Association between prenatal and perinatal factors and the severity of clinical presentation of children with ASD: Report from the ELENA COHORT.

Many studies have suggested that prenatal and perinatal factors increase the risk for autism spectrum disorder (ASD). However, few reports have addressed the question of their influence on the severity of the clinical presentation of children with ASD. Our objective was to determine the prenatal and perinatal factors that are associated with the severity of autistic symptoms and intellectual and adaptive functioning deficits. Data were collected from a subset of 169 children with a confirmed diagnosis of ASD, recruited from the ELENA cohort. A risk of premature delivery was associated, with an increased risk for severe autistic symptoms and placental pathologies and birth complications were associated with an increased risk of communication adaptive deficits, in multivariate analysis. Our results highlight the importance of systematic screening for these pre/perinatal factors, especially in mothers at risk of having a child with ASD.

Investigating the natural history and prognostic factors of ASD in children: the multicEntric Longitudinal study of childrEN with ASD - the ELENA study protocol.

INTRODUCTION: There is global concern about the increasing prevalence of autism spectrum disorders (ASDs), which are early-onset and long-lasting disorders. Although ASDs are considered to comprise a unique syndrome, their clinical presentation and outcome vary widely. Large-scale and long-term cohort studies of well-phenotyped samples are needed to better understand the course of ASDs and their determinants. The primary objective of the multicEntric Longitudinal study of childrEN with ASD (ELENA) study is to understand the natural history of ASD in children and identify the risk and prognostic factors that affect their health and development. METHODS AND ANALYSIS: This is a multicentric, longitudinal, prospective, observational cohort in which 1000 children with ASD diagnosed between 2 and 16 years of age will be recruited by 2020 and followed over 6 years. The baseline follow-up starts with the clinical examination to establish the ASD diagnosis. A battery of clinical tools consisting of the Autism Diagnostic Observation Schedule, the revised version of the Autism Diagnostic Interview, measures of intellectual functioning, as well as large-scale behavioural and developmental measurements will allow us to study the heterogeneity of the clinical presentation of ASD subtypes. Subsequent follow-up at 18 months and at 3, 4.5 and 6 years after the baseline examination will allow us to explore the developmental trajectories and variables associated with the severity of ASD. In addition to the children's clinical and developmental examinations, parents are invited to complete self-reported questionnaires concerning perinatal and early postnatal history, congenital anomalies, genetic factors, lifestyle factors, medical and psychiatric comorbidities, and the socioeconomic environment. As of 1 November 2018, a total of 766 participants have been included. ETHICS AND DISSEMINATION: Ethical approval was obtained through the Marseille Mediterranean Ethics Committee (ID RCB: 2014-A01423-44), France. We aim to disseminate the findings through national and international conferences, international peer-reviewed journals, and social media. TRIAL REGISTRATION NUMBER: NCT02625116; Pre-results.

Proteomic data on the nuclear interactome of human MCM9.

We present data relating to the interactome of MCM9 from the nuclei of human cells. MCM9 belongs to the AAA+ superfamily, and contains an MCM domain and motifs that may confer DNA helicase activity. MCM9 has been shown to bind MCM8, and has been implicated in DNA replication and homologous recombination. However, the mechanistic basis of MCM9's role in DNA repair is poorly understood, and proteins with which it interacts were hitherto unknown. We performed tandem affinity purification of MCM9 and its interacting proteins from nuclear extracts of human cells, followed by proteomic analysis, thereby generating a set of mass spectrometry data corresponding to the MCM9 interactome [1]. The proteomic data set comprises 29 mass spectrometry RAW files, deposited to the ProteomeXchange Consortium, and freely available from the PRIDE partner repository with the data set identifier PXD000212. A set of 22 interacting proteins identified from the proteomic data was used to create an MCM9-centered interactive network diagram, using the Cytoscape program. These data allow the scientific community to access, mine and explore the human nuclear MCM9 interactome.

