Childhood adversity, stress reactivity, and structural brain measures in stress-related/neurodevelopmental disorders, and their comorbidity: A large transdiagnostic cross-sectional study.

Childhood adversity (CA), including childhood adverse life events, increases the risk for development of psychiatric disorders later in life. Both CA and psychiatric disorders are associated with structural brain changes and dysfunctional hypothalamic-pituitary-adrenal-axis. However, many studies investigated single diagnostic and single regions of interest of the brain, and did not take stress reactivity into account. We investigated associations of CA and cortisol levels with gray matter volume and cortical thickness, in a whole-brain manner. Primary analysis constituted of a transdiagnostic approach, followed by a moderation analysis to investigate the influence of diagnosis. Patients with stress-related and/or neurodevelopmental disorders and matched healthy controls underwent an magnetic resonance imaging scan, next to assessing hair cortisol levels and CA/life events. CA was reported by 62-72% of the patients versus 33% of the controls. Primary transdiagnostic linear regression analyses revealed that CA was not associated with gray matter volume, while childhood life events were associated with lower right thalamic volume. Hair cortisol was not associated with any lobe volume. None of the associations were moderated by diagnosis. In conclusion, CA is a risk factor that needs to be taken into account when investigating psychiatric disorders. Yet the relationship with structural brain changes and stress reactivity is less clear than postulated on the basis of more seed-based studies.

Neurodevelopmental Disorder Caused by Deletion of CHASERR, a lncRNA Gene.

CHASERR encodes a human long noncoding RNA (lncRNA) adjacent to CHD2, a coding gene in which de novo loss-of-function variants cause developmental and epileptic encephalopathy. Here, we report our findings in three unrelated children with a syndromic, early-onset neurodevelopmental disorder, each of whom had a de novo deletion in the CHASERR locus. The children had severe encephalopathy, shared facial dysmorphisms, cortical atrophy, and cerebral hypomyelination - a phenotype that is distinct from the phenotypes of patients with CHD2 haploinsufficiency. We found that the CHASERR deletion results in increased CHD2 protein abundance in patient-derived cell lines and increased expression of the CHD2 transcript in cis. These findings indicate that CHD2 has bidirectional dosage sensitivity in human disease, and we recommend that other lncRNA-encoding genes be evaluated, particularly those upstream of genes associated with mendelian disorders. (Funded by the National Human Genome Research Institute and others.).

Deficiency of DDX3X results in neurogenesis defects and abnormal behaviors via dysfunction of the Notch signaling.

The molecular mechanisms underlying the neurodevelopmental disorders (NDDs) caused by DDX3X variants remain poorly understood. In this study, we validated that de novo DDX3X variants are enriched in female developmental delay (DD) patients and mainly affect the evolutionarily conserved amino acids based on a meta-analysis of 46,612 NDD trios. We generated a ddx3x deficient zebrafish allele, which exhibited reduced survival rate, DD, microcephaly, adaptation defects, anxiolytic behaviors, social interaction deficits, and impaired spatial recognitive memory. As revealed by single-nucleus RNA sequencing and biological validations, ddx3x deficiency leads to reduced neural stem cell pool, decreased total neuron number, and imbalanced differentiation of excitatory and inhibitory neurons, which are responsible for the behavioral defects. Indeed, the supplementation of L-glutamate or glutamate receptor agonist ly404039 could partly rescue the adaptation and social deficits. Mechanistically, we reveal that the ddx3x deficiency attenuates the stability of the crebbp mRNA, which in turn causes downregulation of Notch signaling and defects in neurogenesis. Our study sheds light on the molecular pathology underlying the abnormal neurodevelopment and behavior of NDD patients with DDX3X mutations, as well as providing potential therapeutic targets for the precision treatment.

Altered molecular and cellular mechanisms in KIF5A-associated neurodegenerative or neurodevelopmental disorders.

