Spatial gene expression analysis of neuroanatomical differences in mouse models.

MRI is a powerful modality to detect neuroanatomical differences that result from mutations and treatments. Knowing which genes drive these differences is important in understanding etiology, but candidate genes are often difficult to identify. We tested whether spatial gene expression data from the Allen Brain Institute can be used to inform us about genes that cause neuroanatomical differences. For many single-gene-mutation mouse models, we found that affected neuroanatomy was not strongly associated with the spatial expression of the altered gene and there are specific caveats for each model. However, among models with significant neuroanatomical differences from their wildtype controls, the mutated genes had preferential spatial expression in affected neuroanatomy. In mice exposed to environmental enrichment, candidate genes could be identified by a genome-wide search for genes with preferential spatial expression in the altered neuroanatomical regions. These candidates have functions related to learning and plasticity. We demonstrate that spatial gene expression of single-genes is a poor predictor of altered neuroanatomy, but altered neuroanatomy can identify candidate genes responsible for neuroanatomical phenotypes.

Engrailed-2 (En2) deletion produces multiple neurodevelopmental defects in monoamine systems, forebrain structures and neurogenesis and behavior.

Many genes involved in brain development have been associated with human neurodevelopmental disorders, but underlying pathophysiological mechanisms remain undefined. Human genetic and mouse behavioral analyses suggest that ENGRAILED-2 (EN2) contributes to neurodevelopmental disorders, especially autism spectrum disorder. In mouse, En2 exhibits dynamic spatiotemporal expression in embryonic mid-hindbrain regions where monoamine neurons emerge. Considering their importance in neuropsychiatric disorders, we characterized monoamine systems in relation to forebrain neurogenesis in En2-knockout (En2-KO) mice. Transmitter levels of serotonin, dopamine and norepinephrine (NE) were dysregulated from Postnatal day 7 (P7) to P21 in En2-KO, though NE exhibited the greatest abnormalities. While NE levels were reduced ∼35% in forebrain, they were increased 40 -: 75% in hindbrain and cerebellum, and these patterns paralleled changes in locus coeruleus (LC) fiber innervation, respectively. Although En2 promoter was active in Embryonic day 14.5 -: 15.5 LC neurons, expression diminished thereafter and gene deletion did not alter brainstem NE neuron numbers. Significantly, in parallel with reduced NE levels, En2-KO forebrain regions exhibited reduced growth, particularly hippocampus, where P21 dentate gyrus granule neurons were decreased 16%, suggesting abnormal neurogenesis. Indeed, hippocampal neurogenic regions showed increased cell death (+77%) and unexpectedly, increased proliferation. Excess proliferation was restricted to early Sox2/Tbr2 progenitors whereas increased apoptosis occurred in differentiating (Dcx) neuroblasts, accompanied by reduced newborn neuron survival. Abnormal neurogenesis may reflect NE deficits because intra-hippocampal injections of ß-adrenergic agonists reversed cell death. These studies suggest that disruption of hindbrain patterning genes can alter monoamine system development and thereby produce forebrain defects that are relevant to human neurodevelopmental disorders.

p57(KIP2) regulates radial glia and intermediate precursor cell cycle dynamics and lower layer neurogenesis in developing cerebral cortex.

