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α-Synuclein (αSyn), a 140-residue intrinsically disordered protein, comprises the primary proteinaceous component of pathology-associated Lewy body inclusions in Parkinson’s disease (PD). Due to its association with PD, αSyn is studied extensively; however, the endogenous structure and physiological roles of this protein are yet to be fully understood. Here, ion mobility-mass spectrometry and native top-down electron capture dissociation fragmentation have been used to elucidate the structural properties associated with a stable, naturally occurring dimeric species of αSyn. This stable dimer appears in both wild-type (WT) αSyn and the PD-associated variant A53E. Furthermore, we integrated a novel method for generating isotopically depleted protein into our native top-down workflow. Isotope depletion increases signal-to-noise ratio and reduces the spectral complexity of fragmentation data, enabling the monoisotopic peak of low abundant fragment ions to be observed. This enables the accurate and confident assignment of fragments unique to the αSyn dimer to be assigned and structural information about this species to be inferred. Using this approach, we were able to identify fragments unique to the dimer, which demonstrates a C-terminal to C-terminal interaction between the monomer subunits. The approach in this study holds promise for further investigation into the structural properties of endogenous multimeric species of αSyn.

Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.

Parkinson’s disease (PD) is a neurodegenerative condition with several major hallmarks, including loss ofsubstantia nigraneurons, reduction in striatal dopaminergic function, and formation of α-synuclein-rich Lewy bodies. Mutations inSNCA, encoding for α-synuclein, are a known cause of familial PD, and the G51D mutation causes a particularly aggressive form of the condition. CRISPR/Cas9 technology was used to introduce the G51D mutation into the endogenous ratSNCAgene.SNCAG51D/+andSNCAG51D/G51Drats were born in Mendelian ratios and did not exhibit any severe behavourial defects.L-3,4-dihydroxy-6-18F-fluorophenylalanine (18F-DOPA) positron emission tomography (PET) imaging was used to investigate this novel rat model. Wild-type (WT),SNCAG51D/+andSNCAG51D/G51Drats were characterised over the course of ageing (5, 11, and 16 months old) using18F-DOPA PET imaging and kinetic modelling. We measured the influx rate constant (Ki) and effective distribution volume ratio (EDVR) of18F-DOPA in the striatum relative to the cerebellum in WT,SNCAG51D/+andSNCAG51D/G51Drats. A significant reduction inEDVRwas observed inSNCAG51D/G51Drats at 16 months of age indicative of increased dopamine turnover. Furthermore, we observed a significant asymmetry in EDVR between the left and right striatum in agedSNCAG51D/G51Drats. The increased and asymmetric dopamine turnover observed in the striatum of agedSNCAG51D/G51Drats is similar to prodromal PD, which suggests the presence of compensatory mechanisms.SNCAG51Drats represent a novel genetic model of PD, and kinetic modelling of18F-DOPA PET data has identified a highly relevant early disease phenotype.

Mutations in theSNCAgene cause autosomal dominant Parkinson’s disease (PD), with progressive loss of dopaminergic neurons in the substantia nigra, and accumulation of aggregates of α-synuclein. However, the sequence of molecular events that proceed from theSNCAmutation during development, to its end stage pathology is unknown. Utilising human induced pluripotent stem cells (hiPSCs) withSNCAmutations, we resolved the temporal sequence of pathophysiological events that occur during neuronal differentiation in order to discover the early, and likely causative, events in synucleinopathies. We adapted a small molecule-based protocol that generates highly enriched midbrain dopaminergic (mDA) neurons (>80%). We characterised their molecular identity using single-cell RNA sequencing and their functional identity through the synthesis and secretion of dopamine, the ability to generate action potentials, and form functional synapses and networks. RNA velocity analyses confirmed the developmental transcriptomic trajectory of midbrain neural precursors into mDA neurons using our approach, and identified key driver genes in mDA neuronal development. To characterise the synucleinopathy, we adopted super-resolution methods to determine the number, size and structure of aggregates inSNCA-mutant mDA neurons. At one week of differentiation, prior to maturation to mDA neurons of molecular and functional identity, we demonstrate the formation of small aggregates; specifically, β-sheet rich oligomeric aggregates, inSNCA-mutant midbrain immature neurons. The aggregation progresses over time to accumulate phosphorylated aggregates, and later fibrillar aggregates. When the midbrain neurons were functional, we observed evidence of impaired physiological calcium signalling, with raised basal calcium, and impairments in cytosolic and mitochondrial calcium efflux. Once midbrain identity fully developed,SNCA-mutant neurons exhibited bioenergetic impairments, mitochondrial dysfunction and oxidative stress. During the maturation of mDA neurons, upregulation of mitophagy and autophagy occured, and ultimately these multiple cellular stresses lead to an increase in cell death by six weeks post-differentiation. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD, and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease.

