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Impulse control disorders (e.g., pathological gambling, hypersexuality) may develop as adverse reactions to drugs. Pathogenetic hypotheses have mainly focused on D3-receptor agonism, and switching to alternatives with different pharmacologic mechanisms represents a common management strategy. Nonetheless, treatment failure is common and gaining pathophysiological insights is needed.We aimed to identify targets potentially contributing to pathologic impulsivity.We performed a pharmacovigilance-pharmacodynamic study on dopamine agonists and antipsychotics using the Food and Drug Administration Adverse Event Reporting System (January 2004-December 2021). We estimated disproportionate reporting using the Bayesian information component. Using online public databases (IUPHAR, ChEMBL, PDSP, DrugBank), we calculated drug occupancies. To identify the targets potentially contributing to impulsivity, we fitted univariate regression models interpolating information components and occupancies within dopamine agonists and antipsychotics. Sensitivity analyses were performed to check for the robustness of the results.Among 19,887 reports of impulsivity, 5,898 recorded an antipsychotic, and 3,100 a dopamine agonist. The more robust signals concerned aripiprazole (N=3,091; median information component [95% confidence interval] = 4.51[4.45-4.55]) and brexpiprazole (229; 4.00[3.78-4.16]) for antipsychotics, pergolide (105; 5.82[5.50-6.06]) and pramipexole (2009; 5.43[5.36-5.48]) for dopamine agonists. Robust, significant positive associations between drug occupancy and impulsivity reporting were found for D3 within dopamine agonists (beta=1.52; p-value=0.047) and 5-HT1a within antipsychotics (1.92, 0.029).Our results supported the role of D3-receptor agonism in inducing impulsivity in dopamine receptor agonists and identified a potential role of 5-HT1a receptor agonism in antipsychotics. Investigating these receptors may drive towards a better management of drug-induced impulsivity. This article is protected by copyright. All rights reserved.

The cholinergic neurotransmitter system in the brain is crucial in processing information related to cognitive, behavioral, and motor functions. A cholinergic dysfunction has been correctly described as one of the primary causes of neurodegenerative diseases. Differences in levels of acetylcholine or expression and function of receptors in selected brain areas have been indicated as one of the causes of sexual dimorphism in neurotransmission. However, variability in results among studies based on different mice strains could affect conclusions on this topic. Visual evoked potentials (VEPs) of male and female DBA/2J and C57BL/6J mice, which are two of the most common strains backgrounds in use for developing transgenic mice models of neurological diseases, have been studied. Effects induced by a single low dose of physostigmine have also been performed to evaluate the cholinergic system involvement. VEPs responses to luminous stimuli in C57BL/6J mice have shown a consistently lower latency than in DBA/2J, confirming the previous observation of strain differences in cholinergic function. Interestingly, strains present an opposite-sex difference in VEP latency not apparently related to sensitivity to physostigmine. These findings point at paying extreme attention to the choice of the genetic background of the animal model, especially in those basic and pre-clinical experiments that involve visual functioning.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause a rare neurodevelopmental disorder characterized by early-onset seizures and severe cognitive, motor, and visual impairments. To date there are no therapies for CDKL5 deficiency disorder (CDD). In view of the severity of the neurological phenotype of CDD patients it is widely assumed that CDKL5 may influence the activity of a variety of cellular pathways, suggesting that an approach aimed at targeting multiple cellular pathways simultaneously might be more effective for CDD. Previous findings showed that a single-target therapy aimed at normalizing impaired GSK-3β or histone deacetylase (HDAC) activity improved neurodevelopmental and cognitive alterations in a mouse model of CDD. Here we tested the ability of a first-in-class GSK-3β/HDAC dual inhibitor, Compound 11 (C11), to rescue CDD-related phenotypes. We found that C11, through inhibition of GSK-3β and HDAC6 activity, not only restored maturation, but also significantly improved survival of both human CDKL5-deficient cells and hippocampal neurons from Cdkl5 KO mice. Importantly, in vivo treatment with C11 restored synapse development, neuronal survival, and microglia over-activation, and improved motor and cognitive abilities of Cdkl5 KO mice, suggesting that dual GSK-3β/HDAC6 inhibitor therapy may have a wider therapeutic benefit in CDD patients.

