Molecular Autism

FXS is the most common genetic cause of intellectual (ID) and autism spectrum disorders (ASD). FXS is caused by loss of FMRP, an RNA-binding protein involved in the translational regulation of a large number of neuronal mRNAs. Absence of FMRP has been shown to lead to elevated protein synthesis and is thought to be a major cause of the synaptic plasticity and behavioural deficits in FXS. The increase in protein synthesis results in part from abnormal activation of key protein translation pathways downstream of ERK1/2 and mTOR signalling. Pharmacological and genetic interventions that attenuate hyperactivation of these pathways can normalize levels of protein synthesis and improve phenotypic outcomes in animal models of FXS. Several efforts are currently underway to trial this strategy in patients with FXS. To date, elevated global protein synthesis as a result of FMRP loss has not been validated in the context of human neurons. Here, using an isogenic human stem cell-based model, we show that de novo protein synthesis is elevated in FMRP-deficient neural cells. We further show that this increase is associated with elevated ERK1/2 and Akt signalling and can be rescued by metformin treatment. Finally, we examined the effect of normalizing protein synthesis on phenotypic abnormalities in FMRP-deficient neural cells. We find that treatment with metformin attenuates the increase in proliferation of FMRP-deficient neural progenitor cells but not the neuronal deficits in neurite outgrowth. The elevated level of protein synthesis and the normalization of neural progenitor proliferation by metformin treatment were validated in additional control and FXS patient-derived hiPSC lines. Overall, our results validate that loss of FMRP results in elevated de novo protein synthesis in human neurons and suggest that approaches targeting this abnormality are likely to be of partial therapeutic benefit in FXS.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by mutations affecting UBE3A function. AS is characterized by intellectual disability, impaired motor coordination, epilepsy, and behavioral abnormalities including autism spectrum disorder features. The development of treatments for AS heavily relies on the ability to test the efficacy of drugs in mouse models that show reliable, and preferably clinically relevant, phenotypes. We previously described a number of behavioral paradigms that assess phenotypes in the domains of motor performance, repetitive behavior, anxiety, and seizure susceptibility. Here, we set out to evaluate the robustness of these phenotypes when tested in a standardized test battery. We then used this behavioral test battery to assess the efficacy of minocycline and levodopa, which were recently tested in clinical trials of AS. We combined data of eight independent experiments involving 111 Ube3a mice and 120 wild-type littermate control mice. Using a meta-analysis, we determined the statistical power of the subtests and the effect of putative confounding factors, such as the effect of sex and of animal weight on rotarod performance. We further assessed the robustness of these phenotypes by comparing Ube3a mutants in different genetic backgrounds and by comparing the behavioral phenotypes of independently derived Ube3a-mutant lines. In addition, we investigated if the test battery allowed re-testing the same animals, which would allow a within-subject testing design. We find that the test battery is robust across different Ube3a-mutant lines, but confirm and extend earlier studies that several phenotypes are very sensitive to genetic background. We further found that the audiogenic seizure susceptibility phenotype is fully reversible upon pharmacological treatment and highly suitable for dose-finding studies. In agreement with the clinical trial results, we found that minocycline and levodopa treatment of Ube3a mice did not show any sign of improved performance in our test battery. Our study provides a useful tool for preclinical drug testing to identify treatments for Angelman syndrome. Since the phenotypes are observed in several independently derived Ube3a lines, the test battery can also be employed to investigate the effect of specific Ube3a mutations on these phenotypes.

