Oxidative conditions must be generated in the endoplasmic reticulum (ER) to allow disulfide bond formation in secretory proteins. A family of conserved genes, termed EROfor ER oxidoreductins, plays a key role in this process. We have previously described the human gene ERO1-L, which complements several phenotypic traits of the yeast thermo-sensitive mutant ero1-1 (Cabibbo, A., Pagani, M., Fabbri, M., Rocchi, M., Farmery, M. R., Bulleid, N. J., and Sitia, R. (2000)J. Biol. Chem. 275, 4827–4833). Here, we report the cloning and characterization of a novel human member of this family,ERO1-Lβ. Immunofluorescence, endoglycosidase sensitivity, and in vitro translation/translocation assays reveal that the products of the ERO1-Lβ gene are primarily localized in the ER of mammalian cells. The ability to allow growth at 37 °C and to alleviate the “unfolded protein response” when expressed inero1-1 cells indicates that ERO1-Lβ is involved also in generating oxidative conditions in the ER. ERO1-L and ERO1-Lβ display different tissue distributions. Furthermore, only ERO1-Lβ transcripts are induced in the course of the unfolded protein response. Our results suggest a complex regulation of ER redox homeostasis in mammalian cells.

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.

In the endoplasmic reticulum (ER), disulfide bonds are simultaneously formed in nascent proteins and removed from incorrectly folded or assembled molecules. In this compartment, the redox state must be, therefore, precisely regulated. Here we show that both human Ero1‐Lα and Ero1‐Lβ (hEROs) facilitate disulfide bond formation in immunoglobulin subunits by selectively oxidizing PDI. Disulfide bond formation is controlled by hEROs, which stand at a crucial point of an electron‐flow starting from nascent secretory proteins and passing through PDI. The redox state of ERp57, another ER‐resident oxidoreductase, is not affected by over‐expression of Ero1‐Lα, suggesting that parallel and specific pathways control oxidative protein folding in the ER. Mutants in the Ero1‐Lα CXXCXXC motif act as dominant negatives by limiting immunoglobulin oxidation. PDI‐dependent oxidative folding in living cells can thus be manipulated by using hERO variants.

Synapsins (Syns) are synaptic vesicle (SV) phosphoproteins that play a role in neurotransmitter release and synaptic plasticity by acting at multiple steps of exocytosis. Mutation ofSYNgenes results in an epileptic phenotype in mouse and man suggesting a role of Syns in the control of network excitability. We have studied the effects of the genetic ablation of theSYN1gene on inhibitory synaptic transmission in primary hippocampal neurons. Inhibitory neurons lacking SynI showed reduced amplitude of IPSCs evoked by isolated action potentials. The impairment in inhibitory transmission was caused by a decrease in the size of the SV readily releasable pool, rather than by changes in release probability or quantal size. The reduction of the readily releasable pool was caused by a decrease in the number of SVs released by single synaptic boutons in response to the action potential, in the absence of variations in the number of synaptic contacts between couples of monosynaptically connected neurons. The deletion ofSYN1did not affect paired-pulse depression or post-tetanic potentation, but was associated with a moderate increase of synaptic depression evoked by trains of action potentials, which became apparent at high stimulation frequencies and was accompanied by a slow down of recovery from depression. The decreased size of the SV readily releasable pool, coupled with a decreased SV recycling rate and refilling by the SV reserve pool, may contribute to the epileptic phenotype of SynI knock-out mice.

Heterozygous mutations in proline-rich transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders, including epilepsy, kinesigenic dyskinesia, and migraine. Most of the mutations lead to impaired PRRT2 expression, suggesting that loss of PRRT2 function may contribute to pathogenesis. We show that PRRT2 is enriched in presynaptic terminals and that its silencing decreases the number of synapses and increases the number of docked synaptic vesicles at rest. PRRT2-silenced neurons exhibit a severe impairment of synchronous release, attributable to a sharp decrease in release probability and Ca(2+) sensitivity and associated with a marked increase of the asynchronous/synchronous release ratio. PRRT2 interacts with the synaptic proteins SNAP-25 and synaptotagmin 1/2. The results indicate that PRRT2 is intimately connected with the Ca(2+)-sensing machinery and that it plays an important role in the final steps of neurotransmitter release.