KCTD10 regulates brain development by destabilizing brain disorder-associated protein KCTD13.

KCTD10 belongs to the KCTD (potassiumchannel tetramerization domain) family, many members of which are associated with neuropsychiatric disorders. However, the biological function underlying the association with brain disorders remains to be explored. Here, we reveal that Kctd10 is highly expressed in neuronal progenitors and layer V neurons throughout brain development. Kctd10 deficiency triggers abnormal proliferation and differentiation of neuronal progenitors, reduced deep-layer (especially layer V) neurons, increased upper-layer neurons, and lowered brain size. Mechanistically, we screened and identified a unique KCTD10-interacting protein, KCTD13, associated with neurodevelopmental disorders. KCTD10 mediated the ubiquitination-dependent degradation of KCTD13 and KCTD10 ablation resulted in a considerable increase of KCTD13 expression in the developing cortex. KCTD13 overexpression in neuronal progenitors led to reduced proliferation and abnormal cell distribution, mirroring KCTD10 deficiency. Notably, mice with brain-specific Kctd10 knockout exhibited obvious motor deficits. This study uncovers the physiological function of KCTD10 and provides unique insights into the pathogenesis of neurodevelopmental disorders.

The E2A splice variant E47 regulates the differentiation of projection neurons via p57(KIP2) during cortical development.

During corticogenesis, distinct classes of neurons are born from progenitor cells located in the ventricular and subventricular zones, from where they migrate towards the pial surface to assemble into highly organized layer-specific circuits. However, the precise and coordinated transcriptional network activity defining neuronal identity is still not understood. Here, we show that genetic depletion of the basic helix-loop-helix (bHLH) transcription factor E2A splice variant E47 increased the number of Tbr1-positive deep layer and Satb2-positive upper layer neurons at E14.5, while depletion of the alternatively spliced E12 variant did not affect layer-specific neurogenesis. While ChIP-Seq identified a big overlap for E12- and E47-specific binding sites in embryonic NSCs, including sites at the cyclin-dependent kinase inhibitor (CDKI) Cdkn1c gene locus, RNA-Seq revealed a unique transcriptional regulation by each splice variant. E47 activated the expression of the CDKI Cdkn1c through binding to a distal enhancer. Finally, overexpression of E47 in embryonic NSCs in vitro impaired neurite outgrowth, and overexpression of E47 in vivo by in utero electroporation disturbed proper layer-specific neurogenesis and upregulated p57(KIP2) expression. Overall, this study identifies E2A target genes in embryonic NSCs and demonstrates that E47 regulates neuronal differentiation via p57(KIP2).

A mathematical insight into cell labelling experiments for clonal analysis.

Studying the progression of the proliferative and differentiative patterns of neural stem cells at the individual cell level is crucial to the understanding of cortex development and how the disruption of such patterns can lead to malformations and neurodevelopmental diseases. However, our understanding of the precise lineage progression programme at single-cell resolution is still incomplete due to the technical variations in lineage-tracing approaches. One of the key challenges involves developing a robust theoretical framework in which we can integrate experimental observations and introduce correction factors to obtain a reliable and representative description of the temporal modulation of proliferation and differentiation. In order to obtain more conclusive insights, we carry out virtual clonal analysis using mathematical modelling and compare our results against experimental data. Using a dataset obtained with Mosaic Analysis with Double Markers, we illustrate how the theoretical description can be exploited to interpret and reconcile the disparity between virtual and experimental results.

Cortical progenitor biology: key features mediating proliferation versus differentiation.

The cerebral cortex is a highly organized structure whose development depends on diverse progenitor cell types, namely apical radial glia, intermediate progenitors, and basal radial glia cells, which are responsible for the production of the correct neuronal output. In recent years, these progenitor cell types have been deeply studied, particularly basal radial glia and their role in cortical expansion and gyrification. We review here a broad series of factors that regulate progenitor behavior and daughter cell fate. We first describe the different neuronal progenitor types, emphasizing the differences between lissencephalic and gyrencephalic species. We then review key factors shown to influence progenitor proliferation versus differentiation, discussing their roles in progenitor dynamics, neuronal production, and potentially brain size and complexity. Although spindle orientation has been considered a critical factor for mode of division and daughter cell output, we discuss other features that are emerging as crucial for these processes such as organelle and cell cycle dynamics. Additionally, we highlight the importance of adhesion molecules and the polarity complex for correct cortical development. Finally, we briefly discuss studies assessing progenitor multipotency and its possible contribution to the production of specific neuronal populations. This review hence summarizes recent aspects of cortical progenitor cell biology, and pinpoints emerging features critical for their behavior.

