Defective Neural Stem and Progenitor Cell Proliferation in Neurodevelopmental Disorders

1. Introduction

Neurodevelopmental disorders (NDDs) encompass a broad spectrum of conditions including autism spectrum disorder (ASD), intellectual disability (ID), and attention deficit hyperactivity disorder (ADHD) [1]. These disorders affect approximately 1 in 20 children globally [2] and pose significant lifelong challenges for affected individuals and their families [3,4]. Despite their clinical heterogeneity, these disorders are believed to share a common underlying feature: the disruption of early neurodevelopmental processes critical for establishing proper brain architecture and function. [5,6].

Among the earliest processes in brain formation is the proliferation of embryonic cortical neural stem and progenitor cells (NSPCs), which give rise to the neurons and glial cells that populate the central nervous system [5,7,8]. In principle, neural stem cells generate neural progenitors that predominantly give rise to neurons, whereas glial progenitors are thought to exclusively produce astrocytes and oligodendrocytes (Figure 1). However, it has revealed that neural progenitor cells exhibit glial characteristics and are capable of producing not only neurons but also astrocytes and oligodendrocytes [9], thereby blurring the distinction between dedicated glial progenitors and multipotent neural progenitors. Notably, neurogenesis precedes gliogenesis, commencing at approximately embryonic day 10 in mice (gestational week 10 in humans) and shifting toward gliogenesis around embryonic day 16 in mice (gestational week 16 in humans) [10,11,12]. This temporal progression indicates a dynamic change in the developmental potential of neural progenitor cells. Although mounting evidence implicates glial abnormalities in NDDs [13,14], the specific contribution of aberrant gliogenesis remains less well defined. Accordingly, in this review, we highlight defective NSPC proliferation and its relevance to the pathogenesis of NDDs.

NSPCs are highly sensitive to both intrinsic and extrinsic cues that govern their proliferation, survival, and differentiation [15,16,17,18]. Tight regulation of NSPC expansion and lineage specification is indispensable for the proper establishment of brain size, cortical lamination, and neural circuitry [19,20,21,22]. Disruptions in these processes can yield profound neuroanatomical and functional abnormalities, exemplified by microcephaly and macrocephaly [23,24,25], as well as aberrant synaptic organization frequently observed in neurodevelopmental disorders [26] (Figure 2).

Leveraging animal models and pluripotent stem cell–derived NSPCs, recent studies have implicated a diverse spectrum of genetic factors—many of which are mutated in patients with NDDs—in the regulation of NSPC proliferation [27,28]. In parallel, NDD-associated environmental insults during gestation have also been shown to perturb NSPC proliferation [29,30,31,32]. Collectively, these findings underscore defective NSPC proliferation as a critical early pathogenic event in NDDs, emphasizing the necessity of elucidating this process to inform the development of targeted therapeutic strategies. In this review, we summarize current knowledge of the molecular mechanisms underlying aberrant NSPC proliferation, with particular focus on the regulation of the cell division cycle, a fundamental determinant of NSPC expansion.

2. Neural Stem and Progenitor Cells in Normal Brain Development

The earliest NSPCs in the developing brain are neuroepithelial cells, a population of neural stem cells that initially undergo symmetric divisions to expand the progenitor pool during early neurogenesis [7]. As the neural tube matures, these neuroepithelial cells progressively transform into radial glial cells—highly polarized progenitors that extend across the entire thickness of the embryonic cortex. Radial glia generate both neurons and intermediate progenitors through symmetric and asymmetric divisions [33]. Intermediate progenitors (IPs), residing predominantly within the subventricular zone, possess more restricted developmental potential and primarily give rise to neurons via neurogenic divisions [34,35] (Figure 3).

NSPC proliferation is orchestrated by a complex interplay of intrinsic and extrinsic mechanisms. Intrinsic regulation encompasses mitotic spindle orientation, chromatin remodeling, and the expression of lineage-determining transcription factors [36,37]. For instance, the balance between symmetric and asymmetric divisions, essential for self-renewal and differentiation, respectively, is critically shaped by the orientation of the mitotic spindle and the polarized distribution of cell fate determinants [20,38]. Extrinsic signals from the neural stem cell niche—including Notch, Wnt, Sonic Hedgehog (SHH), fibroblast growth factor (FGF), and insulin-like growth factor-I (IGF-I)—further modulate NSPC behavior [39,40,41,42,43,44]. In addition, interactions with the extracellular matrix and local metabolic states play pivotal roles in regulating proliferative capacity and lineage specification [45,46].

