Expression profiles of the autism-related SHANK proteins in the human brain.

BACKGROUND: SHANKs are major scaffolding proteins at postsynaptic densities (PSDs) in the central nervous system. Mutations in all three family members have been associated with neurodevelopmental disorders such as autism spectrum disorders (ASDs). Despite the pathophysiological importance of SHANK2 and SHANK3 mutations in humans, research on the expression of these proteins is mostly based on rodent model organisms. RESULTS: In the present study, cellular and neuropil SHANK2 expression was analyzed by immunofluorescence (IF) staining of post mortem human brain tissue from four male individuals (19 brain regions). Mouse brains were analyzed in comparison to evaluate the degree of phylogenetic conservation. Furthermore, SHANK2 and SHANK3 isoform patterns were compared in human and mouse brain lysates. While isoform expression and subcellular distribution were largely conserved, differences in neuropil levels of SHANK2 were found by IF staining: Maximum expression was concordantly measured in the cerebellum; however, higher SHANK2 expression was detected in the human brainstem and thalamus when compared to mice. One of the lowest SHANK2 levels was found in the human amygdala, a moderately expressing region in mouse. Quantification of SHANK3 IF in mouse brains unveiled a distribution comparable to humans. CONCLUSIONS: In summary, these data show that the overall expression pattern of SHANK is largely conserved in defined brain regions; however, differences do exist, which need to be considered in the translation of rodent studies. The summarized expression patterns of SHANK2 and SHANK3 should serve as a reference for future studies.

Behavioral and Sensory Deficits Associated with Dysfunction of GABAergic System in a Novel shank2-Deficient Zebrafish Model.

Hyper-reactivity to sensory inputs is a common and debilitating symptom of autism spectrum disorder (ASD), but the underlying neural abnormalities remain unclear. Two of three patients in our clinical cohort screen harboring de novo SHANK2 mutations also exhibited high sensitivity to visual, auditory, and tactile stimuli, so we examined whether shank2 deficiencies contribute to sensory abnormalities and other ASD-like phenotypes by generating a stable shank2b-deficient zebrafish model (shank2b-/-). The adult shank2b-/- zebrafish demonstrated reduced social preference and kin preference as well as enhanced behavioral stereotypy, while larvae exhibited hyper-sensitivity to auditory noise and abnormal hyperactivity during dark-to-light transitions. This model thus recapitulated the core developmental and behavioral phenotypes of many previous genetic ASD models. Expression levels of γ-aminobutyric acid (GABA) receptor subunit mRNAs and proteins were also reduced in shank2b-/- zebrafish, and these animals exhibited greater sensitivity to drug-induced seizures. Our results suggest that GABAergic dysfunction is a major contributor to the sensory hyper-reactivity in ASD, and they underscore the need for interventions that target sensory-processing disruptions during early neural development to prevent disease progression.

Genetic influences of autism candidate genes on circuit wiring and olfactory decoding.

Olfaction supports a multitude of behaviors vital for social communication and interactions between conspecifics. Intact sensory processing is contingent upon proper circuit wiring. Disturbances in genetic factors controlling circuit assembly and synaptic wiring can lead to neurodevelopmental disorders, such as autism spectrum disorder (ASD), where impaired social interactions and communication are core symptoms. The variability in behavioral phenotype expression is also contingent upon the role environmental factors play in defining genetic expression. Considering the prevailing clinical diagnosis of ASD, research on therapeutic targets for autism is essential. Behavioral impairments may be identified along a range of increasingly complex social tasks. Hence, the assessment of social behavior and communication is progressing towards more ethologically relevant tasks. Garnering a more accurate understanding of social processing deficits in the sensory domain may greatly contribute to the development of therapeutic targets. With that framework, studies have found a viable link between social behaviors, circuit wiring, and altered neuronal coding related to the processing of salient social stimuli. Here, the relationship between social odor processing in rodents and humans is examined in the context of health and ASD, with special consideration for how genetic expression and neuronal connectivity may regulate behavioral phenotypes.

