Human Neurodevelopmental Disorders Research Articles

Novel compound heterozygous variants in the STIL gene identified in a Chinese family with presentation of foetal microcephaly

Primary microcephaly 7 (MCPH7) is an autosomal recessive human neurodevelopmental disorder characterized by microcephaly, sloping forehead, and prominent midface. The STIL gene encodes a protein that regulates the mitotic spindle checkpoint. STIL is the pathogenic gene of MCPH7. Although more than 25 genes have been reported to cause MCPH, many patients lack a molecular diagnosis. The clinical manifestations and genetic factors of MCPH7 remain to be revealed. This research reported two consecutive microcephalic foetuses from unaffected parents. Prenatal ultrasound examination and pre- and postnatal MRI studies were performed. Whole-genome sequencing (WGS) was performed using blood derived from the umbilical cord, and variants were confirmed by Sanger sequencing on the parents. Ultrasound examination showed that the two foetuses suffered primary microcephaly. Using the WGS approach, novel compound heterozygous variants in STIL (c.2344_2347delTTGC, p. Leu782Thrfs*2 in exon 13; c.3838C > T, p. Arg1280Cys in exon 17) were identified in two foetuses with MCPH7. The MRI results of the two siblings were quite similar. Postnatal MRI confirmed the ultrasound and prenatal examinations. The two foetuses had typical microcephaly. Ultrasound and MRI showed that the two foetuses had a thick skull plate, significantly reduced bilateral frontal lobe, upward rotated cerebellum vermis, and dilated fourth ventricle. Our findings have important implications for prenatal diagnosis and genetic counselling for any patients with MCPH7. We extend both the mutational spectrum in the STIL gene and the clinical spectrum of MCPH7.

Synthetic female gonadal hormones alter neurodevelopmental programming and behavior in F1 offspring

The increased prevalence of neurodevelopmental disorders during the last half-century led us to investigate the potential for intergenerational detrimental neurodevelopmental effects of synthetic female gonadal hormones, typically used in contraceptive pills. We examined 3 separate cohorts of mice over the span of 2 years, a total of 150 female F0 mice and over 300 male and female rodents from their F1 progeny. We demonstrate that F1 male offsprings of female mice previously exposed to the synthetic estrogen 17α-ethinylestradiol (EE2) in combination with the synthetic progestin Norethindrone, exhibit neurodevelopmental and behavioral differences compared to control mice. Because the EE2 + Norethindrone administration resulted in gene expression changes in the exposed F0 mice ovaries persisting after the end of treatment, it is likely that the synthetic hormone treatment caused changes in the germline cells and that led to altered neurodevelopment in the offsprings. An altered gene expression pattern was discovered in the frontal cortex of male mice from the first offspring (F1.1) at infancy and an ADHD-like hyperactive locomotor behavior was exhibited in young male mice from the second offspring (F1.2) of female mice treated with contraceptive pill doses of EE2 + Norethindrone prior to pregnancy. The intergenerational neurodevelopmental effects of EE2 + Norethindrone treatment were sex specific, predominantly affecting males. Our observations in mice support the hypothesis that the use of synthetic contraceptive hormones is a potential environmental factor impacting the prevalence of human neurodevelopmental disorders. Additionally, our results indicate that contraceptive hormone drug safety assessments may need to be extended to F1 offspring.

Lasting and Sex-Dependent Impact of Maternal Immune Activation on Molecular Pathways of the Amygdala.

The prolonged and sex-dependent impact of maternal immune activation (MIA) during gestation on the molecular pathways of the amygdala, a brain region that influences social, emotional, and other behaviors, is only partially understood. To address this gap, we investigated the effects of viral-elicited MIA during gestation on the amygdala transcriptome of pigs, a species of high molecular and developmental homology to humans. Gene expression levels were measured using RNA-Seq on the amygdala for 3-week-old female and male offspring from MIA and control groups. Among the 403 genes that exhibited significant MIA effect, a prevalence of differentially expressed genes annotated to the neuroactive ligand–receptor pathway, glutamatergic functions, neuropeptide systems, and cilium morphogenesis were uncovered. Genes in these categories included corticotropin-releasing hormone receptor 2, glutamate metabotropic receptor 4, glycoprotein hormones, alpha polypeptide, parathyroid hormone 1 receptor, vasointestinal peptide receptor 2, neurotensin, proenkephalin, and gastrin-releasing peptide. These categories and genes have been associated with the MIA-related human neurodevelopmental disorders, including schizophrenia and autism spectrum disorders. Gene network reconstruction highlighted differential vulnerability to MIA effects between sexes. Our results advance the understanding necessary for the development of multifactorial therapies targeting immune modulation and neurochemical dysfunction that can ameliorate the effects of MIA on offspring behavior later in life.

