Effects of aloe emodin on U87MG glioblastoma cell growth: In vitro and in vivo study.

Glioblastoma, the most aggressive and malignant form of glioma, appears to be resistant to various chemotherapeutic agents. Hence other approaches have been investigated to target more pathways involved in glioblastoma development and progression. Here we investigate the anticancer effect of Aloe-Emodin (AE), an anthraquinone compound presents in the leaves of Aloe arborescens, on human glioblastoma cell line U87MG. U87MG were treated with various concentrations of AE (20 and 40 µM) for different times (24, 48, and 72 hr). Cell growth was monitored by daily cell count after treatments. Growth analysis showed that AE significantly decrease proliferation of U87MG in a time and dose dependent manner. FACS analysis demonstrates a block of cell cycle in S and G2/M phase. AE probably induced also apoptosis by releasing of apoptosis-inducing factor: PARP and Lamin activation leading to nuclear shrinkage. In addition, exposure of U87MG to AE reduced pAKT phosphorylation. AE inhibition of U87MG growth is a result of more mechanism together. Here we report that AE has a specific growth inhibition on U87MG also in in vivo. The growth of U87MG, subcutaneously injected in nude mice with severe combined immunodeficiency, is inhibited without any appreciable toxic effects on the animals after AE treatment. AE might represent a conceptually new lead antitumor adjuvant drug.

Immuno-pharmacological characterization of group II metabotropic glutamate receptors controlling glutamate exocytosis in mouse cortex and spinal cord.

BACKGROUND AND PURPOSE: We recently proposed the existence of mGlu3 -preferring autoreceptors in spinal cord terminals and of mGlu2 -preferring autoreceptors in cortical terminals. This study aims to verify our previous conclusions and to extend their pharmacological characterization. EXPERIMENTAL APPROACH: We studied the effect of LY566332, an mGlu2 receptor positive allosteric modulator (PAM), and of LY2389575, a selective mGlu3 receptor negative allosteric (NAM) modulator, on the mGlu2/3 agonist LY379268-mediated inhibition of glutamate exocytosis [measured as KCl-evoked release of preloaded [3 H]-D-aspartate]. The mGlu2 PAM BINA and the mGlu3 NAM ML337, as well as selective antibodies recognizing the N-terminal of the receptor proteins, were used to confirm the pharmacological characterization of the native receptors. KEY RESULTS: Cortical synaptosomes possess LY566332-sensitive autoreceptors that are slightly, although significantly, susceptible to LY2389575. In contrast, LY566332-insensitive and LY2389575-sensitive autoreceptors are present in spinal cord terminals. BINA and ML337 mimicked LY566332 and LY2389575, respectively, in controlling LY379268-mediated inhibition of glutamate exocytosis from both cortical and spinal cord synaptosomes. Incubation of cortical synaptosomes with anti-mGlu2 antibody prevented the LY379268-induced inhibition of glutamate exocytosis, and this response was partially reduced by the anti-mGlu3 antibody. Incubation of spinal cord synaptosomes with the anti-mGlu3 antibody abolished LY379268-mediated reduction of glutamate exocytosis from these terminals, while the anti-mGlu2 antibody was inactive. Western blot analysis and confocal microscopy data were largely consistent with these functional observations. CONCLUSIONS AND IMPLICATIONS: We confirmed that mGlu3 -preferring autoreceptors exist in spinal cord terminals. Differently, cortical glutamatergic terminals possess mGlu2 /mGlu3 heterodimers, whose inhibitory effect is largely mediated by mGlu2 receptors.

Genetic deletion of mGlu2 metabotropic glutamate receptors improves the short-term outcome of cerebral transient focal ischemia.

