THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 19, pp. 12048 –12057, May 8, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Amyloid Precursor Protein Enhances Nav1.6 Sodium Channel Cell Surface Expression* Received for publication, October 13, 2014, and in revised form, March 10, 2015 Published, JBC Papers in Press, March 12, 2015, DOI 10.1074/jbc.M114.617092

Chao Liu‡§¶, Francis Chee Kuan Tan‡§¶, Zhi-Cheng Xiao储**1, and Gavin S. Dawe‡§¶2 From the ‡Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 10 Medical Drive, Singapore 117597, the §Neurobiology and Ageing Programme, Life Sciences Institute and ¶Singapore Institute for Neurotechnology (SINAPSE), Centre for Life Sciences, National University of Singapore, 28 Medical Drive, Singapore 117456, the 储 Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical College, Kunming 650031, China, and the **Shunxi-Monash Immune Regeneration and Neuroscience Laboratories, Department of Anatomy and Developmental Biology, Monash University, Clayton, Melbourne, Victoria 3800, Australia Background: Loss-of-function amyloid precursor protein (APP) and Nav1.6 transgenic mice share similar phenotypes. Results: APP interacts with Nav1.6 and enhances its cell surface expression through a Go-coupled JNK pathway. Conclusion: APP regulates Nav1.6 function, likely through a Go-coupled JNK pathway. Significance: This finding advances the current understanding of APP functions and has implications for novel therapies for neurodegenerative disorders. Amyloid precursor protein (APP) is commonly associated with Alzheimer disease, but its physiological function remains unknown. Nav1.6 is a key determinant of neuronal excitability in vivo. Because mouse models of gain of function and loss of function of APP and Nav1.6 share some similar phenotypes, we hypothesized that APP might be a candidate molecule for sodium channel modulation. Here we report that APP colocalized and interacted with Nav1.6 in mouse cortical neurons. Knocking down APP decreased Nav1.6 sodium channel currents and cell surface expression. APP-induced increases in Nav1.6 cell surface expression were Go protein-dependent, enhanced by a constitutively active Go protein mutant, and blocked by a dominant negative Go protein mutant. APP also regulated JNK activity in a Go protein-dependent manner. JNK inhibition attenuated increases in cell surface expression of Nav1.6 sodium channels induced by overexpression of APP. JNK, in turn, phosphorylated APP. Nav1.6 sodium channel surface expression was increased by T668E and decreased by T668A, mutations of APP695 mimicking and preventing Thr668 phosphorylation, respectively. Phosphorylation of APP695 at Thr-668 enhanced its interaction with Nav1.6. Therefore, we show that APP enhances Nav1.6 sodium channel cell surface expression through a Go-coupled JNK pathway.

Amyloid precursor protein (APP)3 is best known for its involvement in the pathogenesis of Alzheimer disease (AD), in

* This work was supported by the National Medical Research Council, Ministry of Health, Singapore with NMRC NUHS Centre Grant—Memory, Aging, and Cognition Centre Seed Funding, NMRC/CG/013/2013 (to G. S. D.) and in collaboration with Z. C. X. (Grant NMRC/1059/2006). This work was also supported by grants from the Professorial Fellowship of Monash University, Australia, and by the Talent Program of Yunnan Province, China (to Z. C. X.). 1 To whom correspondence may be addressed: Tel.: 61-3-9902-4574; Fax: 61-3-9905-0680; E-mail: [email protected]. 2 To whom correspondence may be addressed: Tel.: 65-6516-8864; Fax: 65-6777-3271; E-mail: [email protected]. 3 The abbreviations used are: APP, amyloid precursor protein; AD, Alzheimer disease; hAPP, human amyloid precursor protein; PTX, pertussis toxin;

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which amyloid plaques consisting of aggregates of the amyloid ␤ peptide released by sequential cleavage of APP are a hallmark of the neuropathology. Despite immense interest in the pathological role of amyloid ␤ peptide, the physiological functions of APP itself are poorly understood, although it has been implicated as a regulator of synapse formation (1), neural plasticity (2), and iron export (3). APP-deficient mice exhibited decreased locomotor activity and forelimb grip strength, indicating compromised neuronal or muscular function (4). In contrast, human amyloid precursor protein (hAPP) transgenic mice exhibit clusters of hyperactive neurons, neuronal network hyperexcitability, and spontaneous epileptiform activity (5–7). APP is an integral membrane glycoprotein consisting of an extracellular domain, a single transmembrane domain, and a short cytoplasmic tail. The APP intracellular domain contains a Go protein-binding domain, His-657 to Lys-676 of APP695 (8 –10). Moreover, an antibody, 22C11, to the extracellular domain of APP can act as a ligand mimetic to activate Go protein, demonstrating that APP can act like a G protein-coupled receptor (8). Neuron-specific phosphorylation of APP695 at Thr-668, within the Go protein-binding domain of APP, can alter APP binding to partners (11). In the brain, phosphorylation of APP695 at Thr-668 is mediated by neuronal CDK5 (12), GSK-3␤ (13), or JNK3 (14). Voltage-gated sodium channels are responsible for action potentials in excitable cells. In addition to producing the large, transient current responsible for the upstroke of the action potential, sodium channels carry smaller currents at subthreshold voltages that contribute to the generation of spontaneous action potentials (15). Four distinct ␣ subunits of sodium channels are expressed in mammalian CNS neurons: Nav1.1, Nav1.2, Nav1.3, and Nav1.6 (16). Nav1.6 (encoded by the SCN8A gene) channels are highly concentrated at nodes of Ranvier in both the peripheral and central nervous systems; are also

DIV, day(s) in vitro; ANOVA, analysis of variance; TfR, transferrin receptor.