A gap exists in our mechanistic understanding of how genetic and environmental risk factors converge at the molecular level to result in the emergence of autism symptoms. We compared blood-based gene expression signatures in identical twins concordant and discordant for autism spectrum condition (ASC) to differentiate genetic and environmentally driven transcription differences, and establish convergent evidence for biological mechanisms involved in ASC. Genome-wide gene expression data were generated using RNA-seq on whole blood samples taken from 16 pairs of monozygotic (MZ) twins and seven twin pair members (39 individuals in total), who had been assessed for ASC and autism traits at age 12. Differential expression (DE) analyses were performed between (a) affected and unaffected subjects (N = 36) and (b) within discordant ASC MZ twin pairs (total N = 11) to identify environmental-driven DE. Gene set enrichment and pathway testing was performed on DE gene lists. Finally, an integrative analysis using DNA methylation data aimed to identify genes with consistent evidence for altered regulation in cis. In the discordant twin analysis, three genes showed evidence for DE at FDR < 10%: IGHG4, EVI2A and SNORD15B. In the case-control analysis, four DE genes were identified at FDR < 10% including IGHG4, PRR13P5, DEPDC1B, and ZNF501. We find enrichment for DE of genes curated in the SFARI human gene database. Pathways showing evidence of enrichment included those related to immune cell signalling and immune response, transcriptional control and cell cycle/proliferation. Integrative methylomic and transcriptomic analysis identified a number of genes showing suggestive evidence for cis dysregulation. Identical twins stably discordant for ASC are rare, and as such the sample size was limited and constrained to the use of peripheral blood tissue for transcriptomic and methylomic profiling. Given these primary limitations, we focused on transcript-level analysis. Using a cohort of ASC discordant and concordant MZ twins, we add to the growing body of transcriptomic-based evidence for an immune-based component in the molecular aetiology of ASC. Whilst the sample size was limited, the study demonstrates the utility of the discordant MZ twin design combined with multi-omics integration for maximising the potential to identify disease-associated molecular signals.

Chromodomain helicase DNA-binding protein 8 (Chd8) is a high-confidence risk gene for autism spectrum disorder (ASD). However, how Chd8 haploinsufficiency impairs gene expression in the brain and impacts behavior at different stages of life is unknown. We generated a mutant mouse line with an ASD-linked loss-of-function mutation in Chd8 (V986*; stop codon mutation). We examined the behavior of Chd8 mutant mice along with transcriptional changes in the cerebral cortex as a function of age, with a focus on one embryonic (E14.5) and three postnatal ages (1, 6, and 12 months). Chd8V986*/+ mutant mice displayed macrocephaly, reduced rearing responses and reduced center time in the open field, and enhanced social novelty preference. Behavioral phenotypes were more evident in Chd8V986*/+ mutant mice at 1 year of age. Pup survival was reduced in wild-type x Chd8V986*/+ crosses when the mutant parent was female. Transcriptomic analyses indicated that pathways associated with synaptic and neuronal projections and sodium channel activity were reduced in the cortex of embryonic Chd8V986*/+ mice and then equalized relative to wild-type mice in the postnatal period. At 12 months of age, expression of genes associated with endoplasmic reticulum (ER) stress, chaperone-mediated protein folding, and the unfolded protein response (UPR) were reduced in Chd8V986*/+ mice, whereas genes associated with the c-MET signaling pathway were increased in expression. It is unclear whether the transcriptional changes observed with age in Chd8V986*/+ mice reflect a direct effect of CHD8-regulated gene expression, or if CHD8 indirectly affects the expression of UPR/ER stress genes in adult mice as a consequence of neurodevelopmental abnormalities. Collectively, these data suggest that UPR/ER stress pathways are reduced in the cerebral cortex of aged Chd8V986*/+ mice. Our study uncovers neurodevelopmental and age-related phenotypes in Chd8V986*/+ mice and highlights the importance of controlling for age when studying Chd8 haploinsufficient mice.

Computer vision combined with human annotation could offer a novel method for exploring facial expression (FE) dynamics in children with autism spectrum disorder (ASD). We recruited 157 children with typical development (TD) and 36 children with ASD in Paris and Nice to perform two experimental tasks to produce FEs with emotional valence. FEs were explored by judging ratings and by random forest (RF) classifiers. To do so, we located a set of 49 facial landmarks in the task videos, we generated a set of geometric and appearance features and we used RF classifiers to explore how children with ASD differed from TD children when producing FEs. Using multivariate models including other factors known to predict FEs (age, gender, intellectual quotient, emotion subtype, cultural background), ratings from expert raters showed that children with ASD had more difficulty producing FEs than TD children. In addition, when we explored how RF classifiers performed, we found that classification tasks, except for those for sadness, were highly accurate and that RF classifiers needed more facial landmarks to achieve the best classification for children with ASD. Confusion matrices showed that when RF classifiers were tested in children with ASD, anger was often confounded with happiness. The sample size of the group of children with ASD was lower than that of the group of TD children. By using several control calculations, we tried to compensate for this limitation. Children with ASD have more difficulty producing socially meaningful FEs. The computer vision methods we used to explore FE dynamics also highlight that the production of FEs in children with ASD carries more ambiguity.

