Impact of alcohol exposure on neural development and network formation in human cortical organoids.

Prenatal alcohol exposure is the foremost preventable etiology of intellectual disability and leads to a collection of diagnoses known as Fetal Alcohol Spectrum Disorders (FASD). Alcohol (EtOH) impacts diverse neural cell types and activity, but the precise functional pathophysiological effects on the human fetal cerebral cortex are unclear. Here, we used human cortical organoids to study the effects of EtOH on neurogenesis and validated our findings in primary human fetal neurons. EtOH exposure produced temporally dependent cellular effects on proliferation, cell cycle, and apoptosis. In addition, we identified EtOH-induced alterations in post-translational histone modifications and chromatin accessibility, leading to impairment of cAMP and calcium signaling, glutamatergic synaptic development, and astrocytic function. Proteomic spatial profiling of cortical organoids showed region-specific, EtOH-induced alterations linked to changes in cytoskeleton, gliogenesis, and impaired synaptogenesis. Finally, multi-electrode array electrophysiology recordings confirmed the deleterious impact of EtOH on neural network formation and activity in cortical organoids, which was validated in primary human fetal tissues. Our findings demonstrate progress in defining the human molecular and cellular phenotypic signatures of prenatal alcohol exposure on functional neurodevelopment, increasing our knowledge for potential therapeutic interventions targeting FASD symptoms.

Neuronal hyperexcitability and ion channel dysfunction in CDKL5-deficiency patient iPSC-derived cortical organoids.

Early epilepsy is a prominent feature in patients with CDKL5-deficiency disorder (CDD). The underlying mechanism for excessive excitability in CDD is largely unknown. The brain organoid model has been recently developed to resemble many critical features of early human brain development. Here, we used a brain organoid model to investigate the cellular electrophysiological basis for hyper-excitability in CDD patients. Our study employed cortical organoids derived from two CDD patients harboring the same CDKL5 mutation (R59X) and two controls from their healthy parents. Whole-cell patch-clamp recordings revealed higher action potential (AP) firing rate and lower rheobase in both CDD organoids, indicating increased intrinsic neuronal excitability. We further found dysfunction of voltage-gated ion channels in CDD neurons that leads to hyperexcitability, including higher Na+ and K+ current densities and a negative shift in Na+ channel activation. In contrast to neuronal properties, we found that glutamatergic neurotransmission and the electrophysiological properties of glial cells were not altered in CDD organoids. In support of our CDD findings, we further discovered similar electrophysiologic properties in cortical organoids derived from a Rett syndrome (RTT) patient, including alterations in AP firings and Na+ and K+ channel function suggesting a convergent mechanism. Together, our study suggests a critical role of intrinsic neuronal hyperexcitability and ion channel dysfunction, seen in early brain development in both CDD and RTT disorders. This investigation provides potential novel drug targets for developing treatments of early epilepsy in such disorders.

Response to Comment on "Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment".

Maricic et al. performed an undisclosed in silico­only whole-exome sequencing analysis of our data and found genomic alterations previously undetected in some clones. Some of the predicted alterations, if true, could change the original genotype of the clones. We failed to experimentally validate all but one of these genomic alterations, which did not affect our previous results or data interpretation.

Cortical organoids model early brain development disrupted by 16p11.2 copy number variants in autism.

Reciprocal deletion and duplication of the 16p11.2 region is the most common copy number variation (CNV) associated with autism spectrum disorders. We generated cortical organoids from skin fibroblasts of patients with 16p11.2 CNV to investigate impacted neurodevelopmental processes. We show that organoid size recapitulates macrocephaly and microcephaly phenotypes observed in the patients with 16p11.2 deletions and duplications. The CNV dosage affects neuronal maturation, proliferation, and synapse number, in addition to its effect on organoid size. We demonstrate that 16p11.2 CNV alters the ratio of neurons to neural progenitors in organoids during early neurogenesis, with a significant excess of neurons and depletion of neural progenitors observed in deletions. Transcriptomic and proteomic profiling revealed multiple pathways dysregulated by the 16p11.2 CNV, including neuron migration, actin cytoskeleton, ion channel activity, synaptic-related functions, and Wnt signaling. The level of the active form of small GTPase RhoA was increased in both, deletions and duplications. Inhibition of RhoA activity rescued migration deficits, but not neurite outgrowth. This study provides insights into potential neurobiological mechanisms behind the 16p11.2 CNV during neocortical development.

All-Optical Electrophysiology in hiPSC-Derived Neurons With Synthetic Voltage Sensors.

