Skip Navigation


Human Molecular Genetics Advance Access originally published online on July 1, 2003
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
12/16/2021    most recent
ddg218v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (28)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Li, H.
Right arrow Articles by Li, X.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, H.
Right arrow Articles by Li, X.-J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics, 2003, Vol. 12, No. 16 2021-2030
DOI: 10.1093/hmg/ddg218
© 2003 Oxford University Press

Abnormal association of mutant huntingtin with synaptic vesicles inhibits glutamate release

He Li1,2, Travis Wyman1, Zhao-Xue Yu1, Shi-Hua Li1 and Xiao-Jiang Li1,*

1Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA and 2Department of Anatomy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China

Received April 16, 2003; Revised June 16, 2003; Accepted June 23, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In Huntington disease (HD), polyglutamine expansion causes the disease protein huntingtin to aggregate and accumulate in the nucleus and cytoplasm. The cytoplasmic huntingtin aggregates are found in axonal terminals and electrophysiological studies show that mutant huntingtin affects synaptic neurotransmission. However, the biochemical basis for huntingtin-mediated synaptic dysfunction is unclear. Using electron microscopy on sections of HD mouse brains, we found that axonal terminals containing huntingtin aggregates often had fewer synaptic vesicles than did normal axonal terminals. Subcellular fractionation and electron microscopy revealed that mutant huntingtin is co-localized with huntingtin-associated protein-1 (HAP1) in axonal terminals in the brains of HD transgenic mice. Mutant huntingtin binds more tightly to synaptic vesicles than does normal huntingtin, and it decreases the association of HAP1 with synaptic vesicles in HD mouse brains. Brain slices from HD transgenic mice that had axonal aggregates showed a significant decrease in [3H]glutamate release, suggesting that neurotransmitter release from synaptic vesicles was impaired. Taken together, these findings suggest that mutant huntingtin has an abnormal association with synaptic vesicles and this association impairs synaptic function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Huntington disease (HD) is characterized by a movement disorder, cognitive impairment and psychiatric abnormalities. In HD, selective neurodegeneration occurs initially in the striatum and extends to other brain regions in later stages of the disease (1–3). Although this neurodegeneration constitutes the pathological basis of HD, considerable evidence has also shown that neuronal dysfunction occurs in the absence of neurodegeneration in the early stages of HD. Cognitive symptoms often appear before the onset of the other classical symptoms (4–7). For example, postmortem studies show the absence of overt neuronal cell loss in early stage patients who had cognitive symptoms (1).

Studies of HD mouse models provide compelling evidence that the N-terminal fragments of mutant huntingtin (htt) with an expanded glutamine repeat (82Q or 150Q) cause neurological symptoms resembling some clinical features of HD (8,9). Mice expressing exon1 HD protein (R6/2 and R6/1) with a highly expanded polyglutamine repeat (147–155 glutamines) develop motor deficiencies, cognitive impairment and impaired spatial cognition (10–12). Similarly, mice expressing the first 171 amino acids with an 82-glutamine repeat (N171-82Q) also show neurological symptoms and early death at the age of 4–5 months (9). These neurological symptoms are apparently associated with the nuclear accumulation of mutant htt and its effect on gene expression (8,13). Another subcellular site for the neuropathology mediated by mutant htt may be the neuronal processes in which the formation of htt aggregates (neuropil aggregates) is also associated with neurological symptoms (14). Consistent with this idea, mutant htt inhibits the glutamate uptake in the HD mouse brain (15) or by isolated synaptic vesicles (16). Mutant htt in axonal terminals may affect a variety of synaptic functions, including neurotransmitter release and uptake. Indeed, electrophysiological studies revealed a variety of abnormalities in neurotransmitter transmission in HD mice (12,17–19). Also, biochemical studies show altered neurotransmitter concentrations in HD mouse brains (20–22). However, the biochemical basis for these abnormalities has not been characterized.

Normal htt is a cytoplasmic protein and is also associated with synaptic vesicles (23,24). Thus, mutant htt may interact with synaptic vesicle membrane proteins or proteins associated with vesicles in axonal terminals. One of these interacting proteins may be htt-associated protein-1 (HAP1), which co-localizes with synaptic vesicles in axonal terminals (25–27). It is possible that polyglutamine expansion of htt causes it to bind abnormally other proteins in axonal terminals, contributing to impaired neurotransmitter transmission. However, it has not been examined whether the abnormal protein interactions could impair synaptic function, such as vesicular uptake or release of neurotransmitters.

To examine the biochemical basis for the effect of mutant htt on synapses, we characterized the association of mutant htt with synaptic vesicles and its effect on the glutamate release from these vesicles. Here we report that mutant htt binds abnormally to synaptic vesicles and decreases the glutamate release in HD brain slices. These findings suggest that the abnormal association of mutant htt with synaptic vesicles affects the uptake and release of neurotransmitters, leading to altered synaptic neurotransmission.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Decreased vesicular density in axonal terminals containing htt aggregates
Our previous studies have revealed that htt aggregates are localized in axonal terminals in the brains of R6/2 and HdhCAG80 HD mice (14,16). However, it remains unclear how this localization is related to synaptic dysfunction in HD. We examined several other HD mouse models using electron immunogold labeling to determine whether there is a common ultrastructural change related to axonal htt aggregates. The HD mice examined included R6/1, which express the HD exon1 protein with 150Q (8); N171-82Q, which express the first 171 amino acids containing 82Q (9); and HD repeat knock-in mice, which express 150Q in the endogenous mouse htt (28). Similar to the HD mouse models we examined previously (14,16), all these HD mice showed htt aggregates in axonal terminals. More importantly, the axonal terminals containing htt aggregates often displayed a lower density of synaptic vesicles than normal axonal terminals (Fig. 1). It was difficult to quantify the number of synaptic vesicles under electron microscopy because of the different planes of ultrathin sections. Thus, our examination focused on those axonal terminals that have a similar size and shape in the same plane. For those axonal terminals that were examined for comparison, we often found that the number of vesicles in htt aggregate-containing axons is <60% of that for axons without aggregates. Such decreased density of synaptic vesicles could be found in ~65% of htt aggregate-containing axonal terminals in our electronic examination. Furthermore, the decreased synaptic vesicle density was also seen in serial sections (180–240 nm interval) of the cortex of 12-week-old R6/2 mice (data not shown). Since this phenomenon was present in various HD mouse brains expressing mutant htt of different sizes and polyglutamine repeats, we concluded that mutant htt, once accumulated in axons, may mediate a common pathological change in axonal terminals.



View larger version (125K):
[in this window]
[in a new window]
  		Figure 1. Electron micrographs of HD mouse brains labeled by EM48 immunogold staining. (A) Htt immunogold particles are associated with synaptic vesicles in the brain cortex of an R6/1 mouse at 7 months of age. (B,C) Htt aggregates (single arrow) in axonal terminals of the cortex (B) and striatum (C) of N171-82Q mice at 4 months of age. Note that the density of synaptic vesicles is lower in the htt aggregate-containing axons than in the normal axons (double arrows). (D) In the striatum of HdhCAG150 knock-in mouse at 9 months of age, a low density of synaptic vesicles is also seen in an axon that contains aggregates associated with dark or degenerated organelles. (E) An htt aggregate almost occupies the entire cross section of an axon in a section of HdhCAG150 mouse striatum. Scale bars=5 µm.

