Spinocerebellar Ataxia (SCA) is a group of inherited autosomal dominant disorders characterized by the loss of coordination in the limbs and atrophy of the cerebellum. SCA progresses gradually and has a diverse presentation of symptoms from debilitating to mild amongst its different forms. There are many genetic causes for SCA, however, they remain unknown in many cases. Although there is no treatment, recent scientific advances have illuminated mechanisms of pathogenesis and potential gene therapies to help patients with SCA. My project in the Raskind Lab contributes to this research by attempting to identify the causal gene for a family with a novel form of autosomal dominant SCA. Whole exome sequencing is currently being conducted on the DNA of three affected members of the family. From the exome sequencing, we will receive all the genetic differences in the protein-coding region from a reference sequence in any of these three subjects; we will first search for variants with genes known for SCA. I will then begin the process of choosing candidate variants for further analysis. I am choosing them by first filtering for variants that are heterozygous in all three exomes and have a prevalence of less than 0.01% in genetic databases and then prioritizing the remaining variants based on the type of mutation, model-predicted effect of the variant, and relevance of the gene function to SCA. For each chosen candidate variant, I will amplify and sequence the DNA from each family member to determine whether it co-segregates by being present in all those who are affected and absent from those who are not. Once a co-segregating variant is identified, other studies will be conducted to support its causality. My research will contribute to our understanding of SCA and neurodegenerative disorders.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder, marked by progressive muscle degeneration due to the absence or impairment of the dystrophin protein. Our laboratory has been developing methods for gene therapy of DMD. The dystrophin gene is the largest known gene, which complicates gene therapy. We have shown that gene delivery vectors based on adeno-associated virus (AAV) can be used to deliver new genes body wide. However, AAV has a ~5 kb carrying capacity, so we have been developing miniaturized dystrophins. These smaller, highly functional dystrophins, which we named micro-dystrophin (µdys) were tested in a transgenic mdx^4cv  mouse model for DMD. Furthermore, we sought to develop a non-invasive method to evaluate treatment efficiency of µdys. In this project, the gait of mdx^4cv, wild type (WT) and transgenic mice were observed to deduce potential benefits of the µdys transgene. We hypothesized that µdys treatment will enable the gait of the transgenic mice to resemble that of WT animals as opposed to mdx^4cv. To test this, gait was assessed at three time points: 1.5, 3 and 6 months of age using the video-based Noldus CatWalk XT. Our results show that the length of a single stride of the transgenic mice hind paws increased over time compared to mdx^4cv. The normal walking pattern of mice consists of placing diagonal paws on the surface, one front and one hind from opposite sides. In the case of mdx^4cv, more paws were placed down for majority of the walk, as a compensatory mechanism for muscle weakness. The transgenic mice were found to walk with a diagonal pattern more frequently, like WT mice. Similarities in the gait of transgenic and WT mice help us conclude the µdys treatment was successfully able to mitigate the effects of DMD as seen by the gait analysis.

Stroke is the leading cause of long-term disability in the USA. Ischemic preconditioning (IPC) is a neuroprotective phenomenon wherein a brief ischemic exposure induces robust neuroprotection against subsequent prolonged ischemia. The Weinstein laboratory has previously demonstrated: (i) a robust type 1 interferon response in cortical microglia following IPC, (ii) type I interferon signaling in microglia is required for IPC-mediated protection and (iii) IPC induces a robust increase in the number of microglia in preconditioned cortex. An initial component of my project was to validate this microglial response by first staining for Iba1 (a microglial marker) alone and then double staining for Iba1 and proliferation marker BrdU. We used immunofluorescent microscopy (IFM) following by quantitative stereology (QS). Our hypothesis was that type I interferon signaling is necessary for IPC-induced microglial proliferation. We carried out IPC on WT and type 1 interferon receptor deficient (IFNAR-/-) mice and quantified cortical microglial number and proliferation as above. Preliminary results were: (i) in naïve WT, 0.643 ± 0.038 (mean ± S.D), Iba1+ cells per position, (ii) in preconditioned WT, 1.113 ± 0.1385, (iii) in naïve IFNAR1-/-, 0.751 ± 0.058 and in preconditioned IFNAR1-/-, 0.903 ± 0.125. Two way ANOVA revealed a significant difference between naïve and IPC-induced cortical microglia numbers [F(1,14)=9.62, p=0.0078], but no significant effect of genotype [F(1,14)=0.258, p=0.619]. IPC induced increases in the number of cortical Iba1+/BrdU+ proliferating microglia in both IFNAR1-/- (0.451 ± 0.107) and WT (0.205 ± 0.069) mice. These results suggest a complex picture in which deficiency in type 1 IFN signaling may not influence IPC-induced microglial proliferation but does attenuate the overall number of cortical microglia. This raises the possibility that type 1 IFN signaling may be required for optimal microglial survival following IPC. More studies will be required to confirm the above findings and explore possible mechanisms.

