Impact of Sustained Exposure to β-Amyloid on Calcium Homeostasis and Neuronal Integrity in Model Nerve Cell System Expressing α4β2 Nicotinic Acetylcholine Receptors*

Background: The consequence of sustained exposure of nicotinic receptors to β-amyloid is unknown. Results: Sustained exposure to β-amyloid potentiated nicotinic receptor function, promoting oxidative stress and cellular toxicity. Conclusion: The presence of nicotinic receptors sensitizes cells to the toxic actions of β-amyloid. Significance: The potent action of β-amyloid on nicotinic receptors may contribute to the cholinergic deficit found in Alzheimer disease. Although the interaction between β-amyloid (Aβ) and nicotinic acetylcholine receptors has been widely studied, the impact of prolonged exposure to Aβ on nAChR expression and signaling is not known. In this study, we employed a neuronal culture model to better understand the impact of sustained exposure of Aβ on the regulation of cellular and synaptic function. The differentiated rodent neuroblastoma cell line NG108-15 expressing exogenous high-affinity α4β2 nAChRs was exposed to soluble oligomeric Aβ for several days. Ca2+ responses, expression levels of α4β2 nAChRs, rate of mitochondrial movement, mitochondrial fission, levels of reactive oxygen species, and nuclear integrity were compared between Aβ-treated and untreated cells, transfected or not (mock-transfected) with α4β2 nAChRs. Sustained exposure of Aβ1–42 to α4β2 nAChR-transfected cells for several days led to increased Ca2+ responses on subsequent acute stimulation with Aβ1–42 or nicotine, paralleled by increased expression levels of α4β2 nAChRs, likely the result of enhanced receptor recycling. The rate of mitochondrial movement was sharply reduced, whereas the mitochondrial fission protein pDrp-1 was increased in α4β2 nAChR-transfected cells treated with Aβ1–42. In addition, the presence of α4β2 nAChRs dramatically enhanced Aβ1–42-mediated increases in reactive oxygen species and nuclear fragmentation, eventually leading to apoptosis. Our data thus show disturbed calcium homeostasis coupled with mitochondrial dysfunction and loss of neuronal integrity on prolonged exposure of Aβ in cells transfected with α4β2 nAChRs. Together, the results suggest that the presence of nAChRs sensitizes neurons to the toxic actions of soluble oligomeric Aβ, perhaps contributing to the cholinergic deficit in Alzheimer disease.

Numerous studies using a wide range of preparations have shown that diffusible oligomeric ␤-amyloid (A␤) 3 assemblies are toxic (1), being specifically responsible for the development of synaptic impairment (2). The impact of soluble A␤ on synaptic function was found to be dependent upon concentration, with impairment typically occurring at micromolar levels of the peptide. In contrast, at picomolar concentrations A␤ enhanced synaptic plasticity, suggesting a neuromodulatory role under physiological conditions (3,4). However, as A␤ accumulates in AD, its action will thus convert to synaptic (and cellular) disruption.
The synapse is likely a major site of action for A␤, as evident from studies showing that the production and release of A␤ from the presynaptic terminal is regulated by synaptic activity (5,6). The average "normal" concentrations of A␤, i.e. concentrations found in nondemented adults, have been found to be in the high picomolar (ϳ250 pM) range in brain (7), and the presynaptic terminal appears to be the dominant source of A␤ production (8) and undergoes synaptic regulation by A␤ (4). A␤ thus appears to be localized to the synapse at concentrations that regulate presynaptic dynamics.
Among an array of different targets at synapses to which A␤ might bind and exert downstream effects, those of notable significance are neuronal nicotinic acetylcholine receptors (nAChRs) and metabotropic glutamate receptors (9). The nAChRs play important modulatory roles in neuronal development and synaptic plasticity, participating in cognitive functions such as learning, memory, and attention (10). The most abundant high affinity nAChR in brain is comprised of ␣4 and ␤2 subunits, whereas the other major nAChR subtype in brain contains ␣7 subunits (11). Activation of ␣7-nAChRs produces a rapid, sharp increase in the intracellular Ca 2ϩ signal, whereas ␣4␤2-nAChRs cause a more delayed but long-lasting signal (11). A␤ activation of different nAChR subtypes will therefore have different impacts on intracellular Ca 2ϩ and hence synaptic signaling.
Acute application of pico-to nanomolar A␤ evokes increases in Ca 2ϩ and neurotransmitter release via presynaptic nAChRs (12)(13)(14). Mutation of a key tyrosine residue in the agonist-binding site of ␣7 nAChRs eliminates the A␤ effect on presynaptic Ca 2ϩ (15), directly confirming the agonist-like action of A␤. At picomolar concentrations, acute A␤ was also found to enhance long-term potentiation and contextual memory in a manner dependent upon presynaptic nAChRs (3,4).
The high-affinity ␣4␤2 nAChRs are significantly up-regulated in animals chronically treated with nicotine on a daily dose basis (16,17). This nicotine-induced up-regulation has been further characterized in a variety of systems ranging from clonal cell lines to primary neurons in culture to mouse models to smokers' brains (18 -26). Up-regulation of ␣4␤2 nAChRs may sensitize cellular targets to the action of nicotine.
