Elsevier

Neurobiology of Disease

Volume 73, January 2015, Pages 163-173
Neurobiology of Disease

The mGluR5 positive allosteric modulator, CDPPB, ameliorates pathology and phenotypic signs of a mouse model of Huntington's disease

https://doi.org/10.1016/j.nbd.2014.08.021Get rights and content

Highlights

  • CDPPB is a potent neuroprotective drug in vitro and in vivo.

  • Chronic treatment of BACHD mice with CDPPB increases AKT and ERK1/2 activation

  • BDNF mRNA expression is increased upon CDPPB chronic treatment.

  • CDPPB chronic treatment of BACHD mice also decreases htt aggregate formation.

  • CDPPB chronic treatment rescues the memory deficit exhibited by BACHD mice.

Abstract

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine expansion in the amino-terminal region of the huntingtin protein (htt), leading to motor dysfunction, cognitive decline, psychiatric alterations, and death. The metabotropic glutamate receptor 5 (mGluR5) has been implicated in HD and we have recently demonstrated that mGluR5 positive allosteric modulators (PAMs) are neuroprotective in vitro. In the present study we demonstrate that the mGluR5 PAM, CDPPB, is a potent neuroprotective drug, in vitro and in vivo, capable of delaying HD-related symptoms. The HD mouse model, BACHD, exhibits many HD features, including neuronal cell loss, htt aggregates, motor incoordination and memory impairment. However, chronic treatment of BACHD mice with CDPPB 1.5 mg/kg s.c. for 18 weeks increased the activation of cell signaling pathways important for neuronal survival, including increased AKT and ERK1/2 phosphorylation and augmented the BDNF mRNA expression. CDPPB chronic treatment was also able to prevent the neuronal cell loss that takes place in the striatum of BACHD mice and decrease htt aggregate formation. Moreover, CDPPB chronic treatment was efficient to partially ameliorate motor incoordination and to rescue the memory deficit exhibited by BACHD mice. Importantly, no toxic effects or stereotypical behavior were observed upon CDPPB chronic treatment. Thus, CDPPB is a potential drug to treat HD, preventing neuronal cell loss and htt aggregate formation and delaying HD symptoms.

Introduction

Huntington's disease (HD) is a neurodegenerative disorder caused by an unstable CAG repeat expansion in the amino-terminal region of the huntingtin protein (htt) (Group, 1993). Clinical manifestations of HD usually initiate between 35 and 50 years of age as involuntary body movement, loss of cognitive function, and psychiatric disturbance, inevitably leading to death (Li and Li, 2004, Vonsattel and DiFiglia, 1998, Young, 2003). Neuropathological analysis reveals selective and progressive neuronal loss in the striatum (DiFiglia, 1990, Vonsattel et al., 1985).

Glutamate-mediated neurotoxicity has been postulated to play an important role in the pathogenesis of HD (Anborgh et al., 2005, Calabresi et al., 1999, DiFiglia, 1990, Ribeiro et al., 2010). Stimulation of metabotropic glutamate receptor 5 (mGluR5) leads to the formation of inositol 1,4,5-trisphosphate (InsP3) and release of intracellular Ca2 + and mutant htt can enhance this cell signaling pathway, leading to toxic levels of intracellular Ca2 + (Ribeiro et al., 2010, Tang et al., 2005, Tang et al., 2003). However, mGluR5 stimulation can also promote activation of neuroprotective cell signaling pathways, involving ERK1/2 and AKT (Doria et al., 2013, Ribeiro et al., 2010). We have recently demonstrated that mGluR5 positive allosteric modulators (PAMs) can promote neuronal survival by activating AKT without triggering Ca2 + release (Doria et al., 2013). Moreover, it has been shown that mGluR5 PAMs have a positive effect on memory, facilitating neurotransmission and improving spatial learning (Ayala et al., 2009) and rescuing pharmacologically induced object recognition memory impairment (Reichel et al., 2011, Uslaner et al., 2009). In contrast, both mGluR5 receptor antagonism and genetic deletion have been shown to cause deleterious effects on learning and memory (Simonyi et al., 2010). Thus, mGluR5 PAMs are potential drugs to treat HD.

