Alpha-synuclein in Parkinson's disease: molecular pathogenesis and development of genome engineering-based silencing approaches

Abstract

The ability to reprogram adult somatic cells into induced pluripotent stem cells (iPSCs) and subsequent development of protocols for their differentiation into disease-relevant cell types have enabled in-depth molecular analyses of multiple disease states as hitherto impossible. Parkinson’s disease (PD) is one such example, in which the dopaminergic neurons that manifest pathology are embedded in an inaccessible region of the midbrain, the substantia nigra pars compacta, making it unfeasible to obtain samples of the affected region from living patients. Overabundance of alpha-synuclein, encoded by the SNCA gene, has long been implicated in the pathogenesis of PD. However, the precise mechanism by which overexpression of alpha-synuclein leads to the demise of dopaminergic neurons remains elusive. Neurons differentiated from PD patient-specific iPSCs carrying multiplications of the SNCA gene may provide a means to recapitulate molecular phenotypes of the disease in vitro. The application of CRISPR/Cas9 to mammalian systems is likewise revolutionizing the utilization of genome editing in the study of molecular contributors to the pathogenesis of numerous diseases, including PD. In this body of work, I have utilized the double-nicking CRISPR/Cas9 system to mediate site-specific mutagenesis of SNCA in PD patient-specific iPSCs harboring a triplication of the SNCA gene locus, resulting in isogenic cells which can be used to phenocopy normal alpha-synuclein expression from two alleles. I have further demonstrated the utility of nuclease null or "dead" Cas9, for transcriptional silencing of alpha-synuclein expression. Finally, I have used these systems to interrogate phenotypic outcomes of SNCA triplication, demonstrating that alpha-synuclein induces endoplasmic reticulum stress and aberrant activation of a highly conserved arm of the unfolded protein response. The tools generated in these studies can be applied in future efforts to dissect the contributions of alpha-synuclein overexpression to PD pathogenesis and as a platform for the discovery of disease-modifying therapeutic approaches for PD.</p