Changes in Oxidised Phospholipids in Response to Oxidative Stress in Microtubule-Associated Protein Tau (MAPT) Mutant Dopamine Neurons.

Microtubule-associated protein Tau (MAPT) is strongly associated with the development of neurodegenerative diseases. In addition to driving the formation of neurofibrillary tangles (NFT), mutations in the MAPT gene can also cause oxidative stress through hyperpolarisation of the mitochondria. This study explores the impact that MAPT mutation is having on phospholipid metabolism in iPSC-derived dopamine neurons, and to determine if these effects are exacerbated by mitochondrial and endoplasmic reticulum stress. Neurons that possessed a mutated copy of MAPT were shown to have significantly higher levels of oxo-phospholipids (Oxo-PL) than wild-type neurons. Oxidation of the hydrophobic fatty acid side chains changes the chemistry of the phospholipid leading to disruption of membrane function and potential cell lysis. In wild-type neurons, both mitochondrial and endoplasmic reticulum stress increased Oxo-PL abundance; however, in MAPT mutant neurons mitochondrial stress appeared to have a minimal effect. Endoplasmic reticulum stress, surprisingly, reduced the abundance of Oxo-PL in MAPT mutant dopamine neurons, and we postulate that this reduction could be modulated through hyperactivation of the unfolded protein response and X-box binding protein 1. Overall, the results of this study contribute to furthering our understanding of the regulation and impact of oxidative stress in Parkinson's disease pathology.

Variant-specific effects of GBA1 mutations on dopaminergic neuron proteostasis.

Glucocerebrosidase 1 (GBA1) mutations are the most important genetic risk factors for Parkinson's disease (PD). Clinically, mild (e.g., p.N370S) and severe (e.g., p.L444P and p.D409H) GBA1 mutations have different PD phenotypes, with differences in age at disease onset, progression, and the severity of motor and non-motor symptoms. We hypothesize that GBA1 mutations cause the accumulation of α-synuclein by affecting the cross-talk between cellular protein degradation mechanisms, leading to neurodegeneration. Accordingly, we tested whether mild and severe GBA1 mutations differentially affect the degradation of α-synuclein via the ubiquitin-proteasome system (UPS), chaperone-mediated autophagy (CMA), and macroautophagy and differentially cause accumulation and/or release of α-synuclein. Our results demonstrate that endoplasmic reticulum (ER) stress and total ubiquitination rates were significantly increased in cells with severe GBA1 mutations. CMA was found to be defective in induced pluripotent stem cell (iPSC)-derived dopaminergic neurons with mild GBA1 mutations, but not in those with severe GBA1 mutations. When examining macroautophagy, we observed reduced formation of autophagosomes in cells with the N370S and D409H GBA1 mutations and impairments in autophagosome-lysosome fusion in cells with the L444P GBA1 mutation. Accordingly, severe GBA1 mutations were found to trigger the accumulation and release of oligomeric α-synuclein in iPSC-derived dopaminergic neurons, primarily as a result of increased ER stress and defective macroautophagy, while mild GBA1 mutations affected CMA, which is mainly responsible for the degradation of the monomeric form of α-synuclein. Overall, our findings provide new insight into the molecular basis of the clinical variability in PD associated with different GBA1 mutations.

Post-translational proteomics platform identifies neurite outgrowth impairments in Parkinson's disease GBA-N370S dopamine neurons.

Variants at the GBA locus, encoding glucocerebrosidase, are the strongest common genetic risk factor for Parkinson's disease (PD). To understand GBA-related disease mechanisms, we use a multi-part-enrichment proteomics and post-translational modification (PTM) workflow, identifying large numbers of dysregulated proteins and PTMs in heterozygous GBA-N370S PD patient induced pluripotent stem cell (iPSC) dopamine neurons. Alterations in glycosylation status show disturbances in the autophagy-lysosomal pathway, which concur with upstream perturbations in mammalian target of rapamycin (mTOR) activation in GBA-PD neurons. Several native and modified proteins encoded by PD-associated genes are dysregulated in GBA-PD neurons. Integrated pathway analysis reveals impaired neuritogenesis in GBA-PD neurons and identify tau as a key pathway mediator. Functional assays confirm neurite outgrowth deficits and identify impaired mitochondrial movement in GBA-PD neurons. Furthermore, pharmacological rescue of glucocerebrosidase activity in GBA-PD neurons improves the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate neurodegeneration-associated pathways and potential drug targets in complex disease models.

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.

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.

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.

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.

