Joana Gonçalves

Methamphetamine‐induced neuroinflammation and neuronal dysfunction in the mice hippocampus: preventive effect of indomethacin

Methamphetamine (METH) causes irreversible damage to brain cells leading to neurological and psychiatric abnormalities. However, the mechanisms underlying life‐threatening effects of acute METH intoxication remain unclear. Indeed, most of the hypotheses focused on intra‐neuronal events, such as dopamine oxidation, oxidative stress and excitotoxicity. Yet, recent reports suggested that glia may contribute to METH‐induced neuropathology. In the present study, we investigated the hippocampal dysfunction induced by an acute high dose of METH (30 mg/kg; intraperitoneal injection), focusing on the inflammatory process and changes in several neuronal structural proteins. For that, 3‐month‐old male wild‐type C57BL/6J mice were killed at different time‐points post‐METH. We observed that METH caused an inflammatory response characterized by astrocytic and microglia reactivity, and tumor necrosis factor (TNF) system alterations. Indeed, glial fibrillary acidic protein (GFAP) and CD11b immunoreactivity were upregulated, likewise TNF‐α and TNF receptor 1 protein levels. Furthermore, the effect of METH on hippocampal neurons was also investigated, and we observed a downregulation in beta III tubulin expression. To clarify the possible neuronal dysfunction induced by METH, several neuronal proteins were analysed. Syntaxin‐1, calbindin D28k and tau protein levels were downregulated, whereas synaptophysin was upregulated. We also evaluated whether an anti‐inflammatory drug could prevent or diminish METH‐induced neuroinflammation, and we concluded that indomethacin (10 mg/kg; i.p.) prevented METH‐induced glia activation and both TNF system and beta III tubulin alterations. In conclusion, we demonstrated that METH triggers an inflammatory process and leads to neuronal dysfunction in the hippocampus, which can be prevented by an anti‐inflammatory treatment.

Methamphetamine‐Induced Early Increase of IL‐6 and TNF‐α mRNA Expression in the Mouse Brain

The mechanisms by which methamphetamine (METH) causes neurotoxicity are not well understood. Recent studies have suggested that METH‐induced neuropathology may result from a multicellular response in which glial cells play a prominent role, and so it is plausible to suggest that cytokines may participate in the toxic effects of METH. Therefore, in the present work we evaluated the effect of an acute administration of METH (30 mg/kg in a single intraperitoneal injection) on the interleukin (IL)‐1β, IL‐6, and tumor necrosis factor (TNF)‐α mRNA expression levels in the hippocampus, frontal cortex, and striatum of mice. We observed that METH did not induce changes in the IL‐1β mRNA expression levels in both hippocampus and striatum, with immeasurable levels in the frontal cortex. Regarding IL‐6, METH induced an increase in the expression levels of this cytokine in the hippocampus and striatum, 1 h and 30 min post injection, respectively. In the frontal cortex, the increase in IL‐6 mRNA levels was more significant and remained high even after 2 h. Moreover, the expression levels of TNF‐α were increased in both hippocampus and frontal cortex 30 min post METH administration, with immeasurable levels in the striatum. We conclude that the pro‐inflammatory cytokines IL‐6 and TNF‐α rapidly increase after METH administration, providing a new insight for understanding the effect of this drug of abuse in the brain.

Methamphetamine transiently increases the blood–brain barrier permeability in the hippocampus: Role of tight junction proteins and matrix metalloproteinase-9

