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Alzheimer’s amyloid peptides mediate hypoxic up-regulation of L-type Ca²⁺ channels

Jason L Scragg, Ian M. Fearon^*, John P. Boyle, Stephen G. Ball, Gyula Varadi and Chris Peers¹

Institute for Cardiovascular Research, The University of Leeds, Leeds, UK.;
^* Department of Biology, McMaster University, Hamilton, ON, Canada; and
Departments of Surgery and Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA

¹ Correspondence: Institute for Cardiovascular Research, The University of Leeds, Leeds LS2 9JT, UK. E-mail: c.s.peers{at}leeds.ac.uk

SPECIFIC AIMS

Periods of prolonged hypoxia predispose individuals to subsequent development of Alzheimer’s disease. This neurodegenerative disorder involves excessive production of amyloid ß peptides (AßPs), which are neurotoxic. Toxicity occurs at least in part through disruption of intracellular Ca²⁺ homeostasis. Since altered Ca²⁺ influx is a fundamental mechanism by which Ca²⁺ homeostasis can be disrupted, our aim was to investigate potential AßP-mediated effects of hypoxia on functional Ca²⁺ channel expression.

PRINCIPAL FINDINGS

1. Hypoxia increases functional expression of recombinant L-type Ca²⁺ channels
We monitored whole-cell Ca²⁺ current density in HEK 293 cells stably expressing the _1C subunit of the human L-type Ca²⁺ channel (in the absence of auxiliary subunits). After 24 h of hypoxia (2.5% O₂), Ca²⁺ current density (measured at +20 mV for all values reported herein) increased significantly from –7.60 ± 1.26pA/pF (n=12 normoxically cultured cells) to –15.99 ± 1.43pA/pF (n=15; P<0.0005). The L-type Ca²⁺ channel blocker nifedipine (2 µM) caused a similar degree of inhibition in normoxic cells (96.6±6.1%, n=5) and hypoxic cells (90.9±2.6%, n=6), indicating that increased current density was attributable to increased functional expression of L-type Ca²⁺ channels rather than induction of novel channel types.

2. AßPs mimic the effects of hypoxia on Ca²⁺ channel currents
Twenty-four hour exposure of cells to AßP_(1-40), the commonest form of amyloid peptide, caused a concentration-dependent increase in Ca²⁺ current density (e.g., at 20 nM, current density significantly increased from –7.53±1.05pA/pF (n=9) to –11.26±0.52pA/pF (n=18, P<0.002)). Nifedipine inhibited the AßP-augmented currents by 98.0 ± 4.8% (n=6). This augmentation was also seen when cells were exposed to AßP_(1-42) (–11.88±0.57pA/pF, n=11, P<0.0001) and the "toxic fragment" AßP_(25-35) (–11.97±0.93pA/pF, n=8, P<0.0001), but not when cells were exposed to the reverse-sequence peptide AßP_(40-1) (–6.08±0.73pA/pF, n=10) used as a control.

3. Inhibition of AßP formation prevents hypoxic enhancement of Ca²⁺ currents
AßPs are produced by the sequential cleavage of amyloid precursor protein (APP) by ß- and -secretase. Selective inhibition of -secretase with -VI (2.5 µM, Fig. 1 A,upper) or ß-secretase (BS-I; 30 nM, Fig. 1A , lower) almost fully reversed the augmentation of currents by hypoxia. The summary bar graph (Fig. 1B ), which plots current densities recorded at +20 mV, shows that either of two separate -secretase inhibitors or the ß-secretase inhibitor prevented current augmentation by hypoxia whereas normoxic currents were unaffected.

View larger version (24K): [in this window] [in a new window]   Figure 1. Secretase inhibition prevents hypoxic augmentation of Ca2+ channel currents. A) Upper plot: mean current density (±SE bars) vs. voltage relationships obtained from hypoxic (CH) cells cultured under hypoxic conditions in the absence (solid symbols, n=15) and presence (open symbols, n=20) of the -secretase inhibitor, -IV (2.5 µM). Lower plot: as upper, except cells were cultured under hypoxic conditions in the absence (solid symbols, n=15) or presence (open symbols, n=17) of the ß-secretase inhibitor BS-I (30 nM). B) Bar graph showing mean (±SE) current densities (determined at a test potential of +20 mV) in control cells (open bars) and in hypoxically cultured (CH) cells (shaded bars) exposed to various secretase inhibitors. Values in bars indicate n numbers.