We have recently shown that pharmacological blockade of mGlu2 metabotropic glutamate receptors protects vulnerable neurons in the 4-vessel occlusion model of transient global ischemia, whereas receptor activation amplifies neuronal death. This raised the possibility that endogenous activation of mGlu2 receptors contributes to the pathophysiology of ischemic neuronal damage. Here, we examined this possibility using two models of transient focal ischemia: (i) the monofilament model of middle cerebral artery occlusion (MCAO) in mice, and (ii) the model based on intracerebral infusion of endothelin-1 (Et-1) in rats. Following transient MCAO, mGlu2 receptor knockout mice showed a significant reduction in infarct volume and an improved short-term behavioural outcome, as assessed by a neurological disability scale and the "grip test". Following Et-1 infusion, Grm2 gene mutated Hannover Wistar rats lacking mGlu2 receptors did not show changes in the overall infarct volume as compared to their wild-type counterparts, although they showed a reduced infarct area in the agranular insular cortex. Interestingly, however, mGlu2 receptor-deficient rats performed better than wild-type rats in the adhesive tape test, in which these rats did not show the laterality preference typically observed after focal ischemia. These findings support the hypothesis that activation of mGlu2 receptors is detrimental in the post-ischemic phase, and support the use of mGlu2 receptor antagonists in the experimental treatment of brain ischemia.

Dickkopf-3 Upregulates VEGF in Cultured Human Endothelial Cells by Activating Activin Receptor-Like Kinase 1 (ALK1) Pathway.

Dkk-3 is a member of the dickkopf protein family of secreted inhibitors of the Wnt pathway, which has been shown to enhance angiogenesis. The mechanism underlying this effect is currently unknown. Here, we used cultured HUVECs to study the involvement of the TGF-ß and VEGF on the angiogenic effect of Dkk-3. Addition of hrDkk-3 peptide (1 or 10 ng/ml) to HUVECs for 6 or 12 h enhanced the intracellular and extracellular VEGF protein levels, as assessed by RTPCR, immunoblotting, immunocytochemistry and ELISA. The increase in the extracellular VEGF levels was associated to the VEGFR2 activation. Pharmacological blockade of VEGFR2 abrogated Dkk-3-induced endothelial cell tubes formation, indicating that VEGF is a molecular player of the angiogenic effects of Dkk-3. Moreover, Dkk-3 enhanced Smad1/5/8 phosphorylation and recruited Smad4 to the VEGF gene promoter, suggesting that Dkk-3 activated ALK1 receptor leading to a transcriptional activation of VEGF. This mechanism was instrumental to the increased VEGF expression and endothelial cell tubes formation mediated by Dkk-3, because both effects were abolished by siRNA-mediated ALK1 knockdown. In summary, we have found that Dkk-3 activates ALK1 to stimulate VEGF production and induce angiogenesis in HUVECs.

Targeting type-2 metabotropic glutamate receptors to protect vulnerable hippocampal neurons against ischemic damage.

BACKGROUND: To examine whether metabotropic glutamate (mGlu) receptors have any role in mechanisms that shape neuronal vulnerability to ischemic damage, we used the 4-vessel occlusion (4-VO) model of transient global ischemia in rats. 4-VO in rats causes a selective death of pyramidal neurons in the hippocampal CA1 region, leaving neurons of the CA3 region relatively spared. We wondered whether changes in the expression of individual mGlu receptor subtypes selectively occur in the vulnerable CA1 region during the development of ischemic damage, and whether post-ischemic treatment with drugs targeting the selected receptor(s) affords neuroprotection. RESULTS: We found that 4-VO caused significantly reduction in the transcript of mGlu2 receptors in the CA1 region at times that preceded the anatomical evidence of neuronal death. Down-regulation of mGlu2 receptors was associated with reduced H3 histone acetylation at the Grm2 promoter. The transcripts of other mGlu receptor subtypes were unchanged in the CA1 region of 4-VO rats. Ischemia did not cause changes in mGlu2 receptor mRNA levels in the resistant CA3 region, which, interestingly, were lower than in the CA1 region. Targeting the mGlu2 receptors with selective pharmacologic ligands had profound effects on ishemic neuronal damage. Post-ischemic oral treatment with the selective mGlu2 receptor NAM (negative allosteric modulator), ADX92639 (30 mg/kg), was highly protective against ischemic neuronal death. In contrast, s.c. administration of the mGlu2 receptor enhancer, LY487379 (30 mg/kg), amplified neuronal damage in the CA1 region and extended the damage to the CA3 region. CONCLUSION: These findings suggest that the mGlu2 receptor is an important player in mechanisms regulating neuronal vulnerability to ischemic damage, and that mGlu2 receptor NAMs are potential candidates in the experimental treatments of disorders characterized by brain hypoperfusion, such as hypovolemic shock and cardiac arrest.

