throughout the ES-derived cultures (f). Nuclei in all pictures were counterstained with DAPI (blue). a,d bar = 100 um, b,c,e,f bar = 20 um  38  Overall, ICC revealed that using the Bain et al protocol generated mixed ES cellderived cultures, comprised of Nestin+ neuronal precursors, TUJ1+ neurons and GFAP+ (glial fibrillary acidic protein) astrocytes. NeuN, a marker for mature neurons (Mullen 1992), was not detected in either of those two cultures. Nonetheless, NeuN staining of ES-derived neurons was later observed in 21 DIC old cultures. The differentiation protocol was then repeated for 14 of the 17 remaining cell lines. All of the selected novel ES cell lines were able to form EBs after 2 to 4 days of culture in LIFfree media and all of these cell lines subsequently generated TUJ1+ neurons. The outcome for different cell lines varied in the proportions of differentiated TUJ1+ expressing neuronal cells; from only a few TUJ1+ cells in some populations, to many TUJ1+ colonies in other cultures. This difference was most likely due to technical issues (the large number of cell lines that had to be maintained). To characterize the mixed cultures of differentiated cells more closely 3 wild-type, 3 YAC128 cell lines and the control-cell line mEMS159 all on the 129 strain were again differentiated and carefully evaluated via ICC. All cell lines derived EBs of similar number, size and appearance, except cell line 16.2Y128-129 which generated only a few EBs. After 21 DIC all cell lines had derived TUJ1/NeuN+ neurons (for 16.2Y128-129 only TUJ1 staining was possible, because only one coverslip with cells was established) as well as GFAP+ astrocytes, independently of their genotype (Figure 9). Interestingly, in comparison to the thin network of neurites of wild-type ES cell and primary neurons (control) YAC128 neurites appeared much thicker. However, no quantitative analysis was performed. An excitatory assay was performed, where cells were exposed to 200 uM NMDA and 50 uM glycine. In this preliminary assay, ES cell-derived differentiated neuronal cells from all genotypes underwent excitotoxic cell death, with no obvious differences in the number of apoptotic nuclei detected after evaluation with TUNEL staining between the cells based on genotype.  39  Figure 9: ES-derived neurons and astrocytes. Panel I shows 8 days old EBs (bar = 100 um). Wild-type and YAC128 cells appear to be similar to the control cell line mEMS159; no differences in morphology of the EBs were detected based on genotype. In panel II are shown ICC results for neuron specific β-III-tubulin (green). In comparison to the thin network of neurites of wild-type ES cell and primary neurons (control) YAC128 neurites appeared much thicker. The NeuN staining (green) of wild-type and YAC128 ES-derived neurons in panel III was very similar to that observed in brain sections (cortex) of wild-type 129 mice. Panel IV shows astrocyte clusters stained with GFAP (red) which were present in all ES-derived neuronal cultures independent of the genotype and appeared to be similar to primary astrocyte cultures (control). ES-derived neuronal cultures were 21 days old. For ICC pictures nuclei were counterstained with DAPI (blue). Panel II-IV: bar = 20 um  40  2.3.5 ES cell differentiation into macrophages Initially, when establishing the ES cell differentiation protocol, I chose to differentiate only the control cell line mEMS159 into macrophages. The procedure by Lindmark et al (2004) contained a selection step on ULC plates, that allowed only the macrophage-like cells to attach and so ensured a homogeneous macrophage culture. As shown in Figure 10, the ES-derived cells stained for the markers F4/80 - a macrophage-restricted cell surface glycoprotein - and CD11b/CD18 (alternatively Macrophage-1 antigen) - a type three complement receptor (CR3) that is expressed on macrophages, granulocytes, NK cells and B-1 cells (Benimetskaya 1997) - similar to primary alveolar macrophages. What was instantly apparent was the cell size difference between the primary and ES-derived macrophages. ES cell derived macrophages (Figure 10b/d) appeared 5 to 10 times larger than alveolar macrophages (Figure 10a/c). I hypothesized that this may be an effect of different duration of time in culture for the primary cells compared to the ES cell-derived macrophages. Establishing ES-cell derived macrophages requires a minimum of 14 DIC while the primary macrophages were stained one day after isolation, following less than 24 hours in culture.  Figure 10: Homogeneous ES-derived cultures stain for the macrophages markers F4/80 (a-b) and CD11b (c-d). All ES-derived cells stained (red) for F4/80 (b) and CD11b (d) in a similar manner as alveolar macrophages (a,c respectively), but were 5 to 10 times larger than the primary cells. Nuclei were counterstained with DAPI (blue). bar = 20 um 41  Figure 11: Culture time influenced the cell size of alveolar macrophages. F4/80 staining (red) revealed the size of primary macrophages increased from 2 DIC (a) and 3 DIC (b) to 10 DIC (c). At the later time points cells appeared more similar in size when compared to ES-derived macrophages (d). bar = 20 um  Figure 11 shows primary alveolar macrophages indeed increased in cell size after 3 to 10 DIC. After 10 days in culture, primary macrophages (Figure 11a-c) have spread out and show a similar somatic size when compared to ES-derived macrophages (Figure 11d). Macrophages respond to lipopolysaccharide (LPS), the toxic component in control standard endotoxin (CSE) found in the outer membrane of Gram-negative bacteria, with the secretion of cytokines such as TNFα and IL-6 (reviewed by Elkins et al 2007). As shown in Figure 12 below, mEMS159-derived macrophages responded to LPS stimulation with TNFα release. The TNFα level in the media was about five-fold less than that of primary alveolar macrophages. Given the very different procedures of establishing the two cultures, I hypothesized that culture time had an influence on activation level of the cells and therefore cytokine release. After 10 days of culture the TNFα response of alveolar macrophages was similar to levels seen in ES-cell derived macrophages and was decreased to 25 % of the initial TNFα level measured in primary macrophages at 3 DIC (data not shown). The Phosphodiesterase IV (PDE4) inhibitor Rolipram, was used to pre-treat ESderived and primary macrophage cultures before LPS stimulation. Rolipram is an antiinflammatory drug developed to treat chronic obstructive pulmonary disease (COPD); it is thought to elevate intracellular cAMP and norepinephrine, suppressing the expression of pro-inflammatory cytokines (reviewed by Wang et al, 2006). As shown in Figure 13, 20 minutes of Rolipram treatment prior to LPS stimulation caused reduction of TNFα release into the media from ES-derived macrophages and alveolar macrophages to 52 % and 68 % respectively, of the initial response without inhibitor. I further show evidence that ES-  42  derived macrophages could be a used for the screening of anti-inflammatory treatments as  TNF α [pg/ ml]  they react in a dose-dependent manner to this inhibitor (Figure 14).  5000  4000  3000  2000  1000  0  Uu  Us  Du  Ds  Mu  Ms  Figure 12: Upon LPS stimulation ES-derived macrophages released TNFα into the culture media. Macrophages generated from mEMS159 ES cells (Ds) and primary alveolar macrophages (Ms), but not undifferentiated ES cells (Us) responded to LPS stimulation with secretion of TNFα. No TNFα was detected in supernatants from un-stimulated undifferentiated ES cells (Uu), ESderived macrophages (Du) or primary macrophages (Mu) cultures. The culture time for ESderived macrophages was > 2 weeks, while primary macrophages were tested after only 1 day in culture.  43  Figure 13: Rolipram treatment reduced TNFα release. Pre-treatment (20 minutes) with the first-generation PDE4 inhibitor Rolipram reduced the initial TNFα response after LPS stimulation in ES-derived macrophages (52 %) cultures similar to primary macrophages (68 %). ‘n’ indicates the number of individual culture wells used to derive the mean TNFα level with according standard deviation.  Figure 14: Rolipram dose-response of ES-derived macrophages. In vitro cultures of ES-derived macrophages, developed over > 2 weeks, responded to Rolipram administration prior to LPS stimulation in a dose-dependent manner. Shown are the mean TNFα levels of a minimum of three independent culture wells with standard deviation. 44  An internalization assay with latex beads was also performed to further functionally characterize the ES-derived macrophages. In Figure 15 I show evidence that these cells take up 2 um particles by forming actin cups, a marker for phagocytosis (Champion 2006, Haberzettl 2007), in a similar manner as primary alveolar macrophages.  Figure 15: ES-derived macrophages (d-f) internalize 2 um latex beads by forming actin cups like primary macrophages (a-c). Picture a. and d. show external beads (green), internal beads (blue) and the F-actin skeleton (red) of macrophages stained with Phallotoxin. In the picture b. and e. actin cups are marked with arrow heads. Picture c. and f. provide a bright-field view of the area. bar = 20 um  After careful characterization of the mEMS159-derived macrophages, 3 wild-type and 3 YAC128 cell lines from the 129 strain were differentiated into EBs and macrophages. On day 11 of differentiation, EBs had formed in cultures from all cell lines. No difference in number and appearance of these EBs was observed based on the genotype of the initial ES cell lines (Figure 16, panel I). By day 18, macrophages had attached to the ULC plates (Figure 16, panel II) and the cell-type was confirmed by F4/80 ICC (Figure 16, panel III). All cells examined stained positive for the macrophages marker, similar to previous results obtain using the control cell line mEMS159. I noted that the 16.2Y128-129 ES cell line generated the most cells, however the two other YAC128 ES cell lines showed no  45  difference in number or appearance of macrophages when compared to their wild-type controls. Following a defined protocol, ES-derived macrophages were harvested, primed with Interferon-γ and stimulated with LPS, while cells in the control condition received only Interferon-γ treatment. 24 hours later I observed elevated TNFα and IL-6 levels in the supernatant of LPS stimulated cells, but not in the control condition. As shown in Figure 17 and 18, for TNFα and IL-6 levels respectively (corrected for the total protein amount per well) all cultures responded to LPS stimulation, but the response was highly variable. It would be premature to assume that the cytokine response in ES-derived macrophages of YAC128 ES cell lines is abnormal when compared to cells of wild-type lines.  Figure 16: Novel ES-derived macrophages. Panel I shows 11 days old EBs. Wild-type and YAC128 cells appear to be similar to the control cell line mEMS159 (CTRL); no differences in morphology of the EBs were detected based on genotype (bar = 100 um). As shown in panel II under the bright-field microscope ES-derived macrophages initially appear similar in size to primary macrophages (CTRL). Panel III shows positive staining for F4/80, a macrophages specific marker, in ES-derived macrophages independent of the genotype and appeared to be similar to primary macrophage cultures (CTRL, 10 DIC). ES-derived macrophage cultures were > 2 weeks old. For ICC pictures nuclei were counterstained with DAPI (blue). Panel II-III: bar = 20 um  46  Figure 17: Upon Interferon-γ/CSE stimulation novel ES-derived macrophages released TNFα into the culture media. Shown are the mean TNFα level in the culture media of a minimum of two individual wells containing ES cell-derived macrophages with standard error of mean. No TNFα was detected in any control culture which received only Interferon-γ (data not shown). All TNFα concentrations are relative to the total protein amount.  Figure 18: Upon Interferon-γ/CSE stimulation novel ES-derived macrophages released IL-6 into the culture media. Shown are the mean IL-6 level of a minimum of two individual wells containing ES cellderived macrophages with standard error of mean. No IL-6 was detected in any control culture which received only Interferon-γ (data not shown). All IL-6 concentrations are relative to the total protein amount.  