Haploinsufficiency of the Shank3 gene, which encodes a scaffolding protein at glutamatergic synapses, is a highly prevalent and penetrant risk factor for autism. Using combined behavioral, electrophysiological, biochemical, imaging and molecular approaches, we find that Shank3-deficient mice exhibit autism-like social deficits and repetitive behaviors, as well as the significantly diminished NMDAR synaptic function and synaptic distribution in prefrontal cortex. Concomitantly, Shank3-deficient mice have a marked loss of cortical actin filaments, which is associated with the reduced Rac1/PAK activity and increased activity of cofilin, the major actin depolymerizing factor. The social deficits and NMDAR hypofunction are rescued by inhibiting cofilin or activating Rac1 in Shank3-deficient mice, and are induced by inhibiting PAK or Rac1 in wild-type mice. These results indicate that the aberrant regulation of synaptic actin filaments and loss of synaptic NMDARs contribute to the manifestation of autism-like phenotypes. Thus, targeting actin regulators provides a novel strategy for autism treatment.

Haploinsufficiency of the SHANK3 gene is causally linked to autism spectrum disorder (ASD), and ASD-associated genes are also enriched for chromatin remodelers. Here, we found that brief treatment with romidepsin, a highly potent class I histone deacetylase (HDAC) inhibitor, alleviated social deficits in Shank3-deficient mice, which persisted for ~3 weeks. HDAC2 transcription was upregulated in these mice, and knockdown of HDAC2 in prefrontal cortex also rescued their social deficits. Nuclear localization of β-catenin, a Shank3-binding protein that regulates cell adhesion and transcription, was increased in Shank3-deficient mice, which induced HDAC2 upregulation and social deficits. At the downstream molecular level, romidepsin treatment elevated the expression and histone acetylation of Grin2a and actin regulatory genes, and restored NMDAR function and actin filaments in Shank3-deficient mice. Taken together, these findings highlight an epigenetic mechanism underlying social deficits linked to Shank3 deficiency, which may suggest potential therapeutic strategies for ASD patients bearing SHANK3 mutations.

No abstract

BackgroundMotor impairments are amongst the earliest and most consistent signs of autism spectrum disorders but are not used as diagnostic criteria. In addition, the relationship between motor and cognitive impairments and their respective neural substrates remain unknown.MethodsHere, we aimed at determining whether a well-acknowledged animal model of autism spectrum disorders, the valproic acid model, displays motor impairments and whether they may correlate with social deficits and neuronal loss within motor brain areas. For this, pregnant female mice (C57BL/6J) received valproic acid (450 mg/kg) at embryonic day 12.5 and offspring underwent a battery of behavioral analyses before being killed for histological correlates in motor cortex, nigrostriatal pathway, and cerebellum.ResultsWe show that while valproic acid male mice show both social and motor impairments, female mice only show motor impairments. Prenatal valproic acid exposure induces specific cell loss within the motor cortex and cerebellum and that is of higher magnitude in males than in females. Finally, we demonstrate that motor dysfunction correlates with reduced social behavior and that motor and social deficits both correlate with a loss of Purkinje cells within the Crus I cerebellar area.ConclusionsOur results suggest that motor dysfunction could contribute to social and communication deficits in autism spectrum disorders and that motor and social deficits may share common neuronal substrates in the cerebellum. A systematic assessment of motor function in autism spectrum disorders may potentially help the quantitative diagnosis of autism spectrum disorders and strategies aimed at improving motor behavior may provide a global therapeutic benefit.

Many of the genes disrupted in autism are identified as histone-modifying enzymes and chromatin remodelers, most prominently those that mediate histone methylation/demethylation. However, the role of histone methylation enzymes in the pathophysiology and treatment of autism remains unknown. To address this, we used mouse models of haploinsufficiency of the Shank3 gene (a highly penetrant monogenic autism risk factor), which exhibits prominent autism-like social deficits. We found that histone methyltransferases EHMT1 and EHMT2, as well as histone lysine 9 dimethylation (specifically catalyzed by EHMT1/2), were selectively increased in the prefrontal cortex (PFC) of Shank3-deficient mice and autistic human postmortem brains. Treatment with the EHMT1/2 inhibitor UNC0642 or knockdown of EHMT1/2 in PFC induced a robust rescue of autism-like social deficits in Shank3-deficient mice, and restored NMDAR-mediated synaptic function. Activity-regulated cytoskeleton-associated protein (Arc) was identified as one of the causal factors underlying the rescuing effects of UNC0642 on NMDAR function and social behaviors in Shank3-deficient mice. UNC0642 treatment also restored a large set of genes involved in neural signaling in PFC of Shank3-deficient mice. These results suggest that targeting histone methylation enzymes to adjust gene expression and ameliorate synaptic defects could be a potential therapeutic strategy for autism.

