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The oligomeric form of the peptide amyloid beta 42 (Abeta42) contributes to the development of synaptic abnormalities and cognitive impairments associated with Alzheimer's disease (AD). To date, there is a gap in knowledge regarding how Abeta42 alters the elementary parameters of GABAergic synaptic function. Here we found that Abeta42 increased the frequency and amplitude of miniature GABAergic currents as well as the amplitude of evoked inhibitory postsynaptic currents. When we focused on paired pulse depression (PPD) to establish whether GABA release probability was affected by Abeta42, we did not observe any significant change. On the other hand, a more detailed investigation of the presynaptic effects induced by Abeta42 by means of multiple probability fluctuation analysis and cumulative amplitude analysis showed an increase in both the size of the readily releasable pool responsible for synchronous release and the number of release sites. We further explored whether ryanodine receptors (RyRs) contributed to exacerbating these changes by stabilizing the interaction between RyRs and the accessory protein calstabin. We observed that the RyR-calstabin interaction stabilizer S107 restored the synaptic parameters to values comparable to those measured in control conditions. In conclusion, our results clarify the mechanisms of potentiation of GABAergic synapses induced by Abeta42. We further suggest that RyRs are involved in the control of synaptic activity during the early stage of AD onset and that their stabilization could represent a new therapeutical approach for AD treatment. KEY POINTS: Accumulation of the peptide amyloid beta 42 (Abeta42) is a key characteristic of Alzheimer's disease (AD) and causes synaptic dysfunctions. To date, the effects of Abeta42 accumulation on GABAergic synapses are poorly understood. The findings reported here suggest that, similarly to what is observed on glutamatergic synapses, Abeta42 modifies GABAergic synapses by targeting ryanodine receptors and causing calcium dysregulation. The GABAergic impairments can be restored by the ryanodine receptor-calstabin interaction stabilizer S107. Based on this research, RyRs stabilization may represent a novel pharmaceutical strategy for preventing or delaying AD.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a devastating rare neurodevelopmental disease without a cure, caused by mutations of the serine/threonine kinase CDKL5 highly expressed in the forebrain. CDD is characterized by early-onset seizures, severe intellectual disabilities, autistic-like traits, sensorimotor and cortical visual impairments (CVI). The lack of an effective therapeutic strategy for CDD urgently demands the identification of novel druggable targets potentially relevant for CDD pathophysiology. To this aim, we studied Class I metabotropic glutamate receptors 5 (mGluR5) because of their important role in the neuropathological signs produced by the lack of CDKL5 in-vivo, such as defective synaptogenesis, dendritic spines formation/maturation, synaptic transmission and plasticity. Importantly, mGluR5 function strictly depends on the correct expression of the postsynaptic protein Homer1bc that we previously found atypical in the cerebral cortex of Cdkl5-/y mice. In this study, we reveal that CDKL5 loss tampers with (i) the binding strength of Homer1bc-mGluR5 complexes, (ii) the synaptic localization of mGluR5 and (iii) the mGluR5-mediated enhancement of NMDA-induced neuronal responses. Importantly, we showed that the stimulation of mGluR5 activity by administering in mice specific positive-allosteric-modulators (PAMs), i.e., 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) or RO6807794, corrected the synaptic, functional and behavioral defects shown by Cdkl5-/y mice. Notably, in the visual cortex of 2 CDD patients we found changes in synaptic organization that recapitulate those of mutant CDKL5 mice, including the reduced expression of mGluR5, suggesting that these receptors represent a promising therapeutic target for CDD.

Although delivery of a wild-type copy of the mutated gene to cells represents the most effective approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for treatment of brain disorders. Herein, we develop a cross-correction-based strategy to enhance the efficiency of a gene therapy for CDKL5 deficiency disorder, a severe neurodevelopmental disorder caused by CDKL5 gene mutations. We created a gene therapy vector that produces an Igk-TATk-CDKL5 fusion protein that can be secreted via constitutive secretory pathways and, due to the cell-penetration property of the TATk peptide, internalized by cells. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to higher CDKL5 protein replacement due to secretion and penetration of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle or AAVPHP.B_CDKL5 vector-treated Cdkl5 KO mice. In conclusion, we provide the first evidence that a gene therapy based on a cross-correction approach is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5, opening avenues for the development of this innovative approach for other monogenic diseases.

