Spinal neuronal activity and neuroinflammatory component in a mouse model of CFA-induced vestibulodynia.

Vestibulodynia is a complex pain disorder characterized by chronic discomfort in the vulvar region, often accompanied by tactile allodynia and spontaneous pain. In patients a depressive behaviour is also observed. In this study, we have used a model of vestibulodynia induced by complete Freund's adjuvant (CFA) focusing our investigation on the spinal cord neurons and microglia. We investigated tactile allodynia, spontaneous pain, and depressive-like behavior as key behavioral markers of vestibulodynia. In addition, we conducted in vivo electrophysiological recordings to provide, for the first time to our knowledge, the characterization of the spinal sacral neuronal activity in the L6-S1 dorsal horn of the spinal cord. Furthermore, we examined microglia activation in the L6-S1 dorsal horn using immunofluorescence, unveiling hypertrophic phenotypes indicative of neuroinflammation in the spinal cord. This represents a novel insight into the role of microglia in vestibulodynia pathology. To address the therapeutic aspect, we employed pharmacological interventions using GABApentin, amitriptyline, and PeaPol. Remarkably, all three drugs, also used in clinic, showed efficacy in alleviating tactile allodynia and depressive-like behavior. Concurrently, we also observed a normalization of the altered neuronal firing and a reduction of microglia hypertrophic phenotypes. In conclusion, our study provides a comprehensive understanding of the CFA-induced model of vestibulodynia, encompassing behavioral, neurophysiological and neuroinflammatory aspects. These data pave the way to investigate spinal cord first pain plasticity in vestibulodynia.

The pharmacological management of asthma in adults: 2023 update.

INTRODUCTION: The pharmacotherapy of asthma is a dynamic process that changes as our knowledge of the underlying pathophysiology and treatment of this disease continues to evolve. This implies the need for continuous revision of the recommendations of asthma guidelines and strategies. AREAS COVERED: This review summarizes the latest key practical information on the pharmacological management of asthma in adults. We provide the background to the 2023 update of the GINA strategy report, focusing on changes and discussing areas of uncertainty. We review current and emerging pharmacotherapy for uncontrolled asthma, including synthetic agents and new biologics, and provide expert perspectives and opinions on the treatment of uncontrolled asthma. EXPERT OPINION: The current pharmacological treatment of asthma, based on a step-by-step, control-based approach, with ICSs, LABAs and LAMAs being the mainstay generally provides good symptom control. Biologic therapies are often effective in treating T2high severe asthma. However, there is still room for improvement, such as the discovery of new molecules that specifically target chronic inflammation and, most importantly, the ability to provide solutions to the various areas of uncertainty that still exist. Also finding solutions to improve the accessibility and affordability of rescue ICS in resource-constrained settings is critical.

Current and emerging treatment modalities for bacterial rhinosinusitis in adults: a comprehensive review.

INTRODUCTION: Rhinosinusitis (RS) is defined as acute when it lasts up to 4 weeks and chronic when it lasts at least 12 weeks. Most acute forms begin with a viral upper respiratory infection that spreads into the paranasal sinuses and is followed by bacterial infection. It is uncertain how bacteria affect chronic rhinosinusitis (CRS). AREA COVERED: We review the current treatment of bacterial rhinosinusitis in adults, referring mainly to the two key documents published by the European Position Paper on Rhinosinusitis and Nasal Polyps 2020 and the International Consensus Statement 2021 on Allergy and Rhinology: Rhinosinusitis. EXPERT OPINION: The routine use of antibiotics should be avoided because most acute RS (ARS) have a viral origin. In patients with persistent/worsening symptoms, the most appropriate empirical therapy is a course of amoxicillin (with or without clavulanate). Macrolides are considered therapy options for CRS mainly because of their anti-inflammatory activity. The best agent, dose, and treatment duration still need to be identified due to a lack of solid evidence. Inflammation and symptoms must also be reduced, mainly by using nasal corticosteroids. Since antibiotic use in bacterial rhinosinusitis is questionable, the research focuses on non-antibiotic antimicrobial treatments.

Clinical characteristics, treatment patterns and adherence in patients with asthma on multiple inhaler triple therapy: a review of findings.