 
Other axonal pathology was also noticeable, including degenerated and dark organelles in axonal terminals (Fig. 1D). Strikingly, large aggregates were found to occupy the majority of space in some axons, suggesting that these aggregates could physically block the axonal transport of organelles and molecules (Fig. 1E). Also, htt aggregates were observed in the postsynaptic region, indicating a dendritic localization of some htt aggregates (data not shown). However, htt aggregates at the axon terminal were observed much more frequently than postsynaptic aggregates.

Axonal distribution of htt and HAP1
To provide biochemical evidence for the association of mutant htt with synaptic vesicles, we used an established method to isolate synaptosomes (29). The synaptosomes isolated from HD transgenic mice contained both the endogenous and transgenic mutant htt (Fig. 2). Notably, aggregated htt, which could be seen in the stacking gel, also appeared in the synaptosomal samples. The isolated synaptosomes were then extracted with 1% Triton-X100 at pH 6. This treatment maintains the intact synaptic junctions and removes soluble synaptic proteins (29). For example, syntaxin, a presynaptic membrane protein, is resistant to extraction whereas the soluble synaptic vesicle protein synaptophysin is removed (29). Huntingtin and HAP1, however, still remained in the extracted synaptosomes, suggesting that they might tightly associate with synaptic scaffold. As expected, insoluble htt aggregates also remained in the extracted synaptosomes. The distribution of synaptophysin in isolated synaptosomes from HD and wild type mouse brains did not appear to be different. We also examined the distribution of several synaptic proteins including synapsin-II and synaptotagmin I but did not find any significant difference in their synaptosomal distribution between wild type and HD mouse brains (data not shown).



View larger version (50K):
[in this window]
[in a new window]
  		Figure 2. Reduced association of HAP1 with synaptic vesicles in HD mouse brains. (A) Western blotting of homogenates (H) and synaptosomes (V) isolated from wild type (WT) and N171-82Q (HD) mouse cortex shows that aggregated htt (Aggr) and soluble transgenic htt (mHtt) were present in the synaptosomes and in the extracted synaptosomes (Extrac) treated with 1% Triton X-100 at pH 6 that partially disrupts isolated synaptosomes and removes soluble pre-synaptic proteins. The endogenous mouse htt (Htt-F) was also detected. The amount of HAP1 was lower in the synaptosomal fraction than that of wild type mouse cortex. The blots were also probed with antibodies against the presynaptic membrane protein syntaxin (Synt), the synaptic vesicle protein synaptophysin (synaph) and tubulin (Tub). (B) Densitometric analysis of the relative amount of HAP1 in synaptosomes from cortical tissues of wild type (WT) or N171-82Q (HD) mice at the age of 4 months. The ratio (mean±SEM, n=3) of HAP1 to syntaxin in each lane was measured from synaptosomes without (-Triton) or with (+Triton) Triton-X100 extraction. *P<0.05 compared to wild type (WT) synaptosomes.

 
The localization of mutant htt in a subset of axonal terminals might not allow us to detect a change of synaptic vesicle proteins that are enriched in almost all synaptosomes. Thus we focused on HAP1, which interacts with mutant htt and is also distributed in the axonal terminals. Compared with wild type mouse brains, HD mouse brains showed a reduced amount of HAP1 in the synaptosomal fraction (Fig. 2A). Endogenous htt appeared to be slightly decreased in HD synaptosomes, which could also contribute to the decreased level of HAP1. To quantify the difference of HAP1 expression, we compared the ratios of HAP1 to syntaxin in the synaptosomal samples from wild type and HD mouse brains. The result also confirmed a decrease in the distribution of HAP1 in HD synaptosomes (Fig. 2B).

The western blots in Figure 2 suggested that aggregation of htt might reduce the association of its interacting proteins with synaptic vesicles. To test this hypothesis, we used electron immunogold labeling to examine HD mouse brains. HAP1 immunogold labeling confirmed that HAP1 was associated with synaptic vesicles in the presynaptic terminals (Fig. 3A). To investigate whether both HAP1 and mutant htt proteins were co-localized in the same axonal terminals, we used a guinea-pig htt antibody and a rabbit HAP1 antibody for double labeling. Huntingtin was then detected by immunogold labeling and HAP1 was detected by DAB staining. Only some axonal terminals contained htt aggregates, perhaps because mutant htt formed aggregates in a fraction of axonal terminals. The negative staining of htt aggregates in some axonal terminals could also be due to the localization of htt aggregates in different ultrathin sections of these axonal terminals. In those axonal terminals labeled by HAP1-DAB and htt immunogold, clustered immunogold particles overlapped with HAP1 immunoreactive products (Fig. 3B and C). This finding suggests that mutant htt aggregates are localized with HAP1 in the same terminals. Importantly, htt aggregate-containing axonal terminals showed less HAP1 immunostaining and fewer synaptic vesicles than those that did not have htt aggregates. This result is consistent with biochemical isolation data (Fig. 2), which also showed a reduced HAP1 distribution in the synaptosomes isolated from HD transgenic mouse brains.



View larger version (202K):
[in this window]
[in a new window]
  		Figure 3. Electron micrographs showing co-localization of mutant htt and HAP1 in axonal terminals. (A) HAP1 immunogold labeling revealed the association of HAP1 with synaptic vesicles. (B) Double labeling of an 11-week-old R6/2 mouse brain using immunogold for htt and DAB for HAP1. Note that htt aggregates (arrow) are localized in a DAB-stained axonal terminal. The intensity of HAP1-DAB staining in this axon is weaker than in other axonal terminals (arrowheads). (C) High magnification image showing decreased HAP1-DAB staining and fewer synaptic vesicles in an axon containing htt aggregates (arrow). A normal axon terminal labeled by HAP1-DAB is also indicated (arrowhead). Scale bars=5 µm.

 
Interaction of N-terminal htt with HAP1 and synaptic vesicles
Since HAP1 is not recruited into htt aggregates (26), the soluble form of htt may bind to HAP1 but aggregated htt may prevent the interaction of HAP1 with synaptic vesicles due to the increased association of aggregated htt with synaptic vesicles. Two sets of experiments were performed to test this hypothesis. In the first experiment, we examined the interaction of HAP1 with the soluble form of mutant htt. GST-htt exon1 fusion proteins were soluble and could be purified with agarose beads containing glutathione. When these beads were incubated with rat brain extracts, we observed that GST-htt with an expanded glutamine repeat (GST-67Q) precipitated more HAP1 than that with a normal glutamine repeat (GST-20Q) (Fig. 4A and B). We have established stably transfected PC12 cells that express the HD exon1 protein with either 150 (150Q) or 20 (20Q) glutamines (30). These cells allowed us to use anti-HAP1 to precipitate endogenous HAP1 and then examine co-immunoprecipitation of htt with EM48. Mutant htt was co-precipitated with HAP1 (Fig. 4C). Overexposure of the blots could reveal weak signals of 20Q protein associated with HAP1 (data not shown). However, mutant htt apparently binds more tightly to HAP1 than does 20Q protein.



