Etuaivoidentiteetillisten ihmisen monikykyisistä kantasoluista johdettujen astrosyyttien tuottaminen

Astrosyytit ovat hermoston tukisoluja, joiden toiminnalliset ja morfologiset ominaisuudet vaihtelevat eri aivoalueilla. Astrosyyttien ominaisuuksien vaihtelun on todettu olevan erityisen suurta ihmisen aivoissa. Ihmisen pluripotentit kantasolut (hPS-solut) mahdollistavat astroglian monimuotoisuutta säätelevien mekanismien tutkimisen. Olemme luoneet menetelmän, joka tuottaa hPS-soluista ihmisen etuaivojen astrosyyttejä, ja kuvanneet tuotettujen astrosyyttien erityispiirteitä. Määritimme hPS-soluista erilaistettujen solujen geenien ilmentymisprofiilin päivänä 0 (D0), neuronaalisen induktion jälkeen D12 sekä solujen kasvutekijöillä monistamisen jälkeen D30 ja D60. Astrosyyttien lopullinen määräytyminen toteutettiin siliaarisella neurotrofisella tekijällä (ciliary neurotrophic factor; CNTF) ja D95-ikäisien astrosyyttien osoitettiin ilmentävän lähes 100 prosenttisesti yleisesti käytössä olevia astrosyyttimarkkereita. Erilaistamisen aikana tehty geeniprofilointi vahvisti solujen etuaivojen identiteetin. Kuvasimme solunsisäisen kalsiumkuvantamisen avulla, että erilaistamamme astrosyytit olivat elinkykyisiä ja antoivat toiminnallisia vasteita ATP:lle. Lisäksi määritimme astrosyyttien perustehtävää eli kykyä säädellä immuunivasteita aivoissa tutkimalla niistä erittyvien sytokiinien määriä. Totesimme D95-astrosyyttien viljelynesteessä merkittäviä pitoisuuksia MCP-1- ja TIMP-2-proteiinia yhteneväisesti näitä proteiineja ilmentävien geenien kohonneisiin mRNA-määriin. Astrosyyttien erilaistamismenetelmä oli toistettavissa usealla hPSC-linjalla, ja tutkimuksemme osoitti, että erilaistamamme etuaivojen astrosyytit tarjoavat uudenlaisen keinon sekä astrosyyttien soluspesifisten ominaisuuksien että yhteisviljelmissä muiden hermoston solujen kanssa hermoston solujen yhteisvaikutusten tutkimiseen. Potilaskohtaisista hPS-soluista erilaistettujen astrosyyttien avulla voidaan selvittää ihmisen astrosyyttien toimintaa myös sairaustiloissa

Generation of the Human Pluripotent Stem-Cell-Derived Astrocyte Model with Forebrain Identity

Astrocytes form functionally and morphologically distinct populations of cells with brainregion-specific properties. Human pluripotent stem cells (hPSCs) offer possibilities to generate astroglia for studies investigating mechanisms governing the emergence of astrocytic diversity. We established a method to generate human astrocytes from hPSCs with forebrain patterning and final specification with ciliary neurotrophic factor (CNTF). Transcriptome profiling and gene enrichment analysis monitored the sequential expression of genes determining astrocyte differentiation and confirmed activation of forebrain differentiation pathways at Day 30 (D30) and D60 of differentiation in vitro. More than 90% of astrocytes aged D95 in vitro co-expressed the astrocytic markers glial fibrillary acidic protein (GFAP) and S100 beta. Intracellular calcium responses to ATP indicated differentiation of the functional astrocyte population with constitutive monocyte chemoattractant protein-1 (MCP-1/CCL2) and tissue inhibitor of metalloproteinases-2 (TIMP-2) expression. The method was reproducible across several hPSC lines, and the data demonstrated the usefulness of forebrain astrocyte modeling in research investigating forebrain pathology.Peer reviewe

Elevated de novo protein synthesis in FMRP-deficient human neurons and its correction by metformin treatment

