Huntingtin associated protein 1 regulates ... - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2012 | 122 | 1010–1022

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doi: 10.1111/j.1471-4159.2012.07845.x

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*Department of Human Physiology and Centre for Neuroscience, Flinders University, Adelaide, SA, Australia Department of Histology and Embryology, Sichuan University, P.R. China àSchool of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA, Australia

Abstract Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer’s disease. It is axonally transported, endocytosed and sorted to different cellular compartments where amyloid beta (Ab) is produced. However, the mechanism of APP trafficking remains unclear. We present evidence that huntingtin associated protein 1 (HAP1) may reduce Ab production by regulating APP trafficking to the non-amyloidogenic pathway. HAP1 and APP are highly colocalized in a number of brain regions, with similar distribution patterns in both mouse and human brains. They are associated with each other, the interacting site is the 371–599 of HAP1. APP is more retained in cis-Golgi, trans-Golgi complex, early endosome and ER-Golgi

intermediate compartment in HAP1)/) neurons. HAP1 deletion significantly alters APP endocytosis and reduces the reinsertion of APP into the cytoplasmic membrane. Amyloid precursor protein-YFP(APP-YFP) vesicles in HAP1)/) neurons reveal a decreased trafficking rate and an increased number of motionless vesicles. Knock-down of HAP1 protein in cultured cortical neurons of Alzheimer’s disease mouse model increases Ab levels. Our data suggest that HAP1 regulates APP subcellular trafficking to the non-amyloidogenic pathway and may negatively regulate Ab production in neurons. Keywords: amyloid beta, amyloid precursor protein, huntingtin associated protein, trafficking. J. Neurochem. (2012) 122, 1010–1022.

Amyloid precursor protein (APP) is a ubiquitous type I transmembrane receptor-like protein molecule involved in the pathogenesis of Alzheimer’s disease (AD) (Goate et al. 1991; Hardy and Allsop 1991; Hardy and Higgins 1992; Haass and Selkoe 1993; Tanzi and Bertram 2005; Peng et al. 2009; Ho et al. 2010). APP is synthesized in the endoplasmic reticulum (ER) and post-translationally modified during its transit from the ER to the plasma membrane. Only a small fraction of nascent APP molecules is present in the plasma membrane, whereas the majority of APP localizes to the Golgi and transGolgi network (TGN) steadily (Thinakaran and Koo 2008). Following endocytosis, APP is delivered to endosomes, and a fraction of the endocytosed molecules is recycled to the cell surface or back to the Golgi (Thinakaran and Koo 2008; Tang 2009). During this trafficking cycle, APP is cleaved by several secretases located in different compartments (Tang 2009). The activity of a-secretase is found primarily at the cell surface and cleaves APP at the a site, resulting in the shedding of the

APPsa ectodomain and a-C-terminal fragment (Parvathy et al. 1999). b-Secretase (BACE) predominantly localizes to the late Golgi/TGN and endosomes, and cleaves APP to produce APPsb and b-C-terminal fragment (Small and Gandy 2006). The c-secretase complex and its activity are found in multiple compartments, including ER, ER-Golgi intermediate compartment, Golgi, TGN, endosomes, and plasma membrane; it can cleave the a- and b- C-terminal fragments into

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Received April 11, 2012; revised manuscript received June 15, 2012; accepted June 15, 2012. Address correspondence and reprint requests to Dr Xin-Fu Zhou, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia. E-mail: [email protected] 1 These authors contributed to this work equally. Abbreviations used: AD, Alzheimer’s disease; APP, amyloid precursor protein; BACE, b-secretase; HAP1, huntingtin associated protein 1; KLC, kinesin light chain; TGN, trans-Golgi network.

2012 The Authors Journal of Neurochemistry 2012 International Society for Neurochemistry, J. Neurochem. (2012) 122, 1010–1022

Huntingtin associated protein 1 regulation | 1011

P3 and Ab and simultaneously release the cytoplasmic polypeptide termed APP intracellular C-terminal domain (Small and Gandy 2006). Therefore, APP and its cleaved fragments are generated in different compartments and then transferred to different places for different functions. The APP ectodomain can participate in cell adhesion, neurite outgrowth, and synaptogenesis (Mattson 1997), whereas the APP intracellular C-terminal domain modulates cell migration, axonal transport, and cell signaling, with its featuring motif that interacts with an array of adaptor proteins (Muller et al. 2008). However, the mechanisms of APP trafficking remain controversial. Growing evidence has demonstrated that the anterograde transport, especially the anterograde transport of APP vesicles is kinesin-dependent. Anterograde transport of APP is mediated by conventional kinesin (Koo et al. 1990; Amaratunga et al. 1993; Kamal et al. 2000; McGuire et al. 2006). Although a direct interaction of the APP C terminal with kinesin light chain (KLC) was proposed (SatputeKrishnan et al. 2006), a report derived from a collaboration among several independent laboratories suggests that APP does not bind directly to kinesin (Lazarov et al. 2005). Nevertheless the interaction of APP and KLCs might be indirectly mediated by c-Jun N-terminal kinase-interacting protein 1b (JIP1b) (Inomata et al. 2003). The kinesin-dependent APP axonal transport is affected by Huntingtin associated protein 1 (HAP1) in neurons and lack of HAP1 decreases the number of cultured olfactory neurons containing mobile APPYFP vesicles in neurite (McGuire et al. 2006). HAP1 is a brain-enriched protein and participates in intracellular trafficking in neurons (Li et al. 1995; Li and Li 2005). It interacts with kinesin light chain (McGuire et al. 2006) and dynactin p150Glued (Engelender et al. 1997; Li et al. 1998), as well as regulates the anterograde and retrograde transport of a number of proteins including probrain-derived neurotrophic factor (proBDNF) (Wu and Zhou 2009; Wu et al. 2010). It is known that HAP1 is involved in axonal trafficking of APP (McGuire et al. 2006), but the underlying mechanism is not clear. It is also not known whether HAP1 affects APP subcellular trafficking and whether HAP1 regulates the production of Ab, a neurotoxic peptide playing a central role in the pathogenesis of AD. Here we report that HAP1 is co-localized with APP in cultured mouse cortical neurons and human brain cortex. Furthermore, HAP1 regulates its subcellular distribution and trafficking, and may affect Ab production by association with APP.

