Ischemic preconditioning (IPC) is a phenomenon in which a brief ischemic event confers protection against subsequent prolonged ischemia, such as stroke. Understanding the mechanisms underlying this endogenous neuroprotective response could lead to advances in stroke therapeutics. Most experimental rodent models of stroke focus on injury to grey matter. However, injury to the white matter (WM) is a significant component of ischemic stroke in human patients and is poorly understood. Our laboratory has shown that microglial innate immune signaling is required for WM IPC-induced protection. The nodes of Ranvier (NoR) are critical WM structures that control conductance and action potential propagation along axons. Recent studies have shown elongation of the NoR in the context of WM injury and disease. The mouse optic nerve (MON) is a fully myelinated central nervous system WM tract. We expose MONs to a brief in vivo ischemic event to induce IPC. Seventy-two hours later, we isolate MONs and expose them to ex vivo oxygen-glucose deprivation (OGD) to mimic stroke. Using MONs from mice that underwent pharmacologic depletion of microglia followed by in vivo IPC and then ex vivo OGD, I performed immunohistochemistry with Nav1.6 and Caspr antibodies to label the NoR and image them with a confocal microscope. NoR size, diameter, and length are quantified with ImageJ in conjunction with an in-house MATLAB script. I hypothesize that IPC will protect and conserve structural parameters of the NoR from OGD-induced injury and that this protection will be eliminated by microglial depletion. Our preliminary data show feasibility of this project (i.e., all the methods to carry out this work are in place) and we can generate precise and reproducible measurements of the NoR in MONs. Findings from this project will provide a better understanding of the mechanisms by which microglia mediate IPC-induced axonal protection in WM.
 

Spinocerebellar ataxias (SCAs) are a group of hereditary diseases that are characterized by slowly progressive incoordination in gait, hand and eye movement, and cerebellum degeneration. Most forms of SCAs are caused by an expansion of short tandem repeats; however, SCA 14 is an autosomal dominant form of the disease associated with mutations in the Protein Kinase C gamma (PRKCG) gene, which encodes for the PKCγ protein, a serine/threonine kinase that plays a role in signaling and regulating cerebellar Purkinje cells' development. Our lab first reported this genetic cause in SCA 14 patients. To understand the pathogenesis of SCA 14, our lab generated mouse models of SCA 14 mutant (H101Y, F643L) and wild type (WT) PKCγ transgenic (Tg) mice using modified human-BAC constructs. By 3 months of age, both mutant Tg lines demonstrated impaired rotarod performance as compared to WT-Tg mice and showed PKCγ aggregates. Here I further examined the pathological changes by quantitatively analyzing the morphology and fluorescent intensity changes of Purkinje cells in mice cerebellum across age groups of 2-months, 6-months, and 12-months. I observed the dendritic arborization abnormality at an early age, and the abnormality is more severe in the Tg-PKCγ-F643L mice. PKCγ intensity in the cell body decreased in 2-month H101Y and F643L mice. These results revealed pathological changes and provided evidence the missense mutations caused an early development of SCA 14 cerebellar disease. It will be interesting to compare and verify these findings from the Tg mice in SCA 14 patients’ autopsy brain tissues as it could provide novel measures to characterize SCA 14 pathology.

Chimeric antigen receptor (CAR) T cell therapy is a highly effective treatment for blood cancers; however, it is correlated with neurotoxicity in about 40% of cases. Neurotoxicity manifests as headaches, delirium, and in more severe cases, seizures, coma, and death. In a mouse model of CD19-directed CAR T cell therapy, we observed that mice developed behavioral changes, as well stalled blood flow in over 10% of brain capillaries. This was caused by circulating leukocytes becoming stuck in the capillaries. My goal for this project was to investigate the underlying mechanism of capillary stalls in the context of CAR T cell therapy. We predicted that capillary stalls are caused by increased expression of adhesion molecules, such as Intercellular Adhesion Molecule-1 (ICAM-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1), by the endothelial cells that comprise the capillary walls. These adhesion molecules grip onto molecules on the surfaces of leukocytes, creating plugs and halting blood flow through the vessels. To test this hypothesis, I used immunohistochemistry to compare the expression of ICAM-1 and VCAM-1 between 4 treatment groups: mock, tumor, CAR T, and tumor+CAR T. Mice from each treatment group were perfused and their brains were collected and sectioned. I incubated mouse brain sections in a primary antibody solution targeting ICAM-1 and VCAM-1, and then in a secondary fluorescently conjugated antibody solution that bound to the previous antibodies, enabling specific labeling of the molecules of interest. I then imaged the sections with a confocal microscope and analyzed them using ImageJ software. I quantified the brightness of fluorescence of ICAM-1 and VCAM-1 to determine the relative expression of these molecules between control mice and those receiving CAR T. The findings from this project have contributed to our overall understanding of the mechanisms of neurotoxicity associated with CAR T cell therapy.