During cerebral cortex development, precise control of precursor cell cycle length and cell cycle exit is required for balanced precursor pool expansion and layer-specific neurogenesis. Here, we defined the roles of cyclin-dependent kinase inhibitor (CKI) p57(KIP2), an important regulator of G1 phase, using deletion mutant mice. Mutant mice displayed macroencephaly associated with cortical hyperplasia during late embryogenesis and postnatal development. Embryonically, proliferation of radial glial cells (RGC) and intermediate precursors (IPC) was increased, expanding both populations, with greater effect on IPCs. Furthermore, cell cycle re-entry was increased during early corticogenesis, whereas cell cycle exit was augmented at middle stage. Consequently, neurogenesis was reduced early, whereas it was enhanced during later development. In agreement, the timetable of early neurogenesis, indicated by birthdating analysis, was delayed. Cell cycle dynamics analyses in mutants indicated that p57(KIP2) regulates cell cycle length in both RGCs and IPCs. By contrast, related CKI p27(KIP1) controlled IPC proliferation exclusively. Furthermore, p57(KIP2) deficiency markedly increased RGC and IPC divisions at E14.5, whereas p27(KIP1) increased IPC proliferation at E16.5. Consequently, loss of p57(KIP2) increased primarily layer 5-6 neuron production, whereas loss of p27(KIP1) increased neurons specifically in layers 2-5. In conclusion, our observations suggest that p57(KIP2) and p27(KIP1) control neuronal output for distinct cortical layers by regulating different stages of precursor proliferation, and support a model in which IPCs contribute to both lower and upper layer neuron generation.

Pro- and anti-mitogenic actions of pituitary adenylate cyclase-activating polypeptide in developing cerebral cortex: potential mediation by developmental switch of PAC1 receptor mRNA isoforms.

During corticogenesis, pituitary adenylate cyclase-activating polypeptide (PACAP; ADCYAP1) may contribute to proliferation control by activating PAC1 receptors of neural precursors in the embryonic ventricular zone. PAC1 receptors, specifically the hop and short isoforms, couple differentially to and activate distinct pathways that produce pro- or anti-mitogenic actions. Previously, we found that PACAP was an anti-mitogenic signal from embryonic day 13.5 (E13.5) onward both in culture and in vivo and activated cAMP signaling through the short isoform. However, we now find that mice deficient in PACAP exhibited a decrease in the BrdU labeling index (LI) in E9.5 cortex, suggesting that PACAP normally promotes proliferation at this stage. To further define mechanisms, we established a novel culture model in which the viability of very early cortical precursors (E9.5 mouse and E10.5 rat) could be maintained. At this stage, we found that PACAP evoked intracellular calcium fluxes and increased phospho-PKC levels, as well as stimulated G1 cyclin mRNAs and proteins, S-phase entry, and proliferation without affecting cell survival. Significantly, expression of hop receptor isoform was 24-fold greater than the short isoform at E10.5, a ratio that was reversed at E14.5 when short expression was 15-fold greater and PACAP inhibited mitogenesis. Enhanced hop isoform expression, elicited by in vitro treatment of E10.5 precursors with retinoic acid, correlated with sustained pro-mitogenic action of PACAP beyond the developmental switch. Conversely, depletion of hop receptor using short-hairpin RNA abolished PACAP mitogenic stimulation at E10.5. These observations suggest that PACAP elicits temporally specific effects on cortical proliferation via developmentally regulated expression of specific receptor isoforms.

Dysregulation of mTOR signaling mediates common neurite and migration defects in both idiopathic and 16p11.2 deletion autism neural precursor cells.

Autism spectrum disorder (ASD) is defined by common behavioral characteristics, raising the possibility of shared pathogenic mechanisms. Yet, vast clinical and etiological heterogeneity suggests personalized phenotypes. Surprisingly, our iPSC studies find that six individuals from two distinct ASD subtypes, idiopathic and 16p11.2 deletion, have common reductions in neural precursor cell (NPC) neurite outgrowth and migration even though whole genome sequencing demonstrates no genetic overlap between the datasets. To identify signaling differences that may contribute to these developmental defects, an unbiased phospho-(p)-proteome screen was performed. Surprisingly despite the genetic heterogeneity, hundreds of shared p-peptides were identified between autism subtypes including the mTOR pathway. mTOR signaling alterations were confirmed in all NPCs across both ASD subtypes, and mTOR modulation rescued ASD phenotypes and reproduced autism NPC-associated phenotypes in control NPCs. Thus, our studies demonstrate that genetically distinct ASD subtypes have common defects in neurite outgrowth and migration which are driven by the shared pathogenic mechanism of mTOR signaling dysregulation.

