Muscarinic Receptors and Alzheimer's Disease: New Perspectives and Mechanisms.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases on a global scale. Historically, this pathology has been linked to cholinergic transmission, and despite the scarcity of effective therapies, numerous alternative processes and targets have been proposed as potential avenues for comprehending this complex illness. Nevertheless, the fundamental pathophysiological mechanisms underpinning AD remain largely enigmatic, with a growing body of evidence advocating for the significance of muscarinic receptors in modulating the brain's capacity to adapt and generate new memories. This review summarizes the current state of the art in the field of muscarinic receptors' involvement in AD. A specific key factor was the relationship between comorbidity and the emergence of new mechanisms.

Complement tunes glutamate release and supports synaptic impairments in an animal model of multiple sclerosis.

BACKGROUND AND PURPOSE: To deepen our knowledge of the role of complement in synaptic impairment in experimental autoimmune encephalomyelitis (EAE) mice, we investigated the distribution of C1q and C3 proteins and the role of complement as a promoter of glutamate release in purified nerve endings (synaptosomes) and astrocytic processes (gliosomes) isolated from the cortex of EAE mice at the acute stage of the disease (21 ± 1 day post-immunization). EXPERIMENTAL APPROACH: EAE cortical synaptosomes and gliosomes were analysed for glutamate release efficiency (measured as release of preloaded [3H]D-aspartate ([3H]D-ASP)), C1q and C3 protein density, and for viability and ongoing apoptosis. KEY RESULTS: In healthy mice, complement releases [3H]D-ASP from gliosomes more efficiently than from synaptosomes. The releasing activity occurs in a dilution-dependent manner and involves the reversal of the excitatory amino acid transporters (EAATs). In EAE mice, the complement-induced releasing activity is significantly reduced in cortical synaptosomes but amplified in cortical gliosomes. These adaptations are paralleled by decreased density of the EAAT2 protein in synaptosomes and increased EAAT1 staining in gliosomes. Concomitantly, PSD95, GFAP, and CD11b, but not SNAP25, proteins are overexpressed in the cortex of the EAE mice. Similarly, C1q and C3 protein immunostaining is increased in EAE cortical synaptosomes and gliosomes, although signs of ongoing apoptosis or altered viability are not detectable. CONCLUSION AND IMPLICATIONS: Our results unveil a new noncanonical role of complement in the CNS of EAE mice relevant to disease progression and central synaptopathy that suggests new therapeutic targets for the management of MS.

Correction: Prophylactic versus Therapeutic Fingolimod: Restoration of Presynaptic Defects in Mice Suffering from Experimental Autoimmune Encephalomyelitis.

[This corrects the article DOI: 10.1371/journal.pone.0170825.].

Progress in metamodulation and receptor-receptor interaction: From physiology to pathology and therapy.

The organization and the role of receptor-receptor interaction (RRI) and metamodulation in physiological conditions have been extensively analyzed and discussed. In this Special Issue of Neuropharmacology, we review recent advances in the understanding of the RRI and the mechanisms underlying its adaptation that could be relevant to the etiopathogenesis of central neuropsychiatric disorders, as well as to the development of new therapeutic approaches to control the activity and to restore the physiological functions, posing the basis for new targeted pharmacological interventions.

Metamodulation of presynaptic NMDA receptors: New perspectives for pharmacological interventions.

Metamodulation shifted the scenario of the central neuromodulation from a simplified unimodal model to a multimodal one. It involves different receptors/membrane proteins physically associated or merely colocalized that act in concert to control the neuronal functions influencing each other. Defects or maladaptation of metamodulation would subserve neuropsychiatric disorders or even synaptic adaptations relevant to drug dependence. Therefore, this "vulnerability" represents a main issue to be deeply analyzed to predict its aetiopathogenesis, but also to propose targeted pharmaceutical interventions. The review focusses on presynaptic release-regulating NMDA receptors and on some of the mechanisms of their metamodulation described in the literature. Attention is paid to the interactors, including both ionotropic and metabotropic receptors, transporters and intracellular proteins, which metamodulate their responsiveness in physiological conditions but also undergo adaptation that are relevant to neurological dysfunctions. All these structures are attracting more and more the interest as promising druggable targets for the treatment of NMDA receptor-related central diseases: these substances would not exert on-off control of the colocalized NMDA receptors (as usually observed with NMDA receptor full agonists/antagonists), but rather modulate their functions, with the promise of limiting side effects that would favor their translation from preclinic to clinic. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".

Anxiety and Metabolic Disorders: The Role of Botanicals.

