Autism-linked Cullin3 germline haploinsufficiency impacts cytoskeletal dynamics and cortical neurogenesis through RhoA signaling

Abstract

E3-ubiquitin ligase Cullin3 (Cul3) is a high confidence risk gene for Neurodevelopmental Disorders (NDD) including Autism Spectrum Disorder (ASD) and Developmental Delay (DD). We generated haploinsufficient Cul3 mouse model to investigate brain anatomy, behavior, molecular, cellular, and circuit-level mechanisms dysregulated by Cul3 mutations. Brain MRI of Cul3 mutant animals found profound abnormalities in half of brain regions, including decreased volume of cortical regions and increased volume of subcortical regions. Cul3 mutant mice exhibited social and cognitive deficits, and hyperactive behavior. Spatiotemporal transcriptomic and proteomic profiling of the brain implicated neurogenesis and cytoskeletal defects as key drivers of Cul3 functional impact. Cortical neurons from mutant mice had reduced dendritic length and loss of filamentous actin puncta, along with reduced spontaneous network activity. Inhibition of small GTPase RhoA, a molecular substrate of Cul3 ligase, rescued dendrite length phenotype. This study identified RhoA signaling as a potential mechanism, through which Cul3 mutation impacts early brain development.introduced a germline 1bp insertion in exon 6 of Cul3, immediately adjacent to G754T (E246X) mutation detected in an ASD patient (O'Roak et al., 2012) (Figure 1B). Extensive characterization of the mutant Cul3 +/mice at multiple levels including brain neuroanatomy, behavior, transcriptomic and proteomic profiling of several brain region at three developmental time points, provided a detailed picture of the functional impact of Cul3 mutation (Figure 1C). Our results demonstrate that Cul3 haploinsufficiency severely impacts the developing brain and leads to social deficits and hyperactivity in mice. Dysregulation of genes and proteins involved in actin, intermediate filament and binding, pointed to cytoskeletal defects. Reduced synaptic connectivity and dendritic length were among most notable neuronal phenotypes observed in Cul3 +/mice. Importantly, upregulation of one of the Cul3 substrates, small GTPase RhoA, a regulator of cytoskeleton, neuronal growth and migration, linked observed molecular defects with Cul3 pathology. Treatment of cortical neurons with RhoA inhibitor Rhosin rescued dendritic length phenotype. ResultsCul3 +/-mice have severe brain anatomical defectsWe introduced a 1bp frame-shifting insertion with CRISPR/Cas9 into exon-6 of Cul3 one base pair upstream from the nucleotide mutated in an ASD patient (O'Roak et al., 2012) to generate Cul3 +/haploinsufficient mouse model on C57BL/6N background (STAR Methods). A heterozygous Cul3 +/founder mouse harboring the insertion was expanded via breeding with wildtype (WT) C57BL/6N mice for at least four generations to eliminate CRISPR off-target effects. In agreement with previous observations (Singer et al., 1999), homozygous Cul3 −/− mutant animals were not viable. The heterozygous Cul3 +/mice were viable, reached a normal lifespan, and were fertile irrespective of sex. However, Cul3 +/mice had reduced body weight (P<0.001 for mal...

Current trends of high-risk gene Cul3 in neurodevelopmental disorders

“…Previous research has highlighted the crucial role of Cul3 in diseases such as breast ( 21) and ovarian cancer (22). However, recent studies have demonstrated its involvement in central nervous system (CNS) diseases, particularly (NDDs) (23)(24)(25)(26).…”

“…To investigate the effects of Cul3 mutations on brain development, Amar et al (23) employed CRISPR/Cas9 genome engineering technology to create a whole-brain Cul3-KO mouse (Cul3 +/− )(Table 2; Figure 3). They introduced a 1-bp frameshift insertion into Cul3 exon 6, which is one base pair upstream of the mutated nucleotide in ASD patients, thereby establishing a Cul3 +/− haploinsufficiency mouse model on a C57BL/6 N background (9,23). Cul3 +/− mice displayed social and cognitive deficits, as well as hyperactivity, accompanied by reduced dendritic growth and cortical neuronal activity.…”

“…Increased RhoA was found both in whole-brain KO mice (CRISPR/Cas9 Cul3-KO) (23) and forebrain cKO (Emx1-Cre;Cul3 f/+ ) (26), which was accompanied by suppressed dendrite length and neurites, reduced total spines, respectively. Administration of Rhosin (23), a pharmacological inhibitor of RhoA, contributes to neurite outgrowth and restores neural network activity caused by Cul3 deficiency.…”

“…To investigate potential downstream molecular targets, most studies have employed proteomics (23,25,41) and RNA-seq (23). These methods have allowed for the screening of several targets, including RhoA (23), Smyd3 (26), and EIF4 (41).…”

“…To investigate potential downstream molecular targets, most studies have employed proteomics (23,25,41) and RNA-seq (23). These methods have allowed for the screening of several targets, including RhoA (23), Smyd3 (26), and EIF4 (41). However, as Cul3 directly modulates protein degradation, any resulting transcriptional changes would be indirect in nature.…”