5-HT(2C) serotonin receptor blockade prevents tau protein hyperphosphorylation and corrects the defect in hippocampal synaptic plasticity caused by a combination of environmental stressors in mice.

Exposure to multimodal sensory stressors is an everyday occurrence and sometimes becomes very intense, such as during rave parties or other recreational events. A growing body of evidence suggests that strong environmental stressors might cause neuronal dysfunction on their own in addition to their synergistic action with illicit drugs. Mice were exposed to a combination of physical and sensory stressors that are reminiscent of those encountered in a rave party. However, this is not a model of rave because it lacks the rewarding properties of rave. A 14-h exposure to environmental stressors caused an impairment of hippocampal long-term potentiation (LTP) and spatial memory, and an enhanced phosphorylation of tau protein in the CA1 and CA3 regions. These effects were transient and critically depended on the activation of 5-HT2C serotonin receptors, which are highly expressed in the CA1 region. Acute systemic injection of the selective 5-HT2C antagonist, RS-102,221 (2 mg/kg, i.p., 2 min prior the onset of stress), prevented tau hyperphosphorylation and also corrected the defects in hippocampal LTP and spatial memory. These findings suggest that passive exposure to a combination of physical and sensory stressors causes a reversible hippocampal dysfunction, which might compromise mechanisms of synaptic plasticity and spatial memory for a few days. Drugs that block 5-HT2C receptors might protect the hippocampus against the detrimental effect of environmental stressors.

Activation of mGlu3 metabotropic glutamate receptors enhances GDNF and GLT-1 formation in the spinal cord and rescues motor neurons in the SOD-1 mouse model of amyotrophic lateral sclerosis.

Enhancement of glial-derived neurotrophic factor (GDNF) is an established therapeutic target for amyotrophic lateral sclerosis (ALS). Activation of group II metabotropic glutamate (mGlu) receptors with the orthosteric agonist, LY379268, enhanced GDNF levels in cultured spinal cord astrocytes from wild-type mice and mGlu2(-/-) mice, but not in astrocytes from mGlu3(-/-) mice. LY379268 protected Sternberger monoclonal incorporated antibody-32 (SMI-32)(+) motor neurons against excitotoxic death in mixed cultures of spinal cord cells, and its action was abrogated by anti-GDNF antibodies. Acute systemic injection of LY379268 (0.5, 1 or 5mg/kg, i.p.) enhanced spinal cord GDNF levels in wild-type and mGlu2(-/-) mice, but not in mGlu3(-/-) mice. No tolerance developed to the GDNF-enhancing effect of LY379268 when the drug was continuously delivered for 28days by means of s.c. osmotic minipumps (0.5-5mg/day). Double fluorescent immunostaining showed a co-localization of GDNF with the astrocyte marker, GFAP, but not with the neuronal marker, Neuronal Nuclear Antigen (NeuN), or with SMI-32. Continuous infusion of LY379268 also enhanced the expression of the glutamate transporter GLT-1, in the spinal cord. These data laid the groundwork for the study of LY379268 in ALS mice. Continuous treatment with 1 or 5mg/kg/day with LY379268 had a beneficial effect on neurological disability in SOD1G93A mice. At day 40 of treatment, LY379268 enhanced spinal cord levels of GDNF and GLT-1, and rescued spinal cord motor neurons, as assessed by stereologic counting of SMI-32(+) cells. LY379268 had no significant effect on the mortality rate of SODG93A. These findings encourage the development of selective mGlu3 receptor agonists/enhancers as neuroprotective agents in ALS.

mGlu1 receptor-induced LTD of NMDA receptor transmission selectively at Schaffer collateral-CA1 synapses mediates metaplasticity.

