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Background: Synucleinopathies are a group of neurodegenerative disorders that are pathologically characterized by intracellular aggregates called Lewy bodies. Lewy bodies are primarily composed of α-synuclein (asyn) protein, which is mostly phosphorylated at serine 129 (pS129) when aggregated and therefore used as a marker for pathology. Currently commercial antibodies against pS129 asyn stain aggregates well but in healthy brains cross react with other proteins, thus making it difficult to specifically detect physiological pS129 asyn. Objective: To develop a staining procedure that detects endogenous and physiological relevant pS129 asyn with high specificity and low background. Methods: We used the fluorescent and brightfield in situ proximity ligation assay (PLA) to specifically detect pS129 asyn in cell culture, mouse, and human brain sections. Results: The pS129 asyn PLA specifically stained physiological and soluble pS129 asyn in cell culture, mouse brain sections, and human brain tissue without significant cross-reactivity or background signal. However, this technique was not successful in detecting Lewy bodies in human brain tissue. Conclusion: We successfully developed a novel PLA method that can, in the future, be used on in vitro and in vivo samples as a tool to explore and better understand the cellular localization and function of pS129 asyn in health and disease.

Parkinson's disease is a common incurable neurodegenerative disease. The identification of genetic variants via genome-wide association studies has considerably advanced our understanding of the Parkinson's disease genetic risk. Understanding the functional significance of the risk loci is now a critical step towards translating these genetic advances into an enhanced biological understanding of the disease. Impaired mitophagy is a key causative pathway in familial Parkinson's disease, but its relevance to idiopathic Parkinson's disease is unclear. We used a mitophagy screening assay to evaluate the functional significance of risk genes identified through genome-wide association studies. We identified two new regulators of PINK1-dependent mitophagy initiation, KAT8 and KANSL1, previously shown to modulate lysine acetylation. These findings suggest PINK1-mitophagy is a contributing factor to idiopathic Parkinson's disease. KANSL1 is located on chromosome 17q21 where the risk associated gene has long been considered to be MAPT. While our data do not exclude a possible association between the MAPT gene and Parkinson's disease, they provide strong evidence that KANSL1 plays a crucial role in the disease. Finally, these results enrich our understanding of physiological events regulating mitophagy and establish a novel pathway for drug targeting in neurodegeneration. ispartof: BRAIN vol:145 issue:12 pages:4349-4367 ispartof: location:England status: published

Alterations in the p38 mitogen-activated protein kinases (MAPKs) play an important role in the pathogenesis of dementia with Lewy bodies (DLB) and Parkinson’s disease (PD). Activation of the p38α MAPK isoform and mislocalization of the p38γ MAPK isoform are associated with neuroinflammation and synaptic degeneration in DLB and PD. Therefore, we hypothesized that p38α might be associated with neuronal p38γ distribution and synaptic dysfunction in these diseases. To test this hypothesis, we treated in vitro cellular and in vivo mouse models of DLB/PD with SKF-86002, a compound that attenuates inflammation by inhibiting p38α/β, and then investigated the effects of this compound on p38γ and neurodegenerative pathology. We found that inhibition of p38α reduced neuroinflammation and ameliorated synaptic, neurodegenerative, and motor behavioral deficits in transgenic mice overexpressing human α-synuclein. Moreover, treatment with SKF-86002 promoted the redistribution of p38γ to synapses and reduced the accumulation of α-synuclein in mice overexpressing human α-synuclein. Supporting the potential value of targeting p38 in DLB/PD, we found that SKF-86002 promoted the redistribution of p38γ in neurons differentiated from iPS cells derived from patients with familial PD (carrying the A53T α-synuclein mutation) and healthy controls. Treatment with SKF-86002 ameliorated α-synuclein–induced neurodegeneration in these neurons only when microglia were pretreated with this compound. However, direct treatment of neurons with SKF-86002 did not affect α-synuclein–induced neurotoxicity, suggesting that SKF-86002 treatment inhibits α-synuclein–induced neurotoxicity mediated by microglia. These findings provide a mechanistic connection between p38α and p38γ as well as a rationale for targeting this pathway in DLB/PD.

