Role of G Protein-coupled Receptor Kinase 4 and β-Arrestin 1 in Agonist-stimulated Metabotropic Glutamate Receptor 1 Internalization and Activation of Mitogen-activated Protein Kinases

The metabotropic glutamate 1 (mGlu(1)) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G protein-coupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu(1) receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu(1) receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Galpha(q). In cells transfected with GRK4 and exposed to agonist, beta-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu(1) receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of beta-arrestin V53D dominant negative mutant, which did not affect the mGlu(1) receptor internalization, reduced by 70-80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu(1) receptor. The agonist-stimulated differential sorting of the mGlu(1) receptor and beta-arrestin as well as the activation of MAP kinases by mGlu(1) agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of beta-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing beta-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu(1) receptor by different mechanisms and that beta-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule

A Large Family with p.Arg554His Mutation in ABCD1: Clinical Features and Genotype/Phenotype Correlation in Female Carriers

X-linked adrenoleukodystrophy (X-ALD, OMIM #300100) is the most common peroxisomal disorder clinically characterized by two main phenotypes: adrenomyeloneuropathy (AMN) and the cerebral demyelinating form of X-ALD (cerebral ALD). The disease is caused by defects in the gene for the adenosine triphosphate (ATP)-binding cassette protein, subfamily D (ABCD1) that encodes the peroxisomal transporter of very-long-chain fatty acids (VLCFAs). The defective function of ABCD1 protein prevents β-oxidation of VLCFAs, which thus accumulate in tissues and plasma, to represent the hallmark of the disease. As in many X-linked diseases, it has been routinely expected that female carriers are asymptomatic. Nonetheless, recent findings indicate that most ABCD1 female carriers become symptomatic, with a motor disability that typically appears between the fourth and fifth decade. In this paper, we report a large family in which affected males died during the first decade, while affected females develop, during the fourth decade, progressive lower limb weakness with spastic or ataxic-spastic gait, tetra-hyperreflexia with sensory alterations. Clinical and genetic evaluations were performed in nine subjects, eight females (five affected and three healthy) and one healthy male. All affected females were carriers of the c.1661G>A (p.Arg554His, rs201568579) mutation. This study strengthens the relevance of clinical symptoms in female carriers of ABCD1 mutations, which leads to a better understanding of the role of the genetic background and the genotype-phenotype correlation. This indicates the relevance to include ABCD1 genes in genetic panels for gait disturbance in women

Activation of group III metabotropic glutamate receptors inhibits the production of RANTES in glial cell cultures

The chemokine RANTES is critically involved in neuroinflammation and has been implicated in the pathophysiology of multiple sclerosis. We examined the possibility that activation of G-protein-coupled metabotropic glutamate (mGlu) receptors regulates the formation of RANTES in glial cells. A 15 hr exposure of cultured astrocytes to tumor necrosis factor-alpha and interferon-gamma induced a substantial increase in both RANTES mRNA and extracellular RANTES levels. These increases were markedly reduced when astrocytes were coincubated with l-2-amino-4-phosphonobutanoate (l-AP-4), 4-phosphonophenylglycine, or l-serine-O-phosphate, which selectively activate group III mGlu receptor subtypes (i.e., mGlu4, -6, -7, and -8 receptors). Agonists of mGlu1/5 or mGlu2/3 receptors were virtually inactive. Inhibition of RANTES release produced by l-AP-4 was attenuated by the selective group III mGlu receptor antagonist (R,S)-alpha-methylserine-O-phosphate or by pretreatment of the cultures with pertussis toxin. Cultured astrocytes expressed mGlu4 receptors, and the ability of l-AP-4 to inhibit RANTES release was markedly reduced in cultures prepared from mGlu4 knock-out mice. This suggests that activation of mGlu4 receptors negatively modulates the production of RANTES in glial cells. We also examined the effect of l-AP-4 on the development of experimental allergic encephalomyelitis (EAE) in Lewis rats. l-AP-4 was subcutaneously infused for 28 d by an osmotic minipump that released 250 nl/hr of a solution of 250 mm of the drug. Detectable levels of l-AP-4 ( approximately 100 nm) were found in the brain dialysate of EAE rats. Infusion of l-AP-4 did not affect the time at onset and the severity of neurological symptoms but significantly increased the rate of recovery from EAE. In addition, lower levels of RANTES mRNA were found in the cerebellum and spinal cord of EAE rats infused with l-AP-4. These results suggest that pharmacological activation of group III mGlu receptors may be useful in the experimental treatment of neuroinflammatory CNS disorders

Protection from angiotensin II–mediated vasculotoxic and hypertensive response in mice lacking PI3Kγ

Hypertension affects nearly 20% of the population in Western countries and strongly increases the risk for cardiovascular diseases. In the pathogenesis of hypertension, the vasoactive peptide of the renin-angiotensin system, angiotensin II and its G protein–coupled receptors (GPCRs), play a crucial role by eliciting reactive oxygen species (ROS) and mediating vessel contractility. Here we show that mice lacking the GPCR-activated phosphoinositide 3-kinase (PI3K)γ are protected from hypertension that is induced by administration of angiotensin II in vivo. PI3Kγ was found to play a role in angiotensin II–evoked smooth muscle contraction in two crucial, distinct signaling pathways. In response to angiotensin II, PI3Kγ was required for the activation of Rac and the subsequent triggering of ROS production. Conversely, PI3Kγ was necessary to activate protein kinase B/Akt, which, in turn, enhanced L-type Ca2+ channel–mediated extracellular Ca2+ entry. These data indicate that PI3Kγ is a key transducer of the intracellular signals that are evoked by angiotensin II and suggest that blocking PI3Kγ function might be exploited to improve therapeutic intervention on hypertension