The NMDARs are key players in both physiological and pathologic synaptic plasticity because of their involvement in many aspects of neuronal transmission as well as learning and memory. The contribution in these events of different types of GluN2A-interacting proteins is still unclear. The p140Cap scaffold protein acts as a hub for postsynaptic complexes relevant to psychiatric and neurologic disorders and regulates synaptic functions, such as the stabilization of mature dendritic spine, memory consolidation, LTP, and LTD. Here we demonstrate that p140Cap directly binds the GluN2A subunit of NMDAR and modulates GluN2A-associated molecular network. Indeed, in p140Cap KO male mice, GluN2A is less associated with PSD95 both in ex vivo synaptosomes and in cultured hippocampal neurons, and p140Cap expression in KO neurons can rescue GluN2A and PSD95 colocalization. p140Cap is crucial in the recruitment of GluN2A-containing NMDARs and, consequently, in regulating NMDARs' intrinsic properties. p140Cap is associated to synaptic lipid-raft (LR) and to soluble postsynaptic membranes, and GluN2A and PSD95 are less recruited into synaptic LR of p140Cap KO male mice. Gated-stimulated emission depletion microscopy on hippocampal neurons confirmed that p140Cap is required for embedding GluN2A clusters in LR in an activity-dependent fashion. In the synaptic compartment, p140Cap influences the association between GluN2A and PSD95 and modulates GluN2A enrichment into LR. Overall, such increase in these membrane domains rich in signaling molecules results in improved signal transduction efficiency. SIGNIFICANCE STATEMENT Here we originally show that the adaptor protein p140Cap directly binds the GluN2A subunit of NMDAR and modulates the GluN2A-associated molecular network. Moreover, we show, for the first time, that p140Cap also associates to synaptic lipid rafts and controls the selective recruitment of GluN2A and PSD95 to this specific compartment. Finally, gated-stimulated emission depletion microscopy on hippocampal neurons confirmed that p140Cap is required for embedding GluN2A clusters in lipid rafts in an activity-dependent fashion. Overall, our findings provide the molecular and functional dissection of p140Cap as a new active member of a highly dynamic synaptic network involved in memory consolidation, LTP, and LTD, which are known to be altered in neurologic and psychiatric disorders.

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.

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder 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 balance between excitation and inhibition. Interestingly, GABAergic transmission mediated by parvalbumin+ interneurons was recently found enhanced in the primary visual cortex of Cdkl5-knockout (KO) mice; this might contribute to the visual deficits characteristic of the CDKL5 disorder. Further studies are needed to clarify the relation between GABAergic abnormalities in the cerebral cortex and the functional deficits observed in these mice. Here we examined the GABAergic network in the perirhinal cortex (PRC), where we previously observed a long-term potentiation (LTP) impairment associated with a visual recognition memory deficit. We found a higher number of vesicular GABA transporter (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 in vitro exposure to the GABAAR antagonist picrotoxin had no positive effects on LTP in PRC slices from Cdkl5-KO mice, exposure to the selective GABABR antagonist CGP55845 increased LTP magnitude, suggesting that exaggerated GABABR-mediated inhibition might contribute to LTP impairment. Moreover, acute in vivo treatment with CGP55845 restored novel object recognition in Cdkl5-KO mice, and increased the number of PSD95+ puncta and number and maturation of dendritic spines. Present data suggest that inhibition of enhanced GABABR-mediated synaptic transmission might be an effective way to improve synaptic plasticity and cognition in CDKL5 deficiency.

No therapy is currently available for CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD), a severe neurodevelopmental disorder caused by mutations in the CDKL5 gene. Although delivery of a wild-type copy of the mutated gene to cells represents the most curative approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for brain gene therapy. Herein, we used a secretable TATk-CDKL5 protein to enhance the efficiency of a gene therapy for CDD. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to a higher CDKL5 protein replacement due to secretion and transduction of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle-treated Cdkl5 KO mice or Cdkl5 KO mice treated with the AAVPHP.B_CDKL5 vector, indicating that a gene therapy based on a secretable recombinant TATk-CDKL5 protein is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5.

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 GABA

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 behavioral responses to whisker-mediated tactile stimulation. Notably, a 2-day paradigm of enriched whisker stimulation rescued both number and configuration of excitatory synapses in Cdkl5-KO mice, restored cortical activity and normalized behavioral responses to wild-type mice levels. Our findings disclose a novel and unsuspected 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. This article is part of a Special Issue entitled: Animal Models of Neurodevelopmental Disorders.

