Dual effects of ARX poly-alanine mutations in human cortical and interneuron development.

Mutations in ARX , an X-linked gene, are implicated in a wide spectrum of neurological disorders including patients who have intellectual disability and epilepsy. Mouse models have shown that Arx is critical for cortical development and interneuron migration, however they do not recapitulate the full phenotype observed in patients. Moreover, the epilepsy in many patients with poly-alanine tract expansion (PAE) mutations in ARX show pharmacoresistance, emphasizing the need to develop new treatments. Here, we used human neural organoid models to study the consequences of PAE mutations, one of the most prevalent mutations in ARX . We found that PAE mutations result in an early increase in radial glia cells and intermediate progenitor cells, and premature differentiation leading to a loss of cortical neurons at later timepoints. Moreover, ARX expression is upregulated in CO derived from patient at 30 DIV which alters the expression of CDKN1C , SFRP1 , DLK1 and FABP7 , among others. We also found a cell autonomously enhanced interneuron migration, which can be rescued by CXCR4 inhibition. Furthermore, ARX PAE assembloids had hyper-activity and synchrony evident from the early stages. These data provide novel insights to the pathogenesis of these and likely related human neurological disorders and identifies a critical window for therapeutic interventions.

Brain IGF-I regulates LTP, spatial memory, and sexual dimorphic behavior.

Insulin-like growth factor-I (IGF-I) exerts multiple actions, yet the role of IGF-I from different sources is poorly understood. Here, we explored the functional and behavioral consequences of the conditional deletion of Igf-I in the nervous system (Igf-I Δ/Δ), and demonstrated that long-term potentiation was impaired in hippocampal slices. Moreover, Igf-I Δ/Δ mice showed spatial memory deficits in the Morris water maze, and the significant sex-dependent differences displayed by Igf-I Ctrl/Ctrl mice disappeared in Igf-I Δ/Δ mice in the open field and rota-rod tests. Brain Igf-I deletion disorganized the granule cell layer of the dentate gyrus (DG), and it modified the relative expressions of GAD and VGLUT1, which are preferentially localized to inhibitory and excitatory presynaptic terminals. Furthermore, Igf-I deletion altered protein modules involved in receptor trafficking, synaptic proteins, and proteins that functionally interact with estrogen and androgen metabolism. Our findings indicate that brain IGF-I is crucial for long-term potentiation, and that it is involved in the regulation of spatial memory and sexual dimorphic behaviors, possibly by maintaining the granule cell layer structure and the stability of synaptic-related protein modules.

Buprenorphine Exposure Alters the Development and Migration of Interneurons in the Cortex.

The misuse of opioids has reached epidemic proportions over the last decade, with over 2.1 million people in the United States suffering from substance use disorders related to prescription opioid pain relievers. This increase in opioid misuse affects all demographics of society, including women of child-bearing age, which has led to a rise in opioid use during pregnancy. Opioid use during pregnancy has been associated with increased risk of obstetric complications and adverse neonatal outcomes, including neonatal abstinence syndrome. Currently, opioid use disorder in pregnant women is treated with long-acting opioid agonists, including buprenorphine. Although buprenorphine reduces illicit opioid use during pregnancy and improves infant outcomes at birth, few long-term studies of the neurodevelopmental consequences have been conducted. The goal of the current experiments was to examine the effects of buprenorphine on the development of the cortex using fetal brain tissue, 3D brain cultures, and rodent models. First, we demonstrated that we can grow cortical and subpallial spheroids, which model the cellular diversity, connectivity, and activity of the developing human brain. Next, we show that cells in the developing human cortex express the nociceptin opioid (NOP) receptor and that buprenorphine can signal through this receptor in cortical spheroids. Using subpallial spheroids to grow inhibitory interneurons, we show that buprenorphine can alter interneuron development and migration into the cortex. Finally, using a rodent model of prenatal buprenorphine exposure, we demonstrate that alterations in interneuron distribution can persist into adulthood. Together, these results suggest that more research is needed into the long-lasting consequences of buprenorphine exposure on the developing human brain.