Autism spectrum disorder (ASD) is characterized by impaired social interactions and repetitive patterns of behavior. Symptoms appear in early life and persist throughout adulthood. Early social stimulation can help reverse some of the symptoms, but the biological mechanisms of these therapies are unknown. By analyzing the effects of early social stimulation on ASD-related behavior in the mouse, we aimed to identify brain structures that contribute to these behaviors. We injected pregnant mice with 600-mg/kg valproic acid (VPA) or saline (SAL) at gestational day 12.5 and evaluated the effect of weaning their offspring in cages containing only VPA animals, only SAL animals, or mixed. We analyzed juvenile play at PD21 and performed a battery of behavioral tests in adulthood. We then used preclinical PET imaging for an unbiased analysis of the whole brain of these mice and studied the function of the piriform cortex by c-Fos immunoreactivity and HPLC. Compared to control animals, VPA-exposed animals play less as juveniles and exhibit a lower frequency of social interaction in adulthood when reared with other VPA mice. In addition, these animals were less likely to investigate social odors in the habituation/dishabituation olfactory test. However, when VPA animals were weaned with control animals, these behavioral alterations were not observed. Interestingly, repetitive behaviors and depression-related behaviors were not affected by social enrichment. We also found that VPA animals present high levels of glucose metabolism bilaterally in the piriform cortex (Pir), a region known to be involved in social behaviors. Moreover, we found alterations in the somatosensory, motor, and insular cortices. Remarkably, these effects were mostly reversed after social stimulation. To evaluate if changes in glucose metabolism in the Pir correlated with changes in neuronal activity, we measured c-Fos immunoreactivity in the Pir and found it increased in animals prenatally exposed to VPA. We further found increased dopamine turnover in the Pir. Both alterations were largely reversed by social enrichment. We show that early social enrichment can specifically rescue social deficits in a mouse model of ASD. Our results identified the Pir as a structure affected by VPA-exposure and social enrichment, suggesting that it could be a key component of the social brain circuitry.

Fragile X syndrome (FXS), a common cause of intellectual disability and autism, results from the expansion of a CGG-repeat tract in the 5′ untranslated region of the FMR1 gene to >200 repeats. Such expanded alleles, known as full mutation (FM) alleles, are epigenetically silenced in differentiated cells thus resulting in the loss of FMRP, a protein important for learning and memory. The timing of repeat expansion and FMR1 gene silencing is controversial. We monitored the repeat size and methylation status of FMR1 alleles with expanded CGG repeats in patient-derived induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) that were grown for extended period of time either as stem cells or differentiated into neurons. We used a PCR assay optimized for the amplification of large CGG repeats for sizing, and a quantitative methylation-specific PCR for the analysis of FMR1 promoter methylation. The FMR1 mRNA levels were analyzed by qRT-PCR. FMRP levels were determined by western blotting and immunofluorescence. Chromatin immunoprecipitation was used to study the association of repressive histone marks with the FMR1 gene in FXS ESCs. We show here that while FMR1 gene silencing can be seen in FXS embryonic stem cells (ESCs), some silenced alleles contract and when the repeat number drops below ~400, DNA methylation erodes, even when the repeat number remains >200. The resultant active alleles do not show the large step-wise expansions seen in stem cells from other repeat expansion diseases. Furthermore, there may be selection against large active alleles and these alleles do not expand further or become silenced on neuronal differentiation. Our data support the hypotheses that (i) large expansions occur prezygotically or in the very early embryo, (ii) large unmethylated alleles may be deleterious in stem cells, (iii) methylation can occur on alleles with >400 repeats very early in embryogenesis, and (iv) expansion and contraction may occur by different mechanisms. Our data also suggest that the threshold for stable methylation of FM alleles may be higher than previously thought. A higher threshold might explain why some carriers of FM alleles escape methylation. It may also provide a simple explanation for why silencing has not been observed in mouse models with >200 repeats.