Background The dopaminergic partial agonism of the so-called third-generation antipsychotics (TGAs; aripiprazole, brexpiprazole, cariprazine) is hypothesized to cause impulse control disorders (ICDs). Relevant warnings by the Food and Drug Administration (FDA) were posted on aripiprazole (2016) and brexpiprazole (2018). Our study investigated the FDA Adverse Event Reporting System and the pharmacodynamic CHEMBL database to further characterize TGA-induced ICDs. Methods We downloaded and pre-processed the FDA Adverse Event Reporting System up to December 2020. We adapted Bradford Hill criteria to assess each TGA’s —and secondarily other antipsychotics’—causal role in inducing ICDs (pathological gambling, compulsive shopping, hyperphagia, hypersexuality), accounting for literature and disproportionality. ICD clinical features were analyzed, and their pathogenesis was investigated using receptor affinities. Results A total of 2708 reports of TGA-related ICDs were found, primarily recording aripiprazole (2545 reports, 94%) among the drugs, and gambling (2018 reports, 75%) among the events. Bradford-Hill criteria displayed evidence for a causal role of each TGA consistent across subpopulations and when correcting for biases. Significant disproportionalities also emerged for lurasidone with compulsive shopping, hyperphagia, and hypersexuality, and olanzapine and ziprasidone with hyperphagia. Time to onset varied between days and years, and positive dechallenge was observed in 20% of cases. Frequently, co-reported events were economic (50%), obsessive-compulsive (44%), and emotional conditions (34%). 5-Hydroxytryptamine receptor type 1a agonism emerged as an additional plausible pathogenetic mechanism. Conclusions We detected an association between TGAs and ICDs and identified a new signal for lurasidone. ICD characteristics are behavior specific and may heavily impact on life. The role of 5-Hydroxytryptamine receptor type 1a agonism should be further explored.

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.

Memory consolidation requires astrocytic microdomains for protein recycling; but whether this lays a mechanistic foundation for long-term information storage remains enigmatic. Here we demonstrate that persistent synaptic strengthening invited astrocytic microdomains to convert initially internalized (pro)-brain-derived neurotrophic factor (proBDNF) into active prodomain (BDNFpro) and mature BDNF (mBDNF) for synaptic re-use. While mBDNF activates TrkB, we uncovered a previously unsuspected function for the cleaved BDNFpro, which increases TrkB/SorCS2 receptor complex at post-synaptic sites. Astrocytic BDNFpro release reinforced TrkB phosphorylation to sustain long-term synaptic potentiation and to retain memory in the novel object recognition behavioral test. Thus, the switch from one inactive state to a multi-functional one of the proBDNF provides post-synaptic changes that survive the initial activation. This molecular asset confines local information storage in astrocytic microdomains to selectively support memory circuits., Beatrice Vignoli et al. examine potential molecular mechanisms of long-term storage information in mice. Their results suggest that astrocytes may help convert neuronal BDNF precursor into active prodomain and mature forms to enhance post-synaptic signaling and memory, providing further insight into the development of memory circuits.

Memory consolidation requires astrocytic microdomains for protein recycling; but whether this lays a mechanistic foundation for long-term information storage remains enigmatic. Here we demonstrate that persistent synaptic strengthening invited astrocytic microdomains to convert initially internalized (pro)-brain-derived neurotrophic factor (proBDNF) into active prodomain (BDNFpro) and mature BDNF (mBDNF) for synaptic re-use. While mBDNF activates TrkB, we uncovered a previously unsuspected function for the cleaved BDNFpro, which increases TrkB/SorCS2 receptor complex at post-synaptic sites. Astrocytic BDNFpro release reinforced TrkB phosphorylation to sustain long-term synaptic potentiation and to retain memory in the novel object recognition behavioral test. Thus, the switch from one inactive state to a multi-functional one of the proBDNF provides post-synaptic changes that survive the initial activation (molecular memory). This molecular asset confines local information storage in astrocytic microdomains to selectively support memory circuits.

Alcohol dependence continues to be an important health concern and animal models are critical to furthering our understanding of this complex disease. A hallmark feature of alcoholism is a significant increase in alcohol drinking over time. While several different animal models of excessive alcohol (ethanol) drinking exist for mice and rats, a growing number of laboratories are using a model that combines chronic ethanol exposure procedures with voluntary ethanol drinking with mice as experimental subjects. In the last years several experimental evidences have shown an involvement of opioid system in alcoholism.

