Role of micrornas in human-specific regulation of neurotrophic factor CDNF and MANF

Abstract

Parkinson’s disease (PD), like most neurological disorders, is becoming an increasing problem in aging populations. In PD patients, degeneration of dopaminergic neurons in the substantia nigra of the brain leads to impairment of motor activities as well as cognitive defects. As promising alternatives to conventional treatments that do not cure the condition, neurotrophic factors which promote neuronal survival and protection against degeneration have been researched for several years. Newly discovered cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are arising as novel options for disease-modifying therapies, as they have shown efficacy in rat and mouse disease models and show better diffusion in brain parenchyma than conventional neurotrophic factors. MicroRNAs are small non-coding RNAs formed by 20–22 nucleotides that regulate post-transcriptionally at least 30% of the human coding genes. MicroRNAs bind to 3’ untranslated region (3’UTR) of RNA transcripts by complementarity, preventing translation. In this study, we explored the role of microRNAs in the regulation of CDNF and MANF expression by using reporter genes in constructs with MANF and CDNF 3’UTR and enzyme-linked immunosorbent assay (ELISA) to detect protein levels after transfection with microRNA mimics. Several microRNAs seemed to downregulate MANF (miR-144 and miR-544a) and CDNF (miR-141, miR-382 and miR-134), although not significant decrease in protein levels was found. Additionally, plasmid-based target protectors were designed and cloned to cover binding sites of microRNAs in MANF and CDNF transcripts, in order to confirm such regulation by microRNAs and potentially serve as therapeutic molecules to treat PD. Transfection with target protectors successfully restored MANF and CDNF expression levels in most cases when treated in combination with microRNA mimics and, what is more, increased the controls’ expression levels by blocking binding of endogenous microRNAs. Even though further research is needed, target protectors are a promising new alternative to restore neurotrophic protection in neurodegenerative diseases.