Several genes predisposing to autism spectrum disorders (ASDs) with or without epilepsy have been identified, many of which are implicated in synaptic function. Here we report a Q555X mutation in synapsin 1 (SYN1), an X-linked gene encoding for a neuron-specific phosphoprotein implicated in the regulation of neurotransmitter release and synaptogenesis. This nonsense mutation was found in all affected individuals from a large French-Canadian family segregating epilepsy and ASDs. Additional mutations in SYN1 (A51G, A550T and T567A) were found in 1.0 and 3.5% of French-Canadian individuals with autism and epilepsy, respectively. The majority of these SYN1 mutations were clustered in the proline-rich D-domain which is substrate of multiple protein kinases. When expressed in synapsin I (SynI) knockout (KO) neurons, all the D-domain mutants failed in rescuing the impairment in the size and trafficking of synaptic vesicle pools, whereas the wild-type human SynI fully reverted the KO phenotype. Moreover, the nonsense Q555X mutation had a dramatic impact on phosphorylation by MAPK/Erk and neurite outgrowth, whereas the missense A550T and T567A mutants displayed impaired targeting to nerve terminals. These results demonstrate that SYN1 is a novel predisposing gene to ASDs, in addition to epilepsy, and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies the pathogenesis of both diseases.

Hepatitis C virus (HCV) readily establishes high-level lifelong persistent infection in the majority of immunocompetent adults with failure of HCV-specific CD8 + CTL to clear viral replication. Virus-induced conditioning of innate immune responses is a possible mechanism that may contribute to the impairment of virus-specific CD8 + CTL responses. Here, we analyzed whether triggering of NK cell receptor expression and function is affected during chronic viremic HCV infection. Flow cytometric analysis of purified resting peripheral NK cells showed no evidence of NK cell activation, while analysis of natural cytotoxicity receptors (NCR) showed that NK cells from HCVinfected patients had selective increased expression of NKp30 and NKp46. NK cells had corresponding conserved cytotoxic activity against all targets with the exception of HepG2 hepatoma cells. Freshly separated NK cells from HCV patients showed significant production of IL-10 and normal concentrations of IFN-c upon cell-mediated triggering. Thus, increased expression of NKp30 during HCV infection with increased IL-10 production could contribute, once NK cells localize in the liver, to a NK-DC crosstalk leading to skewing of subsequent adaptive immune responses and lack of virus control. IntroductionHepatitis C virus (HCV), a human hepatotropic Flaviviridae member not requiring insect vector transmission, may establish chronic replication and is responsible for a heavy burden of long-term disease including chronic hepatitis, cirrhosis, hepatocellular carcinoma, cryoglobulinaemia and vasculitis [1]. The high worldwide prevalence of infection (*170 million cases estimated by the World Health Organization) is associated, at least in part, to the chronic persistent course of infection that ensues in the majority of acutely infected patients (>85%) leading to continuous highlevel viral replication [2]. Both viral and host immune mechanisms contribute to the establishment of chronic infection. Spontaneous resolution of HCV infection has been linked to vigorous and multi-specific T cell responses, while attenuated CD4 + and CD8 + T cell responses have been observed during the chronic phase of viral persistence [3][4][5][6]. Failure to control HCV replication has also been associated with functional defects of virus-specific CD8 + cytotoxic T lymphocytes (CTL) [7][8][9][10] and, most recently, to the appearance of viral escape mutations in immunodominant CD8 + CTL epitopes associated with a lack of or relative defects in HCV-specific CD4 + T cell responses [11][12][13]. Adaptive immune defects during HCV infection are not limited to HCV-specific immune responses and may reflect the broader effects of HCV on the editing of T cell responses also on other antigen specificities [14].Perturbations of the innate immune system, including dendritic cell (DC) and natural killer (NK) cell function, are likely to contribute to the skewed finetuning of anti-HCV CD8 + and CD4 + T cell immune responses leading ultimately to T cell dysfunction [15,16]. Evidence supports an inv...

