RESUMEN
Human telencephalon is an evolutionarily advanced brain structure associated with many uniquely human behaviors and disorders. However, cell lineages and molecular pathways implicated in human telencephalic development remain largely unknown. We produce human telencephalic organoids from stem cell-derived single neural rosettes and investigate telencephalic development under normal and pathological conditions. We show that single neural rosette-derived organoids contain pallial and subpallial neural progenitors, excitatory and inhibitory neurons, as well as macroglial and periendothelial cells, and exhibit predictable organization and cytoarchitecture. We comprehensively characterize the properties of neurons in SNR-derived organoids and identify transcriptional programs associated with the specification of excitatory and inhibitory neural lineages from a common pool of NPs early in telencephalic development. We also demonstrate that neurons in organoids with a hemizygous deletion of an autism- and intellectual disability-associated gene SHANK3 exhibit intrinsic and excitatory synaptic deficits and impaired expression of several clustered protocadherins. Collectively, this study validates SNR-derived organoids as a reliable model for studying human telencephalic cortico-striatal development and identifies intrinsic, synaptic, and clustered protocadherin expression deficits in human telencephalic tissue with SHANK3 hemizygosity.
Asunto(s)
Trastorno Autístico , Trastorno Autístico/genética , Humanos , Proteínas del Tejido Nervioso/metabolismo , Organoides/metabolismo , Protocadherinas , TelencéfaloRESUMEN
Over the past decade, tremendous progress has been made in defining autism spectrum disorder (ASD) as a disorder of brain connectivity. Indeed, whole-brain imaging studies revealed altered connectivity in the brains of individuals with ASD, and genetic studies identified rare ASD-associated mutations in genes that regulate synaptic development and function. However, it remains unclear how specific mutations alter the development of neuronal connections in different brain regions and whether altered connections can be restored therapeutically. The main challenge is the lack of preclinical models that recapitulate important aspects of human development for studying connectivity. Through recent technological innovations, it is now possible to generate patient- or mutation-specific human neurons or organoids from induced pluripotent stem cells (iPSCs) and to study altered connectivity in vitro or in vivo upon xenotransplantation into an intact rodent brain. Here, we discuss how deficits in neurodevelopmental processes may lead to abnormal brain connectivity and how iPSC-based models can be used to identify abnormal connections and to gain insights into underlying cellular and molecular mechanisms to develop novel therapeutics.
Asunto(s)
Trastorno del Espectro Autista , Trastorno Autístico , Células Madre Pluripotentes Inducidas , Trastorno del Espectro Autista/genética , Trastorno Autístico/genética , Encéfalo , Humanos , OrganoidesRESUMEN
The ability to measure changes in neuronal activity in a quantifiable and precise manner is of fundamental importance to understand neuron development and function. Repeated monitoring of neuronal activity of the same population of neurons over several days is challenging and, typically, low-throughput. Here, we describe a new biochemical reporter assay that allows for repeated measurements of neuronal activity in a cell type-specific manner. We coupled activity-dependent elements from the Arc/Arg3.1 gene with a secreted reporter, Gaussia luciferase (Gluc), to quantify neuronal activity without sacrificing the neurons. The reporter predominantly senses calcium and NMDA receptor (NMDAR)-dependent activity. By repeatedly measuring the accumulation of the reporter in cell media, we can profile the developmental dynamics of neuronal activity in cultured neurons from male and female mice. The assay also allows for longitudinal analysis of pharmacological treatments, thus distinguishing acute from delayed responses. Moreover, conditional expression of the reporter allows for monitoring cell type-specific changes. This simple, quantitative, cost-effective, automatable, and cell type-specific activity reporter is a valuable tool to study the development of neuronal activity in normal and disease-model conditions, and to identify small molecules or protein factors that selectively modulate the activity of a specific population of neurons.