MCM9 Is Required for Mammalian DNA Mismatch Repair.

DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects DNA polymerase errors during replication to maintain genomic integrity. In E. coli, the DNA helicase UvrD is implicated in MMR, yet an analogous helicase activity has not been identified in eukaryotes. Here, we show that mammalian MCM9, a protein involved in replication and homologous recombination, forms a complex with MMR initiation proteins (MSH2, MSH3, MLH1, PMS1, and the clamp loader RFC) and is essential for MMR. Mcm9-/- cells display microsatellite instability and MMR deficiency. The MCM9 complex has a helicase activity that is required for efficient MMR since wild-type but not helicase-dead MCM9 restores MMR activity in Mcm9-/- cells. Moreover, MCM9 loading onto chromatin is MSH2-dependent, and in turn MCM9 stimulates the recruitment of MLH1 to chromatin. Our results reveal a role for MCM9 and its helicase activity in mammalian MMR.

Cell-free DNA in Human Follicular Microenvironment: New Prognostic Biomarker to Predict in vitro Fertilization Outcomes.

Cell-free DNA (cfDNA) fragments, detected in blood and in other biological fluids, are released from apoptotic and/or necrotic cells. CfDNA is currently used as biomarker for the detection of many diseases such as some cancers and gynecological and obstetrics disorders. In this study, we investigated if cfDNA levels in follicular fluid (FF) samples from in vitro fertilization (IVF) patients, could be related to their ovarian reserve status, controlled ovarian stimulation (COS) protocols and IVF outcomes. Therefore, 117 FF samples were collected from women (n = 117) undergoing IVF/Intra-cytoplasmic sperm injection (ICSI) procedure and cfDNA concentration was quantified by ALU-quantitative PCR. We found that cfDNA level was significantly higher in FF samples from patients with ovarian reserve disorders (low functional ovarian reserve or polycystic ovary syndrome) than from patients with normal ovarian reserve (2.7 ± 2.7 ng/µl versus 1.7 ± 2.3 ng/µl, respectively, p = 0.03). Likewise, FF cfDNA levels were significant more elevated in women who received long ovarian stimulation (> 10 days) or high total dose of gonadotropins (≥ 3000 IU/l) than in women who received short stimulation duration (7-10 days) or total dose of gonadotropins < 3000 IU/l (2.4 ± 2.8 ng/µl versus 1.5 ± 1.9 ng/µl, p = 0.008; 2.2 ± 2.3 ng/µl versus 1.5 ± 2.1 ng/µl, p = 0.01, respectively). Finally, FF cfDNA level was an independent and significant predictive factor for pregnancy outcome (adjusted odds ratio = 0.69 [0.5; 0.96], p = 0.03). In multivariate analysis, the Receiving Operator Curve (ROC) analysis showed that the performance of FF cfDNA in predicting clinical pregnancy reached 0.73 [0.66-0.87] with 88% specificity and 60% sensitivity. CfDNA might constitute a promising biomarker of follicular micro-environment quality which could be used to predict IVF prognosis and to enhance female infertility management.

The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance.

Loss-of-function mutations in PARK2/PARKIN and PINK1 cause early-onset autosomal recessive Parkinson disease (PD). The cytosolic E3 ubiquitin-protein ligase PARK2 cooperates with the mitochondrial kinase PINK1 to maintain mitochondrial quality. A loss of mitochondrial transmembrane potential (ΔΨ) leads to the PINK1-dependent recruitment of PARK2 to the outer mitochondrial membrane (OMM), followed by the ubiquitination and proteasome-dependent degradation of OMM proteins, and by the autophagy-dependent clearance of mitochondrial remnants. We showed here that blockade of mitochondrial protein import triggers the recruitment of PARK2, by PINK1, to the TOMM machinery. PD-causing PARK2 mutations weakened or disrupted the molecular interaction between PARK2 and specific TOMM subunits: the surface receptor, TOMM70A, and the channel protein, TOMM40. The downregulation of TOMM40 or its associated core subunit, TOMM22, was sufficient to trigger OMM protein clearance in the absence of PINK1 or PARK2. However, PARK2 was required to promote the degradation of whole organelles by autophagy. Furthermore, the overproduction of TOMM22 or TOMM40 reversed mitochondrial clearance promoted by PINK1 and PARK2 after ΔΨ loss. These results indicated that the TOMM machinery is a key molecular switch in the mitochondrial clearance program controlled by the PINK1-PARK2 pathway. Loss of functional coupling between mitochondrial protein import and the neuroprotective degradation of dysfunctional mitochondria may therefore be a primary pathogenic mechanism in autosomal recessive PD.

