Neuroglial Dysregulation in Autism Spectrum Disorder: Pathogenetic Insights, Genetic Threads, and Therapeutic Horizons

Abstract

Background/Objectives: Autism Spectrum Disorder (ASD) is a complex neurodevelopmental condition marked by challenges in social communication, restricted interests, and repetitive behaviors. Recent studies highlight the crucial roles of neuroglial cells—astrocytes, microglia, and oligodendrocytes—in synaptic function, neural connectivity, and neuroinflammation. These findings offer a fresh perspective on ASD pathophysiology. This review synthesizes current knowledge on neuroglial dysfunction in ASD, emphasizing its role in pathophysiological mechanisms, genetic influences, and potential therapeutic strategies. Methods: We conducted a comprehensive literature review, integrating insights from neuroscience, molecular biology, and clinical studies. Special focus was given to glial-mediated neuroinflammatory mechanisms, synaptic plasticity regulation, and the impact of genetic mutations on neuroglial signaling and homeostasis. Results: Neuroglial dysfunction in ASD is evident in abnormal synaptic pruning by microglia, impaired astrocytic glutamate regulation, and defective oligodendrocyte-driven myelination, which collectively disrupt neuronal architecture. Emerging therapies targeting these pathways, including anti-inflammatory drugs, microglial modulators, and cell-based approaches, show promise in alleviating key ASD symptoms. Additionally, advanced interventions such as gene editing and glial progenitor therapy present opportunities to correct underlying neuroglial dysfunction. Conclusions: This review establishes a comprehensive framework for understanding neuroglial contributions to ASD. By integrating insights from diverse disciplines, it enhances our understanding of ASD pathophysiology and paves the way for novel therapeutic strategies targeting neuroglial pathways.

1. Introduction

Autism Spectrum Disorder (ASD) is a pervasive neurodevelopmental condition affecting approximately 1 in 36 children globally, with a rising prevalence due to improved diagnostic tools and increased awareness [1,2]. Males are diagnosed with ASD more frequently than females, with a ratio of approximately 4:1. However, evidence suggests that subtler symptom presentations or masking behaviors may lead to underdiagnosing in females [3,4]. ASD is characterized by clinically considerable heterogeneity. Core symptoms include difficulties with social communication, restricted interests, and repetitive behavior [5]. These core features often present alongside a spectrum of associated comorbidities, such as global developmental delay, intellectual disabilities, various forms of epilepsy, gastrointestinal issues, anxiety, and sleep disturbances [6,7]. The severity of symptoms can range from mild, allowing for independent living and functional communication, to profound, requiring lifelong support [8].

ASD’s clinical manifestations typically emerge by the age of three years, although subtle signs may be observed as early as infancy. Early indicators include delayed speech and language development, reduced eye contact, atypical social interactions, and repetitive motor behaviors such as hand-flapping, rocking, or spinning [9]. Sensory sensitivities, including aversion to certain sounds, textures, or lights, are also common [10,11]. The clinical trajectory varies widely, with some children showing marked improvement with early intervention, while others experience persistent or worsening symptoms [12].

The diagnosis of ASD is clinical and based on criteria specified in the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) [13]. Key diagnostic criteria include persistent deficits in social communication and interaction across multiple contexts, as well as restricted, repetitive patterns of behavior, interest, or activity [14]. Diagnostic tools commonly used include structured interviews like the Autism Diagnostic Interview-Revised (ADI-R) and standardized assessments like the Autism Diagnostic Observation Schedule (ADOS) [12,15]. A multidisciplinary team consisting of pediatricians, neurologists, psychologists, and speech therapists often conducts developmental, behavioral, and cognitive assessments as part of their evaluations [14]. Neuroimaging, laboratory, and genetic testing are not diagnostic but can provide supportive evidence [5,16]. MRI scans of the brain often show structural and functional problems in children with ASD. These problems include different connections in the default mode network and less gray matter in the amygdala and prefrontal cortex [17,18].

Various forms of genetic testing are part of the diagnostic algorithm for ASD. Testing typically follows established clinical guidelines that aim to detect syndromic forms of autism. These syndromes are characterized by well-documented genetic mutations that contribute to ASD symptoms and other associated features [19].

For suspected monogenic syndromes with the clinical presentation of ASD, such as fragile X syndrome, targeted tests like FMR1 gene analysis are often the first choice [20]. The testing algorithm usually proceeds with chromosomal microarray analysis (CMA), which is widely recommended as a first-tier test due to its ability to identify submicroscopic chromosomal deletions or duplications that are frequently associated with ASD [21]. Additionally, next-generation sequencing (NGS), including whole-exome sequencing (WES) or panel testing of autism-related genes, is increasingly used to identify mutations in single genes linked to ASD [22]. While these tests have significantly advanced our understanding and diagnostic capabilities, their overall detection rate for pathogenic variants in children with isolated, non-syndromic forms of ASD remains approximately 10–20% [16,23].

Chromosomal abnormalities, single-gene mutations, and copy number variations (CNV) have been identified as a potential key contributor to ASD, with many of these genetic changes impacting synaptic function, neurogenesis, and neuronal migration [16,19]. A detailed overview of the major genes involved, their functions, and their impact on neurodevelopment is provided in

Table 1. Genetic Factors Associated with Neuroglial Dysfunction in Autism.

Gene	Function	Neuroglial Changes
CNTNAP2	Synaptic adhesion and neuronal communication	Astrocyte and microglial dysfunction
MECP2	Epigenetic regulation of gene expression	Altered expression of GABAergic receptors
PTEN	Regulation of cell growth and survival	Reduced oligodendrocyte proliferation
SHANK3	Synaptic signaling proteins	Disruption in synaptic maintenance

This table summarizes key genes implicated in ASD that affect neuroglial function. The listed genes are involved in various cellular processes, including synaptic adhesion, epigenetic regulation, cell survival, and synaptic signaling. Their dysfunction contributes to astrocytic and microglial abnormalities, altered neurotransmission, and impaired oligodendrocyte proliferation, all of which are relevant to ASD pathophysiology.

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Environmental factors, including advanced parental age, prenatal infections, and exposure to toxins like valproic acid, have been linked to an elevated risk of ASD [24,25,26]. Epigenetic modifications, such as DNA methylation and histone acetylation, provide a valuable link between genetic predisposition and environmental triggers [25,26,27]. These modifications regulate gene expression without altering the underlying DNA sequence, allowing dynamic responses to environmental influences. For instance, changes in DNA methylation patterns can silence or activate key genes involved in neurodevelopmental processes, while histone acetylation alters chromatin structure, influencing the accessibility of transcriptional machinery [27,28,29]. Epigenetic mechanisms like these are a major part of how prenatal environmental exposures affect glial function and neurodevelopment [30]. Emerging evidence suggests that maternal immune activation during pregnancy may alter fetal brain development via cytokine-driven neuroinflammatory mechanisms. These immune-related disruptions are particularly concerning during critical developmental windows, as they may interfere with processes such as synaptic pruning and myelination, potentially leading to long-term neurodevelopmental consequences [31].

Astrocytes, microglia, and oligodendrocytes are vital components of the central nervous system, contributing to both the structural and functional maturation of the brain. These neuroglial cells regulate fundamental processes, including synaptogenesis, immune homeostasis, and neuronal support, all of which are essential for establishing and maintaining neural architecture [32]. Given their diverse roles, disruptions in neuroglial function can significantly impact brain connectivity and plasticity [33,34]. A detailed overview of the key functions of astrocytes, microglia, and oligodendrocytes, along with specific alterations observed in ASD pathophysiology, is provided in

Table 2. Roles of Neuroglial Cells and Changes in ASD Pathophysiology.