INTRODUCTION: The value of treating asthma with the triple regimen of inhaled corticosteroid (ICS), long-acting ß2-agonist (LABA), and long-acting muscarinic antagonist (LAMA) delivered using multiple inhalers (MITT), or a single inhaler (SITT) is supported by a growing body of evidence, although research is still limited regarding the use of MITT. AREAS COVERED: Clinical characteristics, treatment patterns, disease burden, and persistence/adherence associated with MITT use in asthma. The MEDLINE database was searched to identify references from inception until October 2022. EXPERT OPINION: The use of MITT is not very frequent in asthma patients, although it improves lung function and reduces the incidence of severe exacerbations. This may be due to existing concerns about using different devices on adherence and persistence to treatment, with a negative influence on outcomes, and to the fear that the patient will discontinue ICS/LABA but not LAMA. Nevertheless, although the current trend favors the SITT approach, some physicians may be induced to prescribe MITT over SITT because it allows the titration of individual components of triple therapy to be increased or decreased. Therefore, there is an evident need for pragmatic real-life studies to document when to prefer SITT and when MITT should be used.

Long-term neuropathic pain behaviors correlate with synaptic plasticity and limbic circuit alteration: a comparative observational study in mice.

ABSTRACT: Neuropathic pain has long-term consequences in affective and cognitive disturbances, suggesting the involvement of supraspinal mechanisms. In this study, we used the spared nerve injury (SNI) model to characterize the development of sensory and aversive components of neuropathic pain and to determine their electrophysiological impact across prefrontal cortex and limbic regions. Moreover, we evaluated the regulation of several genes involved in immune response and inflammation triggered by SNI. We showed that SNI led to sensorial hypersensitivity (cold and mechanical stimuli) and depressive-like behavior lasting 12 months after nerve injury. Of interest, changes in nonemotional cognitive tasks (novel object recognition and Y maze) showed in 1-month SNI mice were not evident normal in the 12-month SNI animals. In vivo electrophysiology revealed an impaired long-term potentiation at prefrontal cortex-nucleus accumbens core pathway in both the 1-month and 12-month SNI mice. On the other hand, a reduced neural activity was recorded in the lateral entorhinal cortex-dentate gyrus pathway in the 1-month SNI mice, but not in the 12-month SNI mice. Finally, we observed the upregulation of specific genes involved in immune response in the hippocampus of 1-month SNI mice, but not in the 12-month SNI mice, suggesting a neuroinflammatory response that may contribute to the SNI phenotype. These data suggest that distinct brain circuits may drive the psychiatric components of neuropathic pain and pave the way for better investigation of the long-term consequences of peripheral nerve injury for which most of the available drugs are to date unsatisfactory.

Disputes over the production and dissemination of misinformation in the time of COVID-19.

Ultimate coronavirus disease 2019 (COVID-19) mitigation and crisis resolution is dependent on trustworthy data and actionable information. At present time, there is still no cure for COVID-19, although some treatments are being used in severe illness. Regrettably, as the SARS-CoV-2 virus spreads, the lack of cure has been accompanied by an increasing amount of medical misinformation. In particular, there is a lot of misinformation about how to treat patients who have tested positive for SARS-CoV-2 and who are asymptomatic or have mild symptoms and for whom management at home is deemed appropriate. In this editorial, we highlight the risks deriving from this misinformation, which often arises from the publication of studies that are not conceptually and methodologically accurate.

2-Pentadecyl-2-oxazoline ameliorates memory impairment and depression-like behaviour in neuropathic mice: possible role of adrenergic alpha2- and H3 histamine autoreceptors.

Neuropathic pain (NP) remains an untreatable disease due to the complex pathophysiology that involves the whole pain neuraxis including the forebrain. Sensory dysfunctions such as allodynia and hyperalgesia are only part of the symptoms associated with neuropathic pain that extend to memory and affectivity deficits. The development of multi-target molecules might be a promising therapeutic strategy against the symptoms associated with NP. 2-pentadecyl-2-oxazoline (PEA-OXA) is a plant-derived agent, which has shown effectiveness against chronic pain and associated neuropsychiatric disorders. The molecular mechanisms by which PEA-OXA exerts its effects are, however, only partially known. In the current study, we show that PEA-OXA, besides being an alpha2 adrenergic receptor antagonist, also acts as a modulator at histamine H3 receptors, and report data on its effects on sensory, affective and cognitive symptoms associated with the spared nerve injury (SNI) model of neuropathic pain in mice. Treatment for 14 days with PEA-OXA after the onset of the symptoms associated with neuropathic pain resulted in the following effects: (i) allodynia was decreased; (ii) affective/cognitive impairment associated with SNI (depression, spatial, and working memories) was counteracted; (iii) long-term potentiation in vivo in the lateral entorhinal cortex-dentate gyrus (perforant pathway, LPP) was ameliorated, (iv) hippocampal glutamate, GABA, histamine, norepinephrine and dopamine level alterations after peripheral nerve injury were reversed, (v) expression level of the TH positive neurons in the Locus Coeruleus were normalized. Thus, a 16-day treatment with PEA-OXA alleviates the sensory, emotional, cognitive, electrophysiological and neurochemical alterations associated with SNI-induced neuropathic pain.

