N-Methyl-d-aspartate (NMDA) receptor function and excitotoxicity in Huntington's disease

Many lines of evidence support a role for neuronal damage arising as a result of excessive activation of glutamate receptors by excitatory amino acids in the pathogenesis of Huntington disease. The N-methyl-d-aspartate subclass of ionotropic glutamate receptors (NMDARs) is more selective and effective than the other subclasses in mediating this damage. As well, neurons expressing high levels of NMDARs are lost early from the striatum of individuals affected with Huntington's disease (HD), and injection of NMDAR agonists into the striatum of rodents or non-human primates recapitulates the pattern of neuronal damage observed in HD. Altered NMDAR function has been reported in corticostriatal synapses in one mouse model of HD, and NMDAR-mediated current and/or toxicity have been found to be potentiated in striatal neurons from several HD mouse models as well as heterologous cells expressing the mutant huntingtin protein. Changes in NMDAR activity have been correlated with altered calcium homeostasis, mitochondrial membrane depolarization and caspase activation. NMDAR stimulation is also closely linked to mitochondrial activity, as treatment with mitochondrial toxins has been demonstrated to produce striatal damage that can be reversed by the addition of NMDAR antagonists. Recent efforts have focused on the elucidation of molecular pathways linking huntingtin to NMDARs, as well as the mechanisms which underlie the enhancement of NMDAR activity by mutant huntingtin. Here, we review the literature to date and recent findings concerning the role of NMDARs in HD pathogenesis.     

Enhanced striatal NR2B-containing N-methyl-D-aspartate receptor-mediated synaptic currents in a mouse model of Huntington disease

Huntington disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine tract near the N terminus of the protein huntingtin, leading to dramatic loss of striatal medium-sized spiny GABAergic projection neurons (MSNs). Evidence suggests overactivation of N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDARs) contributes to selective degeneration of MSNs in HD. Striatal MSNs are enriched in NR2B, and whole cell current and excitotoxicity mediated predominantly by the NR2B subtype of NMDARs is increased with expression of mutant huntingtin in transfected cell lines and striatal MSNs from mice models. To test whether synaptic NMDAR current is altered by mutant huntingtin expression, we recorded striatal MSN excitatory postsynaptic currents (EPSCs) evoked by stimulation of cortical afferents in corticostriatal slices from YAC72 mice and their wild-type (WT) littermates at age 21-31 days. The ratio of NMDAR- to AMPAR-mediated EPSC amplitude was significantly increased in YAC72 compared to WT mice. Furthermore, using a paired-pulse stimulation protocol as a measure of presynaptic glutamate release probability, we found no significant differences between YAC72 and WT striatal MSN responses. These data suggest selective potentiation of postsynaptic NMDAR activity at corticostriatal synapses in YAC72 mice. Measurements of EPSC decay kinetics, as well as the effects of NR2B-subtype selective antagonists and glycine concentration on EPSC amplitude, are consistent with the majority of postsynaptic NMDARs being triheteromers of NR1/NR2A/NR2B in both WT and YAC72 mice. Together with previous results, our data suggest that enhanced activity of NR2B-containing NMDARs is one of the earliest changes leading to neuronal degeneration in HD.     

Role of NR2B-type NMDA receptors in selective neurodegeneration in Huntington disease

N-Methyl-D-aspartate receptor (NMDAR)-mediated excitotoxicity has been proposed to play a role in Huntington disease (HD), caused by expansion of a polyglutamine tract in the protein huntingtin. HD is characterized by selective neurodegeneration most severely affecting striatal medium-sized spiny projection neurons (MSNs), where expression of the NMDAR subunit NR2B is increased relative to other NR2 subunits. Here, we review our data that NR2B-type NMDAR currents are selectively potentiated by mutant huntingtin in transfected non-neuronal cells and acutely dissociated striatal MSNs from the YAC72 transgenic mouse model of HD. As well, we report increased striatal MSN NMDAR-mediated synaptic currents in corticostriatal slice recordings from YAC72 compared with wild-type mice. This effect was associated with a larger NMDAR- to AMPAR-mediated current ratio, suggesting specific potentiation of postsynaptic NMDARs. Enhanced NMDAR current likely involves increased surface receptor numbers or activity, since we observed no differences between genotypes in striatal NR2B expression. Potentiation of NR2B-containing NMDAR current in striatal MSNs expressing mutant huntingtin may help explain the exquisite vulnerability of these neurons to degeneration in HD.     