Although the clinical presentation of individuals with autism spectrum disorder (ASD) can vary widely, the core features are repetitive behaviors and difficulties with social interactions and communication. In most cases, the cause of autism is unknown. However, in some cases, such as a form of ASD known as 16p11.2 deletion syndrome, specific genetic changes are responsible. Despite this variability in possible causes and clinical manifestations, the similarity of the core behavioral symptoms across different forms of the disorder indicates that there could be a shared biological mechanism. Furthermore, genetic studies suggest that abnormalities in early fetal brain development could be a crucial underlying cause of ASD. In order to form the complex structure of the brain, fetal brain cells must migrate and start growing extensions that ultimately become key structures of neurons. To test for shared biological mechanisms, Prem et al. reprogrammed blood cells from people with either 16p11.2 deletion syndrome or ASD with an unknown cause to become fetal-like brain cells. Experiments showed that both migration of the cells and their growth of extensions were similarly disrupted in the cells derived from both groups of individuals with autism. These crucial developmental changes were driven by alterations to an important signaling molecule in a pathway involved in brain function, known as the mTOR pathway. However, in some cells the pathway was overactive, whereas in others it was underactive. To probe the potential of the mTOR pathway as a therapeutic target, Prem et al. tested drugs that manipulate the pathway, finding that they could successfully reverse the defects in cells derived from people with both types of ASD. The discovery that a shared biological process may underpin different forms of ASD is important for understanding the early brain changes that are involved. A common target, like the mTOR pathway, could offer hope for treatments for a wide range of ASDs. However, to translate these benefits to the clinic, further research is needed to understand whether a treatment that is effective in fetal cells would also benefit people with autism.

Deletion or activation of the aryl hydrocarbon receptor alters adult hippocampal neurogenesis and contextual fear memory.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxin and serves multiple developmental roles. In the adult brain, while we now localize AhR mRNA to nestin-expressing neural progenitor cells in the dentate gyrus (DG) of the hippocampus, its function is unknown. This study tested the hypothesis that AhR participates in hippocampal neurogenesis and associated functions. AhR deletion and activation by the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), adversely impacted neurogenesis and cognition. Adult AhR-deficient mice exhibited impaired hippocampal-dependent contextual fear memory while hippocampal-independent memory remained intact. AhR-deficient mice displayed reduced cell birth, decreased cell survival, and diminished neuronal differentiation in the DG. Following TCDD exposure, wild-type mice exhibited impaired hippocampal-dependent contextual memory, decreased cell birth, reduced neuronal differentiation, and fewer mature neurons in the DG. Glial differentiation and apoptosis were not altered in either TCDD-exposed or AhR-deficient mice. Finally, defects observed in TCDD-exposed mice were dependent on AhR, as TCDD had no negative effects in AhR-deficient mice. Our findings suggest that AhR should be further evaluated as a potential transcriptional regulator of hippocampal neurogenesis and function, although other sites of action may also warrant consideration. Moreover, TCDD exposure should be considered as an environmental risk factor that disrupts adult neurogenesis and potentially related memory processes.

Cocaine-regulated trafficking of dopamine transporters in cultured neurons revealed by a pH sensitive reporter.

Cocaine acts by inhibiting plasma membrane dopamine transporter (DAT) function and altering its surface expression. The precise manner and mechanism by which cocaine regulates DAT trafficking, especially at neuronal processes, are poorly understood. In this study, we engineered and validated the use of DAT-pHluorin for studying DAT localization and its dynamic trafficking at neuronal processes of cultured mouse midbrain neurons. We demonstrate that unlike neuronal soma and dendrites, which contain a majority of the DATs in weakly acidic intracellular compartments, axonal DATs at both shafts and boutons are primarily (75%) localized to the plasma membrane, whereas large varicosities contain abundant intracellular DAT within acidic intracellular structures. We also demonstrate that cocaine exposure leads to a Synaptojanin1-sensitive DAT internalization process followed by membrane reinsertion that lasts for days. Thus, our study reveals the previously unknown dynamics and molecular regulation for cocaine-regulated DAT trafficking in neuronal processes.