Hippocampal CA1 pyramidal neurons receive inputs from entorhinal cortex directly via the temporoammonic (TA) pathway and indirectly via the Schaffer collateral (SC) pathway from CA3. NMDARs at synapses of both pathways are critical for the induction of synaptic plasticity, information processing, and learning and memory. We now demonstrate that, in the rat hippocampus, activity-dependent mGlu1 receptor-mediated LTD (mGlu1-LTD) of NMDAR-mediated transmission (EPSC(NMDA)) at the SC-CA1 input prevents subsequent LTP of AMPAR-mediated transmission. In contrast, there was no activity-dependent mGlu1-LTD of EPSC(NMDA) at the TA-CA1 pathway, or effects on subsequent plasticity of AMPAR-mediated transmission. Therefore, the two major pathways delivering information to CA1 pyramidal neurons are subject to very different plasticity rules.

Changes in mGlu5 receptor-dependent synaptic plasticity and coupling to homer proteins in the hippocampus of Ube3A hemizygous mice modeling angelman syndrome.

Angelman syndrome (AS) is caused by the loss of Ube3A, an ubiquitin ligase that commits specific proteins to proteasomal degradation. How this defect causes autism and other pathological phenotypes associated with AS is unknown. Long-term depression (LTD) of excitatory synaptic transmission mediated by type 5 metabotropic glutamate (mGlu5) receptors was enhanced in hippocampal slices of Ube3A(m-/p+) mice, which model AS. No changes were found in NMDA-dependent LTD induced by low-frequency stimulation. mGlu5 receptor-dependent LTD in AS mice was sensitive to the protein synthesis inhibitor anisomycin, and relied on the same signaling pathways as in wild-type mice, e.g., the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycine pathway, and protein tyrosine phosphatase. Neither the stimulation of MAPK and PI3K nor the increase in Arc (activity-regulated cytoskeleton-associated protein) levels in response to mGlu5 receptor activation were abnormal in hippocampal slices from AS mice compared with wild-type mice. mGlu5 receptor expression and mGlu1/5 receptor-mediated polyphosphoinositide hydrolysis were also unchanged in the hippocampus of AS mice. In contrast, AS mice showed a reduced expression of the short Homer protein isoform Homer 1a, and an increased coupling of mGlu5 receptors to Homer 1b/c proteins in the hippocampus. These findings support the link between Homer proteins and monogenic autism, and lay the groundwork for the use of mGlu5 receptor antagonists in AS.

Gender differences in Parkinson's disease: focus on plasma α-synuclein.

Among promising biological markers proposed for Parkinson's disease (PD) and other disorders related to Lewy bodies, plasma alpha-synuclein assay has provided conflicting results mainly owing to the various laboratory assay techniques used and protein forms assayed. In this observational and exploratory cross-sectional study, using an immunoenzymatic technique, we assayed and compared total plasma alpha-synuclein concentrations in 69 patients with PD and 110 age-matched healthy control subjects. Two previously unreported findings concerned gender. First, plasma alpha-synuclein concentrations measured in the more advanced parkinsonian disease stages decreased in men, but not in women. Second, again only in men, plasma alpha-synuclein concentration was associated with cognitive impairments, hallucinations, and sleep disorders. These findings underline the gender-related differences in parkinsonian patients and indicate plasma alpha-synuclein expression as a potential biological marker for PD progression in men.

Constitutively active group I mGlu receptors and PKMzeta regulate synaptic transmission in developing perirhinal cortex.