Glial Cell Type	Key Physiological Functions	Alterations in ASD	Functional Implications in ASD
Astrocytes	Regulate synaptic transmission, maintain the blood-brain barrier, and supply metabolic support	Increased GFAP expression, reduced glutamate transport, and altered cytokine release	Excitotoxicity, impaired homeostasis, and neuroinflammation
Microglia	Mediate synaptic pruning and immune surveillance	Persistent activation, excessive complement signaling, and overproduction of pro-inflammatory cytokines	Abnormal synaptic connectivity, neuroinflammation, and impaired neurodevelopment
Oligodendrocytes	Myelinate axons to ensure fast and efficient signal transmission	Delayed maturation, reduced myelination, and dysregulated oligodendrocyte precursor cells	Disrupted neuronal communication and decreased brain network efficiency
Ependymal Cells	Facilitate cerebrospinal fluid (CSF) flow and neurogenesis	Altered CSF flow dynamics and potential influence on neurogenic niches	Impaired clearance of metabolites and possible disruptions in neurogenesis
Radial Glia	Guide neuronal migration and differentiation during development	Dysregulated guidance signals and proliferation in early development	Misplacement of neurons and disrupted cortical architectonic

This table provides an overview of the primary physiological roles of different neuroglial cell types and the alterations observed in ASD. Astrocytes, microglia, oligodendrocytes, ependymal cells, and radial glia each play essential roles in maintaining neural homeostasis, synaptic connectivity, and brain development. In ASD, disruptions in these glial functions contribute to altered synaptic transmission, neuroinflammation, impaired myelination, and deficits in neuronal migration. The listed changes highlight the importance of neuroglial dysfunction in the pathophysiology of ASD and its impact on overall brain network organization.

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Astrocytes play a critical role in metabolic exchange by linking the vasculature and neurons through the delivery of glucose and lactate, essential substrates for neuronal function. They play a crucial role in neurovascular coupling by modulating blood flow in the brain in response to neuronal activity [35,36].

Microglia serve as the primary immune effector cells of the central nervous system, playing a vital role in maintaining immune homeostasis. They are integral to synaptic pruning, a developmental process that refines neural networks by eliminating redundant or unnecessary synapses [33,37]. Oligodendrocytes, on the other hand, are essential for preserving axonal functionality and structural integrity. They achieve this by synthesizing myelin sheaths, which facilitate rapid propagation of action potentials and provide metabolic support to axons [38].

The coordinated actions of these neuroglial cells provide the dynamic processes that shape neural architecture, which form the basis for higher cognitive and behavioral functions [32,39]. Neuroglia, once considered merely supportive elements for neurons, are currently considered major players in shaping neural connectivity, regulating synaptic dynamics, and maintaining homeostatic balance within the central nervous system [40].

Recent progress in imaging and molecular biology has provided a new way to look at glial abnormalities in ASD, shedding light on their roles in dysregulated synapses, neuroinflammation, and changed neural circuitry. These findings not only deepen our understanding of ASD pathophysiology but also point to neuroglia as promising targets for novel therapeutic strategies [18,33,41].

The following sections will provide a detailed examination of the distinct roles of astrocytes, microglia, and oligodendrocytes in normal brain development, the disruptions in these functions observed in ASD, and the genetic and molecular pathways linking glial dysfunction to the clinical manifestations of this neurodevelopmental disorder.

2. Role of Neuroglia in Typical Brain Development

2.1. Astrocytes

Astrocytes are fundamental to maintaining homeostasis in the neural environment and play an essential role in supporting normal brain development and function. They regulate synaptic development and function by modulating neurotransmitter uptake, particularly glutamate, and converting it to glutamine to prevent excitotoxicity, a process that can severely damage neurons due to excessive glutamate accumulation in the synaptic cleft. This regulation is crucial during early developmental stages when synaptic over-activity can disrupt network formation [35,36].

During critical periods of neural growth and plasticity, astrocytes ensure metabolic support by transporting glucose and lactate to neurons through specialized transport systems [42]. In addition, astrocytes release trophic factors such as brain-derived neurotrophic factor (BDNF), which is essential for neuronal survival, differentiation, and synaptic maturation [43]. These cells modulate calcium signals within astrocytic networks, enabling coordinated responses to neuronal activity and influencing synaptic plasticity [35].

Astrocytic endfeet, which surround blood vessels, contribute to the blood-brain barrier (BBB), regulating nutrient exchange and protecting the brain from harmful substances [44]. During development, problems with astrocytic functions can cause changes in synaptic connectivity and neurovascular deficits, both of which are linked to neurodevelopmental disorders [35,36].

2.2. Microglia

Microglia are the main immune cells of the brain. They play an indispensable role during neural development that extends beyond immune surveillance to include the critical refinement of neural circuits [37]. Through dynamic interactions with neurons and other glial cells, microglia synchronize an essential process for optimizing neural network architecture called synaptic pruning [33]. This activity is particularly vital during developmentally sensitive periods, as it facilitates the elimination of excessive or suboptimal synapses, thereby strengthening functional connectivity and ensuring the maturation of efficient neural pathways [45].

Microglia dynamically survey the brain’s environment. They respond to molecular signals from neurons and astrocytes to identify and eliminate unnecessary synapses through phagocytosis [46]. Signaling pathways, such as complement proteins (e.g., C1q and C3), tightly regulate this activity by tagging synapses for removal [47]. It also helps neural growth by releasing cytokines and growth factors like interleukin-1β (IL-1β) and insulin-like growth factor-1 (IGF-1) [37]. Microglia play a critical role in maintaining neural homeostasis by removing apoptotic cells and clearing cellular debris. This process is essential for proper neurodevelopment [48]. Dysregulated microglial activity, whether excessive or insufficiently regulated, can lead to abnormal synaptic pruning. Such disruptions may result in either excessive synaptic loss or aberrant connectivity, both of which are implicated in the pathogenesis of developmental brain disorders [33,39].

2.3. Oligodendrocytes

Oligodendrocytes are the primary cells responsible for myelination within the central nervous system. Proper myelination ensures rapid and efficient propagation of electrical signals along neuronal axons [38]. Myelination is a dynamic and tightly regulated process during brain development, essential for the maturation and functionality of neural circuits. Oligodendrocyte precursor cells (OPCs) respond to neuronal activity by migrating toward axons that require myelination. Upon arrival, they differentiate into mature oligodendrocytes and wrap axons with concentric layers of myelin [49].

In addition to speeding up signal transmission, myelination plays a role in maintaining axonal integrity by protecting axons from metabolic stress [50]. Oligodendrocytes also provide metabolic support through lactate transfer to axons, ensuring their long-term viability [51].

The timing and pattern of myelination are essential for cognitive and motor development. Disruptions in oligodendrocyte function or myelination during early development can impair the connectivity of neural networks, resulting in deficits in learning, memory, and behavior [52].

Myelination is increasingly recognized as a modulator of neural plasticity, supporting adaptive changes in brain connectivity throughout life [38]. Activity-dependent myelination enables neural circuits to fine-tune their functionality in response to environmental stimuli, learning experiences, and behavioral demands [53]. This dynamic process highlights the importance of oligodendrocytes not only in early development but also in maintaining cognitive flexibility in adulthood [54,55].

