A GABAB receptor antagonist rescues functional and structural impairments in the perirhinal cortex of a mouse model of CDKL5 deficiency disorder.

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a severe neurodevelopmental encephalopathy characterized by early-onset epilepsy and intellectual disability. Studies in mouse models have linked CDKL5 deficiency to defects in neuronal maturation and synaptic plasticity, and disruption of the excitatory/inhibitory balance. Interestingly, increased density of both GABAergic synaptic terminals and parvalbumin inhibitory interneurons was recently observed in the primary visual cortex of Cdkl5 knockout (KO) mice, suggesting that excessive GABAergic transmission might contribute to the visual deficits characteristic of CDD. However, the functional relevance of cortical GABAergic circuits abnormalities in these mutant mice has not been investigated so far. Here we examined GABAergic circuits in the perirhinal cortex (PRC) of Cdkl5 KO mice, where we previously observed impaired long-term potentiation (LTP) associated with deficits in novel object recognition (NOR) memory. We found a higher number of GABAergic (VGAT)-immunopositive terminals in the PRC of Cdkl5 KO compared to wild-type mice, suggesting that increased inhibitory transmission might contribute to LTP impairment. Interestingly, while exposure of PRC slices to the GABAA receptor antagonist picrotoxin had no positive effects on LTP in Cdkl5 KO mice, the selective GABAB receptor antagonist CGP55845 restored LTP magnitude, suggesting that exaggerated GABAB receptor-mediated inhibition contributes to LTP impairment in mutants. Moreover, acute in vivo treatment with CGP55845 increased the number of PSD95 positive puncta as well as density and maturation of dendritic spines in PRC, and restored NOR memory in Cdkl5 KO mice. The present data show the efficacy of limiting excessive GABAB receptor-mediated signaling in improving synaptic plasticity and cognition in CDD mice.

Synaptic Reshaping and Neuronal Outcomes in the Temporal Lobe Epilepsy.

Temporal lobe epilepsy (TLE) is one of the most common types of focal epilepsy, characterized by recurrent spontaneous seizures originating in the temporal lobe(s), with mesial TLE (mTLE) as the worst form of TLE, often associated with hippocampal sclerosis. Abnormal epileptiform discharges are the result, among others, of altered cell-to-cell communication in both chemical and electrical transmissions. Current knowledge about the neurobiology of TLE in human patients emerges from pathological studies of biopsy specimens isolated from the epileptogenic zone or, in a few more recent investigations, from living subjects using positron emission tomography (PET). To overcome limitations related to the use of human tissue, animal models are of great help as they allow the selection of homogeneous samples still presenting a more various scenario of the epileptic syndrome, the presence of a comparable control group, and the availability of a greater amount of tissue for in vitro/ex vivo investigations. This review provides an overview of the structural and functional alterations of synaptic connections in the brain of TLE/mTLE patients and animal models.

CDKL5 protein substitution therapy rescues neurological phenotypes of a mouse model of 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.

Heterozygous CDKL5 Knockout Female Mice Are a Valuable Animal Model for CDKL5 Disorder.

CDKL5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked CDKL5 (cyclin-dependent kinase-like five) gene. CDKL5 disorder primarily affects girls and is characterized by early-onset epileptic seizures, gross motor impairment, intellectual disability, and autistic features. Although all CDKL5 female patients are heterozygous, the most valid disease-related model, the heterozygous female Cdkl5 knockout (Cdkl5 +/-) mouse, has been little characterized. The lack of detailed behavioral profiling of this model remains a crucial gap that must be addressed in order to advance preclinical studies. Here, we provide a behavioral and molecular characterization of heterozygous Cdkl5 +/- mice. We found that Cdkl5 +/- mice reliably recapitulate several aspects of CDKL5 disorder, including autistic-like behaviors, defects in motor coordination and memory performance, and breathing abnormalities. These defects are associated with neuroanatomical alterations, such as reduced dendritic arborization and spine density of hippocampal neurons. Interestingly, Cdkl5 +/- mice show age-related alterations in protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) signaling, two crucial signaling pathways involved in many neurodevelopmental processes. In conclusion, our study provides a comprehensive overview of neurobehavioral phenotypes of heterozygous female Cdkl5 +/- mice and demonstrates that the heterozygous female might be a valuable animal model in preclinical studies on CDKL5 disorder.

