Metabotropic glutamate receptor 5 positive allosteric modulators are neuroprotective in a mouse model of Huntington's disease

Abstract

Background and purpose: Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein. We have previously demonstrated that the cell signalling of the metabotropic glutamate receptor 5 (mGluR5) is altered in a mouse model of HD. Although mGluR5-dependent protective pathways are more activated in HD neurons, intracellular Ca²⁺ release is also more pronounced, which could contribute to excitotoxicity. In the present study, we aim to investigate whether mGluR5 positive allosteric modulators (PAMs) could activate protective pathways without triggering high levels of Ca²⁺ release and be neuroprotective in HD.

Experimental approach: We performed a neuronal cell death assay to determine which drugs are neuroprotective, Western blot and Ca²⁺ release experiments to investigate the molecular mechanisms involved in this neuroprotection, and object recognition task to determine whether the tested drugs could ameliorate HD memory deficit.

Key results: We find that mGluR5 PAMs can protect striatal neurons from the excitotoxic neuronal cell death promoted by elevated concentrations of glutamate and NMDA. mGluR5 PAMs are capable of activating Akt without triggering increased intracellular Ca²⁺ concentration ([Ca²⁺]i ); and Akt blockage leads to loss of PAM-mediated neuroprotection. Importantly, PAMs' potential as drugs that may be used to treat neurodegenerative diseases is highlighted by the neuroprotection exerted by mGluR5 PAMs on striatal neurons from a mouse model of HD, BACHD. Moreover, mGluR5 PAMs can activate neuroprotective pathways more robustly in BACHD mice and ameliorate HD memory deficit.

Conclusions and implications: mGluR5 PAMs are potential drugs that may be used to treat neurodegenerative diseases, especially HD.

Figure 1

DHPG, but not MPEP, protects against glutamate-induced neuronal cell death. Shown is a representative image for primary-cultured striatal neurons labelled with calcein AM (A, green, live cells) and ethidium homodimer-1 (B, red, dead cells) exhibiting basal neuronal death. Also shown is a representative image for primary-cultured striatal neurons treated with 50.0 μM glutamate for 20 h and labelled with calcein AM (C) and ethidium homodimer-1 (D). (E) Graph shows percentage of neuronal cell death in primary cultured striatal neurons that were treated with 10.0 μM MPEP, 10.0 μM DHPG, 50.0 μM glutamate, 10.0 μM MPEP + 50.0 μM glutamate and 10.0 μM DHPG + 50.0 μM glutamate for 20 h. Data represent the means ± SEM of four independent experiments. * indicates significant difference as compared with neurons treated with glutamate and ^# indicates significant differences as compared with basal neuronal death (P < 0.05).

Figure 2

mGluR5 PAMs do not promote increased intracellular Ca²⁺ concentration. Graph shows intracellular Ca²⁺ concentration in primary-cultured striatal neurons stimulated with 10.0 μM DFB, 10.0 μM VU1545, 10.0 μM CDPPB and 10.0 μM DHPG. Data represent the means ± SEM of six independent experiments expressed as percentage of 30.0 mM KCl-induced Ca²⁺ release. * indicates significant difference as compared with basal Ca²⁺ release (P < 0.05).

Figure 3

DFB, VU1545 and CDPPB promote AKT activation. Shown are representative immunoblots for phospho- (upper panel) and total-Akt expression (lower panel) in primary-cultured striatal neurons that were either untreated (NT) or treated with 10.0 μM DHPG, 10.0 or 100.0 μM DFB (A), 10.0 or 100.0 μM VU1545 (C) and 10.0 or 100.0 μM CDPPB (E) for 5 min. About 100.0 μg of cell lysate was used for each sample. Graphs show the densitometric analysis of phospho-AKT normalized to total-AKT expression in primary-cultured striatal neurons that were either untreated (NT) or treated with 10.0 μM DHPG, 10.0 or 100.0 μM DFB (B), 10.0 or 100.0 μM VU1545 (D) and 10.0 or 100.0 μM CDPPB (F) for 5 min. Data represent the means ± SEM of six independent experiments, expressed as percentage of basal Akt phosphorylation. * indicates significant differences as compared withy untreated neurons (P < 0.05).

Figure 4

VU1545 and CDPPB promote AKT activation even at lower concentrations. Shown are representative immunoblots for phospho- (upper panel) and total-AKT expression (lower panel) in primary-cultured striatal neurons that were either untreated (NT) or treated with 10 μM DHPG, 0.1 or 1.0 μM VU1545 (A) and 0.1 or 1.0 μM CDPPB (C) for 5 min. About 100.0 μg of cell lysate was used for each sample. Graphs show the densitometric analysis of phospho-AKT normalized to total-AKT expression in primary-cultured striatal neurons that were either untreated (NT) or treated with 10.0 μM DHPG, 0.1 or 1.0 μM VU1545 (B) and 0.1 or 1.0 μM CDPPB (D) for 5 min. Data represent the means ± SEM of six independent experiments, expressed as percentage of basal AKT phosphorylation. * indicates significant differences as compared with untreated neurons (P < 0.05).

