Postsynaptic Dysfunction

5.1. Morphological and Gross Membrane Alterations

Altered cellular morphology is well characterized in human HD tissue, consisting of: increased dendritic complexity, as well as spine number and size in moderate HD grades, and truncated, swollen dendritic processes with marked spine loss in advanced cases42. Across many of the HD mouse models, reductions in brain volume and weight are observed when little or no cell death is seen, a finding possibly accounted for by altered cellular morphology, including reduced membrane area and spine numbers, (reviewed in ref. 3). This could be the result of an active process in response to altered afferentation and/or synaptic activity, or a breakdown of the cytoskeletal structure, as has been reported in human HD tissue24.

The basic integrity of the cell membrane and the rapid passage of ions across it are fundamental to the unique properties of excitable cells. Changes to membrane function have direct consequences for neuronal activity: the ability to receive, process, store, and transmit information. Hippocampal pyramidal cell intrinsic membrane properties and basal transmission are near normal in R6/2 mice, even at the terminal phase30. In contrast, the membrane physiology of striatal MSNs is markedly altered in adult R6/2 mice and a subset of cells in CAG knock-in mice43-46. Such changes predict that striatal MSNs are hyperexcitable after the onset of symptoms. Similar alterations in membrane properties are observed across several neuronal types at later, postsymptomatic ages in R6/1 mice (K. Murphy, unpublished observations). In MSNs, potassium (K+) channel conductances maintain marked hyperpolarization, regulate AP firing, and confer unique physiological characteristics; importantly, MSNs from symptomatic R6/2 and HD100 mice have reduced K+ currents, and concomitant decreases in K+ channel protein expression47, findings that may account for altered membrane properties and contribute to latent neuronal vulnerability after the onset of dysfunction.

5.2. Excitotoxicity and Glutamate Receptor Function

Intrastriatal injections of excitotoxins produce lesions that resemble the pattern of neurodegeneration seen in human HD. Highly selective lesions that result in the depletion of MSNs have been produced by injections of compounds that act specifically on NMDA receptors; furthermore, such lesions are blocked by NMDAR antagonists (reviewed in ref. 48). NMDAR signaling is critically involved in a variety of fundamental activity-dependent brain processes; notably gating alterations in synaptic weights, gene transcription, and even cell survival. Many seminal reports provide strong evidence that NMDAR receptor dysfunction or aberrant signaling may underlie neuropathology in HD.

Surprisingly, transgenic mice expressing only exon 1 of the HD gene exhibit reduced sensitivity to excitotoxic lesions as compensatory neuroprotective mechanisms develop, correlating with overt symptoms of disease49,50. For example, basal Ca2+ concentration is higher in R6/2 MSNs, and while quinolinic acid induces larger current changes (Ca2+ influx through NMDARs) in R6/2 than in controls, R6/2 MSNs recover to their basal Ca2+ concentration whereas WT neurons do not50. Such changes would place R6/2 MSNs in a protracted sublethal grade of excitotoxicity that may render them susceptible to further excitotoxic insult over time; in the short term, however, increased Ca2+ influx may be responsible for inducing neuroprotective strategies, such as changes in Ca2+

buffering50 and gene transcription that combat NMDAR overactivation49. An additional explanation for resistance to excitotoxicity may stem from reduced cortical activity, since intact glutamatergic afferentation is required for excitotoxic lesions to be produced in the striatum18. Importantly, intrastriatal injection of quinolinate produces equivalent lesions in WT and larger construct HD100 mice51, while full-length htt YAC72 mice exhibit increased sensitivity52. Thus, the latter models may be representative of earlier disease stages: at the time of, and prior to, induction of neuronal compensatory mechanisms, respectively.