3. Cell Cycle Regulation in NSPC Proliferation

Because terminally differentiated cells have lost their proliferative capacity, disruptions in NSPC proliferation can arise through two principal mechanisms: direct perturbation of cell division or indirect perturbation mediated by aberrant differentiation timing (Figure 4). Consequently, abnormal NSPC proliferation often stems from mutations in genes that compromise the regulation of either cell cycle progression or differentiation.

Proper and timely cell division is tightly regulated through coordinated control of cell cycle progression. During the G1 phase, diploid cells prepare for DNA synthesis, which occurs in the S phase. The resulting tetraploid cells then enter the G2 phase, where they prepare for mitosis, ultimately producing two daughter cells in the M phase [37,47,48] (Figure 5). In response to growth factors described in Section 2, signaling cascades such as the MAPK and PI3K/mTOR pathways are activated, leading to the induction of Cyclin D. Cyclin D subsequently activates CDK4/6, which is required for entry into S phase and DNA replication. Cyclin D–CDK4/6 activity further induces Cyclin E, which activates CDK2 to drive G1 phase progression. Thereafter, the Cyclin A–CDK2 complex plays a pivotal role in initiating DNA replication by activating the replication machinery, while also coordinating the timely activation of the Cyclin B–CDK1 complex, which governs progression into mitosis.

The duration of the cell cycle is a critical determinant of NSPC proliferation and differentiation. As NSPCs progress toward neuronal differentiation, the overall cell cycle lengthens, predominantly due to an extended G1 phase. This prolonged G1 phase is thought to be essential, as it provides sufficient time for the accumulation and activity of fate-determining factors that promote differentiation [37,49]. Consistently, experimental elongation of the G1 phase via CDK inhibitors drives NSPC differentiation [50]. Conversely, artificial shortening of the G1 phase through overexpression of Cyclin D or Cyclin E delays differentiation and maintains NSPCs in a proliferative, undifferentiated state [51,52]. These findings indicate that inappropriate inhibition of cell cycle progression can prematurely trigger differentiation and deplete the NSPC pool, thereby contributing to the pathogenesis of NDDs.

4. Impairment of NSPC Proliferation in Neurodevelopmental Disorders

Abnormal cell cycle progression is largely driven by dysregulated expression of key cell cycle regulators, often as a consequence of disrupted signaling pathways [53,54], aberrant activity of transcription factors, and altered chromatin remodeling [55,56,57]. These perturbations collectively impair NSPC proliferation and compromise normal brain development.

4.1. Genetic Factors

Notch signaling is a master regulator of NSPC proliferation. Upon ligand activation (Jagged, Delta), Notch receptors undergo proteolytic cleavage to release the Notch intracellular domain (NICD), which associates with RBPJ and MAML to drive transcription of target genes, notably the Hes and Hey families [58] (Figure 6a). Hes and Hey proteins repress proneural genes, thereby maintaining NSPCs in an undifferentiated state and preventing premature neuronal differentiation [59]. Mutations in NOTCH2NL (encoding a Notch receptor) and DLL1 (encoding a Delta ligand) have been identified in NDD patients [60,61]. Importantly, NOTCH2NL deletions are associated with microcephaly, whereas duplications result in macrocephaly [60]. Similarly, DLL1 mutations have been linked to microcephaly [61], underscoring the essential role of Notch signaling in NSPC expansion.

The Wnt pathway is also pivotal in regulating NSPC proliferation. In the absence of Wnt ligands, β-catenin is continuously targeted for degradation by the destruction complex [62]. Wnt binding to Frizzled and LRP5/6 recruits Dishevelled (Dvl), sequestering the destruction complex at the membrane and preventing β-catenin degradation. Stabilized β-catenin then accumulates, translocates to the nucleus, and partners with TCF/LEF transcription factors to activate target genes such as Cyclin D, which promote NSPC proliferation [63,64] (Figure 6b). Pathogenic missense variants in ZNRF3, a negative regulator of Wnt signaling that ubiquitinates and inactivates Frizzled, have been identified in NDD patients [65]. Variants clustered in the RING domain impair Frizzled ubiquitination, leading to excessive Wnt activity, aberrant NSPC proliferation, and macrocephaly. Conversely, microcephaly-associated ZNRF3 variants localize to the RSPO-binding domain, conferring resistance to RSPO inhibition and resulting in hyperactive Frizzled degradation, diminished Wnt signaling, reduced NSPC proliferation, and microcephaly [65,66].

