Des-Arg(9) bradykinin as a causal metabolite for autism spectrum disorder.

BACKGROUND: Early diagnosis and therapeutic interventions can greatly enhance the developmental trajectory of children with autism spectrum disorder (ASD). However, the etiology of ASD is not completely understood. The presence of confounding factors from environment and genetics has increased the difficulty of the identification of diagnostic biomarkers for ASD. AIM: To estimate and interpret the causal relationship between ASD and metabolite profile, taking into consideration both genetic and environmental influences. METHODS: A two-sample Mendelian randomization (MR) analysis was conducted using summarized data from large-scale genome-wide association studies (GWAS) including a metabolite GWAS dataset covering 453 metabolites from 7824 European and an ASD GWAS dataset comprising 18381 ASD cases and 27969 healthy controls. Metabolites in plasma were set as exposures with ASD as the main outcome. The causal relationships were estimated using the inverse variant weight (IVW) algorithm. We also performed leave-one-out sensitivity tests to validate the robustness of the results. Based on the drafted metabolites, enrichment analysis was conducted to interpret the association via constructing a protein-protein interaction network with multi-scale evidence from databases including Infinome, SwissTargetPrediction, STRING, and Metascape. RESULTS: Des-Arg(9)-bradykinin was identified as a causal metabolite that increases the risk of ASD (ß = 0.262, SE = 0.064, PIVW = 4.64 × 10-5). The association was robust, with no significant heterogeneity among instrument variables (PMR Egger = 0.663, PIVW = 0.906) and no evidence of pleiotropy (P = 0.949). Neuroinflammation and the response to stimulus were suggested as potential biological processes mediating the association between Des-Arg(9) bradykinin and ASD. CONCLUSION: Through the application of MR, this study provides practical insights into the potential causal association between plasma metabolites and ASD. These findings offer perspectives for the discovery of diagnostic or predictive biomarkers to support clinical practice in treating ASD.

Association among blood BPDE-DNA adduct, serum interleukin-8 (IL-8) and DNA strand breaks for children with pulmonary diseases.

Exposure to benzo[a]pyrene (B[a]P) may be a risk factor for pulmonary diseases. To investigate the correlations among B[a]P exposure level, DNA strand breaks and pulmonary inflammation, we recruited 83 children diagnosed with pulmonary diseases and 63 healthy children from Guangzhou, China. Results showed that the levels of Benzo[a]pyrene diol epoxide (BPDE) DNA adduct in blood and IL-8 in serum in case group were significantly higher than those in control group (p < 0.01). Moreover, levels of atmospheric B[a]P in case group was about twice of those in control group, which was consistent with the levels of BPDE-DNA adduct in blood. Significant positive correlations were observed among the levels of BPDE-DNA adduct, IL-8 and DNA strand breaks (p < 0.05). Our findings indicate that environmental air is an important exposure source of B[a]P and higher B[a]P exposure may contribute to the occurrence of pulmonary inflammation and lead to high health risks.

Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications.

AIMS: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. BACKGROUND: Microtubule polymerization and severing form the basis for neurite outgrowth and branch formation. However, the mechanisms that underlie the dynamic instability of microtubules are unclear. Here, we showed that neurite outgrowth mediated by collapsing response mediator protein 2 (CRMP2) can be enhanced by spastin, which had an effect on the severing of microtubule cytoskeleton. OBJECTIVE: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. METHODS: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. RESULTS: We first demonstrated that CRMP2 interacted with spastin to promote neurite outgrowth and branch formation. Then our results identified that CRMP2 interacted with the microtubule- binding domain of spastin via its C-terminus, and deleting these binding sites inhibited neurite outgrowth and branch formation. In addition, we confirmed one phosphorylation site at S210 of spastin in hippocampal neurons. Spastin phosphorylation at S210 failed to alter the binding affinity of CRMP2 but inhibited its binding to microtubules. Further study showed that phosphorylation spastin at S210 inhibited the neurite outgrowth induced by CRMP2 and spastin interaction through downregulation of microtubule-severing activity. CONCLUSION: Taken together, our data demonstrated that both CRMP2 and spastin interaction and the microtubule-severing activity of spastin were required for neurite outgrowth and branch formation.

Collapsin Response Mediator Protein 2 and Endophilin2 Coordinate Regulation of AMPA Receptor GluA1 Subunit Recycling.

