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The neurodevelopmental disorder Pitt Hopkins syndrome (PTHS) causes clinical symptoms similar to Rett syndrome (RTT) patients. However, RTT is caused by MECP2 mutations whereas mutations in the TCF4 gene lead to PTHS. The mechanistic commonalities underling these two disorders are unknown, but their shared symptomology suggest that convergent pathway-level disruption likely exists. We reprogrammed patient skin derived fibroblasts into induced neuronal progenitor cells. Interestingly, we discovered that MeCP2 levels were decreased in PTHS patient iNPCs relative to healthy controls and that both iNPCs and iAstrocytes displayed defects in function and differentiation in a mutation-specific manner. When Tcf4+/- mice were genetically crossed with mice overexpressing MeCP2, molecular and phenotypic defects were significantly ameliorated, underlining and important role of MeCP2 in PTHS pathology. Importantly, post-natal intracerebroventricular gene replacement therapy with adeno-associated viral vector serotype 9 (AAV9)-expressing MeCP2 (AAV9.P546.MeCP2) significantly improved iNPC and iAstrocyte function and effectively ameliorated histological and behavioral defects in Tcf4+/- mice. Combined, our data suggest a previously unknown role of MeCP2 in PTHS pathology and common pathways that might be affected in multiple neurodevelopmental disorders. Our work highlights potential novel therapeutic targets for PTHS, including upregulation of MeCP2 expression or its downstream targets or, potentially, MeCP2-based gene therapy.
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The AAV9 gene therapy vector presented in this study is safe in mice and non-human primates and highly efficacious without causing overexpression toxicity, a major challenge for clinical translation of Rett syndrome gene therapy vectors to date. Our team designed a new truncated methyl-CpG-binding protein 2 (MECP2) promoter allowing widespread expression of MECP2 in mice and non-human primates after a single injection into the cerebrospinal fluid without causing overexpression symptoms up to 18 months after injection. Additionally, this new vector is highly efficacious at lower doses compared with previous constructs as demonstrated in extensive efficacy studies performed by two independent laboratories in two different Rett syndrome mouse models carrying either a knockout or one of the most frequent human mutations of Mecp2. Overall, data from this multicenter study highlight the efficacy and safety of this gene therapy construct, making it a promising candidate for first-in-human studies to treat Rett syndrome.
Assuntos
Síndrome de Rett , Humanos , Camundongos , Animais , Síndrome de Rett/genética , Síndrome de Rett/terapia , Síndrome de Rett/metabolismo , Primatas/genética , Terapia Genética , MutaçãoRESUMO
Patient diversity and unknown disease cause are major challenges for drug development and clinical trial design for amyotrophic lateral sclerosis (ALS). Transgenic animal models do not adequately reflect the heterogeneity of ALS. Direct reprogramming of patient fibroblasts to neuronal progenitor cells and subsequent differentiation into patient astrocytes allows rapid generation of disease relevant cell types. Thus, this methodology can facilitate compound testing in a diverse genetic background resulting in a more representative population for therapeutic evaluation. Here, we used established co-culture assays with motor neurons and reprogrammed patient skin-derived astrocytes (iAs) to evaluate the effects of (SP-4-2)-[[2,2'-(1,2-dimethyl-1,2-ethanediylidene)bis[N-methylhydrazinecarbothioamidato-κN2 ,κS]](2-)]-copper (CuATSM), currently in clinical trial for ALS in Australia. Pretreatment of iAs with CuATSM had a differential effect on neuronal survival following co-culture with healthy motor neurons. Using this assay, we identified responding and non-responding cell lines for both sporadic and familial ALS (mutant SOD1 and C9ORF72). Importantly, elevated mitochondrial respiration was the common denominator in all CuATSM-responders, a metabolic phenotype not observed in non-responders. Pre-treatment of iAs with CuATSM restored mitochondrial activity to levels comparable to healthy controls. Hence, this metabolic parameter might allow selection of patient subpopulations best suited for CuATSM treatment. Moreover, CuATSM might have additional therapeutic value for mitochondrial disorders. Enhanced understanding of patient-specific cellular and molecular profiles could help improve clinical trial design in the future.