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

PURPOSE: It has been hypothesized that specific early-life stress (ES) procedures on CD-1 male mice produce diabetes-like alterations due to the failure of negative feedback of glucocorticoid hormone in the pituitary. The aim of this study is to investigate the possible mechanism that leads to this pathological model, framing it in a more specific clinical condition.METHODS: Metabolic and hypothalamic-pituitary-adrenal-related hormones of stressed mice (SM) have been analyzed immediately after stress procedures (21 postnatal days, PND) and after 70 days of a peaceful (unstressed) period (90 PND). These data have been compared to parameters from age-matched controls (CTR), and mice treated during ES procedures with oligonucleotide antisense for pro-opiomelanocortin (AS-POMC).RESULTS: At 21 PND, SM presented an increased secretion of hypothalamic CRH and pituitary POMC-derived peptides, as well as higher plasmatic levels of ACTH and corticosterone vs. CTR. At 90 PND, SM showed hyperglycemia, with suppression of hypothalamic CRH, while pituitary and plasmatic ACTH levels, as well as plasma corticosterone, were constantly higher than in CTR. These values are accompanied by a progressive acceleration in gaining total body weight, which became significant vs. CTR at 90 PND together with a higher pituitary weight. Treatment with AS-POMC prevented all hormonal and metabolic alterations observed in SM, both at 21 and 90 PND.CONCLUSIONS: These findings show that these specific ES procedures affect the negative glucocorticoid feedback in the pituitary, but not in the hypothalamus, suggesting a novel model of ACTH-dependent hypercortisolism that can be prevented by silencing the POMC gene.

No therapies currently exist for intellectual disability in Down syndrome (DS). In view of its similarities with DS, including learning and memory (L&M) defects, the Ts65Dn mouse model of DS is widely used for the design of therapy. 7,8-dihydroxyflavone (7,8-DHF), a flavonoid that targets the tropomyosin-related kinase B (TrkB) receptor of brain-derived neurotrophic factor (BDNF), exerts positive effects in various brain disease models. Based on previous demonstration that administration of 7,8-DHF in the postnatal period P3-P15 restores hippocampal neurogenesis and spinogenesis, we sought to establish whether these effects translate into behavioral benefits after treatment cessation. We found that Ts65Dn mice treated with 7,8-DHF (5.0 mg/kg/day) during postnatal days P3-P15 did not show any L&M improvement at one month after treatment cessation, indicating that the effects of 7,8-DHF on the brain are ephemeral. Based on evidence that chronic treatment with 7,8-DHF in juvenile Ts65Dn mice restores L&M, we sought to establish whether a similar effect is elicited in adulthood. We found that Ts65Dn mice treated with 7,8-DHF (5.0 mg/kg/day) for about 40 days starting from 4 months of age did not show any improvement in L&M. The results suggest that timing of therapy with 7,8-DHF is a critical issue for attainment of positive effects on the brain.

von Frey hairs are important tools for the study of mechanisms of cutaneous stimulation-induced sensory input. Mechanical force is exerted via application of a particular hair to the cutaneous receptive field until buckling of the hair occurs. The most commonly used von Frey filaments are productive in evaluating behavioral responses of neuropathic pain in preclinical and clinical research. To reduce the potential experimenter bias, automated instruments are being developed for behavioral assessment.

Alcohol dependence continues to be an important health concern and animal models are critical to furthering our understanding of this complex disease. A hallmark feature of alcoholism is a significant increase in alcohol drinking over time. While several different animal models of excessive alcohol (ethanol) drinking exist for mice and rats, a growing number of laboratories are using a model that combines chronic ethanol exposure procedures with voluntary ethanol drinking with mice as experimental subjects. In the last years several experimental evidences have shown an involvement of opioid system in alcoholism.

von Frey hairs are important tools for the study of mechanisms of cutaneous stimulation-induced sensory input. Mechanical force is exerted via application of a particular hair to the cutaneous receptive field until buckling of the hair occurs. The most commonly used von Frey filaments are productive in evaluating behavioral responses of neuropathic pain in preclinical and clinical research. To reduce the potential experimenter bias, automated instruments are being developed for behavioral assessment.

Several chronic neuroinflammatory diseases, including Parkinson&rsquo, s disease (PD), have the so-called &lsquo, redox imbalance&rsquo, in common, a dynamic system modulated by various factors. Among them, alteration of the mitochondrial functionality can cause overproduction of reactive oxygen species (ROS) with the consequent induction of oxidative DNA damage and apoptosis. Considering the failure of clinical trials with drugs that eliminate ROS directly, research currently focuses on approaches that counteract redox imbalance, thus restoring normal physiology in a neuroinflammatory condition. Herein, we used SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA), a neurotoxin broadly employed to generate experimental models of PD. Cells were pre-treated with the Rho-modulating Escherichia coli cytotoxic necrotizing factor 1 (CNF1), before the addition of 6-OHDA. Then, cell viability, mitochondrial morphology and dynamics, redox profile as well as autophagic markers expression were assessed. We found that CNF1 preserves cell viability and counteracts oxidative stress induced by 6-OHDA. These effects are accompanied by modulation of the mitochondrial network and an increase in macroautophagic markers. Our results confirm the Rho GTPases as suitable pharmacological targets to counteract neuroinflammatory diseases and evidence the potentiality of CNF1, whose beneficial effects on pathological animal models have been already proven to act against oxidative stress through an autophagic strategy.