Cell-Type-Specific Shank2 Deletion in Mice Leads to Differential Synaptic and Behavioral Phenotypes.

Shank2 is an excitatory postsynaptic scaffolding protein implicated in synaptic regulation and psychiatric disorders including autism spectrum disorders. Conventional Shank2-mutant (Shank2-/-) mice display several autistic-like behaviors, including social deficits, repetitive behaviors, hyperactivity, and anxiety-like behaviors. However, cell-type-specific contributions to these behaviors have remained largely unclear. Here, we deleted Shank2 in specific cell types and found that male mice lacking Shank2 in excitatory neurons (CaMKII-Cre;Shank2fl/fl) show social interaction deficits and mild social communication deficits, hyperactivity, and anxiety-like behaviors. In particular, male mice lacking Shank2 in GABAergic inhibitory neurons (Viaat-Cre;Shank2fl/fl) display social communication deficits, repetitive self-grooming, and mild hyperactivity. These behavioral changes were associated with distinct changes in hippocampal and striatal synaptic transmission in the two mouse lines. These results indicate that cell-type-specific deletions of Shank2 in mice lead to differential synaptic and behavioral abnormalities.SIGNIFICANCE STATEMENT Shank2 is an abundant excitatory postsynaptic scaffolding protein implicated in the regulation of excitatory synapses and diverse psychiatric disorders including autism spectrum disorders. Previous studies have reported in vivo functions of Shank2 mainly using global Shank2-null mice, but it remains largely unclear how individual cell types contribute to Shank2-dependent regulation of neuronal synapses and behaviors. Here, we have characterized conditional Shank2-mutant mice carrying the Shank2 deletion in excitatory and inhibitory neurons. These mouse lines display distinct alterations of synaptic transmission in the hippocampus and striatum that are associated with differential behavioral abnormalities in social, repetitive, locomotor, and anxiety-like domains.

Effect of the autism-associated lncRNA Shank2-AS on architecture and growth of neurons.

The pathogenic mechanism of autism is complex, and current research has shown that long noncoding RNAs (lncRNAs) may play important roles in this process. The antisense lncRNA of SH3 and multiple ankyrin repeat domains 2 (Shank2-AS) is upregulated in patients with autism spectrum disorder (ASD), whereas the expression of its sense strand gene Shank2 is downregulated. In neuronal cells, Shank2-AS and Shank2 can form a double-stranded RNA and inhibit Shank2 expression. Overexpression of Shank2-AS decreases neurite numbers and lengths, thereby inhibiting the proliferation of neuronal cells and promoting their apoptosis. Overexpression of Shank2 inhibits the abovementioned effects of Shank2-AS, and transfection of a vector containing the 10th intron of Shank2 (Shank2-AS is reverse-transcribed from this region) also blocks the function of Shank2-AS. Shank2 small interfering RNA plays a role similar to Shank2-AS. Therefore, Shank2-AS is abnormally expressed in patients with ASD and may affect the structure and growth of neurons by regulating Shank2 expression, thereby facilitating the development of ASD.

Differential recruitment of E3 ubiquitin ligase complexes regulates RET isoform internalization.

The RET receptor tyrosine kinase is implicated in normal development and cancer. RET is expressed as two isoforms, RET9 and RET51, with unique C-terminal tail sequences that recruit distinct protein complexes to mediate signals. Upon activation, RET isoforms are internalized with distinct kinetics, suggesting differences in regulation. Here, we demonstrate that RET9 and RET51 differ in their abilities to recruit E3 ubiquitin ligases to their unique C-termini. RET51, but not RET9, interacts with, and is ubiquitylated by CBL, which is recruited through interactions with the GRB2 adaptor protein. RET51 internalization was not affected by CBL knockout but was delayed in GRB2-depleted cells. In contrast, RET9 ubiquitylation requires phosphorylation-dependent changes in accessibility of key RET9 C-terminal binding motifs that facilitate interactions with multiple adaptor proteins, including GRB10 and SHANK2, to recruit the NEDD4 ubiquitin ligase. We showed that NEDD4-mediated ubiquitylation is required for RET9 localization to clathrin-coated pits and subsequent internalization. Our data establish differences in the mechanisms of RET9 and RET51 ubiquitylation and internalization that may influence the strength and duration of RET isoform signals and cellular outputs.This article has an associated First Person interview with the first authors of the paper.