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Esclerose Lateral Amiotrófica , Animais , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Astrócitos/metabolismo , Neurônios Motores , Técnicas de Cocultura , Superóxido Dismutase-1/metabolismoRESUMO
The recently discovered neurological disorder NEDAMSS is caused by heterozygous truncations in the transcriptional regulator IRF2BPL. Here, we reprogram patient skin fibroblasts to astrocytes and neurons to study mechanisms of this newly described disease. While full-length IRF2BPL primarily localizes to the nucleus, truncated patient variants sequester the wild-type protein to the cytoplasm and cause aggregation. Moreover, patient astrocytes fail to support neuronal survival in coculture and exhibit aberrant mitochondria and respiratory dysfunction. Treatment with the small molecule copper ATSM (CuATSM) rescues neuronal survival and restores mitochondrial function. Importantly, the in vitro findings are recapitulated in vivo, where co-expression of full-length and truncated IRF2BPL in Drosophila results in cytoplasmic accumulation of full-length IRF2BPL. Moreover, flies harboring heterozygous truncations of the IRF2BPL ortholog (Pits) display progressive motor defects that are ameliorated by CuATSM treatment. Our findings provide insights into mechanisms involved in NEDAMSS and reveal a promising treatment for this severe disorder.
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ER stress signaling is linked to the pathophysiological and clinical disease manifestations in amyotrophic lateral sclerosis (ALS). Here, we have investigated ER stress-induced adaptive mechanisms in C9ORF72-ALS/FTD, focusing on uncovering early endogenous neuroprotective mechanisms and the crosstalk between pathological and adaptive responses in disease onset and progression. We provide evidence for the early onset of ER stress-mediated adaptive response in C9ORF72 patient-derived motoneurons (MNs), reflected by the elevated increase in GRP75 expression. These transiently increased GRP75 levels enhance ER-mitochondrial association, boosting mitochondrial function and sustaining cellular bioenergetics during the initial stage of disease, thereby counteracting early mitochondrial deficits. In C9orf72 rodent neurons, an abrupt reduction in GRP75 expression coincided with the onset of UPR, mitochondrial dysfunction and the emergence of PolyGA aggregates, which co-localize with GRP75. Similarly, the overexpression of PolyGA in WT cortical neurons or C9ORF72 patient-derived MNs led to the sequestration of GRP75 within PolyGA inclusions, resulting in mitochondrial calcium (Ca2+) uptake impairments. Corroborating these findings, we found that PolyGA aggregate-bearing human post-mortem C9ORF72 hippocampal dentate gyrus neurons not only display reduced expression of GRP75 but also exhibit GRP75 sequestration within inclusions. Sustaining high GRP75 expression in spinal C9orf72 rodent MNs specifically prevented ER stress, normalized mitochondrial function, abrogated PolyGA accumulation in spinal MNs, and ameliorated ALS-associated behavioral phenotype. Taken together, our results are in line with the notion that neurons in C9ORF72-ALS/FTD are particularly susceptible to ER-mitochondrial dysfunction and that GRP75 serves as a critical endogenous neuroprotective factor. This neuroprotective pathway, is eventually targeted by PolyGA, leading to GRP75 sequestration, and its subsequent loss of function at the MAM, compromising mitochondrial function and promoting disease onset.