Regulation of receptor expression also depends on steadystate control via endocytosis and membrane recycling, along with degradation via lysosomes. Changes in AMPA-type glutamate receptor expression in postsynaptic membranes in the context of synaptic plasticity, for example, have been shown to involve changes in receptor recycling. Rab proteins, members of the Ras family of small GTPases, have been shown to play a key role at various steps in endocytic, recycling, and degradative pathways. Specifically, Rab5 expression levels are higher in plasma membrane, recycling endosomes, and clathrin-coated pits (27,28) and regulates early endosomal fusion and recycling. Rab11 is mainly found in the trans-Golgi network and recycling endosomes (29,30) from where it regulates trafficking of proteins to the plasma membrane. The extent to which nAChRs are regulated by these processes in response to sustained agonist exposure remains to be determined.
An important downstream consequence of A␤ pathology is mitochondrial toxicity (31). As synapses are high energy demanding sites, mitochondria play critical roles in maintaining synaptic function, and mitochondria toxicity likely contributes to synaptic loss in AD (32). It has been reported that brains of AD patients show ultrastructural alterations in mitochondrial morphology, such as reduced number, increased size, and broken internal membrane cristae (33). A␤ may also affect mitochondrial dynamics (fission/fusion equilibrium) and distribution in the axon and synapse. Recent studies using an in vivo fly model overexpressing A␤ showed that depletion of presynaptic and axonal mitochondria was one of the earliest detectable deficits, preceding A␤-induced presynaptic deficits in motor function (34,35). A␤-induced mitochondrial mislocalization was also confirmed in hippocampal neurons (36). Such mitochondrial trafficking disruption in AD could potentially compromise normal synaptic function. However, it is still not clear what role presynaptic receptors play in mitotoxicity induced by A␤ at the synapse.
Here, we addressed the impact of sustained exposure to A␤ on a model neuronal system expressing exogenous ␣4␤2 nAChRs on [Ca 2ϩ ] i , receptor regulation, axonal mitochondrial dynamics, oxidative stress, nuclear and cellular integrity, and endocytosis/recycling. The results suggest that the presence of ␣4␤2 nAChRs may sensitize neurons to the effects of sustained exposure to A␤.

EXPERIMENTAL PROCEDURES
Cell Culture-NG108-15 hybrid neuroblastoma cells were plated on Cell-Tak-coated coverslips (Warner) in 35-mm dishes in Dulbecco's modified Eagle's medium (DMEM) containing 15% fetal bovine serum (FBS) (37). The cells were induced to differentiate in media containing 1% FBS and 1 mM cyclic AMP for 48 -72 h, elaborating long neurites with varicosities. These varicosities display features found for presynaptic elements in the brain, like voltage-gated calcium channels (38), ER-based Ca 2ϩ stores, vesicles, mitochondria, and synaptic proteins (39) and are capable of forming fully functional cholinergic synapses with target cells (40). As they do not express functional nAChRs themselves, mouse sequences for ␣4 and ␤2 nAChR subunits housed in pcDNA3.1 expression vectors (courtesy of Dr. Jerry Stitzel, University of Colorado) were transiently transfected at a ratio of 1:4, respectively, into differentiated NG108-15 cells using FuGENE HD, a lipid-based transfection reagent, for 48 h. This ratio was found to yield maximal expression of functional receptors. Mock-transfected NG108-15 cells, containing only FuGENE HD and no plasmid DNA were used as controls.
Live-cell Calcium Imaging-To measure changes in Ca 2ϩ levels in individual varicosities, the transfected, differentiated cells were loaded for 40 min at 37°C with fluorescent Ca 2ϩ indicator dye Fluo-4/AM (Invitrogen) at 5 M in HEPES-buffered saline (HBS: containing (in mM) 10 HEPES, 142 NaCl, 2.4 KCl, 1.2 K 2 PO 4 , 1 MgCl 2 , 1 CaCl 2 , 5 D-glucose, and 100 nM tetrodotoxin, pH 7.4, saturated with O 2 ) in preparation for confocal imaging, as described (41). Cells were mounted in a rapidexchange Warner open perfusion chamber for confocal imaging using a Zeiss LSM 5 Pascal imaging system (excitation: 488 nm; emission: 515-565 nm band-pass; ϫ40/1.3 NA epifluorescence, oil-immersion Plan-Neofluar objective). Imaging was started and after obtaining a baseline series of four images, reagents (agonists; antagonists) were applied by rapid switching via a Warner six-channel valve-controlled perfusion system (VC-66CS, Warner Instruments) using DN series constant flow reservoirs. Image series consisted of a time course of 200 -400 s (typically to the peak of the response). For each run, a minimum of four to eight varicosities in each successive image were selected as regions of interest and their associated fluorescence intensities were determined for all 30 frames using ImageJ. Time series responses were normalized to baseline, corrected for photobleaching, and presented as F/F 0 .
