Translational studies of glucocerebrosidase in Parkinson´s disease

Abstract

A number of genetic variants have been linked to Parkinson’s disease (PD). Among these, mutations in the GBA1 gene are identified as one of the most common risk factors for developing PD. The connection between increased PD risk and individuals with GBA1 mutations was first recognized in the 1990s, but even today, the mechanism remains unclear. GBA1 gene encodes glucocerebrosidase (GCase), a lysosomal hydrolase degrading glucosylceramide (GlcCer). The enzyme activity of GCase is reduced in PD patients, particularly among GBA1 mutation carriers. However, compromised GCase activity alone does not lead to disease development, adding complexity to its contribution to the greater risk of PD. Therefore, this thesis aims to untangle the intricate connection between GBA1 variants and PD pathogenesis.

In this thesis, we take advantage of patient-derived induced pluripotent stem cells (iPSCs) as a tool for exploring the role of the GBA1 mutations in PD pathogenesis. Firstly, we develop an optimized protocol for efficiently generating midbrain dopaminergic (mDA) neurons from iPSCs. The established protocol is validated to produce mDA neurons with high reproducibility across several iPS cell lines.

Using GBA1-PD patient-derived iPSCs as a reference for human samples, we investigate GBA1-specific secretome alterations in the cerebrospinal fluid (CSF) of GBA1-PD patients. The comparison of CSF and iPSC-derived mDA neurons allows us to excerpt the CSF proteins attributed to mDA neuronal populations. Notably, the most significantly altered protein in the CSF of GBA1-PD patients, FKBP4, is upregulated in the GBA1-PD mDA neurons. Our identification of FKBP4, a member of the immunophilin protein family, suggests an involvement of immune systems in GBA1-associated PD.

Glycosphingolipids (GSLs) are brain-enriched lipids metabolized by lysosomal glycosidases, including GCase. Herein, we report that GBA1-PD mDA neurons exhibit decreased levels of α-2,3SpG, a neolacto-series GSL, compared with its isogenic control neurons. Also, we show a 1.39-fold elevation of α-synuclein release in GBA1-PD mDA neurons. These results highlight the alterations in GSL and α-synuclein secretion specific in GBA1-PD mDA neurons.

Prosaposin (PSAP) is a precursor protein of saposin C, an essential activator of GCase. To investigate the potential of PSAP/saposin C as a therapeutic target for PD, we employ gene overexpression systems and evaluate the role of PSAP in α-synuclein pathology. Human neuroblastoma SH-SY5Y cells stably overexpressing PSAP display enhanced GCase activities with a concomitant decrease in intracellular/extracellular α-synuclein levels. small interfering RNA-mediated knockdown of endogenous PSAP shows the opposite effect on α-synuclein levels, indicating the involvement of PSAP in α-synuclein regulation. Furthermore, we demonstrate that saposin C detaches α-synuclein from an artificial lipid bilayer membrane containing GlcCer, leading us to postulate that observed α-synuclein regulation of PSAP might be attributed to saposin C’s ability to interfere with α-synuclein-to-lipid membrane interaction.

To conclude, this thesis contributes to elucidating the missing link in our understanding of the molecular mechanisms underlying the elevated risk of PD among GBA1 mutation carriers, providing valuable insights into the pathogenesis of GBA1-associated PD.