Synaptic transmission is essential for early development of the central nervous system. However, the mechanisms that regulate early synaptic transmission in the cerebral cortex are unclear. PKMζ is a kinase essential for the maintenance of LTP. We show for the first time that inhibition of PKMζ produces a profound depression of basal synaptic transmission in neonatal, but not adult, rat perirhinal cortex. This suggests that synapses in early development are in a constitutive LTP-like state. Furthermore, basal synaptic transmission in immature, but not mature, perirhinal cortex relies on persistent activity of metabotropic glutamate (mGlu) receptor, PI3Kinase and mammalian target of rapamycin (mTOR). Thus early in development, cortical synapses exist in an LTP-like state maintained by tonically active mGlu receptor-, mTOR- and PKMζ- dependent cascades. These results provide new understanding of the molecular mechanisms that control synapses during development and may aid our understanding of developmental disorders such as autism and schizophrenia. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Fingolimod protects cultured cortical neurons against excitotoxic death.

Fingolimod (FTY720), a novel drug approved for the treatment of relapsing-remitting multiple sclerosis, activates different sphingosine-1-phosphate receptor (S1PR) subtypes. Its primary mechanism of action is to reduce the egress of T lymphocytes from secondary lymphoid organs, thus restraining neuroinflammation and autoimmunity. However, recent evidence suggests that the action of FTY720 involves S1PRs expressed by cells resident in the CNS, including neurons. Here, we examined the effect of FTY720, its active metabolite, FTY720-P, and sphingosine-1-phosphate (S1P) on neuronal viability using a classical in vitro model of excitotoxic neuronal death. Mixed cultures of mouse cortical cells were challenged with toxic concentrations of N-methyl-d-aspartate (NMDA) for 10 min, and neuronal death was assessed 20 h later. FTY720, FTY720-P, and S1P were all neuroprotective when applied 18-20 h prior to the NMDA pulse. Neuroprotection was attenuated by pertussis toxin, and inhibited by the selective type-1 S1PR (S1P1R) antagonist, W146, and by inhibitors of the mitogen associated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PtdIns-3-K) pathways. Both FTY720 and FTY720-P retained their protective activity in pure cultures of mouse or rat cortical neurons. These data offer the first direct demonstration that FTY720 and its active metabolite protect neurons against excitotoxic death.

Increased serum Dickkopf-1 levels in drug-abusing psychotic patients.

BACKGROUND: Dickkopf-1 (DKK1) is an inhibitor of the canonical Wnt pathway, which is known to be impaired in both psychotic and neurodegenerative disorders. Here, we examined serum DKK1 levels as an indicator of ongoing neurodegeneration in psychotic patients, with or without a recent or current history of drug abuse. METHODS: We measured serum DKK1 levels by ELISA in 22 inpatients with psychosis and no history of drug abuse, 22 with psychosis and drug abuse, and 16 controls. We rated psychopathology using the following rating scales: the Positive and Negative Syndrome Scale (PANSS); the Clinical Global Impressions (CGI) severity scale; and the Global Assessment of Functioning (GAF) scale. Extrapyramidal motor symptoms were assessed by the Simpson-Angus Neurological Rating Scale (NRS). RESULTS: Inpatients with psychosis and comorbid substance abuse showed significantly higher serum DKK1 levels than inpatients with psychosis and no comorbid substance abuse or controls. Comorbid patients had earlier onset, longer duration of psychosis, and more severe extrapyramidal motor symptoms. However, we did not find any significant correlation between DKK1 levels and rating scale scores. CONCLUSION: Psychosis led to elevated serum DKK1 levels, and substance abuse led to a further increase. Knowing that there is a correlation between brain and blood levels of DKK1, we speculate that the observed increase in DKK1 levels reflects drug-induced neurotoxicity in our patients.

Induction of the Wnt antagonist Dickkopf-1 is involved in stress-induced hippocampal damage.

