Lipotype acquisition during neural development is not recapitulated in stem cell-derived neurons.

During development, different tissues acquire distinct lipotypes that are coupled to tissue function and homeostasis. In the brain, where complex membrane trafficking systems are required for neural function, specific glycerophospholipids, sphingolipids, and cholesterol are highly abundant, and defective lipid metabolism is associated with abnormal neural development and neurodegenerative disease. Notably, the production of specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery during development, but when and how this occurs is unclear. To address this, we used high-resolution MSALL lipidomics to generate an extensive time-resolved resource of mouse brain development covering early embryonic and postnatal stages. This revealed a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero, whereas cholesterol attains its characteristic high levels after birth. Using the resource as a reference, we next examined to which extent this can be recapitulated by commonly used protocols for in vitro neuronal differentiation of stem cells. Here, we found that the programming of the lipid metabolic machinery is incomplete and that stem cell-derived cells can only partially acquire a neural lipotype when the cell culture media is supplemented with brain-specific lipid precursors. Altogether, our work provides an extensive lipidomic resource for early mouse brain development and highlights a potential caveat when using stem cell-derived neuronal progenitors for mechanistic studies of lipid biochemistry, membrane biology and biophysics, which nonetheless can be mitigated by further optimizing in vitro differentiation protocols.

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.

Identification of bioactive metabolites in human iPSC-derived dopaminergic neurons with PARK2 mutation: Altered mitochondrial and energy metabolism.

PARK2 (parkin) mutations cause early-onset Parkinson's disease (PD). Parkin is an ubiquitin E3 ligase that participates in several cellular functions, including mitochondrial homeostasis. However, the specific metabolomic changes caused by parkin depletion remain unknown. Here, we used isogenic human induced pluripotent stem cells (iPSCs) with and without PARK2 knockout (KO) to investigate the effect of parkin loss of function by comparative metabolomics supplemented with ultrastructural and functional analyses. PARK2 KO neurons displayed increased tricarboxylic acid (TCA) cycle activity, perturbed mitochondrial ultrastructure, ATP depletion, and dysregulation of glycolysis and carnitine metabolism. These perturbations were combined with increased oxidative stress and a decreased anti-oxidative response. Key findings for PARK2 KO cells were confirmed using patient-specific iPSC-derived neurons. Overall, our data describe a unique metabolomic profile associated with parkin dysfunction and show that combining metabolomics with an iPSC-derived dopaminergic neuronal model of PD is a valuable approach to obtain novel insight into the disease pathogenesis.

Microglia-Secreted Factors Enhance Dopaminergic Differentiation of Tissue- and iPSC-Derived Human Neural Stem Cells.

Microglia have recently been established as key regulators of brain development. However, their role in neuronal subtype specification remains largely unknown. Using three different co-culture setups, we show that microglia-secreted factors enhance dopaminergic differentiation of somatic and induced pluripotent stem cell-derived human neural stem cells (NSCs). The effect was consistent across different NSC and microglial cell lines and was independent of prior microglial activation, although restricted to microglia of embryonic origin. We provide evidence that the effect is mediated through reduced cell proliferation and decreased apoptosis and necrosis orchestrated in a sequential manner during the differentiation process. tumor necrosis factor alpha, interleukin-1ß, and insulinlike growth factor 1 are identified as key mediators of the effect and shown to directly increase dopaminergic differentiation of human NSCs. These findings demonstrate a positive effect of microglia on dopaminergic neurogenesis and may provide new insights into inductive and protective factors that can stimulate in vitro derivation of dopaminergic neurons.

The Validity of Intracerebral Hemorrhage Diagnoses in the Danish Patient Registry and the Danish Stroke Registry.

PURPOSE: To establish the validity of intracerebral hemorrhage (ICH) diagnoses in the Danish Stroke Registry (DSR) and the Danish National Patient Registry (DNPR). PATIENTS AND METHODS: Based on discharge summaries and brain imaging reports, we estimated the positive predictive value (PPV) of a first-ever diagnosis code for ICH (ICD-10, code I61) for all patients in the Region of Southern Denmark (1.2 million) during 2009-2017 according to either DNPR or DSR. We estimated PPVs for any non-traumatic ICH (a-ICH) and spontaneous ICH (s-ICH) alone (ie, without underlying structural cause). We also calculated the sensitivity of these diagnoses in each of the registers. Finally, we classified the location of verified s-ICH. RESULTS: A total of 3,956 patients with ICH diagnosis codes were studied (DSR only: 87; DNPR only: 1,513; both registries: 2,356). In the DSR, the PPVs were 86.5% (95% CI=85.1-87.8) for a-ICH and 81.8% (95% CI=80.2-83.3) for s-ICH. The PPVs in DNPR (discharge code, primary diagnostic position) were 76.2% (95% CI=74.7-77.6) for a-ICH and 70.2% (95% CI=68.6-71.8) for s-ICH. Sensitivity for a-ICH and s-ICH was 76.4% (95% CI=74.8-78.0) and 78.7% (95% CI=77.1-80.2) in DSR, and 87.3% (95% CI=86.0-88.5) and 87.7% (95% CI=86.3-88.9) in DNPR. The location of verified s-ICH was lobar (39%), deep (33.6%), infratentorial (13.2%), large unclassifiable (11%), isolated intraventricular (1.9%), or unclassifiable due to insufficient information (1.3%). CONCLUSION: The validity of a-ICH diagnoses is high in both registries. For s-ICH, PPV was higher in DSR, while sensitivity was higher in DNPR. The location of s-ICH was similar to distributions seen in other populations.