Mutations in the gene glucosidase, beta acid 1 (GBA) are not only the strongest genetic risk factor for Parkinson’s Disease (PD), but also accelerate the progression of PD. We hypothesize that GBA mutations accelerate disease progression by promoting propagation of Lewy pathology from cell to cell via dysregulated extracellular vesicles (EVs). To investigate this, we developed a Drosophila model of GBA deficiency (GBA^del) manifesting neurodegeneration and accelerated protein aggregation. We also developed a human neuronal model by generating human induced pluripotent stem cells (iPSCs) from an individual with PD heterozygous for a null GBA mutation (GBA^IVS PD). Neurons were differentiated from GBA^IVS PD iPSCs, isogenic GBA^WT PD iPSCs, and iPSCs from an age- and sex-matched healthy control. I performed immunocytochemistry and western blots to evaluate protein aggregation within neurons. Additionally, I isolated neuronal EVs by size exclusion chromatography and analyzed them using a ZetaView nanoparticle analyzer. We previously found the expression of wildtype GBA in muscles of GBA^del mutant flies rescued levels of protein aggregation in the brain. This non-cell autonomous rescue was accompanied by normalization of alterations observed in EVs from GBA^del flies. Similar to our fly model, I found human GBA^IVS PD neurons and EVs have increased ubiquitinated proteins when compared to GBA^WT PD or healthy control neurons and EVs collected from these neurons. Our results suggest that GBA deficiency mediates PD pathogenesis by accelerating propagation of pathogenic protein aggregation through the alteration of EV protein cargo. We are now further investigating how GBA influences endolysosomal trafficking and EV biogenesis and I will now test whether GBA^IVS PD EVs can propagate protein aggregation faster in recipient neurons than control EVs. Understanding mechanisms regulating the spread of protein aggregates could reveal novel therapeutic targets to slow the rate of progression of neurodegeneration.

GNE myopathy (GNEM) is an autosomal recessive inherited disease that causes progressive muscle weakness starting around ages 20 - 40, which leads to physical disability. GNE myopathy is caused by a mutation in the GNE gene, which encodes the enzyme glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase. This enzyme is responsible for sialic acid (SA) production. SA is a sugar that is required by all cells, including muscle cells, to produce energy. SA is also an important component of the cell membrane. Thus, lacking SA causes muscle wasting. Interestingly, sialic acid produced in the liver could be used by skeletal muscle. Thus, our lab is developing new promoters for GNEM gene therapy that combine liver and muscle expression. By using PCR and Hifi-assembly, I cloned three control promoters--one muscle-specific enhancer/promoter (Ck8e), one liver-specific enhancer/promoter (ApoE-hAAT), and one constitutive promoter (CMV)--into three reporter plasmids ahead of firefly luciferase. These reporter plasmids also have CMV-renilla luciferase as an internal control. Now I am transfecting six tandem expression cassettes plasmids (already cloned) and the three plasmids that I cloned into the culture of liver cells (HepG2) to observe the expression of the genes based on the ratio of firefly and renilla luciferase. In this way, we can compare expression in liver cells from the novel promoters and the control promoters. Another member of the lab will be transfecting the plasmids into muscle cells (C2C12), so we will be able to compare our results. This research is important to figure out the most optimal promoter to express the GNE gene in both muscle and liver cells. Thus, after treatment, the cells could produce SA by themselves to maintain their functions and prevent muscle wasting for patients with GNEM.

Alzheimer’s Disease (AD), despite its prevalence, remains a much-researched yet incurable condition. Most prominently characterized by cognitive decline with advanced age, AD is associated with beta-amyloid plaques, tau protein tangles, and neurodegeneration in the brain. Previous research has shown that a relevant contributor of AD is neuroinflammation in the brain and spinal cord. Neuroinflammation is driven by cells such as microglia, brain immune cells, and astrocytes, which regulate the blood-brain barrier and homeostasis. However, our understanding of cell-type specific changes in their gene expression is lacking. We hypothesize alterations in gene expression in microglia that result in a more inflammatory phenotype in AD compared to controls. We also expect to observe alterations in the prevalence and inflammation of astrocytes in AD brain. Single-nucleus RNA sequencing (snRNAseq) provides a more detailed analysis of cells, allowing us to examine expression differences between cells in the same brain region. Applying this technology to compare phenotypic differences in specific cell types of AD patients and non-affected controls has yielded a more comprehensive look at differences in cell types that influence AD. This study examines a snRNAseq dataset generated by a cohort of 20 individuals, using brain tissue acquired post-mortem. This tissue is separated into individual cells, which are analysed using our lab's sequencing pipeline in R, allowing us to determine gene expression for each cell. My role in the project involves piloting this pipeline and analysing the various outputs to identify differentially expressed genes and how they are characterized between groups (control vs AD) for specific cell types such as microglia. I have also begun to validate these findings with protein expression assays such as Western blots, as well as in cultured microglia and astrocytes. This study enables us to determine and characterize the alterations in gene expression in AD, and hopefully identify potential therapeutic targets in the future.