Anna Corradi

SYN1 loss-of-function mutations in autism and partial epilepsy cause impaired synaptic function

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.

Hypogonadotropic hypogonadism and peripheral neuropathy in Ebf2-null mice

Olf/Ebf transcription factors have been implicated in numerous developmental processes, ranging from B-cell development to neuronal differentiation. We describe mice that carry a targeted deletion within the Ebf2 (O/E3) gene. In Ebf2-null mutants, because of defective migration of gonadotropin releasing hormone-synthesizing neurons, formation of the neuroendocrine axis (which is essential for pubertal development) is impaired, leading to secondary hypogonadism. In addition, Ebf2-/- peripheral nerves feature defective axon sorting, hypomyelination, segmental dysmyelination and axonal damage, accompanied by a sharp decrease in motor nerve conduction velocity. Ebf2-null mice reveal a novel genetic cause of hypogonadotropic hypogonadism and peripheral neuropathy in the mouse, disclosing an important role for Ebf2 in neuronal migration and nerve development.

Phosphorylation of Synapsin I by cAMP-Dependent Protein Kinase Controls Synaptic Vesicle Dynamics in Developing Neurons

In developing neurons, synaptic vesicles (SVs) undergo cycles of exo-endocytosis along isolated axons. However, it is currently unknown whether SV exocytosis is regulated before synaptogenesis. Here, we show that cAMP-dependent pathways affect SV distribution and recycling in the axonal growth cone and that these effects are mediated by the SV-associated phosphoprotein synapsin I. The presence of synapsin I on SVs is necessary for the correct localization of the vesicles in the central portion of the growth cone. Phosphorylation of synapsin I by cAMP-dependent protein kinase (protein kinase A) causes the dissociation of the protein from the SV membrane, allowing diffusion of the vesicles to the periphery of the growth cone and enhancing their rate of recycling. These results provide new clues as to the bases of the well known activity of synapsin I in synapse maturation and indicate that molecular mechanisms similar to those operating at mature nerve terminals are active in developing neurons to regulate the SV life cycle before synaptogenesis.

A key role for the HLH transcription factor EBF2COE2,O/E-3 in Purkinje neuron migration and cerebellar cortical topography

Early B-cell factor 2 (EBF2) is one of four mammalian members of an atypical helix-loop-helix transcription factor family (COE). COE proteins have been implicated in various aspects of nervous and immune system development. We and others have generated and described mice carrying a null mutation of Ebf2, a gene previously characterized in the context of Xenopus laevis primary neurogenesis and neuronal differentiation. In addition to deficits in neuroendocrine and olfactory development, and peripheral nerve maturation, Ebf2 null mice feature an ataxic gait and obvious motor deficits associated with clear-cut abnormalities of cerebellar development. The number of Purkinje cells (PCs) in the Ebf2 null is markedly decreased, resulting in a small cerebellum with notable foliation defects, particularly in the anterior vermis. We show that this stems from the defective migration of a molecularly defined PC subset that subsequently dies by apoptosis. Part of the striped cerebellar topography is disrupted due to cell death and, in addition, many of the surviving PCs, that would normally adopt a zebrin II-negative phenotype, transdifferentiate to Zebrin II-positive, an unprecedented finding suggesting that Ebf2 is required for the establishment of a proper cerebellar cortical map.

Synapsin-I- and synapsin-II-null mice display an increased age-dependent cognitive impairment

Synapsin I (SynI) and synapsin II (SynII) are major synaptic vesicle (SV) proteins that function in the regulation of the availability of SVs for release in mature neurons. SynI and SynII show a high level of sequence similarity and share many functions in vivo, although distinct physiological roles for the two proteins have been proposed. Both SynI–/– and SynII–/– mice have a normal lifespan, but exhibit a decreased number of SVs and synaptic depression upon high-frequency stimulation. Because of the role of the synapsin proteins in synaptic organization and plasticity, we studied the long-lasting effects of synapsin deletion on the phenotype of SynI–/– and SynII–/– mice during aging. Both SynI–/– and SynII–/– mice displayed behavioural defects that emerged during aging and involved emotional memory in both mutants, and spatial memory in SynII–/– mice. These abnormalities, which were more pronounced in SynII–/– mice, were associated with neuronal loss and gliosis in the cerebral cortex and hippocampus. The data indicate that SynI and SynII have specific and non-redundant functions, and that synaptic dysfunctions associated with synapsin mutations negatively modulate cognitive performances and neuronal survival during senescence.