The dynamic trafficking of AMPA receptors (AMPARs), which enables the endocytosis, recycling, and exocytosis of AMPARs, is crucial for synaptic plasticity. Endophilin2, which directly interacts with the GluA1 subunit of AMPARs, plays an important role in AMPAR endocytosis. Collapsin response mediator protein 2 (CRMP2) promotes the maturation of the dendritic spine and can transfer AMPARs to the membrane. Although the mechanisms of AMPAR endocytosis and exocytosis are well known, the exact molecular mechanisms underlying AMPAR recycling remain unclear. Here, we report a unique interaction between CRMP2 and endophilin2. Our results showed that overexpression of CRMP2 and endophilin2 increased the amplitude and frequency of miniature excitatory synaptic currents (mEPSCs) and modestly enhanced AMPAR levels in hippocampal neurons. Furthermore, the CRMP2 and endophilin2 overexpression phenotype failed to occur when the interaction between these two proteins was inhibited. Further analysis revealed that this interaction was regulated by CRMP2 phosphorylation. The phosphorylation of CRMP2 inhibited its interaction with endophilin2; this was mainly affected by the phosphorylation of Thr514 and Ser518 by glycogen synthase kinase (GSK) 3ß. CRMP2 phosphorylation increased degradation and inhibited the surface expression of AMPAR GluA1 subunits in cultured hippocampal neurons. However, the dephosphorylation of CRMP2 inhibited degradation and promoted the surface expression of AMPAR GluA1 subunits in cultured hippocampal neurons. Taken together, our data demonstrated that the interaction between CRMP2 and endophilin2 was conductive to the recycling of AMPA receptor GluA1 subunits in hippocampal neurons.

Association between 10 urinary heavy metal exposure and attention deficit hyperactivity disorder for children.

Attention deficit hyperactivity disorder (ADHD) is associated with heavy metal exposure during adolescent development. However, the direct clinical evidence is limited. To investigate the possible association between environmental heavy metal exposure and ADHD, a case-control study was conducted with children aged 6-14 years in Guangzhou, China. Results showed that median concentrations of chromium (Cr), manganese (Mn), cobalt (Co), nickel (Ni), copper (Cu), molybdenum (Mo), tin (Sn), barium (Ba), and lead (Pb) in the urine of the case group were significantly higher than those of the control group. Children with ADHD had significantly higher levels of 8-OHdG and MDA compared with those from the control group. In addition, correlations between urinary Co, Ni, Cu, Mo, and Sn were significantly correlated with 8-OHdG and MDA concentrations in urine. After the case and control groups were combined together and the first quartile was used as the reference category, odds ratios (ORs) of ADHD for children increased significantly with the quartile increasing of urinary Co, Cu, and Sn. Our study provides a clinical evidence that Co, Cu, and Sn exposure, particularly Sn exposure, may be an environmental risk of the incurrence of ADHD for children. Furthermore, Co, Ni, Cu, Mo, and Sn exposures were significantly correlated with DNA and lipid damage.

A nuclear localization signal is required for the nuclear translocation of Fign and its microtubule­severing function.

It is commonly known that the specific function of a given ATPase associated with diverse cellular activities protein (i.e., a member of the AAA superfamily of proteins) depends primarily on its subcellular location. The microtubule­severing protein fidgetin (Fign) possesses a nuclear localization signal (NLS) that facilitates its translocation to the nucleus, where its assembly is finalized; here, Fign contributes to the regulation of microtubule configuration by cutting and trimming microtubule polymers. In the present study, Fign was found to be a nuclear protein, whose N­terminal sequence (SSLKRKAFYM; residues 314­323) acts as an NLS. Following substitution (KR to NN; 317­318) or deletion (NT; 314­323) mutations within the NLS, Fign, which is predominantly expressed in the nucleus, was found to reside in the cytoplasm of transfected cells. Furthermore, Fign was found to have an essential role in microtubule severing by preferentially targeting highly­tyrosinated microtubules (tyr­MTs). Mutation of the Fign NLS did not affect its microtubule­severing function or the cleavage of tyr­MTs, but did affect the cellular distribution of the Fign protein itself. Taken altogether, an NLS for Fign was identified, and it was demonstrated that the basic amino acids K317 and R318 are necessary for regulating its entry into the nucleus, whereas an increase in Fign in the cytosol due to mutations of the NLS did not affect its cleavage function.

Endophilin 1 knockdown prevents synaptic dysfunction induced by oligomeric amyloid ß.