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

Fabry disease (FD) is a X-linked lysosomal storage disorder caused by deficient function of the alpha-galactosidase A (α-GalA) enzyme. α-GalA deficiency leads to multisystemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide (Gb3). A hallmark symptom of FD patients is neuropathic pain that appears in the early stage of the disease as a result of peripheral small fiber damage. Previous studies have shown that Acetyl-L-carnitine (ALC) has neuroprotective, neurotrophic, and analgesic activity in animal models of neuropathic pain. To study the action of ALC on neuropathic pain associated with FD, we treated α-GalA gene null mice (α-GalA(-/0)) with ALC for 30 days. In α-Gal KO mice, ALC treatment induced acute and long-lasting analgesia, which persisted 1 month after drug withdrawal. This effect was antagonized by single administration of LY341495, an orthosteric antagonist of mGlu2/3 metabotropic glutamate receptors. We also found an up-regulation of mGlu2 receptors in cultured DRG neurons isolated from 30-day ALC-treated α-GalA KO mice. However, the up-regulation of mGlu2 receptors was no longer present in DRG neurons isolated 30 days after the end of treatment. Taken together, these findings suggest that ALC induces analgesia in an animal model of FD by up-regulating mGlu2 receptors, and that analgesia is maintained by additional mechanisms after ALC withdrawal. ALC might represent a valuable pharmacological strategy to reduce pain in FD patients.

The major genetic risk factor for Alzheimer’s disease (AD), apolipoprotein E4 (ApoE4), has been suggested to have detrimental effects on neurons, including direct toxicity via apoptosis. Thioredoxin-1 (Trx1) is an endogenous antioxidant protein important for redox regulation and participates in the regulation of apoptosis through the inhibition of apoptosis signal-regulating kinase-1 (Ask-1). In this study, we have investigated the effects of ApoE on Trx1 in the brain. Our results showed that the protein levels of Trx1 were reduced in the hippocampus of ApoE4 targeted replacement (TR) mice compared to ApoE3 TR mice. The reduction was also seen in vitro after treatment of both human primary cortical neurons and neuroblastoma cells with human recombinant ApoE4 (rApoE4). Furthermore, ApoE4 caused a disruption of lysosomal integrity and a shift in the localization of Cathepsin D, an enzyme known to degrade Trx1. ApoE4 treatment induced in addition apoptosis through translocation of Death-domain associated protein-6 (Daxx) from the nucleus to the cytosol, suggesting an activation of the Ask-1 pathway. This toxicity was prevented by overexpression of Trx1 and other endogenous Ask-1 inhibitors. Our data suggests that down-regulation of Trx1 is involved in the toxicity caused by ApoE4. An activated ASK-1 pathway might indeed make cells more vulnerable to other insults such as amyloid-β, which could partially explain the mechanism behind the strongest genetic risk factor for AD.

Neuronal and synaptic loss are the best pathological correlates for memory decline in Alzheimer's disease (AD). Soluble beta-amyloid oligomers (AβO) are considered to putatively play a crucial role in the early synapse loss and cognitive impairment observed in AD. Evidence suggests that oxidative stress and apoptosis are involved in the mechanism of Aβ-induced neurotoxicity and AD pathogenesis. This study aimed to explore the molecular mechanisms that contribute to the early memory deficits induced by intracerebroventricular injection of AβO in mice. Ten days after a single AβO injection memory impairments were observed, as measured by Morris water maze and novel object recognition tests. Cognitive decline was associated with increased oxidative stress, caspase-9 activation, and decreased hippocampal synaptophysin immunoreactivity. Furthermore, GSH levels were significantly higher in AβO-injected mice than in sham mice, showing that a protective mechanism might develop due to oxidative stress. Additionally, AβO-induced toxicity was aligned with an increment of the activation of Akt and ERK1/2, and reduced activity of GSK3. These findings suggest that AβO injection triggers a cascade of events that mimic the key neuropathological hallmarks of AD. Aβ acute injection helps to better understand how this peptide impairs specific signaling pathways leading to synaptic and memory dysfunctions. Thus, this model is a valid tool for investigating AD and may suggest a new way to develop neuroprotective therapies at such early stages of the disease.