Infections during gestation and the consequent maternal immune activation (MIA) increase the risk of developing neuropsychiatric disorders in infants and throughout life, including autism spectrum disorders (ASD). ASD is a neurodevelopmental disorder that affects three times more males than females and is mainly characterized by deficits in social communication and restricted interests. Consistent findings also indicate that ASD patients suffer from movement disorders, although these symptoms are not yet considered as diagnosis criteria. Here we used the double-stranded RNA analog polyinosinic:polycytidylic acid (poly I:C) MIA animal model of ASD in mice and explored its effects in males and females on social and motor behavior. We then investigated brain areas implicated in controlling and coordinating movements, namely the nigro-striatal pathway, motor cortex and cerebellum. We show that male mice are more affected by this treatment than females as they show reduced social interactions as well as motor development and coordination deficits. Reduced numbers of Purkinje cells in the cerebellum was found more widespread and within distinct lobules in males than in females. Moreover, a reduced number of neurons was found in the motor cortex of males only. These results suggest that females are better protected against developmental insults leading to ASD symptoms in mice. They also point to brain areas that may be targeted to better manage social and motor consequences of ASD.

Background Contrasting findings were reported in several animal models with a Shank3 mutation used to induce various autism spectrum disorder (ASD) symptoms. Here, we aimed at investigating behavioral, cellular, and molecular consequences of a C-terminal (frameshift in exon 21) deletion in Shank3 protein in mice, a mutation that is also found in clinical conditions and which results in loss of major isoforms of Shank3. A special focus was made on cerebellar related parameters. Methods All three genotypes were analyzed [wild type (WT), heterozygote (Shank3+/ΔC) and homozygote (Shank3 ΔC/ΔC)] and males and females were separated into two distinct groups. Motor and social behavior, gait, Purkinje cells (PC) and glutamatergic protein levels were determined. Behavioral and cellular procedures used here were previously validated using two environmental animal models of ASD. ANOVA and post-hoc analysis were used for statistical analysis. Results Shank3 ΔC/ΔC mice showed significant impairments in social novelty preference, stereotyped behavior and gait. These were accompanied by a decreased number of PC in restricted cerebellar sub-regions and decreased cerebellar expression of mGluR5. Females Shank3 ΔC/ΔC were less affected by the mutation than males. Shank3+/ΔC mice showed impairments only in social novelty preference, grooming, and decreased mGluR5 expression and that were to a much lesser extent than in Shank3 ΔC/ΔC mice. Limitations As Shank3 mutation is a haploinsufficiency, it is of interest to emphasize that Shank3+/ΔC mice showed only mild to no deficiencies compared to Shank3 ΔC/ΔC. Conclusions Our findings indicate that several behavioral, cellular, and molecular parameters are affected in this animal model. The reported deficits are more pronounced in males than in females. Additionally, male Shank3 ΔC/ΔC mice show more pronounced alterations than Shank3+/ΔC. Together with our previous findings in two environmental animal models of ASD, our studies indicate that gait dysfunction constitutes a robust set of motor ASD symptoms that may be considered for implementation in clinical settings as an early and quantitative diagnosis criteria.

Autism is a neurodevelopmental disorder characterized by social deficits and repetitive behaviors. Genetic screening has identified synaptic, transcriptional, and chromatin genes disrupted in autistic patients. Haploinsufficiency of Shank3, which encodes a scaffold protein at glutamatergic synapses, is causally linked to autism. Using a Shank3-deficient mouse model that exhibits prominent autism-like phenotypes, we have found that histone acetylation in the prefrontal cortex (PFC) is abnormally low, which can be reversed by MS-275 (also known as Entinostat, SNDX-275), a class I histone deacetylase (HDAC) inhibitor that is selectively potent in PFC. A brief (3-day) treatment with MS-275 (i.p.) led to the sustained (11 days) rescue of autistic social preference deficits in Shank3-deficient mice, without altering locomotion, motor coordination, anxiety, or the increased grooming. MS-275 treatment also rescued the diminished NMDAR surface expression and NMDAR function induced by Shank3 deficiency. Moreover, F-actin at synapses was restored and the transcription of actin regulators was elevated by MS-275 treatment of Shank3-deficient mice, which may contribute to the recovery of actin-based NMDAR synaptic delivery. Taken together, these results suggest that MS-275 treatment could normalize the aberrant epigenetic regulation of genes, leading to the amelioration of synaptic and social deficits associated with autism.