May 18, 2018 Mary Gates Hall

POSTER SESSION 1 SESSION 1E MGH 206, Easel 166 11:00 AM to 1:00 PM FROM VIRAL PATHOGENESIS TO Identification of a Causal for a Novel Form of GENETIC DISEASESTO BUILDINGA Spinocerebellar Ataxia BETTER KIDNEY Olga Sarby Cherepakhin, Senior, Biology (Molecular, Cellular & Developmental), Anthropology: Medical Anth & Session Moderator: Michael Lagunoff, Microbiology Global Hlth MGH 231 Mary Gates Scholar 12:30 PM to 2:15 PM Mentor: Dong-Hui Chen, Neurology * Note: Titles in order of presentation.

Spinocerebellar Ataxia (SCA) is a group of inherited autoso- Gait Evaluation of mdx4cv Mice Expressing Micro mal dominant disorders characterized by the loss of coordi- Transgene nation in the limbs and atrophy of the cerebellum. SCA pro- Indu Tejasa Vanteru, Senior, Biology (Molecular, Cellular & gresses gradually and has a diverse presentation of symptoms Developmental) from debilitating to mild amongst its different forms. There Mary Gates Scholar, UW Honors Program are many genetic causes for SCA, however, they remain un- Mentor: Jeffrey S Chamberlain, Neurology known in many cases. Although there is no treatment, recent Mentor: Katrin Hollinger, Neurology scientific advances have illuminated mechanisms of patho- genesis and potential gene therapies to help patients with Duchenne muscular dystrophy (DMD) is an X-linked reces- SCA. My project in the Raskind Lab contributes to this re- sive genetic disorder, marked by progressive muscle degener- search by attempting to identify the causal gene for a family ation due to the absence or impairment of the dystrophin pro- with a novel form of autosomal dominant SCA. Whole exome tein. Our laboratory has been developing methods for gene sequencing is currently being conducted on the DNA of three therapy of DMD. The dystrophin gene is the largest known affected members of the family. From the exome sequenc- gene, which complicates gene therapy. We have shown that ing, we will receive all the genetic differences in the - gene delivery vectors based on adeno-associated virus (AAV) coding region from a reference sequence in any of these three can be used to deliver new body wide. However, AAV subjects; we will first search for variants with genes known has a ˜5 kb carrying capacity, so we have been developing for SCA. I will then begin the process of choosing candidate miniaturized . These smaller, highly functional variants for further analysis. I am choosing them by first fil- dystrophins, which we named micro-dystrophin (µdys) were 4 tering for variants that are heterozygous in all three exomes tested in a transgenic mdx cvmouse model for DMD. Further- and have a prevalence of less than 0.01% in genetic databases more, we sought to develop a non-invasive method to eval- and then prioritizing the remaining variants based on the type uate treatment efficiency of µdys. In this project, the gait 4 of , model-predicted effect of the variant, and rele- of mdx cv, wild type (WT) and transgenic mice were ob- vance of the gene function to SCA. For each chosen candidate served to deduce potential benefits of the µdys transgene. We variant, I will amplify and sequence the DNA from each fam- hypothesized that µdys treatment will enable the gait of the ily member to determine whether it co-segregates by being transgenic mice to resemble that of WT animals as opposed 4 present in all those who are affected and absent from those to mdx cv. To test this, gait was assessed at three time points: who are not. Once a co-segregating variant is identified, other 1.5, 3 and 6 months of age using the video-based Noldus Cat- studies will be conducted to support its causality. My research Walk XT. Our results show that the length of a single stride will contribute to our understanding of SCA and neurodegen- of the transgenic mice hind paws increased over time com- 4 erative disorders. pared to mdx cv. The normal walking pattern of mice con- sists of placing diagonal paws on the surface, one front and one hind from opposite sides. In the case of mdx4cv, more paws were placed down for majority of the walk, as a com-

Undergraduate Research Program 1 www.uw.edu/undergradresearch pensatory mechanism for muscle weakness. The transgenic of cortical microglia. This raises the possibility that type 1 mice were found to walk with a diagonal pattern more fre- IFN signaling may be required for optimal microglial survival quently, like WT mice. Similarities in the gait of transgenic following IPC. More studies will be required to confirm the and WT mice help us conclude the µdys treatment was suc- above findings and explore possible mechanisms. cessfully able to mitigate the effects of DMD as seen by the gait analysis. POSTER SESSION 2 MGH 241, Easel 136 SESSION 1I 1:00 PM to 2:30 PM

