The Electrophysiological Phenotype of HD: Impact of the NMDA Receptor

September 20-21, 2003

Los Angeles, California

Prepared by Marina Chicurel, Ph.D.

Abstract

Several studies have implicated alterations in the responses of excitatory receptors, particularly NMDA receptors, in the pathology of Huntington’s disease (HD). Yet the chain of events that link these disruptions to HD’s primary mutation on the one hand, and to clinical symptoms, on the other, remains unclear. Workshop participants made progress towards generating an integrated view of HD pathology and identifying new directions for future study by analyzing recent results generated by a variety of approaches. They discussed several receptor abnormalities and their potential relationships, including alterations in NMDA receptors’ sensitivity to magnesium, abnormal receptor phosphorylation, disruption of postsynaptic density scaffolds, aberrant IP₃ signaling, and altered receptor trafficking. They also underscored the need to understand the function of wildtype huntingtin, given that loss-of-function effects seem to be important  in HD pathology. To explain the selective vulnerability of striatal neurons, participants noted their unique complement of NMDA receptor subunits—identifying the NR2B subunit as a potentially key determinant of susceptibility—as well as potassium channel subtypes, dopamine receptors and phosphatases. Of particular interest was the convergence of several lines of evidence on dopamine modulation as an important player in HD pathology.

A person with HD highlights the need to elucidate HD’s mechanisms of disease

Despite the many drugs in neurologists’ armamentaria, physicians remain fundamentally powerless to control HD’s inexorable progression. They can ameliorate some of the late-stage manifestations of the disease, but they cannot wage a frontal attack on the disease’s core pathology. Illustrating this situation, a woman with HD and her husband generously shared their experiences with workshop participants. They recounted how she has been treated with carbamazepine and valproate as anticonvulsants, clonazepam to control seizures and dystonia, lorazepam to counter anxiety, and high doses of fluoxetine to stave off depression and improve her speech. Yet as was painfully clear from the wheelchair-bound woman’s difficulty moving and communicating, the best these compounds can do falls distressingly short of a successful treatment. Participants were made keenly aware of the urgent need to understand the mechanisms of HD to develop treatments that go beyond the patching of secondary symptoms and combat HD’s core dysfunctions. By identifying approaches to dissect the molecular and electrophysiological underpinnings of the disease, participants took an important step towards realizing this goal.

1. Mechansims of toxicity: How does mutant huntingtin cause disease?

            Because mutant huntingtin is associated with a wide spectrum of cellular and electrophysiological alterations, a major challenge in the field is distinguishing primary from secondary and compensatory effects. Focusing on how synaptic transmission is altered in HD, workshop participants made inroads towards resolving this issue. They discussed phenomenological observations of disruptions in synaptic transmission in mouse models of HD, as well as mechanistic experiments that implicate presynaptic, postsynaptic, or both pre- and postsynaptic elements in HD pathology.

1.1 Synaptic transmission is abnormal in HD

As noted by Michael Levine, several studies indicate that synaptic transmission in HD is abnormal and changes with disease progression. In the early stages of disease, Levine has observed NMDA hypersensitivity in a subset of striatal cells in R6/2 mice. But this hypersensitivity appears to be transient. A study by Brundin and colleagues, for example, showed that, at later stages, R6/1 mice are strongly protected from acute striatal excitotoxic lesions induced by quinolinic acid, and Levine has observed that at approximately 5 weeks of age, the striata of R6/2 mice show progressive reductions in spontaneous excitatory currents, as if the two structures have been disconnected. Spine density and synaptic markers, including PSD-95 and synaptophysin, are decreased.

Anne Young suggested extending these findings by repeating an experiment originally performed in wildtype mice in which animals became insensitive to excitotoxic drugs after decortication. Interestingly, these animals regained their sensitivity when drug exposure was coupled to metabotropic receptor stimulation.

