SARS-CoV-2 Spike Protein Induces Time-Dependent CTSL Upregulation in HeLa Cells and Alveolarspheres.

Autophagy and lysosomal pathways are involved in the cell entry of SARS-CoV-2 virus. To infect the host cell, the spike protein of SARS-CoV-2 binds to the cell surface receptor angiotensin-converting enzyme 2 (ACE2). To allow the fusion of the viral envelope with the host cell membrane, the spike protein has to be cleaved. One possible mechanism is the endocytosis of the SARS-CoV-2-ACE2 complex and subsequent cleavage of the spike protein, mainly by the lysosomal protease cathepsin L. However, detailed molecular and dynamic insights into the role of cathepsin L in viral cell entry remain elusive. To address this, HeLa cells and iPSC-derived alveolarspheres were treated with recombinant SARS-CoV-2 spike protein, and the changes in mRNA and protein levels of cathepsins L, B, and D were monitored. Additionally, we studied the effect of cathepsin L deficiency on spike protein internalization and investigated the influence of the spike protein on cathepsin L promoters in vitro. Furthermore, we analyzed variants in the genes coding for cathepsin L, B, D, and ACE2 possibly associated with disease progression using data from Regeneron's COVID Results Browser and our own cohort of 173 patients with COVID-19, exhibiting a variant of ACE2 showing significant association with COVID-19 disease progression. Our in vitro studies revealed a significant increase in cathepsin L mRNA and protein levels following exposure to the SARS-CoV-2 spike protein in HeLa cells, accompanied by elevated mRNA levels of cathepsin B and D in alveolarspheres. Moreover, an increase in cathepsin L promoter activity was detected in vitro upon spike protein treatment. Notably, the knockout of cathepsin L resulted in reduced internalization of the spike protein. The study highlights the importance of cathepsin L and lysosomal proteases in the SARS-CoV-2 spike protein internalization and suggests the potential of lysosomal proteases as possible therapeutic targets against COVID-19 and other viral infections.

The hexosamine biosynthetic pathway rescues lysosomal dysfunction in Parkinson's disease patient iPSC derived midbrain neurons.

Disrupted glucose metabolism and protein misfolding are key characteristics of age-related neurodegenerative disorders including Parkinson's disease, however their mechanistic linkage is largely unexplored. The hexosamine biosynthetic pathway utilizes glucose and uridine-5'-triphosphate to generate N-linked glycans required for protein folding in the endoplasmic reticulum. Here we find that Parkinson's patient midbrain cultures accumulate glucose and uridine-5'-triphosphate, while N-glycan synthesis rates are reduced. Impaired glucose flux occurred by selective reduction of the rate-limiting enzyme, GFPT2, through disrupted signaling between the unfolded protein response and the hexosamine pathway. Failure of the unfolded protein response and reduced N-glycosylation caused immature lysosomal hydrolases to misfold and accumulate, while accelerating glucose flux through the hexosamine pathway rescued hydrolase function and reduced pathological α-synuclein. Our data indicate that the hexosamine pathway integrates glucose metabolism with lysosomal activity, and its failure in Parkinson's disease occurs by uncoupling of the unfolded protein response-hexosamine pathway axis. These findings offer new methods to restore proteostasis by hexosamine pathway enhancement.

Late-onset Krabbe disease presenting as spastic paraplegia - implications of GCase and CTSB/D.