The Future of Neuroscience: Flexible and Wireless Implantable Neural Electronics.

Neurological diseases are a prevalent cause of global mortality and are of growing concern when considering an ageing global population. Traditional treatments are accompanied by serious side effects including repeated treatment sessions, invasive surgeries, or infections. For example, in the case of deep brain stimulation, large, stiff, and battery powered neural probes recruit thousands of neurons with each pulse, and can invoke a vigorous immune response. This paper presents challenges in engineering and neuroscience in developing miniaturized and biointegrated alternatives, in the form of microelectrode probes. Progress in design and topology of neural implants has shifted the goal post toward highly specific recording and stimulation, targeting small groups of neurons and reducing the foreign body response with biomimetic design principles. Implantable device design recommendations, fabrication techniques, and clinical evaluation of the impact flexible, integrated probes will have on the treatment of neurological disorders are provided in this report. The choice of biocompatible material dictates fabrication techniques as novel methods reduce the complexity of manufacture. Wireless power, the final hurdle to truly implantable neural interfaces, is discussed. These aspects are the driving force behind continued research: significant breakthroughs in any one of these areas will revolutionize the treatment of neurological disorders.

Age-Related Cognitive and Motor Decline in a Mouse Model of CDKL5 Deficiency Disorder is Associated with Increased Neuronal Senescence and Death.

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 ß-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.

Murine cerebral organoids develop network of functional neurons and hippocampal brain region identity.

Brain organoids are in vitro three-dimensional (3D) self-organized neural structures, which can enable disease modeling and drug screening. However, their use for standardized large-scale drug screening studies is limited by their high batch-to-batch variability, long differentiation time (10-20 weeks), and high production costs. This is particularly relevant when brain organoids are obtained from human induced pluripotent stem cells (iPSCs). Here, we developed, for the first time, a highly standardized, reproducible, and fast (5 weeks) murine brain organoid model starting from embryonic neural stem cells. We obtained brain organoids, which progressively differentiated and self-organized into 3D networks of functional neurons with dorsal forebrain phenotype. Furthermore, by adding the morphogen WNT3a, we generated brain organoids with specific hippocampal region identity. Overall, our results showed the establishment of a fast, robust and reproducible murine 3D in vitro brain model that may represent a useful tool for high-throughput drug screening and disease modeling.

Pharmacotherapy with sertraline rescues brain development and behavior in a mouse model of CDKL5 deficiency disorder.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause a severe neurodevelopmental disorder, CDKL5 deficiency disorder (CDD). CDKL5 is fundamental for correct brain development and function, but the molecular mechanisms underlying aberrant neurologic dysfunction in CDD are incompletely understood. Here we show a dysregulation of hippocampal and cortical serotonergic (5-HT) receptor expression in heterozygous Cdkl5 knockout (KO) female mice, suggesting that impaired 5-HT neurotransmission contributes to CDD. We demonstrate that targeting impaired 5-HT signaling via the selective serotonin reuptake inhibitor (SSRI) sertraline rescues CDD-related neurodevelopmental and behavioral defects in heterozygous Cdkl5 KO female mice. In particular, chronic treatment with sertraline normalized locomotion, stereotypic and autistic-like features, and spatial memory in Cdkl5 KO mice. These positive behavioral effects were accompanied by restored neuronal survival, dendritic development and synaptic connectivity. At a molecular level, sertraline increased brain-derived neurotrophic factor (BDNF) expression and restored abnormal phosphorylation levels of tyrosine kinase B (TrkB) and its downstream target the extracellular signal-regulated kinase (ERK1/2). Since sertraline is an FDA-approved drug with an extensive safety and tolerability data package, even for children, our findings suggest that sertraline may improve neurodevelopment in children with CDD. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'.

