Lessons from the physiological role of guanosine in neurodegeneration and cancer: Toward a multimodal mechanism of action?

Neurodegenerative diseases and brain tumours represent important health challenges due to their severe nature and debilitating consequences that require substantial medical care. Interestingly, these conditions share common physiological characteristics, namely increased glutamate, and adenosine transmission, which are often associated with cellular dysregulation and damage. Guanosine, an endogenous nucleoside, is safe and exerts neuroprotective effects in preclinical models of excitotoxicity, along with cytotoxic effects on tumour cells. However, the lack of well-defined mechanisms of action for guanosine hinders a comprehensive understanding of its physiological effects. In fact, the absence of specific receptors for guanosine impedes the development of structure-activity research programs to develop guanosine derivatives for therapeutic purposes. Alternatively, given its apparent interaction with the adenosinergic system, it is plausible that guanosine exerts its neuroprotective and anti-tumorigenic effects by modulating adenosine transmission through undisclosed mechanisms involving adenosine receptors, transporters, and purinergic metabolism. Here, several potential molecular mechanisms behind the protective actions of guanosine will be discussed. First, we explore its potential interaction with adenosine receptors (A1R and A2AR), including the A1R-A2AR heteromer. In addition, we consider the impact of guanosine on extracellular adenosine levels and the role of guanine-based purine-converting enzymes. Collectively, the diverse cellular functions of guanosine as neuroprotective and antiproliferative agent suggest a multimodal and complementary mechanism of action.

Neuroprotective and Antioxidant Role of Oxotremorine-M, a Non-selective Muscarinic Acetylcholine Receptors Agonist, in a Cellular Model of Alzheimer Disease.

Alzheimer disease (AD) is a multifactorial and age-dependent neurodegenerative disorder, whose pathogenesis, classically associated with the formation of senile plaques and neurofibrillary tangles, is also dependent on oxidative stress and neuroinflammation chronicization. Currently, the standard symptomatic therapy, based on acetylcholinesterase inhibitors, showed a limited therapeutic potential, whereas disease-modifying treatment strategies are still under extensive research. Previous studies have demonstrated that Oxotremorine-M (Oxo), a non-selective muscarinic acetylcholine receptors agonist, exerts neurotrophic functions in primary neurons, and modulates oxidative stress and neuroinflammation phenomena in rat brain. In the light of these findings, in this study, we aimed to investigate the neuroprotective effects of Oxo treatment in an in vitro model of AD, represented by differentiated SH-SY5Y neuroblastoma cells exposed to Aß1-42 peptide. The results demonstrated that Oxo treatment enhances cell survival, increases neurite length, and counteracts DNA fragmentation induced by Aß1-42 peptide. The same treatment was also able to block oxidative stress and mitochondria morphological/functional impairment associated with Aß1-42 cell exposure. Overall, these results suggest that Oxo, by modulating cholinergic neurotransmission, survival, oxidative stress response, and mitochondria functionality, may represent a novel multi-target drug able to achieve a therapeutic synergy in AD. Illustration of the main pathological hallmarks and mechanisms underlying AD pathogenesis, including neurodegeneration and oxidative stress, efficiently counteracted by treatment with Oxo, which may represent a promising therapeutic molecule. Created with BioRender.com under academic license.

Guanosine modulates K+ membrane currents in SH-SY5Y cells: involvement of adenosine receptors.

Guanosine (GUO), widely considered a key signaling mediator, is implicated in the regulation of several cellular processes. While its interaction with neural membranes has been described, GUO still is an orphan neuromodulator. It has been postulated that GUO may eventually interact with potassium channels and adenosine (ADO) receptors (ARs), both particularly important for the control of cellular excitability. Accordingly, here, we investigated the effects of GUO on the bioelectric activity of human neuroblastoma SH-SY5Y cells by whole-cell patch-clamp recordings. We first explored the contribution of voltage-dependent K+ channels and, besides this, the role of ARs in the regulation of GUO-dependent cellular electrophysiology. Our data support that GUO is able to specifically modulate K+-dependent outward currents over cell membranes. Importantly, administering ADO along with GUO potentiates its effects. Overall, these results suggested that K+ outward membrane channels may be targeted by GUO with an implication of  ADO receptors in SH-SY5Y cells, but also support the hypothesis of a functional interaction of the two ligands. The present research runs through the leitmotif of the deorphanization of GUO, adding insight on the interplay with adenosinergic signaling and suggesting GUO as a powerful modulator of SH-SY5Y excitability.

