Tks5 Regulates Synaptic Podosome Formation and Stabilization of the Postsynaptic Machinery at the Neuromuscular Junction.

Currently, the etiology of many neuromuscular disorders remains unknown. Many of them are characterized by aberrations in the maturation of the neuromuscular junction (NMJ) postsynaptic machinery. Unfortunately, the molecular factors involved in this process are still largely unknown, which poses a great challenge for identifying potential therapeutic targets. Here, we identified Tks5 as a novel interactor of αdystrobrevin-1, which is a crucial component of the NMJ postsynaptic machinery. Tks5 has been previously shown in cancer cells to be an important regulator of actin-rich structures known as invadosomes. However, a role of this scaffold protein at a synapse has never been studied. We show that Tks5 is crucial for remodeling of the NMJ postsynaptic machinery by regulating the organization of structures similar to the invadosomes, known as synaptic podosomes. Additionally, it is involved in the maintenance of the integrity of acetylcholine receptor (AChR) clusters and regulation of their turnover. Lastly, our data indicate that these Tks5 functions may be mediated by its involvement in recruitment of actin filaments to the postsynaptic machinery. Collectively, we show for the first time that the Tks5 protein is involved in regulation of the postsynaptic machinery.

Modulation of glutamatergic functional connectivity by a prototypical antipsychotic: Translational inference from a postsynaptic density immediate-early gene-based network analysis.

BACKGROUND: Although extensively studied, the effect of antipsychotics is not completely understood at a network level. We tested the hypothesis that acute administration of haloperidol would modulate functional connectivity of brain regions relevant to schizophrenia pathophysiology. To assess putative changes in brain network properties and regional interactivity, we studied the expression of Homer1a, an Immediate Early Gene (IEG) demonstrated to be induced by antipsychotic administration and coding for a protein involved in glutamatergic synapses remodeling. METHODS: Sprague-Dawley rats (n = 26) assigned to vehicle (VEH; NaCl 0.9%) or haloperidol (HAL; 0.8 mg/kg) were included in the network analysis. Homer1a mRNA induction was evaluated by in situ hybridization. Signal intensity analysis was performed in 33 Regions of Interest (ROIs) in the cortex, the caudate putamen, and the nucleus accumbens. A signal correlation analysis was performed, computing all possible pairwise Pearson correlations among ROIs in the two groups. Two networks were generated for HAL and VEH groups, and their properties and topography were explored. RESULTS: VEH and HAL networks showed qualitative differences in global efficiency and clustering coefficient. The HAL network showed enhanced interactivity between cortical and striatal regions, and within caudate putamen subdivisions. On the other hand, it exhibited reduced inter-correlations between cingulate cortex and anterior insula and caudate putamen and nucleus accumbens. Moreover, haloperidol was able to modulate centrality of crucial functional hubs. These preclinical results corroborate and expand the clinical evidence that antipsychotics may modulate specific brain network properties and disease-related circuits' interactivity.

Small noncoding vault RNA modulates synapse formation by amplifying MAPK signaling.

The small noncoding vault RNA (vtRNA) is a component of the vault complex, a ribonucleoprotein complex found in most eukaryotes. Emerging evidence suggests that vtRNAs may be involved in the regulation of a variety of cellular functions when unassociated with the vault complex. Here, we demonstrate a novel role for vtRNA in synaptogenesis. Using an in vitro synapse formation model, we show that murine vtRNA (mvtRNA) promotes synapse formation by modulating the MAPK signaling pathway. mvtRNA is transported to the distal region of neurites as part of the vault complex. Interestingly, mvtRNA is released from the vault complex in the neurite by a mitotic kinase Aurora-A-dependent phosphorylation of MVP, a major protein component of the vault complex. mvtRNA binds to and activates MEK1 and thereby enhances MEK1-mediated ERK activation in neurites. These results suggest the existence of a regulatory mechanism of the MAPK signaling pathway by vtRNAs as a new molecular basis for synapse formation.