Acrylamide exposure represses neuronal differentiation, induces cell apoptosis and promotes tau hyperphosphorylation in hESC-derived 3D cerebral organoids

Acrylamide (ACR) is a common food contaminant with neurotoxic effects that are formed in the Maillard browning reaction during the heat processing of food. Importantly, pregnant women are also exposed to ACR in food during pregnancy and thus, the fetus is likely affected. However, the mechanisms of ACR-caused neurotoxicity on human brain development are still unclear. Many recent studies employed cerebral organoids based on human embryonic stem cells (hESC) for investigating human neurodevelopmental disorders and toxicity. Here, we generated hESC-derived cerebral organoids to evaluate the neurodevelopmental toxicity of ACR. The results indicated that exposure to ACR significantly altered the transcriptional profile, increased nuclear factor erythroid 2-related factor 2 (NRF2)-mediated gene expression, induced cell apoptosis, repressed neuronal differentiation, and promoted tau hyperphosphorylation in cerebral organoids, which may contribute to ACR-induced neurodevelopmental toxicity. These results indicate that the risk of transplacental exposure of the fetus to ACR should be evaluated and pregnant mothers should limit their exposure to ACR.

General population ZBTB18 missense variants influence DNA binding and transcriptional regulation.

Genetic variation of the multi-zinc finger BTB domain transcription factor ZBTB18 can cause a spectrum of human neurodevelopmental disorders, but the underlying mechanisms are not well understood. Recently, we reported that pathogenic, de novo ZBTB18 missense mutations alter its DNA-binding specificity and gene regulatory functions, leading to human neurodevelopmental disease. However, the functional impact of the general population ZBTB18 missense variants is unknown. Here, we investigated such variants documented in the Genome Aggregation Database (gnomAD) to discover that ZBTB gene family members are intolerant to loss-of-function and missense mutations, but not synonymous mutations. We studied ZBTB18 as a protein-DNA complex to find that general population missense variants are rare, and disproportionately map to non-DNA-contact residues, in contrast to the majority of disease-associated variants that map to DNA-contact residues, essential to motif binding. We studied a selection of variants (n = 12), which spans the multi-zinc finger region to find 58.3% (7/12) of variants displayed altered DNA binding, 41.6% (5/12) exhibited altered transcriptional activity in a luciferase reporter assay, 33.3% (4/12) exhibited altered DNA binding and transcriptional activity, whereas 33.3% (4/12) displayed a negligible functional impact. Our results demonstrate that general population variants, while rare, can influence ZBTB18 function, with potential consequences for neurodevelopment, homeostasis, and disease.

Aberrant sorting of hippocampal complex pyramidal cells in type I lissencephaly alters topological innervation.

Layering has been a long-appreciated feature of higher order mammalian brain structures but the extent to which it plays an instructive role in synaptic specification remains unknown. Here we examine the formation of synaptic circuitry under cellular heterotopia in hippocampal CA1, using a mouse model of the human neurodevelopmental disorder Type I Lissencephaly. We identify calbindin-expressing principal cells which are mispositioned under cellular heterotopia. Ectopic calbindin-expressing principal cells develop relatively normal morphological features and stunted intrinsic physiological features. Regarding network development, a connectivity preference for cholecystokinin-expressing interneurons to target calbindin-expressing principal cells is diminished. Moreover, in vitro gamma oscillatory activity is less synchronous across heterotopic bands and mutants are less responsive to pharmacological inhibition of cholecystokinin-containing interneurons. This study will aid not only in our understanding of how cellular networks form but highlight vulnerable cellular circuit motifs that might be generalized across disease states.

