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PCDH19 encodes for protocadherin-19 (PCDH19), a cell-adhesion molecule of the cadherin superfamily preferentially expressed in the brain. PCDH19 mutations cause a neurodevelopmental syndrome named epileptic encephalopathy, early infantile, 9 (EIEE9) characterized by seizures associated with cognitive and behavioral deficits. We recently reported that PCDH19 binds the alpha subunits of GABAA receptors (GABAARs), modulating their surface availability and miniature inhibitory postsynaptic currents (mIPSCs). Here, we investigated whether PCDH19 regulatory function on GABAARs extends to the extrasynaptic receptor pool that mediates tonic current. In fact, the latter shapes neuronal excitability and network properties at the base of information processing. By combining patch-clamp recordings in whole-cell and cell-attached configurations, we provided a functional characterization of primary hippocampal neurons from embryonic rats of either sex expressing a specific PCDH19 short hairpin (sh)RNA. We first demonstrated that PCDH19 downregulation reduces GABAAR-mediated tonic current, evaluated by current shift and baseline noise analysis. Next, by single-channel recordings, we showed that PCDH19 regulates GABAARs kinetics without altering their conductance. In particular, GABAARs of shRNA-expressing neurons preferentially exhibit brief openings at the expense of long ones, thus displaying a flickering behavior. Finally, we showed that PCDH19 downregulation reduces the rheobase and increases the frequency of action potential firing, thus indicating neuronal hyperexcitability. These findings establish PCDH19 as a critical determinant of GABAAR-mediated tonic transmission and GABAARs gating, and provide the first mechanistic insights into PCDH19-related hyperexcitability and comorbidities.

A disintegrin and metalloproteinase 10 (ADAM10) is a synaptic enzyme that has been previously shown to limit amyloid-β1–42 (Aβ1–42) peptide formation in Alzheimer’s disease (AD). Furthermore, ADAM10 participates to spine shaping through the cleavage of adhesion molecules and its activity is under the control of synaptic plasticity events. In particular, long-term depression (LTD) promotes ADAM10 synaptic localization triggering its forward trafficking to the synapse, while long-term potentiation elicits ADAM10 internalization. Here, we show that a short-term in vitro exposure to Aβ1–42 oligomers, at a concentration capable of inducing synaptic depression and spine loss, triggers an increase in ADAM10 synaptic localization in hippocampal neuronal cultures. However, the Aβ1–42 oligomers-induced synaptic depression does not foster ADAM10 delivery to the synapse, as the physiological LTD, but impairs ADAM10 endocytosis. Moreover, Aβ1–42 oligomers-induced inhibition of ADAM10 internalization requires neuronal activity and the activation of the NMDA receptors. These data suggest that, at the synaptic level, Aβ1–42 oligomers trigger an aberrant plasticity mechanism according to which Aβ1–42 oligomers can downregulate Aβ generation through the modulation of ADAM10 synaptic availability. Moreover, the increased activity of ADAM10 towards its synaptic substrates could also affect the structural plasticity phenomena. Overall, these data shed new lights on the strict and complex relationship existing between synaptic activity and the primary mechanisms of AD pathogenesis. Electronic supplementary material The online version of this article (10.1007/s12035-019-1583-5) contains supplementary material, which is available to authorized users.

Cyclase-associated protein (CAP) has been discovered three decades ago in budding yeast as a protein that associates with the cyclic adenosine monophosphate (cAMP)-producing adenylyl cyclase and that suppresses a hyperactive RAS2 variant. Since that time, CAP has been identified in all eukaryotic species examined and it became evident that the activity in RAS-cAMP signaling is restricted to a limited number of species. Instead, its actin binding activity is conserved among eukaryotes and actin cytoskeleton regulation emerged as its primary function. However, for many years, the molecular functions as well as the developmental and physiological relevance of CAP remained unknown. In the present article, we will compile important recent progress on its molecular functions that identified CAP as a novel key regulator of actin dynamics, i.e., the spatiotemporally controlled assembly and disassembly of actin filaments (F-actin). These studies unraveled a cooperation with ADF/Cofilin and Twinfilin in F-actin disassembly, a nucleotide exchange activity on globular actin monomers (G-actin) that is required for F-actin assembly and an inhibitory function towards the F-actin assembly factor INF2. Moreover, by focusing on selected model organisms, we will review current literature on its developmental and physiological functions, and we will present studies implicating CAP in human pathologies. Together, this review article summarizes and discusses recent achievements in understanding the molecular, developmental and physiological functions of CAP, which led this protein emerge as a novel CAPt'n of actin dynamics.