Distinct Effects of BDNF and NT-3 on the Dendrites and Presynaptic Boutons of Developing Olfactory Bulb GABAergic Interneurons In Vitro.

Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) are known to regulate neuronal morphology and the formation of neural circuits, yet the neuronal targets of each neurotrophin are still to be defined. To address how these neurotrophins regulate the morphological and synaptic differentiation of developing olfactory bulb (OB) GABAergic interneurons, we analyzed the effect of BDNF and NT-3 on GABA+-neurons and on different subtypes of these neurons: tyrosine hydroxylase (TH+); calretinin (Calr+); calbindin (Calb+); and parvalbumin (PVA+). These cells were generated from cultured embryonic mouse olfactory bulb neural stem cells (eOBNSCs) and after 14 days in vitro (DIV), when the neurons expressed TrkB and/or TrkC receptors, BDNF and NT-3 did not significantly change the number of neurons. However, long-term BDNF treatment did produce a longer total dendrite length and/or more dendritic branches in all the interneuron populations studied, except for PVA+-neurons. Similarly, BDNF caused an increase in the cell body perimeter in all the interneuron populations analyzed, except for PVA+-neurons. GABA+- and TH+-neurons were also studied at 21 DIV, when BDNF produced significantly longer neurites with no clear change in their number. Notably, these neurons developed synaptophysin+ boutons at 21 DIV, the size of which augmented significantly following exposure to either BDNF or NT-3. Our results show that in conditions that maintain neuronal survival, BDNF but not NT-3 promotes the morphological differentiation of developing OB interneurons in a cell-type-specific manner. In addition, our findings suggest that BDNF and NT-3 may promote synapse maturation by enhancing the size of synaptic boutons.

Neural stem cells in the adult olfactory bulb core generate mature neurons in vivo.

Although previous studies suggest that neural stem cells (NSCs) exist in the adult olfactory bulb (OB), their location, identity, and capacity to generate mature neurons in vivo has been little explored. Here, we injected enhanced green fluorescent protein (EGFP)-expressing retroviral particles into the OB core of adult mice to label dividing cells and to track the differentiation/maturation of any neurons they might generate. EGFP-labeled cells initially expressed adult NSC markers on days 1 to 3 postinjection (dpi), including Nestin, GLAST, Sox2, Prominin-1, and GFAP. EGFP+ -doublecortin (DCX) cells with a migratory morphology were also detected and their abundance increased over a 7-day period. Furthermore, EGFP-labeled cells progressively became NeuN+ neurons, they acquired neuronal morphologies, and they became immunoreactive for OB neuron subtype markers, the most abundant representing calretinin expressing interneurons. OB-NSCs also generated glial cells, suggesting they could be multipotent in vivo. Significantly, the newly generated neurons established and received synaptic contacts, and they expressed presynaptic proteins and the transcription factor pCREB. By contrast, when the retroviral particles were injected into the subventricular zone (SVZ), nearly all (98%) EGFP+ -cells were postmitotic when they reached the OB core, implying that the vast majority of proliferating cells present in the OB are not derived from the SVZ. Furthermore, we detected slowly dividing label-retaining cells in this region that could correspond to the population of resident NSCs. This is the first time NSCs located in the adult OB core have been shown to generate neurons that incorporate into OB circuits in vivo.

HDAC1 Regulates Neuronal Differentiation.