Oxygen Levels Regulate the Development of Human Cortical Radial Glia Cells.

The oxygen (O2) concentration is a vital parameter for controlling the survival, proliferation, and differentiation of neural stem cells. A prenatal reduction of O2 levels (hypoxia) often leads to cognitive and behavioral defects, attributable to altered neural development. In this study, we analyzed the effects of O2 levels on human cortical progenitors, the radial glia cells (RGCs), during active neurogenesis, corresponding to the second trimester of gestation. Small changes in O2 levels profoundly affected RGC survival, proliferation, and differentiation. Physiological hypoxia (3% O2) promoted neurogenesis, whereas anoxia (<1% O2) and severe hypoxia (1% O2) arrested the differentiation of human RGCs, mainly by altering the generation of glutamatergic neurons. The in vitro activation of Wnt-ß-catenin signaling rescued the proliferation and neuronal differentiation of RGCs subjected to anoxia. Pathologic hypoxia (≤1% O2) also exerted negative effects on gliogenesis, by decreasing the number of O4+ preoligodendrocytes and increasing the number of reactive astrocytes derived from cortical RGCs. O2-dependent alterations in glutamatergic neurogenesis and oligodendrogenesis can lead to significant changes in cortical circuitry formation. A better understanding of the cellular effects caused by changes in O2 levels during human cortical development is essential to elucidating the etiology of numerous neurodevelopmental disorders.

Pleiotropic Hes-1 Concomitant with its Differential Activation Mediates Neural Stem Cell Maintenance and Radial Glial Propensity in Developing Neocortex.

Notch signaling pathway and its downstream effector Hes-1 are well known for their role in cortical neurogenesis. Despite the canonical activation of Hes-1 in developing neocortex, recent advances have laid considerable emphasis on Notch/CBF1-independent Hes-1 (NIHes-1) expression with poor understanding of its existence and functional significance. Here, using reporter systems and in utero electroporation, we could qualitatively unravel the existence of NIHes-1 expressing neural stem cells from the cohort of dependent progenitors throughout the mouse neocortical development. Though Hes-1 expression is maintained in neural progenitor territory at all times, a simple shift from Notch-independent to -dependent state makes it pleiotropic as the former maintains the neural stem cells in a non-dividing/slow-dividing state, whereas the latter is very much required for maintenance and proliferation of radial glial cells. Therefore, our results provide an additional complexity in neural progenitor heterogeneity regarding differential Hes-1 expression in the germinal zone during neo-cortical development.

Lhx2 regulates the timing of ß-catenin-dependent cortical neurogenesis.

The timing of cortical neurogenesis has a major effect on the size and organization of the mature cortex. The deletion of the LIM-homeodomain transcription factor Lhx2 in cortical progenitors by Nestin-cre leads to a dramatically smaller cortex. Here we report that Lhx2 regulates the cortex size by maintaining the cortical progenitor proliferation and delaying the initiation of neurogenesis. The loss of Lhx2 in cortical progenitors results in precocious radial glia differentiation and a temporal shift of cortical neurogenesis. We further investigated the underlying mechanisms at play and demonstrated that in the absence of Lhx2, the Wnt/ß-catenin pathway failed to maintain progenitor proliferation. We developed and applied a mathematical model that reveals how precocious neurogenesis affected cortical surface and thickness. Thus, we concluded that Lhx2 is required for ß-catenin function in maintaining cortical progenitor proliferation and controls the timing of cortical neurogenesis.

Integrin αvß3 and thyroid hormones promote expansion of progenitors in embryonic neocortex.