Single nuclear RNA-sequencing was performed on human post-mortem brain tissue from four distinct nuclei that have implications for neurodegenerative diseases, including Alzheimer's and Parkinson’s diseases. Younger, aged 20s to 30s, and middle-aged and older, aged 60s to 80s, adults were used to identify changes in cell populations and transcriptomes during aging. Files included: aging.glmmtmb_age_diffs.csv.gz - All results modeled using glmmTMB aging.glmmtmb_age_diffs_fdr.csv.gz - glmmTMB results with a statistically significant result Column headers for the glmmtmb results are: feature - gene name intercept - regression intercept estimate - regression coefficient std.error - standard error statistic - regression test statistic p.value - regression p-value tissue - the brain region or cell-type (depending on 'type' column) type - type for 'tissue' column, brain_region, specific or broad cell-type fdr_bh - B&H FDR multiple-test corrected p-value aging.diffxpy_age_diffs.csv.gz - All results using diffxpy differential expression aging.diffxpy_age_diffs_fdr.csv.gz - diffxpy results with a statistically significant result Column headers for the diffxpy results are: gene - gene name pval - p-value for differential expression qval - q-value for differential expression; multiple-test corrected p-value log2fc - log base 2 fold change in expression mean - zero_mean - zero_variance - tissue - the brain region or cell-type (depending on 'type' column) type - type for 'tissue' column, brain_region, specific or broad cell-type fdr_bh - B&H FDR cmultiple-test orrected p-value &nbsp

Objective To define the transcriptomic changes responsible for the histologic alterations in skeletal muscle and their progression in collagen VI‐related muscular dystrophy (COL6‐RD). Methods COL6‐RD patient muscle biopsies were stratified into three groups based on the overall level of pathologic severity considering degrees of fibrosis, muscle fiber atrophy, and fatty replacement of muscle tissue. Using microarray and RNA‐Seq, we then performed global gene expression profiling on the same muscle biopsies and compared their transcriptome with age‐ and sex‐matched controls. Results COL6‐RD muscle biopsy transcriptomes as a group revealed prominent upregulation of muscle extracellular matrix component genes and the downregulation of skeletal muscle and mitochondrion‐specific genes. Upregulation of the TGFβ pathway was the most conspicuous change across all biopsies and was fully evident even in the mildest/earliest histological group. There was no difference in the overall transcriptional signature between the different histologic groups but polyserial analysis identified relative changes along with COL6‐RD histological severity. Interpretation Overall, our study establishes the prominent dysregulation of extracellular matrix genes, TGFβ signaling, and its downstream cellular pathways at the transcriptomic level in COL6‐RD muscle.

Lysosomes are dynamic organelles that can remodel their membrane as an adaptive response to various cell signaling events including membrane damage. Recently, we have discovered that damaged lysosomes form and sort tubules into moving vesicles. We named this process LYTL for LYsosomal Tubulation/sorting driven by LRRK2, as the Parkinson's disease protein LRRK2 promotes tubulation by recruiting the motor adaptor protein JIP4 to lysosomes via phosphorylated RAB proteins. Here we use spinning-disk microscopy combined with superresolution to further characterize LYTL after membrane damage with LLOMe (l-leucyl-l-leucine methyl ester). We identified the endoplasmic reticulum (ER) colocalizing with sites of fission of lysosome-derived tubules. In addition, modifying the morphology of the ER by reducing ER tubules leads to a decrease in LYTL sorting, suggesting that contact with tubular ER is necessary for lysosomal membrane sorting. Given the central roles of LRRK2 and lysosomal biology in Parkinson's disease, these discoveries are likely relevant to disease pathology and highlight interactions between organelles in this model.