Immunocytochemistry-Cell cultures were fixed with freshly prepared 4% paraformaldehyde in HBS at room temperature for 30 min and rinsed with phosphate-buffered saline (PBS) for 30 min. The cultures were then incubated in Tris-buffered saline (TBS) containing 5% bovine serum albumin (BSA) and 10% normal goat serum for 30 min to block nonspecific binding. Affinity-purified antibodies (1:200) were then added to the cultures and incubated overnight at 4°C. The cultures were then washed with 10% goat serum in TBS for 30 min, and incubated with the FITC-conjugated secondary antibodies (IgGs, typically at 1:500) for 30 min at room temperature. The coverslips were finally washed with 10% normal goat serum and TBS, plated onto glass microscope slides, and sealed for imaging via confocal microscopy. To detect the mitochondrial fission protein Drp-1 (dynamin-related protein-1) and the Rab proteins, cells were permeabilized before incubation with blocking buffer. For Drp-1, cells were then co-stained with rabbit anti-phospho-Drp-1 and mouse anti-Drp-1. Anti-rabbit Alexa-488 and antimouse Alexa-633 were used as secondary antibodies, respectively. The immunostained preparations were subsequently visualized using a Zeiss LSM 5 Pascal confocal imaging system on a Zeiss Axiovert 200M microscope with appropriate fluorescence filters via a ϫ40 Plan-Neofluar objective. Immunostaining for the ␣4 and ␤2 nAChR subunits was visualized on a Nikon Cameleon AOTF confocal microscopic system via a ϫ40 objective.
Reactive Oxygen Species (ROS)/Hoechst Staining-The Image iT live Reactive Oxygen Species Detection kit (Invitrogen) was used to determine the extent of oxidative stress in response to daily treatment with A␤ 1-42 in NG108-15 cells transfected with ␣4␤2-nAChRs as compared with mock-transfected (control) cells, exchanging the culture medium each day. After treatment, the cells were rinsed with warm HBS containing Ca 2ϩ and Mg 2ϩ and incubated with carboxy-H 2 DCFDA (component A) at 37°C for 30 min. During the last 5 min of incubation, 2 g/ml of Hoechst stain (component B) was added. The cells were washed twice with HBS and visualized using an Olympus IX71 epifluorescence microscope at excitation/emission of 495/529 nm (ROS) and 350/461 nm (Hoechst), respectively, linked to a Macrofire camera.
Mitochondrial Size and Movement Along the Neurite-Differentiated NG108-15 cells transfected with ␣4␤2-nAChRs were treated daily with A␤  or not (control) for 3 days, exchanging the culture medium each day. On day 3, the cells were incubated with BacMam Mito-RFP reagent (Invitrogen) for another 16 h to label the mitochondria. The cells were then washed for 30 min and imaged for the live movement of individual RFPlabeled mitochondria in the axonal neurites. A total of 30 images were recorded over a period of 720 s using confocal imaging (see previous methods) under standardized settings. Using Image J, the x and y coordinates of each individual mitochondrion were measured to determine the relative distance traversed by that particular mitochondrion along a given neurite. In addition, the size of individual mitochondria was determined (diameter in pixels) across the 3-day time course. Mocktransfected cells were used as separate controls.
Cell Surface Labeling and Recycling-Differentiated NG108-15 cells transfected with ␣4␤2-nAChRs and treated (or not) with A␤ 1-42 daily for 3 days, exchanging the culture medium each day, were surface labeled at 4°C using a cleavable biotinylation reagent from the Pierce Cell Surface Protein Isolation kit (Thermo Scientific). After various treatment conditions, the cells were lysed using the lysis reagent and the biotinylated proteins were pulled down using NeutrAvidin-agarose beads. These were then mixed with SDS sample buffer and loaded onto 4 -20% Tris-HCl gels, transferred onto a nitrocellulose membrane and probed for ␣4-nAChR subunits using a highly specific rat monoclonal (299) antibody.
Quantitative RT-PCR-Total RNA was isolated from mocktransfected and ␣4␤2-nAChR-transfected cells treated daily for 3 days with A␤  or not (untreated), exchanging the culture medium each day, using the RNeasy Mini Kit (Qiagen). The total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), following the manufacturer's instructions, and quantitative real-time PCR was performed according to standard protocols. For data analysis, 18S rRNA was used as an endogenous control. Data are expressed as relative expression for each individual gene normalized to their corresponding controls. The following primer sequences were used for amplification of the mouse ␣4-nAChR cDNA (NM015730, GenBank TM ) at an annealing temperature of 61°C: forward, 5Ј-ACTTTGCAGTCACCCA-CCTAACCAAA-3Ј; reverse, 5Ј-ATGAGCAGCAGAAGACG-GTGAGAGAAA-3Ј. This yielded a 481-bp PCR product.
Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) Assay-NG108-15 cells were seeded on coverslips in 24-well plates (6 ϫ 10 4 cells/well). Cells were either mock-or ␣4␤2-nAChR transfected as previously described (Cell Culture). After transfection, cells were treated daily with A␤ 1-42 or A␤ 42-1 (control peptide) for 5 days, exchanging the culture medium each day. The TUNEL assay was performed using Click-iT TUNEL Alexa Fluor Imaging Kit (Invitrogen) in accordance with the manufacturer's protocol. In brief, cell cultures were fixed with freshly prepared 4% paraformaldehyde in PBS at room temperature for 20 min and permeabilized with Triton X-100 (0.25% in PBS) for another 20 min. The cultures were then washed twice and incubated with 50 l of terminal deoxynucleotidyl transferase reaction buffer (Component A) for 10 min at room temperature. The buffer was removed and the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase was added, incubating the cultures in a humidified chamber at 37°C for 60 min. After treatment, cells were washed three times with 3% BSA in PBS for 2 min each and then incubated with 50 l of Click-iT reaction mixture (containing Alexa 488 azide) for 30 min at room temperature, while being protected from light exposure. The cells were again washed with 3% BSA in PBS and the cell nuclei were counterstained with Hoechst 33342 for 15 min at room temperature, protected from light. The coverslips were washed twice with PBS before mounting onto a slide with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Negative and positive controls were included in each experiment. For the negative control, cells were processed using a reaction mixture that did not contain terminal deoxynucleotidyl transferase. For the positive control, cells were incubated with DNase I (3 units/ml) for 30 min to induce DNA strand breaks. Labeled nuclei were detected by fluorescence microscopy on a Zeiss Pascal confocal imaging system on a Zeiss Axiovert 200 microscope, equipped with appropriate fluorescence filters and objectives. The TUNELpositive cells were counted in eight different, random fields for each well.
A␤-A␤ 1-42 and the control reverse peptide A␤ 42-1 were purchased from American Peptide. A stock solution of the peptides were prepared at 0.1 mM by dissolving solid peptide in double deionized water (see Ref. 37). A␤  (and A␤ 42-1 ) for each experiment was diluted from 1 pM to 100 nM from the 0.1 mM stock into oxygenated HBS and vortexed to assure full suspension. Under these conditions, the A␤ is largely present as stable oligomers, as assessed by SDS-PAGE and native gel analysis (36).
Chemicals-DMEM was from Mediatech, Inc. FBS was from Invitrogen. Cell-Tak was from BD Biosciences. Fluo 4/AM, Image-iT Live ROS Detection kit, and BacMam Mito-RFP were purchased from Invitrogen. BSA, nicotine tartrate citrate, and Rab5 antibody were purchased from Sigma. Nicotine (1 M final) was freshly prepared by dissolving in oxygenated HBS before each experiment. Tetrodotoxin was from Calbiochem. The polyclonal anti-␣4-nAChR and anti-␤2-nAChR antibodies were purchased from Millipore. Monoclonal rat anti-␣4 nAChR and Rab11 antibodies were from Abcam. The anti-Drp-1 and anti-phospho-Drp-1 antibodies were from Cell Signaling. The FITC-labeled and peroxidase-conjugated secondary antibodies (goat anti-rabbit and goat anti-mouse) were from Jackson ImmunoResearch. The Alexa-488 and Alexa-633 secondary antibodies were purchased from Invitrogen. IRDye 680 goat anti-rabbit secondary antibodies were from Li-Cor. The RT-PCR kit was from Qiagen. The Pierce surface biotinylation kit was from Thermo Scientific. All common reagents (e.g. buffers, salts, etc.) were either from Fisher or Sigma.
Statistical Analysis-Multiple comparisons were subjected to analysis of variance, followed by Bonferroni post hoc tests. Pairwise comparisons were made using Student's t tests. A minimum of p Ͻ 0.05 was used to establish a significant difference. All experiments were replicated at least 3 times.

Ca 2ϩ
Responses Mediated by ␣4␤2-nAChRs following Acute Treatment with A␤-To test the effect of A␤ 1-42 on ␣4␤2-nAChRs, exogenous mouse ␣4-nAChR and ␤2-nAChR sequences in pcDNA3.1 vectors were transfected into differentiated NG108-15 cells, whereas mock-transfected cells were used as controls. Ca 2ϩ changes in the axonal varicosities of the NG108-15 cells, as relative changes in fluorescence intensities (F/F 0 ) were determined in cells loaded with the Ca 2ϩ indicator Fluo-4 ( Fig. 1A) as test for synaptic regulation (37). As shown in Fig. 1B, left, ␣4␤2-nAChR-transfected cells displayed apparent increases in [Ca 2ϩ ] i on stimulation with 100 nM A␤ 1-42 (p Ͻ 0.05 relative to mock-transfected cells) and 1 M nicotine (p Ͻ 0.005 relative to mock-transfected cells), whereas mock cells did not display significant Ca 2ϩ changes to A␤ 1-42 or nicotine over baseline. Ca 2ϩ changes were also apparent in select cell bodies. Ca 2ϩ responses to depolarization induced by elevated KCl (HK) were recorded after each drug/agonist treatment to assess the cell integrity. There were no significant differences in the responses in the varicosities to elevated KCl following various treatments as compared with control. These results indicate that A␤ 1-42 has an agonist-like effect on ␣4␤2-nAChRs in axonal varicosities, which is consistent with the idea that A␤ 1-42 can serve as physiological regulator of presynaptic nAChRs (3,4,37). Prior treatment with 1 M dihydro-␤erythroidine (DH␤E), an ␣4-selective antagonist, for 10 min attenuated Ca 2ϩ responses in the varicosities to acute stimulation with both A␤ (p Ͻ 0.05) and nicotine (p Ͻ 0.05) (Fig.  1B, right). These latter results confirm that the increased calcium levels elicited by A␤ are indeed mediated by ␣4␤2-nAChRs.