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APP Enhances Sodium Channel Cell Surface Expression present in unmyelinated axons, including axons in the retina and parallel fibers in the cerebellum; and are prominent in dendrites of cerebral cortical pyramidal cells and cerebellar Purkinje cells (17). Previous studies have demonstrated that Nav1.6 is a key determinant of neuronal excitability in vivo. Haploinsufficiency is associated with impaired cognition (18, 19), whereas hyperactivity can result in epilepsy (20). A hypomorphic allele of SCN8A results in severe muscle weakness and dystonic postures (21). SCN8Amedj has only 12% of transcripts correctly spliced. This low level of expression prevents paralysis, but homozygotes have severe muscle weakness and cannot support their own weight. Reduced repetitive firing has been observed consistently in cerebellar Purkinje cells, granule neurons, trigeminal mesencephalic neurons, and retinal ganglion cells from Scn8a mutant mice (22–25). A Nav1.6 gain-of-function mutant, SCN8A-p.N1768D, was discovered in a child with an early-onset, debilitating epileptic encephalopathy. The biophysical properties of the mutant channel include an increase in persistent sodium current, incomplete channel inactivation, and a depolarizing shift in the voltage dependence of steady-state fast inactivation (20). Transfection of mouse hippocampal neurons with the mutant cDNA resulted in increased spontaneous and induced firing, characteristic of neuronal hyperexcitability, consistent with the dominant expression of seizures in the heterozygous patient. Because gain of function and loss of function of APP and Nav1.6 share similar phenotypes in mouse models, we hypothesized that APP might be a candidate molecule for sodium channel modulation. We investigated the localization of APP and Nav1.6 in cultured cortical neurons. Next we investigated the interaction of APP with Nav1.6 sodium channels and found that APP promotes the surface expression of sodium channels by a Go protein-coupled JNK pathway.

EXPERIMENTAL PROCEDURES Antibodies, Inhibitors, and Activators—Antibodies against APP were as follows: catalog no. APP Y188 (rabbit monoclonal, Abcam), catalog no. APP MAB348 (mouse monoclonal, Millipore), and catalog no. APP A8717 (rabbit polyclonal, Sigma). Antibodies against Nav1.6 were as follows: catalog no. Nav1.6 ACS-009 (rabbit polyclonal, Alomone Laboratories), catalog no. Nav1.6 WH00063334M4 (mouse monoclonal, Sigma), and catalog no. Nav1.6 sc-81884 (mouse monoclonal, Santa Cruz Biotechnology). Monoclonal antibodies against ankyrin G (catalog no. sc-31778, goat polyclonal, Santa Cruz Biotechnology), transferrin receptor (catalog no. 13-6800, Invitrogen), G␣o (catalog no. sc-13532, Santa Cruz Biotechnology), ␥-tubulin (catalog no. T6557, Sigma), and ␤3-tubulin (catalog no. MMS435P, Covance) and polyclonal antibodies against phosphoAPP (Thr-668 in APP695 or the corresponding position in other isoforms, catalog no. 3823, Cell Signaling Technology) and phospho-c-Jun (Ser-63, catalog no. 2361, Cell Signaling Technology) were obtained from the respective companies. Normal mouse IgG (catalog no. sc-2025) was purchased from Santa Cruz Biotechnology, and normal rabbit IgG (catalog no. 2729) was from Cell Signaling Technology. Pertussis toxin (PTX) (Sigma), mastoparan (Tocris Bioscience), JNK inhibitor MAY 8, 2015 • VOLUME 290 • NUMBER 19

III (Calbiochem), 1-azakenpaullone (Calbiochem), and roscovitine (Calbiochem) were purchased from the respective commercial sources. Animals—The APP null (4) mice were as described previously. The Institutional Animal Care and Use Committee of the National University of Singapore approved all experiments involving mice. Primary Cortical Culture—Cerebral hemispheres from postnatal day 0 –1 mice were extracted, and meninges and blood vessels were carefully removed under a stereoscope in dissection medium consisting of Hanks’ balanced salt solution (Invitrogen) supplemented with 0.01 M HEPES (Invitrogen). The cortex was isolated and cut into small pieces with sharp scissors, and tissue pieces were transferred to a 1.5-ml tube with 1 ml of digestion medium (1.5 mM CaCl2, 0.2 mg/ml L-cysteine, 0.5 mM EDTA, 20 units/ml DNase I, and 15 units/ml papain in dissection medium). The brain tissue was then incubated in a 37 °C water bath for 30 min with frequent swirling. An equal amount of DMEM with 10% (v/v) FBS (Invitrogen) was then added to stop the papain activity. The pelleted tissues and cells were washed twice with 2 ml of culture medium (Neurobasal medium with 2% (v/v) B-27 supplements and 2 mM GlutaMAX, 33 mM D-(⫹)-glucose, and 1⫻ penicillin/streptomycin; Invitrogen) and resuspended with 2 ml of culture medium. The tissue was dissociated further by passing it through a 1-ml tip five times. Dissociated cells were then filtered through a 40-␮m nylon cell strainer (BD Falcon), transferred to a new 50-ml tube, counted, and plated onto poly-L-lysine (Sigma)-coated glass coverslips or a plastic 6-cm plate. Immunocytochemistry—14 DIV mouse primary cortical cultures were rinsed with Hanks’ balanced salt solution and fixed with 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer for 10 min. After three washes with PBS, the cortical cultures were incubated overnight with mouse antibody against Nav1.6 (1:250, Sigma) and rabbit antibody Y188 against APP (1:250, Abcam) in primary antibody dilution buffer (1% v/v BSA, 0.3% v/v Triton X-100, and 0.01% w/v NaN3 in PBS) at 4 °C. After three washes with PBS, the cells were incubated with a mixture of Alexa Fluor 488 donkey anti-Mouse IgG (H⫹L) antibody (1:200, Invitrogen) and Alexa Fluor 555 donkey anti-rabbit IgG (H⫹L) antibody (1:200, Invitrogen) for 40 min at room temperature. After three washes with PBS, the cells were mounted and dried overnight in the dark. The cells were visualized and photographed using a confocal microscope system (LSM 510, Zeiss Microimaging GmbH). Duolink in Situ Interaction Assay—The interaction between Nav1.6 and APP in cortical cultures was detected in situ using the Duolink II secondary antibodies and detection kit (catalog nos. 92001, 92005, and 92008; Olink Bioscience, Uppsala, Sweden) according to the instructions of the manufacturer. Briefly, primary antibody incubation against Nav1.6 (catalog no. sc-81884, Santa Cruz Biotechnology) and APP (catalog no. Y188, Abcam) were applied using the same conditions as immunocytochemistry staining. Duolink secondary antibodies against the primary antibodies were then added. These secondary antibodies were provided as conjugates to oligonucleotides that were able to form a closed circle via base pairing and ligation using Duolink ligation solution when the antibodies were JOURNAL OF BIOLOGICAL CHEMISTRY