Increased glutamate excitotoxicity could result from increased release, decreased uptake, increased receptor responsiveness, altered downstream receptor signaling, or impaired Ca2+ homeostasis following repeated receptor activation. Evidence for a contribution by most of these mechanisms has been demonstrated. Altered release and uptake are implicated at the HD synapse (see Section 4). As well, mitochondrial function (which provides the major Ca2+ buffering reservoir following NMDAR stimulation) also appears to be directly impaired by mhtt, while intracellular Ca2+ release, in response to mGluR activation, is augmented by altered interaction between the IP3 receptor and expanded htt (reviewed in ref. 53). On the other hand, mGluR desensitization is facilitated by mhtt expression via an interaction between mhtt and the mGluR inhibitory protein optineurin, which could dampen the effects of enhanced IP3 signaling54. In addition to these mechanisms that may increase excitotoxic vulnerability, it is increasingly clear that mhtt augments NMDAR activity in striatal MSNs.

Striatal MSNs have high levels of NR2B subunit-containing NMDARs55, and NMDAR-mediated whole cell currents in cell lines transfected with NR1/NR2B are specifically increased by the presence of mhtt56. As one might predict from the study of Chen and colleagues (1999), MSNs in acute slices prepared from R6/2 and CAG knock-in mice exhibit enhanced responsiveness to NMDA application prior to the onset of overt symptoms43,46, as do a population of MSNs in slices from adult YAC72 and HD100 transgenic mice43,57. In cultured striatal MSNs, prepared from YAC mice soon after birth, NMDA-induced whole cell current density and apoptotic cell death are enhanced in a polyQ length-dependent manner52,58,59. Also, NMDAR EPSCs are specifically enhanced, whereas AMPA receptor currents are not, at cortico-striatal synapses in slices prepared from 1-month-old YAC72 mice, long before onset of phenotype40. The NMDAR activity underlying increased current density, excitotoxicity, and EPSCs is largely mediated by NR2B-containing NMDARs40,52,58.

Increased NMDAR current may result from increased channel open probability or conductance; however, these channel properties are similar for NR1/NR2B expressed in HEK293 cells with htt containing expanded (138) and normal (15) polyQ56. It is possible such findings may not directly correlate with neuronal systems, where the presence of mhtt might alter the channel properties of NR2B-containing heteromeric (NR1/NR2A/NR2B) receptors. Alternatively, increased current could result from increased receptor expression, and increased NR1/NR2B mRNA levels have been observed (prior to large decreases; associated with cell death) in HD post mortem tissue37. Striatal immunohistochemistry in symptomatic R6/2 mice has demonstrated increased NR1 NMDAR subunit numbers, but this was accompanied by a decrease in NR2A/B subunits43. Other studies have demonstrated no change in striatal NMDAR mRNA and a trend toward decreased membrane bound NR2 in R6/2 mice (see ref. 60). Moreover, expression levels of NR1 and NR2B in the striatum of 2-month-old YAC46 and YAC72 mice (when increased currents are apparent but symptoms are not) are similar to WT controls61. Therefore it would appear that mhtt-induced alterations other than transcription or translation of NR1 or NR2B underlie augmented NMDAR current, leaving the possibility that subcellular distribution and/or post-translational modification of NMDARs is altered by mhtt.

5.3. NMDA Receptor Trafficking and Surface Expression

A variety of post-translational modifications and protein interactions govern NMDAR function and localization. Recent studies indicate that NMDAR delivery to, and endocytosis from, the surface membrane is regulated by NR2 subunit composition, NR1 splice variants, phosphorylation of specific sites within the C-terminus of NR2B, and NMDAR interactions with SAP 102, and postsynaptic density protein-95 (PSD-95), both of which are members of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins (reviewed in ref. 62).

