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1.
Neurobiol Dis ; 86: 41-51, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26626080

ABSTRACT

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by dyskinesia, cognitive impairment and emotional disturbances, presenting progressive neurodegeneration in the striatum and intracellular mutant Huntingtin (mHTT) aggregates in various areas of the brain. Recombinant Adeno Associated Viral (rAAV) vectors have been successfully used to transfer foreign genes to the brain of adult animals. In the present study we report a novel in vivo rat HD model obtained by stereotaxic injection of rAAV serotype2/9 containing Exon1-Q138 mHTT (Q138) and Exon1-Q17 wild type HTT (Q17; control), respectively in the right and in the left striatum, and expressed as C-terminal GFP fusions to facilitate detection of infected cells and aggregate production. Immunohistochemical analysis of brain slices from animals sacrificed twenty-one days after viral infection showed that Q138 injection resulted in robust formation of GFP-positive aggregates in the striatum, increased GFAP and microglial activation and neurodegeneration, with little evidence of any of these events in contralateral tissue infected with wild type (Q17) expressing construct. Differences in the relative metabolite concentrations (N-Acetyl Aspartate/Creatine and Myo-Inositol/Creatine) were observed by H1 MR Spectroscopy. By quantitative RT-PCR we also demonstrated that mHTT induced changes in the expression of genes previously shown to be altered in other rodent HD models. Importantly, administration of reference compounds previously shown to ameliorate the aggregation and neurodegeneration phenotypes in preclinical HD models was demonstrated to revert the mutant HTT-dependent effects in our model. In conclusion, the AAV2/9-Q138/Q17 exon 1 HTT stereotaxic injection represents a useful first-line in vivo preclinical model for studying the biology of mutant HTT exon 1 in the striatum and to provide early evidence of efficacy of therapeutic approaches.


Subject(s)
Corpus Striatum/metabolism , Corpus Striatum/virology , Dependovirus/genetics , Disease Models, Animal , Drug Discovery/methods , Genetic Vectors/administration & dosage , Huntington Disease/genetics , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Animals , Corpus Striatum/pathology , Encephalitis/metabolism , Encephalitis/virology , Exons , Female , Green Fluorescent Proteins/metabolism , Huntingtin Protein , Huntington Disease/metabolism , Neuroglia/metabolism , Neurons/pathology , Neurons/virology , Rats , Rats, Wistar , Recombinant Proteins/metabolism
3.
J Med Chem ; 66(8): 5622-5656, 2023 04 27.
Article in English | MEDLINE | ID: mdl-37017110

ABSTRACT

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease characterized by lung fibrosis leading to an irreversible decline of lung function. Current antifibrotic drugs on the market slow down but do not prevent the progression of the disease and are associated with tolerability issues. The involvement of lysophosphatidic acid receptor 2 (LPA2) in IPF is supported by LPA2 knockdown studies. To further validate the role of LPA2 receptors in modulating IPF and potentially other fibrotic processes, a potent and selective LPA2 receptor antagonist with a good pharmacokinetic (PK) profile is needed. Herein, we report the medicinal chemistry exploration that led to the discovery of a new class of highly potent and selective LPA2 antagonists. Among them, compound 58 exhibits excellent potency, selectivity, and oral PK profile, making it a suitable tool for probing the involvement of LPA2 receptors in IPF and other fibrotic processes.


Subject(s)
Idiopathic Pulmonary Fibrosis , Receptors, Lysophosphatidic Acid , Humans , Lysophospholipids
4.
Ann Clin Lab Sci ; 52(4): 677-683, 2022 Jul.
Article in English | MEDLINE | ID: mdl-36197772

ABSTRACT

The devastating COVID-19 outbreak posed serious challenges for the diagnostics laboratories, facing global shortage of reagents and equipment. This study aimed at evaluating an additional RNA extraction method respect to those already recommended by WHO and CDC. A new protocol for RNA extraction from nasopharyngeal swab was set up, adapting a Qiagen kit, and validated on a set of 96 clinical samples. The analysis showed a sensitivity of 94% and a specificity of 97%, but considering samples with Ct<36.5, the sensitivity and the specificity increased to 100%. The adapted method was also able to detect samples with very low viral load (Ct>38), indicating that the two approaches can be considered equivalent for the SARS-CoV-2 diagnostics. This extraction method can help in increasing the throughput for SARS-CoV-2 molecular test, even in a low automation setting.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Testing , Clinical Laboratory Techniques/methods , Humans , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
5.
J Neurochem ; 104(4): 1020-31, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17995933

ABSTRACT

The Group C G protein-coupled receptors include the metabotropic glutamate receptors (mGluRs), the GABA(B) receptor, the calcium sensor and several taste receptors, most of which are obligate dimers, indeed recent work has shown that dimerization is necessary for the activation of these receptors. Consequently factors that regulate their ability to homo- or heterodimerize are important. The Group 1 mGluRs include mGluR1 and mGluR5 both of which have splice variants with altered C-termini. In this study, we show that mGluR1b is a dimer and that it does not efficiently heterodimerize with mGluR1a, unlike the two splice variants of mGluR5 that can heterodimerize. Mutation of a positively charged motif (RRKK) at the C-terminus of the mGluR1b tail permits mGluR1b to heterodimerize with mGluR1a. Co-expression of mGluR1a and mGluR1b in COS-7 cells results in the accumulation of mGluR1b in intracellular inclusions that do not contain mGluR1a. This behaviour is mimicked by a chimera of the lymphocyte antigen CD2 with the C-terminus of mGluR1b (pCD1b) and depends on the presence of the RRKK motif. These accumulations are immunoreactive for endoplasmic reticulum (ER) markers, but not Golgi and ERGIC markers. This segregation of mGluR1b from other ER proteins may contribute to its failure to dimerize with mGluR1a.


