Phosphorylation by PKA and Cdk5 Mediates the Early Effects of Synapsin III in Neuronal Morphological Maturation.

Synapsin III (SynIII) is a neuron-specific phosphoprotein that plays a unique role in neuronal development. SynIII is phosphorylated by cAMP-dependent protein kinase (PKA) at a highly conserved phosphorylation site and by cyclin-dependent kinase-5 (Cdk5) at a newly described site. Although SynIII is known to be involved in axon elongation in vitro, the role of its phosphorylation by PKA and Cdk5 in the modulation of this process is unknown. We expressed either wild-type (WT) or phosphorylation-site mutants of SynIII in primary SynIII knock-out (KO) mouse neurons at early stages of in vitro development. Whereas the neurite elongation phenotype of SynIII KO neurons was fully rescued by the expression of WT SynIII, the expression of nonphosphorylatable and pseudo-phosphorylated PKA mutants was ineffective. Also, the nonphosphorylatable Cdk5 mutant was unable to rescue the neurite elongation phenotype of SynIII KO neurons. By contrast, the pseudo-phosphorylated mutant rescued the delay in neuronal maturation and axonal elongation, revealing a Cdk5-dependent regulation of SynIII function. Interestingly, SynIII KO neurons also exhibited decreased survival that was fully rescued by the expression of WT SynIII, but not by its phosphorylation mutants, and was associated with increased activated caspase3 and altered tropomyosin receptor kinase B isoform expression. These results indicate that PKA and Cdk5 phosphorylation is required for the physiological action of SynIII on axon specification and neurite outgrowth and that the expression of a functional SynIII is crucial for cell survival. Significance statement: Synapsin III is an atypical member of the synapsin family of synaptic vesicle-associated phosphoproteins that is precociously expressed in neurons and is downregulated afterward. Although experimental evidence suggests a specific role for Synapsin III in neuronal development, the molecular mechanisms are still largely unknown. We found that Synapsin III plays a central role in early stages of neuronal development involving neuronal survival, polarization, and neuritic growth and that these effects are dependent on phosphorylation by cAMP-dependent protein kinase and cyclin-dependent protein kinase-5. These results explain the recently described neurodevelopmental defects in the migration and orientation of Synapsin III-depleted cortical neurons and support the potential association of Synapsin III with neurodevelopmental disorders such as schizophrenia.

Effects of phosphorylation and neuronal activity on the control of synapse formation by synapsin I.

Synapsins are synaptic vesicle (SV)-associated proteins that regulate synaptic transmission and neuronal differentiation. At early stages, Syn I and II phosphorylation at Ser9 by cAMP-dependent protein kinase (PKA) and Ca(2+)/calmodulin-dependent protein kinase I/IV modulates axon elongation and SV-precursor dynamics. We evaluated the requirement of Syn I for synapse formation by siRNA-mediated knockdown as well as by overexpression of either its wild-type (WT) form or its phosphorylation mutants. Syn1 knockdown at 14 days in vitro caused a decrease in the number of synapses, accompanied by a reduction of SV recycling. Although overexpression of WT Syn I was ineffective, overexpression of its phosphorylation mutants resulted in a complex temporal regulation of synapse density. At early stages of synaptogenesis, phosphomimetic Syn I S9E significantly increased the number of synapses. Conversely, dephosphomimetic Syn I S9A decreased synapse number at more advanced stages. Overexpression of either WT Syn I or its phosphomimetic S9E mutant rescued the decrease in synapse number caused by chronic treatment with tetrodotoxin at early stages, suggesting that Syn I participates in an alternative PKA-dependent mechanism that can compensate for the impairment of the activity-dependent synaptogenic pathway. Altogether these results indicate that Syn I is an important regulator of synapse formation, which adjusts synapse number in response to extracellular signals.

A rat model of a focal mosaic expression of PCDH19 replicates human brain developmental abnormalities and behaviours.