Several studies support the relation between leptin and Alzheimer’s disease (AD). We show that leptin levels in CSF are unchanged as subjects progress to AD. However, in AD hippocampus, leptin signalling was decreased and leptin localization was shifted, being more abundant in reactive astrocytes and less in neurons. Similar translocation of leptin was found in brains from Tg2576 and apoE4 mice. Moreover, an enhancement of leptin receptors was found in hippocampus of young Tg2576 mice and in primary astrocytes and neurons treated with Ab1-42. In contrast, old Tg2576 mice showed decreased leptin receptors levels. Similar findings to those seen in Tg2576 mice were found in apoE4, but not in apoE3 mice. These results suggest that leptin levels are intact, but leptin signalling is impaired in AD. Thus, Ab accumulation and apoE4 genotype result in a transient enhancement of leptin signalling that might lead to a leptin resistance state over time.

Cognitive disability is an unavoidable feature of Down syndrome (DS), a genetic disorder due to the triplication of human chromosome 21. DS is associated with alterations of neurogenesis, neuron maturation and connectivity that are already present at prenatal life stages. Recent evidence shows that pharmacotherapies can have a large impact on the trisomic brain provided that they are administered perinatally. Epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, performs many actions in the brain, including inhibition of DYRK1A, a kinase that is over-expressed in the DS brain and contributes to the DS phenotype. Young adults with DS treated with EGCG exhibit some cognitive benefits, although these effects disappear with time. We deemed it extremely important, however, to establish whether treatment with EGCG at the initial stages of brain development leads to plastic changes that outlast treatment cessation. In the current study, we exploited the Ts65Dn mouse model of DS in order to establish whether pharmacotherapy with EGCG during peak of neurogenesis in the hippocampal dentate gyrus (DG) enduringly restores hippocampal development and memory performance. Euploid and Ts65Dn mice were treated with EGCG from postnatal day 3 (P3) to P15. The effects of treatment were examined at its cessation (at P15) or after one month (at P45). We found that at P15 treated trisomic pups exhibited restoration of neurogenesis, total hippocampal granule cell number and levels of pre- and postsynaptic proteins in the DG, hippocampus and neocortex. However, at P45 none of these effects were still present, nor did treated Ts65Dn mice exhibit any improvement in hippocampus-dependent tasks. These findings show that treatment with EGCG carried out in the neonatal period rescues numerous trisomy-linked brain alterations. However, even during this, the most critical time window for hippocampal development, EGCG does not elicit enduring effects on the hippocampal physiology.

Evidence is accumulating that the main chronic diseases of aging Alzheimer’s disease (AD) and type-2 diabetes mellitus (T2DM) share common pathophysiological mechanisms. This study aimed at applying systems biology approaches to increase the knowledge of the shared molecular pathways underpinnings of AD and T2DM. We analysed transcriptomic data of post-mortem AD and T2DM human brains to obtain disease signatures of AD and T2DM and combined them with protein-protein interaction information to construct two disease-specific networks. The overlapping AD/T2DM network proteins were then used to extract the most representative Gene Ontology biological process terms. The expression of genes identified as relevant was studied in two AD models, 3xTg-AD and ApoE3/ApoE4 targeted replacement mice. The present transcriptomic data analysis revealed a principal role for autophagy in the molecular basis of both AD and T2DM. Our experimental validation in mouse AD models confirmed the role of autophagy-related genes. Among modulated genes, Cyclin-Dependent Kinase Inhibitor 1B, Autophagy Related 16-Like 2, and insulin were highlighted. In conclusion, the present investigation revealed autophagy as the central dys-regulated pathway in highly co-morbid diseases such as AD and T2DM allowing the identification of specific genes potentially involved in disease pathophysiology which could become novel targets for therapeutic intervention.

Background Fabry disease (FD) is a hereditary X-linked metabolic storage disorder characterized by deficient or absent lysosomal α-galactosidase A (α-Gal A) activity. This deficiency causes progressive accumulation of glycosphingolipids, primarily globotriaosylceramide (Gb3), in nearly all organ systems. Gastrointestinal (GI) symptoms can be very debilitating and are among the most frequent and earliest of the disease. As the pathophysiology of these symptoms is poorly understood, we carried out a morphological and molecular characterization of the GI tract in α-Gal A knockout mice colon in order to reveal the underlying mechanisms. Methods Here, we performed the first morphological and biomolecular characterization of the colon wall structure in the GI tract of the α-Gal A knock-out mouse (α-Gal A -/0), a murine model of FD. Key results Our data show a greater thickness of the gastrointestinal wall in α-Gal A (-/0) mice due to enlarged myenteric plexus' ganglia. This change is paralleled by a marked Gb3 accumulation in the gastrointestinal wall and a decreased and scattered pattern of mucosal nerve fibers. Conclusions and inferences The observed alterations are likely to be a leading cause of gut motor dysfunctions experienced by FD patients and imply that the α-Gal A (-/0) male mouse represents a reliable model for translational studies on enteropathic pain and GI symptoms in FD.