47  Altogether, the results confirm that the 17 out of the initial 24 isolated ES lines are pluripotent and capable of sustained propagation. 129 mouse strain  129 x FVB F1 hybrid  4 ES cell lines  3 ES cell lines  YAC 18  5 ES cell lines (Colum Connolly)  2 ES cell lines  YAC128  6 ES cell lines  2 ES cell lines  wild-type  Table 4: Overview of all established novel ES cell lines. Altogether, 7 wild-type cell lines, 2 YAC18 cell lines and 8 YAC128 cell lines have been developed on two genetic backgrounds.  2.4 Discussion The efficiency with which ES cell lines are generated is greatly dependent on the genetic background of the mouse strain that is used, as well as the derivation-protocol. The derivation of 10 novel stable ES cell lines from 129S1/SvIm1 mice supports that the 129 mouse strain is particularly well suited for the generation of ES cell lines (Simpson 1997). The HD-like phenotype - progressive striatal volume loss and motor dysfunction - of YAC128 mice is penetrant on different genetic backgrounds. However, Van Raamsdonk et al (2007a) demonstrated that the severity of the phenotype is altered on various mouse strains. The FVB/N mouse strain was identified as the genetic background where YAC128 show the most severe phenotype. Unfortunately, we were unable to establish and maintain any ES cell line from crosses of FVB/N mice. It is well known that inbred FVB/N mice are unsuitable for ES cell generation and culture. Nevertheless, using a more ideal genetic background of the 129 mouse strain crossed with FVB/N mice, we generated seven 129/FVB F1 hybrid ES cell lines. Overall, we were able to generate and confirm 17 novel ES cell lines from 108 collected mouse embryos, an efficiency of 16%. Establishment of the newly described serum-replacement method by Cheng et al (2004) in the Leavitt laboratory and maintenance of strict characterization criteria were contributing factors to this derivationefficiency. Consistent with the existing YAC model of HD, no significant differences in the number of established wild-type and transgenic ES cell lines were observed.  48  Furthermore, in the YAC mouse model it has been shown that the transgene is stably integrated into the genome. Initial genotyping confirmed the existence of the YAC construct in transgenic ES cell lines. Nonetheless, abnormal karyotypes, such as the loss of sex chromosomes (Sugawara 2006) and trisomy 8 (Liu 1997) are common in most ES cell lines and have been documented at higher passages in the control cell line mEMS159 as well as one novel wild-type cell line (see Appendix Figure 31). Therefore, all novel ES cell lines should be genotyped at later passage numbers to assure that the YAC transgene is not lost over long periods of culture. In addition, karyotype analysis should be performed to evaluate the chromosomal integrity of the established cell lines. During embryogenesis expression of Oct4, a member of the POU family of transcriptions factors and endogenous AP activity is limited to pluripotent cells of the inner cell mass (ICM) that contribute to the formation of all fetal cell types. Of 24 initially isolated cell lines 17 exhibited AP activity similar to the control ES cell line. In the same cell lines that expressed AP, I observed nuclear expression of Oct4, confirming their pluripotent potential. In comparison MEF feeder layers did not show staining for these markers. I chose to examine the expression of Oct4 via RT-PCR, to verify results generated through ICC. In addition to Oct4 we analyzed the presence of Nanog, a homeodomain protein. Both, Oct4 and Nanog are essential transcription factors that act with Sox2 to suppress functional expression and activity of lineage specification factors in a dominant manner (reviewed by Niwa 2007). Moreover, it has been shown that forced expression of these transcription factors can reinstate pluripotency in embryonic and adult mouse fibroblast cultures (Takahashi 2006) generating iPS (induced pluripotent stem) cells. Therefore, these factors provided a precise tool for the phenotypic assessment of novel ES cell lines, confirming my ICC results. ES cell lines have been shown to proliferate for long periods of time in an undifferentiated state when cultured in LIF containing media. However, it has become more apparent that ES cells are not homogeneous cultures and hold different developmental potential (Silva 2008). It was highly evident that the undifferentiated state, especially of the 129-FVB F1 hybrid cell lines, could only be maintained under strict culture conditions. The culture without MEF feeder layers was found to be sub-optimal as it induced heterogeneity, differentiation and Nestin expressing cells. The self-renewal potential of our established ES cell lines was demonstrated by high Oct4 expression after serial cultures to up to 50 passages followed by the differentiation into neurons and macrophages. During these serial cultures, cells maintained the typical ES cell morphology, however, Oct4 together  49  with Sox2, direct the expression of FGF4 acting as an autoinductive stimulus driving general lineage specification in ES cells (Kunath 2007). Constitutive expression of Nanog reverses this action and should therefore be investigated as well. We were able to generate EBs, Nestin+ neuronal precursors and neuron-like cells expressing β-III-tubulin and NeuN as well as astrocytes in mixed cultures from 14 novel ES cell lines, as previously described by Li et al (1998). Mixed cultures appeared to contain similar numbers of neurons and astrocytes, independent of the genotype. In fact, in an initial analysis of my ES-derived neuronal cultures, I did not see a difference in the neuronal cell death response to NMDA, a compound that induces increased apoptosis in cultures of primary YAC128 medium spiny neurons (MSN) - the most affected neuronal population in HD patients - when compared to YAC18 (Shehadeh 2006). This could be due to do technical matters, such as NMDA dosage and exposure time. The ES-derived neurons in these cultures may also not express mature NMDA-type glutamate receptors. It is evident that, the ES-derived neuronal cultures need to be characterized in more detail. I did not determine what type of neurons I had derived nor have I examined how functional these cells were. These cultures should ideally contain MSNs in high numbers, which can be identified with markers such as DARPP32 (dopamine-and cyclic AMPregulated phosphoprotein, Mr=32000). Therefore, the chosen differentiation protocol was good to show the pluripotent potential of our ES cell lines, but was not ideal to examine the derived neuronal cells for any possible genotype dependent HD pathology. Future studies will focus on defining methods to differentiate ES cells more efficiently into neurons. One possibility that is already being pursued in collaboration with CHDI will be to generate neural stem cell lines from these novel ES cell lines, which can be used to generate pure neuronal cultures with high-efficiency (Spiliotopoulos 2009). Additionally, the pluripotent status of 6 ES cell lines on the 129 background was confirmed by deriving F4/80+-macrophages. In doing so I demonstrated that these cell lines were able to differentiate into cells of different germlayers, ectoderm (neurons) and endoderm (macrophages). Secondly, cells of the innate immune system, microglia in the CNS and macrophages/ monocytes in the periphery, have shown abnormal patterns of immune-activation in HD patients and various mouse models (Björkqvist 2008). I therefore studied the TNFα and IL-6 release of ES-derived macrophages in response to LPS. Although, all cell lines showed elevation of TNFα and IL-6 levels in the supernatant after LPS stimulation in comparison to un-stimulated cells, the extent of response was highly variable in these initial studies across all cell lines independently of their genotype. In order  50  to reduce variability, I used INF-γ initially priming the cells and ‘Control Standard Endotoxin’-quality LPS. Moreover, I determined the total protein amount of each culture, to ensure that the difference in response was not observed due to uneven cell density. Nevertheless, at this state it is unclear if macrophages derived from YAC128 ES cell lines differ in their response to LPS when compared to their wild-type counterparts. The epigenetic status of each ES cell line, transcription of regulatory genes, and abnormal karyotype in consequence of in vitro culture may in part cause the variability among these cell lines and would need to be investigated before coming to a conclusion. Even if an altered response in YAC128 derived cells would be observed in the future, ESderived macrophages of YAC18-129 cell lines are needed to assure that this is not an effect of the YAC transgene itself. Furthermore, other functional assays such as Rolipram inhibition or phagocytosis assay could provide options with less variability. Also, the quality and developmental potential of the newly established ES cell lines may not be equal. In comparison to wild-type and YAC128 cell lines on both genetic backgrounds, both of the YAC18 ES cell lines and their wild-type controls show slightly different morphology and appeared to differentiate more rapidly in media containing LIF. Despite this, these cell lines were able to generate neurons in a similar efficiency to the other ES cell lines. Recently, Colum Connolly (a new Graduate student in the Leavitt Lab) established an additional 5 wild-type and 5 YAC18 ES cell lines from blastocysts of the 129 background. These cells need to be carefully evaluated and compared to YAC18 on the F1 hybrid background. Overall, the characterization experiments I have performed were useful to examine the basic ‘stem cell’ properties of our various ES cell lines, nevertheless all of these experiments were in vitro assays. Ultimately, to prove self-renewal and pluripotency, these cells should be able to establish chimeric mice and undergo germline transmission. Thus far, we have not pursued these final blastocyst injection experiments, as they are expensive and these transgenic mice already exist. This type of study may be pursued in the future if mutations altering the HD phenotype were identified or introduced in these ES cells. Other possible in vivo experiments to further characterize our ES cell lines would include the induction of teratoma carcinomas after the subcutaneous injection of ES cells, and the serial transfer of ES cells from one mouse to another. The induction of teratoma carcinomas would be simple to perform and not expensive, but would not provide much additional insight. As for in vivo dilution experiments, cells would need to be stabily  51  transfected with a reporter gene such as LacZ or GFP, to be able to identify these cells in situ and would be rather time- and cost-intensive. My work has characterized the stem cell nature of the various ES cell lines I generated, but has not addressed whether these ES cell lines will be useful as a novel cellular model for HD. This project has successfully generated ES cell lines of 3 different genotypes - wildtype, YAC18 and YAC128 - on 2 different genetic backgrounds from a well established fulllength mouse model of HD. The simplicity of an in vitro model derived from these ES cell lines compared to the existing animal model could provide faster less expensive answers when screening drugs as well as gene-gene-, gene-protein-, and protein-proteininteractions and could help to further understand HD pathogenesis. 