Levodopa Therapy for Parkinson's Disease: History, Current Status and Perspectives.

Parkinson's Disease (PD) is a neurodegenerative disorder characterized by a preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta. This results in a profound decrease of striatal dopamine (DA) levels, which in turn leads to the cardinal motor symptoms of PD; muscle rigidity, hypo- and bradykinesia and resting tremor. Even 50 years after its initial use, the DA precursor levodopa (L-dopa), is still the most effective medical therapy for the symptomatic treatment of PD. Long-term L-dopa treatment is however, unfortunately associated with undesirable side effects such as motor fluctuations and dyskinesias. Furthermore, despite the disease alleviating effects of L-dopa, it is still discussed whether L-dopa has a neurotoxic or neuroprotective effect on dopaminergic neurons. Here we review the history of L-dopa, including its discovery, development and current use in the treatment of PD. We furthermore review current evidence of the L-dopa-induced side effects and perspectives of L-dopa treatment in PD compared to other established treatments such as DA-agonists and the inhibitors of catechol-o-methyltransferase and monoamine oxidase B.

Lysosomal perturbations in human dopaminergic neurons derived from induced pluripotent stem cells with PARK2 mutation.

Mutations in the PARK2 gene encoding parkin, an E3 ubiquitin ligase, are associated with autosomal recessive early-onset Parkinson's disease (PD). While parkin has been implicated in the regulation of mitophagy and proteasomal degradation, the precise mechanism leading to neurodegeneration in both sporadic and familial PD upon parkin loss-of-function remains unknown. Cultures of isogenic induced pluripotent stem cell (iPSC) lines with and without PARK2 knockout (KO) enable mechanistic studies of the effect of parkin deficiency in human dopaminergic neurons. We used such cells to investigate the impact of PARK2 KO on the lysosomal compartment and found a clear link between parkin deficiency and lysosomal alterations. PARK2 KO neurons exhibited a perturbed lysosomal morphology with enlarged electron-lucent lysosomes and an increased lysosomal content, which was exacerbated by mitochondrial stress and could be ameliorated by antioxidant treatment. We also found decreased lysosomal enzyme activity and autophagic perturbations, suggesting an impairment of the autophagy-lysosomal pathway in parkin-deficient cells. Interestingly, activity of the GBA-encoded enzyme, ß-glucocerebrosidase, was increased, suggesting the existence of a compensatory mechanism. In conclusion, our data provide a unique characterization of the morphology, content, and function of lysosomes in PARK2 KO neurons and reveal an important new connection between mitochondrial dysfunction and lysosomal dysregulation in PD pathogenesis.

Perturbations in RhoA signalling cause altered migration and impaired neuritogenesis in human iPSC-derived neural cells with PARK2 mutation.

Mutations in parkin, encoded by the PARK2 gene, causes early-onset familial Parkinson's disease (PD), but dysfunctional parkin has also been implicated in sporadic PD. By combining human isogenic induced pluripotent stem cells (iPSCs) with and without PARK2 knockout (KO) and a novel large-scale mass spectrometry based proteomics and post-translational modification (PTM)-omics approach, we have mapped changes in protein profiles and PTMs caused by parkin deficiency in neurons. Our study identifies changes to several proteins previously shown to be dysregulated in brains of sporadic PD patients. Pathway analysis and subsequent in vitro assays reveal perturbations in migration and neurite outgrowth in the PARK2 KO neurons. We confirm the neurite defects using long-term engraftment of neurons in the striatum of immunosuppressed hemiparkinsonian adult rats. The GTP-binding protein RhoA was identified as a key upstream regulator, and RhoA activity was significantly increased in PARK2 KO neurons. By inhibiting RhoA signalling the migration and neurite outgrowth phenotypes could be rescued. Our study provides new insight into the pathogenesis of PD and demonstrates the broadly applicable potential of proteomics and PTMomics for elucidating the role of disease-causing mutations.

PARK2 Mutation Causes Metabolic Disturbances and Impaired Survival of Human iPSC-Derived Neurons.