To directly monitor the expression pattern of nicotinic receptors on transfected NG108-15 cells, immunostaining for ␣4␤2 nAChRs was performed using specific antibodies for the ␣4 and ␤2 subunits. ␣4␤2-nAChR-transfected cells and mocktransfected cells were compared. As shown in Fig. 1C, ␣4␤2-nAChR transfected cells were strongly immunopositive for both ␣4 and ␤2 subunits when compared with mock-transfected cells immunostained for the ␣4 subunit. By comparison, the overall signal for mock cells is evenly weak, similar to the nonspecific binding of secondary antibody alone to the cell surface, whereas there is no clear signal on the varicosities. The immunostaining results provide direct evidence of efficient receptor expression, consistent with the functional responses.
A␤-induced Ca 2ϩ Responses Mediated by ␣4␤2-nAChRs: Impact of Sustained Exposure to A␤-To determine the consequences of sustained exposure to A␤ 1-42 on subsequent acute stimulation by A␤ 1-42 or nicotine, calcium imaging was performed as previously described. Differentiated NG108-15 cells expressing ␣4␤2-nAChRs were subjected to 100 nM A␤ 1-42 for 1, 2, and 3 days, exchanging the culture media every 24 h with media containing fresh oligomeric A␤  . On the day of calcium imaging, the culture medium containing A␤ 1-42 was removed and cells were washed with HBS for 15 min, followed by Fluo-4 loading. After an extensive second wash period, perfusion was started and Ca 2ϩ responses elicited by acute stimulation with A␤ 1-42 (100 nM) and nicotine (1 M) were measured for control and A␤ 1-42 -treated cells. As shown in Fig. 2, [Ca 2ϩ ] i responses in the varicosities were unchanged after 1 or 2 days but increased significantly after 3 days of A␤ 1-42 treatment in response to both A␤ 1-42 and nicotine as compared with control cells (Fig. 2, A-C; p Ͻ 0.05). There was no effect of sustained A␤ 1-42 treatment on mock-transfected cells (Fig. 2E). There was also no effect of sustained treatment with the control reverse peptide, A␤ 42-1 (Fig. 2F). These results are consistent with previous work demonstrating that chronic agonist exposure (nicotine) leads to an alteration in the expression and function of presynaptic ␣4␤2-nAChRs (42). Similarly chronic nicotine exposure resulted in significantly increased levels of Ca 2ϩ in ␣4␤2 nAChR-transfected cells in response to acute stimulation by A␤ 1-42 as well as nicotine ( Fig. 2D; p Ͻ 0.01) as compared with control cells. As found for untreated NG108-15 cells expressing ␣4␤2-nAChRs, the ␣4-selective antagonist DH␤E fully attenuated Ca 2ϩ responses in the varicosities to acute stimulation with A␤ or nicotine in NG108-15 cultures treated with A␤ for 3 days (Fig. 2G). In addition, co-treatment with DH␤E for 3 days fully attenuated the acute A␤-or nicotineinduced increases in Ca 2ϩ responses in the varicosities found following sustained treatment with A␤ 1-42 for 3 days (Fig. 2H), confirming that the action of A␤ 1-42 was specifically through the nAChRs. Together, these results support a role for ␣4␤2-nAChRs in mediating changes in calcium homeostasis on sustained exposure to A␤.

Sustained Exposure to A␤ Caused Receptor Up-regulation in the Cell Bodies and Varicosities of Neurites of NG108-15 cells Expressing ␣4␤2 nAChRs in a Manner Similar to Chronic
Nicotine Treatment-The surface expression levels of ␣4␤2-nAChRs in mock-and ␣4␤2-nAChR transfected cells were compared with those in cells treated with A␤ 1-42 and nicotine by immunostaining using a selective anti-␣4-nAChR antibody (Fig. 3A). ␣4␤2-nAChR-transfected cells treated with A␤ 1-42 and nicotine for 3 days showed apparent increased expression of ␣4␤2-nAChRs as compared with untreated cells (varicosities and cell bodies, Fig. 3B). Co-treatment with the antagonist DH␤E blocked the apparent up-regulation of the ␣4-nAChRs, although the effect was only clear for the cell bodies. To demonstrate that this up-regulation was post-transcriptional, we   . Up-regulation of ␣4␤2-nAChRs in response to prolonged A␤ treatment. A, the expression levels of ␣4␤2-nAChRs in mock-and ␣4␤2-nAChRtransfected cells were compared with those in cells treated with 100 nM A␤ 1-42 (with or without DH␤E) or nicotine by immunostaining for the ␣4-nAChR subunit, counterstained with DAPI (nuclear stain). B, fluorescent signals from cell bodies and varicosities were selected and averaged to calculate the mean signal intensity, normalized to that obtained for mock-transfected cells (n ϭ 5). ␣4␤2-nAChR-transfected cells treated with A␤ 1-42 or nicotine showed apparent increased expression of ␣4␤2-nAChRs as compared with untreated cells. *, p Ͻ 0.05 when compared with controls. C, real-time PCR was performed to determine the changes in mRNA levels of ␣4-nAChRs on prolonged treatment with A␤  . No difference was detected in transcript expression between control and 3-day A␤ 1-42 -treated cells transfected with ␣4␤2-nAChRs (n ϭ 4).
performed quantitative RT-PCR on ␣4-nAChR cDNA obtained following RNA isolation from control and 3-day A␤ 1-42treated cells. There was no significant difference in transcript levels in A␤-treated cells as compared with control cells (Fig. 3C).