N-acetyl cysteine treatment reduces mercury-induced neurotoxicity in the developing rat hippocampus.

Mercury is an environmental toxicant that can disrupt brain development. However, although progress has been made in defining its neurotoxic effects, we know far less about available therapies that can effectively protect the brain in exposed individuals. We previously developed an animal model in which we defined the sequence of events underlying neurotoxicity: Methylmercury (MeHg) injection in postnatal rat acutely induced inhibition of mitosis and stimulated apoptosis in the hippocampus, which later resulted in intermediate-term deficits in structure size and cell number. N-acetyl cysteine (NAC) is the N-acetyl derivative of L-cysteine used clinically for treatment of drug intoxication. Here, based on its known efficacy in promoting MeHg urinary excretion, we evaluated NAC for protective effects in the developing brain. In immature neurons and precursors, MeHg (3 µM) induced a >50% decrease in DNA synthesis at 24 hr, an effect that was completely blocked by NAC coincubation. In vivo, injection of MeHg (5 µg/g bw) into 7-day-old rats induced a 22% decrease in DNA synthesis in whole hippocampus and a fourfold increase in activated caspase-3-immunoreactive cells at 24 hr and reduced total cell numbers by 13% at 3 weeks. Treatment of MeHg-exposed rats with repeated injections of NAC abolished MeHg toxicity. NAC prevented the reduction in DNA synthesis and the marked increase in caspase-3 immunoreactivity. Moreover, the intermediate-term decrease in hippocampal cell number provoked by MeHg was fully blocked by NAC. Altogether these results suggest that MeHg toxicity in the perinatal brain can be ameliorated by using NAC, opening potential avenues for therapeutic intervention.

The cyclin-dependent kinase inhibitor p57Kip2 regulates cell cycle exit, differentiation, and migration of embryonic cerebral cortical precursors.

Mounting evidence indicates cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family, including p57(Kip2) and p27(Kip1), control not only cell cycle exit but also corticogenesis. Nevertheless, distinct activities of p57(Kip2) remain poorly defined. Using in vivo and culture approaches, we show p57(Kip2) overexpression at E14.5-15.5 elicits precursor cell cycle exit, promotes transition from proliferation to neuronal differentiation, and enhances process outgrowth, while opposite effects occur in p57(Kip2)-deficient precursors. Studies at later ages indicate p57(Kip2) overexpression also induces precocious glial differentiation, suggesting stage-dependent effects. In embryonic cortex, p57(Kip2) overexpression advances cell radial migration and alters postnatal laminar positioning. While both CKIs induce differentiation, p57(Kip2) was twice as effective as p27(Kip1) in inducing neuronal differentiation and was not permissive to astrogliogenic effects of ciliary neurotrophic factor, suggesting that the CKIs differentially modulate cell fate decisions. At molecular levels, although highly conserved N-terminal regions of both CKIs elicit cycle withdrawal and differentiation, the C-terminal region of p57(Kip2) alone inhibits in vivo migration. Furthermore, p57(Kip2) effects on neurogenesis and gliogenesis require the N-terminal cyclin/CDK binding/inhibitory domains, while previous p27(Kip1) studies report cell cycle-independent functions. These observations suggest p57(Kip2) coordinates multiple stages of corticogenesis and exhibits distinct and common activities compared with related family member p27(Kip1).

Exposure to deltamethrin at the NOAEL causes ER stress and disruption of hippocampal neurogenesis in adult mice.