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APP Enhances Sodium Channel Cell Surface Expression in close proximity (26) at a distance estimated to be ⬍40 nm (27). The detection of the signals was conducted by rolling circle amplification using DNA polymerase incorporating fluorescently labeled nucleotides into the amplification products. The resulting positive signals were visualized as bright fluorescent dots, with each dot representing one interaction event. The specificity of this assay was assessed by staining APP KO primary cortical cultures (these cultures do not express APP; therefore, no positive signals are obtained from APP/Nav1.6 interactions). The cells were visualized using a confocal microscope system (LSM 510, Zeiss). Cell Culture and Transfection—HEK293 cells stably expressing Nav1.6 were obtained from Dr. J. J. Clare (28) and grown in DMEM supplemented with 10% (v/v) FBS and 400 ␮g/ml G418 (Invitrogen). HEK293 Nav1.6 cells were transfected with various plasmids using Effectene transfection reagent (Qiagen) or with siRNA using Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the instructions of the manufacturer. Two days after transfection, the cells were used for experiments. Electrophysiological Recording in HEK293 Nav1.6 Cells— HEK293 Nav1.6 cells grown on glass coverslips were placed in bath solution containing 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4). All recordings were performed at room temperature (20 –24 °C) within 2 h after taking the cells out of the incubator. Current signals from HEK293 Nav1.6 cells recorded in whole cell voltage clamp mode were sampled at 20 kHz and filtered at 5 kHz using a MultiClamp 700A amplifier in conjunction with a Digidata 1322A interface and pClamp 8.1 software (Axon Instruments). Micropipettes were pulled from borosilicate glasses (World Precision Instruments) with a Flaming Brown micropipette puller (catalog no. P2000, Sutter Instruments) to an electrode resistance ranging from 2–5 M⍀. The pipette solution contained 115 mM potassium gluconate, 4 mM NaCl, 1.5 mM MgCl2, 20 mM HEPES, and 0.5 mM EGTA (pH 7.4). The pipette potential was zeroed before seal formation, and the voltages were not corrected for liquid junction potentials. The leakage current was digitally subtracted online using hyperpolarizing control pulses, applied before the test pulse, of one-fourth test pulse amplitude (P/4 procedure). Plasmids and siRNA—pcDNA3-FLAG-hAPP695 was a gift from Dr. C. Schmidt. pcDNA3-FLAG-hAPP695 T668E was from Dr. T. Suzuki. pcDNA3-FLAG-hAPP695 T668A was from Dr. S. Itohara. pcDNA3.1(⫹)-G␣o G203T and pcDNA3.1(⫹)G␣o Q205L were purchased from the Missouri University of Science and Technology cDNA Resource Centre. The sequences of APP siRNA were as follows: 5⬘-CCAACCAACCAGUGACCAU[dT][dT] and 5⬘-AUGGUCACUGGUUGGUUGG[dT][dT], synthesized by Sigma. Western Blot Analysis—To prepare total cell lysate, cultured cells were rinsed with PBS and lysed in a lysis buffer (150 mM NaCl, 30 mM HEPES, 10 mM NaF, 1% v/v Triton X-100, 0.01% w/v SDS, and complete protease inhibitor mixtures (pH 7.5)). After centrifugation (16,000 ⫻ g, 4 °C, 10 min), the supernatants were collected, mixed with 4⫻ protein loading buffer, and stored at ⫺20 °C for future use. Equal amounts of protein were separated on acrylamide gels and transferred onto nitrocellu-

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lose membranes. Western blotting was performed under standard conditions, applying antibodies against target proteins or a loading control. Either anti-mouse IgG or anti-rabbit IgG peroxidase-conjugated secondary antibodies were applied at 1:10,000, and blots were visualized with West Pico (Thermo Scientific) or Luminata Forte (Millipore) ECL detection kits. Cell Surface Biotinylation—Cell surface expression of protein was determined according to our protocol published previously (29), with modifications. Briefly, the cultured neurons or treated HEK293 Nav1.6 cells were incubated with SulfoNHS-LC-Biotin (Pierce) in Ca2⫹/Mg2⫹ PBS for 45 min at 4 °C. After stopping the reaction with 10 mM glycine, tissues or cells were lysed in lysis buffer (150 mM NaCl, 30 mM HEPES, 10 mM NaF, 1% v/v Triton X-100, 0.01% w/v SDS, and protease inhibitors (pH 7.5)) for 2 h. After centrifugation (16,000 ⫻ g, 4 °C, 10 min), the supernatants were precipitated overnight with Immunopure Immobilized Neutravidin (Pierce). After washing, the beads were incubated for 15 min at 50 °C in SDS-PAGE loading buffer. PTX (100 ng/ml), mastoparan (10 ␮M), and JNK inhibitor III (10 ␮g/ml) were applied in the assay of cell surface biotinylation. Coimmunoprecipitation—The method described by Cousins et al. (30) was applied with minor modifications. Briefly, adult WT mouse brain was harvested, cut into several pieces, and homogenized in ice-cold lysis buffer (150 mM NaCl, 30 mM HEPES, 10 mM NaF, 1% v/v Triton X-100, 0.01% w/v SDS, and complete protease inhibitor mixtures (pH 7.5)). HEK293 cells were harvested and lysed in the same lysis buffer. The lysates were rotated for 2 h at 4 °C and centrifuged at 100,000 ⫻ g for 40 min. The detergent-soluble supernatants were incubated overnight at 4 °C with each antibody as described in the figure legends, followed by incubation with protein G-Sepharose 4 Fast Flow (GE Healthcare) for 3 h at 4 °C. The immunoprecipitates were washed efficiently with lysis buffer and analyzed by Western blotting. Each experiment was repeated at least three times. Statistics—Data are presented as mean ⫾ S.E. The density of the Western blot bands were normalized to the internal loading control and then normalized to control plasmids or treatments. Student’s t test was used for two group comparisons, and oneway ANOVA was used for multigroup comparison, followed by appropriate post hoc tests.