MAPK/Erk-dependent phosphorylation of synapsin mediates formation of functional synapses and short-term homosynaptic plasticity.

MAPK/Erk is a protein kinase activated by neurotrophic factors involved in synapse formation and plasticity, which acts at both the nuclear and cytoplasmic level. Synapsin proteins are synaptic-vesicle-associated proteins that are well known to be MAPK/Erk substrates at phylogenetically conserved sites. However, the physiological role of MAPK/Erk-dependent synapsin phosphorylation in regulating synaptic formation and function is poorly understood. Here, we examined whether synapsin acts as a physiological effector of MAPK/Erk in synaptogenesis and plasticity. To this aim, we developed an in vitro model of soma-to-soma paired Helix B2 neurons, that establish bidirectional excitatory synapses. We found that the formation and activity-dependent short-term plasticity of these synapses is dependent on the MAPK/Erk pathway. To address the role of synapsin in this pathway, we generated non-phosphorylatable and pseudo-phosphorylated Helix synapsin mutants at the MAPK/Erk sites. Overexpression experiments revealed that both mutants interfere with presynaptic differentiation, synapsin clustering, and severely impair post-tetanic potentiation, a form of short-term homosynaptic plasticity. Our findings show that MAPK/Erk-dependent synapsin phosphorylation has a dual role both in the establishment of functional synaptic connections and their short-term plasticity, indicating that some of the multiple extranuclear functions of MAPK/Erk in neurons can be mediated by the same multifunctional presynaptic target.

Phosphorylation of Synapsin I by Cyclin-Dependent Kinase-5 Sets the Ratio between the Resting and Recycling Pools of Synaptic Vesicles at Hippocampal Synapses

Cyclin-dependent kinase-5 (Cdk5) was reported to downscale neurotransmission by sequestering synaptic vesicles (SVs) in the release-reluctant resting pool, but the molecular targets mediating this activity remain unknown. Synapsin I (SynI), a major SV phosphoprotein involved in the regulation of SV trafficking and neurotransmitter release, is one of the presynaptic substrates of Cdk5, which phosphorylates it in its C-terminal region at Ser549 (site 6) and Ser551 (site 7). Here we demonstrate that Cdk5 phosphorylation of SynI fine tunes the recruitment of SVs to the active recycling pool and contributes to the Cdk5-mediated homeostatic responses. Phosphorylation of SynI by Cdk5 is physiologically regulated and enhances its binding to F-actin. The effects of Cdk5 inhibition on the size and depletion kinetics of the recycling pool, as well as on SV distribution within the nerve terminal, are virtually abolished in mouse SynI knock-out (KO) neurons or in KO neurons expressing the dephosphomimetic SynI mutants at sites 6,7 or site 7 only. The observation that the single site-7 mutant phenocopies the effects of the deletion of SynI identifies this site as the central switch in mediating the synaptic effects of Cdk5 and demonstrates that SynI is necessary and sufficient for achieving the effects of the kinase on SV trafficking. The phosphorylation state of SynI by Cdk5 at site 7 is regulated during chronic modification of neuronal activity and is an essential downstream effector for the Cdk5-mediated homeostatic scaling.