Tumor necrosis factor-like weak inducer of apoptosis induces astrocyte proliferation through the activation of transforming-growth factor-α/epidermal growth factor receptor signaling pathway.

Reactive astrogliosis is beneficial in many aspects; however, it is also detrimental in some pathological states such as the development of lethal brain tumors. It is therefore crucial to understand the mechanisms regulating astrocyte proliferation. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor family, was shown to stimulate astrocyte proliferation in vitro. Herein, we further characterize the mitogenic potential of TWEAK on central nervous system cells. Among these cells, astrocytes express the highest level of TWEAK and Fn14 transcripts, suggesting that they are particularly sensitive to TWEAK stimulation. Using in vitro model systems, we found that TWEAK was as potent as epidermal growth factor (EGF) (a prototypical astrocyte mitogen) in mediating astrocyte proliferation. However, its mitogenic activity was delayed compared with that of EGF, suggesting distinct mechanisms of action. Using cell signaling pathway inhibitors, neutralizing antibodies, and protein assays, we further show that the mitogenic activity of TWEAK on primary astrocytes requires stimulation of the transforming growth factor-α (TGF-α) and of the epidermal growth factor receptor (EGFR) signaling pathway through extracellular signal-regulated kinase and p38 mitogen-activated protein kinase activation. In aggregates, our data demonstrate that TWEAK acts as a potent astrocyte mitogen through the induction of a TGF-α/EGFR signaling pathway. We anticipate that description of such a mechanism may allow novel approaches to human pathologies associated with astrocyte proliferation.

MCM8- and MCM9-deficient mice reveal gametogenesis defects and genome instability due to impaired homologous recombination.

We generated knockout mice for MCM8 and MCM9 and show that deficiency for these genes impairs homologous recombination (HR)-mediated DNA repair during gametogenesis and somatic cells cycles. MCM8(-/-) mice are sterile because spermatocytes are blocked in meiotic prophase I, and females have only arrested primary follicles and frequently develop ovarian tumors. MCM9(-/-) females also are sterile as ovaries are completely devoid of oocytes. In contrast, MCM9(-/-) testes produce spermatozoa, albeit in much reduced quantity. Mcm8(-/-) and Mcm9(-/-) embryonic fibroblasts show growth defects and chromosomal damage and cannot overcome a transient inhibition of replication fork progression. In these cells, chromatin recruitment of HR factors like Rad51 and RPA is impaired and HR strongly reduced. We further demonstrate that MCM8 and MCM9 form a complex and that they coregulate their stability. Our work uncovers essential functions of MCM8 and MCM9 in HR-mediated DSB repair during gametogenesis, replication fork maintenance, and DNA repair.

Leucine-rich repeat kinase 2 is associated with the endoplasmic reticulum in dopaminergic neurons and accumulates in the core of Lewy bodies in Parkinson disease.