4. AßPs physically associate with Ca²⁺ channels to increase their surface expression
Since transcription of the Ca²⁺ channel subunit was under control of a foreign promoter in this recombinant expression system, we investigated the possibility that amyloid-mediated hypoxic enhancement of currents involved a post-transcriptional mechanism. Dual immunofluorescence studies (Fig. 2 A)indicated that, in control cells (upper images), while AßPs could be detected (red) along with Ca²⁺ channel subunits (green) their colocalization (yellow) was limited; clear areas lacking colocalization were evident. Line scan analysis of pixel intensity (shown below) emphasizes this limited colocalization. In marked contrast, the association of AßPs and Ca²⁺ channel subunits was much stronger in hypoxic cells (lower images) and was strikingly evident at the cell periphery, as emphasized by the associated line scan analysis. The possibility that AßPs might be physically associated with _1C subunits was explored using immunoprecipitation and Western blotting. Figure 2B shows blots taken from homogenates precipitated using the 3D6 antibody probed with the anti-_1C antibody. In control cells, the signal was consistently extremely weak (left) and was not apparent at all in untransfected cells (not shown). However, a clear band of appropriate molecular weight was detected in hypoxic cells (right). These results (representative of six paired experiments), were supported in experiments where homogenates were precipitated with the anti-_1C antibody, then probed using the 3D6 antibody. In this case, AßPs were detected in both control (Fig. 2B , left) and hypoxic (right) cell samples, evidence of physical interaction under both conditions, but band intensity was consistently (n=6 experiments) greater in hypoxic samples. Exposure of cells to the 3D6 antibody for 1 h fully reversed the augmentation of Ca²⁺ currents caused by chronic hypoxia or AßP_1-40 exposure (Fig. 2C ). This effect of the 3D6 antibody was not due to a current-reducing effect of the antibody per se, since when control cells were incubated in the presence of 3D6 no change in current density was seen (data not shown).

View larger version (44K): [in this window] [in a new window]   Figure 2. Colocalization of amyloid peptides and 1C Ca2+ channel subunits. A) Upper: Immunofluorescent images of the same 2 clusters of HEK 293 cells cultured under normoxic conditions. Cells were permeabilized and stained using primary antibodies against AßPs (monoclonal 3D6, left, red) and 1C subunits (middle, green). Right-hand panel shows the overlay of the other 2 panels, indicating (in yellow) limited colocalization of the two proteins. Colocalization is examined in the graph below, which plots pixel intensity taken from the scan line (A, right panel) for AßP staining (red) and 1C subunit staining (green). Lower: as upper, but images taken from hypoxic cells. B) Upper: Western blots probed with the anti-1C antibody after immunoprecipitation of cell homogentates using the anti-AßP monoclonal antibody 3D6. Note marked immunodetection in chronically hypoxic (CH) but not normoxic cells. Representative of 6 experiments. Lower: detection of AßPs in samples of control and hypoxic cell homogenates, immunoprecipitated using the anti-1C antibody as indicated. Blots were probed using the 3D6 antibody. Representative of 6 experiments. C) Bar graph plotting mean (±SE) current density observed at a test potential of +20 mV in control cells, cells exposed to 20 nM AßP1-40 for 24 h, and cells exposed to chronic hypoxia without (open bars) or with (shaded bars) subsequent exposure to the anti-amyloid peptide monoclonal antibody 3D6 (5 µg/mL). Values in bars indicate n numbers.

CONCLUSIONS AND SIGNIFICANCE

These data provide strong evidence for a novel and important role for AßPs in mediating the increase in Ca²⁺ channel activity after prolonged hypoxia. The effects of hypoxia were mimicked by exposure of cells to AßPs, and prevention of AßP formation prevented the augmenting effects of hypoxia on Ca²⁺ currents. Our use of a recombinant expression system excludes, for the first time, the possible actions of hypoxia on channel transcription, since no hypoxia response elements were present upstream of the coding sequence introduced into HEK 293 cells. Instead, our findings show that AßPs act post-transcriptionally to promote _1C subunit insertion into (and/or retention within) the plasma membrane, as summarized in Fig. 3 .This was most likely due to physical interaction of amyloid peptide with the Ca²⁺ channel subunit, as indicated by our colocalization and coimmunoprecipitation experiments (Fig. 2) . The remarkable reversal of current augmentation by the anti-AßP monoclonal antibody 3D6 (Fig. 2C ) is reminiscent of the actions of autoantibodies present in the plasma of individuals suffering from Lambert-Eaton myasthenic syndrome, in which muscular weakness occurs due to insufficient acetylcholine release from the neuromuscular junction because autoantibodies cross-link and so down-regulate presynaptic Ca²⁺ channels. If a similar mechanism underlies the effects of the 3D6 antibody as shown here, this would provide further support for direct interaction of AßPs with Ca²⁺ channels after hypoxia. Such an action of AßPs to augment Ca²⁺ influx will likely contribute to the Ca²⁺ dyshomeostasis of Alzheimer’s disease, and may contribute to the mechanisms underlying the known increased incidence of this neurodegenerative disease after hypoxic episodes.

View larger version (14K): [in this window] [in a new window]   Figure 3. Schematic indicating that hypoxia promotes amyloidogenic processing of APP to generate increased levels of AßPs that increase Ca2+ current density by promoting trafficking to (and insertion in) the plasma membrane. A possible additional effect to prevent or impede channel retrieval has not yet been discounted.

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