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Esclerose Lateral Amiotrófica , Estresse do Retículo Endoplasmático , Demência Frontotemporal , Esclerose Lateral Amiotrófica/patologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Cálcio/metabolismo , Demência Frontotemporal/genética , Proteínas de Choque Térmico HSP70 , Humanos , Proteínas de Membrana , Neurônios Motores/patologia , PolirribonucleotídeosRESUMO
Amyotrophic lateral sclerosis (ALS) is a degenerative disease that progressively destroys motor neurons (MNs). Earlier studies identified EphA4, a receptor tyrosine kinase, as a possible disease-modifying gene. The complex interplay between the EphA4 receptor and its ephrin ligands in motor neurons and astrocytes has not yet been fully elucidated and includes a putative pro-apoptotic activity of the unbound receptor compared to ephrin-bound receptor. We recently reported that astrocytes from patients with ALS induce cell death in co-cultured MNs. Here we found that first-generation synthetic EphA4 agonistic agent 123C4, effectively protected MNs when co-cultured with reactive astrocytes from patients with ALS from multiple subgroups (sALS and mutant SOD1). Newer generation and more potent EphA4 agonistic agents 150D4, 150E8, and 150E7 provided effective protection at a lower therapeutic dose. Combined, the data suggest that the development of EphA4 agonistic agents provides potentially a promising therapeutic strategy for patients with ALS.
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A lack of stratification methods in patients with amyotrophic lateral sclerosis (ALS) is likely implicated in therapeutic failures. Regional diversities and pathophysiological abnormalities in astrocytes from mice with SOD1 mutations (mSOD1-ALS) can now be explored in human patients using somatic cell reprogramming. Here, fibroblasts from four sporadic (sALS) and three mSOD1-ALS patients were transdifferentiated into induced astrocytes (iAstrocytes). ALS iAstrocytes were neurotoxic toward HB9-GFP mouse motor neurons (MNs) and exhibited subtype stratification through GFAP, CX43, Ki-67, miR-155 and miR-146a expression levels. Up- (two cases) and down-regulated (three cases) miR-146a values in iAstrocytes were recapitulated in their secretome, either free or as cargo in small extracellular vesicles (sEVs). We previously showed that the neuroprotective phenotype of depleted miR-146 mSOD1 cortical astrocytes was reverted by its mimic. Thus, we tested such modulation in the most miR-146a-depleted patient-iAstrocytes (one sALS and one mSOD1-ALS). The miR-146a mimic in ALS iAstrocytes counteracted their reactive/inflammatory profile and restored miR-146a levels in sEVs. A reduction in lysosomal activity and enhanced synaptic/axonal transport-related genes in NSC-34 MNs occurred after co-culture with miR-146a-modulated iAstrocytes. In summary, the regulation of miR-146a in depleted ALS astrocytes may be key in reestablishing their normal function and in restoring MN lysosomal/synaptic dynamic plasticity in disease sub-groups.
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Esclerose Lateral Amiotrófica , MicroRNAs , Síndromes Neurotóxicas , Esclerose Lateral Amiotrófica/genética , Animais , Astrócitos , Modelos Animais de Doenças , Fibroblastos , Humanos , Camundongos , MicroRNAs/genéticaRESUMO
The diffusion-limited reaction of nitric oxide (NO) and superoxide (O2-) produces peroxynitrite (ONOO-), a biological oxidant that has been implicated in a number of pathological conditions, including neurodegenerative disorders. We previously reported that incubation of PC12 cells with peroxynitrite triggers apoptosis by simultaneously inhibiting the PI3K/Akt survival pathway, and activating the p38 and JNK MAP kinase pathways. We also reported that peroxynitrite-treated Heat Shock Protein 90 (Hsp90) stimulates PC12 cell death. Here, we show that nitrated Hsp90 mediates peroxynitrite-induced apoptosis by regulating specific signaling pathways triggered by activation of the purine receptor P2X7 (P2X7R) and downstream activation of PTEN. Intracellular delivery of peroxynitrite-treated Hsp90 was sufficient to stimulate PC12 cell death. In contrast, intracellular delivery of peroxynitrite-treated Hsp90 in which the five tyrosine (Tyr) residues susceptible to nitration were replaced by nitration-resistant phenylalanine had no effect on PC12 cell survival. Further, only nitration of Hsp90 at Tyr 56 was necessary and sufficient to stimulate PC12 cell apoptosis, and incubation of PC12 cells with peroxynitrite resulted in Hsp90 nitration at Tyr 56. Inhibition of P2X7R or downstream inhibition of PTEN prevented PC12 cell death stimulated by both incubation with peroxynitrite and nitrated Hsp90 (Hsp90NY). Peroxynitrite, Hsp90NY, and P2X7R activation all increased p38 and JNK MAP kinases activity, while inhibiting the Akt survival pathway. These results suggest that, in undifferentiated PC12 cells, peroxynitrite triggers apoptosis via nitration of Hsp90 at Tyr 56, which in turn activates P2X7R and PTEN. These results contrast with observations in motor neurons where the nitration of either Tyr 33 or Tyr 56 in Hsp90 stimulates apoptosis, suggesting that the targets of peroxynitrite may be different in different cell types. However, uncovering the pathways through which peroxynitrite triggers cell death in neurodegenerative conditions will provide new potential targets for therapeutic treatment.