Mutation of the leucine-rich repeat kinase 2 (LRRK2) gene is the most frequent genetic cause of Parkinson disease (PD). To understand the role of LRRK2 in the neuropathology of PD, we investigated the protein expression in a healthy brain and brains from patients with PD and its subcellular localization in dopaminergic neurons. LRRK2 was found to be widely expressed in healthy adult brain, including areas involved in PD. By double fluorescent staining, we found that endogenous LRRK2 is colocalized with the endoplasmic reticulum (ER) markers Neurotrace and KDEL in human dopaminergic neurons. Labeling of brain sections with anti-LRRK2 and anti-α-synuclein antibodies revealed localization of LRRK2 in the core of 24% of Lewy bodies (LBs) in the substantia nigra and 11% of LBs in the locus coeruleus in idiopathic PD patients. The percentage was increased to 50% in both areas in a patient with the G2019S LRRK2 mutation. The finding of ER localization suggests the possibility that LRRK2 is involved in the ER stress response and could account for the susceptibility to neuronal degeneration of LRRK2 mutation carriers. The localization of LRRK2 protein in the core of a subset of LBs demonstrates the contribution of LRRK2 to LB formation and disease pathogenesis.

Parkin deficiency delays motor decline and disease manifestation in a mouse model of synucleinopathy.

In synucleinopathies, including Parkinson's disease, partially ubiquitylated alpha-synuclein species phosphorylated on serine 129 (P(S129)-alpha-synuclein) accumulate abnormally. Parkin, an ubiquitin-protein ligase that is dysfunctional in autosomal recessive parkinsonism, protects against alpha-synuclein-mediated toxicity in various models.We analyzed the effects of Parkin deficiency in a mouse model of synucleinopathy to explore the possibility that Parkin and alpha-synuclein act in the same biochemical pathway. Whether or not Parkin was present, these mice developed an age-dependent neurodegenerative disorder preceded by a progressive decline in performance in tasks predictive of sensorimotor dysfunction. The symptoms were accompanied by the deposition of P(S129)-alpha-synuclein but not P(S87)-alpha-synuclein in neuronal cell bodies and neuritic processes throughout the brainstem and the spinal cord; activation of caspase 9 was observed in 5% of the P(S129)-alpha-synuclein-positive neurons. As in Lewy bodies, ubiquitin-immunoreactivity, albeit less abundant, was invariably co-localized with P(S129)-alpha-synuclein. During late disease stages, the disease-specific neuropathological features revealed by ubiquitin- and P(S129)-alpha-synuclein-specific antibodies were similar in mice with or without Parkin. However, the proportion of P(S129)-alpha-synuclein-immunoreactive neuronal cell bodies and neurites co-stained for ubiquitin was lower in the absence than in the presence of Parkin, suggesting less advanced synucleinopathy. Moreover, sensorimotor impairment and manifestation of the neurodegenerative phenotype due to overproduction of human alpha-synuclein were significantly delayed in Parkin-deficient mice.These findings raise the possibility that effective compensatory mechanisms modulate the phenotypic expression of disease in parkin-related parkinsonism.

Gem associates with Ezrin and acts via the Rho-GAP protein Gmip to down-regulate the Rho pathway.

Gem is a protein of the Ras superfamily that plays a role in regulating voltage-gated Ca2+ channels and cytoskeletal reorganization. We now report that GTP-bound Gem interacts with the membrane-cytoskeleton linker protein Ezrin in its active state, and that Gem binds to active Ezrin in cells. The coexpression of Gem and Ezrin induces cell elongation accompanied by the disappearance of actin stress fibers and collapse of most focal adhesions. The same morphological effect is elicited when cells expressing Gem alone are stimulated with serum and requires the expression of ERM proteins. We show that endogenous Gem down-regulates the level of active RhoA and actin stress fibers. The effects of Gem downstream of Rho, i.e., ERM phosphorylation as well as disappearance of actin stress fibers and most focal adhesions, require the Rho-GAP partner of Gem, Gmip, a protein that is enriched in membranes under conditions in which Gem induced cell elongation. Our results suggest that Gem binds active Ezrin at the plasma membrane-cytoskeleton interface and acts via the Rho-GAP protein Gmip to down-regulate the processes dependent on the Rho pathway.

