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【关键词】 Up-Regulation
The aim of the present study was to investigate whether KV3.4 channel subunits are involved in neuronal death induced by neurotoxic -amyloid peptides (A). In particular, to test this hypothesis, three main questions were addressed: 1) whether the A peptide can up-regulate both the transcription/translation and activity of KV3.4 channel subunit and its accessory subunit, MinK-related peptide 2 (MIRP2); 2) whether the increase in KV3.4 expression and activity can be mediated by the nuclear factor-B (NF-B) family of transcriptional factors; and 3) whether the specific inhibition of KV3.4 channel subunit reverts the A peptide-induced neurodegeneration in hippocampal neurons and nerve growth factor (NGF)-differentiated PC-12 cells. We found that A1–42 treatment induced an increase in KV3.4 and MIRP2 transcripts and proteins, detected by reverse transcription-polymerase chain reaction and Western blot analysis, respectively, in NGF-differentiated PC-12 cells and hippocampal neurons. Patch-clamp experiments performed in whole-cell configuration revealed that the A peptide caused an increase in IA current amplitude carried by KV3.4 channel subunits, as revealed by their specific blockade with blood depressing substance-I (BDS-I) in both hippocampal neurons and NGF-differentiated PC-12 cells. The inhibition of NF-B nuclear translocation with the cell membrane-permeable peptide SN-50 prevented the increase in KV3.4 protein and transcript expression. In addition, the SN-50 peptide was able to block A1–42-induced increase in KV3.4 K+ currents and to prevent cell death caused by A1–42 exposure. Finally, BDS-I produced a similar neuroprotective effect by inhibiting the increase in KV3.4 expression. As a whole, our data indicate that KV3.4 channels could be a novel target for Alzheimer's disease pharmacological therapy.
Alteration of neuronal K+ channel function, which is involved in the regulation of membrane excitability—including setting the resting potential, keeping action potentials short, and timing interspike intervals (Birnbaum et al., 2004)—leads to remarkable perturbations in neuronal function. In particular, intracellular potassium concentrations ([K+]i) play a key role in cell survival. Indeed, a decrease in cytoplasmic [K+]i, mainly caused by an increased activity of plasma membrane voltage-gated potassium (KV) channels, triggers cell death (Yu et al., 1997, 1999a,b; Lauritzen et al., 2003), possibly by activating nucleases (Bortner et al., 1997) and caspases (Hughes and Cidlowski, 1999) that, in turn, propagate and amplify death signals. The involvement of K+ currents in neuronal apoptosis (Yu, 2003) is also supported by the fact that the inhibition of K+ efflux, induced by a wide range of K+ channel blockers or by the increase in extracellular K+ concentration, fully prevents cell death (Pike et al., 1996; Colom et al., 1998). Recent research has demonstrated that changes in K+ channel activity play a major pathogenetic role in many neurodegenerative disorders, including Alzheimer's disease (AD). In fact, -amyloid fragments (A), generated upon processing of the integral membrane amyloid precursor protein (Suh and Checler, 2002), alter the properties of K+ currents in mammalian neurons (Jalonen et al., 1997; Yu et al., 1998; Jhamandas et al., 2001; Ramsden et al., 2001; Pannaccione et al., 2005). Furthermore, a study performed on SN56 cells, a cholinergic cell line from rat, reported that treatment with A increased the amount of delayed rectifier K+ currents and increased cell death (Colom et al., 1998). Among the different KV currents exhibiting distinct biophysical properties, fast-inactivating K+ currents (IA), which are known to contribute to action potential repolarization (Rudy and McBain, 2001) and to regulate interspike interval, have been suggested to participate in neurodegenerative processes (Birnbaum et al., 2004). The genes encoding for several KV channel subunits that give rise to IA currents have been cloned. Among these are KV4.1, 4.2, and 4.3 of the Shal family, KV3.3 and 3.4 of the Shaw family, and KV1.4 of the Shaker subfamily. These channel subunits differ in their pharmacology and biophysical characteristics. In particular, the expression and modulation of the KV3 subfamily play a relevant role in axons and presynaptic terminal electrophysiological control. In fact, these channels are able to keep action potentials short and to facilitate recovery of processes that accumulate during repetitive action potentials. Furthermore, they can also limit the frequency of action potential transmission. One of the components of KV3 family, namely KV3.4, besides being present in skeletal muscle and in sympathetic ganglia (Rudy et al., 1999), is also prominently expressed in the neurons of the adult dentate gyrus and in the mossy fibers of the hippocampus. In these neurons, KV3.4 subunits seem to be predominantly expressed on axons and nerve terminals (Heinemann et al., 1996; Rudy et al., 1999). Moreover, KV3.4 subunits can form stable complexes with MinK-related peptide 2 (MIRP2). Such an interaction modifies the voltage-dependence of activation, speeds the recovery from inactivation, reduces the cumulative inactivation, and finally, affects unitary conductance of KV3.4 channels (Abbott et al., 2001, 2006). In addition, KV3.4 channel subunits can form heteromeric channels by coassembling with other members of the KV3 subfamily, such as KV3.1, KV3.2, and KV3.3 channel subunits (Rudy et al., 1999; Rudy and McBain, 2001).