CDKL5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked CDKL5 (cyclin-dependent kinase-like five) gene. CDKL5 disorder primarily affects girls and is characterized by early-onset epileptic seizures, gross motor impairment, intellectual disability, and autistic features. Although all CDKL5 female patients are heterozygous, the most valid disease-related model, the heterozygous femaleCdkl5knockout (Cdkl5+/−) mouse, has been little characterized. The lack of detailed behavioral profiling of this model remains a crucial gap that must be addressed in order to advance preclinical studies. Here, we provide a behavioral and molecular characterization of heterozygousCdkl5+/− mice. We found thatCdkl5+/− mice reliably recapitulate several aspects of CDKL5 disorder, including autistic-like behaviors, defects in motor coordination and memory performance, and breathing abnormalities. These defects are associated with neuroanatomical alterations, such as reduced dendritic arborization and spine density of hippocampal neurons. Interestingly,Cdkl5+/− mice show age-related alterations in protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) signaling, two crucial signaling pathways involved in many neurodevelopmental processes. In conclusion, our study provides a comprehensive overview of neurobehavioral phenotypes of heterozygous femaleCdkl5+/− mice and demonstrates that the heterozygous female might be a valuable animal model in preclinical studies on CDKL5 disorder.

Cyclin-dependent kinase like-5 (CDKL5) disorder is a rare neurodevelopmental disease caused by mutations in the CDKL5 gene. The consequent misexpression of the CDKL5 protein in the nervous system leads to a severe phenotype characterized by intellectual disability, motor impairment, visual deficits, and early-onset epilepsy. No therapy is available for CDKL5 disorder. It has been reported that a protein transduction domain (TAT) is able to deliver macromolecules into cells and even into the brain when fused to a given protein. We demonstrate that TAT-CDKL5 fusion protein is efficiently internalized by target cells and retains CDKL5 activity. Intracerebroventricular infusion of TAT-CDKL5 restored hippocampal development, hippocampus-dependent memory and breathing pattern in Cdkl5-null mice. Notably, systemically-administered TAT-CDKL5 protein passed the blood-brain-barrier, reached the CNS, and rescued various neuroanatomical and behavioral defects, including breathing pattern and visual responses. Our results suggest that CDKL5 protein therapy may be an effective clinical tool for the treatment of CDKL5 disorder.

Mutations in the X-linked cyclin-dependent kinase-like 5 ( CDKL5 ) gene have been identified in a rare neurodevelopmental disorder characterized by early-onset seizures, severe developmental delay, intellectual disability and Rett syndrome-like features. CDKL5 is highly expressed in the brain during early postnatal stages, suggesting its importance for brain maturation. Using a newly-generated Cdkl5 knockout ( Cdkl5 −/Y) mouse, we recently found that loss of Cdkl5 impairs postnatal hippocampal development with a reduction in neuronal precursor survival and maturation. These defects were accompanied by increased activity of the glycogen synthase kinase 3β (GSK3β) a crucial inhibitory regulator of many neurodevelopmental processes. The goal of the current study was to establish whether inhibition of GSK3β corrects hippocampal developmental defects due to Cdkl5 loss. We found that treatment with the GSK3β inhibitor SB216763 restored neuronal precursor survival, dendritic maturation, connectivity and hippocampus-dependent learning and memory in the Cdkl5 −/Y mouse. Importantly, these effects were retained one month after treatment cessation. At present, there are no therapeutic strategies to improve the neurological defects of subjects with CDKL5 disorder. Current results point at GSK3β inhibitors as potential therapeutic tools for the improvement of abnormal brain development in CDKL5 disorder.