Aggregation of α-Synuclein (α-Syn) drives Parkinson’s disease, although the initial stages of self-assembly and structural conversion have not been captured inside neurons. We track the intracellular conformational states of α-Syn utilizing a single-molecule FRET biosensor, and show that α-Syn converts from its monomeric state to form two distinct oligomeric states in neurons in a concentration dependent, and sequence specific manner. 3D FRET-CLEM reveals the structural organization, and location of aggregation hotspots inside the cell. Notably multiple intracellular seeding events occur preferentially on membrane surfaces, especially mitochondrial membranes. The mitochondrial lipid, cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid-protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial ROS generation, which accelerates the oligomerization of A53T α-Syn, and ultimately causes permeabilization of mitochondrial membranes, and cell death. Patient iPSC derived neurons harboring A53T mutations exhibit accelerated oligomerization that is dependent on mitochondrial ROS, early mitochondrial permeabilization and neuronal death. Our study highlights a mechanism of de novo oligomerization at the mitochondria and its induction of neuronal toxicity.

Background: First-in-human studies to test the efficacy and safety of human embryonic stem cells (hESC)-derived dopaminergic cells in the treatment of Parkinson’s disease (PD) are imminent. Pre-clinical studies using hESC-derived dopamine neuron transplants in rat models have indicated that the benefits parallel those shown with fetal tissue but have thus far failed to consider how ongoing L-DOPA administration might impact on the graft. Objective: To determine whether L-DOPA impacts on survival and functional recovery following grafting of hESC-derived dopaminergic neurons. Methods: Unilateral 6-OHDA lesioned rats were administered with either saline or L-DOPA prior to, and for 18 weeks following surgical implantation of dopaminergic neural progenitors derived from RC17 hESCs according to two distinct protocols in independent laboratories. Results: Grafts from both protocols elicited reduction in amphetamine-induced rotations. Reduced L-DOPA-induced dyskinesia preceded the improvement in amphetamine-induced rotations. Furthermore, L-DOPA had no effect on overall survival (HuNu) or dopaminergic neuron content of the graft (TH positive cells) but did lead to an increase in the number of GIRK2 positive neurons. Conclusion: Critically, we found that L-DOPA was not detrimental to graft function, potentially enhancing graft maturation and promoting an A9 phenotype. Early improvement of L-DOPA-induced dyskinesia suggests that grafts may support the handling of exogenously supplied dopamine earlier than improvements in amphetamine-induced behaviours indicate. Given that one of the protocols will be employed in the production of cells for the European STEM-PD clinical trial, this is vital information for the management of patients and achieving optimal outcomes following transplantation of hESC-derived grafts for PD.

Protein aggregation and abnormal lipid homeostasis are both implicated in neurodegeneration through unknown mechanisms. Here we demonstrate that aggregate-membrane interaction is critical to induce a form of cell death called ferroptosis. Importantly, the aggregate-membrane interaction that drives ferroptosis depends both on the conformational structure of the aggregate, as well as the oxidation state of the lipid membrane. We generated human stem cell-derived models of synucleinopathy, characterized by the intracellular formation of α-synuclein aggregates that bind to membranes. In human iPSC-derived neurons with SNCA triplication, physiological concentrations of glutamate and dopamine induce abnormal calcium signaling owing to the incorporation of excess α-synuclein oligomers into membranes, leading to altered membrane conductance and abnormal calcium influx. α-synuclein oligomers further induce lipid peroxidation. Targeted inhibition of lipid peroxidation prevents the aggregate-membrane interaction, abolishes aberrant calcium fluxes, and restores physiological calcium signaling. Inhibition of lipid peroxidation, and reduction of iron-dependent accumulation of free radicals, further prevents oligomer-induced toxicity in human neurons. In summary, we report that peroxidation of polyunsaturated fatty acids underlies the incorporation of β-sheet-rich aggregates into the membranes, and that additionally induces neuronal death. This suggests a role for ferroptosis in Parkinson’s disease, and highlights a new mechanism by which lipid peroxidation causes cell death.

