Interaction of Huntingtin-associated Protein-1 with Kinesin Light Chain

Huntingtin-associated protein-1 (HAP1)² was the first protein identified to interact with huntingtin (htt), the Huntington disease (HD) protein (1, 2). Htt contains a polyglutamine (polyQ) stretch in its N terminus, and expansion of this glutamine repeat (>37 glutamines) causes selective neurodegeneration. However, the underlying mechanisms of the specific neuropathology of HD remain unclear, especially in light of the widespread expression of htt. It is believed that the expanded polyQ confers an abnormal protein conformation and affects the function of other neuronal proteins (3). This idea, or the theory of gain of function, is strongly supported by the fact that polyQ expansion causes htt to abnormally interact with other proteins (4, 5). HAP1 is a good candidate for htt-mediated pathology, because its binding to the N-terminal region of htt is enhanced by expanded polyQ tracts, and its expression is enriched in the brain (1).

The critical role of HAP1 in neuronal function has been demonstrated in HAP1 knock-out mice. Deletion of the mouse HAP1 gene leads to retarded growth, depressed feeding behavior, and postnatal death of these mice (6-8). This phenotype may be caused by the degeneration of hypothalamic neurons that control feeding behavior (7). Several studies have suggested that HAP1 is involved in neuronal transport of organelles or molecules. HAP1 is required for vesicular transport of brain-derived neurotrophic factor along microtubules, and mutant htt impairs this transport concomitant with its increased interactions with HAP1 and dynactin p150 (9). HAP1 might also be involved in endocytotic trafficking of membrane receptors. Evidence to support this idea includes the interaction of HAP1 with Hrs, which plays a critical role in the endocytosis of epidermal growth factor receptor (10). A recent study by Kittler et al. (11) provides further evidence that HAP1 is involved in the internalization and recycling of the GABA_A receptor and that HAP1 overexpression leads to an increase in the level of GABA_A (11). In addition, HAP1 interacts with the type 1 inositol (1, 4, 5)-triphosphate receptor (InsP3R1), forming an InsP3R1-HAP1-A-htt ternary complex in which mutant htt enhances the sensitivity of InsPR1 to IP3 (12, 13).

The biochemical evidence to support the role of HAP1 in intracellular trafficking includes the interaction of HAP1 with dynactin p150 Glued (dynactin p150), a dynein-associated protein (14, 15). p150 Glued and dynein drive retrograde transport in neuronal cells, whereas kinesin directs plus-end (anterograde) movement (16, 17). The cotransport of HAP1 and htt in both anterograde and retrograde directions (18) and the sequestration by mutant htt of soluble dynein and kinesin (19, 20) further raise the possibility that HAP1 participates in kinesin-mediated transport in neurons.

The native conventional kinesin complex (379-386 kDa) consists of two heavy chains (KHC) (110-120 kDa) and two light chains (KLC-1 and KLC-2) (60-70 kDa) (21, 22). The N-terminal heads of KHC move along microtubules. The C-terminal tails are linked with light chains (KLC) that are thought to interact with membrane-bound organelles and regulate KHC activity (23-26). A number of associated proteins participate in linking motor proteins to various cargos (17). Here we demonstrate that HAP1 interacts with the kinesin motor complex and that suppressing the expression of HAP1 inhibits kinesin-associated transport in neurons, providing the biochemical basis for the involvement of HAP1 in anterograde transport in neuronal cells.

MATERIALS AND METHODS

Reagents—The following antibodies were used: rabbit polyclonal antibodies against HAP1-A, HAP1-B, and HAP1 (27); a mouse antibody against kinesin heavy chain (Chemicon); and a rabbit antibody against kinesin light chain-2 was generated using GST fusion proteins containing a rat KLC-2 fragment (amino acids 124-411) by Covance Inc. Other antibodies used in the study were those against tubulin (Sigma) and the hemagglutinin epitope 12CA5 (Cell Signaling).