In the present study we demonstrate that the mGluR5 PAM, 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), can ameliorate pathology and phenotypic signs exhibited by a mouse model of HD, BACHD. We performed a concentration–response curve using CDPPB and we found that this mGluR5 PAM is a very potent neuroprotective drug as it was capable of protecting neurons against glutamate insult even at low concentrations. We also investigated the effect of CDPPB chronic administration and we found that BACHD mice treated with 1.5 mg/kg CDPPB s.c. for 18 weeks exhibited higher levels of AKT and ERK1/2 activation, as well as enhanced brain-derived neurotrophic factor (BDNF) mRNA expression. Moreover, treatment with CDPPB was capable of reducing the neurodegeneration and htt aggregate formation that take place in BACHD brain. Electron microscopy analyses showed that there was a decrease in the number of vesicles at the pre-synaptic active zone of BACHD mice and that CDPPB chronic treatment normalized this deficit. Finally, our behavioral tests demonstrated that CDPPB treatment partially improved motor coordination and normalized memory deficit in BACHD mice. Thus, our results indicate that CDPPB chronic treatment has the potential to prevent the neuronal loss and ameliorate the motor and cognitive symptoms observed in a HD mouse model.

Section snippets

Materials

Neurobasal medium, N2 and B27 supplements, GlutaMAX (50.0 mg/ml penicillin and 50.0 mg/ml streptomycin), Live/Dead viability assay, TRIzol, Nuclease-Free Water, and Power SYBR® Green PCR Master Mix were purchased from Life Technologies (Foster City, CA, USA). Mouse anti-Huntingtin EM48 (Cat# MAB5374, RRID: AB_177645) and mouse anti-NeuN (Cat# MAB377, RRID: AB_2298772) monoclonal antibodies were purchased from Millipore (Billerica, MA, USA). 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide

Results

Glutamate is considered the major neurotransmitter involved in HD-related neuronal cell death and we have previously demonstrated that CDPPB can protect against glutamate-induced neuronal death in vitro (Doria et al., 2013). Thus, we decided to investigate whether CDPPB exhibits efficacy in vivo. To determine optimal CDPPB dose for in vivo studies we performed a concentration–response experiment. Primary cultured striatal neurons stimulated with 50.0 μM glutamate for 20 h exhibited higher levels

Discussion

The high prevalence of neurodegenerative diseases worldwide poses a major public health problem as incidence rate increases exponentially with age. Currently, the prevalence is estimated to amount to 24 million and predicted to be quadruple by the year of 2050 (Reitz and Mayeux, 2014). Thus, drugs that could modify disease course are an urgent unmet need. In the present study we demonstrate that the mGluR5 positive allosteric modulator, CDPPB, can ameliorate various aspects related to HD

Abbreviations

    HD

    Huntington's disease

    Htt

    huntingtin

    InsP3

    inositol-1,4,5-triphosphate

    PAM

    positive allosteric modulator

    mGluR

    metabotropic glutamate receptor

    CDPPB

    3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide

    ERK

    extracellular signal-regulated kinase

    AKT

    Protein kinase B

    PFA

    paraformaldehyde

    HBSS

    Hank's balanced salt solution

    PBS

    Phosphate buffered saline

    DIV

    days in vitro

    BSA

    Bovine serum albumin;

    DMSO

    dimethyl sulfoxide

    qPCR

    quantitative polymerase chain reaction

    RT-PCR

    reverse transcriptase PCR

    BDNF

    Brain-derived neurotrophic factor

Conflict of interest

The authors declare no competing financial interests.

Acknowledgments

This work was supported by CNPq (490191/2012-6) and FAPEMIG grants to F. M. R. (PPM-00015-13) and by the grant from the Ministry of Education, Science, Research and Sport of the Slovak Republic to T. D., DB Biotech, Slovakia. We thank L. R. Souza for the technical assistance. We also thank the Center of Acquisition and Processing of Images (CAPI) – ICB – Universidade Federal de Minas Gerais and Microscopy Center at Universidade Federal de Minas Gerais for providing the equipment and technical

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