Moderate early life stress improves adult zebrafish (Danio rerio) working memory but does not affect social and anxiety-like responses.

Early life stress (ELS) is defined as a short or chronic period of trauma, environmental or social deprivation, which can affect different neurochemical and behavioral patterns during adulthood. Zebrafish (Danio rerio) have been widely used as a model system to understand human neurodevelopmental disorders and display translationally relevant behavioral and stress-regulating systems. In this study, we aimed to investigate the effects of moderate ELS by exposing young animals (6-weeks postfertilization), for 3 consecutive days, to three stressors, and analyzing the impact of this on adult zebrafish behavior (16-week postfertilization). The ELS impact in adults was assessed through analysis of performance on tests of unconditioned memory (free movement pattern Y-maze test), exploratory and anxiety-related task (novel tank diving test), and social cohesion (shoaling test). Here, we show for the first time that moderate ELS increases the number of alternations in turn-direction compared to repetitions in the unconditioned Y-maze task, suggesting increased working memory, but has no effect on shoal cohesion, locomotor profile, or anxiety-like behavior. Overall, our data suggest that moderate ELS may be linked to adaptive flexibility which contributes to build "resilience" in adult zebrafish by improving working memory performance.

Mutations in the KIF21B kinesin gene cause neurodevelopmental disorders through imbalanced canonical motor activity

KIF21B is a kinesin protein that promotes intracellular transport and controls microtubule dynamics. We report three missense variants and one duplication in KIF21B in individuals with neurodevelopmental disorders associated with brain malformations, including corpus callosum agenesis (ACC) and microcephaly. We demonstrate, in vivo, that the expression of KIF21B missense variants specifically recapitulates patients’ neurodevelopmental abnormalities, including microcephaly and reduced intra- and inter-hemispheric connectivity. We establish that missense KIF21B variants impede neuronal migration through attenuation of kinesin autoinhibition leading to aberrant KIF21B motility activity. We also show that the ACC-related KIF21B variant independently perturbs axonal growth and ipsilateral axon branching through two distinct mechanisms, both leading to deregulation of canonical kinesin motor activity. The duplication introduces a premature termination codon leading to nonsense-mediated mRNA decay. Although we demonstrate that Kif21b haploinsufficiency leads to an impaired neuronal positioning, the duplication variant might not be pathogenic. Altogether, our data indicate that impaired KIF21B autoregulation and function play a critical role in the pathogenicity of human neurodevelopmental disorder.

Waking up quiescent neural stem cells: Molecular mechanisms and implications in neurodevelopmental disorders.

Neural stem cells (NSCs) are crucial for development, regeneration, and repair of the nervous system. Most NSCs in mammalian adult brains are quiescent, but in response to extrinsic stimuli, they can exit from quiescence and become reactivated to give rise to new neurons. The delicate balance between NSC quiescence and activation is important for adult neurogenesis and NSC maintenance. However, how NSCs transit between quiescence and activation remains largely elusive. Here, we discuss our current understanding of the molecular mechanisms underlying the reactivation of quiescent NSCs. We review recent advances on signaling pathways originated from the NSC niche and their crosstalk in regulating NSC reactivation. We also highlight new intrinsic paradigms that control NSC reactivation in Drosophila and mammalian systems. We also discuss emerging evidence on modeling human neurodevelopmental disorders using NSCs.

Zebrafish as a tool to study schizophrenia-associated copy number variants.