Cellular α-synuclein pathology is associated with bioenergetic dysfunction in Parkinson's iPSC-derived dopamine neurons.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and a central role for α-synuclein (αSyn; SNCA) in disease aetiology has been proposed based on genetics and neuropathology. To better understand the pathological mechanisms of αSyn, we generated induced pluripotent stem cells (iPSCs) from healthy individuals and PD patients carrying the A53T SNCA mutation or a triplication of the SNCA locus and differentiated them into dopaminergic neurons (DAns). iPSC-derived DAn from PD patients carrying either mutation showed increased intracellular αSyn accumulation, and DAns from patients carrying the SNCA triplication displayed oligomeric αSyn pathology and elevated αSyn extracellular release. Transcriptomic analysis of purified DAns revealed perturbations in expression of genes linked to mitochondrial function, consistent with observed reduction in mitochondrial respiration, impairment in mitochondrial membrane potential, aberrant mitochondrial morphology and decreased levels of phosphorylated DRP1Ser616. Parkinson's iPSC-derived DAns showed increased endoplasmic reticulum stress and impairments in cholesterol and lipid homeostasis. Together, these data show a correlation between αSyn cellular pathology and deficits in metabolic and cellular bioenergetics in the pathology of PD.

Variant U1 snRNAs are implicated in human pluripotent stem cell maintenance and neuromuscular disease.

The U1 small nuclear (sn)RNA (U1) is a multifunctional ncRNA, known for its pivotal role in pre-mRNA splicing and regulation of RNA 3' end processing events. We recently demonstrated that a new class of human U1-like snRNAs, the variant (v)U1 snRNAs (vU1s), also participate in pre-mRNA processing events. In this study, we show that several human vU1 genes are specifically upregulated in stem cells and participate in the regulation of cell fate decisions. Significantly, ectopic expression of vU1 genes in human skin fibroblasts leads to increases in levels of key pluripotent stem cell mRNA markers, including NANOG and SOX2. These results reveal an important role for vU1s in the control of key regulatory networks orchestrating the transitions between stem cell maintenance and differentiation. Moreover, vU1 expression varies inversely with U1 expression during differentiation and cell re-programming and this pattern of expression is specifically de-regulated in iPSC-derived motor neurons from Spinal Muscular Atrophy (SMA) type 1 patient's. Accordingly, we suggest that an imbalance in the vU1/U1 ratio, rather than an overall reduction in Uridyl-rich (U)-snRNAs, may contribute to the specific neuromuscular disease phenotype associated with SMA.

ER Stress and Autophagic Perturbations Lead to Elevated Extracellular α-Synuclein in GBA-N370S Parkinson's iPSC-Derived Dopamine Neurons.

Heterozygous mutations in the glucocerebrosidase gene (GBA) represent the strongest common genetic risk factor for Parkinson's disease (PD), the second most common neurodegenerative disorder. However, the molecular mechanisms underlying this association are still poorly understood. Here, we have analyzed ten independent induced pluripotent stem cell (iPSC) lines from three controls and three unrelated PD patients heterozygous for the GBA-N370S mutation, and identified relevant disease mechanisms. After differentiation into dopaminergic neurons, we observed misprocessing of mutant glucocerebrosidase protein in the ER, associated with activation of ER stress and abnormal cellular lipid profiles. Furthermore, we observed autophagic perturbations and an enlargement of the lysosomal compartment specifically in dopamine neurons. Finally, we found increased extracellular α-synuclein in patient-derived neuronal culture medium, which was not associated with exosomes. Overall, ER stress, autophagic/lysosomal perturbations, and elevated extracellular α-synuclein likely represent critical early cellular phenotypes of PD, which might offer multiple therapeutic targets.

Cellular reprogramming: a new approach to modelling Parkinson's disease.

iPSCs (induced pluripotent stem cells) offer an unparalleled opportunity to generate and study physiologically relevant cell types in culture. iPSCs can be generated by reprogramming almost any somatic cell type using pluripotency factors such as Oct4, SOX2, Nanog and Klf4. By reprogramming cells from patients carrying disease-associated mutations, and subsequent differentiation into the cell type of interest, researchers now have the opportunity to study disease-specific cell types which were previously inaccessible. In the case of PD (Parkinson's disease), reprogramming is advancing rapidly, and cell lines have been generated from patients carrying mutations in several disease-associated genes, including SNCA (α-synuclein), PARK2 (parkin), PINK1 (phosphatase and tensin homologue deleted on chromosome 10-induced putative kinase 1), PARK7 (DJ-1) and LRRK2 (leucine-rich repeat kinase 2), as well as idiopathic cases. Functional dopaminergic neurons have been differentiated from these cells and their physiology has been compared with control neurons. Human dopaminergic neurons had been previously inaccessible until post-mortem, when the disease is generally highly progressed into pathology. In comparison, iPSCs provide a living cell model with the potential to study early molecular changes which accumulate in cells and ultimately result in neurodegeneration. Although clear phenotypes have not yet been unambiguously identified in patient-derived dopaminergic neurons, there are suggested aberrations in cellular pathways involved in neurodegeneration. Overall, these cells offer a unique opportunity to study dopaminergic neurons carrying a 'Parkinsonian genome'. The present review discusses the advances in cellular reprogramming technologies and studies that have been carried out on PD-derived iPSCs and differentiated dopaminergic neurons.