Alterations in microtubule-dependent trafficking and certain signaling pathways in neuronal cells represent critical pathogenesis in neurodegenerative diseases. Huntingtin (Htt)-associated protein-1 (Hap1) is a brain-enriched protein and plays a key role in the trafficking of neuronal surviving and differentiating cargos. Lack of Hap1 reduces signaling through tropomyosin-related kinases including extracellular signal regulated kinase (ERK), resulting in inhibition of neurite outgrowth, hypothalamic dysfunction and postnatal lethality in mice. To examine how Hap1 is involved in microtubule-dependent trafficking and neuronal differentiation, we performed a proteomic analysis using taxol-precipitated microtubules from Hap1-null and wild-type mouse brains. Breakpoint cluster region protein (Bcr), a Rho GTPase regulator, was identified as a Hap1-interacting partner. Bcr was co-immunoprecipitated with Hap1 from transfected neuro-2a cells and co-localized with Hap1A isoform more in the differentiated than in the nondifferentiated cells. The Bcr downstream effectors, namely ERK and p38, were significantly less activated in Hap1-null than in wild-type mouse hypothalamus. In conclusion, Hap1 interacts with Bcr on microtubules to regulate neuronal differentiation.
The prevalence of neurodegenerative diseases is increasing due to an increase in aging population, but the molecular mechanism remains elusive. Neurodegenerative diseases such as Alzheimer’s disease (AD) and Huntington’s disease (HD) are characterized by progressive neuropsychiatric dysfunction and loss of specific subtypes of neurons. Although there are distinct neuropathology and clinical profiles among these diseases, many similarities in pathological pathways were reported . One of the common pathogenic events in neurodegenerative diseases is impaired intracellular trafficking in neuronal cells. The critical role of intracellular trafficking in neurodegenerative diseases is also supported by mounting evidence that mutations of proteins involved in microtubule-dependent trafficking often induce neuronal pathologies including neurodegeneration .
Huntingtin (Htt)-associated protein-1 (Hap1), the first identified Htt-interacting protein, participates in microtubule-dependent trafficking [3–5]. Hap1 associates with microtubule-dependent motor proteins kinesin KIF5, kinesin light chain-2 and dynactin p150Glued (p150) [6–8], and is involved in the transport of various proteins such as brain-derived neurotrophin factor, AD-related amyloid precursor protein and kalirin-7, a guanine nucleotide exchange factor (GEF), [6, 9–12]. Like Htt, which is critical for embryonic development, Hap1 is also essential for animal survival, as deletion of the Hap1 gene in mouse leads to feeding defect, retarded growth, and early postnatal death [13–15]. Suppressing Hap1 expression reduces signaling through tropomyosin-related kinases, extracellular signal regulated kinase (ERK) and protein kinase B (or Akt), resulting in an inhibition of neurite outgrowth [16, 17]. However, unlike Htt that is ubiquitously expressed, Hap1 is enriched in the brain regions such as hypothalamus. Loss of Hap1 in mice leads to hypothalamic neuronal degeneration, and reductions of food-intake and body weight, which can be found in HD patients at late stages [13, 15, 18]. These findings suggest a fundamental role of Hap1 in hypothalamic neuron survival.
In murine brains, Hap1 consists of two isoforms (Hap1A and Hap1B) that differ in their C-terminal sequences . The major Hap1 isoform in primate brain is more similar to murine Hap1A than to Hap1B . Previous report showed the different subcellular localizations of the two isoforms . Hap1A is enriched in the growth cones and neuritic puncta of developing neurons, while Hap1B is diffusely distributed within the cytoplasm. Cells overexpressing Hap1A develop more extended neurites than do those overexpressing Hap1B, indicating a dominant role for Hap1A in neuronal differentiation.
