Search

Ageing is the main risk factor common to most primary neurodegenerative disorders. Indeed, age-related brain alterations have been long considered to predispose to neurodegeneration. Although protein misfolding and the accumulation of toxic protein aggregates have been contemplated as causative events in neurodegeneration, several biological pathways affected by brain ageing are also contributing to pathogenesis. Here, we discuss the evidence showing the involvement of the mechanisms controlling neuronal structure, gene expression, autophagy, cell metabolism, and neuroinflammation in the onset and progression of neurodegenerative disorders. Furthermore, we review the therapeutic strategies currently under development or as future approaches designed to normalize these pathways, which may then boost brain resilience to cope with toxic protein species. Therefore, in addition to therapies targeting the insoluble protein aggregates specifically associated with each neurodegenerative disorder, these novel pharmacological approaches may be part of combined therapies designed to rescue brain function.

Intranasal (IN) drug delivery is a non-invasive and effective route for the administration of drugs to the brain at pharmacologically relevant concentrations, bypassing the blood–brain barrier (BBB) and minimizing adverse side effects. IN drug delivery can be particularly promising for the treatment of neurodegenerative diseases. The drug delivery mechanism involves the initial drug penetration through the nasal epithelial barrier, followed by drug diffusion in the perivascular or perineural spaces along the olfactory or trigeminal nerves, and final extracellular diffusion throughout the brain. A part of the drug may be lost by drainage through the lymphatic system, while a part may even enter the systemic circulation and reach the brain by crossing the BBB. Alternatively, drugs can be directly transported to the brain by axons of the olfactory nerve. To improve the effectiveness of drug delivery to the brain by the IN route, various types of nanocarriers and hydrogels and their combinations have been proposed. This review paper analyzes the main biomaterials-based strategies to enhance IN drug delivery to the brain, outlining unsolved challenges and proposing ways to address them.

The vast majority of excitatory synapses are formed on small dendritic protrusions termed dendritic spines. Dendritic spines vary in size and density that are crucial determinants of excitatory synaptic transmission. Aberrations in spine morphogenesis can compromise brain function and have been associated with neuropsychiatric disorders. Actin filaments (F-actin) are the major structural component of dendritic spines and therefore actin-binding proteins (ABP) that control F-actin dis-/assembly moved into the focus as critical regulators of brain function. Studies of the past decade identified the ABP cofilin1 as a key regulator of spine morphology, synaptic transmission and behavior, and they emphasized the necessity for a tight control of cofilin1 to ensure proper brain function. Here, we report spine enrichment of cyclase-associated protein 1 (CAP1), a conserved multidomain protein with largely unknown physiological functions. Super-resolution microscopy and live cell imaging of CAP1-deficient hippocampal neurons revealed impaired synaptic F-actin organization and dynamics associated with alterations in spine morphology. Mechanistically, we found that CAP1 cooperates with cofilin1 in spines and that its helical folded domain is relevant for this interaction. Moreover, our data proved functional interdependence of CAP1 and cofilin1 in control of spine morphology. In summary, we identified CAP1 as a novel regulator of the postsynaptic actin cytoskeleton that is essential for synaptic cofilin1 activity.

Novel bone substitutes such as highly porous ceramic scaffolds can serve as platforms for delivering active molecules. A common problem is to control the release of the drug, therefore, it is beneficial to use a drug-functionalized polymer coating. In this study, β-tricalcium phosphate-based porous scaffolds were obtained and coated with diclofenac-functionalized biopolymer – poly(3-hydroxyoctanoate) – P(3HO). To the best of our knowledge, studies using P(3HO) as a component in ceramic-polymer based drug delivery system for bone tissue regeneration have not yet been reported. Presented materials were comprehensively investigated by various techniques such as powder X-ray diffraction, scanning electron microscopy with energy dispersive spectroscopy, hydrostatic weighing and compression tests, pH and ionic conductivity measurements, high-performance liquid chromatography and in vitro cytotoxicity studies. The obtained diclofenac-loaded composite was not only characterised by controlled and sustained drug release, but also possessed improved mechanical properties. Moreover, the precipitation of apatite-like forms on its surface was observed after incubation in simulated body fluid, which indicates its bioactive potential. After 24 hours no cytotoxic effect on MC3T3-E1 mouse preosteoblastic cells was confirmed using indirect cytotoxicity studies. Thus, this promising multifunctional composite scaffold can be a promising candidate as an anti-inflammatory drug-delivery system in bone tissue engineering.