RESULTS APP Is Colocalized with Nav1.6 in Mouse Cortical Neurons— To explore the potential for functionally relevant interactions between APP and Nav1.6, we first investigated whether the two proteins colocalize in cultured mouse cortical neurons. By 14 DIV, cortical cultures reach a peak in synaptic density and neuronal cell connectivity (31), which reflects maturation of the neuronal network, paralleled by maturation of electrophysiological properties (32, 33). We used the Y188 rabbit monoclonal antibody, which is highly specific for APP (34), and antiNav1.6 antibodies to stain 14 DIV cortical neuron cultures from WT mice and APP KO mice. Two different anti-Nav1.6 antibodies from different sources showed similar results in the immunostaining, and later also in the Duolink experiments, suggesting that the results obtained are Nav1.6-specific. In agreement with previous reports, APP was predominantly VOLUME 290 • NUMBER 19 • MAY 8, 2015

APP Enhances Sodium Channel Cell Surface Expression

FIGURE 1. APP interacts with Nav1.6 in vivo and in vitro. A, confocal images of 14 DIV WT and APP KO primary cortical cultures costained with anti-Nav1.6 (Sigma) and anti-APP (Y188, Abcam) antibodies (representative images from 21 cells from three independent cultures). The insets show ⫻4 lower magnification. Scale bar ⫽ 10 ␮m. B, confocal images showing Duolink fluorescence alone (APP/Nav1.6 interaction, red, left column), differential interference contrast (DIC, center column), and merged with DAPI (right column) in 14 DIV WT and APP KO primary cortical cultures (representative images from 24 cells from three independent cultures). The red dots are positive Duolink signals showing the APP/Nav1.6 interaction. The insets show ⫻2 lower magnification. Scale bar ⫽ 10 ␮m. C, APP interacts with Nav1.6 in vivo. Adult WT mouse brains were homogenized and solubilized in lysis buffer, and then the soluble extracts were collected by centrifugation at 100,000 ⫻ g, immunoprecipitated (IP) using rabbit antibody against the APP C terminus (A8717, Sigma) or normal rabbit IgG control (Cell Signaling Technology), and analyzed by Western blotting using antibodies against Nav1.6, Nav1.2, Nav1.1 (Alomone Laboratories), or APP (MAB348, Millipore) (representative blots from three mice). IB, immunoblot. D, APP interacts with Nav1.6 in vitro. Lysates of HEK293 Nav1.6 cells transfected with pcDNA3-FLAG-APP695 or pcDNA3 collected by centrifugation at 100,000 ⫻ g were immunoprecipitated using mouse monoclonal antibody against FLAG and analyzed by Western blotting using antibodies against Nav1.6 (Alomone Laboratories) or FLAG (Sigma) (representative blots from three independent transfections). Input, 5% of total cell lysate; IP, 50% of IP fraction. The arrows indicate the target bands in the multi-band lanes.

expressed in neurons (34), and Nav1.6 staining was present mostly in the somata and dendrites in mouse cortical neuroglial cultures (35). Colocalization of APP and Nav1.6 was observed in cortical neuron cultures from WT mice but not APP KO mice (Fig. 1A). Colocalized APP and Nav1.6 was observed throughout the somata and proximal neurites. Therefore, we show that APP colocalized with Nav1.6 in cultured mouse cortical neurons. APP Interacts with Nav1.6 in Vitro and in Vivo—Because APP and Nav1.6 are colocalized in cortical neurons, we next investigated whether they interact with each other. The Duolink in situ interaction detection assay can detect proximal proteins in their normal cellular context at normal expression levels (26). We used the anti-APP and anti-Nav1.6 antibodies to Duolink-immunostain 14 DIV cortical neuron cultures. Additionally, we performed Duolink together with immunostaining for Ankyrin G (data not shown) as a neuronal marker (36). Strong Duolink signals were observed in WT cortical neuron cultures, whereas no signals were detected in APP KO neuron MAY 8, 2015 • VOLUME 290 • NUMBER 19

cultures (Fig. 1B). The Duolink staining was distributed throughout the soma and proximal neurites. We also used coimmunoprecipitation assays to detect the interaction of APP with Nav1.6, Nav1.2, and Nav1.1 (the major three sodium channel isoforms in adult mouse brain) in the mouse brain lysates (Fig. 1C) and APP and Nav1.6 in transfected HEK293 Nav1.6 cells (Fig. 1D). Nav1.6, Nav1.2, and Nav1.1 coimmunoprecipitated with APP in mouse brain, and Nav1.6 coimmunoprecipitated with FLAG-APP695 in HEK293 cells as well. However, the coimmunoprecipitation assay cannot distinguish between direct or indirect interactions. Therefore, we show that APP interacts with Nav1.6 in vitro and in vivo. Sodium Currents Are Reduced in APP Knockdown HEK293 Nav1.6 Cells—Given that APP and sodium channels interact in cortical neurons, we hypothesized that APP might modulate the function of sodium channels. To investigate the effects of APP on sodium currents, we performed whole cell patch clamp recordings to measure Nav1.6 currents. Because CNS neurons express Nav1.1, Nav1.2, Nav1.3, and Nav1.6 (16), it is difficult to JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 2. APP enhances Nav1. 6 currents and cell surface expression. A, whole cell recordings of Nav1.6 sodium currents in siRNA control or APP siRNA-transfected HEK293 Nav1.6 cells in which cells were depolarized to a variety of potentials (⫺80 to ⫹20 mV) from a holding potential of ⫺120 mV. n is indicated in the columns. B–D, total protein and cell surface protein isolated from HEK293 Nav1.6 cells transfected with siRNA control or APP siRNA (B), 14 DIV WT and APP KO primary cortical cultures (C), and mock-transfected and FLAG-APP695 overexpressing Nav1.6 HEK293 cells (D) were blotted using antibodies against Nav1.6, APP, TfR, ␥-tubulin (␥-Tub), and ␤3-tubulin (␤3Tub). TfR, ␥-tubulin, and ␤3-tubulin served as loading controls for cell surface or total cell lysate fraction, respectively (29). The bar charts show quantitative analysis of relative surface Nav1.6 (sNav1.6) and total Nav1.6 (tNav1.6) expression. The density of the Western blot bands were normalized to the internal loading control and then normalized to control plasmids or treatments. E, representative full immunoblots for the antibodies against TfR, ␥-tubulin, and ␤3-tubulin. Data were analyzed by unpaired Student’s t test (A) and paired Student’s t tests (B–D). Representative blots from at least three independent experiments were used for statistics. *, p ⬍ 0.05; **, p ⬍ 0.01. Error bars represent mean ⫾ S.E.

