Genome-wide CRISPR/Cas9 screen shows that loss of GET4 increases mitochondria-endoplasmic reticulum contact sites and is neuroprotective.

Organelles form membrane contact sites between each other, allowing for the transfer of molecules and signals. Mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are cellular subdomains characterized by close apposition of mitochondria and ER membranes. They have been implicated in many diseases, including neurodegenerative, metabolic, and cardiac diseases. Although MERCS have been extensively studied, much remains to be explored. To uncover novel regulators of MERCS, we conducted a genome-wide, flow cytometry-based screen using an engineered MERCS reporter cell line. We found 410 genes whose downregulation promotes MERCS and 230 genes whose downregulation decreases MERCS. From these, 29 genes were selected from each population for arrayed screening and 25 were validated from the high population and 13 from the low population. GET4 and BAG6 were highlighted as the top 2 genes that upon suppression increased MERCS from both the pooled and arrayed screens, and these were subjected to further investigation. Multiple microscopy analyses confirmed that loss of GET4 or BAG6 increased MERCS. GET4 and BAG6 were also observed to interact with the known MERCS proteins, inositol 1,4,5-trisphosphate receptors (IP3R) and glucose-regulated protein 75 (GRP75). In addition, we found that loss of GET4 increased mitochondrial calcium uptake upon ER-Ca2+ release and mitochondrial respiration. Finally, we show that loss of GET4 rescues motor ability, improves lifespan and prevents neurodegeneration in a Drosophila model of Alzheimer's disease (Aß42Arc). Together, these results suggest that GET4 is involved in decreasing MERCS and that its loss is neuroprotective.

Unravelling cell type-specific responses to Parkinson's Disease at single cell resolution.

Parkinson's Disease (PD) is the second most common neurodegenerative disorder. The pathological hallmark of PD is loss of dopaminergic neurons and the presence of aggregated α-synuclein, primarily in the substantia nigra pars compacta (SNpc) of the midbrain. However, the molecular mechanisms that underlie the pathology in different cell types is not currently understood. Here, we present a single nucleus transcriptome analysis of human post-mortem SNpc obtained from 15 sporadic Parkinson's Disease (PD) cases and 14 Controls. Our dataset comprises ∼84K nuclei, representing all major cell types of the brain, allowing us to obtain a transcriptome-level characterization of these cell types. Importantly, we identify multiple subpopulations for each cell type and describe specific gene sets that provide insights into the differing roles of these subpopulations. Our findings reveal a significant decrease in neuronal cells in PD samples, accompanied by an increase in glial cells and T cells. Subpopulation analyses demonstrate a significant depletion of tyrosine hydroxylase (TH) enriched astrocyte, microglia and oligodendrocyte populations in PD samples, as well as TH enriched neurons, which are also depleted. Moreover, marker gene analysis of the depleted subpopulations identified 28 overlapping genes, including those associated with dopamine metabolism (e.g., ALDH1A1, SLC6A3 & SLC18A2). Overall, our study provides a valuable resource for understanding the molecular mechanisms involved in dopaminergic neuron degeneration and glial responses in PD, highlighting the existence of novel subpopulations and cell type-specific gene sets.

Single-cell transcriptomics identifies perturbed molecular pathways in midbrain organoids using α-synuclein triplication Parkinson's disease patient-derived iPSCs.

Three-dimensional (3D) brain organoids provide a platform to study brain development, cellular coordination, and disease using human tissue. Here, we generate midbrain dopaminergic (mDA) organoids from induced pluripotent stem cells (iPSC) from healthy and Parkinson's Disease (PD) donors and assess them as a human PD model using single-cell RNAseq. We characterize cell types in our organoid cultures and analyze our model's Dopamine (DA) neurons using cytotoxic and genetic stressors. Our study provides the first in-depth, single-cell analysis of SNCA triplication and shows evidence for molecular dysfunction in oxidative phosphorylation, translation, and ER protein-folding in DA neurons. We perform an in-silico identification of rotenone-sensitive DA neurons and characterization of corresponding transcriptomic profiles associated with synaptic signalling and cholesterol biosynthesis. Finally, we show a novel chimera organoid model from healthy and PD iPSCs allowing the study of DA neurons from different individuals within the same tissue.

HNRNPH1 regulates the neuroprotective cold-shock protein RBM3 expression through poison exon exclusion.

Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 cold induction remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator. Splicing analysis revealed that moderate hypothermia significantly represses the inclusion of a poison exon, which, when retained, targets the mRNA for nonsense-mediated decay. Importantly, we show that HNRNPH1 mediates this cold-dependent exon skipping via its thermosensitive interaction with a G-rich motif within the poison exon. Our study provides novel mechanistic insights into the regulation of RBM3 and provides further targets for neuroprotective therapeutic strategies.

CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death.

Accumulation of aggregated and misfolded proteins, leading to endoplasmic reticulum stress and activation of the unfolded protein response, is a hallmark of several neurodegenerative disorders, including Alzheimer's and Parkinson's disease. Genetic screens are powerful tools that are proving invaluable in identifying novel modulators of disease associated processes. Here, we performed a loss-of-function genetic screen using a human druggable genome library, followed by an arrayed-screen validation, in human iPSC-derived cortical neurons. We identified and genetically validated 13 genes, whose knockout was neuroprotective against Tunicamycin, a glycoprotein synthesis inhibitor widely used to induce endoplasmic reticulum stress. We also demonstrated that pharmacological inhibition of KAT2B, a lysine acetyltransferase identified by our genetic screens, by L-Moses, attenuates Tunicamycin-mediated neuronal cell death and activation of CHOP, a key pro-apoptotic member of the unfolded protein response in both cortical and dopaminergic neurons. Follow-up transcriptional analysis suggested that L-Moses provided neuroprotection by partly reversing the transcriptional changes caused by Tunicamycin. Finally, L-Moses treatment attenuated total protein levels affected by Tunicamycin, without affecting their acetylation profile. In summary, using an unbiased approach, we identified KAT2B and its inhibitor, L-Moses, as potential therapeutic targets for neurodegenerative diseases.

A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

Synapse loss strongly correlates with cognitive decline in Alzheimer's disease (AD), but the underlying mechanisms are poorly understood. Deficient Wnt signaling contributes to synapse dysfunction and loss in AD. Consistently, a variant of the LRP6 receptor, (LRP6-Val), with reduced Wnt signaling, is linked to late-onset AD. However, the impact of LRP6-Val on the healthy and AD brain has not been examined. Knock-in mice, generated by gene editing, carrying this Lrp6 variant develop normally. However, neurons from Lrp6-val mice do not respond to Wnt7a, a ligand that promotes synaptic assembly through the Frizzled-5 receptor. Wnt7a stimulates the formation of the low-density lipoprotein receptor-related protein 6 (LRP6)-Frizzled-5 complex but not if LRP6-Val is present. Lrp6-val mice exhibit structural and functional synaptic defects that become pronounced with age. Lrp6-val mice present exacerbated synapse loss around plaques when crossed to the NL-G-F AD model. Our findings uncover a previously unidentified role for Lrp6-val in synapse vulnerability during aging and AD.

Mitochondrial and Endoplasmic Reticulum Stress Trigger Triglyceride Accumulation in Models of Parkinson's Disease Independent of Mutations in MAPT.

The metabolic basis of Parkinson's disease pathology is poorly understood. However, the involvement of mitochondrial and endoplasmic reticulum stress in dopamine neurons in disease aetiology is well established. We looked at the effect of rotenone- and tunicamycin-induced mitochondrial and ER stress on the metabolism of wild type and microtubule-associated protein tau mutant dopamine neurons. Dopamine neurons derived from human isolated iPSCs were subjected to mitochondrial and ER stress using RT and TM, respectively. Comprehensive metabolite profiles were generated using a split phase extraction analysed by reversed phase lipidomics whilst the aqueous phase was measured using HILIC metabolomics. Mitochondrial and ER stress were both shown to cause significant dysregulation of metabolism with RT-induced stress producing a larger shift in the metabolic profile of both wild type and MAPT neurons. Detailed analysis showed that accumulation of triglycerides was a significant driver of metabolic dysregulation in response to both stresses in both genotypes. Whilst the consequence is similar, the mechanisms by which triglyceride accumulation occurs in dopamine neurons in response to mitochondrial and ER stress are very different. Thus, improving our understanding of how these mechanisms drive the observed triglyceride accumulation can potentially open up new therapeutic avenues.

Derivation of nociceptive sensory neurons from hiPSCs with early patterning and temporally controlled NEUROG2 overexpression.

Despite development of protocols to differentiate human pluripotent stem cells (hPSCs), those used to produce sensory neurons remain difficult to replicate and result in heterogenous populations. There is a growing clinical burden of chronic pain conditions, highlighting the need for relevant human cellular models. This study presents a hybrid differentiation method to produce nociceptive sensory neurons from hPSCs. Lines harboring an inducible NEUROG2 construct were patterned toward precursors with small molecules followed by NEUROG2 overexpression. Neurons expressed key markers, including BRN3A and ISL1, with single-cell RNA sequencing, revealing populations of nociceptors expressing SCN9A and TRP channels. Physiological profiling with multi-electrode arrays revealed that neurons responded to noxious stimuli, including capsaicin. Finally, we modeled pain-like states to identify genes and pathways involved in pain transduction. This study presents an optimized method to efficiently produce nociceptive sensory neurons and provides a tool to aid development of chronic pain research.