OBJECTIVE: Krabbe disease (KD) is a multisystem neurodegenerative disorder with severe disability and premature death, mostly with an infancy/childhood onset. In rare cases of late-onset phenotypes, symptoms are often milder and difficult to diagnose. We here present a translational approach combining diagnostic and biochemical analyses of a male patient with a progressive gait disorder starting at the age of 44 years, with a final diagnosis of late-onset KD (LOKD). METHODS: Additionally to cerebral MRI, protein structural analyses of the ß-galactocerebrosidase protein (GALC) were performed. Moreover, expression, lysosomal localization, and activities of ß-glucocerebrosidase (GCase), cathepsin B (CTSB), and cathepsin D (CTSD) were analyzed in leukocytes, fibroblasts, and lysosomes of fibroblasts. RESULTS: Exome sequencing revealed biallelic likely pathogenic variants: GALC exons 11-17: 33 kb deletion; exon 4: missense variant (c.334A>G, p.Thr112Ala). We detected a reduced GALC activity in leukocytes and fibroblasts. While histological KD phenotypes were absent in fibroblasts, they showed a significantly decreased activities of GCase, CTSB, and CTSD in lysosomal fractions, while expression levels were unaffected. INTERPRETATION: The presented LOKD case underlines the age-dependent appearance of a mildly pathogenic GALC variant and its interplay with other lysosomal proteins. As GALC malfunction results in reduced ceramide levels, we assume this to be causative for the here described decrease in CTSB and CTSD activity, potentially leading to diminished GCase activity. Hence, we emphasize the importance of a functional interplay between the lysosomal enzymes GALC, CTSB, CTSD, and GCase, as well as between their substrates, and propose their conjoined contribution in KD pathology.

Activation and Purification of ß-Glucocerebrosidase by Exploiting its Transporter LIMP-2 - Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease.

Genetic variants of GBA1 can cause the lysosomal storage disorder Gaucher disease and are among the highest genetic risk factors for Parkinson's disease (PD). GBA1 encodes the lysosomal enzyme beta-glucocerebrosidase (GCase), which orchestrates the degradation of glucosylceramide (GluCer) in the lysosome. Recent studies have shown that GluCer accelerates α-synuclein aggregation, exposing GCase deficiency as a major risk factor in PD pathology and as a promising target for treatment. This study investigates the interaction of GCase and three disease-associated variants (p.E326K, p.N370S, p.L444P) with their transporter, the lysosomal integral membrane protein 2 (LIMP-2). Overexpression of LIMP-2 in HEK 293T cells boosts lysosomal abundance of wt, E326K, and N370S GCase and increases/rescues enzymatic activity of the wt and E326K variant. Using a novel purification approach, co-purification of untagged wt, E326K, and N370S GCase in complex with His-tagged LIMP-2 from cell supernatant of HEK 293F cells is achieved, confirming functional binding and trafficking for these variants. Furthermore, a single helix in the LIMP-2 ectodomain is exploited to design a lysosome-targeted peptide that enhances lysosomal GCase activity in PD patient-derived and control fibroblasts. These findings reveal LIMP-2 as an allosteric activator of GCase, suggesting a possible therapeutic potential of targeting this interaction.

Gut-to-brain spreading of pathology in synucleinopathies: A focus on molecular signalling mediators.

Synucleinopathies are a group of neurodegenerative disorders, classically characterized by the accumulation of aggregated alpha synuclein (aSyn) in the central nervous system. Parkinson's disease (PD) and multiple system atrophy (MSA) are the two prominent members of this family. Current treatment options mainly focus on the motor symptoms of these diseases. However, non-motor symptoms, including gastrointestinal (GI) symptoms, have recently gained particular attention, as they are frequently associated with synucleinopathies and often arise before motor symptoms. The gut-origin hypothesis has been proposed based on evidence of an ascending spreading pattern of aggregated aSyn from the gut to the brain, as well as the comorbidity of inflammatory bowel disease and synucleinopathies. Recent advances have shed light on the mechanisms underlying the progression of synucleinopathies along the gut-brain axis. Given the rapidly expanding pace of research in the field, this review presents a summary of the latest findings on the gut-to-brain spreading of pathology and potential pathology-reinforcing mediators in synucleinopathies. Here, we focus on 1) gut-to-brain communication pathways, including neuronal pathways and blood circulation, and 2) potential molecular signalling mediators, including bacterial amyloid proteins, microbiota dysbiosis-induced alterations in gut metabolites, as well as host-derived effectors, including gut-derived peptides and hormones. We highlight the clinical relevance and implications of these molecular mediators and their possible mechanisms in synucleinopathies. Moreover, we discuss their potential as diagnostic markers in distinguishing the subtypes of synucleinopathies and other neurodegenerative diseases, as well as for developing novel individualized therapeutic options for synucleinopathies.