HAP1 constructs were generated in our previous studies (15). We also fused GFP or RFP to the N terminus of HAP1-A or HAP1-B in the PRK vector for examining its dynamic movement in cultured cells. The C-terminal region of HAP1-A at the BglII site of HAP1 cDNA was removed, resulting in a truncated HAP1 encoding 1-473 amino acids of HAP1 (27). The full-length KLC-2 cDNA was isolated by screening a rat brain cDNA library (Stratagene) and inserted into the PRK expression vector. Amyloid precursor protein (APP)-YFP cDNA was a gift from Dr. Lawrence S. B. Goldstein at University of California in San Diego (28). PC12 cell lines stably expressing antisense HAP1 RNA were generated in our previous studies (7).

Yeast Two-hybrid Assays—HAP1 (1-599 amino acids) fused to the GAL-4 DNA binding domain was used as a bait to screen a rat brain cDNA library (1). Two cDNAs were identified to contain a partial KLC-2 (amino acids 80-396) fragment that was able to interact with HAP1 in yeast. Filter and liquid β-galactosidase assays with different HAP1 fragments were performed using the same method as described previously (1).

Protein Interaction Assays and Western Blotting—In vitro binding assays were performed as described (1, 15). Protein A-Sepharose (Sigma) was added to the mixture for an additional 1 h incubation. The beads were precipitated by gravity and washed twice in lysis buffer, which were then resolved by SDS-PAGE and detected by Western blotting. For immunoprecipitation of HAP1 from mouse brain, hypothalamus was dissected from postnatal day one pups. Three to four hypothalami from wild type or HAP1 knock-out pups were combined and homogenized with a handheld Kontes pellet pestle motorized homogenizer in 0.2% Triton X-100 in a phosphate-buffered saline solution. Samples were then sonicated for 10 s at low power and were gently rocked for 15 min at 4 °C, followed by centrifugation at 4 °C at 16,000 × g for 10 min. Samples were adjusted to 500 μl of protein at 1.5 μg/μl and preabsorbed by protein A-agarose beads (25 μl, Sigma 1406-5G) for 1 h at 4 °C with gentle rocking. Supernatants were collected, and 20 μl of rabbit anti-HAP1-A (EM31) were added for incubation at 4 °C for 4 h. Next, 12.5 μl of protein A beads were added and incubated with gentle rocking at 4 °C for 1 h. Samples were spun in a tabletop microcentrifuge for 10-15 s. Beads were washed three times with lysis buffer and finally resuspended in 80 μl of lysis buffer for Western blotting. For immunoprecipitation of PC12 cells, PC12 cells were lysed in a Nonidet P-40-containing buffer. Anti-HAP1-A (10 μl) was then added to lysates (500 μl of 1 μg/μl solution) to incubate overnight at 4 °C with gentle rocking. Protein A beads (15 μl) were then added to the tube for immunoprecipitation.

For Western blots, cultured cells or brain tissues were solubilized in 1% SDS, resuspended in SDS sample buffer, and sonicated for 10 s. The total lysate was used for Western blotting with the ECL kit (Amersham Biosciences).

Neuronal Culture and Imaging Analysis—Primary neurons were cultured using the method similar to that described previously (29). Olfactory bulb neurons isolated from wild type and HAP1 knock-out mice at postnatal day one were plated on lysine-coated coverglass in two-well chamber slides from Nalge Nunc International Lab-Tek (catalog number 155379). Cells were cultured in B27-supplemented neurobasal medium (Invitrogen). PC12 cells were cultured in Dulbecco's modified Eagle's medium with 5% fetal bovine serum and 10% horse serum containing 50-100 ng/ml nerve growth factor (NGF) for 3 days. Control cells were cultured without NGF.

For immunofluorescence light microscopy, light micrographs were taken using a 63× objective lens (LD-Achroplan 63×/0.75) on a Zeiss microscope (Axiovert 200 MOT) attached to a digital camera (Hamamatsu Orca-100). Micrographs were taken using Openlab software (Improvision Inc). Confocal image analysis was performed with a Ziess LSM 510 NLO confocal microscope system.