Cofilin is one of the major regulators of actin dynamics in spines where it is required for structural synaptic plasticity. However, our knowledge of the mechanisms controlling Cofilin activity in spines remains still fragmented. Here, we describe the cyclase-associated protein 2 (CAP2) as a novel master regulator of Cofilin localization in spines. The formation of CAP2 dimers through its Cys32 is important for CAP2 binding to Cofilin and for normal spine actin turnover. The Cys32-dependent CAP2 homodimerization and association to Cofilin are triggered by long-term potentiation (LTP) and are required for LTP-induced Cofilin translocation into spines, spine remodeling and the potentiation of synaptic transmission. This mechanism is specifically affected in the hippocampus, but not in the superior frontal gyrus, of both Alzheimer’s Disease (AD) patients and APP/PS1 mice, where CAP2 is down-regulated and CAP2 dimer synaptic levels are reduced. In AD hippocampi, Cofilin preferentially associates with CAP2 monomer and is aberrantly localized in spines. Taken together, these results provide novel insights into structural plasticity mechanisms that are defective in AD.

Background Recent studies support the therapeutic utility of repetitive transcranial magnetic stimulation in Parkinson's disease (PD), whose progression is correlated with loss of corticostriatal long-term potentiation and long-term depression. Glial cell activation is also a feature of PD that is gaining increasing attention in the field because astrocytes play a role in chronic neuroinflammatory responses but are also able to manage dopamine (DA) levels. Methods Intermittent theta-burst stimulation protocol was applied to study the effect of therapeutic neuromodulation on striatal DA levels measured by means of in vivo microdialysis in 6-hydroxydopamine-hemilesioned rats. Effects on corticostriatal synaptic plasticity were studied through in vitro intracellular and whole-cell patch clamp recordings while stepping test and CatWalk were used to test motor behavior. Immunohistochemical analyses were performed to analyze morphological changes in neurons and glial cells. Results Acute theta-burst stimulation induced an increase in striatal DA levels in hemiparkinsonian rats, 80 minutes post-treatment, correlated with full recovery of plasticity and amelioration of motor performances. With the same timing, immediate early gene activation was restricted to striatal spiny neurons. Intense astrocytic and microglial responses were also significantly reduced 80 minutes following theta-burst stimulation. Conclusion Taken together, these results provide a first glimpse on physiological adaptations that occur in the parkinsonian striatum following intermittent theta-burst stimulation and may help to disclose the real potential of this technique in treating PD and preventing DA replacement therapy-associated disturbances. © 2017 International Parkinson and Movement Disorder Society.

In this paper, we designed, synthesized and tested a small set of three new derivatives potentially targeting the D3R-nAChR heteromer, a receptor complex recently identified and characterized as the molecular entity that, in dopaminergic neurons, mediates the neurotrophic effects of nicotine. By means of a partially rigidified spacer of variable length, we incorporated in the new compounds (1a-c) the pharmacophoric substructure of a known β2-subunit-containing nAChR agonist (A-84543) and that of the D2/D3R agonist drug ropinirole. All the compounds retained the ability to bind with high affinity both β2-subunit-containing nAChR and D3R. Compound 1a, renamed HyNDA-1, which is characterized by the shortest linker moiety, was the most interesting ligand. We found, in fact, that HyNDA-1 significantly modulated structural plasticity on both mice and human dopaminergic neurons, an effect strongly prevented by co-incubating this ligand with either nAChR or D3R antagonists. Moreover, the neurotrophic effects of HyNDA-1 were specifically lost by disrupting the complex with specific interfering peptides. Interestingly, by using the Bioluminescence Resonance Energy Transfer 2 (BRET2) assay in HEK-293 transfected cells, we also found that HyNDA-1 has the ability to increase the affinity of interaction between nAChR and D3R. Overall, our results indicate that the neurotrophic effects of HyNDA-1 are mediated by activation of the D3R-nAChR heteromeric complex specifically expressed on dopaminergic neurons.