Behavioral, Biochemical and Electrophysiological Changes in Spared Nerve Injury Model of Neuropathic Pain.

Neuropathic pain is a pathological condition induced by a lesion or disease affecting the somatosensory system, with symptoms like allodynia and hyperalgesia. It has a multifaceted pathogenesis as it implicates several molecular signaling pathways involving peripheral and central nervous systems. Affective and cognitive dysfunctions have been reported as comorbidities of neuropathic pain states, supporting the notion that pain and mood disorders share some common pathogenetic mechanisms. The understanding of these pathophysiological mechanisms requires the development of animal models mimicking, as far as possible, clinical neuropathic pain symptoms. Among them, the Spared Nerve Injury (SNI) model has been largely characterized in terms of behavioral and functional alterations. This model is associated with changes in neuronal firing activity at spinal and supraspinal levels, and induces late neuropsychiatric disorders (such as anxious-like and depressive-like behaviors, and cognitive impairments) comparable to an advanced phase of neuropathy. The goal of this review is to summarize current findings in preclinical research, employing the SNI model as a tool for identifying pathophysiological mechanisms of neuropathic pain and testing pharmacological agent.

Ultra-micronized palmitoylethanolamide rescues the cognitive decline-associated loss of neural plasticity in the neuropathic mouse entorhinal cortex-dentate gyrus pathway.

Chronic pain is associated with cognitive deficits. Palmitoylethanolamide (PEA) has been shown to ameliorate pain and pain-related cognitive impairments by restoring glutamatergic synapses functioning in the spared nerve injury (SNI) of the sciatic nerve in mice. SNI reduced mechanical and thermal threshold, spatial memory and LTP at the lateral entorhinal cortex (LEC)-dentate gyrus (DG) pathway. It decreased also postsynaptic density, volume and dendrite arborization of DG and increased the expression of metabotropic glutamate receptor 1 and 7 (mGluR1 and mGluR7), of the GluR1, GluR1s845 and GluR1s831 subunits of AMPA receptor and the levels of glutamate in the DG. The level of the endocannabinoid 2-arachidonoylglycerol (2-AG) was instead increased in the LEC. Chronic treatment with PEA, starting from when neuropathic pain was fully developed, was able to reverse mechanical allodynia and thermal hyperalgesia, memory deficit and LTP in SNI wild type, but not in PPARα null, mice. PEA also restored the level of glutamate and the expression of phosphorylated GluR1 subunits, postsynaptic density and neurogenesis. Altogether, these results suggest that neuropathic pain negatively affects cognitive behavior and related LTP, glutamatergic synapse and synaptogenesis in the DG. In these conditions PEA treatment alleviates pain and cognitive impairment by restoring LTP and synaptic maladaptative changes in the LEC-DG pathway. These outcomes open new perspectives for the use of the N-acylethanolamines, such as PEA, for the treatment of neuropathic pain and its central behavioural sequelae.

Ketones and pain: unexplored role of hydroxyl carboxylic acid receptor type 2 in the pathophysiology of neuropathic pain.