Laboratory (San Diego, CA, USA); EEA1 (2411, early endosome marker) was purchased from Cell Signalling Technology (Beverly, MA, USA); CD71 (sc-7087, endosome marker) and Golgi97 (sc73619, trans-Golgi marker) were purchased from (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Mouse anti-HAP1 antibody (MA1-46412) was purchased from Thermo Scientific (Rockford, IL, USA); rabbit anti-APP c-terminal antibody (A8717) was purchased from Sigma Aldrich (St Louis, MO, USA); Anti-APP (22c11, MAB348) antibody was purchased from Millipore (Bedford, MA, USA), SEC22b (OSC-224) and beclin 1 antibodies from Osenses (Flagstaff, SA, Australia).

Materials and methods

Coimmunoprecipitation (Co-IP) and GST pulldown assay Co-IP studies were performed as described previously (Park et al. 2008). Briefly, cell lysates were prepared from HEK293 cells cotransfected with APP-YFP and HAP1-CFP plasmids. Human cortex was homogenized in the lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100,0.5% Sodium deoxycholate, 0.1 mg/mL PMSF,1 lg/mL Aprotinin,1 lg/mL Leupeptin,

Antibodies The following antibody were used: Lamp1 (ab62562, lysosome marker), Giantin (ab24586, cis-Golgi marker) and GRP78 (ab21685, ER marker) were purchased from Abcam (Cambridge, UK); GM130 (610823, cis-Golgi marker) was purchased from BD transduction

Animals and human brain tissue All procedures involving animals were approved by the Animal Welfare Committee of Flinders University and undertaken according to the guidelines of the National Health and Medical Research Council of Australia. HAP1 knock-out mice were generated previously (Li et al. 2003). APPSwe/PS1dE9 transgenic mice (AD mouse model) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All animals were kept under standardized barrier breeding conditions (12-h light/12-h dark cycle) with free access to water and food. Human brain samples (two males, age 68 and 73) without clinical history of neurological disorders and no significant neuropathology were obtained from the Flinders University Brain Bank and approved by Flinders Human Ethic Committee. The postmortem time is 15 and 18 h respectively. Immunocytochemical and immunohistochemical staining and fluorescence microscopy Primary cortical neurons of postnatal day 1 HAP1+/+ (wild type) and HAP1)/)(knockout) litter mates from mice crossed between HAP1+/) were grown on glass coverslips coated with poly-D-lysine (Sigma, St Louis, MO, USA) as described previously (Hilgenberg and Smith 2007; Cui et al. 2011). 6 days after seeding, neurons were fixed with 4% paraformaldehyde solution in phosphate-buffered saline; Frozen sections of human brain were immersed in the citronic anhydride solution (0.05%, pH7.4) and incubated at 26C for 1 week. The primary cortical neurons and frozen sections were then used for immunoassaying. Leica (Wetzlar, Germany) SP5 confocal fluorescence microscopy was performed for imaging. Fo¨rster resonance energy transfer (FRET) analysis FRET was performed as described previously (Yang et al. 2011). Briefly, HEK293 cells were transiently cotransfected with HAP1-CFP and APP-YFP (a gift from LS Goldstein, University California) plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). APP-YFP/pECFP, HAP1-CFP/pEYFP and p75NTR-CFPYFP (a gift from Dr. E. Coulson, University of Queensland, Chancellors Place, QLD 4067, Australia) were used as negative and positive control, respectively. FRET efficiency was measured using SP5 Leica confocal microscopy.


1012 | G.-Z. Yang et al.