Reciprocal effects of alpha-synuclein aggregation and lysosomal homeostasis in synucleinopathy models.

BACKGROUND: Lysosomal dysfunction has been implicated in a number of neurodegenerative diseases such as Parkinson's disease (PD). Various molecular, clinical and genetic studies have highlighted a central role of lysosomal pathways and proteins in the pathogenesis of PD. Within PD pathology the synaptic protein alpha-synuclein (αSyn) converts from a soluble monomer to oligomeric structures and insoluble amyloid fibrils. The aim of this study was to unravel the effect of αSyn aggregates on lysosomal turnover, particularly focusing on lysosomal homeostasis and cathepsins. Since these enzymes have been shown to be directly involved in the lysosomal degradation of αSyn, impairment of their enzymatic capacity has extensive consequences. METHODS: We used patient-derived induced pluripotent stem cells and a transgenic mouse model of PD to examine the effect of intracellular αSyn conformers on cell homeostasis and lysosomal function in dopaminergic (DA) neurons by biochemical analyses. RESULTS: We found impaired lysosomal trafficking of cathepsins in patient-derived DA neurons and mouse models with αSyn aggregation, resulting in reduced proteolytic activity of cathepsins in the lysosome. Using a farnesyltransferase inhibitor, which boosts hydrolase transport via activation of the SNARE protein ykt6, we enhanced the maturation and proteolytic activity of cathepsins and thereby decreased αSyn protein levels. CONCLUSIONS: Our findings demonstrate a strong interplay between αSyn aggregation pathways and function of lysosomal cathepsins. It appears that αSyn directly interferes with the enzymatic function of cathepsins, which might lead to a vicious cycle of impaired αSyn degradation. Lysosomal trafficking of cathepsin D (CTSD), CTSL and CTSB is disrupted when alpha-synuclein (αSyn) is aggregated. This results in a decreased proteolytic activity of cathepsins, which directly mediate αSyn clearance. Boosting the transport of the cathepsins to the lysosome increases their activity and thus contributes to efficient αSyn degradation.

Proteolysis of CD44 at the cell surface controls a downstream protease network.

The cell surface receptor cluster of differentiation 44 (CD44) is the main hyaluronan receptor of the human body. At the cell surface, it can be proteolytically processed by different proteases and was shown to interact with different matrix metalloproteinases. Upon proteolytic processing of CD44 and generation of a C-terminal fragment (CTF), an intracellular domain (ICD) is released after intramembranous cleavage by the γ-secretase complex. This intracellular domain then translocates to the nucleus and induces transcriptional activation of target genes. In the past CD44 was identified as a risk gene for different tumor entities and a switch in CD44 isoform expression towards isoform CD44s associates with epithelial to mesenchymal transition (EMT) and cancer cell invasion. Here, we introduce meprin ß as a new sheddase of CD44 and use a CRISPR/Cas9 approach to deplete CD44 and its sheddases ADAM10 and MMP14 in HeLa cells. We here identify a regulatory loop at the transcriptional level between ADAM10, CD44, MMP14 and MMP2. We show that this interplay is not only present in our cell model, but also across different human tissues as deduced from GTEx (Gene Tissue Expression) data. Furthermore, we identify a close relation between CD44 and MMP14 that is also reflected in functional assays for cell proliferation, spheroid formation, migration and adhesion.

From Lysosomal Storage Disorders to Parkinson's Disease - Challenges and Opportunities.