The SHH pathway likewise regulates NSPC proliferation. SHH binding to the PTCH1 receptor relieves its inhibition of Smoothened (SMO), thereby enabling SMO activation [67]. Activated SMO promotes nuclear translocation of GLI transcription factors, which induce expression of proliferation-associated genes, including Cyclin D and Cyclin E [67] (Figure 6c). Consistently, mutations in SHH or PTCH1 have been identified in patients with microcephalic NDDs [68,69].

FGF and IGF-I also critically regulate NSPC proliferation. These ligands bind to their receptors (FGFR and IGF-IR, respectively), activating shared downstream pathways—RAS-MAPK (RAS → RAF → MEK → MAPK) and PI3K-mTOR (PI3K → AKT → mTOR)—that are most extensively characterized [70,71,72]. The MAPK cascade phosphorylates transcription factors that upregulate cell cycle drivers such as Cyclin D [73,74], while mTOR promotes progression by degrading ARF [75], and repressing CDK inhibitors p21 and p27 [76] (Figure 6d). PI3K-mediated phosphorylation of PIP2 to PIP3 activates AKT, whereas PTEN antagonizes this pathway by dephosphorylating PIP3 back to PIP2, thereby acting as a brake on NSPC proliferation [77]. The pathological relevance of these signaling cascades to NDDs is underscored by numerous genetic findings: loss-of-function mutations in FGFR1 [78], FGFR3 [79], FGF8 [80,81], IGF-1 [82,83,84], IGF-IR [85,86], and AKT3 [87] have been linked to microcephalic NDDs. Conversely, loss-of-function mutations in PTEN [88,89,90,91], stabilizing mutations in Cyclin D2 [92], and activating mutations in PI3K [91,93,94], AKT3 [87,93,94], and mTOR [91,93,95] are consistently associated with macrocephalic NDDs.

In addition to the signaling pathways described above, several transcriptional regulators critically modulate NSPC proliferation. Loss of function of the transcription factor ARX has been associated with NDDs accompanied by microcephaly [96]. ARX deficiency leads to upregulation of p57, a Cip/Kip family CDK inhibitor (Figure 6e), which is normally repressed by ARX. This results in premature cell cycle exit and depletion of progenitor pools [97]. Moreover, ARX loss elevates expression of the transcription factor OLIG2 [98], which in turn reduces neurogenesis and contributes to overall brain size reduction [99]. Mechanistically, OLIG2 overexpression downregulates PAX6, a key transcription factor governing both proliferation and differentiation of cortical progenitors [98,99].

PAX6 positively regulates several cell cycle–promoting genes, including CDK2, CDK4, and Cyclin E, thereby enhancing NSPC proliferation during embryonic brain development [100] (Figure 6e). Consistently, compound heterozygous PAX6 mutations are associated with microcephaly in human patients [101,102]. Interestingly, PAX6 also functions as a repressor of NSPC cell cycle progression, as evidenced by studies in mice carrying Pax6 mutations [103] or conditional Pax6 deletions in developing neurons [104]. This inhibitory effect is likely mediated by suppression of CDK6 expression. Supporting evidence includes PAX6 binding to regulatory regions surrounding the Cdk6 locus [104], rapid upregulation of Cdk6 following acute Pax6 loss [105], and reduced NSPC proliferation upon CDK6 inhibition or gene knockout [104,106]. Consistently, CDK6 mutations have been linked to microcephaly [107].

Notably, PAX6 simultaneously promotes neuronal differentiation by activating neurogenic gene expression, suggesting a dual role in balancing NSPC proliferation and neurogenesis [100]. Indeed, experimental evidence from Pax6-mutant mice demonstrates that Pax6 loss reduces NSPC proliferation, whereas Pax6 overexpression accelerates neuronal differentiation at the expense of progenitor expansion [100]. Thus, both upregulation and downregulation of Pax6 ultimately converge on a common outcome—microcephaly. This sensitivity is partly managed by its antagonistic interactions within a network of neurogenic transcription factors such as Neurog2, Ascl1, Hes1 [100]. These findings suggest that the seemingly paradoxical roles of PAX6—in both promoting and restraining NSPC proliferation—are governed by context-dependent mechanisms, including gene dosage effects and interactions with other transcriptional regulators.