In adult hippocampal neurogenesis, chromatin modification plays an important role in neural stem cell self-renewal and differentiation by regulating the expression of multiple genes. Histone deacetylases (HDACs), which remove acetyl groups from histones, create a non-permissive chromatin that prevents transcription of genes involved in adult neurogenesis. HDAC inhibitors have been shown to promote adult neurogenesis and have also been used to treat nervous system disorders, such as epilepsy. However, most HDAC inhibitors are not specific and may have other targets. Therefore, it is important to decipher the role of individual HDACs in adult hippocampal neurogenesis. HDACs 1, 2, and 3 have been found expressed at different cellular stages during neurogenesis. Conditional deletion of HDAC2 in neural stem cells impairs neuronal differentiation in adult hippocampus. HDAC3 supports proliferation of adult hippocampal neural stem/progenitor cells. The role of HDAC1 in adult neurogenesis remains still open. Here, we used a conditional knock-out mouse to block HDAC1 expression in neural stem cells (Nestin+ cells) during hippocampal neurogenesis. Our results showed that both HDAC1 and HDAC2 are expressed in all cellular stages during hippocampal neurogenesis. Moreover, we found that deletion of HDAC1 by viral infection of neural stem cells is sufficient to compromise neuronal differentiation in vitro. However, we were unable to reduce the expression of HDAC1 in vivo using Nestin-CreERT2 mice. Understanding the role of HDAC1 may lead to ways to control stem cell proliferation and neuronal regeneration in the adult hippocampus, and to more specific HDAC therapeutics for neurological disorders.

Human Brain Organoid Models of Developmental Epilepsies.

Epilepsy is a common neurological disorder characterized by recurrent and unprovoked seizures due to neuronal hyperactivity. A large proportion of epilepsy cases begin during childhood. Causes of epilepsy include stroke, infections, brain injury, genetic factors, or other factors that alter brain structure and development, but in up to 50% of cases the cause is unknown. Approximately 35% of patients have refractory seizures that do not respond to medication. Animal models and in vitro cultures have contributed to our understanding of epilepsy, but there is a clear need for better models to explore the human brain in normal and pathological conditions. Human pluripotent stem cell (PSC) technologies opened the door for new models for analyzing brain development and disease, especially conditions with a genetic component. Initially, PSCs were differentiated into 2-dimensional cultures of a homogenous population of neural cells, such as glutamatergic excitatory or γ-aminobutyric acidergic inhibitory neurons, as well as glial cells. Nevertheless, these cultures lacked the structure and complexity of a human brain. In the last decade, PSC technology has advanced to the next level through the development of 3-dimensional culture, called organoids. These organoids recapitulate features of the human brain that are missing in animal models, enabling a deeper study of the human brain. In this review, we will summarize the current status of organoid research and its application to epilepsy.

CHD2: One Gene, Many Roles.

Mutations in the chromodomain helicase DNA-binding 2 (CHD2) gene have been found in patients with a range of neurodevelopmental disorders. In this issue of Neuron, Kim et al. (2018) showed that Chd2 haploinsufficiency compromises cortical development, synaptic function, and memory in mice.

Retinoblastoma protein controls growth, survival and neuronal migration in human cerebral organoids.

The tumor suppressor retinoblastoma protein (RB) regulates S-phase cell cycle entry via E2F transcription factors. Knockout (KO) mice have shown that RB plays roles in cell migration, differentiation and apoptosis, in developing and adult brain. In addition, the RB family is required for self-renewal and survival of human embryonic stem cells (hESCs). Since little is known about the role of RB in human brain development, we investigated its function in cerebral organoids differentiated from gene-edited hESCs lacking RB. We show that RB is abundantly expressed in neural stem and progenitor cells in organoids at 15 and 28 days of culture. RB loss promoted S-phase entry in DCX+ cells and increased apoptosis in Sox2+ neural stem and progenitor cells, and in DCX+ and Tuj1+ neurons. Associated with these cell cycle and pro-apoptotic effects, we observed increased CCNA2 and BAX gene expression, respectively. Moreover, we observed aberrant Tuj1+ neuronal migration in RB-KO organoids and upregulation of the gene encoding VLDLR, a receptor important in reelin signaling. Corroborating the results in RB-KO organoids in vitro, we observed ectopically localized Tuj1+ cells in RB-KO teratomas grown in vivo Taken together, these results identify crucial functions for RB in the cerebral organoid model of human brain development.