Cerebellar Shank2 Regulates Excitatory Synapse Density, Motor Coordination, and Specific Repetitive and Anxiety-Like Behaviors.

Shank2 is a multidomain scaffolding protein implicated in the structural and functional coordination of multiprotein complexes at excitatory postsynaptic sites as well as in psychiatric disorders, including autism spectrum disorders. While Shank2 is strongly expressed in the cerebellum, whether Shank2 regulates cerebellar excitatory synapses, or contributes to the behavioral abnormalities observed in Shank2-/- mice, remains unexplored. Here we show that Shank2-/- mice show reduced excitatory synapse density in cerebellar Purkinje cells in association with reduced levels of excitatory postsynaptic proteins, including GluD2 and PSD-93, and impaired motor coordination in the Erasmus test. Shank2 deletion restricted to Purkinje cells (Pcp2-Cre;Shank2fl/fl mice) leads to similar reductions in excitatory synapse density, synaptic protein levels, and motor coordination. Pcp2-Cre;Shank2fl/fl mice do not recapitulate autistic-like behaviors observed in Shank2-/- mice, such as social interaction deficits, altered ultrasonic vocalizations, repetitive behaviors, and hyperactivity. However, Pcp2-Cre;Shank2fl/fl mice display enhanced repetitive behavior in the hole-board test and anxiety-like behavior in the light-dark test, which are not observed in Shank2-/- mice. These results implicate Shank2 in the regulation of cerebellar excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors. SIGNIFICANCE STATEMENT: The postsynaptic side of excitatory synapses contains multiprotein complexes, termed the postsynaptic density, which contains receptors, scaffolding/adaptor proteins, and signaling molecules. Shank2 is an excitatory postsynaptic scaffolding protein implicated in the formation and functional coordination of the postsynaptic density and has been linked to autism spectrum disorders. Using Shank2-null mice and Shank2-conditional knock-out mice with a gene deletion restricted to cerebellar Purkinje cells, we explored functions of Shank2 in the cerebellum. We found that Shank2 regulates excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors, but is not associated with autistic-like social deficits or repetitive behaviors.

A critical role of spinal Shank2 proteins in NMDA-induced pain hypersensitivity.

Background Self-injurious behaviors (SIBs) are devastating traits in autism spectrum disorder (ASD). Although deficits in pain sensation might be one of the contributing factors underlying the development of SIBs, the mechanisms have yet to be addressed. Recently, the Shank2 synaptic protein has been considered to be a key component in ASD, and mutations of SHANK2 gene induce the dysfunction of N-methyl-D-aspartate (NMDA) receptors, suggesting a link between Shank2 and NMDA receptors in ASD. Given that spinal NMDA receptors play a pivotal role in pain hypersensitivity, we investigated the possible role of Shank2 in nociceptive hypersensitivity by examining changes in spontaneous pain following intrathecal NMDA injection in S hank2-/- ( Shank2 knock-out, KO) mice. Results Intrathecal NMDA injection evoked spontaneous nociceptive behaviors. These NMDA-induced nociceptive responses were significantly reduced in Shank2 KO mice. We also observed a significant decrease of NMDA currents in the spinal dorsal horn of Shank2 KO mice. Subsequently, we examined whether mitogen-activated protein kinase or AKT signaling is involved in this reduced pain behavior in Shank2 KO mice because the NMDA receptor is closely related to these signaling molecules. Western blotting and immunohistochemistry revealed that spinally administered NMDA increased the expression of a phosphorylated form of extracellular signal-regulated kinase (p-ERK) which was significantly reduced in Shank2 KO mice. However, p38, JNK, or AKT were not changed by NMDA administration. The ERK inhibitor, PD98059, decreased NMDA-induced spontaneous pain behaviors in a dose-dependent manner in wild-type mice. Moreover, it was found that the NMDA-induced increase in p-ERK was primarily colocalized with Shank2 proteins in the spinal cord dorsal horn. Conclusion Shank2 protein is involved in spinal NMDA receptor-mediated pain, and mutations of Shank2 may suppress NMDA-ERK signaling in spinal pain transmission. This study provides new clues into the mechanisms underlying pain deficits associated with SIB and deserves further study in patients with ASD.