Lysosomes are specialized organelles with an acidic pH that act as recycling hubs for intracellular and extracellular components. They harbour numerous different hydrolytic enzymes to degrade substrates like proteins, peptides, and glycolipids. Reduced catalytic activity of lysosomal enzymes can cause the accumulation of these substrates and loss of lysosomal integrity, resulting in lysosomal dysfunction and lysosomal storage disorders (LSDs). Post-mitotic cells, such as neurons, seem to be highly sensitive to damages induced by lysosomal dysfunction, thus LSDs often manifest with neurological symptoms. Interestingly, some LSDs and Parkinson's disease (PD) share common cellular pathomechanisms, suggesting convergence of aetiology of the two disease types. This is further underlined by genetic associations of several lysosomal genes involved in LSDs with PD. The increasing number of lysosome-associated genetic risk factors for PD makes it necessary to understand functions and interactions of lysosomal proteins/enzymes both in health and disease, thereby holding the potential to identify new therapeutic targets. In this review, we highlight genetic and mechanistic interactions between the complex lysosomal network, LSDs and PD, and elaborate on methodical challenges in lysosomal research.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) is localized in lysosomes and mediates resistance against kinase inhibitors.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3), a splice variant of the hepatic uptake transporter OATP1B3 (liver-type; Lt-OATP1B3), is expressed in several tumor entities including colorectal carcinoma (CRC) and breast cancer. In CRC, high OATP1B3 expression has been associated with reduced progression-free and overall survival. Several kinase inhibitors used for antitumor treatment are substrates and/or inhibitors of OATP1B3 (e.g. encorafenib, vemurafenib). The functional importance of Ct-OATP1B3 has not been elucidated so far. HEK293 cells stably overexpressing Ct-OATP1B3 protein were established and compared with control cells. Confocal laser scanning microscopy, immunoblot, and proteomics-based expression analysis demonstrated that Ct-OATP1B3 protein is intracellularly localized in lysosomes of stably-transfetced cells. Cytotoxicity experiments showed that cells recombinantly expressing the Ct-OATP1B3 protein were more resistant against the kinase inhibitor encorafenib compared to control cells [e.g. encorafenib (100 µM) survival rates: 89.5% vs. 52.8%]. In line with these findings, colorectal cancer DLD1 cells endogenously expressing Ct-OATP1B3 protein had poorer survival rates when the OATP1B3 substrate bromosulfophthalein (BSP) was coincubated with encorafenib or vemurafenib compared to the incubation with the kinase inhibitor alone. This indicates a competitive inhibition of Ct-OATP1B3-mediated uptake into lysosomes by BSP. Accordingly, mass spectrometry-based drug analysis of lysosomes showed a reduced lysosomal accumulation of encorafenib in DLD1 cells additionally exposed to BSP. These results demonstrate that Ct-OATP1B3 protein is localized in the lysosomal membrane and can mediate transport of certain kinase inhibitors into lysosomes revealing a new mechanism of resistance. Significance Statement We describe the characterization of a splice variant of the liver-type uptake transporter OATP1B3 expressed in several tumor entities. This variant is localized in lysosomes mediating resistance against kinase inhibitors which are substrates of this transport protein by transporting them into lysosomes and thereby reducing the cytoplasmic concentration of these antitumor agents. Therefore, the expression of the Ct-OATP1B3 protein is associated with a better survival of cells revealing a new mechanism of drug resistance.

The Quest for Anti-α-Synuclein Antibody Specificity-Lessons Learnt From Flow Cytometry Analysis.