Cholesterol determines the cytosolic entry and seeded aggregation of tau.

Assemblies of tau can transit between neurons, seeding aggregation in a prion-like manner. To accomplish this, tau must cross cell-limiting membranes, a process that is poorly understood. Here, we establish assays for the study of tau entry into the cytosol as a phenomenon distinct from uptake, in real time, and at physiological concentrations. The entry pathway of tau is cell type specific and, in neurons, highly sensitive to cholesterol. Depletion of the cholesterol transporter Niemann-Pick type C1 or extraction of membrane cholesterol renders neurons highly permissive to tau entry and potentiates seeding even at low levels of exogenous tau assemblies. Conversely, cholesterol supplementation reduces entry and almost completely blocks seeded aggregation. Our findings establish entry as a rate-limiting step to seeded aggregation and demonstrate that dysregulated cholesterol, a feature of several neurodegenerative diseases, potentiates tau aggregation by promoting entry of tau assemblies into the cell interior.

The transcription factor BCL11A defines distinct subsets of midbrain dopaminergic neurons.

Midbrain dopaminergic (mDA) neurons are diverse in their projection targets, effect on behavior, and susceptibility to neurodegeneration. Little is known about the molecular mechanisms establishing this diversity during development. We show that the transcription factor BCL11A is expressed in a subset of mDA neurons in the developing and adult murine brain and in a subpopulation of pluripotent-stem-cell-derived human mDA neurons. By combining intersectional labeling and viral-mediated tracing, we demonstrate that Bcl11a-expressing mDA neurons form a highly specific subcircuit within the murine dopaminergic system. In the substantia nigra, the Bcl11a-expressing mDA subset is particularly vulnerable to neurodegeneration upon α-synuclein overexpression or oxidative stress. Inactivation of Bcl11a in murine mDA neurons increases this susceptibility further, alters the distribution of mDA neurons, and results in deficits in skilled motor behavior. In summary, BCL11A defines mDA subpopulations with highly distinctive characteristics and is required for establishing and maintaining their normal physiology.

MicroExonator enables systematic discovery and quantification of microexons across mouse embryonic development.

BACKGROUND: Microexons, exons that are ≤ 30 nucleotides, are a highly conserved and dynamically regulated set of cassette exons. They have key roles in nervous system development and function, as evidenced by recent results demonstrating the impact of microexons on behaviour and cognition. However, microexons are often overlooked due to the difficulty of detecting them using standard RNA-seq aligners. RESULTS: Here, we present MicroExonator, a novel pipeline for reproducible de novo discovery and quantification of microexons. We process 289 RNA-seq datasets from eighteen mouse tissues corresponding to nine embryonic and postnatal stages, providing the most comprehensive survey of microexons available for mice. We detect 2984 microexons, 332 of which are differentially spliced throughout mouse embryonic brain development, including 29 that are not present in mouse transcript annotation databases. Unsupervised clustering of microexons based on their inclusion patterns segregates brain tissues by developmental time, and further analysis suggests a key function for microexons in axon growth and synapse formation. Finally, we analyse single-cell RNA-seq data from the mouse visual cortex, and for the first time, we report differential inclusion between neuronal subpopulations, suggesting that some microexons could be cell type-specific. CONCLUSIONS: MicroExonator facilitates the investigation of microexons in transcriptome studies, particularly when analysing large volumes of data. As a proof of principle, we use MicroExonator to analyse a large collection of both mouse bulk and single-cell RNA-seq datasets. The analyses enabled the discovery of previously uncharacterized microexons, and our study provides a comprehensive microexon inclusion catalogue during mouse development.

Using Reactome to build an autophagy mechanism knowledgebase.

The 21st century has revealed much about the fundamental cellular process of autophagy. Autophagy controls the catabolism and recycling of various cellular components both as a constitutive process and as a response to stress and foreign material invasion. There is considerable knowledge of the molecular mechanisms of autophagy, and this is still growing as new modalities emerge. There is a need to investigate autophagy mechanisms reliably, comprehensively and conveniently. Reactome is a freely available knowledgebase that consists of manually curated molecular events (reactions) organized into cellular pathways (https://reactome.org/). Pathways/reactions in Reactome are hierarchically structured, graphically presented and extensively annotated. Data analysis tools, such as pathway enrichment, expression data overlay and species comparison, are also available. For customized analysis, information can also be programmatically queried. Here, we discuss the curation and annotation of the molecular mechanisms of autophagy in Reactome. We also demonstrate the value that Reactome adds to research by reanalyzing a previously published work on genome-wide CRISPR screening of autophagy components.Abbreviations: CMA: chaperone-mediated autophagy; GO: Gene Ontology; MA: macroautophagy; MI: microautophagy; MTOR: mechanistic target of rapamycin kinase; SQSTM1: sequestosome 1.

