Balance between synaptic versus extrasynaptic NMDA receptor activity influences inclusions and neurotoxicity of mutant huntingtin

Abstract

Huntington's disease is caused by an expanded CAG repeat in the gene encoding huntingtin (HTT), resulting in loss of striatal and cortical neurons. Given that the gene product is widely expressed, it remains unclear why neurons are selectively targeted. Here we show the relationship between synaptic and extrasynaptic activity, inclusion formation of mutant huntingtin protein (mtHtt) and neuronal survival. Synaptic N-methyl-D-aspartate-type glutamate receptor (NMDAR) activity induces mtHtt inclusions via a T complex-1 (TCP-1) ring complex (TRiC)-dependent mechanism, rendering neurons more resistant to mtHtt-mediated cell death. In contrast, stimulation of extrasynaptic NMDARs increases the vulnerability of mtHtt-containing neurons to cell death by impairing the neuroprotective cyclic AMP response element-binding protein (CREB)-peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) cascade and increasing the level of the small guanine nucleotide-binding protein Rhes, which is known to sumoylate and disaggregate mtHtt. Treatment of transgenic mice expressing a yeast artificial chromosome containing 128 CAG repeats (YAC128) with low-dose memantine blocks extrasynaptic (but not synaptic) NMDARs and ameliorates neuropathological and behavioral manifestations. By contrast, high-dose memantine, which blocks both extrasynaptic and synaptic NMDAR activity, decreases neuronal inclusions and worsens these outcomes. Our findings offer a rational therapeutic approach for protecting susceptible neurons in Huntington's disease.

Figure 1

Suppression of excitatory NMDAR synaptic transmission ameliorates inclusion formation in mtHtt-expressing neurons. (a, b) Immunostaining and quantification of inclusion bodies in rat primary cortical neurons transfected with wtHtt (Myc-wtHtt-N63-18Q) or mtHtt (Myc-mtHtt-N63-148Q) after treatment with AMPA receptor antagonist CNQX (10 μM) or with NMDAR antagonists memantine (10 μM or 30 μM), d-APV (150 μM), or ifenprodil (10 μM), a relatively selective inhibitor of NR2B subunits of the NMDAR. Values are mean ± s.e.m. (n ≥ 1,200). Scale bar, 10 μm. *, P < 0.01 by ANOVA. (c) Inclusion formation was quantified in rat primary neurons transfected with wtHtt or mtHtt and treated with synaptic activity suppressors (0.2 μM TTX or 100 μM NO-711). Values are mean ± s.e.m. *, P < 0.01 by ANOVA.

Figure 2

Pharmacology of NMDAR-mediated sEPSCs and whole-cell currents recorded from wtHtt- and mtHtt-transfected neurons. (a-e) sEPSCs (left-hand panels) and charge transfer (right-hand panels), normalized for each cell (see Supplementary Methods). Both wtHtt- and mtHtt-transfected neurons were treated with TTX (a), NO-711 (b), memantine (c), or ifenprodil (d). Recording solutions in (a-e) contained 20 μM glycine; for (c. d), CNQX and bicuculline (10 μM each) were also added. Values are mean ± s.e.m. (n ≥ 4 cells in each case); *, P < 0.03 (paired t-test on raw data). (f) Effects of memantine and ifenprodil on extrasynaptic NMDAR-mediated currents. Incomplete reversal was observed because of slow washout in this system. Recordings were performed in the presence of 0.1-1 μM TTX. NMDA currents were evoked by co-application of 100 μM NMDA and 20 μM glycine. (g) Charge transfer (fC/pF) for NMDAR-sEPSCs in mtHtt- and wtHtt-transfected neurons.

Figure 3

Involvement of the chaperonin TRiC TCP1 subunit in synaptic activity-mediated inclusion formation. (a) Immunoblotting demonstrating the level of TCP1 protein expression in neurons exposed to TTX (0.2 μM) or control conditions for 24 h. Actin served as a loading control. (b) Inclusion formation was quantified in neurons transfected with mtHtt plus two different small hairpin vectors for TCP1 or control vector. Values are mean ± s.e.m. for n ≥ 300. *, P ≤ 0.001 by ANOVA.