For transfection of PC12 cells and imaging analysis of live transfected cells, cells were plated on two-chambered coverglass (Lab-Tek 155379) and transfected with GFP-HAP1-A or GFP-HAP1-B by Lipofectamine 2000 (Invitrogen 18324-020) for 5 h. Cells were then loaded for 10-15 min with red MitoTracker (Molecular Probes) at a concentration of 1:1000 in CO₂-independent medium from Invitrogen (18045-088) at 37 °C. After two washes with CO₂-independent medium, CO₂-independent medium containing 100 ng/ml NGF (Sigma) was added to stimulate differentiation. Cells were then immediately placed in a heated stage and imaged using the Zeiss LSM 510 confocal microscope with a 40x objective lens. Images were scanned at 512 × 512 resolution, scan speed of 7, and a mean of 4 scans for an overall scan time of 3.93 s per image. The mitotracker dye (Molecular Probe) was imaged at a laser wavelength of 514 nm; the GFP fusion proteins were imaged at 488 nm. Scans were taken every 3 min over a course of 3.5 h. Movies were created using Metamorph software showing 6 frames/s.

For vesicular movement assays, cultured olfactory bulb neurons or PC12 cells were plated in 35-mm culture dishes. After culturing PC12 cells for 24 h and primary neurons for 96 h, cells were transfected with APP-YFP DNA using Lipofectamine 2000. Wild type cells served as a control, and the identity of the cells was blinded. Cells were examined 24-36 h later in CO₂-independent medium using the 63× objective lens of the fluorescence light microscope. Cells were observed for 1 min to score those having visible vesicle movement in neurites. For each culture dish, 20 cells were randomly selected. The number of cells showing vesicular movement was divided by the total number of transfected cells examined to obtain a percentage of transfected cells having vesicle movement. For each cell type, three plates were assayed, and the data are expressed as mean ± S.E.

Adenoviral HAP1-siRNA Preparation—The testing of small interfering RNA (siRNA) sequences revealed that one siRNA (GAAGTATGTCCTCCAGCAA of 1695-1713 nucleotides of the rat HAP1 gene) was able to effectively inhibit the expression of HAP1. This siRNA was inserted into an adenoviral vector that independently expresses GFP under the control of the CMV promoter. A vector that expresses GFP alone or a scrambled HAP1 siRNA (GCGCGCTTTGTAGGATTCG) served as the control. Recombinant adenoviruses were generated and purified by Welgen Inc. (Worcester, MA). The viral titer was determined by measuring the number of infected HEK293 cells expressing GFP. All viral stocks were adjusted to 1 × 10⁸ plaque-forming units/μl before their use.

Fluorescence Recovery after Photobleaching (FRAP) Assay—Cultured olfactory neurons were transfected with APP-YFP DNA construct. After 18-36 h, cells were incubated with CO₂-independent medium. Chamber slides were then placed into a heated microscope stage and maintained at 37 °C. Cells were imaged using a Zeiss LSM 510 confocal microscope microimaging system (Zeiss LSM 510 Meta from Carl Zeiss Microimaging) and software (LSM 510 version 302 SP2). All imaging was done with a 63× oil immersion objective lens. For imaging, the Zeiss Argon laser (maximum power of 30 milliwatt) was adjusted to a wavelength of 488 nm. Laser power was set to 75%, and transmittance was set at 4%. Transfected cells were then selected by the presence of YFP fluorescent puncta. A time series of images was collected. Total scanning time was set at 3.93 s with a wait time of 3 s between scans. Three scans were taken before photobleaching using the conditions listed above. The distal one-third to one-quarter of the longest process (in an effort to select the tips of axons over dendrites) was outlined as a region of interest to be photobleached. To photobleach cells, the laser transmittance was adjusted to 100% for both the 477 nm and 488 nm line of the argon laser. Seventy bleaching iterations were performed, lasting a total of ∼10-15s. Immediately after photobleaching, scans were taken as described above for up to 5 min. Regions of interest were also established for an unbleached cell region and for a region outside of cells at each time point. These values were used to correct background and for overall fluorescence loss because of scanning. For each wild type or HAP1 knock-out animal, 3 cells were examined and their fluorescence data averaged at each time point. A total of 13 cells from four mice were examined and the data averaged for each genotype.