ULTIDISCIPLINARY PPROACHES Isolating Murine Microglia Progenitors and Identifying M A Senescence Marker Expression in vitro TO MEDICAL RESEARCH Lewis Wenbo Yin Luo, Senior, Business Administration Session Moderator: Gwenn Garden, Neurology (Finance), Neurobiology MGH 248 Mary Gates Scholar 12:30 PM to 2:15 PM Mentor: Gwenn Garden, Neurology * Note: Titles in order of presentation. Mentor: Katherine Prater, Neurology Microglia are the resident immune cells of the CNS and are Type 1 Interferon Signaling Modulates Microglial hypothesized to influence aging in the . Like somatic Response to Ischemic Preconditioning cells, microglia can be replaced by self-renewal. Recently, Jasmine Shen, Senior, Neurobiology some studies have suggested that new microglia derive from Mary Gates Scholar, UW Honors Program asymmetric cell division of a progenitor population. Mi- Mentor: Jonathan Weinstein, Neurology croglia progenitor cells have been difficult to study due to a Stroke is the leading cause of long-term disability in the lack of specific molecular markers of this population. How- USA. Ischemic preconditioning (IPC) is a neuroprotective ever, the Garden lab has recently identified novel candidate phenomenon wherein a brief ischemic exposure induces ro- markers. We hypothesize that in neurodegenerative disorders bust neuroprotection against subsequent prolonged ischemia. associated with advanced age, microglia progenitor senes- The Weinstein laboratory has previously demonstrated: (i) a cence may contribute to disease pathology. To efficiently robust type 1 interferon response in cortical microglia fol- study the senescence of microglia progenitors, we turned to lowing IPC, (ii) type I interferon signaling in microglia is neonatal mixed glia cultures, in which the presence of mi- required for IPC-mediated protection and (iii) IPC induces croglia progenitors has long been inferred. In these cultures, a robust increase in the number of microglia in precondi- microglia are harvested from cells floating above a mono- tioned cortex. An initial component of my project was to layer culture of mixed neonatal glial cells. The size of each validate this microglial response by first staining for Iba1 (a microglia harvest generally decreases with successive har- microglial marker) alone and then double staining for Iba1 vests. This suggests that microglia progenitors in the attached and proliferation marker BrdU. We used immunofluorescent monolayer may become senescent after multiple rounds of microscopy (IFM) following by quantitative stereology (QS). the cell cycle, leading to stagnation in the generation of new Our hypothesis was that type I interferon signaling is neces- floating microglia. We evaluated microglia progenitor senes- sary for IPC-induced microglial proliferation. We carried out cence in neonatal mixed-glia cultures by labeling with BrdU, IPC on WT and type 1 interferon receptor deficient (IFNAR- a thymidine analog taken up by proliferating cells and re- /-) mice and quantified cortical microglial number and prolif- maining in their daughters. Microglia harvested from these eration as above. Preliminary results were: (i) in na¨ıve WT, cultures weekly were assessed for BrdU incorporation using 0.643 ± 0.038 (mean ± S.D), Iba1+ cells per position, (ii) in flow cytometry and immunofluorescent microscopy. We co- preconditioned WT, 1.113 ± 0.1385, (iii) in na¨ıve IFNAR1- labeled floating microglia and dissociated monolayer mixed /-, 0.751 ± 0.058 and in preconditioned IFNAR1-/-, 0.903 glia cultures with antibodies directed against a progenitor ± 0.125. Two way ANOVA revealed a significant difference marker (CD133), a microglia marker (Iba1), and BrdU. The between na¨ıve and IPC-induced cortical microglia numbers attached mixed-glia cell layer was also labeled for SA-ß-Gal, [F(1,14)=9.62, p=0.0078], but no significant effect of geno- an indicator of cellular senescence. Progenitor senescence type [F(1,14)=0.258, p=0.619]. IPC induced increases in the will be detected by a decrease in CD133/BrdU-positive cells number of cortical Iba1+/BrdU+ proliferating microglia in and an increase in CD133/ SA-ß-Gal positive cells. both IFNAR1-/- (0.451 ± 0.107) and WT (0.205 ± 0.069) mice. These results suggest a complex picture in which defi- ciency in type 1 IFN signaling may not influence IPC-induced microglial proliferation but does attenuate the overall number