PSD-95 binds NR2 subunits, forming clusters in the postsynaptic membrane, in addition to linking NMDARs with cytoskeletal and signaling proteins, including protein kinases and phosphatases. The interaction of PSD-95 with receptors is, unsurprisingly, heavily implicated in the regulation of synaptic plasticity63,64, and the suppression of PSD-95 attenuates NMDAR-mediated excitotoxicity, despite having no effect on NMDAR current or Ca2+ influx65. Together these studies demonstrate the importance of physical interactions of NMDARs with other proteins for appropriate downstream responses to their activation. Htt has been shown to associate with both PSD-95 and NMDARs in human cortical tissue, suggesting that htt/PSD-95/NMDA complexes occur66. Furthermore, htt/PSD-95 binding is reduced by polyQ expansion and is also reduced in HD patient cortex66. The same study found that expression of mhtt in cell lines increased NMDA-mediated excitotoxicity and that the increase was reduced by addition of WT htt. Sun and co-workers (2001) propose that htt/PSD-95 binding negatively regulates NMDAR signaling and that mhtt disrupts this negative regulation. However, it is also possible that mhtt interferes with PSD-95 transcription and that reduced htt/PSD-95 binding is attributable to reduced PSD-95, as has been observed in HD

transgenic mice .

Tyrosine phosphorylation of NMDARs (by nonreceptor Src-family protein tyrosine kinases) increases NMDAR currents, shifts NMDARs from intracellular pools to the surface membrane, and increases NMDAR localization to the postsynaptic membrane (reviewed in ref. 62). Co-expression of mhtt and PSD-95 in cell lines results in increased PSD-95 surface expression and levels of activated Src. Moreover, co-expression of mhtt, PSD-95, NR1, and NR2B results in increased NMDAR-mediated excitotoxicity as well as tyrosine phosphorylation of NR2B; Src inhibitors decreased this phosphorylation and excitotoxicity67. As a note of caution, these data may not directly correlate with neurons in situ, as PSD-95 is normally targeted not just to the membrane surface, but specifically to synapses by palmitoylation (reviewed in ref. 68), and it has been shown that nonsynaptic NMDARs activate an apoptotic pathway whereas synaptic NMDA receptors preferentially activate a prosurvival pathway69. Also, recent studies have demonstrated no increase (but a trend toward decrease) of phosphorylated NR2 in symptomatic HD mouse striatum49,60.

Serine phosphorylation of NMDARs is also altered in MSNs from HD transgenic mice. Progressive reductions in NR1 phosphorylation at serine 897 (a candidate site for cAMP-induced increases in NMDA activity) were observed in N171 mice49, which may be related to latent neuroprotective strategies. On the other hand, levels of phosphorylated serine 897 are increased in R6/2 mice at the age at which they exhibit reduced excitotoxicity70. Thus, there is no clear correlation between NR1 Ser897 phosphorylation and NMDAR current and excitotoxicity.

NMDARs are internalized by clathrin-dependent mechanisms and HIP1 provides a link between htt and clathrin endocytosis71. Moreover, htt/HIP-1 binding is disrupted by polyQ expansion33,34, and a breakdown in clathrin interactions may contribute to altered NMDAR surface expression or synaptic localization. The binding between another huntingtin-interacting protein (HIP14) and htt is also disrupted by polyglutamine expansion. HIP14 shares sequence homology with proteins involved in endocytosis and is also a palmitoyl acyl-transferase that modulates the trafficking and membrane association of a variety of proteins, including postsynaptic density protein-95 (PSD-95), synaptosomal-associated protein-25 (SNAP-25), and htt itself (reviewed in ref. 68). Altered mhtt/HIP14 function may affect not only endocytosis of NT receptors, but also the trafficking of htt and other synaptic proteins.

