The novel potent GSK3 inhibitor AF3581 reverts fragile X syndrome phenotype.

Glycogen-synthase kinase 3 (GSK3) is a kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK3 has been linked to several disease conditions such as fragile X syndrome (FXS). Recent evidences demonstrating an increased activity of GSK3 in murine models of FXS, suggest that dysregulation/hyperactivation of the GSK3 path should contribute to FXS development. A likely possibility could be that in FXS there is a functional impairment of the upstream inhibitory input over GSK3 thus making overactive the kinase. Since GSK3 signaling is a central regulatory node for critical neurodevelopmental pathways, understanding the contribution of GSK3 dysregulation to FXS, may provide novel targets for therapeutic interventions for this disease. In this study we used AF3581, a potent GSK3 inhibitor that we recently discovered, in an in vivo FXS mouse model to elucidate the crucial role of GSK3 in specific behavioral patterns (locomotor activity, sensorimotor gating and social behavior) associated with this disease. All the behavioral alterations manifested by Fmr1 knockout mice were reverted after a chronic treatment with our GSK3 inhibitor, confirming the importance of this pathway as a therapeutic target.

Inhibition of N-acylethanolamine-hydrolyzing acid amidase reduces T cell infiltration in a mouse model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), in which myeloid cells sustain inflammation, take part in priming, differentiation, and reactivation of myelin-specific T cells, and cause direct myelin damage. N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a proinflammatory enzyme induced by phlogosis and overexpressed in macrophages and microglia of EAE mice. Targeting these cell populations by inhibiting NAAA may be a promising pharmacological strategy to modulate the inflammatory aspect of MS and manage disease progression. To address this goal, we used ARN16186, a small molecule specifically designed and synthesized as a pharmacological tool to inhibit NAAA. We assessed whether enzyme inhibition affected the severity of neurological symptoms and modulated immune cell infiltration into the central nervous system of EAE mice. We found that preventive chronic treatment with ARN16186 was efficacious in slowing disease progression and preserving locomotor activity in EAE mice. Furthermore, NAAA inhibition reduced the number of immune cells infiltrating the spinal cord and modulated the overactivation of NF-kB and STAT3 transcription factors, leading to less expansion of Th17 cells over the course of the disease.

Novel fragile X syndrome 2D and 3D brain models based on human isogenic FMRP-KO iPSCs.

Fragile X syndrome (FXS) is a neurodevelopmental disorder, characterized by intellectual disability and sensory deficits, caused by epigenetic silencing of the FMR1 gene and subsequent loss of its protein product, fragile X mental retardation protein (FMRP). Delays in synaptic and neuronal development in the cortex have been reported in FXS mouse models; however, the main goal of translating lab research into pharmacological treatments in clinical trials has been so far largely unsuccessful, leaving FXS a still incurable disease. Here, we generated 2D and 3D in vitro human FXS model systems based on isogenic FMR1 knock-out mutant and wild-type human induced pluripotent stem cell (hiPSC) lines. Phenotypical and functional characterization of cortical neurons derived from FMRP-deficient hiPSCs display altered gene expression and impaired differentiation when compared with the healthy counterpart. FXS cortical cultures show an increased number of GFAP positive cells, likely astrocytes, increased spontaneous network activity, and depolarizing GABAergic transmission. Cortical brain organoid models show an increased number of glial cells, and bigger organoid size. Our findings demonstrate that FMRP is required to correctly support neuronal and glial cell proliferation, and to set the correct excitation/inhibition ratio in human brain development.

Optimization of Indazole-Based GSK-3 Inhibitors with Mitigated hERG Issue and In Vivo Activity in a Mood Disorder Model.

Bipolar disorders still represent a global unmet medical need and pose a requirement for novel effective treatments. In this respect, glycogen synthase kinase 3ß (GSK-3ß) aberrant activity has been linked to the pathophysiology of several disease conditions, including mood disorders. Therefore, the development of GSK-3ß inhibitors with good in vivo efficacy and safety profile associated with high brain exposure is required. Accordingly, we have previously reported the selective indazole-based GSK-3 inhibitor 1, which showed excellent efficacy in a mouse model of mania. Despite the favorable preclinical profile, analog 1 suffered from activity at the hERG ion channel, which prevented its further progression. Herein, we describe our strategy to improve this off-target liability through modulation of physicochemical properties, such as lipophilicity and basicity. These efforts led to the potent inhibitor 14, which possessed reduced hERG affinity, promising in vitro ADME properties, and was very effective in a mood stabilizer in vivo model.