Calpain and STriatal-Enriched protein tyrosine phosphatase (STEP) activation contribute to extrasynaptic NMDA receptor localization in a Huntington's disease mouse model

In Huntington's disease (HD), the mutant huntingtin (mhtt) protein is associated with striatal dysfunction and degeneration. Excitotoxicity and early synaptic defects are attributed, in part, to altered NMDA receptor (NMDAR) trafficking and function. Deleterious extrasynaptic NMDAR localization and signalling are increased early in yeast artificial chromosome mice expressing full-length mhtt with 128 polyglutamine repeats (YAC128 mice). NMDAR trafficking at the plasma membrane is regulated by dephosphorylation of the NMDAR subunit GluN2B tyrosine 1472 (Y1472) residue by STriatal-Enriched protein tyrosine Phosphatase (STEP). NMDAR function is also regulated by calpain cleavage of the GluN2B C-terminus. Activation of both STEP and calpain is calcium-dependent, and disruption of calcium homeostasis occurs early in the HD striatum. Here, we show increased calpain cleavage of GluN2B at both synaptic and extrasynaptic sites, and elevated extrasynaptic total GluN2B expression in the YAC128 striatum. Calpain inhibition significantly reduced extrasynaptic GluN2B expression in the YAC128 but not wild-type striatum. Furthermore, calpain inhibition reduced whole-cell NMDAR current and the surface/internal GluN2B ratio in co-cultured striatal neurons, without affecting synaptic GluN2B localization. Synaptic STEP activity was also significantly higher in the YAC128 striatum, correlating with decreased GluN2B Y1472 phosphorylation. A substrate-trapping STEP protein (TAT-STEP C-S) significantly increased VGLUT1-GluN2B colocalization, as well as increasing synaptic GluN2B expression and Y1472 phosphorylation. Moreover, combined calpain inhibition and STEP inactivation reduced extrasynaptic, while increasing synaptic GluN2B expression in the YAC128 striatum. These results indicate that increased STEP and calpain activation contribute to altered NMDAR localization in an HD mouse model, suggesting new therapeutic targets for HD.     

Mutant huntingtin directly increases susceptibility of mitochondria to the calcium-induced permeability transition and cytochrome c release

Huntington's disease (HD) is initiated by an abnormally expanded polyglutamine stretch in the huntingtin protein, conferring a novel property on the protein that leads to the loss of striatal neurons. Defects in mitochondrial function have been implicated in the pathogenesis of HD. Here, we have examined the hypothesis that the mutant huntingtin protein may directly interact with the mitochondrion and affect its function. In human neuroblastoma cells and clonal striatal cells established from HdhQ7 (wild-type) and HdhQ111 (mutant) homozygote mouse knock-in embryos, huntingtin was present in a purified mitochondrial fraction. Subfractionation of the mitochondria and limited trypsin digestion of the organelle demonstrated that huntingtin was associated with the outer mitochondrial membrane. We further demonstrated that a recombinant truncated mutant huntingtin protein, but not a wild-type, directly induced mitochondrial permeability transition (MPT) pore opening in isolated mouse liver mitochondria, an effect that was prevented completely by cyclosporin A (CSA) and ATP. Importantly, the mutant huntingtin protein significantly decreased the Ca2+ threshold necessary to trigger MPT pore opening. We found a similar increased susceptibility to the calcium-induced MPT in liver mitochondria isolated from a knock-in HD mouse model. The mutant huntingtin protein-induced MPT pore opening was accompanied by a significant release of cytochrome c, an effect completely inhibited by CSA. These findings suggest that the development of specific MPT inhibitors may be an interesting therapeutic avenue to delay the onset of HD.     