Compartmentalization of adenosine metabolism in cancer cells and its modulation during acute hypoxia.

Extracellular adenosine mediates diverse anti-inflammatory, angiogenic and vasoactive effects, and has become an important therapeutic target for cancer, which has been translated into clinical trials. This study was designed to comprehensively assess adenosine metabolism in prostate and breast cancer cells. We identified cellular adenosine turnover as a complex cascade, comprising (1) the ectoenzymatic breakdown of ATP via sequential ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1, officially known as ENPP1), ecto-5'-nucleotidase (CD73, also known as NT5E), and adenosine deaminase reactions, and ATP re-synthesis through a counteracting adenylate kinase and members of the nucleoside diphosphate kinase (NDPK, also known as NME/NM23) family; (2) the uptake of nucleotide-derived adenosine via equilibrative nucleoside transporters; and (3) the intracellular adenosine phosphorylation into ATP by adenosine kinase and other nucleotide kinases. The exposure of cancer cells to 1% O2 for 24 h triggered an ∼2-fold upregulation of CD73, without affecting nucleoside transporters, adenosine kinase activity and cellular ATP content. The ability of adenosine to inhibit the tumor-initiating potential of breast cancer cells via a receptor-independent mechanism was confirmed in vivo using a xenograft mouse model. The existence of redundant pathways controlling extracellular and intracellular adenosine provides a sufficient justification for reexamination of the current concepts of cellular purine homeostasis and signaling in cancer.This article has an associated First Person interview with the first author of the paper.

Purinergic Signaling in Oral Tissues.

The role of the purinergic signal has been extensively investigated in many tissues and related organs, including the central and peripheral nervous systems as well as the gastrointestinal, cardiovascular, respiratory, renal, and immune systems. Less attention has been paid to the influence of purines in the oral cavity, which is the first part of the digestive apparatus and also acts as the body's first antimicrobial barrier. In this review, evidence is provided of the presence and possible physiological role of the purinergic system in the different structures forming the oral cavity including teeth, tongue, hard palate, and soft palate with their annexes such as taste buds, salivary glands, and nervous fibers innervating the oral structures. We also report findings on the involvement of the purinergic signal in pathological conditions affecting the oral apparatus such as Sjögren's syndrome or following irradiation for the treatment of head and neck cancer, and the use of experimental drugs interfering with the purine system to improve bone healing after damage. Further investigations are required to translate the results obtained so far into the clinical setting in order to pave the way for a wider application of purine-based treatments in oral diseases.

Low-Density Lipoprotein Receptor-Related Protein 8 at the Crossroad between Cancer and Neurodegeneration.

The low-density-lipoprotein receptors represent a family of pleiotropic cell surface receptors involved in lipid homeostasis, cell migration, proliferation and differentiation. The family shares common structural features but also has significant differences mainly due to tissue-specific interactors and to peculiar proteolytic processing. Among the receptors in the family, recent studies place low-density lipoprotein receptor-related protein 8 (LRP8) at the center of both neurodegenerative and cancer-related pathways. From one side, its overexpression has been highlighted in many types of cancer including breast, gastric, prostate, lung and melanoma; from the other side, LRP8 has a potential role in neurodegeneration as apolipoprotein E (ApoE) and reelin receptor, which are, respectively, the major risk factor for developing Alzheimer's disease (AD) and the main driver of neuronal migration, and as a γ-secretase substrate, the main enzyme responsible for amyloid formation in AD. The present review analyzes the contributions of LDL receptors, specifically of LRP8, in both cancer and neurodegeneration, pointing out that depending on various interactions and peculiar processing, the receptor can contribute to both proliferative and neurodegenerative processes.

