Regular articleComplex alteration of NMDA receptors in transgenic Huntington's disease mouse brain: analysis of mRNA and protein expression, plasma membrane association, interacting proteins, and phosphorylation
Introduction
Huntington's disease (HD) is a progressive neurodegenerative disease characterized clinically by movement, cognitive, and psychiatric difficulties (Harper 1996). Pathologically, the striatum (caudate-putamen) is preferentially involved, with striatal projection neurons showing more vulnerability than striatal interneurons Ferrante et al., 1985, Vonsattel et al., 1985. Pathological damage is not limited to the striatum, as cell loss has also been described in cortex, thalamus, subthalamic nucleus, and hippocampus (reviewed in Vonsattel and DiFiglia, 1998). HD is inherited as an autosomal dominant disease and is caused by an unstable CAG trinucleotide repeat expansion within the IT15 gene (Huntington's Disease Collaborative Research Group, 1993). The CAG region encodes an expanded polyglutamine repeat within the huntingtin protein.
Dysregulation of glutamatergic signaling has long been hypothesized to play a role in HD (DiFiglia, 1990). This hypothesis is supported by the pathological mimicry of intrastriatal injections of excitatory amino acid compounds (Coyle and Schwarcz, 1976), particularly those acting selectively at the N-methyl-d-aspartate (NMDA) receptor (Beal et al., 1986), in experimental animal models. NMDA receptors are multimeric complexes composed of NR1 (ζ1 in the mouse) and NR2 (ε in the mouse) subunits. In cortex and striatum, NR2A (ε1) and NR2B (ε2) predominate, while NR2C (ε3) and NR2D (ε4) are expressed in small amounts. NMDA receptor function is known to be regulated at many levels, including subunit stoichiometry, phosphorylation, and anchoring (Sheng and Pak, 2000). Evidence directly linking the HD mutation to glutamatergic neurotransmission is limited, however.
Since the cloning of the HD gene, transgenic mice for all or selected portions of the gene carrying the CAG expansion have been created Mangiarini et al., 1996, Hodgson et al., 1999, Reddy et al., 1999, Schilling et al., 1999. Assessments of NMDA receptor function in mouse models of HD have yielded varied results, with some reports showing enhanced NMDA receptor excitability Levine et al., 1999, Cepeda et al., 2001, Zeron et al., 2002, others showing decreases in NMDA receptor-dependent functions Hansson et al., 1999, Murphy et al., 2000, and one report describing age-dependent phases of both enhanced and reduced function of NMDA receptor-dependent processes (Hodgson et al., 1999).
To systematically assess the regulation of NMDA receptors by mutant huntingtin at the molecular level, we have quantitated NMDA receptor subunit mRNAs and proteins in multiple brain regions in transgenic mice expressing a mutant N-terminal fragment of huntingtin (R6/2 mice). To determine whether the trafficking of NMDA receptors may be affected by the N-terminal portion of mutant huntingtin, we have assessed the abundance of NMDA subunit proteins in plasma membrane fractions, quantitated the expression of the NMDA receptor interacting proteins α-actinin-2 and PSD-95, and assessed the tyrosine phosphorylation state of ε1 and ε2 subunits in these animals. Our results reveal a complex relationship between mutant huntingtin and the expression and targeting of NMDA receptors. NMDA receptor alterations vary not only in level of regulation (e.g., mRNA vs protein) but also by brain region (e.g., hippocampus vs striatum). Such complex alterations are predicted to have significant effects on neural function.
Section snippets
Brain tissue from R6/2 mice
R6/2 mice express human huntingtin exon 1 protein with a large polyglutamine repeat expansion of approximately 150 glutamines (Mangiarini et al., 1996). These animals display a progressive neurological phenotype starting at approximately 3–4 weeks of age and ending in premature death at approximately 12–17 weeks Mangiarini et al., 1996, Davies et al., 1997, Lione et al., 1999, Carter et al., 2000. While R6/2 mice have been reported to develop diabetes mellitus Hurlbert et al., 1999, Luesse et
Array-based expression profiling of R6/2 mouse brain
The expression levels of 588 known genes were profiled in whole brains from 12-week-old R6/2 and wild-type mice using Atlas Mouse Expression Arrays. Separate experiments were performed for three individual mouse brains of each genotype. To minimize variation between filters two pairs of filters were used; one pair was probed twice with replicate cDNA samples, then probed a third time with the opposite genotype sample (control vs R6/2). For our cutoff, only cDNA signals that had intensity values
Discussion
Mutant huntingtin has been shown to cause changes in the expression of certain mRNAs Cha et al., 1998, Cha, 2000, Li et al., 1999, Bibb et al., 2000, Denovan-Wright and Robertson, 2000, Luthi-Carter et al., 2000, Menalled et al., 2000, Kusakabe et al., 2001. In some cases, decreased RNA levels correspond to decreased protein abundance (e.g., DARPP-32 mRNA and protein (Bibb et al., 2000)); while in other cases, there is a mismatch (e.g., preproenkephalin mRNA versus enkephalin peptide (Menalled
Acknowledgements
We thank Ben Woodman and Anj Mahal for generation and genotyping of R6/2 mice. This work was supported by National Institutes of Neurological Disorders and Stroke 34361 (D.G.S.), 38106 (J.-H.J.C.), and 10800 (R.L.-C.), National Institute of Aging 13617 (A.B.Y.), Wellcome Trust (G.P.B.), Human Frontiers Science Program (G.P.B., L.M.T.), Parkinson Disease Foundation (A.W.D.), Hereditary Disease Foundation Cure HD Initiative (R.L.-C., A.B.Y., L.M.T.), Glendorn Foundation (J.-H.J.C.), and
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These authors contributed equally to this work.