Neonatal Isoflurane Anesthesia or Disruption of Postsynaptic Density-95 Protein Interactions Change Dendritic Spine Densities and Cognitive Function in Juvenile Mice.

BACKGROUND: Experimental evidence shows postnatal exposure to anesthesia negatively affects brain development. The PDZ2 domain, mediating protein-protein interactions of the postsynaptic density-95 protein, serves as a molecular target for several inhaled anesthetics. The authors hypothesized that early postnatal disruption of postsynaptic density-95 PDZ2 domain interactions has persistent effects on dendritic spines and cognitive function. METHODS: One-week-old mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active postsynaptic density-95 wild-type PDZ2 peptide along with their respective controls. A subset of these mice also received 4 mg/kg of the nitric oxide donor molsidomine. Hippocampal spine density, long-term potentiation, novel object recognition memory, and fear learning and memory were evaluated in mice. RESULTS: Exposure of 7-day-old mice to isoflurane or postsynaptic density-95 wild-type PDZ2 peptide relative to controls causes: (1) a long-term decrease in mushroom spines at 7 weeks (mean ± SD [spines per micrometer]): control (0.8 ± 0.2) versus isoflurane (0.4 ± 0.2), P < 0.0001, and PDZ2MUT (0.7 ± 0.2) versus PDZ2WT (0.4 ± 0.2), P < 0.001; (2) deficits in object recognition at 6 weeks (mean ± SD [recognition index]): naïve (70 ± 8) versus isoflurane (55 ± 14), P = 0.010, and control (65 ± 13) versus isoflurane (55 ± 14), P = 0.045, and PDZ2MUT (64 ±11) versus PDZ2WT (53 ± 18), P = 0.045; and (3) deficits in fear learning at 7 weeks and memory at 8 weeks (mean ± SD [% freezing duration]): Learning, control (69 ± 12) versus isoflurane (52 ± 13), P < 0.0001, and PDZ2MUT (65 ± 14) versus PDZ2WT (55 ± 14) P = 0.011, and Memory, control (80 ± 17) versus isoflurane (56 ± 23), P < 0.0001 and PDZ2MUT (73 ± 18) versus PDZ2WT (44 ± 19) P < 0.0001. Impairment in long-term potentiation has fully recovered here at 7 weeks (mean ± SD [% baseline]): control (140 ± 3) versus isoflurane (137 ± 8), P = 0.560, and PDZ2MUT (136 ± 17) versus PDZ2WT (128 ± 11), P = 0.512. The isoflurane induced decrease in mushroom spines was preventable by introduction of a nitric oxide donor. CONCLUSIONS: Early disruption of PDZ2 domain-mediated protein-protein interactions mimics isoflurane in decreasing mushroom spine density and causing learning and memory deficits in mice. Prevention of the decrease in mushroom spine density with a nitric oxide donor supports a role for neuronal nitric oxide synthase pathway in mediating this cellular change associated with cognitive impairment.

The Effects of Antipsychotics on the Synaptic Plasticity Gene Homer1a Depend on a Combination of Their Receptor Profile, Dose, Duration of Treatment, and Brain Regions Targeted.

BACKGROUND: Antipsychotic agents modulate key molecules of the postsynaptic density (PSD), including the Homer1a gene, implicated in dendritic spine architecture. How the antipsychotic receptor profile, dose, and duration of administration may influence synaptic plasticity and the Homer1a pattern of expression is yet to be determined. METHODS: In situ hybridization for Homer1a was performed on rat tissue sections from cortical and striatal regions of interest (ROI) after acute or chronic administration of three antipsychotics with divergent receptor profile: Haloperidol, asenapine, and olanzapine. Univariate and multivariate analyses of the effects of topography, treatment, dose, and duration of antipsychotic administration were performed. RESULTS: All acute treatment regimens were found to induce a consistently higher expression of Homer1a compared to chronic ones. Haloperidol increased Homer1a expression compared to olanzapine in striatum at the acute time-point. A dose effect was also observed for acute administration of haloperidol. CONCLUSIONS: Biological effects of antipsychotics on Homer1a varied strongly depending on the combination of their receptor profile, dose, duration of administration, and throughout the different brain regions. These molecular data may have translational valence and may reflect behavioral sensitization/tolerance phenomena observed with prolonged antipsychotics.

