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

Quantitative real-time PCR analysis of GDNF mRNA in mouse striatum (A) and cortex (B) at 3, 6 or 12 h after systemic treatment with saline, LY379268 (3 mg/kg, i.p.), or LY379268 (3 mg/kg, i.p.)+LY341495 (1 mg/kg, i.p.).

<p>Values were normalized with respect to the amount of β-actin mRNA. Values are mean+S.E.M. of four determinations (each from triplicates). *<i>p</i><0.05 (One-way ANOVA+Fisher's PLSD) vs. saline-treated mice.</p

Basal GDNF levels in cultured mouse striatal neurons and in cultured astrocytes (A).

<p>Expression of phosphoERK1/2 and phospho-Akt in cultured striatal neurons treated with LY379268 (1 µM), LY341495 (1 µM) and LY379268+LY341495 for 15 min (B). Densitometric values are means+S.E.M. of 3–4 determinations. *p<0.05 (One-Way ANOVA+Fisher's PLSD) vs. basal values, #p<0.05 vs. LY379268 values. Treatment of cultured neurons with 1 µM LY379268 enhanced GDNF levels 24 h later (C), and it was abrogated by the co-application of the MEK inhibitor, PD98059, or the PI-3-K inhibitor, LY294002 (C). Application of LY379268 to astrocytes made “reactive” by several passages in culture and by the G5 supplement in the medium did not affect GDNF levels (D).</p

Immunohistochemical analysis of TH in the pars compacta of substantia nigra of mice injected with a single i.p. dose of 30 mg/kg of MPTP, alone or combined with LY379268 (0.25 or 3 mg/kg in a single i.p. injection, 30 min prior to MPTP injection or 0.25 mg/kg/7 days once a day, i.p.).

<p>Scale bar = 100 µm. Stereological TH-positive cell counts are also shown. Values (means+S.E.M.) were calculated from 7–8 mice per group (10 sections - 10 µm thick, cut every 100 µm, per animal were used for the calculation of the density of TH-positive neurons in the pars compacta of the substantia nigra). *<i>p</i><0.05 (One-way ANOVA+Fisher's PLSD) vs. mice treated with MPTP alone.</p

Western blot analysis of striatal GDNF expression in wild-type, <i>mGlu2^−/−</i> or <i>mGlu3^−/−</i> mice in basal conditions (A) and after treatment with LY379268, 3 mg/kg, i.p. (B).

<p>Animals were killed 24 h later. Densitometric data of GDNF are shown and are the mean+S.E.M. of 3 animals performed two times.*<i>p</i><0.05 (One-way ANOVA+Fisher's PLSD) vs. saline-treated mice.</p

LY379268 fails to protects against MPTP toxicity in mice unilaterally implanted with anti-GDNF antibodies.

<p>Mice were implanted with a gelfoam (Spongostan) pre-soaked with saline alone (A,B) or a saline solution containing 5 µg of neutralizing anti-GDNF antibodies (A,B) in the left caudate nucleus. Stereological counts of TH-positive neurons in the substantia nigra pars compact in the implantation side (left) or contralateral side (right) in response to i.p. injection of saline, LY379268 (3 mg/kg), MPTP alone (30 mg/kg) or MPTP+LY379268 (injected 30 min prior to MPTP injection). Drugs were administered 24 h after the gelfoam implantation. Mice were killed 7 days after MPTP injection. Values (means+S.E.M.) were calculated from 6 mice per group. <i>p</i><0.05 (One-way ANOVA+Fisher's PLSD) vs. the corresponding values in mice treated with saline (*) or vs. the MPTP values of the right side (#).</p

ELISA analysis of GDNF expression in the striatum of mice after treatment with saline, LY379268 (3 mg/kg, i.p.), MPTP (30 mg/kg, i.p.), or MPTP+LY379268.

<p>Animals were killed 1,2,3 or 7 days after treatments. Data of GDNF are the mean+S.E.M. of 8 animals. <i>p</i><0.05 (One-way ANOVA+Fisher's PLSD) vs. the corresponding groups of mice treated with saline (*) or with MPTP or LY379268 alone (#).</p

Double immunolabeling for GDNF and NeuN or GFAP in striatal cells showing the labelling of GDNF within neuronal cells (A).