Although it has been demonstrated recently that A1–42 can potentiate the IA (Pannaccione et al., 2005), the molecular identity of K+ channel subunit(s) modulated by the neurotoxic peptide has not yet been determined. Thus, we investigated whether KV3.4 channel subunits and its accessory subunit MIRP2 are involved in A-induced IA modulation. In addition, the hypothesis that the overexpression and hyperactivity of KV3.4 channels could be related to the apoptotic process induced by A was evaluated by the inhibition of KV3.4 channel and by the blockade of the nuclear translocation of NF-B, the transcription factor involved in A neurotoxicity.
The data obtained showed that the A peptide induced an increase in IA currents through an up-regulation of KV3.4 channel subunit and MIRP2. Thus, the increase in KV3.4 functional activity is involved in A neurotoxicity.
Materials
-Amyloid peptide 1–42 (A1–42) and all other unmentioned materials were from Sigma Chemical (St. Louis, MO). Primary antibodies against KV3.4 and KV3.3 channel subunits and MIRP2 -subunits, as well as nerve growth factor (NGF 2.5S), tetrodotoxin, and blood depressing substance-I (BDS-I) were from Alomone Labs (Jerusalem, Israel). Primary antibody against caspase-3 and anti-rabbit IgG secondary antibody were purchased from Invitrogen (Carlsbad, CA). SN-50 and Protease Inhibitor Cocktail II were purchased from Calbiochem (San Diego, CA). RPMI 1640 medium, horse serum, fetal bovine serum, and phosphate-buffered saline (PBS) were from Invitrogen. L-Glutamine, fetal calf serum, Earle's balanced salt solution, TRIzol lysis buffer, and SuperScript III were from Invitrogen (Carlsbad, CA). Primers for KV were from Primm (Milan, Italy). AmpliTaq DNA Polymerase was from Eppendorf (Milan, Italy), and RNAase-free DNAase I was purchased from Stratagene (Milan, Italy). The marker Ladder, 100 bp, was purchased from New England Biolabs (Milan, Italy).
Cell Culture
PC-12 Cells. Rat pheochromocytoma cells (PC-12 cells) were grown as described previously (Pannaccione et al., 2005). Cells were seeded at low density on glass coverslips coated with poly(L-lysine) (50 µg/ml). Differentiation of PC-12 cells was achieved by treatment with NGF 2.5S (50 ng/ml) for 7 to 9 days (Greene and Tischler, 1976).
Rat Hippocampal Neuronal Cultures. Hippocampal neurons were obtained from the brains of 18-day-old Wistar rat embryos (Charles River, Sulzfeld, Germany) as described previously (Scorziello et al., 2001). Cytosine arabinoside at 5 µM was added within 48 h of plating to prevent the growth of non-neuronal cells. In all experiments, neurons were cultured in a humidified atmosphere at 37°C with 5% carbonic anhydrase (CO2) and used after 8 days of culturing.
Electrophysiology
K+ currents were recorded from NGF-differentiated PC-12 cells and primary rat hippocampal neurons at 20°–22°C using a commercially available amplifier (Axopatch 200A; Molecular Devices, Sunnyvale, CA), as described previously (Pannaccione et al., 2005). In most experiments, the patch-clamp technique in whole-cell configuration was performed using glass micropipettes of 2.5 to 4 M resistance. Currents were filtered at 5 kHz and digitized using a Digidata 1200 interface (Axon Instruments). Data were acquired and analyzed using the pClamp software (version 6.0.4; Axon Instruments). The pipette solution contained the following: 140 mM KCl, 2 mM MgCl2, 10 mM HEPES, 10 mM glucose, 10 mM EGTA, and 1 mM Mg-ATP adjusted at pH 7.4 with KOH. The extracellular solution contained the following: 150 mM NaCl, 5.4 mM KCl, 3 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, adjusted at pH 7.4 with NaOH. The extracellular solution for all experiments contained 50 nM tetrodotoxin.
