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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.

PCDH19 gene-related epilepsy or epileptic encephalopathy, early infantile, 9 (EIEE9) is an infantile onset epilepsy syndrome characterized by psychiatric (including autistic) sensory and cognitive impairment of varying degrees. EIEE9 is caused by X-linked PCDH19 protein loss of function. Due to random X-chromosome inactivation, EIEE9-affected females present a mosaic population of healthy and Pcdh19-mutant cells. Unfortunately, no mouse models recapitulate to date both the brain histological and behavioural deficits present in people with EIEE9. Thus, the search for a proper understanding of the disease, and possible future treatment is hampered. By inducing a focal mosaicism of Pcdh19 expression using in utero electroporation in rat, we found here that Pcdh19 signaling in specific brain areas is implicated in neuronal migration, as well as in core behaviors related to autism and cognitive function.

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.

Inherited retinal dystrophies are a large and heterogeneous group of degenerative diseases caused by mutations in various genes. Given the favourable anatomical and immunological characteristics of the eye, gene therapy holds great potential for their treatment. We used a tailored CRISPR/Cas9-based gene editing system to prevent retinal photoreceptor death in the Rd10 mouse model of retinitis pigmentosa. We tested the gene editing toolin vitroand then usedin vivosubretinal 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.

Since the dawn of neuroscience, genetic alterations causing macroscopic brain defects have attracted the attention of scientists. However, in recent years, the increasing understanding of the biological causes of neurological disorders such as Autism Spectrum Disorder or Schizophrenia have indicated that affected patients do not display gross structural alterations in the brain. These findings have shifted the attention of scientists toward the study of genetic alterations that may cause subtle defects in brain architecture. The investigation on such microscopic brain alterations is currently favored by the progress in experimental techniques for brain research. In Perlini et al., 2015 [1], we took advantage of the in utero electroporation technique coupled with RNA interference to examine the role of Synapsin III (SynIII) in the developing neocortex. SynIII belongs to the Syn family, which comprises three phosphoproteins abundantly expressed in the brain. Whereas a large body of literature indicates that both SynI and SynII are mainly involved in synaptic transmission and plasticity in the adult brain, the available data suggest an involvement of SynIII in brain development. First, SynIII is expressed early during mammalian brain formation and is involved in early stages of neuronal development in vitro, while it is downregulated in the adult brain [2, 3]. Second, SynIII participates in adult neurogenesis [3], and often proteins involved in adult neurogenesis are also players in brain development. Third, SynIII SNPs have been implicated in neurodevelopmental disorders such as schizophrenia, and SynIII knockout (KO) mice display some behavioral traits of schizophrenia [4]. Finally, according to evolutionary studies, the Syn family originated from a single primordial Syn gene that was expressed in the brain since early development [5]. As in higher vertebrates SynI and II are expressed mostly in the adult brain, SynIII probably carries out the functions performed during development by the primitive single Syn. Interestingly, the emergence of a distinct SynIII gene correlates with the appearance of the telencephalic region during evolution [6]. Despite the evidence suggesting a developmental role for SynIII, the initial analysis of SynIII KO mice revealed a mild behavioral phenotype with no gross alterations in brain development. We hypothesized that the alterations caused by SynIII absence could be very subtle and difficult to detect with conventional histochemical techniques. In Perlini et al., 2015 [1], we demonstrated that SynIII KO brains do have aberrant brain development with defects in neuronal orientation as visualized by in utero transfection of a fluorescent (GFP) reporter. As the migration and orientation phenotypes were milder in SynIII KO mice than in animals where SynIII was acutely downregulated by RNA interference, we hypothesized that a partial compensation phenomenon could be triggered by the chronic depletion of the protein in KO animals. Interestingly, also animals with in utero upregulation of SynIII levels presented defects in radial migration with the appearance of ectopic cells at late stages. Thus, SynIII plays a key role in the orchestration of cortical development, and a tight regulation of its expression is essential for this function. In Perlini et al., 2015 [1], we also demonstrated that the migration and orientation defects upon SynIII depletion involved the Sema3A/CDK5 signaling cascade. Indeed, Sema3A stimulation caused SynIII phosphorylation by CDK5 on Ser404 and such CDK5-dependent phosphorylation was essential for Sema3A-induced SynIII activity. It would be interesting to investigate whether SynIII participates in other developmental events mediated by Sema3A. Indeed, Sema3A is also involved in axon/dendrite initiation during the early phase of neuronal polarization through its negative regulation of PKA activity. Interestingly, SynIII also carries a highly conserved phosphorylation site targeted by PKA (Ser9), and defects in neuronal polarization consequent to SynIII downregulation were recently observed [7]. Besides the findings in Perlini et al., 2015 [1] demonstrating that SynIII acts downstream Sema3A, the existence of multiple phosphorylation sites on SynIII indicates that SynIII may be potentially downstream to other signaling pathways. Interestingly, during development, migrating cortical neurons encounter an increasing gradient of both Sema3A and Reelin around the marginal zone, and mice defective in Sema3A- and Reelin-signaling share common phenotypic features, such as misoriented neurons, also found in SynIII-downregulated brains. This suggests that Sema3A/SynIII and Reelin signaling interact at least in part. Notably, this interaction might be important for proper brain development and function, as an increase in Sema3A with a concomitant decrease of Reelin was observed in brains from schizophrenic patients. Moreover, both Sema3A and Reelin activate the Src-family kinase Fyn. The presence of a phosphorylation site for Src-family kinases (Tyr301) on SynIII puts this protein again downstream of Sema3A, but potentially also downstream of Reelin signaling, as a potential link through these two pivotal pathways for brain development. Therefore, despite the recent advancements in the comprehension of the role of SynIII in brain development, many questions on other potential functions of this protein remain open.

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 elongationin 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 ofin vitrodevelopment. 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 STATEMENTSynapsin 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.