Methamphetamine (METH) is a powerful stimulant drug of abuse that has steadily gained popularity worldwide. It is known that METH is highly neurotoxic and causes irreversible damage of brain cells leading to neurological and psychiatric abnormalities. Recent studies suggested that METH-induced neurotoxicity might also result from its ability to compromise blood-brain barrier (BBB) function. Due to the crucial role of BBB in the maintenance of brain homeostasis and protection against toxic molecules and pathogenic organisms, its dysfunction could have severe consequences. In this study, we investigated the effect of an acute high dose of METH (30mg/kg) on BBB permeability after different time points and in different brain regions. For that, young adult mice were sacrificed 1h, 24h or 72h post-METH administration. METH increased BBB permeability, but this effect was detected only at 24h after administration, being therefore a transitory effect. Interestingly, we also found that the hippocampus was the most susceptible brain region to METH, comparing to frontal cortex and striatum. Moreover, in an attempt to identify the key players in METH-induced BBB dysfunction we further investigated potential alterations in tight junction (TJ) proteins and matrix metalloproteinase-9 (MMP-9). METH was able to decrease the protein levels of zonula occludens (ZO)-1, claudin-5 and occludin in the hippocampus 24h post-injection, and increased the activity and immunoreactivity of MMP-9. The pre-treatment with BB-94 (30mg/kg), a matrix metalloproteinase inhibitor, prevented the METH-induced increase in MMP-9 immunoreactivity in the hippocampus. Overall, the present data demonstrate that METH transiently increases the BBB permeability in the hippocampus, which can be explained by alterations on TJ proteins and MMP-9.

Histamine modulates microglia function

BackgroundHistamine is commonly acknowledged as an inflammatory mediator in peripheral tissues, leaving its role in brain immune responses scarcely studied. Therefore, our aim was to uncover the cellular and molecular mechanisms elicited by this molecule and its receptors in microglia-induced inflammation by evaluating cell migration and inflammatory mediator release.MethodsFirstly, we detected the expression of all known histamine receptor subtypes (H1R, H2R, H3R and H4R), using a murine microglial cell line and primary microglia cell cultures from rat cortex, by real-time PCR analysis, immunocytochemistry and Western blotting. Then, we evaluated the role of histamine in microglial cell motility by performing scratch wound assays. Results were further confirmed using murine cortex explants. Finally, interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels were evaluated by ELISA measurements to determine the role of histamine on the release of these inflammatory mediators.ResultsAfter 12 h of treatment, 100 μM histamine and 10 μg/ml histamine-loaded poly (lactic-co-glycolic acid) microparticles significantly stimulated microglia motility via H4R activation. In addition, migration involves α5β1 integrins, and p38 and Akt signaling pathways. Migration of microglial cells was also enhanced in the presence of lipopolysaccharide (LPS, 100 ng/ml), used as a positive control. Importantly, histamine inhibited LPS-stimulated migration via H4R activation. Histamine or H4R agonist also inhibited LPS-induced IL-1β release in both N9 microglia cell line and hippocampal organotypic slice cultures.ConclusionsTo our knowledge, we are the first to show a dual role of histamine in the modulation of microglial inflammatory responses. Altogether, our data suggest that histamine per se triggers microglia motility, whereas histamine impedes LPS-induced microglia migration and IL-1β release. This last datum assigns a new putative anti-inflammatory role for histamine, acting via H4R to restrain exacerbated microglial responses under inflammatory challenge, which could have strong repercussions in the treatment of CNS disorders accompanied by microglia-derived inflammation.

The Angiogenic Factor Angiopoietin-1 Is a Proneurogenic Peptide on Subventricular Zone Stem/Progenitor Cells

In the adult mammalian brain, the subventricular zone (SVZ) hosts stem cells constantly generating new neurons. Angiopoietin-1 (Ang-1) is an endothelial growth factor with a critical role in division, survival, and adhesion of endothelial cells via Tie-2 receptor activity. Expression of Tie-2 in nonendothelial cells, especially neurons and stem cells, suggests that Ang-1 may be involved in neurogenesis. In the present work, we investigated the putative role of Ang-1 on SVZ neurogenesis. Immature cells from SVZ-derived neurospheres express Ang-1 and Tie-2 mRNA, suggesting a role for the Ang-1/Tie-2 system in the neurogenic niche. Moreover, we also found that Tie-2 protein expression is retained on differentiation in neurons and glial cells. Ang-1 triggered proliferation via activation of the ERK1/2 (extracellular signal-regulated kinase 1/2) mitogen-activated protein kinase (MAPK) kinase pathway but did not induce cell death. Accordingly, coincubation with an anti-Tie-2 neutralizing antibody prevented the pro-proliferative effect of Ang-1. Furthermore, Ang-1 increased the number of NeuN (neuronal nuclear protein)-positive neurons in cultures treated for 7 d, as well as the number of functional neurons, as assessed by monitoring [Ca2+]i rises after application of specific stimuli for neurons and immature cells. The proneurogenic effect of Ang-1 is mediated by Tie-2 activation and subsequent mTOR (mammalian target of rapamycin kinase) mobilization. In agreement, neuronal differentiation significantly decreased after exposure to an anti-Tie-2 neutralizing antibody and to rapamycin. Moreover, Ang-1 elicited the activation of the SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase) MAPK, involved in axonogenesis. Our work shows a proneurogenic effect of Ang-1, highlighting the relevance of blood vessel/stem cell cross talk in health and disease.