Kerry Murphy also discussed findings indicating abnormalities in HD synaptic transmission. Studying long-term depression (LTD) and potentiation (LTP) in HD mice, he has observed deficiencies in the hippocampus and perirhinal cortex. Murphy found that the magnitude of LTP is reduced and its threshold increased in the hippocampi of three different mouse models of HD (R6, knock-in, YAC). On the other hand, LTD, a phenomenon that in the hippocampi of normal mice occurs only during the first month of life, is strikingly robust in R6/2 and R6/1 hippocampi, apparently persisting throughout the animals’ life. To further investigate this alteration, Murphy studied the perirhinal cortex, in which LTD can be elicited in normal adult mice. Surprisingly, in this brain region, LTD could not be induced in R6/1 mice.

            Murphy suggested his data may reflect an imbalance between the activities of kinases and phosphatases. But he also acknowledged that much work remained to be done to clarify his results. Still unresolved, for example, are the distinct roles of pre- and post-synaptic mechanisms in the reduction in LTP. Some experiments (including studies of the rate of block by MK801) implicate presynaptic mechanisms, whereas others (including the study of post-tetanic potentiation (PTP) in isolation using AP5) implicate postsynaptic mechanisms. Participants suggested various experiments to examine this issue further. David Lovinger, for example, proposed monitoring the responses of postsynaptic cells loaded with chelators to block NMDA signaling and obtaining frequency curves to examine LTD alterations, and Matthew Dalva suggested testing multiple tetani to determine whether stimulus saturation was affecting Murphy’s PTP results. Murphy also noted that he may test LTP in the mossy fiber-CA3 synapse which is known to involve purely pre-synaptic mechanisms.

Dopamine modulation may be altered in HD

Participants were most interested, however, in the apparent role of dopamine in Murphy’s experiments. Murphy noted that exposing wildtype mice to a D2 receptor antagonist disrupted LTD, leading to facilitation instead of depression, an effect which is similar to the behavior observed in the perirhinal cortices of HD mice. He suggested that the HD defect may be a decrease in dopaminergic input from the ventral tegmental area. He also noted that alterations in D1 receptors may be implicated in the disrupted behavior of HD hippocampi.

Such alterations in dopamine transmission are consistent with observations from others. For example, Anne Young noted that dopamine, tyrosine hydroxylase activity, and D1 and D2 receptors are reduced in HD, and her group has observed alterations in the activation of adenylyl cyclases by dopamine receptors. In addition, Murphy pointed out that Elizabeth Abercrombie has reported a loss of dopaminergic fibers in R6/2 mice, and Marjorie Ariano and Levine have found that, except for the D5 receptor, all other dopamine receptors are reduced in R6/2 striata. Young added that whereas D2 responses are decreased in HD as expected, D1 responses seem to be exaggerated. In patients, D2 dopamine antagonists seem to decrease the quality of living, said Young. Treatments to increase dopamine responses have not been tested, noted Lynn Raymond,  because they are expected to worsen patients’ chorea. However, given the results in animal models, they may improve patients’ cognitive abilities. Levine agreed, noting that specific brain regions are affected differentially by the disease, so that experiments and treatments should be designed accordingly.

Based on this collection of intriguing observations, participants encouraged Murphy to examine the effects of dopamine in his system more carefully. For example, Dalva suggested testing whether the activation of dopamine receptors can rescue the perirhinal phenotype observed in R6/1 mice. In addition, James Surmeier said he would analyze the dopaminergic regulation of enkephalinergic medium spiny neurons in HD mice (see “Regional Vulnerability” below).

Other modulators of synaptic transmission may be altered in HD

Participants also discussed the potential importance of alterations in metabotropic glutamate receptor and cannabinoid receptor signaling. Young noted that there is a downregulation of mRNA and protein levels of mGluR2 glutamate receptors in the striata of HD mice, as well as a reduction in CB1 cannabinoid receptors. As described above, she also noted that stimulation of metabotropic receptors can re-sensitize decorticated animals to the effects of excitotoxic drugs. On the other hand, because mGluR2 receptors act to decrease synaptic transmission, Lovinger said that their downregulation could contribute to excitotoxicity.