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Assessment of macrophages and microglia from YAC128 mice in a model of acute inflammation2 3.1 Introduction Huntington disease (HD) is a neurodegenerative disorder characterized by progressive motor dysfunction, cognitive impairment and behavioural abnormalities and associated with selective loss of specific subsets of neurons within caudate, putamen, cortex and other brain regions. Historically, HD research has focused on neuropathology since core symptoms are thought to be directly derived from brain dysfunction. However, despite the regional neurodegeneration seen in this disorder, huntingtin is ubiquitously expressed throughout the body (Trottier 1995, Hodgson 1996) and throughout embryonic development (Dure 1994, Buraczynska 1995). The expression pattern of huntingtin does not explain the selective neurodegeneration instead suggests a more widespread cellular function of the wild-type protein. Supporting this idea is the observation that huntingtin null mice (Htt-/-) are embryonic lethal (Nasir 1995, Duyao 1995, Zeitlin 1995) and recently nonneuronal pathology (changes other than selective neuronal degeneration) have been demonstrated in human HD as well as in animal models of HD (reviewed in Van der Burg et al, 2009). Increased numbers of astroglial cells have been identified in post-mortem HD brain tissue with Vonsattel grades 2 to 4 (Vonsattel 1985). Astrocytes are thought to provide metabolic support to neurons and play a key role in the repair and scarring processes following traumatic injuries (Stichel 1994). The astrocytosis observed in postmortem HD brain tissue was initially thought to be a consequence of neuronal loss in these tissues. However, additional support for an important role of glia in HD pathogenesis comes from studies of a new line of transgenic mice expressing N-terminal mutant huntingtin fragments under the GFAP promoter and therefore exclusively in astrocytes (Bradford 2009). These mice exhibit decreased expression of glutamate transporters, and hence decreased glutamate up-take by increased binding to the transcription factor Sp1. These mice also show an age-dependent HD-like phenotype, with loss of body weight, motor function deficits, and shorter survival when compared to wild-type or control transgenic mice.  2  A version of this chapter will be submitted for publication. Thiele J, Connolly C, Mazarei G, Gao Y, Lu G, Inoue K and Leavitt BR (2010) Assessment of microglia from YAC128 mice in a model of acute inflammation.  57  In addition to increased numbers of reactive astrocytes, microglia appear to be activated early in HD and increase in number over the course of the disease. Microglia normally represent 10–20% of the glial cell population in the brain, are a key part of the innate immune system of the CNS, and play a key role in neuroinflammatory responses to infection and cell injury. These glia cells respond rapidly to subtle changes in the local microenvironment by entering an activated state that is characterized by distinct morphological changes. Upon activation and morphologic differentiation, they remove dying cells through phagocytosis, secrete proteases and a variety of pro- and antiinflammatory cytokines, much like their counterparts in the periphery, macrophages and monocytes. A growing body of evidence suggests that activated microglia might play a protective role in the CNS as they produce neurotrophic and/or neuroprotective molecules (reviewed by Nakajima et al, 2004). Töpper et al (1993) recognized that microglial activation accompanies neuronal injury in some situations. During excitotoxic striatal injury, caused by direct injection of the Nmethyl-D-aspartate (NMDA) receptor agonist quinolinic acid, microglia activation influenced the degeneration of striatal projection neurons. However the authors proposed that other non-direct mechanisms must account for the rapid transsynaptic microglial activation seen in the thalamus in this model of acute neuronal injury. Examining grade 3 to grade 4 HD post-mortem brain tissues, Singhrao et al (1999) have also suggested that reactive microglial become activated in response to early neuronal damage and produce complement factors which activate on the membranes of neurons and subsequently may lead to increased neuronal cell death via proinflammatory mechanisms. At this point most scientists believed that the response of microglia was an effect secondary to the ongoing neuronal damage, similar to the astrocytosis observed. Until Sapp et al hypothesised a direct role for activated microglia in HD pathogenesis. The researchers studied human post-mortem brain tissue and stated that the grade-dependent increase in activated microglia and their close proximity to degenerating neurons in the striatum and cortex of HD brains may be related to the disease process and not to random pre-morbid events (Sapp 2001). This hypothesis was further supported by the results of a combined clinical and positron emission tomography (PET) study of 11 human HD patients at various stages of the disease (Pavese 2006). Pavese et al demonstrated that binding of [11C](R)-PK11195, a selective in vivo marker for activated microglia, correlated with disease progression measured by Unified Huntington's Disease Rating Scale scores. Levels of activated microglia correlated inversely with [11C]Raclopride binding, which was  58  employed as marker of striatal cell function. Interestingly, further PET analysis revealed that increased microglial activation can already be detected in pre-symptomatic HD gene carriers, suggesting that microglial activation is an early process in the HD pathology (Tai 2007). So far not much is known about microglial activation in mouse models of HD. Microglia in the brains of late stage R6/2 mice appear dystrophic and show increased expression of ferritin, similar to microglia of human HD subjects (Simmons 2007). On the contrary, an age-dependent reduction in size and density of R6/2 microglia compared to microglia from age-matched wild-type mice has also been reported (Ma 2003). Moreover, altered levels of a number of proteins involved in the regulation of the innate immune system such as interleukin 6 (IL-6), have been observed in studies performing proteomic profiling of CSF and plasma samples from human HD patients and plasma samples from R6/2 mice (Dalrymple 2007). Astrocytes and microglia can be activated in response to glutamate excitotoxicity. It is believed that the neuronal excitotoxicity occurring in HD, is predominately mediated through NMDA-gated ion channels (Zeron 2001 and 2002). Once activated, NMDA-gated ion channels allow rapid calcium influx that leads to the activation of several enzymes, including neuronal nitric oxide synthase (NOS; Hahn 1988, Alagarsamy 1994). Nitric oxide (NO) in turn increases neuronal glutamate release (Meffert 1994, Montague 1994) and inhibits glial glutamate up-take (Pogun 1994, Trotti 1996) which subsequently results in more excitotoxic cell death. Activated microglia in turn, release inducible nitric oxide synthase (iNOS), which has also been shown to contribute to neuronal death following NMDA treatment in rat mixed glial cultures (Chao 1992, Tikka 2001). Minocycline, an antiinflammatory compound which inhibits the IL1β-converting enzyme and prevents the upregulation of iNOS, increases neuronal survival in these cultures (Tikka 2001). Furthermore, the scientists reported that NMDA treatment alone induced microglial proliferation that preceeds neuronal death. In the R6/2 transgenic mouse model of HD combined treatment of Coenzyme 10 and Minocycline significantly extended survival and improved rotarod performance (Stack 2006). Minocycline treatment combined with pyruvate has also been shown to be protective in an excitotoxic rat model of HD (Ryu 2006). In addition to the role microglia may play in excitotoxic events, the production of proinflammatory cytokines (e.g. TNFα and IL-1β) and reactive oxygen species subsequently results in activation of additional microglia, causing a self-propagating inflammatory circuit. The exact biochemical mechanisms by which mutant huntingtin expression alters microglia  59  activation remain unclear. However, activated microglia have been implicated in the pathogenesis of other neurodegenerative diseases as well, including Alzheimer’s disease (reviewed by Cameron et al, 2009) and Parkinson’s disease (reviewed by Lee et al, 2009). Little is known about the state of astrocytes and microglia in the YAC128 mouse model of HD. For this project I hypothesize that abnormal immune activation takes place in the YAC128 model of HD similar to the human condition. Furthermore, these changes can be found not only in microglia in the CNS, but also in macrophages in the periphery. I aim to evaluate the altered immune activation by assessing the response of primary microglia and macrophage cultures in an endotoxin stimulation assay as well as the quantification of microglia activity after striatal injections of endotoxin.  60  3.2 Materials and methods All animal procedures were performed following the guidelines of the Canadian Council on Animal Care.  3.2.1 Ex vivo studies Isolation of alveolar macrophages 12 month old wild-type, YAC18, and YAC128 mice, all maintained on a pure FVB/N strain background, were used to isolate primary macrophage cultures. Animals received an overdose of 10 mg avertine via i.p. injection, before the respiratory tract was dissected. Alveolar macrophages were extracted by intra-tracheal infusion of ice-cold PBS (Invitrogen) followed by centrifugation and re-suspension of extracted cells. Cells were counted and seeded at a density of 1.5x105 cells/ ml onto 96-well gelatin-coated plates or glass coverslips and cultured in 5 % culture medium (RPMI 1640 [Invitrogen], 5% FBS [PAA], and 1% penicillin/ streptomycin [Invitrogen]).  Isolation of primary glia cells Whole brains were obtained from post-natal day 1 to 3 old wild-type, YAC18 and YAC128 mouse pups on the FVB/N strain background and placed in Hanks Balanced Salt Solution (HBSS; Invitrogen) on ice. A piece of tail tissue was collected from each animal for genotyping. Surgery tools were dipped into ethanol in between animals to avoid cross contamination. Meninges were removed carefully, the remaining brain tissue was placed into growth medium (DMEM [Invitrogen], 10 % FBS [PAA], 1 % L-glutamine [Invitrogen], 1 % penicillin/ streptomycin [Invitrogen]) and homogenized using a 5 ml pipette. Cells of each brain were pelleted, re-suspend in growth medium and transferred into a T150 flask, cultured at 37 ºC with 5 % CO2. Growth medium was replaced after 24 hours and then every 7 days. After 18 to 21 days in culture (DIC), loosely attached microglia were harvested and seeded at 1.4x105 cells/ ml with their own conditioned media into 96-well PRIMARIATM tissue culture plates (BD Falcon) or glass coverslips.  Immunocytochemistry (ICC) Cells were cultured on glass-coverslips. Culture media was aspirated; cells were washed once with PBS and fixed with 3 % paraformaldehyde for 10 minutes at room temperature (RT). Cells were then washed and permeabilized three times 5 minutes with  61  PBS-T (T = Triton X-100; 0.3 % in PBS) for, followed by a block step with 2 % normal goat serum (NGS) in PBS-T for 30 minutes, both at RT. Primary antibodies, diluted in PBS-T and 2 % NGS were added and left overnight at 4 °C. Table 5 below provides a complete list of all primary antibodies used. About 15 hours later cells were washed three times 5 minutes with PBS before fluorescent secondary antibodies (diluted in PBS) were added. All following steps were performed at RT with minimum light exposure. After 1 hour cells were washed twice for 3 minutes with PBS and then counter-stained with 200 nM 4',6-diamidino-2-phenylindole (DAPI) solution for 5 minutes. Afterwards, cells were washed once more for 5 minutes with PBS. The coverslips were transferred out of the 24well plate and air-dried. Each coverslip was dipped into xylene before being mounted with DePeX MOUNTING MEDIUM (Electron Microscopy Sciences) onto glass slides.  