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Browsing by Subject "dopaminergic neurons"

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  • Their, Anna (2021)
    The contact site between the endoplasmic reticulum and mitochondria, also known as the mitochondria endoplasmic reticulum contact sites (MERCS), have a crucial role in maintaining the homeostasis within the cell. Across the MERCS multiple functions, such as regulation of calcium (Ca2+) homeostasis, lipid metabolism, ER stress, mitochondrial quality control (MQC) and regulation of unfolded protein response (UPR) take place. These processes have been shown to be implicated in numerous different neurodegenerative diseases, such as Parkinson’s disease. Parkinson’s disease is the second most common neurodegenerative disease that at the moment has no cure. The main obstacle in developing a neuroprotective treatment for the disease is the limited understanding of the key molecular events leading to neurodegeneration. One of the things in Parkinson’s disease that has eluded scientists for years is the selective death of the dopaminergic (DA) neurons in substantia nigra pars compacta. One hypothesis that could explain the selective death is the Ca2+ hypothesis, looking at the Ca2+ vulnerability of SNpc DA neurons as a plausible cause leading to the selective cell death. This project focused looking at the protein level and morphological changes of the ER and MERCS in stressed neurons, hypothesizing these as possible sites that contribute to the neuron vulnerability, as they are known to be the key modulators of the intracellular Ca2+ homeostasis. This study looked closer at two MERC proteins, GRP75 and BAP31, and one ER protein, SERCA2, to see how they are affected in stressed dopamine-like neurons. Firstly, the in vitro model was established by differentiating SH-SY5Y neuroblastoma cells to dopamine-like neurons expressing tyrosine hydroxylase. Three different molecular compounds were tested as possible stressors affecting the Ca2+ homeostasis within the neurons, and we concluded that thapsigargin, a commonly used stressor to model PD like pathology, leads to the highest measurable ER Ca2+ depletion. Lastly, we quantitatively and qualitatively analyzed the effect of 24-hour treatment with each stressor on the differentiated SH-SY5Y neurons. Thapsigargin treatment lead to an increased level of GRP75 and SERCA2. A slight increase in BAP31 was also detected after thapsigargin treatment, but no apparent changes of the ER morphology were detected. The results, together with previous research, show GRP75 to be a possible contributor to the pathology of the disease, but further research is needed to see if it could be a possible target for treatment.
  • Sandelin, Amanda (2022)
    Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved protein with pleiotropic therapeutic effects in several disease models, including Parkinson’s disease (PD), diabetes and stroke. PD is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and many GWAS-based genes predisposing for PD are involved in oxidative stress. MANF has been shown to alleviate oxidative stress in PD models, however, the role of MANF in the antioxidant defense and mitochondrial respiration is not fully understood. By performing bulk RNA sequencing on wildtype and MANF knockout (MANF-KO) human embryonic stem cells (hESCs), we uncovered several genes involved in antioxidant defense to be up- or downregulated in MANF-KO hESC. Here we report that MANF-KO hESCs do not express the evolutionary conserved antioxidant enzyme catalase. We show that the loss of catalase makes the MANF-KO hESCs more vulnerable to hydrogen peroxide indued oxidative stress, and that MANF-KO hESCs have a reduced maximal respiration and spare respiratory capacity. Additionally, we examined if the loss of catalase in MANF-KO hESCs inhibits the differentiation of the cells to human dopaminergic neurons in vitro. We show that MANF-KO hESCs differentiate to TH+/MAP2+ cells despite a sustained deficiency of catalase, but the MANF-KO DA cultures tend to have a reduced spare respiratory capacity and higher basal glycolytic activity. To elucidate the structure-to-function relationship of MANF we utilize molecular dynamics simulations in combination with spin relaxation data from nuclear magnetic resonance spectroscopy. By examining the two-domain nature of MANF in different intracellular conditions we provide insight of the biological relevance of MANF interactions. Here we show that MANF conformational ensemble is more compact than previously reported. By simulating MANF in the presence of calcium and ATP, in neutral and low pH, we observed competitive binding of ATP and calcium to MANF. This study provides novel evidence of a regulatory role of MANF in the cellular antioxidant defense and explores the biological relevance of ATP and calcium binding to MANF.