pH7.5). The protein concentration of the lysates was determined using bicinchoninic acid protein assay kit (Thermo Scientific). The cell lysate and brain homogenates were pre-cleared by adding protein G Sepharose 4 Fast Flow (GE Healthcare Bio Science AB, Uppsala, CA, USA) in lysis buffer, the mixture was rotated for 30 min in 4C, and then spin 9391 g, keep the supernatant for co-IP experiment. The pre-cleaned lysates were incubated with 1 lg of rabbit anti-APP antibody at 4C for 12 h and then incubated with protein G beads for 1 h. The beads were washed five times with the lysis buffer and boiled in loading buffer for western blot using antiHAP1. For negative control, rabbit IgG was used for validation. GST-Huntingtin associated protein-1 (GST-HAP1) constructs (HAP1 153–599, HAP1 215–599,HAP1 240–599, HAP1 328–599 and HAP1 371–599) were gifts from JT Kittler from University of London(Twelvetrees et al. 2010).GST-HAP1 fusion proteins were produced in E.coli BL21 (Invitrogen) and purified with glutathioneagarose beads (Sigma). The APP-YFP lysates were incubated with GST-HAP1 fusion protein (20 lg) coupled to 40 lL of glutathione agarose beads at 4C for 2 h. After washing five times, the proteins bound to the beads were subjected to western blot analysis probed with anti-APP antibody (22C11, MAB348). Sucrose density gradient fraction and Western blotting Sucrose density gradient fraction analysis was performed as described previously (Chang et al. 2003; Lin et al. 2010). HAP1+/+ and HAP1)/) mouse brains were homogenized and centrifuged. Fractions of 200 lL were collected for Western blot. APP internalization assay in primary cortical neurons APP internalization assay was performed as described previously (Huang et al. 2009; Okada et al. 2010). Briefly, mouse neonatal cortical neurons were cultured on glass coverslips. The cells were incubated with 6E10 and fixed with 4% paraformaldehyde. CY5 was used for labeling of APP on the cell surface, and CY3 for the internalized APP. Images were taken by using (Olympus, Tokyo, Japan) BX50 Fluorescence Microscopy, and the intensity of the images was analyzed by ImageJ (NIH). Percentage of APP endocytosis was calculated by the formula Ie/(Is + Ie) · 100, where Ie was the intensity value of Cy3 representing internalized APP and Is was the intensity value of CY5 representing APP on the cell surface. APP biotinylation assays APP biotinylation assays were performed as described previously (Ko et al. 1998; Huang et al. 2009; Zhao et al. 2009; Twelvetrees et al. 2010). Briefly, cultured neurons were incubated with 0.5 mg/ mL EZ-Link-Sulfo-ss-Biotin (Thermo Scientific) for 30 min, and were respectively subjected to incubation at 4C for the surface APP biotinylation, and at 37C for the internalization of biotinylated APP, and then incubation in the reducing buffer [50 mM glutathione, 5 mM NaCl, 1% bovine serum albumin (pH7.4)] to remove the remaining biotin on the cell surface. For determining the reinsertion of APP, neurons were incubated at 37C for 30 min, the biotin recycled back to the cell surface was quenched again in the reducing buffer. All neurons were lysed in radioimmunoprecipitation assay buffer [50 mM Tris (pH7.4) 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.5% sodium deoxycholate, protease inhibitor cocktail]. After sonication and centrifugation, the supernatant of samples was used as an input control and was also incubated with streptavidin agarose at 4C for 2 h. The agarose was then washed

five times with ristocetin-induced platelet agglutination buffer and analyzed by western blotting for biotinylated APP. Video microscopy Live cell time-lapse microscopy was carried out according to method described previously (Szodorai et al. 2009). Cortical neurons were transfected with APP-YFP or APP-YFP/HAP1-CFP DNA plasmids and were videoed by using a BioStation (Nikon, Japan). The picture was taken every 3 s within 2 min and analyzed using AR3.1 software. On the first photo, 10 APP-YFP vesicles were selected randomly and analyzed their movement and direction according to the records. Three independent experiments were performed and 10 neurons were analyzed for each group. Fluorescence recovery after photobleaching analysis Fluorescence recovery after photobleaching (FRAP) was performed as described previously (Giese et al. 2003; McGuire et al. 2006; Rasmussen et al. 2010). Briefly, Mouse cortical neurons were cotransfected with APP-YFP alone or APP-YFP and HAP1-CFP. The fluorescence of YFP was assessed before and after photobleaching in a time series. A total of 30 scans were performed at 3 s intervals after bleaching. For each genotype, six neurons were examined, and the fluorescent data were averaged at each time point. RNA interference knockdown of APP and quantification of Ab by ELISA Cortical neurons from APPSwe/PS1dE9 transgenic neonatal mice were cultured in 24-well plates. When reaching 60% confluence, neurons were transfected with nucleotides mixture 5¢-GUGU CUUGAUGGAGGA GAATT-3¢/5¢-UUCUCCUCCAUCAAGAC ACTT-3¢ and 5-CGAAGAGGAGGAACGAGAAT-3/5-UUCUCG UUCCUCCUCUUCGTT-3 (Genepharma company, Shanghai, China) at 80 nM and 40 nM by Magnetofection kit (NeuroMag, OZ Bioscience, Marseille, France). Nucleotides for negative control were 5¢-UUCUCCGAACGUGUCACGUTT-3¢/ 5¢-ACGUGACACGUUCGGAGAATT-3¢. Neuronal lysates and culture medium were prepared and collected after 72 h transfection, respectively. Cell lysates were used to analyze the interfering efficiency by western blot, Culture media were used to measure Ab by ELISA according to the manufacturer’s instructions (Covance, Dedham, MA, USA). Statistical analysis All data were expressed as mean ± SEM. Statistical results were analyzed by SPSS13.0, and statistical significance (p < 0.05) was assessed using Student’s t-test or one-way analysis of variance (ANOVA), followed by a post-hoc analysis using Tukey’s test when appropriate.