isolate pure Nav1.6 currents. Therefore, we recorded Nav1.6 currents in HEK293 cells stably expressing human Nav1.6 (28), which also have robust expression of endogenous APP. Fast sodium currents were activated by stepped depolarization. Sodium current densities were significantly decreased in HEK293 Nav1.6 cells transfected with APP-targeting siRNA compared with control siRNA (Fig. 2A). More than 90% of endogenous APP was knocked down, as determined by Western blotting (data not shown). Therefore, these observations indicate that APP increases sodium current density in mammalian cells. APP Increases Cell Surface Expression but Not Total Protein Expression of Nav1.6 —To investigate whether the effect of APP on sodium currents reflects an increased expression of sodium channels in the cell membrane, we manipulated APP expression and performed cell surface biotinylation and immunoblotting assays. First, reduced APP levels were investigated by knockdown with APP-targeting siRNA in HEK293 Nav1.6 cells and by culturing the cortical neurons from postnatal days 0 –1 APP KO mice and WT littermates. Surface, but not total, lysate

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FIGURE 3. APP promotes Nav1.6 cell surface expression in a Go proteindependent manner. A–E, total protein and cell surface proteins were isolated from HEK293 Nav1.6 cells transfected with a control plasmid or Go Q205L (A); treated with vehicle or mastoparan (MP, 10 ␮M, B); transfected with a control plasmid or Go G203T (C); treated with vehicle or PTX (100 ng/ml, D); transfected with control plasmids, FLAG-APP695 alone, or FLAG-APP695 plus Go 203T (E) were blotted using antibodies against Nav1.6, APP, TfR, and Go. Right panels represent quantitative analysis of relative surface Nav1.6 (sNav1.6) and total Nav1.6 (tNav1.6) expression. ␥-tub, ␥-tubulin. F, representative full immunoblot for the antibody against Go. Representative blots from at least three independent experiments were used for statistics. A–D, paired Student’s t test. E, one-way ANOVA followed by post hoc Bonferroni test. *, p ⬍ 0.05; **, p ⬍ 0.01. Error bars represent mean ⫾ S.E.

expression of Nav1.6 was decreased significantly in APP knockdown versus control HEK293 Nav1.6 cells (Fig. 2B) and APP KO versus WT mice, whereas transferrin receptor (TfR), the internal loading control for the cell surface fraction, did not change significantly (Fig. 2C). Next, we analyzed Nav1.6 cell surface expression in HEK293 Nav1.6 cells overexpressing FLAG-APP695. Surface, but not total, expression of Nav1.6 was increased significantly in FLAG-APP695 overexpression HEK293 Nav1.6 cells versus mock-transfected cells, whereas TfR was not changed significantly (Fig. 2D). Full immunoblots for the antibodies used against Nav1.6, APP (Fig. 1C), ␥-tubulin, ␤3-tubulin, and TfR (Fig. 2E) indicated that the antibodies were reasonably specific. These results demonstrate that APP modulates membrane expression, but not total expression, of Nav1.6 sodium channels. APP-mediated Increases in Nav1.6 Cell Surface Expression Are G Protein-dependent—Various sodium channels are modulated by G proteins (37–39). To investigate whether a Go protein-linked mechanism could contribute to the increase in Nav1.6 surface expression on overexpression of APP, we conducted experiments in HEK293 Nav1.6 cells, which have strong endogenous expression of APP. Both expression of the dominant active Go Q205L (Fig. 3A) and treatment with the Go activator mastoparan (Fig. 3B) significantly up-regulated surface, but not total, expression of Nav1.6, whereas both expression of VOLUME 290 • NUMBER 19 • MAY 8, 2015

