Browsing by Subject "PFF"

Selective degeneration and dysregulation of specific neuronal populations is a common hallmark shared by neurodegenerative diseases affecting the aging population. Parkinson’s disease (PD) is one of the most prevalent neurodegenerative diseases with debilitating clinical manifestations that follow a chronic and progressive course. Pathological hallmarks of PD involve gradual and specific loss of DA (DA) neurons and widespread presence of Lewy body (LB) inclusions that consist of aggregated presynaptic protein, α-Synuclein (αSyn). Treatment of PD remains to be at symptomatic management as the underlying mechanisms that trigger neurodegeneration are still not fully elucidated. Over the past two decades, microRNAs (miRNAs) have become a major area of interest within biomedical fields and gained increasing momentum in the context of neurodegenerative diseases. In recent developments, changes in mature miRNA profiles have been reported in aging tissue and many age-related diseases, including PD. More recently, a number of studies have found that the most essential enzyme in the miRNA biogenesis pathway, Dicer, exhibits reduced expression with aging. To these ends, a genetic mouse model based on heterozygous knockout of Dicer (DicerHET) was introduced to simulate Dicer downregulation. Initial investigations identified the DicerHET model as a promising tool for studying the relationship between disrupted miRNA biogenesis and neurodegeneration associated with PD. To facilitate future investigations and speed up screening of potential therapeutic compounds using this genetic model, in the current work, we aimed to produce a DicerHET in vitro model with a practical and convenient genetic engineering approach. The main focus of this work was to validate the model and establish a standardized reproducible approach suitable for research that addresses the role of miRNA biogenesis in PD. The desired DicerHET genotype was generated in vitro by employing traditional Cre/loxP system in conjunction with a virally mediated Cre expression. More specifically, primary cortical cultures, derived from Dicer flox/+ mice embryos, were transduced with Cre expressing lentiviral vectors (lenti-hSYN-T2A-Cre) to delete the “floxed” Dicer allele. To establish optimal parameters for the procedure, we analysed recombination efficiency under different transduction conditions. The most efficient recombination was achieved after 5 days of induction in cultures. However, we observed that DicerHET genotype did not attenuate survival of the cells, as assessed by immunohistochemistry. Further, as a proof of concept, we exposed the DicerHET cultures to pre-formed fibrils (PFFs) - a PD related stressor that causes αSyn aggregation. pSer129-αSyn-positive LB-like aggregates were detected in all the PFF-treated cultures, however, with a greater accumulation in the DicerHET cultures. Interestingly, increased aggregation was not accompanied by increased cell death, suggesting that DicerHET genotype does not increase vulnerability of cortical neurons to pSer129-αSyn aggregation. Based on our earlier studies we presume that DA neurons may bear a specific vulnerability towards the age-related Dicer depletion. More conclusive evidence on this intriguing relationship could be provided in future research using the DicerHET model that can be readily applied to primary DA cultures.