Breakpoint cluster region (Bcr) protein is enriched in neurons and involved in neural activities [21–23]. It contains tandem DH-PH which has GEF activity, and GTPase activating protein (GAP) domains to regulate Rho GTPases [24, 25]. Originally Bcr was identified as a Bcr-Abl fusion protein via Philadelphia chromosomal translocation and induces chronic myeloid leukemia . Recently researchers found that Bcr-Abl fusion protein interacts with Abelson helper integration site 1 (Ahi1) , a protein that forms a stable complex with Hap1 in neurons , implicating a close relationship of Hap1 and Bcr in neuronal activities. Early studies suggested the involvement of Bcr in neural development and regulation of long-term potentiation and memory formation . In Bcr-null mice, the number of dendrites was increased, indicating a dysregulation of its GAP and GEF activities in their brains . In support of this, the Bcr downstream effectors are found to be the mitogen-activated protein (MAP) kinases including p38, extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), which are implicated in cellular processes such as regulation of cell cycle, differentiation and morphogenesis [28, 29]. In addition, the activation of p21-activated kinase (PAK) was reported to correlate with Rac1 GTPase activity and is increased in Bcr-null mice . In the present study, we hypothesize that Hap1 interacts with Bcr for hypothalamic neuron development.
Hap1-null mice were generated via the conventional gene knock-out approach as described . The first two exons of the Hap1 gene were deleted and replaced with a neomycin gene. Before brain excision, neonates were anesthetized on ice. All animal procedures were approved by the Institutional Animal Care and Use Committee in Taipei Medical University (LAC-99–0143).
Neuro-2a mouse neuroblastoma cell line (ATCC, CCL-131) was maintained as monolayer cultures in 90% minimum essential medium (Eagle) with 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1.5g/L sodium bicarbonate and 1.0 mM sodium pyruvate (Life Technologies Co., U.S.A), and supplemented with 10% heat inactivated fetal bovine serum (Life Technologies Co., U.S.A), 100 units/mL penicillin, 100 μg/mL streptomycin, and 2.5 μg/mL amphotericin B (Life Technologies Co., U.S.A). Cultures were maintained at 37°C with 5% CO2in a humidified incubator. For immunofluorescence experiment, the cells were cultured in slide chambers (Merck Millipore, U.S.A.). The cells were transfected with 1 μg of DNA according to the manufacturer protocol of TurboFect (Thermo Scientific, U.S.A.) as required.
Plasmids used in this study were a green fluorescent protein (GFP) fusion vector, pEGFP-C3 (BD Biosciences Clontech, U.S.A.), GFP-fused rat Hap1 constructs, pEGFP-rHap1A and pEGFP-rHap1B , and myc-tagged mouse Bcr construct, pCMV6-mBcr-myc (OriGene, U.S.A.).
Rabbit anti-JNK polyclonal, rabbit anti-p38 polyclonal, mouse anti-phosphorylated JNK (p-JNK) Thr183 and Tyr185 monoclonal, mouse anti-PAK monoclonal, mouse anti-p-p38 Tyr182 monoclonal, and goat anti-Hap1 (N-18) polyclonal antibodies were purchased from Santa Cruz, U.S.A. Rabbit anti—Bcr polyclonal, anti-p-PAK1/2 Ser144/141 polyclonal, anti-p-ERK1/2 Thr202/204 monoclonal, and mouse anti-ERK1/2 monoclonal antibodies were purchased from Cell Signaling, U.S.A. Mouse anti—Myc antibody was purchased from OriGene Technologies, U.S.A. Rabbit anti-GFP polyclonal and secondary antibodies conjugated with HRP, Dylight 488 and Dylight 594 were purchased from GeneTex, Taiwan.
A wild-type C57BL/6 female mouse was fed with heavy isotope-labeled chow containing [13C615N4] arginine and [13C6] lysine before pregnancy. Its offspring kept the same diet to adulthood. Brain lysates from the isotope-labeled wild-type (WT) adult mouse and a non-labeled newborn mouse at P1, with either WT or Hap1-null genotype, were mixed at 1:1 ratio in a buffer containing 50 mM PIPES pH 6.8, 1 mM EGTA, 1 mM MgSO4 and complete protease inhibitor cocktail (Roche). Microtubule-associated proteins were polymerized by taxol (Sigma) as described previously . Briefly, 200 μl of the mixed brain lysates were centrifuged at 18,000 × g for 20 min, and the supernatant (S2) was further centrifuged at 120,000 × g for 30 min. The resulting supernatant (S3) was treated with 1 mM GTP and 20 μM taxol at room temperature for 30 min. The polymerized microtubules were pelleted by centrifugation at 120,000 × g for 30 min. The pellets were washed once with PIPES buffer and resuspended in 100 μl of RIPA buffer containing 10 mM dithiothreitol. The samples were resolved on a 10% polyacrylamide SDS gel. After staining with Coomassie G-250, each gel lane was cut into pieces and subjected to in-gel digestion and LC-MS/MS analysis as described . Another set of microtubule sedimentation without isotope-labeled WT adult mouse brain was performed and analyzed by Western blotting to confirm the findings in mass spectrometry. Each sample with the same volume (10 μl) instead of mass was analyzed to reflect a similar level of microtubules in the lanes on the blot. S2 supernatant was used as input.