Anxiety and anxiety-related disorders are becoming more evident every day, affecting an increasing number of people around the world. Metabolic disorders are often associated with anxiety. Furthermore, anxiety branches into metabolic disorders by playing multiple roles as a cofactor, symptom, and comorbidity. Taken together, these considerations open the possibility of integrating the therapy of metabolic disorders with specific drugs for anxiety control. However, anxiolytic compounds often cause disabling effects in patients. The main goal could be to combine therapeutic protocols with compounds capable of reducing side effects while performing multiple beneficial effects. In this article we propose a group of bioactive ingredients called botanicals as a healthy supplement for the treatment of metabolic disorders related to anxiety.

Central nervous system interaction and crosstalk between nAChRs and other ionotropic and metabotropic neurotransmitter receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in both the peripheral and the central nervous systems. nAChRs exert a crucial modulatory influence on several brain biological processes; they are involved in a variety of neuronal diseases including Parkinson's disease, Alzheimer's disease, epilepsy, and nicotine addiction. The influence of nAChRs on brain function depends on the activity of other neurotransmitter receptors that co-exist with nAChRs on neurons. In fact, the crosstalk between receptors is an important mechanism of neurotransmission modulation and plasticity. This may be due to converging intracellular pathways but also occurs at the membrane level, because of direct physical interactions between receptors. In this line, this review is dedicated to summarizing how nAChRs and other ionotropic and metabotropic receptors interact and the relevance of nAChRs cross-talks in modulating various neuronal processes ranging from the classical modulation of neurotransmitter release to neuron plasticity and neuroprotection.

Presynaptic 5-HT2A-mGlu2/3 Receptor-Receptor Crosstalk in the Prefrontal Cortex: Metamodulation of Glutamate Exocytosis.

The glutamatergic nerve endings of a rat prefrontal cortex (PFc) possess presynaptic 5-HT2A heteroreceptors and mGlu2/3 autoreceptors, whose activation inhibits glutamate exocytosis, and is measured as 15 mM KCl-evoked [3H]D-aspartate ([3H]D-asp) release (which mimics glutamate exocytosis). The concomitant activation of the two receptors nulls their inhibitory activities, whereas blockade of the 5-HT2A heteroreceptors with MDL11,939 (1 µM) strengthens the inhibitory effect elicited by the mGlu2/3 receptor agonist LY329268 (1 µM). 5-HT2A receptor antagonists (MDL11,939; ketanserin; trazodone) amplify the impact of low (3 nM) LY379268. Clozapine (0.1-10 µM) mimics the 5-HT2A agonist (±) DOI and inhibits the KCl-evoked [3H]D-asp overflow in a MDL11,939-dependent fashion, but does not modify the (±) DOI-induced effect. mGlu2 and 5-HT2A proteins do not co-immunoprecipitate from synaptosomal lysates, nor does the incubation of PFc synaptosomes with MDL11,939 (1 µM) or clozapine (10 µM) modify the insertion of mGlu2 subunits in synaptosomal plasma membranes. In conclusion, 5-HT2A and mGlu2/3 receptors colocalize, but do not physically associate, in PFc glutamatergic terminals, where they functionally interact in an antagonist-like fashion to control glutamate exocytosis. The mGlu2/3-5-HT2A metamodulation could be relevant to therapy for central neuropsychiatric disorders, including schizophrenia, but also unveil cellular events accounting for their development, which also influence the responsiveness to drugs regimens.

The Anti-Aggregative Peptide KLVFF Mimics Aß1-40 in the Modulation of Nicotinic Receptors: Implications for Peptide-Based Therapy.

In recent years, the inhibition of beta-amyloid (Aß) aggregation has emerged as a potential strategy for Alzheimer's disease. KLVFF, a small peptide corresponding to the aminoacidic sequence 16-20 of Aß, reduces Aß fibrillation dose dependently. Therefore, the toxic and functional characterization of its brain activity is fundamental for clarifying its potential therapeutic role. Accordingly, we studied the modulatory role of KLVFF on the cholinergic receptors regulating dopamine and noradrenaline release in rat synaptosomes. Nicotinic receptors on dopaminergic nerve terminals in the nucleus acccumbens are inhibited by KLVFF, which closely resembles full-length Aß1-40. Moreover, KLVFF entrapped in synaptosomes does not modify the nicotinic receptor's function, suggesting that external binding to the receptor is required for its activity. The cholinergic agent desformylflustrabromine counteracts the KLVFF effect. Remarkably, muscarinic receptors on dopaminergic terminals and nicotinic receptors regulating noradrenaline release in the hippocampus are completely insensitive to KLVFF. Based on our findings, KLVFF mimics Aß1-40 as a negative modulator of specific nicotinic receptor subtypes affecting dopamine transmission in the rat brain. Therefore, new pharmacological strategies using the anti-aggregative properties of KLVFF need to be evaluated for potential interference with nicotinic receptor-mediated transmission.