Statistical Analysis—Error bars in the vesicle movement assays reflect S.E., as computed for three trials of 20 cells/trial. Differences were found to be statistically significant using the Student's t test for both primary neurons (p = 0.02) and antisense RNA expressing PC12 cells (p = 0.02). Error bars in the FRAP assays reflect the S.E. for 13 cells from four wild type and four knock-out mice.

RESULTS

Interaction of HAP1 with Kinesin—Rodent HAP1 consists of two isoforms, HAP1-A and HAP1-B, which have different C-terminal sequences (1). Yeast two-hybrid analysis revealed that a C-terminal fragment of HAP1 present in both rodent isoforms binds kinesin light chain-2 (KLC-2, Fig. 1), a subtype of KLC that is highly homologous to kinesin light chain-1 (KLC-1) with 71.1% amino acid identity (30). The coiled-coil domains of HAP1 do not appear to mediate the binding of HAP1 to KLC, as HAP1 fragments that contained coiled-coil domains but lacked C-terminal amino acids failed to yield a positive interaction (Fig. 1B). To confirm the interaction between HAP1 and KLC, we performed a GST binding assay in which GST or GST-HAP1 fusion proteins were incubated with HEK293 cell lysates containing transfected KLC-2. A GST fusion protein consisting of a HAP1 fragment (160-599 amino acids), but not 160-445 amino acids, precipitated KLC-2, confirming the yeast two-hybrid results (Fig. 1C).

To examine the in vivo interaction of HAP1 with KLC, we performed HAP1 immunoprecipitation of mouse brains. We used hypothalamic tissue for HAP1 immunoprecipitation, as it is enriched in HAP1. The hypothalamic tissue of HAP1 knock-out mice we previously created (7) was used as a control to confirm that precipitation of kinesin is dependent on the presence of HAP1. We generated a rabbit anti-KLC using the conserved region (124-411 amino acids) of KLC-2 as an antigen. This antibody reacts with both KLC-1 and KLC-2 in the brain. Western blotting revealed that KLC-1 is the major form of KLC in the hypothalamus and was coprecipitated by the antibody to HAP1. Only when HAP1 was present did HAP1 immunoprecipitation coprecipitate kinesin (Fig. 1D), indicating that KLC associates with HAP1 in vivo.

Colocalization and Association of HAP1-A with Kinesin—To investigate whether HAP1 colocalizes with kinesin in cells, we examined PC12 cells that show elongated neurites in response to NGF. Without NGF treatment, KHC and KLC were mainly distributed in the cell body or soma (data not shown). After NGF treatment, PC12 cells developed long neurites. Consistent with a previous report (31), KHC and KLC were distributed in the growing tips of cellular processes (Fig. 2A). We have shown previously that HAP1-A is enriched in neurite tips, and HAP1-B is largely confined to the cell body (32). Therefore, we wanted to know whether kinesin and HAP1-A are colocalized in neurite tips. We used mouse anti-KHC and rabbit antibodies to HAP1 in immunofluorescence double labeling. KHC and HAP1-A were clearly colocalized in the same neurite tips (Fig. 2B). In contrast, most HAP1-B remained in the body of PC12 cells (Fig. 2B). Using immunoprecipitation, we then asked whether HAP1-A preferentially binds to kinesin. It is known that HAP1-A interacts with HAP1-B to form a dimer in cells (27). As expected, immunoprecipitation of either HAP1-A or HAP1-B from PC12 cells pulled down both HAP1-A and HAP1-B. Because PC12 cells express more HAP1-B than HAP1-A, the HAP1-B immunoprecipitation yielded a much greater amount of HAP1-B than HAP1-A. Importantly, more KLC was precipitated by anti-HAP1-A than by anti-HAP1-B. Because KLC binds KHC to form an active motor complex, we probed the same blot with anti-KHC. The increased level of KHC in the HAP1-A immunoprecipitation as compared with the HAP1-B immunoprecipitation also supports the idea that HAP1-A is more likely than HAP1-B to associate with the kinesin complex (Fig. 2C).