ABSTRACTSchizophrenia remains one of the most debilitating human neurodevelopmental disorders, with few effective treatments and striking consequences felt by individuals, communities and society as a whole. As such, there remains a critical need for further investigation into the mechanistic underpinnings of schizophrenia so that novel therapeutic targets can be identified. Because schizophrenia is a highly heritable disorder, genetic risk factors remain an attractive avenue for this research. Given their clear molecular genetic consequences, recurrent microdeletions and duplications, or copy number variants (CNVs), represent one of the most tractable genetic entry points to elucidating these mechanisms. To date, eight CNVs have been shown to significantly increase the risk of schizophrenia. Although rodent models of these CNVs that exhibit behavioral phenotypes have been generated, the underlying molecular mechanisms remain largely elusive. Over the past decades, the zebrafish has emerged as a powerful vertebrate model that has led to fundamental discoveries in developmental neurobiology and behavioral genetics. Here, we review the attributes that make zebrafish exceptionally well suited to investigating individual and combinatorial gene contributions to CNV-mediated brain dysfunction in schizophrenia. With highly conserved genetics and neural substrates, an ever-expanding molecular genetic and imaging toolkit, and ability to perform high-throughput and high-content genetic and pharmacologic screens, zebrafish is poised to generate deep insights into the molecular genetic mechanisms of schizophrenia-associated neurodevelopmental and behavioral deficits, and to facilitate the identification of therapeutic targets.

Modeling Bainbridge-Ropers Syndrome in Xenopus laevis Embryos.

The Additional sex combs-like (ASXL1-3) genes are linked to human neurodevelopmental disorders. The de novo truncating variants in ASXL1-3 proteins serve as the genetic basis for severe neurodevelopmental diseases such as Bohring-Opitz, Shashi-Pena, and Bainbridge-Ropers syndromes, respectively. The phenotypes of these syndromes are similar but not identical, and include dramatic craniofacial defects, microcephaly, developmental delay, and severe intellectual disability, with a loss of speech and language. Bainbridge-Ropers syndrome resulting from ASXL3 gene mutations also includes features of autism spectrum disorder. Human genomic studies also identified missense ASXL3 variants associated with autism spectrum disorder, but lacking more severe Bainbridge-Ropers syndromic features. While these findings strongly implicate ASXL3 in mammalian brain development, its functions are not clearly understood. ASXL3 protein is a component of the polycomb deubiquitinase complex that removes mono-ubiquitin from Histone H2A. Dynamic chromatin modifications play important roles in the specification of cell fates during early neural patterning and development. In this study, we utilize the frog, Xenopus laevis as a simpler and more accessible vertebrate neurodevelopmental model system to understand the embryological cause of Bainbridge-Ropers syndrome. We have found that ASXL3 protein knockdown during early embryo development highly perturbs neural cell fate specification, potentially resembling the Bainbridge-Ropers syndrome phenotype in humans. Thus, the frog embryo is a powerful tool for understanding the etiology of Bainbridge-Ropers syndrome in humans.

Nestlet Shredding and Nest Building Tests to Assess Features of Psychiatric Disorders in Mice.

Mimicking the various facets of human psychiatric and neurodevelopmental disorders in animal models is a challenging task. Nevertheless, mice have emerged as a widely used model system to study pathophysiology and treatment strategies for these diseases. However, the corresponding behavioral tests are often elaborate and require extensive experience in behavioral testing. Here, we present protocols for two simple assays, nest building and nestlet shredding, that can serve as a starting point for the behavioral phenotyping of mouse models with (potential) features of psychiatric disorders. Both tests have been reported previously and we extend prior descriptions by including adaptations and refinements derived from our practical experience, like the use of the home cage instead of a fresh cage for nestlet shredding. Summarized, we provide ready-to-use protocols for two behavioral assays that allow the generation of robust data with minimal time and cost expenditure and enable an initial assessment of features of psychiatric or neurodevelopmental disorders in mouse models of these diseases.

Plasma levels of glial cell marker S100B in children with autism.

Autism spectrum disorder (ASD) is a neurodevelopmental condition with increasing incidence. Recent evidences suggest glial cells involvement in autism pathophysiology. S100B is a calcium binding protein, mainly found in astrocytes and therefore used as a marker of their activity. In our study, children with autism had higher plasma concentrations of S100B compared to non-autistic controls. No association of S100B plasma levels with behavioral symptoms (ADI-R and ADOS-2 scales) was found. Plasma S100B concentration significantly correlated with urine serotonin, suggesting their interconnection. Correlation of plasma S100B levels with stool calprotectin concentrations was found, suggesting not only brain astrocytes, but also enteric glial cells may take part in autism pathogenesis. Based on our findings, S100B seems to have a potential to be used as a biomarker of human neurodevelopmental disorders, but more investigations are needed to clarify its exact role in pathomechanism of autism.