α-Synuclein (αSyn) is a small, disordered protein that becomes aggregated in Lewy body diseases, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Human induced pluripotent stem cells (hiPSCs) potentially provide a tractable disease model to monitor early molecular changes associated with PD/DLB. We and others have previously derived hiPSC lines from patients with duplication and triplication of the SNCA gene, encoding for αSyn. It is now recognised that to perform meaningful disease modelling with these hiPSC lines, it is critical to generate isogenic control cell lines that lack the disease causing mutations. In order to complement the existing and emerging hiPSC models for PD/DLB, we have generated an allelic series of αSyn over-expressing hESC lines on the same isogenic background. An unresolved question is whether pluripotent stem cell lines, with elevated levels of αSyn, can undergo efficient differentiation into dopaminergic and cortical neurons to model PD and DLB, respectively. We took advantage of our isogenic collection of hESC lines to determine if increased expression of αSyn affects neural induction and neuronal differentiation. Clonal hESC lines with significantly different levels of αSyn expression proliferated normally and maintained expression of pluripotent markers, such as OCT4. All cell lines efficiently produced PAX6+ neuroectoderm and there was no correlation between αSyn expression and neural induction efficiency. Finally, global transcriptomic analysis of cortical differentiation of hESC lines with low or high levels of αSyn expression demonstrated robust and similar induction of cortical neuronal expression profiles. Gene expression differences observed were unrelated to neural induction and neuronal differentiation. We conclude that elevated expression of αSyn in human pluripotent stem cells does not adversely affect their neuronal differentiation potential and that collections of isogenic cell lines with differing levels of αSyn expression are valid and suitable models to investigate synucleinopathies.

Aggregation of alpha-synuclein (α-Syn) drives Parkinson’s disease (PD), although the initial stages of self-assembly and structural conversion have not been directly observed inside neurons. In this study, we tracked the intracellular conformational states of α-Syn using a single-molecule Förster resonance energy transfer (smFRET) biosensor, and we show here that α-Syn converts from a monomeric state into two distinct oligomeric states in neurons in a concentration-dependent and sequence-specific manner. Three-dimensional FRET-correlative light and electron microscopy (FRET-CLEM) revealed that intracellular seeding events occur preferentially on membrane surfaces, especially at mitochondrial membranes. The mitochondrial lipid cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid–protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial reactive oxygen species (ROS) generation, which accelerates the oligomerization of A53T α-Syn and causes permeabilization of mitochondrial membranes and cell death. These processes were also observed in induced pluripotent stem cell (iPSC)–derived neurons harboring A53T mutations from patients with PD. Our study highlights a mechanism of de novo α-Syn oligomerization at mitochondrial membranes and subsequent neuronal toxicity.

SNCA, the first gene associated with Parkinson’s disease, encodes the α-synuclein protein, the predominant component within pathological inclusions termed Lewy bodies. The presence of Lewy bodies is one of the classical hallmarks found in the brain of patients with Parkinson’s disease, and Lewy bodies have also been observed in patients with other synucleinopathies. However, the study of α-synuclein pathology in cells has relied largely on two-dimensional culture models, which typically lack the cellular diversity and complex spatial environment found in the brain. Here, to address this gap, we use three-dimensional midbrain organoids, differentiated from human-induced pluripotent stem cells derived from patients carrying a triplication of the SNCA gene and from CRISPR/Cas9 corrected isogenic control iPSCs. These human midbrain organoids recapitulate key features of α-synuclein pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication human midbrain organoids express elevated levels of α-synuclein and exhibit an age-dependent increase in α-synuclein aggregation, manifested by the presence of both oligomeric and phosphorylated forms of α-synuclein. These phosphorylated α-synuclein aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, human midbrain organoids from patients carrying SNCA gene multiplication can reliably model key pathological features of Parkinson’s disease and provide a powerful system to study the pathogenesis of synucleinopathies.

