A personalized medicine approach for the study of dopaminergic neuron degeneration in parkinson’s disease

Parkinson’s disease (PD) is caused by the loss of dopaminergic neurons (DA neurons) in the substantia nigra pars compacta (SNpc). The cause of DA neuron degeneration was investigated in many fronts though it remained inconclusive. An association of PD with genetic mutations was revealed by a genome wide association study (GWAS) showing that the GBA1 and SNCA genes to be the most common and penetrant mutations found in PD patients, respectively. SNCA encodes for α-synuclein (SNCA) which when mutated, accumulates in the affected DA neurons. GBA1 encodes the enzyme glucocerebrosidase (GCase) in the lysosome which catabolizes glucosylceramide and glucosylsphingosine to glucose and ceramide or sphingosine, respectively. In a previous work in the lab, the high level of phosphorylated α-synuclein (P-SNCA) in the presence of SNCA mutation, was shown to be reduced when treated with a recombinant enzyme GCase (rGCase) in DA neurons differentiated from PD patient-derived induced-pluripotent stem cells (iPSCs). The reverse was also true as shown by an increase of P-SNCA when GCase was abolished by GBA1 deletion. This was supported by evidence showing that the accumulation of SNCA impeded lysosomal maturation and therefore GCase activity. Reduced GCase function on the other hand, raised the level of glucosylceramide which can stabilize soluble oligomeric intermediates of SNCA fibrils. Breaking down this self-propagating positive feedback loop by raising GCase or reducing SNCA levels could potentially stop this pathogenic cycle in the affected DA neurons; this may present a cure to PD.
To further investigate the effects of GBA1 or SNCA mutations on DA neurons, extracellular network electrophysiology of DA neuron cultures were recorded. The result showed that the network firing activities were reduced in the presence of SNCA or GBA1 mutations, each with distinctive functional characteristics. In the morphological perspective, DA neurons with both mutations had shorter neurite lengths and smaller soma sizes. In a rescue approach, we raised the GCase activity of GBA1 mutant DA neurons by rGCase treatment. We also reduced the SNCA level of SNCA mutant DA neurons by RNA interference with locked nucleic acid (LNA) treatment. Both treatments resulted in the rescue of DA neurons neurite lengths. We further identified that the aberrant GCase activity resulted in higher amount of activated calcineurin (CaN), resulting in the nuclear localization of transcription factor EB (TFEB). We validated that by reducing CaN activation via rGCase treatment or by blocking CaN activation using FK506 in GBA1 mutant DA neurons, TFEB nuclear localization can be rescued. Taken together, these approaches suggest a promising treatment for PD patients. This part of the project was shown in the first manuscript.
To identify and validate the molecular pathways involved in aberrant GCase or SNCA conditions, a transcriptomics/proteomics experiment was planned. Owing to the heterogeneity of cell types in the differentiated midbrain DA neuron cultures, human iPSC lines were generated with a reporter system to identify the presence of DA neurons when differentiated. These lines also contained the GBA1 and SNCA mutations. To generate these knocked-in human iPSC lines, an mCherry coding sequence was knocked-in into the tyrosine hydroxlase (TH) locus, a gene that is exclusively expressed by DA neurons. In the presence of an internal ribosomal entry sequence (IRES), mCherry was expressed bicistronically under the TH regulatory element. Differentiation of these knocked-in cell lines into midbrain DA neuron cultures showed expression of mCherry exclusively in DA neurons. These mCherry-positive DA neurons can be purified by florescence-activated cell sorting (FACS) and survived replating post-FACS, forming pure mature DA neuron networks. Ultimately, these reporter lines can be used to obtain purified DA neurons for -omics studies, which have not been done by the PD research community to date. Besides for purification purpose, these lines can also be used to identify the positions of DA neurons on high density microelectrode arrays (HD-MEAs) used in the network electrophysiology recordings in the first manuscript.
To understand DA neuron firing activity changes caused by the SNCA or GBA1 mutations, the ability to control DA neuron activity is vital. Therefore, channel-rhodopsin and human M3 muscarinic receptor knocked-in human iPSC lines were also generated using the same approach. The knock-in strategy and methodology used in this part of the project were documented in the second manuscript of the Thesis.