The neuronal scaffold protein p140Cap was investigated during hippocampal network formation. p140Cap is present in presynaptic GABAergic terminals and its genetic depletion results in a marked alteration of inhibitory synaptic activity. p140Cap-/- cultured neurons display higher frequency of miniature inhibitory postsynaptic currents (mIPSCs) with no changes of their mean amplitude. Consistent with a potential presynaptic alteration of basal GABA release, p140Cap-/- neurons exhibit a larger synaptic vesicle readily releasable pool, without any variation of single GABAA receptor unitary currents and number of postsynaptic channels. Furthermore, p140Cap-/- neurons show a premature and enhanced network synchronization and appear more susceptible to 4-aminopyridine-induced seizures in vitro and to kainate-induced seizures in vivo. The hippocampus of p140Cap-/- mice showed a significant increase in the number of both inhibitory synapses and of parvalbumin- and somatostatin-expressing interneurons. Specific deletion of p140Cap in forebrain interneurons resulted in increased susceptibility to in vitro epileptic events and increased inhibitory synaptogenesis, comparable to those observed in p140Cap-/- mice. Altogether, our data demonstrate that p140Cap finely tunes inhibitory synaptogenesis and GABAergic neurotransmission, thus regulating the establishment and maintenance of the proper hippocampal excitatory/inhibitory balance.

Cell signalling mechanisms are central to neuronal activity and their dysregulation may lead to neurodegenerative processes and associated cognitive decline. So far, a major effort has been directed toward the dissection of disease specific pathways with the still unmet promise to develop precision medicine strategies. With a different approach, here we show that a selective genetic potentiation of neuronal ERK signalling prevents cell death in vitro and in vivo in the mouse brain while ERK attenuation does the opposite. This neuroprotective effect can also be induced pharmacologically by a cell permeable peptide mimicking the loss of ERK1 MAP kinase, leading to a selective enhancement of ERK2 mediated nuclear cell signalling. The drug treatment prevents neurodegeneration in mouse models of Huntington's (HD), Alzheimer's (AD), and Parkinson's disease (PD). Importantly, the selective potentiation of ERK2 signalling facilitates both structural and synaptic plasticity, enhances cognition in healthy mice and rescues mild cognitive impairments in both models of AD and HD. Altogether, our observation truly represents a remarkable example of a shared molecular mechanism across multiple neurodegenerative disorders and a potentially valuable therapeutic target for neuro-enhancement.

Background CDKL5 (cyclin-dependent kinase-like 5) is mutated in many severe neurodevelopmental disorders, including atypical Rett syndrome. CDKL5 was shown to interact with synaptic proteins, but an in vivo analysis of the role of CDKL5 in dendritic spine dynamics and synaptic molecular organization is still lacking. Methods In vivo two-photon microscopy of the somatosensory cortex of Cdkl 5 −/y mice was applied to monitor structural dynamics of dendritic spines. Synaptic function and plasticity were measured using electrophysiological recordings of excitatory postsynaptic currents and long-term potentiation in brain slices and assessing the expression of synaptic postsynaptic density protein 95 (PSD-95). Finally, we studied the impact of insulin-like growth factor 1 (IGF-1) treatment on CDKL5 null mice to restore the synaptic deficits. Results Adult mutant mice showed a significant reduction in spine density and PSD-95-positive synaptic puncta, a reduction of persistent spines, and impaired long-term potentiation. In juvenile mutants, short-term spine elimination, but not formation, was dramatically increased. Exogenous administration of IGF-1 rescued defective rpS6 phosphorylation, spine density, and PSD-95 expression. Endogenous cortical IGF-1 levels were unaffected by CDKL5 deletion. Conclusions These data demonstrate that dendritic spine stabilization is strongly regulated by CDKL5. Moreover, our data suggest that IGF-1 treatment could be a promising candidate for clinical trials in CDKL5 patients.