In Alzheimer’s disease (AD) patients, apopoliprotein (APOE) polymorphism is the main genetic factor associated with more aggressive clinical course. However, the interaction between cerebrospinal fluid (CSF) tau protein levels and APOE genotype has been scarcely investigated. A possible key mechanism invokes the dysfunction of synaptic plasticity. We investigated how CSF tau interacts with APOE genotype in AD patients. We firstly explored whether CSF tau levels and APOE genotype influence disease progression and long-term potentiation (LTP)-like cortical plasticity as measured by transcranial magnetic stimulation (TMS) in AD patients. Then, we incubated normal human astrocytes (NHAs) with CSF collected from sub-groups of AD patients to determine whether APOE genotype and CSF biomarkers influence astrocytes survival. LTP-like cortical plasticity differed between AD patients with apolipoprotein E4 (APOE4) and apolipoprotein E3 (APOE3) genotype. Higher CSF tau levels were associated with more impaired LTP-like cortical plasticity and faster disease progression in AD patients with APOE4 but not APOE3 genotype. Apoptotic activity was higher when cells were incubated with CSF from AD patients with APOE4 and high tau levels. CSF tau is detrimental on cortical plasticity, disease progression and astrocyte survival only when associated with APOE4 genotype. This is relevant for new therapeutic approaches targeting tau.

Cyclin-dependent kinase-like 5 (CDKL5) disorder is a severe neurodevelopmental disorder characterized by early-onset epileptic seizures, severe developmental delay, and intellectual disability. To date, no effective pharmacological treatments are available to improve the neurological phenotype that is due to mutations in the CDKL5 gene. Murine models of CDKL5 disorder have recently been generated, making the preclinical testing of pharmacological interventions possible. Using a Cdkl5 knockout (KO) mouse model, we recently demonstrated that deficiency of Cdkl5 causes defects in postnatal hippocampal development and hippocampus-dependent learning and memory. These defects were accompanied by an increased activity of GSK3β, an important inhibitory regulator of many neuronal functions. Pharmacological inhibition of GSK3β activity was able to recover hippocampal defects and cognitive performance in juvenile Cdkl5 KO mice, suggesting that GSK3β inhibitors might be a potential therapeutic option for CDKL5 disorder. As GSK3β inhibitors have been shown to have differential medication responses in young people and adults, this study was designed to examine whether GSK3β is a possible therapeutic target both in juvenile and in adult CDKL5 patients. We found that treatment with the GSK3β inhibitor Tideglusib during the juvenile period improved hippocampal development and hippocampus-dependent behaviors in Cdkl5 KO mice, while treatment later on in adulthood had no positive effects. These results suggest that pharmacological interventions aimed at normalizing impaired GSK3β activity might have different age-dependent outcomes in CDKL5 disorder. This is of utmost importance in the development of therapeutic approaches in CDKL5 patients and in the design of rational clinical trials.

Intellectual disability is the unavoidable hallmark of Down syndrome (DS), with a heavy impact on public health. Reduced neurogenesis and impaired neuron maturation are considered major determinants of altered brain function in DS. Since the DS brain starts at a disadvantage, attempts to rescue neurogenesis and neuron maturation should take place as soon as possible. The brain-derived neurotrophic factor (BDNF) is a neurotrophin that plays a key role in brain development by specifically binding to tropomyosin-related kinase receptor B (TrkB). Systemic BDNF administration is impracticable because BDNF has a poor blood-brain barrier penetration. Recent screening of a chemical library has identified a flavone derivative, 7,8-dihydroxyflavone (7,8-DHF), a small-molecule that crosses the blood-brain barrier and binds with high affinity and specificity to the TrkB receptor. The therapeutic potential of TrkB agonists for neurogenesis improvement in DS has never been examined. The goal of our study was to establish whether it is possible to restore brain development in the Ts65Dn mouse model of DS by targeting the TrkB receptor with 7,8-DHF. Ts65Dn mice subcutaneously injected with 7,8-DHF in the neonatal period P3-P15 exhibited a large increase in the number of neural precursor cells in the dentate gyrus and restoration of granule cell number, density of dendritic spines and levels of the presynaptic protein synaptophysin. In order to establish the functional outcome of treatment, mice were treated with 7,8-DHF from P3 to adolescence (P45-50) and were tested with the Morris Water Maze. Treated Ts65Dn mice exhibited improvement of learning and memory, indicating that the recovery of the hippocampal anatomy translated into a functional rescue. Our study in a mouse model of DS provides novel evidence that treatment with 7,8-DHF during the early postnatal period restores the major trisomy-linked neurodevelopmental defects, suggesting that therapy with 7,8-DHF may represent a possible breakthrough for Down syndrome.

