Targeting Neuroinflammation with Abscisic Acid Reduces Pain Sensitivity in Females and Hyperactivity in Males of An ADHD Mice Model

Attention deficit/hyperactivity disorder (ADHD) is a neurodevelopmental syndrome characterized by dopaminergic dysfunction. In this study, we aimed to demonstrate that there is a link between dopaminergic deficit and neuroinflammation that underlies ADHD symptoms. We used a validated ADHD mice model involving perinatal 6-OHDA lesions. The animals received abscisic acid (ABA), an anti-inflammatory phytohormone, at a concentration of 20 mg/L (drinking water) for one month. We tested a battery of behavior tests, learning and memory, anxiety, social interactions, and pain thresholds in female and male mice (control and lesioned, with or without ABA treatment). Postmortem, we analyzed microglia morphology and Ape1 expression in specific brain areas related to the descending pain inhibitory pathway. In females, the dopaminergic deficit increased pain sensitivity but not hyperactivity. In contrast, males displayed hyperactivity but showed no increased pain sensitivity. In females, pain sensitivity was associated with inflammatory microglia and lower Ape1 levels in the anterior cingulate cortex (ACC) and posterior insula cortex (IC). In addition, ABA treatment alleviated pain sensitivity concomitant with reduced inflammation and normalized APE1. In males, ABA reduced hyperactivity but had no significant effect on inflammation in these areas. This is the first study proving a sex-dependent association between dopamine dysfunction and inflammation in specific brain areas, hence leading to different behavioral outcomes in a mouse model of ADHD. These findings provide new clues for potential treatments for ADHD

AAV delivery of shRNA against IRS1 in GABAergic neurons in rat hippocampus impairs spatial memory in females and male rats

This is a pre-print of an article published in Brain Structure and Function. The final authenticated version is available online at: https://doi.org/10.1007/s00429-020-02155-xBrain insulin resistance is a major factor leading to impaired cognitive function and it is considered as the onset of Alzheimer´s disease. Insulin resistance is intimately linked to inflammatory conditions, many studies have revealed how pro-inflammatory cytokines lead to insulin resistance, by inhibiting IRS1 function. Thus, the dysfunction of insulin signaling is concomitant with inflammatory biomarkers. However, the specific effect of IRS1 impaired function in otherwise healthy brain has not been dissected out. So, we decided in our study, to study the specific role of IRS1 in the hippocampus, in the absence of comorbidities. To that end, shRNA against rat and human IRS1 was designed and tested in cultured HEK cells to evaluate mRNA levels and specificity. The best candidate sequence was encapsulated in an AAV vector (strain DJ8) under the control of the cytomegalovirus promoter and together with the green fluorescent protein gene as a reporter. AAV-CMV-shIRS1-EGFP and control AAV-CMV-EGFP were inoculated into the dorsal hippocampus of female and male Wistar rats. One month later, animals undertook a battery of behavioral paradigms evaluating spatial and social memory and anxiety. Our results suggest that females displayed increased susceptibility to AAV-shIRS1 in the novel recognition object paradigm; whereas both females and males show impaired performance in the T maze when infected with AAV-shIRS1 compared to control. Anxiety parameters were not affected by AAV-shIRS1 infection. We observed specific fluorescence within the hilum of the dentate gyrus, in immuno-characterized parvalbumin and somatostatin neurons. AAV DJ8 did not enter astrocytes. Intense green fibers were found in the fornix, mammillary bodies, and in the medial septum indicating that hippocampal efferent had been efficiently targeted by the AAV DJ8 infection. We observed that AAV-shIRS1 reduced significantly synaptophysin labeling in hippocampal-septal projections compared to controls. These results support that, small alterations in the insulin/IGF1 pathway in specific hippocampal circuitries can underlie alterations in synaptic plasticity and affect behavior, in the absence of inflammatory condition

Can We Treat Neuroinflammation in Alzheimer's Disease?