Changes in the blood-based RNA transcriptome have the potential to inform biomarkers of Parkinson’s disease (PD) progression. Here we sequenced a discovery set of whole-blood RNA species in 4,871 longitudinally collected samples from 1,570 clinically phenotyped individuals from the Parkinson’s Progression Marker Initiative (PPMI) cohort. Samples were sequenced to an average of 100 million read pairs to create a high-quality transcriptome. Participants with PD in the PPMI had significantly altered RNA expression (>2,000 differentially expressed genes), including an early and persistent increase in neutrophil gene expression, with a concomitant decrease in lymphocyte cell counts. This was validated in a cohort from the Parkinson’s Disease Biomarkers Program (PDBP) consisting of 1,599 participants and by alterations in immune cell subtypes. This publicly available transcriptomic dataset, coupled with available detailed clinical data, provides new insights into PD biological processes impacting whole blood and new paths for developing diagnostic and prognostic PD biomarkers. The authors report whole-blood RNA-seq for 4,871 samples from 1,570 participants in the Parkinson Progression Marker Initiative. This Resource documents blood-based transcriptomic changes associated with PD, including early increases in neutrophil gene expression with a decrease in lymphocytes.

Genetic variation at the leucine-rich repeat kinase 2 ( LRRK2 ) locus contributes to an enhanced risk of familial and sporadic Parkinson’s disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2 + lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments.

Protein coding mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD), and noncoding variations around the gene increase the risk of developing sporadic PD. It is generally accepted that pathogenic LRRK2 mutations increase LRRK2 kinase activity, resulting in a toxic hyperactive protein that is inferred to lead to the PD phenotype. LRRK2 has long been linked to different membrane trafficking events, but the specific role of LRRK2 in these events has been difficult to resolve. Recently, several papers have reported the activation and translocation of LRRK2 to cellular organelles under specific conditions, which suggests that LRRK2 may influence intracellular membrane trafficking. Here, we review what is known about the role of LRRK2 at various organelle compartments.

Studies of multiple neurodegenerative disorders have identified many genetic variants that are associated with risk of disease throughout a lifetime. For example, Parkinson’s disease (PD) risk is attributed in part to both coding mutations in the leucine-rich repeat kinase 2 (LRRK2) gene and to a common noncoding variation in the 5′ region of theLRRK2locus, as identified by genome-wide association studies (GWAS). However, the mechanisms linking GWAS variants to pathogenicity are largely unknown. Here, we found that the influence of PD-associated noncoding variation onLRRK2expression is specifically propagated through microglia and not by other cell types that expressLRRK2in the human brain. We find microglia-specific regulatory chromatin regions that modulate theLRRK2expression in human frontal cortex and substantia nigra and confirm these results in a human-induced pluripotent stem cell–derived microglia model. We showed, using a large-scale clustered regularly interspaced short palindromic repeats interference (CRISPRi) screen, that a regulatory DNA element containing the single-nucleotide variant rs6581593 influences theLRRK2expression in microglia. Our study demonstrates that cell type should be considered when evaluating the role of noncoding variation in disease pathogenesis and sheds light on the mechanism underlying the association of the 5′ region of LRRK2 with PD risk.

Human DJ-1 is a cytoprotective protein whose absence causes Parkinson's disease and is also associated with other diseases. DJ-1 has an established role as a redox-regulated protein that defends against oxidative stress and mitochondrial dysfunction. Multiple studies have suggested that DJ-1 is also a protein/nucleic acid deglycase that plays a key role in the repair of glycation damage caused by methylglyoxal (MG), a reactive α-keto aldehyde formed by central metabolism. Contradictory reports suggest that DJ-1 is a glyoxalase but not a deglycase and does not play a major role in glycation defense. Resolving this issue is important for understanding how DJ-1 protects cells against insults that can cause disease. We find that DJ-1 reduces levels of reversible adducts of MG with guanine and cysteine in vitro. The steady-state kinetics of DJ-1 acting on reversible hemithioacetal substrates are fitted adequately with a computational kinetic model that requires only a DJ-1 glyoxalase activity, supporting the conclusion that deglycation is an apparent rather than a true activity of DJ-1. Sensitive and quantitative isotope-dilution mass spectrometry shows that DJ-1 modestly reduces the levels of some irreversible guanine and lysine glycation products in primary and cultured neuronal cell lines and whole mouse brain, consistent with a small but measurable effect on total neuronal glycation burden. However, DJ-1 does not improve cultured cell viability in exogenous MG. In total, our results suggest that DJ-1 is not a deglycase and has only a minor role in protecting neurons against methylglyoxal toxicity.