The mechanisms underlying neuropathic pain are poorly understood. Here we show the unexplored role of the hydroxyl carboxylic acid receptor type 2 (HCAR2) in 2 models of neuropathic pain. We used an oral treatment with dimethyl fumarate and the HCAR2 endogenous ligand ß-hydroxybutyrate (BHB) in wild-type (WT) and HCAR2-null mice. We found an up-regulation of the HCAR2 in the sciatic nerve and the dorsal root ganglia in neuropathic mice. Accordingly, acute and chronic treatment with dimethylfumarate (DMF) and BHB reduced the tactile allodynia. This effect was completely lost in the HCAR2-null mice after a 2-d starvation protocol, in which the BHB reached the concentration able to activate the HCAR2-reduced tactile allodynia in female WT mice, but not in the HCAR2-null mice. Finally, we showed that chronic treatment with DMF reduced the firing of the ON cells (cells responding with an excitation after noxious stimulation) of the rostral ventromedial medulla. Our results pave the way for investigating the mechanisms by which HCAR2 regulates neuropathic pain plasticity.-Boccella, S., Guida, F., De Logu, F., De Gregorio, D., Mazzitelli, M., Belardo, C., Iannotta, M., Serra, N., Nassini, R., de Novellis, V., Geppetti, P., Maione, S., Luongo, L. Ketones and pain: unexplored role of hydroxyl carboxylic acid receptor type 2 in the pathophysiology of neuropathic pain.

Metabotropic glutamate receptor subtype 7 in the dorsal striatum oppositely modulates pain in sham and neuropathic rats.

The study investigated the role of the metabotropic glutamate receptor subtype 7 (mGluR7) in pain signalling in the dorsal striatum of sham and neuropathic rats. Supraspinal circuitries involved in the dorsal striatum control of pain were also explored. In the sham rats, microinjection of N,N'-bis(diphenylmethyl)-1,2-ethanediamine (AMN082), a selective mGluR7 positive allosteric modulator, into the dorsal striatum, facilitated pain, increased the activity of the ON cells and inhibited the activity of the OFF cells in the rostral ventromedial medulla, and decreased glutamate levels in the dorsal striatum. Conversely, AMN082 inhibited pain and the activity of the ON cells while increased the activity of the OFF cells in rats with spared nerve injury (SNI) of the sciatic nerve. AMN082 also decreased glutamate levels in the dorsal striatum of SNI rats. The effect of AMN082 on mechanical allodynia and glutamate release was blocked by 6-(2,4-dimethylphenyl)-2-ethyl-6,7-dihydro-4(5H)-benzoxazolone (ADX71743), a selective mGluR7 negative allosteric modulator. Moreover, in the sham rats, AMN082 increased the activity of total nociceptive convergent neurons in the dorsal reticular nucleus while in the SNI rats, such activity was decreased. The administration of lidocaine into the subthalamic nucleus abolished the effect of AMN082 on the total nociceptive convergent neurons in the sham rats but not in the SNI rats. Thus, the dual effect of mGluR7 in facilitating or inhibiting pain responses may be due to the recruitment of different pathways of the basal ganglia, the indirect or direct pathway, in physiological or pathological conditions, respectively.

Spared Nerve Injury as a Long-Lasting Model of Neuropathic Pain.

This chapter describes surgical procedures for the induction of neuropathic pain using an animal model (rat or mouse) of spared nerve injury. In addition to technical details of the surgical technique, details of anesthesia and perioperative care are also included.

Preclinical evaluation of the urokinase receptor-derived peptide UPARANT as an anti-inflammatory drug.

BACKGROUND: Inflammation plays a key role in the pathogenesis of several chronic diseases. The urokinase plasminogen activator receptor (uPAR) exerts a plethora of functions in both physiological and pathological processes, including inflammation. OBJECTIVE AND DESIGN: In this study, we evaluated the anti-inflammatory effect of a novel peptide ligand of uPAR, UPARANT, in different animal models of inflammation. SUBJECTS AND TREATMENT: Rats and mice were divided in different groups (n = 5) for single or repeated administration of vehicle (9% DMSO in 0.9% NaCl), UPARANT (6, 12 and 24 mg/kg) or dexamethasone (2 mg/kg). Animals were subjected to carrageenan-induced paw oedema or zymosan-induced peritonitis. METHODS: UPARANT effects were tested on: (1) the carrageenan-induced paw oedema volume, (2) the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the nitrite/nitrate (NOx) levels in the paw exudates, (3) cells recruitment into the peritoneal cavity after zymosan injection and (4) NOx levels in the peritoneal lavage. RESULTS: UPARANT (12 and 24 mg/kg) reduced inflammation in both experimental paradigms. Analysis of pro-inflammatory enzymes revealed that administration of UPARANT reduced iNOS, COX2 and NO over-production. CONCLUSIONS: Our study provides a solid evidence that UPARANT reduces the severity of inflammation in diverse animal models, thus representing a novel anti-inflammatory drug with potential advantages with respect to the typical steroidal agents.

d-Aspartic acid ameliorates painful and neuropsychiatric changes and reduces ß-amyloid Aß1-42 peptide in a long lasting model of neuropathic pain.