Subject(s)
Peptide Fragments/physiology , Receptors, Metabotropic Glutamate/metabolism , Receptors, Metabotropic Glutamate/physiology , Animals , COS Cells , Cerebellum/physiology , Chlorocebus aethiops , Dimerization , Humans , Peptide Fragments/chemistry , Peptide Fragments/genetics , Rats , Receptors, Metabotropic Glutamate/chemistry , Receptors, Metabotropic Glutamate/genetics
6.
ISRN Mol Biol ; 2012: 823875, 2012.
Article in English | MEDLINE | ID: mdl-27398238

ABSTRACT

Background. DKK1 antagonizes canonical Wnt signalling through high-affinity binding to LRP5/6, an essential component of the Wnt receptor complex responsible for mediating downstream canonical Wnt signalling. DKK1 overexpression is known for its pathological implications in osteoporosis, cancer, and neurodegeneration, suggesting the interaction with LRP5/6 as a potential therapeutic target. Results. We show that the small-molecule NCI8642 can efficiently displace DKK1 from LRP6 and block DKK1 inhibitory activity on canonical Wnt signalling, as shown in binding and cellular assays, respectively. We further characterize NCI8642 binding activity on LRP6 by Surface Plasmon Resonance (SPR) technology. Conclusions. This study demonstrates that the DKK1-LRP6 interaction can be the target of small molecules and unlocks the possibility of new therapeutic tools for diseases associated with DKK1 dysregulation.

7.
J Biol Chem ; 282(16): 12143-53, 2007 Apr 20.
Article in English | MEDLINE | ID: mdl-17311919

ABSTRACT

Receptors for neurotransmitters require scaffolding proteins for membrane microdomain targeting and for regulating receptor function. Using a yeast two-hybrid screen, alpha-actinin-1, a major F-actin cross-linking protein, was identified as a binding partner for the C-terminal domain of metabotropic glutamate receptor type 5b (mGlu(5b) receptor). Co-expression, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between mGlu(5b) receptor and alpha-actinin-1 in both transfected HEK-293 cells and rat striatum. The interaction of alpha-actinin-1 with mGlu(5b) receptor modulated the cell surface expression of the receptor. This was dependent on the binding of alpha-actinin-1 to the actin cytoskeleton. In addition, the alpha-actinin-1/mGlu(5b) receptor interaction regulated receptor-mediated activation of the mitogen-activated protein kinase pathway. Together, these findings indicate that there is an alpha-actinin-1-dependent mGlu(5b) receptor association with the actin cytoskeleton modulating receptor cell surface expression and functioning.


Subject(s)
Actinin/metabolism , Cell Membrane/metabolism , Gene Expression Regulation , Receptors, Metabotropic Glutamate/metabolism , Actins/metabolism , Amino Acid Sequence , Animals , Brain/metabolism , Cytoskeleton/metabolism , Humans , Mice , Models, Biological , Molecular Sequence Data , Receptor, Metabotropic Glutamate 5 , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino Acid
8.
Synapse ; 50(4): 269-76, 2003 Dec 15.
Article in English | MEDLINE | ID: mdl-14556231

ABSTRACT

The mechanisms through which blockade of metabotropic glutamate receptors 5 (mGluR5) results in anxiolytic and antidepressant effects are currently unknown. In the present study, we therefore hypothesized that the anxiolytic- and antidepressant-like profile of the noncompetitive mGluR5 receptor antagonist 2-ethyl-6-(phenylethynyl)-pyridine (MPEP) may be mediated by inhibition of the norepinephrine transporter (NET). Accordingly, we first examined the potency of MPEP to bind to or inhibit uptake at the NET as well as the dopamine and serotonin transporters (DAT and SERT, respectively). We also examined the simultaneous in vivo effects of MPEP and desipramine (DMI) on both NE-like oxidation current in the amygdala (AMY) and cell firing in the locus coeruleus (LC) by means of differential pulse voltammetry (DPV) coupled with electrophysiology. MPEP completely displaced the binding of [3H]-nisoxetine on human NET with a pKi of 6.63 +/- 0.02. In addition, MPEP was able to inhibit [3H]-NE uptake in LLCPK cells expressing human NET, with a pIC50 of 5.55 +/- 0.09. In vivo DPV data revealed that both MPEP (30 mg/kg i.p.) and DMI (10 mg/kg i.p.) significantly increased NE-like voltammetric responses levels in the AMY, whereas both compounds also significantly decreased cell firing monitored concomitantly from the second microelectrode in the LC. Collectively, the results of the present study provide potential new mechanisms through which MPEP exerts its anxiolytic and antidepressant effects.


Subject(s)
Amygdala/drug effects , Excitatory Amino Acid Antagonists/pharmacology , Fluoxetine/analogs & derivatives , Locus Coeruleus/drug effects , Pyridines/pharmacology , Receptors, Kainic Acid/antagonists & inhibitors , Symporters/antagonists & inhibitors , Action Potentials/drug effects , Action Potentials/physiology , Adrenergic Uptake Inhibitors/pharmacology , Amygdala/metabolism , Animals , Antidepressive Agents, Tricyclic/pharmacology , Binding, Competitive , Desipramine/pharmacology , Electrochemistry/methods , Fluoxetine/metabolism , Humans , Imipramine/pharmacology , In Vitro Techniques , Locus Coeruleus/metabolism , Male , Microelectrodes , Norepinephrine Plasma Membrane Transport Proteins , Radioligand Assay/methods , Rats , Time Factors
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