Asunto(s)
Neuronas , Receptores de N-Metil-D-Aspartato , Animales , Femenino , Genes Reporteros , Luciferasas/genética , Masculino , Ratones , Neuronas/metabolismo , Transducción de SeñalRESUMEN
Genetic abnormalities in synaptic proteins are common in individuals with autism; however, our understanding of the cellular and molecular mechanisms disrupted by these abnormalities is limited. SHANK3 is a postsynaptic scaffolding protein of excitatory synapses that has been found mutated or deleted in most patients with 22q13 deletion syndrome and about 2% of individuals with idiopathic autism and intellectual disability. Here, we generated CRISPR/Cas9-engineered human pluripotent stem cells (PSCs) with complete hemizygous SHANK3 deletion (SHANK3+/-), which is the most common genetic abnormality in patients, and investigated the synaptic and morphological properties of SHANK3-deficient PSC-derived cortical neurons engrafted in the mouse prefrontal cortex. We show that human PSC-derived neurons integrate into the mouse cortex by acquiring appropriate cortical layer identities and by receiving and sending anatomical projections from/to multiple different brain regions. We also demonstrate that SHANK3-deficient human neurons have reduced AMPA-, but not NMDA- or GABA-mediated synaptic transmission and exhibit impaired dendritic arbors and spines, as compared to isogenic control neurons co-engrafted in the same brain region. Together, this study reveals specific synaptic and morphological deficits caused by SHANK3 hemizygosity in human cortical neurons at different developmental stages under physiological conditions and validates the use of co-engrafted control and mutant human neurons as a new platform for studying connectivity deficits in genetic neurodevelopmental disorders associated with autism.
Asunto(s)
Proteínas del Tejido Nervioso , Transmisión Sináptica , Animales , Humanos , Ratones , Proteínas de Microfilamentos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Corteza Prefrontal/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiónicoRESUMEN
Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.
Asunto(s)
Ventrículos Cerebrales/citología , Células-Madre Neurales/trasplante , Animales , Diferenciación Celular/genética , Movimiento Celular , Femenino , Masculino , Ratones , Neuritas , Neuronas/fisiología , Ingeniería de TejidosRESUMEN
The architecture of neocortical projection neurons is subject of a complex gene control. Here we demonstrated that Foxg1, a transcription factor gene which patterns the early rostral brain and sets the pace of telencephalic neuronogenesis, specifically stimulates dendrite elongation. This phenomenon occurs in vivo like in vitro, and it is detectable even upon moderate changes of Foxg1 expression levels. We found that Foxg1 acts by stimulating Hes1, which in turn upregulates pCreb1, a well-known pro-dendritogenic effector, and downregulates Syt and Ndr1, namely two established antagonizers of dendrite elongation. Moreover, Foxg1-driven pCreb1 upregulation requires PKA and AKT, and correlates with reduced PP1 and PP2A phosphatase activity. These findings contribute to clarify normal neurodevelopmental and activity-related regulation of neuritogenesis. They further suggest that an abnormal sizing of the dendritic tree of neocortical projection neurons may occur in West and Rett syndrome patients with anomalous FOXG1 allele dosages and contribute to their neurolopathological profiles.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Dendritas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Neocórtex/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células Piramidales/metabolismo , Factor de Transcripción HES-1/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo , Femenino , Regulación de la Expresión Génica , Masculino , Ratones , Fosforilación , Transducción de Señal , Regulación hacia ArribaRESUMEN
Colorectal cancer (CRC) transcriptional subtypes have been recently identified by gene expression profiling. Here we describe an analytical pipeline, microRNA master regulator analysis (MMRA), developed to search for microRNAs potentially driving CRC subtypes. Starting from a microRNA-mRNA tumour expression data set, MMRA identifies candidate regulator microRNAs by assessing their subtype-specific expression, target enrichment in subtype mRNA signatures and network analysis-based contribution to subtype gene expression. When applied to a CRC data set of 450 samples, assigned to subtypes by 3 different transcriptional classifiers, MMRA identifies 24 candidate microRNAs, in most cases downregulated in the stem/serrated/mesenchymal (SSM) poor prognosis subtype. Functional validation in CRC cell lines confirms downregulation of the SSM subtype by miR-194, miR-200b, miR-203 and miR-429, which share target genes and pathways mediating this effect. These results show that, by combining statistical tests, target prediction and network analysis, MMRA effectively identifies microRNAs functionally associated to cancer subtypes.