Hypothalamic Monoaminergic Pathology in a Neurodevelopmental Rat Model Showing Prenatal 5-Bromo-2′-Deoxyuridine Treatment-Induced Hyperactivity and Hyporeproductivity

Objective: Prenatal treatment of rats with 5-bromo-2′-deoxyuridine (BrdU) is a neurodevelopmental model showing hyperactivity and impaired sexual activity. Human neurodevelopmental disorders, such as autism, exhibit sex-related pathology, but sex-related neurodevelopment has not been fully investigated in this model. We conducted this study to facilitate the understanding of the pathophysiology of neurodevelopmental disorders. Methods: Pregnant rats received 50 mg/kg BrdU on gestational days 9–15. The tissue content of dopamine (DA), serotonin (5-HT), and their metabolites dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid were measured in male and female offspring at 3 weeks (juveniles) and 10 weeks (adults) of age. Results: Prenatally BrdU-treated rats had reduced DA metabolism or DA content in the hypothalamus from the juvenile through the adult period without sex differences, but sex-specific striatal DA abnormalities emerged after maturation. A reduction in 5-HT metabolism was measured in the hypothalamus without sex differences throughout development. Developmental alterations in the striatal 5-HT states were sex-dependent. Temporal changes in DA or 5-HT metabolism were found in the frontal cortex and midbrain. Conclusion: The sex-specific influence of a genotoxic factor on the development of the DA and 5-HT systems was clarified in the hypothalamus and striatum. The results suggest that the observed sex dependence and region specificity are related to the pathology of social dysfunction in neurodevelopmental disorders.

Early Actions of Neurotransmitters During Cortex Development and Maturation of Reprogrammed Neurons.

The development of the brain is shaped by a myriad of factors among which neurotransmitters play remarkable roles before and during the formation and maturation of synaptic circuits. Cellular processes such as neurogenesis, morphological development, synaptogenesis and maturation of synapses are temporary and spatially regulated by the local or distal influence of neurotransmitters in the developing cortex. Thus, research on this area has contributed to the understanding of fundamental mechanisms of brain development and to shed light on the etiology of various human neurodevelopmental disorders such as autism and Rett syndrome (RTT), among others. Recently, the field of neuroscience has been shaken by an explosive advance of experimental approaches linked to the use of induced pluripotent stem cells and reprogrammed neurons. This new technology has allowed researchers for the first time to model in the lab the unique events that take place during early human brain development and to explore the mechanisms that cause synaptopathies. In this context, the role of neurotransmitters during early stages of cortex development is beginning to be re-evaluated and a revision of the state of the art has become necessary in a time when new protocols are being worked out to differentiate stem cells into functional neurons. New perspectives on reconsidering the function of neurotransmitters include opportunities for methodological advances, a better understanding of the origin of mental disorders and the potential for development of new treatments.

Kv2.1 voltage-gated potassium channels in developmental perspective.

Kv2.1 voltage-gated potassium channels consist of two types of α-subunits: (a) electrically-active Kcnb1 α-subunits and (b) silent or modulatory α-subunits plus β-subunits that, similar to silent α-subunits, also regulate electrically-active subunits. Voltage-gated potassium channels were traditionally viewed, mainly by electrophysiologists, as regulators of the electrical activity of the plasma membrane in excitable cells, a role that is performed by transmembrane protein domains of α-subunits that form the electric pore. Genetic studies revealed a role for this region of α-subunits of voltage-gated potassium channels in human neurodevelopmental disorders, such as epileptic encephalopathy. The N- and C-terminal domains of α-subunits interact to form the cytoplasmic subunit of heterotetrameric potassium channels that regulate electric pores. Subsequent animal studies revealed the developmental functions of Kcnb1-containing voltage-gated potassium channels and illustrated their role during brain development and reproduction. These functions of potassium channels are discussed in this review in the context of regulatory interactions between electrically-active and regulatory subunits.