Figure 4

Excitatory synaptic versus extrasynaptic activity in HD-related neuronal cell death. (a) Excessive glutamate insult (50 μM for 40 h) led to neuronal cell death in mtHtt-transfected cortical neurons in the presence of inclusion formation (scale bar, 10 μm). Neurons transfected with mtHtt contained inclusions and displayed healthy nuclei. (b, c) Cell death analysis after transfection with the N-terminal fragment of mtHtt (b) and full-length mtHtt (c). Values represent mean ± s.e.m. for n ≥ 1,200. *, P < 0.01 by ANOVA. (d) Memantine attenuated neuronal cell death induced by blockade of physiological excitatory synaptic activity with NO-711 or TTX in mtHtt-transfected neurons. Co-transfection of PGC-1α mitigated neuronal cell death induced by blockade of synaptic activity with TTX. Values represent mean ± s.e.m. for n ≥ 1,200. *, P < 0.05; **, P < 0.01 by ANOVA. (e) Cell death was quantified in neurons transfected with wtHtt or mtHtt plus two different small hairpin vectors for TCP1 or a control vector. Values are mean ± s.e.m. for n ≥ 300. *, P ≤ 0.01 by ANOVA. (f) Low concentration (5 μM) memantine abrogated the decrease in CREB activity induced by blockade of physiological excitatory synaptic activity with TTX in mtHtt-transfected neurons. Neurons were transfected with a CRE-luciferase reporter construct and wtHtt or mtHtt. Ordinate axis represents relative luciferase activity. Values represent mean ± s.e.m. *, P < 0.01 by ANOVA. (g) Low concentration memantine ameliorated the decrease in PGC-1α levels induced by blockade of synaptic activity with TTX in mtHtt-transfected neurons. PGC-1α levels were quantified by immunofluorescence under deconvolution microscopy in wtHtt- or mtHtt-transfected neurons. Values represent mean ± s.e.m. for n ≥ 300. *, P < 0.01 by ANOVA.

Figure 5

Long-term treatment with memantine affects neuropathology and motor function in a dose-specific manner in transgenic YAC128 HD animals. (a) The extent of inclusion formation was assessed using unbiased densitometry of striatal neurons stained with EM48. (b) Representative immunohistological photographs of EM48-stained striata from WT, untreated YAC128 animals, and YAC128 animals treated with 1 or 30 mg kg^-1 memantine. The photomicrographs were taken using an 100x objective (scale bar, 50 μm). (c) Striatal volumes were determined by tracing the perimeter of the striatum in serial sections spanning the striatum. (d) Effects of low-dose or high-dose memantine on the fixed-speed (left) or accelerating rotarod task (right) in YAC128 animals. Values represent the mean change from baseline latency to fall. Data represent mean + s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6

Schematic model showing the role of physiological synaptic vs. excessive extrasynaptic NMDAR activity in the neurodegeneration of Huntington’s disease. Physiological synaptic NMDAR activity promotes neuroprotection, in part by facilitating non-toxic aggregation of mtHtt via the chaperonin TRiC. Otherwise toxic mtHtt would interfere with the neuroprotective CREB-PGC-1α pathway. In contrast, extrasynaptic NMDAR activity promotes neuronal cell injury and death, in part by increasing the relative level of Rhes, and, in conjunction with soluble/toxic mtHtt, by contributing to transcriptional deregulation of the CREB-PGC-1α cascade. Drugs inhibiting extrasynaptic or synaptic activity are indicated. Note that most NMDAR antagonists, as well as high concentrations of memantine, block both synaptic and extrasynaptic NMDAR-mediated currents, while low concentrations of memantine block predominantly the extrasynaptic component, thus relatively sparing synaptic activity and promoting neuroprotection.