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Mutations in the Contactin-associated protein-like 2 (CNTNAP2) gene are associated with autism spectrum disorder (ASD), and ectodomain shedding of the CNTNAP2 protein plays a role in its function. However, key enzymes involved in the C-terminal cleavage of CNTNAP2 remain largely unknown, and the effect of ASD-associated mutations on this process and its role in ASD pathogenesis remain elusive. In this report we showed that CNTNAP2 undergoes sequential cleavages by furin, ADAM10/17-dependent α-secretase and presenilin-dependent γ-secretase. We identified that the cleavage sites of ADAM10 and ADAM17 in CNTNAP2 locate at its C-terminal residue I79 and L96, and the main α-cleavage product C79 by ADAM10 is required for the subsequent γ-secretase cleavage to generate CNTNAP2 intracellular domain (CICD). ASD-associated CNTNAP2 mutations impair the α-cleavage to generate C79, and the inhibition leads to ASD-like repetitive and social behavior abnormalities in the Cntnap2-I1254T knock-in mice. Finally, exogenous expression of C79 improves autism-like phenotypes in the Cntnap2-I1254T knock-in and Cntnap2-/- knockout mice. This data demonstrates that the α-secretase is essential for CNTNAP2 processing and its function. Our study indicates that inhibition of the cleavage by pathogenic mutations underlies ASD pathogenesis, and upregulation of its C-terminal fragments could have therapeutical potentials for ASD treatment.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Animais , Camundongos , Secretases da Proteína Precursora do Amiloide/genética , Transtorno do Espectro Autista/genética , Mutação/genética , Camundongos Knockout , Contactinas/genética , Fenótipo , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genéticaRESUMO
As the most prevalent neurodevelopmental disorders in children, autism spectrum disorders (ASD) are characterized by deficits in language development, social interaction, and repetitive behaviors or inflexible interests. Contactin associated protein like 2 (CNTNAP2), encoding a single transmembrane protein (CNTNAP2) with 1331 amino acid residues, is a widely validated ASD-susceptible gene. Cntnap2-deficient mice also show core autism-relevant behaviors, including the social deficits and repetitive behavior. However, the cellular mechanisms underlying dysfunction CNTNAP2 and ASD remain elusive. In this study, we found a motif within the transmembrane domain of CNTNAP2 was highly homologous to the γ-secretase cleavage site of amyloid-ß precursor protein (APP), suggesting that CNTNAP2 may undergo proteolytic cleavage. Further biochemical analysis indicated that CNTNAP2 is cleaved by γ-secretase to produce the CNTNAP2 intracellular domain (CICD). Virally delivery of CICD to the medial prefrontal cortex (mPFC) in Cntnap2-deficient (Cntnap2-/-) mice normalized the deficit in the ASD-related behaviors, including social deficit and repetitive behaviors. Furthermore, CICD promoted the nuclear translocation of calcium/calmodulin-dependent serine protein kinase (CASK) to regulate the transcription of genes, such as Prader Willi syndrome gene Necdin. Whereas Necdin deficiency led to reduced social interaction in mice, virally expression of Necdin in the mPFC normalized the deficit in social preference of Cntnap2-/- mice. Our results thus reveal a critical function of CICD and highlight a role of the CNTNAP2-CASK-Necdin signaling pathway in ASD.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Camundongos , Animais , Transtorno Autístico/genética , Secretases da Proteína Precursora do Amiloide , Transtorno do Espectro Autista/genética , Transdução de Sinais , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genéticaRESUMO
Introduction: Hypoxia is an environmental risk factor for many disorders throughout life. Perinatal hypoxia contributes to autism spectrum disorder (ASD), while hypoxic conditions in the elderly facilitate memory deficits. However, the effects of hypoxia on adolescence remains elusive. CNTNAP2 is a critical molecule in ASD pathogenesis with undefined mechanisms. We investigate hypoxia's impact on adolescence and the underlying mechanism related to CNTNAP2. Methods: Three-chamber social approach test, Y maze, Morris Water Maze and Open Field Test were applied to evaluate behavioral alterations. Immunoblotting, 5'- RACE and dual-luciferase reporter assay were performed to examine CNTNAP2 protein expression, transcription start site (TSS) of human CNTNAP2 gene and CNTNAP2 promoter activity, respectively. Results: Intermittent hypoxia treatment improved social behaviors and working memory in adolescent mice. CNTNAP2 was increased in the brains of hypoxia-treated mice. The sequencing results identified the TSS at 518 bp upstream of the translation start site ATG. Hypoxia upregulated CNTNAP2 by interacting with functional hypoxia response elements in CNTNAP2 promoter. Conclusion: Intermittent hypoxia enhanced sociability and working memory associated with CNTNAP2 upregulation. Our study provides novel insights into intermittent hypoxia's impact on development and the interaction between genetic and environmental risk factors in ASD pathogenesis.