Bidirectional Control between Cholesterol Shuttle and Purine Signal at the Central Nervous System.

Recent studies have highlighted the mechanisms controlling the formation of cerebral cholesterol, which is synthesized in situ primarily by astrocytes, where it is loaded onto apolipoproteins and delivered to neurons and oligodendrocytes through interactions with specific lipoprotein receptors. The "cholesterol shuttle" is influenced by numerous proteins or carbohydrates, which mainly modulate the lipoprotein receptor activity, function and signaling. These molecules, provided with enzymatic/proteolytic activity leading to the formation of peptide fragments of different sizes and specific sequences, could be also responsible for machinery malfunctions, which are associated with neurological, neurodegenerative and neurodevelopmental disorders. In this context, we have pointed out that purines, ancestral molecules acting as signal molecules and neuromodulators at the central nervous system, can influence the homeostatic machinery of the cerebral cholesterol turnover and vice versa. Evidence gathered so far indicates that purine receptors, mainly the subtypes P2Y2, P2X7 and A2A, are involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer's and Niemann-Pick C diseases, by controlling the brain cholesterol homeostasis; in addition, alterations in cholesterol turnover can hinder the purine receptor function. Although the precise mechanisms of these interactions are currently poorly understood, the results here collected on cholesterol-purine reciprocal control could hopefully promote further research.

Adipose Stromal/Stem Cell-Derived Extracellular Vesicles: Potential Next-Generation Anti-Obesity Agents.

Over the last decade, several compounds have been identified for the treatment of obesity. However, due to the complexity of the disease, many pharmacological interventions have raised concerns about their efficacy and safety. Therefore, it is important to discover new factors involved in the induction/progression of obesity. Adipose stromal/stem cells (ASCs), which are mostly isolated from subcutaneous adipose tissue, are the primary cells contributing to the expansion of fat mass. Like other cells, ASCs release nanoparticles known as extracellular vesicles (EVs), which are being actively studied for their potential applications in a variety of diseases. Here, we focused on the importance of the con-tribution of ASC-derived EVs in the regulation of metabolic processes. In addition, we outlined the advantages/disadvantages of the use of EVs as potential next-generation anti-obesity agents.

Pros and Cons of Pharmacological Manipulation of cGMP-PDEs in the Prevention and Treatment of Breast Cancer.

The cyclic nucleotides, cAMP and cGMP, are ubiquitous second messengers responsible for translating extracellular signals to intracellular biological responses in both normal and tumor cells. When these signals are aberrant or missing, cells may undergo neoplastic transformation or become resistant to chemotherapy. cGMP-hydrolyzing phosphodiesterases (PDEs) are attracting tremendous interest as drug targets for many diseases, including cancer, where they regulate cell growth, apoptosis and sensitization to radio- and chemotherapy. In breast cancer, PDE5 inhibition is associated with increased intracellular cGMP levels, which is responsible for the phosphorylation of PKG and other downstream molecules involved in cell proliferation or apoptosis. In this review, we provide an overview of the most relevant studies regarding the controversial role of PDE inhibitors as off-label adjuvants in cancer therapy.

Adult mesenchymal stem cells: is there a role for purine receptors in their osteogenic differentiation?

The role played by mesenchymal stem cells (MSCs) in contributing to adult tissue homeostasis and damage repair thanks to their differentiation capabilities has raised a great interest, mainly in bone regenerative medicine. The growth/function of these undifferentiated cells of mesodermal origin, located in specialized structures (niches) of differentiated organs is influenced by substances present in this microenvironment. Among them, ancestral and ubiquitous molecules such as adenine-based purines, i.e., ATP and adenosine, may be included. Notably, extracellular purine concentrations greatly increase during tissue injury; thus, MSCs are exposed to effects mediated by these agents interacting with their own receptors when they act/migrate in vivo or are transplanted into a damaged tissue. Here, we reported that ATP modulates MSC osteogenic differentiation via different P2Y and P2X receptors, but data are often inconclusive/contradictory so that the ATP receptor importance for MSC physiology/differentiation into osteoblasts is yet undetermined. An exception is represented by P2X7 receptors, whose expression was shown at various differentiation stages of bone cells resulting essential for differentiation/survival of both osteoclasts and osteoblasts. As well, adenosine, usually derived from extracellular ATP metabolism, can promote osteogenesis, likely via A2B receptors, even though findings from human MSCs should be implemented and confirmed in preclinical models. Therefore, although many data have revealed possible effects caused by extracellular purines in bone healing/remodeling, further studies, hopefully performed in in vivo models, are necessary to identify defined roles for these compounds in favoring/increasing the pro-osteogenic properties of MSCs and thereby their usefulness in bone regenerative medicine.