Trafficking of HAP1 and Neurite Outgrowth—Microtubule-dependent transport is required for neurite outgrowth. We have previously reported that overexpression of HAP1-A significantly increases the number of PC12 cells containing extended neurites (29.4%) as compared with HAP-1B overexpression (9.7%) and vector transfection (4.7%) (32). However, it remains unclear whether this neurite promotion is related to the trafficking function of HAP1. To test this hypothesis, we first examined the effect of HAP1 on live NGF-stimulated PC12 cells. We expressed GFP- or RFP-linked HAP1-A or HAP1-B in these cells and then examined the dynamic subcellular localization of the different HAP1 isoforms. We observed that RFP-HAP1-A was able to move anterogradely from the cell body to neurite terminal (supplemental movie 1). Time-lapse imaging clearly shows that cells expressing GFP-HAP1-A are able to extend neurites. More importantly, GFP-HAP1-A is localized in the extended neurites and growth cone tips (Fig. 3A). However, GFP-HAP1-B remained in the cell body of PC12 cells and seemed to inhibit neurite outgrowth when compared with nontransfected cells labeled with a red mitochondrial staining (Fig. 3A). We also expressed a truncated HAP1 (amino acids 1-473), which lacks the C-terminal kinesin light chain binding domain. Overexpression of this mutated HAP1 was unable to promote neurite outgrowth of PC12 cells (Fig. 3B). As HAP1-B and HAP1-A are known to form heterodimers, it is possible that overexpression of HAP1-B sequesters all of the free endogenous HAP1-A of the cell, preventing cells from extending neurites. These results are consistent with the finding that HAP1-A preferentially binds kinesin and support the idea that the interaction of HAP1-A with kinesin may be involved in neurite outgrowth in some neurons.

Altering the Expression of HAP1 in Neurons via siRNA—We next wanted to test the idea that HAP1-associated trafficking is indeed involved in the neurite outgrowth in PC12 cells. To do so, we used siRNA to reduce the expression of endogenous HAP1 in PC12 cells. We identified one siRNA (see sequences under “Materials and Methods”) that was able to effectively inhibit the expression of HAP1. This siRNA was inserted into an adenoviral vector that also independently expresses GFP. Thus, GFP-positive cells are likely to express HAP1 siRNA. Infection of PC12 cells with this adenoviral siRNA effectively reduced endogenous levels of HAP1 in PC12 cells (Fig. 4A). Consequently, HAP1 siRNA reduced the number PC12 cells showing neurites longer than two cell bodies in the presence of NGF (Fig. 4B). This reduction by adenoviral HAP1 siRNA (20.95% ± 0.18, n = 3) is statistically significant (p < 0.01) as compared with adenoviral GFP infection (47.4% ± 1.94, n = 3), confirming the role of HAP1 in neurite growth of PC12 cells. We also tested PC12 cells with a scrambled siRNA in the same adenoviral vector and did not find any significant effect of this control siRNA on neurite outgrowth of PC12 cells (data not shown). The inhibitory effect of HAP1 siRNA on neurite outgrowth is not likely due to cell death, as we did not find nuclear DNA fragmentation or other obvious morphological features of apoptosis (data not shown). As siRNA would also decrease the level of free HAP1-A in the cell, these results are consistent with the possibility raised earlier that the interaction of HAP1-A with the kinesin complex plays a role in neurite outgrowth.