Alzheimer’s disease (AD) represents a global burden in the economics of healthcare systems. Amyloid-β (Aβ) peptides are formed by amyloid-β precursor protein (AβPP) cleavage, which can be processed by two pathways. The cleavage by the α-secretase A Disintegrin And Metalloprotease 10 (ADAM10) releases the soluble portion (sAβPPα) and prevents senile plaques. This pathway remains largely unknown and ignored, mainly regarding pharmacological approaches that may act via different signaling cascades and thus stimulate non-amyloidogenic cleavage through ADAM10. This review emphasizes the effects of natural compounds on ADAM10 modulation, which eventuates in a neuroprotective mechanism. Moreover, ADAM10 as an AD biomarker is revised. New treatments and preventive interventions targeting ADAM10 regulation for AD are necessary, considering the wide variety of ADAM10 substrates.

Actin-based remodeling underlines spine morphogenesis and plasticity and is crucially involved in the processes that constantly reshape the circuitry of the adult brain in response to external stimuli, leading to learning and memory formation and supporting cognitive functions. Hence spine morphology and synaptic strength are tightly linked and indeed abnormalities in spine number and morphology have been described in a number of neurological disorders such as autism spectrum disorders (ASDs), schizophrenia and intellectual disabilities. We have recently demonstrated that the actin regulating protein, Epidermal growth factor receptor pathway substrate 8 (Eps8), is essential for spine growth and long term potentiation. Indeed, mice lacking Eps8 display immature filopodia-like spines, which are unable to undergo potentiation, and are impaired in cognitive functions. Furthermore, reduced levels of Eps8 have been found in the brain of a cohort of patients affected by ASD compared to controls. Here we investigated whether the lack of Eps8, which is also part of the N-methyl-d-aspartate (NMDA) receptor complex, affects the functional maturation of the postsynaptic compartment. Our results demonstrate that Eps8 knock out mice (Eps8 KO) neurons display altered synaptic expression and subunit composition of NMDA receptors (i.e., increased GluN2B-, decreased GluN2A-containing receptors) and impaired GluN2B to GluN2A subunit shift. Indeed Eps8 KO neurons display increased content of GluN2B containing NMDA receptors both at the synaptic and extrasynaptic level. Furthermore, Eps8 KO neurons display an increased content of extra-synaptic GluN2B-containing receptors, suggesting that also the synaptic targeting of NMDA receptors is affected by the lack of Eps8. These data demonstrate that, besides regulation of spine morphogenesis, Eps8 also regulates the synaptic balance of NMDA receptors subunits GluN2A and GluN2B.

In the central nervous system a disintegrin and metalloproteinase 10 (ADAM10) controls several functions such as neurodevelopment, synaptic plasticity and dendritic spine morphology thanks to its activity towards a high number of substrates, including the synaptic cell adhesion molecules as the Amyloid Precursor Protein, N-cadherin, Notch and Ephrins. In particular, ADAM10 plays a key role in the modulation of the molecular mechanisms responsible for dendritic spine formation, maturation and stabilization and in the regulation of the molecular organization of the glutamatergic synapse. Consequently, an alteration of ADAM10 activity is strictly correlated to the onset of different types of synaptopathies, ranging from neurodevelopmental disorders, i.e. autism spectrum disorders, to neurodegenerative diseases, i.e. Alzheimer's Disease. Areas covered: We describe the most recent discoveries in understanding of the role of ADAM10 activity at the glutamatergic excitatory synapse and its involvement in the onset of neurodevelopmental and neurodegenerative disorders. Expert opinion: A progress in the understanding of the molecular mechanisms driving ADAM10 activity at synapses and its alterations in brain disorders is the first step before designing a specific drug able to modulate ADAM10 activity.