Endothelial cells cope with hypoxia-induced depletion of ATP via activation of cellular purine turnover and phosphotransfer networks.

Intravascular ATP and adenosine have emerged as important regulators of endothelial barrier function, vascular remodeling and neovascularization at various pathological states, including hypoxia, inflammation and oxidative stress. By using human umbilical vein endothelial cells (HUVEC) and bovine vasa vasorum endothelial cells (VVEC) as representatives of macro- and microvessel phenotypes, this study was undertaken to evaluate cellular mechanisms contributing to physiological adaptation of vascular endothelium to hypoxia, with a particular emphasis on ectoenzymatic purine-converting activities and their link to intracellular ATP homeostasis and signaling pathways. Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), ecto-5'-nucleotidase/CD73 and ecto-adenylate kinase activities were determined by thin-layer chromatography (TLC) with 3H-labelled nucleotide substrates. Exposure of HUVEC and VVEC to 1% O2 for 4-24 h triggered rather moderate activation of ATP breakdown into adenosine via the CD39-CD73 axis. Additional TLC analysis of salvage pathways revealed the enhanced ability of hypoxic HUVEC to convert cell-incorporated [3H]adenosine into [3H]ADP/ATP. Furthermore, following a period of hypoxia, HUVEC underwent concurrent changes in intracellular signaling manifested in the depletion of putative ATP stores and targeted up-regulation of phospho-p53, p70S6K/mTOR and other tyrosine kinases. The revealed complex implication of both extrinsic and intrinsic mechanisms into a tuned hypoxia-induced control of purine homeostasis and signaling may open up further research for the development of pharmacological treatments to improve endothelial cell function under disease conditions associated with a loss of cellular ATP during oxygen deprivation.

Evidence for purine nucleoside phosphorylase (PNP) release from rat C6 glioma cells.

Intracellular purine turnover is mainly oriented to preserving the level of triphosphate nucleotides, fundamental molecules in vital cell functions that, when released outside cells, act as receptor signals. Conversely, high levels of purine bases and uric acid are found in the extracellular milieu, even in resting conditions. These compounds could derive from nucleosides/bases that, having escaped to cell reuptake, are metabolized by extracellular enzymes similar to the cytosolic ones. Focusing on purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of purine (deoxy)-nucleosides/bases, we found that it is constitutively released from cultured rat C6 glioma cells into the medium, and has a molecular weight and enzyme activity similar to the cytosolic enzyme. Cell exposure to 10 µM ATP or guanosine triphosphate (GTP) increased the extracellular amount of all corresponding purines without modifying the levels/activity of released PNP, whereas selective activation of ATP P2Y1 or adenosine A2A metabotropic receptors increased PNP release and purine base formation. The reduction to 1% in oxygen supply (2 h) to cells decreased the levels of released PNP, leading to an increased presence of extracellular nucleosides and to a reduced formation of xanthine and uric acid. Conversely, 2 h cell re-oxygenation enhanced the extracellular amounts of both PNP and purine bases. Thus, hypoxia and re-oxygenation modulated in opposite manner the PNP release/activity and, thereby, the extracellular formation of purine metabolism end-products. In conclusion, extracellular PNP and likely other enzymes deputed to purine base metabolism are released from cells, contributing to the purinergic system homeostasis and exhibiting an important pathophysiological role.