FXS is the most common genetic cause of intellectual (ID) and autism spectrum disorders (ASD). FXS is caused by loss of FMRP, an RNA-binding protein involved in the translational regulation of a large number of neuronal mRNAs. Absence of FMRP has been shown to lead to elevated protein synthesis and is thought to be a major cause of the synaptic plasticity and behavioural deficits in FXS. The increase in protein synthesis results in part from abnormal activation of key protein translation pathways downstream of ERK1/2 and mTOR signalling. Pharmacological and genetic interventions that attenuate hyperactivation of these pathways can normalize levels of protein synthesis and improve phenotypic outcomes in animal models of FXS. Several efforts are currently underway to trial this strategy in patients with FXS. To date, elevated global protein synthesis as a result of FMRP loss has not been validated in the context of human neurons. Here, using an isogenic human stem cell-based model, we show that de novo protein synthesis is elevated in FMRP-deficient neural cells. We further show that this increase is associated with elevated ERK1/2 and Akt signalling and can be rescued by metformin treatment. Finally, we examined the effect of normalizing protein synthesis on phenotypic abnormalities in FMRP-deficient neural cells. We find that treatment with metformin attenuates the increase in proliferation of FMRP-deficient neural progenitor cells but not the neuronal deficits in neurite outgrowth. The elevated level of protein synthesis and the normalization of neural progenitor proliferation by metformin treatment were validated in additional control and FXS patient-derived hiPSC lines. Overall, our results validate that loss of FMRP results in elevated de novo protein synthesis in human neurons and suggest that approaches targeting this abnormality are likely to be of partial therapeutic benefit in FXS.Peer reviewe

Elevated de novo protein synthesis in FMRP-deficient human neurons and its correction by metformin treatment

FXS is the most common genetic cause of intellectual (ID) and autism spectrum disorders (ASD). FXS is caused by loss of FMRP, an RNA-binding protein involved in the translational regulation of a large number of neuronal mRNAs. Absence of FMRP has been shown to lead to elevated protein synthesis and is thought to be a major cause of the synaptic plasticity and behavioural deficits in FXS. The increase in protein synthesis results in part from abnormal activation of key protein translation pathways downstream of ERK1/2 and mTOR signalling. Pharmacological and genetic interventions that attenuate hyperactivation of these pathways can normalize levels of protein synthesis and improve phenotypic outcomes in animal models of FXS. Several efforts are currently underway to trial this strategy in patients with FXS. To date, elevated global protein synthesis as a result of FMRP loss has not been validated in the context of human neurons. Here, using an isogenic human stem cell-based model, we show that de novo protein synthesis is elevated in FMRP-deficient neural cells. We further show that this increase is associated with elevated ERK1/2 and Akt signalling and can be rescued by metformin treatment. Finally, we examined the effect of normalizing protein synthesis on phenotypic abnormalities in FMRP-deficient neural cells. We find that treatment with metformin attenuates the increase in proliferation of FMRP-deficient neural progenitor cells but not the neuronal deficits in neurite outgrowth. The elevated level of protein synthesis and the normalization of neural progenitor proliferation by metformin treatment were validated in additional control and FXS patient-derived hiPSC lines. Overall, our results validate that loss of FMRP results in elevated de novo protein synthesis in human neurons and suggest that approaches targeting this abnormality are likely to be of partial therapeutic benefit in FXS.Peer reviewe

Transcriptional changes in Huntington disease identified using genome-wide expression profiling and cross-platform analysis

Evaluation of transcriptional changes in the striatum may be an effective approach to understanding the natural history of changes in expression contributing to the pathogenesis of Huntington disease (HD). We have performed genome-wide expression profiling of the YAC128 transgenic mouse model of HD at 12 and 24 months of age using two platforms in parallel: Affymetrix and Illumina. The data from these two powerful platforms were integrated to create a combined rank list, thereby revealing the identity of additional genes that proved to be differentially expressed between YAC128 and control mice. Using this approach, we identified 13 genes to be differentially expressed between YAC128 and controls which were validated by quantitative real-time PCR in independent cohorts of animals. In addition, we analyzed additional time points relevant to disease pathology: 3, 6 and 9 months of age. Here we present data showing the evolution of changes in the expression of selected genes: Wt1, Pcdh20 and Actn2 RNA levels change as early as 3 months of age, whereas Gsg1l, Sfmbt2, Acy3, Polr2a and Ppp1r9a RNA expression levels are affected later, at 12 and 24 months of age. We also analyzed the expression of these 13 genes in human HD and control brain, thereby revealing changes in SLC45A3, PCDH20, ACTN2, DDAH1 and PPP1R9A RNA expression. Further study of these genes may unravel novel pathways contributing to HD pathogenesis. DDBJ/EMBL/GenBank accession no: GSE1967

Claudin-11 in health and disease: implications for myelin disorders, hearing, and fertility

Claudin-11 plays a critical role in multiple physiological processes, including myelination, auditory function, and spermatogenesis. Recently, stop-loss mutations in CLDN11 have been identified as a novel cause of hypomyelinating leukodystrophy (HLD22). Understanding the multifaceted roles of claudin-11 and the potential pathogenic mechanisms in HLD22 is crucial for devising targeted therapeutic strategies. This review outlines the biological roles of claudin-11 and the implications of claudin-11 loss in the context of the Cldn11 null mouse model. Additionally, HLD22 and proposed pathogenic mechanisms, such as endoplasmic reticulum stress, will be discussed