Rett syndrome (RTT) is caused in most cases by loss-of-function mutations in the X-linked gene encoding methyl CpG-binding protein 2 (MECP2). Understanding the pathological processes impacting sensory-motor control represents a major challenge for clinical management of individuals affected by RTT, but the underlying molecular and neuronal modifications remain unclear. We find that symptomatic male Mecp2 knockout (KO) mice show atypically elevated parvalbumin (PV) expression in both somatosensory (S1) and motor (M1) cortices together with excessive excitatory inputs converging onto PV-expressing interneurons (INs). In accordance, high-speed voltage-sensitive dye imaging shows reduced amplitude and spatial spread of synaptically induced neuronal depolarizations in S1 of Mecp2 KO mice. Moreover, motor learning-dependent changes of PV expression and structural synaptic plasticity typically occurring on PV+ INs in M1 are impaired in symptomatic Mecp2 KO mice. Finally, we find similar abnormalities of PV networks plasticity in symptomatic female Mecp2 heterozygous mice. These results indicate that in Mecp2 mutant mice the configuration of PV+ INs network is shifted toward an atypical plasticity state in relevant cortical areas compatible with the sensory-motor dysfunctions characteristics of RTT. Rett syndrome (RTT) is caused in most cases by loss-of-function mutations in the X-linked gene encoding methyl CpG-binding protein 2 (MECP2). Understanding the pathological processes impacting sensory-motor control represents a major challenge for clinical management of individuals affected by RTT, but the underlying molecular and neuronal modifications remain unclear. We find that symptomatic male Mecp2 knockout (KO) mice show atypically elevated parvalbumin (PV) expression in both somatosensory (S1) and motor (M1) cortices together with excessive excitatory inputs converging onto PV-expressing interneurons (INs). In accordance, high-speed voltage-sensitive dye imaging shows reduced amplitude and spatial spread of synaptically induced neuronal depolarizations in S1 of Mecp2 KO mice. Moreover, motor learning-dependent changes of PV expression and structural synaptic plasticity typically occurring on PV + INs in M1 are impaired in symptomatic Mecp2 KO mice. Finally, we find similar abnormalities of PV networks plasticity in symptomatic female Mecp2 heterozygous mice. These results indicate that in Mecp2 mutant mice the configuration of PV + INs network is shifted toward an atypical plasticity state in relevant cortical areas compatible with the sensory-motor dysfunctions characteristics of RTT.

Rett syndrome (RTT) is caused in most cases by loss-of-function mutations in the X-linked gene encoding methyl CpG-binding protein 2 (

SHANK3 (also called PROSAP2) genetic haploinsufficiency is thought to be the major cause of neuropsychiatric symptoms in Phelan-McDermid syndrome (PMS). PMS is a rare genetic disorder that causes a severe form of intellectual disability (ID), expressive language delays and other autistic features. Furthermore, a significant number of SHANK3 mutations have been identified in patients with autism spectrum disorders (ASD), and SHANK3 truncating mutations are associated with moderate to profound ID. The Shank3 protein is a scaffold protein that is located in the postsynaptic density (PSD) of excitatory synapses and is crucial for synapse development and plasticity. In this study, we investigated the molecular mechanisms associated with the ASD-like behaviors observed in Shank3Δ11-/- mice, in which exon 11 has been deleted. Our results indicate that Shank3 is essential to mediating metabotropic glutamate receptor 5 (mGlu5)-receptor signaling by recruiting Homer1b/c to the PSD, specifically in the striatum and cortex. Moreover, augmenting mGlu5-receptor activity by administering 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide ameliorated the functional and behavioral defects that were observed in Shank3Δ11-/- mice, suggesting that pharmaceutical treatments that increase mGlu5 activity may represent a new approach for treating patients that are affected by PMS and SHANK3 mutations.

Stressful events evoke molecular adaptations of neural circuits through chromatin remodeling and regulation of gene expression. However, the identity of the molecular pathways activated by stress in experimental models of depression is not fully understood. We investigated the effect of acute forced swimming (FS) on the phosphorylation of the extracellular signal-regulated kinase (ERK)1/2 (pERK) and histone H3 (pH3) in limbic brain areas of genetic models of vulnerability (RLA, Roman low-avoidance rats) and resistance (RHA, Roman high-avoidance rats) to stress-induced depression-like behavior. We demonstrate that FS markedly increased the density of pERK-positive neurons in the infralimbic (ILCx) and the prelimbic area (PrLCx) of the prefrontal cortex (PFCx), the nucleus accumbens, and the dorsal blade of the hippocampal dentate gyrus to the same extent in RLA and RHA rats. In addition, FS induced a significant increase in the intensity of pERK immunoreactivity (IR) in neurons of the PFCx in both rat lines. However, RHA rats showed stronger pERK-IR than RLA rats in the ILCx both under basal and stressed conditions. Moreover, the density of pH3-positive neurons was equally increased by FS in the PFCx of both rat lines. Interestingly, pH3-IR was higher in RHA than RLA rats in PrLCx and ILCx, either under basal conditions or upon FS. Finally, colocalization analysis showed that in the PFCx of both rat lines, almost all pERK-positive cells express pH3, whereas only 50% of the pH3-positive neurons is also pERK-positive. Moreover, FS increased the percentage of neurons that express exclusively pH3, but reduced the percentage of cells expressing exclusively pERK. These results suggest that (i) the distinctive patterns of FS-induced ERK and H3 phosphorylation in the PFCx of RHA and RLA rats may represent molecular signatures of the behavioural traits that distinguish the two lines and (ii) FS-induced H3 phosphorylation is, at least in part, ERK-independent.

