Title

 

Authors

V. Chiodi¹, L. Ferraro², M. Uchigashima³, A. Martire¹, S. Beggiato², M. Armida¹, A. Ferrante¹, R. L. Potenza¹, M. Watanabe³, S. Tanganelli², M. R. Domenici¹, P. Popoli<sup>1

1</sup>Dept of Therapeutic Research and Medicine Evaluation, Istituto Superiore di Sanità, Rome, Italy; ²Dept of Experimental and Clinical Medicine, Section of Pharmacology, University of Ferrara, Italy; ³Dept of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan.

 

Abstract

Huntington’s disease (HD) is a genetic neurodegenerative disorder that predominantly affects the striatum. In spite of the well known genetic basis of the disease (a CAG repeat expansion in the promoter region of the huntingtin gene), the pathogenesis of the selective neuronal degeneration is still unclear (The Huntington's Disease Collaborative Research Group, 1993). The involvement of type 1 cannabinoid receptors (CB1Rs) in the pathogenesis of HD has been long postulated and, indeed, one of the earliest neurochemical alterations observed in both HD patients and transgenic mouse models of HD is a downregulation of CB1Rs in the basal ganglia, an alteration that precedes the development of neuropsychiatric symptoms and neuronal degeneration (Fernandez-Ruiz and Gonzales 2005; Scotter et al., 2010). The aim of our study was to evaluate, in the R6/2 mouse model of HD, the effect of CB1R stimulation on synaptic transmission, neurotransmitter release and motor activity. Additionally, since CB1 receptors are expressed on both GABAergic and glutamatergic neurons, we verified whether the two neuronal subtypes were differentially affected by the downregulation of CB1Rs demonstrated in HD. Synaptic transmission was investigated in corticostriatal slices (300 µm) of 12 week old R6/2 and wild type (WT) mice by recording extracellular field potential (FP) in the dorsal striatum after stimulation of the white matter. The application of CB1R agonist WIN 55,212-2 (2 μM) induced a marked depression of synaptic transmission, which was significantly more pronounced in R6/2 than in WT mice (30.6 ± 8.2% and 54.37 ± 5.1% of the basal values, respectively, p<0.05 Mann Whitney U-test), and which was prevented by the CB₁R antagonist AM251 (2 µM). The increased response to CB1 agonist was also demonstrated in a motor activity test. The systemic administration of WIN (2.5 mg/kg)in 8 week old mice produced a significant hypolocomotor effect that was more pronounced in R6/2 than in WT mice. In order to explore how CB1R agonist influenced neurotransmitter release, we measured spontaneous and evoked [³H]GABA and endogenous glutamate efflux in synaptosomes prepared from WT and R6/2 striata. Spontaneous [³H]GABA efflux was significantly higher in R6/2 mice than in synaptosomes from WT mice (39 ± 3.3 and 23 ± 2.3 pmol/min/mg protein respectively, p<0.05) while no differences were observed in endogenous glutamate release. WIN (1 µM) produced a significant inhibition of the K⁺-evoked [³H]GABA and glutamate efflux in both WT and R6/2 mice. In R6/2, however, the inhibition of [³H]GABA and glutamate were significantly reduced and increased, respectively, withrespect to WT. In western blotting experiments a strong reduction of the expression of CB1 in the striatum of R6/2 mice with respect to WT was found (0.27 ± 0.046% and 0.65 ± 0.065%; in R6/2 and WT, respectively, n=4, p<0.05). In particular, with double immunofluorescence staining (using VGluT1 and VIAAT, glutamatergic and GABAergic terminal markers, respectively), and with immunoelectron microscopy we found that CB1 labelling was drastically downregulated in GABAergic terminals of R6/2 mice with respect to WT, while VGluT1-positive excitatory terminals appeared to show no significant difference in CB1 expression between the two genotypes.
Thus, we showed for the first time that HD induces a downregulation of CB1 expressed on GABAergic neurons and an impairment of CB1-induced reduction of GABA release. On the contrary, the expression of CB1 on glutamatergic neurons does not change and CB1-mediated glutamatergic functions are increased. In conclusion, HD affects CB1 receptor functions differently, according to the site of expression of the receptor.  
The Huntington's Disease Collaborative Research Group (1993). Cell  72, 971-83.
Fernandez-Ruiz J. and Gonzàles S.(2005). Handb Exp Pharmacol.168, 479-507.
Scotter E.L., Abood M.E. and  Glass M. (2010). Br. J. Pharmacol. 160, 480–498.