In addition to age and traumatic brain injury, environmental exposure to pesticides is a potential risk factor for neurodegenerative diseases and cognitive impairments in humans. Deltamethrin is a type II pyrethroid insecticide widely used in agriculture and homes for pest control. Previously, we reported that repeated exposure of mice to 3 mg/kg deltamethrin for 30 or 60 days caused a marked increase in the endoplasmic reticulum (ER) stress and reduced adult hippocampal neurogenesis that was accompanied by impaired learning and memory. However, it is unknown whether an acute exposure to low doses of deltamethrin elicits similar effects. Here, we sought to characterize the dose-related effects of deltamethrin on ER stress and hippocampal neurogenesis at different time points following acute exposure. Following oral administration of 0, 0.3, 1, or 3 mg/kg deltamethrin, doses below, at, and above the acute NOAEL, mice were euthanized at 24 h, 48 h, 7 d, or 14 d to assess the acute and intermediate-term effects of deltamethrin on neural progenitor cells (NPCs). Deltamethrin at both 1 and 3 mg/kg elicited ER stress response and activation of apoptotic signaling. Data revealed that a dose as low as 1 mg/kg of deltamethrin, considered the acute NOAEL, produced a significant reduction in BrdU+ and Ki-67+ neural stem cells in the subgranular zone of the dentate gyrus of the hippocampus as early as 48 h after exposure. Furthermore, mice treated with 1 and 3 mg/kg deltamethrin exhibited a decreased number of immature neurons, determined by counting DCX-positive cells 7 days after exposure. These data establish that 0.3 mg/kg should be considered a NOAEL and that the previously established acute NOAEL of 1 mg/kg shows significant effects on ER stress and apoptotic pathways accompanied by deficits in aspects of adult hippocampal neurogenesis.

Autism NPCs from both idiopathic and CNV 16p11.2 deletion patients exhibit dysregulation of proliferation and mitogenic responses.

Neural precursor cell (NPC) dysfunction has been consistently implicated in autism. Induced pluripotent stem cell (iPSC)-derived NPCs from two autism groups (three idiopathic [I-ASD] and two 16p11.2 deletion [16pDel]) were used to investigate if proliferation is commonly disrupted. All five individuals display defects, with all three macrocephalic individuals (two 16pDel, one I-ASD) exhibiting hyperproliferation and the other two I-ASD subjects displaying hypoproliferation. NPCs were challenged with bFGF, and all hyperproliferative NPCs displayed blunted responses, while responses were increased in hypoproliferative cells. mRNA expression studies suggest that different pathways can result in similar proliferation phenotypes. Since 16pDel deletes MAPK3, P-ERK was measured. P-ERK is decreased in hyperproliferative but increased in hypoproliferative NPCs. While these P-ERK changes are not responsible for the phenotypes, P-ERK and bFGF response are inversely correlated with the defects. Finally, we analyzed iPSCs and discovered that 16pDel displays hyperproliferation, while idiopathic iPSCs were normal. These data suggest that NPC proliferation defects are common in ASD.

Engrailed-2 is a cell autonomous regulator of neurogenesis in cultured hippocampal neural stem cells.

Abnormalities in genes that regulate early brain development are known risk factors for neurodevelopmental disorders. Engrailed-2 (En2) is a homeodomain transcription factor with established roles in cerebellar patterning. En2 is highly expressed in the developing mid-hindbrain region, and En2 knockout (KO) mice exhibit major deficits in mid-hindbrain structures. However, En2 is also expressed in forebrain regions including the hippocampus, but its function is unknown. Previous studies have shown that the hippocampus of En2-KO mice exhibits reductions in its volume and cell numbers due to aberrant neurogenesis. Aberrant neurogenesis is due, in part, to noncell autonomous effects, specifically, reductions of innervating norepinephrine fibers from the locus coeruleus. In this study, we investigate possible cell autonomous roles of En2 in hippocampal neurogenesis. We examine proliferation, survival, and differentiation using cultures of hippocampal neurospheres of P7 wild-type (WT) and En2-KO hippocampal neural progenitor cells (NPCs). At 7 days, En2-KO neurospheres were larger on average than WT spheres and exhibited 2.5-fold greater proliferation and 2-fold increase in apoptotic cells, similar to in vivo KO phenotype. Further, En2-KO cultures exhibited 40% less cells with neurite projections, suggesting decreased differentiation. Lastly, reestablishing En2 expression in En2-KO NPCs rescued excess proliferation. These results indicate that En2 functions in hippocampal NPCs by inhibiting proliferation and promoting survival and differentiation in a cell autonomous manner. More broadly, this study suggests that En2 impacts brain structure and function in diverse regions outside of the mid-hindbrain.