Activity-dependent redistribution of CaMKII in the postsynaptic compartment of hippocampal neurons.

Calcium/calmodulin-dependent protein kinase II (CaMKII), an abundant protein in neurons, is involved in synaptic plasticity and learning. CaMKII associates with multiple proteins located at or near the postsynaptic density (PSD), and CaMKII is known to translocate from cytoplasm to PSD under excitatory conditions. The present study examined the laminar distribution of CaMKII at the PSD by immunogold labeling in dissociated hippocampal cultures under low calcium (EGTA or APV), control, and stimulated (depolarization with high K+ or NMDA) conditions. The patterns of CaMKII distribution are classified with particular reference to the two layers of the PSD: (1) the PSD core, a layer within ~ 30-40 nm to the postsynaptic membrane, and (2) the PSD pallium, a deeper layer beyond the PSD core, ~ 100-120 nm from the postsynaptic membrane. Under low calcium conditions, a subpopulation (40%) of synapses stood out with no CaMKII labeling at the PSD, indicating that localization of CaMKII at the PSD is sensitive to calcium levels. Under control conditions, the majority (~ 60-70%) of synapses had label for CaMKII dispersed evenly in the spine, including the PSD and the nearby cytoplasm. Upon stimulation, the majority (60-75%) of synapses had label for CaMKII concentrated at the PSD, delineating the PSD pallium from the cytoplasm. Median distance of label for CaMKII to postsynaptic membrane was higher in low calcium samples (68-77 nm), than in control (59-63 nm) and stimulated samples (49-53 nm). Thus, upon stimulation, not only more CaMKII translocated to the PSD, but they also were closer to the postsynaptic membrane. Additionally, there were two relatively infrequent labeling patterns that may represent intermediate stages of CaMKII distribution between basal and stimulated conditions: (1) one type showed label preferentially localized near the PSD core where CaMKII may be binding to NR2B, an NMDA receptor concentrated at the PSD core, and (2) the second type showed label preferentially in the PSD pallium, where CaMKII may be binding to Shank, a PSD scaffold protein located in the PSD pallium. Both of these distribution patterns may portray the initial stages of CaMKII translocation upon synaptic activation. In addition to binding to PSD proteins, the concentrated CaMKII labeling at the PSD under heightened excitatory conditions could also be formed by self-clustering of CaMKII molecules recruited to the PSD. Most importantly, these accumulated CaMKII molecules do not extend beyond the border of the PSD pallium, and are likely held in the pallium by binding to Shank under these conditions.

Translating preclinical findings in clinically relevant new antipsychotic targets: focus on the glutamatergic postsynaptic density. Implications for treatment resistant schizophrenia.

There is a growing interest in new molecular targets for antipsychotic therapy. Multiple signal transduction systems have been recently implicated in the pathophysiology of schizophrenia. However, the weight of each specific mechanism remains controversial. A need for a more vigorous approach to the pharmacotherapy of schizophrenia arises from the bedside: about 30-40% of patients do not respond to antipsychotic therapy. Postsynaptic Density (PSD) proteins have recently attracted attention for their role in signal transduction modulation and for their potential implication in psychosis and cognition. The involvement of PSD in the pathophysiology of schizophrenia is supported by post mortem studies, preclinical animal models, modulation by antipsychotics, and association of PSD genes with schizophrenia in GWAS. Taken together, these studies underline the role of PSD modulation, its effects on striatal function and its relationship with motor, executive- and cognitive-like functions suggesting a potential role of PSD proteins as a l target of novel intervention in the treatment of refractory psychosis.

Neurobiology and therapeutic utility of neurotoxins targeting postsynaptic mechanisms of neuromuscular transmission.