The large apolar tunnel traversing the mini-hemoglobin from Cerebratulus lacteus (CerHb) has been examined by xray crystallography, ligand binding kinetics, and molecular dynamic simulations. The addition of 10 atm of xenon causes loss of diffraction in wild-type (wt) CerHbO 2 crystals, but Leu-86(G12)Ala CerHbO 2 , which has an increased tunnel volume, stably accommodates two discrete xenon atoms: one adjacent to Leu-86(G12) and another near Ala-55(E18). Molecular dynamics simulations of ligand migration in wt CerHb show a low energy pathway through the apolar tunnel when Leu or Ala, but not Phe or Trp, is present at the 86(G12) position. The addition of 10 -15 atm of xenon to solutions of wt CerHbCO and L86A CerHbCO causes 2-3-fold increases in the fraction of geminate ligand recombination, indicating that the bound xenon blocks CO escape. This idea was confirmed by L86F and L86W mutations, which cause even larger increases in the fraction of geminate CO rebinding, 2-5-fold decreases in the bimolecular rate constants for ligand entry, and large increases in the computed energy barriers for ligand movement through the apolar tunnel. Both the addition of xenon to the L86A mutant and oxidation of wt CerHb heme iron cause the appearance of an out Gln-44(E7) conformer, in which the amide side chain points out toward the solvent and appears to lower the barrier for ligand escape through the E7 gate. However, the observed kinetics suggest little entry and escape (<25%) through the E7 pathway, presumably because the in Gln-44(E7) conformer is thermodynamically favored. Although molecular dynamics (MD)7 simulations suggest multiple interior pathways for O 2 entry into and exit from globins, most experiments with mammalian myoglobins (Mbs) and hemoglobins (Hbs) suggest a well defined single pathway involving a short channel between the heme propionates and the heme iron atom that is gated by the distal E7 histidine (1). To search for and define an interior ligand migration trajectory, we chose to use the neuronal mini-hemoglobin from Cerebratulus lacteus as a model globin system to examine its long apolar tunnel that leads from the distal portion of the heme pocket to an exit point between the C-terminal regions of the E and H helices of the tertiary fold, a pathway that is roughly 180°opposite the E7 channel and appears to be a major route for ligand entry (2). This model globin provides a well defined system to examine both experimentally and theoretically the effects of xenon binding, mutagenesis, and conformational heterogeneity on the competition between movement through the E7 gate versus an internal apolar pathway.Nerve tissue Hbs occur in both vertebrates and invertebrates (3). Among these, the nerve Hb from the nemertean worm C. lacteus (CerHb) is the smallest functional globin known, being composed of 109 amino acids instead of the ϳ140 -160 residues typical of most monomeric globins (4, 5). Analysis of the three-dimensional structure of CerHb has shown a markedly edited 3-over-3-globin fold with deletion of

SummarySynapsins are synaptic vesicle (SV)-associated phosphoproteins involved in the regulation of neurotransmitter release. Synapsins reversibly tether SVs to the cytoskeleton and their phosphorylation by serine/threonine kinases increases SV availability for exocytosis by impairing their association with SVs and/or actin. We recently showed that synapsin I, through SH3-or SH2-mediated interactions, activates Src and is phosphorylated by the same kinase at Tyr301. Here, we demonstrate that, in contrast to serine phosphorylation, Src-mediated tyrosine phosphorylation of synapsin I increases its binding to SVs and actin, and increases the formation of synapsin dimers, which are both potentially involved in SV clustering. Synapsin I phosphorylation by Src affected SV dynamics and was physiologically regulated in brain slices in response to depolarization. Expression of the non-phosphorylatable (Y301F) synapsin I mutant in synapsin-I-knockout neurons increased the sizes of the readily releasable and recycling pools of SVs with respect to the wild-type form, which is consistent with an increased availability of recycled SVs for exocytosis. The data provide a mechanism for the effects of Src on SV trafficking and indicate that tyrosine phosphorylation of synapsins, unlike serine phosphorylation, stimulates the reclustering of recycled SVs and their recruitment to the reserve pool.

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