Neocortex expansion during evolution is associated with the enlargement of the embryonic subventricular zone, which reflects an increased self-renewal and proliferation of basal progenitors. In contrast to human, the vast majority of mouse basal progenitors lack self-renewal capacity, possibly due to lack of a basal process contacting the basal lamina and downregulation of cell-autonomous production of extracellular matrix (ECM) constituents. Here we show that targeted activation of the ECM receptor integrin αvß3 on basal progenitors in embryonic mouse neocortex promotes their expansion. Specifically, integrin αvß3 activation causes an increased cell cycle re-entry of Pax6-negative, Tbr2-positive intermediate progenitors, rather than basal radial glia, and a decrease in the proportion of intermediate progenitors committed to neurogenic division. Interestingly, integrin αvß3 is the only known cell surface receptor for thyroid hormones. Remarkably, tetrac, a thyroid hormone analog that inhibits the binding of thyroid hormones to integrin αvß3, completely abolishes the intermediate progenitor expansion observed upon targeted integrin αvß3 activation, indicating that this expansion requires the binding of thyroid hormones to integrin αvß3. Convergence of ECM and thyroid hormones on integrin αvß3 thus appears to be crucial for cortical progenitor proliferation and self-renewal, and hence for normal brain development and the evolutionary expansion of the neocortex.

Altered cholesterol biosynthesis causes precocious neurogenesis in the developing mouse forebrain.

We previously reported a mutation in the cholesterol biosynthesis gene, hydroxysteroid (17-beta) dehydrogenase 7 (Hsd17b7(rudolph)), that results in striking embryonic forebrain dysgenesis. Here we describe abnormal patterns of neuroprogenitor proliferation in the mutant forebrain, namely, a decrease in mitotic cells within the ventricular zone (VZ) and an increase through the remainder of the cortex by E11.5. Further evidence suggests mutant cells undergo abnormal interkinetic nuclear migration (IKNM). Furthermore, intermediate progenitors are increased at the expense of apical progenitors by E12.5, and post-mitotic neurons are expanded by E14.5. In vitro primary neuron culture further supports our model of accelerated cortical differentiation in the mutant. Combined administration of a statin and dietary cholesterol in utero achieved partial reversal of multiple developmental abnormalities in the Hsd17b7(rudolph) embryo, including the forebrain. These results suggest that abnormally increased levels of specific cholesterol precursors in the Hsd17b7(rudolph) embryo cause cortical dysgenesis by altering patterns of neurogenesis.

Mutant huntingtin affects cortical progenitor cell division and development of the mouse neocortex.

A polyglutamine expansion in huntingtin (HTT) causes the specific death of adult neurons in Huntington's disease (HD). Most studies have thus focused on mutant HTT (mHTT) toxicity in adulthood, and its developmental effects have been largely overlooked. We found that mHTT caused mitotic spindle misorientation in cultured cells by altering the localization of dynein, NuMA, and the p150(Glued) subunit of dynactin to the spindle pole and cell cortex and of CLIP170 and p150(Glued) to microtubule plus-ends. mHTT also affected spindle orientation in dividing mouse cortical progenitors, altering the thickness of the developing cortex. The serine/threonine kinase Akt, which regulates HTT function, rescued the spindle misorientation caused by the mHTT, by serine 421 (S421) phosphorylation, in cultured cells and in mice. Thus, cortical development is affected in HD, and this early defect can be rescued by HTT phosphorylation at S421.

Role for Lhx2 in corticogenesis through regulation of progenitor differentiation.

