RESUMO
Huntington's disease (HD) causes selective degeneration of striatal and cortical neurons, resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells, grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids, confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons, while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity, trajectories, synaptic density, and communication pathways upon cell-cell contact, while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells, emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment.
Assuntos
Doença de Huntington , Organoides , Fenótipo , Telencéfalo , Doença de Huntington/patologia , Doença de Huntington/genética , Doença de Huntington/metabolismo , Organoides/patologia , Organoides/metabolismo , Animais , Telencéfalo/patologia , Telencéfalo/citologia , Telencéfalo/metabolismo , Humanos , Camundongos , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/patologia , Análise de Célula Única , Comunicação Celular , Mosaicismo , Neurônios/metabolismo , Neurônios/patologiaRESUMO
Huntington's disease is caused by a pathologically long (>35) CAG repeat located in the first exon of the Huntingtin gene (HTT). While pathologically expanded CAG repeats are the focus of extensive investigations, non-pathogenic CAG tracts in protein-coding genes are less well characterized. Here, we investigated the function and evolution of the physiological CAG tract in the HTT gene. We show that the poly-glutamine (polyQ) tract encoded by CAGs in the huntingtin protein (HTT) is under purifying selection and subjected to stronger selective pressures than CAG-encoded polyQ tracts in other proteins. For natural selection to operate, the polyQ must perform a function. By combining genome-edited mouse embryonic stem cells and cell assays, we show that small variations in HTT polyQ lengths significantly correlate with cells' neurogenic potential and with changes in the gene transcription network governing neuronal function. We conclude that during evolution natural selection promotes the conservation and purity of the CAG-encoded polyQ tract and that small increases in its physiological length influence neural functions of HTT. We propose that these changes in HTT polyQ length contribute to evolutionary fitness including potentially to the development of a more complex nervous system.
Assuntos
Doença de Huntington , Peptídeos , Animais , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/patologia , Camundongos , Neurônios/metabolismo , Peptídeos/genética , Peptídeos/metabolismoRESUMO
RUES2 cell lines represent the first collection of isogenic human embryonic stem cells (hESCs) carrying different pathological CAG lengths in the HTT gene. However, their neuronal differentiation potential has yet to be thoroughly evaluated. Here, we report that RUES2 during ventral telencephalic differentiation is biased towards medial ganglionic eminence (MGE). We also show that HD-RUES2 cells exhibit an altered MGE transcriptional signature in addition to recapitulating known HD phenotypes, with reduced expression of the neurodevelopmental regulators NEUROD1 and BDNF and increased cleavage of synaptically enriched N-cadherin. Finally, we identified the transcription factor SP1 as a common potential detrimental co-partner of muHTT by de novo motif discovery analysis on the LGE, MGE, and cortical genes differentially expressed in HD human pluripotent stem cells in our and additional datasets. Taken together, these observations suggest a broad deleterious effect of muHTT in the early phases of neuronal development that may unfold through its altered interaction with SP1.
Assuntos
Biomarcadores Tumorais/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes/citologia , Receptores Imunológicos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/patologia , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismoRESUMO
The Huntingtin (HTT) gene contains a CAG repeat in exon 1, whose expansion beyond 39 repeats consistently leads to Huntington's disease (HD), whereas normal-to-intermediate alleles seemingly modulate brain structure, function and behavior. The role of the CAG repeat in Autism Spectrum Disorder (ASD) was investigated applying both family-based and case-control association designs, with the SCA3 repeat as a negative control. Significant overtransmission of "long" CAG alleles (≥17 repeats) to autistic children and of "short" alleles (≤16 repeats) to their unaffected siblings (all p < 10-5 ) was observed in 612 ASD families (548 simplex and 64 multiplex). Surprisingly, both 193 population controls and 1,188 neurological non-HD controls have significantly lower frequencies of "short" CAG alleles compared to 185 unaffected siblings and higher rates of "long" alleles compared to 548 ASD patients from the same families (p < .05-.001). The SCA3 CAG repeat displays no association. "Short" HTT alleles seemingly exert a protective effect from clinically overt autism in families carrying a genetic predisposition for ASD, while "long" alleles may enhance autism risk. Differential penetrance of autism-inducing genetic/epigenetic variants may imply atypical developmental trajectories linked to HTT functions, including excitation/inhibition imbalance, cortical neurogenesis and apoptosis, neuronal migration, synapse formation, connectivity and homeostasis.
