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

© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society). 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.This research was supported by the Danish Parkinson Association, IMK Almene Fond, Hørslev-Fonden, Kirsten og Freddy Johansens Fond, Grosserer Brogaard og Hustrus Mindefond and Fonden for Lægevidenskabens Fremme.Peer Reviewe

Lysosomal perturbations in human dopaminergic neurons derived from induced pluripotent stem cells with <i>PARK2</i> 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, β-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.Innovation Fund Denmark (BrainStem; 4108–00008 A), H. Lundbeck A/S (R167-2013-15778), the Danish Parkinson Foundation (3687), the A.P. Møller Foundation for the Advancement of Medical Science (15–396, 14–427), and the Faculty of Health Sciences at University of Southern Denmark. Work at the AMS group at the CBMSO-Madrid was supported by grants from MINECO (SAF-2017-83241-R), and ISC-III RETICS TerCel (RD16/0011/0032)

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

In this article, Schmidt and colleagues show that differentiating human NSCs in co-culture with microglia enhance dopaminergic differentiation. The effect is consistent across different NSC and microglial cell lines but restricted to microglia of embryonic origin. TNFα, IL-1β, and IGF1 are identified as key mediators of the effect, providing new insights into factors stimulating dopaminergic differentiation.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-1β, 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.Innovation Fund Denmark (BrainStem; www.brainstem.dk), the Lundbeck Foundation, the Danish Parkinson Foundation, the Jascha Foundation, IMK Almene Fond, the A.P. Møller Foundation, and the Faculty of Health Sciences, University of Southern Denmar

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

Abstract 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+/CD29low sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM+/CD29low 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+/CD29low 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.</jats:p

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