The accumulation of alpha-synuclein (aSyn) is the hallmark of a group of neurodegenerative conditions termed synucleopathies. Physiological functions of aSyn, including those outside of the CNS, remain elusive. However, a reliable and reproducible evaluation of aSyn protein expression in different cell types and especially in low-expressing cells is impeded by the existence of a huge variety of poorly characterized anti-aSyn antibodies and a lack of a routinely used sensitive detection methods. Here, we developed a robust flow cytometry-based workflow for aSyn detection and antibody validation. We test our workflow using three commercially available antibodies (MJFR1, LB509, and 2A7) in a variety of human cell types, including induced pluripotent stem cells, T lymphocytes, and fibroblasts, and provide a cell- and antibody-specific map for aSyn expression. Strikingly, we demonstrate a previously unobserved unspecificity of the LB509 antibody, while the MJFR1 clone revealed specific aSyn binding however with low sensitivity. On the other hand, we identified an aSyn-specific antibody clone 2A7 with an optimal sensitivity for detecting aSyn in a range of cell types, including those with low aSyn expression. We further utilize our workflow to demonstrate the ability of the 2A7 antibody to distinguish between physiological differences in aSyn expression in neuronal and non-neuronal cells from the cortical organoids, and in neural progenitors and midbrain dopaminergic neurons from healthy controls and in patients with Parkinson's disease who have aSyn gene locus duplication. Our results provide a proof of principle for the use of high-throughput flow cytometry-based analysis of aSyn and highlight the necessity of rigorous aSyn antibody validation to facilitate the research of aSyn physiology and pathology.

Detection of neuron-derived pathological α-synuclein in blood.

To date, no reliable clinically applicable biomarker has been established for Parkinson's disease. Our results indicate that a long anticipated blood test for Parkinson's disease may be realized. Following the isolation of neuron-derived extracellular vesicles of Parkinson's disease patients and non-Parkinson's disease individuals, immunoblot analyses were performed to detect extracellular vesicle-derived α-synuclein. Pathological α-synuclein forms derived from neuronal extracellular vesicles could be detected under native conditions and were significantly increased in all individuals with Parkinson's disease and clearly distinguished disease from the non-disease state. By performing an α-synuclein seeding assay these soluble conformers could be amplified and seeding of pathological protein folding was demonstrated. Amplified α-synuclein conformers exhibited ß-sheet-rich structures and a fibrillary appearance. Our study demonstrates that the detection of pathological α-synuclein conformers from neuron-derived extracellular vesicles from blood plasma samples has the potential to evolve into a blood-biomarker of Parkinson's disease that is still lacking so far. Moreover, the distribution of seeding-competent α-synuclein within blood exosomes sheds a new light of pathological disease mechanisms in neurodegenerative disorders.

The role of triggering receptor expressed on myeloid cells 2 in Parkinson's disease and other neurodegenerative disorders.

Parkinson's disease (PD) is a progressive neurological disorder marked by cardinal clinical symptoms such as rigor, tremor, and akinesia. Albeit a loss of dopaminergic neurons from the substantia nigra pars compacta is causative for the movement impairments found in patients, molecular reasoning for this loss is still incomplete. In recent years, triggering factor expressed on myeloid cells (TREM2) gained attention in the field of neurodegeneration as it could be associated with different neurodegenerative disorders. Primarily identified as a risk factor in Alzheimer's disease, variants in TREM2 were linked to PD and multiple sclerosis, too. Expressed on phagocytic cells, such as macrophages and microglia, TREM2 puts the focus on inflammation associated conditions in PD and provides a molecular target that could at least partly explain the role of immune cells in PD. Here, we summarize expression patterns and molecular functions of TREM2, recapitulate on its role in inflammation, phagocytosis and cell survival, before turning to neurodegenerative disorders with an emphasis on PD.

Molecular dynamics simulations of the delta and omicron SARS-CoV-2 spike - ACE2 complexes reveal distinct changes between both variants.