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Ácido Peroxinitroso , Tirosina , Animais , Morte Celular , Proteínas de Choque Térmico HSP90 , Células PC12 , PTEN Fosfo-Hidrolase , Ácido Peroxinitroso/metabolismo , Fosfatidilinositol 3-Quinases , Ratos , Receptores Purinérgicos P2X7 , Tirosina/metabolismoRESUMO
In this paper, we applied an innovative nuclear magnetic resonance (NMR)-guided screening and ligand design approach, named focused high-throughput screening by NMR (fHTS by NMR), to derive potent, low-molecular-weight ligands capable of mimicking interactions elicited by ephrin ligands on the receptor tyrosine kinase EphA4. The agents bind with nanomolar affinity, trigger receptor activation in cellular assays with motor neurons, and provide remarkable motor neuron protection from amyotrophic lateral sclerosis (ALS) patient-derived astrocytes. Structural studies on the complex between EphA4 ligand-binding domain and a most active agent provide insights into the mechanism of the agents at a molecular level. Together with preliminary in vivo pharmacology studies, the data form a strong foundation for the translation of these agents for the treatment of ALS and potentially other human diseases.
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Aminoácidos/farmacologia , Esclerose Lateral Amiotrófica/tratamento farmacológico , Desenho de Fármacos , Fluorenos/farmacologia , Receptor EphA4/agonistas , Aminoácidos/química , Esclerose Lateral Amiotrófica/metabolismo , Animais , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Fluorenos/química , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Estrutura Molecular , Receptor EphA4/metabolismo , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.
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Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Animais , Astrócitos , Diferenciação Celular , Fibroblastos , Humanos , Camundongos , NeurôniosRESUMO
Riluzole is the only FDA approved drug for the treatment of amyotrophic lateral sclerosis (ALS). However, the drug affords moderate protection to ALS patients, extending life for a few months by a mechanism that remains controversial. In the presence of riluzole, astrocytes increase the production of factors protective to motor neurons. The stimulation of trophic factor production by motor neuron associated cells may contribute to riluzole's protective effect in ALS. Here, we investigated the effects of media conditioned by astrocytes and Schwann cells acutely or chronically incubated with riluzole on trophic factor-deprived motor neuron survival. While acute riluzole incubation induced CT-1 secretion by astrocytes and Schwann cells, chronic treatment stimulated a significant decrease in trophic factor production compared to untreated cultures. Accordingly, conditioned media from astrocytes and Schwann cells acutely treated with riluzole protected motor neurons from trophic factor deprivation-induced cell death. Motor neuron protection was prevented by incubation with CT-1 neutralizing antibodies. In contrast, conditioned media from astrocytes and Schwann cells chronically treated with riluzole was not protective. Acute and chronic treatment of mice with riluzole showed opposite effects on trophic factor production in spinal cord, sciatic nerve and brain. There was an increase in the production of CT-1 and GDNF in the spinal cord and CT-1 in the sciatic nerve during the first days of treatment with riluzole, but the levels dropped significantly after chronic treatment with the drug. Similar results were observed in brain for CT-1 and BDNF while there was no change in GDNF levels after riluzole treatment. Our results reveal that riluzole regulates long-lasting processes involving protein synthesis, which may be relevant for riluzole therapeutic effects. Changing the regimen of riluzole administration to favor the acute effect of the drug on trophic factor production by discontinuous long-term treatment may improve the outcome of ALS patient therapy.