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.

Dendritic spines are postsynaptic domains that shape structural and functional properties of neurons. Upon neuronal activity, Ca2+ transients trigger signaling cascades that determine the plastic remodeling of dendritic spines, which modulate learning and memory. Here, we study in mice the role of the intracellular Ca2+ channel Ryanodine Receptor 2 (RyR2) in synaptic plasticity and memory formation. We demonstrate that loss of RyR2 in pyramidal neurons of the hippocampus impairs maintenance and activity-evoked structural plasticity of dendritic spines during memory acquisition. Furthermore, post-developmental deletion of RyR2 causes loss of excitatory synapses, dendritic sparsification, overcompensatory excitability, network hyperactivity and disruption of spatially tuned place cells. Altogether, our data underpin RyR2 as a link between spine remodeling, circuitry dysfunction and memory acquisition, which closely resemble pathological mechanisms observed in neurodegenerative disorders.

Schizophrenia (SCZ) is a mental illness characterized by aberrant synaptic plasticity and connectivity. A large bulk of evidence suggests genetic and functional links between postsynaptic abnormalities and SCZ. Here, we performed quantitative PCR and Western blotting analysis in the dorsolateral prefrontal cortex (DLPFC) and hippocampus of SCZ patients to investigate the mRNA and protein expression of three key spine shapers: the actin-binding protein cyclase-associated protein 2 (CAP2), the sheddase a disintegrin and metalloproteinase 10 (ADAM10), and the synapse-associated protein 97 (SAP97). Our analysis of the SCZ post-mortem brain indicated increased DLG1 mRNA in DLPFC and decreased CAP2 mRNA in the hippocampus of SCZ patients, compared to non-psychiatric control subjects, while the ADAM10 transcript was unaffected. Conversely, no differences in CAP2, SAP97, and ADAM10 protein levels were detected between SCZ and control individuals in both brain regions. To assess whether DLG1 and CAP2 transcript alterations were selective for SCZ, we also measured their expression in the superior frontal gyrus of patients affected by neurodegenerative disorders, like Parkinson’s and Alzheimer’s disease. Interestingly, also in Parkinson’s disease patients, we found a selective reduction of CAP2 mRNA levels relative to controls but unaltered protein levels. Taken together, we reported for the first time altered CAP2 expression in the brain of patients with psychiatric and neurological disorders, thus suggesting that aberrant expression of this gene may contribute to synaptic dysfunction in these neuropathologies.

The synapse is the locus of plasticity where short-term alterations in synaptic strength are converted to long-lasting memories. In addition to the presynaptic terminal and the postsynaptic compartment, a more holistic view of the synapse includes the astrocytes and the extracellular matrix to form a tetrapartite synapse. All these four elements contribute to synapse health and are crucial for synaptic plasticity events and, thereby, for learning and memory processes. Synaptic dysfunction is a common pathogenic trait of several brain disorders. In Alzheimer's Disease, the degeneration of synapses can be detected at the early stages of pathology progression before neuronal degeneration, supporting the hypothesis that synaptic failure is a major determinant of the disease. The synapse is the place where amyloid-β peptides are generated and is the target of the toxic amyloid-β oligomers. All the elements constituting the tetrapartite synapse are altered in Alzheimer's Disease and can synergistically contribute to synaptic dysfunction. Moreover, the two main hallmarks of Alzheimer's Disease, i.e., amyloid-β and tau, act in concert to cause synaptic deficits. Deciphering the mechanisms underlying synaptic dysfunction is relevant for the development of the next-generation therapeutic strategies aimed at modifying the disease progression.