Tonic NMDA receptor-mediated current in prefrontal cortical pyramidal cells and fast-spiking interneurons

Tonically activated neuronal currents mediated by N-methyl-d-aspartate receptors (NMDARs) have been hypothesized to contribute to normal neuronal function as well as to neuronal pathology resulting from excessive activation of glutamate receptors (e.g., excitotoxicity). Whereas cortical excitatory cells are very vulnerable to excitotoxic insult, the data regarding resistance of inhibitory cells (or interneurons) are inconsistent. Types of neurons with more pronounced tonic NMDAR current potentially associated with the activation of extrasynaptic NMDARs could be expected to be more vulnerable to excessive activation by glutamate. In this study, we compared tonic activation of NMDARs in excitatory pyramidal cells and inhibitory fast-spiking interneurons in prefrontal cortical slices. We assessed tonic NMDAR current by measuring holding current shift as well as noise reduction following NMDAR blockade after removal of spontaneous glutamate release. In addition, we compared NMDAR miniature excitatory postsynaptic currents (EPSCs) in both cell types. We have demonstrated for the first time that tonic NMDAR currents are present in inhibitory fast-spiking interneurons. We found that the magnitude of tonic NMDAR current is similar in pyramidal cells and fast-spiking interneurons, and that quantal release of glutamate does not significantly impact tonic NMDAR current.     

Characterization of neuron-specific huntingtin aggregates in human huntingtin knock-in mice

Huntington's disease (HD) is caused by a mutation causing expanded polyglutamine tracts in the N-terminal fragment of huntingtin. A pathological hallmark of HD is the formation of aggregates in the striatal neurons. Here we report that ageing human huntingtin knock-in mice expressing mutant human huntingtin contained neuronal huntingtin aggregates, as revealed by immunohistochemical analysis. In heterozygous knock-in mice with 77 CAG repeats, aggregates of N-terminal fragments of huntingtin were specifically formed in nuclei and neuropils in the striatal projection neurons, and in neuropils in their projection regions. This aggregate formation progressed depending on age, became interacted with proteolytic or chaperone proteins, and occurred most prominently in the nucleus accumbens. These mutant mice demonstrated abnormal aggressive behavior. In homozygous knock-in mice, heavy deposits of intranuclear and neuropil aggregates were detected, which extended to other regions; and characteristic large perikaryal aggregates were also found in the affected neurons. However, cell death was not observed among the striatal and affected neurons of these mutant mice. Our results indicate that the polyglutamine aggregates do not necessarily correlate with neuronal death. These human huntingtin knock-in mice should be useful to provide an effective therapeutic approach against HD.     

HD CAG repeat implicates a dominant property of huntingtin in mitochondrial energy metabolism

The 'expanded' HD CAG repeat that causes Huntington's disease (HD) encodes a polyglutamine tract in huntingtin, which first targets the death of medium-sized spiny striatal neurons. Mitochondrial energetics, related to N-methyl-d-aspartate (NMDA) Ca2+-signaling, has long been implicated in this neuronal specificity, implying an integral role for huntingtin in mitochondrial energy metabolism. As a genetic test of this hypothesis, we have looked for a relationship between the length of the HD CAG repeat, expressed in endogenous huntingtin, and mitochondrial ATP production. In STHdhQ111 knock-in striatal cells, a juvenile onset HD CAG repeat was associated with low mitochondrial ATP and decreased mitochondrial ADP-uptake. This metabolic inhibition was associated with enhanced Ca2+-influx through NMDA receptors, which when blocked resulted in increased cellular [ATP/ADP]. We then evaluated [ATP/ADP] in 40 human lymphoblastoid cell lines, bearing non-HD CAG lengths (9-34 units) or HD-causing alleles (35-70 units). This analysis revealed an inverse association with the longer of the two allelic HD CAG repeats in both the non-HD and HD ranges. Thus, the polyglutamine tract in huntingtin appears to regulate mitochondrial ADP-phosphorylation in a Ca2+-dependent process that fulfills the genetic criteria for the HD trigger of pathogenesis, and it thereby determines a fundamental biological parameter--cellular energy status, which may contribute to the exquisite vulnerability of striatal neurons in HD. Moreover, the evidence that this polymorphism can determine energy status in the non-HD range suggests that it should be tested as a potential physiological modifier in both health and disease.     