Neuroimaging studies have identified widespread disruptions in white matter integrity associated with impaired oligodendrocyte function and aberrant myelination in ASD [17]. Reduced fractional anisotropy in major white matter tracts, including the corpus callosum and the superior longitudinal fasciculus, has been linked to the social and cognitive deficits characteristic of ASD [56,57]. Furthermore, experimental evidence indicates that genetic variants influencing oligodendrocyte differentiation, such as those involving OLIG2 and CNTNAP2, exacerbate these disruptions [58,59].

2.4. Neurone-Glia Interactions

The cooperative interaction between neurons and glial cells synchronises synaptic plasticity, neuronal migration, and overall brain maturation [40]. Astrocytes, for example, release thrombospondins and hevin, which promote the formation of new synapses [35]. Microglia refine these connections by pruning redundant synapses through complementary system activity [47]. In turn, oligodendrocytes support long-distance neuronal communication by insulating axonal pathways with myelin, ensuring efficient information transfer between brain regions [49].

Neuronal activity further modulates this complex interplay by providing feedback to glial cells, directing their functions to areas of high demand. For instance, increased neuronal firing can recruit astrocytic support for ion buffering and metabolic exchange or signal oligodendrocytes to augment myelination in active circuits [52,60].

These feedback mechanisms are crucial for adaptive plasticity during learning and development. Disruptions in neurone–glia interactions can have cascading effects on brain development, leading to impaired synapse formation, reduced myelination, or excessive inflammation. These deficits contribute to the pathogenesis of various neurodevelopmental disorders, including ASD [61].

3. Results

This section summarizes key findings on neuroglial dysfunction in ASD, focusing on cellular, molecular, and genetic alterations that contribute to the disorder.

4. Neuroglial Dysregulation in Autism Spectrum Disorder

Dysregulation of neuroglial function in ASD profoundly affects brain development and activity. Impaired glial cell function contributes to core features of ASD, including deficits in social communication, repetitive behaviors, and cognitive impairments [39]. Recent research highlights reactive astrogliosis, hyperactive microglial pruning, and impaired myelination as critical glial abnormalities contributing to ASD pathophysiology [62]. These glial changes alter neuronal signaling and network dynamics, leading to impaired cognitive and behavioral outcomes [39,62]. A summary of key molecular changes observed in astrocytes, microglia, and oligodendrocytes in ASD is presented in

Table 3. Key Molecular Changes in Neuroglia in Autism.

Molecular Change	Glial Cell Type	Functional Consequences
Increased pro-inflammatory cytokines	Microglia	Leads to sustained neuroinflammation and impaired synaptic pruning
Reduced glutamate uptake	Astrocytes	Causes excitotoxicity and disruption of the excitatory–inhibitory balance
Altered complement Signaling	Microglia	Impairs synapse elimination, contributing to abnormal connectivity
Dysregulated GFAP Expression	Astrocytes	Indicates reactive gliosis, which can disturb neuronal support and homeostasis
Defective myelination processes	Oligodendrocytes	Slows neuronal signaling and disrupts communication between brain regions
Epigenetic changes (e.g., DNA methylation, histone modifications)	All glial types	Alters gene expression involved in neurodevelopment and synaptic maintenance

This table summarizes major molecular alterations in neuroglial cells observed in ASD. These changes include dysregulation of cytokine production, glutamate uptake, complement signaling, and GFAP expression, each of which contributes to disruptions in synaptic pruning, neuronal homeostasis, and the excitatory–inhibitory balance. Additionally, defects in oligodendrocyte-mediated myelination and epigenetic modifications across multiple glial cell types further influence brain connectivity and neurodevelopment. Understanding these molecular abnormalities provides insight into the mechanisms underlying neuroglial dysfunction in ASD.

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Astrocytes are essential for preserving synaptic homeostasis, providing neuronal support, and maintaining general cerebral equilibrium [36,42]. In ASD, astrocytic dysfunction disrupts these processes, contributing to pathophysiological changes observed in the condition [36,62].

Astrocytes express Glial Fibrillary Acidic Protein (GFAP), a cytoskeletal protein indicative of their reactive state [63]. Post-mortem analyses of ASD brains reveal elevated GFAP levels, consistent with chronic astrogliosis and associated neuroinflammation [64]. Reactive astrocytes demonstrate altered calcium signaling, disrupting their ability to respond appropriately to synaptic activity. This dysregulation impairs critical functions, including neurotransmitter recycling (e.g., glutamate–glutamine cycling) and ion homeostasis, leading to excitatory–inhibitory imbalances in neural circuits [65].

Emerging evidence suggests that distinct reactive astrocyte subtypes, such as A1 (neurotoxic) and A2 (neuroprotective), may differentially contribute to ASD pathophysiology. Such changes further exacerbate the disruption of synaptic plasticity, neuronal connectivity, and overall network stability [65].

Astrocytes play a crucial role in the glutamate–glutamine cycle by converting synaptic glutamate into glutamine, thereby preventing excitotoxicity and maintaining synaptic homeostasis [66]. In ASD, disruptions in glutamate uptake have been associated with reduced expression of excitatory amino acid transporter 2 (EAAT2), the primary astrocytic glutamate transporter. This reduction leads to elevated extracellular glutamate levels, resulting in excitotoxic neuronal injury and altered neural circuit functionality. Such dysregulation of glutamate signaling has been implicated in the emergence of core ASD features, including repetitive behaviors and sensory processing abnormalities [67,68].

Neurovascular coupling is successfully mediated by the coordinated activity of astrocytes. They mediate communication between neurons and blood vessels to regulate cerebral blood flow and meet metabolic demands [42,44]. In ASD, astrocytic dysfunction reduces cerebral blood flow, particularly in regions critical for higher cognitive and social functions [69,70]. This impaired neurovascular coupling compromises metabolic support to neurons, further exacerbating neural circuit dysregulation [60]. Neuroimaging studies consistently report hypoperfusion in key areas such as the prefrontal cortex and anterior cingulate cortex, regions associated with executive function and social cognition [71].

Beyond astrocytic dysfunction, microglia also exhibit profound abnormalities in ASD [33]. These cells play a critical role in synaptic pruning, immune surveillance, and inflammatory regulation, yet in ASD, they often display chronic activation and excessive complement-mediated synapse elimination [33,45]. The growing body of research suggests that microglial hyperactivity contributes to altered neuronal circuit formation, excessive synaptic connectivity, and persistent neuroinflammation, all of which are hallmarks of ASD neuropathology [33].

Microglial pruning of synaptic connections is tightly regulated by complement proteins (e.g., C1q and C3). Dysregulation of this pathway in ASD leads to excessive or insufficient pruning, resulting in either hyperconnectivity or synaptic deficits [47]. The latest study investigated the role of complement in ASD development using multiple reaction monitoring (MRM). The study has successfully identified 16 out of 33 proteins within these pathways as differentially expressed in the plasma of children with ASD compared to controls. Notably, CFHR3, C4BPB, C4BPA, CFH, C9, SERPIND1, C8A, F9, and F11 were reported as altered in ASD plasma for the first time. Among these, SERPIND1 expression showed a positive correlation with ASD severity, as measured by the Childhood Autism Rating Scale (CARS). Furthermore, machine learning analysis identified a panel of 12 differentially expressed proteins with potential diagnostic utility for ASD [72].