Results Distribution and co-localization of HAP1 and APP in the human brain and cultured mouse neuron In order to investigate the roles of HAP1 in APP trafficking, we first examined the distribution and co-localization of APP with HAP1. Our results demonstrated that APP was present in cultured mice cortical neurons (Fig. 1a) and cortical neurons of human brain (Fig. 1b). Colocalization data showed that



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Fig. 1 Distribution and co-localization of huntingtin associated protein 1 (HAP1) and amyloid precursor protein (APP) in the cultured mouse neuron and human brain. (a–c) Immunochemistry analyzed distribution and co-localization (72.47%) of APP and HAP1 in primary cortical neurons from wild type mice of HAP1 litter mate (a) and frozen section of human brain cortex (81.06%)with high magnification (b) and low

magnification (c). (d) Human brain cortex homogenates were subjected to western blot analysis probed with APP and HAP1 antibodies respectively for demonstrating the distribution of endogenous APP and HAP1 in human brains cortex. The two bands of APP 95100 kD represent glycosylated mature (upper band) and unmature APP (lower band). Scale bar 10 lm.

APP and HAP1 were highly colocalized in both mice cultured cortex neurons (72.47%) and human brain cortex (81.06%) (Fig. 1a and b). Insets showed that both HAP1 and APP were present in granular structures of neurons. APP and HAP1 were also co-distributed in different brain structures such as the human cortex (Fig. 1c and d), hippocampus, hypothalamus and other brain structures (data not shown). Taken together, our data demonstrate that APP and HAP1 are co-distributed and co-localized in neuronal tissue, suggesting that they may be a functional partner of each other.

(HAP1 153–599, HAP1 215–599, HAP1 240–599, HAP1 328–599 and HAP1 371–599) as described by Twelvetrees et al. (2010) were purified and incubated with APP-containing cell lysates. All HAP1 fragments pulled down APP (Fig. 2d), but HAP1 371–599 showed the strongest interaction, suggesting that the binding site is within aa 371–599 of HAP1. The negative control GST did not pull down APP.

HAP1 is associated with APP To confirm the hypothesis that APP interacts with HAP1 physically, we performed FRET analysis and co-immunoprecipitation (Co-IP) assay (Fig. 2a). The results showed that the negative controls generated a background FRET efficiency below 2%. The positive control of the fusion protein p75-CFP-YFP produced a FRET efficiency of 25%. With the same plasmid concentration as the negative control used for transfection, we found that the pair of APP-YFP and HAP1CFP showed a higher FRET efficiency (30%) than the positive control, suggesting APP and HAP1 are in close proximity. The co-IP studies further confirm that HAP1 can be immunoprecipitated by anti-APP antibody from the lysates of HEK293 cells cotransfected with APP-YFP/ HAP1-CFP (Fig. 2b) and the homogenates of human brains (Fig. 2c), indicating they are in the same complex in neurons. To see whether HAP1 directly interacts with APP and to identify the APP-binding sites in HAP1, we performed a GST-HAP1 pulldown assay. Five GST-HAP1 fusion protein

HAP1 modulates APP intracellular distribution Because HAP1 interacts with APP and is involved in vesicular trafficking of BDNF and proBDNF (Yang et al. 2011), we hypothesized that it may modulate APP intracellular trafficking. To test this hypothesis, we analyzed the co-localization of APP with organelles markers in HAP1+/+ and HAP1)/) cultured neurons. Our data showed that APP had a higher co-localization with cis-Golgi (giantin: cisGolgi markers), trans-Golgi (Golgi97: a trans-Golgi marker), early endosome (EEA1: early endosome marker) and SEC22b (an ER-Golgi intermediate compartment marker) in HAP1)/) neurons (Fig. 3a–h) than in HAP1+/+ neurons. By contrast, APP had a lower co-localization with autophagy marker beclin1 in HAP1)/) neurons (Fig. 3i and j). These co-distribution data suggest that APP is retained in the cis-Golgi, trans-Golgi complex, early endosome and ER-Golgi intermediate compartment in HAP1)/) neurons (Table 1). To further confirm the altered APP distribution, we performed sucrose gradient fractionation assay as described (Chang et al. 2003; Lin et al. 2010). To determine whether APP and organelle markers are equally expressed in the brain



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Fig. 2 Interaction of huntingtin associated protein 1 (HAP1) and amyloid precursor protein (APP) in cotransfected HEK293 cells and human brain cortex. (a) FRET assay was performed for analyzing interaction of HAP1 and APP. FRET ratio was measured by SP5 Leica confocal microscope in HEK293 cells cotransfected with APP-YFP / pECFP and HAP1-CFP/pEYFP plasmids respectively as negative control (ii and iii), with P75-CFP-YFP plasmid as positive control (iv), and with HAP1-CFP /APP-YFP plasmids (i) as test. CFP was showed in blue and YFP in red. Results of FRET ratio were quantified as histograms. (b and c) Co-IP of APP and HAP1. (b) Cell lysate from HEK293 cells cotransfected APP-YFP and HAP1-CFP plasmids was incubated with APP antibody (rabbit APP c-terminal polyclonal antibody) for immunoprecipitation. HAP1 and APP in the Co-IP samples were detected by western blots, respectively. (c) Co-IP of APP from human brain homogenate. APP from human cortical lysates was immunoprecipitated by rabbit anti-APP antibodies and both APP and HAP1 in the samples were probed by APP and HAP1 antibodies respectively. In parallel samples, pre-immune rabbit IgG was added for negative controls and non-precipitated samples were loaded for input positive controls. (d) The five GST-HAP1 constructs (HAP1 153–599, HAP1 215–599, HAP1 240–599, HAP1 328–599 and HAP1 371–599) (upper). APP-YFP lysate was used for incubation with all GST-HAP1 fusion proteins in GST-pulldown assays. APP-YFP is indicated by an arrow from pulled down and input samples (bottom), which was blotted with mouse anti-APP antibody. The APP binding region is indicated between dashed lines.