Use of an Animal Model to Evaluate Anxiolytic Effects of Dietary Supplementation with Tilia tomentosa Moench Bud Extracts.

Anxiety disorders are common and complex psychiatric syndromes affecting a broad spectrum of patients. On top of that, we know that aging produces an increase in anxiety vulnerability and sedative consumption. Moreover, stress disorders frequently show a clear gender susceptibility. Currently, the approved pharmacological strategies have severe side effects such as hallucinations, addiction, suicide, insomnia, and loss of motor coordination. Dietary integration with supplements represents an intriguing strategy for improving the efficacy and the safety of synthetic anxiolytics. Accordingly, a recent article demonstrated that glyceric bud extracts from Tilia tomentosa Moench (TTBEs) exert effects that are consistent with anxiolytic activity. However, the effects of these compounds in vivo are unknown. To examine this question, we conducted behavioral analysis in mice. A total of 21 days of oral supplements (vehicle and TTBEs) were assessed by Light Dark and Hole Board tests in male and female mice (young, 3 months; old, 24 months). Interestingly, the principal component analysis revealed gender and age-specific behavioral modulations. Moreover, the diet integration with the botanicals did not modify the body weight gain and the daily intake of water. Our results support the use of TTBEs as dietary supplements for anxiolytic purposes and unveil age and gender-dependent responses.

Bud-Derivatives, a Novel Source of Polyphenols and How Different Extraction Processes Affect Their Composition.

The use of herbal food supplements, as a concentrate form of vegetable extracts, increased so much over the past years to count them among the relevant sources of dietetic polyphenols. Bud-derivatives are a category of botanicals perceived as a "new entry" in this sector since they are still poorly studied. Due to the lack of a manufacturing process specification, very different products can be found on the market in terms of their polyphenolic profile depending on the experimental conditions of manufacturing. In this research two different manufacturing processes, using two different protocols, and eight species (Carpinus betulus L., Cornus mas L., Ficus carica L., Fraxinus excelsior L., Larix decidua Mill., Pinus montana Mill., Quercus petraea (Matt.) Liebl., Tilia tomentosa Moench), commonly used to produce bud-derivatives, have been considered as a case study. An untargeted spectroscopic fingerprint of the extracts, coupled to chemometrics, provide to be a useful tool to identify these botanicals. The targeted phytochemical fingerprint by HPLC provided a screening of the main bud-derivatives polyphenolic classes highlighting a high variability depending on both method and protocol used. Nevertheless, ultrasonic extraction proved to be less sensitive to the different extraction protocols than conventional maceration regarding the extract polyphenolic profile.

Neuroinflammation in Aged Brain: Impact of the Oral Administration of Ellagic Acid Microdispersion.

The immune system and the central nervous system message each other to preserving central homeostasis. Both systems undergo changes during aging that determine central age-related defects. Ellagic acid (EA) is a natural product which is beneficial in both peripheral and central diseases, including aging. We analyzed the impact of the oral administration of a new oral ellagic acid micro-dispersion (EAm), that largely increased the EA solubility, in young and old mice. Oral EAm did not modify animal weight and behavioral skills in young and old mice, but significantly recovered changes in "ex-vivo, in vitro" parameters in old animals. Cortical noradrenaline exocytosis decreased in aged mice. EAm administration did not modify noradrenaline overflow in young animals, but recovered it in old mice. Furthermore, GFAP staining was increased in the cortex of aged mice, while IBA-1 and CD45 immunopositivities were unchanged when compared to young ones. EAm treatment significantly reduced CD45 signal in both young and old cortical lysates; it diminished GFAP immunopositivity in young mice, but failed to affect IBA-1 expression in both young and old animals. Finally, EAm treatment significantly reduced IL1beta expression in old mice. These results suggest that EAm is beneficial to aging and represents a nutraceutical ingredient for elders.

Ellagic acid a multi-target bioactive compound for drug discovery in CNS? A narrative review.

