Mitochondrial localization and function of the lysosomal enzyme glucocerebrosidase

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dc.contributor.advisor Deleidi, Michela (Prof. Dr. Dr.) Baden, Pascale 2023-08-14T10:31:32Z 2023-08-14T10:31:32Z 2023-08-14
dc.identifier.uri de_DE
dc.description.abstract Biallelic mutations in the GBA1 gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase), cause the lysosomal storage disease Gaucher's disease (GD). These mutations have been recently identified as the strongest risk factor for Parkinson’s disease (PD) and other synucleinopathies. In the lysosome, GCase catalyzes the hydrolysis of glucosylceramide (GlcCer), a membrane glycosphingolipid (GSL), to ceramide and glucose and of glucosylsphingosine to glucose and sphingosine. Interestingly, a reduction in GCase activity has also been observed in sporadic PD as well as with ageing. The mechanisms leading to neurodegeneration in GBA1 carriers remain unclear. To explore the mechanisms involved in GBA1-linked neurodegeneration, we assessed the interactome of wild-type (WT) and mutant GCase. To this end, we developed an inducible T-Rex HEK cell model overexpressing V5-Flag-tagged WT, p.E326K, or p.L444P mutant GCase. We chose these two mutants as p.L444P is a severe mutation leading to a neuropathic form of GD and as it is one of the most common severe GBA1-PD mutations. p.E326K is another common mutation in GBA1-PD. However, unlike most other GBA1 mutations, p.E326K homozygous mutations on their own are not linked to GD. The overexpression model was validated by the analysis of GCase expression and protein level, as well as co-localization with the lysosomal marker LAMP1. We performed Flag co-immunoprecipitation and analyzed the eluates by quantitative tandem mass tag liquid chromatography mass spectrometry. Interestingly, we found that 13.3% of GCase interactors are mitochondrial proteins. As mitochondrial dysfunction has previously been linked to GBA1, we further explored a potential direct link between GCase and mitochondrial dysfunction. First, we validated the mitochondrial localization of GCase by split-GFP experiments, in which the interaction between mitochondrial matrix targeted GFP1-10 and WT and mutant GBA1-GFP-S11ß lead to green fluorescence. We demonstrated that the import of GCase into the mitochondrial matrix is HSC70, translocase of outer mitochondrial membrane (TOM)-, and translocase of inner mitochondrial membrane (TIM)-complex dependent. Removal of the internal mitochondrial targeting like sequences prevented import of GCase into mitochondria. In addition, we found an increased interaction between HSP60 and LONP1 with mutant GCase, suggesting their potential role in the refolding or degradation of mutant GCase in mitochondria. Furthermore, we found a decreased interaction of mutant GCase with the complex I (CI) assembly factor TIMMDC1 and the CI subunit NDUFA10. To validate these results in a model relevant to GBA1-PD, we generated induced pluripotent stem cells (iPSCs) from p.L444P and p.E326K heterozygous PD patients’ fibroblasts. Next we employed genome editing (zinc finger nucleases and CRISPR-Cas9) to generate corresponding isogenic gene-corrected controls. Gene-correction rescued GCase protein level and activity. Mutant and isogenic controls did not show differences in midbrain neuron differentiation potential. The interaction with HSP60 and LONP1 was validated by co-immunoprecipitation with endogenous GCase in neural precursor cell (NPC) lysates. Preliminary data point towards CI assembly defects in mutant and GBA1 KO NPCs as well as midbrain neurons. These data confirm the potential involvement of GCase in CI assembly. Improving GCase trafficking to mitochondria could be a potential therapeutic target as overexpression of GCase improved mitochondrial dysfunction in T-Rex HEK cells. Our data provide evidence for a novel mitochondrial role of GCase, showing its potential involvement in the maintenance of CI integrity by modulating the stability of the assembly factor TIMMDC1. en
dc.language.iso en de_DE
dc.publisher Universität Tübingen de_DE
dc.rights ubt-podok de_DE
dc.rights.uri de_DE
dc.rights.uri en
dc.subject.ddc 500 de_DE
dc.subject.ddc 570 de_DE
dc.subject.ddc 610 de_DE
dc.title Mitochondrial localization and function of the lysosomal enzyme glucocerebrosidase en
dc.type PhDThesis de_DE
dcterms.dateAccepted 2023-06-07
utue.publikation.fachbereich Medizin de_DE
utue.publikation.fakultaet 4 Medizinische Fakultät de_DE
utue.publikation.noppn yes de_DE


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