mouse homogenates APP distributed near the bottom of the gradient (Fig. 4b), indicating HAP1 may also regulate the APP distribution. Thus, our data suggest that the APP intracellular distribution in HAP1)/) mice is indeed altered as compared to HAP1+/+ mice. Interestingly, it appears that a small proportion of APP in the HAP1)/) knockout cells is distributed in the fractions of the lowest density, which is consistent with plasma membrane/lipid raft and endocytic vesicle localization. This phenomenon is not selective to APP, as it is also observed for the cis-Golgi marker GM130 and ER marker GRP78. Conversely, the early endosome marker EEA1 redistributes to denser fractions. These results were reproducible as we can observe the changes in all three separate experiments. Our data suggest that HAP1 may play roles in sorting and transporting these important markers.

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homogenates of HAP1+/+ and HAP1)/) mice, we examined APP and organelles markers including EEA1, GM130, GRP78,and LAMP1 by using western blot and did not find any difference in the expression level (Fig. 4a). However, the results from sucrose gradient fractionation showed that the APP distribution was altered. In HAP1)/) brain, APP was codistributed with cis-Golgi and ER, whereas in the HAP1+/+

HAP1 affects APP endocytosis and cytoplasmic membrane re-insertion To further investigate the mechanism underlying the alteration of APP intracellular distribution, we analyzed APP internalization and cytoplasmic membrane re-insertion by internalization and biotinylation assays. The monoclonal antibody 6E10 was used for the APP internalization assay as described (Huang et al. 2009; Okada et al. 2010). 6E10 binds cell surface APP and can be internalized into the cytoplasm when cells are incubated at 37C for different time (also called activation time). Staining intensity of cell surface or intracellular 6E10 with an immunocytochemical technique would generate the ratio of



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Fig. 3 Co-localization of amyloid precursor protein (APP) and organelle marker proteins in primary cortical neurons of HAP1+/+ and HAP1)/) mice. Cortical neurons from neonatal mice were isolated and cultured for 6 days in serum free medium. Neurons on coverslips were stained immunocytochemically with respective antibodies. All confocal images and co-localization rates were performed on Leica SP5 Spectral Confocal Microscopy. The bar graphs on the right panels showed the co-localization rates corresponding to the markers shown on the left panels. The co-localization rates were recorded by microscope with the same threshold set-ups and analyzed as described in Materials and Methods. All data are present as a mean ± SE, n ‡ 8 in each group, Student’s t-test.

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APP internalization. Our results showed there was a significant difference in APP internalization between HAP1+/+ and HAP1)/) mice. For example, HAP1+/+ neurons showed very low internalized APP during the earlier period of the activation (0–10 min) (Fig. 5a), and APP endocytosis increased abruptly at 10–20 min after the activation, and then remained steady between 20 min and 30 min. In contrast with HAP1+/+ neurons, APP internali-

zation was immediately observed after activation and increased slowly during 30 min in HAP1)/) neurons. Furthermore, the ratio of internalization was also lower in HAP1)/) neurons than in HAP1+/+ mice at 20 and 30 min post activation (Fig. 5a). To see whether the internalized APP is colocalized with HAP1, we performed double labeling. The data showed that the internalized APP and HAP1 were partially co-localized (Fig. 5b).



Table 1 Summary of APP and organelle marker protein co-localization in primary cortical neurons between from HAP1+/+ and HAP1)/) mice Co-localization rate %

Organelle marker proteins

HAP1+/+

HAP1)/)

p-value

Relative to HAP1+/+, APP subcellular distribution in HAP1)/)

cis-Golgi complex(GM130) cis-Golgi complex(giantin) trans-Golgi complex(Golgi97) Early endosome(EEA1) ER-Golgi intermediate compartment (SEC22b) Autophage(beclin1) Endoplasmic reticulum(GRP78) Late endosome(CD71) Lysosome(Lamp1) Retromer (Vps35)

10.82 52.26 50.86 60.7 51.36 88.04 83.08 54.33 80.37 64.27

16.43 69.38 66.44 70.37 84.93 68.47 75.31 65.26 78.07 54.98

0.000 0.032 0.000 0.028 0.044 0.001 0.059 0.352 0.650 0.183

› › › › › ﬂ – – – –

The table shows the names of organelle following the marker protein in the brackets, co-localization rate, p value analyzed by SPSS13.0 software and alteration trend judged according to p value. Statistical significance (p < 0.05) was assessed using the Student’s t-test. APP, amyloid precursor protein; HAP1, huntingtin associated protein 1.

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Fig. 4 Effects of huntingtin associated protein 1 (HAP1) knockout on amyloid precursor protein (APP) distribution in subcellular fractions. (a) Brain lysates from HAP1+/+ and HAP1)/) mice were detected by western blot using antibodies of APP, EEA1 (early endosome), GM130 (cis-Golgi marker), GRP78 (endoplasmic reticulum marker), LAMP1 (lysosome marker) and b-actin. No significant difference in the expression of these markers and APP was seen. (b) The membranous organelles from the brain of HAP1+/+ and HAP1)/)

mice were fractionated by sucrose gradient centrifugation and different fractions were subjected for immunoblotting for HAP1 (1 and 2), APP (3 and 4), EEA1 (early endosome marker, 5 and 6), GM130 (cis-Golgi marker, 7 and 8), integrin (cell membrane marker, 9 and 10) and GRP78 (endoplasmic reticulum marker, 11 and 12). The triangle on the top of panels marks the sucrose gradient fractions from the bottom to the top. Stars mark the APP distribution peaks in HAP1)/) mice.