The protein alpha-synuclein is predominantly expressed in neurons and is associated with neurodegenerative diseases like Parkinson’s disease and dementia with Lewy bodies. However, the normal function of alpha-synuclein in neurons is not clearly defined. We have previously shown that mice lacking alpha-synuclein expression exhibit markedly increased viral growth in the brain, increased mortality and increased neuronal cell death, implicating alpha-synuclein in the neuronal innate immune response. To investigate the mechanism of alpha-synuclein-induced immune responses to viral infections in the brain, we challenged alpha-synuclein knockout mice and human alpha-synuclein knockout dopaminergic neurons with RNA virus infection and discovered that alpha-synuclein is required for neuronal expression of interferon-stimulated genes. Furthermore, human alpha-synuclein knockout neurons treated with type 1 interferon failed to induce a broad range of interferon stimulated genes, implying that alpha-synuclein interacts with type 1 interferon signalling. We next found that alpha-synuclein accumulates in the nucleus of interferon-treated human neurons after interferon treatment and we demonstrated that interferon-mediated phosphorylation of STAT2 is dependent on alpha-synuclein expression in human neurons. Next, we found that activated STAT2 co-localizes with alpha-synuclein following type 1 interferon stimulation in neurons. Finally, we found that brain tissue from patients with viral encephalitis expresses increased levels of phospho-serine129 alpha-synuclein in neurons. Taken together, our results show that alpha-synuclein expression supports neuron-specific interferon responses by localizing to the nucleus, supporting STAT2 activation, co-localizing with phosphorylated STAT2 in neurons and supporting expression of interferon-stimulated genes. These data provide a novel mechanism that links interferon activation and alpha-synuclein function in neurons.

Many proteins that self-assemble into amyloid and amyloid-like fibres can adopt polymorphic forms. These forms have been observed both in vitro and in vivo and can arise through variations in the steric-zipper interactions between β-sheets, variations in the arrangements between protofilaments, and differences in the number of protofilaments that make up a given fibre class. Different polymorphs which arise from the same precursor molecule not only exhibit different levels of toxicity, but importantly can contribute to different disease conditions. In this work, we show that in the presence of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine, a highly abundant lipid in the plasma membrane of neurons, the aggregation of α-synuclein is markedly accelerated and yields a diversity of polymorphic forms under identical experimental conditions. This morphological diversity includes thin and curly amyloid fibrils, helical and twisted ribbons, nanotubes and flat sheets. TEM analysis of fibrils sampled from the early stage of the growth phase shows the presence of helical and twisted ribbons, indicating that these morphological variants form at the early stages of aggregation. Total internal reflection fluorescence microscopy (TIRFM) indicated the presence of lipids collocated with the mature fibrils. This finding has important implication as the presence of α-synuclein with co-localized high lipid content has been reported in Lewy bodies, the pathological hallmark of Parkinson’s disease and other synucleinopathies. Thus, the present study demonstrates that an interface, such as that provided by a lipid membrane, can not only modulate the kinetics of α-synuclein amyloid aggregation but also plays an important role in the formation of morphological variants by incorporating lipid molecules in the process of amyloid fibril formation.

Summary During mammalian development, liver differentiation is driven by signals that converge on multiple transcription factor networks. The hepatocyte nuclear factor signaling network is known to be essential for hepatocyte specification and maintenance. In this study, we have generated deletion and point mutants of hepatocyte nuclear factor-4alpha (HNF4α) to precisely evaluate the function of protein domains during hepatocyte specification from human pluripotent stem cells. We demonstrate that nuclear HNF4α is essential for hepatic progenitor specification, and the introduction of point mutations in HNF4α′s Small Ubiquitin-like Modifier (SUMO) consensus motif leads to disrupted hepatocyte differentiation. Taking a multiomics approach, we identified key deficiencies in cell biology, which included dysfunctional metabolism, substrate adhesion, tricarboxylic acid cycle flux, microRNA transport, and mRNA processing. In summary, the combination of genome editing and multiomics analyses has provided valuable insight into the diverse functions of HNF4α during pluripotent stem cell entry into the hepatic lineage and during hepatocellular differentiation., Graphical Abstract, Highlights • Deletion of HNF4α's DNA-binding domain blocks hepatic progenitor formation • HNF4α′s C-terminal SUMO consensus motif plays roles in hepatocyte specification • Multiomics analyses reveal various processes regulated by HNF4α during differentiation, Biological Sciences; Cell Biology; Developmental Biology; Stem Cells Research

