Browsing by Subject "MANF"

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.

Parkinson's disease causes changes in the basal ganglia GABAergic neurotransmission in addition to the well-known dopaminergic changes. These GABAergic modulations may cause somed of the symptoms not responding well to the standard dopaminergic medication. Neurotrophic factors are a group of endogenous proteins showing promise as a future treatment for Parkinson's disease. They are known to have neuroprotective and neurorestorative effects on the dopaminergic cells. Their effects to the GABAergic cells are still mostly unknown. Intrastriatal injection of GDNF to rats caused significantly slower weight gain compared to CDNF, MANF one week after stereotaxic operation (p=0,002 for CDNF vs. GDNF and p<0,001 for MANF vs. GDNF). Difference to the vehicle (phosphate buffered saline) used as a negative control was not statistically significant (p=0,055). Three weeks after the operation the differences between the treatment groups were no longer statistically significant. Because of problems with the separation in analysis, microdialysis samples remain still to be analysed. To help the analysis of GABA in the future we determined the analytical parameters of the analytical apparatus. We also defined differences in probe permeability between 1 mm and 2 mm probes and between old and new batches. GABA analysis was performed with a HPLC-fluorometric detection of o-phtaldialdehyde-derived GABA. Detection limit for old apparatus was 7,2 nM and for new apparatus 6,2 nM in a sample of 15 µl (0,11 pmol and 93 fmol respectively). Quantification limits defined were 22 nM and 19 nM (0,33 pmol and 0,28 pmol) for the old and the new apparatus, respectively. Upper limit of quantification was estimated to be 246 nM (3,7 pmol). Probes had significant differences in permeability between 1 mm and 2 mm probes, as well as between batches. The variance of permeability of 1 mm probes was estimated to be approximately twofold compared to the 2 mm probes. Furthermore the permeability of 1 mm probes varied between batches significantly. An average of permeability of the old batch was 34 % lower than that of a new batch (p<0,001).