Immunohistochemistry (IHC) Mounted brain sections were blocked for 1.5 hours at RT with PBS containing 5 % NGS and 3 % of a 10 % Triton-X solution in tris-buffered saline. Primary antibodies, diluted in blocking solution were added and left overnight at 4 °C. In Table 5 below is provided a complete list of the primary antibodies used. About 15 hours later brain sections were washed six times 10 minutes with PBS before fluorescent secondary antibodies (diluted in PBS) were added. All following steps were performed at RT with minimum light exposure. After 1 hour sections were washed 6 times for 10 minutes with PBS, and at the second last wash nuclei were counter-stained with 200 nM DAPI solution for 5 minutes. Afterwards, slides were air-dried and dipped into xylene for about 1 minute before being cover-slipped with DePeX MOUNTING MEDIUM (Electron Microscopy Sciences). Designation  Clone and/or catalogue # (company)  F4/80  BM8, # MF48000 (Caltag Labs- Invitrogen)  CD11b  M1/70.15 (Caltag Labs- Invitrogen)  CD18  M18/2.A.12.7 (Springer TA, Centre for Blood Research)  CD68  FA-11, # MCA1957 (AbD Serotec)  IBA-1  # 019-19741 (Wako)  GFAP  # AB5040 (Chemicon - Millipore)  NeuN  A60, # MAB377 (Chemicon - Millipore)  Table 5: List of primary antibodies used for ICC and IHC.  62  Phagocytosis assay Latex beads (Sigma-Aldrich) were rinsed according to manufacturer’s instructions and opsonised with 1 mg/ ml human immunoglobulin G (IgG; Sigma) for 1 hour at 37 °C and 5 % CO2 The beads were then washed three times to remove excess IgG and re-suspended in PBS. Alveolar macrophages were seeded onto coverslips (24-well plate) one day prior and were pre-cooled for 15 minutes to 4 °C and then incubated 30 minutes with IgGlabelled beads to allow binding. Approximately 200 beads per cell were applied. The cold media was replaced by preheated (37 °C) media. After 1 hour incubation at 37 °C and 5 % CO2 cells were washed three times with ice-cold PBS and stained with Alexa Fluor®488 goat anti-human IgG (Invitrogen) for 5 minutes on ice. Cells were fixed then cells using 2 % paraformaldehyde in PBS for 10 minutes at RT, followed by 3 washes with PBS. The cells were then permeabilized in PBS-T for 3 minutes at RT, washed again three times with PBS and subsequently stained with, 2.5 Units/ ml PBS Alexa Fluor®594 phalloidin (Invitrogen) for 15 minutes at RT. Cells were washed once more for 5 minutes with PBS. The coverslips were transferred out of the 24-well plate and air-dried. Each coverslip was dipped into xylene before being mounted it with DePeX MOUNTING MEDIUM (Electron Microscopy Sciences) onto glass slides.  Control standard endotoxin (CSE) stimulation After the initial isolation primary macrophages and microglia were seeded at a density of 1.5x105 cells/ ml onto 96-well gelatin-coated plates or 1.4x105 cells/ ml into 96well PRIMARIATM tissue culture plates respectively. About 24 hours later the culture media was replaced by growth medium containing 1 % FBS. Functional studies were performed after an additional 24 hours. Medium, containing Interferon-γ (INF-γ; final concentration 10 ng/ ml; R&D Systems) with or without CSE (a widely used standard for endotoxin testing, purified from Escherichia coli O113:H10, final concentration 100 ng/ ml; Associates of Cape Cod) or plain growth medium was added. Supernatants were collected after 20 to 24 hours, and stored at -20 °C. Cells were lysed and total protein levels were determined using the microBCA kit (Thermo Scientific). Cytokine concentrations were quantified in supernatants from independent wells from each animal for each condition, using the mouse IL-6 or TNFα ELISA kit (eBioscience) or using Meso Scale Discovery (MSD) assays as per the manufacturer’ s protocol and analyzed with a SECTOR 2400 instrument (MSD). Numbers of animals used in each experiment are given in Table 6 below. Statistical analysis was performed using the SPSS software.  63  number of animals used per condition control  INF-γ  ATP  CSE  IL-6 release from primary microglia after CSE stimulation wt/YAC18: wild-type  1  4  3  4  YAC18  2  7  4  7  IL-6 release from primary microglia after CSE stimulation wt/YAC128: wild-type  4  6  5  6  YAC128  3  5  4  5  cytokine release from primary microglia after CSE stimulation wt/YAC18: wild-type  3  3  N/A  3  YAC18  3  3  N/A  3  cytokine release from primary microglia after CSE stimulation wt/YAC128: wild-type  4  4  N/A  4  YAC128  4  4  N/A  4  Table 6: Numbers of animals used to isolate primary microglia in each ex vivo CSE experiment.  3.2.2 In vivo study Direct intra-striatal injections of CSE 9 month old wild-type and YAC128 littermates on a pure FVB/N background received unilateral striatal injections with 0.5 ul of 2 ug CSE/ ul (n=4 per genotype) or PBS (n=2 per genotype). After seven days mice were perfused with 3 % paraformaldehyde, brains collected, cryo-sectioned and IHC was performed as described above.  64  3.3 Results 3.3.1 Ex vivo studies Alveolar macrophages were isolated from 12 month old wild-type, YAC18 and YAC128 mice on a pure FVB/N background. The primary wild-type cultures were examine via ICC for F4/80, a macrophage-restricted cell surface glycoprotein, and CD11b/ CD18 which together form Mac-1, a type three complement receptor (CR3), to ensure pure primary macrophage cultures. My ICC results suggest that the primary cells cultures were homogenous as the macrophage-characteristic glycoproteins were present in nearly all cells isolated (Figure 19a-c). In addition, I demonstrated CD68 expression in primary cultures of alveolar macrophages from C57BL-6J mice (Figure 19d). Cultures of FVB/N wild-type macrophages were able to engulf human IgG-coated 2-3 um latex beads (Figure 20), demonstrating that these cells are functional under in vitro conditions.  Figure 19: Primary cells isolated from the lung expressed glycoproteins characteristic for macrophages. The murine cell-surface markers F4/80 (a), CD11b (b) and CD18 (c) were observed in macrophage cultures from wild-type FVB/N mice. CD68 (d) expression was shown in wild-type C57BL-6J macrophages. Nuclei were counter-stained with DAPI (blue). bar = 50 um  Figure 20: Primary macrophages isolated from the lungs of wild-type FVB/N mice were able to engulf latex particles. After one hour incubation, the macrophage had taken up a number of latex beads. Fully engulfed beads appear in blue, while particles attached to the surface of the cells were labelled green (a). The F-actin skeleton (red) shows the formation of actin-cups (arrow head, b). bar = 10 um 65  Figure 21: HD monocytes, macrophages, and microglia were overactive when stimulated. Only upon LPS stimulation, IL-6 was detected in supernatants of human monocytes (A), murine macrophages (B-C) and microglia (D) cultures. IL-6 levels were significantly higher in supernatants of premanifest HD subjects, YAC128 and R6/2 cells when compared to their controls. Alveolar macrophages from YAC18 mice did not differ in their IL-6 levels from wildtype controls. Graphs show the mean concentration with standard error bars. ND, not detected. NS, not significant. Unpaired t tests: *, P < 0.05; **, P < 0.01.This figure was published as ‘Figure 6’ in: “A novel pathogenic pathway of immune activation detectable before clinical onset in Huntington’s disease.” Björkqvist et al (2008).  I then investigated the functional response of wild-type, YAC18 and YAC128 alveolar macrophages to control standard endotoxin (CSE) stimulation. Un-stimulated macrophage cultures and macrophages primed with Interferon-γ (INF-γ) alone, did not release detectable levels of IL-6 (Figure 21) or TNFα. However, macrophages stimulated with CSE responded with a pronounced release of cytokines. The IL-6 response of YAC128 macrophages was found to be significantly higher (T-test, p<0.05), than the response of 66  corresponding wild-type cells. Macrophages from YAC18 mice, which differ from YAC128 in the number of CAG repeats, exhibited similar levels of IL-6 when compared to wild-type macrophages. Furthermore, together with our collaborators we demonstrated that monocytes isolated from premanifest HD subjects and microglia from R6/2 mice were also over-active and responded with significantly elevated IL-6 release following endotoxin exposure, similar to YAC128 macrophages (Figure 21). This provides evidence that these changes occur in the periphery and the CNS of HD subjects. The combined data were published in 2008 by Björkqvist et al. In the study we noticed that although, serum levels of cytokines were elevated, they appeared to be slightly different among various HD mouse models. I therefore proceeded to examine whether YAC128 microglia, just as microglia from R6/2 mice, are overactive when stimulated with CSE. Mixed glia cultures where isolated from the whole brain of 1 to 3 days old, wild-type, YAC18 and YAC128 mice on a pure FVB/N background. Figure 22 shows an example of such mixed culture. Once the primary microglia cultures reached confluency, they appeared to be multi-layered, consistent of mainly microglia and astrocytes at various activation stages. A layer of loosely attached microglia grew on top of astrocytes. In addition, colonies of microglia, fully attached to the plastic plates grew among astrocyte colonies.  Figure 22: Mixed glia cultures appeared to be multi-layered consistent of microglia and astrocytes at different activation levels. The schematic (a) and bright-field picture (b) show how these primary cultures organized, loosely attached microglia (white arrow heads) sat on top of astrocytes (blue arrow heads), while a second layer of microglia (black arrow heads) fully attached to the plastic culture surface. bar = 100 um  67  To obtain pure microglial cultures from the mixed glial cultures, the flasks were tapped gently to collect the loosely attached microglia while the astrocyte layer remained intact. Given the opportunity to attach to the surface, re-seeded microglia exhibited similar size and shape of those fully attached microglia in mixed cultures. In these relatively pure microglia cultures 98.8 % of the cells stained for IBA-1 (ionized calcium binding adapter molecule 1), a 147-amino-acid calcium-binding protein and marker for microglia and most macrophages. The astrocytic marker GFAP (glial fibrillary acidic protein) was found to be expressed almost exclusively in mixed glia cultures (Figure 23) and was not found to be expressed in the pure microglial cultures. Since the mixed glia cultures were established from whole brains, I examined whether neurons remained within the culture. Using an antibody against NeuN, a neuron-specific nuclear protein, I did not detect any neurons in the primary glia cultures. Primary neuronal cultures require special care; it is likely that after 18 DIC these cells did not survive under the chosen culture conditions (Figure 23).  Figure 23: Primary glial cultures expressed common markers of microglia and astrocytes, but not neurons. Pure microglia (a-c) and mixed glial cultures (d-f) stained for the microglia marker IBA-1 (a,d). GFAP was used to label astrocytes, these cells were not present in pure (b) but in mixed glia cultures (e). The nuclear neuronal marker NeuN was not observed in any of the cultures (c,f). Nuclei were counter-stained with DAPI (blue) for all conditions. bar = 50 um  68  After successfully establishing pure microglia cultures from wild-type, YAC18 and YAC128 mice I proceeded to examine their response to CSE stimulation. Consistent with data generated from my primary macrophage cultures, IL-6 and TNFα (later data not shown) were only detected in supernatants from cultures stimulated with CSE, but not from cultures treated with only INF-γ or plain media (Figure 24 and 25). In comparison to YAC18 microglia, YAC128 microglia produced significantly more IL-6 than their corresponding wild-type controls (T-test, p<0.05). This result suggests that microglia isolated from YAC128 mice are over-active when stimulated with CSE, similar to findings using R6/2 microglial cultures. Furthermore, I was able to show changes of the innate immune response occur in YAC128 mice in both CNS and the periphery. In this study, cultures were controlled for total protein amount to ensure that differences seen in IL-6 levels were not caused by irregular cell density.  