The protein parkin, encoded by the PARK2 gene, is vital for mitochondrial homeostasis, and although it has been implicated in Parkinson's disease (PD), the disease mechanisms remain unclear. We have applied mass spectrometry-based proteomics to investigate the effects of parkin dysfunction on the mitochondrial proteome in human isogenic induced pluripotent stem cell-derived neurons with and without PARK2 knockout (KO). The proteomic analysis quantified nearly 60% of all mitochondrial proteins, 119 of which were dysregulated in neurons with PARK2 KO. The protein changes indicated disturbances in oxidative stress defense, mitochondrial respiration and morphology, cell cycle control, and cell viability. Structural and functional analyses revealed an increase in mitochondrial area and the presence of elongated mitochondria as well as impaired glycolysis and lactate-supported respiration, leading to an impaired cell survival in PARK2 KO neurons. This adds valuable insight into the effect of parkin dysfunction in human neurons and provides knowledge of disease-related pathways that can potentially be targeted for therapeutic intervention.

Activation of Group II Metabotropic Glutamate Receptors Increases Proliferation but does not Influence Neuronal Differentiation of a Human Neural Stem Cell Line.

The multiple functions of glutamate include regulation of neural development and stem cells. While the importance of the ionotropic glutamate receptors is well-established, less is known about the role of metabotropic glutamate receptors (mGluRs). In this study, we examined the effects of pharmacological activation and inhibition of mGluR2/3 on proliferation, differentiation and viability of a human neural stem cell line. Immunofluorescence staining revealed the presence of mGluR2/3 receptors on both proliferating and differentiating stem cells, including cells differentiated into ß-tubulin III-positive immature neurons and glial fibrillary acidic protein-positive astrocytes. Stimulation of mGluR2/3 receptors during cell propagation using the agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl) glycine (DCG-IV) increased total cell numbers significantly (60% compared to untreated controls). This effect could be inhibited by the specific antagonist (2S)-2-Amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495). The antagonist alone had no effect. No significant decrease in cell death was found following mGluR2/3 stimulation, suggesting that the observed elevation in cell number was not related to cell viability. Subsequent differentiation of the cells resulted in a slight decrease in ß-tubulin III-positive neurons (5.2-3.2% of total cells) for DCG-IV pre-treated cultures. Treatment with DCG-IV and LY342495 during cell differentiation alone had no such effect. Western blot analysis revealed that the active, dimeric form of mGluR2/3 was mainly present on the proliferating cells, which may explain our findings. This study emphasizes the importance of glutamate and mGluRs on regulation of human neural stem cells and suggests a significant role of mGluR2/3 during cell proliferation.

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.

Group I metabotropic glutamate receptors: a potential target for regulation of proliferation and differentiation of an immortalized human neural stem cell line.

Human neural stem cells (NSCs) from the developing embryo or the subventricular zone of the adult brain can potentially elicit brain repair after injury or disease, either via endogenous cell proliferation or by cell transplantation. Profound knowledge of the diverse signals affecting these cells is, however, needed to realize their therapeutic potential. Glutamate and group I metabotropic glutamate receptors (mGluRs) affect proliferation and survival of rodent NSCs both during embryonic and post-natal development. To investigate the role of group I mGluRs (mGluR1 and mGluR5) on human NSCs, we differentiated an immortalized, forebrain-derived stem cell line in the presence or absence of glutamate and with addition of either the group I mGluR agonist DHPG or the selective antagonists, MPEP (mGluR5) and LY367385 (mGluR1). Characterization of differentiated cells revealed that both mGluR1 and mGluR5 were present on the cells. Addition of glutamate to the growth medium significantly increased cell proliferation and reduced cell death, resulting in increased cell numbers. In the presence of glutamate, selective activation of group I mGluRs reduced gliogenesis, whereas selective inhibition of group I mGluRs reduced neurogenesis. Our results substantiate the importance of glutamate signalling in the regulation of human NSCs and may as such be applied to promote proliferation and neuronal differentiation.

Improved cell therapy protocols for Parkinson's disease based on differentiation efficiency and safety of hESC-, hiPSC-, and non-human primate iPSC-derived dopaminergic neurons.

The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results, NCAM(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM(+) /CD29(low) DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM(+) /CD29(low) DA neurons were able to restore motor function of 6-hydroxydopamine (6-OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue, with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation, the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future.

Pharmacological rescue of mitochondrial deficits in iPSC-derived neural cells from patients with familial Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder caused by genetic and environmental factors that results in degeneration of the nigrostriatal dopaminergic pathway in the brain. We analyzed neural cells generated from induced pluripotent stem cells (iPSCs) derived from PD patients and presymptomatic individuals carrying mutations in the PINK1 (PTEN-induced putative kinase 1) and LRRK2 (leucine-rich repeat kinase 2) genes, and compared them to those of healthy control subjects. We measured several aspects of mitochondrial responses in the iPSC-derived neural cells including production of reactive oxygen species, mitochondrial respiration, proton leakage, and intraneuronal movement of mitochondria. Cellular vulnerability associated with mitochondrial dysfunction in iPSC-derived neural cells from familial PD patients and at-risk individuals could be rescued with coenzyme Q(10), rapamycin, or the LRRK2 kinase inhibitor GW5074. Analysis of mitochondrial responses in iPSC-derived neural cells from PD patients carrying different mutations provides insight into convergence of cellular disease mechanisms between different familial forms of PD and highlights the importance of oxidative stress and mitochondrial dysfunction in this neurodegenerative disease.