The mood stabilizing properties of AF3581, a novel potent GSK-3ß inhibitor.

Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine protein kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK-3ß has been linked to several disease conditions. There is now large evidence on the role of GSK-3ß in the pathophysiology of mood disturbances with special regard to bipolar disorders. In the present study we further investigated the role of GSK-3ß in bipolar disorders by studying AF3581, the prototype of a novel class of ATP-competitive GSK-3ß inhibitors having the common N-[(1- alkylpiperidin-4-yl) methyl]-1H-indazole-3-carboxamide scaffold. Based on previous studies, AF3581 inhibits GSK-3ß in the nanomolar range on purified human enzyme and highly selective with respect to other kinases. Current study demonstrates that the compound has efficacy both in the chronic mild stress paradigm of depression (mimicking the down phase of bipolar disorder) and on mice aggressiveness in the resident intruder model (mimicking the up phase). These findings underline the importance of aberrant GSK-3ß activity in the development/ maintenance of mood oscillation in this peculiar pathological condition. Moreover, the present work also suggests a therapeutic potential for selective GSK-3 ß inhibitors in the management of bipolar disorders patients. Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine protein kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK-3ß has been linked to several disease conditions. There is now large evidence on the role of GSK-3ß in the pathophysiology of mood disturbances with special regard to bipolar disorders. In the present study we further investigated the role of GSK-3ß in bipolar disorders by studying AF3581, the prototype of a novel class of ATP-competitive GSK-3ß inhibitors having the common N-[(1- alkylpiperidin-4-yl) methyl]-1H-indazole-3-carboxamide scaffold. Based on previous studies, AF3581 inhibits GSK-3ß in the nanomolar range on purified human enzyme and highly selective with respect to other kinases. Current study demonstrates that the compound has efficacy both in the chronic mild stress paradigm of depression (mimicking the down phase of bipolar disorder) and on mice aggressiveness in the resident intruder model (mimicking the up phase). These findings underline the importance of aberrant GSK-3ß activity in the development/ maintenance of mood oscillation in this peculiar pathological condition. Moreover, the present work also suggests a therapeutic potential for selective GSK-3 ß inhibitors in the management of bipolar disorders patients. Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine protein kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK-3ß has been linked to several disease conditions. There is now large evidence on the role of GSK-3ß in the pathophysiology of mood disturbances with special regard to bipolar disorders. In the present study we further investigated the role of GSK-3ß in bipolar disorders by studying AF3581, the prototype of a novel class of ATP-competitive GSK-3ß inhibitors having the common N-[(1- alkylpiperidin-4-yl) methyl]-1H-indazole-3-carboxamide scaffold. Based on previous studies, AF3581 inhibits GSK-3ß in the nanomolar range on purified human enzymeand highly selective with respect to other kinases. Current study demonstrates that the compound has efficacy both in the chronic mild stress paradigm of depression (mimicking the down phase of bipolar disorder) and on mice aggressiveness in the resident intruder model (mimicking the up phase). These findings underline the importance of aberrant GSK-3ß activity in the development/ maintenance of mood oscillation in this peculiar pathological condition. Moreover, the present work also suggests a therapeutic potential for selective GSK-3 ß inhibitors in the management of bipolar disorders patients. Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine protein kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK-3ß has been linked to several disease conditions. There is now large evidence on the role of GSK-3ß in the pathophysiology of mood disturbances with special regard to bipolar disorders. In the present study we further investigated the role of GSK-3ß in bipolar disorders by studying AF3581, the prototype of a novel class of ATP-competitive GSK-3ß inhibitors having the common N-[(1- alkylpiperidin-4-yl) methyl]-1H-indazole-3-carboxamide scaffold. Based on previous studies, AF3581 inhibits GSK-3ß in the nanomolar range on purified human enzyme and highly selective with respect to other kinases. Current study demonstrates that the compound has efficacy both in the chronic mild stress paradigm of depression (mimicking the down phase of bipolar disorder) and on mice aggressiveness in the resident intruder model (mimicking the up phase). These findings underline the importance of aberrant GSK-3ß activity in the development/ maintenance of mood oscillation in this peculiar pathological condition. Moreover, the present work also suggests a therapeutic potential for selective GSK-3 ß inhibitors in the management of bipolar disorders patients. Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine protein kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK-3ß has been linked to several disease conditions. There is now large evidence on the role of GSK-3ß in the pathophysiology of mood disturbances with special regard to bipolar disorders. In the present study we further investigated the role of GSK-3ß in bipolar disorders by studying AF3581, the prototype of a novel class of ATP-competitive GSK-3ß inhibitors having the common N-[(1- alkylpiperidin-4-yl) methyl]-1H-indazole-3-carboxamide scaffold. Based on previous studies, AF3581 inhibits GSK-3ß in the nanomolar range on purified human enzyme and highly selective with respect to other kinases. Current study demonstrates that the compound has efficacy both in the chronic mild stress paradigm of depression (mimicking the down phase of bipolar disorder) and on mice aggressiveness in the resident intruder model (mimicking the up phase). These findings underline the importance of aberrant GSK-3ß activity in the development/ maintenance of mood oscillation in this peculiar pathological condition. Moreover, the present work also suggests a therapeutic potential for selective GSK-3 ß inhibitors in the management of bipolar disorders patients. Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine protein kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK-3ß has been linked to several disease conditions. There is now large evidence on the role of GSK-3ß in the pathophysiology of mood disturbances with special regard to bipolar disorders. In the present study we further investigated the role of GSK-3ß in bipolar disorders by studying AF3581, the prototype of a novel class of ATP-competitive GSK-3ß inhibitors having the common N-[(1- alkylpiperidin-4-yl) methyl]-1H-indazole-3-carboxamide scaffold. Based on previous studies, AF3581 inhibits GSK-3ß in the nanomolar range on purified human enzyme and highly selective with respect to other kinases. Current study demonstrates that the compound has efficacy both in the chronic mild stress paradigm of depression (mimicking the down phase of bipolar disorder) and on mice aggressiveness in the resident intruder model (mimicking the up phase). These findings underline the importance of aberrant GSK-3ß activity in the development/ maintenance of mood oscillation in this peculiar pathological condition. Moreover, the present work also suggests a therapeutic potential for selective GSK-3 ß inhibitors in the management of bipolar disorders patients. Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine protein kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK-3ß has been linked to several disease conditions. There is now large evidence on the role of GSK-3ß in the pathophysiology of mood disturbances with special regard to bipolar disorders. In the present study we further investigated the role of GSK-3ß in bipolar disorders by studying AF3581, the prototype of a novel class of ATP-competitive GSK-3ß inhibitors having the common N-[(1- alkylpiperidin-4-yl) methyl]-1H-indazole-3-carboxamide scaffold. Based on previous studies, AF3581 inhibits GSK-3ß in the nanomolar range on purified human enzyme and highly selective with respect to other kinases. Current study demonstrates that the compound has efficacy both in the chronic mild stress paradigm of depression (mimicking the down phase of bipolar disorder) and on mice aggressiveness in the resident intruder model (mimicking the up phase). These findings underline the importance of aberrant GSK-3ß activity in the development/ maintenance of mood oscillation in this peculiar pathological condition. Moreover, the present work also suggests a therapeutic potential for selective GSK-3 ß inhibitors in the management of bipolar disorders patients.