Brain Insulin-Like Growth Factor-I Directs the Transition from Stem Cells to Mature Neurons During Postnatal/Adult Hippocampal Neurogenesis.

The specific actions of insulin-like growth factor-I (IGF-I) and the role of brain-derived IGF-I during hippocampal neurogenesis have not been fully defined. To address the influence of IGF-I on the stages of hippocampal neurogenesis, we studied a postnatal/adult global Igf-I knockout (KO) mice (Igf-I(-/-) ) and a nervous system Igf-I conditional KO (Igf-I(Δ/Δ) ). In both KO mice we found an accumulation of Tbr2(+) -intermediate neuronal progenitors, some of which were displaced in the outer granule cell layer (GCL) and the molecular layer (ML) of the dentate gyrus (DG). Similarly, more ectopic Ki67(+) - cycling cells were detected. Thus, the GCL was disorganized with significant numbers of Prox1(+) -granule neurons outside this layer and altered morphology of radial glial cells (RGCs). Dividing progenitors were also generated in greater numbers in clonal hippocampal stem cell (HPSC) cultures from the KO mice. Indeed, higher levels of Hes5 and Ngn2, transcription factors that maintain the stem and progenitor cell state, were expressed in both HPSCs and the GCL-ML from the Igf-I(Δ/Δ) mice. To determine the impact of Igf-I deletion on neuronal generation in vivo, progenitors in Igf-I(-/-) and Igf-I(+/+) mice were labeled with a GFP-expressing vector. This revealed that in the Igf-I(-/-) mice more GFP(+) -immature neurons were formed and they had less complex dendritic trees. These findings indicate that local IGF-I plays critical roles during postnatal/adult hippocampal neurogenesis, regulating the transition from HPSCs and progenitors to mature granule neurons in a cell stage-dependent manner. Stem Cells 2016;34:2194-2209.

IGF-I: A Key Growth Factor that Regulates Neurogenesis and Synaptogenesis from Embryonic to Adult Stages of the Brain.

The generation of neurons in the adult mammalian brain requires the activation of quiescent neural stem cells (NSCs). This activation and the sequential steps of neuron formation from NSCs are regulated by a number of stimuli, which include growth factors. Insulin-like growth factor-I (IGF-I) exert pleiotropic effects, regulating multiple cellular processes depending on their concentration, cell type, and the developmental stage of the animal. Although IGF-I expression is relatively high in the embryonic brain its levels drop sharply in the adult brain except in neurogenic regions, i.e., the hippocampus (HP) and the subventricular zone-olfactory bulb (SVZ-OB). By contrast, the expression of IGF-IR remains relatively high in the brain irrespective of the age of the animal. Evidence indicates that IGF-I influences NSC proliferation and differentiation into neurons and glia as well as neuronal maturation including synapse formation. Furthermore, recent studies have shown that IGF-I not only promote adult neurogenesis by regulating NSC number and differentiation but also by influencing neuronal positioning and migration as described during SVZ-OB neurogenesis. In this article we will revise and discuss the actions reported for IGF-I signaling in a variety of in vitro and in vivo models, focusing on the maintenance and proliferation of NSCs/progenitors, neurogenesis, and neuron integration in synaptic circuits.

Pax6 is essential for the maintenance and multi-lineage differentiation of neural stem cells, and for neuronal incorporation into the adult olfactory bulb.