Mutations targeting distinct domains of the neuron-specific kinesin KIF5A associate with different neurodegenerative/neurodevelopmental disorders, but the molecular bases of this clinical heterogeneity are unknown. We characterised five key mutants covering the whole spectrum of KIF5A-related phenotypes: spastic paraplegia (SPG, R17Q and R280C), Charcot-Marie-Tooth disease (CMT, R864*), amyotrophic lateral sclerosis (ALS, N999Vfs*40), and neonatal intractable myoclonus (NEIMY, C975Vfs*73) KIF5A mutants. CMT-R864*-KIF5A and ALS-N999Vfs*40-KIF5A showed impaired autoinhibition and peripheral localisation accompanied by altered mitochondrial distribution, suggesting transport competence disruption. ALS-N999Vfs*40-KIF5A formed SQSTM1/p62-positive inclusions sequestering WT-KIF5A, indicating a gain of toxic function. SPG-R17Q-KIF5A and ALS-N999Vfs*40-KIF5A evidenced a shorter half-life compared to WT-KIF5A, and proteasomal blockage determined their accumulation into detergent-insoluble inclusions. Interestingly, SPG-R280C-KIF5A and ALS-N999Vfs*40-KIF5A both competed for degradation with proteasomal substrates. Finally, NEIMY-C975Vfs*73-KIF5A displayed a similar, but more severe aberrant behaviour compared to ALS-N999Vfs*40-KIF5A; these two mutants share an abnormal tail but cause disorders on the opposite end of KIF5A-linked phenotypic spectrum. Thus, our observations support the pathogenicity of novel KIF5A mutants, highlight abnormalities of recurrent variants, and demonstrate that both unique and shared mechanisms underpin KIF5A-related diseases.

[The contribution of cerebral organoids to the understanding and treatment of rare genetic diseases with neurodevelopmental disorders]. / Des organoïdes cérébraux pour la compréhension et la thérapie des maladies génétiques rares avec troubles neurodéveloppementaux.

Rare genetic diseases with neurodevelopmental disorders (NDDs) encompass several heterogeneous conditions (autism spectrum disorder (ASD), intellectual disability (ID), attention deficit hyperactivity disorder (ADHD), specific learning disorder (SLD), among others). Currently, few treatments are available for these patients. The difficulty in accessing human brain samples and the discrepancies between human and animal models highlight the need for new research approaches. One promising approach is the use of the cerebral organoids. These 3D, self-organized structures, generated from induced pluripotent stem cells (iPSCs), enable the reproduction of the stages of human brain development, from the proliferation of neural stem cells to their differentiation into neurons, oligodentrocytes, and astrocytes. Cerebral organoids hold great promise in understanding brain development and in the search for treatments.

Title: Des organoïdes cérébraux pour la compréhension et la thérapie des maladies génétiques rares avec troubles neurodéveloppementaux. Abstract: Les maladies génétiques associées à des troubles neurodéveloppementaux (TND) regroupent plusieurs maladies pour lesquelles peu de traitements sont proposés. L'impossibilité d'accéder à des échantillons de cerveaux humains pour des études ex vivo, et les divergences entre l'homme et les modèles animaux rendent nécessaires de nouvelles approches de recherche. L'organoïde cérébral, une structure en trois dimensions, auto-organisée, et générée à partir de cellules souches pluripotentes induites, permet de reproduire les étapes de développement du cerveau humain, de la prolifération des cellules souches neurales à leur différenciation en neurones, en oligodendrocytes, ou en astrocytes. L'intérêt de ce modèle est désormais prouvé pour la compréhension du développement cérébral et pour la recherche de traitements. Après une présentation des cellules souches pluripotentes induites et des organoïdes, nous exposerons comment cette technique est actuellement déployée, en particulier pour étudier les mécanismes physiopathologiques résultant de variations génétiques pathogènes de gènes candidats de TND.

Dataset factors associated with age-related changes in brain structure and function in neurodevelopmental conditions.

With brain structure and function undergoing complex changes throughout childhood and adolescence, age is a critical consideration in neuroimaging studies, particularly for those of individuals with neurodevelopmental conditions. However, despite the increasing use of large, consortium-based datasets to examine brain structure and function in neurotypical and neurodivergent populations, it is unclear whether age-related changes are consistent between datasets and whether inconsistencies related to differences in sample characteristics, such as demographics and phenotypic features, exist. To address this, we built models of age-related changes of brain structure (regional cortical thickness and regional surface area; N = 1218) and function (resting-state functional connectivity strength; N = 1254) in two neurodiverse datasets: the Province of Ontario Neurodevelopmental Network and the Healthy Brain Network. We examined whether deviations from these models differed between the datasets, and explored whether these deviations were associated with demographic and clinical variables. We found significant differences between the two datasets for measures of cortical surface area and functional connectivity strength throughout the brain. For regional measures of cortical surface area, the patterns of differences were associated with race/ethnicity, while for functional connectivity strength, positive associations were observed with head motion. Our findings highlight that patterns of age-related changes in the brain may be influenced by demographic and phenotypic characteristics, and thus future studies should consider these when examining or controlling for age effects in analyses.