Common and rare variants in TMEM175 gene concur to the pathogenesis of Parkinson’s Disease in Italian patients

Parkinson’s disease (PD) represents the most common neurodegenerative movement disorder. We recently identified 16 novel genes associated with PD. In this study, we focused the attention on the common and rare variants identified in the lysosomal K+ channel TMEM175. The study includes a detailed clinical and genetic analysis of 400 cases and 300 controls. Molecular studies were performed on patient-derived fibroblasts. The functional properties of the mutant channels were assessed by patch-clamp technique and co-immunoprecipitation. We have found that TMEM175 was highly expressed in dopaminergic neurons of the substantia nigra pars compacta and in microglia of the cerebral cortex of the human brain. Four common variants were associated with PD, including two novel variants rs2290402 (c.-10C > T) and rs80114247 (c.T1022C, p.M341T), located in the Kozak consensus sequence and TM3II domain, respectively. We also disclosed 13 novel highly penetrant detrimental mutations in the TMEM175 gene associated with PD. At least nine of these mutations (p.R35C, p. R183X, p.A270T, p.P308L, p.S348L, p. L405V, p.R414W, p.P427fs, p.R481W) may be sufficient to cause the disease, and the presence of mutations of other genes correlated with an earlier disease onset. In vitro functional analysis of the ion channel encoded by the mutated TMEM175 gene revealed a loss of the K+ conductance and a reduced channel affinity for Akt. Moreover, we observed an impaired autophagic/lysosomal proteolytic flux and an increase expression of unfolded protein response markers in patient-derived fibroblasts. These data suggest that mutations in TMEM175 gene may contribute to the pathophysiology of PD

SIRT1 pharmacological activation rescues vascular dysfunction and prevents thrombosis in MTHFR deficiency

Beyond well-assessed risk factors, cardiovascular events could be also associated with the presence of epigenetic and genetic alterations, such as the methylenetetrahydrofolate-reductase (MTHFR) C677T polymorphism. This gene variant is related to increased circulating levels of homocysteine (Hcy) and cardiovascular risk. However, heterozygous carriers have an augmented risk of cardiovascular accidents independently from normal Hcy levels, suggesting the presence of additional deregulated processes in MTHFR C677T carriers. Here, we hypothesize that targeting Sirtuin 1 (SIRT1) could be an alternative mechanism to control the cardiovascular risk associated to MTHFR deficiency condition. Flow Mediated Dilatation (FMD) and light transmission aggregometry assay were performed in subjects carrying MTHFR C677T allele after administration of resveratrol, the most powerful natural clinical usable compound that owns SIRT1 activating properties. MTHFR C677T carriers with normal Hcy levels revealed endothelial dysfunction and enhanced platelet aggregation associated with SIRT1 downregulation. SIRT1 activity stimulation by resveratrol intake was able to override these abnormalities without affecting Hcy levels. Impaired endothelial function, bleeding time, and wire-induced thrombus formation were rescued in a heterozygous Mthfr-deficient (Mthfr+/-) mouse model after resveratrol treatment. Using a cell-based high-throughput multiplexed screening (HTS) assay, a novel selective synthetic SIRT1 activator, namely ISIDE11, was identified. Ex vivo and in vivo treatment of Mthfr+/- mice with ISIDE11 rescues endothelial vasorelaxation and reduces wire-induced thrombus formation, effects that were abolished by SIRT1 inhibitor. Moreover, platelets from MTHFR C677T allele carriers treated with ISIDE11 showed normalization of their typical hyper-reactivity. These results candidate SIRT1 activation as a new therapeutic strategy to contain cardio and cerebrovascular events in MTHFR carriers

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

Mouse hepatocytes lacking mGlu5 metabotropic glutamate receptors are less sensitive to hypoxic damage

Endogenous activation of type-5 metabotropic glutamate receptors contributes to the development of hypoxia-induced liver cell injury. We have strengthened this hypothesis using glutamate mGlu5 receptor knockout mice. Hepatocytes isolated from knockout mice were less sensitive to hypoxic cell damage than hepatocytes from wild-type mice as assessed by lactate dehydrogenase release and formation of reactive oxygen species. The mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP) also protect hepatocytes against hypoxic damage. © 2004 Elsevier B.V. All rights reserved

Brain MRI fiber-tracking reveals white matter alterations in hypertensive patients without damage at conventional neuroimaging

Hypertension is one of the main risk factor for dementia. The subtle damage provoked by chronic high blood pressure in the brain is usually evidenced by conventional magnetic resonance imaging (MRI), in terms of white matter (WM) hyperintensities or cerebral atrophy. However, it is clear that by the time brain damage is visible, it may be too late hampering neurodegeneration. Aim of this study was to characterize a signature of early brain damage induced by hypertension, before the neurodegenerative injury manifests