Protocadherin 19 gene-related epilepsy or protocadherin 19 clustering epilepsy is an infantile-onset epilepsy syndrome characterized by psychiatric (including autism-related), sensory, and cognitive impairment of varying degrees. Protocadherin 19 clustering epilepsy is caused by X-linked protocadherin 19 protein loss of function. Due to random X-chromosome inactivation, protocadherin 19 clustering epilepsy-affected females present a mosaic population of healthy and protocadherin 19-mutant cells. Unfortunately, to date, no current mouse model can fully recapitulate both the brain histological and behavioural deficits present in people with protocadherin 19 clustering epilepsy. Thus, the search for a proper understanding of the disease and possible future treatment is hampered. By inducing a focal mosaicism of protocadherin 19 expression using in utero electroporation in rats, we found here that protocadherin 19 signalling in specific brain areas is implicated in neuronal migration, heat-induced epileptic seizures, core/comorbid behaviours related to autism and cognitive function.

Rescuing Over-activated Microglia Restores Cognitive Performance in Juvenile Animals of the Dp(16) Mouse Model of Down Syndrome.

Microglia are brain-resident immune cells and regulate mechanisms essential for cognitive functions. Down syndrome (DS), the most frequent cause of genetic intellectual disability, is caused by a supernumerary chromosome 21, containing also genes related to the immune system. In the hippocampus of the Dp(16) mouse model of DS and DS individuals, we found activated microglia, as assessed by their morphology; activation markers; and, for DS mice, electrophysiological profile. Accordingly, we found increased pro-inflammatory cytokine levels and altered interferon signaling in Dp(16) hippocampi. DS mice also showed decreased spine density and activity of hippocampal neurons and hippocampus-dependent cognitive behavioral deficits. Depletion of defective microglia or treatment with a commonly used anti-inflammatory drug rescued the neuronal spine and activity impairments and cognitive deficits in juvenile Dp(16) mice. Our results suggest an involvement of microglia in Dp(16)-mouse cognitive deficits and identify a new potential therapeutic approach for cognitive disabilities in DS individuals.

Gene Editing Preserves Visual Functions in a Mouse Model of Retinal Degeneration.

Inherited retinal dystrophies (IRDs) are a large and heterogeneous group of degenerative diseases caused by mutations in various genes. Given the favorable anatomical and immunological characteristics of the eye, gene therapy holds great potential for their treatment. Our goal is to validate the preservation of visual functions by viral-free homology directed repair (HDR) in an autosomal recessive loss of function mutation. We used a tailored gene editing system based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to prevent retinal photoreceptor death in the retinal degeneration 10 (Rd10) mouse model of retinitis pigmentosa. We tested the gene editing tool in vitro and then used in vivo subretinal electroporation to deliver it to one of the retinas of mouse pups at different stages of photoreceptor differentiation. Three months after gene editing, the treated eye exhibited a higher visual acuity compared to the untreated eye. Moreover, we observed preservation of light-evoked responses both in explanted retinas and in the visual cortex of treated animals. Our study validates a CRISPR/Cas9-based therapy as a valuable new approach for the treatment of retinitis pigmentosa caused by autosomal recessive loss-of-function point mutations.

Synapsin III acts downstream of semaphorin 3A/CDK5 signaling to regulate radial migration and orientation of pyramidal neurons in vivo.

Synapsin III (SynIII) is a phosphoprotein that is highly expressed at early stages of neuronal development. Whereas in vitro evidence suggests a role for SynIII in neuronal differentiation, in vivo evidence is lacking. Here, we demonstrate that in vivo downregulation of SynIII expression affects neuronal migration and orientation. By contrast, SynIII overexpression affects neuronal migration, but not orientation. We identify a cyclin-dependent kinase-5 (CDK5) phosphorylation site on SynIII and use phosphomutant rescue experiments to demonstrate its role in SynIII function. Finally, we show that SynIII phosphorylation at the CDK5 site is induced by activation of the semaphorin-3A (Sema3A) pathway, which is implicated in migration and orientation of cortical pyramidal neurons (PNs) and is known to activate CDK5. Thus, fine-tuning of SynIII expression and phosphorylation by CDK5 activation through Sema3A activity is essential for proper neuronal migration and orientation.