A major challenge in the neuroscience field is the identification of molecules and pathways that control synaptic plasticity and memory. Dendritic spines play a pivotal role in these processes, as the major sites of excitatory synapses in neuronal communication. Previous studies have shown that the scaffold protein p140Cap localizes into dendritic spines and that its knockdown negatively modulates spine shape in culture. However, so far, there is no information on itsin vivorelevance. By using a knock-out mouse model, we here demonstrate that p140Cap is a key element for both learning and synaptic plasticity. Indeed,p140Cap−/−mice are impaired in object recognition test, as well as in LTP and in LTD measurements. Thein vivoeffects of p140Cap loss are presumably attenuated by noncell-autonomous events, since primary neurons obtained fromp140Cap−/−mice show a strong reduction in number of mushroom spines and abnormal organization of synapse-associated F-actin. These phenotypes are most likely caused by a local reduction of the inhibitory control of RhoA and of cortactin toward the actin-depolymerizing factor cofilin. These events can be controlled by p140Cap through its capability to directly inhibit the activation of Src kinase and by its binding to the scaffold protein Citron-N. Altogether, our results provide new insight into how protein associated with dynamic microtubules may regulate spine actin organization through interaction with postsynaptic density components.

Epigenetic changes such as covalent modifications of histone proteins represent complex molecular signatures that provide a cellular memory of previously experienced stimuli without irreversible changes of the genetic code. In this study we show that new gene expression induced in vivo by morphine withdrawal occurs with concomitant epigenetic modifications in brain regions critically involved in drug-dependent behaviors. We found that naloxone-precipitated withdrawal, but not chronic morphine administration, caused a strong induction of phospho-histone H3 immunoreactivity in the nucleus accumbens (NAc) shell/core and in the lateral septum (LS), a change that was accompanied by augmented H3 acetylation (lys14) in neurons of the NAc shell. Morphine withdrawal induced the phosphorylation of the epigenetic factor methyl-CpG-binding protein 2 (MeCP2) in Ser421 both in the LS and the NAc shell. These epigenetic changes were accompanied by the activation of members of the ERK pathway as well as increased expression of the immediate early genes (IEG) c-fos and activity-regulated cytoskeleton-associated protein (Arc/Arg3.1). Using a pharmacological approach, we found that H3 phosphorylation and IEG expression were partially dependent on ERK activation, while MeCP2 phosphorylation was fully ERK-independent. These findings provide new important information on the role of the ERK pathway in the regulation of epigenetic marks and gene expression that may concur to regulate in vivo the cellular changes underlying the onset of the opioid withdrawal syndrome.

Vascular normalizing strategies, aimed at ameliorating blood vessel perfusion and lessening tissue hypoxia, are treatments that may improve the outcome of cancer patients. Secreted class 3 semaphorins (SEMA3), which are thought to directly bind neuropilin (NRP) co-receptors that, in turn, associate with and elicit plexin (PLXN) receptor signaling, are effective normalizing agents of the cancer vasculature. Yet, SEMA3A was also reported to trigger adverse side effects via NRP1. We rationally designed and generated a safe, parenterally deliverable, and NRP1-independent SEMA3A point mutant isoform that, unlike its wild-type counterpart, binds PLXNA4 with nanomolar affinity and has much greater biochemical and biological activities in cultured endothelial cells. In vivo, when parenterally administered in mouse models of pancreatic cancer, the NRP1-independent SEMA3A point mutant successfully normalized the vasculature, inhibited tumor growth, curbed metastatic dissemination, and effectively improved the supply and anticancer activity of chemotherapy. Mutant SEMA3A also inhibited retinal neovascularization in a mouse model of age-related macular degeneration. In summary, mutant SEMA3A is a vascular normalizing agent that can be exploited to treat cancer and, potentially, other diseases characterized by pathological angiogenesis.

International audience; Loss of function mutations in human Oligophrenin1 (OPHN1) gene are responsible for syndromic intellectual disability (ID) associated with cerebellar hypoplasia and cerebral ventricles enlargement. Functional studies in rodent models suggest that OPHN1 linked ID is a consequence of abnormal synaptic transmission and shares common pathophysiological mechanisms with other cognitive disorders. Variants of this gene have been also identified in autism spectrum disorder and schizophrenia. The advanced understanding of the mechanisms underlying OPHN1-related ID, allowed us to develop a therapeutic approach targeting the Ras homolog gene family, member A (RHOA) signalling pathway and repurpose Fasudil-a well-tolerated Rho Kinase (ROCK) and Protein Kinase A (PKA) inhibitor-as a treatment of ID. We have previously shown ex-vivo its beneficial effect on synaptic transmission and plasticity in a mouse model of the OPHN1 loss of function. Here, we report that chronic treatment in adult mouse with Fasudil, is able to counteract vertical and horizontal hyperactivities, restores recognition memory and limits the brain ventricular dilatation observed in Ophn1 À/y. However, deficits in working and spatial memories are partially or not rescued by the treatment. These results highlight the potential of Fasudil treatment in synaptopathies and also the need for multiple therapeutic approaches especially in adult where brain plasticity is reduced. †