ER-mitochondria contact sites in neurodegeneration: genetic screening approaches to investigate novel disease mechanisms.

Mitochondria-ER contact sites (MERCS) are known to underpin many important cellular homoeostatic functions, including mitochondrial quality control, lipid metabolism, calcium homoeostasis, the unfolded protein response and ER stress. These functions are known to be dysregulated in neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease (AD) and amyloid lateral sclerosis (ALS), and the number of disease-related proteins and genes being associated with MERCS is increasing. However, many details regarding MERCS and their role in neurodegenerative diseases remain unknown. In this review, we aim to summarise the current knowledge regarding the structure and function of MERCS, and to update the field on current research in PD, AD and ALS. Furthermore, we will evaluate high-throughput screening techniques, including RNAi vs CRISPR/Cas9, pooled vs arrayed formats and how these could be combined with current techniques to visualise MERCS. We will consider the advantages and disadvantages of each technique and how it can be utilised to uncover novel protein pathways involved in MERCS dysfunction in neurodegenerative diseases.

Development and Application of High-Throughput Single Cell Lipid Profiling: A Study of SNCA-A53T Human Dopamine Neurons.

Advances in single cell genomics and transcriptomics have shown that at tissue level there is complex cellular heterogeneity. To understand the effect of this inter-cell heterogeneity on metabolism it is essential to develop a single cell lipid profiling approach that allows the measurement of lipids in large numbers of single cells from a population. This will provide a functional readout of cell activity and membrane structure. Using liquid extraction surface analysis coupled with high-resolution mass spectrometry we have developed a high-throughput method for untargeted single cell lipid profiling. This technological advance highlighted the importance of cellular heterogeneity in the functional metabolism of individual human dopamine neurons, suggesting that A53T alpha-synuclein (SNCA) mutant neurons have impaired membrane function. These results demonstrate that this single cell lipid profiling platform can provide robust data that will expand the frontiers in biomedical research.

Single-Cell Transcriptomics of Parkinson's Disease Human In Vitro Models Reveals Dopamine Neuron-Specific Stress Responses.

The advent of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized Parkinson's disease (PD) research, but single-cell transcriptomic analysis suggests unresolved cellular heterogeneity within these models. Here, we perform the largest single-cell transcriptomic study of human iPSC-derived dopaminergic neurons to elucidate gene expression dynamics in response to cytotoxic and genetic stressors. We identify multiple neuronal subtypes with transcriptionally distinct profiles and differential sensitivity to stress, highlighting cellular heterogeneity in dopamine in vitro models. We validate this disease model by showing robust expression of PD GWAS genes and overlap with postmortem adult substantia nigra neurons. Importantly, stress signatures are ameliorated using felodipine, an FDA-approved drug. Using isogenic SNCA-A53T mutants, we find perturbations in glycolysis, cholesterol metabolism, synaptic signaling, and ubiquitin-proteasomal degradation. Overall, our study reveals cell type-specific perturbations in human dopamine neurons, which will further our understanding of PD and have implications for cell replacement therapies.

Author Correction: Felodipine induces autophagy in mouse brains with pharmacokinetics amenable to repurposing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Felodipine induces autophagy in mouse brains with pharmacokinetics amenable to repurposing.

Neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Huntington's disease manifest with the neuronal accumulation of toxic proteins. Since autophagy upregulation enhances the clearance of such proteins and ameliorates their toxicities in animal models, we and others have sought to re-position/re-profile existing compounds used in humans to identify those that may induce autophagy in the brain. A key challenge with this approach is to assess if any hits identified can induce neuronal autophagy at concentrations that would be seen in humans taking the drug for its conventional indication. Here we report that felodipine, an L-type calcium channel blocker and anti-hypertensive drug, induces autophagy and clears diverse aggregate-prone, neurodegenerative disease-associated proteins. Felodipine can clear mutant α-synuclein in mouse brains at plasma concentrations similar to those that would be seen in humans taking the drug. This is associated with neuroprotection in mice, suggesting the promise of this compound for use in neurodegeneration.