It is well-known that the health properties attributed to several fruits, herbs, seeds and their processed foods/beverages are due to an important group of natural polyphenols classified as hydrolysable tannins (HT) named ellagitannins (ETs), that encompass both one or more gallic acid (GA) units and one or more hexahydroxydiphenoic acid (HHDP) units, ester-connected with a sugar residue. In vivo, ETs are rather not absorbed and in gastrointestinal tract (GIT), they are hydrolysed providing mainly ellagic acid (EA). Due to its trivial water-solubility, first pass effect, metabolism in GIT, or irreversible binding to cellular DNA and proteins, EA has a very low bioavailability. Some authors are studying methods to increase EA water-solubility and thus to improve its bioavailability. At the same, EA metabolism to urolithins (UROs), whose concentration and activity is inter-individual and intra-individual dependent, is still under study and not completely elucidate. Numerous in vitro and in vivo studies have been carried out to define the molecular and cellular events underlying the beneficial effects that this compound and its metabolites exert in pathological conditions. The anti-inflammatory and the antioxidant properties of EA attracted the interest of researchers for its potential health benefits in humans, including anti-cancer, anti-diabetes activities and cardio-protection. Nevertheless, lately the attention paid to EA is focusing on its potential protective action towards several neurodegenerative disorders. Thus, EA is investigated as a potential "lead compound" endowed with multi-target pharmacological properties on CNS. Since the identification of the pharmacophore(s) responsible for both health benefits and collateral effects of this compound is crucial in drug discovery, this review aims to provide an all-round updated analysis of the literature concerning EA involvement in several CNS disorders, hoping that such information will be useful to researchers involved in multi-target drug design for CNS.

Beta-amyloid short- and long-term synaptic entanglement.

Beta-amyloid (Aß) is a peptide that derives from the proteolytic cleavage of the amyloid precursor protein (APP) by several secretases. Since its isolation and sequencing from Alzheimer's disease (AD) brains, Aß has been intensively investigated in the context of AD as the main pathogenic marker responsible for neurodegenerative processes. During the last three decades, results from several independent studies have converged to form the so-called amyloid cascade hypothesis of AD and several therapeutic strategies designed to modulate the APP amyloidogenic pathway have been developed. However, none of the clinical trials targeting Aß culminated in a significant clinical outcome, thus challenging the concept that targeting Aß, at least within the time window so far explored in clinical trials, may have a therapeutic effect. However, besides its presence in AD brains, brain cells produce Aß, thus suggesting that, under normal conditions, the peptide may have a role in the regulation of brain functions, which is consistent with its ubiquitous presence and normal synthesis. Taking into account that Aß has been found to exhibit a dual role strictly correlated with its concentration (neuromodulatory/neuroprotective vs neurotoxic), we discuss emerging evidence indicating that physiological concentrations of Aß peptide modulate synaptic activity. The review examines the physiological effects of Aß on acute synaptic activities and the functional interplay existing between Aß and different neurotransmitter systems, i.e. cholinergic, glutamatergic, GABAergic, catecholaminergic, serotoninergic, and peptidergic. The review also provides an insight into the different mechanisms through which Aß affects synaptic activity, focusing in particular on Aß interaction with the key synaptic proteins that regulate the neurotransmitter release machinery. These interactions may help to identify or recognize alterations in neurotransmitter activity and correlated behaviors as predictive signs for the development of AD and to understand the limitations of current interventions and the failure so far of amyloid targeted therapies.

5-HT2A-mGlu2/3 receptor complex in rat spinal cord glutamatergic nerve endings: A 5-HT2A to mGlu2/3 signalling to amplify presynaptic mechanism of auto-control of glutamate exocytosis.

Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the finding that LY379268 inhibited the 15 mM KCl-evoked release of [3H]D-aspartate ([3H]D-Asp) in a LY341495-sensitive manner. Spinal cord glutamatergic nerve terminals also possess presynaptic release-regulating 5-HT2A heteroreceptors. Actually, the 15 mM KCl-evoked [3H]D-Asp exocytosis from spinal cord synaptosomes was reduced by the 5-HT2A agonist (±)DOI, an effect reversed by the 5-HT2A antagonists MDL11,939, MDL100907, ketanserin and trazodone (TZD). We investigated whether mGlu2/3 and 5-HT2A receptors colocalize and cross-talk in these terminals and if 5-HT2A ligands modulate the mGlu2/3-mediated control of glutamate exocytosis. Western blot analysis and confocal microscopy highlighted the presence of mGlu2/3 and 5-HT2A receptor proteins in spinal cord VGLUT1 positive synaptosomes, where mGlu2/3 and 5-HT2A receptor immunoreactivities largely colocalize. Furthermore, mGlu2/3 immunoprecipitates from spinal cord synaptosomes were also 5-HT2A immunopositive. Interestingly, the 100 pM LY379268-induced reduction of the 15 mM KCl-evoked [3H]D-Asp overflow as well as its inhibition by 100 nM (±)DOI became undetectable when the two agonists were concomitantly added. Conversely, 5-HT2A antagonists (MDL11,939, MDL100907, ketanserin and TZD) reinforced the release-regulating activity of mGlu2/3 autoreceptors. Increased expression of mGlu2/3 receptor proteins in synaptosomal plasmamembranes paralleled the gain of function of the mGlu2/3 autoreceptors elicited by 5-HT2A antagonists. Based on these results, we propose that in spinal cord glutamatergic terminals i) mGlu2/3 and 5-HT2A receptors colocalize and interact one each other in an antagonist-like manner, ii) 5-HT2A antagonists are indirect positive allosteric modulator of mGlu2/3 autoreceptors controlling glutamate exocytosis.