Modulation of the TGF-ß1-induced epithelial to mesenchymal transition (EMT) mediated by P1 and P2 purine receptors in MDCK cells.

Epithelial to mesenchymal transition (EMT) occurs during embryogenesis or under pathological conditions such as hypoxia, injury, chronic inflammation, or tissue fibrosis. In renal tubular epithelial cells (MDCK), TGF-ß1 induces EMT by reducing or increasing epithelial or mesenchymal marker expression, respectively. In this study, we confirmed that the cAMP analogues, 8-CPT-cAMP or N6-Ph-cAMP, inhibited the TGF-ß1-driven overexpression of the mesenchymal markers ZEB-1, Slug, Fibronectin, and α-SMA. Furthermore, we showed that A1, A2A, P2Y1, P2Y11, and P2X7 purine receptor agonists modulated the TGF-ß1-induced EMT through the involvement of PKA and/or MAPK/ERK signaling. The stimulation of A2A receptor reduced the overexpression of the EMT-related markers, mainly through the cAMP-dependent PKA pathway, as confirmed by cell pre-treatment with Myr-PKI. Both A1 and P2Y1 receptor stimulation exacerbated the TGF-ß1-driven effects, which were reduced by cell pre-treatment with the MAPK inhibitor PD98059, according to the increased ERK1/2 phosphorylation upon receptor activation. The effects induced by P2Y11 receptor activation were oppositely modulated by PKA or MAPK inhibition, in line with the dual nature of the Gs- and Gq-coupled receptor. Differently, P2X7 receptor induced, per se, similar and not additive effects compared to TGF-ß1, after prolonged cell exposure to BzATP. These results suggest a putative role of purine receptors as target for anti-fibrotic agents.

Potentiation of temozolomide antitumor effect by purine receptor ligands able to restrain the in vitro growth of human glioblastoma stem cells.

Glioblastoma multiforme (GBM), the most common and aggressive brain tumor in humans, comprises a population of stem-like cells (GSCs) that are currently investigated as potential target for GBM therapy. Here, we used GSCs isolated from three different GBM surgical specimens to examine the antitumor activity of purines. Cultured GSCs expressed either metabotropic adenosine P1 and ATP P2Y receptors or ionotropic P2X7 receptors. GSC exposure for 48 h to 10-150 µM ATP, P2R ligand, or to ADPßS or MRS2365, P2Y1R agonists, enhanced cell expansion. This effect was counteracted by the PY1R antagonist MRS2500. In contrast, 48-h treatment with higher doses of ATP or UTP, which binds to P2Y2/4R, or 2'(3')-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), P2X7R agonist, decreased GSC proliferation. Such a reduction was due to apoptotic or necrotic cell death but mostly to growth arrest. Accordingly, cell regrowth and secondary neurosphere formation were observed 2 weeks after the end of treatment. Suramin, nonselective P2R antagonist, MRS1220 or AZ11645373, selective A3R or P2X7R antagonists, respectively, counteracted ATP antiproliferative effects. AZ11645373 also abolished the inhibitory effect of Bz-ATP low doses on GSC growth. These findings provide important clues on the anticancer potential of ligands for A3R, P2Y1R, and P2X7R, which are involved in the GSC growth control. Interestingly, ATP and BzATP potentiated the cytotoxicity of temozolomide (TMZ), currently used for GBM therapy, enabling it to cause a greater and long-lasting inhibitory effect on GSC duplication when readded to cells previously treated with purine nucleotides plus TMZ. These are the first findings identifying purine nucleotides as able to enhance TMZ antitumor efficacy and might have an immediate translational impact.

A Proteomic Approach Identified TFEB as a Key Player in the Protective Action of Novel CB2R Bitopic Ligand FD22a against the Deleterious Effects Induced by ß-Amyloid in Glial Cells.