SARS-CoV-2, the virus causing the COVID-19 pandemic, changes frequently through the appearance of mutations constantly leading to new variants. However, only few variants evolve as dominating and will be considered as "Variants of Concern" (VOCs) by the world health organization (WHO). At the end of 2020 the alpha (B.1.1.7) variant appeared in the United Kingdom and dominated the pandemic situation until mid of 2021 when it was substituted by the delta variant (B.1.617.2) that first appeared in India as predominant. At the end of 2021, SARS-CoV-2 omicron (B.1.1.529) evolved as the dominating variant. Here, we use in silico modeling and molecular dynamics (MD) simulations of the receptor-binding domain of the viral spike protein and the host cell surface receptor ACE2 to analyze and compare the interaction pattern between the wild type, delta and omicron variants. We identified residue 493 in delta (glutamine) and omicron (arginine) with altered binding properties towards ACE2.

Recombinant pro-CTSD (cathepsin D) enhances SNCA/α-Synuclein degradation in α-Synucleinopathy models.

Parkinson disease (PD) is a neurodegenerative disorder characterized by the abnormal intracellular accumulation of SNCA/α-synuclein. While the exact mechanisms underlying SNCA pathology are not fully understood, increasing evidence suggests the involvement of autophagy as well as lysosomal deficiencies. Because CTSD (cathepsin D) has been proposed to be the major lysosomal protease involved in SNCA degradation, its deficiency has been linked to the presence of insoluble SNCA conformers in the brain of mice and humans as well as to the transcellular transmission of SNCA aggregates. We here postulate that SNCA degradation can be enhanced by the application of the recombinant human proform of CTSD (rHsCTSD). Our results reveal that rHsCTSD is efficiently endocytosed by neuronal cells, correctly targeted to lysosomes and matured to an enzymatically active protease. In dopaminergic neurons derived from induced pluripotent stem cells (iPSC) of PD patients harboring the A53T mutation within the SNCA gene, we confirm the reduction of insoluble SNCA after treatment with rHsCTSD. Moreover, we demonstrate a decrease of pathological SNCA conformers in the brain and within primary neurons of a ctsd-deficient mouse model after dosing with rHsCTSD. Boosting lysosomal CTSD activity not only enhanced SNCA clearance in human and murine neurons as well as tissue, but also restored endo-lysosome and autophagy function. Our findings indicate that CTSD is critical for SNCA clearance and function. Thus, enzyme replacement strategies utilizing CTSD may also be of therapeutic interest for the treatment of PD and other synucleinopathies aiming to decrease the SNCA burden.Abbreviations: aa: amino acid; SNCA/α-synuclein: synuclein alpha; APP: amyloid beta precursor protein; BBB: blood brain barrier; BF: basal forebrain; CBB: Coomassie Brilliant Blue; CLN: neuronal ceroid lipofuscinosis; CNL10: neuronal ceroid lipofuscinosis type 10; Corr.: corrected; CTSD: cathepsin D; CTSB: cathepsin B; DA: dopaminergic; DA-iPSn: induced pluripotent stem cell-derived dopaminergic neurons; dox: doxycycline; ERT: enzyme replacement therapy; Fx: fornix, GBA/ß-glucocerebrosidase: glucosylceramidase beta; h: hour; HC: hippocampus; HT: hypothalamus; i.c.: intracranially; IF: immunofluorescence; iPSC: induced pluripotent stem cell; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LSDs: lysosomal storage disorders; MAPT: microtubule associated protein tau; M6P: mannose-6-phosphate; M6PR: mannose-6-phosphate receptor; MB: midbrain; mCTSD: mature form of CTSD; neurofil.: neurofilament; PD: Parkinson disease; proCTSD: proform of CTSD; PRNP: prion protein; RFU: relative fluorescence units; rHsCTSD: recombinant human proCTSD; SAPC: Saposin C; SIM: structured illumination microscopy; T-insol: Triton-insoluble; T-sol: Triton-soluble; TEM: transmission electron microscopy, TH: tyrosine hydroxylase; Thal: thalamus.

The role of lysosomal cathepsins in neurodegeneration: Mechanistic insights, diagnostic potential and therapeutic approaches.