Activity and circadian rhythm influence synaptic Shank3 protein levels in mice.

Various recent studies revealed that the proteins of the Shank family act as major scaffold organizing elements in the post-synaptic density of excitatory synapses and that their expression level is able to influence synapse formation, maturation and ultimately brain plasticity. An imbalance in Shank3 protein levels has been associated with a variety of neuropsychological and neurodegenerative disorders including autism spectrum disorders and Phelan-McDermid syndrome. Given that sleep disorders and low melatonin levels are frequently observed in autism spectrum disorders, and that circadian rhythms may be able to modulate Shank3 signaling and thereby synaptic function, here, we performed in vivo studies on CBA mice using protein biochemistry to investigate the synaptic expression levels of Shank3α during the day in different brain regions. Our results show that synaptic Shank3 protein concentrations exhibit minor oscillations during the day in hippocampal and striatal brain regions that correlate with changes in serum melatonin levels. Furthermore, as circadian rhythms are tightly connected to activity levels in mice, we increased physical activity using running wheels. The expression of Shank3α increases rapidly by induced activity in thalamus and cortex, but decreases in striatum, superimposing the circadian rhythms of different brain regions. We conclude that synaptic Shank3 proteins build highly dynamic platforms that are modulated by the light:dark cycles but even more so driven by activity. Using wild-type CBA mice, we show that Shank3 is a highly dynamic and activity-regulated protein at synapses. In the hippocampus, changes in synaptic Shank3 levels are influenced by circadian rhythm/melatonin concentration, while running activity increases and decreases levels of Shank3 in the cortex and striatum respectively.

Shank3 is localized in axons and presynaptic specializations of developing hippocampal neurons and involved in the modulation of NMDA receptor levels at axon terminals.

Autism-related Shank1, Shank2, and Shank3 are major postsynaptic scaffold proteins of excitatory glutamatergic synapses. A few studies, however, have already indicated that within a neuron, the presence of Shank family members is not limited to the postsynaptic density. By separating axons from dendrites of developing hippocampal neurons in microfluidic chambers, we show that RNA of all three Shank family members is present within axons. Immunostaining confirms these findings as all three Shanks are indeed found within separated axons and further co-localize with well-known proteins of the presynaptic specialization in axon terminals. Therefore, Shank proteins might not only serve as postsynaptic scaffold proteins, but also play a crucial role during axonal outgrowth and presynaptic development and function. This is supported by our findings that shRNA-mediated knockdown of Shank3 results in up-regulation of the NMDA receptor subunit GluN1 in axon terminals. Taken together, our findings will have major implications for the future analysis of neuronal Shank biology in both health and disease. Shank1, Shank2, and Shank3 are major postsynaptic scaffold proteins of excitatory glutamatergic synapses strongly related to several neuropsychiatric disorders. However, a few studies have already implicated a functional role of the Shanks beyond the postsynaptic density (PSD). We here show that all three Shanks are localized in both axons and pre-synaptic specializiations of developing hippocampal neurons in culture. We further provide evidence that Shank3 is involved in the modulation of NMDA receptor levels at axon terminals. Taken together, our study will open up novel avenues for the future analysis of neuronal Shank biology in both health and disease.