Protocadherin-19 (PCDH19) is a synaptic cell-adhesion molecule encoded by X-linked PCDH19, a gene linked with epilepsy. Here, we report a synapse-to-nucleus signaling pathway through which PCDH19 bridges neuronal activity with gene expression. In particular, we describe the NMDA receptor (NMDAR)-dependent proteolytic cleavage of PCDH19, which leads to the generation of a PCDH19 C-terminal fragment (CTF) able to enter the nucleus. We demonstrate that PCDH19 CTF associates with chromatin and with the chromatin remodeler lysine-specific demethylase 1 (LSD1) and regulates expression of immediate-early genes (IEGs). Our results are consistent with a model whereby PCDH19 favors maintenance of neuronal homeostasis via negative feedback regulation of IEG expression and provide a key to interpreting PCDH19-related hyperexcitability.

Toxic aggregates of α-synuclein (αsyn) are considered key drivers of Parkinson's disease (PD) pathology. In early PD, αsyn induces synaptic dysfunction also modulating the glutamatergic neurotransmission. However, a more detailed understanding of the molecular mechanisms underlying αsyn-triggered synaptic failure is required to design novel therapeutic interventions. Here, we described the role of Rabphilin-3A (Rph3A) as novel target to counteract αsyn-induced synaptic loss in PD. Rph3A is a synaptic protein interacting with αsyn and involved in stabilizing dendritic spines and in promoting the synaptic retention of NMDA-type glutamate receptors. We found that in vivo intrastriatal injection of αsyn-preformed fibrils in mice induces the early loss of striatal synapses associated with decreased synaptic levels of Rph3A and impaired Rph3A/NMDA receptors interaction. Modulating Rph3A striatal expression or interfering with the Rph3A/αsyn complex with a small molecule prevented dendritic spine loss and rescued associated early motor defects in αsyn-injected mice. Notably, the same experimental approaches prevented αsyn-induced synaptic loss in vitro in primary hippocampal neurons. Overall, these findings indicate that approaches aimed at restoring Rph3A synaptic functions can slow down the early synaptic detrimental effects of αsyn aggregates in PD.

Cyclase-associated protein (CAP) is an actin binding protein that has been initially described as partner of the adenylyl cyclase in yeast. In all vertebrates and some invertebrate species, two orthologs, named CAP1 and CAP2, have been described. CAP1 and CAP2 are characterized by a similar multidomain structure, but different expression patterns. Several molecular studies clarified the biological function of the different CAP domains, and they shed light onto the mechanisms underlying CAP-dependent regulation of actin treadmilling. However, CAPs are crucial elements not only for the regulation of actin dynamics, but also for signal transduction pathways. During recent years, human genetic studies and the analysis of gene-targeted mice provided important novel insights into the physiological roles of CAPs and their involvement in the pathogenesis of several diseases. In the present review, we summarize and discuss recent progress in our understanding of CAPs’ physiological functions, focusing on heart, skeletal muscle and central nervous system as well as their involvement in the mechanisms controlling metabolism. Remarkably, loss of CAPs or impairment of CAPs-dependent pathways can contribute to the pathogenesis of different diseases. Overall, these studies unraveled CAPs complexity highlighting their capability to orchestrate structural and signaling pathways in the cells.