Brain organoids: A new tool for modelling of neurodevelopmental disorders.

Neurodevelopmental disorders are mostly studied using mice as models. However, the mouse brain lacks similar cell types and structures as those of the human brain. In recent years, emergence of three-dimensional brain organoids derived from human embryonic stem cells or induced pluripotent stem cells allows for controlled monitoring and evaluation of early neurodevelopmental processes and has opened a window for studying various aspects of human brain development. However, such organoids lack original anatomical structure of the brain during maturation, and neurodevelopmental maturation processes that rely on unique cellular interactions and neural network connections are limited. Consequently, organoids are difficult to be used extensively and effectively while modelling later stages of human brain development and disease progression. To address this problem, several methods and technologies have emerged that aim to enhance the sophisticated regulation of brain organoids developmental processes through bioengineering approaches, which may alleviate some of the current limitations. This review discusses recent advances and application areas of human brain organoid culture methods, aiming to generalize optimization strategies for organoid systems, improve the ability to mimic human brain development, and enhance the application value of organoids.

A Novel De Novo STAG1 Variant in Monozygotic Twins with Neurodevelopmental Disorder: New Insights in Clinical Heterogeneity.

BACKGROUND: The STAG1 gene encodes a component of the cohesin complex, involved in chromosome segregation and DNA repair. Variants in genes of the cohesin complex determine clinical conditions characterized by facial dysmorphisms, upper limb anomalies, intellectual disability, and other neurological deficits. However, to date, the STAG1-related clinical phenotype has been poorly investigated (around 20 cases reported). METHODS AND RESULTS: We report, for the first time, two twins affected by a syndromic neurodevelopmental disorder associated with a de novo variant in the STAG1 gene. Although both the twins showed a neurodevelopmental delay, one of them showed a more severe phenotype with greater behavioral problems, speech defects and limb apraxia. CGH array showed a 15q13.3 microduplication, inherited from an unaffected mother. CONCLUSIONS: We found different degrees of behavioral, speech and cognitive impairment in two twins affected by a neurodevelopmental disorder associated with a STAG1 variant. These findings highlight the variability of the STAG1-associated phenotype or a probable role of associated variants (like the discovered 15q13.3 microduplication) in modulating the clinical features.

Cardiovascular abnormalities in patients with SHANK3 pathogenic variants: Beyond neurodevelopmental disorders and epilepsy.

Neurodevelopmental disorders have been linked to numerous genes, particularly pathogenic variants in genes encoding postsynaptic scaffolding proteins, like SHANK3. This study aims to provide insights into the cardiovascular profile of patients with pathogenic SHANK3 variants, expanding beyond the well-established associations with neurodevelopmental disorders and epilepsy. We conducted a prospective study involving patients affected by neurodevelopmental disorders with pathogenic SHANK3 variants. Comprehensive cardiovascular assessments were performed and molecular genetic testing included chromosomal microarray followed by clinical exome sequencing. We identified five patients with de novo SHANK3 variants, all of whom exhibited cardiac involvement, including myocardial dysfunction, congenital heart disease (patent ductus arteriosus), and a case of postictal atrial fibrillation. Our findings emphasize an elevated risk of cardiovascular abnormalities in patients with SHANK3 pathogenic variants compared to prior reports. Despite their young age, these patients displayed significant cardiac abnormalities. The study highlights the necessity of integrating cardiac evaluation and ongoing cardiovascular monitoring into multidisciplinary care, facilitating early detection of heart failure and assessment of the risk of sudden unexpected death in epilepsy (SUDEP). Further research is needed to elucidate the underlying mechanisms of cardiac manifestations in SHANK3 mutation carriers.

The Phenotypic Spectrum of 16p11.2 Recurrent Chromosomal Rearrangements.