The neuromuscular junction (NMJ) is the principal site for the translation of motor neurochemical signals to muscle activity. Therefore, the release and sensing machinery of acetylcholine (ACh) along with muscle contraction are two of the main targets of natural toxins and pathogens, causing paralysis. Given pharmacology and medical advances, the active ingredients of toxins that target postsynaptic mechanisms have become of major interest, showing promise as drug leads. Herein, we review key facets of prevalent toxins modulating the mechanisms of ACh sensing and generation of the postsynaptic response, with muscle contraction. We consider the correlation between their outstanding selectivity and potency plus effects on motor function, and discuss emerging data advocating their usage for the development of therapies alleviating neuromuscular dysfunction.

Transgenerational Bisphenol A Causes Deficits in Social Recognition and Alters Postsynaptic Density Genes in Mice.

Bisphenol A (BPA) is a ubiquitous endocrine-disrupting chemical. Developmental exposure produces changes in behavior and gene expression in the brain. Here, we examined social recognition behaviors in mice from the third familial generation (F3) after exposure to gestational BPA. Second-generation mice were bred in one of four mating combinations to reveal whether characteristics in F3 were acquired via maternal or paternal exposures. After repeated habituation to the same mouse, offspring of dams from the BPA lineage failed to display increased investigation of a novel mouse. Genes involved in excitatory postsynaptic densities (PSDs) were examined in F3 brains using quantitative PCR. Differential expression of genes important for function and stability of PSDs were assessed at three developmental ages. Several related PSD genes-SH3 and multiple ankyrin repeat domains 1 (Shank1), Homer scaffolding protein 1c (Homer1c), DLG associated protein 1 (Gkap), and discs large MAGUK scaffold protein 4 (PSD95)-were differentially expressed in control- vs BPA-lineage brains. Using a second strain of F3 inbred mice exposed to BPA, we noted the same differences in Shank1 and PSD95 expression in C57BL/6J mice. In sum, transgenerational BPA exposure disrupted social interactions in mice and dysregulated normal expression of PSD genes during neural development. The fact that the same genetic effects were found in two different mouse strains and in several brain regions increased potential for translation. The genetic and functional relationship between PSD and abnormal neurobehavioral disorders is well established, and our data suggest that BPA may contribute in a transgenerational manner to neurodevelopmental diseases.

Ultra-micronized palmitoylethanolamide rescues the cognitive decline-associated loss of neural plasticity in the neuropathic mouse entorhinal cortex-dentate gyrus pathway.

Chronic pain is associated with cognitive deficits. Palmitoylethanolamide (PEA) has been shown to ameliorate pain and pain-related cognitive impairments by restoring glutamatergic synapses functioning in the spared nerve injury (SNI) of the sciatic nerve in mice. SNI reduced mechanical and thermal threshold, spatial memory and LTP at the lateral entorhinal cortex (LEC)-dentate gyrus (DG) pathway. It decreased also postsynaptic density, volume and dendrite arborization of DG and increased the expression of metabotropic glutamate receptor 1 and 7 (mGluR1 and mGluR7), of the GluR1, GluR1s845 and GluR1s831 subunits of AMPA receptor and the levels of glutamate in the DG. The level of the endocannabinoid 2-arachidonoylglycerol (2-AG) was instead increased in the LEC. Chronic treatment with PEA, starting from when neuropathic pain was fully developed, was able to reverse mechanical allodynia and thermal hyperalgesia, memory deficit and LTP in SNI wild type, but not in PPARα null, mice. PEA also restored the level of glutamate and the expression of phosphorylated GluR1 subunits, postsynaptic density and neurogenesis. Altogether, these results suggest that neuropathic pain negatively affects cognitive behavior and related LTP, glutamatergic synapse and synaptogenesis in the DG. In these conditions PEA treatment alleviates pain and cognitive impairment by restoring LTP and synaptic maladaptative changes in the LEC-DG pathway. These outcomes open new perspectives for the use of the N-acylethanolamines, such as PEA, for the treatment of neuropathic pain and its central behavioural sequelae.