The transition from hedonic alcohol drinking to problematic drinking is a hallmark of alcohol use disorder that occurs only in a subset of drinkers. This transition is known to require long-lasting changes in the synaptic drive and the activity of striatal neurons expressing dopamine D1 receptor (D1R). The molecular mechanisms that generate vulnerability in some individuals to undergo the transition are less understood. Here, we report that the Parkinson’s-related protein leucine-rich repeat kinase 2 (LRRK2) modulates striatal D1R function to affect the behavioral response to alcohol and the likelihood that mice transition to heavy, persistent alcohol drinking. Deletion of the Lrrk2 gene specifically from D1R-expressing neurons potentiates D1R signaling at the cellular and synaptic level, enhancing alcohol-related behaviors and drinking. Mice with cell-specific deletion of Lrrk2 are more prone to heavy alcohol drinking and consumption is insensitive to punishment. These findings identify a novel role for LRRK2 function in the striatum in promoting resilience against heavy and persistent alcohol drinking.

Background: Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson’s disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10. Objective: To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols. Methods: Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers. Results: Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated. Conclusion: Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution.

Human DJ-1 is a cytoprotective protein whose absence causes Parkinson’s disease and is also associated with other diseases. DJ-1 has an established role as a redox-regulated protein that defends against oxidative stress and mitochondrial dysfunction. Multiple studies have suggested that DJ-1 is also a protein/nucleic acid deglycase that plays a key role in the repair of glycation damage caused by methylglyoxal (MG), a reactive α-keto aldehyde formed by central metabolism. Contradictory reports suggest that DJ-1 is a glyoxalase but not a deglycase and does not play a major role in glycation defense. Resolving this issue is important for understanding how DJ-1 protects cells against insults that can cause disease. We find that DJ-1 reduces levels of reversible adducts of MG with guanine and cysteine in vitro. The steady-state kinetics of DJ-1 acting on reversible hemithioacetal substrates are fitted adequately with a computational kinetic model that requires only a DJ-1 glyoxalase activity, supporting the conclusion that deglycation is an apparent rather than a true activity of DJ-1. Sensitive and quantitative isotope-dilution mass spectrometry shows that DJ-1 modestly reduces the levels of some irreversible guanine and lysine glycation products in primary and cultured neuronal cell lines and whole mouse brain, consistent with a small but measurable effect on total neuronal glycation burden. However, DJ-1 does not improve cultured cell viability in exogenous MG. In total, our results suggest that DJ-1 is not a deglycase and has only a minor role in protecting neurons against methylglyoxal toxicity.

The progressive neuropathological damage seen in Parkinson's disease (PD) is thought to be related to the spreading of aggregated forms of α‐synuclein. Clearance of extracellular α‐synuclein released by degenerating neurons may be therefore a key mechanism to control the concentration of α‐synuclein in the extracellular space. Several molecular chaperones control misfolded protein accumulation in the extracellular compartment. Among these, clusterin, a glycoprotein associated with Alzheimer's disease, binds α‐synuclein aggregated species and is present in Lewy bodies, intraneuronal aggregates mainly composed by fibrillary α‐synuclein. In this study, using murine primary astrocytes with clusterin genetic deletion, human‐induced pluripotent stem cell (iPSC)‐derived astrocytes with clusterin silencing and two animal models relevant for PD we explore how clusterin affects the clearance of α‐synuclein aggregates by astrocytes. Our findings showed that astrocytes take up α‐synuclein preformed fibrils (pffs) through dynamin‐dependent endocytosis and that clusterin levels are modulated in the culture media of cells upon α‐synuclein pffs exposure. Specifically, we found that clusterin interacts with α‐synuclein pffs in the extracellular compartment and the clusterin/α‐synuclein complex can be internalized by astrocytes. Mechanistically, using clusterin knock‐out primary astrocytes and clusterin knock‐down hiPSC‐derived astrocytes we observed that clusterin limits the uptake of α‐synuclein pffs by cells. Interestingly, we detected increased levels of clusterin in the adeno‐associated virus‐ and the α‐synuclein pffs‐ injected mouse model, suggesting a crucial role of this chaperone in the pathogenesis of PD. Overall, our observations indicate that clusterin can limit the uptake of extracellular α‐synuclein aggregates by astrocytes and, hence, contribute to the spreading of Parkinson pathology., Main Points Extracellular clusterin binds α‐synuclein fibrils thus limiting their uptake by murine and human astrocytes.Reduced levels of clusterin improve α‐synuclein aggregates clearance by astrocytes.