Voltage imaging and "all-optical electrophysiology" in human induced pluripotent stem cell (hiPSC)-derived neurons have opened unprecedented opportunities for high-throughput phenotyping of activity in neurons possessing unique genetic backgrounds of individual patients. While prior all-optical electrophysiology studies relied on genetically encoded voltage indicators, here, we demonstrate an alternative protocol using a synthetic voltage sensor and genetically encoded optogenetic actuator that generate robust and reproducible results. We demonstrate the functionality of this method by measuring spontaneous and evoked activity in three independent hiPSC-derived neuronal cell lines with distinct genetic backgrounds.

MeCP2 controls neural stem cell fate specification through miR-199a-mediated inhibition of BMP-Smad signaling.

Rett syndrome (RTT) is a severe neurological disorder, with impaired brain development caused by mutations in MECP2; however, the underlying mechanism remains elusive. We know from previous work that MeCP2 facilitates the processing of a specific microRNA, miR-199a, by associating with the Drosha complex to regulate neuronal functions. Here, we show that the MeCP2/miR-199a axis regulates neural stem/precursor cell (NS/PC) differentiation. A shift occurs from neuronal to astrocytic differentiation of MeCP2- and miR-199a-deficient NS/PCs due to the upregulation of a miR-199a target, Smad1, a downstream transcription factor of bone morphogenetic protein (BMP) signaling. Moreover, miR-199a expression and treatment with BMP inhibitors rectify the differentiation of RTT patient-derived NS/PCs and development of brain organoids, respectively, suggesting that facilitation of BMP signaling accounts for the impaired RTT brain development. Our study illuminates the molecular pathology of RTT and reveals the MeCP2/miR-199a/Smad1 axis as a potential therapeutic target for RTT.

Altered network and rescue of human neurons derived from individuals with early-onset genetic epilepsy.

Early-onset epileptic encephalopathies are severe disorders often associated with specific genetic mutations. In this context, the CDKL5 deficiency disorder (CDD) is a neurodevelopmental condition characterized by early-onset seizures, intellectual delay, and motor dysfunction. Although crucial for proper brain development, the precise targets of CDKL5 and its relation to patients' symptoms are still unknown. Here, induced pluripotent stem cells derived from individuals deficient in CDKL5 protein were used to generate neural cells. Proteomic and phosphoproteomic approaches revealed disruption of several pathways, including microtubule-based processes and cytoskeleton organization. While CDD-derived neural progenitor cells have proliferation defects, neurons showed morphological alterations and compromised glutamatergic synaptogenesis. Moreover, the electrical activity of CDD cortical neurons revealed hyperexcitability during development, leading to an overly synchronized network. Many parameters of this hyperactive network were rescued by lead compounds selected from a human high-throughput drug screening platform. Our results enlighten cellular, molecular, and neural network mechanisms of genetic epilepsy that could ultimately promote novel therapeutic opportunities for patients.

Autism-linked Cullin3 germline haploinsufficiency impacts cytoskeletal dynamics and cortical neurogenesis through RhoA signaling.

E3-ubiquitin ligase Cullin3 (Cul3) is a high confidence risk gene for autism spectrum disorder (ASD) and developmental delay (DD). To investigate how Cul3 mutations impact brain development, we generated a haploinsufficient Cul3 mouse model using CRISPR/Cas9 genome engineering. Cul3 mutant mice exhibited social and cognitive deficits and hyperactive behavior. Brain MRI found decreased volume of cortical regions and changes in many other brain regions of Cul3 mutant mice starting from early postnatal development. Spatiotemporal transcriptomic and proteomic profiling of embryonic, early postnatal and adult brain implicated neurogenesis and cytoskeletal defects as key drivers of Cul3 functional impact. Specifically, dendritic growth, filamentous actin puncta, and spontaneous network activity were reduced in Cul3 mutant mice. Inhibition of small GTPase RhoA, a molecular substrate of Cul3 ligase, rescued dendrite length and network activity phenotypes. Our study identified defects in neuronal cytoskeleton and Rho signaling as the primary targets of Cul3 mutation during brain development.

Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment.

The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 (NOVA1) plays a key role in neural development and function. NOVA1 also includes a protein-coding difference between the modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version of NOVA1 Moreover, levels of synaptic markers and synaptic protein coassociations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution in NOVA1, which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species' evolution.

Pharmacological reversal of synaptic and network pathology in human MECP2-KO neurons and cortical organoids.