An important issue to investigate, he added, is the relative timing of the decrease in mGluR2 and CB1 receptors compared to the degeneration of corresponding terminals to determine whether the receptor reductions are a result of terminal degeneration.

1.2 Presynaptic mechanisms implicated in HD

            As summarized by Levine, HD-associated changes in synaptic release have been observed for both cortico-striatal and hippocampal synapses. But because of variability in the timing of experiments and the selection of animal models, consistent alterations have been hard to pin down.

Nevertheless, changes at the molecular level indicate that mechanisms of synaptic release are altered in HD. Michael Edwardson described collaborating with Jenny Morton on immunoblotting experiments showing that levels of the synaptic protein complexin II drop 60% throughout the brain of R6/2 mice by 18 weeks of age. They also observed that the protein begins appearing in inclusions. Furthermore, exocytosis is impaired in PC12 cells when an inducible mutant form of huntingtin exon 1 is expressed. The disruption can be specifically rescued by complexin II transfection. Although a complexin II knockout appears to be normal, at least superficially, Edwardson noted that Jenny Morton has shown that these animals suffer from a progressive learning deficit. He now intends to extend his HD studies in the PC12 cell system.

1.3 Postsynaptic mechanisms implicated in HD

            Several studies have addressed the behavior of NMDARs in HD because of their relevance to the “excitotoxic hypothesis”. For example, Raymond described experiments showing that evoked synaptic currents mediated by NMDARs, but not AMPARs, were enhanced in YAC72 striatal cells. Consistent with this effect being post-, rather than pre-synaptic, she  observed that glutamate release at corticostriatal synapses of juvenile YAC72 mice was indistinguishable from controls as assessed by paired pulse experiments.

Various mechanisms were discussed to explain disruptions in NMDAR responses, including alterations in the receptors’ sensitivity to magnesium, abnormal receptor phosphorylation, disruption of postsynaptic density (PSD) scaffolds, aberrant modulation by metabotropic or dopaminergic receptors that activate IP₃ signaling, and altered receptor trafficking.

Altered NMDAR magnesium sensitivity

Levine noted that he has identified a subpopulation of neurons in the striata of young R6/2 mice which have NMDARs that are less sensitive to magnesium than their wildtype counterparts and are thus hyper-responsive to NMDA. In contrast, in the cortex he found pyramidal neurons with NMDARs with increased magnesium sensitivity. Dalva noted that NR2A subunits are more sensitive to magnesium, which Levine said was consistent with the relatively lower levels of NR2A expression he observes in striatal cells.

Altered NMDAR phosphorylation

Salter pointed out that post-translational modifications, such as phosphorylation by Src kinases or CaM kinase II, could also change NMDAR magnesium sensitivity, in addition to regulating channel gating, surface turnover, and the receptors’ association with  signaling molecules. Such effects are particularly interesting in the context of Liu and colleagues’ findings indicating that mutated huntingtin induces tyrosine phosphorylation of NR2B subunits. Raymond noted, however, that her experiments have not revealed evidence for tyrosine phosphorylation. Dalva recommended immunoprecipitating the NMDAR subunits before assessing phosphorylation and noted that Michael Greenberg’s lab has phospho-specific antibodies for NMDAR subunits.

Abnormalities at the PSD

            Edoardo Marcora, on the other hand, noted that he has found normal huntingtin and the huntingtin-associated protein HAP1 in PSDs. He believes that normal huntingtin may act together with PSD-95 (which has been shown to bind wildtype, but not mutant, huntingtin) as a scaffold protein. Huntingtin may help link receptors to various signaling proteins, including the kinase MLK2 (which, like PSD95, interacts with wildtype, but not mutant, huntingtin), kalirin-7, a GDP/GTP exchange factor for Rac1 which binds to HAP-1, and the transcription factor NeuroD. By playing a role in the formation of such multi-functional complexes, huntingtin would be expected to have several pathological effects when mutated, and to act differently depending on its cellular context.