The epigenetic changes of the chromatin represent an attractive molecular substrate for adaptation to the environment. We examined here the role of CREB-binding protein (CBP), a histone acetyltransferase involved in mental retardation, in the genesis and maintenance of long-lasting systemic and behavioural adaptations to environmental enrichment (EE). Morphological and behavioural analyses demonstrated that EE ameliorates deficits associated to CBP deficiency. However, CBP-deficient mice also showed a strong defect in environment-induced neurogenesis and impaired EE-mediated enhancement of spatial navigation and pattern separation ability. These defects correlated with an attenuation of the transcriptional programme induced in response to EE and with deficits in histone acetylation at the promoters of EE-regulated, neurogenesis-related genes. Additional experiments in CBP restricted and inducible knockout mice indicated that environment-induced adult neurogenesis is extrinsically regulated by CBP function in mature granule cells. Overall, our experiments demonstrate that the environment alters gene expression by impinging on activities involved in modifying the epigenome and identify CBP-dependent transcriptional neuroadaptation as an important mediator of EE-induced benefits, a finding with important implications for mental retardation therapeutics.

Rett syndrome (RTT) is a neurodevelopmental disorder with no efficient treatment that is caused in the majority of cases by mutations in the gene methyl-CpG binding-protein 2 (MECP2). RTT becomes manifest after a period of apparently normal development and causes growth deceleration, severe psychomotor impairment and mental retardation. Effective animal models for RTT are available and show morphofunctional abnormalities of synaptic connectivity. However, the molecular consequences of MeCP2 disruption leading to neuronal and synaptic alterations are not known. Protein synthesis regulation via the mammalian target of the rapamycin (mTOR) pathway is crucial for synaptic organization, and its disruption is involved in a number of neurodevelopmental diseases. We investigated the phosphorylation of the ribosomal protein (rp) S6, whose activation is highly dependent from mTOR activity. Immunohistochemistry showed that rpS6 phosphorylation is severely affected in neurons across the cortical areas of Mecp2 mutants and that this alteration precedes the severe symptomatic phase of the disease. Moreover, we found a severe defect of the initiation of protein synthesis in the brain of presymptomatic Mecp2 mutant that was not restricted to a specific subset of transcripts. Finally, we provide evidence for a general dysfunction of the Akt/mTOR, but not extracellular-regulated kinase, signaling associated with the disease progression in mutant brains. Our results indicate that defects in the AKT/mTOR pathway are responsible for the altered translational control in Mecp2 mutant neurons and disclosed a novel putative biomarker of the pathological process. Importantly, this study provides a novel context of therapeutic interventions that can be designed to successfully restrain or ameliorate the development of RTT.

Long-term synaptic plasticity requires both gene expression in the nucleus and local protein synthesis at synapses. The effector proteins that link molecular events in the cell body with local maintenance of synaptic strength are not known. We now show that treatment with serotonin (5-HT) that produces long-term facilitation induces the Aplysia eukaryotic translation elongation factor 1α (Ap-eEF1A) as a late gene that might serve this coupling function in sensory neurons. Although the translation factor is induced, it is not transported into axon processes when the stimulation with 5-HT was restricted to the cell body. In contrast, its mRNA is transported when 5-HT was applied to both cell body and synapses. Intracellular injection of antisense oligonucleotides or antibodies that block the induction and expression of Ap-eEF1A do not affect the initial expression of long-term facilitation but do block its maintenance beyond 24 h. The transport of eEF1A protein and its mRNA to nerve terminals suggests that the translation factor plays a role in the local protein synthesis that is essential for maintaining newly formed synapses.

Excitatory and inhibitory inputs converge on single neurons and are integrated into a coherent output. Although much is known about short-term integration, little is known about how neurons sum opposing signals for long-term synaptic plasticity and memory storage. In Aplysia, we find that when a sensory neuron simultaneously receives inputs from the facilitatory transmitter 5-HT at one set of synapses and the inhibitory transmitter FMRFamide at another, long-term facilitation is blocked and synapse-specific long-term depression dominates. Chromatin immunoprecipitation assays show that 5-HT induces the downstream gene C/EBP by activating CREB1, which recruits CBP for histone acetylation, whereas FMRFa leads to CREB1 displacement by CREB2 and recruitment of HDAC5 to deacetylate histones. When the two transmitters are applied together, facilitation is blocked because CREB2 and HDAC5 displace CREB1-CBP, thereby deacetylating histones.