[Image: see text] α-Synuclein fibrils are considered a hallmark of Parkinson’s disease and other synucleinopathies. However, small oligomers that formed during the early stages of α-synuclein aggregation are thought to be the main toxic species causing disease. The formation of α-synuclein oligomers has proven difficult to follow, because of the heterogeneity and transient nature of the species formed. Here, a novel bead-based aggregation assay for monitoring the earliest stages of α-synuclein oligomerization, α-Synuclein–Confocal Nanoscanning (ASYN-CONA), is presented. The α-synuclein A91C single cysteine mutant is modified with a trifunctional chemical tag, which allows simultaneous fluorescent labeling with a green dye (tetramethylrhodamine, TMR) and attachment to microbeads. Beads with bound TMR-labeled α-synuclein are then incubated with a red dye (Cy5)-labeled variant of α-synuclein A91C, and EtOH (20%) to induce aggregation. Aggregation is detected by confocal scanning imaging, below the equatorial plane of the beads, which is known as the CONA technique. On-bead TMR-labeled α-synuclein and aggregated Cy5-labeled α-synuclein from the solution are quantitatively monitored in parallel by detection of fluorescent halos or “rings”. α-Synuclein on-bead oligomerization results in a linear increase of red bead ring fluorescence intensity over a period of 5 h. Total internal reflection fluorescence microscopy was performed on oligomers cleaved from the beads, and it revealed that (i) oligomers are sufficiently stable in solution to investigate their composition, consisting of 6 ± 1 monomer units, and (ii) oligomers containing a mean of 15 monomers bind Thioflavin-T. Various known inhibitors of α-synuclein aggregation were used to validate the ASYN-CONA assay for drug screening. Baicalein, curcumin, and rifampicin showed concentration-dependent inhibition of the α-synuclein aggregation and the IC(50) (the concentration of the compound at which the maxiumum intensity was reduced by one-half) were calculated.

SNCA, the first gene associated with Parkinson’s disease, encodes the α-synuclein protein, the predominant component within pathological inclusions termed Lewy bodies. The presence of Lewy bodies is one of the classical hallmarks found in the brain of patients with Parkinson’s disease, and Lewy bodies have also been observed in patients with other synucleinopathies. However, the study of α-synuclein pathology in cells has relied largely on two-dimensional culture models, which typically lack the cellular diversity and complex spatial environment found in the brain. Here, to address this gap, we use three-dimensional midbrain organoids, differentiated from human-induced pluripotent stem cells derived from patients carrying a triplication of the SNCA gene and from CRISPR/Cas9 corrected isogenic control iPSCs. These human midbrain organoids recapitulate key features of α-synuclein pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication human midbrain organoids express elevated levels of α-synuclein and exhibit an age-dependent increase in α-synuclein aggregation, manifested by the presence of both oligomeric and phosphorylated forms of α-synuclein. These phosphorylated α-synuclein aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, human midbrain organoids from patients carrying SNCA gene multiplication can reliably model key pathological features of Parkinson’s disease and provide a powerful system to study the pathogenesis of synucleinopathies., Mohamed et al. investigate synucleinopathy in human midbrain organoids, derived from patients carrying a triplication of the SNCA gene. Human midbrain organoids recapitulate pathology observed in the brains of patients: an elevated level of α-synuclein, an age-dependent increase in aggregation and a selective reduction in dopaminergic neurons., Graphical Abstract Graphical Abstract

SNCA, the first gene associated with Parkinson′s disease, encodes the α-synuclein (α-syn) protein, the predominant component within pathological inclusions termed Lewy bodies (LBs). The presence of LBs is one of the classical hallmarks found in the brain of patients with Parkinson′s disease, and LBs have also been observed in patients with other synucleinopathies. However, the study of α-syn pathology in cells has relied largely on two-dimensional culture models, which typically lack the cellular diversity and complex spatial environment found in the brain. Here, to address this gap, we use 3D midbrain organoids (hMOs), differentiated from human induced pluripotent stem cells derived from patients carrying a triplication of the SNCA gene and from CRISPR/Cas9 corrected isogenic control iPSCs. These hMOs recapitulate key features of α-syn pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication hMOs express elevated levels of α-syn and exhibit an age-dependent increase in α-syn aggregation, manifested by the presence of both oligomeric and phosphorylated forms of α-syn. These phosphorylated α-syn aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, hMOs from patients carrying SNCA gene multiplication can reliably model key pathological features of Parkinson′s disease and provide a powerful system to study the pathogenesis of synucleinopathies.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that does not currently have a robust clinical diagnostic test. Nonmotor symptoms such as skin disorders have long since been associated with the disease, and more recently a characteristic odor emanating from the skin of people with Parkinson’s has been identified. Here, dynamic head space (DHS) thermal desorption (TD) gas chromatography–mass spectrometry (GC-MS) is implemented to directly measure the volatile components of sebum on swabs sampled from people with Parkinson’s—both drug naïve and those on PD medications (n = 100) and control subjects (n = 29). Supervised multivariate analyses of data showed 84.4% correct classification of PD cases using all detected volatile compounds. Variable importance in projection (VIP) scores were generated from these data, which revealed eight features with VIP > 1 and p < 0.05 which all presented a downregulation within the control cohorts. Purified standards based on previously annotated analytes of interest eicosane and octadecanal did not match to patient sample data, although multiple metabolite features are annotated with these compounds all with high spectral matches indicating the presence of a series of similar structured species. DHS-TD-GC-MS analysis of a range of lipid standards has revealed the presence of common hydrocarbon species rather than differentiated intact compounds which are hypothesized to be breakdown products of lipids. This replication study validates that a differential volatile profile between control and PD cohorts can be measured using an analytical method that measures volatile compounds directly from skin swabs., We validate our previously published findings (cohort of 64) that distinct metabolites in sebum differentiate a person with Parkinson’s from matched controls with an improved (84−86%) correct classification rate.