View larger version (57K):
[in this window]
[in a new window]
  		Figure 4. Interaction of HAP1 with N-terminal htt. (A) Coomassie staining of purified GST-htt fusion proteins containing the exon1 htt with 20 (GST-20Q) or 67 (GST-67Q) glutamines. GST protein was included as a control. (B) GST fusion proteins were incubated with homogenized synaptic vesicles isolated from rat brain cortex. Note that HAP1 proteins were bound to GST-67Q, but not GST or GST-20Q. (C) Co-immunoprecipitation of HAP1 and the exon1 htt containing 20 (20Q) or 150 (150Q) glutamines from stably htt transfected PC12 cells. Extracts of stably transfected PC12 cells were immunoprecipitated with anti-HAP1, and the blot was probed with anti-htt (EM48).

 
In the second experiment, we examined whether aggregated htt bound to synaptic vesicles using an established assay as described previously (31,32). Since addition of a small epitope onto the N-terminus of htt does not affect the aggregation of mutant htt, we produced His-tagged fusion proteins containing the first 171 amino acids with 23 (His-23Q) or 120 (His-120Q) glutamines. Western blots showed that His-120Q displayed different forms; one was the monomer that represented a soluble htt and another was the aggregated form that remained in the stacking gel. Dimerized fusion proteins were seen on the blots sometimes (Fig. 5A, arrowheads), suggesting that the dimerized proteins were unstable. When these fusion proteins were incubated with the isolated synaptic vesicles, His-23Q was not detected in the vesicle precipitates, whereas both soluble and aggregated His-120Q proteins were bound to the vesicles. However, more aggregated htt bound to the vesicles than did the soluble form, as revealed by the ratios of the amount of the bound protein to synaptophysin. After stripping the vesicles with 0.1 M sodium carbonate (pH 11) to remove proteins associated with synaptic vesicles, the binding of htt to the synaptic vesicles was greatly reduced (Fig. 5A). Thus, the association of htt with synaptic vesicles appeared to depend on the presence of other vesicle associated proteins.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 5. The binding of mutant htt to synaptic vesicles. (A) His-tagged htt (input, the first 171 amino acids containing 23 or 120 glutamines) was incubated with isolated rat synaptic vesicles (SV). Input is His-tagged htt without incubation with SV. The vesicles were precipitated by centrifugation and analysed by western blotting with EM48 (see detail in the Materials and Methods section). Note that more aggregated htt (in the stacking gel indicated with bracket) than soluble mutant htt bound to the precipitated vesicles. Arrowheads indicate htt that might be dimerized 23Q (60K, lane 1 in A) and 120Q (110K, lane 1 in B) htt. Stripping the vesicles with 0.1 M sodium carbonate (pH 11) greatly reduced this association. The blot was also probed with an antibody against synaptophysin (Synaph), an integral membrane protein of synaptic vesicles, to verify the presence of synaptic vesicles. (B) Adding anti-HAP1 antibody to the incubation containing synaptic vesicle fraction (SV) and His-htt (input) attenuated the association of soluble htt with vesicles. Anti-htt antibody almost completely abolished the binding of htt to vesicles. Anti-synaptophysin (Synaph) was also used to probe the blots. (C) Densitometric analysis of the relative binding (ratio of htt to synaptophysin, mean±SEM, n=3) of aggregated and monomer htt to the vesicles. Control is the binding in the absence of rabbit IgG or rabbit antibodies to HAP1 or htt. (D) Filter trap assay of SDS resistant insoluble pellets from brain cortical tissues of wild type (WT) and R6/2 (HD) mice at the age of 12 weeks. The filter members were probed with EM48 (anti-htt) and anti-HAP1 antibodies.

 
To examine whether HAP1 is involved in the association of the soluble mutant htt with synaptic vesicles, we added anti-HAP1 antibody in the incubation to block the interaction of HAP1 with htt. Less soluble htt than aggregated htt bound to the vesicles after anti-HAP1 treatment, suggesting that more soluble htt than aggregated htt interacts with HAP1 to associate with synaptic vesicles. Thus, the antibody preferentially affected the association of soluble mutant htt with synaptic vesicles. Anti-htt antibody almost completely eliminated the binding of both soluble and aggregated htt to the vesicles (Fig. 5B).

To confirm that aggregated htt does not bind to HAP1, we used a filter trap assay to examine aggregated htt in the cortex of R6/2 mice at the age of 12 weeks. The cortical tissue of wild type mice was used as a control. Filter trap assay clearly revealed aggregated htt on the membrane, which was negative to anti-HAP1 staining. This result also suggests that aggregated htt was unable to bind HAP1.

The accumulation of mutant htt in the neuropil and decreased [3H]-glutamine release in HD brain slices
Our previous studies have shown that neuropil aggregates are htt aggregates that are frequently localized in axonal terminals (14,16). Thus, the density of neuropil aggregates reflects the accumulation of mutant htt in axonal terminals. If axonal terminal htt affects neuronal function, we should observe a relationship between neuropil aggregate density and this neuronal dysfunction. To assess neuronal function, we used a well-established in vitro assay (33–36) to measure [3H]glutamate release from cortical-striatal slices of HD mouse brains. Brain slices maintain their axonal structure well enough to allow examination of the effect of mutant htt on neurotransmitter release. Also, because the striatum receives cortical glutamatergic inputs and because htt forms aggregates in the glutamatergic axonal terminals (14) and affects synaptic function of the corticostriatal pathway (17–19) in HD mice, we were able to measure the effect of mutant htt on the release of glutamate from synaptic vesicles. This assay involves incubating brain slices with [3H]glutamate and then measuring its release from synaptic vesicles after depolarization with potassium (33–36). We examined R6/1 and N171-82Q mouse brains, as these mice survive for more than 4 months, allowing us to characterize the relationship between the axonal accumulation of mutant htt and glutamate release from synaptic vesicles in adult animals.

EM48 immunostaining revealed htt aggregates in the nuclei and neuropil in both R6/1 and N171-82Q mice. More neuropil aggregates were seen in brains of R6/1 than N171-82Q mice (Fig. 6A). We then measured the glutamate release from brain slices of these HD mice. In the brain slices from the age-matched littermates, we observed amounts of KCL-induced [3H]glutamate efflux (158–183%) that were very similar to those reported previously (34,35). However, HD brain slices showed significantly less [3H]glutamate release (102–140%) (Fig. 6B). Less [3H]glutamate release was seen in R6/1 slices than in N171-82Q slices, consistent with the greater neuropil aggregates in R6/1 brains. We observed that N171-82Q mice showed a similar density of nuclear inclusions, but different densities in the neuropil aggregates at the ages of 12 and 20 weeks (Fig. 6A). The number of neuropil aggregates in the cortex in these mice increased from 83.2±14/per image at 12 weeks of age to 141.7±43 (mean±SD, n=4, P<0.05) at 20 weeks of age. This allowed us to further examine whether the increased neuropil aggregates are associated with the decreased glutamate release in the slices from N171-82Q mice. No significant decrease in [3H]glutamate was observed in the brain slices from the 12-week-old mice in which neuropil aggregates were not prominent (Fig. 6B). However, in the 20-week-old mice, significantly less [3H]glutamate was released, concurrent with the increase in neuropil aggregates. These findings suggest that the accumulation of htt aggregates in the neuropil or axonal terminals is associated with the decrease in glutamate release.