We further investigated the effect of HAP1 on APP internalization by cell surface biotinylation assays. We analyzed APP biotinylation at three phases: (i) incubation with biotin and endocytosis at 0C, as a negative control; (ii) 30 min activation for endocytosis at 37C after incubation (endocytosed APP); and (iii) after incubation, activation at 37C for 30 min, and then the cell surface biotin was stripped and activated again for 30 min at 37C to measure re-inserted

APP (APP re-insertion). Our results showed that the endocytosed biotinylated APP levels were similar between HAP1+/+ and HAP1)/) neurons at 4C (inactivation) (Fig. 5c, lane 1). The endocytosed biotinylated APP in the cytoplasm was also indistinguishable between HAP1+/+ and HAP1)/) neurons after activation in 37C for 30 min (Fig. 5c, lane 2). After cell surface biotin stripped and cells activated again to allow the biotinylated APP re-cycling and



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Fig. 5 The effect of huntingtin associated protein 1 (HAP1) knockout on amyloid precursor protein (APP) endocytosis and re-insertion in primary neurons. (a) APP antibody 6E10 internalization in primary cortical neurons from HAP1+/+ and HAP1)/) mice, as described in Material and Methods. Endocytosed 6E10 relative to total 6E10 was measured in neurons incubated at 37C medium (activation) for 0 min (1st group), 10 min (2nd group), 20 min (3rd group) and 30 min (4th group). Internalization ratio of activation minus ratio at 0 min as relative internalization ratio for statistics. All the data are presented as means ± SE. Three independent experiments were performed (n ‡ 15 cells for each condition per experiment, **p < 0.01, *p < 0.05, one-way analysis of variance (ANOVA). (b) Confocal images for co-localization of endocyto-

sed 6E10 and HAP1. (c) Biotinylation assay analyzing APP endocytosis and re-insertion by using streptavidin agarose pull-down and western blot. Lane 1 is negative control, for neurons which were kept on ice (inactive) after incubation with EZ-Link-Sulfo-ss-Biotin; lane 2 is endocytosed APP, for neurons which were activated in 37C medium before the surface biotin was bleached; and lane 3 is measurement of APP reinsertion, for neurons which were subjected for the treatment as those in lane 2, but activated again for endocytosed APP re-inserting into cell membrane and subjected for stripping. (d) Quantitative analysis for ratio of biotinylated APP relative to input APP in c using imageJ. All data are presented as a mean ± SE. Three independent experiments were performed, *p < 0.05, one-way analysis of variance (ANOVA).

re-inserting into cytoplasm, HAP1+/+ and HAP1)/) neurons were significantly different (Fig. 5c, lane 3). The biotinylated APP left in cytoplasm of HAP1)/) neurons was more than that in HAP1+/+ neurons, suggesting that the absence of HAP1 reduced APP re-cycling and re-insertion into the cytoplasm membrane.

vesicles trafficking, including anterograde and retrograde transport (Fig. 6a). To confirm these findings, primary neurons were transfected with APP-YFP, and the trafficking rate of APP-YFP containing vesicles was quantified using the FRAP assay. In HAP1+/+ neurons, the fluorescence was recovered in about 30 s, but in HAP1)/) neurons it recovered in nearly 60 s (Fig. 6b and c). When HAP1)/) neurons were cotransfected with HAP1-CFP plasmid, the recovery rate of APP-YFP vesicles was increased to the level of the control (Fig. 6b). These data suggest that HAP1 promotes APP vesicles trafficking.

HAP1 increases the ratio and rate of APP trafficking To elucidate the roles of HAP1 in APP trafficking, we performed live imaging and FRAP. The live imaging data showed significant differences in three aspects of vesicle movements (anterograde transport, retrograde transport and motionless). In HAP1+/+ neurons the percentage of moving APP-vesicles was more than that in HAP1)/) neurons. Among the moving APP vesicles, both anterograde and retrograde transport APP vesicles were reduced in HAP1)/) mice, suggesting HAP1 promotes the APP

HAP1 knockdown increases the Ab production Ab is the metabolic product of APP, and the alteration of APP vesicles trafficking may affect Ab production, especially when APP mainly locates in endosomes and Golgi complexes (Koo and Squazzo 1994; Pasternak et al. 2004;



Small and Gandy 2006). In order to identify whether HAP1 regulates the Ab levels, neurons from APPSwe/PS1dE9 transgenic neonatal mice were cultured and the HAP1 gene was knockdown using siRNA mixture, and then the level of Ab production was measured with an ELISA assay. Western blot results confirmed that HAP1 protein was down-regulated by 50–60%, and inhibitory effect of siRNA mixture was dose-dependent (Fig. 7a and b) whereas the siRNA mixture treatment had no effect on APP expression (Fig. 7c and d). However, the Ab levels in the medium from neurons, after HAP1 knockdown, were higher than the control neurons (Fig. 7e). These results indicate that down regulation of HAP1 increases Ab production of neurons.