Human embryonic stem cell (hESC) – derived midbrain dopamine (DA) neurons stand out as a cell source for transplantation with their sustainability and consistency superior to the formerly used fetal tissues. However, multiple studies of DA neurons in culture failed to register action potential (AP) generation in response to synaptic input. To test, whether this is due to deficiency of NMDA receptor (NMDAR) co-agonists Glycine and D-Serine released from astroglia in living tissue, we studied the functional properties of neural receptors in hESC-derived DA neuronal cultures. We find that, apart of insufficient amount of co-agonists, lack of interneuronal crosstalk is caused by hypofunction of synaptic NMDARs due to their direct inhibition by synaptically released dopamine. This inhibitory tone is independent from DA receptors and affects NMDARs via the modulation of co-agonist binding site.

A metabolomics profiling approach was conducted to identify diagnostic biomarkers of PD from sebum, a non-invasively available biofluid. In this study, we used liquid chromatography-mass spectrometry (LC-MS) to analyse 274 samples from participants (80 drug naïve PD, 138 medicated PD and 56 well matched control subjects) and detected metabolites that could predict PD phenotype. Partial least squares-discriminant analysis (PLS-DA) models based on this sebum metabolome had correct classification rates of 70.4% and 69.7% to distinguish between drug naïve PD and medicated PD from control, respectively. Variable importance in projection (VIP) scores indicate compounds with significance belonged to sphingolipid, triacylglycerol and fatty acid/ester lipid classes. Pathway enrichment analysis showed alterations in lipid metabolism and mitochondrial dysfunction viz. the carnitine shuttle, sphingolipid metabolism and arachidonic acid metabolism. This study unveiled novel diagnostic sebum-based biomarkers for PD, and provides insight towards our current understanding of the pathogenesis of PD.

The protein alpha-synuclein (asyn) is predominantly expressed in neurons and is associated with neurodegenerative diseases like Parkinson’s disease (PD); yet, a functional role for asyn in neurons is not clearly established. We have previously shown that asyn expression is up-regulated following viral infection in neurons and is critical for host immune responses to RNA virus infections. Here, we investigate the mechanisms underlying asyn-dependent immune responses to RNA virus infection in the brain. Using asyn knock-out (KO) mice and human neuronal models, we show that asyn is required for expression of the full repertoire of interferon-stimulated genes (ISGs) in neurons following acute RNA virus infection. Furthermore, treatment of asyn KO human neurons with poly I:C or type I interferon also fail to induce expression of the full complement of ISGs suggesting that asyn plays an important role in modulating neuronal innate immune responses. In brain tissue, asyn-dependent ISG expression is independent of microglia activation and supports activation of infiltrating lymphocytes following viral challenge. We also show that virus infections lead to accumulation of phosphorylated S129 asyn in human and non-human primate neuronal tissues. In a model of pS129 asyn pathology, we found that infection with West Nile virus increases microglia activation but does not significantly alter pS129 asyn pathology in the mouse model. Taken together, our results establish asyn as a novel, neuron-specific modulator of innate immunity by a mechanism that promotes interferon-stimulated gene expression and links responses to virus infection with formation of phosphorylated S129-asyn in neuronal tissue.

