Cyclin-dependent kinase-like 5 (CDKL5) mutations are found in severe neurodevelopmental disorders, including the Hanefeld variant of Rett syndrome (RTT; CDKL5 disorder). CDKL5 loss-of-function murine models recapitulate pathological signs of the human disease, such as visual attention deficits and reduced visual acuity. Here we investigated the cellular and synaptic substrates of visual defects by studying the organization of the primary visual cortex (V1) of Cdkl5−/y mice. We found a severe reduction of c-Fos expression in V1 of Cdkl5−/y mutants, suggesting circuit hypoactivity. Glutamatergic presynaptic structures were increased, but postsynaptic PSD-95 and Homer were significantly downregulated in CDKL5 mutants. Interneurons expressing parvalbumin, but not other types of interneuron, had a higher density in mutant V1, and were hyperconnected with pyramidal neurons. Finally, the developmental trajectory of pavalbumin-containing cells was also affected in Cdkl5−/y mice, as revealed by fainter appearance perineuronal nets at the closure of the critical period (CP). The present data reveal an overall disruption of V1 cellular and synaptic organization that may cause a shift in the excitation/inhibition balance likely to underlie the visual deficits characteristic of CDKL5 disorder. Moreover, ablation of CDKL5 is likely to tamper with the mechanisms underlying experience-dependent refinement of cortical circuits during the CP of development.

Stress and genetic predisposition are two of the major risk factors for a variety of psychiatric illnesses. Inbred mouse strains are considered useful tools in dissecting the genetic basis of complex disorders. Indeed, mice of the C57BL/6 and BALB/c strains, differing markedly in anxiety behaviours, are among the most widely used in psychopharmacological research. However, there is a paucity of studies investigating the impact of social stress in these two strains. Moreover, whether these two mouse strains exhibit different sensitivities to chronic social defeat stress remains poorly studied. Thus in this study we compared the impact of repeated (10 days) social defeat stress on a variety of behavioural and endocrine parameters including social interaction, locomotor activity, plasma corticosterone, body weight and stress-related physiological parameters in both mouse strains. Given that the duration of stress exposure may differentially affect such responses we also compared stressors of short (Social Defeat-Short; SD-S) and of long (Social Defeat-Long; SD-L) duration. Our results show that although mice from both strains were defeated in both social defeat paradigms, only BALB/c mice displayed social interaction impairments following SD-S, whereas both strains were behaviourally sensitive to SD-L. Moreover, both strains also differed in some of the physiological alterations induced by social defeat stress. Specifically, SD-S did not induce any change in corticosterone levels in either of the two strains, whereas SD-L was able to induce significant changes in C57BL/6 mice only. SD-S induced differential effects on bodyweight gain in both strains, increasing it in C57BL/6 and decreasing it in BALB/c mice, whereas SD-L had no effect. On the other hand, exposure to SD-S resulted in cardiac hypertrophy in C57BL/6 mice and SD-L induced spleen hypertrophy and thymus atrophy in BALB/c mice in addition to decreasing faecal output. Overall, the innately anxious BALB/c mice were more sensitive to social stress than C57BL/6, with differential behavioural and physiological alterations emerging as a function of stress severity. These data suggest different coping strategies to social interaction stress between the two mouse strains. The genetic basis of this stress-resilience/susceptibility warrants further investigation.

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Highlights • CDKL5 deficiency disrupts the synaptic organization of thalamo-cortical (TC) and cortico-cortical (CC) connections in the barrel cortex (BC) • CDKL5 deficiency leads to BC hypoactivation • CDKL5 deficiency causes atypical whisker-mediated behavioural responses • CDKL5 deficiency does not prevent TC circuitry to undergo experience-dependent structural plasticity • Enhanced sensory stimulation restores cortical connectivity, BC activation levels and whisker-related behavioural responses ABSTRACT Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene cause CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental syndrome where patients exhibit early-onset seizures, intellectual disability, stereotypies, limited or absent speech, autism-like symptoms and sensory impairments. Mounting evidences indicate that disrupted sensory perception and processing represent core signs also in mouse models of CDD, however we have very limited knowledge on their underlying causes. In this study, we investigated how CDKL5 deficiency affects synaptic organization and experience-dependent plasticity in the thalamo-cortical (TC) pathway carrying whisker-related tactile information to the barrel cortex (BC). By using synapse-specific antibodies and confocal microscopy, we found that Cdkl5-KO mice display a lower density of TC synapses in the BC that was paralleled by a reduction of cortico-cortical (CC) connections compared to wild-type mice. These synaptic defects were accompanied by reduced BC activation, as shown by a robust decrease of c-fos immunostaining, and atypical behavioural responses to whisker-mediated tactile stimulation. Notably, a two-day paradigm of enriched whisker stimulation rescued both number and configuration of TC and CC synapses in Cdkl5-KO mice, and restored cortical activity as well as behavioural responses to control levels. Our findings disclose an important role of CDKL5 in controlling the organization and experience-induced modifications of excitatory connections in the BC and indicate how mutations of CDKL5 produce failures in higher-order processing of somatosensory stimuli.

CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental disease caused by mutations in the X-linked CDKL5 gene. Children affected by CDD display a clinical phenotype characterized by early-onset epilepsy, intellectual disability, motor impairment, and autistic-like features. Although the clinical aspects associated with CDKL5 mutations are well described in children, adults with CDD are still under-characterized. Similarly, most animal research has been carried out on young adult Cdkl5 knockout (KO) mice only. Since age represents a risk factor for the worsening of symptoms in many neurodevelopmental disorders, understanding age differences in the development of behavioral deficits is crucial in order to optimize the impact of therapeutic interventions. Here, we compared young adult Cdkl5 KO mice with middle-aged Cdkl5 KO mice, at a behavioral, neuroanatomical, and molecular level. We found an age-dependent decline in motor, cognitive, and social behaviors in Cdkl5 KO mice, as well as in breathing and sleep patterns. The behavioral decline in older Cdkl5 KO mice was not associated with a worsening of neuroanatomical alterations, such as decreased dendritic arborization or spine density, but was paralleled by decreased neuronal survival in different brain regions such as the hippocampus, cortex, and basal ganglia. Interestingly, we found increased β-galactosidase activity and DNA repair protein levels, γH2AX and XRCC5, in the brains of older Cdkl5 KO mice, which suggests that an absence of Cdkl5 accelerates neuronal senescence/death by triggering irreparable DNA damage. In summary, this work provides evidence that CDKL5 may play a fundamental role in neuronal survival during brain aging and suggests a possible worsening with age of the clinical picture in CDD patients.

BackgroundThe m-AAA (ATPases Associated with a variety of cellular Activities) is an evolutionary conserved metalloprotease complex located in the internal mitochondrial membrane. In the mouse, it is a hetero-oligomer variably formed by the Spg7, Afg3l1, and Afg3l2 encoded proteins, or a homo-oligomer formed by either Afg3l1 or Afg3l2. In humans, AFG3L2 and SPG7 genes are conserved, whereas AFG3L1 became a pseudogene. Both AFG3L2 and SPG7 are involved in a neurodegenerative disease, namely the autosomal dominant spinocerebellar ataxia SCA28 and a recessive form of spastic paraplegia, respectively.ResultsUsing quantitative RT-PCR, we measured the expression levels of Spg7, Afg3l1, and Afg3l2 in the mouse brain. In all regions Afg3l2 is the most abundant transcript, followed by Spg7, and Afg3l1, with a ratio of approximately 5:3:1 in whole-brain mRNA. Using in-situ hybridization, we showed that Spg7, Afg3l1 and Afg3l2 have a similar cellular pattern of expression, with high levels in mitral cells, Purkinje cells, deep cerebellar nuclei cells, neocortical and hippocampal pyramidal neurons, and brainstem motor neurons. However, in some neuronal types, differences in the level of expression of these genes were present, suggesting distinct degrees of contribution of their proteins.ConclusionsNeurons involved in SCA28 and hereditary spastic paraplegia display high levels of expression, but similar or even higher expression is also present in other types of neurons, not involved in these diseases, suggesting that the selective cell sensitivity should be attributed to other, still unknown, mechanisms.

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Background Rett syndrome (RTT) is a monogenic X-linked neurodevelopmental disorder characterized by loss-of-function mutations in the MECP2 gene, which lead to structural and functional changes in synapse communication, and impairments of neural activity at the basis of cognitive deficits that progress from an early age. While the restoration of MECP2 in animal models has been shown to rescue some RTT symptoms, gene therapy intervention presents potential side effects, and with gene- and RNA-editing approaches still far from clinical application, strategies focusing on signaling pathways downstream of MeCP2 may provide alternatives for the development of more effective therapies in vivo. Here, we investigate the role of the c-Jun N-terminal kinase (JNK) stress pathway in the pathogenesis of RTT using different animal and cell models and evaluate JNK inhibition as a potential therapeutic approach. Results We discovered that the c-Jun N-terminal kinase (JNK) stress pathway is activated in Mecp2-knockout, Mecp2-heterozygous mice, and in human MECP2-mutated iPSC neurons. The specific JNK inhibitor, D-JNKI1, promotes recovery of body weight and locomotor impairments in two mouse models of RTT and rescues their dendritic spine alterations. Mecp2-knockout presents intermittent crises of apnea/hypopnea, one of the most invalidating RTT pathological symptoms, and D-JNKI1 powerfully reduces this breathing dysfunction. Importantly, we discovered that also neurons derived from hiPSC-MECP2 mut show JNK activation, high-phosphorylated c-Jun levels, and cell death, which is not observed in the isogenic control wt allele hiPSCs. Treatment with D-JNKI1 inhibits neuronal death induced by MECP2 mutation in hiPSCs mut neurons. Conclusions As a summary, we found altered JNK signaling in models of RTT and suggest that D-JNKI1 treatment prevents clinical symptoms, with coherent results at the cellular, molecular, and functional levels. This is the first proof of concept that JNK plays a key role in RTT and its specific inhibition offers a new and potential therapeutic tool to tackle RTT.