Of the many genetic mutations known to increase the risk of autism spectrum disorder, a large proportion cluster upon synaptic proteins. One such family of presynaptic proteins are the neurexins (NRXN), and recent genetic and mouse evidence has suggested a causative role for NRXN2 in generating altered social behaviours. Autism has been conceptualised as a disorder of atypical connectivity, yet how single-gene mutations affect such connectivity remains under-explored. To attempt to address this, we have developed a quantitative analysis of microstructure and structural connectivity leveraging diffusion tensor MRI (DTI) with high-resolution 3D imaging in optically cleared (CLARITY) brain tissue in the same mouse, applied here to the Nrxn2α knockout (KO) model. Fixed brains of Nrxn2α KO mice underwent DTI using 9.4 T MRI, and diffusion properties of socially relevant brain regions were quantified. The same tissue was then subjected to CLARITY to immunolabel axons and cell bodies, which were also quantified. DTI revealed increases in fractional anisotropy in the amygdala (including the basolateral nuclei), the anterior cingulate cortex, the orbitofrontal cortex and the hippocampus. Axial diffusivity of the anterior cingulate cortex and orbitofrontal cortex was significantly increased in Nrxn2α KO mice, as were tracts between the amygdala and the orbitofrontal cortex. Using CLARITY, we find significantly altered axonal orientation in the amygdala, orbitofrontal cortex and the anterior cingulate cortex, which was unrelated to cell density. Our findings demonstrate that deleting a single neurexin gene (Nrxn2α) induces atypical structural connectivity within socially relevant brain regions. More generally, our combined within-subject DTI and CLARITY approach presents a new, more sensitive method of revealing hitherto undetectable differences in the autistic brain.

In the last decade, accumulative evidence has shown that oxytocin can modulate social perception in typically developed individuals and individuals diagnosed with autism. While several studies show that oxytocin (OT) modulates neural activation in social-related neural regions, the mechanism that underlies OT effects in ASD is not fully known yet. Despite evidence from animal studies on connections between the oxytocinergic system and excitation/inhibition neural balance, the influence of OT on oscillatory responses among individuals with ASD has been rarely examined. To bridge these gaps in knowledge, we investigated the effects of OT on both social and non-social stimuli while focusing on its specific influence on the neural connectivity between three socially related neural regions—the left and right fusiform and the medial frontal cortex. Twenty-five adolescents with ASD participated in a wall-established social task during a randomized, double-blind placebo-controlled MEG and OT administration study. Our main task was a social-related task that required the identification of social and non-social-related pictures. We hypothesized that OT would modulate the oscillatory connectivity between three pre-selected regions of interest to be more adaptive to social processing. Specifically, we focused on alpha and gamma bands which are known to play an important role in face processing and top-down/bottom-up balance. Compared to placebo, OT reduced the connectivity between the medial frontal cortex and the fusiform in the low gamma more for social stimuli than for non-social ones, a reduction that was correlated with individuals’ performance in the task. Additionally, for both social and non-social stimuli, OT increased the connectivity in the alpha and beta bands. Sample size was determined based on sample sizes previously reported in MEG in clinical populations, especially OT administration studies in combination with neuroimaging in ASD. We were limited in our capability to recruit for such a study, and as such, the sample size was not based on a priori power analysis. Additionally, we limited our analyses to specific neural bands and regions. To validate the current results, future studies may be needed to explore other parameters using whole-brain approaches in larger samples. These results suggest that OT influenced social perception by modifying the communication between frontal and posterior regions, an attenuation that potentially impacts both social and non-social early perception. We also show that OT influences differ between top-down and bottom-up processes, depending on the social context. Overall, by showing that OT influences both social-related perception and overall attention during early processing stages, we add new information to the existing understanding of the impact of OT on neural processing in ASD. Furthermore, by highlighting the influence of OT on early perception, we provide new directions for treatments for difficulties in early attentional phases in this population. Trial registration Registered on October 27, 2021—Retrospectively registered, https://clinicaltrials.gov/ct2/show/record/NCT05096676 (details on clinical registration can be found in www.clinicalTrial.gov , unique identifier: NCT05096676 ).