Figure 5

mGluR5 PAMs protect against glutamate-induced neuronal cell death. Graph shows percentage of neuronal cell death induced by either 50.0 or 100.0 μM glutamate in primary-cultured striatal neurons that were either untreated (no PAM) or treated with DFB, VU1545 and CDPPB at the concentrations of 10.0 μM (A) and 100.0 nM (B) for 20 h. Data represent the means ± SEM of four independent experiments. * indicates significant differences as compared with neurons treated with glutamate in the absence of PAMs (P < 0.05).

Figure 6

VU1545 is a very potent neuroprotective drug and this neuroprotection is dependent on Akt activation. (A) Graph shows the effect of VU1545 at the concentrations of 1.0, 5.0, 10.0, 50.0, 100.0 and 10 000.0 nM on the percentage of neuronal cell death in primary-cultured striatal neurons that were either untreated (NT) or treated with 50.0 μM glutamate for 20 h. Data represent the means ± SEM of four independent experiments. * indicates significant differences as compared with neurons treated with glutamate in the absence of VU1545 (P < 0.05). (B) Graph shows percentage of neuronal cell death induced by NMDA at the concentrations of 0.1, 1.0 or 10.0 μM in primary-cultured striatal neurons that were either NT (no VU1545) or treated with VU1545 100.0 nM for 20 h. Data represent the means ± SEM of three independent experiments. * indicates significant differences as compared with neurons treated with 10.0 μM NMDA in the absence of VU1545 (P < 0.05). (C) Graph shows percentage of neuronal cell death in primary-cultured striatal neurons that were treated with 50.0 μM glutamate, 100.0 nM VU1545 + 50.0 μM glutamate, 100.0 nM VU1545 + 50.0 μM glutamate + 10.0 μM MPEP and 100.0 nM VU1545 for 20 h. Data represent the means ± SEM of three independent experiments. * indicates significant difference as compared with neurons treated with glutamate (P < 0.05). (D) Graph shows percentage of neuronal cell death in primary-cultured striatal neurons that were treated with 50.0 μM glutamate, 25.0 μM LY294002 + 50.0 μM glutamate, 1.0 μM VU1545 + 50.0 μM glutamate and 1.0 μM VU1545 + 25.0 μM LY294002 + 50.0 μM glutamate for 20 h. Data represent the means ± SEM of four independent experiments. * indicates significant difference as compared with neurons treated with glutamate (P < 0.05).

Figure 7

mGluR5 PAMs protect BACHD neurons from glutamate-induced neuronal cell death. Graphs show percentage of neuronal cell death induced by 50.0 μM glutamate in either BACHD or wild-type (WT) primary cultured striatal neurons in the presence or absence of 1.0 μM DFB (A), 1.0 μM VU1545 (B) and 1.0 μM CDPPB (C) for 20 h. Data represent the means ± SEM of four to six independent experiments. * indicates significant differences as compared with neurons treated with glutamate in the absence of PAMs (P < 0.05).

Figure 8

VU1545 activates Akt more robustly in BACHD than in wild-type (WT) neurons. (A) Shown is a representative immunoblot for phospho- (upper panel) and total-Atk expression (lower panel) in primary cultured striatal neurons from either WT or BACHD mice that were either untreated (NT) or treated with 10.0 μM DHPG or 10.0 μM VU1545. About 100.0 μg of cell lysate was used for each sample. (B) Graph shows the densitometric analysis of phospho-Akt normalized to total-Akt expression in primary-cultured striatal neurons from either WT or BACHD mice that were either NT or treated with 10.0 μM DHPG or 10.0 μM VU1545. Data represent the means ± SEM of six independent experiments, expressed as percentage of basal Akt phosphorylation. * indicates significant difference as compared with matched treated WT neurons (P < 0.05).

Figure 9

CDPPB ameliorates BACHD deficit in novel object recognition memory. Graph shows percentage of time of novel object exploration calculated as an index between time spent exploring a novel object × 100/time with both objects. A score of 50% indicates no preference. Two weeks after untreated (NT) wild-type (WT, n = 6) and BACHD (n = 6) mice were submitted to object recognition test, both WT and BACHD were treated with CDPPB i.p. 5 mg·kg⁻¹ for 7 days and reassessed for recognition memory. Data represent the means ± SEM, expressed as percentage of time exploring both objects. * indicates significant differences from chance exploration (50%) (P < 0.05).