Lack of Rhes Increases MDMA-Induced Neuroinflammation and Dopamine Neuron Degeneration: Role of Gender and Age.

Ras homolog enriched in striatum (Rhes) is a protein that exerts important physiological functions and modulates psychostimulant drug effects. On this basis, the object of this study was to assess 3,4-methylenedioxymethamphetamine (MDMA) effects on microglial (CD11b) and astroglial (GFAP) activation and on dopamine neuron degeneration (TH) in wild-type (WT) and Rhes knockout (KO) male and female mice of different ages. Motor activity was also evaluated. Adult (3 months) MDMA-treated mice displayed an increase in GFAP-positive cells in striatum (STR), whereas the substantia nigra pars compacta (SNc) was affected only in male mice. In these mice, the increase of CD11b was more extensive including STR, SNc, motor cortex (CTX), ventral tegmental area (VTA), and nucleus accumbens (NAc). MDMA administration also affected TH immunoreactivity in both STR and SNc of male but not female WT and Rhes KO mice. In middle-aged mice (12 months), MDMA administration further increased GFAP and CD11b and decreased TH immunoreactivity in STR and SNc of all mice. Finally, MDMA induced a higher increase of motor activity in adult Rhes KO male, but not female mice. The results show that Rhes protein plays an important role on MDMA-mediated neuroinflammation and neurodegeneration dependent on gender and age, and confirm the important role of Rhes protein in neuroinflammatory and neurodegenerative processes.

Rhes Counteracts Dopamine Neuron Degeneration and Neuroinflammation Depending on Gender and Age.

We have recently shown that male Rhes knockout (KO) mice develop a mild form of spontaneous Parkinson's disease (PD)-like phenotype, characterized by motor impairment and a decrease in nigrostriatal dopamine (DA) neurons. Experimental evidence has implicated neuroinflammation in PD progression, and the presence of activated glial cells has been correlated with DA neuron degeneration. Despite this, several factors, such as gender, have been found to affect DAergic neuron degeneration and influence neuroinflammation, explaining the differences between men and women in the etiology of PD. On these basis, we studied age and gender differences in DA neuron degeneration and gliosis in the nigrostriatal system of adult (3-month-old) and middle aged (12-month-old) male and female Rhes wild-type (WT) and KO mice. Through immunohistochemistry, tyrosine hydroxylase (TH), microglial (complement type 3 receptor [CD11b]) and astroglial (glial fibrillary acid protein [GFAP]) increase, were evaluated. Adult male Rhes KO mice showed a decrease in TH and an increase in CD11b, both in the caudate putamen (CPu) and substantia nigra pars compacta (SNc), and an increase in GFAP in the CPu. In contrast, adult female Rhes KO mice showed only a decrease in TH in the SNc, whereas no modifications to the levels of GFAP and CD11b were observed in the CPu or SNc. Middle aged male Rhes KO mice showed a decrease in TH in the CPu and SNc, and an increase in GFAP and CD11b in the SNc. Middle aged female Rhes KO mice showed a decrease in TH in the CPu and SNc and an increase in CD11b only in the CPu, but no modifications to GFAP levels. The more marked DA neuron degeneration and neuroinflammation in male compared with female Rhes KO mice, while confirming the role of Rhes as an important protein for DA neuron survival, gives support to Rhes KO mice as a valuable preclinical model for studying the vulnerability factors of DA neuron degeneration as in PD.

Widespread reduction of dopamine cell bodies and terminals in adult rats exposed to a low dose regimen of MDMA during adolescence.

Although MDMA (3,4-methylendioxymethamphetamine, ecstasy) neurotoxicity in serotonin neurons is largely recognized in a wide variety of species including man, neurotoxicity in dopamine (DA) neurons is thought to be species-specific. MDMA is mainly consumed by adolescents, often in conjunction with caffeine (Energy Drinks) and this association has been reported to exacerbate MDMA toxic effects. In order to model these aspects of MDMA use, vis-à-vis their impact on DA neurons, we investigated the effects of adolescent exposure to low doses of MDMA (5 mg/kg for 10 days), alone or in combination with caffeine (10 mg/kg) on neuronal and functional DA indices and on recognition memory in adult rats. MDMA reduced density of tyrosine hydroxylase (TH) positive neurons in the ventral tegmental area and in the substantia nigra pars compacta, and immunoreactivity of TH and DA transporter in the nucleus accumbens (NAc) shell and core, and caudate-putamen. This same treatment caused a reduction of basal dialysate DA in the NAc core. MDMA-pretreated rats also showed behavioral sensitization to a MDMA challenge at adulthood and potentiation of MDMA-induced increase of dialysate DA in the NAc core, but not in the NAc shell. In addition, MDMA-treated rats displayed a deficit in recognition memory. Caffeine co-administration did not affect the above outcomes. Our results show that adolescent exposure of rats to low doses of MDMA induces long-lasting and widespread reduction of DA neurons indicative of a neurotoxic effect on DA neurons and suggestive of a degeneration of the same neurons.