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TREM2 encoding the transmembrane receptor protein TREM2 is a risk gene of Alzheimer's disease (AD), and the impairment of TREM2 functions in microglia due to mutations in TREM2 may significantly increase the risk of AD by promoting AD pathologies. However, how the expression of TREM2 is regulated and the transcription factors required for TREM2 expression are largely unknown. By luciferase assay, DNA pull-down, and in silico predictions, we identified Yin Yang 1(YY1) as a binding protein of the minimal promoter of the TREM2 gene, and the binding was further confirmed by EMSA and DNA pull-down assay. shRNA-mediated YY1 silencing significantly reduced the activity of the TREM2 minimal promoter and TREM2 protein levels in the microglial cell line BV2 and the neuroblastoma Neuro2A. Furthermore, we found that the levels of TREM2 and YY1 were both downregulated in lipopolysaccharide-treated BV2 cells and in the brain of AD model mice. These results demonstrated that YY1 plays a crucial role in the regulation of TREM2 expression. Our study suggests that microglial YY1 could be targeted to maintain TREM2 expression for AD prevention and therapy.
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Doença de Alzheimer , Receptores Imunológicos , Fator de Transcrição YY1 , Animais , Camundongos , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Linhagem Celular , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
Contactin-associated protein-like 2 (CNTNAP2) gene, located on chromosome 7q35, is one of the largest genes in the human genome. CNTNAP2 protein is a type-I transmembrane protein specifically expressed in the nervous system, with versatile roles in the axonal organization, synaptic functions, neuronal migration, and functional connectivity. CNTNAP2 has been widely investigated as a risk gene for autism spectrum disorder (ASD), and recent studies also implicated CNTNAP2 in Alzheimer's disease (AD). Knowledge of the regulations on CNTNAP2's life cycle is necessary for understanding the related physiological functions and pathological conditions. However, the mechanisms underlying CNTNAP2 protein degradation remain elusive. Therefore, we systematically investigated the half-life and degradation pathway of the human CNTNAP2 protein. We discovered that CNTNAP2 has C-terminal fragments (CTF), which may have essential physiological functions. Our results demonstrated that CNTNAP2 full-length protein and CTF have a short half-life of about 3-4 h. CNTNAP2 proteins are degraded by the ubiquitin-proteasome system and the macroautophagy-lysosome pathway, while the lysosome pathway is more common for CNTNAP2 degradation. This study will provide novel insights and valuable tools for CNTNAP2 functional research in physiological and pathological scenarios.
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Transtorno do Espectro Autista , Complexo de Endopeptidases do Proteassoma , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Macroautofagia , Transtorno do Espectro Autista/genética , Lisossomos/metabolismo , Proteínas de Membrana , Proteínas do Tecido NervosoRESUMO
Autism spectrum disorder (ASD) is a highly variable and heritable neurodevelopmental disease (NDD) with strong genetic underpinnings. In this issue of the JCI, Chen et al. analyzed 2 previously reported, large-scale sequenced ASD cohorts and reported that GIGYF1 is the second most mutated among ASD risk genes. In this issue of the JCI, Chen et al. used a conditional mouse model combined with molecular technologies based on human genetic analyses to determine the critical role of GIGYF1 in ASD. GIGYF1-deficiency affected the recycling of IGF-1R, thereby suppressing the IGF-1R/ERK signaling pathway. Disruption of GIGYF1 in the developing mouse brain led to social deficits and cognitive impairments. These findings extend our understanding of ASD pathogenesis and provide an avenue for developing potentially effective preventions and treatments for patients with ASD.