<p>Arrows in the left panel, NeuN-positive cells containing GDNF mRNA black grains; arrow in the right panel, GDNF mRNA black grains, arrow head in the right panel, GFAP-positive cell. Immunohistochemical analysis of GDNF in the striatum of mice treated with a single injection of LY379268 (3 mg/kg, i.p.) and killed 24 h later (B). In both control mice and mice treated with LY379268, GDNF immunoreactivity is exclusively localized in neurons (note the absence of co-localization between GDNF and GFAP), and the extent of immunostaining increases after drug treatment. GDNF immunostaining in the striatum of mice treated 7 days before with MPTP, 20 mg/kg, i.p., x 3, two h apart (C). This treatment led to reactive gliosis in the striatum, as a result of the degeneration of nigro-striatal dopaminergic neurons. Under these conditions, GDNF immunostaining is localized both in neurons and reactive astrocytes. A single injection of LY379268 (3 mg/kg, i.p.) 7 days following MPTP injection did not enhance GDNF immunoreactivity in reactive astrocytes, but still enhanced immunoreactivity in neurons. Interestingly, the number of GDNF-positive reactive astrocytes is lower 24 h following LY379268 injection. Scale bar = 50 and 10 µm.</p

<i>In situ</i> hybridization of sagittal sections at basal ganglia level showing the expression of mRNA encoding GDNF (A) or NGF (B).

<p>Autoradiogram showing GDNF expression in the striatum of saline-treated mice or LY379268 (0.25 mg/kg, i.p.)-treated mice (A). The inserts show representative GDNF mRNA labeled cells (black grains) with increased levels of labeling in LY379268-treated mice. Autoradiogram showing NGF expression of saline-treated mice or LY379268 (0.25 mg/kg, i.p.)-treated mice (B). Dose-response curve of GDNF mRNA levels in the striatum of mice treated with saline or LY379268 (0.1, 0.25, 1, 3 or 4 mg/kg, i.p) (C) and time-course of GDNF mRNA levels in the striatum of mice after a single injection of LY379268 (0.25 mg/kg, i.p.) (D); values are means±S.E.M (n = 4–5, animals per group; three independent experiments). Striatal GDNF mRNA levels in mice treated with saline, LY379268 (0.25 mg/kg, i.p), LY341495 (1 mg/kg, i.p) or LY379268+LY341495 (E); value are means±S.E.M (n = 4, animals per group; three independent experiments). *<i>p</i><0.05; **<i>p</i><0.01 (One-way ANOVA+Fisher's PLSD) vs. control mice. Scale bar: A–B = 4 mm. Str, striatum; Ctx, cortex; Hipp, hippocampus.</p

Fisiologia umana. Fondamenti. Con e-book. Con espansione online

Lo studio della fisiologia è fondamentale e caratterizzante in diversi corsi universitari: propedeutico alle discipline cliniche, ha come obiettivo principale la conoscenza del funzionamento degli organismi viventi a tutti i livelli dell’organizzazione biologica, dalle molecole fino ai sistemi d’organo attraverso l’integrazione delle nozioni e dei concetti acquisiti in altre materie di base. Nell’opera si è cercato di mantenere un equilibrio tra le due anime in cui è tradizionalmente suddivisa la fisiologia, cioè la fisiologia cellulare e quella d’organo e di sistema. In particolare è stata proposta una visione integrata dei processi fisiologici. L’impostazione grafica e la struttura del libro sono state concepite per favorire e stimolare il percorso didattico dello studente. Ogni capitolo è preceduto da un riassunto elaborato graficamente e da tabelle per comprenderne immediatamente il contenuto e l’organizzazione. All’interno di ciascun capitolo gli argomenti sono strutturati in sezioni introdotte da un breve, ma dettagliato, sommario. Nell’area web – Virtual campus: Esercizi di autovalutazione; Percorsi guidati e laboratori interattivi; Accesso alla versione digitale del libro