Developmental Role of Adenosine Kinase in the Cerebellum.

Adenosine acts as a neuromodulator and metabolic regulator of the brain through receptor dependent and independent mechanisms. In the brain, adenosine is tightly controlled through its metabolic enzyme adenosine kinase (ADK), which exists in a cytoplasmic (ADK-S) and nuclear (ADK-L) isoform. We recently discovered that ADK-L contributes to adult hippocampal neurogenesis regulation. Although the cerebellum (CB) is a highly plastic brain area with a delayed developmental trajectory, little is known about the role of ADK. Here, we investigated the developmental profile of ADK expression in C57BL/6 mice CB and assessed its role in developmental and proliferative processes. We found high levels of ADK-L during cerebellar development, which was maintained into adulthood. This pattern contrasts with that of the cerebrum, in which ADK-L expression is gradually downregulated postnatally and largely restricted to astrocytes in adulthood. Supporting a functional role in cell proliferation, we found that the ADK inhibitor 5-iodotubericine (5-ITU) reduced DNA synthesis of granular neuron precursors in a concentration-dependent manner in vitro In the developing CB, immunohistochemical studies indicated ADK-L is expressed in immature Purkinje cells and granular neuron precursors, whereas in adulthood, ADK is absent from Purkinje cells, but widely expressed in mature granule neurons and their molecular layer (ML) processes. Furthermore, ADK-L is expressed in developing and mature Bergmann glia in the Purkinje cell layer, and in astrocytes in major cerebellar cortical layers. Together, our data demonstrate an association between neuronal ADK expression and developmental processes of the CB, which supports a functional role of ADK-L in the plasticity of the CB.

Engrailed 2 deficiency and chronic stress alter avoidance and motivation behaviors.

Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder characterized by impairments in social interaction, cognition, and communication, as well as the presence of repetitive or stereotyped behaviors and interests. ASD is most often studied as a neurodevelopmental disease, but it is a lifelong disorder. Adults with ASD experience more stressful life events and greater perceived stress, and frequently have comorbid mood disorders such as anxiety and depression. It remains unclear whether adult exposure to chronic stress can exacerbate the behavioral and neurodevelopmental phenotypes associated with ASD. To address this issue, we first investigated whether adult male and female Engrailed-2 deficient (En2-KO, En2-/-) mice, which display behavioral disturbances in avoidance tasks and dysregulated monoaminergic neurotransmitter levels, also display impairments in instrumental behaviors associated with motivation, such as the progressive ratio task. We then exposed adult En2-KO mice to chronic environmental stress (CSDS, chronic social defeat stress), to determine if stress exacerbated the behavioral and neuroanatomical effects of En2 deletion. En2-/- mice showed impaired instrumental acquisition and significantly lower breakpoints in a progressive ratio test, demonstrating En2 deficiency decreases motivation to exert effort for reward. Furthermore, adult CSDS exposure increased avoidance behaviors in En2-KO mice. Interestingly, adult CSDS exposure also exacerbated the deleterious effects of En2 deficiency on forebrain-projecting monoaminergic fibers. Our findings thus suggest that adult exposure to stress may exacerbate behavioral and neuroanatomical phenotypes associated with developmental effects of genetic En2 deficiency.

Insulin-like growth factor-1 promotes G(1)/S cell cycle progression through bidirectional regulation of cyclins and cyclin-dependent kinase inhibitors via the phosphatidylinositol 3-kinase/Akt pathway in developing rat cerebral cortex.