Animal models demonstrate that imbalances in microglial activity contribute to behavioral rigidity and sensory hypersensitivity, trades that are commonly observed in ASD patients [73]. Interestingly, human postmortem and imaging studies have also reported alterations in microglial activation patterns in ASD, further bridging preclinical and clinical findings [74].

In addition to astrocytes and microglia, oligodendrocytes—the myelinating cells of the central nervous system—also exhibit significant dysfunction in ASD [75]. Proper oligodendrocyte maturation and myelination are essential for efficient neuronal communication, yet studies indicate that ASD is associated with delayed oligodendrocyte precursor maturation, impaired myelin production, and reduced white matter integrity [38,49,52]. Diffusion tensor imaging (DTI) studies have consistently demonstrated structural abnormalities in major white matter tracts, further supporting the role of oligodendrocyte dysfunction in the pathophysiology of ASD [76].

Hypomyelination is a prominent feature observed in ASD brains [50,62]. Studies indicate delayed or reduced oligodendrocyte differentiation and myelination in key brain regions, such as the corpus callosum and prefrontal cortex [77]. Longitudinal neuroimaging studies show that impaired white matter integrity often appears early in ASD and persists throughout development [78]. The convergence of neuroimaging and genetic studies highlights a critical role for disrupted myelination in ASD, further expanding our understanding of how neuroglial dysfunction contributes to altered brain connectivity. These deficits correlate with impairments in executive function, social behavior, and sensory processing, linking structural abnormalities to core behavioral features of ASD [77,79].

Furthermore, neuroimaging studies of individuals with ASD consistently report reduced fractional anisotropy (FA), a key measure of white matter integrity that reflects microstructural disruptions in neural connectivity. These alterations are particularly pronounced in major white matter tracts, including the corpus callosum, which is crucial for interhemispheric communication, and the superior longitudinal fasciculus, which facilitates long-range connectivity between frontal, parietal, and temporal regions. Reduced FA in these pathways has been linked to deficits in social communication, executive function, and sensory processing, further underscoring the role of white matter abnormalities in ASD pathophysiology. Such findings align with the broader hypothesis that disrupted neural connectivity is a core feature of ASD, contributing to atypical information processing and cognitive function [80,81].

5. Integrative Insights: Dysregulated Pathways and Genetic Base of Neuroglial Dysfunction in ASD

Dysregulated signaling pathways, chronic neuroinflammation, and genetic and epigenetic factors intricately interact to shape neuroglial dysfunction in ASD. These interconnected mechanisms disrupt synaptic homeostasis, neuronal–glial communication, and overall brain connectivity, contributing to the hallmark features of ASD [27,62,65].

Among these pathways, the mammalian target of the rapamycin (mTOR) signaling pathway and the wingless-related integration site/β-catenin (Wnt/β-catenin) signaling pathway signaling cascades emerge as crucial regulators of neurodevelopmental processes that are often altered in ASD [82,83]. The mammalian target of the rapamycin (mTOR) pathway, a central controller of cellular metabolism, homeostasis, integrating signals from nutrients, growth factors, energy availability, and synaptic plasticity, is highly sensitive to environmental and genetic cues that shape early brain development. It functions through two distinct complexes, the mammalian target of rapamycin complex 1 and 2 (mTORC1 and mTORC2), each with specific physiological roles. mTORC1 controls protein synthesis, lipid metabolism, and autophagy by phosphorylating downstream effectors such as S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1), promoting anabolic processes crucial for neuronal and glial function. It is highly active in synaptic plasticity, regulating dendritic spine formation and the remodeling of synaptic connections. mTORC2, in contrast, governs cytoskeletal organization and cell survival through the activation of Akt and protein kinase C (PKC), playing a role in neuronal differentiation and migration. In neuroglial physiology, mTOR signaling modulates astrocytic metabolic support, microglial immune responses, and oligodendrocyte-driven myelination, ensuring proper neuronal connectivity and brain network stability [84].

Persistent hyperactivation of mTOR in ASD has been associated with excessive synaptogenesis, disrupted neuronal connectivity, and impaired synaptic pruning. This dysregulation affects both neurons and glial cells, leading to an imbalance in excitatory and inhibitory neurotransmission [85,86]. Microglial dysfunction, driven by unchecked mTOR signaling, alters synaptic remodeling by either failing to eliminate redundant synapses or excessively pruning functional connections, both of which contribute to the atypical network architecture characteristic of ASD. Additionally, astrocytes, which play a vital role in maintaining synaptic homeostasis and metabolic support, exhibit abnormal glutamate clearance, altered calcium signaling, and disrupted neurovascular coupling due to mTOR dysregulation. The net effect is a hyperconnected but functionally inefficient neural circuitry, manifesting as the core behavioral and cognitive symptoms of ASD [87,88].

In parallel, the Wnt/β-catenin pathway plays an essential role in orchestrating neurodevelopmental processes, including synaptic assembly, glial differentiation, and myelination [89,90]. The pathway is initiated when Wnt proteins, a family of secreted glycoproteins, bind to Frizzled receptors and low-density lipoprotein receptor-related proteins (LRP5/6) on the cell membrane. This interaction inhibits the activity of the β-catenin destruction complex, which includes adenomatous polyposis coli (APC), axin, and glycogen synthase kinase-3β (GSK-3β). As a result, β-catenin accumulates in the cytoplasm and translocates into the nucleus, where it interacts with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors to regulate the expression of genes involved in cell proliferation, differentiation, and survival [91,92].

In the central nervous system, Wnt/β-catenin signaling is essential for oligodendrocyte differentiation and myelination, as it modulates the balance between precursor cell proliferation and maturation. It influences axonal guidance and neuronal migration during early development, ensuring the proper establishment of cortical layers and functional neuronal networks. Additionally, Wnt/β-catenin signaling plays a crucial role in dendritic arborization, regulating synaptic stability and adaptability in response to environmental stimuli. Astrocytic function is also modulated by this pathway, as Wnt/β-catenin signaling controls astrocyte-mediated glutamate uptake, ion homeostasis, and trophic support for neurons. Furthermore, Wnt/β-catenin signaling regulates microglial activity, particularly in synaptic pruning and neuroinflammatory responses, helping maintain synaptic efficiency and circuit refinement [85,93].

Beyond its developmental functions, Wnt/β-catenin signaling remains active in the adult brain, contributing to synaptic maintenance, plasticity, and repair mechanisms in response to injury. Proper modulation of this pathway is critical for maintaining white matter integrity and overall brain connectivity, underscoring its significance in both neurodevelopment and ongoing neuronal function [94].

Altered Wnt/β-catenin activity in ASD has been associated with impaired white matter integrity, as evidenced by diffusion tensor imaging (DTI) studies, which reveal disrupted connectivity in major associative tracts such as the corpus callosum and superior longitudinal fasciculus [80]. Dysfunctional Wnt/β-catenin signaling also impacts astrocytic function, diminishing their ability to regulate synapse formation and plasticity while concurrently influencing microglial activation states [95]. Additionally, disruptions in Wnt/β-catenin signaling have been linked to the dysregulation of neural progenitor cell fate determination, further exacerbating the structural and functional abnormalities characteristic of ASD [96].