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Transtorno do Espectro Autista , Receptor IGF Tipo 1/metabolismo , Animais , Transtorno do Espectro Autista/genética , Proteínas de Transporte/genética , Modelos Animais de Doenças , Humanos , Camundongos , Transdução de SinaisRESUMO
Toxoplasma gondii is an obligate intracellular protozoan parasite, which can infect almost all warm-blooded animals, including humans, leading to toxoplasmosis. Currently, the effective treatment for human toxoplasmosis is the combination of sulphadiazine and pyrimethamine. However, both drugs have serious side-effects and toxicity in the host. Therefore, there is an urgent need for the discovery of new anti-T. gondii drugs with high potency and less or no side-effects. Our findings suggest that lumefantrine exerts activity against T. gondii by inhibiting its proliferation in Vero cells in vitro without being toxic to Vero cells (P ≤ 0.01). Lumefantrine prolonged mice infected with T. gondii from death for 3 days at the concentration of 50 µg L-1 than negative control (phosphate-buffered saline treated only), and reduced the parasite burden in mouse tissues in vivo (P ≤ 0.01; P ≤ 0.05). In addition, a significant increase in interferon gamma (IFN-γ) production was observed in high-dose lumefantrine-treated mice (P ≤ 0.01), whereas interleukin 10 (IL-10) and IL-4 levels increased in low-dose lumefantrine-treated mice (P ≤ 0.01). The results demonstrated that lumefantrine may be a promising agent to treat toxoplasmosis, and more experiments on the protective mechanism of lumefantrine should be undertaken in further studies.
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Lumefantrina/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasmose/tratamento farmacológico , Animais , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Humanos , Camundongos , Células VeroRESUMO
Host cell invasion by Toxoplasma gondii is crucial for the survival and proliferation of parasite. The process of T. gondii tachyzoite invasion requires interaction between parasite proteins and receptors on the surface of host cells. Sialic acid is one of the important receptors for host cell invasion by T. gondii. However, the parasite-derived proteins interacting with sialic acid have not been well characterized. In this study, a novel protein named putative TCP-1 chaperonin (TGME49_318410) in T. gondii (TgTCP-1) was targeted and characterized. TgTCP-1 protein colocalized with MIC3 protein, which could be secreted from T. gondii tachyzoites, and this protein showed a specific binding activity to sialic acid, and DC and Vero cells in vitro. The binding of TgTCP-1 protein to DC and Vero cells were inhibited by either pre-incubation with free sialic acid or neuraminidase treatment of the cells. Moreover, a significant reduction of T. gondii invasion in Vero cells was observed after pre-incubation of the cells with recombinant TgTCP-1 protein. These results illustrated that TgTCP-1 is an important molecule involved in sialic acid-dependent host cell invasion by T. gondii.
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Many obligate intracellular apicomplexan parasites have adapted a distinct invasion mechanism involving a close interaction between the parasite ligands and the sialic acid (SA) receptor. We found that sialic acid binding protein-1 (SABP1), localized on the outer membrane of the zoonotic parasite Toxoplasma gondii, readily binds to sialic acid on the host cell surface. The binding was sensitive to neuraminidase treatment. Cells preincubated with recombinant SABP1 protein resisted parasite invasion in vitro. The parasite lost its invasion capacity and animal infectivity after the SABP1 gene was deleted, whereas complementation of the SABP1 gene restored the virulence of the knockout strain. These data establish the critical role of SABP1 in the invasion process of T. gondii. The previously uncharacterized protein, SABP1, facilitated T. gondii attachment and invasion via sialic acid receptors.
Assuntos
Proteínas de Transporte/genética , Interações Hospedeiro-Parasita , Infecções/genética , Ácido N-Acetilneuramínico/metabolismo , Toxoplasma/genética , Toxoplasmose/genética , Virulência/genética , Animais , Infecções/fisiopatologia , Modelos Animais , Ácido N-Acetilneuramínico/genética , Toxoplasmose/fisiopatologiaRESUMO
Toxoplasma gondii is an obligate intracellular zoonotic parasite, infecting warm-blood animals including humans. Previous serological surveys of T. gondii infection have focused on people of different occupations and special groups, such as slaughterhouse workers, AIDS patients and pregnant women. To investigate the potential impact of T. gondii infection on the health of young students, the prevalence of T. gondii infection and associated risk factors among the newly enrolled undergraduates and postgraduate students were investigated. A total of 3,569 newly enrolled students (age range: 15- to 37-years-old, median 26 years) from various regions of China were recruited in this study. The serum samples were tested for the presence of T. gondii specific IgG by the modified agglutination test (MAT). Questionnaires were used to collect information on risk factors for T. gondii infection. Sixty-five (1.82%) out of 3,569 participants were seropositive for IgG antibodies to T. gondii by MAT (titer≥1:20). Four variables were found to be positively associated with T. gondii infection, including primary geographical location, living in rural areas, gardening or agriculture, and drinking unboiled water by the univariate logistic regression, and only gardening or agriculture was the independent risk factor for T. gondii positivity by using multivariate logistic regression in this study, which may provide information to guide future research and control policies.