The human 16p11.2 chromosomal region is rich in segmental duplications which mediate the formation of recurrent CNVs. CNVs affecting the 16p11.2 region are associated with an increased risk for developing neuropsychiatric disorders, including autism spectrum disorder (ASD), schizophrenia, and intellectual disability (ID), as well as abnormal body weight and head circumference and dysmorphic features, with marked phenotypic variability and reduced penetrance. CNVs affecting the 16p11.2 region mainly affect a distal interval of ~220 Kb, between Breakpoints 2 and 3 (BP2-BP3), and a proximal interval of ~593 Kb (BP4-BP5). Here, we report on 15 patients with recurrent 16p11.2 rearrangements that were identified among a cohort of 1600 patients (0.9%) with neurodevelopmental disorders. A total of 13 deletions and two duplications were identified, of which eight deletions included the proximal 16p11.2 region (BP4-BP5) and five included the distal 16p11.2 region (BP2-BP3). Of the two duplications that were identified, one affected the proximal and one the distal 16p11.2 region; however, both patients had additional CNVs contributing to phenotypic severity. The features observed and their severity varied greatly, even between patients within the same family. This article aims to further delineate the clinical spectrum of patients with 16p11.2 recurrent rearrangements in order to aid the counselling of patients and their families.

Deleterious ZNRF3 germline variants cause neurodevelopmental disorders with mirror brain phenotypes via domain-specific effects on Wnt/ß-catenin signaling.

Zinc and RING finger 3 (ZNRF3) is a negative-feedback regulator of Wnt/ß-catenin signaling, which plays an important role in human brain development. Although somatically frequently mutated in cancer, germline variants in ZNRF3 have not been established as causative for neurodevelopmental disorders (NDDs). We identified 12 individuals with ZNRF3 variants and various phenotypes via GeneMatcher/Decipher and evaluated genotype-phenotype correlation. We performed structural modeling and representative deleterious and control variants were assessed using in vitro transcriptional reporter assays with and without Wnt-ligand Wnt3a and/or Wnt-potentiator R-spondin (RSPO). Eight individuals harbored de novo missense variants and presented with NDD. We found missense variants associated with macrocephalic NDD to cluster in the RING ligase domain. Structural modeling predicted disruption of the ubiquitin ligase function likely compromising Wnt receptor turnover. Accordingly, the functional assays showed enhanced Wnt/ß-catenin signaling for these variants in a dominant negative manner. Contrarily, an individual with microcephalic NDD harbored a missense variant in the RSPO-binding domain predicted to disrupt binding affinity to RSPO and showed attenuated Wnt/ß-catenin signaling in the same assays. Additionally, four individuals harbored de novo truncating or de novo or inherited large in-frame deletion variants with non-NDD phenotypes, including heart, adrenal, or nephrotic problems. In contrast to NDD-associated missense variants, the effects on Wnt/ß-catenin signaling were comparable between the truncating variant and the empty vector and between benign variants and the wild type. In summary, we provide evidence for mirror brain size phenotypes caused by distinct pathomechanisms in Wnt/ß-catenin signaling through protein domain-specific deleterious ZNRF3 germline missense variants.

Neurodevelopmental and Neuropsychiatric Disorders.

This chapter will focus on microglial involvement in neurodevelopmental and neuropsychiatric disorders, particularly autism spectrum disorder (ASD), schizophrenia and major depressive disorder (MDD). We will describe the neuroimmune risk factors that contribute to the etiopathology of these disorders across the lifespan, including both in early life and adulthood. Microglia, being the resident immune cells of the central nervous system, could play a key role in triggering and determining the outcome of these disorders. This chapter will review preclinical and clinical findings where microglial morphology and function were examined in the contexts of ASD, schizophrenia and MDD. Clinical evidence points out to altered microglial morphology and reactivity, as well as increased expression of pro-inflammatory cytokines, supporting the idea that microglial abnormalities are involved in these disorders. Indeed, animal models for these disorders found altered microglial morphology and homeostatic functions which resulted in behaviours related to these disorders. Additionally, as microglia have emerged as promising therapeutic targets, we will also address in this chapter therapies involving microglial mechanisms for the treatment of neurodevelopmental and neuropsychiatric disorders.

Human microglial cells as a therapeutic target in a neurodevelopmental disease model.