Fingolimod, also known as FTY720, is an analogue of the sphingolipid sphingosine, which has been proved to be neuroprotective in rodent models of Alzheimer’s disease (AD). Several cellular and molecular targets underlying the neuroprotective effects of FTY720 have been recently identified. However, whether the drug directly protects neurons from toxicity of amyloid-beta (Aβ) still remains poorly defined. Using a combination of biochemical assays, live imaging and electrophysiology we demonstrate that FTY720 induces a rapid increase in GLUN2A-containing neuroprotective NMDARs on the surface of dendritic spines in cultured hippocampal neurons. In addition, the drug mobilizes extrasynaptic GLUN2B-containing NMDARs, which are coupled to cell death, to the synapses. Altered ratio of synaptic/extrasynaptic NMDARs decreases calcium responsiveness of neurons to neurotoxic soluble Aβ 1–42 and renders neurons resistant to early alteration of calcium homeostasis. The fast defensive response of FTY720 occurs through a Sphingosine-1-phosphate receptor (S1P-R) -dependent mechanism, as it is lost in the presence of S1P-R1 and S1P-R3 antagonists. We propose that rapid synaptic relocation of NMDARs might have direct impact on amelioration of cognitive performance in transgenic APPswe/PS1dE9 AD mice upon sub-chronic treatment with FTY720.

NMDA receptor (NMDAR) composition and synaptic retention represent pivotal features in the physiology and pathology of excitatory synapses. Here, we identify Rabphilin 3A (Rph3A) as a new GluN2A subunit-binding partner. Rph3A is known as a synaptic vesicle-associated protein involved in the regulation of exo- and endocytosis processes at presynaptic sites. We find that Rph3A is enriched at dendritic spines. Protein–protein interaction assays reveals that Rph3A N-terminal domain interacts with GluN2A(1349–1389) as well as with PSD-95(PDZ3) domains, creating a ternary complex. Rph3A silencing in neurons reduces the surface localization of synaptic GluN2A and NMDAR currents. Moreover, perturbing GluN2A/Rph3A interaction with interfering peptides in organotypic slices or in vivo induces a decrease of the amplitude of NMDAR-mediated currents and GluN2A density at dendritic spines. In conclusion, Rph3A interacts with GluN2A and PSD-95 forming a complex that regulates NMDARs stabilization at postsynaptic membranes., Rabphilin 3A is a synaptic vesicle-associated protein involved in membrane trafficking at presynaptic sites. Here Stanic et al. show that Rabphilin 3A binds to the NMDA receptor GluN2A and stabilises its surface localisation at postsynaptic sites in dendritic spines.

International audience; A disintegrin and metalloproteinase 10 (ADAM10), a disintegrin and metalloproteinase that resides in the postsynaptic densities (PSDs) of excitatory synapses, has previously been shown to limit β-amyloid peptide (Aβ) formation in Alzheimer's disease (AD). ADAM10 also plays a critical role in regulating functional membrane proteins at the synapse. Using human hippocampal homogenates, we found that ADAM10 removal from the plasma membrane was mediated by clathrin-dependent endocytosis. Additionally, we identified the clathrin adaptor AP2 as an interacting partner of a previously uncharacterized atypical binding motif in the ADAM10 C-terminal domain. This domain was required for ADAM10 endocytosis and modulation of its plasma membrane levels. We found that the ADAM10/AP2 association was increased in the hippocampi of AD patients compared with healthy controls. Long-term potentiation (LTP) in hippocampal neuronal cultures induced ADAM10 endocytosis through AP2 association and decreased surface ADAM10 levels and activity. Conversely, long-term depression (LTD) promoted ADAM10 synaptic membrane insertion and stimulated its activity. ADAM10 interaction with the synapse-associated protein-97 (SAP97) was necessary for LTD-induced ADAM10 trafficking and required for LTD maintenance and LTD-induced changes in spine morphogenesis. These data identify and characterize a mechanism controlling ADAM10 localization and activity at excitatory synapses that is relevant to AD pathogenesis.