Although survival-promoting effects of insulin-like growth factor-1 (IGF-1) during neurogenesis are well characterized, mitogenic effects remain less well substantiated. Here, we characterize cell cycle regulators and signaling pathways underlying IGF-1 effects on embryonic cortical precursor proliferation in vitro and in vivo. In vitro, IGF-1 stimulated cell cycle progression and increased cell number without promoting cell survival. IGF-1 induced rapid increases in cyclin D1 and D3 protein levels at 4 h and cyclin E at 8 h. Moreover, p27(KIP1) and p57(KIP2) expression were reduced, suggesting downregulation of negative regulators contributes to mitogenesis. Furthermore, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway specifically underlies IGF-1 activity, because blocking this pathway, but not MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal-regulated kinase), prevented mitogenesis. To determine whether mechanisms defined in culture relate to corticogenesis in vivo, we performed transuterine intracerebroventricular injections. Whereas blockade of endogenous factor with anti-IGF-1 antibody decreased DNA synthesis, IGF-1 injection stimulated DNA synthesis and increased the number of S-phase cells in the ventricular zone. IGF-1 treatment increased phospho-Akt fourfold at 30 min, cyclins D1 and E by 6 h, and decreased p27(KIP1) and p57(KIP2) expression. Moreover, blockade of the PI3K/Akt pathway in vivo decreased DNA synthesis and cyclin E, increased p27(KIP1) and p57(KIP2) expression, and prevented IGF-1-induced cyclin E mRNA upregulation. Finally, IGF-1 injection in embryos increased postnatal day 10 brain DNA content by 28%, suggesting a role for IGF-1 in brain growth control. These results demonstrate a mitogenic role for IGF-1 that tightly controls both positive and negative cell cycle regulators, and indicate that the PI3K/Akt pathway mediates IGF-1 mitogenic signaling during corticogenesis.

Dysregulation of Neurite Outgrowth and Cell Migration in Autism and Other Neurodevelopmental Disorders.

Despite decades of study, elucidation of the underlying etiology of complex developmental disorders such as autism spectrum disorder (ASD), schizophrenia (SCZ), intellectual disability (ID), and bipolar disorder (BPD) has been hampered by the inability to study human neurons, the heterogeneity of these disorders, and the relevance of animal model systems. Moreover, a majority of these developmental disorders have multifactorial or idiopathic (unknown) causes making them difficult to model using traditional methods of genetic alteration. Examination of the brains of individuals with ASD and other developmental disorders in both post-mortem and MRI studies shows defects that are suggestive of dysregulation of embryonic and early postnatal development. For ASD, more recent genetic studies have also suggested that risk genes largely converge upon the developing human cerebral cortex between weeks 8 and 24 in utero. Yet, an overwhelming majority of studies in autism rodent models have focused on postnatal development or adult synaptic transmission defects in autism related circuits. Thus, studies looking at early developmental processes such as proliferation, cell migration, and early differentiation, which are essential to build the brain, are largely lacking. Yet, interestingly, a few studies that did assess early neurodevelopment found that alterations in brain structure and function associated with neurodevelopmental disorders (NDDs) begin as early as the initial formation and patterning of the neural tube. By the early to mid-2000s, the derivation of human embryonic stem cells (hESCs) and later induced pluripotent stem cells (iPSCs) allowed us to study living human neural cells in culture for the first time. Specifically, iPSCs gave us the unprecedented ability to study cells derived from individuals with idiopathic disorders. Studies indicate that iPSC-derived neural cells, whether precursors or "matured" neurons, largely resemble cortical cells of embryonic humans from weeks 8 to 24. Thus, these cells are an excellent model to study early human neurodevelopment, particularly in the context of genetically complex diseases. Indeed, since 2011, numerous studies have assessed developmental phenotypes in neurons derived from individuals with both genetic and idiopathic forms of ASD and other NDDs. However, while iPSC-derived neurons are fetal in nature, they are post-mitotic and thus cannot be used to study developmental processes that occur before terminal differentiation. Moreover, it is important to note that during the 8-24-week window of human neurodevelopment, neural precursor cells are actively undergoing proliferation, migration, and early differentiation to form the basic cytoarchitecture of the brain. Thus, by studying NPCs specifically, we could gain insight into how early neurodevelopmental processes contribute to the pathogenesis of NDDs. Indeed, a few studies have explored NPC phenotypes in NDDs and have uncovered dysregulations in cell proliferation. Yet, few studies have explored migration and early differentiation phenotypes of NPCs in NDDs. In this chapter, we will discuss cell migration and neurite outgrowth and the role of these processes in neurodevelopment and NDDs. We will begin by reviewing the processes that are important in early neurodevelopment and early cortical development. We will then delve into the roles of neurite outgrowth and cell migration in the formation of the brain and how errors in these processes affect brain development. We also provide review of a few key molecules that are involved in the regulation of neurite outgrowth and migration while discussing how dysregulations in these molecules can lead to abnormalities in brain structure and function thereby highlighting their contribution to pathogenesis of NDDs. Then we will discuss whether neurite outgrowth, migration, and the molecules that regulate these processes are associated with ASD. Lastly, we will review the utility of iPSCs in modeling NDDs and discuss future goals for the study of NDDs using this technology.