Amyloid ß (Aß) has been reported to have an important role in the cognitive deficits of Alzheimer's disease (AD), as oligomeric Aß promotes synaptic dysfunction and triggers neuronal death. Recent evidence has associated an endocytosis protein, endophilin 1, with AD, as endophilin 1 levels have been reported to be markedly increased in the AD brain. The increase in endophilin 1 levels in neurons is associated with an increase in the activation of the stress kinase JNK, with subsequent neuronal death. In the present study, whole­cell patch­clamp recording demonstrated that oligomeric Aß caused synaptic dysfunction and western blotting revealed that endophilin 1 was highly expressed prior to neuronal death of cultured hippocampal neurons. Furthermore, RNA interference and electrophysiological recording techniques in cultured hippocampal neurons demonstrated that knockdown of endophilin 1 prevented synaptic dysfunction induced by Aß. Thus, a potential role for endophilin 1 in Aß­induced postsynaptic dysfunction has been identified, indicating a possible direction for the prevention of postsynaptic dysfunction in cognitive impairment and suggesting that endophilin may be a potential target for the clinical treatment of AD.

Spastin Interacts with CRMP5 to Promote Neurite Outgrowth by Controlling the Microtubule Dynamics.

Changing the microtubule dynamics is sufficient to alter the axon and dendrite specification and development. Spastin participates in the growth and regeneration of neurites by severing microtubules into small segments, and collapsin response mediator protein 5 (CRMP5) provides structural support and serves as a track for cargo transport by promoting microtubule polymerization. Nevertheless, how spastin and CRMP5 cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. In our present study, spastin interacted with CRMP5 in vitro and in vivo. The binding domains for the spastin and CRMP5 interaction were the N-terminal fragment of spastin (residues 270-328) and the C-terminal fragment of CRMP5 (residues 472-564). Spastin and its truncation mutants, including the microtubule-binding domain (MTBD) and ATPases associated with diverse cellular activities (AAA) domain, were necessary for the severing of microtubules. Furthermore, we demonstrated that microtubule polymerization of CRMP5 interfered with the microtubule-severing function of spastin. Knocking down either spastin or CRMP5 inhibited neurite outgrowth in hippocampal neurons. However, co-transfected spastin and CRMP5 promoted the outgrowth of neurites including dendrites and axons. Taken together, our data support a model in which the spastin interaction with CRMP5 promotes neurite outgrowth by controlling the microtubule dynamics.

Phosphorylation and SUMOylation of CRMP2 regulate the formation and maturation of dendritic spines.

The posttranslational modifications of CRMP2 play an important role in axon outgrowth, cell polarization and dendritic morphogenesis. However, whether CRMP2 and its posttranslational modifications are involved in dendritic spine development specifically is not completely clear. Here, we show that CRMP2 can promote the formation and maturation of dendritic spines in cultured hippocampal neurons. Overexpression of CRMP2 results in an increase in the density of spines especially the mushroom-shape spines. The amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) are both enhanced and the intensity of PSD95 is strengthened in the neurons with CRMP2 overexpression. Furthermore, dephosphorylation of CRMP2 at Thr514 and deSUMOylation at Lys374 can further promote the formation and maturation of dendritic spines, whereas, no cross-talk is found between these two posttranslational modifications in the regulation of dendritic spine formation and maturation. Taken together, our data support a model in which phosphorylation and SUMOylation modification of CRMP2 independently promote the formation and maturation of dendritic spines and participate in the process of dendritic spine plasticity.

Relationship between bisphenol A exposure and attention-deficit/ hyperactivity disorder: A case-control study for primary school children in Guangzhou, China.