View larger version (48K):
[in this window]
[in a new window]
  		Figure 6. Increased formation of neuropil aggregates and decreased [3H]glutamate release from brain slices in HD mice. (A) EM48 immunostaining of the cortex from wild type (WT, 7-months-old), R6/1 (7-months-old) and N171-82Q mice (12 or 20-weeks-old). Note that neuropil aggregates (arrows) are prominent in R6/1 mice. More neuropil aggregates were seen in N171-82Q mice at 20 weeks than at 12 weeks. Scale bar=5 µm. (B) [3H]glutamate release from corticostriatal slices from WT, R6/1 and N171-82Q mice at the same ages as in (A). Slices were stimulated with 40 mM KCl for 5 min, and the efflux of [3H]glutamate was measured at 5, 10 and 15 min. The basal levels of [3H]glutamate were the radioactivity in the collected efflux after [3H]glutamate uptake by the slices and before KCl stimulation. The data are presented as mean±SE and obtained from 4–6 mice for each group. *P<0.05; **P<0.01 compared with age-matched littermates (WT).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Several new findings in the present study suggest that mutant htt abnormally binds to synaptic vesicles to affect synaptic function. Firstly, the axons containing htt aggregates have fewer synaptic vesicles than do normal axons. Secondly, aggregated htt binds tightly to synaptic vesicles and reduces the association of HAP1 with synaptic vesicles. Thirdly, brain slices from HD mice containing axonal aggregates have reduced glutamate release from synaptic vesicles.

While previous studies by us and others have identified htt aggregates in axonal terminals (14,16,37), the present study, by comparing various HD mouse models, revealed a common axonal pathology in HD brains that displayed neuropil aggregates. This axonal pathology includes a low density of synaptic vesicles in htt aggregate-containing axonal terminals and reduced glutamate release from HD brain slices. The reduced density of synaptic vesicles may contribute to the decrease in some presynaptic proteins, such as complexin II, which can be observed in R6/2 mice at the late stage of HD (38). The decreased release of [3H]glutamate from brain slices reflects early defective synaptic transmission caused by axonal mutant htt. Consistent with this idea, mutant htt affects a variety of neurotransmitter transmission in various HD mouse models that have been found to contain axonal aggregates (12,17–19,39). Also, altered concentrations of neurotransmitters were documented in the brains of HD transgenic mice (20–22,40). For example, microdialysis of extracellular glutamate level showed that basal striatal glutamate levels were reduced by 43% in R6/1 mice at the age of 16 weeks (20). However, microdialysis investigations of glutamate release generate inconsistent results. Since the concentration of glutamate in the brain is also largely dependent on its uptake by astrocytes, it is unclear whether impairment of synaptic vesicles or of glial cells accounts for the altered neurotransmitter concentrations. By measuring the glutamate that is directly released from synaptic vesicles in brain slices, we found a decreased release of glutamate from the vesicles in HD mouse brains. Evidence supporting this defect also includes the correlation of the increased neuropil aggregates in HD mouse brains with the decreased release of glutamate. Furthermore, the axonal localization of htt aggregates and their association with the decreased numbers of synaptic vesicles also suggest that axonal htt aggregates may affect glutamate release. The decreased release of glutamate from synaptic vesicles is consistent with findings that HD exon1 transgenic mice have reduced glutamatergic synaptic activity (17) and are resistant to the glutamatergic excitotoxicity of NMDA and quinolinic acid (20,41).

Electron microscopy revealed a significant amount of aggregated protein clustered in axonal terminals. The EM48 immunogold labeling might only be able to detect aggregated htt in the fixed brain sections. Using biochemical assays, we observed the presence of both soluble and aggregated htt in axonal terminals. Moreover, aggregated htt bound more tightly to synaptic vesicles than did the soluble form of mutant htt, as evidenced by the increased amount of aggregated htt associated with synaptic vesicles in the subcellular fractionation and in vitro binding assays.

Given the limited space within axons and the fairly large size of polyglutamine aggregates, these aggregates could readily create a physical barrier that interferes with organellar transport and vesicle recycling in axons (42,43). This idea is supported by the result in Figure 1C, in which a large htt aggregate occupies almost the entire axon. In addition, through their association with synaptic vesicles, axonal terminal aggregates could affect the synaptic vesicle recycling to inhibit the regeneration of synaptic vesicles. Such an effect could alter the release and uptake of various neurotransmitters and could lead to axonal degeneration. Overt axonal degeneration is not observed in HD transgenic mice that have a rapid disease progression and early death; however, obvious axonal degeneration was found to associate with axonal htt aggregates in old HD repeat knock-in mice (>17 months) (42). Thus, axonal degeneration appears to be age-dependent. Axonal degeneration is an early pathologic feature in a number of age-dependent neurodegenerative diseases (44) and may be an early pathological change in HD patients, which contributes to impaired memory, reduced learning ability, uncontrolled body movement and other early symptoms (4–7).

Based on our observations, we proposed a diagram showing the interactions of different htt forms with synaptic vesicles and their associated proteins (Fig. 7). Both htt and HAP1 may function as scaffold proteins and dynamically associate with synaptic vesicles in axonal terminals. In HD, the association of soluble mutant htt with synaptic vesicles may depend on its interacting proteins including HAP1 and HIP-1. HIP1 is a protein that is important for clathrin-mediated endocytosis (45–47). HAP1 is expressed predominantly in neurons, and deletion of its expression causes a feeding defect and postnatal death in mice (48,49). The reduced association of HAP1 with synaptic vesicles in HD mice may be due to the enhanced interaction of soluble mutant htt with HAP1, which in turn prevents HAP1 from interacting with other synaptic vesicle proteins. Aggregated htt, on the other hand, may bind synaptic vesicle membrane tightly and affects the release and uptake of glutamate and perhaps other neurotransmitters as well. The increased binding of mutant htt to the vesicle membrane could also reduce the association of HAP1 and/or other proteins with synaptic vesicles. Whether and how htt binds to other synaptic vesicle proteins to affect synaptic function remains to be further investigated.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 7. Model depicting interactions of htt with synaptic vesicles. Glutamate is released from and uptaken by (black block arrows) synaptic vesicles (SV). Htt and HAP1 may dynamically associate with synaptic vesicles in axonal terminals. In HD, soluble mutant htt binds tightly to HAP1 and reduces HAP1's association with synaptic vesicles. Aggregated mutant htt also binds synaptic vesicle membranes and inhibits their uptake and release of glutamate (white block arrows). Such effects could also affect synaptic vesicular release or uptake of other neurotransmitters.