Mismatch repair gene Msh2 modifies the timing of early disease in Hdh(Q111) striatum

Somatic instability of expanded HD CAG repeats that encode the polyglutamine tract in mutant huntingtin has been implicated in the striatal selectivity of Huntington's disease (HD) pathology. Here in Hdh(Q111) mice, we have tested whether a genetic background deficient in Msh2, expected to eliminate the unstable behavior of the 109 CAG array inserted into the murine HD gene, would alter the timing or striatal specificity of a dominant disease phenotype that predicts late-onset neurodegeneration. Our analyses of Hdh(Q111/+):Msh2(+/+) and Hdh(Q111/+): Msh2(-/-) progeny revealed that, while inherited instability involved Msh2-dependent and -independent mechanisms, lack of Msh2 was sufficient to abrogate progressive HD CAG repeat expansion in striatum. The absence of Msh2 also eliminated striatal mutant huntingtin with somatically expanded glutamine tracts and caused an approximately 5 month delay in nuclear mutant protein accumulation, but did not alter the striatal specificity of this early phenotype. Thus, somatic HD CAG instability appears to be a consequence of a striatal-selective disease process that accelerates the timing of an early disease phenotype, via expansion of the glutamine tract in mutant huntingtin. Therefore Msh2, as a striking modifier of early disease onset in a precise genetic HD mouse model, provides a novel target for the development of pharmacological agents that aim to slow pathogenesis in man.     

Striatal neurons expressing full-length mutant huntingtin exhibit decreased N-cadherin and altered neuritogenesis

The expanded CAG repeat that causes striatal cell vulnerability in Huntington's disease (HD) encodes a polyglutamine tract in full-length huntingtin that is correlated with cellular [ATP] and [ATP/ADP]. Since striatal neurons are vulnerable to energy deficit, we have investigated, in Hdh CAG knock-in mice and striatal cells, the hypothesis that decreased energetics may affect neuronal (N)-cadherin, a candidate energy-sensitive adhesion protein that may contribute to HD striatal cell sensitivity. In vivo, N-cadherin was sensitive to ischemia and to the effects of full-length mutant huntingtin, progressively decreasing in Hdh(Q111) striatum with age. In cultured striatal cells, N-cadherin was decreased by ATP depletion and STHdh(Q111) striatal cells exhibited dramatically decreased N-cadherin, due to decreased Cdh2 mRNA and enhanced N-cadherin turnover, which was partially normalized by adenine supplementation to increase [ATP] and [ATP/ADP]. Consistent with decreased N-cadherin function, STHdh(Q111) striatal cells displayed profound deficits in calcium-dependent N-cadherin-mediated cell clustering and cell-substratum adhesion, and primary Hdh(Q111) striatal neuronal cells exhibited decreased N-cadherin and an abundance of immature neurites, featuring diffuse, rather than clustered, staining for N-cadherin and synaptic vesicle markers, which was partially rescued by adenine treatment. Thus, mutant full-length huntingtin, via energetic deficit, contributes to decreased N-cadherin levels in striatal neurons, with detrimental effects on neurite maturation, strongly suggesting that N-cadherin-mediated signaling merits investigation early in the HD pathogenic disease process.     

Early mitochondrial calcium defects in Huntington's disease are a direct effect of polyglutamines

Huntington's disease (HD) is caused by an expansion of exonic CAG triplet repeats in the gene encoding huntingtin protein (Htt), but the mechanisms by which this mutant protein causes neurodegeneration remain unknown. Here we show that lymphoblast mitochondria from patients with HD have a lower membrane potential and depolarize at lower calcium loads than do mitochondria from controls. We found a similar defect in brain mitochondria from transgenic mice expressing full-length mutant huntingtin, and this defect preceded the onset of pathological or behavioral abnormalities by months. By electron microscopy, we identified N-terminal mutant huntingtin on neuronal mitochondrial membranes, and by incubating normal mitochondria with a fusion protein containing an abnormally long polyglutamine repeat, we reproduced the mitochondrial calcium defect seen in human patients and transgenic animals. Thus, mitochondrial calcium abnormalities occur early in HD pathogenesis and may be a direct effect of mutant huntingtin on the organelle.     