The identification of mechanisms that mediate stress-induced hippocampal damage may shed new light into the pathophysiology of depressive disorders and provide new targets for therapeutic intervention. We focused on the secreted glycoprotein Dickkopf-1 (Dkk-1), an inhibitor of the canonical Wnt pathway, involved in neurodegeneration. Mice exposed to mild restraint stress showed increased hippocampal levels of Dkk-1 and reduced expression of ß-catenin, an intracellular protein positively regulated by the canonical Wnt signalling pathway. In adrenalectomized mice, Dkk-1 was induced by corticosterone injection, but not by exposure to stress. Corticosterone also induced Dkk-1 in mouse organotypic hippocampal cultures and primary cultures of hippocampal neurons and, at least in the latter model, the action of corticosterone was reversed by the type-2 glucocorticoid receptor antagonist mifepristone. To examine whether induction of Dkk-1 was causally related to stress-induced hippocampal damage, we used doubleridge mice, which are characterized by a defective induction of Dkk-1. As compared to control mice, doubleridge mice showed a paradoxical increase in basal hippocampal Dkk-1 levels, but no Dkk-1 induction in response to stress. In contrast, stress reduced Dkk-1 levels in doubleridge mice. In control mice, chronic stress induced a reduction in hippocampal volume associated with neuronal loss and dendritic atrophy in the CA1 region, and a reduced neurogenesis in the dentate gyrus. Doubleridge mice were resistant to the detrimental effect of chronic stress and, instead, responded to stress with increases in dendritic arborisation and neurogenesis. Thus, the outcome of chronic stress was tightly related to changes in Dkk-1 expression in the hippocampus. These data indicate that induction of Dkk-1 is causally related to stress-induced hippocampal damage and provide the first evidence that Dkk-1 expression is regulated by corticosteroids in the central nervous system. Drugs that rescue the canonical Wnt pathway may attenuate hippocampal damage in major depression and other stress-related disorders.

Targeting group II metabotropic glutamate (mGlu) receptors for the treatment of psychosis associated with Alzheimer's disease: selective activation of mGlu2 receptors amplifies beta-amyloid toxicity in cultured neurons, whereas dual activation of mGlu2 and mGlu3 receptors is neuroprotective.

Dual orthosteric agonists of metabotropic glutamate 2 (mGlu2) and mGlu3 receptors are being developed as novel antipsychotic agents devoid of the adverse effects of conventional antipsychotics. Therefore, these drugs could be helpful for the treatment of psychotic symptoms associated with Alzheimer's disease (AD). In experimental animals, the antipsychotic activity of mGlu2/3 receptor agonists is largely mediated by the activation of mGlu2 receptors and is mimicked by selective positive allosteric modulators (PAMs) of mGlu2 receptors. We investigated the distinct influence of mGlu2 and mGlu3 receptors in mixed and pure neuronal cultures exposed to synthetic ß-amyloid protein (Aß) to model neurodegeneration occurring in AD. The mGlu2 receptor PAM, N-4'-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride (LY566332), devoid of toxicity per se, amplified Aß-induced neurodegeneration, and this effect was prevented by the mGlu2/3 receptor antagonist (2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl)glycine (LY341495). LY566332 potentiated Aß toxicity regardless of the presence of glial mGlu3 receptors, but it was inactive when neurons lacked mGlu2 receptors. The dual mGlu2/3 receptor agonist, (-)-2-oxa-4-aminobicyclo[3.1.0]exhane-4,6-dicarboxylic acid (LY379268), was neuroprotective in mixed cultures via a paracrine mechanism mediated by transforming growth factor-ß1. LY379268 lost its protective activity in neurons grown with astrocytes lacking mGlu3 receptors, indicating that protection against Aß neurotoxicity was mediated entirely by glial mGlu3 receptors. The selective noncompetitive mGlu3 receptor antagonist, (3S)-1-(5-bromopyrimidin-2-yl)-N-(2,4-dichlorobenzyl)pyrrolidin-3-amine methanesulfonate hydrate (LY2389575), amplified Aß toxicity on its own, and, interestingly, unmasked a neurotoxic activity of LY379268, which probably was mediated by the activation of mGlu2 receptors. These data indicate that selective potentiation of mGlu2 receptors enhances neuronal vulnerability to Aß, whereas dual activation of mGlu2 and mGlu3 receptors is protective against Aß-induced toxicity.