The neocortex represents the brain region that has undergone a major increase in its relative size during the course of mammalian evolution. The larger cortex results from a corresponding increase in progenitor cell number. The progenitors giving rise to neocortex are located in the ventricular zone of the dorsal telencephalon and highly express Lhx2, a LIM-homeodomain transcription factor. The neocortex fails to form in the Lhx2 constitutive knockout, indicating a role for Lhx2 in corticogenesis, but mid-embryonic lethality of the Lhx2 knockout requires the use of conditional strategies for further studies. Therefore, to explore Lhx2 function in neocortical progenitors, we generated mice with Lhx2 conditionally deleted from cortical progenitors at the onset of neurogenesis. We find that Lhx2 is critical for maintaining the proliferative state of neocortical progenitors during corticogenesis. In the conditional knockouts, the neocortex is formed but is significantly smaller than wild type. We find that deletion of Lhx2 leads to significantly decreased numbers of cortical progenitors and premature neuronal differentiation. A likely mechanism is indicated by our findings that Lhx2 is required for the expression of Hes1 in cortical progenitors, a key effector in the Notch signaling pathway that maintains the proliferative progenitor state. We conclude that Lhx2 regulates the balance between proliferation and differentiation in cortical progenitors and through this mechanism Lhx2 controls cortical size.

BMP7 expression in mammalian cortical radial glial cells increases the length of the neurogenic period.

The seat of human intelligence is the human cerebral cortex, which is responsible for our exceptional cognitive abilities. Identifying principles that lead to the development of the large-sized human cerebral cortex will shed light on what makes the human brain and species so special. The remarkable increase in the number of human cortical pyramidal neurons and the size of the human cerebral cortex is mainly because human cortical radial glial cells, primary neural stem cells in the cortex, generate cortical pyramidal neurons for more than 130 days, whereas the same process takes only about 7 days in mice. The molecular mechanisms underlying this difference are largely unknown. Here, we found that bone morphogenic protein 7 (BMP7) is expressed by increasing the number of cortical radial glial cells during mammalian evolution (mouse, ferret, monkey, and human). BMP7 expression in cortical radial glial cells promotes neurogenesis, inhibits gliogenesis, and thereby increases the length of the neurogenic period, whereas Sonic Hedgehog (SHH) signaling promotes cortical gliogenesis. We demonstrate that BMP7 signaling and SHH signaling mutually inhibit each other through regulation of GLI3 repressor formation. We propose that BMP7 drives the evolutionary expansion of the mammalian cortex by increasing the length of the neurogenic period.

The Principle of Cortical Development and Evolution.

Human's robust cognitive abilities, including creativity and language, are made possible, at least in large part, by evolutionary changes made to the cerebral cortex. This paper reviews the biology and evolution of mammalian cortical radial glial cells (primary neural stem cells) and introduces the concept that a genetically step wise process, based on a core molecular pathway already in use, is the evolutionary process that has molded cortical neurogenesis. The core mechanism, which has been identified in our recent studies, is the extracellular signal-regulated kinase (ERK)-bone morphogenic protein 7 (BMP7)-GLI3 repressor form (GLI3R)-sonic hedgehog (SHH) positive feedback loop. Additionally, I propose that the molecular basis for cortical evolutionary dwarfism, exemplified by the lissencephalic mouse which originated from a larger gyrencephalic ancestor, is an increase in SHH signaling in radial glia, that antagonizes ERK-BMP7 signaling. Finally, I propose that: (1) SHH signaling is not a key regulator of primate cortical expansion and folding; (2) human cortical radial glial cells do not generate neocortical interneurons; (3) human-specific genes may not be essential for most cortical expansion. I hope this review assists colleagues in the field, guiding research to address gaps in our understanding of cortical development and evolution.

ESCRT-I protein UBAP1 controls ventricular expansion and cortical neurogenesis via modulating adherens junctions of radial glial cells.

Intricate cerebral cortex formation is orchestrated by the precise behavior and division dynamics of radial glial cells (RGCs). Endocytosis functions in the recycling and remodeling of adherens junctions (AJs) in response to changes in RGC activity and function. Here, we show that conditional disruption of ubiquitin-associated protein 1 (UBAP1), a component of endosomal sorting complex required for transport (ESCRT), causes severe brain dysplasia and prenatal ventriculomegaly. UBAP1 depletion disrupts the AJs and polarity of RGCs, leading to failure of apically directed interkinetic nuclear migration. Accordingly, UBAP1 knockout or knockdown results in reduced proliferation and precocious differentiation of neural progenitor cells. Mechanistically, UBAP1 regulates the expression and surface localization of cell adhesion molecules, and ß-catenin over-expression significantly rescues the phenotypes of Ubap1 knockdown in vivo. Our study reveals a critical physiological role of the ESCRT machinery in cortical neurogenesis by regulating AJs of RGCs.