Microglia are highly motile phagocytic cells that infiltrate and take up residence in the developing brain, where they are thought to provide a surveillance and scavenging function. However, although microglia have been shown to engulf and clear damaged cellular debris after brain insult, it remains less clear what role microglia play in the uninjured brain. Here, we show that microglia actively engulf synaptic material and play a major role in synaptic pruning during postnatal development in mice. These findings link microglia surveillance to synaptic maturation and suggest that deficits in microglia function may contribute to synaptic abnormalities seen in some neurodevelopmental disorders.

Mutations in cyclin-dependent kinase-like 5 (CDKL5) cause early-onset epileptic encephalopathy, a neurodevelopmental disorder with similarities to Rett Syndrome. Here we describe the physiological, molecular, and behavioral phenotyping of a Cdkl5 conditional knockout mouse model of CDKL5 disorder. Behavioral analysis of constitutive Cdkl5 knockout mice revealed key features of the human disorder, including limb clasping, hypoactivity, and abnormal eye tracking. Anatomical, physiological, and molecular analysis of the knockout uncovered potential pathological substrates of the disorder, including reduced dendritic arborization of cortical neurons, abnormal electroencephalograph (EEG) responses to convulsant treatment, decreased visual evoked responses (VEPs), and alterations in the Akt/rpS6 signaling pathway. Selective knockout of Cdkl5 in excitatory and inhibitory forebrain neurons allowed us to map the behavioral features of the disorder to separable cell-types. These findings identify physiological and molecular deficits in specific forebrain neuron populations as possible pathological substrates in CDKL5 disorder. © 2014 Amendola et al. Mutations in cyclin-dependent kinase-like 5 (CDKL5) cause early-onset epileptic encephalopathy, a neurodevelopmental disorder with similarities to Rett Syndrome. Here we describe the physiological, molecular, and behavioral phenotyping of a Cdkl5 conditional knockout mouse model of CDKL5 disorder. Behavioral analysis of constitutive Cdkl5 knockout mice revealed key features of the human disorder, including limb clasping, hypoactivity, and abnormal eye tracking. Anatomical, physiological, and molecular analysis of the knockout uncovered potential pathological substrates of the disorder, including reduced dendritic arborization of cortical neurons, abnormal electroencephalograph (EEG) responses to convulsant treatment, decreased visual evoked responses (VEPs), and alterations in the Akt/rpS6 signaling pathway. Selective knockout of Cdkl5 in excitatory and inhibitory forebrain neurons allowed us to map the behavioral features of the disorder to separable cell-types. These findings identify physiological and molecular deficits in specific forebrain neuron populations as possible pathological substrates in CDKL5 disorder.

The synaptic organization of the accessory olfactory bulb (AOB) was studied in the rat with antibodies against the excitatory neurotransmitter glutamate (Glu) and the inhibitory neurotransmitter γ-aminobutyric acid (GABA). To a large extent, the immunoreactivity patterns produced by the two antibodies were complementary. Glu-like immunoreactivity (-LI) was observed in the glomerular neuropil, in the mitral cells, and in large neurons located in the periglomerular region. Immunogold electron microscopy revealed particularly high levels of Glu-LI in the axon terminals of vomeronasal neurons. GABA-LI was present in granule and periglomerular cells and in their processes. The dendritic spines of granule cells, which were presynaptic to mitral cells, were strongly labelled by the antiserum against GABA. Labelling of serial semithin sections showed that the GABA-positive and Glu-positive neurons of the periglomerular region are generally distinct, and colocalization of Glu and GABA occurred only in a few cells. These results are consistent with electrophysiological studies indicating that the synaptic organization of the AOB is similar to that of the main olfactory bulb. In both systems, Glu is the neurotransmitter used by primary afferents and output neurons, whereas GABA is involved in the circuits underlying lateral and feed-back inhibition. J. Comp. Neurol. 408:61–72, 1999. © 1999 Wiley-Liss, Inc.