APP Enhances Sodium Channel Cell Surface Expression dominant negative Go G203T (Fig. 3C) and treatment with the Go inhibitor PTX (Fig. 3D) significantly down-regulated surface, but not total, expression of Nav1.6. Importantly, expression of the dominant negative Go G203T together with FLAGAPP695 prevented the increase in surface, but not total, expression of Nav1.6 seen following transfection with FLAGAPP695 alone (Fig. 3E). The cell surface loading control TfR was not changed significantly by any of these treatments (Fig. 3). Full immunoblots for the antibody against G␣o indicated that it was reasonably specific (Fig. 3F). These results, obtained by both genetic and chemical approaches, demonstrate that Go protein is involved in the APP-dependent cell surface expression of Nav1.6. APP Activates JNK in a Go Protein-dependent Manner—JNK has been implicated as a downstream signal transducer of Go in APP-mediated cell death (40). The mechanism of JNK activation involves dual phosphorylation on the TPY motif. Activated JNK phosphorylates several downstream targets, such as c-Jun, ATF2, and Elk-1. However, c-Jun is the major substrate that mediates JNK-induced neurotoxicity in AD. JNK potentiates c-Jun activity by phosphorylation of Ser-63 and Ser-73 in the activation domain (41, 42). It has been reported that p-c-Jun(Ser-63) colocalizes with intracellular neurofibrillary tangles in AD brains (43). We therefore investigated whether APP activated JNK by assaying the phosphorylation of c-Jun at Ser-63 in HEK293 Nav1.6 cells. Phosphorylation of c-Jun at Ser-63 was up-regulated by APP695 overexpression and down-regulated by siRNA knockdown of APP (Fig. 4A). The effect of APP695 overexpression on c-Jun Ser-63 phosphorylation was prevented by the Go inhibitor PTX (Fig. 4B). Treatment with the Go activator mastoparan or expression of the dominant active Go Q205L increased c-Jun Ser-63 phosphorylation, whereas expression of the dominant negative Go G203T or treatment with PTX decreased c-Jun Ser-63 phosphorylation (Fig. 4, C and D). Full immunoblots for the antibody against p-c-Jun(Ser63) indicated that it was reasonably specific (Fig. 4E). These data indicate that APP activates JNK via a Go protein-dependent pathway. JNK Is Involved in APP-enhanced Nav1.6 Cell Surface Expression through Go Protein-dependent Phosphorylation of APP— Because APP increases Nav1.6 sodium channel surface expression in a Go protein-dependent manner, we next investigated whether JNK is involved in the regulation of Nav1.6 cell surface expression. HEK293 Nav1.6 cells treated with JNK inhibitor III showed significantly down-regulated surface, but not total, expression of Nav1.6, with no significant changes in TfR (Fig. 4F). Treatment of cells transfected with FLAG-APP695 with JNK inhibitor III prevented the increase in surface, but not total, expression of Nav1.6 seen after transfection with FLAGAPP695 alone (Fig. 4G). These data indicate that JNK is involved in Nav1.6 sodium channel currents and cell surface expression. Because we found that JNK, which is known to phosphorylate APP695 at Thr-668 (14), is activated by a G protein-dependent pathway, we next investigated whether APP phosphorylation is G protein-dependent. HEK293 cells were transfected with Go Q205L or Go Q203T (Fig. 5A) or treated with mastoparan or PTX (Fig. 5B). Expression of the dominant active Go MAY 8, 2015 • VOLUME 290 • NUMBER 19

FIGURE 4. APP activates JNK in a Go-protein-dependent manner, and JNK modulates Nav1.6 cell surface expression. A and B, total proteins isolated from HEK293 Nav1.6 cells transfected with APP695 cDNA to induce APP overexpression (APP OE, A), APP siRNA, or control and transfected with APP695 cDNA (APP OE) or control and treated with PTX (100 ng/ml) or vehicle (B) were blotted using antibodies against p-c-Jun Ser-63 (p-c-Jun63), APP, and ␥-tubulin (␥-tub). C and D, total proteins isolated from HEK293 Nav1.6 cells transfected with Go Q205L or Go G203T or mock-transfected (C) and treated with vehicle control (cont.), mastoparan (MP, 10 ␮M) or PTX (100 ng/ml) (D) were blotted using antibodies against p-c-Jun63, APP, Go, and ␥-tubulin. E, representative full immunoblot for the antibody against p-c-Jun Ser-63. F and G, total proteins and cell surface proteins isolated from HEK293 Nav1.6 cells treated with vehicle or JNK inhibitor III (10 ␮g/ml, F) or transfected with control plasmids, FLAG-APP695, or FLAG-APP695 and treated with JNK inhibitor III (10 ␮g/ml, G) were blotted using antibodies against Nav1.6, FLAG, TfR, and ␥-tubulin. The bottom panels of A–D represent quantitative analyses of relative p-c-Jun Ser-63 expression by one-way ANOVA followed by post hoc Bonferroni test. The bottom panels of F and G represent quantitative analyses of relative surface Nav1.6 (sNav1.6) and total Nav1.6 (tNav1.6) expression by paired Student’s t test or one-way ANOVA followed by post hoc Bonferroni test. Representative blots from at least three independent experiments were used for statistics. *, p ⬍ 0.05; **, p ⬍ 0.01. Error bars represent mean ⫾ S.E.

Q205L or treatment with the Go activator mastoparan increased APP phosphorylation, whereas expression of the dominant negative Go G203T or treatment with PTX decreased APP phosphorylation (Fig. 5, A and B). These data indicate that phosphorylation of APP is activated by a Go protein-dependent mechanism. We next investigated whether JNK is involved in this Go protein-activated phosphorylation of APP by activating JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 5. Go-protein-dependent Thr-668 phosphorylation of APP695 is mediated by JNK and enhances Nav1.6 sodium channel cell surface expression. A–C, total proteins isolated from HEK293 Nav1.6 cells transfected with Go Q205L or Go G203T or mock-transfected (A); treated with vehicle, mastoparan (MP, 10 ␮M) or PTX (100 ng/ml) (B); and treated with vehicle or mastoparan (10 ␮M) or mastoparan (10 ␮M) plus JNK inhibitor III (JNKi, 10 ␮g/ml) (C) were blotted using antibodies against p-APP, APP, Go, and ␥-tubulin (␥-tub). cont., control. D, to show the specificity of rabbit polyclonal antibody against p-APP, lysates of HEK293 Nav1.6 cells transfected with pcDNA3-FLAG-APP695 T668A, treated with vehicle or mastoparan (10 ␮M), were immunoprecipitated (IP) using normal mouse IgG or mouse monoclonal antibody against FLAG and analyzed by Western blotting using antibody against pAPP. E, representative full immunoblot for the antibody against p-APP. The bar charts in A–C represent quantitative analyses of the relative amount of p-APP by one-way ANOVA followed by post hoc Bonferroni test. Representative blots from at least three independent experiments were used for statistics. *, p ⬍ 0.05; **, p ⬍ 0.01. Error bars represent mean ⫾ S.E.