Alzheimer’s disease (AD), considered the most common type of dementia, is characterized by a progressive loss of memory, visuospatial, language and complex cognitive abilities. In addition, patients often show comorbid depression and aggressiveness. Aging is the major factor contributing to AD; however, the initial cause that triggers the disease is yet unknown. Scientific evidence demonstrates that AD, especially the late onset of AD, is not the result of a single event, but rather it appears because of a combination of risk elements with the lack of protective ones. A major risk factor underlying the disease is neuroinflammation, which can be activated by different situations, including chronic pathogenic infections, prolonged stress and metabolic syndrome. Consequently, many therapeutic strategies against AD have been designed to reduce neuro-inflammation, with very promising results improving cognitive function in preclinical models of the disease. The literature is massive; thus, in this review we will revise the translational evidence of these early strategies focusing in anti-diabetic and anti-inflammatory molecules and discuss their therapeutic application in humans. Furthermore, we review the preclinical and clinical data of nutraceutical application against AD symptoms. Finally, we introduce new players underlying neuroinflammation in AD: the activity of the endocannabinoid system and the intestinal microbiota as neuroprotectors. This review highlights the importance of a broad multimodal approach to treat successfully the neuroinflammation underlying AD

Abscisic Acid Supplementation Rescues High Fat Diet-Induced Alterations in Hippocampal Inflammation and IRSs Expression

Accumulated evidence indicates that neuroinflammation induces insulin resistance in the brain. Moreover, both processes are intimately linked to neurodegenerative disorders, including Alzheimer’s disease. Potential mechanisms underlying insulin resistance include serine phosphorylation of the insulin receptor substrate (IRS) or insulin receptor (IR) misallocation. However, only a few studies have focused on IRS expression in the brain and its modulation in neuroinflammatory processes. This study used the high-fat diet (HFD) model of neuroinflammation to study the alterations of IR, an insulin-like growth factor receptor (IGF1R) and IRS expressions in the hippocampus. We observed that HFD effectively reduced mRNA and protein IRS2 expression. In contrast, a HFD induced the upregulation of the IRS1 mRNA levels, but did not alter an IR and IGF1R expression. As expected, we observed that a HFD increased hippocampal tumor necrosis factor alpha (TNFα) and amyloid precursor protein (APP) levels while reducing brain-derived neurotrophic factor (BDNF) expression and neurogenesis. Interestingly, we found that TNFα correlated positively with IRS1 and negatively with IRS2, whereas APP levels correlated positively only with IRS1 but not IRS2. These results indicate that IRS1 and IRS2 hippocampal expression can be affected differently by HFD-induced neuroinflammation. In addition, we aimed to establish whether abscisic acid (ABA) can rescue hippocampal IRS1 and IRS2 expression, as we had previously shown that ABA supplementation prevents memory impairments and improves neuroinflammation induced by a HFD. In this study, ABA restored HFD-induced hippocampal alterations, including IRS1 and IRS2 expression, TNFα, APP, and BDNF levels and neurogenesis. In conclusion, this study highlights different regulations of hippocampal IRS1 and IRS2 expression using a HFD, indicating the important differences of these scaffolding proteins, and strongly supports ABA therapeutic effects

Central relaxin-3 receptor (RXFP3) activation increases ERK phosphorylation in septal cholinergic neurons and impairs spatial working memory.

The medial septum/diagonal band (MS/DB) is a relay region connecting the hypothalamus and brainstem with the hippocampus, and both the MS/DB and dorsal/ventral hippocampus receive strong topographic GABA/peptidergic projections from the nucleus incertus of the pontine tegmentum. The neuropeptide relaxin-3, released by these neurons, is the cognate ligand for a Gi/o-protein-coupled receptor, RXFP3, which is highly expressed within the MS/DB, and both cholinergic and GABAergic neurons in this region of rat brain receive relaxin-3 positive terminals/boutons. Comprehensive in vitro studies have demonstrated that a range of cell signaling pathways can be altered by RXFP3 stimulation, including inhibition of forskolin-activated cAMP levels and activation of ERK phosphorylation. In this study we investigated whether intracerebroventricular (icv) injection of RXFP3-A2, a selective relaxin-3 receptor agonist, altered ERK phosphorylation levels in the MS/DB of adult male rats. In addition, we assessed the neurochemical phenotype of phosphorylated (p) ERK-positive neurons in MS/DB after RXFP3-A2 administration by dual-label immunostaining for pERK and key neuronal markers. RXFP3-A2 injection significantly increased pERK levels in MS/DB, compared to vehicle at 20 and 90 min post-injection. In addition, icv injection of RXFP3-A2 increased the number of cells expressing pERK in the MS/DB after 90 min, with increases detected in cholinergic, but not GABAergic neurons. Moreover, we found that septal cholinergic neurons express RXFP3 and that icv infusions of RXFP3-A2 impaired alternation in a spatial working memory behavioral paradigm. The presence of the receptor and the specific RXFP3-related activation of the MAPK/ERK pathway in MS/DB cholinergic neurons identifies them as a key target of ascending relaxin-3 projections with implications for the acute and chronic inhibition of cholinergic neuron activity/function by relaxin-3/RXFP3 signaling.This research was supported by a predoctoral fellowship (FPI-UJI: PREDOC/2014/35) to HAG; a traineeship fellowship (UJI P1·1A2014-06) to AGA; the FP7-PEOPLE-IRSES PIRSES-GA-2012-318997 NEUREN project to ALG and FEO-B; NHMRC (Australia) project grants (1027522, 1026939) and a Brain and Behavior Research Foundation (USA) NARSAD Independent Investigator Award to ALG; Generalitat Valenciana (AICO/2015/042) project grant and Universitat Jaume I (P1·1A2014-06) project grant to AMS