The interplay between these signaling pathways underscores the complexity of neuroglial dysfunction in ASD. Emerging evidence suggests that these pathways interact at multiple levels, with mTOR influencing Wnt/β-catenin activity and vice versa [86,95]. Dysregulation within one pathway may cascade into alterations in the other, compounding neurodevelopmental impairments. Understanding these intricate molecular interactions will be essential for advancing knowledge of ASD pathophysiology and identifying potential therapeutic targets. Given the central role of these pathways in regulating neuroglial homeostasis, targeting their dysregulation has become a promising avenue for therapeutic intervention [97]. Experimental studies demonstrate that pharmacological modulation of these pathways can restore glial function and improve behavioral outcomes in ASD models [98].

Besides dysregulation of the key signaling pathways, chronic neuroinflammation is another complex pathophysiological element of ASD. The intricate crosstalk between immune signaling and neuroglial function further complicates the pathophysiology of the disorder [99,100]. A growing body of evidence suggests that persistent activation of microglia and astrocytes contributes to aberrant synaptic pruning, impaired neuronal plasticity, and disruptions in excitatory/inhibitory balance, which are hallmark features of ASD [101]. Elevated levels of pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1β not only disrupt neuroglial communication but also exacerbate synaptic dysfunction by altering glutamatergic and GABAergic signaling, leading to cognitive and behavioral impairments [65,102].

Another form of the neuroinflammatory process connected with the pathogenesis of ASD is prenatal exposure to maternal immune activation (MIA) [31]. This exposure has been shown to induce long-term neuroglial abnormalities and ASD-like behavior in offspring, with experimental models demonstrating that maternal infection, autoimmunity, or other immune insults can lead to persistent alterations in microglial morphology and function. These immune-mediated changes are accompanied by an increased susceptibility to environmental stressors, further amplifying neurodevelopmental vulnerabilities [103,104].

However, neuroinflammation does not act in isolation but rather intersects with genetic and epigenetic mechanisms that govern neuroglial function [101]. Inflammatory signaling can influence gene expression patterns by modifying epigenetic landscapes, while genetic mutations in key ASD-related genes further exacerbate glial dysregulation [27,33].

Studies have shown that mutations in genes like PTEN, MECP2, and CNTNAP2 disrupt critical processes such as synaptic pruning, inflammation regulation, and myelination [105]. These genetic predispositions do not fully account for the complexity of ASD pathology [106]. Increasing evidence suggests that epigenetic mechanisms serve as a critical interface between genetic predispositions and environmental factors, fine-tuning neuroglial gene expression [107]. Modifications such as DNA methylation and histone acetylation dynamically regulate transcription in response to developmental cues, potentially mediating the long-term impact of prenatal exposures on neuroglial integrity [108].

Epigenetic mechanisms such as DNA methylation and histone acetylation modulate gene expression in response to environmental factors, linking prenatal exposures to long-term neuroglial deficits [29]. DNA methylation, primarily occurring at CpG islands, is a key epigenetic modification that typically leads to transcriptional silencing by altering chromatin structure and preventing transcription factor binding. In contrast, histone acetylation relaxes chromatin and facilitates gene transcription by neutralizing the positive charge of histone proteins, thereby allowing greater accessibility of regulatory elements [109].

These modifications are highly dynamic and responsive to external stimuli, including maternal stress, dietary factors, infections, inflammations and exposure to environmental toxins during critical periods of neurodevelopment. Disruptions in these epigenetic processes can lead to persistent changes in the gene expression involved in synaptic plasticity, glial differentiation, and neuroinflammation, thereby contributing to long-lasting alterations in brain structure and function. Understanding this delicate interplay between epigenetic regulation and neuroglial integrity provides valuable insight into how early-life environmental influences shape neurodevelopmental trajectories [27,29].

Advances in genome-editing tools have enabled precise investigations into these genetic contributions, allowing researchers to dissect the functional impact of specific epigenetic modifications and their role in neurodevelopmental processes. Techniques such as Clustered Regularly Interspaced Short Palindromic Repeats- Cas9 (CRISPR-Cas9) and base editing provide unprecedented control over gene regulation, facilitating the development of targeted therapeutic approaches. By refining our ability to manipulate epigenetic landscapes, these tools hold promise for correcting aberrant gene expression patterns associated with neuroglial dysfunction, paving the way for precision medicine strategies in neurological disorders [110].

6. Discussion

The findings presented above described the intricate role of neuroglial dysfunction in ASD, revealing how abnormalities in astrocytes, microglia, and oligodendrocytes contribute to altered synaptic connectivity, neuroinflammation, and impaired neuronal communication. These pathophysiological disruptions suggest that ASD is not solely a disorder of neuronal dysfunction, but rather a condition in which glial cells play a crucial regulatory role in shaping brain development and function [62].

Due to this complex interplay of neuroglial dysfunction in ASD, multiple therapeutic approaches have been explored [111]. These interventions target different aspects of glial pathology, ranging from neuroinflammation to synaptic remodeling and myelination support [112]. Below, we discuss key pharmacological and biological strategies that have shown promise in modulating neuroglial activity in ASD. A visual summary of these therapeutic strategies and their mechanisms of action is provided in

Table 4. Comprehensive Therapeutic Approaches Targeting Neuroglia in Autism.

Therapeutic Approach	Targeted Glial Cell	Mechanism of Action	Current Limitations	Future Prospects
Anti-inflammatory drugs	M	Reduces neuroinflammation and synaptic Remodeling	Limited efficacy in heterogeneous populations	Development of microglia-specific modulators
Glial progenitor transplants	O	Restores myelination and improves axonal functionality	Requires invasive procedures and long-term monitoring	Refinement of stem cell delivery techniques
Modulation of mTOR signaling	M/A	Limits hyperactivation and cell metabolism	Non-specific targeting can affect multiple pathways	Precision medicine approaches for targeted modulation
Cannabidiol (CBD)	A/M	Reduces neuroinflammation and modulates excitatory–inhibitory balance	Unclear long-term effects in pediatric populations	Research into optimized formulations for ASD
Epigenetic therapy	A	Reverses gene expression changes linked to glial dysfunction	Lack of specificity in targeting neuroglial cells	Tailored therapies based on individual epigenetic profiles

This table outlines various therapeutic strategies aimed at modulating neuroglial dysfunction in ASD. The listed approaches target microglia, oligodendrocytes, and astrocytes through mechanisms such as reducing neuroinflammation, restoring myelination, and modulating signaling pathways. Each intervention is associated with specific limitations, such as the need for invasive procedures or non-specific targeting of cellular processes. Future prospects include the refinement of delivery techniques, precision medicine approaches, and the development of more selective neuroglial modulators to enhance therapeutic efficacy in ASD. Abbreviations: M—Microglia; O—Oligodendrocytes; A—Astrocytes; M/A—Microglia/Astrocytes; A/M—Astrocytes/Microglia; A—Astrocytes.

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6.1. Anti-Inflammatory Agents

Chronic neuroinflammation is increasingly recognized as a key factor in ASD pathophysiology, with excessive activation of microglia and astrocytes contributing to disrupted synaptic pruning, excitotoxicity, and impaired neuronal network function [99]. This has led to growing interest in anti-inflammatory agents as potential therapeutic strategies to mitigate neuroglial dysfunction and restore homeostasis in the developing neural structures [112,113]. Several classes of anti-inflammatory drugs have been explored in both preclinical and early clinical studies, with varying degrees of success [114].

