Alzheimer's disease (AD) is the leading cause of dementia, and its precise cause is still unknown. Mutations in the two Presenilin (PS) genes, Presenilin 1 and Presenilin 2 (PS2), cause AD. Understanding how AD mutations impact PS function may help us understand AD pathogenesis. One function of PS involves calcium (Ca2+) transfer between the endoplasmic reticulum (ER) and mitochondria. Presenilin 2 (PS2) and mutant PS2 (mtPS2) in particular have been shown to increase this transfer, possibly causing mitochondrial Ca2+ overload. This could lead to metabolic dysfunction, which has been suggested as a key pathogenic mechanism in AD. Establishing a link between PS mutations and mitochondrial dysfunction would support this hypothesis. One symptom of mitochondrial dysfunction is changes in mitochondrial fragmentation and localization. "Fragmented," rather than interconnected, "branchlike" mitochondrial networks are associated with increased ER-mitochondria Ca2+-transfer and apoptosis. Furthermore, while healthy mitochondria move towards energy-demanding neuronal processes, mitochondria with reduced ATP production stay near the nucleus. To determine the effects of PS2 and mtPS2 on mitochondrial fragmentation and localization, wild-type (wt) and mtPS2 (N141I) transgenic mouse cortical neurons were infected with a lentiviral vector expressing mitochondrial targeted red fluorescent protein (DsRed). Fixed cultures were imaged using confocal microscopy and analyzed using Huygens software. Currently, we have plated, infected, and analyzed sample preparations for mitochondrial length, sphericity, and other characteristics associated with fragmentation, as well as mitochondrial volume as a function of distance from the soma. Using this paradigm, we will also challenge cultured wt and mtPS2 neurons with camptothecin and staurosporine, inducers of mitochondrial fission and fusion, respectively. If mtPS2 induces mitochondrial dysfunction, mtPS2-expressing neurons should exhibit more mitochondrial fragmentation and mitochondria localized nearer to the soma. This would provide evidence for mitochondrial dysfunction in AD, supporting further research into mitochondrial changes in AD and a better understanding of AD’s etiology.

Spinocerebellar ataxia type 7 (SCA7) belongs to a family of neurodegenerative diseases characterized by degeneration of Purkinje cell neurons (PC) within the cerebellum, which leads to motor dysfunction. The disease results from a CAG tract expansion mutation of the ataxin-7 gene. This type of expansion is common within the SCA family along with other neurological disorders like Huntington’s disease. Recent research has provided evidence for a non-cell autonomous degeneration of PCs, which means that other cell types cause the PCs to degenerate. One population of cells that may be involved is the inferior olive (IO). The IO, which is known to degenerate in humans, is made up of neurons and is located within the brainstem. The axons of the IO, known as climbing fibers, extend to the cerebellum and synapse with PCs. These climbing fibers transport neurotrophic factors to the PCs. We hypothesize that loss of neurotrophic factors due to the degeneration of climbing fibers may contribute to the disease state of SCA7.
To support this belief, we seek to characterize the climbing fibers by immunohistochemistry. I have stained cerebellar sections of non-transgenic and SCA7 positive mice for VGLUT2, a marker of climbing fiber terminals. In order to understand whether a change in climbing fibers occurs before or after presentation of the disease I have also stained sections from multiple time points. To analyze the differences between the groups I have focused on measuring the amount of VGLUT2 positive puncta in regions proximal and distal to the Purkinje cell soma. Preliminary results indicate that a change in distribution of puncta occurs in mice in the late stages of disease.
 

Spinocerebellar ataxia 7 (SCA7) is a heritable disease caused by a mutation in the gene for the protein ataxin-7. SCA7 results in progressive neurodegeneration in the cerebellum, brain stem, and retina, which causes patients to lose motor control and vision. The disease disrupts the intricate circuitry of the cerebellum, impacting cells such as Purkinje cell (PC) neurons and the Bergmann glia (BG) that support them. BG and PC function is interdependent such that dysfunction in one cell type causes damage in the other. I set out to characterize how BG function is altered in SCA7 by examining brain tissue from 40 week old mice with or without the ataxin-7 mutation. The tissue was analyzed for various markers of BG function and morphology. I addressed whether mutant ataxin-7 expression in BG leads to changes in their morphology and identity as radial glia using markers for radial glia such as Sox9. I also used the marker for GLAST, a glutamate transporter, to study changes in their ability to support proper PC neurotransmission through glutamate uptake from the synapses. BG distribution in the cerebellum was studied using the markers Calbindin and S100β to mark PC and BG respectively. The APC/β-catenin intracellular signaling pathway in BG was studied using markers for APC and β-catenin, as this pathway may impact BG’s ability to maintain its structure and thus, its function. The results of this study may suggest which BG functions are affected by SCA7. These functional changes may then provide insight into possible mechanisms for cerebellar neurodegeneration in SCA7.