The effect of abscisic acid chronic treatment on neuroinflammatory markers and memory in a rat model of high-fat diet induced neuroinflammation

Background Western diet and lifestyle are associated with overweight, obesity, and type 2 diabetes, which, in turn, are correlated with neuroinflammation processes. Exercise and a healthy diet are important in the prevention of these disorders. However, molecules inhibiting neuroinflammation might also be efficacious in the prevention and/or treatment of neurological disorders of inflammatory etiology. The abscisic acid (ABA) is a phytohormone involved in hydric-stress responses. This compound is not only found in plants but also in other organisms, including mammals. In rodents, ABA can play a beneficial role in the regulation of peripheral immune response and insulin action. Thus, we hypothesized that chronic ABA administration might exert a protective effect in a model of neuroinflammation induced by high-fat diet (HFD). Methods Male Wistar rats were fed with standard diet or HFD with or without ABA in the drinking water for 12 weeks. Glucose tolerance test and behavioral paradigms were performed to evaluate the peripheral and central effects of treatments. One-Way ANOVA was performed analyzed statistical differences between groups. Results The HFD induced insulin resistance peripherally and increased the levels of proinflammatory markers in in the brain. We observed that ABA restored glucose tolerance in HFD-fed rats, as expected. In addition, chronic ABA treatment rescued cognitive performance in these animals, while not affecting control diet fed animals. Moreover, it counteracted the changes induced by HFD in the hypothalamus; microglia activations and TNFα mRNA levels. Conclusion These results suggest that ABA might become a new therapeutic molecule improving the neuroinflammatory status and insulin resistance.This work was supported by Plan Propi Universitat Jaume I P1.1A2014-06 and GVA AICO/2015/042 to AMSP

Neuroinflammation and insulin resistance underly cognitive impairment. Sex differences and potential treatments

Neuroinflammation and insulin resistance are two correlated processes. Abscisic acid(ABA) is a phytohormone also found in mammals. In a model of neuroinflammation induced by high fat diet(HFD), ABA administration rescued memory alterations and prevented pro-inflammatory markers’ increase. Furthermore, ABA restored the HFD-induced changes in insulin receptor substrates, IRS1 and IRS2. Inflammation has been proposed a contributing factor in attention-deficit-hyperactivity-disorder(ADHD). In a pilot study, female and male mice seem to display differences in ADHD symptomatology. ABA administration appeared to also affect differentially males and females. The role of IRS1 and IRS2 isolated from inflammation has not been fully established.We used AAV expressing shRNA targeting IRS1 into hippocampus of female and male rats. Female rats showed spatial and recognition memory deficits but males only showed spatial memory alterations. In medial septum, we observed reduced synaptophysin levels on shIRS1-AAV fibers, suggesting impaired synaptic plasticity. Thus, IRS1 is required for proper neuronal activity.Neuroinflamación y resistencia a insulina son dos procesos correlacionados. Ácido abscísico(ABA) es una fitohormona que también está en mamíferos. En un modelo de neuroinflamación inducida por dieta alta en grasa, ABA mejora alteraciones en memoria y previene el aumento de marcadores proinflamatorios. Además, ABA restaura los cambios en sustratos-del-receptor-de-insulina, IRS1 e IRS2. La inflamación se ha propuesto como factor que contribuye al trastorno-por-déficit-de-atención-e-hiperactividad. En un estudio piloto, ratones hembra y macho parecen presentar diferencias en sintomatología. ABA parece afectar también de forma distinta ambos sexos. El papel de IRS1/IRS2 en ausencia de inflamación se desconoce. Inyectamos un AAV expresando shARN anti-IRS1 en hipocampo de ratas macho y hembra. Las hembras mostraron déficits de memoria espacial y de reconocimiento;los machos únicamente de memoria espacial. En septum medial, observamos niveles reducidos de sinaptofisina en fibras shIRS1-AAV, indicando alteraciones en plasticidad sináptica. Por tanto, IRS1 se requiere para una correcta actividad neuronal.Programa de Doctorat en Ciències Biomèdiques i Salu