Among the most widely studied are nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen and celecoxib, which act by inhibiting cyclooxygenase (COX) enzymes and reducing prostaglandin-mediated inflammation [115,116]. Preclinical models suggest that COX-2 inhibition may attenuate microglial activation and modulate neuroinflammatory cascades, leading to improvements in synaptic plasticity and behavioral outcomes [117]. However, clinical trials with NSAIDs in ASD have produced mixed results, with some studies reporting mild improvements in social behavior, while others indicate limited or inconsistent effects [114,118]. One of the primary challenges in using NSAIDs for ASD treatment is their limited ability to cross the blood–brain barrier, as well as the potential for systemic side effects, including gastrointestinal disturbances and increased cardiovascular risk [116].

Another agent that has gained attention is minocycline, a tetracycline antibiotic with notable anti-inflammatory and neuroprotective properties. In addition to its antimicrobial effects, minocycline is known to suppress microglial activation, inhibit matrix metalloproteinases (MMPs), and modulate neuroinflammatory pathways implicated in ASD [116]. Studies in animal models suggest that minocycline administration can reduce neuroinflammatory markers, enhance synaptic remodeling, and improve social interaction deficits [119]. However, despite these promising preclinical findings, its clinical application remains uncertain due to concerns regarding long-term safety, mitochondrial toxicity, and the risk of antibiotic resistance with prolonged use [120].

More targeted approaches to neuroinflammation have involved the use of cytokine-modulating biologics, including monoclonal antibodies that inhibit pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Given that elevated levels of these cytokines have been detected in some individuals with ASD, it has been hypothesized that suppressing their activity may help alleviate neuroinflammation and improve behavioral outcomes. While these therapies have shown considerable success in treating autoimmune and inflammatory conditions, their application in ASD is still in the experimental stages [114,121]. One of the primary concerns is that broad immunosuppression may interfere with essential neurodevelopmental processes, particularly in young children, making it necessary to develop strategies that selectively target pathological inflammation while preserving physiological immune functions [122].

Despite the potential benefits of anti-inflammatory interventions, several key challenges limit their widespread clinical application. One of the most significant barriers is the heterogeneity of ASD, as not all individuals exhibit the same degree of neuroinflammation [99]. This variability makes it difficult to identify which subgroups may benefit most from anti-inflammatory treatments and complicates the development of standardized therapeutic protocols. Additionally, many existing anti-inflammatory agents exert systemic effects on peripheral immune function, increasing the risk of infections, metabolic disturbances, and gastrointestinal complications with long-term use [116]. Another major limitation is the lack of specificity in targeting neuroglial cells, as most currently available drugs modulate inflammation in a broad manner without selectively affecting microglial or astrocytic function [123].

To address these challenges, ongoing research is focused on developing next-generation microglia-specific modulators that can precisely regulate aberrant activation states while preserving essential homeostatic functions. There is also increasing interest in identifying reliable biomarkers of neuroinflammation, such as elevated levels of IL-6, TNF-α, and C-reactive protein (CRP), to facilitate personalized treatment strategies. By refining our understanding of the relationship between neuroinflammation and ASD, these advances may ultimately lead to more targeted and effective therapeutic interventions, minimizing adverse effects while maximizing clinical benefits [124].

6.2. Microglial Modulators

Modulating microglial activation represents a promising therapeutic strategy for addressing neuroinflammatory dysregulation in ASD [125]. Given that excessive microglial activation is associated with aberrant synaptic pruning, excitotoxicity, and chronic inflammation, several pharmacological approaches have been explored to selectively regulate microglial responses while preserving their essential homeostatic functions [33].

Beyond broad-spectrum anti-inflammatory agents, more targeted approaches have focused on the modulation of complement system signaling, particularly through the inhibition of key regulators such as C1q and C3, which are involved in microglia-mediated synaptic pruning. The complement system plays a crucial role in tagging excess synapses for elimination during early brain development, a process that is often dysregulated in ASD, leading to abnormal connectivity patterns. In preclinical models, excessive complement activation has been linked to hyperconnectivity in cortical circuits, a finding that aligns with neuroimaging studies in individuals with ASD. Complement inhibitors designed to block C1q or C3 activity have been proposed as a means to restore the balance of synaptic pruning, preventing excessive synapse elimination without broadly suppressing microglial function [33].

Experimental interventions, such as the sushi domain protein SRPX2, have been shown to selectively interfere with C1q function, effectively protecting against complement-mediated synapse loss and preserving synaptic homeostasis [126].

Given the close interplay between immune dysregulation and gut microbiota imbalance in ASD, anti-inflammatory modulatory therapies and microbiota-targeted interventions represent complementary strategies that converge on common pathophysiological pathways. Modulation of the gut microbiota influences ASD by altering neuroimmune interactions, gut-brain axis signaling, and metabolic pathways [127]. Changes in microbiota composition can impact neurotransmitter levels, inflammation, and intestinal permeability, contributing to ASD-associated behavioral and cognitive symptoms. Notably, dysbiosis-driven immune activation can influence microglial function, leading to aberrant synaptic pruning, neuroinflammation, and disrupted neuronal connectivity, further exacerbating ASD-related phenotypes [128].

Therapeutic strategies targeting gut microbiota in ASD aim to restore microbial balance and improve neurobehavioral outcomes through various interventions. Probiotics introduce beneficial bacterial strains, such as Lactobacillus and Bifidobacterium, which may enhance gut integrity, reduce inflammation, and modulate neurotransmitter production. Prebiotics, including dietary fibers and oligosaccharides, support the growth of beneficial microbes and influence short-chain fatty acid production, which has neuroprotective effects. Fecal microbiota transplantation (FMT), though still experimental, has shown promising results in improving both gastrointestinal and behavioral symptoms by directly reconstituting a more diverse and healthier gut microbiome. Dietary interventions, such as gluten-free, casein-free, ketogenic, or fiber-rich diets, may influence microbiota composition and metabolic signaling, potentially alleviating some ASD-associated symptoms. These approaches highlight the gut–brain axis as a key therapeutic target in ASD, though further large-scale studies are needed to establish standardized treatments [129].

While these findings provide a strong rationale for microglia-targeted interventions, several challenges remain. One of the primary concerns is ensuring precise modulation of microglial activity, as excessive suppression of their function could impair their essential roles in immune surveillance, debris clearance, and synaptic maintenance. Additionally, given the heterogeneity of ASD, not all individuals exhibit the same degree of microglial dysfunction, necessitating the development of reliable biomarkers to identify patient subgroups most likely to benefit from complement modulation or other microglia-targeted therapies. Future research will need to focus on refining these interventions to achieve selective microglial regulation without compromising broader neuroimmune function, ultimately paving the way for more personalized and effective therapeutic strategies in ASD [33].

6.3. Cannabinoid-Based Therapies

Cannabidiol (CBD), a non-psychoactive cannabinoid derived from the Cannabis sativa plant, has garnered significant interest for its potential neuroprotective and anti-inflammatory properties in ASD. CBD interacts with the endocannabinoid system, primarily by modulating CB1 and CB2 receptors, which are widely expressed in both neurons and glial cells. By influencing these pathways, CBD has been shown to reduce neuroinflammation, regulate neurotransmitter release, and modulate the excitatory–inhibitory balance, which is often disrupted in ASD [130].