Duplication or deficiency of the X-linked MECP2 gene reliably produces profound neurodevelopmental impairment. MECP2 mutations are almost universally responsible for Rett syndrome (RTT), and particular mutations and cellular mosaicism of MECP2 may underlie the spectrum of RTT symptomatic severity. No clinically approved treatments for RTT are currently available, but human pluripotent stem cell technology offers a platform to identify neuropathology and test candidate therapeutics. Using a strategic series of increasingly complex human stem cell-derived technologies, including human neurons, MECP2-mosaic neurospheres to model RTT female brain mosaicism, and cortical organoids, we identified synaptic dysregulation downstream from knockout of MECP2 and screened select pharmacological compounds for their ability to treat this dysfunction. Two lead compounds, Nefiracetam and PHA 543613, specifically reversed MECP2-knockout cytologic neuropathology. The capacity of these compounds to reverse neuropathologic phenotypes and networks in human models supports clinical studies for neurodevelopmental disorders in which MeCP2 deficiency is the predominant etiology.

RNA processing in neurological tissue: development, aging and disease.

Gene expression comprises a diverse array of enzymes, proteins, non-coding transcripts, and cellular structures to guide the transfer of genetic information to its various final products. In the brain, the coordination among genes, or lack thereof, characterizes individual brain regions, mediates a variety of brain-related disorders, and brings light to fundamental differences between species. RNA processing, occurring between transcription and translation, controls an essential portion of gene expression through splicing, editing, localization, stability, and interference. The machinery to regulate transcripts must operate with precision serving as a blueprint for proteins and non-coding RNAs to derive their identity. Therefore, RNA processing has a broad scope of influence in the brain, as it modulates cell morphogenesis during development and underlies mechanisms behind certain neurological diseases. Here, we present these ideas through recent findings on RNA processing in development and post-developmental maturity to advance therapeutic discoveries and the collective knowledge of the RNA life cycle.

Methadone Suppresses Neuronal Function and Maturation in Human Cortical Organoids.

Accumulating evidence has suggested that prenatal exposure to methadone causes multiple adverse effects on human brain development. Methadone not only suppresses fetal neurobehavior and alters neural maturation, but also leads to long-term neurological impairment. Due to logistical and ethical issues of accessing human fetal tissue, the effect of methadone on brain development and its underlying mechanisms have not been investigated adequately and are therefore not fully understood. Here, we use human cortical organoids which resemble fetal brain development to examine the effect of methadone on neuronal function and maturation during early development. During development, cortical organoids that are exposed to clinically relevant concentrations of methadone exhibited suppressed maturation of neuronal function. For example, organoids developed from 12th week till 24th week have an about 7-fold increase in AP firing frequency, but only half and a third of this increase was found in organoids exposed to 1 and 10 µM methadone, respectively. We further demonstrated substantial increases in I Na (4.5-fold) and I KD (10.8-fold), and continued shifts of Na+ channel activation and inactivation during normal organoid development. Methadone-induced suppression of neuronal function was attributed to the attenuated increase in the densities of I Na and I KD and the reduced shift of Na+ channel gating properties. Since normal neuronal electrophysiology and ion channel function are critical for regulating brain development, we believe that the effect of prolonged methadone exposure contributes to the delayed maturation, development fetal brain and potentially for longer term neurologic deficits.

Modeling Rett Syndrome With Human Patient-Specific Forebrain Organoids.

Engineering brain organoids from human induced pluripotent stem cells (hiPSCs) is a powerful tool for modeling brain development and neurological disorders. Rett syndrome (RTT), a rare neurodevelopmental disorder, can greatly benefit from this technology, since it affects multiple neuronal subtypes in forebrain sub-regions. We have established dorsal and ventral forebrain organoids from control and RTT patient-specific hiPSCs recapitulating 3D organization and functional network complexity. Our data revealed a premature development of the deep-cortical layer, associated to the formation of TBR1 and CTIP2 neurons, and a lower expression of neural progenitor/proliferative cells in female RTT dorsal organoids. Moreover, calcium imaging and electrophysiology analysis demonstrated functional defects of RTT neurons. Additionally, assembly of RTT dorsal and ventral organoids revealed impairments of interneuron's migration. Overall, our models provide a better understanding of RTT during early stages of neural development, demonstrating a great potential for personalized diagnosis and drug screening.

Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin αvß5 Axis.

Zika virus (ZIKV) causes microcephaly by killing neural precursor cells (NPCs) and other brain cells. ZIKV also displays therapeutic oncolytic activity against glioblastoma (GBM) stem cells (GSCs). Here we demonstrate that ZIKV preferentially infected and killed GSCs and stem-like cells in medulloblastoma and ependymoma in a SOX2-dependent manner. Targeting SOX2 severely attenuated ZIKV infection, in contrast to AXL. As mechanisms of SOX2-mediated ZIKV infection, we identified inverse expression of antiviral interferon response genes (ISGs) and positive correlation with integrin αv (ITGAV). ZIKV infection was disrupted by genetic targeting of ITGAV or its binding partner ITGB5 and by an antibody specific for integrin αvß5. ZIKV selectively eliminated GSCs from species-matched human mature cerebral organoids and GBM surgical specimens, which was reversed by integrin αvß5 inhibition. Collectively, our studies identify integrin αvß5 as a functional cancer stem cell marker essential for GBM maintenance and ZIKV infection, providing potential brain tumor therapy.