The phenotypic differentiation of locus ceruleus noradrenergic neurons mediated by brain-derived neurotrophic factor is enhanced by corticotropin releasing factor through the activation of a cAMP-dependent signaling pathway.

We have developed a model system of locus ceruleus (LC) neurons in culture, in which brain-derived neurotrophic factor (BDNF) induces the emergence of noradrenergic neurons attested by the presence of tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase and the absence of phenylethanolamine N-methyl-transferase. Although inactive in itself, the neuropeptide corticotropin releasing factor (CRF) strongly amplified the effect of BDNF, increasing the number of cells expressing TH and the active accumulation of noradrenaline by a factor of 2 to 3 via a mechanism that was nonmitogenic. CRF also acted cooperatively with neurotrophin-4, which like BDNF is a selective ligand of the TrkB tyrosine kinase receptor. The effect of CRF but not that of BDNF was prevented by astressin, a nonselective CRF-1/CRF-2 receptor antagonist. However, only CRF-1 receptor transcripts were detectable in LC cultures, suggesting that this receptor subtype mediated the effect of CRF. Consistent with the positive coupling of CRF-1 receptors to adenylate cyclase, the trophic action of CRF was mimicked by cAMP elevating agents. Epac, a guanine nucleotide exchange factor directly activated by cAMP, contributed to the effect of CRF through the stimulation of extracellular signal-regulated kinases (ERKs) 1/2. However, downstream of ERK1/2 activation by CRF, the phenotypic induction of noradrenergic neurons relied upon the stimulation of the phosphatidylinositol-3-kinase/Akt transduction pathway by BDNF. Together, our results suggest that CRF participates to the phenotypic differentiation of LC noradrenergic neurons during development. Whether similar mechanisms account for the high degree of plasticity of these neurons in the adult brain remains to be established.

Substance P, neurokinins A and B, and synthetic tachykinin peptides protect mesencephalic dopaminergic neurons in culture via an activity-dependent mechanism.

We evaluated the neuroprotective potential of tachykinin peptides using a model system in which mesencephalic dopaminergic (DA) neurons die spontaneously and selectively as they mature. The three native tachykinins, substance P (SP), neurokinin (NK) A, and NKB afforded substantial protection against DA cell demise. The selective NK1 receptor antagonist [D-Pro9,[spiro-gamma-lactam] Leu10,Trp11]substance P (GR71251) was sufficient in itself to suppress the effect of SP, whereas a cotreatment with GR71251 and the NK3 receptor antagonist (R)-N-[alpha-(methoxycarbonyl)benzyl]-2-phenylquinoline-4-carboxamide (SB218795) was required to prevent the effects of both NKA and NKB. Consistent with these results, D-Ala-[L-Pro9,Me-Leu8]substance P(7-11) (GR73632), a selective agonist of NK1 receptors and [pro7]-NKB, a selective agonist of NK3 receptors, conferred protection to DA neurons, whereas (Lys3, Gly8-R-gamma-lactam-Leu9)neurokinin A(3-10) (GR64349), which activates specifically NK2 receptors, did not. DA neurons rescued by tachykinins accumulated [3H]DA efficiently, which suggests that they were also totally functional. Neuroprotection by tachykinins was highly selective for DA neurons, rapidly reversed upon treatment withdrawal, and reproduced by but independent of glial cell line-derived neurotrophic factor. Survival promotion by tachykinins was abolished by blocking voltage-gated Na+ channels with tetrodotoxin or N-type voltage-gated Ca2+ channels with omega-conotoxin-MVIIA, which indicates that an increase in neuronal excitability was crucially involved in this effect. Together, these data further support the notion that the survival of mesencephalic DA neurons during development depends largely on excitatory inputs, which may be provided in part by tachykinins.

Granulocyte colony-stimulating factor is not protective against selective dopaminergic cell death in vitro.