Several clinical studies have explored the effects of CBD on ASD-related behavioral symptoms, with some reporting improvements in social interaction, reductions in repetitive behaviors, and alleviation of anxiety and irritability [131]. Additionally, preliminary findings suggest that CBD may dampen excessive microglial activation, thereby mitigating chronic neuroinflammation that contributes to synaptic dysfunction. In a recent study, children with ASD who received CBD-enriched treatments exhibited enhanced social responsiveness and decreased frequency of self-stimulatory behaviors, supporting the hypothesis that cannabinoids may exert beneficial effects on neural circuits implicated in ASD symptomatology [132].

Despite these promising results, the widespread clinical adoption of CBD for ASD remains limited due to several unresolved challenges. One of the primary concerns is the lack of large-scale randomized controlled trials to establish definitive efficacy and safety profiles. While small-scale studies suggest potential benefits, variability in dosage, purity, and formulation complicates the interpretation of results. Additionally, long-term effects of chronic CBD administration in pediatric populations remain unclear, particularly regarding its impact on neurodevelopmental processes. Another challenge lies in the optimal dosing regimen, as CBD exhibits a biphasic effect, where low and high doses can produce different physiological responses [133].

To address these concerns, future research must focus on conducting well-controlled, large-scale clinical trials to determine appropriate therapeutic windows, long-term safety, and individualized dosing strategies. Additionally, advances in CBD formulations, such as lipid-based carriers or nanoemulsions, may improve bioavailability and precision in targeting neuroglial dysfunction. While CBD presents a compelling avenue for ASD therapy, further investigations are required to refine its clinical application and establish its role in personalized treatment approaches [130,133].

6.4. Cell-Based Therapies

Stem cell-based therapies represent a frontier in ASD treatment. Mesenchymal stem cells (MSCs), known for their immunomodulatory and neurotrophic properties, show potential to reduce neuroinflammation and repair synaptic networks [134]. Clinical trials with glial progenitor cell transplantation demonstrate promising outcomes in enhancing myelination and restoring neural connectivity. Stem cell-based therapies represent a promising frontier in ASD treatment, offering a potential means of modulating neuroinflammation, repairing synaptic networks, and restoring disrupted neuroglial function. Among the most studied stem cell types are mesenchymal stem cells (MSCs), multipotent progenitors derived from bone marrow, adipose tissue, and umbilical cord blood. MSCs are particularly attractive for neurodevelopmental applications due to their immunomodulatory, anti-inflammatory, and neurotrophic properties. These cells can secrete a variety of cytokines and growth factors, including brain-derived neurotrophic factor (BDNF) and transforming growth factor-beta (TGF-β), which play essential roles in promoting neuronal survival, synaptic plasticity, and astrocyte function. By regulating immune responses and promoting repair mechanisms, MSCs have shown the potential to attenuate chronic neuroinflammation, reduce oxidative stress, and support the regeneration of neural circuits affected in ASD. Preclinical studies have demonstrated that MSC transplantation can lead to improvements in synaptic remodeling, reduced microglial overactivation, and enhanced neurogenesis, resulting in behavioral improvements in animal models of ASD. These findings have provided the rationale for early-phase clinical trials investigating the therapeutic potential of MSCs in individuals with ASD. Some preliminary studies have reported encouraging results, including improvements in social communication, decreased repetitive behaviors, and enhanced cognitive function following MSC administration [135]. However, despite these promising early findings, the clinical application of MSC therapy remains in its infancy, with several significant challenges that need to be addressed before it can be widely implemented [136].

One of the primary obstacles in translating MSC therapy into a standardized ASD treatment is the lack of uniform protocols for cell preparation, administration, and dosing. Variability in the source, expansion methods, and delivery routes of MSCs can lead to inconsistent therapeutic outcomes. Furthermore, while MSCs are generally considered safe, the long-term effects of stem cell transplantation remain largely unknown, raising concerns about potential tumorigenicity, immune rejection, and unintended differentiation into non-neuronal cell types. Additionally, given the heterogeneity of ASD, it is unclear which subgroups of patients may benefit the most from stem cell therapy, necessitating the development of biomarkers that can predict treatment responsiveness [137].

Beyond MSCs, glial progenitor cell transplantation has also emerged as a promising avenue for ASD treatment, particularly in addressing myelination deficits and white matter abnormalities commonly observed in affected individuals. Glial progenitor cells have the capacity to differentiate into functional oligodendrocytes, the myelinating cells of the central nervous system, thereby facilitating the restoration of disrupted neuronal connectivity. Early studies in animal models suggest that transplantation of glial progenitor cells may enhance axonal conduction velocity, promote neural repair, and improve behavioral outcomes. While preliminary human studies have suggested potential benefits, glial progenitor cell therapy presents similar challenges to MSC transplantation, including the need for optimized delivery methods, long-term monitoring for safety, and precise control over cell fate differentiation [138].

As stem cell-based therapies continue to advance, future research will need to focus on refining protocols to enhance efficacy and ensure safety. Strategies such as preconditioning stem cells with neurotrophic factors, engineering cells to enhance their survival and integration, and using exosome-based approaches to deliver regenerative factors without direct cell transplantation may help overcome some of the current limitations. Additionally, combining stem cell therapy with other neuroprotective interventions, such as anti-inflammatory drugs or gene-editing technologies, may further enhance therapeutic outcomes. While stem cell-based therapies hold immense potential for addressing the complex neurobiological underpinnings of ASD, their clinical application remains an area of ongoing investigation, requiring rigorous testing in well-controlled, large-scale trials before they can be considered a viable treatment option [139].

6.5. Gene Therapy and Emerging Directions

Advances in gene editing have opened new possibilities for precise genetic modifications aimed at correcting mutations linked to neuroglial dysfunction in ASD [140]. The development of CRISPR-Cas9 technology has revolutionized molecular medicine by enabling targeted modifications of specific genomic sequences with unprecedented accuracy. This system, which utilizes a guide RNA to direct the Cas9 endonuclease to a particular DNA region, allows for the correction of pathogenic mutations, the introduction of protective genetic variants, or the regulation of gene expression in affected neural circuits. Preclinical studies have demonstrated that CRISPR-based interventions can restore synaptic balance by correcting disruptions in genes involved in neuronal connectivity, excitatory–inhibitory signaling, and glial homeostasis. In particular, gene editing approaches targeting astrocytic and microglial dysfunction have shown potential in modulating neuroinflammation, improving synaptic remodeling, and enhancing the overall stability of neural networks [141].

Despite these promising findings, significant challenges remain in translating gene editing techniques into clinical applications, including concerns over off-target effects, immune responses to CRISPR components, and ethical considerations surrounding germline modifications. Future efforts will need to focus on refining gene delivery methods, improving specificity, and ensuring safety before these approaches can be considered viable therapeutic strategies for ASD [110,137,142].

In addition to gene editing technologies, pharmacological interventions targeting key signaling pathways implicated in neuroglial dysfunction are under active investigation. Small-molecule inhibitors and modulators of the mTOR, NF-κB, and Wnt/β-catenin pathways have been explored for their ability to regulate neuroinflammation, synaptic plasticity, and glial-mediated neural repair. The mTOR pathway, which governs cellular metabolism and synaptic homeostasis, has been a major focus of drug development, with rapamycin and related compounds showing promise in preclinical ASD models. By reducing hyperactive mTOR signaling, these agents help restore proper synaptic pruning and prevent excitatory overconnectivity, thereby mitigating behavioral abnormalities [140].