Mutations in CHD8, a gene encoding a chromatin remodeler, have been strongly associated with macrocephaly, suggesting that CHD8 acts as a transcriptional brake on NSPC proliferation. Consistently, haploinsufficiency of CHD8—commonly observed in NDD patients—shortens the cell cycle and enhances NSPC proliferation [108]. This phenotype has been attributed to the upregulation of Cyclin E, a key driver of S-phase entry [108] (Figure 6e). Similarly, germline Chd8 haploinsufficiency in mice accelerates NSPC proliferation, accompanied by dysregulated expression of cell cycle–associated genes [109]. Although these findings support a role for CHD8 as a negative regulator of cell proliferation, other studies have reported that CHD8 promotes NSPC proliferation at both cellular and organismal levels [110,111]. These discrepancies may reflect methodological differences, as studies reporting pro-proliferative roles primarily employed mRNA knockdown rather than genetic knockout models, suggesting that the degree of CHD8 reduction critically influences NSPC outcomes.

Gene dosage effects on chromatin remodeling also extend to components of the BAF (SWI/SNF) complex. Duplication of ARID1A (also known as BAF250A), a core subunit of the BAF complex, has been associated with microcephaly [112]. Conversely, haploinsufficiency of ARID1B (also known as BAF250B), a mutually exclusive paralog of ARID1A, similarly results in microcephaly [113,114,115], implying antagonistic roles in NSPC proliferation—ARID1A-BAF functioning as a repressor and ARID1B-BAF as an activator. Supporting this notion, conditional deletion of Arid1a in murine NSPCs enhances proliferation and upregulates cell cycle–related genes [116] (Figure 6e). By contrast, the role of ARID1B in NSPC proliferation remains largely unexplored. Nonetheless, Arid1b-deficient murine embryonic stem cells, as well as patient-derived fibroblasts with ARID1B haploinsufficiency, exhibit reduced proliferation accompanied by downregulation of CDC20, a key positive regulator of cell cycle progression [117,118]. Whether NSPC proliferation is similarly attenuated by ARID1B reduction remains an open question warranting further investigation.

Another gene whose mutation disrupts NSPC proliferation through transcriptional dysregulation is SETD5, an epigenetic regulator that modulates rDNA transcription and translation of key cell cycle proteins such as Cyclin D [119] (Figure 6e). Haploinsufficiency of SETD5 leads to reduced rRNA synthesis and impaired NSPC proliferation [119]. In humans, heterozygous loss-of-function mutations in SETD5 cause a neurodevelopmental disorder termed IDD23 (also known as MRD23) [120]. Intriguingly, pathogenic SETD5 variants have also been identified in patients presenting with KBG-like phenotypes—syndromes that phenotypically overlap with KBG syndrome [121,122].

Haploinsufficiency of another epigenetic regulator, ANKRD11, is recognized as the primary genetic cause of KBG syndrome, suggesting a functional interplay between ANKRD11 and SETD5 [120]. Supporting this notion, recent studies have shown that ANKRD11 positively regulates SETD5 expression, thereby sustaining translational capacity and neuronal proliferation—processes essential for mitigating NDD pathogenesis [123] (Figure 6e). Notably, both IDD23 and KBG syndrome patients frequently exhibit microcephaly, underscoring the crucial role of SETD5- and ANKRD11-mediated control of NSPC proliferation in maintaining proper brain development [124,125,126].

Key regulators that directly control NSPC proliferation also include centrosomal and spindle apparatus proteins such as ASPM, CDK5RAP2, WDR62, and MCPH1, mutations in which are causatively linked to microcephaly in humans [127,128]. Defects in these genes disrupt mitotic spindle orientation and impair symmetric cell divisions, resulting in premature depletion of the progenitor pool and consequent reduction in cortical size [127,128] (Figure 6f).