The paired type homeobox 6 (Pax6) transcription factor (TF) regulates multiple aspects of neural stem cell (NSC) and neuron development in the embryonic central nervous system. However, less is known about the role of Pax6 in the maintenance and differentiation of adult NSCs and in adult neurogenesis. Using the +/Sey(Dey) mouse, we have analyzed how Pax6 heterozygosis influences the self-renewal and proliferation of adult olfactory bulb stem cells (aOBSCs). In addition, we assessed its influence on neural differentiation, neuronal incorporation, and cell death in the adult OB, both in vivo and in vitro. Our results indicate that the Pax6 mutation alters Nestin(+)-cell proliferation in vivo, as well as self-renewal, proliferation, and survival of aOBSCs in vitro although a subpopulation of +/Sey(Dey) progenitors is able to expand partially similar to wild-type progenitors. This mutation also impairs aOBSC differentiation into neurons and oligodendrocytes, whereas it increases cell death while preserving astrocyte survival and differentiation. Furthermore, Pax6 heterozygosis causes a reduction in the variety of neurochemical interneuron subtypes generated from aOBSCs in vitro and in the incorporation of newly generated neurons into the OB in vivo. Our findings support an important role of Pax6 in the maintenance of aOBSCs by regulating cell death, self-renewal, and cell fate, as well as in neuronal incorporation into the adult OB. They also suggest that deregulation of the cell cycle machinery and TF expression in aOBSCs which are deficient in Pax6 may be at the origin of the phenotypes observed in this adult NSC population.

Transcriptional regulation of olfactory bulb neurogenesis.

The neurons in the olfactory bulb originate from molecularly defined and spatially distinct proliferative regions. Glutamatergic projection neurons are generated during the embryonic period in the local ventricular zone of the olfactory bulb, a territory in the dorsal telencephalon in which the transcription factor Pax6 is expressed. Some cells in this zone also express Tbr1, a marker of glutamatergic neurons. By contrast, embryonic olfactory bulb interneurons are derived from Gsx2 expressing cells in the dorsal lateral ganglionic eminence of the ventral telencephalon, and from progenitors outside the dorsal lateral ganglionic eminence, including the olfactory bulb neuroepithelium. Postnatally, interneurons arise from the subventricular zone of the lateral ventricle, although the rostral migratory stream and the olfactory bulb also appear to serve as a source of neurons. Transcription factors are crucial to generate all classes of neurons and glia in the olfactory bulb, both during development and adulthood. In this article, we discuss and propose models on how the spatial and temporal regulation of transcription factor expression controls the self-renewal, proliferation and cell fate of neural stem cells and progenitors, which finally leads to the generation of distinct functional subtypes of neurons in the developing and adult olfactory bulb.

A global transcriptome analysis reveals molecular hallmarks of neural stem cell death, survival, and differentiation in response to partial FGF-2 and EGF deprivation.

Neurosphere cell culture is a commonly used model to study the properties and potential applications of neural stem cells (NSCs). However, standard protocols to culture NSCs have yet to be established, and the mechanisms underlying NSC survival and maintenance of their undifferentiated state, in response to the growth factors FGF-2 and EGF are not fully understood. Using cultures of embryonic and adult olfactory bulb stem cells (eOBSCs and aOBSCs), we analyzed the consequences of FGF-2 and EGF addition at different intervals on proliferation, cell cycle progression, cell death and differentiation, as well as on global gene expression. As opposed to cultures supplemented daily, addition of FGF-2 and EGF every 4 days significantly reduced the neurosphere volume and the total number of cells in the spheres, mainly due to increased cell death. Moreover, partial FGF-2 and EGF deprivation produced an increase in OBSC differentiation during the proliferative phase. These changes were more evident in aOBSC than eOBSC cultures. Remarkably, these effects were accompanied by a significant upregulation in the expression of endogenous Fgf-2 and genes involved in cell death and survival (Cryab), lipid catabolic processes (Pla2g7), cell adhesion (Dscaml1), cell differentiation (Dscaml1, Gpr17, S100b, Ndrg2) and signal transduction (Gpr17, Ndrg2). These findings support that a daily supply of FGF-2 and EGF is critical to maintain the viability and the undifferentiated state of NSCs in culture, and they reveal novel molecular hallmarks of NSC death, survival and the initiation of differentiation.