Polygenic inheritance plays a central role in Parkinson disease (PD). A priority in elucidating PD etiology lies in defining the biological basis of genetic risk. Unraveling how risk leads to disruption will yield disease-modifying therapeutic targets that may be effective. Here, we utilized a high-throughput and hypothesis-free approach to determine biological processes underlying PD using the largest currently available cohorts of genetic and gene expression data from International Parkinson’s Disease Genetics Consortium (IPDGC) and the Accelerating Medicines Partnership-Parkinson’s disease initiative (AMP-PD), among other sources. We applied large-scale gene-set specific polygenic risk score (PRS) analyses to assess the role of common variation on PD risk focusing on publicly annotated gene sets representative of curated pathways. We nominated specific molecular sub-processes underlying protein misfolding and aggregation, post-translational protein modification, immune response, membrane and intracellular trafficking, lipid and vitamin metabolism, synaptic transmission, endosomal–lysosomal dysfunction, chromatin remodeling and apoptosis mediated by caspases among the main contributors to PD etiology. We assessed the impact of rare variation on PD risk in an independent cohort of whole-genome sequencing data and found evidence for a burden of rare damaging alleles in a range of processes, including neuronal transmission-related pathways and immune response. We explored enrichment linked to expression cell specificity patterns using single-cell gene expression data and demonstrated a significant risk pattern for dopaminergic neurons, serotonergic neurons, hypothalamic GABAergic neurons, and neural progenitors. Subsequently, we created a novel way of building de novo pathways by constructing a network expression community map using transcriptomic data derived from the blood of PD patients, which revealed functional enrichment in inflammatory signaling pathways, cell death machinery related processes, and dysregulation of mitochondrial homeostasis. Our analyses highlight several specific promising pathways and genes for functional prioritization and provide a cellular context in which such work should be done. Electronic supplementary material The online version of this article (10.1007/s00401-020-02181-3) contains supplementary material, which is available to authorized users.

The ε4 allele of apolipoprotein E (APOE) is the dominant genetic risk factor for late-onset Alzheimer’s disease (AD). However, the reason for the association between APOE4 and AD remains unclear. While much of the research has focused on the ability of the apoE4 protein to increase the aggregation and decrease the clearance of Aβ, there is also an abundance of data showing that APOE4 negatively impacts many additional processes in the brain, including bioenergetics. In order to gain a more comprehensive understanding of APOE4′s role in AD pathogenesis, we performed a transcriptomics analysis of APOE4 vs. APOE3 expression in the entorhinal cortex (EC) and primary visual cortex (PVC) of aged APOE mice. This study revealed EC-specific upregulation of genes related to oxidative phosphorylation (OxPhos). Follow-up analysis utilizing the Seahorse platform showed decreased mitochondrial respiration with age in the hippocampus and cortex of APOE4 vs. APOE3 mice, but not in the EC of these mice. Additional studies, as well as the original transcriptomics data, suggest that multiple bioenergetic pathways are differentially regulated by APOE4 expression in the EC of aged APOE mice in order to increase the mitochondrial coupling efficiency in this region. Given the importance of the EC as one of the first regions to be affected by AD pathology in humans, the observation that the EC is susceptible to differential bioenergetic regulation in response to a metabolic stressor such as APOE4 may point to a causative factor in the pathogenesis of AD.