Striatal cells from mutant huntingtin knock-in mice are selectively vulnerable to mitochondrial complex II inhibitor-induced cell death through a non-apoptotic pathway

Extensive striatal neuronal loss occurs in Huntington's disease (HD), which is caused by an expanded polyglutamine tract in huntingtin (htt). Evidence suggests that mutant htt directly or indirectly compromises mitochondrial function, contributing to the neuronal loss. To determine the role of compromised mitochondrial function in the neuronal cell death in HD, immortalized striatal cells established from Hdh(Q7) (wild-type) and Hdh(Q111) (mutant) mouse knock-in embryos were treated with 3-nitropropionic acid (3-NP), a mitochondrial complex II toxin. 3-NP treatment caused significantly greater cell death in mutant striatal cells compared with wild-type cells. In contrast, the extent of cell death induced by rotenone, a complex I inhibitor, was similar in both cell lines. Although evidence of apoptosis was present in 3-NP-treated wild-type striatal cells, it was absent in 3-NP-treated mutant cells. 3-NP treatment caused a greater loss of mitochondrial membrane potential (deltapsim) in mutant striatal cells compared with wild-type cells. Cyclosporine A, an inhibitor of mitochondrial permeability transition pore (PTP), and ruthenium red, an inhibitor of the mitochondrial calcium uniporter, both rescued mutant striatal cells from 3-NP-induced cell death and prevented the loss of deltapsim. These data show that mutant htt specifically increases cell vulnerability to mitochondrial complex II inhibition and further switched the type of cell death induced by complex II inhibition from apoptosis to a non-apoptotic form, caused by mitochondrial membrane depolarization, probably initiated by mitochondrial calcium overload and subsequent PTP opening. These findings suggest that impaired mitochondrial complex II function in HD may contribute to non-apoptotic neuronal cell death.     

Early phenotypes that presage late-onset neurodegenerative disease allow testing of modifiers in Hdh CAG knock-in mice

In Huntington's disease (HD), CAG repeats extend a glutamine tract in huntingtin to initiate the dominant loss of striatal neurons and chorea. Neuropathological changes include the formation of insoluble mutant N-terminal fragment, as nuclear/neuropil inclusions and filter-trap amyloid, which may either participate in the disease process or be a degradative by-product. In young Hdh knock-in mice, CAGs that expand the glutamine tract in mouse huntingtin to childhood-onset HD lengths lead to nuclear accumulation of full-length mutant huntingtin and later accumulation of insoluble fragment. Here we report late-onset neurodegeneration and gait deficits in older Hdh(Q111) knock-in mice, demonstrating that the nuclear phenotypes comprise early stages in a disease process that conforms to genetic and pathologic criteria determined in HD patients. Furthermore, using the early nuclear-accumulation phenotypes as surrogate markers, we show in genetic experiments that the disease process, initiated by full-length mutant protein, is hastened by co-expression of mutant fragment; therefore, accrual of insoluble-product in already compromised neurons may exacerbate pathogenesis. In contrast, timing of early disease events was not altered by normal huntingtin or by mutant caspase-1, two proteins shown to reduce inclusions and glutamine toxicity in other HD models. Thus, potential HD therapies in man might be directed at different levels: preventing the disease-initiating mechanism or slowing the subsequent progression of pathogenesis.     

Identification of a presymptomatic molecular phenotype in Hdh CAG knock-in mice

The hallmark striatal neurodegeneration of Huntington's disease (HD) is first triggered by a dominant property of the expanded glutamine tract in mutant huntingtin that increases in severity with glutamine size. Indeed 111-glutamine murine huntingtin leads to a dominant cascade of phenotypes in Hdh(Q111) mice, although these abnormalities are not manifest in Hdh(Q50) mice, with 50-glutamine mutant protein. Therefore, to identify phenotypes that might reflect events closer to the fundamental trigger mechanism, and that can be measured as a consequence of adult-onset HD mutant huntingtin, we have screened for altered expression of genes conserved in evolution, which are likely to encode essential proteins. Probes generated from Hdh(Q111) homozygote and wild-type striatal RNAs were hybridized to human gene segments on filter arrays, disclosing a mutant-specific increase in hybridization to Rrs1, encoding a ribosomal protein. Subsequent, quantitative RT-PCR assays demonstrated increased Rrs1 mRNA from 3 weeks of age in homozygous and heterozygous Hdh(Q111) striatum and increased Rrs1 mRNA expression with a single copy's worth of 50-glutamine mutant huntingtin in Hdh(Q50) striatum. Moreover, quantitative RT-PCR assays for the human homologue demonstrated elevated Rrs1 mRNA in HD compared with control postmortem brain. These findings, therefore, support a chronic impact of mutant huntingtin on an essential ribosomal regulatory gene to be investigated for its role very early in HD pathogenesis.