Early defect of transforming growth factor ß1 formation in Huntington's disease.

A defective expression or activity of neurotrophic factors, such as brain- and glial-derived neurotrophic factors, contributes to neuronal damage in Huntington's disease (HD). Here, we focused on transforming growth factor-ß (TGF-ß(1) ), a pleiotropic cytokine with an established role in mechanisms of neuroprotection. Asymptomatic HD patients showed a reduction in TGF-ß(1) levels in the peripheral blood, which was related to trinucleotide mutation length and glucose hypometabolism in the caudate nucleus. Immunohistochemical analysis in post-mortem brain tissues showed that TGF-ß(1) was reduced in cortical neurons of asymptomatic and symptomatic HD patients. Both YAC128 and R6/2 HD mutant mice showed a reduced expression of TGF-ß(1) in the cerebral cortex, localized in neurons, but not in astrocytes. We examined the pharmacological regulation of TGF-ß(1) formation in asymptomatic R6/2 mice, where blood TGF-ß(1) levels were also reduced. In these R6/2 mice, both the mGlu2/3 metabotropic glutamate receptor agonist, LY379268, and riluzole failed to increase TGF-ß(1) formation in the cerebral cortex and corpus striatum, suggesting that a defect in the regulation of TGF-ß(1) production is associated with HD. Accordingly, reduced TGF-ß(1) mRNA and protein levels were found in cultured astrocytes transfected with mutated exon 1 of the human huntingtin gene, and in striatal knock-in cell lines expressing full-length huntingtin with an expanded glutamine repeat. Taken together, our data suggest that serum TGF-ß(1) levels are potential biomarkers of HD development during the asymptomatic phase of the disease, and raise the possibility that strategies aimed at rescuing TGF-ß(1) levels in the brain may influence the progression of HD.

Activation of mGlu3 receptors stimulates the production of GDNF in striatal neurons.

Metabotropic glutamate (mGlu) receptors have been considered potential targets for the therapy of experimental parkinsonism. One hypothetical advantage associated with the use of mGlu receptor ligands is the lack of the adverse effects typically induced by ionotropic glutamate receptor antagonists, such as sedation, ataxia, and severe learning impairment. Low doses of the mGlu2/3 metabotropic glutamate receptor agonist, LY379268 (0.25-3 mg/kg, i.p.) increased glial cell line-derived neurotrophic factor (GDNF) mRNA and protein levels in the mouse brain, as assessed by in situ hybridization, real-time PCR, immunoblotting, and immunohistochemistry. This increase was prominent in the striatum, but was also observed in the cerebral cortex. GDNF mRNA levels peaked at 3 h and declined afterwards, whereas GDNF protein levels progressively increased from 24 to 72 h following LY379268 injection. The action of LY379268 was abrogated by the mGlu2/3 receptor antagonist, LY341495 (1 mg/kg, i.p.), and was lost in mGlu3 receptor knockout mice, but not in mGlu2 receptor knockout mice. In pure cultures of striatal neurons, the increase in GDNF induced by LY379268 required the activation of the mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathways, as shown by the use of specific inhibitors of the two pathways. Both in vivo and in vitro studies led to the conclusion that neurons were the only source of GDNF in response to mGlu3 receptor activation. Remarkably, acute or repeated injections of LY379268 at doses that enhanced striatal GDNF levels (0.25 or 3 mg/kg, i.p.) were highly protective against nigro-striatal damage induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice, as assessed by stereological counting of tyrosine hydroxylase-positive neurons in the pars compacta of the substantia nigra. We speculate that selective mGlu3 receptor agonists or enhancers are potential candidates as neuroprotective agents in Parkinson's disease, and their use might circumvent the limitations associated with the administration of exogenous GDNF.

Memantine treatment reduces the expression of the K(+)/Cl(-) cotransporter KCC2 in the hippocampus and cerebral cortex, and attenuates behavioural responses mediated by GABA(A) receptor activation in mice.