The protective mechanism underlying phenylethanoid glycosides (PHG) actions on synaptic plasticity in rat Alzheimer's disease model induced by beta amyloid 1-42.

Phenylethanoid glycosides (PHG), derived from Herba cistanche, were found to exert protective effects on cognitive dysfunctions by improving synaptic plasticity in Alzheimer's disease (AD) rat model. However, the mechanisms underlying these effects of PHG on synaptic plasticity remain to be determined. Thus the aim of this study was to examine the influence of PHG on synaptic plasticity in male AD rat model induced by bilateral central nervous system ventricle injections of beta amyloid 1-42 oligomers (Aß1-42). The following parameters were measured: (1) number of intact pyramidal cells in hippocampal CA1 region by Nissl staining, (2) post synaptic density 95 (PSD-95), phosphorylated N-methyl-D-aspartate receptor-1(p-NMDAR1) and (3) phosphorylated Tau protein (p-Tau) by immunohistochemistry and western blot. In addition, the content of malondialdehyde (MDA) and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined. Aß1-42 lowered the number of intact pyramidal cells in hippocampal CA1 region. In contrast, treatment with PHG significantly elevated this cell number. Aß1-42 significantly diminished protein expression levels of PSD-95 accompanied by elevated protein expression levels of p-NMDAR1 and p-Tau. PHG markedly increased protein expression levels of PSD-95, but significantly reduced protein expression levels of p-NMDAR1 and p-Tau. Further, Aß1-42 markedly increased MDA content concomitantly with reduced activities of SOD and GSH-Px. PHG significantly decreased MDA content accompanied by elevated activities of SOD and GSH-Px. Data suggest that the protective effects of PHG on synaptic plasticity may involve inhibition of cytotoxicity-mediated by Aß-1-42 administration and reduction of oxidant stress.

Tissue-type plasminogen activator protects the postsynaptic density in the ischemic brain.

Cerebral ischemia causes the presynaptic release of tissue-type plasminogen activator (tPA). The postsynaptic density (PSD) is a postsynaptic structure that provides a matrix where signaling transduction of excitatory synapses takes place. The postsynaptic density protein-95 (PSD-95) is the most abundant scaffolding protein in the postsynaptic density (PSD), where it modulates the postsynaptic response to the presynaptic release of glutamate by regulating the anchoring of glutamate receptors to the PSD. We found that tPA induces the local translation of PSD-95 mRNA and the subsequent recruitment of PSD-95 protein to the PSD, via plasminogen-independent activation of TrkB receptors. Our data show that PSD-95 is removed from the PSD during the early stages of cerebral ischemia, and that this effect is abrogated by either the release of neuronal tPA, or intravenous administration of recombinant tPA (rtPA). We report that the effect of tPA on PSD-95 is associated with inhibition of the phosphorylation and recruitment of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors to the PSD, known to amplify the effect of the excitotoxic injury, and that this is followed by TrkB-mediated protection of dendritic spines from the harmful effects of the hypoxic insult. These data reveal that tPA is a synaptic protector in the ischemic brain.

Nicotine and caffeine modulate haloperidol-induced changes in postsynaptic density transcripts expression: Translational insights in psychosis therapy and treatment resistance.