Microglia are the resident immune cells of the CNS and are hypothesized to influence aging in the brain. Like somatic cells, microglia can be replaced by self-renewal. Recently, some studies have suggested that new microglia derive from asymmetric cell division of a progenitor population. Microglia progenitor cells have been difficult to study due to a lack of specific molecular markers of this population. However, the Garden lab has recently identified novel candidate markers. We hypothesize that in neurodegenerative disorders associated with advanced age, microglia progenitor senescence may contribute to disease pathology. To efficiently study the senescence of microglia progenitors, we turned to neonatal mixed glia cultures, in which the presence of microglia progenitors has long been inferred. In these cultures, microglia are harvested from cells floating above a monolayer culture of mixed neonatal glial cells. The size of each microglia harvest generally decreases with successive harvests. This suggests that microglia progenitors in the attached monolayer may become senescent after multiple rounds of the cell cycle, leading to stagnation in the generation of new floating microglia. We evaluated microglia progenitor senescence in neonatal mixed-glia cultures by labeling with BrdU, a thymidine analog taken up by proliferating cells and remaining in their daughters. Microglia harvested from these cultures weekly were assessed for BrdU incorporation using flow cytometry and immunofluorescent microscopy. We co-labeled floating microglia and dissociated monolayer mixed glia cultures with antibodies directed against a progenitor marker (CD133), a microglia marker (Iba1), and BrdU. The attached mixed-glia cell layer was also labeled for SA-ß-Gal, an indicator of cellular senescence. Progenitor senescence will be detected by a decrease in CD133/BrdU-positive cells and an increase in CD133/ SA-ß-Gal positive cells.

Mutations in the Presenilin 2 gene, (PSEN2), cause early onset familial Alzheimer’s disease (FAD). PSEN2 encodes the presenilin 2 protein which forms the catalytic subunit of the gamma-secretase complex, responsible for cleaving amyloid precursor protein into fragments. One of the resulting fragments is Aβ_1-42, a major component of brain amyloid plaques and a clinical hallmark of Alzheimer’s disease. Microglia are the resident innate immune cells in the brain clearing the brain of Aß plaque and debris through phagocytosis. Without presenilin 2 protein present, levels of secreted cytokines increase implying a higher immune response, and recent data suggest that hyper-activation of an inflammation state can contribute to development of Alzheimer's disease. We hypothesized that FAD mutations can impair normal presenilin 2 protein function and therefore promote AD through dysregulation of immune function. We have developed a mouse model to study the impact of the most common FAD PSEN2 mutation, the PSEN2N141I, also known as the “Volga German” mutation. Previous experiments in the lab have shown that the absence of wild type PSEN2KO disrupts normal microglial phagocytosis of neuronal debris. My work involves measuring phagocytosis of apoptotic bodies by mouse primary neonatal microglia expressing the FAD PSEN2 mutation to determine if AD associated mutations contribute to AD through altering microglia phagocytosis behavior. Microglia from wild type mice, mice with presenilin 2 knocked out, and mice heterozygous for the human N141I mutation are collected and co-cultured with apoptotic cell bodies. Microglia 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.

Microglia are the innate immune cells of the central nervous system that exhibit a sustained pro-inflammatory response in the Alzheimer’s disease (AD) brain. Regulation of inflammatory gene expression in microglia by microRNA miR-155 modulates transition between distinct phases of the inflammatory response. Though altered expression profiles of miR-155 is seen in other neurodegenerative disorders, the precise role of this microRNA in modulating inflammation and downstream behavioral deficits in mouse models of AD remains unknown. We hypothesize that microglia specific deletion of miR-155 will alter neuroinflammation and behavioral phenotypes in transgenic mice expressing human mutant amyloid precursor protein and presenilin 1 (APP/PS1), an AD model that exhibits Aβ pathology and memory impairments. We generated trigenic (Cx3cr1-Cre^+/-/Floxed-miR155^+/+/APP/PS1^+/-) to acutely induce microglia specific Cx3cr1 driven Cre-mediated deletion of floxed miR-155 alleles in the APP/PS1 mouse AD model. Changes in inflammatory gene and microRNA expression in microglia 6 and 9 months post miR-155 deletion were assessed by qPCR. We expect that conditional deletion of miR-155 leads to anti-inflammatory gene expression and thus improve cognitive performance. To measure anxiety, spatial memory, and spatial learning, we employ open field chambers with and without novel object recognition and T-maze assessments. Preliminary results support the hypothesis that conditional miR-155 deletion specifically in microglia alters innate immune gene expression and behavioral phenotypes in the APP/PS1 mouse model of AD, further elucidating the impact of the molecular regulators in neuroinflammation in AD.