            Because HD is an inherited autosomal dominant disease, expressing similarly in heterozygotes and homozygotes, however, the huntingtin mutation has commonly been expected to act as a gain-of-function, rather than a loss-of-function, mutation. But Mary Kennedy noted that increasing evidence indicates that both gain- and loss-of-function effects may be important in HD pathology. Such dual effects are not unprecedented. As pointed out by Young, in spinocerebellar ataxia type 1 the androgen receptor gene carries expanded CAG repeats which result in both types of alterations. Marcora proposed that mutant huntingtin may have a primarily dominant-negative effect, either sequestering the wildtype protein, or forming dysfunctional complexes with other proteins that normally interact with wildtype huntingtin. To investigate this further, Lovinger underscored the need to study what happens to wildtype huntingtin in the presence of mutated huntingtin.

            In addition, participants proposed expanding the scope of studies regarding huntingtin’s interactions with PSD proteins. Kennedy said she was interested in studying a synaptic protein that is highly homologous to PSD-95, synaptic-associated protein 102 (SAP102). SAP102 is abundant in fetal synaptic and extrasynaptic membranes and binds the cytoplasmic tail of NR2B NMDAR subunits. Robert Wenthold added that SAP102 may be involved in the delivery of NMDARs to the cell surface, associating with NMDAR subunits in the endoplasmic reticulum, while PSD-95 is involved in anchoring receptors at the synapse. Based on these observations and Raymond’s findings implicating the NR2B subunit in the selective neurodegeneration of medium spiny cells (see “Regional vulnerability” section below), SAP102 may be even more relevant to HD than PSD-95.

            Another post-synaptic density protein discussed by participants was the ephrin receptor (EphR). Dalva explained that these receptors may be involved in synaptogenesis. Consistent with this possibility, he recently found that when the Eph ligand is added to cells expressing EphRs in culture, the EphRs cluster and recruit NMDARs. He also observed that EphRs can mediate the phosphorylation of NMDARs through a Src kinase. In addition, Kennedy noted that NMDAR signaling through SynGAP, a Ras GTPase activating protein that is extremely abundant in PSDs, may converge with EphR signaling in the activation of Ras.

Although there is no direct evidence linking huntingtin to EphRs, they could interact through PSD-95. In addition, as pointed out by Marcora, kalirin-7 binds to HAP1 and activates Rac-1 in response to EphB2 stimulation. Dalva noted that the interactions between EphRs and other PSD components are dynamic, such that if mutated huntingtin is stickier than the wildtype protein, as suggested by some participants, it could cause NMDARs to be abnormally phosphorylated and lead to altered calcium influx.

Alterations in IP₃ signaling

In addition to exerting pathological effects at the PSD that could result in altered calcium influx through NMDARs, mutant huntingtin may disrupt calcium release from internal stores. Ilya Bezprovzanny performed a two-hybrid screen using the carboxy-terminus of the IP₃ receptor (IP₃-R) and discovered that HAP-1 is one of its binding partners. He also discovered that mutant, but not wildtype, huntingtin can bind directly to the IP₃-R, bypassing HAP-1.

To assess the functional effects of huntingtin binding, Bezprozvanny then conducted experiments in which IP₃-Rs were inserted into lipid bilayers and exposed to various concentrations of IP₃ in the presence of either wildtype or mutant huntingtin. Receptors incubated with mutant huntingtin were much more sensitive to IP₃, a finding that was confirmed in cells co-expressing IP₃-Rs and huntingtin constructs. In addition, Bezprozvanny has found that when mGluR1 receptors, which activate the IP₃ signaling pathway, are stimulated in medium spiny cells expressing huntingtin constructs, calcium release from internal stores is enhanced in a glutamine repeat-dependent manner. Thus, Bezprozvanny’s current model is that mutant huntingtin causes cytoplasmic calcium to increase abnormally, both by enhancing NMDAR currents and shifting the sensitivity of IP₃-Rs, which results in increased mitochondrial calcium uptake, and ultimately the induction of apoptosis.