 
Identification of the association of axonal htt aggregates with defective glutamate release from synaptic vesicles implies that cytoplasmic mutant htt could mediate cytotoxicity at a specific subcellular site. Like the nucleus, neuronal processes may have lower activity of chaperones and/or proteasomes and are thus favorable for the aggregation of mutant htt. If the limited space in axons and their terminals are unable to accommodate large htt aggregates, neuronal dysfunction could occur in the absence of cell body degeneration. The morphological and biochemical evidence in the present study provides a basis for the early pathological changes in HD mouse brains and would also be important for the development of effective therapeutic interventions for HD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
HD mice
R6/1 and R6/2 mice [B6CBA-TgN (HDexon1) 61 and 62], which express exon1 of the human mutant HD gene containing 115–150 CAGs (50) and N171-82Q mice [B6C3F1/TgN(HD82Gln)81Dbo], which express the first 171 amino acids with 82 glutamines (9), were obtained from the Jackson Laboratory. Breeding pairs of HD repeat knock-in mice (HdhCAG150) expressing full-length mouse htt with an expanded polyglutamine repeat (150Q) were provided by Dr Peter Detloff (28). HD transgenic mice were bred and maintained in the animal facility at Emory University. Genotyping of transgenic mice was performed using methods described previously (9,28,50).

Antibodies
EM48 and EM73 are rabbit and guinea-pig polyclonal antibodies, respectively, which were generated using GST fusion proteins containing amino acids 1–256 of human htt, as previously described (16). This fusion protein was also used to generate mouse monoclonal antibody mEM48 as described previously (51). Rabbit anti-HAP1 was generated from our previous studies (25). Other antibodies used included mouse monoclonal antibody 2166 against htt (Chemicon), mouse monoclonal antibodies against syntaxin (Sigma), gamma tubulin (Sigma), synaptophysin (Transduction Laboratories), rabbit antibodies against synaptotagmin and Rab3A (StressGen) and rabbit anti-synapsin I (Sigma).

Immunocytochemistry
For electron microscopy, mice were anesthetized by intraperi-toneal injection of chloral hydrate (400 mg/kg body weight), and then fixed by intracardial perfusion with 0.1 M sodium phosphate buffer (PB, pH 7.3) containing 4% paraformaldehyde and 0.2% glutaraldehyde. After perfusion, the brains were removed, postfixed with 4% paraformaldehyde in PB for 6–8 h, and then sectioned using a vibratome.

For immunogold single labeling, brain sections were incubated with EM48 in PBS containing 4% normal goat serum (NGS) for 24–48 h at 4°C. After washes with PBS, sections were incubated overnight at 4°C in PBS with 4% NGS and Fab fragments of goat anti-rabbit secondary antibody conjugated to 1.4 nm gold particles (1 : 200; Nanoprobes, Stony Brook, NY). After rinsing in PBS, sections were fixed again in 2% glutaraldehyde in PB for 1 h, and silver intensified using the IntenSEM kit (Amersham International, Buckinghamshire, UK) for 5–10 min on ice.

Double labeling of htt and HAP1 in mouse brain sections was performed as described previously (52). Briefly, tissues were incubated sequentially, first with the guinea pig antibody EM73 (1 : 500) and then with the rabbit anti-HAP1A antibody (1 : 4000). Each incubation was performed in PBS containing 4% NGS at 4°C for 24–48 h. After washes, the sections were incubated at 4°C overnight with goat anti-guinea-pig IgG Fab fragment conjugated with 1.4 nm gold particles (1 : 100) for htt labeling and biotinylated goat anti-rabbit IgG (1 : 200; Vector, Burlingame, CA) for HAP1 labeling, followed by the incubation with avidin-biotin complex (1 : 100; Vector ABC Elite, Burlingame, CA) at room temperature for 2 h. After the brain sections were incubated with reagents from the IntenSEM kit for 5–10 min on ice, the sections were treated with 0.05% 3,3'-diaminobenzidine (DAB, Sigma, St Louis, MO) and 0.01% hydrogen peroxide in 0.05 M Tris buffer (pH 7.6) at room temperature for 10 min.

All sections were osmicated in 1% OsO4 in PB, dehydrated in ascending concentrations of ethanol and propylene oxide/Eponate 12 (1 : 1) and then embedded in Eponate12 (Ted Pella, Redding, CA). Under a dissection microscope, the cortex and striatum were removed, and then cut into ultrathin sections (60 nm) on a Leica Ultracut S ultramicrotome. The ultrathin sections were counterstained with 5% aqueous uranyl acetate for 5 min followed by Reynolds lead citrate for 5 min and examined using an Hitachi H-7500 electron microscope.

Light microscopy was used to quantify neuropil aggregates as described previously (14). The numbers of neuropil aggregates per image (650x) were obtained from four mice for each group and analyzed with a Student t-test. Two mice of each group were used for immunogold labeling so 8 HD mouse brains were analyzed by electron microscopy. Ages of the HD mice examined are indicated in the figure legends.

Synaptosome preparation
Synaptosomes were prepared using a previously described method (29). Cortices from control littermates and N171-82Q mice at the age of 4–5 months were homogenized in 15 ml of homogenization solution (0.32 M sucrose, 0.1 mM CaCl2, 1 mM MgCl2, 0.1 mM PMSF) at 4°C using a Teflon-glass homogenizer. The homogenate was brought to a final sucrose concentration of 1.25 M and then overlaid with 10 ml of 1.0 M sucrose, 0.1 mM CaCl2 and 5 ml of homogenization solution. After centrifugation at 100 000g for 3 h at 4°C, a band representing synaptosomal membranes was collected at the 1.25 M/1.0 M sucrose interface.

Synaptosomes were also extracted with 1% Triton-X100 at pH 6.0 as described previously (29). This treatment removed soluble or non-synaptic proteins and maintained the intact synaptic junction. After washes with the extraction buffer, the pellet was resuspended and the protein concentrations were determined using a protein assay kit (Pierce, Rockford, IL). Equal amounts of protein (100 µg) were resolved by SDS electrophoresis and western blotting with antibodies 2166 (1 : 2000), anti-syntaxin (1 : 5000), EM48 (1 : 500), anti-HAP1 (1 : 1000), anti-synaptophysin (1 : 200) and anti-tubulin (1 : 3000).

In vitro interaction and co-immunoprecipitation
To examine the interaction of htt and HAP1, GST fusion proteins containing exon1 HD protein with 20 (20Q) or 67 (67Q) glutamines were produced and purified as described previously (16). GST-htt linked with glutathione-agarose beads was incubated with rat brain cortical lysates in PBS with 1% Triton X-100 and protease inhibitor mix (Sigma). The reaction was kept on a rocking platform at 4°C for 3 h. After the beads were precipitated at a low speed (900g) and washed three times with PBS, the beads were resuspended in SDS sample buffer for western blot analysis.

To precipitate HAP1 protein complexes from HD cells, we used stably transfected PC12 cell lines that expressed the htt exon1 protein containing either 20Q or 150Q (30). Cell extracts (0.1 g/ml) in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM PMSF and protease inhibitor cocktail (1x; Sigma P8340)] were clarified by centrifugation at 10 000g for 15 min. The supernatant (500 µl at 1 mg protein/ml) was subjected to immunoprecipitation with 10 µl of anti-HAP1 serum for 2 h at 4°C. Protein A Sepharose (15 µl of 50% slurry) was added to the mixture and incubated for an additional 1 h. The protein A beads were precipitated by centrifugation (2000g) for 1 min, washed three times with lysis buffer, resolved by 4–12% SDS–PAGE and analyzed with mEM48 (1 : 100 dilution) and anti-HAP1 (1 : 1000 dilution).