APP phosphorylation with the Go activator mastoparan and applying JNK inhibitor III. As before, mastoparan increased APP phosphorylation, and JNK inhibitor III prevented this increase in phosphorylation (Fig. 5C). Neither the CDK5 inhibitor roscovitine nor the GSK-3␤ inhibitor 1-azakenpaullone was able to prevent the phosphorylation of APP (data not shown). Because T668A mutation of APP695 prevents phosphorylation (44), the specificity of the rabbit polyclonal antibody against p-APP was demonstrated by showing that it did not react with FLAG-APP T668A (Fig. 5D). Moreover, mastoparan treatment did not result in phosphorylation of the FLAGAPP T668A mutant so that it was recognized by the antibody. Full immunoblots for the antibody against p-APP indicated that it was reasonably specific (Fig. 5E). Phosphorylation of APP695 at Thr-668 Increases Nav1.6 Cell Surface Expression—Together, the data above suggest that APP colocalizes with Nav1.6 sodium channels and increases Nav1.6 sodium channel currents by increasing cell surface expression of Nav1.6 sodium channels in a Go protein-dependent manner. APP activates JNK via a Go protein-dependent mechanism and

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JNK then phosphorylates APP. Because JNK3 phosphorylates APP695 at Thr-668 (14), we next investigated whether Thr-668 phosphorylation of APP695 was sufficient to increase surface expression of Nav1.6 sodium channels. APP695 T668E mutation mimics phosphorylation of APP695 at Thr-668, whereas T668A mutation of APP695 prevents phosphorylation (44). In HEK293 Nav1.6 cells, expression of APP695 T668E up-regulated surface, but not total, expression of Nav1.6, whereas expression of APP695 T668A down-regulated surface, but not total, expression of Nav1.6 (Fig. 6A). TfR was not changed significantly by these treatments (Fig. 6A). These data indicate that phosphorylation of APP695 at Thr-668 increases surface expression of Nav1.6 sodium channels. Phosphorylation of APP Enhanced Its Interaction with Nav1.6 —How does phosphorylation of APP increase Nav1.6 sodium channel surface expression? We first investigated whether the surface expression of APP was related to its phosphorylation state. The surface expression of the phosphorylation mimic form T668E and the dephosphorylated form T668A (in APP695) was detected, and no significant difference was found between them (Fig. 6B). Next we investigated the interaction between Nav1.6 and differently phosphorylated forms of APP. We found that APP695 T668E interacted with Nav1.6 much more strongly than APP695 T668A (Fig. 6C). These data suggested that phosphorylation of APP695 at Thr-668 increased Nav1.6 cell surface expression by enhancing its interaction with Nav1.6.

DISCUSSION Our results indicate that increased expression of APP results in increased Go protein-mediated activation of JNK. This leads to phosphorylation of APP695 at Thr-668, which increases association of APP with Nav1.6 sodium channels. This leads to increased cell surface expression of Nav1.6 and increased sodium currents (Fig. 7). APP has been a key focus of AD research for many years. However, despite extensive studies on APP biology for more than 20 years, a thorough understanding of the physiological function of APP has remained elusive. This study shows that APP interacts with and regulates Nav1.6, the major type of voltage-gated sodium channels in the CNS. Our findings supply a novel addition to the growing list of APP functions in neurons. It has been reported previously that APP associates with NMDA receptor subunits and alters their synaptic cell surface levels (30, 45). Demonstration of these interactions suggests that it may be fruitful to investigate whether APP, likewise, regulates other ion channels or receptors by similar mechanisms. Moreover, because sodium channels are crucial for neuronal action potential generation and propagation, the implications of the interaction of APP with Nav1.6 for mechanisms underlying symptomatology or disease progression in AD may deserve further investigation. To investigate the functional relevance of APP and Nav1.6, we studied the localization and interaction of these two proteins in 14 DIV cultured mouse cortical neurons. Previous studies showed that APP is largely localized to the Golgi complex and late endosomes (34) and that Nav1.6 is mostly localized in the soma and dendrites in mouse cortical neuroglial cultures VOLUME 290 • NUMBER 19 • MAY 8, 2015

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FIGURE 7. Schematic of the interaction between APP and Nav1.6. Increased expression of APP led to Go protein-dependent activation of JNK. Increased activation of JNK, in turn, led to increased phosphorylation of APP695 at Thr-668. Phosphorylation of APP695 at Thr-668 increased association of APP with Nav1.6, which led to increased cell surface expression of Nav1.6 and, likely, explains greater sodium currents in HEK293 Nav1.6 cells with endogenous APP expression compared with HEK293 Nav1.6 cells in which APP was knocked down.

FIGURE 6. Thr-668 mutants of APP695 influence Nav1.6 surface expression through different interactions with Nav1.6. A, total proteins and cell surface proteins isolated from HEK293 Nav1.6 cells transfected with wild-type APP695 (WT), APP695 T668E (TE, Thr-668 phosphorylation mimic) or APP695 T668A (TA, Thr-668 dephosphorylation mimic) were blotted using antibodies against Nav1.6, APP, TfR, and ␥-tubulin (␥-tub). The right panel shows a quantitative analysis of relative surface Nav1.6 and total Nav1.6 expression by oneway ANOVA followed by post hoc Bonferroni test. Representative blots from at least three independent experiments were used for statistics. *, p ⬍ 0.05. Error bars represent mean ⫾ S.E. B, total proteins and cell surface proteins isolated from HEK293 Nav1.6 cells transfected with APP T668E (TE) or APP T668A (TA) were blotted using antibodies against FLAG, TfR, and ␥-tubulin. The right panel shows a quantitative analysis of relative surface APP (sAPP). Representative blots from at least three independent experiments were used for statistics (paired Student’s t test, p ⬎ 0.05). Error bars represent mean ⫾ S.E. C, APP695 T668E interacts with Nav1.6 more strongly than APPT 668A. HEK293 Nav1.6 cells transfected with wild-type APP695 (WT), APP695 T668E, or APP695 T668A were solubilized in lysis buffer. The soluble extracts were collected by centrifugation at 100,000 ⫻ g and immunoprecipitated using mouse monoclonal antibody against FLAG (Sigma) or normal mouse IgG (Santa Cruz Biotechnology) added in the same volume mixture of wild-type APP695, APP695 T668E, and APP695 T668A (mix) and analyzed by Western blotting using antibodies against Nav1.6 (Alomone Laboratories) or FLAG (Sigma). The bottom panel shows a quantitative analysis of the relative ratio (normalized to the wild type) of coimmunoprecipitated Nav1.6 versus immunoprecipitated wild-type APP695, APP695 T668E, or APP695 T668A by oneway ANOVA followed by post hoc Bonferroni test. **, p ⬍ 0.01. Error bars represent mean ⫾ S.E. Input, 5% of total cell lysate; IP, 50% of immunoprecipitation fraction. Representative blots from at least three independent experiments were used for statistics.