Based on this hypothesis, Bezprozvanny predicts that blocking receptors that activate IP₃ signaling—including mGluR5 and dopamine D2 receptors—should prevent mutant huntingtin from causing neurodegeneration. He is currently testing this prediction in the YAC128 model of HD. Participants also suggested testing the effects of inhibitors of the IP₃-R. Bezprozvanny noted there were no specific inhibitory compounds, but that he was considering using a blocking peptide. Carl Johnson suggested using siRNAs, which Peter Reinhart said he could do using his biolistics HD model system (see “New techniques to probe HD pathology” section below). In the future, Bezprozvanny plans to extend his observations to other poly-glutamine diseases.

Potential involvement of huntingtin in receptor trafficking

Participants also discussed the possibility that HD pathology involves alterations in receptor trafficking. Several studies have implicated huntingtin in vesicular trafficking. Cell fractionation and immunostaining experiments, for example, indicate that both wildtype and mutant huntingtin associate with clathrin-coated vesicles. In addition, huntingtin interacting protein 1 (HIP1) has been characterized as a clathrin-associated protein whose overexpression disrupts trafficking of some membrane proteins. Thus, as suggested by Wenthold, cells that are particularly dependent on trafficking, such as neurons, may be uniquely vulnerable to disruptions caused by mutated huntingtin.

Wenthold recently identified sec-8 as a protein that interacts with NMDAR PDZ domains using a yeast two-hybrid screen. Sec-8 is a component of the exocyst, a complex involved in exocytosis and vesicle trafficking. When the researchers expressed a dominant-negative form of sec-8, NMDARs were trapped in the endoplasmic reticulum, unable to reach the cell surface. Wenthold is now interested in elucidating whether wildtype huntingtin is involved in this pathway, and if so, at what point. In addition, he is interested in assessing if huntingtin interacts at different points for the transport of different proteins.

Salter also expressed interest in huntingtin’s role in trafficking but in the context of   NMDAR signaling. Based on recent experiments from his lab, Salter believes that NMDARs may not only mediate signaling through channel opening, but through receptor internalization. Salter observed that when he repeatedly puffed saturating amounts of NMDA and glycine onto acutely dissociated cells, the synaptic responses gradually declined. If the cells were exposed to inhibitors of clathrin-mediated endocytosis, however, the decline did not occur. Salter has further shown that the effect is independent of calcium influx, and mediated by glycine binding. His working hypothesis is that, at certain concentrations, glycine can trigger endocytosis of NMDARs which activates signaling systems within the cell, possibly leading to cell death. To investigate the potential role of huntingtin in this internalization process, Young suggested monitoring NMDAR internalization in cells expressing conditional huntingtin constructs. In addition, Marcora suggested testing for the presence of huntingtin in immunoprecipitations using anti-NMDAR antibodies.

2. Regional vulnerability

One of the key mysteries of HD is the large variability in the damage suffered by different cell types and, in particular, the vulnerability of striatal medium spiny cells. Raymond presented a compelling set of experiments, using both acutely dissociated cells and slices from the YAC model of HD, implicating NR2B-type NMDARs. She noted that the relative expression of NR2B to NR2A is higher in the striatum than in other brain regions. In addition, NMDAR currents from YAC72 medium spiny cells are larger than those from wildtype animals and can be blocked to a large extent by the NR2B-selective antagonist ifenprodil, which also protects the cells from death. Furthermore, the enhanced sensitivity to NMDAR excitotoxicity in YAC72 striatal cells was not observed in YAC72 cerebellar cells which express NR2A and NR2C, but almost no NR2B subunits. 

Raymond is now investigating whether the regulation of NMDARs by mutant huntingtin depends on the receptors’ subcellular localization. Extra-synaptic receptors are known to contain NR2B subunits, but the subunits’ presence and association with other subunits at synaptic sites is uncertain. To help resolve this issue, Raymond analyzed the behavior of synaptic currents in medium spiny cells using ifenprodil and different concentrations of glycine, given that NR1/NR2B receptors and NR1/NR2A/NR2B heterotrimeric receptors differ in their glycine sensitivities. The results indicated that the majority of synaptic NMDARs are most likely NR1/NR2A/NR2B. Consistent with these findings, conantokin G, which inhibits NMDARs containing NR2B, suppressed NMDAR EPSC amplitudes. Whether synaptic receptors, extra-synaptic receptors, or both are mediating cell death, however, remains uncertain. Raymond cited work indicating that extrasynaptic signals in hippocampal cells trigger cell death, but Salter noted that other studies, also in hippocampal cells, indicate that cell death is mediated by synaptic signals.