Depressive symptoms and other neuropsychiatric dysfunctions are common in neurodegenerative disorders, including chronic pain and dementia. A correlation between the ß-amyloid protein accumulation and the development of depression has been suggested, however the underlying mechanisms are unknown. d-Aspartate (d-Asp) is a free d-amino acid found in the mammalian brain and involved in neurological and psychiatric processes, such as cognition and affective disorders. In this study we have investigated the effects of a repeated treatment with d-Asp in a long-lasting (12 months) model of neuropathic pain, the spared nerve injury (SNI), in mice. Specifically, we evaluated i) the pain sensitivity and related emotional/cognitive dysfunctions induced by SNI, ii) possible changes in the ß-amyloid protein accumulation in specific brain regions involved in pain mechanisms ii) possible changes in steroids level in neuropathic animals with or without d-Asp in the same brain areas. SNI mice showed an increase of the insoluble form of Aß1-42 at hippocampal level and displayed cognitive impairments, stereotypical and depressive-like behaviours. d-Asp treatment reduced abnormal behaviours and normalized the ß-amyloid protein expression. Moreover, d-Asp dramatically increased steroids level measured in the prefrontal cortex and in the hippocampus. Our findings provide new insights into pain mechanisms and suggest a possible role of ß-amyloid protein in neuropsychiatric dysfunctions associated with chronic pain.

Palmitoylethanolamide Reduces Neuropsychiatric Behaviors by Restoring Cortical Electrophysiological Activity in a Mouse Model of Mild Traumatic Brain Injury.

Traumatic brain injury (TBI) represents a major public health problem, which is associated with neurological dysfunction. In severe or moderate cases of TBI, in addition to its high mortality rate, subjects may encounter diverse behavioral dysfunctions. Previous reports suggest that an association between TBI and chronic pain syndromes tends to be more common in patients with mild forms of brain injury. Despite causing minimal brain damage, mild TBI (mTBI) often leads to persistent psychologically debilitating symptoms, which can include anxiety, various forms of memory and learning deficits, and depression. At present, no effective treatment options are available for these symptoms, and little is known about the complex cellular activity affecting neuronal activity that occurs in response to TBI during its late phase. Here, we used a mouse model to investigate the effect of Palmitoylethanolamide (PEA) on both the sensorial and neuropsychiatric dysfunctions associated with mTBI through behavioral, electrophysiological, and biomolecular approaches. Fourteen-day mTBI mice developed anxious, aggressive, and reckless behavior, whilst depressive-like behavior and impaired social interactions were observed from the 60th day onward. Altered behavior was associated with changes in interleukin 1 beta (IL-1ß) expression levels and neuronal firing activity in the medial prefrontal cortex. Compared with vehicle, PEA restored the behavioral phenotype and partially normalized the biochemical and functional changes occurring at the supraspinal level. In conclusion, our findings reveal some of the supraspinal modifications responsible for the behavioral alterations associated with mTBI and suggest PEA as a pharmacological tool to ameliorate neurological dysfunction induced by the trauma.

Nociception modulation by supraspinal group III metabotropic glutamate receptors.

The modulatory actions of glutamate, the main excitatory neurotransmitter in the central nervous system (CNS), are exerted through the activation of metabotropic glutamate receptors (mGluRs). Of the eight known mGluRs (mGluR1-8), group III mGluRs (mGluR4, mGluR6, mGluR7, and mGluR8) are less understood because of the lack of selective ligands. Except for mGluR6, group III mGluRs are widely distributed throughout the CNS. They are mainly located on presynaptic terminals where they inhibit neurotransmitter release at glutamatergic and γ-aminobutyric acid (GABA)ergic synapses. Their location at certain synapses is considered critical for normal CNS function, which makes them potential targets in neurological and psychiatric treatments. Novel ligands that are selective for group III mGluR subtypes have recently been developed. These compounds, which mainly target allosteric sites and act as positive or negative allosteric modulators (PAMs or NAMs) of glutamate transmission, are contributing to the understanding of the functional roles of group III mGluRs in a number of pathological conditions, such as epilepsy, anxiety, neurodegenerative diseases, and chronic pain. Moreover, the presence of group III mGluRs throughout the entire pain neuraxis and particularly in the descending system suggests that these endogenous substrates that extend from the cortex to the first spinal synapse are candidates for pain control. Recent data on chronic pain alleviation by group III mGluR ligands encourage further studies as pathological pain is one of the most troublesome diseases because of the current lack of satisfactory therapy. This review summarizes recent studies on group III mGluRs in animal models of chronic pain, which evidence an opposite modulation of mGluR7 and mGluR8 on pain responses and their capability to affect pain responses only in pathological states. This article is part of the special article series "Pain".