Differentiation of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) into midbrain dopaminergic (mDA) neurons is described in this protocol. This protocol has been adapted from published protocols from three laboratories, Lorenz Studer (New York), Malin Parmar/Agnete Kirkeby (Lund), and Su-Chun Zhang (Madison). The relevant papers from these labs are PMIDs: 20362538, 22056989, 22813745, 28094017, 28858290, 22696177. This protocol instructs hESCs and hiPSCs to adopt a midbrain floor plate radial glia-like identity before maturation into mDA neurons with mixed substania nigra pars compacta and ventral tegemental area identity. It has been applied to more than 10 hESC and hiPSC lines in our laboratory, with some cell lines producing exceptional results, and others less so. Our version the protocol is published here PMID: 30472757.

Recent advancements in protocols to differentiate human pluripotent stem cells into midbrain dopaminergic (mDA) neurons has improved the ability to model Parkinson’s disease (PD) in a dish, and has provided a scalable source of donor cells for emerging PD cell replacement therapy (CRT). However, to facilitate reproducibility, collaboration, and clinical trials it would be highly beneficial to cryopreserve committed mDA neural precursors cells in a ready-to-use format. In terms of cell manufacturing for PD CRT trials, a cryopreserved transplantation-ready mDA cell product would provide a critical opportunity for quality control, efficacy testing, and safety assessments. To address this challenge, we have compared six (6) different clinical-grade cryopreservation media and different freezing conditions for mDA neural precursor cells differentiated from two human embryonic stem cell (ESC) lines, MasterShef7 and RC17. Significant differences in cell viability were observed at 24h post-thawing, but no differences were observed immediately upon thawing. This highlights the need to check cell viability over the first 24h after thawing, and that viability of freshly thawed cells is insufficient to gauge the success of a cryopreservation protocol. Considerable apoptosis occurs in the first 24h post-thawing, and significant differences between cryopreservation procedures were only revealed during this time period. The presence of ROCK inhibitors improved cell viability at 24h for all conditions tested. A faster cooling rate of 1-2°C/min was significantly better than 0.5°C/min for all conditions tested, while rapid thawing at 37°C was not always superior to slow thawing at 4°C. Indeed, the optimal cryopreservation and thawing conditions in this study, as determined by 24h post-thaw viability, were cells frozen in PSC Cryopreservation medium at a cooling rate of 1°C/min and slow thawing at 4°C. These conditions permitted recovery of 60%-70% live cells at 24h with respect to the starting number of cryopreserved cells. Importantly, cryopreservation of mDA neural precursor cells did not alter their potential to resume differentiation into mDA neurons.HighlightsFirst systematic comparison of multiple clinical-grade cryopreservation media for human ESC-derived mDA neural precursor cellsDifferences in cell viability were observed at 24h after thawing, but not immediately upon thawingCooling rates of 1°C/min or 2°C/min were significantly better than 0.5°C/min for all cryopreservation conditions testedA slow thawing condition at 4°C was significantly better than quick thawing at 37°C for cells frozen in PSC Cryopreservation mediumCryopreservation of mDA cells does not significantly alter their potential to differentiate into mDA neurons

A metabolomics profiling approach was conducted to identify diagnostic biomarkers of PD from sebum, a non-invasively available biofluid. In this study, we used liquid chromatography-mass spectrometry (LC-MS) to analyse 274 samples from participants (80 drug naïve PD, 138 medicated PD and 56 well matched control subjects) and detected metabolites that could predict PD phenotype. Partial least squares-discriminant analysis (PLS-DA) models based on this sebum metabolome had correct classification rates of 70.4% and 69.7% to distinguish between drug naïve PD and medicated PD from control, respectively. Variable importance in projection (VIP) scores indicate compounds with significance belonged to sphingolipid, triacylglycerol and fatty acid/ester lipid classes. Pathway enrichment analysis showed alterations in lipid metabolism and mitochondrial dysfunction viz. the carnitine shuttle, sphingolipid metabolism and arachidonic acid metabolism. This study unveiled novel diagnostic sebum-based biomarkers for PD, and provides insight towards our current understanding of the pathogenesis of PD.