HAP1 Deficiency Affects Kinesin-dependent APP Transport—Microtubule motors transport a variety of molecules over a long range along neuronal processes and also over a short range in endocytosis and vesicle recycling at nerve terminals. It is known that amyloid precursor protein (APP) is inserted into the membranes of vesicles that are transported along microtubules (28, 33). APP is found to interact with KLC-1 (28), although one study has failed to show this interaction (34). Nevertheless, it is evident that the transport of APP vesicles is dependent on kinesin (28), and anterograde transport of APP has been described in axons of neurons in both the peripheral and central nervous systems (33, 35-37). The anterograde movement of APP reflects kinesin-dependent transport of a subset of vesicles in axons, as has been observed by a number of investigators (28, 33, 38-40). We therefore used a fusion protein of APP to yellow fluorescent protein (YFP) to assay for kinesin-based vesicular transport in live cells. If HAP1 is indeed involved in transport of APP-YFP vesicles, we should see that the two proteins are colocalized to the same vesicles. We therefore tagged RFP to HAP1-A and coexpressed both APP-YFP and RFP-HAP1-A in PC12 cells. RFP-HAP1-A showed a strong colocalization with APP-YFP vesicles (Fig. 5A). Moreover, there was dynamic movement of APP-YFP vesicles that also contained RFP-HAP1-A (see supplemental movie 2). This data further suggests a role for HAP1-A in vesicular transport in PC12 cells.

We have shown that a lack of HAP1 selectively causes some hypothalamic neurons to degenerate in the brain (7), suggesting that HAP1 is more critical to hypothalamic neurons than other types of neurons for survival. We found that olfactory neurons also express a high level of HAP1, although less than hypothalamic neurons. Olfactory neurons, unlike hypothalamic neurons, from HAP1 knock-out mice can still be cultured, allowing us to examine the effect of lack of HAP1 on the transport of transfected APP-YFP. We chose those HAP1 knock-out neurons that show normal neuritic morphology so that we could minimize the potential influence of abnormal neurites on the transport of APP vesicles. HAP1 immunostaining confirmed that wild type olfactory neurons express HAP1 whereas HAP1 knock-out neurons lack the staining (Fig. 5B). Because each cell contained a large number of vesicles that move at widely different speeds in both anterograde and retrograde directions, it would have been difficult to calculate an average rate of APP-YFP vesicle movement. Therefore, we measured the number of transfected primary neurons containing moving APP-YFP vesicles in their neurites and confirmed that wild type neurons more frequently contained moving vesicles than HAP1 knock-out neurons (Fig. 5, C and D). The same result was observed in HAP1 antisense RNA expressing PC12 cells, which have decreased HAP1 expression (Fig. 5, E and F, and Ref. 7).

FRAP analysis recently has been used to measure the dynamic movement (41-45) or transport (46) of small fluorescent particles or molecules. APP-YFP transfection results in cells containing a wide array of vesicles, from large immobile vesicles to vesicles smaller than can be easily visualized using confocal microscopy. Our use of FRAP allows for a quantitative assay of the mass trafficking of the chosen mobile of the neurite APP-YFP vesicles. APP-YFP intensity seemed weaker in the neurites of some HAP1 knock-out neurons than in wild type neuronal neurites (Fig. 6A), suggesting that the lack of HAP1 decreases the transport of APP-YFP vesicles from the cell body to neurites. To examine the vesicle movement from the cell body outward, we bleached only the distal tips of neurons. The recovery of fluorescence after photobleaching of the APP-YFP vesicles is apparently faster in wild type neurons than in HAP1 knock-out neurons (Fig. 6, B and C), suggesting that anterograde trafficking of APP-YFP to neurite tips is impaired in the absence of HAP1. Thus, using both conventional time-lapse imaging and FRAP, we demonstrated that lack of HAP1 can significantly affect APP-YFP vesicle movement in neurites.

DISCUSSION

We have used biochemical and functional analyses to show the involvement of HAP1 in kinesin-associated transport in neurons. First, HAP1 binds kinesin light chain in vitro and in vivo and is colocalized with kinesin in neurite tips. Second, reducing the expression of HAP1 affects neurite outgrowth. Third, the lack of HAP1 decreases kinesin-dependent vesicular transport in cultured neurons.