Caffeine and nicotine are widely used by schizophrenia patients and may worsen psychosis and affect antipsychotic therapies. However, they have also been accounted as augmentation strategies in treatment-resistant schizophrenia. Despite both substances are known to modulate dopamine and glutamate transmission, little is known about the molecular changes induced by these compounds in association to antipsychotics, mostly at the level of the postsynaptic density (PSD), a site of dopamine-glutamate interplay. Here we investigated whether caffeine and nicotine, alone or combined with haloperidol, elicited significant changes in the levels of both transcripts and proteins of the PSD members Homer1 and Arc, which have been implicated in synaptic plasticity, schizophrenia pathophysiology, and antipsychotics molecular action. Homer1a mRNA expression was significantly reduced by caffeine and nicotine, alone or combined with haloperidol, compared to haloperidol. Haloperidol induced significantly higher Arc mRNA levels than both caffeine and caffeine plus haloperidol in the striatum. Arc mRNA expression was significantly higher by nicotine plus haloperidol vs. haloperidol in the cortex, while in striatum gene expression by nicotine was significantly lower than that by both haloperidol and nicotine plus haloperidol. Both Homer1a and Arc protein levels were significantly increased by caffeine, nicotine, and nicotine plus haloperidol. Homer1b mRNA expression was significantly increased by nicotine and nicotine plus haloperidol, while protein levels were unaffected. Locomotor activity was not significantly affected by caffeine, while it was reduced by nicotine. These data indicate that both caffeine and nicotine trigger relevant molecular changes in PSD sites when given in association with haloperidol.

The actions of volatile anesthetics: a new perspective.

This article reviews recent work in applying neutron and X-ray scattering towards the elucidation of the molecular mechanisms of volatile anesthetics. Experimental results on domain mixing in ternary lipid mixtures, and the influence of volatile anesthetics and hydrostatic pressure are placed in the contexts of ion-channel function and receptor trafficking at the postsynaptic density.

Effects of tamoxifen on neuronal morphology, connectivity and biochemistry of hypothalamic ventromedial neurons: Impact on the modulators of sexual behavior.

Tamoxifen (TAM) is a selective estrogen receptor modulator, widely used in the treatment and prevention of estrogen-dependent breast cancer. Although with great clinical results, women on TAM therapy still report several side effects, such as sexual dysfunction, which impairs quality of life. The anatomo-functional substrates of the human sexual behavior are still unknown; however, these same substrates are very well characterized in the rodent female sexual behavior, which has advantage of being a very simple reflexive response, dependent on the activation of estrogen receptors (ERs) in the ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl). In fact, in the female rodent, the sexual behavior is triggered by increasing circulation levels of estradiol that changes the nucleus neurochemistry and modulates its intricate neuronal network. Therefore, we considered of notice the examination of the possible neurochemical alterations and the synaptic plasticity impairment in VMNvl neurons of estradiol-primed female rats treated with TAM that may be in the basis of this neurological disorder. Accordingly, we used stereological and biochemical methods to study the action of TAM in axospinous and axodendritic synaptic plasticity and on ER expression. The administration of TAM changed the VMNvl neurochemistry by reducing ERα mRNA and increasing ERß mRNA expression. Furthermore, present results show that TAM induced neuronal atrophy and reduced synaptic connectivity, favoring electrical inactivity. These data suggest that these cellular and molecular changes may be a possible neuronal mechanism of TAM action in the disruption of the VMNvl network, leading to the development of behavioral disorders.

Heterogeneous Association of Alzheimer's Disease-Linked Amyloid-ß and Amyloid-ß Protein Precursor with Synapses.

Alzheimer's disease (AD) is increasingly viewed as a disease of synapses. Loss of synapses correlates better with cognitive decline than amyloid plaques and neurofibrillary tangles, the hallmark neuropathological lesions of AD. Soluble forms of amyloid-ß (Aß) have emerged as mediators of synapse dysfunction. Aß binds to, accumulates, and aggregates in synapses. However, the anatomical and neurotransmitter specificity of Aß and the amyloid-ß protein precursor (AßPP) in AD remain poorly understood. In addition, the relative roles of Aß and AßPP in the development of AD, at pre- versus post-synaptic compartments and axons versus dendrites, respectively, remain unclear. Here we use immunogold electron microscopy and confocal microscopy to provide evidence for heterogeneity in the localization of Aß/AßPP. We demonstrate that Aß binds to a subset of synapses in cultured neurons, with preferential binding to glutamatergic compared to GABAergic neurons. We also highlight the challenge of defining pre- versus post-synaptic localization of this binding by confocal microscopy. Further, endogenous Aß42 accumulates in both glutamatergic and GABAergic AßPP/PS1 transgenic primary neurons, but at varying levels. Moreover, upon knock-out of presenilin 1 or inhibition of γ-secretase AßPP C-terminal fragments accumulate both pre- and post-synaptically; however earlier pre-synaptically, consistent with a higher rate of AßPP processing in axons. A better understanding of the synaptic and anatomical selectivity of Aß/AßPP in AD can be important for the development of more effective new therapies for this major disease of aging.