Brain Organoids as Tools for Modeling Human Neurodevelopmental Disorders.

Brain organoids recapitulate in vitro the specific stages of in vivo human brain development, thus offering an innovative tool by which to model human neurodevelopmental disease. We review here how brain organoids have been used to study neurodevelopmental disease and consider their potential for both technological advancement and therapeutic development.

Haploinsufficiency of the Notch Ligand DLL1 Causes Variable Neurodevelopmental Disorders

Notch signaling is an established developmental pathway for brain morphogenesis. Given that Delta-like 1 (DLL1) is a ligand for the Notch receptor and that a few individuals with developmental delay, intellectual disability, and brain malformations have microdeletions encompassing DLL1, we hypothesized that insufficiency of DLL1 causes a human neurodevelopmental disorder. We performed exome sequencing in individuals with neurodevelopmental disorders. The cohort was identified using known Matchmaker Exchange nodes such as GeneMatcher. This method identified 15 individuals from 12 unrelated families with heterozygous pathogenic DLL1 variants (nonsense, missense, splice site, and one whole gene deletion). The most common features in our cohort were intellectual disability, autism spectrum disorder, seizures, variable brain malformations, muscular hypotonia, and scoliosis. We did not identify an obvious genotype-phenotype correlation. Analysis of one splice site variant showed an in-frame insertion of 12bp. In conclusion, heterozygous DLL1 pathogenic variants cause a variable neurodevelopmental phenotype and multi-systemic features. The clinical and molecular data support haploinsufficiency as a mechanism for the pathogenesis of this DLL1-related disorder and affirm the importance of DLL1 in human brain development.

Plasticity of dendritic spines: Molecular function and dysfunction in neurodevelopmental disorders

Dendritic spines are tiny postsynaptic protrusions from a dendrite that receive most of the excitatory synaptic input in the brain. Functional and structural changes in dendritic spines are critical for synaptic plasticity, a cellular model of learning and memory. Conversely, altered spine morphology and plasticity are common hallmarks of human neurodevelopmental disorders, such as intellectual disability and autism. The advances in molecular and optical techniques have allowed for exploration of dynamic changes in structure and signal transduction at single-spine resolution, providing significant insights into the molecular regulation underlying spine structural plasticity. Here, I review recent findings on: how synaptic stimulation leads to diverse forms of spine structural plasticity; how the associated biochemical signals are initiated and transmitted into neuronal compartments; and how disruption of single genes associated with neurodevelopmental disorders can lead to abnormal spine structure in human and mouse brains. In particular, I discuss the functions of the Ras superfamily of small GTPases in spatiotemporal regulation of the actin cytoskeleton and protein synthesis in dendritic spines. Multiple lines of evidence implicate disrupted Ras signaling pathways in the spine structural abnormalities observed in neurodevelopmental disorders. Both deficient and excessive Ras activities lead to disrupted spine structure and deficits in learning and memory. Dysregulation of spine Ras signaling, therefore, may play a key role in the pathogenesis of multiple neurodevelopmental disorders with distinct etiologies.

Analysis of Synapses in Cerebral Organoids

Cerebral organoids are an emerging cutting-edge technology to model human brain development and neurodevelopmental disorders, for which mouse models exhibit significant limitations. In the human brain, synaptic connections define neural circuits, and synaptic deficits account for various neurodevelopmental disorders. Thus, harnessing the full power of cerebral organoids for human brain modeling requires the ability to visualize and analyze synapses in cerebral organoids. Previously, we devised an optimized method to generate human cerebral organoids, and showed that optimal organoids express mature-neuron markers, including synaptic proteins and neurotransmitter receptors and transporters. Here, we give evidence for synaptogenesis in cerebral organoids, via microscopical visualization of synapses. We also describe multiple approaches to quantitatively analyze synapses in cerebral organoids. Collectively, our work provides sufficient evidence for the possibility of modeling synaptogenesis and synaptic disorders in cerebral organoids, and may help advance the use of cerebral organoids in molecular neuroscience and studies of neurodevelopmental disorders such as autism.