Neuro-2a cells were cultured in 4-well slide chamber (Merck Millipore, U.S.A.) at 10,000 cells per well. Cells were co-transfected with pCMV6-Bcr-myc and pEGFP-C3, pEGFP-Hap1A or pEGFP—Hap1B. After transfection, cells were washed with 1X PBS and fixed using 4% paraformaldehyde for 5 min at room temperature. Following washes, cells were permeablized and blocked with 0.1% Triton X-100, 3% BSA in PBS for 30–60 min at room temperature. Afterward the cells were incubated with primary mouse anti-Myc antibody overnight. The cells were then washed and incubated with a fluorophore-conjugated secondary antibody at 1:5000 for 2 hours at room temperature. After washes, the cells were incubated with 300 ng/ml DAPI in 1X PBS for 10 min at room temperature. With sufficient washes, the chamber was disassembled, and the slide was air-dried and mounted. The slide was covered and sealed, and then visualized using TCS SP5 Confocal Spectral Microscope Imaging System (Leica, Germany) at Taipei Medical University Instrument Center. Images of co-localized area were generated using Image J 1.48v (Wayne Rasband, NIH, U.S.A), and image densities were quantified using Un-Scan-It 6.1 (Silk Scientific Crop., U.S.A.).
Neuro-2a cells were harvested 48 h after transfection, and homogenized in 50 mM PIPES pH 6.8, 1 mM EGTA, 1 mM MgSO4, 100 μg/ml PMSF, 1X protease inhibitors and phosphatase inhibitors (Merck Millipore, U.S.A.). The homogenate was centrifuged at 15,000 rpm for 15 min at 4°C. Anti-GFP antibody was added to protein A magnetic beads (Recenttec, Taiwan) and incubated for 2 h at 4°C to immobilize the antibody. Then 300 μg of the centrifuged supernatant was incubated with immobilized antibody overnight with continuous mixing at 4°C. The immunoprecipitates were washed five times with the PIPES buffer containing 0.1% NP-40 and then eluted by boiling in SDS—PAGE sample loading buffer. The proteins were resolved on a 10% SDS-PAGE and transferred onto PVDF membrane (BioRad) for immunoblotting.
Three different sets of wild type and Hap1-null newborn mouse (P1) hypothalamus were excised on ice. The excised tissues were homogenized and sonicated in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, 1 mM DTT, 1X protease inhibitors and phosphatase inhibitors; Merck Millipore, U.S.A.) at 4°C and kept on ice through all the process.
Protein concentrations of cell lysates were determined by DC protein assay kit protocol (BioRad, U.S.A.). About 20 μg of proteins were analyzed in 10% SDS-PAGE at a constant voltage of 80 V until full separation. The proteins were transferred to PVDF membrane under 300 Am for 2 h at 4°C. After transfer, the membrane was blocked using 5% nonfat milk in 1X PBS/0.05% tween-20 (PBST) for 1 h at room temperature. The membrane was washed and incubated with specific primary antibody diluted in 3% BSA PBST for overnight at 4°C. After washes, the membrane was incubated with HRP-conjugated secondary antibody diluted in 5% nonfat milk PBST for 1 h at room temperature. The membrane was washed and developed using ECL chemiluminescence kit (Thermo Scientific, U.S.A.). Quantitative analysis was done using Un-Scan-It 6.1 (Silk Scientific Crop., U.S.A.).
All experiments were performed at least three times. Results were expressed as mean ± standard error, and analyzed through Student’s t test or one-way ANOVA using SigmaPlot 12.5 software. Values of p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) were considered statistically significant.