Schizophrenia (SCZ) is a mental illness characterized by aberrant synaptic plasticity and connectivity. A large bulk of evidence suggests genetic and functional links between postsynaptic abnormalities and SCZ. Here, we performed quantitative PCR and Western blotting analysis in the dorsolateral prefrontal cortex (DLPFC) and hippocampus of SCZ patients to investigate the mRNA and protein expression of three key spine shapers: the actin-binding protein cyclase-associated protein 2 (

Background Synaptic degeneration and accumulation of amyloid β-peptides (Aβ) are hallmarks of the Alzheimer diseased brain. Aβ is synaptotoxic and produced by sequential cleavage of the amyloid precursor protein (APP) by the β-secretase BACE1 and by γ-secretase. If APP is instead cleaved by the α-secretase ADAM10, Aβ will not be generated. Although BACE1 is considered to be a presynaptic protein and ADAM10 has been reported to mainly localize to the postsynaptic density, we have previously shown that both ADAM10 and BACE1 are highly enriched in synaptic vesicles of rat brain and mouse primary hippocampal neurons. Results Here, using brightfield proximity ligation assay, we expanded our previous result in primary neurons and investigated the in situ synaptic localization of ADAM10 and BACE1 in rat and human adult brain using both pre- and postsynaptic markers. We found that ADAM10 and BACE1 were in close proximity with both the presynaptic marker synaptophysin and the postsynaptic marker PSD-95. The substrate APP was also detected both pre- and postsynaptically. Subcellular fractionation confirmed that ADAM10 and BACE1 are enriched to a similar degree in synaptic vesicles and as well as in the postsynaptic density. Conclusions We show that the α-secretase ADAM10 and the β-secretase BACE1 are located in both the pre- and postsynaptic compartments in intact brain sections. These findings increase our understanding of the regulation of APP processing, thereby facilitating development of more specific treatment strategies.

The Ring Finger Protein 10 [RNF10] is a novel synapse-to-nucleus signaling protein that specifically links activation of synaptic NMDA receptors to modulation of gene expression. RNF10 dissociation from the GluN2A subunit of the NMDA receptor represents the first step of its synaptonuclear transport and it is followed by an importin-dependent translocation into the nucleus. Here, we have identified protein kinase C [PKC]-dependent phosphorylation of RNF10 Ser31 as a key step for RNF10 detachment from NMDA receptor and its subsequent trafficking to the nucleus. We show that pSer31-RNF10 plays a role both in synaptonuclear signaling and in neuronal morphology. In particular, the prevention of Ser31 RNF10 phosphorylation induces a decrease in spine density, neuronal branching, and CREB signaling, while opposite effects are obtained by mimicking a stable RNF10 phosphorylation at Ser31. Overall, these results add novel information about the functional and structural role of synaptonuclear protein messengers in shaping dendritic architecture in hippocampal neurons.

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.

Alzheimer’s disease (AD) is a neurodegenerative disorder representing the most common form of dementia. It is biologically characterized by the deposition of extracellular amyloid-β (Aβ) senile plaques and intracellular neurofibrillary tangles, constituted by hyperphosphorylated tau protein. The key protein in AD pathogenesis is the amyloid precursor protein (APP), which is cleaved by secretases to produce several metabolites, including Aβ and APP intracellular domain (AICD). The greatest genetic risk factor associated with AD is represented by the Apolipoprotein E ε4 (APOE ε4) allele. Importantly, all of the above-mentioned molecules that are strictly related to AD pathogenesis have also been described as playing roles in the cell nucleus. Accordingly, evidence suggests that nuclear functions are compromised in AD. Furthermore, modulation of transcription maintains cellular homeostasis, and alterations in transcriptomic profiles have been found in neurodegenerative diseases. This report reviews recent advancements in the AD players-mediated gene expression. Aβ, tau, AICD, and APOE ε4 localize in the nucleus and regulate the transcription of several genes, part of which is involved in AD pathogenesis, thus suggesting that targeting nuclear functions might provide new therapeutic tools for the disease.