(a)

(b)

Discussion (c)

Fig. 6 Roles of huntingtin associated protein 1 (HAP1) in APP-YFP trafficking in primary neurons from HAP1+/+ and HAP1)/) mice by using BioStation live imaging and FRAP assays. (a) Live image analysis, primary cortical neurons were transfected with the APP-YFP plasmid after seeding 5–6 days. After 18–24 h, photos on positive neurons were taken every 3 s within 2 min on BioStation. From the first photo, the vesicles containing APP-YFP were randomly marked and the numbers of anterograde or retrograde movement of marked vesicles or stationary vesicles (motionless) were recorded using a AR3.1 software. The data are mean ± SE (n = 60.Three independent experiments were performed per cell type *p < 0.05, **p < 0.01). (b) FRAP assay of APP-YFP trafficking in primary cortical neurons. Three groups of neurons were analyzed: HAP1+/+ neurons transfected with APP-YFP (HAP1+/+), HAP1)/) neurons transfected with APP-YFP (HAP1)/)), HAP1)/) neurons cotransfected HAP1-CFP and APP-YFP (HAP1)/), rescue). After bleaching, the mean fluorescence intensity recovered relevant to time after photobleaching were recorded and compared among the three groups. The data presented are average recovery rate (mean ± SE, n ‡ 8 cells per group. *p < 0.05). (c) FRAP images show time course including prephotobleaching, photobleaching and after-photobleaching fluorescence recovery. The arrows indicate the site bleached.

HAP1 is the cytoplasmic protein that interacts with many proteins such as Huntingtin, p150Glued, KLC, TrkA, TATAbinding protein, proBDNF, and plays roles in microtubuledependent transport, vesicular trafficking, nerve growth, signal transduction and transcriptional regulation (Li et al. 1998; McGuire et al. 2006; Rong et al. 2006; Takeshita et al. 2006; Wu et al. 2010). Using APP vesicular trafficking as a model, McGuire et al. (McGuire et al. 2006) found that HAP1 interacts with KLC and is required for APP trafficking. However, how HAP1 regulates APP trafficking and the significance underlying the HAP1 regulation of APP trafficking are not clear. We have shown here, for the first time, that endogenous APP and HAP1 are highly co-localized in primary cortical neurons of mice and in the human brain cortex, which is similar to that in PC12 cells cotransfected with APP and HAP1 DNA constructs (McGuire et al. 2006), suggesting that HAP1 may play physiological roles in APP process. To elucidate the issue, we have found that HAP1 and APP are physically associated with each other. Our data support this notion:(i) high FRET efficiency results from APP-YPF and HAP1-CFP pair, indicating these two proteins are very close to each other in vivo; (ii) HAP1 can be immunoprecipitated by anti-APP antibody from lysates of cotransfected HEK293 cells and human cerebral cortex and GST-HAP1 fragments between 153 and 599 aa can pull down APP from cell lysate, and (iii) APP and HAP1 are highly colocalized in human brain. These experiments eliminate any possible artifacts and suggest that HAP1 and APP indeed form a complex in physiological conditions of human brains. We further mapped the binding sites between APP and HAP1, showing that HAP1 amino acids 371–559 contain the key binding site for APP. However, our data cannot conclude the possibility that these two proteins directly interact with each other as we only used APP cell lysates in our experiments. It is likely that HAP1 indirectly interacts with APP via other adaptor proteins such as Mint/X11 (Hilgenberg and Smith 2007), Fe65 (Wang et al. 2009) and kinesin light Chain (McGuire et al. 2006) which are associated with APP, Our



(a)

(b)

(c)

(d)

(e)

Fig. 7 Effect of huntingtin associated protein 1 (HAP1) knockdown on Ab level in medium of primary neurons from APPSwe/PS1dE9 transgenic neonatal mice. (a) HAP1 expression was interfered with HAP1 siRNA mixture in different concentrations. No treating means blank control; negative control refers to the treatment with the scrambled siRNA; and HAP1 siRNA refers to the treatment with different concentrations of specific interference RNA mixture to HAP1. Western blot detect the HAP1 expression (upper panel) and b-actin (lower

panel). (b) Histogram of HAP1 expression after treatment with siRNA mixture. Using HAP1 expression in blank control as 100%, quantitative analysis was performed accordingly. (c and d) western blot analyzed the APP expression in APPSwe/PS1dE9 transgenic neurons, in which HAP1 was knockdown (c), (d) Quantitative analysis of APP levels after different treatments as shown in c. (e) Ab level in the media of APPSwe/PS1dE9 transgenic neurons knockdown with HAP1 siRNA mixture (n = 8, *p < 0.05, **p < 0.01 one-way analysis of variance).

future studies will elucidate whether these possible proteins are present in the HAP1/APP complex and whether HAP1 expression in AD brain is altered. Thus, these morphological and biochemical data strongly suggest that HAP1 may participate in APP sorting, trafficking and processing. As APP traverses both the exocytic and endocytic pathways (Thinakaran and Koo 2008; Tang 2009), it is essential to determine whether HAP1 regulates anterograde trafficking of APP. In the present study, we found that APP is significantly accumulated in the cis-Golgi, TGN, endosomes, and ER-Golgi intermediate compartments, and reduced in autophagies in HAP1 knockout mice. These data suggest that HAP1 normally promotes the anterograde transport of APP containing vesicles to the cytoplasmic membrane. Indeed, the biotinylation assays

showed that the re-insertion of APP to the cell membrane is reduced in HAP1 knockout neurons (biotinylated APP increased inside HAP1)/) neurons). These data clearly demonstrate that HAP1 regulates the anterograde trafficking and cytoplasmic membrane insertion of APP in neurons. Our live imaging data has showed that the number of anterograde transported APP vesicles is significantly decreased but the number of motionless APP-vesicles is increased in HAP1)/) neurons, further supporting the concept that HAP1 regulates the anterograde trafficking of APP. Since both HAP1 and APP are involved in the neurite growth (Hung et al. 1992; Rossjohn et al. 1999; McGuire et al. 2006; Rong et al. 2006, 2007) which requires anterograde transport of membrane vesicles and membrane insertion, it is likely that the interaction of