Prophylactic versus Therapeutic Fingolimod: Restoration of Presynaptic Defects in Mice Suffering from Experimental Autoimmune Encephalomyelitis.

Fingolimod, the first oral, disease-modifying therapy for MS, has been recently proposed to modulate glutamate transmission in the central nervous system (CNS) of mice suffering from Experimental Autoimmune Encephalomyelitis (EAE) and in MS patients. Our study aims at investigating whether oral fingolimod recovers presynaptic defects that occur at different stages of disease in the CNS of EAE mice. In vivo prophylactic (0.3 mg/kg for 14 days, from the 7th day post immunization, d.p.i, the drug dissolved in the drinking water) fingolimod significantly reduced the clinical symptoms and the anxiety-related behaviour in EAE mice. Spinal cord inflammation, demyelination and glial cell activation are markers of EAE progression. These signs were ameliorated following oral fingolimod administration. Glutamate exocytosis was shown to be impaired in cortical and spinal cord terminals isolated from EAE mice at 21 ± 1 d.p.i., while GABA alteration emerged only at the spinal cord level. Prophylactic fingolimod recovered these presynaptic defects, restoring altered glutamate and GABA release efficiency. The beneficial effect occurred in a dose-dependent, region-specific manner, since lower (0.1-0.03 mg/kg) doses restored, although to a different extent, synaptic defects in cortical but not spinal cord terminals. A delayed reduction of glutamate, but not of GABA, exocytosis was observed in hippocampal terminals of EAE mice at 35 d.p.i. Therapeutic (0.3 mg/kg, from 21 d.p.i. for 14 days) fingolimod restored glutamate exocytosis in the cortex and in the hippocampus of EAE mice at 35 ± 1 d.p.i. but not in the spinal cord, where also GABAergic defects remained unmodified. These results improve our knowledge of the molecular events accounting for the beneficial effects elicited by fingolimod in demyelinating disorders.

Long-term hippocampal glutamate synapse and astrocyte dysfunctions underlying the altered phenotype induced by adolescent THC treatment in male rats.

Cannabis use has been frequently associated with sex-dependent effects on brain and behavior. We previously demonstrated that adult female rats exposed to delta-9-tetrahydrocannabinol (THC) during adolescence develop long-term alterations in cognitive performances and emotional reactivity, whereas preliminary evidence suggests the presence of a different phenotype in male rats. To thoroughly depict the behavioral phenotype induced by adolescent THC exposure in male rats, we treated adolescent animals with increasing doses of THC twice a day (PND 35-45) and, at adulthood, we performed a battery of behavioral tests to measure affective- and psychotic-like symptoms as well as cognition. Poorer memory performance and psychotic-like behaviors were present after adolescent THC treatment in male rats, without alterations in the emotional component. At cellular level, the expression of the NMDA receptor subunit, GluN2B, as well as the levels of the AMPA subunits, GluA1 and GluA2, were significantly increased in hippocampal post-synaptic fractions from THC-exposed rats compared to controls. Furthermore, increases in the levels of the pre-synaptic marker, synaptophysin, and the post-synaptic marker, PSD95, were also present. Interestingly, KCl-induced [(3)H]D-ASP release from hippocampal synaptosomes, but not gliosomes, was significantly enhanced in THC-treated rats compared to controls. Moreover, in the same brain region, adolescent THC treatment also resulted in a persistent neuroinflammatory state, characterized by increased expression of the astrocyte marker, GFAP, increased levels of the pro-inflammatory markers, TNF-α, iNOS and COX-2, as well as a concomitant reduction of the anti-inflammatory cytokine, IL-10. Notably, none of these alterations was observed in the prefrontal cortex (PFC). Together with our previous findings in females, these data suggest that the sex-dependent detrimental effects induced by adolescent THC exposure on adult behavior may rely on its ability to trigger different region-dependent changes in glutamate synapse and glial cells. The phenotype observed in males is mainly associated with marked dysregulations in the hippocampus, whereas the prevalence of alterations in the emotional sphere in females is associated with profound changes in the PFC.