In the present study, we evaluated the potential neuroprotective effect of granulocyte colony-stimulating factor (G-CSF), a hematopoietic growth factor in two different culture models in which dopaminergic (DA) neurons die selectively: first, in a culture model in which death of DA neurons occurs spontaneously and second, in a toxin-based paradigm, the in vitro 1-methyl-4-phenylpyridinium model of PD. In neither of the two models, a treatment with G-CSF, could prevent or halt the progressive neurodegeneration. However, we cannot rule out that G-CSF might exert neuroprotective or even deleterious effects in in vivo models of PD, based on the significant increase in the number of microglial cells observed after G-CSF treatment.

The neurotransmitter noradrenaline rescues septal cholinergic neurons in culture from degeneration caused by low-level oxidative stress.

We have developed a model system in which rat basal forebrain cholinergic neurons degenerate progressively when maintained in culture conditions that make them susceptible to low-level oxidative stress. In this study, we showed that cholinergic neurons identified by acetylcholinesterase cytochemistry or choline acetyl transferase immunocytochemistry are rescued efficiently by the neurotransmitter noradrenaline (NA). The effect of NA required neither adrenoceptor activation nor intracellular accumulation. NA operated via a mechanism that precluded activation of a cell death pathway in which reactive oxygen species (ROS) and proapoptotic caspases were crucially involved. It is noteworthy that NA remained protective even when applied late in the degenerative process but before intracellular ROS began to increase. The high efficacy of iron chelators and catalase in preventing the death of cholinergic neurons in this model suggested that NA neutralized the effects of hydroxyl radicals produced through a Fenton-type reaction. Pyrocatechol [the diphenolic moiety of NA] was sufficient in itself to prevent ROS production and cholinergic cell demise, indicating that the catechol structure was instrumental for the neuroprotective function of NA. Therefore, the noncatecholic neurotransmitter GABA failed to prevent neurodegeneration. Nerve growth factor and brain derived neurotrophic factor, two trophic peptides for septal cholinergic neurons, did not afford protection by themselves and did not improve neuroprotection provided by NA. However, in the presence of NA, they both retained their efficacy to stimulate cholinergic parameters. These data indicate that NA-based therapeutic strategies may be of interest in such neurodegenerative conditions as Alzheimer's disease, where progressive cholinergic deficits occur.

The RGS (regulator of G-protein signalling) and GoLoco domains of RGS14 co-operate to regulate Gi-mediated signalling.

RGS (regulator of G-protein signalling) proteins stimulate the intrinsic GTPase activity of the a subunits of heterotrimeric G-proteins, and thereby negatively regulate G-protein-coupled receptor signalling. RGS14 has been shown previously to stimulate the GTPase activities of Ga(o) and Ga(i) subunits through its N-terminal RGS domain, and to down-modulate signalling from receptors coupled to G(i). It also contains a central domain that binds active Rap proteins, as well as a C-terminal GoLoco/G-protein regulatory motif that has been shown to act in vitro as a GDP-dissociation inhibitor for Ga(i). In order to elucidate the respective contributions of the three functional domains of RGS14 to its ability to regulate G(i) signalling, we generated RGS14 mutants invalidated in each of its domains, as well as truncated molecules, and assessed their effects on G(i) signalling via the bg pathway in a stable cell line ectopically expressing the G(i)-coupled M2 muscarinic acetylcholine receptor (HEK-m2). We show that the RGS and GoLoco domains of RGS14 are independently able to inhibit signalling downstream of G(i). Targeting of the isolated GoLoco domain to membranes, by myristoylation/palmitoylation or Rap binding, enhances its inhibitory activity on G(i) signalling. Finally, in the context of the full RGS14 molecule, the RGS and GoLoco domains co-operate to confer maximal activity on RGS14. We therefore propose that RGS14 combines the inhibition of G(i) activation or coupling to receptors via its GoLoco domain with stimulation of the GTPase activity of Ga(i)-GTP via its RGS domain to negatively regulate signalling downstream of G(i).