HAP1 and APP is involved in neurite growth and membrane insertion. However, HAP1 also regulates anterograde transport and membrane insertion, or release, of other molecules such as GABAA receptors (Kittler et al. 2004; Twelvetrees et al. 2010), proBDNF (Wu et al. 2010) and trkA (Rong et al. 2006), which control neurite growth and nerve synaptic activities. HAP1 may also regulate the retrograde trafficking of APP. It is well known that APP can be endocytosed and retrogradely transported from cell membrane to Golgi (von Arnim et al. 2006; Wahle et al. 2006). Retrograde transport of APP from endosomes to Golgi is regulated by retromers (Andersen et al. 2005; Rogaeva et al. 2007; Okada et al. 2010). Downregulation of retromers leads to the accumulation of APP in endosomes and increases the production of Ab (He et al. 2005; Small et al. 2005; Rogaeva et al. 2007; Muhammad et al. 2008; Small 2008). In the present study we have found that the surface APP internalization is disregulated in HAP1 knockout neurons, as shown by the APP antibody imaging assay being increased at 0C during the inactivation stage whereas activated internalization were reduced. Although the biotinylation assay did not show any difference in APP internalization between HAP1+/+ and HAP1)/) neurons during the 30 min activation period, the APP recycling was significantly reduced in HAP1 knockout neurons. We have not found a significant difference in APP co-localization with the retromer marker VPS35 in HAP1 knockout neurons (data not shown), suggesting that HAP1 may not be involved in the APP retrograde trafficking from the retromer to Golgi. Nevertheless, the number of retrogradely moving APP containing vesicles is reduced in HAP1 knockout neurons, indicating that HAP1 may be involved in the retrograde trafficking between different types of vesicles. For example, the reduction of APP in autophagies in HAP1 knockout neurons may be due to the defect in APP retrograde trafficking. HAP1 may decrease the Ab production by regulating APP inter-organelles trafficking. As APP traverses among the subcellular compartments accompanied by proteolytic processing, APP and its secretases are sorted through the plasma membrane and membrane of organelles, including the transGolgi network (TGN) and endosomes (Small and Gandy 2006). When APP and secretases are sorted into the same intracellular compartments, the secretases may play roles in APP proteolysis and regulate the Ab production. Ab is the product of BACE and c-secretase processing on APP. BACE predominantly localizes to endosomes and the TGN (Huse et al. 2000), whereas the c-secretase complex and enzyme activity are in multiple compartments, including the ER-Golgi intermediate compartment, Golgi, TGN, endosomes and plasma membrane (Thinakaran and Koo 2008). Our data showed that APP has high co-localization ratio in cis- and trans-Golgi compartments, ER-Golgi intermediate compartments and early endosomes after HAP1 deletion, suggesting APP retains and spends more residential time in these

compartments which, by coincidence, contains the molecular machinery necessary for both b- and c-cleavages (Tang 2009). Our ELISA assay data illustrates that the Ab level in the culture media of HAP1 knockdown neurons was significantly increased, whereas the levels of APP protein expression did not altered in HAP1 knockdown neurons. This data support our notion that HAP1 may reduce Ab production by facilitating anterograde transport and membrane insertion of APP and directing APP into the non-amyloidogenic pathway. An alternate explanation for increased Ab level in HAP1 knockdown neurons may be altered trafficking of APP into autophagies. Beclin1 is a protein with a key role in autophagy, which is decreased in affected brain regions of patients with Alzheimer disease early in the disease process (Pickford et al. 2008) and reduction of Beclin 1 expression in AD mice increases Ab intracellular accumulation and extracellular deposition, and causes neurodegeneration (Lee and Gao 2008; Pickford et al. 2008; Jaeger et al. 2010). The reduction in the co-localization of APP and Beclin 1 in HAP1 knockout neurons found in the present study suggests that HAP1 may promote the trafficking of APP into autophagy vacuoles and increase degradation of APP and the metabolites in the organelles, subsequently reducing Ab.

Conclusion In summary, our findings suggest that HAP1 co-localizes and associates with APP in physiological conditions of mouse and human brain. HAP1 knockout leads to the accumulation of APP in ER-cis Golgi intermediate compartments, TGN, endosomes and reduction in autophagies, reduction in APP membrane recycling and alteration in APP endocytosis. HAP1 knockout also causes the defect in APP movement in neurons and increases Ab levels. This means that HAP1 may promote the trafficking of APP into the non-amyloidogenic pathway and reduces production of Ab. Thus regulation of HAP1 expression may help to control Ab production and affect the development of Alzheimer’s disease.

Acknowledgements We thank Dr. Xiaojiang Li of Emory University for the gift of HAP1 knockout mice, Dr. LS Goldstein of University California for the gift of APP-YFP plasmid, Dr. JT Kittler of University of College London for the GST-HAP1 fragment plasmids and Dr. Patrick Falckh of Flinders University for reading and criticising the manuscript. This work was supported by NHMRC grants. Authors declare no conflict of interest for this study.

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