The apolipoprotein E4 (apoE4) allele is consistently associated with increased risk for Alzheimer’s disease (AD). We investigated the molecular mechanism of this susceptibility by analyzing the levels of genes involved in AD pathogenesis in transgenic mice expressing human apoE3 or apoE4 isoforms. mRNA and protein levels of Pin1, Sirtuin 1 (Sirt1), Presenilin 1 (PS1), and pro-Brain-derived Neurotrophic Factor (BDNF) were analyzed in brain regions affected by neuropathological changes in AD. Pin1 mRNA was significantly higher in the hippocampus of apoE4 mice than in apoE3 controls, whereas lower expression was detected in the entorhinal and parietal cortices. Reduced Pin1 levels may increase neurofibrillary degeneration and amyloidogenic processes, while compensatory mechanisms may take place in the hippocampus to balance spatial memory deficits. Sirt1 levels were significantly reduced in the frontal cortex of apoE4 mice. Sirt1 reduction may hinder its protective role against the formation of plaques and tangles and diminish its anti-inflammatory actions. Sirt1 decrease may also play a role in apoE4-associated memory impairments. Moreover, in apoE4 mice PS1 mRNA levels were lower in the frontal cortex. Lower PS1 expression may hamper γ-secretase function, thus affecting amyloid precursor protein processing. Pro-BDNF mRNA levels did not differ between apoE3 and apoE4 mice in any region analyzed. This study showed dysregulated expression of Pin1, Sirt1, and PS1 genes in different cerebral areas of apoE4 mice, suggesting that these changes may play a role in the mechanism of AD vulnerability.

Von Frey hairs are important tools for the study of mechanisms of cutaneous stimulation-induced sensory input. Mechanical force is exerted via application of a particular hair to the cutaneous receptive field until buckling of the hair occurs. The most commonly used Von Frey filaments are productive in evaluating behavioral responses of neuropathic pain in preclinical and clinical research. To reduce the potential experimenter bias, automated instruments are being developed for behavioral assessment.

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in the brain. Mutations of the CDKL5 gene lead to CDKL5 disorder, a neurodevelopmental pathology that shares several features with Rett Syndrome and is characterized by severe intellectual disability. The phosphorylation targets of CDKL5 are largely unknown, which hampers the discovery of therapeutic strategies for improving the neurological phenotype due to CDKL5 mutations. Here, we show that the histone deacetylase 4 (HDAC4) is a direct phosphorylation target of CDKL5 and that CDKL5-dependent phosphorylation promotes HDAC4 cytoplasmic retention. Nuclear HDAC4 binds to chromatin as well as to MEF2A transcription factor, leading to histone deacetylation and altered neuronal gene expression. By using a Cdkl5 knockout (Cdkl5 -/Y) mouse model, we found that hypophosphorylated HDAC4 translocates to the nucleus of neural precursor cells, thereby reducing histone 3 acetylation. This effect was reverted by re-expression of CDKL5 or by inhibition of HDAC4 activity through the HDAC4 inhibitor LMK235. In Cdkl5 -/Y mice treated with LMK235, defective survival and maturation of neuronal precursor cells and hippocampus-dependent memory were fully normalized. These results demonstrate a critical role of HDAC4 in the neurodevelopmental alterations due to CDKL5 mutations and suggest the possibility of HDAC4-targeted pharmacological interventions.

Fabry disease is a X-linked lysosomal storage disorder caused by deficient function of the alpha-galactosidase A (α-GalA) enzyme. α-GalA deficiency leads to multisystemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide (Gb3) in the endothelium and vascular smooth muscles. A hallmark symptom of Fabry disease patients is neuropathic pain that appears in the early stage of the disease as a result of peripheral small fiber damage. The α-GalA gene null mouse model (α-GalA(-/0)) has provided molecular evidence for the molecular alterations in small type-C nociceptors in Fabry disease that may underlie their hyperexcitability, although the specific mechanism remains elusive. Here, we have addressed this question and report that small type-C nociceptors from α-GalA(-/0) mice exhibit a significant increase in the expression and function of the TRPV1 channel, a thermoTRP channel implicated in painful heat sensation. Notably, male α-GalA(-/0) mice displayed a ≈2-fold higher heat sensitivity than wild-type animals, consistent with the augmented expression levels and activity of TRPV1 in α-GalA(-/0) nociceptors. Intriguingly, blockade of neuronal exocytosis with peptide DD04107, a process that inhibits among others the algesic membrane recruitment of TRPV1 channels in peptidergic nociceptors, virtually eliminated the enhanced heat nociception of α-GalA(-/0) mice. Together, these findings suggest that the augmented expression of TRPV1 in α-GalA(-/0) nociceptors may underly at least in part their increased heat sensitivity, and imply that blockade of peripheral neuronal exocytosis may be a valuable pharmacological strategy to reduce pain in Fabry disease patients, increasing their quality of life.