In the future, Raymond plans to identify proteins that interact specifically with the carboxy tail of NR2B subunits using the yeast two-hybrid system. She is also interested in examining whether the increased NMDAR currents in HD spiny cells might be explained, at least in part, by a defect in endocytosis which leaves an overabundance of receptors at the cell surface. To further investigate synaptic transmission in slices and avoid the potentially complicating effects of transporters, Salter encouraged Raymond to extend some of her studies with analyses of miniature excitatory postsynaptic currents (mEPSCs). In addition, Surmeier suggested examining different striatal regions.

Differences in the expression of inward-rectifier channels (Kir channels) may also help explain medium spiny cell vulnerability. As noted by Surmeier, enkephalinergic spiny cells express a unique subset of Kir 2 channels in their dendrites that slowly inactivate, and which may make them more susceptible to excitatory inputs. Levine pointed out that some of these channels appear to be reduced in R6/2 mice. Whether the reduction precedes or follows the neurodegenerative changes observed in these cells’ dendrites, however, remains uncertain. It is possible, as noted by Surmeier, that the alterations in channel expression are simply the result of dendrite loss.

Another distinguishing characteristic of these vulnerable neurons is their expression of dopamine D2 receptors which modulate a variety of cellular functions, including the activities of sodium and L-type calcium channels, IP₃ signaling, and NMDA responses. Surmeier intends to monitor various electrophysiological behaviors which may be affected by alterations in these functions, including pace-making capacity, burst firing, and dopamine-mediated regulation of NMDA receptor (NMDAR) activity.

Yet another factor that may contribute to medium spiny cell vulnerability is the distinct composition of these cells’ NMDA signaling pathways. As noted by Marina Chicurel, medium spiny cells are particularly enriched in STEP, a tyrosine phosphatase that regulates the duration of ERK signaling mediated by NMDA stimulation. The duration of ERK signaling critically determines a cell’s response to stimulation: transient activation, mediated by NMDA receptors, results in specific transcriptional changes that differ from those induced by sustained activation, as mediated by KCl-induced membrane depolarization and calcium influx. Thus, the enrichment of STEP in medium spiny cells may be an important determinant of the cells’ responses to both normal and altered NMDA stimulation.

3. New techniques to probe HD pathology

Participants discussed several techniques of importance for future HD research. They agreed that methods to distinguish cell types in the striata of living animals are sorely needed.  In addition,  they noted the importance of systems to study calcium signaling. Of particular interest was Adam Carter's presentation of a two-photon imaging (TPI) system for the study of calcium dynamics in striatal slices. This TPI system makes use of two-photon laser scanning microscopy (TPLSM) and two-photon laser uncaging (TPLU). Carter pointed out that TPSLM is less harmful to cells and can penetrate more deeply than confocal microscopy. Moreover, TPLU has the same advantages as TPLSM, and can be used to excite small subcellular regions using caged compounds. For example, Carter has used this technique to uncage glutamate and mimic miniature EPSCs at single dendritic spines. He can use whole-cell recordings to hold the cells at different potentials and monitor the EPSCs while tracking changes in calcium concentrations at individual spines using calcium-sensitive dyes.

Carter noted there were several ways in which these techniques could be applied to the study of HD. In particular, they could be used to examine NMDAR conductances and the effects of dopamine in HD models. Surmeier added they could be used to correlate morphological changes in spines and dendrites with changes in electrophysiology and calcium dynamics. Levine suggested starting with a transgenic model, such as the R6/2 mouse, because these are well characterized and express more severe pathology than the knock-in models.

