Mutations in GBA1, the gene encoding the lysosomal enzyme β-glucocerebrosidase (GCase), which cause Gaucher’s disease, are the most frequent genetic risk factor for Parkinson’s disease (PD). Here, we employ global proteomic and single-cell genomic approaches in stable cell lines as well as induced pluripotent stem cell (iPSC)-derived neurons and midbrain organoids to dissect the mechanisms underlying GCase-related neurodegeneration. We demonstrate that GCase can be imported from the cytosol into the mitochondria via recognition of internal mitochondrial targeting sequence-like signals. In mitochondria, GCase promotes the maintenance of mitochondrial complex I (CI) integrity and function. Furthermore, GCase interacts with the mitochondrial quality control proteins HSP60 and LONP1. Disease-associated mutations impair CI stability and function and enhance the interaction with the mitochondrial quality control machinery. These findings reveal a mitochondrial role of GCase and suggest that defective CI activity and energy metabolism may drive the pathogenesis of GCase-linked neurodegeneration.

Mitochondrial disorders (MD) comprise a group of heterogeneous clinical disorders for which non-invasive diagnosis remains a challenge. Two protein biomarkers have so far emerged for MD detection, FGF-21 and GDF-15, but the identification of additional biomarkers capable of improving their diagnostic accuracy is highly relevant. Previous studies identified Gelsolin as a regulator of cell survival adaptations triggered by mitochondrial defects. Gelsolin presents a circulating plasma isoform (pGSN), whose altered levels could be a hallmark of mitochondrial dysfunction. Therefore, we investigated the diagnostic performance of pGSN for MD relative to FGF-21 and GDF-15. Using ELISA assays, we quantified plasma levels of pGSN, FGF-21, and GDF-15 in three age- and gender-matched adult cohorts: 60 genetically diagnosed MD patients, 56 healthy donors, and 41 patients with unrelated neuromuscular pathologies (non-MD). Clinical variables and biomarkers’ plasma levels were compared between groups. Discrimination ability was calculated using the area under the ROC curve (AUC). Optimal cut-offs and the following diagnostic parameters were determined: sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios, and efficiency. Comprehensive statistical analyses revealed significant discrimination ability for the three biomarkers to classify between MD and healthy individuals, with the best diagnostic performance for the GDF-15/pGSN combination. pGSN and GDF-15 preferentially discriminated between MD and non-MD patients under 50 years, whereas FGF-21 best classified older subjects. Conclusion: pGSN improves the diagnosis accuracy for MD provided by FGF-21 and GDF-15.

Mitochondrial oxidative phosphorylation (OXPHOS) defects are the primary cause of inborn errors of energy metabolism. Despite considerable progress on their genetic basis, their global pathophysiological consequences remain undefined. Previous studies reported that OXPHOS dysfunction associated with complex III deficiency exacerbated the expression and mitochondrial location of cytoskeletal gelsolin (GSN) to promote cell survival responses. In humans, besides the cytosolic isoform, GSN presents a plasma isoform secreted to extracellular environments. We analyzed the interplay between both GSN isoforms in human cellular and clinical models of OXPHOS dysfunction. Regardless of its pathogenic origin, OXPHOS dysfunction induced the physiological upregulation of cytosolic GSN in the mitochondria (mGSN), in parallel with a significant downregulation of plasma GSN (pGSN) levels. Consequently, significantly high mGSN-to-pGSN ratios were associated with OXPHOS deficiency both in human cells and blood. In contrast, control cells subjected to hydrogen peroxide or staurosporine treatments showed no correlation between oxidative stress or cell death induction and the altered levels and subcellular location of GSN isoforms, suggesting their specificity for OXPHOS dysfunction. In conclusion, a high mitochondrial-to-plasma GSN ratio represents a useful cellular indicator of OXPHOS defects, with potential use for future research of a wide range of clinical conditions with mitochondrial involvement.

Mutations in the lysosomal enzyme β-glucocerebrosidase (GCase), which cause Gaucher's disease, are the most frequent genetic risk factor for Parkinson’s disease (PD). Here, we employed global proteomic and single-cell genomic approaches in stable cell lines as well as induced pluripotent stem cell (iPSC)-derived neurons and midbrain organoids to dissect the mechanisms underlying GCase-related neurodegeneration. We demonstrate that a fraction of GCase can be imported from the cytosol into the mitochondria via the translocase of the outer mitochondrial membrane (TOM) import machinery via recognition of internal mitochondrial targeting sequence-like signals. In mitochondria, GCase promotes the assembly and function of mitochondrial complex I (CI). Furthermore, GCase interacts with the mitochondrial quality control proteins HSP60 and LONP1. Disease-associated mutations impair the assembly and function of CI and enhance the interaction with the mitochondrial quality control machinery. These findings reveal a previously unknown function of GCase in mitochondria and suggest that defective CI activity and energy metabolism may drive the pathogenesis of GCase-driven neurodegeneration.