Amphetamine-related drugs neurotoxicity in humans and in experimental animals: Main mechanisms.

Amphetamine-related drugs, such as 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (METH), are popular recreational psychostimulants. Several preclinical studies have demonstrated that, besides having the potential for abuse, amphetamine-related drugs may also elicit neurotoxic and neuroinflammatory effects. The neurotoxic potentials of MDMA and METH to dopaminergic and serotonergic neurons have been clearly demonstrated in both rodents and non-human primates. This review summarizes the species-specific cellular and molecular mechanisms involved in MDMA and METH-mediated neurotoxic and neuroinflammatory effects, along with the most important behavioral changes elicited by these substances in experimental animals and humans. Emphasis is placed on the neuropsychological and neurological consequences associated with the neuronal damage. Moreover, we point out the gap in our knowledge and the need for developing appropriate therapeutic strategies to manage the neurological problems associated with amphetamine-related drug abuse.

Effect of crowding, temperature and age on glia activation and dopaminergic neurotoxicity induced by MDMA in the mouse brain.

3,4-methylenedyoxymethamphetamine (MDMA or "ecstasy"), a recreational drug of abuse, can induce glia activation and dopaminergic neurotoxicity. Since MDMA is often consumed in crowded environments featuring high temperatures, we studied how these factors influenced glia activation and dopaminergic neurotoxicity induced by MDMA. C57BL/6J adolescent (4 weeks old) and adult (12 weeks old) mice received MDMA (4×20mg/kg) in different conditions: 1) while kept 1, 5, or 10×cage at room temperature (21°C); 2) while kept 5×cage at either room (21°C) or high (27°C) temperature. After the last MDMA administration, immunohistochemistry was performed in the caudate-putamen for CD11b and GFAP, to mark microglia and astroglia, and in the substantia nigra pars compacta for tyrosine hydroxylase, to mark dopaminergic neurons. MDMA induced glia activation and dopaminergic neurotoxicity, compared with vehicle administration. Crowding (5 or 10 mice×cage) amplified MDMA-induced glia activation (in adult and adolescent mice) and dopaminergic neurotoxicity (in adolescent mice). Conversely, exposure to a high environmental temperature (27°C) potentiated MDMA-induced glia activation in adult and adolescent mice kept 5×cage, but not dopaminergic neurotoxicity. Crowding and exposure to a high environmental temperature amplified MDMA-induced hyperthermia, and a positive correlation between body temperature and activation of either microglia or astroglia was found in adult and adolescent mice. These results provide further evidence that the administration setting influences the noxious effects of MDMA in the mouse brain. However, while crowding amplifies both glia activation and dopaminergic neurotoxicity, a high environmental temperature exacerbates glia activation only.

Metformin Prevented Dopaminergic Neurotoxicity Induced by 3,4-Methylenedioxymethamphetamine Administration.

Metformin, a well-known antidiabetic drug, has recently been proposed to promote neurogenesis and to have a neuroprotective effect on the neurodegenerative processes induced by the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in models of Parkinson's disease. Interestingly, metformin has antioxidant properties and is involved in regulating the production of cytokines released during the neuroinflammatory process. Several studies have reported that 3,4-methylenedioxymethamphetamine (MDMA), a recreational drug mostly consumed by young adults, produces a persistent loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and caudate putamen (CPu) of mice. The aim of this study was to investigate the potential neuroprotective effect of metformin against short- and long-term neurotoxicity induced by MDMA and its role on MDMA-induced hyperthermia. Adult mice received metformin (2 × 200 mg/kg, 11-h intervals, administered orally), MDMA (4 × 20 mg/kg, 2-h interval, administered intraperitoneally), or MDMA plus metformin (2 × 200 mg/kg, 1 h before the first MDMA administration and 4 h after the last). On the second and third day, mice were treated with vehicle or metformin (1 × 200 mg/kg) and sacrificed 48 h and 7 days after the last MDMA administration. The neuroprotective effect of metformin on MDMA-induced dopaminergic damage was evaluated by dopamine transporter (DAT) and tyrosine hydroxylase (TH) immunohistochemistry in SNc and CPu. Metformin prevented the MDMA-induced loss of TH-positive neurons in the SNc and TH- and DAT-positive fibers in CPu, both at 48 h and 7 days after the last MDMA administration. These results show that metformin is neuroprotective against the short- and long-lasting dopaminergic neurodegeneration induced by MDMA.

