Hickey, Miriam Ann, juhendajaJakobson, Maili, juhendajaEltalb, Imane Muhammad HigazyTartu Ülikool. Loodus- ja täppisteaduste valdkondTartu Ülikool. Tehnoloogiainstituut2023-07-142023-07-142023https://hdl.handle.net/10062/91418Background: Parkinson’s disease (PD) is associated with tremor, slowness of movement and stiffness, the latter two of which are caused by the loss of dopaminergic neurons of the substantia nigra pars compacta. Interestingly, although olfactory impairment is now well recognised in PD, skin symptoms differentiate individuals who go on to develop PD even better than olfactory impairment. However, little is known of the cause of this skin pathology, which includes proteinaceous aggregates and loss of peripheral neurites. Recently, several excitatory ion channels were found to be upregulated in PD patient skin. Here, we have examined the effect of overexpression of these channels in a sensory neuronal cell model of PD. Methods: The 50B11 cell line, which is an immortalised DRG rat sensory neuronal cell line (kind gift from Dr Höke, Johns Hopkins University), was used. Familial PD was modelled by transfection with eGFP-alpha synuclein. Sporadic PD was modelled by treating with rotenone, a well-validated risk factor for PD and inhibitor of complex I. In our first series of experiments, cells overexpressing eGFP-alpha-synuclein or GFP (control plasmid) were treated with rotenone or DMSO and were then fixed for imaging and cytotoxicity analysis. In our second series, cells were co-transfected with alpha synuclein and HCN1 and then treated with rotenone or vehicle, then fixed for imaging and analysis of cytotoxicity. In ourfinal series,cells were transfected with HCN1 or control plasmid, treated with rotenone or vehicle and then imaged live for mitochondrial membrane potential (MMP) and cell morphology. Results: In our first series of experiments,the density of alpha synuclein-overexpressing cells was increased following rotenone exposure. However, in our second series, co-transfection of HCN1 with alpha synuclein resulted in lowered density of cells treated with rotenone. In our final series of experiments, rotenonecaused a small reduction in MMP. HCN1 overexpressionalone reducedMMP to a much greater degree. HCN1 overexpression exacerbated the effect of rotenone on MMP. This combination of HCN1 overexpression with rotenone changed the morphology of sensory neurons to a smaller, more rounded shape. Conclusions: HCN1 is an important regulator of activity in sensory neurons and its overactivity is involved in several types of neuropathic pain. PD patients show loss of peripheral neurons and recently, HCN1was found to be overexpressed in skin samples of PD patients. Although the normal function of alpha synuclein is unclear, it may induce proliferation and also may increase expression of anti-oxidant genes, and here, we found that alpha synuclein protected against rotenone toxicity. However, co-expression of HCN1 prevented this effect and resulted in increased cell death. In separate experiments, the combination of overexpression of HCN1 and rotenone resulted in exacerbated loss of mitochondrial membrane potential, and a change in cell shape indicative of toxicity. Thus, HCN1 overexpression may precipitate toxicity in parkinsonian sensory neurons and contribute to peripheral neuropathy in PD.engopenAccessAttribution-NonCommercial-NoDerivatives 4.0 Internationalsensory neuropathyHCN1alpha synucleinrotenonedorsal root ganglion cellsfamilial Parkinson’s diseasesporadic Parkinson’s diseasemagistritöödThesis