Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies1, which involve human-specific mechanisms2-5 that cannot be directly studied in animal models. Here, to explore the emergence and consequences of TDP-43 pathologies, we generated induced pluripotent stem cell-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors6. Single-cell transcriptomics and comparison to independent neural stem cells7 showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks (which we designate iNets). Neuronal and glial maturation in iNets was similar to that of cortical organoids8. Overexpression of wild-type TDP-43 in a minority of neurons within iNets led to progressive fragmentation and aggregation of the protein, resulting in a partial loss of function and neurotoxicity. Single-cell transcriptomics revealed a novel set of misregulated RNA targets in TDP-43-overexpressing neurons and in patients with TDP-43 proteinopathies exhibiting a loss of nuclear TDP-43. The strongest misregulated target encoded the synaptic protein NPTX2, the levels of which are controlled by TDP-43 binding on its 3' untranslated region. When NPTX2 was overexpressed in iNets, it exhibited neurotoxicity, whereas correcting NPTX2 misregulation partially rescued neurons from TDP-43-induced neurodegeneration. Notably, NPTX2 was consistently misaccumulated in neurons from patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby revealing a TDP-43-dependent pathway of neurotoxicity.
- MeSH
- amyotrofická laterální skleróza * metabolismus patologie MeSH
- C-reaktivní protein * metabolismus MeSH
- DNA vazebné proteiny * nedostatek metabolismus MeSH
- frontotemporální lobární degenerace * metabolismus patologie MeSH
- lidé MeSH
- nervová síť * metabolismus patologie MeSH
- nervové kmenové buňky cytologie MeSH
- neuroglie cytologie MeSH
- neurony * cytologie metabolismus MeSH
- proteiny nervové tkáně * metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Toxic dipeptide-repeat (DPR) proteins are produced from expanded G4C2 repeats in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥20 repeats inhibit the ribosome's peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryogenic electron microscopy (cryo-EM) reveals that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center (PTC). Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with poly-PR and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.
- MeSH
- amyotrofická laterální skleróza * genetika metabolismus MeSH
- dipeptidy metabolismus MeSH
- elektronová kryomikroskopie MeSH
- frontotemporální demence * genetika metabolismus MeSH
- lidé MeSH
- protein C9orf72 genetika metabolismus MeSH
- proteiny genetika metabolismus MeSH
- ribozomy metabolismus MeSH
- transferasy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Amyotrophic lateral sclerosis (ALS) is a fatal non-cell-autonomous neurodegenerative disease characterized by the loss of motor neurons (MNs). Mutations in CRMP4 are associated with ALS in patients, and elevated levels of CRMP4 are suggested to affect MN health in the SOD1G93A -ALS mouse model. However, the mechanism by which CRMP4 mediates toxicity in ALS MNs is poorly understood. Here, by using tissue from human patients with sporadic ALS, MNs derived from C9orf72-mutant patients, and the SOD1G93A -ALS mouse model, we demonstrate that subcellular changes in CRMP4 levels promote MN loss in ALS. First, we show that while expression of CRMP4 protein is increased in cell bodies of ALS-affected MN, CRMP4 levels are decreased in the distal axons. Cellular mislocalization of CRMP4 is caused by increased interaction with the retrograde motor protein, dynein, which mediates CRMP4 transport from distal axons to the soma and thereby promotes MN loss. Blocking the CRMP4-dynein interaction reduces MN loss in human-derived MNs (C9orf72) and in ALS model mice. Thus, we demonstrate a novel CRMP4-dependent retrograde death signal that underlies MN loss in ALS.