Figure 24: Microglia isolated from neonatal mice released IL-6 when stimulated with CSE. The figure shows mean IL-6 concentrations detected in the culture-media after 24 hours of CSE stimulation with standard error of mean. All concentrations derived are relative to the total protein amount of each individual culture. No IL-6 was detectable in the supernatant of microglia in the un-stimulated (WT n=2, YAC18 n=3) state, after priming with IFN-γ (WT n=8, YAC18 n=14) or after ATP stimulation (WT n=7, YAC18 n=9). Microglia stimulated by the addition of both IFN-γ and CSE released IL-6, no significant difference was identify between wild-type (n=12) and YAC18 (n=21) cultures. ‘ND’ not detected; ‘NS’ not significant in T-test; ‘n’ indicate the number of individual wells quantified.  69  IL-6 conc./ total protein  Figure 25: YAC128 primary microglia were over-active when stimulated with CSE. The figure shows mean concentrations of IL-6 detected in the culture-media after 24 hours of CSE stimulation with standard error of mean. All concentrations derived are relative to the total protein amount of each individual culture. No IL-6 was detectable in the supernatant of microglia in the un-stimulated (WT n=8, YAC128 n=5) state, after priming with IFN-γ (WT n=12, YAC128 n=10) or after ATP stimulation (WT n=14, YAC128 n=11). Microglia stimulated by the addition of both IFN-γ and CSE expressed IL-6, YAC128 (n=15) cultures released significantly more IL-6 than wild-type (n=18) cultures. ‘ND’ not detected; * indicates p value < 0.05 in T-test; ‘n’ indicate the number of individual wells quantified.  Interestingly, stimulation with 1-2 mM ATP, a dose-range shown to cause cytokine release in wild-type rat microglia (Hide 2000), did not stimulate mouse microglia to release detectable levels of IL-6 or TNFα. This could be due to technical matters, such as dose and exposure time may vary in mice, and needs therefore be investigated before coming to a conclusion. I performed two completely separate CSE stimulation experiments (including microglia isolation) with YAC18 and wild-type controls as well as three experiments with YAC128 and wild-type controls. In both YAC18 experiments no difference in IL-6 release was detected when compared to the response of wild-type microglial cells. However, all three YAC128 experiments showed significantly elevated levels of IL-6 after 24 hours of CSE stimulation compared to wild-type microglia. Combining these data, I show that YAC128 cultures generated a 1.4-fold higher IL-6 response to CSE, while YAC18 microglia responded similar to wild-type cells (Figure 26).  70  IL-6 release relative to wild-type microglia  1.5  1.0  0.5  0.0  WT  YAC18  YAC128  Figure 26: YAC128 microglia exhibited elevated IL-6 levels in response to 24 hours of CSE exposure when compared to wild-type and YAC18 microglia. The figure represents the mean IL-6 concentrations normalized to the corresponding wildtype microglia with standard error of mean derived from five individual experiments (wildtype/YAC18 n= 2 experiments, wild-type/YAC128 n= 3 experiments). In the two YAC18 experiments no significant difference in IL-6 release was detected, while each of the three YAC128 experiments showed significantly increased levels of IL-6 after CSE stimulation when compared to wild-type controls.  The culture supernatant from all the cells for one YAC18 and one YAC128 microglia stimulation experiment were sent to our collaborators in the UK. Multiplex ELISA analysis revealed that YAC128 released higher concentrations of various cytokines than YAC18 microglia (Figure 27), with the major differences seen in TNFα and mKC, a mouse functional homologue of IL-8. This result is consistent with changes seen in human plasma samples, providing further evidence that primary microglia from YAC128 mice are overactive to CSE stimulation. In this case individual cytokine concentrations per culture were not adjusted for the total protein amount and need to be repeated for confirmation.  71  cytokine concentration YAC/ wt  YAC18 1.5  YAC128  1.0  0.5  0.0 TNFa  IL-6  IL-10  mKC  IL-12p70  IL-1b  Figure 27: YAC128 microglia release higher levels of cytokines than YAC18 after 20 hours of CSE exposure. The figure shows mean cytokine concentration in culture media of YAC18 and YAC128 microglia cultures normalized to their wild-type controls. The results were derived from two separate experiments, one wild-type/YAC18 and one wild-type/YAC128.TNFα and mKC, a mouse functional homologue of IL-8 show the largest changes.  3.3.2 In vivo study To further investigate our in vitro findings, I examined the in vivo response of brain microglial to direct endotoxin stimulation and injected 10 ug CSE or equal volumes of PBS bilaterally into the striatum of wild-type mice on a pure FVB/N background in a pilot study. This dose of CSE caused a dramatic increase in the number of activated microglia within the injected hemisphere and also induced large areas of cell loss as shown by IBA-1 staining 7 days post injection in Figure 28. To establish an optimal dose for examining possibly differences in microglia activation between wild-type and YAC128 mice, I injected CSE unilateral at doses of 1 ug, 2.5 ug and 5 ug per 0.5 ul into the striatum of 9 month old wild- mice on a pure FVB/N background. After IHC for IBA-1, F4/80, GFAP, NeuN I found elevated numbers of activated microglia and astrocytes even at the lowest CSE dose when compared to PBS injected animals.  72  Figure 28: Injections of CSE into the striatum of FVB/N mice caused up-regulation of activated microglia. The bright-field picture show brain sections of wild-type FVB/N mice stain for IBA-1 in combination with DAB and counterstained with Cresyl-violet at a magnification of 2.5x (bigger pictures above) and 20x (smaller pictures). Bilateral injections of 1 ul PBS (left, green) or 1 ul CSE at 10 ug/ ul (right, red) into the mouse striatum resulted in dramatic upregulation of IBA-1 signal and areas of possibly cell loss, when compared to PBS injected mice, seven days post injection.  Based on this preliminary data, I injected 9 month old wild-type and YAC128 FVB/N littermates unilaterally with 1 ug/ 0.5 ul CSE and quantified the IBA-1 signal per striatum (Figure 29). The IBA-1 signal of PBS injected animals was significantly lower than that of the CSE injected groups. An almost 4-fold higher IBA-1 signal was detected in YAC128 when compared to wild-type mice, but the difference was not significant due to high variability within the injection groups. At this point it remains unclear whether the increase in IBA-1 signal was due to higher numbers of microglia or the cell size (amoeboid) or both.  73  Figure 29: Striatal injections of CSE caused a 4-fold increase of IBA-1 signal in YAC128 mice compared to wild-type mice. The figure shows the mean integrated optical density (IOD) for IBA-1 fluorescent signal per investigated area derived form 5x pictures using the Metamorph software. 9 month old wild-type and YAC128 FVB/N animals were injected with 0.5 ul PBS or CSE at 1 ug/ ul and dissected seven days after the striatal injection. Perfused brain sections were stained with the IBA-1 antibody. ‘n’ indicates the numbers of animals used.  74  3.4 Discussion In HD patients, selective neurodegeneration in the striatum and cortex occurs early in the disease and preceeds motor, cognitive, and behavioural dysfunction. However, huntingtin (htt) is expressed ubiquitously throught the body and mutant huntingtin has been shown to have toxic effects outside of the CNS including muscle (Farrer 1985) and testis (Van Raamsdonk 2007). We were able to demonstrate that primary cultures of macrophages and monocytes, part of the innate immune system of the periphery, reflect effects seen by microglia when stimulated with CSE. In presymtomatic subjects and various mouse models of HD, immune cells expressing the mutant HD gene were significantly over-active when releasing the cytokine IL-6. It is thought that IL-6 does not cross through the blood-brain barrier, although it cannot be ruled out (Björkqvist 2008). Therefore, it is possible that changes occurring in both compartments, the periphery and the CNS, can impact the severity of HD. Interestingly, these peripheral changes in HD plasma can be measured with a mean of 16 years before predicted onset of motor symptoms (Björkqvist 2008). It was proposed that different combinations of cytokines (early: IL-6, IL-8; late: IL-4, IL-10) could be used as biomarkers of HD progression. Thus, unlike studies using the UHDRS as a therapeutic endpoint which is insensitive during the pre-manifest period, plasma-based biomarker endpoints could enable early therapeutic intervention studies in early and pre-manifest HD. Of course, this will only be possible if treatment outcomes in HD are mirrored in the cytokine response. Using serum samples and blood monocytes would be an easy accessible source when compared to taking cerebrospinal fluid samples. An advantage of using the YAC mouse of HD model for my studies is in the availability of the YAC18 mice, which express human huntingtin and vary from YAC128 mice only in the length of the CAG repeat length and can be used to control for the expression of the human full-length transgene in mice. If we want to use the CSE stimulation system to understand HD pathogenic mechanisms, we first need to fully understand how our mouse model responds in vitro and in vivo. Therefore, I examined pure primary microglia cultures from wild-type, YAC18 and YAC128 neonatal mice. Even though the difference is small, I found a 1.4-fold increase in IL-6 release in YAC128 microglia in response to CSE when compared to wild-type and YAC18, but it was reproduced in three individual YAC128 experiments. The mice used to establish these cultures were one to three days old. This is months prior to any observed HD-phenotype in these mice. In general the YAC128 mouse  75  first shows motor-symptoms at 4 months and is thought to be similar to the phenotype of early HD patients. Given this time-frame, it is striking to see elevated IL-6 levels in these microglia cultures from early post-natal mice. The fact that YAC18 microglia show similar IL-6 levels to wild-type microglia after CSE stimulation provides support for the hypothesis that the expression of the full-length mutant HTT gene causes the elevated IL-6 levels. IL-6, is a pro-inflammatory and anti-inflammatory cytokine, and is produced by a variety of lymphoid and non-lymphoid cells, like macrophages and monocytes, but also T cells, B cells, fibroblasts, as well as several kinds of tumour cells (reviewed by Benveniste 1992 and Heinrich 2003). Furthermore, IL-6 has a wide range of biological activities on various target cells, inducing the differentiation of T cells and immune globulin production of B cells. It is unknown how the mutant HTT gene modifies IL-6 release. In supernatants of stimulated YAC128 microglia other pro-inflammatory cytokines, like mKC and TNFα were also elevated in comparison to YAC18 microglia. We will also need to examine how the cytokine level in serum of wild-type, YAC18 and YAC128 animals changes throughout disease progression to draw parallels to human patients and possible treatment options. In addition to CSE stimulation, I tested whether exposure to 1-2 mM ATP, a range shown to activate rat microglia, can induce IL-6 and TNFα release in primary microglial from our YAC mice. It is thought that ATP can be released into the extracellular space from nerve terminals, activated immune cells, damaged or dying cells. Extracellular ATP is rapidly cleaved by nucleases and cleared (reviewed by Inoue, 2002) and appears to act as a neurotransmitter or a cell-to-cell mediator through binding to P2 purinoceptors. Activated microglia exhibit chemotaxis towards ATP (Honda 2001, Davalos 2005). However, I was not able to induce significant IL-6 or TNFα release in response to ATP stimulation. That may be due technical issues, such as dose and exposure time and needs to be investigated before drawing any definite conclusions. To further evaluate our mouse model, I proceeded to an in vivo model system of acute CNS inflammation via CSE injections. The results presented here are preliminary, and need to be further studied. I was able to induce activated microglia and astrocytes via injections of CSE and the effect seemed to be dose-dependent. At 1 ug/ 0.5 ul injected, I observed a trend towards increased IBA-1 staining in YAC128 animals, but the variability among CSE injected animals was large and the test groups were very small. Furthermore, IHC was not an ideal method to quantify IBA-1 signal as the analysis was very dependent on the quality of the picture. Quantitative real time-PCR (qRT-PCR), western blots, or ELISA assays are more accurate methods and should be pursued. Together with my  76  collaborators, I have shown evidence for an altered peripheral and CNS immune response in Huntington’s disease (HD) patients and HD mouse models. The increased IL-6 plasma levels were seen in HD gene carriers estimated to be on average16 years prior to disease onset. Although we were able to demonstrate over-reactive microglia and macrophages in the CNS and periphery of HD subjects compared to normal individuals, the pathways by which this contributes to neuronal death and disease pathology remain unclear.  