Bisphenol A (BPA) is an endocrine-disrupting chemical. Studies have shown that the exposure to BPA is associated with attention-deficit/hyperactivity disorder (ADHD) during adolescent development. However the direct clinical evidence is limited. To investigate the possible association between environmental BPA exposure and the altered behavior of children, a case-control study was conducted with children aged 6-12 years in Guangzhou, China. Two hundred fifteen children diagnosed with ADHD and 253 healthy children from Guangzhou were recruited as the case and control groups, respectively. Urinary BPA and 8-hydroxy-2'-deoxyguanosine (8-OHdG, a biomarker of oxidative DNA damage) concentrations were determined by high-performance liquid chromatography/tandem spectrometry. The results showed that concentrations of urinary BPA for the case group were significantly higher than those for the control group (3.44 vs 1.70 µg/L; 4.63 vs 1.71 µg/g Crt. p < .001). A stepwise increase in the odds ratios for ADHD was observed with the increasing quartiles of children's urinary BPA (first quartile: reference category; second quartile adjusted OR: 1.79, 95% CI: 0.95-3.37; third quartile adjusted OR: 7.44, 95% CI: 3.91-14.1; fourth quartile adjusted OR: 9.41, 95% CI: 4.91-18.1). When the BPA levels were stratified by gender, the odds of ADHD among boys and girls increased significantly with urinary BPA concentrations (adjusted OR: 4.58, 95% CI: 2.84-7.37; adjusted OR: 2.83, 95% CI: 1.17-6.84). Urinary 8-OHdG concentrations in the ADHD children were significantly higher than those in the control group. Furthermore, the linear regression analysis results indicated that a significant relationship existed between BPA exposure and 8-OHdG levels (R = 0.257, p < .001). Our findings provide direct evidence that childhood BPA exposure may be related to ADHD and 8-OHdG concentrations for children. Moreover, BPA exposure could increase the higher occurrence of ADHD for boy than for girls.

CRMP4 regulates dendritic growth and maturation via the interaction with actin cytoskeleton in cultured hippocampal neurons.

CRMP family proteins (CRMPs) are critical for neurite outgrowth and maturation in the developing nervous system. However, the distinct roles of CRMP isoforms remain to be elucidated, especially in dendritic development. Here, we show that CRMP4 is sufficient and necessary for dendritic growth and maturation in cultured hippocampal neurons. Overexpression of CRMP4 promotes and genetic knockdown of CRMP4 inhibits the amount of dendritic tips, total dendritic length, spine density, and the frequency but not amplitude of miniature excitatory synaptic current. By GST-pulldown assay, we reveal that CRMP4 interacts with actin cytoskeleton by its C-terminal region, but not by N-terminal. Overexpression of actin-interacting region of CRMP4 promoted dendritic growth and maturation as CRMP4 wildtype. Taken together, these results suggest that CRMP4 is involved in dendritic development via the interaction with actin cytoskeleton in hippocampal neurons.

CRMP4 and CRMP2 Interact to Coordinate Cytoskeleton Dynamics, Regulating Growth Cone Development and Axon Elongation.

Cytoskeleton dynamics are critical phenomena that underpin many fundamental cellular processes. Collapsin response mediator proteins (CRMPs) are highly expressed in the developing nervous system, mediating growth cone guidance, neuronal polarity, and axonal elongation. However, whether and how CRMPs associate with microtubules and actin coordinated cytoskeletal dynamics remain unknown. In this study, we demonstrated that CRMP2 and CRMP4 interacted with tubulin and actin in vitro and colocalized with the cytoskeleton in the transition-zone in developing growth cones. CRMP2 and CRMP4 also interacted with one another coordinately to promote growth cone development and axonal elongation. Genetic silencing of CRMP2 enhanced, whereas overexpression of CRMP2 suppressed, the inhibitory effects of CRMP4 knockdown on axonal development. In addition, knockdown of CRMP2 or overexpression of truncated CRMP2 reversed the promoting effect of CRMP4. With the overexpression of truncated CRMP2 or CRMP4 lacking the cytoskeleton interaction domain, the promoting effect of CRMP was suppressed. These data suggest a model in which CRMP2 and CRMP4 form complexes to bridge microtubules and actin and thus work cooperatively to regulate growth cone development and axonal elongation.

[Mental health state of parents of children with autism].

OBJECTIVE: To study the mental health state of parents of children with autism. METHODS: The mental health state was evaluated by conducting the Symptom Checklist (SCL-90) on parents of 34 children with autism and of 35 healthy children. RESULTS: The SCL-90 total scores in the fathers (162.5±34.0) and mothers of autistic children (175.1±51.0) were significantly higher than those in healthy children's parents (142.4±42.8 and 152.3±40.6, respectively) (P<0.05). The SCL-90 scores of obsessive-compulsive symptoms, depression, anxiety and paranoia in the fathers of autistic children were significantly higher than those in the fathers of healthy children (P<0.05). The SCL-90 scores of obsessive-compulsive symptoms, interpersonal sensitivity, depression, anxiety, paranoia, psychotic symptoms, hostility and sleep/diet were significantly higher in the mothers of autistic children than those in the mothers of healthy children (P<0.05). The mothers of autistic children presented higher SCL-90 factor scores in interpersonal sensitivity, anxiety and psychotic symptoms than the fathers (P<0.05). CONCLUSIONS: We should pay more attention to the mental health of parents of autistic children.