- MeSH
- amyotrofická laterální skleróza genetika metabolismus MeSH
- axonální transport * MeSH
- axony metabolismus MeSH
- buněčná smrt MeSH
- buněčné linie MeSH
- dyneiny metabolismus MeSH
- kultivované buňky MeSH
- motorické neurony metabolismus patologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- proteiny nervové tkáně genetika metabolismus MeSH
- signální transdukce MeSH
- superoxiddismutasa 1 genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
AIMS: Mesenchymal stem cells (MSCs) have recently been tested in clinical trials to treat severe diseases, including amyotrophic lateral sclerosis (ALS). Since autologous MSCs are frequently used for therapy, we aimed to evaluate the possible influence of the disease on characteristics and function of these cells. METHODS: MSCs were isolated from the bone marrow of patients with ALS and compared with MSCs from healthy controls (HC). The cells were tested for phenotype, growth properties, differentiation ability, metabolic activity, secretory potential, expression of genes for immunomodulatory molecules and for the ability to regulate proliferation of mitogen-stimulated peripheral blood leucocytes. MSCs from patients with ALS and HC were either unstimulated or treated with proinflammatory cytokines for 24 hours before testing. RESULTS: MSCs isolated from patients with ALS have a higher differentiation potential into adipocytes, express elevated levels of mRNA for interleukin-6, but produce less hepatocyte growth factor than MSCs from HC. On the other hand, there were no significant differences between MSCs from patients with ALS and HC in the expression of phenotypic markers, growth properties, metabolic activity, osteogenic differentiation potential and immunoregulatory properties. CONCLUSIONS: The results suggest that, in spite of some differences in cytokine production, MSCs from patients with ALS can be useful as autologous cells in therapy of ALS.
- MeSH
- adipogeneze MeSH
- aktivace lymfocytů MeSH
- amyotrofická laterální skleróza imunologie metabolismus patologie MeSH
- biologické markery metabolismus MeSH
- buňky kostní dřeně imunologie metabolismus patologie MeSH
- energetický metabolismus MeSH
- fenotyp MeSH
- kokultivační techniky MeSH
- kultivované buňky MeSH
- lidé středního věku MeSH
- lidé MeSH
- mezenchymální kmenové buňky imunologie metabolismus patologie MeSH
- osteogeneze MeSH
- proliferace buněk MeSH
- separace buněk metody MeSH
- studie případů a kontrol MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Astrocyte responses to neuronal injury may be beneficial or detrimental to neuronal recovery, but the mechanisms that determine these different responses are poorly understood. Here we show that ephrin type-B receptor 1 (EphB1) is upregulated in injured motor neurons, which in turn can activate astrocytes through ephrin-B1-mediated stimulation of signal transducer and activator of transcription-3 (STAT3). Transcriptional analysis shows that EphB1 induces a protective and anti-inflammatory signature in astrocytes, partially linked to the STAT3 network. This is distinct from the response evoked by interleukin (IL)-6 that is known to induce both pro inflammatory and anti-inflammatory processes. Finally, we demonstrate that the EphB1-ephrin-B1 pathway is disrupted in human stem cell derived astrocyte and mouse models of amyotrophic lateral sclerosis (ALS). Our work identifies an early neuronal help-me signal that activates a neuroprotective astrocytic response, which fails in ALS, and therefore represents an attractive therapeutic target.
- MeSH
- amyotrofická laterální skleróza metabolismus MeSH
- antiflogistika farmakologie MeSH
- astrocyty cytologie metabolismus MeSH
- axony metabolismus MeSH
- interleukin-6 metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- motorické neurony metabolismus MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nervus ischiadicus metabolismus MeSH
- neurony metabolismus MeSH
- neuroprotekce MeSH
- receptor EphB1 metabolismus MeSH
- signální transdukce MeSH
- transkripční faktor STAT3 metabolismus MeSH
- transkriptom MeSH
- zánět MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. In a recent study by Steinberg and colleagues, 2 recessive missense mutations were identified in the Cav3.2 T-type calcium channel gene (CACNA1H), in a family with an affected proband (early onset, long duration ALS) and 2 unaffected parents. We have introduced and functionally characterized these mutations using transiently expressed human Cav3.2 channels in tsA-201 cells. Both of these mutations produced mild but significant changes on T-type channel activity that are consistent with a loss of channel function. Computer modeling in thalamic reticular neurons suggested that these mutations result in decreased neuronal excitability of thalamic structures. Taken together, these findings implicate CACNA1H as a susceptibility gene in amyotrophic lateral sclerosis.