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Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Sistema Nervoso/crescimento & desenvolvimento , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Riluzol/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Embrião de Mamíferos , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sistema Nervoso/metabolismo , Neuroglia/metabolismo , Ratos , Ratos Sprague-Dawley , Células de Schwann/química , Células de Schwann/efeitos dos fármacos , Fatores de TempoRESUMO
Peroxynitrite production and tyrosine nitration are present in several pathological conditions, including neurodegeneration, stroke, aging, and cancer. Nitration of the pro-survival chaperone heat shock protein 90 (Hsp90) in position 33 and 56 induces motor neuron death through a toxic gain-of-function. Here we show that nitrated Hsp90 regulates mitochondrial metabolism independently of the induction of cell death. In PC12 cells, a small fraction of nitrated Hsp90 was located on the mitochondrial outer membrane and down-regulated mitochondrial membrane potential, oxygen consumption, and ATP production. Neither endogenous Hsp90 present in the homogenate nor unmodified and fully active recombinant Hsp90 was able to compete with the nitrated protein for the binding to mitochondria. Moreover, endogenous or recombinant Hsp90 did not prevent the decrease in mitochondrial activity but supported nitrated Hsp90 mitochondrial gain-of-function. Nitrotyrosine in position 33, but not in any of the other four tyrosine residues prone to nitration in Hsp90, was sufficient to down-regulate mitochondrial activity. Thus, in addition to induction of cell death, nitrated Hsp90 can also regulate mitochondrial metabolism, suggesting that depending on the cell type, distinct Hsp90 nitration states regulate different aspects of cellular metabolism. This regulation of mitochondrial homeostasis by nitrated Hsp90 could be of particular relevance in cancer cells.
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Proteínas de Choque Térmico HSP90/metabolismo , Mitocôndrias/metabolismo , Processamento de Proteína Pós-Traducional , Tirosina/análogos & derivados , Trifosfato de Adenosina/biossíntese , Animais , Metabolismo Energético , Células PC12 , Transporte Proteico , Ratos , Tirosina/metabolismoRESUMO
Tyrosine nitration is a biomarker for the production of peroxynitrite and other reactive nitrogen species. Nitrotyrosine immunoreactivity is present in many pathological conditions including several cardiac diseases. Because the events observed during heart failure may recapitulate some aspects of development, we tested whether nitrotyrosine is present during normal development of the rat embryo heart and its potential relationship in cardiac remodeling through apoptosis. Nitric oxide production is highly dynamic during development, but whether peroxynitrite and nitrotyrosine are formed during normal embryonic development has received little attention. Rat embryo hearts exhibited strong nitrotyrosine immunoreactivity in endocardial and myocardial cells of the atria and ventricles from E12 to E18. After E18, nitrotyrosine staining faded and disappeared entirely by birth. Tyrosine nitration in the myocardial tissue coincided with elevated protein expression of nitric oxide synthases (eNOS and iNOS). The immunoreactivity for these NOS isoforms remained elevated even after nitrotyrosine had disappeared. Tyrosine nitration did not correlate with cell death or proliferation of cardiac cells. Analysis of tryptic peptides by MALDI-TOF showed that nitration occurs in actin, myosin, and the mitochondrial ATP synthase α chain. These results suggest that reactive nitrogen species are not restricted to pathological conditions but may play a role during normal embryonic development.