Altered Endocytosis-receptor Recycling on Sustained Exposure to A␤: Rab5, and Rab11-To consider the possibility that up-regulation of ␣4␤2-nAChRs in response to sustained exposure to A␤ results from an alteration in the endocytotic-receptor recycling pathway, we first tested for the endosomal marker Rab5 and the recycling marker Rab11. Relative changes in the expression levels of Rab proteins were detected using immunocytochemistry of differentiated NG108-15 cells treated or not with A␤ 1-42 for 3 days. ␣4␤2-nAChR-transfected cells showed increased levels of Rab5 as compared with that in mock-transfected cells (Fig. 4A, top series). Sustained exposure to A␤  further increased Rab5 levels in ␣4␤2-nAChR-transfected cells. Similarly, intracellular Rab11 levels were also found to be increased in ␣4␤2-nAChR-transfected cells treated with A␤ to a significant extent as compared with that in mock-transfected cells (Fig. 4A, bottom series). The results from co-immunostaining with rabbit anti-␣4-nAChR and rat anti-Rab5 or anti-Rab11 antibodies (Fig. 4C) and Western blots to detect the total levels of Rab5 and Rab11 (Fig. 4D) provided further support that the levels of endosomal/recycling markers are altered in a time-dependent manner in response to A␤ 1-42 treatment. Rab5, in particular, was substantially increased in response to A␤ treatment at 2-3 days by over 3-fold, as assessed by immunoblot analysis (390 Ϯ 25% of day 0; p Ͻ 0.01; n ϭ 6).
To address directly whether changes in the expression levels of nAChRs on prolonged exposure to A␤ resulted, at least in part, from a change in the endocytotic/recycling pathway, we Regulation of Rab5 and Rab11 in ␣4-nAChR-expressing cells in response to A␤ 1-42 treatment across the 3-day time course was verified in co-immunolabeling (C) and Western blots (D). E, after surface labeling with cleavable sulfo-NHS-SS-biotin, cells were allowed to undergo endocytosis at 37°C followed by DTT treatment at 4°C to remove biotin from the remaining surface protein, demonstrating an increased level of expression of the receptor following 3-day A␤ treatment, when probed with rat monoclonal anti-␣4-nAChR antibody, as compared with untreated or mock-transfected cells (first to third lanes). (The molecular mass of the ␣4-nAChR band was confirmed relative to the protein standards by regression analysis as 72 kDa. The lower bands are nonspecific background also present in mock-transfected cells.) After the labeled cells were allowed to recycle the biotin-labeled receptors, the A␤-treated cells displayed predominately DTT-sensitive, biotin-labeled ␣4-containing nAChRs, indicating substantial recycling of the receptor (lane 6 versus 9). *, p Ͻ 0.05; ***, p Ͻ 0.001 when compared with controls.
used surface labeling with biotin to follow the fate of the nAChRs. Following biotinylation at 4°C, endocytosis of the surface proteins of ␣4␤2-nAChR-transfected cells was allowed to proceed by shifting the cells to 37°C for 30 min, thereafter treating with DTT at 4°C to remove biotin from the remaining surface proteins. As shown in Fig. 4E (lanes 1-3), the ␣4␤2-nAChR-transfected cells treated with A␤ for 3 days showed a markedly increased surface expression of receptor as compared with untreated ␣4␤2-nAChR-transfected cells or mock-transfected cells, when probed with a rat monoclonal anti-␣4-nAChR antibody. The cells were then shifted back to 37°C for 45 min to allow for receptor recycling. Reappearance of surface DTT-sensitive receptors was assessed as a measure of recycling. The A␤-treated, ␣4␤2-nAChR-transfected cells had substantially more surface expression of the receptor as compared with controls. The results suggest that receptor up-regulation upon prolonged exposure to A␤ is due, in part, to increased receptor recycling.
