Novel Targets for Parkinson`s Disease Research – The Flcn Gene and its Role in Dopaminergic Neurons
Parkinson`s disease (PD) has been and is intensively studied by scientists worldwide, but the etiology of PD is still not clearly defined. Studies of genes associated with familial PD have provided us with a plethora of insights into cellular mechanisms involved in the disease. However, there are still many unanswered questions and conflicting results that slow the progress of research. Current treatments only alleviate symptoms and therefore, there is still an unmet need to provide new therapeutic targets. New targets are continually emerging, either through recent genome-wide association studies or, as in our case, from an unbiased CRISPR-based, genome-wide screen. Our primary objective was to identify novel target genes that enhance the viability of dopaminergic neurons when exposed to oxidative stress. Through our screening process, we successfully pinpointed a list of target genes that conferred increased survival resistance when their expression was silenced. One of our top candidate genes is Flcn. The neurotoxin Rotenone, when applied to dopaminergic neurons in vitro, induces oxidative stress, resulting in the death of these neurons. However, this detrimental effect was mitigated by the knockout of the Flcn gene. The Flcn protein is implicated in the mTOR pathway, which is known to modulate the autophagy-lysosome pathway. Lysosomal function and cellular capacity to degrade unwanted material is of paramount importance, with several PD risk genes being linked to the autophagy-lysosome pathway. In fact, the mechanisms involved in the maintenance and degradation of Lewy bodies in PD are still under intense investigation and highlight the importance of research directed towards the autophagy-lysosome pathway.
We used two different models to validate and further investigate the mechanisms involved in the neuroprotective effects of Flcn knock-out (KO, no functional protein). First, we knocked out Flcn in dopamine neurons in the mouse brain and used viral expression of human alpha-synuclein (aSyn) to model PD. Removing Flcn in dopamine neurons ameliorates the motor deficits induced by aSyn overexpression, specifically when testing for grip strength and overall motor coordination. Furthermore, Flcn removal rescues the degeneration of dopamine neurons in the midbrain and their axon terminals in the striatum induced by aSyn. We also observed an increase of mTOR activation, as suggested by the literature. Most importantly, Flcn KO also reduces the levels of pathological aSyn in neurons.
In parallel to our experiments conducted in mice, we tested the neuroprotective effect of Flcn KO in human dopaminergic neurons derived from stem cells. We are using stem cells that have been derived from a PD patient with the triplication of the aSyn encoding gene SNCA (3xSNCA), and the isogenic control cells (normal expression levels of aSyn). We differentiate the stem cells into dopaminergic neurons for 60 days, after which time the 3xSNCA neurons display deficits in different cellular processes, including mitochondrial function and the autophagy-lysosome pathway. Similar to the mouse model, Flcn KO in human dopaminergic neurons leads to an increase of mTORC1 activation, reduces reactive oxygen species and modulates the autophagy-lysosome pathway. Our results suggest that the beneficial effects of knocking out Flcn depend on the translocation of transcription factor EB (TFEB) to the nucleus of the cell, where it induces the transcription of lysosome-related genes and therefore increases lysosomal degradation in the cell. TFEB is also known as the master regulator of lysosome biogenesis and autophagy and has already been intensively studied in PD.
We used an unbiased screening method to identify new potential targets for neuroprotection in PD. Our investigation has identified Flcn as a novel neuroprotective target, providing insight into the importance of autophagy-lysosome pathway in PD. We are now planning to explore the impact of Flcn-modulating compounds on disease onset and progression. The results of this study will contribute to the immediate advancement of knowledge of the research community. In the long term the goal is to introduce new therapeutics into clinical trials, to ultimately improve the life of people living with PD.
Julia Obergasteiger is a Postdoctoral Researcher at the CERVO Brain research centre, in Québec, Canada. She presented her research in Barcelona.
Ideas and opinions expressed in this post reflect that of the author solely. They do not reflect the opinions or positions of the World Parkinson Coalition®