CDKL5 disorder is a neurodevelopmental disorder still without a cure. Murine models of CDKL5 disorder have been recently generated raising the possibility of preclinical testing of treatments. However, unbiased, quantitative biomarkers of high translational value to monitor brain function are still missing. Moreover, the analysis of treatment is hindered by the challenge of repeatedly and non-invasively testing neuronal function. We analyzed the development of visual responses in a mouse model of CDKL5 disorder to introduce visually evoked responses as a quantitative method to assess cortical circuit function. Cortical visual responses were assessed in CDKL5 null male mice, heterozygous females, and their respective control wild-type littermates by repeated transcranial optical imaging from P27 until P32. No difference between wild-type and mutant mice was present at P25-P26 whereas defective responses appeared from P27-P28 both in heterozygous and homozygous CDKL5 mutant mice. These results were confirmed by visually evoked potentials (VEPs) recorded from the visual cortex of a different cohort. The previously imaged mice were also analyzed at P60–80 using VEPs, revealing a persistent reduction of response amplitude, reduced visual acuity and defective contrast function. The level of adult impairment was significantly correlated with the reduction in visual responses observed during development. Support vector machine showed that multi-dimensional visual assessment can be used to automatically classify mutant and wt mice with high reliability. Thus, monitoring visual responses represents a promising biomarker for preclinical and clinical studies on CDKL5 disorder.

Cyclin-dependent kinase like-5 (CDKL5) disorder is a rare neurodevelopmental disease caused by mutations in the CDKL5 gene. The consequent misexpression of the CDKL5 protein in the nervous system leads to a severe phenotype characterized by intellectual disability, motor impairment, visual deficits and early-onset epilepsy. No therapy is available for CDKL5 disorder. It has been reported that a protein transduction domain (TAT) is able to deliver macromolecules into cells and even into the brain when fused to a given protein. We demonstrate that TAT-CDKL5 fusion protein is efficiently internalized by target cells and retains CDKL5 activity. Intracerebroventricular infusion of TAT-CDKL5 restored hippocampal development, hippocampus-dependent memory and breathing pattern in Cdkl5-null mice. Notably, systemically administered TAT-CDKL5 protein passed the blood-brain-barrier, reached the CNS, and rescued various neuroanatomical and behavioral defects, including breathing pattern and visual responses. Our results suggest that CDKL5 protein therapy may be an effective clinical tool for the treatment of CDKL5 disorder.

CDKL5 deficiency disorder (CDD) is a neurodevelopmental disorder characterized by a severe global developmental delay and early-onset seizures. Notably, patients show distinctive visual abnormalities often clinically diagnosed as cortical visual impairment. However, the involvement of cerebral cortical dysfunctions in the origin of the symptoms is poorly understood. CDD mouse models also display visual deficits, and cortical visual responses can be used as a robust biomarker in CDKL5 mutant mice. A deeper understanding of the circuits underlying the described visual deficits is essential for directing preclinical research and translational approaches. Here, we addressed this question in two ways: first, we performed an in-depth morphological analysis of the visual pathway, from the retina to the primary visual cortex (V1), of CDKL5 null mice. We found that the lack of CDKL5 produced no alteration in the organization of retinal circuits. Conversely, CDKL5 mutants showed reduced density and altered morphology of spines and decreased excitatory synapse marker PSD95 in the dorsal lateral geniculate nucleus and in V1. An increase in the inhibitory marker VGAT was selectively present in V1. Second, using a conditional CDKL5 knockout model, we showed that selective cortical deletion of CDKL5 from excitatory cells is sufficient to produce abnormalities of visual cortical responses, demonstrating that the normal function of cortical circuits is dependent on CDKL5. Intriguingly, these deficits were associated with morphological alterations of V1 excitatory and inhibitory synaptic contacts. In summary, this work proposes cortical circuit structure and function as a critically important target for studying CDD.

Perineuronal nets (PNNs) are condensed structures in the extracellular matrix that mainly surround GABA-ergic parvalbumin-positive interneurons in the adult brain. Previous studies revealed a parallel between PNNs formation and the closure of the critical period. Moreover, ocular dominance plasticity is enhanced in response to PNN manipulations in adult animals. However, the mechanisms through which perineuronal nets modulate plasticity are still poorly understood. Recent work indicated that perineuronal nets may convey molecular signals by binding, and storing proteins with important roles in cellular communication. Here we report that Semaphorin 3A (Sema-3A), a chemorepulsive axon guidance cue known to bind to important perineuronal net components, is necessary to dampen ocular dominance plasticity in adult rats. First, we showed that the accumulation of Sema-3A in PNNs in the visual cortex correlates with critical period closure, following the same time course of perineuronal nets maturation. Second, the accumulation of Sema-3A in perineuronal nets was significantly reduced by rearing animals in the dark in the absence of any visual experience. Finally, we developed and characterized a tool to interfere with Sema-3A signaling by means of AAV-mediated expression of receptor bodies, soluble proteins formed by the extracellular domain of the endogenous Sema-3A receptor (neuropilin1) fused to a human IgG Fc fragment. By using this tool to antagonize Sema-3A signaling in the adult rat visual cortex, we found that the specific inhibition of Sema-3A promoted ocular dominance plasticity. Thus, Sema-3A accumulates in perineuronal nets in an experience-dependent manner and its presence in the mature visual cortex inhibit plasticity. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems CorporationPisa, December 10th, 2018Dear Editor,We have received the review of our manuscript "Inactivation of Semaphorin-3A promotes ocular dominance plasticity in the adult rat visual cortex." We are glad that both reviewers were positive about our manuscript. For rev #4 the manuscript was already acceptable for publication, while the issues raised by rev #3 were answered by new experimental results. These novel results show that:1. interfering with Sema3A signaling by AAV-mediated expression of soluble receptor bodies does not significantly contribute to modify the structural relationship between PNNs and Sema-3A; 2. The time course of WFA positive and Sema-3A positive PNNs progresses in parallel during postnatal development. 3. All the receptor bodies used in the study have the expected molecular weight and are effectively released in the culture medium, as shown by western blot analysis. These results, together with our data showing functional interference in growth cone collapse assays, give a better characterization of the molecular tool employed to counteract Semaphorin-3A. We hope that now the paper is suitable for publication. A point-to-point rebuttal follows Best regards, Tommaso Pizzorusso Ref #3Major points...