5.4. Dopamine Receptor Signaling

Dopamine controls the efficacy of fast synaptic transmission by regulating NT release and postsynaptic responsiveness via multiple receptors, second messengers, kinases and phosphatases (reviewed in ref. 72). The striatum receives dopaminergic input from the substantia nigra, and MSN projection pathways are distinguished by heterogeneous dopamine receptor (DAR) expression and projection targets. MSNs of the direct striatonigral pathway express substance P and D1-type DARs, and decreased activity of this pathway (that normally increases thalamocortical excitation) would lead to rigidity and bradykinesia. MSNs of the indirect striatopallidal pathway express met-enkephalin and D2-type DARs, and diminished function of this pathway (that normally reduces thalamocortical excitation) would produce random involuntary movements, including chorea (reviewed in ref. 73). Many post mortem studies suggest that it is the indirect pathway that first shows evidence of depletion in HD (reviewed in ref. 74), and noninvasive brain imaging of living humans has demonstrated reduced DAR labeling correlated with disease duration, cell loss, and cognitive impairment in symptomatic HD patients (reviewed in ref. 75). Moreover, reduced striatal (and cortical) DAR receptor binding has also been reported in asymptomatic HD gene carriers, demonstrating that dopaminergic dysfunction can be detected before clinical diagnosis and cell death75. Investigations across many mouse models of HD, after phenotypic onset, demonstrate reductions in striatal DAR protein and mRNA expression as well as diminished met-enkephalin staining with unaltered substance P staining21,49,60,76,77. Moreover, specific reductions in striatal D2 binding are seen in knock-in mice that do not exhibit an overt phenotype78, findings reminiscent of that observed in asymptomatic human carriers and possibly related to dysfunction of the indirect pathway (reviewed in ref. 75).

DAR activity modulates multiple synaptic and plasticity-related proteins (including NMDARs and several molecules that regulate NMDAR localization) via varied cascades, e.g., dopamine and cAMP-regulated phosphoprotein 32kDa (DARPP-32) gating of PKA, PKC, and MAP kinase pathways. Interestingly, D1 receptor agonism and forskolin treatment have been shown to induce the redistribution of htt, HIP1 and clathrin to the membrane71, which may play a role in regulating surface expression of NMDARs. Notably, DARPP-32 mRNA

labeling is decreased in R6/1 and R6/2 mice at about the time of phenotypic onset, and at the same time as D1/DARPP-32 signaling and dopamine regulation of Ca2+ channel, GABA receptor, and AMPA receptor function is lost in R6/2 mice21,79. Moreover, perturbation of any one of the steps in the dopamine pathway that regulate the activity or subcellular localization of NMDARs may contribute to early enhancement of NMDARs in HD mice.

5.5. GABA Receptors and Signaling

Striatal MSNs exert their influence upon target structures through the release of GABA, the major inhibitory NT in the brain. MSNs also receive GABAergic inhibition, primarily from interneurons, but also from other MSNs (reviewed in ref. 80). Stimulated (NMDA-induced) GABA release is increased in R6/1 mice, at about the time of phenotype onset39. At the time of symptom onset, striatal MSNs in R6/2 and R6/1 mice exhibit an increased frequency of spontaneous GABAa receptor (GABAAR)-mediated current; this effect is likely due to elevated presynaptic firing, since miniature IPSC frequencies recorded in tetrodotoxin were not significantly different44. Additionally, decay kinetics of GABAaR currents in R6/2 striatum are also affected, suggesting altered receptor composition. In support of these findings, whole cell GABAAR-mediated current densities and GABAA a1 subunit labeling are increased44. Thus, pre- and postsynaptic GABAergic function appears altered in symptomatic R6 mice. On the postsynaptic side, GABAAR undergo rapid constitutive endocytosis, and internalized receptors are either recycled back to the cell surface or targeted for lysosomal degradation. Notably, the fate of internalized receptors is regulated by a direct interaction between GABAARs and HAP181. Overexpression of HAP1 decreases receptor degradation and increases recycling rates, in addition to increasing GABAAR current amplitude and surface numbers. Thus, HAP1 may play a critical role in the control of fast synaptic inhibition. Direct investigation of the effects of mhtt on GABAAR cycling may clarify whether increased MSN GABAAR current in R6/2 mice44 is attributable to altered htt/HAP1 binding, or other (possibly compensatory) phenomena.

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