77  3.5 References Alagarsamy S, Lonart G, Johnson KM, Regulation of nitric oxide synthase activity in cortical slices by excitatory amino acids and calcium, Journal of Neuroscience Research 38(6): 648-653 (1994) Benveniste EN, Inflammatory cytokines within the central nervous system: sources, function, and mechanism of action, Am J Physiol. 263(1 Pt 1): C1-16 (1992) Björkqvist M, Wild EJ, Thiele J et al, A novel pathogenic pathway of immune activation detectable before clinical onset in Huntington's disease, The Journal of Experimental Medicine 205(8): 1869-1877 (2008) Bradford J, Shin JY, Roberts M et al, Expression of mutant huntingtin in mouse brain astrocytes causes age-dependent neurological symptoms, The Proceedings of the National Academy of Sciences USA 106(52): 22480-22485 (2009) Buraczynska MJ, Van Keuren ML, Buraczynska KM et al, Construction of human embryonic cDNA libraries: HD, PKD1 and BRCA1 are transcribed widely during embryogenesis, Cytogenetics and cell genetics 71(2): 197-202 (1995) Cameron B, Landreth GE, Inflammation, microglia, and alzheimer's disease, Neurobiology of Disease (2009, Epub ahead of print) Chao CC, Hu S, Molitor TW et al, Activated microglia mediate neuronal cell injury via a nitric oxide mechanism, Journal of Immunology 149(8): 2736-2741 (1992) Dalrymple A, Wild EJ, Joubert R et al, Proteomic profiling of plasma in Huntington's disease reveals neuroinflammatory activation and biomarker candidates, Journal of Proteome Research 6(7): 2833-2840 (2007) Davalos D, Grutzendler J, Yang G et al, ATP mediates rapid microglial response to local brain injury in vivo, Nature Neuroscience 8(6): 752-758 (2005) Dure LS 4th, Landwehrmeyer GB, Golden J et al, IT15 gene expression in fetal human brain, Brain Research 659(1-2): 33-41 (1994) Duyao MP, Auerbach AB, Ryan A et al, Inactivation of the mouse Huntington's disease gene homolog Hdh, Science 269(5222): 407-410 (1995) Farrer LA, Meaney FJ, An anthropometric assessment of Huntington's disease patients and families, American Journal of Physical Anthropology 67(3): 185-194 (1985) Hahn JS, Aizenman E, Lipton SA, Central mammalian neurons normally resistant to glutamate toxicity are made sensitive by elevated extracellular Ca2+: toxicity is blocked by the N-methyl-D-aspartate antagonist MK-801, The Proceedings of the National Academy of Sciences USA 85(17): 6556-6560 (1988) Heinrich PC, Behrmann I, Haan S et al, Principles of interleukin (IL)-6-type cytokine signalling and its regulation, Biochemical Journal 374(Pt 1): 1-20 (2003) Hide I, Tanaka M, Inoue A et al, Extracellular ATP triggers tumor necrosis factor-alpha release from rat microglia, Journal of Neurochemistry 75(3): 965-972 (2000)  78  Hodgson JG, Smith DJ, McCutcheon K et al, Human huntingtin derived from YAC transgenes compensates for loss of murine huntingtin by rescue of the embryonic lethal phenotype, Human Molecular Genetics 5(12): 1875-1885 (1996) Honda S, Sasaki Y, Ohsawa K et al, Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors, The Journal of Neuroscience 21(6): 1975-1982 (2001) Inoue K, Microglial activation by purines and pyrimidines, Glia 40(2): 156-163 (2002) Lee JK, Tran T, Tansey MG, Review - Neuroinflammation in Parkinson's disease, Journal of Neuroimmune Pharmacology 4(4): 419-429 (2009) Ma L, Morton AJ, Nicholson LF, Microglia density decreases with age in a mouse model of Huntington's disease, Glia 43(3): 274-280 (Glia) Meffert MK, Premack BA, Schulman H, Nitric oxide stimulates Ca(2+)-independent synaptic vesicle release, Neuron 12(6): 1235-1244 (1994) Montague PR, Gancayco CD, Winn MJ et al, Role of NO production in NMDA receptormediated neurotransmitter release in cerebral cortex, Science 263(5149): 973-977 (1994) Nakajima K, Kohsaka S, Microglia: neuroprotective and neurotrophic cells in the central nervous system, Current Drug Targets - Cardiovascular & Haematological Disorders 4(1): 65-84 (2004) Nasir J, Floresco SB, O'Kusky JR et al, Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes, Cell 81(5): 811-823 (1995) Pavese N, Gerhard A, Tai YF et al, Microglial activation correlates with severity in Huntington disease: a clinical and PET study, Neurology 66(11): 1638-1643 (2006) Pogun S, Dawson V, Kuhar MJ, Nitric oxide inhibits 3H-glutamate transport in synaptosomes, Synapse 18(1): 21-26 (1994) Ryu JK, Choi HB, McLarnon JG, Combined minocycline plus pyruvate treatment enhances effects of each agent to inhibit inflammation, oxidative damage, and neuronal loss in an excitotoxic animal model of Huntington's disease, Neuroscience 141(4): 1835-1848 (2006) Sapp E, Kegel KB, Aronin N et al, Early and progressive accumulation of reactive microglia in the Huntington disease brain, Journal of Neuropathology & Experimental Neurology 60(2): 161-172 (2001) Simmons DA, Casale M, Alcon B et al, Ferritin accumulation in dystrophic microglia is an early event in the development of Huntington's disease, Glia 55(10): 1074-1084 (2007) Singhrao SK, Neal JW, Morgan BP et al, Increased complement biosynthesis by microglia and complement activation on neurons in Huntington's disease, Experimental Neurology 159(2): 362-376 (1999) Stack EC, Smith KM, Ryu H et al, Combination therapy using minocycline and coenzyme Q10 in R6/2 transgenic Huntington's disease mice, Biochimica et Biophysica Acta 1762(3): 373-380 (2006)  79  Stichel CC, Müller HW, Relationship between injury-induced astrogliosis, laminin expression and axonal sprouting in the adult rat brain, Journal of Neurocytology 23(10): 615-630 (1994) Tai YF, Pavese N, Gerhard A et al, Microglial activation in presymptomatic Huntington's disease gene carriers, Brain 130(Pt 7): 1759-1766 (2007) Töpper R, Gehrmann J, Schwarz M et al, Remote microglial activation in the quinolinic acid model of Huntington's disease, Expimental Neurology 123(2): 271-283 (1993) Trotti D, Rossi D, Gjesdal O et al, Peroxynitrite inhibits glutamate transporter subtypes, The Journal of Biological Chemistry 271(11): 5976-5979 (1996) Trottier Y, Devys D, Imbert G et al, Cellular localization of the Huntington's disease protein and discrimination of the normal and mutated form, Nature Genetics 10(1): 104-110 (1995) van der Burg JM, Björkqvist M, Brundin P, Beyond the brain: widespread pathology in Huntington's disease, Lancet Neurology 8(8): 765-774 (2009) Zeitlin S, Liu JP, Chapman DL et al, Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue, Nature Genetics 11(2): 155163 (1995) Zeron MM, Chen N, Moshaver A et al, Mutant huntingtin enhances excitotoxic cell death, Molecular and Cellular Neuroscience 17(1): 41-53 (2001) Zeron MM, Hansson O, Chen N et al, Increased sensitivity to N-methyl-D-aspartate receptor-mediated excitotoxicity in a mouse model of Huntington's disease, Neuron 33(6): 849-860 (2002)  80  4. Conclusion 4.1 Summary The ability to model Huntington disease (HD) in animals has provided scientists new insights into disease pathogenesis. Simplified in vitro models are also important as tools to dissect specific pathogenic pathways, the cellular interactions in these pathways and to identify and test novel therapeutic targets that offer the greatest hope of a cure. An ideal system would be genetically faithful to the human condition, and reflect the characteristics of the most affected cell types. While mouse embryonic stem (ES) cells most often are used to generate gene targeted and transgenic mice they hold enormous potential as an in vitro system to evaluate early normal development, to model abnormal development and disease processes, to study potential cell replacement therapies, and to perform compoundscreening and toxicity assays. The unique characteristics of ES cells allow, under defined conditions, prolonged in vitro culture without immortalization procedures and ultimately provide the potential for derivation of any somatic cell type from one of the three embryonic germlayers. To utilize these potentials, I therefore aimed to investigate whether embryonic stem (ES) cells derived from wild-type, YAC18 and YAC128 mice can be used to develop a simplified model of Huntington disease (HD) that could be useful for studies of disease pathogenesis and compound screening. To answer the research question, I set the milestones for this project as followed i) to establish wild-type, YAC18 and YAC128 ES cell lines from mice on the 129 and 129/FVB F1 hybrid backgrounds, ii) to characterized their stem cell properties, and iii) to evaluate neurons and macrophages derived from selected cell lines. 17 novel ES cell lines were generated, 7 wild-type, 2 YAC18 and 8 YAC128 ES cell lines. To my knowledge this has been the first time that ES cell lines have been generated from these mice. These novel cell lines exhibited typical mouse ES cell morphology and expressed alkaline phosphatase, Oct4 and Nanog, well-established markers of undifferentiated ES cells. At this point, however, it is unknown whether there are differences in the level of expression of these markers between our novel ES cell lines or within one cell line after prolonged sub-culture as I have not performed any quantitative  81  analysis. Furthermore, all novel ES cells were able to some degree to differentiate into cells with a defined neuronal profile, even after prolonged sub-culture. Medium spiny neurons (MSNs) isolated from YAC128 mice have previously been shown to undergo increased NMDA-induced cell death compared to wild-type and YAC18 MSNs, and that this sensitivity occurs prior to obvious phenotypic changes (Shehadeh 2006, Graham 2009). Due to the similarity of in vitro differentiated ES cells to primary cell cultures as experimental system, I sought to test if our newly derived ES cells are able to reproduce the HD phenotype observed in these cultures. In a pilot experiment, neurons derived from 3 wild-type and 3 YAC128 ES cell lines on the 129 mouse background were exposed to NMDA induced excitotoxicity. However, there was no difference in the number of TUNEL-labeled nuclei observed between the un-stimulated and NMDA stimulated cultures. Under the chosen experimental conditions, I was not able to induce significant excitotoxic cell death in any of the ES-cell derived neuronal cultures and thus it remains unanswered whether or not YAC128 ES-derived neurons are more sensitive to NMDA. Despite progressive neuronal cell death, various cells of the innate immune system have been suggested to play a pathogenic role not only in HD (Vonsattle 1985, Sapp 2001, Pavese 2006) but also in other neurodegenerative diseases such as Alzheimer disease (reviewed by Cameron et al, 2009) and Parkinson disease (reviewed by Lee et al, 2009; Saijo 2009). During the progressive course of HD, the number of activated microglia and astrocytes increases in the brains of affected individuals, and recent findings have linked activation of these cells to HD pathogenesis (Sapp 2001, Pavese 2006). Little is currently known about CNS immune activity in mouse models of HD. Using 3 wild-type and 3 YAC128 ES cell lines on the 129 mouse background, I generated cultures of ES cellderived macrophages. All cells displayed expression of the macrophages marker F4/80 (observed via ICC) and appeared to be functional macrophages as they responded to endotoxin stimulation with the release of IL-6 and TNFα. Macrophages