A 7-day treatment with memantine (25 mg/kg, i.p.), a drug that is currently prescribed for the treatment of Alzheimer's disease, increased the levels of brain-derived neurotrophic factor (BDNF) and reduced the expression of the neuron-specific K(+)/Cl(-) co-transporter, KCC2, in the hippocampus and cerebral cortex of mice. Knowing that KCC2 maintains low intracellular Cl(-) concentrations, which drive Cl(-) influx in response to GABA(A) receptor activation, we monitored the behavioural response to the GABA(A) receptor enhancer, diazepam, in mice pre-treated for 7 days with saline or 25 mg/kg of memantine. Memantine treatment substantially attenuated motor impairment induced by an acute challenge with diazepam (6 mg/kg, i.p.), as assessed by the rotarod test and the horizontal wire test. We suggest that a prolonged treatment with memantine induces changes in the activity of GABA(A) receptors that might contribute to the therapeutic and/or toxic effects of the drug.

Enhanced tau phosphorylation in the hippocampus of mice treated with 3,4-methylenedioxymethamphetamine ("Ecstasy").

3,4-Methylenedioxymethamphetamine (MDMA) ("Ecstasy") produces neurotoxic effects, which result into an impairment of learning and memory and other neurological dysfunctions. We examined whether MDMA induces increases in tau protein phosphorylation, which are typically associated with Alzheimer's disease and other chronic neurodegenerative disorders. We injected mice with MDMA at cumulative doses of 10-50 mg/kg intraperitoneally, which are approximately equivalent to doses generally consumed by humans. MDMA enhanced the formation of reactive oxygen species and induced reactive gliosis in the hippocampus, without histological evidence of neuronal loss. An acute or 6 d treatment with MDMA increased tau protein phosphorylation in the hippocampus, revealed by both anti-phospho(Ser(404))-tau and paired helical filament-1 antibodies. This increase was restricted to the CA2/CA3 subfields and lasted 1 and 7 d after acute and repeated MDMA treatment, respectively. Tau protein was phosphorylated as a result of two nonredundant mechanisms: (1) inhibition of the canonical Wnt (wingless-type MMTV integration site family) pathway, with ensuing activation of glycogen synthase kinase-3beta; and (2) activation of type-5 cyclin-dependent kinase (Cdk5). MDMA induced the expression of the Wnt antagonist, Dickkopf-1, and the expression of the Cdk5-activating protein, p25. In addition, the increase in tau phosphorylation was attenuated by strategies that rescued the Wnt pathway or inhibited Cdk5. Finally, an impairment in hippocampus-dependent spatial learning was induced by doses of MDMA that increased tau phosphorylation, although the impairment outlasted this biochemical event. We conclude that tau hyperphosphorylation in the hippocampus may contribute to the impairment of learning and memory associated with MDMA abuse.

GABAergic drugs become neurotoxic in cortical neurons pre-exposed to brain-derived neurotrophic factor.

A 24-h pretreatment with BNDF enhanced excitotoxic neuronal death in cultured mouse cortical cells challenged with NMDA in the presence of extracellular Mg(2+). The GABA(A) receptor antagonist, bicuculline, enhanced NMDA toxicity in control cultures but, unexpectedly, became neuroprotective in cultures pretreated with BDNF. In contrast, drugs that activate GABA(A) receptors (e.g. muscimol, benzodiazepines, or phenobarbital) or drugs that indirectly enhance GABAergic transmission were protective in control cultures but amplified NMDA toxicity after pretreatment with BDNF. The atypical behaviour of GABAergic drugs in cultures pretreated with BDNF depended on changes in the anion reversal potential because (i) increases in extracellular Cl(-) concentrations abolished the neurotoxic action of muscimol; (ii) muscimol stimulated (36)Cl(-) efflux after pretreatment with BDNF; and (iii) exposure to BDNF reduced the expression of the neuronal K(+)/Cl(-) co-transporter, KCC2. Our data raise the concern that GABAergic drugs may become neurotoxic under conditions associated with increases in brain BDNF levels.