Neurodegenerative diseases (NDDs) are progressive multifactorial disorders of the nervous system sharing common pathogenic features, including intracellular misfolded protein aggregation, mitochondrial deficit, and inflammation. Taking into consideration the multifaceted nature of NDDs, development of multitarget-directed ligands (MTDLs) has evolved as an attractive therapeutic strategy. Compounds that target the cannabinoid receptor type II (CB2R) are rapidly emerging as novel effective MTDLs against common NDDs, such as Alzheimer's disease (AD). We recently developed the first CB2R bitopic/dualsteric ligand, namely FD22a, which revealed the ability to induce neuroprotection with fewer side effects. To explore the potential of FD22a as a multitarget drug for the treatment of NDDs, we investigated here its ability to prevent the toxic effect of ß-amyloid (Aß25-35 peptide) on human cellular models of neurodegeneration, such as microglia (HMC3) and glioblastoma (U87-MG) cell lines. Our results displayed that FD22a efficiently prevented Aß25-35 cytotoxic and proinflammatory effects in both cell lines and counteracted ß-amyloid-induced depression of autophagy in U87-MG cells. Notably, a quantitative proteomic analysis of U87-MG cells revealed that FD22a was able to potently stimulate the autophagy-lysosomal pathway (ALP) by activating its master transcriptional regulator TFEB, ultimately increasing the potential of this novel CB2R bitopic/dualsteric ligand as a multitarget drug for the treatment of NDDs.

Management and Rehabilitative Treatment in Osteoarthritis with a Novel Physical Therapy Approach: A Randomized Control Study.

Knee osteoarthritis (KOA) is a chronic degenerative disease characterized by progressive joint damage leading to significant disability. Although rehabilitative treatment methods for KOA have been widely implemented, the optimal integrated instrumental physical therapy approach remains unclear. Therefore, this study aimed to analyze the effect of Quantum Molecular Resonance (QMR) on pain reduction as the primary outcome and the functional improvement in activity daily living (ADL) as a secondary outcome. The study was designed as a double-blind, randomized, controlled trial in an outpatient setting. Fifty-four (N = 54) patients were enrolled and then randomized into three groups according to a simple randomization list: Group 1 (intensive protocol, N = 22), Group 2 (extensive protocol, N = 21), and a Sham group (N = 11). Patients were evaluated over time with scales assessing pain and function. Treatment was performed with the QMR model electro-medical device, which generates alternating electric currents characterized by high frequency (4-64 MHz). The results showed that QMR had a positive effect with respect to the Sham group in terms of pain and function (p < 0.01), and intensive treatment was more effective than the extensive treatment in terms of "speed of response" to the treatment (p < 0.05). In conclusion, QMR in KOA could be effective in slowing the progression of clinical symptoms and improving patients' pain and functionality and thus quality of life. Future studies will be necessary to investigate further treatment algorithms and therapeutic associations with rehabilitative exercise.

Influence of Guanine-Based Purines on the Oxidoreductive Reactions Involved in Normal or Altered Brain Functions.

The production of reactive oxygen species (ROS) in the brain is homeostatically controlled and contributes to normal neural functions. Inefficiency of control mechanisms in brain aging or pathological conditions leads to ROS overproduction with oxidative neural cell damage and degeneration. Among the compounds showing therapeutic potential against neuro-dysfunctions induced by oxidative stress are the guanine-based purines (GBPs), of which the most characterized are the nucleoside guanosine (GUO) and the nucleobase guanine (GUA), which act differently. Indeed, the administration of GUO to in vitro or in vivo models of acute brain injury (ischemia/hypoxia or trauma) or chronic neurological/neurodegenerative disorders, exerts neuroprotective and anti-inflammatory effects, decreasing the production of reactive radicals and improving mitochondrial function via multiple molecular signals. However, GUO administration to rodents also causes an amnesic effect. In contrast, the metabolite, GUA, could be effective in memory-related disorders by transiently increasing ROS production and stimulating the nitric oxide/soluble guanylate cyclase/cGMP/protein kinase G cascade, which has long been recognized as beneficial for cognitive function. Thus, it is worth pursuing further studies to ascertain the therapeutic role of GUO and GUA and to evaluate the pathological brain conditions in which these compounds could be more usefully used.