Metabotropic Glutamate Receptor 7: From Synaptic Function to Therapeutic Implications.

Metabotropic glutamate receptor 7 (mGluR7) is localized presynaptically at the active zone of neurotransmitter release. Unlike mGluR4 and mGluR8, which share mGluR7's presynaptic location, mGluR7 shows low affinity for glutamate and is activated only by high glutamate concentrations. Its wide distribution in the central nervous system (CNS) and evolutionary conservation across species suggest that mGluR7 plays a primary role in controlling excitatory synapse function. High mGluR7 expression has been observed in several brain regions that are critical for CNS functioning and are involved in neurological and psychiatric disorder development. Until the recent discovery of selective ligands for mGluR7, techniques to elucidate its role in neural function were limited to the use of knockout mice and gene silencing. Studies using these two techniques have revealed that mGluR7 modulates emotionality, stress and fear responses. N,N`-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082) was reported as the first selective mGluR7 allosteric agonist. Pharmacological effects of AMN082 have not completely confirmed the mGluR7-knockout mouse phenotype; this has been attributed to rapid receptor internalization after drug treatment and to the drug's apparent lack of in vivo selectivity. Therefore, the more recently developed mGluR7 negative allosteric modulators (NAMs) are crucial for understanding mGluR7 function and for exploiting its potential as a target for therapeutic interventions. This review presents the main findings regarding mGluR7's effect on modulation of synaptic function and its role in normal CNS function and in models of neurologic and psychiatric disorders.

D-Aspartate drinking solution alleviates pain and cognitive impairment in neuropathic mice.

D-Aspartate (D-Asp) is a free D-amino acid detected in multiple brain regions and putative precursor of endogenous N-methyl-D-aspartate (NMDA) acting as agonist at NMDA receptors. In this study, we investigated whether D-Asp (20 mM) in drinking solution for 1 month affects pain responses and pain-related emotional, and cognitive behaviour in a model of neuropathic pain induced by the spared nerve injury (SNI) of the sciatic nerve in mice. SNI mice developed mechanical allodynia and motor coordination impairment 30 days after SNI surgery. SNI mice showed cognitive impairment, anxiety and depression-like behaviour, reduced sociability in the three chamber sociability paradigm, increased expression of NR2B subunit of NMDA receptor and Homer 1a in the medial prefrontal cortex (mPFC). The expression of (post synaptic density) PSD-95 and Shank 1was instead unaffected in the mPFC of the SNI mice. Treatment with D-Asp drinking solution, started right after the SNI (day 0), alleviated mechanical allodynia, improved cognition and motor coordination and increased social interaction. D-Asp also restored the levels of extracellular D-Asp, Homer 1a and NR2B subunit of the NMDA receptor to physiological levels and reduced Shank1 and PSD-95 protein levels in the mPFC. Amitriptyline, a tricyclic antidepressant used also to alleviate neuropathic pain in humans, reverted mechanical allodynia and cognitive impairment, and unlike D-Asp, was effective in reducing depression and anxiety-like behaviour in the SNI mice and increased PSD protein level. Altogether these findings demonstrate that D-Asp improves sensorial, motor and cognitive-like symptoms related to chronic pain possibly through glutamate neurotransmission normalization in neuropathic mice.

Age-Related Changes in D-Aspartate Oxidase Promoter Methylation Control Extracellular D-Aspartate Levels and Prevent Precocious Cell Death during Brain Aging.

The endogenous NMDA receptor (NMDAR) agonist D-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebral D-aspartate levels is due to the concomitant onset of D-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylic D-amino acids. In the present work, we show that d-aspartate content in the mouse brain drastically decreases after birth, whereas Ddo mRNA levels concomitantly increase. Interestingly, postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knock-out mice (Ddo(-/-)), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of d-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of D-aspartate levels in Ddo(-/-) brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in Ddo(-/-) brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free D-aspartate.