2 Linking Inflammatory microRNAs to Behavioral Deficits POSTER SESSION 2 in a Mouse Model of Alzheimer’s Disease MGH 241, Easel 144 Rachael Annie Hu, Senior, Biology (Physiology) Mary Gates Scholar, Undergraduate Research 1:00 PM to 2:30 PM Conference Travel Awardee Presenilin 2 Mutation Impacts on Phagocytosis in Mentor: Gwenn Garden, Neurology Alzheimer’s Disease Mentor: Macarena Aloi, Pathology Leah Ariel Osnis, Senior, Biochemistry Microglia are the innate immune cells of the central ner- UW Honors Program vous system that exhibit a sustained pro-inflammatory re- Mentor: Suman Jayadev, Neurology sponse in the Alzheimer’s disease (AD) brain. Regulation in the Presenilin 2 gene, (PSEN2), cause early of inflammatory in microglia by microRNA onset familial Alzheimer’s disease (FAD). PSEN2 encodes miR-155 modulates transition between distinct phases of the the presenilin 2 protein which forms the catalytic subunit of inflammatory response. Though altered expression profiles the gamma-secretase complex, responsible for cleaving amy- of miR-155 is seen in other neurodegenerative disorders, the loid precursor protein into fragments. One of the resulting precise role of this microRNA in modulating inflammation fragments is Aβ1 − 42, a major component of brain amy- and downstream behavioral deficits in mouse models of AD loid plaques and a clinical hallmark of Alzheimer’s disease. remains unknown. We hypothesize that microglia specific Microglia are the resident innate immune cells in the brain deletion of miR-155 will alter neuroinflammation and behav- clearing the brain of Aßplaque and debris through phagocy- ioral phenotypes in transgenic mice expressing human mu- tosis. Without presenilin 2 protein present, levels of secreted tant amyloid precursor protein and presenilin 1 (APP/PS1), cytokines increase implying a higher immune response, and an AD model that exhibits Aβ pathology and memory im- recent data suggest that hyper-activation of an inflammation pairments. We generated trigenic (Cx3cr1-Cre+/−/Floxed- state can contribute to development of Alzheimer’s disease. miR155+/+/APP/PS1+/−) to acutely induce microglia spe- We hypothesized that FAD mutations can impair normal pre- cific Cx3cr1 driven Cre-mediated deletion of floxed miR-155 senilin 2 protein function and therefore promote AD through alleles in the APP/PS1 mouse AD model. Changes in in- dysregulation of immune function. We have developed a flammatory gene and microRNA expression in microglia 6 mouse model to study the impact of the most common FAD and 9 months post miR-155 deletion were assessed by qPCR. PSEN2 mutation, the PSEN2N141I, also known as the “Volga We expect that conditional deletion of miR-155 leads to anti- German” mutation. Previous experiments in the lab have inflammatory gene expression and thus improve cognitive shown that the absence of wild type PSEN2KO disrupts nor- performance. To measure anxiety, spatial memory, and spa- mal microglial phagocytosis of neuronal debris. My work in- tial learning, we employ open field chambers with and with- volves measuring phagocytosis of apoptotic bodies by mouse out novel object recognition and T-maze assessments. Prelim- primary neonatal microglia expressing the FAD PSEN2 mu- inary results support the hypothesis that conditional miR-155 tation to determine if AD associated mutations contribute to deletion specifically in microglia alters innate immune gene AD through altering microglia phagocytosis behavior. Mi- expression and behavioral phenotypes in the APP/PS1 mouse croglia from wild type mice, mice with presenilin 2 knocked model of AD, further elucidating the impact of the molecular out, and mice heterozygous for the human N141I mutation regulators in neuroinflammation in AD. are collected and co-cultured with apoptotic cell bodies. Mi- croglia and apoptotic cell bodies are membrane-labeled and FACS analysis is used to measure percent phagocytosis. My studies will further our understanding of how familial AD mutations alter the normal cellular behavior of brain immune cells and may highlight new areas in pathways appropriate for therapeutic targeting.

POSTER SESSION 2 MGH 241, Easel 137 1:00 PM to 2:30 PM

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