Persistent Lin28 Expression Impairs Neurite Outgrowth and Cognitive Function in the Developing Mouse Neocortex.

Many neurodevelopmental disorders feature learning and memory difficulties. Regulation of neurite outgrowth during development is critical for neural plasticity and memory function. Here, we show a novel regulator of neurite outgrowth during cortical neurogenesis, Lin28, which is an RNA-binding protein. Persistent Lin28 upregulation by in utero electroporation at E14.5 resulted in neurite underdevelopment during cortical neurogenesis. We also showed that Lin28-overexpressing cells had an attenuated response to excitatory inputs and altered membrane properties including higher input resistance, slower action potential repolarization, and smaller hyperpolarization-activated cation currents, supporting impaired neuronal functionality in Lin28-electroporated mice. When we ameliorated perturbed Lin28 expression by siRNA, Lin28-induced neurite underdevelopment was rescued with reduction of Lin28-downstream molecules, high mobility group AT-Hook 2, and insulin-like growth factor 1 receptor. Finally, Lin28-electroporated mice showed significant memory deficits as assessed by the Morris water maze test. Taken together, these findings demonstrate a new role and the essential requirement of Lin28 in developmental control of neurite outgrowth, which has an impact on synaptic plasticity and spatial memory. These findings suggest that targeting Lin28 may attenuate intellectual disabilities by correction of impaired dendritic complexity, providing a novel therapeutic candidate for treating neurodevelopmental disorders.

Tight junction protein occludin regulates progenitor Self-Renewal and survival in developing cortex.

Occludin (OCLN) mutations cause human microcephaly and cortical malformation. A tight junction component thought absent in neuroepithelium after neural tube closure, OCLN isoform-specific expression extends into corticogenesis. Full-length and truncated isoforms localize to neuroprogenitor centrosomes, but full-length OCLN transiently localizes to plasma membranes while only truncated OCLN continues at centrosomes throughout neurogenesis. Mimicking human mutations, full-length OCLN depletion in mouse and in human CRISPR/Cas9-edited organoids produce early neuronal differentiation, reduced progenitor self-renewal and increased apoptosis. Human neural progenitors were more severely affected, especially outer radial glial cells, which mouse embryonic cortex lacks. Rodent and human mutant progenitors displayed reduced proliferation and prolonged M-phase. OCLN interacted with mitotic spindle regulators, NuMA and RAN, while full-length OCLN loss impaired spindle pole morphology, astral and mitotic microtubule integrity. Thus, early corticogenesis requires full-length OCLN to regulate centrosome organization and dynamics, revealing a novel role for this tight junction protein in early brain development.

Saturated fatty acid alters embryonic cortical neurogenesis through modulation of gene expression in neural stem cells.

A perturbed maternal metabolic environment such as chronically elevated circulating free fatty acids have been shown to affect stem cell fate during embryonic neurogenesis. However, molecular mechanisms behind this are not well defined, especially in human. Here in using directed differentiation of human embryonic stem cells (hESCs) into cortical neurons as model, we show that chronically elevated saturated fatty acid (palmitate) results in decreased proliferation of neural stem cells and increased differentiation into neurons. This phenotype could be due to palmitate mediated increased expression of key genes needed for neuronal differentiation such as EOMES, TBR1, NEUROD1 and RELN and reduced expression of SREBP regulated lipogenic genes at early stages of cortical differentiation. Furthermore, palmitate treatment increased histone acetylation globally and at select gene promoters among affected genes. We also found differential expression of several lncRNAs associated with cellular stress and metabolic diseases in the presence of palmitate including BDNF-AS suggesting the contribution of additional epigenetic regulatory mechanisms. Together, our results show that saturated fatty acid affects developmental neurogenesis through modulation of gene expression and through epigenetic regulatory mechanisms.

The Impact of APP on Alzheimer-like Pathogenesis and Gene Expression in Down Syndrome iPSC-Derived Neurons.