New method for the simultaneous identification of cow, sheep, goat, and water buffalo in dairy products by analysis of short species-specific mitochondrial DNA targets.

A novel method is presented here as an analytical tool for food control and authentication of dairy products manufactured from the milk of cow, sheep, goat, and buffalo. The method is based on multiplex polymerase chain reaction (PCR) of species-specific mitochondrial DNA (mtDNA) targets followed by fragment size analysis by capillary electrophoresis. The method includes (a) simultaneous detection of four species, (b) internal control for DNA extraction and PCR, (c) mtDNA as a target for PCR, (d) amplicons of <200 bp, and (e) flexibility in the electrophoresis and fragment size detection method. Species identification proved to be straightforward, efficient, sensitive, and robust. The method is sensitive to an at least 1% (v/v) relative proportion of milk in binary mixtures. A survey of commercial products showed that 12.5% failed to conform to the description of the contents, by either the introduction or absence of listed species, thus demonstrating the relevance of this type of testing.

Brain Injury Associated with Widely Abused Amphetamines: Neuroinflammation, Neurogenesis and Blood-Brain Barrier

Over the course of the 20(th) century, it became increasingly clear that amphetamine-like psychostimulants carried serious abuse liability that has resulted in sociological use patterns that have been described as epidemics. In fact, drug addiction is a brain disease with a high worldwide prevalence, and is considered the most expensive of the neuropsychiatric disorders. This review goes beyond the previously well-documented evidence demonstrating that amphetamines cause neuronal injury. Cellular and molecular mechanisms involved in the neurotoxicity of psychostimulants drugs have been extensively described giving particular attention to the role of oxidative stress and metabolic compromise. Recently, it was shown that the amphetamine class of drugs of abuse triggers an inflammatory process, emerging as a critical concept to understand the toxic effects of these drugs. Moreover, it has been suggested that psychostimulants compromise the capacity of the brain to generate new neurons (neurogenesis), and can also lead to blood-brain barrier (BBB) dysfunction. Together, these effects may contribute to brain damage, allowing the entry of pathogens into the brain parenchyma and thus decreasing the endogenous brain repair resources. The overall objective of this review is to highlight experimental evidence in an attempt to clarify the role of neuroinflammation in amphetamines-induced brain dysfunction and the effect of these drugs on both neurogenesis and BBB integrity.

Neuropeptide Y promotes neurogenesis and protection against methamphetamine-induced toxicity in mouse dentate gyrus-derived neurosphere cultures

Methamphetamine (METH) is a psychostimulant drug of abuse that causes severe brain damage. However, the mechanisms responsible for these effects are poorly understood, particularly regarding the impact of METH on hippocampal neurogenesis. Moreover, neuropeptide Y (NPY) is known to be neuroprotective under several pathological conditions. Here, we investigated the effect of METH on dentate gyrus (DG) neurogenesis, regarding cell death, proliferation and differentiation, as well as the role of NPY by itself and against METH-induced toxicity. DG-derived neurosphere cultures were used to evaluate the effect of METH or NPY on cell death, proliferation or neuronal differentiation. Moreover, the role of NPY and its receptors (Y(1), Y(2) and Y(5)) was investigated under conditions of METH-induced DG cell death. METH-induced cell death by both apoptosis and necrosis at concentrations above 10 nM, without affecting cell proliferation. Furthermore, at a non-toxic concentration (1 nM), METH decreased neuronal differentiation. NPYs protective effect was mainly due to the reduction of glutamate release, and it also increased DG cell proliferation and neuronal differentiation via Y(1) receptors. In addition, while the activation of Y(1) or Y(2) receptors was able to prevent METH-induced cell death, the Y(1) subtype alone was responsible for blocking the decrease in neuronal differentiation induced by the drug. Taken together, METH negatively affects DG cell viability and neurogenesis, and NPY is revealed to be a promising protective tool against the deleterious effects of METH on hippocampal neurogenesis.