The development of new therapeutic avenues that target the early stages of Alzheimer's disease (AD) is urgently necessary. A disintegrin and metalloproteinase domain 10 (ADAM10) is a sheddase that is involved in dendritic spine shaping and limits the generation of amyloid-β. ADAM10 endocytosis increases in the hippocampus of AD patients, resulting in the decreased postsynaptic localization of the enzyme. To restore this altered pathway, we developed a cell-permeable peptide (PEP3) with a strong safety profile that is able to interfere with ADAM10 endocytosis, upregulating the postsynaptic localization and activity of ADAM10. After extensive validation, experiments in a relevant animal model clarified the optimal timing of the treatment window. PEP3 administration was effective for the rescue of cognitive defects in APP/PS1 mice only if administered at an early disease stage. Increased ADAM10 activity promoted synaptic plasticity, as revealed by changes in the molecular compositions of synapses and the spine morphology. Even though further studies are required to evaluate efficacy and safety issues of long-term administration of PEP3, these results provide preclinical evidence to support the therapeutic potential of PEP3 in AD.

ADAM10 is the main α-secretase that participates in the non-amyloidogenic cleavage of amyloid precursor protein (APP) in neurons, inhibiting the production of β-amyloid peptide (Aβ) in Alzheimer’s disease (AD). Strong recent evidence indicates the importance of the localization of ADAM10 for its activity as a protease. In this study, we investigated ADAM10 activity in plasma and CSF samples of patients with amnestic mild cognitive impairment (aMCI) and mild AD compared with cognitively healthy controls. Our results indicated that plasma levels of soluble ADAM10 were significantly increased in the mild AD group, and that in these samples the protease was inactive, as determined by activity assays. The same results were observed in CSF samples, indicating that the increased plasma ADAM10 levels reflect the levels found in the central nervous system. In SH-SY5Y neuroblastoma cells, ADAM10 achieves its major protease activity in the fraction obtained from plasma membrane lysis, where the mature form of the enzyme is detected, confirming the importance of ADAM10 localization for its activity. Taken together, our results demonstrate the potential of plasma ADAM10 to act as a biomarker for AD, highlighting its advantages as a less invasive, easier, faster, and lower-cost processing procedure, compared to existing biomarkers.

Background In experimental models of Parkinson's disease (PD), different degrees of degeneration to the nigrostriatal pathway produce distinct profiles of synaptic alterations that depend on progressive changes in N-methyl-D-aspartate receptors (NMDAR)-mediated functions. Repetitive transcranial magnetic stimulation (rTMS) induces modifications in glutamatergic and dopaminergic systems, suggesting that it may have an impact on glutamatergic synapses modulated by dopamine neurotransmission. However, no studies have so far explored the mechanisms of rTMS effects at early stages of PD. Objectives We tested the hypothesis that in vivo application of rTMS with intermittent theta-burst stimulation (iTBS) pattern alleviates corticostriatal dysfunctions by modulating NMDAR-dependent plasticity in a rat model of early parkinsonism. Methods Dorsolateral striatal spiny projection neurons (SPNs) activity was studied through ex vivo whole-cell patch-clamp recordings in corticostriatal slices obtained from 6-hydroxydopamine-lesioned rats, subjected to a single session (acute) of iTBS and tested for forelimb akinesia with the stepping test. Immunohistochemical analyses were performed to analyze morphological correlates of plasticity in SPNs. Results Acute iTBS ameliorated limb akinesia and rescued corticostriatal long-term potentiation (LTP) in SPNs of partially lesioned rats. This effect was abolished by applying a selective inhibitor of GluN2B-subunit-containing NMDAR, suggesting that iTBS treatment could be associated with an enhanced activation of specific NMDAR subunits, which are major regulators of structural plasticity during synapse development. Morphological analyses of SPNs revealed that iTBS treatment reverted dendritic spine loss inducing a prevalence of thin-elongated spines in the biocytin-filled SPNs. Conclusions Taken together, our data identify that an acute iTBS treatment produces a series of plastic changes underlying striatal compensatory adaptation in the parkinsonian basal ganglia circuit. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Nowadays, regenerative medicine faces a major challenge in providing new, functional materials that will meet the characteristics desired to replenish and grow new tissue. Therefore, this study presents new ceramic-polymer composites in which the matrix consists of tricalcium phosphates covered with blends containing a chemically bounded diclofenac with the biocompatible polymer&mdash, poly(3-hydroxyoctanoate), P(3HO). Modification of P(3HO) oligomers was confirmed by NMR, IR and XPS. Moreover, obtained oligomers and their blends were subjected to an in-depth characterisation using GPC, TGA, DSC and AFM. Furthermore, we demonstrate that the hydrophobicity and surface free energy values of blends decreased with the amount of diclofenac modified oligomers. Subsequently, the designed composites were used as a substrate for growth of the pre-osteoblast cell line (MC3T3-E1). An in vitro biocompatibility study showed that the composite with the lowest concentration of the proposed drug is within the range assumed to be non-toxic (viability above 70%). Cell proliferation was visualised using the SEM method, whereas the observation of cell penetration into the scaffold was carried out by confocal microscopy. Thus, it can be an ideal new functional bone tissue substitute, allowing not only the regeneration and restoration of the defect but also inhibiting the development of chronic inflammation.