Sodium channel subtypes are differentially localized to pre- and post-synaptic sites in rat hippocampus.

Voltage-gated Na+ channels (Nav ) modulate neuronal excitability, but the roles of the various Nav subtypes in specific neuronal functions such as synaptic transmission are unclear. We investigated expression of the three major brain Nav subtypes (Nav 1.1, Nav 1.2, Nav 1.6) in area CA1 and dentate gyrus of rat hippocampus. Using light and electron microscopy, we found labeling for all three Nav subtypes on dendrites, dendritic spines, and axon terminals, but the proportion of pre- and post-synaptic labeling for each subtype varied within and between subregions of CA1 and dentate gyrus. In the central hilus (CH) of the dentate gyrus, Nav 1.1 immunoreactivity was selectively expressed in presynaptic profiles, while Nav 1.2 and Nav 1.6 were expressed both pre- and post-synaptically. In contrast, in the stratum radiatum (SR) of CA1, Nav 1.1, Nav 1.2, and Nav 1.6 were selectively expressed in postsynaptic profiles. We next compared differences in Nav subtype expression between CH and SR axon terminals and between CH and SR dendrites and spines. Nav 1.1 and Nav 1.2 immunoreactivity was preferentially localized to CH axon terminals compared to SR, and in SR dendrites and spines compared to CH. No differences in Nav 1.6 immunoreactivity were found between axon terminals of CH and SR or between dendrites and spines of CH and SR. All Nav subtypes in both CH and SR were preferentially associated with asymmetric synapses rather than symmetric synapses. These findings indicate selective presynaptic and postsynaptic Nav expression in glutamatergic synapses of CH and SR supporting neurotransmitter release and synaptic plasticity.

Rabphilin 3A: A novel target for the treatment of levodopa-induced dyskinesias.

N-methyl-d-aspartate receptor (NMDAR) subunit composition strictly commands receptor function and pharmacological responses. Changes in NMDAR subunit composition have been documented in brain disorders such as Parkinson's disease (PD) and levodopa (L-DOPA)-induced dyskinesias (LIDs), where an increase of NMDAR GluN2A/GluN2B subunit ratio at striatal synapses has been observed. A therapeutic approach aimed at rebalancing NMDAR synaptic composition represents a valuable strategy for PD and LIDs. To this, the comprehension of the molecular mechanisms regulating the synaptic localization of different NMDAR subtypes is required. We have recently demonstrated that Rabphilin 3A (Rph3A) is a new binding partner of NMDARs containing the GluN2A subunit and that it plays a crucial function in the synaptic stabilization of these receptors. Considering that protein-protein interactions govern the synaptic retention of NMDARs, the purpose of this work was to analyse the role of Rph3A and Rph3A/NMDAR complex in PD and LIDs, and to modulate Rph3A/GluN2A interaction to counteract the aberrant motor behaviour associated to chronic L-DOPA administration. Thus, an array of biochemical, immunohistochemical and pharmacological tools together with electron microscopy were applied in this study. Here we found that Rph3A is localized at the striatal postsynaptic density where it interacts with GluN2A. Notably, Rph3A expression at the synapse and its interaction with GluN2A-containing NMDARs were increased in parkinsonian rats displaying a dyskinetic profile. Acute treatment of dyskinetic animals with a cell-permeable peptide able to interfere with Rph3A/GluN2A binding significantly reduced their abnormal motor behaviour. Altogether, our findings indicate that Rph3A activity is linked to the aberrant synaptic localization of GluN2A-expressing NMDARs characterizing LIDs. Thus, we suggest that Rph3A/GluN2A complex could represent an innovative therapeutic target for those pathological conditions where NMDAR composition is significantly altered.