CDKL5 deficiency predisposes neurons to cell death through the deregulation of SMAD3 signaling.

CDKL5 deficiency disorder (CDD) is a rare encephalopathy characterized by early onset epilepsy and severe intellectual disability. CDD is caused by mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene, a member of a highly conserved family of serine-threonine kinases. Only a few physiological substrates of CDKL5 are currently known, which hampers the discovery of therapeutic strategies for CDD. Here, we show that SMAD3, a primary mediator of TGF-ß action, is a direct phosphorylation target of CDKL5 and that CDKL5-dependent phosphorylation promotes SMAD3 protein stability. Importantly, we found that restoration of the SMAD3 signaling through TGF-ß1 treatment normalized defective neuronal survival and maturation in Cdkl5 knockout (KO) neurons. Moreover, we demonstrate that Cdkl5 KO neurons are more vulnerable to neurotoxic/excitotoxic stimuli. In vivo treatment with TGF-ß1 prevents increased NMDA-induced cell death in hippocampal neurons from Cdkl5 KO mice, suggesting an involvement of the SMAD3 signaling deregulation in the neuronal susceptibility to excitotoxic injury of Cdkl5 KO mice. Our finding reveals a new function for CDKL5 in maintaining neuronal survival that could have important implications for susceptibility to neurodegeneration in patients with CDD.

Functional and Structural Impairments in the Perirhinal Cortex of a Mouse Model of CDKL5 Deficiency Disorder Are Rescued by a TrkB Agonist.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodevelopmental encephalopathy caused by mutations in the CDKL5 gene and characterized by early-onset epilepsy and intellectual and motor impairments. No cure is currently available for CDD patients, as limited knowledge of the pathology has hindered the development of therapeutics. Cdkl5 knockout (KO) mouse models, recently created to investigate the role of CDKL5 in the etiology of CDD, recapitulate various features of the disorder. Previous studies have shown alterations in synaptic plasticity and dendritic pattern in the cerebral cortex and in the hippocampus, but the knowledge of the molecular substrates underlying these alterations is still limited. Here, we have examined for the first time synaptic function and plasticity, dendritic morphology, and signal transduction pathways in the perirhinal cortex (PRC) of this mouse model. Being interconnected with a wide range of cortical and subcortical structures and involved in various cognitive processes, PRC provides a very interesting framework for examining how CDKL5 mutation leads to deficits at the synapse, circuit, and behavioral level. We found that long-term potentiation (LTP) was impaired, and that the TrkB/PLCγ1 pathway could be mechanistically involved in this alteration. PRC neurons in mutant mice showed a reduction in dendritic length, dendritic branches, PSD-95-positive puncta, GluA2-AMPA receptor levels, and spine density and maturation. These functional and structural deficits were associated with impairment in visual recognition memory. Interestingly, an in vivo treatment with a TrkB agonist (the 7,8-DHF prodrug R13) to trigger the TrkB/PLCγ1 pathway rescued defective LTP, dendritic pattern, PSD-95 and GluA2-AMPA receptor levels, and restored visual recognition memory in Cdkl5 KO mice. Present findings demonstrate a critical role of TrkB signaling in the synaptic development alterations due to CDKL5 mutation, and suggest the possibility of TrkB-targeted pharmacological interventions.

Adenosine Promotes Endplate nAChR Channel Activity in Adult Mouse Skeletal Muscle Fibers via Low Affinity P1 Receptors.