The tubulin-binding protein gephyrin is essential for the formation of postsynaptic glycine-receptor clusters in cultured spinal neurons. In addition, there is increasing evidence that gephyrin can also be present at nonglycinergic synapses. Here we analyzed immunocytochemically the subcellular localization of gephyrin in the main olfactory bulb of the rat and compared its distribution with that of gamma-aminobutyric acid (GABA) and of two major GABA(A)-receptor subunits. Gephyrin was selectively localized to the postsynaptic side of symmetric synaptic junctions, where the presynaptic terminals contained GABA. Moreover, gephyrin colocalized extensively with the alpha1 and gamma2 subunits of the GABA(A) receptor. In contrast, gephyrin was not detected at presumed glutamatergic synapses. These results indicate that gephyrin is not uniquely associated with glycine receptors, but can also be found at distinct GABAergic synapses. Thus, they raise the possibility that gephyrin is involved in anchoring certain GABA(A)-receptor subtypes in the postsynaptic membrane.

It is well-established that neuronal intracellular signaling governed by the extracellular signal-regulated kinase (ERK/MAPK) plays a crucial role in long-term adaptive changes that occur during cognitive processes. ERK is a downstream component of a conserved signaling module that is activated by the serine/threonine kinase, Raf, which activates the MAPK/ERK kinase (MEK)1/2 protein kinases, which, in turn, activate ERK1/2. This signaling pathway has been reported to be activated in numerous physiological conditions due to a variety of stimuli, ranging from the activation of ionotropic glutamatergic receptors to metabotropic dopaminergic receptors and neurotrophin receptors. Interestingly, activated ERK can have early and late downstream effects at both the nuclear and synaptic levels. Locally, ERK signaling results in transient changes in the efficacy of synaptic transmission by modifying both pre- and post-synaptic targets. Once translocated into the nucleus, ERK signaling may control transcription by targeting several different regulators of gene expression such as transcription factors and histone proteins. ERK function is considered fundamental in processes such as long-term memory storage and drug addiction, by means of its role in activity-dependent epigenetic modifications that occur in the brain. In this review, we summarize the current understanding of ERK action in the neuroepigenetic processes underlying physiological responses, cognitive processes and drug addiction. This article is part of the Special Issue entitled ‘Neuroepigenetic Disorders’.

In this study, we analysed the molecular heterogeneity and synaptic localization of the N-methyl-D-aspartate receptor subunit 1 and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit 1 in the olfactory bulb glomerular synaptic circuitry. Semiquantitative reverse transcriptase polymerase chain reaction showed that approximately 40% of the N-methyl-D-aspartate receptor subunit 1 messenger RNA splice variants contain the N1 exon, which conveys specific functional properties on the channel. In other forebrain and hindbrain regions that we examined, the ratio of the N1-containing (receptor subunit 1(1XX)) to N1-lacking (receptor subunit 1(0XX)) N-methyl-D-aspartate receptor subunit 1 messenger RNAs varied considerably. The cellular and subcellular distribution of N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1 was investigated with antibodies generated against the C-terminal domain of the individual subunits [Petralia R. S. et al. (1994) J. Neurosci. 14, 667 696; Wenthold R. J. et al. (1992) J. biol Chem. 267, 501 507]. Both N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1 were localized to the postsynaptic density of asymmetric synapses established by olfactory receptor neuron terminals with the dendrites of mitral and tufted cells. Not all of these synapses, however, were labelled. These results are consistent with the notion that glutamate is the neurotransmitter at the olfactory nerve to mitral and tufted cell synapses, and suggest a high heterogeneity in the expression of the postsynaptic glutamate receptors.

In September of 2011, the National Institute of Neurological Disorders and Stroke (NINDS), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), the International Rett Syndrome Foundation (IRSF) and the Rett Syndrome Research Trust (RSRT) convened a workshop involving a broad cross-section of basic scientists, clinicians and representatives from the National Institutes of Health (NIH), the US Food and Drug Administration (FDA), the pharmaceutical industry and private foundations to assess the state of the art in animal studies of Rett syndrome (RTT). The aim of the workshop was to identify crucial knowledge gaps and to suggest scientific priorities and best practices for the use of animal models in preclinical evaluation of potential new RTT therapeutics. This review summarizes outcomes from the workshop and extensive follow-up discussions among participants, and includes: (1) a comprehensive summary of the physiological and behavioral phenotypes of RTT mouse models to date, and areas in which further phenotypic analyses are required to enhance the utility of these models for translational studies; (2) discussion of the impact of genetic differences among mouse models, and methodological differences among laboratories, on the expression and analysis, respectively, of phenotypic traits; and (3) definitions of the standards that the community of RTT researchers can implement for rigorous preclinical study design and transparent reporting to ensure that decisions to initiate costly clinical trials are grounded in reliable preclinical data.