Lysosomes are ubiquitous organelles with a fundamental role in maintaining cellular homeostasis by mediating degradation and recycling processes. Cathepsins are the most abundant lysosomal hydrolyses and are responsible for the bulk degradation of various substrates. A correct autophagic function is essential for neuronal survival, as most neurons are post-mitotic and thus susceptible to accumulate cellular components. Increasing evidence suggests a crucial role of the lysosome in neurodegeneration as a key regulator of aggregation-prone and disease-associated proteins, such as α-synuclein, ß-amyloid and huntingtin. Particularly, alterations in lysosomal cathepsins CTSD, CTSB and CTSL can contribute to the pathogenesis of neurodegenerative diseases as seen for neuronal ceroid lipofuscinosis, synucleinopathies (Parkinson's disease, Dementia with Lewy Body and Multiple System Atrophy) as well as Alzheimer's and Huntington's disease. In this review, we provide an overview of recent evidence implicating CTSD, CTSB and CTSL in neurodegeneration, with a special focus on the role of these enzymes in α-synuclein metabolism. In addition, we summarize the potential role of lysosomal cathepsins as clinical biomarkers in neurodegenerative diseases and discuss potential therapeutic approaches by targeting lysosomal function.

Interaction of Alpha Synuclein and Microtubule Organization Is Linked to Impaired Neuritic Integrity in Parkinson's Patient-Derived Neuronal Cells.

Parkinson's disease (PD) is neuropathologically characterized by the loss of dopaminergic neurons and the deposition of aggregated alpha synuclein (aSyn). Mounting evidence suggests that neuritic degeneration precedes neuronal loss in PD. A possible underlying mechanism could be the interference of aSyn with microtubule organization in the neuritic development, as implied by several studies using cell-free model systems. In this study, we investigate the impact of aSyn on microtubule organization in aSyn overexpressing H4 neuroglioma cells and midbrain dopaminergic neuronal cells (mDANs) generated from PD patient-derived human induced pluripotent stem cells (hiPSCs) carrying an aSyn gene duplication (SNCADupl). An unbiased mass spectrometric analysis reveals a preferential binding of aggregated aSyn conformers to a number of microtubule elements. We confirm the interaction of aSyn with beta tubulin III in H4 and hiPSC-derived mDAN cell model systems, and demonstrate a remarkable redistribution of tubulin isoforms from the soluble to insoluble fraction, accompanied by a significantly increased insoluble aSyn level. Concordantly, SNCADupl mDANs show impaired neuritic phenotypes characterized by perturbations in neurite initiation and outgrowth. In summary, our findings suggest a mechanistic pathway, through which aSyn aggregation interferes with microtubule organization and induces neurite impairments.

Rescue of α-synuclein aggregation in Parkinson's patient neurons by synergistic enhancement of ER proteostasis and protein trafficking.

Neurodegenerative disorders are characterized by a collapse in proteostasis, as shown by the accumulation of insoluble protein aggregates in the brain. Proteostasis involves a balance of protein synthesis, folding, trafficking, and degradation, but how aggregates perturb these pathways is unknown. Using Parkinson's disease (PD) patient midbrain cultures, we find that aggregated α-synuclein induces endoplasmic reticulum (ER) fragmentation and compromises ER protein folding capacity, leading to misfolding and aggregation of immature lysosomal ß-glucocerebrosidase. Despite this, PD neurons fail to initiate the unfolded protein response, indicating perturbations in sensing or transducing protein misfolding signals in the ER. Small molecule enhancement of ER proteostasis machinery promotes ß-glucocerebrosidase solubility, while simultaneous enhancement of trafficking improves ER morphology, lysosomal function, and reduces α-synuclein. Our studies suggest that aggregated α-synuclein perturbs the ability of neurons to respond to misfolded proteins in the ER, and that synergistic enhancement of multiple proteostasis branches may provide therapeutic benefit in PD.