Objective: We firstly aim to identify modifiers of β-glucocerebrosidase (GCase) activity and expression in Parkinson’s disease (PD) relevant cell models. We secondly aim to ascertain whether GCase could function in an auto-regulatory manner, influencing its own expression and activity.

Background: Mutations in GBA are the greatest numerical risk factor of PD, however, the pathological mechanisms underpinning this association are not understood. A lysosomal network, comprising progranulin (GRN), prosaposin (PSAP), and cathepsins B and D (CTSB/D), has been implicated in GBA-associated PD. We aim to investigate this lysosomal network in both healthy and PD cell models.

Method: In SH-SY5Y cells, we used siRNA technology to genetically knockdown (KD) each network component to determine any interdependency of expression, as well as evaluate any effect on GCase activity.
In the human iPSC system, we used a novel protocol to generate midbrain dopaminergic (mDA) neurons from healthy controls and patients with GBA mutations (heterozygous and homozygous p.N370S). In the mature neurons, we measured the gene and protein expression of the lysosomal network and investigated cellular phenotypes using live-cell imaging.

Results: In the SH-SY5Ys, we found that KD of prosaposin reduced GCase levels, likely due to the stabilising effect of its peptide, Saposin C, on GCase. This was mirrored by a reduction in GCase activity. However, we did not observe any effects on GCase activity upon other pathway components, potentially due to the in vitro nature of this assay.
In the GBA mutant iPSC-derived neurons, we observed an increase in lysosome number and swelling, as well as mitochondrial fragmentation. Under basal conditions, we found no changes in the expression of any members of the lysosomal network, except for GCase itself – which was reduced in a dosage-dependent manner. This was corroborated by a reduction in GCase activity.

Conclusion: We have corroborated the role for prosaposin in GCase activation in our SH-SY5Y models using in vitro assays and are currently using in vivo assays to ascertain any influence from other lysosomal network members.
We successfully generated iPSC-derived mDA neurons from GBA mutant iPSCs. Although we observed cellular phenotypes, we did not observe changes in the lysosomal network under basal conditions. This model may require additional stressors, such as α-synuclein aggregation, to induce such changes.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/investigation-of-modifiers-of-%ce%b2-glucocerebrosidase-in-cell-models-of-parkinsons-disease/