            Participants also discussed new models of HD. In addition to the wide variety of animal models currently available—Levine estimated over 25 different mouse models—Johnson described new ones. Christopher Ross’s group, for example, has made an inducible mouse model that expresses full length huntingtin under the control of a prion promoter. In addition, there are various transgenic HD mice expressing full length mutant huntingtin, and researchers in Germany have created transgenic HD rats. Models expressing transgenic huntingtin under either striatal or cortical-specific promoters are also under development.

Of particular interest was Reinhart’s presentation of a biolistics-based model of HD which provides a relatively high throughput system for conducting mechanistic studies, as well as for testing the effects of small molecules or potentially neuroprotective genes. Reinhart uses biolistics to deposit beads carrying huntingtin constructs into neurons in living brain slices. Transfection and inclusion formation can be easily monitored because the huntingtin constructs, as well as the beads, carry fluorescent tags. In addition, co-transfections of multiple genes are easily performed by loading multiple DNA constructs onto single beads--as many as 96 different constructs can be currently co-transfected. Also, expression levels can be titrated because they correlate well with the amount of DNA loaded onto the beads.

As an example, Reinhart described particles loaded with genes coding for yellow fluorescent protein, huntingtin exon 1, and an intra-body developed by Paul Patterson that has a dominant-negative effect on NF-kappa B. Reinhart also noted that he could include DNAs coding for small interfering RNAs in his particles. As a proof-of-principle, he has co-transfected mutated huntingtin exon 1 with siRNA constructs against huntingtin, and has thus prevented the formation of inclusions, cell death and neurite retraction. In a similar manner, he could knock down the expression of other proteins, such as specific NMDAR subunits.

Under baseline conditions, Reinhart can maintain the slices with neurons firing normally for 7-9 days. The efficiency of transfection provides 1000-2000 transfected cells per cortical slice and 300-400 per striatal slice. This ensures that individually transfected cells contain a single bead allowing for expression titration. It also ensures that transfected cells are far enough apart from each other to allow clear visualization of their somas and neurites.

            Reinhart is using the system to monitor cell loss, inclusion formation, electrophysiological alterations, and neurite degeneration. He has shown that cell loss in both striatal and cortical slices is dependent on huntingtin and poly-glutamine length, and that prolines enhance the toxicity of exon 1. Kennedy recommended using a test of caspase-3 activation to detect apoptosis unambiguously.

            In the future, Reinhart expects to increase the applications of his system. He hopes to use it to examine the temporal progression of HD, and investigate whether the damage seen in the striatum is mediated by mutated huntingtin’s direct effects on striatal cells (‘suicide’), or alternatively, by the protein’s effects on cortical cells that interact with the striatum (‘murder’). He is particularly interested in investigating presynaptic mechanisms. In addition, he hopes to increase his involvement in the search for HD therapies. Johnson encouraged participants to use Reinhart’s system to test therapeutic candidates, noting that an important bottleneck in therapy development is target validation.

4. Looking ahead

            Illustrating the close link between understanding the mechanisms underlying HD and designing new therapies, some of the workshop discussions that initially centered around mechanisms yielded ideas for the development of new therapies. In particular, results from Salter’s and Dalva’s labs suggested that peptides might prove therapeutically useful. Dalva has generated a peptide that binds to NMDARs and blocks their interaction with EphRs resulting in an inhibition of EphR-mediated enhancement of calcium influx. Such an inhibition provides an extracellular means of regulating NMDARs and a more subtle way of regulating the receptors as opposed to blocking their channels. Using cultured cerebellar cells, Dalva has found that the peptide is neuroprotective.

            Salter, on the other hand, has generated peptides that affect NMDARs from the inside of the cell, blocking the receptors’ interactions with PSD-95. The peptides are composed of the C-terminus of NR2B subunits, including PSD-95 association domains, various regions of PSD-95, and a peptide derived from the HIV Tat protein that can be used as a carrier to introduce proteins and DNA into the cells of living animals.  The Tat peptide can readily traverse cell membranes, such that it can be used to deliver proteins or DNA to the brain within minutes by simply injecting it intravenously.  Salter explained that he originally envisioned these peptides, which he believes uncouple NMDARs from the cell death machinery, as a potential therapy for stroke. However, as noted by Young, similar peptides may be useful for HD. Salter agreed but cautioned that toxicity might be an issue if the peptides were used as a long-term therapy. He also noted that patients may produce antibodies against the peptides and that the peptides might degrade over time. Even if the delivery is not very efficient, however, a subtle effect at the molecular level may be clinically important, said Young.