Altered Mitochondrial Dynamics in Axonal Neurites of NG108-15 Cells following Sustained A␤ Treatment-A␤ was previously found to alter mitochondrial dynamics (36,43,44). To examine the potential disruptive effect of sustained exposure to A␤ on mitochondrial transport, differentiated NG108-15 cells were transfected with ␣4␤2-nAChRs and then treated with A␤ 1-42 or not for 3 days. On the third day, the cells were incubated with BacMam Mito-RFP reagent for another 16 h to label the mitochondria. The ␣4␤2-nAChR-transfected cells treated with A␤ 1-42 showed a significantly reduced rate of movement of mitochondria in the axonal neurites as compared with that found in untreated cells (Fig. 5, B-G) or mock cells FIGURE 5. ␣4␤2-transfected cells exposed to A␤ show reduced mitochondrial movement in neurites. A, a representative kymograph showing the movement of mitochondria in a neurite of an untreated, ␣4␤2-nAChR-transfected NG108-15 cell. The arrows show the relative movement of mitochondria along a given neurite. B and E show the instantaneous rate of movement of individual mitochondria over 720 s in neurites of control and A␤-treated cells, respectively. C and F depict the cumulative rate, whereas D and G show the average rate of movement of 6 different randomly selected mitochondria in ␣4␤2-transfected cells in neurites of control and A␤-treated cells, respectively. There was no significant difference between rate of mitochondrial movement in untreated mock and untreated ␣4␤2-transfected cells (not shown). However, on chronic exposure to 100 nM A␤ 1-42 , there was a significant reduction in both the rate of movement of mitochondria and total distance covered along a given neurite. APRIL 19, 2013 • VOLUME 288 • NUMBER 16

JOURNAL OF BIOLOGICAL CHEMISTRY 11183
(data not shown) in live-cell imaging. In addition, the average size of mitochondria was significantly reduced when the cells were exposed to A␤ 1-42 for various time points (days 1-3) as compared with that observed in untreated cells (Fig. 6, A and  B). However, this reduction in size was observed starting at day 1, suggesting altered mitochondrial dynamics as an early consequence of A␤ toxicity. This effect seemed to be largely independent of ␣4␤2-nAChR up-regulation, which was not evident until 3 days of treatment, in view of a similar effect on mitochondrial size in mock-transfected cells (Fig. 6B).
To further test whether the reduced size of mitochondria was a result of disturbed fission-fusion equilibrium, immunostaining and Western blot for the fission-inducing protein Drp-1 was performed using phospho-Drp-1 and total anti-Drp-1 antibodies. Phospho-Drp-1 was significantly increased in cells treated with A␤ for 3 days (Fig. 6, C-F), indicating an enhanced rate of mitochondrial fission.
Sustained Exposure to A␤ Induced Increases in the Levels of ROS and Nuclear Disintegration in NG108-15 Cells Sensitized by the Presence of ␣4␤2-nAChRs-To investigate the impact of A␤ 1-42 through ␣4␤2 nAChRs on oxidative stress, mock-transfected and ␣4␤2-nAChR-transfected cells were incubated with carboxy-H 2 DCFDA (ROS) and Hoechst (nuclear) stain (Fig.  7A). Exposure of ␣4␤2-nAChR-transfected cells to 100 nM A␤ 1-42 resulted in a time-dependent increase in the levels of ROS (p Ͻ 0.0001) as compared with mock-transfected cells exposed to the same concentration of the peptide (Fig. 7, C, black bars). In mock-transfected cells, there was only a slight increase in ROS staining on sustained exposure of nanomolar levels of A␤ 1-42 , whereas exposure to 1 M A␤ 1-42 did increase ROS levels to a significant extent (Fig. 7C, open bars), indicating that the presence of nAChRs sensitized the cells to the toxic action of A␤.
A similar trend was seen for nuclear disintegration as assessed by Hoechst staining. Application of nanomolar A␤  to mock-transfected cells showed round, intact nuclei without any notable evidence of nuclear disruption, whereas the ␣4␤2-nAChR-transfected cells treated with A␤ 1-42 for 3 days had a significant (p Ͻ 0.0001) level of nuclear disintegration (Fig. 7, A and D), similar to that found with cells undergoing apoptosis.
Direct cell counting experiments showed that by day 4, a large portion of the ␣4␤2-nAChR-transfected cells treated with A␤ 1-42 had indeed died (Fig. 9), with the remaining gone by day 5. There was a very small effect of A␤ 1-42 treatment on mocktransfected cells at day 5, but there was no significant effect of the control peptide A␤ 42-1 over the same time course. Treatment of ␣4␤2-nAChR-transfected cells or control cells with nicotine over the same time course had no significant impact on cell number (not shown).

DISCUSSION
A␤ has been shown to be released at synaptic sites in a highly dynamic fashion (5,6), leading to fluctuations in local concentration in response to synaptic activity. This would, in turn, implicate A␤ in activity-coupled synaptic regulation through target receptors. In the absence of AD, A␤ appears to be present in the high picomolar range (7) and previous work has demonstrated a direct agonist-like action of soluble picomolar-nanomolar A␤ via nAChRs at presynaptic sites (12,15,37,45,46). Acute picomolar A␤ was also found to enhance synaptic plasticity in a nAChR-dependent fashion (3,4). However, the interaction between nAChRs and long-term exposure to A␤ has not yet been clearly elucidated. The objective of the present study was to evaluate the extent to which nAChRs contribute to the effects mediated by sustained exposure to A␤, addressing the impact of dynamic changes in the levels of soluble A␤ at cellular and presynaptic sites using an in vitro model. The primary advantages of the in vitro neuroblastoma model are that epigenetic expression of the receptors avoids transcriptional regulation and allows for a defined, reconstituted neuronal system (37).