derived from one of the YAC128 ES cell lines (16.2Y128-129) exhibited a 2-fold increase in TNFα and IL-6 release when compared to cells of the wild-type cell line with the highest cytokine release (21.2wt128-129). However, the 2 remaining YAC128-derived cultures as well as the other 2 wild-type cell lines responded with less cytokine release than the above described cell lines. Due to the high variability in cytokine release, no significant difference or trends could be detected between wild-type and YAC128 derived cells. Interestingly, the wild-type ES cell lines 21.1wt128-129 and 21.2wt128-129, originated from one blastocyst, but once differentiated into macrophages exhibited very different IL-6  82  and TNFα levels in response to endotoxin stimulation. Possible explanations for this result could be related to technical issues involved in the specific cultures (i.e. incomplete differentiation, contamination), but could also reflect genetic issues such as chromosomal alterations in the different cell lines or line-specific mutations resulting in up- or downregulation of modifying genes. This project is the first attempt to investigate ES-derived macrophages from an HD mouse model. The results obtained from these cultures are preliminary and serve to highlight the fact that more detailed information about the developmental potential (i.e. quantification of transcripts regulating pluripotency) and the genetic status (i.e. karyotype analysis) of these ES cell lines is necessary. However, the results from these initial experiments sparked our interest to investigate potential neuroinflammatory changes in the YAC128 model of HD. Unlike the results attained from ES-derived macrophages, primary alveolar macrophages and microglia cultures isolated from YAC128 mice produced significantly more IL-6 than their wild-type and YAC18 counterparts. In comparison, at the same endotoxin concentration, YAC18 primary macrophages and microglia, which express full-length human wild-type huntingtin but differ from YAC128 cells in the number of CAG repeats, released similar amounts of IL-6 compared to wild-type cells. This outcome suggests that the over-activity in the YAC128 is caused by the mutant HTT transgene. The difference in IL-6 levels observed in wild-type and YAC18 macrophages in comparison to YAC128 after endotoxin stimulation is modest; however it was repeatedly detected in our primary in vitro cultures. Our observation is supported by studies of primary microglia from R6/2 mice and human monocytes isolated from premanifest HD subjects, which are similarly hyper-active post endotoxin stimulation producing significantly more IL6 than the corresponding wild-type cells (Björkqvist 2008). The isolation and evaluation of primary macrophages and microglia from wild-type, YAC18 and YAC128 mice, initially undertaken to clarify the results obtained from ESderived macrophages, has granted us insight into a novel potentially pathogenic pathway in HD. While this finding is robust, the cellular mechanisms by which mutant huntingtin modulates the IL-6 response of macrophages to endotoxin stimulation however remains unclear.  83  4.2 Advantages and disadvantages of the chosen models To screen potential treatments in ES-cell based assays offers significant advantages. First, the cell lines I derived are from a well-established and carefully characterized mouse model of HD. They express full-length wild-type or mutant human HTT and can be differentiated into cell types known to be affected in HD. Because of the direct biological relevance, therapeutic compounds indentified to be effective in these novel ES cell lines should have a high likelihood of being effective in YAC128 mice and ultimately in humans when compared to other cellular assays (i.e. immortalized cell lines). Secondly, reproducing test results employing primary cells isolated separately each time can be challenging. Using ES cells, which have the ability to self-renew and can therefore be maintained in culture for long periods of time, can eliminate variability resultant from separate sets of primary cells. Thirdly, each of these novel cell lines is genetically unique and may model different aspects of HD pathogenesis. In addition, they could be further modified, through homologous recombination, to enable one to evaluate the efficiency by which a particular cell line differentiates or to easily quantify the effect of drugs. For example, the development of an ES cell line engineered to express a reporter protein (i.e. green fluorescent protein - GFP) under a cell-type specific promoter (i.e. DARPP32 for MSNs) could facilitate the establishment of efficient differentiation protocols. Furthermore, an ES cell lines modified to express GFP under for example, the apoptosis-inducing factor (AIF2) promoter - associated with neuronal cell death (Hangen 2010) – could be utilized in compound screens and potentially identify molecules that promote the survival of ES derived neurons. Lastly, testing potential treatments in vitro requires significantly less time and test compound than in vivo assays, which in turn leads to earlier prioritization between drug leads. Utilizing the full potential of the ES cell lines I have generated will be highly dependent on advances in our knowledge about the cellular and molecular conditions that regulate self-renewal and lineage specification of ES cells in culture. Furthermore, these cell lines ideally need to be chromosomally stable since karyotypic changes can alter the phenotypic outcome and therefore represent a pitfall for future applications. It is known that ES cells often lose chromosome integrity during long periods in culture leading to an accumulation of mutations. Sugawara et al (2006) examined 540 mouse ES cell lines from institutes in  84  Japan of which 27.8 % showed abnormal chromosomal numbers and only 66.5 % exhibited normal chromosomal numbers. Moreover, when karyotyping 88 of these cell lines the scientists identified 53 cell lines (60.2 %) with normal diploid karyotypes (52 lines XY, 1 line XX). However, 35 ES cell lines appeared to have abnormal karyotypes, including trisomy 8 (88.6 %), trisomy 11 (17.1 %) and loss of one sex chromosome (25.7 %). Interestingly, trisomy 8 in mouse ES cell lines has been shown to result in a selective growth advantage of these cells which subsequently leads to depletion and loss of the normal ES cell population during sub-culturing (Liu 1997). It was also noted that ES cells with trisomy 8 appear to have a lower probability of contributing to the germ line in chimeric mice (Liu 1997). Recently, researchers have investigated additional genomic alterations such as copy number variations, which contribute to genetic variability in ES cell lines and consequently in transgenic animals (Liang 2008). Some evidence suggests that these genetic changes predominantly arise by adaptation to culture conditions and are influenced by culture duration. Differential vulnerability of ES cell lines to aneuplody due to inherent stem cell properties has been proposed (Catalina 2008). Differences in the developmental potential of ES cell lines will result in unequal differentiation and subsequently lead to high variability when assessing the derived cells in future studies. I believe that the systematic evaluation of chromosomal alterations as well as expression profiles for key regulators of pluripotency (i.e. Oct4, Nanog, Sox2) is necessary before using our novel mouse ES cell lines to study the influence of the mutant huntingtin transgene  and  potential  consequential  phenotypes  in  ES-derived  neurons  and  macrophages. In order to utilize wild-type, YAC18 and YAC128 ES cells to screen compounds, we need to be able to reliably measure phenotypic differences. The fact that I could not identify changes between wild-type and YAC128 ES-derived neurons and macrophages resultant of the mutant transgene represents a major setback and needs to addressed. As the YAC mouse model of HD already exists, the ES cell lines I have generated will be used mainly to enable rapid in vitro studies. However, if any chromosomal alterations leading for example to a more severe HD-like phenotype would be identified in one of these cell lines the possibility remains, in theory, to establish a novel transgenic mouse line. Despite the unclear genetic status of our ES cell lines, another potential disadvantage of this particular in vitro system could be the chosen mouse model itself. The YAC mouse model of HD expresses two endogenous copies of mouse huntingtin together with the human wild-type (18 polyglutamine repeats) or mutant (128 polyglutamine repeats)  85  transgene. Interestingly, a protective function of wild-type huntingtin was suggested (Leavitt 2001, Ho 2001) which in turn could potentially reduce a toxic effect of the mutant YAC128 transgene in the ES cells and their derivatives. A decline in motor coordination, hypoactivity, testicular degeneration and impaired lifespan was shown in YAC128 mice that do not express wild-type huntingtin (YAC128-/-) but express the same amount of mutant HTT as normal YAC128 mice (Van Raamsdonk 2005). Under the given circumstances that I was not able to identify a HD-like phenotype between wild-type and YAC128 ES-derived neurons and macrophages, ES cells generated from YAC128-/- mice may exhibit a more sever and potentially detectable phenotype. Although, the response of primary macrophages and microglia to CSE stimulation resulted only in a subtle increase in IL-6 production in YAC128 over wild-type cells, this change was documented repeatedly and has been confirmed in primary cultures of R6/2 microglia and human monocytes. A reliable phenotype and the fact that chromosomal changes discussed above are less likely to emerge in short term cultures of primary cells, represents the strengths of this test system. It appears that cells isolated from YAC18 mice behave similar to wild-type primary macrophages and microglia in response to CSE, providing strong evidence that the mutant HTT transgene itself influences inflammatory reactions in YAC128 mice. Moreover, in the case of YAC128 primary microglia these alterations can be detected in pure cultures, separate from dying neurons, and long before obvious motor symptoms are present, indicating that they are not secondary to neuronal degeneration. It is advantageous that YAC128 macrophages mirrored the actions of microglia, holding the promise to measure the identified markers such as IL-6 in an easier accessible environment than the CNS. The compound screening process would be time and cost efficient due to the shorter periods for establishing these cultures and assessing drug performance. Furthermore, less amount of the test substances are required. So far, I have been able to measure differences in vitro, however I have no sufficient information on how macrophages or microglia react to CSE stimulation in vivo. The in vitro culture of pure microglia cells has a disadvantage of not investigating interactions with other cells of the CNS. It is unclear, if and how microglia expressing mutant huntingtin influence astrocytes and what consequence this may have for striatal neurons. Furthermore, how relevant is endotoxin stimulation to the HD disease process itself? Lipopolysaccharide (LPS), the toxic component of control standard endotoxin, is part of the outer membrane of Gram-negative bacteria. By using LPS initially I was able to un-mask the hyper-activity of YAC128 macrophages and microglia. However, bacterial infection of  86  the brain does not cause HD and thus LPS stimulation may not be ideal to identify the underlying biochemical mechanisms. Extracellular nucleotides, such as ATP/ADP (Honda 2001, Davalos 2005) and UDP (Koizumi 2007) released from injured or dying neurons have been demonstrated previously in vitro and in vivo to trigger microglia chemotaxis and phagocytosis respectively. Therefore, stimulation by these molecules may be more true to the HD condition. I have started to investigate ATP stimulation in microglia cultures, however, was not able to measure corresponding IL-6 or TNFα release most likely due to the experimental set up. I believe it is necessary to perform experiments concerning the time course and dose-response to ATP/ADP and UDP prior to further investigations of these stimulants in microglia.  