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.

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.

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.

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.

Methadone interrupts neural growth and function in human cortical organoids.

Prenatal opioids exposure can lead to both neonatal abstinence syndrome in newborns and neurological deficits later in life. Although opioids have been well studied in general, the cellular and molecular mechanisms by which opioids affect human fetal brain development has not been well understood. In this work, we have taken advantage of a human 3D-brain cortical organoid (hCO) that facilitated enormously the investigation of early human brain development. Using imaging, immunofluorescence, multi-electrode array (MEA) and patch clamp recording techniques, we have investigated the effect of methadone, a frequently used opioid during pregnancy, on early neural development, including neuronal growth, neural network activity and synaptic transmission in hCOs. Our results demonstrated that methadone dose-dependently halted the growth of hCOs and induced organoid disintegration after a prolonged exposure. In addition, methadone dose-dependently suppressed the firing of spontaneous action potentials in hCOs and this suppression could be reversed upon methadone withdrawal in hCOs treated with lower dosages. Further investigation using patch clamp whole cell configuration revealed that, at clinically relevant concentrations, methadone decreased the frequency and amplitude of excitatory postsynaptic currents in neurons, indicating a critical role of methadone in weakening synaptic transmission in neural networks in hCOs. In addition, methadone significantly attenuated the voltage-dependent Na+ current in hCOs. We conclude that methadone interrupts neural growth and function in early brain development.

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.

Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics.

Loss-of-function mutations in CDKL5 kinase cause severe neurodevelopmental delay and early-onset seizures. Identification of CDKL5 substrates is key to understanding its function. Using chemical genetics, we found that CDKL5 phosphorylates three microtubule-associated proteins: MAP1S, EB2 and ARHGEF2, and determined the phosphorylation sites. Substrate phosphorylations are greatly reduced in CDKL5 knockout mice, verifying these as physiological substrates. In CDKL5 knockout mouse neurons, dendritic microtubules have longer EB3-labelled plus-end growth duration and these altered dynamics are rescued by reduction of MAP1S levels through shRNA expression, indicating that CDKL5 regulates microtubule dynamics via phosphorylation of MAP1S. We show that phosphorylation by CDKL5 is required for MAP1S dissociation from microtubules. Additionally, anterograde cargo trafficking is compromised in CDKL5 knockout mouse dendrites. Finally, EB2 phosphorylation is reduced in patient-derived human neurons. Our results reveal a novel activity-dependent molecular pathway in dendritic microtubule regulation and suggest a pathological mechanism which may contribute to CDKL5 deficiency disorder.

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.

Evidence of nuclei-encoded spliceosome mediating splicing of mitochondrial RNA.

Mitochondria are thought to have originated as free-living prokaryotes. Mitochondria organelles have small circular genomes with substantial structural and genetic similarity to bacteria. Contrary to the prevailing concept of intronless mitochondria, here we present evidence that mitochondrial RNA transcripts (mtRNA) are not limited to policystronic molecules, but also processed as nuclei-like transcripts that are differentially spliced and expressed in a cell-type specific manner. The presence of canonical splice sites in the mtRNA introns and of core components of the nuclei-encoded spliceosome machinery within the mitochondrial organelle suggest that nuclei-encoded spliceosome can mediate splicing of mtRNA.

Bradykinin promotes neuron-generating division of neural progenitor cells through ERK activation.

During brain development, cells proliferate, migrate and differentiate in highly accurate patterns. In this context, published results indicate that bradykinin functions in neural fate determination, favoring neurogenesis and migration. However, mechanisms underlying bradykinin function are yet to be explored. Our findings indicate a previously unidentified role for bradykinin action in inducing neuron-generating division in vitro and in vivo, given that bradykinin lengthened the G1-phase of the neural progenitor cells (NPC) cycle and increased TIS21 (also known as PC3 and BTG2) expression in hippocampus from newborn mice. This role, triggered by activation of the kinin-B2 receptor, was conditioned by ERK1/2 activation. Moreover, immunohistochemistry analysis of hippocampal dentate gyrus showed that the percentage of Ki67(+) cells markedly increased in bradykinin-treated mice, and ERK1/2 inhibition affected this neurogenic response. The progress of neurogenesis depended on sustained ERK phosphorylation and resulted in ERK1/2 translocation to the nucleus in NPCs and PC12 cells, changing expression of genes such as Hes1 and Ngn2 (also known as Neurog2). In agreement with the function of ERK in integrating signaling pathways, effects of bradykinin in stimulating neurogenesis were reversed following removal of protein kinase C (PKC)-mediated sustained phosphorylation.

Effects of ATP and NGF on Proliferation and Migration of Neural Precursor Cells.

Purinergic receptors belong to the most ancient neurotransmitter system. While their relevance in neurotransmission is well characterized, it has become clear that they have many other cellular functions. During development, they participate in regulation of proliferation and differentiation of stem cells. Here, we used rat embryonic telencephalon neurosphere cultures to detect purinergic P2 receptor subtype expression and possible synergistic actions of these receptors with NGF. Neurospheres proliferate in the presence of EGF and FGF-2; however, upon depletion of these growth factors, they migrate and differentiate into neurons and glial phenotypes. Expression patterns of P2X and P2Y receptors changed along neural differentiation. Gene expression of P2X2-7 and P2Y1,2,4,6,12,14 receptors was confirmed in undifferentiated and neural-differentiated neurospheres, with an up-regulation of P2X2 and P2X6 subtypes, together with a down-regulation of P2X4, P2X7 and P2Y subtypes upon induction to differentiation. BrdU-labeling and subsequent flow cytometry analysis was used to measure cell proliferation, which was increased by chronic exposure to NGF and increasing concentrations of ATP, in line with the expression levels of PCNA. Furthermore, a synergistic effect on proliferation was observed in conditions of co-incubation with ATP and NGF. While ATP and NGF independently promoted neural migration, no inter-relation between these factors was detected for this cellular process. As conclusion, an unknown synergism of ATP and NGF in proliferation is described. Future efforts may elucidate the underlying mechanisms of the interrelationship of ATP and NGF during neurogenesis.