SYN2 is an autism predisposing gene: loss-of-function mutations alter synaptic vesicle cycling and axon outgrowth

An increasing number of genes predisposing to autism spectrum disorders (ASDs) has been identified, many of which are implicated in synaptic function. This ‘synaptic autism pathway’ notably includes disruption of SYN1 that is associated with epilepsy, autism and abnormal behavior in both human and mice models. Synapsins constitute a multigene family of neuron-specific phosphoproteins (SYN1-3) present in the majority of synapses where they are implicated in the regulation of neurotransmitter release and synaptogenesis. Synapsins I and II, the major Syn isoforms in the adult brain, display partially overlapping functions and defects in both isoforms are associated with epilepsy and autistic-like behavior in mice. In this study, we show that nonsense (A94fs199X) and missense (Y236S and G464R) mutations in SYN2 are associated with ASD in humans. The phenotype is apparent in males. Female carriers of SYN2 mutations are unaffected, suggesting that SYN2 is another example of autosomal sex-limited expression in ASD. When expressed in SYN2  knockout neurons, wild-type human Syn II fully rescues the SYN2 knockout phenotype, whereas the nonsense mutant is not expressed and the missense mutants are virtually unable to modify the SYN2 knockout phenotype. These results identify for the first time SYN2  as a novel predisposing gene for ASD and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies ASD.

Synapsin Phosphorylation by Src Tyrosine Kinase Enhances Src Activity in Synaptic Vesicles

Synapsins are synaptic vesicle-associated phosphoproteins implicated in the regulation of neurotransmitter release. Synapsin I is the major binding protein for the SH3 domain of the kinase c-Src in synaptic vesicles. Its binding leads to stimulation of synaptic vesicle-associated c-Src activity. We investigated the mechanism and role of Src activation by synapsins on synaptic vesicles. We found that synapsin is tyrosine phosphorylated by c-Src in vitro and on intact synaptic vesicles independently of its phosphorylation state on serine. Mass spectrometry revealed a single major phosphorylation site at Tyr301, which is highly conserved in all synapsin isoforms and orthologues. Synapsin tyrosine phosphorylation triggered its binding to the SH2 domains of Src or Fyn. However, synapsin selectively activated and was phosphorylated by Src, consistent with the specific enrichment of c-Src in synaptic vesicles over Fyn or n-Src. The activity of Src on synaptic vesicles was controlled by the amount of vesicle-associated synapsin, which is in turn dependent on synapsin serine phosphorylation. Synaptic vesicles depleted of synapsin in vitro or derived from synapsin null mice exhibited greatly reduced Src activity and tyrosine phosphorylation of other synaptic vesicle proteins. Disruption of the Src-synapsin interaction by internalization of either the Src SH3 or SH2 domains into synaptosomes decreased synapsin tyrosine phosphorylation and concomitantly increased neurotransmitter release in response to Ca2+-ionophores. We conclude that synapsin is an endogenous substrate and activator of synaptic vesicle-associated c-Src and that regulation of Src activity on synaptic vesicles participates in the regulation of neurotransmitter release by synapsin.

Phosphorylation of synapsin domain A is required for post-tetanic potentiation

Post-tetanic potentiation (PTP) is a form of homosynaptic plasticity important for information processing and short-term memory in the nervous system. The synapsins, a family of synaptic vesicle (SV)-associated phosphoproteins, have been implicated in PTP. Although several synapsin functions are known to be regulated by phosphorylation by multiple protein kinases, the role of individual phosphorylation sites in synaptic plasticity is poorly understood. All the synapsins share a phosphorylation site in the N-terminal domain A (site 1) that regulates neurite elongation and SV mobilization. Here, we have examined the role of phosphorylation of synapsin domain A in PTP and other forms of short-term synaptic enhancement (STE) at synapses between cultured Helix pomatia neurons. To this aim, we cloned H. pomatia synapsin (helSyn) and overexpressed GFP-tagged wild-type helSyn or site-1-mutant helSyn mutated in the presynaptic compartment of C1-B2 synapses. We found that PTP at these synapses depends both on Ca2+/calmodulin-dependent and cAMP-dependent protein kinases, and that overexpression of the non-phosphorylatable helSyn mutant, but not wild-type helSyn, specifically impairs PTP, while not altering facilitation and augmentation. Our findings show that phosphorylation of site 1 has a prominent role in the expression of PTP, thus defining a novel role for phosphorylation of synapsin domain A in short-term homosynaptic plasticity.