5. List of action items

1. NMDAR function / synaptic transmission

*Extend LTP and LTD studies (Murphy): obtain LTD frequency curves (Lovinger suggested), test multiple tetani in PTP experiments (Dalva suggested), monitor postsynaptic responses using chelators (Lovinger suggested), examine LTP at mossy fiber-CA3 synapse (Murphy)

*Examine NMDAR conductances and calcium dynamics in HD mice using TPI (Carter) and correlate with morphological dendritic changes (Surmeier suggested).

*Assess whether increased NMDAR currents in HD are due to elevated numbers of receptors due to abnormal endocytosis (Raymond).

*Determine whether changes in NMDAR magnesium sensitivity are related to subunit composition (Levine)

*Probe NMDAR subunit phosphorylation (Raymond, Murphy, Dalva suggestions)

*Determine relationship between NMDARs, SAP102, and huntingtin (Kennedy)

*Determine whether synaptic or extrasynaptic NMDARs trigger cell death (Raymond, Salter)

*Use in vitro models to extend complexin II studies (Edwardson)

2. Modulation of synaptic transmission

*Extend studies of dopamine in R6/2 cortico-striatal slices (Levine)

*Extend analysis of dopamine effects in LTP and LTD experiments (Murphy)

*Monitor dopamine effects on enkephalinergic medium spiny cells, including pace-making capacity, burst firing, and NMDAR activity (Surmeier)

*Use TPI to investigate the effects of dopamine modulation in HD striatal cells (Carter)

*Test whether blocking mGluR5 and dopamine D2 receptors blocks neurodegeneration

*Assess effects of inhibiting the IP₃-R using a blocking peptide (Bezprozvanny) or siRNA (Reinhart)

*Extend studies on IP₃ signaling to other polyglutamine diseases (Bezprozvanny).

*Test effects of mGluR stimulation on excitotoxicity sensitivity of decorticated animals (Young suggested)

*Assess the relative timing of the decrease in mGluR2 and CB1 receptors compared to the degeneration of corresponding terminals (Lovinger suggested)

3. Selective vulnerability

* Identify proteins that interact with the carboxy tail of NR2B subunits using the yeast two-hybrid system (Raymond).

*Extend studies of the role of NR2B subunits by analyzing mEPSCs (Salter suggested) and examining different striatal regions (Surmeier suggested)

* Extend studies of Kir channel expression in medium spiny cells and compare timing of changes with dendrite degeneration (Surmeier).

*Determine whether STEP enrichment and its role in NMDAR signaling contribute to medium spiny cell vulnerability (Chicurel suggested)

*Investigate early events of pathology and assess whether it is due to “murder or suicide” using the biolistics model system (Reinhart)

4. Function of wildtype huntingtin

*Analyze function of wildtype huntingtin at the PSD (Marcora)

*Evaluate fate of wildtype huntingtin in the presence of mutated huntingtin (Lovinger suggested)

*Determine wildtype huntingtin’s potential role in protein trafficking, particularly NMDAR trafficking (Wenthold)

* Investigate potential role of huntingtin in NMDAR internalization (Salter). Use  conditional huntingtin constructs (Young suggested) and perform immunoprecipitations using anti-NMDAR antibodies (Marcora suggested)

5. Therapeutic directions

*Use biolistics system for target validation (Reinhart, Johnson suggested). Use caspase-3 activation as a marker for apoptosis (Kennedy suggested)

*Investigate the use of NMDAR-modulating peptides, including HIV Tat peptides (Salter) and extracellular-acting peptides (Dalva), as research and therapeutic tools for HD (Young suggested).