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Background Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinson’s disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Methods Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines, induced pluripotent stem cell-derived dopaminergic neurons and midbrain organoids and assessed lysosomal activity and the handling of aggregated synuclein fibrils. Results We find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1 ) activity, and leads to an accumulation of lysosomal content. In cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. In midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. Conclusions These results indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

Background Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R. Methods Serial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. Results The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 ( P2RY12 ) expression, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam 3 CSK 4 . Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1R WT/KO and CSF1R WT/E633K iMGL compared to their respective isogenic controls. Conclusions We optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory, phagocytic and inflammatory activities. Graphical Abstract

In Parkinson′s disease and other synucleinopathies, α-synuclein (α-Syn) misfolds and forms Ser129-phosphorylated aggregates (pSyn129). The factors controlling this process are largely unknown. Here, we used arrayed CRISPR-mediated gene activation and ablation to discover new pSyn129 modulators. Using quadruple-guide RNAs (qgRNAs) and Cas9, or an inactive Cas9 version fused to a synthetic transactivator, we ablated 2304 and activated 2428 human genes related to mitochondrial, trafficking and motility function in HEK293 cells. After exposure of cells to α-Syn fibrils, pSyn129 signals were recorded by high-throughput fluorescence microscopy and aggregates were identified by image analysis. We found that pSyn129 was increased by activating the mitochondrial protein OXR1, which decreased ATP levels and altered the mitochondrial membrane potential. Instead, pSyn129 was reduced by ablation of the endoplasmic reticulum (ER)-associated protein EMC4, which enhanced ER-driven autophagic flux and lysosomal clearance. OXR1 activation preferentially modulated cellular reactions to fibrils derived from multiple system atrophy (MSA) patients, whereas EMC4 ablation broadly reduced pSyn129 across diverse α-Syn polymorphs. These findings were confirmed in human iPSC-derived cortical and dopaminergic neurons, where OXR1 preferentially promoted somatic aggregation and EMC4 reduced both somatic and neuritic aggregates. These results uncover previously unrecognized roles for OXR1 and EMC4 in α-Syn aggregation, thereby broadening our mechanistic understanding of synucleinopathies.

Variants in the gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinson's disease (PD). However, neither the specific variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Here, we conducted genetic analyses of the association between common and rare variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines and induced pluripotent stem cell-derived dopaminergic neurons and assessed lysosomal activity and the handling of aggregated synuclein fibrils. We find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by ) activity, and leads to an accumulation of lysosomal content. In cell lines, reduction of gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas gene activation enhances fibril clearance. In midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. These results indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

The α-synuclein protein, encoded by SNCA gene, is a major constituent of pathological intracellular inclusions such as Lewy bodies found in the brains of patients with Parkinson’s disease and other synucleinopathies. Whereas α-synuclein phosphorylation has been much studied, comparatively less work has been devoted to other post-translational modifications such as acetylation, especially given that N-terminally acetylated α-synuclein is the most abundant endogenous form of the protein in the brain. In this study, using multiple in vitro and in vivo models, we sought to better understand the role of N-terminal acetylation in the pathogenesis of synucleinopathies. We found that N-terminal acetylation slowed aggregation of both α-synuclein monomers and pre-formed fibrils in vitro. Uptake of acetylated α-synuclein pre-formed fibrils into both immortalized cell lines and iPSC-derived dopamine neurons was also slowed compared non-acetylated fibrils. In addition, exposure to acetylated pre-formed fibrils induced less seeding of endogenous α-synuclein, as measured by the accumulation of Serine129-phosphorylated α-synuclein inclusions in both iPSC-derived dopamine neurons and mouse brain. Finally, mice injected with N-terminally acetylated α-synuclein pre-formed fibrils survived significantly longer than mice injected with non-acetylated fibrils. Taken together, our study indicates that N-terminal acetylation reduces α-synuclein aggregation, uptake into cells, seeding of endogenous α-synuclein, and toxicity in vivo, suggesting that this prevalent post-translational modification represents a potent, physiologically relevant protective mechanism, which has thus far largely not been taken into consideration in most experimental paradigms of Parkinson’s disease and synucleinopathies.

Mutations in PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive early onset Parkinson disease (PD). PINK1 is a Ser/Thr kinase that regulates mitochondrial quality control by triggering mitophagy mediated by the ubiquitin ligase Parkin. Upon mitochondrial damage, PINK1 accumulates on the outer mitochondrial membrane (OMM) forming a high molecular weight complex with the translocase of the outer membrane (TOM). PINK1 then phosphorylates ubiquitin, which enables recruitment and activation of Parkin followed by autophagic clearance of the damaged mitochondrion. Thus, Parkin-dependent mitophagy hinges on the stable accumulation of PINK1 on the TOM complex. Yet, the mechanism linking mitochondrial stressors to PINK1 accumulation and whether the translocases of the inner membrane (TIMs) are also involved, remain unclear. Herein, we demonstrate that mitochondrial stress induces the formation of a PINK1-TOM-TIM23 supercomplex in human cultured cell lines, dopamine neurons, and midbrain organoids. Moreover, we show that PINK1 is required to stably tether the TOM to TIM23 complexes in response to stress, such that the supercomplex fails to accumulate in cells lacking PINK1. This tethering is dependent on an interaction between the PINK1 NT-CTE module and the cytosolic domain of the Tom20 subunit of the TOM complex, the disruption of which, by either designer or PD-associated PINK1 mutations, inhibits downstream mitophagy. Together, the findings provide key insight into how PINK1 interfaces with the mitochondrial import machinery, with important implications for the mechanisms of mitochondrial quality control and PD pathogenesis.