- MeSH
- amyotrofická laterální skleróza genetika metabolismus patofyziologie MeSH
- buněčné linie MeSH
- lidé MeSH
- missense mutace * MeSH
- neurony fyziologie MeSH
- thalamus cytologie fyziologie MeSH
- transfekce MeSH
- vápníkové kanály - typ T genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53β are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53β, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53β overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53β expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases.
- MeSH
- alternativní sestřih MeSH
- Alzheimerova nemoc metabolismus patologie MeSH
- amyotrofická laterální skleróza metabolismus patologie MeSH
- astrocyty cytologie účinky léků metabolismus MeSH
- autofagie účinky léků MeSH
- genetické vektory genetika metabolismus MeSH
- interleukin-6 genetika metabolismus MeSH
- kokultivační techniky MeSH
- kultivované buňky MeSH
- leupeptiny farmakologie MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- mozek metabolismus patologie MeSH
- nádorový supresorový protein p53 antagonisté a inhibitory genetika metabolismus MeSH
- neurony cytologie metabolismus MeSH
- neuroprotekce fyziologie MeSH
- protein - isoformy antagonisté a inhibitory genetika metabolismus MeSH
- RNA interference MeSH
- sekvestosom 1 antagonisté a inhibitory genetika metabolismus MeSH
- serin-arginin sestřihové faktory antagonisté a inhibitory genetika metabolismus MeSH
- stárnutí buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder resulting in a lethal outcome. We studied changes in ventral horn perineuronal nets (PNNs) of superoxide dismutase 1 (SOD1) rats during the normal disease course and after the intrathecal application (5 × 10(5) cells) of human bone marrow mesenchymal stromal cells (MSCs) postsymptom manifestation. We found that MSCs ameliorated disease progression, significantly improved motor activity, and prolonged survival. For the first time, we report that SOD1 rats have an abnormal disorganized PNN structure around the spinal motoneurons and give different expression profiles of chondroitin sulfate proteoglycans (CSPGs), such as versican, aggrecan, and phosphacan, but not link protein-1. Additionally, SOD1 rats had different profiles for CSPG gene expression (Versican, Hapln1, Neurocan, and Tenascin-R), whereas Aggrecan and Brevican profiles remained unchanged. The application of MSCs preserved PNN structure, accompanied by better survival of motorneurons. We measured the concentration of cytokines (IL-1α, MCP-1, TNF-α, GM-CSF, IL-4, and IFN-γ) in the rats' cerebrospinal fluid and found significantly higher concentrations of IL-1α and MCP-1. Our results show that PNN and cytokine homeostasis are altered in the SOD1 rat model of ALS. These changes could potentially serve as biological markers for the diagnosis, assessment of treatment efficacy, and prognosis of ALS. We also show that the administration of human MSCs is a safe procedure that delays the loss of motor function and increases the overall survival of symptomatic ALS animals, by remodeling the recipients' pattern of gene expression and having neuroprotective and immunomodulatory effects.
- MeSH
- amyotrofická laterální skleróza metabolismus MeSH
- buněčná diferenciace fyziologie MeSH
- chondroitinsulfát proteoglykany metabolismus MeSH
- extracelulární matrix metabolismus MeSH
- faktor stimulující granulocyto-makrofágové kolonie metabolismus MeSH
- krysa rodu rattus MeSH
- mezenchymální kmenové buňky cytologie MeSH
- mícha metabolismus MeSH
- nervová síť cytologie MeSH
- neurony cytologie MeSH
- TNF-alfa metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