Assuntos
Transtorno Autístico/genética , Proteína Huntingtina/genética , Adulto , Alelos , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Transtorno Autístico/metabolismo , Encéfalo , Estudos de Casos e Controles , Criança , Pré-Escolar , Família , Feminino , Frequência do Gene/genética , Humanos , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Neurogênese , Penetrância , Fatores de Risco , Expansão das Repetições de Trinucleotídeos/genética , Repetições de Trinucleotídeos/genéticaRESUMO
The regulation of the proliferation and polarity of neural progenitors is crucial for the development of the brain cortex. Animal studies have implicated glycogen synthase kinase 3 (GSK3) as a pivotal regulator of both proliferation and polarity, yet the functional relevance of its signaling for the unique features of human corticogenesis remains to be elucidated. We harnessed human cortical brain organoids to probe the longitudinal impact of GSK3 inhibition through multiple developmental stages. Chronic GSK3 inhibition increased the proliferation of neural progenitors and caused massive derangement of cortical tissue architecture. Single-cell transcriptome profiling revealed a direct impact on early neurogenesis and uncovered a selective role of GSK3 in the regulation of glutamatergic lineages and outer radial glia output. Our dissection of the GSK3-dependent transcriptional network in human corticogenesis underscores the robustness of the programs determining neuronal identity independent of tissue architecture.
Assuntos
Córtex Cerebral/citologia , Quinase 3 da Glicogênio Sintase/metabolismo , Neurogênese , Neurônios/citologia , Organoides/citologia , Linhagem Celular , Proliferação de Células , Córtex Cerebral/metabolismo , Deleção de Genes , Quinase 3 da Glicogênio Sintase/genética , Humanos , Neurônios/metabolismo , Organoides/metabolismo , TranscriptomaRESUMO
DACH1 is the human homolog of the Drosophila dachshund gene, which is involved in the development of the eye, nervous system, and limbs in the fly. Here, we systematically investigate DACH1 expression patterns during human neurodevelopment, from 5 to 21 postconceptional weeks. By immunodetection analysis, we found that DACH1 is highly expressed in the proliferating neuroprogenitors of the developing cortical ventricular and subventricular regions, while it is absent in the more differentiated cortical plate. Single-cell global transcriptional analysis revealed that DACH1 is specifically enriched in neuroepithelial and ventricular radial glia cells of the developing human neocortex. Moreover, we describe a previously unreported DACH1 expression in the human striatum, in particular in the striatal medium spiny neurons. This finding qualifies DACH1 as a new striatal projection neuron marker, together with PPP1R1B, BCL11B, and EBF1. We finally compared DACH1 expression profile in human and mouse forebrain, where we observed spatio-temporal similarities in its expression pattern thus providing a precise developmental description of DACH1 in the 2 mammalian species.
Assuntos
Corpo Estriado/embriologia , Corpo Estriado/metabolismo , Proteínas do Olho/metabolismo , Neocórtex/embriologia , Neocórtex/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Feto Abortado/embriologia , Feto Abortado/metabolismo , Células Ependimogliais/metabolismo , Idade Gestacional , Humanos , Ventrículos Laterais/embriologia , Ventrículos Laterais/metabolismo , Células-Tronco Neurais/metabolismo , Células Neuroepiteliais/metabolismo , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , Especificidade da EspécieRESUMO
Amyotrophic lateral sclerosis is heterogeneous with high variability in the speed of progression even in cases with a defined genetic cause such as superoxide dismutase 1 (SOD1) mutations. We reported that SOD1(G93A) mice on distinct genetic backgrounds (C57 and 129Sv) show consistent phenotypic differences in speed of disease progression and life-span that are not explained by differences in human SOD1 transgene copy number or the burden of mutant SOD1 protein within the nervous system. We aimed to compare the gene expression profiles of motor neurons from these two SOD1(G93A) mouse strains to discover the molecular mechanisms contributing to the distinct phenotypes and to identify factors underlying fast and slow disease progression. Lumbar spinal motor neurons from the two SOD1(G93A) mouse strains were isolated by laser capture microdissection and transcriptome analysis was conducted at four stages of disease. We identified marked differences in the motor neuron transcriptome between the two mice strains at disease onset, with a dramatic reduction of gene expression in the rapidly progressive (129Sv-SOD1(G93A)) compared with the slowly progressing mutant SOD1 mice (C57-SOD1(G93A)) (1276 versus 346; Q-value ≤ 0.01). Gene ontology pathway analysis of the transcriptional profile from 129Sv-SOD1(G93A) mice showed marked downregulation of specific pathways involved in mitochondrial function, as well as predicted deficiencies in protein degradation and axonal transport mechanisms. In contrast, the transcriptional profile from C57-SOD1(G93A) mice with the more benign disease course, revealed strong gene enrichment relating to immune system processes compared with 129Sv-SOD1(G93A) mice. Motor neurons from the more benign mutant strain demonstrated striking complement activation, over-expressing genes normally involved in immune cell function. We validated through immunohistochemistry increased expression of the C3 complement subunit and major histocompatibility complex I within motor neurons. In addition, we demonstrated that motor neurons from the slowly progressing mice activate a series of genes with neuroprotective properties such as angiogenin and the nuclear factor (erythroid-derived 2)-like 2 transcriptional regulator. In contrast, the faster progressing mice show dramatically reduced expression at disease onset of cell pathways involved in neuroprotection. This study highlights a set of key gene and molecular pathway indices of fast or slow disease progression which may prove useful in identifying potential disease modifiers responsible for the heterogeneity of human amyotrophic lateral sclerosis and which may represent valid therapeutic targets for ameliorating the disease course in humans.