Reduced acute nociception and chronic pain in Shank2-/- mice.

Autism spectrum disorder is a debilitating mental illness and social issue. Autism spectrum disorder patients suffer from social isolation, cognitive deficits, compulsive behavior, and sensory deficits, including hyposensitivity to pain. However, recent studies argued that autism spectrum disorder patients show physiological pain response and, in some cases, even extremely intense pain response to harmless stimulation. Recently, Shank gene family was reported as one of the genetic risk factors of autism spectrum disorder. Thus, in this study, we used Shank2(-) (/) (-) (Shank2 knock-out, KO) mice to investigate the controversial pain sensitivity issue and found that Shank2 KO mice showed reduced tactile perception and analgesia to chronic pain.

Leveraging FAM features to predict the prognosis of LGG patients and immunotherapy outcome.

Heterogeneity at biological and transcriptomic levels poses a challenge in defining and typing low-grade glioma (LGG), leading to a critical need for specific molecular signatures to enhance diagnosis, therapy, and prognostic evaluation of LGG. This study focused on fatty acid metabolism (FAM) related genes and prognostic features to investigate the mechanisms and treatment strategies for LGG cell metastasis and invasion. By screening 158 FAM-related genes and clustering 512 LGG samples into two subtypes (C1 and C2), differential gene expression analysis and functional enrichment were performed. The immune cell scores and prognosis were compared between the two subtypes, with C1 showing poorer outcomes and higher immune scores. A four-gene signature (PHEX, SHANK2, HOPX, and LGALS1) was identified and validated across different datasets, demonstrating a stable predictive effect. Cellular experiments confirmed the roles of LGALS1 and HOPX in promoting tumor cell proliferation, migration, and invasion, while SHANK2 exhibited a suppressive effect. This four-gene signature based on FAM-related genes offers valuable insights for understanding the pathogenesis and clinical management of LGG.

Probing cognitive flexibility in Shank2-deficient mice: Effects of D-cycloserine and NMDAR signaling hub dynamics.

Neurodevelopmental disorders such as autism spectrum disorder (ASD) have a heterogeneous etiology but are largely associated with genetic factors. Robust evidence from recent human genetic studies has linked mutations in the Shank2 gene to idiopathic ASD. Modeling these Shank2 mutations in animal models recapitulates behavioral changes, e.g. impaired social interaction and repetitive behavior of ASD patients. Shank2-deficient mice exhibit NMDA receptor (NMDAR) hypofunction and associated behavioral deficits. Of note, NMDARs are strongly implicated in cognitive flexibility. Their hypofunction, e.g. observed in schizophrenia, or their pharmacological inhibition leads to impaired cognitive flexibility. However, the association between Shank2 mutations and cognitive flexibility is poorly understood. Using Shank2-deficient mice, we explored the role of Shank2 in cognitive flexibility measured by the attentional set shifting task (ASST) and whether ASST performance in Shank2-deficient mice can be modulated by treatment with the partial NMDAR agonist D-cycloserine (DCS). Furthermore, we investigated the effects of Shank2 deficiency, ASST training, and DCS treatment on the expression level of NMDAR signaling hub components in the orbitofrontal cortex (OFC), including NMDAR subunits (GluN2A, GluN2B, GluN2C), phosphoglycerate dehydrogenase and serine racemase. Surprisingly, Shank2 deficiency did not affect ASST performance or alter the expression of the investigated NMDAR signaling hub components. Importantly, however, DCS significantly improved ASST performance, demonstrating that positive NMDAR modulation facilitates cognitive flexibility. Furthermore, DCS increased the expression of GluN2A in the OFC, but not that of other NMDAR signaling hub components. Our findings highlight the potential of DCS as a pharmacological intervention to improve cognitive flexibility impairments downstream of NMDAR modulation and substantiate the key role of NMDAR in cognitive flexibility.

Transcriptional determinism and stochasticity contribute to the complexity of autism-associated SHANK family genes.