CDKL5 controls postsynaptic localization of GluN2B-containing NMDA receptors in the hippocampus and regulates seizure susceptibility.

Mutations in the Cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders accompanied by intractable epilepsies, i.e. West syndrome or atypical Rett syndrome. Here we report generation of the Cdkl5 knockout mouse and show that CDKL5 controls postsynaptic localization of GluN2B-containing N-methyl-d-aspartate (NMDA) receptors in the hippocampus and regulates seizure susceptibility. Cdkl5 -/Y mice showed normal sensitivity to kainic acid; however, they displayed significant hyperexcitability to NMDA. In concordance with this result, electrophysiological analysis in the hippocampal CA1 region disclosed an increased ratio of NMDA/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (EPSCs) and a significantly larger decay time constant of NMDA receptor-mediated EPSCs (NMDA-EPSCs) as well as a stronger inhibition of the NMDA-EPSCs by the GluN2B-selective antagonist ifenprodil in Cdkl5 -/Y mice. Subcellular fractionation of the hippocampus from Cdkl5 -/Y mice revealed a significant increase of GluN2B and SAP102 in the PSD (postsynaptic density)-1T fraction, without changes in the S1 (post-nuclear) fraction or mRNA transcripts, indicating an intracellular distribution shift of these proteins to the PSD. Immunoelectron microscopic analysis of the hippocampal CA1 region further confirmed postsynaptic overaccumulation of GluN2B and SAP102 in Cdkl5 -/Y mice. Furthermore, ifenprodil abrogated the NMDA-induced hyperexcitability in Cdkl5 -/Y mice, suggesting that upregulation of GluN2B accounts for the enhanced seizure susceptibility. These data indicate that CDKL5 plays an important role in controlling postsynaptic localization of the GluN2B-SAP102 complex in the hippocampus and thereby regulates seizure susceptibility, and that aberrant NMDA receptor-mediated synaptic transmission underlies the pathological mechanisms of the CDKL5 loss-of-function.

Chronic stress-induced dendritic reorganization and abundance of synaptosomal PKA-dependent CP-AMPA receptor in the basolateral amygdala in a mouse model of depression.

Chronic stress is a precipitating factor for disorders including depression. The basolateral amygdala (BLA) is a critical substrate that interconnects with stress-modulated neural networks to generate emotion- and mood-related behaviors. The current study shows that 3 h per day of restraint stress for 14 days caused mice to exhibit long-term depressive behaviors, manifested by disrupted sociality and despair levels, which were rescued by fluoxetine. These behavioral changes corresponded with morphological and molecular changes in BLA neurons, including chronic stress-elicited increases in arborization, dendritic length, and spine density of BLA principal neurons. At the molecular level, calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (CP-AMPARs) within the synaptosome exhibited an increased GluR1:GluR2 subunit ratio. We also observed increased GluR1 phosphorylation at Ser 845 and enhanced cyclic AMP-dependent protein kinase (PKA) activity in the BLA. These molecular changes reverted to the basal state post-treatment with fluoxetine. The expression of synaptophysin (SYP) and postsynaptic density protein 95 (PSD-95) at BLA neuronal synapses was also enhanced by chronic stress, which was reversed post-treatment. Finally, chronic stress-provoked depressive behavior was overcome by local blockage of CP-AMPARs in the BLA via stereotaxic injection (IEM-1460). Chronic stress-elicited depressive behavior may be due to hypertrophy of BLA neuronal dendrites and increased of PKA-dependent CP-AMPAR levels in BLA neurons. Furthermore, fluoxetine can reverse chronic stress-triggered cytoarchitectural and functional changes of BLA neurons. These findings provide insights into depression-linked structural and functional changes in BLA neurons.