In vitro binding of htt to isolated synaptic vesicles was performed using the methods as described (31,32). Since His-tagged htt is more prone to aggregation than GST-htt, we used His-tagged htt to examine the association of aggregated htt with synaptic vesicles. Generation of His-htt containing the first 171 amino acids of htt with 23Q or 120Q was described in our previous study (51). A fraction enriched in synaptic vesicles (LP2 fraction) was prepared by first isolating synaptosomes from rat brain, followed by hypotonic shock and differential centrifugation (53). Purified His-htt was incubated with the isolated synaptic vesicles at room temperature for 1 h. The reaction mixture (200 µl) contained 50 µg synaptic vesicle proteins and 50 ng purified His-htt in PBS with protease inhibitor mix (Sigma). Bound htt was separated from unbound htt by ultracentrifugation at 100 000g at 4°C for 30 min. After washes with the binding buffer, the synaptosomal precipitates were resolved by western blots with EM48. To strip the vesicles, the vesicles were incubated with 0.1 M sodium carbonate (pH 11) for 5 min and centrifuged to remove associated proteins. The precipitated pellets were washed with PBS before binding to His-htt.

Western blots and filter trap assay
Homogenates of brain tissue or cultured cells were resuspended in PBS with protease inhibitor cocktail (1X P8340, Sigma), PMSF (100 µg/ml) and 1% Triton X-100. Western blotting was performed using 4–12% or 4–20% polyacrylamide Tris–Glycine gels (Invitrogen) and ECL kits (Amersham). Filter trap assay was performed as described in our previous study (54). Lysates of cortex from 12-week-old R6/2 mice were precipitated by centrifugation at 13 000g for 15 min. The precipitated pellet was treated with 2% SDS at RT for 15 min, sonicated for 10 s and filtered through a cellulose acetate membrane (Schleicher and Schuell, 0.2 µm pore size). A dot blot filtration unit was used for filter trap assays. The SDS insoluble aggregates that were retained on the filters were detected by incubation with EM48 and anti-HAP1, followed by ECL detection.

Glutamate release assay
In vitro glutamate release from brain slices was measured using established assays (33–36). Cortical-striatal slices (4 slices of 300 µm thick) prepared from each mouse were placed in a 20 ml chromatography column and constantly perfused with 37°C oxygenated (95% O2 and 5% CO2) Krebs–Ringer buffer (in mM: 11.5 glucose, 25 NaHCO3, 1.2 MgCl2, 1.2 NaH2PO4, 118 NaCl, 4.8 KCl, 2.5 CaCl2 and 0.004 Na2EDTA, pH 7.4) at room temperature for 20 min. Slices were incubated with [3H]glutamine (48.2 Ci/mmol; Perkin-Elmer Life Sciences, Boston, MA) for 30 min at a final concentration of 0.2 µM. After extensive rinsing, slices were treated with 40 mM KCl for 5 min to evoke the release of [3H]glutamate. Fractions were collected every 5 min after adding 40 mM KCl. The net efflux of tritium in the fraction right before (basal value) and after KCl depolarization was measured in triplicate using a scintillation counter (Beckman LS6500). The results were expressed as the percentage increase in [3H]glutamine release above the basal level using the following equation: [(K+-evoked-basal release)/basal release]x100%. Data for each group were obtained from 4 to 6 mice.

Statistical analysis
Data are expressed as mean±SEM. Statistical analysis of data used variance followed by the Student t-test. Differences were considered significant if P<0.05.


   ACKNOWLEDGEMENTS
 
We thank Joy Evans and Aja Pillarisetti for their assistance in the genotyping of mice. This work was supported by NIH grants NS36232 and AG19206 (X.J.L.) and National Natural Science Foundation of China (30070248 and 30225024 for H.L.).


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Human Genetics, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA. Tel: +1 4047273290; Fax: +1 4047273949; Email: xiaoli{at}genetics.emory.edu Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Vonsattel, J.P., Myers, R.H., Stevens, T.J., Ferrante, R.J., Bird, E.D. and Richardson, E.P. Jr (1985) Neuropathological classification of Huntington's disease. J. Neuropathol. Exp. Neurol., 44, 559–577.[ISI][Medline]

  2. Cudkowicz, M. and Kowall, N.W. (1990) Degeneration of pyramidal projection neurons in Huntington's disease cortex. Ann. Neurol., 27, 200–204.[CrossRef][ISI][Medline]

  3. Kremer, H.P., Roos, R.A., Dingjan, G., Marani, E. and Bots, G.T. (1990) Atrophy of the hypothalamic lateral tuberal nucleus in Huntington's disease. J. Neuropathol. Exp. Neurol., 49, 371–382.[ISI][Medline]

  4. Mohr, E., Brouwers, P., Claus, J.J., Mann, U.M., Fedio, P. and Chase, T.N. (1991) Visuospatial cognition in Huntington's disease. Mov. Disord., 6, 127–132.[CrossRef][ISI][Medline]

  5. Foroud, T., Siemers, E., Kleindorfer, D., Bill, D.J., Hodes, M.E., Norton, J.A., Conneally, P.M. and Christian, J.C. (1995) Cognitive scores in carriers of Huntington's disease gene compared to noncarriers. Ann. Neurol., 37, 657–664.[CrossRef][ISI][Medline]

  6. Lange, K.W., Sahakian, B.J., Quinn, N.P., Marsden, C.D. and Robbins, T.W. (1995) Comparison of executive and visuospatial memory function in Huntington's disease and dementia of Alzheimer type matched for degree of dementia. J. Neurol. Neurosurg. Psychiatry, 58, 598–606.[Abstract]

  7. Lawrence, A.D., Sahakian, B.J., Hodges, J.R., Rosser, A.E., Lange, K.W. and Robbins, T.W. (1996) Executive and mnemonic functions in early Huntington's disease. Brain, 119, 1633–1645.[Abstract/Free Full Text]

  8. Davies, S.W., Turmaine, M., Cozens, B.A., DiFiglia, M., Sharp, A.H., Ross, C.A., Scherzinger, E., Wanker, E.E., Mangiarini, L. and Bates, G.P. (1997) Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell, 90, 537–548.[CrossRef][ISI][Medline]

  9. Schilling, G., Becher, M.W., Sharp, A.H., Jinnah, H.A., Duan, K., Kotzuk, J.A., Slunt, H.H., Ratovitski, T., Cooper, J.K., Jenkins, N.A. et al. (1999) Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum. Mol. Genet., 8, 397–407.[Abstract/Free Full Text]

  10. Carter, R.J., Lione, L.A., Humby, T., Mangiarini, L., Mahal, A., Bates, G.P., Dunnett, S.B. and Morton, A.J. (1999) Characterization of progressive motor deficits in mice transgenic for the human Huntington's disease mutation. J. Neurosci., 19, 3248–3257.[Abstract/Free Full Text]

  11. Lione, L.A., Carter, R.J., Hunt, M.J., Bates, G.P., Morton, A.J. and Dunnett, S.B. (1999) Selective discrimination learning impairments in mice expressing the human Huntington's disease mutation. J. Neurosci., 19, 10428–10437.[Abstract/Free Full Text]