Functional integrity of the E3-ubiquitin ligase CHIP is essential for healthy aging in the brain. By virtue of its association with the core molecular chaperone machinery, studies into the physiological role(s) of CHIP have been dominated by its involvement in the quality control (QC) and degradation of unfolded, aggregated and/or mutated proteins. To elucidate the dominant changes in protein steady state levels in human neurons where CHIP function was ablated, we conducted an unbiased proteomic analysis in genetically engineered CHIP knockout compared to wild-type iPSC-derived cortical neurons. Rather than a broad effect on protein homeostasis, loss of CHIP function impacted on a focused cohort of proteins that analysis defined as actin cytoskeleton signalling and membrane integrity pathways. In support of the pathway analysis, CHIP KO cell-lines had enhanced sensitivity to mechanical and chemical membrane damage. Consistent with recent studies on the identification of a non-canonical HSP-independent E3-activity for CHIP, the lack of general QC defects in knock-out cells suggests that the major readout of CHIP function in cortical neurons is the regulation of native folded substrates involved in maintaining cellular ‘health’. Thus, CHIP-mediated regulation of the of cytoskeletal- and membrane-related proteins is likely to make a vital contribution to the neuroprotective activity of CHIP.

Summary CHIP is an E3-ubiquitin ligase that contributes to healthy aging and has been characterized as neuroprotective. To elucidate dominant CHIP-dependent changes in protein steady-state levels in a patient-derived human neuronal model, CHIP function was ablated using gene-editing and an unbiased proteomic analysis conducted to compare knock-out and wild-type isogenic induced pluripotent stem cell (iPSC)-derived cortical neurons. Rather than a broad effect on protein homeostasis, loss of CHIP function impacted on a focused cohort of proteins from actin cytoskeleton signaling and membrane integrity networks. In support of the proteomics, CHIP knockout cells had enhanced sensitivity to induced membrane damage. We conclude that the major readout of CHIP function in cortical neurons derived from iPSC of a patient with elevate α-synuclein, Parkinson's disease and dementia, is the modulation of substrates involved in maintaining cellular “health”. Thus, regulation of the actin cytoskeletal and membrane integrity likely contributes to the neuroprotective function(s) of CHIP., Graphical abstract, Highlights • Cortical neurons were generated from iPSC of a patient with Parkinson's disease with ablated CHIP • SWATH-MS identified a focused cohort of CHIP modulated proteins • CHIP loss impacts actin cytoskeleton signaling and membrane integrity networks • CHIP is protective for induced membrane damage, Cell biology; Organizational aspects of cell biology; Bioinformatics; Omics; Proteomics

Human induced pluripotent stem cells (hiPSCs) are invaluable to study developmental processes and disease mechanisms particularly in the brain. hiPSCs can be differentiated into mature and functional dopaminergic (DA) neurons. Having robust protocols for the generation of differentiated DA neurons from pluripotent cells is a prerequisite for the use of hiPSCs to study disease mechanisms, for drug discovery, and eventually for cell replacement therapy. Here, we describe a protocol for generating and expanding large numbers of homogeneous midbrain floor plate progenitors (mFPPs) that retain efficient DA neurogenic potential over multiple passages and can be cryobanked. We demonstrate that expanded mFPPs have increased DA neuron potential and differentiate more efficiently and rapidly than progenitors generated by standard protocols. In addition, this novel method results in increased numbers of DA neurons that in vitro show characteristic electrophysiological properties of nigrostriatal DA neurons, produce high levels of dopamine, and integrate into host mice when grafted in vivo. Thus, we describe a robust method for producing human mesencephalic DA neurons from hiPSCs.

Parkinson's disease (PD) is a common neurodegenerative condition affecting more than 8 million people worldwide. Although, the majority of PD cases are sporadic in nature, there are a growing number of monogenic mutations identified to cause PD in a highly penetrant manner. Many of these familial mutations give rise to a condition that is clinically and neuropathologically similar, if not identical, to sporadic PD. Mutations in genes such as SNCA cause PD in an autosomal dominant manner and patients have motor and non-motor symptoms that are typical for sporadic PD. With the advent of reprogramming technology it is now possible to capture these mutations in induced pluripotent stem cells (iPSCs) to establish models of PD in a dish. There are multiple neuronal subtypes affected in PD including the midbrain dopaminergic (mDA) neurons of the substantia nigra. Robust neuronal differentiation into mDA or other relevant neural cell types are critical to accurately model the disease and ensure the findings are relevant to understanding the disease process. Another challenge for establishing accurate models of PD is being met by the generation of isogenic control iPSC lines with precise correction of mutations using advanced gene editing technology. The contributions of ageing and environmental factors present further challenges to this field, but significant progress is being made in these areas to establish highly relevant and robust models of PD. These human neuronal models, used in conjunction with other model systems, will vastly improve our understanding of the early stages of the PD, which will be key to identifying disease-modifying and preventative treatments.