Wnt signaling networks in autism spectrum disorder and intellectual disability

The strongest single candidate gene for non-syndromic ASDs is chromodomain helicase DNA binding protein 8 (CHD8) [24‐30]. There are multiple de novo, truncating, or missense mutations discovered in CHD8 in individuals with ASDs [27‐29, 31‐34]. CHD8 is found at active transcription sites with histone modifications H3K4me3 or H3K27ac, and it is thought to directly activate genes by binding near the transcriptional start site and promoting transcription factor activity or recruitment. It can also indirectly impact transcription by interacting with modified histone sites and other co-regulators to make chromatin more assessable [24, 34‐36]. Interestingly, one of the major pathways regulated by CHD8 is canonical Wnt signaling [37, 38]. Previous work characterized CHD8 as a negative regulator of canonical Wnt signaling, which fits with the hypothesis that elevated canonical Wnt signaling activity causes excessive proliferation of embryonic neural progenitor cells (NPCs) in the brain and may in part explain the macrocephaly (“big brain”) phenotype observed in patients [27]. Furthermore, recent studies in human neural progenitors lacking one copy of CHD8 support this notion, as it revealed many target genes controlled by CHD8 that are involved in the regulation of spine head size [34, 39, 40]. However a recent study discovered that CHD8 is in fact a positive regulator of Wnt/β-catenin signaling NPCs, while simultaneously demonstrating that it negatively regulates the pathway in non-neuronal cell lines [41]. Given this unexpected finding, this suggests that CHD8 regulates Wnt signaling in a cell-specific manner, and the possibility that some of the CHD8 mutations may not be as simple as loss-of-function for Wnt signaling. Further work is needed to clarify how CHD8 regulates Wnt signaling in different cell types in the brain, and how patients with CHD8 mutations acquire macrocephaly. It is also important to note that Wnt signaling is only one neurodevelopmental pathway regulated by CHD8, and recent studies have identified many others (e.g., chromatin remodeling). Therefore, future work needs to determine the precise mechanisms and time points during which CHD8 regulates Wnt signaling during neurodevelopment. This is important to better comprehend how prenatal brain development could be compromised in individuals with CHD8 mutations.

β-catenin is a central player in the canonical Wnt signaling pathway and works with co-factors to initiate Wnt-dependent gene transcription (Fig. 1). It has directly been implicated in ASDs due to the identification of de novo mutations in the CTNNB1 gene in patients with ASD using exome sequencing [25, 28, 29, 42]. Given the core nature of this gene in canonical Wnt signaling, this strongly places aberrations in Wnt signaling as one of the main networks in ASD pathogenesis. Network analysis from gene expression data also indicates that β-catenin exists in a protein network, including CHD8 and other ASD or ID associated genes [25]. The relationship between Wnt signaling and chromatin remodeling factors demonstrates that proper interplay between these pathways is important for appropriate levels of canonical Wnt-dependent gene transcription. CHD8 regulates β-catenin-mediated canonical Wnt signaling, which could occur by CHD8 directly by binding to β-catenin or indirectly by inhibiting the recruitment of co-factors required for transcription at promoter sequences. Future work will need to determine precisely which neural progenitor sub-populations are most sensitive to disruptions in canonical Wnt/β-catenin signaling during prenatal brain development. For example, previous studies indicated that global stabilization of β-catenin in the cortex, which elevates canonical Wnt signaling, leads to brain overgrowth due to increased cycling neural progenitor cells and production of neurons [43]. In contrast, deletion of β-catenin from parvalbumin-expressing inhibitory neurons leads to ASD-like defects in neuronal activation and behavior, such as social interaction and object recognition impairments [44].