  12. Murphy, K.P., Carter, R.J., Lione, L.A., Mangiarini, L., Mahal, A., Bates, G.P., Dunnett, S.B. and Morton, A.J. (2000) Abnormal synaptic plasticity and impaired spatial cognition in mice transgenic for exon 1 of the human Huntington's disease mutation. J. Neurosci., 20, 5115–5123.[Abstract/Free Full Text]

  13. Luthi-Carter, R., Strand, A., Peters, N.L., Solano, S.M., Hollingsworth, Z.R., Menon, A.S., Frey, A.S., Spektor, B.S., Penney, E.B., Schilling, G. et al. (2000) Decreased expression of striatal signaling genes in a mouse model of Huntington's disease. Hum. Mol. Genet., 9, 1259–1271.[Abstract/Free Full Text]

  14. Li, H., Li, S.H., Cheng, A.L., Mangiarini, L., Bates, G.P. and Li, X.J. (1999) Ultrastructural localization and progressive formation of neuropil aggregates in Huntington's disease transgenic mice. Hum. Mol. Genet., 8, 1227–1236.[Abstract/Free Full Text]

  15. Lievens, J.C., Woodman, B., Mahal, A., Spasic-Boscovic, O., Samuel, D., Kerkerian-Le Goff, L. and Bates, G.P. (2001) Impaired glutamate uptake in the R6 Huntington's disease transgenic mice. Neurobiol. Dis., 8, 807–821.[CrossRef][ISI][Medline]

  16. Li, H., Li, S.H., Johnston, H., Shelbourne, P.F. and Li, X.J. (2000) Amino-terminal fragments of mutant huntingtin show selective accumulation in striatal neurons and synaptic toxicity. Nat. Genet., 25, 385–389.[CrossRef][ISI][Medline]

  17. Cepeda, C., Hurst, R.S., Calvert, C.R., Hernandez-Echeagaray, E., Nguyen, O.K., Jocoy, E., Christian, L.J., Ariano, M.A. and Levine, M.S. (2003) Transient and progressive electrophysiological alterations in the corticostriatal pathway in a mouse model of Huntington's disease. J. Neurosci., 23, 961–969.[Abstract/Free Full Text]

  18. Klapstein, G.J., Fisher, R.S., Zanjani, H., Cepeda, C., Jokel, E.S., Chesselet, M.F. and Levine, M.S. (2001) Electrophysiological and morphological changes in striatal spiny neurons in R6/2 Huntington's disease transgenic mice. J. Neurophysiol., 86, 2667–2677.[Abstract/Free Full Text]

  19. Laforet, G.A., Sapp, E., Chase, K., McIntyre, C., Boyce, F.M., Campbell, M., Cadigan, B.A., Warzecki, L., Tagle, D.A., Reddy, P.H. et al. (2001) Changes in cortical and striatal neurons predict behavioral and electrophysiological abnormalities in a transgenic murine model of Huntington's disease. J. Neurosci., 21, 9112–9123.[Abstract/Free Full Text]

  20. Nicniocaill, B., Haraldsson, B., Hansson, O., O'Connor, W.T. and Brundin, P. (2001) Altered striatal amino acid neurotransmitter release monitored using microdialysis in R6/1 Huntington transgenic mice. Eur. J. Neurosci., 13, 206–210.[CrossRef][ISI][Medline]

  21. Reynolds, G.P., Dalton, C.F., Tillery, C.L., Mangiarini, L., Davies, S.W. and Bates, G.P. (1999) Brain neurotransmitter deficits in mice transgenic for the Huntington's disease mutation. J. Neurochem., 72, 1773–1776.[CrossRef][ISI][Medline]

  22. Behrens, P.F., Franz, P., Woodman, B., Lindenberg, K.S. and Landwehrmeyer, G.B. (2002) Impaired glutamate transport and glutamate-glutamine cycling: downstream effects of the Huntington mutation. Brain, 125, 1908–1922.[Abstract/Free Full Text]

  23. DiFiglia, M., Sapp, E., Chase, K., Schwarz, C., Meloni, A., Young, C., Martin, E., Vonsattel, J.P., Carraway, R., Reeves, S.A. et al. (1995) Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons. Neuron, 14, 1075–1081.[CrossRef][ISI][Medline]

  24. Sharp, A.H., Loev, S.J., Schilling, G., Li, S.H., Li, X.J., Bao, J., Wagster, M.V., Kotzuk, J.A., Steiner, J.P., Lo, A. et al. (1995) Widespread expression of Huntington's disease gene (IT15) protein product. Neuron, 14, 1065–1074.[CrossRef][ISI][Medline]

  25. Li, X.J., Sharp, A.H., Li, S.H., Dawson, T.M., Snyder, S.H. and Ross, C.A. (1996) Huntingtin-associated protein (HAP1): discrete neuronal localizations in the brain resemble those of neuronal nitric oxide synthase. Proc. Natl Acad. Sci. USA, 93, 4839–4844.[Abstract/Free Full Text]

  26. Gutekunst, C.A., Li, S.H., Yi, H., Ferrante, R.J., Li, X.J. and Hersch, S.M. (1998) The cellular and subcellular localization of huntingtin-associated protein 1 (HAP1): comparison with huntingtin in rat and human. J. Neurosci., 18, 7674–7686.[Abstract/Free Full Text]

  27. Martin, E.J., Kim, M., Velier, J., Sapp, E., Lee, H.S., Laforet, G., Won, L., Chase, K., Bhide, P.G., Heller, A., Aronin, N. and Difiglia, M. (1999) Analysis of Huntingtin-associated protein 1 in mouse brain and immortalized striatal neurons. J. Comp. Neurol., 403, 421–430.[CrossRef][ISI][Medline]

  28. Lin, C.H., Tallaksen-Greene, S., Chien, W.M., Cearley, J.A., Jackson, W.S., Crouse, A.B., Ren, S., Li, X., Albin, R.L. and Detloff, P.J. (2001) Neurological abnormalities in a knock-in mouse model of Huntington's disease. Hum. Mol. Genet., 10, 137–144.[Abstract/Free Full Text]

  29. Phillips, G.R., Huang, J.K., Wang, Y., Tanaka, H., Shapiro, L., Zhang, W., Shan, W.S., Arndt, K., Frank, M., Gordon, R.E. et al. (2001) The presynaptic particle web: ultrastructure, composition, dissolution, and reconstitution. Neuron, 32, 63–77.[CrossRef][ISI][Medline]

  30. Li, S.H., Cheng, A.L., Li, H. and Li, X.J., (1999) Cellular defects and altered gene expression in PC12 cells stably expressing mutant huntingtin. J. Neurosci., 19, 5159–5172.[Abstract/Free Full Text]

  31. Thiel, G., Sudhof, T.C. and Greengard, P. (1990) Synapsin II. Mapping of a domain in the NH2-terminal region which binds to small synaptic vesicles. J. Biol. Chem., 265, 16527–16533.[Abstract/Free Full Text]

  32. Chou, J.H. and Jahn, R. (2000) Binding of Rab3A to synaptic vesicles. J. Biol. Chem., 275, 9433–9440.[Abstract/Free Full Text]

  33. Jennings, P. and Collard, K.J. (1995) Postnatal development of the calcium-dependency of glutamate release from rat cortical synaptosomes: comparison with 5-hydroxytryptamine release. Brain Res. Dev. Brain Res., 89, 120–123.[CrossRef][Medline]