(35), which is consistent with our observation. To confirm the interaction of APP and Nav1.6, we used conventional coimmunoprecipitation assays as well as the Duolink assay, which uses the proximity ligation method to examine the subcellular localization of protein-protein interactions at single molecule resolution in situ. There is considerable amino acid sequence similarity between voltage-gated sodium channel ␣ subunits, with MAY 8, 2015 • VOLUME 290 • NUMBER 19

Nav1.6 being considered phylogenetically close to Nav1.2 and Nav1.1 (46). Coimmunoprecipitation also demonstrated the potential for APP to interact with Nav1.2 and Nav1.1. Therefore, we show that APP interacts with Nav1.6 in vitro and in vivo. Our results show that APP knockdown decreased Nav1.6 current density in HEK293 Nav1.6 cells and that these changes in sodium currents were due to decreased surface expression of Nav1.6. It has been reported previously that total expression of Nav1.6 was down-regulated in the parietal cortex of 4- to 7-month-old B6.Cg-Tg(PDGFB-APPSwInd)20Lms/2JMmJax or line J20 (hAPPJ20) mice (47, 48). However, we did not observe significant changes in total Nav1.6 expression in 14 DIV cultured cortical neurons from WT versus APP KO mice or in APP knockdown or overexpression HEK293 Nav1.6 cells. This discrepancy may be due to differences in the brain regions and cell types investigated, the function of hAPP with Swedish and Indiana familial Alzheimer disease mutations overexpressed in J20 mice compared with the WT hAPP used in our study, or gene expression in adult mice and the 14 DIV cultured cortical neurons investigated in our study. The APP-induced increase in cell surface expression of Nav1.6 was Go protein-dependent. The Go protein binding domain in the intracellular domain of APP is known to selectively activate the heterotrimeric G protein Go (8 –10). Although G protein modulation of Nav1.6 has not been reported previously, our findings are consistent with G proteinmediated modulation of various other sodium channels (37– 39). The increase in cell surface expression was dependent upon phosphorylation of APP695 at Thr-668. We found that APP triggers a Go protein-dependent activation of the phosphorylation of c-Jun at Ser-63, indicating an activation of JNK. JNK3 has been reported to phosphorylate APP695 at Thr-668 (49). We found that the APP-dependent increases in cell surface expression of Nav1.6 required JNK and could be replicated by T668E mutation of APP695 to mimic phosphorylation. Our data also revealed that phosphorylation of APP695 at Thr-668 enhanced its interaction with Nav1.6. It has been reported previously that the trafficking of some proteins was regulated by phosphorylation. For example, in the case of receptor tyrosine JOURNAL OF BIOLOGICAL CHEMISTRY

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APP Enhances Sodium Channel Cell Surface Expression kinase, a Tyr(P) motif on the activated receptor binds to the SH2 domain of the E3 protein-ubiquitin ligase c-Cbl, leading to internalization of the receptor (50 –52). We therefore speculate that the phosphorylation of APP at Thr-668 increased Nav1.6 cell surface expression by enhancing its interaction with Nav1.6. It has been reported that the cytoplasmic N-terminal domain of Nav1.6 is required for anterograde transport from the Golgi complex to the plasma membrane (53) and that the microtubule-associated protein MAP1B interacts with Nav1.6 and facilitates its trafficking to the cell surface (54). It remains to be elucidated whether APP enhances the surface expression of Nav1.6 by promoting forward trafficking or by decreasing the endocytosis of sodium channels from the cell surface. The role of Nav1.6 in regulating neuronal excitability may be related to three properties of Nav1.6: its role in persistent and resurgent currents; its voltage-dependent activation; and its subcellular localization at the axon initial segment, the site of initiation of action potentials (55). Persistent currents are important for the generation of repetitive firing in neurons as occurs, for example, in cerebellar Purkinje cells (23) and cultured hippocampal CA1 pyramidal cells (56). Membranes containing Nav1.6 are more excitable than those containing only Nav1.1 and Nav1.2, and loss of Nav1.6 results in a higher threshold for the initiation of action potentials (57). Whether these Nav1.6 functions are regulated by APP and whether these cellular phenotypes are changed in APP KO or APP-overexpressing mice deserves further exploration. Nav1.6 is the predominant sodium channel at adult rat and mouse nodes of Ranvier (17). We recently reported colocalization of APP and sodium channels at nodes of Ranvier in the CNS (58). It remains to be determined whether APP modulates the surface expression of sodium channels at nodes of Ranvier. Although expression of APP has been reported to be predominantly neuronspecific (34), non-neuronal cells, including microglia, have also been reported to express APP (59). An investigation of interactions between APP and sodium channels in microglia may be of interest because sodium channels, in particular Nav1.6, are important in regulating microglial activity (60, 61). Acknowledgments—We thank Drs. K. Hsiao and H. M. Mohajeri for APP null transgenic mice, Dr. C. Schmidt for human APP695 cDNA, Dr. T. Suzuki for APPT668E cDNA, Dr. S. Itohara for APPT668A cDNA, Dr. J. J. Clare for the HEK293 Nav1.6 cell line, and Dr. L. Bao for helpful comments on the manuscript.

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