Precision of transcription is critical because transcriptional dysregulation is disease causing. Traditional methods of transcriptional profiling are inadequate to elucidate the full spectrum of the transcriptome, particularly for longer and less abundant mRNAs. SHANK3 is one of the most common autism causative genes. Twenty-four Shank3-mutant animal lines have been developed for autism modeling. However, their preclinical validity has been questioned due to incomplete Shank3 transcript structure. We apply an integrative approach combining cDNA-capture and long-read sequencing to profile the SHANK3 transcriptome in humans and mice. We unexpectedly discover an extremely complex SHANK3 transcriptome. Specific SHANK3 transcripts are altered in Shank3-mutant mice and postmortem brain tissues from individuals with autism spectrum disorder. The enhanced SHANK3 transcriptome significantly improves the detection rate for potential deleterious variants from genomics studies of neuropsychiatric disorders. Our findings suggest that both deterministic and stochastic transcription of the genome is associated with SHANK family genes.

Downregulation of the Autism Spectrum Disorder Gene Shank2 Decreases Bone Mass in Male Mice.

Mutations of the postsynaptic scaffold protein Shank2 lead to autism spectrum disorders (ASD). These patients frequently suffer from higher fracture risk. Here, we investigated whether Shank2 directly regulates bone mass. We show that Shank2 is expressed in bone and that Shank2 levels are increased during osteoblastogenesis. Knockdown of Shank2 by siRNA targeting the encoding regions for PDZ and SAM domain inhibits osteoblastogenesis of primary murine calvarial osteoblasts. Shank2 knockout mice (Shank2 -/-) have a decreased bone mass due to reduced osteoblastogenesis and bone formation, whereas bone resorption remains unaffected. Induced pluripotent stem cells (iPSCs)-derived osteoblasts from a loss-of-function Shank2 mutation in a patient showed a significantly reduced osteoblast differentiation potential. Moreover, silencing of known Shank2 interacting proteins revealed that a majority of them promote osteoblast differentiation. From this we conclude that Shank2 and interacting proteins known from the central nervous system are decisive regulators in osteoblast differentiation. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Increased social interaction in Shank2-deficient mice following acute social isolation.

Autism spectrum disorder (ASD) is neuropsychiatric disorder with a gender specific risk. Although social impairment in ASD is one of the well characterized phenotypes, loneliness issue resides in patients with ASD and emerging reports show gender distribution in symptoms. Acute social isolation increases the motivation to socially interact in a gender-dependent manner, as only the male mice show increase in sociability following isolation. However, it remains to be explored whether the effects of loneliness in ASD differ between genders. Here, we used Shank2-deficient (Shank2-/-) mice, one of the animal models of ASD, to examine the sociability changes after acute social isolation. While only the male wild-type (WT) mice display increased sociability following 24-h isolation, both sexes of Shank2-/- mice show an increase in social interaction following isolation. These observations provide evidence that animal models of ASD have the sensitivity to acute social isolation and further show the motivation to socially interact.

Identification of novel SHANK2 variants in two Chinese families via exome and RNA sequencing.

Background: SHANK2 encodes a postsynaptic scaffolding protein involved in synapse formation, stabilization and homeostasis. Variations or microdeletions in the SHANK2 gene have been linked to a variety of neurodevelopmental disorders, including autism spectrum disorders (ASD) and mild to moderate intellectual disability (ID) in human. However, the number of reported cases with SHANK2 defects remains limited, with only 14 unrelated patients documented worldwide. Methods: In this study, we investigated four patients from three families with ID. Whole-exome sequencing (WES) was performed to explore the genetic causes, while Sanger sequencing was used to confirm the identified variants. Furthermore, RNA sequencing and functional enrichment analysis were performed on patients with likely pathogenic variants to gain further insights into the molecular landscape associated with these variants. Results: Two novel variants in the SHANK2 gene: a heterozygous splicing substitution (NM_012309.5:c.2198-1G>A p.Pro734Glyfs*22) in Family 1, and a heterozygous nonsense variant [NM_012309.5:c.2310dupT p.(Lys771*)] in Family 2 were identified by WES and confirmed by Sanger sequencing. RNA sequencing and cohort analysis identified a total of 1,196 genes exhibiting aberrant expression in three patients. Functional enrichment analysis revealed the involvement of these genes in protein binding and synaptic functions. Conclusion: We identified two novel loss of function variants that broadens the spectrum of SHANK2 variants. Furthermore, this study enhances our understanding of the molecular mechanisms underlying SHANK2-related disorders.