Novel (Hetero)arylalkenyl propargylamine compounds are protective in toxin-induced models of Parkinson's disease.

BACKGROUND: Mitochondrial dysfunction, oxidative stress and their interplay are core pathological features of Parkinson's disease. In dopaminergic neurons, monoamines and their metabolites provide an additional source of reactive free radicals during their breakdown by monoamine oxidase or auto-oxidation. Moreover, mitochondrial dysfunction and oxidative stress have a supraadditive impact on the pathological, cytoplasmic accumulation of dopamine and its subsequent release. Here we report the effects of a novel series of potent and selective MAO-B inhibitory (hetero)arylalkenylpropargylamine compounds having protective properties against the supraadditive effect of mitochondrial dysfunction and oxidative stress. RESULTS: The (hetero)arylalkenylpropargylamines were tested in vitro, on acute rat striatal slices, pretreated with the complex I inhibitor rotenone and in vivo, using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced acute, subchronic, and chronic experimental models of Parkinson's disease in mice. The compounds exhibited consistent protective effects against i) in vitro oxidative stress induced pathological dopamine release and the formation of toxic dopamine quinone in the rat striatum and rescued tyrosine hydroxylase positive neurons in the substantia nigra after rotenone treatment; ii) in vivo MPTP-induced striatal dopamine depletion and motor dysfunction in mice using acute and subchronic, delayed application protocols. One compound (SZV558) was also examined and proved to be protective in a chronic mouse model of MPTP plus probenecid (MPTPp) administration, which induces a progressive loss of nigrostriatal dopaminergic neurons. CONCLUSIONS: Simultaneous inhibition of MAO-B and oxidative stress induced pathological dopamine release by the novel propargylamines is protective in animal models and seems a plausible strategy to combat Parkinson's disease.

Influence of caffeine on 3,4-methylenedioxymethamphetamine-induced dopaminergic neuron degeneration and neuroinflammation is age-dependent.

Previous studies have demonstrated that caffeine administration to adult mice potentiates glial activation induced by 3,4-methylenedioxymethamphetamine (MDMA). As neuroinflammatory response seems to correlate with neurodegeneration, and the young brain is particularly vulnerable to neurotoxicity, we evaluated dopamine neuron degeneration and glial activation in the caudate-putamen (CPu) and substantia nigra pars compacta (SNc) of adolescent and adult mice. Mice were treated with MDMA (4 × 20 mg/kg), alone or with caffeine (10 mg/kg). Interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, neuronal nitric oxide synthase (nNOS) were evaluated in CPu, whereas tyrosine hydroxylase (TH), glial fibrillary acidic protein, and CD11b were evaluated in CPu and SNc by immunohistochemistry. MDMA decreased TH in SNc of both adolescent and adult mice, whereas TH-positive fibers in CPu were only decreased in adults. In CPu of adolescent mice, caffeine potentiated MDMA-induced glial fibrillary acidic protein without altering CD11b, whereas in SNc caffeine did not influence MDMA-induced glial activation. nNOS, IL-1ß, and TNF-α were increased by MDMA in CPu of adults, whereas in adolescents, levels were only elevated after combined MDMA plus caffeine. Caffeine alone modified only nNOS. Results suggest that the use of MDMA in association with caffeine during adolescence may exacerbate the neurotoxicity and neuroinflammation elicited by MDMA. Previous studies have demonstrated that caffeine potentiated glial activation induced by 3,4-methylenedioxymethamphetamine (MDMA) in adult mice. In this study, caffeine was shown to potentiate MDMA-induced dopamine neuron degeneration in substantia nigra pars compacta, astrogliosis, and TNF-α levels in caudate-putamen of adolescent mice. Results suggest that combined use of MDMA plus caffeine during adolescence may worsen the neurotoxicity and neuroinflammation elicited by MDMA.