Early-onset Alzheimer disease (AD)-like pathology in Down syndrome is commonly attributed to an increased dosage of the amyloid precursor protein (APP) gene. To test this in an isogenic human model, we deleted the supernumerary copy of the APP gene in trisomic Down syndrome induced pluripotent stem cells or upregulated APP expression in euploid human pluripotent stem cells using CRISPRa. Cortical neuronal differentiation shows that an increased APP gene dosage is responsible for increased ß-amyloid production, altered Aß42/40 ratio, and deposition of the pyroglutamate (E3)-containing amyloid aggregates, but not for several tau-related AD phenotypes or increased apoptosis. Transcriptome comparisons demonstrate that APP has a widespread and temporally modulated impact on neuronal gene expression. Collectively, these data reveal an important role for APP in the amyloidogenic aspects of AD but challenge the idea that increased APP levels are solely responsible for increasing specific phosphorylated forms of tau or enhanced neuronal cell death in Down syndrome-associated AD pathogenesis.

3D Culture Method for Alzheimer's Disease Modeling Reveals Interleukin-4 Rescues Aß42-Induced Loss of Human Neural Stem Cell Plasticity.

Neural stem cells (NSCs) constitute an endogenous reservoir for neurons that could potentially be harnessed for regenerative therapies in disease contexts such as neurodegeneration. However, in Alzheimer's disease (AD), NSCs lose plasticity and thus possible regenerative capacity. We investigate how NSCs lose their plasticity in AD by using starPEG-heparin-based hydrogels to establish a reductionist 3D cell-instructive neuro-microenvironment that promotes the proliferative and neurogenic ability of primary and induced human NSCs. We find that administration of AD-associated Amyloid-ß42 causes classical neuropathology and hampers NSC plasticity by inducing kynurenic acid (KYNA) production. Interleukin-4 restores NSC proliferative and neurogenic ability by suppressing the KYNA-producing enzyme Kynurenine aminotransferase (KAT2), which is upregulated in APP/PS1dE9 mouse model of AD and in postmortem human AD brains. Thus, our culture system enables a reductionist investigation of regulation of human NSC plasticity for the identification of potential therapeutic targets for intervention in AD.

The Histamine H1 Receptor Participates in the Increased Dorsal Telencephalic Neurogenesis in Embryos from Diabetic Rats.

Increased neuron telencephalic differentiation during deep cortical layer formation has been reported in embryos from diabetic mice. Transitory histaminergic neurons within the mesencephalon/rhombencephalon are responsible for fetal histamine synthesis during development, fibers from this system arrives to the frontal and parietal cortex at embryo day (E) 15. Histamine is a neurogenic factor for cortical neural stem cells in vitro through H1 receptor (H1R) which is highly expressed during corticogenesis in rats and mice. Furthermore, in utero administration of an H1R antagonist, chlorpheniramine, decreases the neuron markers microtubuline associated protein 2 (MAP2) and forkhead box protein 2. Interestingly, in the diabetic mouse model of diabetes induced with streptozotocin, an increase in fetal neurogenesis in terms of MAP2 expression in the telencephalon is reported at E11.5. Because of the reported effects on cortical neuron differentiation of maternal diabetes in one hand and of histamine in the other, here the participation of histamine and H1R on the increased dorsal telencephalic neurogenesis was explored. First, the increased neurogenesis in the dorsal telencephalon at E14 in diabetic rats was corroborated by immunohistochemistry and Western blot. Then, changes during corticogenesis in the level of histamine was analyzed by ELISA and in H1R expression by qRT-PCR and Western blot and, finally, we tested H1R participation in the increased dorsal telencephalic neurogenesis by the systemic administration of chlorpheniramine. Our results showed a significant increase of histamine at E14 and in the expression of the receptor at E12. The administration of chlorpheniramine to diabetic rats at E12 prevented the increased expression of ßIII-tubulin and MAP2 mRNAs (neuron markers) and partially reverted the increased level of MAP2 protein at E14, concluding that H1R have an important role in the increased neurogenesis within the dorsal telencephalon of embryos from diabetic rats. This study opens new perspective on the participation of HA and H1R receptor in early corticogenesis in health and disease.