Alpha-synuclein SNCA has been implicated in the etiology of Parkinson’s disease (PD); however, the normal function of alpha-synuclein protein and the pathway that mediates its pathogenic effect is yet to be discovered. We investigated the mechanistic role of SNCA in the nucleus utilizing isogenic human-induced pluripotent stem cells-derived neurons from PD patients with autosomal dominant mutations, A53T and SNCA-triplication, and their corresponding corrected lines by genome- and epigenome-editing. Comparisons of shape and integrity of the nuclear envelope and its resistance to stresses found that both mutations result in similar nuclear envelope perturbations that were reversed in the isogenic mutation-corrected cells. Further mechanistic studies showed that SNCA mutation has adverse effects on the nucleus by trapping Ras-related nuclear protein (RAN) and preventing it from transporting key nuclear proteins such as, DNMT3A, for maintaining normal nuclear function. For the first time, we proposed that α-syn interacts with RAN and normally functions in the nucleocytoplasmic transport while exerts its pathogenic effect by sequestering RAN. We suggest that defects in the nucleocytoplasmic transport components may be a general pathomechanistic driver of neurodegenerative diseases.

Background APOEe4 is well‐established genetic risk factor for late onset Alzheimer’s disease (LOAD). It has been demonstrated that DNA methylation plays an important role in controlling the levels of APOEe4 expression. Furthermore, the differential methylation levels were reported between disease‐ and naïve‐ individuals. It also has been shown that reduction of APOEe4 levels may alleviate LOAD pathology. Here we aimed to develop a novel approach for specific and accurate modulation of APOEe4 gene expression. Method Here, we took advantage of a newly engineered VRER‐Cas9/gRNA system which specifically and accurately recognized a novel protospacer adjacent motif (PAM) created with SNP‐rs429358 T‐C transition in the APOEe4 gene. The system is based on an all‐in‐one lentiviral vector harboring gRNA/VRER‐dCas9‐DNMT3A. Using this vector, we were able to achieve an efficient enhancement in DNA methylation and the associated reduction in the expression of APOEe4 allele. The system has been validated on the isogeneic APOE‐human induced pluripotent stem cell (hiPSC)‐derived excitatory neurons. Result We were able to specifically and efficiently downregulate expression of APOEe4 using isogeneic APOE‐human induced pluripotent stem cell (hiPSC)‐derived excitatory neurons as the experimental model. The reduction in the gene expression was linked to robust and specific DNA methylation. Conclusion The developed epigenome‐editing platform highlights the novel approach towards the development of “smart” therapeutics for LOAD.