Polyhydroxyalkanoates (PHA) are natural biopolymers and are polyesters of hydroxyalkanoic acids, stored in the cytoplasm of many prokaryotes as intracellular granules. PHA are one of the best-studied biomaterials due to their wide-ranging desirable properties, including variation in mechanical and thermal properties. Several in vitro studies have confirmed PHA as biocompatible and bioresorbable and they have also been reported to be immunologically inert, thereby confirming a high potential for biomedical applications. In the last decade, preliminary experiments conducted in the authors’ lab and in other groups, have demonstrated the high prospects of developing PHA-based medical prototypes for the repair and regeneration of damaged peripheral nerves, cardiac, bone, cartilage, skin, and pancreatic tissue, for medical device development such as coronary artery stents and nerve guidance conduits, as well as for drug delivery. In this chapter, the production of PHA from non-infectious bacteria via fermentation, and how the polymer is further processed for medical application in the areas mentioned above, are discussed. The key issues related to the development of medical devices from PHA are also highlighted in the chapter. In addition, the current use of PHA-based devices in the clinic or in clinical trials, and predictions on the future of PHA in the biomedical field are reviewed. The ultimate aim is to assess the suitability of PHA as a biomaterial for the production of efficient smart devices that will provide an efficient solution for long-standing problems related to organ or tissue transplantation and regeneration.

Recent studies identified cyclase-associated proteins (CAPs) as important regulators of actin dynamics that control assembly and disassembly of actin filaments (F-actin). While these studies significantly advanced our knowledge of their molecular functions, the physiological relevance of CAPs largely remained elusive. Gene targeting in mice implicated CAP2 in heart physiology and skeletal muscle development. Heart defects in CAP2 mutant mice were associated with altered activity of serum response factor (SRF), a transcription factor involved in multiple biological processes including heart function, but also skeletal muscle development. By exploiting mouse embryonic fibroblasts (MEFs) from CAP2 mutant mice, we aimed at deciphering the CAP2-dependent mechanism relevant for SRF activity. Reporter assays and mRNA quantification by qPCR revealed reduced SRF-dependent gene expression in mutant MEFs. Reduced SRF activity was associated with moderately increased G-actin levels and reduced nuclear levels of myocardin-related transcription factor A (MRTF-A), a transcriptional SRF coactivator that is shuttled out of the nucleus and, hence, inhibited upon G-actin binding. Moreover, pharmacological actin manipulation with jasplakinolide (JASP) restored MRTF-A distribution in mutant MEFs. Our data are in line with a model in which CAP2 controls the MRTF-SRF pathway in an actindependent manner. While MRTF-A localization and SRF activity was impaired under basal conditions, serum stimulation induced nuclear MRTF-A translocation and SRF activity in mutant MEFs similar to controls. In summary, our data revealed that in MEFs CAP2 controls basal MRTF-A localization and SRF activity, while it was dispensable for serum-induced nuclear MRTF-A translocation and SRF stimulation.

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. Electronic supplementary material The online version of this article (10.1007/s12035-020-02099-7) contains supplementary material, which is available to authorized users.