The findings of the present study provide a biochemical basis for the involvement of HAP1 in kinesin-based anterograde transport. HAP1 and htt are reported to move via fast and retrograde transport in axons (18). Also, htt is associated with the kinesin and dynein complexes in neuronal cells (9, 19, 20). Although anterograde transport is mainly driven by kinesin motors, a variety of adaptors or associated proteins are required to link the kinesin complex to different cargos during transport (17). HAP1 may serve as an adaptor to link kinesin motors to some neuronal cargos. This idea is also supported by immunocytochemical studies showing that HAP1 is colocalized with different types of organelles and vesicles in neuronal cells (47, 48). HAP1 could be particularly important for intracellular trafficking in certain types of neurons, because, unlike other microtubule transporter associated proteins that are ubiquitously expressed, HAP1 is expressed at various levels in subsets of neurons, with the highest expression in the hypothalamus. Genetic deletion of HAP1 in mice leads to a selective degeneration of hypothalamic neurons, which may underlie the severely depressed feeding behavior, and early postnatal death of knock-out mice (7), indicating a critical role for HAP1 in neuronal function. All of these findings suggest that the role of HAP1 in intracellular trafficking is critical to the proper development of at least some regions of the brain.

Intracellular trafficking plays a fundamental role in a vast array of cellular processes. In neurons, intracellular trafficking is essential in maintaining the proper localization of molecules and vesicles from the cell body to the distant nerve terminals. The effect of HAP1 gene deletion on kinesin-dependent transport of APP vesicles supports the role of HAP1 in intracellular transport along axons. There is also a short range transport at nerve terminals, which is especially critical for the internalization and recycling of membrane proteins and probably is very active during neurite outgrowth and differentiation. This short range transport involves kinesin and other microtubule-dependent mechanisms (49-51). An inhibition of kinesin activity suppresses neurite outgrowth (31, 52). Consistently, reducing HAP1 inhibits neurite outgrowth, suggesting that HAP1 is required for the transport of various cargos to nerve terminals.

It has been found that HAP1 interacts with dynactin p150 (14, 15), a member of the dynein motor complex known to be involved in retrograde microtubule-based transport. This raises the interesting issue of whether and how HAP1 participates in bidirectional transport. It is possible that posttranslational modification, such as phosphorylation, could regulate the association of HAP1 with kinesin and dynactin p150 to move cargos either anterogradely or retrogradely. Another possibility is that the ratio of one HAP1 isoform to the other can regulate the association of HAP1 with its interactors. For example, we found that HAP1-A preferentially binds KLC as compared with HAP1-B. The interesting possibility that was raised earlier that HAP1-B could regulate the binding of HAP1-A to other proteins by sequestering it via dimerization remains to be tested.

What is the potential role of the interaction of HAP1 with KLC in HD pathology? As described by several groups, htt is involved in axonal transport of vesicles containing neurotrophins or other molecules (9, 19, 20, 53). The abnormal interaction of mutant htt with HAP1 (1, 9) may interfere with this transport. In fact Gauthier et al. (9) demonstrated that the association of huntingtin with dynactin p150 is dependent on the presence of HAP1. The same group showed that increasing the cellular level of wild type huntingtin increases transport of brain-derived neurotrophic factor vesicles but that this increase was suppressed when the expression of HAP1 is inhibited. It remains to be investigated whether mutant htt disrupts kinesin-mediated axonal transport via its effect on HAP1. Another possibility is that mutant htt affects HAP1-associated trafficking of internalized membrane proteins or growth factor receptors at nerve terminals. The role of HAP1 in the endocytosis of membrane receptors has been reported (10, 11), but whether this function is impaired in HD remains to be investigated.

Unlike htt that is ubiquitously expressed, HAP1 is expressed at various levels in different brain regions. The specific function of HAP1 and its associated intracellular transport may depend on its cell-type-specific expression levels, the presence of interacting proteins, and cell-type-specific posttranslational modification. This may explain why a lack of HAP1 causes the degeneration of some hypothalamic neurons but not other types of neurons (7). Given the high level of HAP1 in the hypothalamus (6, 7, 47) and the degeneration of hypothalamic neurons in HD (54), the intracellular transport function of HAP1 may be particularly important for the normal function of hypothalamic neurons and other neurons that also express a relatively high level of HAP1. Accordingly, HAP1 may be involved in the pathology of hypothalamic neurons in HD.