Shank2 identifies a subset of glycinergic neurons involved in altered nociception in an autism model.

BACKGROUND: Autism Spectrum Disorders (ASD) patients experience disturbed nociception in the form of either hyposensitivity to pain or allodynia. A substantial amount of processing of somatosensory and nociceptive stimulus takes place in the dorsal spinal cord. However, many of these circuits are not very well understood in the context of nociceptive processing in ASD. METHODS: We have used a Shank2-/- mouse model, which displays a set of phenotypes reminiscent of ASD, and performed behavioural and microscopic analysis to investigate the role of dorsal horn circuitry in nociceptive processing of ASD. RESULTS: We determined that Shank2-/- mice display increased sensitivity to formalin pain and thermal preference, but a sensory specific mechanical allodynia. We demonstrate that high levels of Shank2 expression identifies a subpopulation of neurons in murine and human dorsal spinal cord, composed mainly by glycinergic interneurons and that loss of Shank2 causes the decrease in NMDAR in excitatory synapses on these inhibitory interneurons. In fact, in the subacute phase of the formalin test, glycinergic interneurons are strongly activated in wild type (WT) mice but not in Shank2-/- mice. Consequently, nociception projection neurons in laminae I are activated in larger numbers in Shank2-/- mice. LIMITATIONS: Our investigation is limited to male mice, in agreement with the higher representation of ASD in males; therefore, caution should be applied to extrapolate the findings to females. Furthermore, ASD is characterized by extensive genetic diversity and therefore the findings related to Shank2 mutant mice may not necessarily apply to patients with different gene mutations. Since nociceptive phenotypes in ASD range between hyper- and hypo-sensitivity, diverse mutations may affect the circuit in opposite ways. CONCLUSION: Our findings prove that Shank2 expression identifies a new subset of inhibitory interneurons involved in reducing the transmission of nociceptive stimuli and whose unchecked activation is associated with pain hypersensitivity. We provide evidence that dysfunction in spinal cord pain processing may contribute to the nociceptive phenotypes in ASD.

Remote ischemic conditioning alleviates chronic cerebral hypoperfusion-induced cognitive decline and synaptic dysfunction via the miR-218a-5p/SHANK2 pathway.

Vascular cognitive impairment (VCI) due to chronic cerebral hypoperfusion (CCH), is the second leading cause of dementia. Although synaptic impairment plays a critical role in VCI, its exact mechanism remains unknown. Our previous research revealed that remote ischemic conditioning (RIC) could alleviate cognitive decline resulting from CCH, however, its effects on synaptic impairment remain unclear. In this study, we confirmed that RIC alleviated both cognitive decline and its associated synaptic dysfunction caused by CCH. RNA sequencing revealed that CCH increased in miR-218a-5p expression, which was decreased by RIC. Elevated miR-218a-5p levels limited the benefits of RIC, however, inhibiting miR-218a-5p in hippocampal CA1 neurons rescued synaptic dysfunction. Additionally, we found that SHANK2 is a downstream target of miR-218a-5p, and inhibiting SHANK2 expression reduced the alleviation caused by hypoxic conditioning in synaptic impairment in vitro. In conclusion, our results suggested that RIC alleviated synaptic impairment via the miR-218a-5p/SHANK2 pathway, which could be a potential biomarker or therapeutic target for cognitive impairment caused by CCH.