Sustained exposure of neuroblastoma cells expressing ␣4␤2 nAChRs to nanomolar A␤ was found in the present study to induce an apparent up-regulation in agonist-triggered Ca 2ϩ responses. Changes in axonal varicosities were monitored as an indication of presynaptic Ca 2ϩ and hence synaptic regulation; however, the impact of A␤ was not restricted to the varicosities. Overall, the results are consistent with studies showing that functional responses of ␣4␤2 nAChRs are increased on longterm exposure to agonist, such as nicotine (47,48). A wide range of studies has shown that this functional up-regulation typically results from up-regulation of ␣4␤2 nAChR expression (e.g. Ref. 49), although alternative mechanisms exist (50). Accordingly, our immunostaining results show an increased expression of ␣4␤2 nAChRs in cell bodies and varicosities in cultures subjected to prolonged exposure to A␤ or nicotine. Although receptor expression was defined in this cell system using plasmids, our quantitative RT-PCR results for ␣4 transcripts rule out any effect on the plasmid promoters or the endogenous receptor genes. Moreover, previous work examining the impact of chronic nicotine demonstrated that up-regulation of ␣4␤2 nAChRs occurs at  the post-translational level (51). Thus, sustained exposure to A␤ leads to up-regulation of nAChRs either at the level of receptor assembly, sorting, targeting, and transport or local endocytosis/recycling/degradation.
As there was no evidence for any detectable differences in up-regulation at the level of the cell somata as compared with the axonal varicosities, it would appear that assembly, sorting, targeting, and transport are likely not major contributors to the up-regulation observed here. Consequently, we focused on the endocytotic/recycling pathway. Our results for endocytotic and recycling markers indicate that an up-regulation of this pathway did occur, which was confirmed in a functional assay of nAChR recycling, suggesting that an increase in endocytosis/ receptor recycling may account, in part, for the observed receptor up-regulation upon prolonged exposure to A␤. How this up-regulation in recycling is induced will be the subject of future studies, focusing, in particular, on calcium-regulated pathways (52) and A␤ internalization (53). Nonetheless, it is proposed that up-regulation of the nAChRs with sustained, elevated levels of A␤ leads to a feed-forward dysregulation of calcium homeostasis.
As sustained exposure to high nanomolar-micromolar A␤ has consistently been found to be neurotoxic (e.g. Refs. 1 and 2), we next addressed the consequence of the A␤-induced up-regulation of nAChRs on mitochondrial and neuronal integrity. Mitochondria are dynamic organelles and play significant roles in intracellular calcium homeostasis and reactive oxygen species production under conditions of oxidative stress. Mitochondrial dysfunction and oxidative damage occur in the earliest phase of AD, namely before significant plaque deposition FIGURE 8. Prolonged exposure to A␤ Induced apoptosis in ␣4␤2-nAChR-transfected cells. A, to evaluate the induction of apoptosis, TUNEL was performed on either mock-transfected or ␣4␤2-nAChR-transfected cells exposed to 100 nM A␤ 1-42 for 0 -5 days. The arrows indicate TUNEL-positive cells identified by HOECHST staining. B, quantification of the percentage of TUNEL-positive nuclei in mock-transfected and ␣4␤2-nAChR-transfected cells exposed to 100 nM A␤ 1-42 for 0 -5 days. C, top panels, positive control using DNase I; bottom panels, negative (untreated) control. D, TUNEL staining on treatment with the control reverse peptide A␤ 42-1 . (43,54). Although these changes take years to develop in AD, because of attenuating if not mitigating processes in brain (e.g. clearance, antioxidative processes, competing neurotransmitter, and synaptic activities), the use of in vitro nerve cell models allows assessment of the impact of elevated A␤ in isolation. Exposure to micromolar A␤ was previously shown to alter mitochondrial dynamics and transport in cultured hippocampal neurons in a manner involving the microtubule-associating protein Tau (36). Here, we found that sustained exposure to nanomolar A␤ also results in altered mitochondrial dynamics and transport. Of particular note is the increased expression of phospho-Drp-1, which is a key player in coupling A␤ to mitochondrial fission via nitric oxide (55,56), consistent with our findings of decreased mitochondria size following A␤ treat-ment. As for mitochondrial transport, Tau has been found to contribute to A␤-induced reductions in general axonal transport (57), and thus it will also be important to consider the role of Tau in the effects of A␤ in the presence of nAChRs. We also found significant production of ROS as well as nuclear fragmentation, followed by apoptosis, all of which were greatly enhanced by the presence of the ␣4␤2 nAChRs, with nuclear fragmentation and apoptosis following receptor up-regulation. Taken together, the results indicate that the presence of nAChRs sensitizes neurons to A␤ such that sustained picomolar to nanomolar levels of the peptide can now disrupt calcium regulation and neuronal integrity. As mitochondrial function is essential for cellular and synaptic integrity, the neurotoxic effect of accumulating A␤ will thus be magnified for cholino- . Sustained exposure to A␤ led to accelerated cell death in ␣4␤2-nAChR-transfected cultures. Mock-transfected and ␣4␤2-nAChR-transfected cells were exposed to daily treatment with 100 nM A␤ 1-42 (A) and the control reverse peptide A␤ 42-1 (B) for 0 -5 days, and cell counts were performed on several random fields. A significant acceleration of cell loss was noted by day 4 in cells expressing ␣4␤2-nAChRs treated with A␤. ceptive cells, possibly contributing to the cholinergic deficit in AD.