Extinción de memorias condicionadas asociadas a la administración de drogas de abuso

Treball Final de Grau en Psicologia. Codi: PS1048. Curs 2013-2014Las señales contextuales previamente asociadas al consumo de una droga juegan un papel fundamental en el “craving” o deseo de volver a consumir y, por tanto, en las recaídas. Se han desarrollado estrategias, como la facilitación de la extinción o la disrupción de la reconsolidación de la memoria, para prevenir las recaídas en sujetos abstinentes. Su objetivo es reducir o bloquear la capacidad de las memorias patológicas relacionadas con los estímulos ambientales asociados a la droga de promover la búsqueda y consumo de la droga. A nivel farmacológico, uno de los sistemas objeto de estudio es el sistema noradrenérgico y, concretamente, los receptores β-adrenérgicos. En este estudio, hemos examinado si un estímulo contextual (tipo de suelo -barras o agujeros- de las cajas de actividad) es capaz de adquirir, tras su asociación con cocaína durante un procedimiento de sensibilización locomotora, las propiedades motivadoras y reforzantes de la droga convirtiéndose así en estímulo condicionado (EC).Exposure to contextual cues previously associated with drugs of abuse often induces craving and relapse in abstinent subjects. Different treatments and strategies, such as facilitation of extinction or disruption of memory reconsolidation, have been developed to interfere with drug-associated pathological memories related to environmental stimuli. Pharmacological manipulations targeting the noradrenergic system have been use to increase the efficacy of the behavioral strategies. The current study examined whether the floor type (grid or hole) of the activity chambers could acquire conditioned stimulus properties by its association with cocaine during a locomotor sensitization procedure

Targeting Neuroinflammation with Abscisic Acid Reduces Pain Sensitivity in Females and Hyperactivity in Males of an ADHD Mice Model

Attention deficit/hyperactivity disorder (ADHD) is a neurodevelopmental syndrome characterized by dopaminergic dysfunction. In this study, we aimed to demonstrate that there is a link between dopaminergic deficit and neuroinflammation that underlies ADHD symptoms. We used a validated ADHD mice model involving perinatal 6-OHDA lesions. The animals received abscisic acid (ABA), an anti-inflammatory phytohormone, at a concentration of 20 mg/L (drinking water) for one month. We tested a battery of behavior tests, learning and memory, anxiety, social interactions, and pain thresholds in female and male mice (control and lesioned, with or without ABA treatment). Postmortem, we analyzed microglia morphology and Ape1 expression in specific brain areas related to the descending pain inhibitory pathway. In females, the dopaminergic deficit increased pain sensitivity but not hyperactivity. In contrast, males displayed hyperactivity but showed no increased pain sensitivity. In females, pain sensitivity was associated with inflammatory microglia and lower Ape1 levels in the anterior cingulate cortex (ACC) and posterior insula cortex (IC). In addition, ABA treatment alleviated pain sensitivity concomitant with reduced inflammation and normalized APE1. In males, ABA reduced hyperactivity but had no significant effect on inflammation in these areas. This is the first study proving a sex-dependent association between dopamine dysfunction and inflammation in specific brain areas, hence leading to different behavioral outcomes in a mouse model of ADHD. These findings provide new clues for potential treatments for ADHD.This article belongs to the Special Issue The Signaling and Cellular Mechanisms of Pain.Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)</p