Adenosine is a powerful modulator of skeletal neuromuscular transmission, operating via inhibitory or facilitatory purinergic-type P1 receptors. To date, studies have been focused mainly on the effect of adenosine on presynaptic P1 receptors controlling transmitter release. In this study, using two-microelectrode voltage-clamp and single-channel patch-clamp recording techniques, we have explored potential postsynaptic targets of adenosine and their modulatory effect on nicotinic acetylcholine receptor (nAChR)-mediated synaptic responses in adult mouse skeletal muscle fibers in vitro. In the whole-mount neuromuscular junction (NMJ) preparation, adenosine (100 µM) significantly reduced the frequency of the miniature endplate currents (MEPCs) and slowed their rising and decay time. Consistent with a postsynaptic site of action, adenosine and the potent P1 receptor agonist NECA significantly increased the open probability, the frequency and the open time of single nAChR channels, recorded at the endplate region. Using specific ligands for the P1 receptor subtypes, we found that the low-affinity P1 receptor subtype A2B was responsible for mediating the effects of adenosine on the nAChR channel openings. Our data suggest that at the adult mammalian NMJ, adenosine acts not only presynaptically to modulate acetylcholine transmitter release, but also at the postsynaptic level, to enhance the activity of nAChRs. Our findings open a new scenario in understanding of purinergic regulation of nAChR activity at the mammalian endplate region.

Xenopus laevis Oocytes as a Model System for Studying the Interaction Between Asbestos Fibres and Cell Membranes.

The mode of interaction of asbestos fibres with cell membranes is still debatable. One reason is the lack of a suitable and convenient cellular model to investigate the causes of asbestos toxicity. We studied the interaction of asbestos fibres with Xenopus laevis oocytes, using electrophysiological and morphological methods. Oocytes are large single cells, with a limited ability to endocytose molecular ligands; we therefore considered these cells to be a good model for investigating the nature of asbestos/membrane interactions. Electrophysiological recordings were performed to compare the passive electrical membrane properties, and those induced by applying positive or negative voltage steps, in untreated oocytes and those exposed to asbestos fibre suspensions. Ultrastructural analysis visualized in detail, any morphological changes of the surface membrane caused by the fibre treatment. Our results demonstrate that Amosite and Crocidolite-type asbestos fibres significantly modify the properties of the membrane, starting soon after exposure. Cells were routinely depolarized, their input resistance decreased, and the slow outward currents evoked by step depolarizations were dramatically enhanced. Reducing the availability of surface iron contained in the structure of the fibres with cation chelators, abolished these effects. Ultrastructural analysis of the fibre-exposed oocytes showed no evidence of phagocytic events. Our results demonstrate that asbestos fibres modify the oocyte membrane, and we propose that these cells represent a viable model for studying the asbestos/cell membrane interaction. Our findings also open the possibly for finding specific competitors capable of hindering the asbestos-cell membrane interaction as a means of tackling the long-standing asbestos toxicity problem.

Reactive oxygen species contribute to the promotion of the ATP-mediated proliferation of mouse skeletal myoblasts.

Reactive oxygen species (ROS) and extracellular adenosine 5'-triphosphate (ATP) participate in autocrine and paracrine regulation in skeletal muscle. However, the link between these two signaling systems is not well established. Here, we studied cell proliferation as a possible consequence of the trophic effect of ATP in cultured skeletal mouse myoblasts and we tested the possibility that low concentrations of ROS represent the intermediate signaling molecule mediating this effect. Exposure to 10 µM ATP increased proliferation of mouse myoblasts by ~20%. ATP also induced intracellular Ca(2+) oscillations, which were independent of extracellular Ca(2+). Both effects of ATP were prevented by suramin, a broad-spectrum purinergic P2 receptor antagonist. In contrast, the adenosine receptor blocker CGS-15943 did not modify the ATP-mediated effects. Consistent with this, adenosine per se did not change myoblast growth, indicating the direct action of ATP via P2 receptor activation. The proliferative effect of ATP was prevented after depletion of hydrogen peroxide (H(2)O(2)) by the peroxidase enzyme catalase. Low-micromolar concentrations of exogenous H(2)O(2) mimicked the stimulatory effect of ATP on myoblast growth. DCF imaging revealed ATP-induced catalase and DPI-sensitive ROS production in myoblasts. In conclusion, our results indicate that extracellular ATP controls mouse myoblast proliferation via induction of ROS generation.