Although microglia are macrophages of the central nervous system, their involvement is not limited to immune functions. The roles of microglia during development in humans remain poorly understood due to limited access to fetal tissue. To understand how microglia can impact human neurodevelopment, the methyl-CpG binding protein 2 (MECP2) gene was knocked out in human microglia-like cells (MGLs). Disruption of the MECP2 in MGLs led to transcriptional and functional perturbations, including impaired phagocytosis. The co-culture of healthy MGLs with MECP2-knockout (KO) neurons rescued synaptogenesis defects, suggesting a microglial role in synapse formation. A targeted drug screening identified ADH-503, a CD11b agonist, restored phagocytosis and synapse formation in spheroid-MGL co-cultures, significantly improved disease progression, and increased survival in MeCP2-null mice. These results unveil a MECP2-specific regulation of human microglial phagocytosis and identify a novel therapeutic treatment for MECP2-related conditions.

Neurodevelopmental disorder-associated CYFIP2 regulates membraneless organelles and eIF2α phosphorylation via protein interactors and actin cytoskeleton.

De novo variants in the Cytoplasmic FMR1-interacting protein 2 (CYFIP2) have been repeatedly associated with neurodevelopmental disorders and epilepsy, underscoring its critical role in brain development and function. While CYFIP2's role in regulating actin polymerization as part of the WAVE regulatory complex (WRC) is well-established, its additional molecular functions remain relatively unexplored. In this study, we performed unbiased quantitative proteomic analysis, revealing 278 differentially expressed proteins (DEPs) in the forebrain of Cyfip2 knock-out embryonic mice compared to wild-type mice. Unexpectedly, these DEPs, in conjunction with previously identified CYFIP2 brain interactors, included not only other WRC components but also numerous proteins associated with membraneless organelles (MLOs) involved in mRNA processing and translation within cells, including the nucleolus, stress granules, and processing bodies. Additionally, single-cell transcriptomic analysis of the Cyfip2 knock-out forebrain revealed gene expression changes linked to cellular stress responses and MLOs. We also observed morphological changes in MLOs in Cyfip2 knock-out brains and CYFIP2 knock-down cells under basal and stress conditions. Lastly, we demonstrated that CYFIP2 knock-down in cells, potentially through WRC-dependent actin regulation, suppressed the phosphorylation levels of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α), thereby enhancing protein synthesis. These results suggest a physical and functional connection between CYFIP2 and various MLO proteins and also extend CYFIP2's role within the WRC from actin regulation to influencing eIF2α phosphorylation and protein synthesis. With these dual functions, CYFIP2 may fine-tune the balance between MLO formation/dynamics and protein synthesis, a crucial aspect of proper mRNA processing and translation.

Establishment of an iPSC line from a NDD patient with a heterozygous mutation in the CTNNB1 gene.

CTNNB1 encodes beta-catenin, which plays a crucial role in Wnt signaling pathway. Mutations in CTNNB1 involve in tumor developing, Primary Aldosteronism, Neurodevelopmental disorders (NDDs), etc. NDDs is a class of disorders that impact brain development and function, manifesting symptom including autism spectrum disorder (ASD), intellectual disability (ID), schizophrenia (SCZ), and epilepsy. Here, we generated an iPSC line (CTUi005-A) from a patient diagnosed with NDDs, carrying a heterozygous mutation of the CTNNB1 gene. CTUi005-A exhibits typical iPSC characteristics, and holds potential as a cellular tool for investigating the pathogenic mechanisms underlying NDDs.

Implications of altered pyramidal cell morphology on clinical symptoms of neurodevelopmental disorders.

The prevalence of pyramidal cells (PCs) in the mammalian cerebral cortex underscore their value as they play a crucial role in various brain functions, ranging from cognition, sensory processing, to motor output. PC morphology significantly influences brain connectivity and plays a critical role in maintaining normal brain function. Pathological alterations to PC morphology are thought to contribute to the aetiology of neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia. This review explores the relationship between abnormalities in PC morphology in key cortical areas and the clinical manifestations in schizophrenia and ASD. We focus largely on human postmortem studies and provide evidence that dendritic segment length, complexity and spine density are differentially affected in these disorders. These morphological alterations can lead to disruptions in cortical connectivity, potentially contributing to the cognitive and behavioural deficits observed in these disorders. Furthermore, we highlight the importance of investigating the functional and structural characteristics of PCs in these disorders to illuminate the underlying pathogenesis and stimulate further research in this area.

Mitochondrial regulation of adult hippocampal neurogenesis: Insights into neurological function and neurodevelopmental disorders.