β-caxtenin also functions in pathways other than canonical Wnt signaling, such as cell adhesion at the plasma membrane. β-catenin interacts with cadherins to regulate dendritic spine growth and synaptic competition during the process of postnatal period of dendritic pruning [45]. A recent study identified that dominant mutations in CTNNB1 from ID patients when expressed in mice have a reduced affinity for membrane-associated cadherins [46]. This is associated with a decrease in cadherin interaction and decreased intrahemispheric connections, with deficits in dendritic branching, long-term potentiation, and cognitive function [46]. Therefore, it is possible that mutations in β-catenin could lead to aberrant Wnt signaling developmentally, concurrent with disruption of plasma membrane signaling at synaptic sites, both leading to disrupted gene transcription programs and abnormal synaptic plasticity. Furthermore, both of these pathogenic events would increase disease risk. In this regard, these pathways are also likely connected given that β-catenin signaling at the membrane impacts nuclear Wnt-dependent transcription [47].

Another high-risk autism candidate gene that has roles in Wnt signaling is PTEN due to the discovery of numerous individuals with mutations [29, 48‐50]. PTEN has multiple functions but is best known for its role as a negative regulator of the PI3K-Akt-mTOR pathway. Individuals with heterozygous mutations in PTEN are also at risk for macrocephaly, indicating that PTEN regulates brain size, which is thought to be an impact on certain individuals with ASD [51‐59]. Multiple mouse models of Pten have further strengthened the notion that it is an important regulator of different neural circuits associated with ASDs and cognition. For example, an early study identified that global Pten +/- mice have impairments in social interaction behaviors [52]. Knocking out Pten in the cerebellar Purkinje cells led to impaired sociability, repetitive behavior, and deficits in motor learning. Additionally, knocking out Pten in a subset of cortical excitatory neurons results in profound synaptic signaling changes [60], while ASD-associated Pten alleles expressed in inhibitory neurons cause excitatory/inhibitory imbalances [54]. Interestingly, PTEN has recently been identified to function with β-catenin to regulate normal brain growth, implicating PTEN in Wnt signaling. It was discovered that β-catenin signaling is elevated in a mouse model of Pten (Pten +/-), and a heterozygous mutation in β-catenin suppresses the excessive cortical brain growth in Pten +/- mice [53]. This indicates that multiple ASD risk factors likely converge upon neural progenitor proliferation during embryonic brain development, potentially through regulation of canonical Wnt signaling.

Recent studies have identified mutations in CTNNB1 within individuals with developmental delay and ID who do not have an ASD [46, 61, 62]. Additional support comes from another ASD exome sequencing study that identified de novo loss of function variants in TCF7L2 (transcription factor 7-like 2 (T-cell specific, HMG-box)) [63, 64], which also goes by the name TCF4, and is confused with transcription factor 4 (TCF4) as they share the same symbol. Importantly, TCF7L2 is a key player in canonical Wnt signaling as it helps to initiate gene transcriptional response when Wnt ligands bind their receptors on the membrane, and the signal is transduced to the nucleus (Fig. 1). These genetic studies directly implicate de novo missense variants in TCF4 in ASD, suggesting that perturbations to the core canonical signaling complex play a pathogenic role. However, the role of TCF4 in brain development and which time points and cell types it regulates is not well known and needs to be identified in future studies.

A recent study identified de novo mutations in DEAD-box helicase 3 , X-linked (DDX3X) in a population of unexplained ID [65]. DDX3X was identified as a regulator of the Wnt-β-catenin network, via regulation of the kinase activity of CK1ε, to promote phosphorylation of Dvl, both critical factors in canonical Wnt signaling (Fig. 1). Moreover, in model systems testing the Wnt pathway, DDX3X was found to be required for Wnt-β-catenin signaling in mammalian cells through loss of function studies [66]. Therefore, in addition to ASD, Wnt-β-catenin signaling may be disrupted in generalized ID and developmental disorders (DD), further demonstrating the importance of this pathway in proper neurodevelopment. However, a critical question that remains is how do mutations in Wnt signaling molecules give rise to different phenotypes, such as ASD versus ID/DD? This insight would provide tremendous clinical utility, as it will allow caregivers and clinicians to plan therapies according to the future outcome.