  34. Maneuf, Y.P. and McKnight, A.T. (2001) Calcitonin gene-related peptide-mediated increase in K(+)-induced [(3)H]-dopamine release from rat caudal striatal slices. Neurosci. Lett., 310, 73–76.[CrossRef][ISI][Medline]

  35. Patel, D.R., Young, A.M. and Croucher, M.J. (2001) Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor-mediated stimulation of glutamate and GABA release in the rat striatum in vivo: a dual-label microdialysis study. Neuroscience, 102, 101–111.[CrossRef][ISI][Medline]

  36. Zhang, W., Yamada, M., Gomeza, J., Basile, A.S. and Wess, J. (2002) Multiple muscarinic acetylcholine receptor subtypes modulate striatal dopamine release, as studied with M1-M5 muscarinic receptor knock-out mice. J. Neurosci., 22, 6347–6352.[Abstract/Free Full Text]

  37. DiFiglia, M., Sapp, E., Chase, K.O., Davies, S.W., Bates, G.P., Vonsattel, J.P. and Aronin, N. (1997). Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science, 277, 1990–1993.[Abstract/Free Full Text]

  38. Morton, A.J. and Edwardson, J.M. (2001) Progressive depletion of complexin II in a transgenic mouse model of Huntington's disease. J. Neurochem., 76, 166–172.[CrossRef][ISI][Medline]

  39. Usdin, M.T., Shelbourne, P.F., Myers, R.M. and Madison, D.V. (1999) Impaired synaptic plasticity in mice carrying the Huntington's disease mutation. Hum. Mol. Genet., 8, 839–846.[Abstract/Free Full Text]

  40. Jenkins, B.G., Klivenyi, P., Kustermann, E., Andreassen, O.A., Ferrante, R.J., Rosen, B.R. and Beal, M.F. (2000) Nonlinear decrease over time in N-acetyl aspartate levels in the absence of neuronal loss and increases in glutamine and glucose in transgenic Huntington's disease mice. J. Neurochem., 74, 2108–2119.[CrossRef][ISI][Medline]

  41. Hansson, O., Petersen, A., Leist, M., Nicotera, P., Castilho, R.F. and Brundin, P. (1999) Transgenic mice expressing a Huntington's disease mutation are resistant to quinolinic acid-induced striatal excitotoxicity. Proc. Natl Acad. Sci. USA, 96, 8727–8732.[Abstract/Free Full Text]

  42. Li, H., Li, S.H., Yu, Z.X., Shelbourne, P. and Li, X.J. (2001) Huntingtin aggregate-associated axonal degeneration is an early pathological event in Huntington's disease mice. J. Neurosci., 21, 8473–8481.[Abstract/Free Full Text]

  43. Parker, J.A., Connolly, J.B., Wellington, C., Hayden, M., Dausset, J. and Neri, C. (2001) Expanded polyglutamines in Caenorhabditis elegans cause axonal abnormalities and severe dysfunction of PLM mechanosensory neurons without cell death. PG - 13318-23. Proc. Natl Acad. Sci. USA, 98, 13318–13323.[Abstract/Free Full Text]

  44. Raff, M.C., Whitmore, A.V. and Finn, J.T (2002) Axonal self-destruction and neurodegeneration. Science, 296, 868–871.[Abstract/Free Full Text]

  45. Metzler, M., Legendre-Guillemin, V., Gan, L., Chopra, V., Kwok, A., McPherson, P.S. and Hayden, M.R. (2001) HIP1 functions in clathrin-mediated endocytosis through binding to clathrin and adaptor protein 2. J. Biol. Chem., 276, 39271–39276.[Abstract/Free Full Text]

  46. Rao, D.S., Chang, J.C., Kumar, P.D., Mizukami, I., Smithson, G.M., Bradley, S.V., Parlow, A.F. and Ross, T.S. (2001) Huntingtin interacting protein 1 Is a clathrin coat binding protein required for differentiation of late spermatogenic progenitors. Mol. Cell. Biol., 21, 7796–7806.[Abstract/Free Full Text]

  47. Waelter, S., Scherzinger, E., Hasenbank, R., Nordhoff, E., Lurz, R., Goehler, H., Gauss, C., Sathasivam, K., Bates, G.P., Lehrach, H. and Wanker, E.E. (2001) The huntingtin interacting protein HIP1 is a clathrin and alpha-adaptin-binding protein involved in receptor-mediated endocytosis. Hum. Mol. Genet., 10, 1807–1817.[Abstract/Free Full Text]

  48. Chan, E.Y., Nasir, J., Gutekunst, C.A., Coleman, S., Maclean, A., Maas, A., Metzler, M., Gertsenstein, M., Ross, C.A., Nagy, A. and Hayden, M.R. (2002) Targeted disruption of Huntingtin-associated protein-1 (Hap1) results in postnatal death due to depressed feeding behavior. Hum. Mol. Genet., 11, 945–959.[Abstract/Free Full Text]

  49. Li, S.-H., Yu, Z.-X., Li, C.-L., Nguyen, H.-P., Zhou, Y.-X., Deng, C.X. and Li, X.-J. (2003) Lack of huntingtin-associated protein-1 causes neuronal death resembling hypothalamic degeneration in Huntington disease. J. Neurosci. (in press).

  50. Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S.W. and Bates, G.P. (1996) Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell, 87, 493–506.[CrossRef][ISI][Medline]

  51. Li, S.H., Cheng, A.L., Zhou, H., Lam, S., Rao, M., Li, H. and Li, X.J. (2002) Interaction of Huntington disease protein with transcriptional activator Sp1. Mol. Cell. Biol., 22, 1277–1287.[Abstract/Free Full Text]

  52. Li, H., Ohishi, H., Kinoshita, A., Shigemoto, R., Nomura, S. and Mizuno, N. (1997) Localization of a metabotropic glutamate receptor, mGluR7, in axon terminals of presumed nociceptive, primary afferent fibers in the superficial layers of the spinal dorsal horn: an electron microscope study in the rat. Neurosci. Lett., 223, 153–156.[CrossRef][ISI][Medline]

  53. Huttner, W.B., Schiebler, W., Greengard, P. and De Camilli, P. (1983) Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation. J. Cell. Biol., 96, 1374–1388.[Abstract/Free Full Text]

  54. Cao, F., Levine, J.J., Li, S.H. and Li, X.J. (2001) Nuclear aggregation of huntingtin is not prevented by deletion of chaperone Hsp104. Biochim. Biophys. Acta., 1537, 158–166.[Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Neurosci.Home page
A. L. Orr, S. Li, C.-E. Wang, H. Li, J. Wang, J. Rong, X. Xu, P. G. Mastroberardino, J. T. Greenamyre, and X.-J. Li
N-Terminal Mutant Huntingtin Associates with Mitochondria and Impairs Mitochondrial Trafficking
J. Neurosci., March 12, 2008; 28(11): 2783 - 2792.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
A. J. Milnerwood and L. A. Raymond
Corticostriatal synaptic function in mouse models of Huntington's disease: early effects of huntingtin repeat length and protein load
J. Physiol., December 15, 2007; 585(3): 817 - 831.
[Abstract] [Full Text] [PDF]