Proteomic Profiling Reveals Specific Molecular Hallmarks of the Pig Claustrum.

The present study, employing a comparative proteomic approach, analyzes the protein profile of pig claustrum (CLA), putamen (PU), and insula (IN). Pig brain is an interesting model whose key translational features are its similarities with cortical and subcortical structures of human brain. A greater difference in protein spot expression was observed in CLA vs PU as compared to CLA vs IN. The deregulated proteins identified in CLA resulted to be deeply implicated in neurodegenerative (i.e., sirtuin 2, protein disulfide-isomerase 3, transketolase) and psychiatric (i.e., copine 3 and myelin basic protein) disorders in humans. Metascape analysis of differentially expressed proteins in CLA vs PU comparison suggested activation of the α-synuclein pathway and L1 recycling pathway corroborating the involvement of these anatomical structures in neurodegenerative diseases. The expression of calcium/calmodulin-dependent protein kinase and dihydropyrimidinase like 2, which are linked to these pathways, was validated using western blot analysis. Moreover, the protein data set of CLA vs PU comparison was analyzed by Ingenuity Pathways Analysis to obtain a prediction of most significant canonical pathways, upstream regulators, human diseases, and biological functions. Interestingly, inhibition of presenilin 1 (PSEN1) upstream regulator and activation of endocannabinoid neuronal synapse pathway were observed. In conclusion, this is the first study presenting an extensive proteomic analysis of pig CLA in comparison with adjacent areas, IN and PUT. These results reinforce the common origin of CLA and IN and suggest an interesting involvement of CLA in endocannabinoid circuitry, neurodegenerative, and psychiatric disorders in humans.

Alterations in rat prefrontal cortex kynurenic acid levels are involved in the enduring cognitive dysfunctions induced by tetrahydrocannabinol exposure during the adolescence.

Introduction: Cannabis abuse during adolescence is a risk factor for cognitive impairments in psychiatric disorders later in life. To date, the possible causal relationship between cannabinoids, kynurenic acid (KYNA; i.e., a neuroactive metabolite of tryptophan degradation) and cognition has not been investigated in adolescence. Early exposure to delta 9-tetrahydrocannabinol (THC; i.e., the main psychotropic component of cannabis) causes enduring cognitive deficits, which critically involve impaired glutamatergic function in the prefrontal cortex (PFC). In addition, prenatal cannabis exposure results in enduring increases in PFC KYNA levels. Based on these findings, the effects of chronic THC exposure in rats, during another critical period of neurodevelopment particularly sensitive to perturbation by exogenous stimuli, such as adolescence, have been investigated. Methods: Male Wistar rats were chronically treated with vehicle or ascending intraperitoneal (i.p.) doses of THC starting on postnatal day (PND) 35 until PND 45. In adulthood (PND 75), cognitive assessment (Y-maze) and extracellular KYNA/glutamate levels were measured in the PFC by in vivo microdialysis, before and after a challenge with KYN (5 mg/kg i.p., the biological precursor of KYNA). By using the selective, brain-penetrable KAT II inhibitor PF-04859989, we then examined whether blockade of KYNA neosynthesis prevents the cognitive impairment. Results: Compared to vehicle-treated controls, extracellular basal KYNA levels were higher in the PFC of adult rats chronically exposed to THC in adolescence (p < 0.01). No changes were observed in extracellular glutamate levels. Following a challenge with KYN, extracellular KYNA levels similarly increased in both groups (i.e., vehicle- and THC-treated; p < 0.001 and p < 0.01, respectively). Chronic adolescent THC exposure negatively affected short-term memory (reduced spontaneous alternation), in adult animals (p < 0.001), while PF-04859989 (30 mg/kg i.p.) restored the cognitive impairment (p < 0.05). Discussion: We propose that the observed alterations in PFC KYNA signaling might be involved in the cognitive dysfunction induced by the exposure to THC during the adolescence. In the translational realm, these experiments raise the prospect of prevention of KYNA neosynthesis as a possible novel approach to counteract some of the detrimental long-term effects of adolescence cannabis use.