Complex Oscillatory Waves Emerging from Cortical Organoids Model Early Human Brain Network Development.

Structural and transcriptional changes during early brain maturation follow fixed developmental programs defined by genetics. However, whether this is true for functional network activity remains unknown, primarily due to experimental inaccessibility of the initial stages of the living human brain. Here, we developed human cortical organoids that dynamically change cellular populations during maturation and exhibited consistent increases in electrical activity over the span of several months. The spontaneous network formation displayed periodic and regular oscillatory events that were dependent on glutamatergic and GABAergic signaling. The oscillatory activity transitioned to more spatiotemporally irregular patterns, and synchronous network events resembled features similar to those observed in preterm human electroencephalography. These results show that the development of structured network activity in a human neocortex model may follow stable genetic programming. Our approach provides opportunities for investigating and manipulating the role of network activity in the developing human cortex.

Brain Organoids and the Study of Neurodevelopment.

Brain organoids are 3D self-assembled structures composed of hundreds of thousands to millions of cells that resemble the cellular organization and transcriptional and epigenetic signature of a developing human brain. Advancements using brain organoids have been made to elucidate the genetic basis of certain neurodevelopmental disorders, such as microcephaly and autism; and to investigate the impact of environmental factors to the brain, such as during Zika virus infection. It remains to be explored how far brain organoids can functionally mature and process external information. An improved brain organoid model might reproduce important aspects of the human brain in a more reproducible and high-throughput fashion. This novel and complementary approach in the neuroscience toolbox opens perspectives to understand the fundamental features of the human neurodevelopment, with implications to personalize therapeutic opportunities for neurological disorders.

Direct Generation of Human Cortical Organoids from Primary Cells.

The study of variations in human neurodevelopment and cognition is limited by the availability of experimental models. While animal models only partially recapitulate the human brain development, genetics, and heterogeneity, human-induced pluripotent stem cells can provide an attractive experimental alternative. However, cellular reprogramming and further differentiation techniques are costly and time-consuming and therefore, studies using this approach are often limited to a small number of samples. In this study, we describe a rapid and cost-effective method to reprogram somatic cells and the direct generation of cortical organoids in a 96-well format. Our data are a proof-of-principle that a large cohort of samples can be generated for experimental assessment of the human neural development.

Stem cell contributions to neurological disease modeling and personalized medicine.

Human induced pluripotent stem cells (iPSCs) represent a revolutionary tool for disease modeling and drug discovery. The generation of tissue-relevant cell types exhibiting a patient's genetic and molecular background offers the ability to develop individual and effective therapies. In this review, we present some major achievements in the neuroscience field using iPSCs and discuss promising perspectives in personalized medicine. In addition to disease modeling, the understanding of the cellular and molecular basis of neurological disorders is explored, including the discovery of new targets and potential drugs. Ultimately, we highlight how iPSC technology, together with genome editing approaches, may bring a deep impact on pre-clinical trials by reducing costs and increasing the success of treatments in a personalized fashion.

Modeling of TREX1-Dependent Autoimmune Disease using Human Stem Cells Highlights L1 Accumulation as a Source of Neuroinflammation.

Three-prime repair exonuclease 1 (TREX1) is an anti-viral enzyme that cleaves nucleic acids in the cytosol, preventing accumulation and a subsequent type I interferon-associated inflammatory response. Autoimmune diseases, including Aicardi-Goutières syndrome (AGS) and systemic lupus erythematosus, can arise when TREX1 function is compromised. AGS is a neuroinflammatory disorder with severe and persistent intellectual and physical problems. Here we generated a human AGS model that recapitulates disease-relevant phenotypes using pluripotent stem cells lacking TREX1. We observed abundant extrachromosomal DNA in TREX1-deficient neural cells, of which endogenous Long Interspersed Element-1 retrotransposons were a major source. TREX1-deficient neurons also exhibited increased apoptosis and formed three-dimensional cortical organoids of reduced size. TREX1-deficient astrocytes further contributed to the observed neurotoxicity through increased type I interferon secretion. In this model, reverse-transcriptase inhibitors rescued the neurotoxicity of AGS neurons and organoids, highlighting their potential utility in therapeutic regimens for AGS and related disorders.