Urokinase plasminogen activator mediates changes in human astrocytes modeling fragile X syndrome

Astrocyte function intertwines with the extracellular matrix, whose glial cell-derived components shape neuronal plasticity. Astrocyte abnormalities are found in the brain of the mouse model for fragile X syndrome (FXS), the most common cause of inherited intellectual disability, and a monogenic cause of autism spectrum disorder. We generated human induced pluripotent stem cell-derived FXS and control astrocytes and we found that several pathways associated with urokinase plasminogen activator (uPA) that modulates degradation of extracellular matrix were activated in FXS astrocytes compared with controls. Expression of uPA was increased in FXS astrocytes and levels of uPA were also increased in conditioned medium collected from FXS astrocyte cultures. Levels of uPA correlated inversely with intracellular Ca2+ responses to activation of L-type voltage-gated calcium channels in human astrocytes. Increased uPA augmented neuronal phosphorylation of TrkB, indicating effects of uPA on neuronal plasticity. FXS-specific changes of gene expression during neuronal differentiation preceding astrogenesis likely contributed to altered properties of FXS astrocytes. Our results identified uPA as an important regulator of astrocyte function and demonstrated that increased uPA in human FXS astrocytes modulated astrocytic responses and neuronal plasticity.Peer reviewe

Widespread dysregulation of mRNA splicing implicates RNA processing in the development and progression of Huntington's disease

Background In Huntington's disease (HD), a CAG repeat expansion mutation in the Huntingtin (HTT) gene drives a gain-of-function toxicity that disrupts mRNA processing. Although dysregulation of gene splicing has been shown in human HD post-mortem brain tissue, post-mortem analyses are likely confounded by cell type composition changes in late-stage HD, limiting the ability to identify dysregulation related to early pathogenesis. Methods To investigate gene splicing changes in early HD, we performed alternative splicing analyses coupled with a proteogenomics approach to identify early CAG length-associated splicing changes in an established isogenic HD cell model. Findings We report widespread neuronal differentiation stage- and CAG length-dependent splicing changes, and find an enrichment of RNA processing, neuronal function, and epigenetic modification-related genes with mutant HTT-associated splicing. When integrated with a proteomics dataset, we identified several of these differential splicing events at the protein level. By comparing with human post-mortem and mouse model data, we identified common patterns of altered splicing from embryonic stem cells through to post-mortem striatal tissue. Interpretation We show that widespread splicing dysregulation in HD occurs in an early cell model of neuronal development. Importantly, we observe HD-associated splicing changes in our HD cell model that were also identified in human HD striatum and mouse model HD striatum, suggesting that splicing-associated pathogenesis possibly occurs early in neuronal development and persists to later stages of disease. Together, our results highlight splicing dysregulation in HD which may lead to disrupted neuronal function and neuropathology. Funding This research is supported by the Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Nanyang Assistant Professorship Start-Up Grant, the Singapore Ministry of Education under its Singapore Ministry of Education Academic Research Fund Tier 1 (RG23/22), the BC Children's Hospital Research Institute Investigator Grant Award (IGAP), and a Scholar Award from the Michael Smith Health Research BC

Early Increase in Extrasynaptic NMDA Receptor Signaling and Expression Contributes to Phenotype Onset in Huntington's Disease Mice

SummaryN-methyl-D-aspartate receptor (NMDAR) excitotoxicity is implicated in the pathogenesis of Huntington's disease (HD), a late-onset neurodegenerative disorder. However, NMDARs are poor therapeutic targets, due to their essential physiological role. Recent studies demonstrate that synaptic NMDAR transmission drives neuroprotective gene transcription, whereas extrasynaptic NMDAR activation promotes cell death. We report specifically increased extrasynaptic NMDAR expression, current, and associated reductions in nuclear CREB activation in HD mouse striatum. The changes are observed in the absence of dendritic morphological alterations, before and after phenotype onset, correlate with mutation severity, and require caspase-6 cleavage of mutant huntingtin. Moreover, pharmacological block of extrasynaptic NMDARs with memantine reversed signaling and motor learning deficits. Our data demonstrate elevated extrasynaptic NMDAR activity in an animal model of neurodegenerative disease. We provide a candidate mechanism linking several pathways previously implicated in HD pathogenesis and demonstrate successful early therapeutic intervention in mice