4.2. Environmental Factors

Environmental factors can also profoundly impact NSPC proliferation. Maternal infection with the Zika virus has been firmly established as a cause of congenital microcephaly and NDDs in offspring [129,130,131,132]. Consistent with these observations, Zika virus infection disrupts NSPC proliferation in developing mouse brains [133,134]], as well as in human three-dimensional brain organoids [135,136]. Although numerous cell cycle–related genes are downregulated [137,138], accumulating evidence indicates that premature activation of CDK1 and unscheduled mitotic entry, together with centrosomal amplification, lead to aberrant chromosome segregation, resulting in NSPC proliferation defects and cell death [137,138,139,140] (Figure 7a). This cascade is considered a primary mechanism underlying the proliferation defects observed in Zika virus–infected NSPCs.

In utero drug exposure also contributes to NDDs associated with microcephaly. Valproic acid (VPA), a widely used antiepileptic and mood stabilizer, has been shown to increase the risk of NDDs with microcephaly [141,142]. Experimental studies in mice have demonstrated that embryonic exposure to VPA induces microcephaly and behavioral phenotypes resembling NDDs [143,144,145], ruling out the possibility that maternal psychiatric conditions alone account for the observed abnormalities. Mechanistically, VPA exposure impairs NSPC proliferation in mice through upregulation of several cell cycle inhibitors [145] (Figure 7b). In human brain organoids, ARF—but not other CDK inhibitors—was significantly induced following VPA treatment, implicating ARF as a key mediator of NSPC proliferation defects [145] (Figure 7b).

Another environmental and metabolic risk factor for microcephalic NDDs is phenylketonuria (PKU), arising from either maternal or inborn deficiency [146,147]. Most PKU cases result from pathogenic loss-of-function variants in PAH, encoding phenylalanine hydroxylase, the enzyme responsible for converting phenylalanine to tyrosine. This defect leads to elevated phenylalanine levels in both blood and brain [146,147]. Studies using human iPSC-derived brain organoids have demonstrated that high phenylalanine concentrations selectively impair NSPC populations, while mature neurons and glia remain largely unaffected, highlighting the heightened vulnerability of NSPCs to metabolic stress [148]. Transcriptomic profiling further demonstrated a broad downregulation of genes essential for cell cycle progression, including CDT1 and MCMs involved in the S phase, as well as ASPM and CDK5RAP2 associated with the M phase [148] (Figure 7c). These findings suggest that impaired NSPC proliferation, driven by defective cell cycle regulation, directly contributes to microcephalic NDD in PKU.

5. Future Directions and Therapeutic Perspectives

The growing body of evidence implicating defective NSPC proliferation in the etiology of NDDs highlights the urgent need to translate mechanistic insights into therapeutic strategies. Future research should aim to delineate how distinct genetic mutations, epigenetic dysregulation, and environmental exposures converge on shared cell cycle and signaling pathways in NSPCs. Integration of single-cell transcriptomics, proteomics, and epigenomics with brain organoid models and in vivo systems will enable the identification of temporal and lineage-specific vulnerabilities that underlie aberrant proliferation. Such high-resolution approaches may reveal common “molecular bottlenecks” in NSPC regulation, offering tractable targets for intervention.

From a therapeutic standpoint, several avenues hold promise. Pharmacological modulation of core signaling cascades—such as Notch, Wnt, SHH, and PI3K–mTOR—could restore balanced NSPC proliferation, provided that interventions are carefully titrated to avoid exacerbating either hypo- or hyperplastic states. Small-molecule inhibitors of CDKs or pathway-specific agonists/antagonists represent potential candidates, some of which are already under clinical investigation for oncology and may be repurposed for NDDs. Epigenetic regulators such as CHD8, SETD5, and ANKRD11, whose dysfunction directly impairs NSPC proliferation, are also attractive therapeutic nodes, particularly as pharmacological modulators of chromatin structure and transcriptional machinery continue to expand.

Ultimately, future efforts will benefit from interdisciplinary approaches that link developmental neurobiology, stem cell biology, and translational neuroscience. By bridging the gap between mechanistic understanding and therapeutic application, these studies have the potential not only to uncover predictive biomarkers of disease severity but also to lay the groundwork for preventive and restorative interventions in NDDs. The challenge ahead is to transform knowledge of NSPC biology into tangible therapies that can improve neurodevelopmental trajectories and quality of life for affected individuals.