Prevention of methamphetamine-induced microglial cell death by TNF-α and IL-6 through activation of the JAK-STAT pathway

BackgroundIt is well known that methamphetamine (METH) is neurotoxic and recent studies have suggested the involvement of neuroinflammatory processes in brain dysfunction induced by misuse of this drug. Indeed, glial cells seem to be activated in response to METH, but its effects on microglial cells are not fully understood. Moreover, it has been shown that cytokines, which are normally released by activated microglia, may have a dual role in response to brain injury. This led us to study the toxic effect of METH on microglial cells by looking to cell death and alterations of tumor necrosis factor-alpha (TNF-α) and interleukine-6 (IL-6) systems, as well as the role played by these cytokines.MethodsWe used the N9 microglial cell line, and cell death and proliferation were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and incorporation of bromodeoxyuridine, respectively. The TNF-α and IL-6 content was quantified by enzyme-linked immunosorbent assay, and changes in TNF receptor 1, IL-6 receptor-alpha, Bax and Bcl-2 protein levels by western blotting. Immunocytochemistry analysis was also performed to evaluate alterations in microglial morphology and in the protein expression of phospho-signal transducer and activator of transcription 3 (pSTAT3).ResultsMETH induced microglial cell death in a concentration-dependent manner (EC50 = 1 mM), and also led to significant morphological changes and decreased cell proliferation. Additionally, this drug increased TNF-α extracellular and intracellular levels, as well as its receptor protein levels at 1 h, whereas IL-6 and its receptor levels were increased at 24 h post-exposure. However, the endogenous proinflammatory cytokines did not contribute to METH-induced microglial cell death. On the other hand, exogenous low concentrations of TNF-α or IL-6 had a protective effect. Interestingly, we also verified that the anti-apoptotic role of TNF-α was mediated by activation of IL-6 signaling, specifically the janus kinase (JAK)-STAT3 pathway, which in turn induced down-regulation of the Bax/Bcl-2 ratio.ConclusionsThese findings show that TNF-α and IL-6 have a protective role against METH-induced microglial cell death via the IL-6 receptor, specifically through activation of the JAK-STAT3 pathway, with consequent changes in pro- and anti-apoptotic proteins.

Protective role of neuropeptide Y Y₂ receptors in cell death and microglial response following methamphetamine injury.

It has been reported that the hippocampus is very susceptible to methamphetamine (METH) and that neuropeptide Y (NPY) is an important neuroprotective agent against hippocampal excitotoxicity. However, there is very little information regarding the role of the NPYergic system in this brain region under conditions of METH toxicity. To clarify this issue, we investigated the role of NPY and its receptors against METH‐induced neuronal cell death in hippocampal organotypic slice cultures. Our data show that NPY (1 μm) is neuroprotective in DG, CA3 and CA1 subregions via Y2 receptors. Moreover, the selective activation of Y1 receptors (1 μm [Leu31,Pro34]NPY) partially prevented the toxicity induced by METH in DG and CA3 subfields, but completely blocked its toxicity in the CA1 pyramidal cell layer. Regarding Y2 receptors, its activation (300 nm NPY13–36) completely prevented METH‐induced toxicity in all subregions analysed, which involved changes in levels of pro‐ and anti‐apoptotic proteins Bcl‐2 and Bax, respectively. Besides neuronal cell death, we also showed that METH triggers a microglial response in the mouse hippocampus which was attenuated by Y2 receptor activation. To better clarify the effect of METH and the NPY system on microglial cells, we further used the N9 microglial cell line. We found that both NPY and the Y2 receptor agonist were able to protect microglia against METH‐induced cell death. Overall, our data demonstrate that METH is toxic to both neurons and microglial cells, and that NPY, mainly via Y2 receptors, has an important protective role against METH‐induced cell death and microgliosis.