Longitudinal multi-dimensional biological datasets are ubiquitous and highly abundant. These datasets are essential to understanding disease progression, identifying subtypes, and drug discovery. Discovering meaningful patterns or disease pathophysiologies in these datasets is challenging due to their high dimensionality, making it difficult to visualize hidden patterns. Several methods have been developed for dimensionality reduction, but they are limited to cross-sectional datasets. Recently proposed Aligned-UMAP, an extension of the UMAP algorithm, can visualize high-dimensional longitudinal datasets. In this work, we applied Aligned-UMAP on a broad spectrum of clinical, imaging, proteomics, and single-cell datasets. Aligned-UMAP reveals time-dependent hidden patterns when color-coded with the metadata. We found that the algorithm parameters also play a crucial role and must be tuned carefully to utilize the algorithm’s potential fully.Altogether, based on its ease of use and our evaluation of its performance on different modalities, we anticipate that Aligned-UMAP will be a valuable tool for the biomedical community. We also believe our benchmarking study becomes more important as more and more high-dimensional longitudinal data in biomedical research becomes available.Highlights-explored the utility of Aligned-UMAP in longitudinal biomedical datasets-offer insights on optimal uses for the technique-provide recommendations for best practicesIn BriefHigh-dimensional longitudinal data is prevalent yet understudied in biological literature. High-dimensional data analysis starts with projecting the data to low dimensions to visualize and understand the underlying data structure. Though few methods are available for visualizing high dimensional longitudinal data, they are not studied extensively in real-world biological datasets. A recently developed nonlinear dimensionality reduction technique, Aligned-UMAP, analyzes sequential data. Here, we give an overview of applications of Aligned-UMAP on various biomedical datasets. We further provide recommendations for best practices and offer insights on optimal uses for the technique.

An intrinsically disordered neuronal protein α-synuclein (αSyn) is known to cause mitochondrial dysfunction, contributing to loss of dopaminergic neurons in Parkinson's disease. Through yet poorly defined mechanisms, αSyn crosses mitochondrial outer membrane and targets respiratory complexes leading to bioenergetics defects. Here, using neuronally differentiated human cells overexpressing wild-type αSyn, we show that the major metabolite channel of the outer membrane, the voltage-dependent anion channel (VDAC), is a pathway for αSyn translocation into the mitochondria. Importantly, the neuroprotective cholesterol-like synthetic compound olesoxime inhibits this translocation. By applying complementary electrophysiological and biophysical approaches, we provide mechanistic insights into the interplay between αSyn, VDAC, and olesoxime. Our data suggest that olesoxime interacts with VDAC β-barrel at the lipid-protein interface thus hindering αSyn translocation through the VDAC pore and affecting VDAC voltage gating. We propose that targeting αSyn translocation through VDAC could represent a key mechanism for the development of new neuroprotective strategies.

Modeling human disease in human stem cells requires precise, scarless editing of single nucleotide variants (SNV) on one or both chromosomes. Here we describe improved conditions for Cas9 RNP editing of SNVs that yield high rates of biallelic homology-directed repair. To recover both heterozygous and homozygous SNV clones, catalytically inactive ‘dCas9’was added to moderate high activity Cas9 RNPs. dCas9 can also block re-cutting and damage to SNV alleles engineered with non-overlapping guide RNAs.

Fully automated proteomic pipelines have the potential to achieve deep coverage of cellular proteomes with high throughput and scalability. However, it is important to evaluate performance, including both reproducibility and ability to provide meaningful levels of biological insight. Here, we present an approach combining high field asymmetric waveform ion mobility spectrometer (FAIMS) interface and data independent acquisition (DIA) proteomics approach developed as part of the induced pluripotent stem cell (iPSC) Neurodegenerative Disease Initiative (iNDI), a large-scale effort to understand how inherited diseases may manifest in neuronal cells. Our FAIMS-DIA approach identified more than 8000 proteins per mass spectrometry (MS) acquisition as well as superior total identification, reproducibility, and accuracy compared to other existing DIA methods. Next, we applied this approach to perform a longitudinal proteomic profiling of the differentiation of iPSC-derived neurons from the KOLF2.1J parental line used in iNDI. This analysis demonstrated a steady increase in expression of mature cortical neuron markers over the course of neuron differentiation. We validated the performance of our proteomics pipeline by comparing it to single cell RNA-Seq datasets obtained in parallel, confirming expression of key markers and cell type annotations. An interactive webapp of this temporal data is available for aligned-UMAP visualization and data browsing (https://share.streamlit.io/anant-droid/singlecellumap). In summary, we report an extensively optimized and validated proteomic pipeline that will be suitable for large-scale studies such as iNDI.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.