4.3 Future directions and significance My hypothesis, that novel ES cell lines from YAC128 mice will be useful as tools for compound screening, remains incompletely assessed. Although, ES cell lines have been generated and characterized, I was not able to demonstrate that any cell types derived from these cultures exhibited a differential response that could be determined to be a result of the mutant HTT transgene alone. Considering all of my results attained thus far, I do not believe that these cell lines model HD pathogenesis accurately and more detailed research on their karyotype and developmental potential as well as improved and more specific differentiation protocols are a necessity before addressing the hypothesis specifically. Recently, a number of human HD ES cell lines have been developed and are currently under investigation (Verlinsky 2005, Mateizel 2006, Niclis 2009). Nonetheless, the novel mouse ES cell lines I have generated may still be valuable to further characterize underlying pathogenesis in the YAC mouse model of HD. Furthermore, each of them is genetically unique and may model different aspects of HD pathogenesis. All novel ES cell lines generated will be made available to HD community. At this point of the study, karyotype analysis of selected cell lines is actively being pursued by GlobalStem through CHDI. Once the optimal genetically stable cell lines for each genotype are identified from the entire collection I generated, GlobalStem intents to derive neural stem (NS) cells. Like ES cells, NS cells can be cultured in vitro indefinitely under defined conditions. The advantage of using NS cells over ES cells however is their potential to derive pure neuronal cultures much more efficiently (Conti 2005, Spiliotopoulos 2009). I was able to derived cells with neuronal properties from our novel ES cell lines 87  using the Bain et al protocol (1995). However these cultures also included neural precursors and non-neural cells and were not ideal for functional studies. To reliably perform compound screening assays large numbers of homogenous neuronal cultures for each genotype are necessary and are difficult to generate using pluripotent ES cells. Utilizing multipotent neural stem cells will be a faster, more efficient procedure to establish pure neuronal cultures. As for the investigation of inflammatory changes in YAC128 mice, primary macrophages and pure microglia cultures were established and characterized. The IL-6 response to CSE treatment is significantly different in YAC128 primary cultures and other cytokines seem to be up-regulated as well. Although, these results have yet to be confirmed in vivo, they suggest that altered responses of the innate immune system are present in the CNS and the periphery of YAC128 mice. Further investigation of these responses in mice is required to evaluate the role of neuroinflammation in HD in vivo. Together with my collaborators, we have used this model to identify a novel pathogenic aspect of mutant huntingtin that is observable long before the classical diagnosis of HD symptoms in mice and human. The identification of IL-6 up-regulation in YAC128 microglial and macrophage cultures holds promise for the development of a potential biochemical biomarker in plasma or cerebrospinal fluid in HD patients. So far, clinicians use the Unified Huntington's Disease Rating Scale (UHDRS) to assess clinical performance and functional capacity of HD patients. Although the UHDRS is internationally accepted and used as the ‘standard’ for the clinical assessment of HD patients, it has limitations. One such limitation is that, as a clinician rated scale, it is a subjective measure of the progression of HD pathology. By examining four domains - motor function, cognitive function, behavioral abnormalities, and functional capacity – the UHDRS allows clinical researchers to measure disease progression in symptomatic (manifest) HD (Huntington Study Group 1996). However, there is now a large body of evidence that suggests that at clinical diagnosis significant neurodegeneration has already occurred. Therefore, there is a need for easily accessible and objective biochemical biomarkers that can predict the true onset of neurodegeneration in HD and enable therapeutic intervention before actual harm is done. This is the focus of recent multi-center observational studies such as Predict-HD (Paulsen 2008) and TRACK-HD (Tabrizi 2009). Future directions of this project will include the quantification of microglia specific transcripts through quantitative RT-PCR, as well as protein studies such as western blotting of mouse brain tissue after CSE injection for wild-type, YAC18 and YAC128 mice.  88  Replication of the transgene-depended cytokine up-regulation first identified in in vitro experiments needs to be performed in vivo. To study the interactions of HD microglia with other cell types, astrocytes and neurons co-cultures need to be investigated for cytokine release after CSE stimulation. Furthermore, experiments examining ATP/ADP induced chemotaxis and phagocytosis possibly stimulated via UTP should be undertaken to identify potential pathogenic pathways and the mechanism by which mutant huntingtin influences them. As the population is aging the number of people affected by neurodegenerative brain diseases such as Alzheimer, Parkinson, and Huntington disease is rising. In 2004, the world health organization (WHO) ranked Alzheimer disease and other dementias as the 6th leading cause of death in high-income countries. Despite great effort and recent advances in our understanding of the pathogenesis of these devastating disorders, there are currently no effective early prevention/treatment strategies in place. In HD, profound disability and consequently the loss of independence caused by cognitive decline and motor dysfunction affects people in the prime of life. These changes often go along with depression, psychosis and suicide. I believe that the work performed in my thesis will eventually help to develop new treatments that prevent or slow down the progression of HD, and translate into improved quality of life and greater self-sufficiency.  89  4.4 References Björkqvist M, Wild EJ, Thiele J et al, A novel pathogenic pathway of immune activation detectable before clinical onset in Huntington's disease, The Journal of Experimental Medicine 205(8): 1869-1877 (2008) Cameron B, Landreth GE, Inflammation, microglia, and alzheimer's disease, Neurobiology of Disease (2009, Epub ahead of print) Catalina P, Montes R, Ligero G et al, Human ESCs predisposition to karyotypic instability: Is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties?, Molecular Cancer 7:76 (2008) Conti L, Pollard SM, Gorba T et al, Niche-independent symmetrical self-renewal of a mammalian tissue stem cell, Public Library of Science Biology 3(9):e283 (2005) Davalos D, Grutzendler J, Yang G et al, ATP mediates rapid microglial response to local brain injury in vivo, Nature Neuroscience 8(6): 752-758 (2005) Graham RK, Pouladi MA, Joshi P et al, Differential susceptibility to excitotoxic stress in YAC128 mouse models of Huntington disease between initiation and progression of disease, The Journal of Neuroscience 29(7): 2193-2204 (2009) Hangen E, De Zio D, Bordi M et al, A brain-specific isoform of mitochondrial apoptosisinducing factor: AIF2, Cell Death and Differentiation (2010, Epub ahead of print) Ho LW, Brown R, Maxwell M et al, Wild type Huntingtin reduces the cellular toxicity of mutant Huntingtin in mammalian cell models of Huntington's disease. Journal of Medical Genetics 38(7): 450-452 (2001) Honda S, Sasaki Y, Ohsawa K et al, Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors, The Journal of Neuroscience 21(6): 1975-1982 (2001) Huntington Study Group, Unified Huntington’s Disease Rating Scale: Reliability and Consistency, Movement Disorders 11(2): 136-142 (1996) Koizumi S, Shigemoto-Mogami Y, Nasu-Tada K et al, UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis, Nature 446(7139): 1091-1095 (2007) Leavitt BR, Guttman JA, Hodgson JG et al, Wild-type huntingtin reduces the cellular toxicity of mutant huntingtin in vivo, The American Journal of Human Genetics 68(2): 313324 (2001) Lee JK, Tran T, Tansey MG, Review - Neuroinflammation in Parkinson's disease, Journal of Neuroimmune Pharmacology 4(4): 419-429 (2009) Liang Q, Conte N, Skarnes WC et al, Extensive genomic copy number variation in embryonic stem cells, The Proceedings of the National Academy of Sciences USA 105(45): 17453–17456 (2008) Liu X, Wu H, Loring J et al, Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission, Developmental Dynamics 209(1): 85-91 (1997) 90  Mateizel I, De Temmerman N, Ullmann U et al, Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders, Human Reproduction 21(2): 503-511 (2006) Paulsen JS, Langbehn DR, Stout JC et al, Detection of Huntington’s disease decades before diagnosis: the Predict-HD study, Journal of Neurology, Neurosurgery & Psychiatry 79(8): 874-880 (2008) Pavese N, Gerhard A, Tai YF et al, Microglial activation correlates with severity in Huntington disease: a clinical and PET study, Neurology 66(11): 1638-1643 (2006) Saijo K, Winner B, Carson CT et al, A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death, Cell 137(1): 47-59 (2009) Sapp E, Kegel KB, Aronin N et al, Early and progressive accumulation of reactive microglia in the Huntington disease brain, Journal of Neuropathology & Experimental Neurology 60(2): 161-172 (2001) Shehadeh J, Fernandes HB, Zeron Mullins MM et al, Striatal neuronal apoptosis is preferentially enhanced by NMDA receptor activation in YAC transgenic mouse model of Huntington disease, Neurobiology of Disease 21(2): 392-403 (2006) Spiliotopoulos D, Goffredo D, Conti L et al, An optimized experimental strategy for efficient conversion of embryonic stem (ES)-derived mouse neural stem (NS) cells into a nearly homogeneous mature neuronal population, Neurobiology of Disease 34(2): 320-331 (2009) Sugawara A, Goto K, Sotomaru Y et al,Current status of chromosomal abnormalities in mouse embryonic stem cell lines used in Japan, Comparative Medicine 56(1): 31-34 (2006) Tabrizi SJ, Langbehn DR, Leavitt BR et al, Biological and clinical manifestations of Huntington’s disease in the longitudinal TRACK-HD study: cross-sectional analysis of baseline data, Lancet Neurology 8(9): 791-801 (2009) Van Raamsdonk JM, Pearson J, Rogers DA et al, Loss of wild-type huntingtin influences motor dysfunction and survival in the YAC128 mouse model of Huntington disease, Human Molecular Genetics 14(10): 1379-1392 (2005) Verlinsky Y, Strelchenko N, Kukharenko V et al, Human embryonic stem cell lines with genetic disorders, Reproductive Biomedicine Online 10(1): 105-110 (2005) Vonsattel JP, Myers RH, Stevens TJ et al, Neuropathological classification of Huntington's disease, Journal of Neuropathology & Experimental Neurology 44(6): 559-577 (1985)  91  Appendix  Figure 30: Novel ES cell lines but not MEFs or whole brain samples express known ES cell markers shown by RT-PCR. Panel I shows the expression of the Pou5f1 gene (Oct4 protein) in ES cell lines (lanes 1-5). In comparison, Pou5f1 is not expressed in MEF feeder layers (lane 7) or whole mouse brain (lane 8). Panel II shows the expression of the Nanog gene. Actin is expressed in all samples expect in the ‘no-RT’ control (lane 6) as shown in panel III. Samples were loaded as followed: (1) mEMS159 (2) 14.2Y128-129 (3) 15.2wt-129 (4) 16.2Y128-129 (5) 21.1wt-129 (6) ‘no RT’ control (7) MEF feeder layers (8) whole mouse brain. (L) MassRulerTM DNA ladder, Low Range  Figure 31: A representative karyotype of an ES cell line after prolonged culture. Spectral karyotyping (SKY) analysis of the control ES cell line mEMS159 performed in the Mai laboratory (University of Manitoba) revealed a tendency towards trisomy 8 and trisomy 11, as well as high prevalence of missing sex chromosomes. 92  

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