Mitochondria are essential regulators of cellular energy metabolism and play a crucial role in the maintenance and function of neuronal cells. Studies in the last decade have highlighted the importance of mitochondrial dynamics and bioenergetics in adult neurogenesis, a process that significantly influences cognitive function and brain plasticity. In this review, we examine the mechanisms by which mitochondria regulate adult neurogenesis, focusing on the impact of mitochondrial function on the behavior of neural stem/progenitor cells and the maturation and plasticity of newborn neurons in the adult mouse hippocampus. In addition, we explore the link between mitochondrial dysfunction, adult hippocampal neurogenesis and genes associated with cognitive deficits in neurodevelopmental disorders. In particular, we provide insights into how alterations in the transcriptional regulator NR2F1 affect mitochondrial dynamics and may contribute to the pathophysiology of the emerging neurodevelopmental disorder Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS). Understanding how genes involved in embryonic and adult neurogenesis affect mitochondrial function in neurological diseases might open new directions for therapeutic interventions aimed at boosting mitochondrial function during postnatal life.

Role of cell metabolism in the pathophysiology of brain size-associated neurodevelopmental disorders.

Cell metabolism is a key regulator of human neocortex development and evolution. Several lines of evidence indicate that alterations in neural stem/progenitor cell (NPC) metabolism lead to abnormal brain development, particularly brain size-associated neurodevelopmental disorders, such as microcephaly. Abnormal NPC metabolism causes impaired cell proliferation and thus insufficient expansion of NPCs for neurogenesis. Therefore, the production of neurons, which is a major determinant of brain size, is decreased and the size of the brain, especially the size of the neocortex, is significantly reduced. This review discusses recent progress understanding NPC metabolism, focusing in particular on glucose metabolism, fatty acid metabolism and amino acid metabolism (e.g., glutaminolysis and serine metabolism). We provide an overview of the contributions of these metabolic pathways to brain development and evolution, as well as to the etiology of neurodevelopmental disorders. Furthermore, we discuss the advantages and disadvantages of various experimental models to study cell metabolism in the developing brain.

CNV Analysis through Exome Sequencing Reveals a Large Duplication Involved in Sex Reversal, Neurodevelopmental Delay, Epilepsy and Optic Atrophy.

BACKGROUND: Duplications on the short arm of chromosome X, including the gene NR0B1, have been associated with gonadal dysgenesis and with male to female sex reversal. Additional clinical manifestations can be observed in the affected patients, depending on the duplicated genomic region. Here we report one of the largest duplications on chromosome X, in a Lebanese patient, and we provide the first comprehensive review of duplications in this genomic region. CASE PRESENTATION: A 2-year-old female patient born to non-consanguineous Lebanese parents, with a family history of one miscarriage, is included in this study. The patient presents with sex reversal, dysmorphic features, optic atrophy, epilepsy, psychomotor and neurodevelopmental delay. Single nucleotide variants and copy number variants analysis were carried out on the patient through exome sequencing (ES). This showed an increased coverage of a genomic region of around 23.6 Mb on chromosome Xp22.31-p21.2 (g.7137718-30739112) in the patient, suggestive of a large duplication encompassing more than 60 genes, including the NR0B1 gene involved in sex reversal. A karyotype analysis confirmed sex reversal in the proband presenting with the duplication, and revealed a balanced translocation between the short arms of chromosomes X and 14:46, X, t(X;14) (p11;p11) in her/his mother. CONCLUSIONS: This case highlights the added value of CNV analysis from ES data in the genetic diagnosis of patients. It also underscores the challenges encountered in announcing unsolicited incidental findings to the family.

Single cell spatial biology over developmental time can decipher pediatric brain pathologies.

Pediatric low grade brain tumors and neurodevelopmental disorders share proteins, signaling pathways, and networks. They also share germline mutations and an impaired prenatal differentiation origin. They may differ in the timing of the events and proliferation. We suggest that their pivotal distinct, albeit partially overlapping, outcomes relate to the cell states, which depend on their spatial location, and timing of gene expression during brain development. These attributes are crucial as the brain develops sequentially, and single-cell spatial organization influences cell state, thus function. Our underlying premise is that the root cause in neurodevelopmental disorders and pediatric tumors is impaired prenatal differentiation. Data related to pediatric brain tumors, neurodevelopmental disorders, brain cell (sub)types, locations, and timing of expression in the developing brain are scant. However, emerging single cell technologies, including transcriptomic, spatial biology, spatial high-resolution imaging performed over the brain developmental time, could be transformational in deciphering brain pathologies thereby pharmacology.