Similarly, NF-κB inhibitors are being investigated for their potential to suppress excessive neuroinflammatory responses, given that chronic activation of this pathway has been linked to increased microglial reactivity and synaptic dysfunction. Modulators of Wnt/β-catenin signaling are also being explored as a means of enhancing neuronal differentiation, stabilizing synaptic connections, and improving myelination processes in ASD [143].

While initial preclinical studies suggest that these pharmacological agents can lead to behavioral and cognitive improvements, their translation into clinical use is still in the early stages. Further research is needed to optimize drug specificity, minimize side effects, and determine the long-term safety of these interventions before they can be widely adopted as therapeutic options for ASD [140].

7. Limitations and Future Directions

Despite significant advances in understanding the neuroglial contributions to ASD, several limitations hinder the direct translation of these findings into clinical practice. One of the most pressing challenges is the substantial heterogeneity of ASD, both in terms of clinical presentation and underlying pathophysiological mechanisms. This variability complicates the development of universal therapeutic strategies, as individuals with ASD exhibit diverse genetic, epigenetic, and neurobiological profiles that influence their response to treatment. Current interventions, while promising, often yield highly variable outcomes, underscoring the need for biomarker-driven precision medicine [144].

A major obstacle in achieving targeted ASD therapies is the lack of reliable biomarkers that can effectively stratify patients into clinically meaningful subgroups. Biomarkers derived from genomic, epigenetic, proteomic, or neuroimaging data hold the potential for improving diagnostic specificity, monitoring treatment efficacy, and minimizing adverse effects. However, many candidate biomarkers fail to demonstrate consistent predictive value across different studies and patient cohorts, limiting their clinical applicability. Future research should prioritize large-scale, multicenter validation studies to establish reproducible and clinically relevant biomarkers [145].

Another emerging approach with transformative potential is the application of artificial intelligence (AI) and machine learning (ML) in ASD research. These advanced computational tools can analyze high-dimensional datasets to identify novel correlations between genetic mutations, neuroglial dysfunction, and clinical phenotypes. AI-driven models may refine individualized treatment predictions, optimize therapeutic strategies, and uncover previously unrecognized molecular targets for intervention. However, translating these methods into clinical practice requires robust validation, standardized methodologies, and interdisciplinary collaboration between data scientists, neuroscientists, and clinicians [146,147].

Beyond scientific and technical challenges, logistical, ethical, and financial constraints present significant barriers to implementing emerging ASD therapies. Glial-targeted treatments, such as stem cell-based interventions and gene-editing approaches, require rigorous safety and efficacy evaluations, as well as ethical frameworks that balance innovation with patient safety. Additionally, the high cost of personalized medicine approaches raises concerns about equitable access, necessitating policy-driven initiatives to ensure that these therapies benefit a broad spectrum of individuals rather than a privileged few [148].

Finally, one of the greatest translational challenges is the difficulty of bridging the gap between preclinical models and human neuroglial function. Many ASD studies rely on rodent models, which fail to fully recapitulate the complexity of human neurodevelopment. The development of induced pluripotent stem cell (iPSC) models and organoid systems offers a more relevant human-specific platform for testing glial-targeted treatments before clinical translation. Future research should expand the use of these advanced models to improve the predictability of therapeutic responses in ASD patients [149].

To overcome these challenges and accelerate the translation of neuroglial-targeted therapies into clinical practice, future research must prioritize several key areas. First, the development and validation of reproducible biomarkers will be critical for predicting treatment response and stratifying ASD subtypes based on distinct neurobiological signatures. Advances in genomic, epigenetic, and neuroimaging-based biomarkers may enable more precise patient classification, ultimately guiding the selection of personalized therapeutic interventions [150].

Additionally, long-term clinical trials are necessary to assess the sustained neurodevelopmental impact of emerging neuroglial-targeted therapies. While several experimental treatments have shown promise in preclinical models, their efficacy and safety over extended periods remain largely unknown. Future studies should incorporate comprehensive longitudinal designs, evaluating not only immediate symptom relief but also long-term cognitive, behavioral, and functional outcomes in individuals with ASD [151].

Another crucial step is the refinement of human-based models that better replicate neuroglial interactions in ASD pathology. Induced pluripotent stem cell (iPSC)-derived neuroglia and brain organoid systems provide promising platforms for studying patient-specific glial dysfunction and screening potential therapeutic compounds. Expanding the use of these advanced models will improve translational validity, bridging the gap between basic research and clinically applicable treatments [152].

In parallel, multi-modal therapeutic strategies should be explored to address the complex and multifactorial nature of ASD. Future interventions should integrate anti-inflammatory, neurotrophic, and synaptic-modulating approaches, potentially combining pharmacological agents, immunotherapies, and neuromodulation techniques to enhance treatment efficacy. Combinatorial strategies may prove particularly beneficial in tailoring interventions to the specific glial dysfunction profiles present in different ASD subtypes [144].

Finally, as the field moves toward more advanced and individualized treatments, it is imperative to establish ethical and regulatory frameworks that guide the responsible implementation of stem cell-based and gene-editing therapies in ASD. Considerations surrounding genetic privacy, patient safety, long-term monitoring, and equitable access must be addressed to ensure that cutting-edge interventions benefit a broad spectrum of individuals rather than a select few. Close collaboration between scientists, clinicians, bioethicists, and policymakers will be essential in developing responsible, widely accessible therapeutic solutions that align with the highest standards of medical ethics and patient care [153].

By systematically addressing these challenges, the field is moving toward a more precise and effective neuroglial-targeted treatment landscape, offering new hope for individuals with ASD through scientifically rigorous, ethically sound, and clinically translatable therapeutic innovations.

8. Conclusions

The growing recognition of neuroglial dysfunction as a key component of ASD pathophysiology has opened new avenues for therapeutic intervention. The interplay between astrocytes, microglia, and oligodendrocytes in shaping neural connectivity, synaptic plasticity, and neuroimmune regulation underscores the complexity of ASD and the need for multifaceted treatment strategies. While current research has identified several promising molecular targets, including mTOR, Wnt/β-catenin, and NF-κB pathways, significant work remains in refining these approaches to ensure safety, specificity, and long-term efficacy [96,97].

The advent of precision medicine in ASD represents a paradigm shift in how we approach diagnosis and treatment. The integration of biomarkers, neuroimaging tools, and AI-driven analytics holds the potential to tailor interventions to individual patients, moving beyond the one-size-fits-all model. Advances in stem cell therapy, gene editing, and pharmacological modulation of neuroglial function offer hope for targeted interventions that address the underlying biological mechanisms of ASD rather than merely alleviating symptoms [134,139].

However, translating these innovations into widespread clinical use will require overcoming scientific, logistical, and ethical hurdles. Collaborative efforts among researchers, clinicians, industry leaders, and policymakers will be essential to bridge the gap between bench-side discoveries and real-world applications. Ensuring equitable access to emerging therapies, particularly in underserved populations, must remain a priority to avoid widening healthcare disparities [144,150].

As research continues to unravel the complexities of ASD, the future of treatment lies in integrative, multidisciplinary approaches that leverage cutting-edge technology, translational research, and personalized medicine. By addressing existing limitations and fostering international collaboration, the field is poised to enter a new era where therapies are tailored to the unique neurobiological profiles of individuals with ASD, ultimately improving outcomes and quality of life for affected individuals and their families.