Using iPSC-Based Models to Understand the Signaling and Cellular Phenotypes in Idiopathic Autism and 16p11.2 Derived Neurons.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that is remarkably heterogeneous at the clinical, neurobiological, and genetic levels. ASD can also affect language, a uniquely human capability, and is caused by abnormalities in brain development. Traditionally obtaining biologically relevant human cells to study ASD has been extremely difficult, but new technologies including iPSC-derived neurons and high-throughput omic techniques now provide new, exciting tools to uncover the cellular and signaling basis of ASD etiology.

Granule cell survival is deficient in PAC1-/- mutant cerebellum.

PACAP exerts neuroprotective effects during development, especially in the cerebellum where PAC1 receptor and ligand are both expressed. However, while previous studies using PACAP injections in postnatal animals defined trophic effects of exogenous peptide, the role of endogenous PACAP remains unexplored. Here, we used PAC1(-/-) mice to investigate the role of PACAP receptor signaling in postnatal day 7 cerebellum. There was no difference in DNA synthesis in the cerebellar EGL of PAC1(-/-) compared to wild type animals, assessed using thymidine incorporation and BrdU immunohistochemistry. In contrast, we found that a significant proportion of newly generated neurons were eliminated before they successfully differentiated in the granule cell layer. In aggregate, these results suggest that endogenous PACAP plays an important role in cell survival during cerebellar development, through the activation of the PAC1 receptor.

Altered salt taste response and increased tongue epithelium Scnna1 expression in adult Engrailed-2 null mice.

Sensory impairments are critical for diagnosing and characterizing neurodevelopmental disorders. Taste is a sensory modality often not well characterized. Engrailed-2 (En2) is a transcription factor critical for neural development, and mice lacking En2 (En2-/-) display signs of impaired social interaction, cognitive processes (e.g., learning and memory, conditioned fear), and neurodevelopmental alterations. As such, En2-/- mice display the behavioral deficits and neural impairments characteristic of the core symptoms associated with autism spectrum disorder (ASD). The objective of this study was to characterize the taste function in En2-/- compared with En2+/+ in adult male mice. Measuring taste responsiveness by an automated gustometer, En2 null mice had decreased lick responses for 1.6 M fructose, whereas they demonstrated an increased taste responsivity (i.e., relative to water) at 0.3 M sodium chloride and 1 M monosodium glutamate. In a separate cohort of mice, En2-/- mice had an increased preference for sodium chloride over a range of concentrations (0.032-0.3 M) compared with En2+/+ mice. Regional gene expression of the tongue epithelium demonstrated an increase in Scnn1a, T2R140, T1R3, and Trpm5 and a decrease in Pkd1l3 in En2 null mice. Taken together, such data indicate that deficits in En2 can produce sensory impairments that can have a measurable impact on taste, particularly salt taste.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs).

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.