Cell & Molecular Biology

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/29922

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    ER-associated Regulation of Astrocyte Mitochondrial Function during METH Exposure
    (2020) Borgmann, Kathleen; Proulx, Jessica
    Purpose: Astrocytes are key regulators of central nervous system (CNS) health and neuronal function. However, astrocyte mitochondrial dysfunction, such as induced by (METH)amphetamine, threatens the ability of astrocytes to provide the essential metabolic and antioxidant support to neurons. This study examined the endoplasmic reticulum (ER)-mitochondrial interface in response to METH to characterize changes in mitochondrial bioenergetics, the unfolded protein response (UPR), calcium signaling, and the regulation of mitochondria associated membranes (MAMs). We hypothesized that the ER regulates astrocyte mitochondrial function via calcium and UPR signaling during METH exposure. Methods: The effects of METH on astrocyte mitochondrial function were examined under both acute and chronic paradigms in primary human astrocytes. Mitochondrial bioenergetics was assessed using Seahorse assay while expression of UPR/MAM mediators were determined using RT-PCR and protein expression assays. Calcium signaling was measured by confocal microscopy using a genetically encoded calcium sensor. Finally, pharmacological inhibition of calcium and the UPR pathways were used to delineate the regulatory mechanisms mediating the changes on mitochondrial bioenergetics. Results: Our results show both acute and chronic METH increased Ca2+ flux, up-regulated the expression of UPR/MAM mediators, and altered mitochondrial bioenergetics in astrocytes. We further implicate a role for UPR signaling on mitochondrial activity. Conclusion: These findings illustrate novel cellular mechanisms in regulating astrocyte mitochondrial function during METH exposure. Additional studies will be required to assess whether these mechanisms can be therapeutically targeted to optimize the metabolic and antioxidant coupling between astrocytes and neurons to promote neuronal survival during neurodegenerative pathologies.
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    Store-Operated Calcium Entry regulated IL-6 expression and matrix protein changes in glomerular mesangial cells.
    (2020) Ma, Rong; Tao, Yu; Yazdizadeh Shotorbani, Parisa; Chaudhari, Sarika
    IL-6 can function as a pro or anti-inflammatory cytokine, depending on the cell type or pathology. Cytokines contribute to the early changes and histological impairment in chronic kidney diseases like diabetic nephropathy (DN). Glomerular mesangial cell (MC) is a major cell type in glomerulus to produce cytokines in response to diabetes and a major contributor to mesangial matrix expansion in DN. This study aimed to determine whether and how Store-Operated Calcium Entry (SOCE) regulated IL-6 production and downstream effects in MCs. Experiments were performed in cultured human MCs. The knockdown of proteins was achieved using a targeted siRNA delivery system, while overexpression of protein was obtained with specific human IL-6 plasmid in MCs. The expression of target proteins was examined either in culture media and cell lysates using ELISA and Western blot analysis, respectively. Thapsigargin (an activator of store-operated Ca2+ entry, SOCE) significantly increased IL-6 level, and this effect was attenuated by GSK 7975-A (a selective inhibitor of SOCE). Silencing Orai1 (the channel protein mediating SOCE) significantly decreased IL-6 protein expression. Inhibition of NFκB pharmacologically and genetically significantly reduced SOCE-induced IL-6 production. Thapsigargin also stimulated the nuclear translocation of the p65 subunit of NFκB. Overexpressing IL-6 and its receptor decreased the expression of fibronectin and collagen IV in MCs. Our results suggest that IL-6 production is positively regulated by Orai1-mediated SOCE via the NFκB pathway in MCs and inhibits ECM protein production by MCs.
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    Safety, efficacy, and availability of genetic therapies to treat neuromuscular disorders
    (2020) Ssentamu, Frank; Sloan, Joshua; Sparks, Chase
    Genetic mutation underlies the etiology of many debilitating neuromuscular disorders. Diseases such as spinal muscular atrophy and Duchenne muscular dystrophy have profound impact on quality of life, and often begin to manifest in infancy or childhood. Identification of the causative genetic mutations for diseases such as spinal muscular atrophy and Duchenne muscular dystrophy provides opportunities for the development of targeted genetic therapies that aim to correct protein deficiencies resulting from these mutations. Clinical trials have demonstrated some efficacy, but only in small studies. Purpose: The primary objective of this review was to determine the safety, efficacy, and availability of gene therapy treatments for neuromuscular diseases compared to standard of care or placebo. The secondary objective was to review clinical trials that are in progress to evaluate therapies currently in development. Methods: A systematic review was performed using databases Pubmed, Medline, Cochrane, and ClinicalTrials.gov. The search terms "gene therapy" and "neurological or muscular" were utilized. The search results from ClincalTrials.gov were limited to completed trials. Results: Published trials detail the utilization of several treatment modalities, including single gene replacement via vector-mediated delivery, transfection and infusion of autologous stem cells, and modification of protein translation by exon skipping. Conclusions: Many treatments demonstrate promise in slowing the progression of disease and exhibit mild side effect profiles. Larger studies are needed to ascertain superiority over standard of care procedures, but favorable tolerability potentially justifies expansion of clinical trials to larger cohorts.