To discriminate K+ current components having distinct inactivation properties (namely an inactivating component IA and a delayed-rectifier noninactivating component IDR), appropriate electrophysiological protocols were used. The total outward K+ current (IK = IA + IDR) was measured by applying, from a holding potential of –80 mV, depolarizing voltage steps of 250-ms duration ranging from –80 to +40 mV. These were preceded by conditioning pulses at –100 mV lasting for 1.5 s to allow full recovery from IA inactivation. Then, after conditioning pulses delivered at –40 mV lasting for 1.5 s fully inactivated IA,IDR was isolated by stepping from –80 to +40 mV for 250 ms. The IA component was thus obtained by subtracting the isolated IDR component from the total K+ current. Current amplitudes were measured for IA and IDR at +40 mV, either at the peak or at the end of the depolarizing pulse, respectively. Possible changes in cell size occurring upon specific pharmacological treatments were calculated by monitoring the membrane capacitance of each cell, which is directly related to membrane surface area, and by expressing the current amplitude data as current densities (in picoamperes per picofarad). Capacitive currents were elicited by 5-mV depolarizing pulses from –80 mV and acquired at a sampling rate of 50 kHz. The capacitance of the membrane was calculated according to the following equation: Cm = c · Io/Em(1 – I/Io), where Cm is membrane capacitance, c is the time constant of the membrane capacitance, Io is the maximum capacitance current value, Em is the amplitude of the voltage step, and I is the amplitude of the steady-state current.
Assessment of Nuclear Morphology
Nuclear morphology was evaluated by using the fluorescent DNA binding dye bis-Benzimide H 33258 (Hoechst 33258; Aventis, Strasbourg, France). To this aim, cells were fixed in 4% paraformaldehyde and incubated for 5 min in PBS containing 1 µg/ml Hoechst at 37°C. Coverslips were mounted on glass slides and observed with a fluorescence microscopy on a Nikon Eclipse E400 microscope (Nikon, Torrance, CA). Digital images were taken with a CoolSnap camera (Media Cybernetics Inc, Silver Spring, MD), stored on the hard-disk of a Pentium III computer, and analyzed with the Image-Pro Plus 4.5 software (Media Cybernetics Inc). Pathological nuclei were characterized by chromatin condensation (pyknosis), fragmentation, or by a decrease in size.
mRNA Analysis by Reverse-Transcription Polymerase Chain Reaction
Possible changes in RNA expression induced by A1–42 treatment were evaluated by semiquantitative RT-PCR. In brief, total RNA was extracted from NGF-differentiated (control) or A1–42 treated PC-12 cells using TRIzol lysis buffer (Invitrogen). To avoid contamination by genomic DNA, the extracted RNA was treated with 10 U/µl RNase-free DNAase I for 1 h at 37°C. The purity and integrity of the RNA was checked by denaturing agarose gel electrophoresis. Total RNA (2 µg) was reverse-transcribed by SuperScript III reverse transcriptase (Invitrogen) for 1 h at 50°C using oligo(dt) primers. The retrotranscribed cDNAs were amplified in an MJ Research Minicycler (MJ Reseach PTC 2000 Thermal Cycler; CELBIO, Milano, Italy) by the polymerase chain reaction using primers directed against several cDNAs (Table 1). Primers directed against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA were used as internal control. The amplification protocol (30 cycles) was the following: 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min. Each 50-µl reaction contained 1.25 U of AmpliTaq DNA Polymerase (Eppendorf) and 10 pmol of each primer. The amplification products were visualized on agarose (2%) gel electrophoresis by loading approximately half (25 µl) of each reaction per lane. The acquisition and densitometric analysis of the RT-PCR products were realized using a ChemiDoc station (Bio-Rad, Milan, Italy).
TABLE 1 mRNA primer sequences
Western Blot Analysis
Cells were washed in PBS and collected by gentle scraping in ice-cold lysis buffer to detect KV3.4, KV3.3, and MIRP2 proteins (Secondo et al., 2003) or immunoprecipitation assay buffer to detect caspase-3 protein expressions (Amoroso et al., 2002). Both lysis buffers contained Protease Inhibitor Cocktail II (Roche Diagnostics, Monza, Italy). To evaluate KV3.4, KV3.3, and MIRP2 expressions, proteins (100 µg per lane) were separated by SDS-polyacrylamide gel electrophoresis on 8% polyacrylamide gels and transferred onto nitrocellulose membranes (Hybond-ECL; GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). KV3.4, KV3.3, and MIRP2 expressions were analyzed with anti-KV3.4-, KV3.3-, and MIRP2-polyclonal antibodies. For caspase-3 expression, proteins (50 µg per lane) were separated by SDS-polyacrylamide gel electrophoresis on 12% polyacrylamide gels and transferred onto nitrocellulose membranes (Hybond-ECL; Amersham). Caspase-3 expression was analyzed with anti caspase-3-polyclonal antibody. The chemiluminescence detection was performed by using ECL Western Detection Kit (Amersham). Nonspecific binding sites were blocked by incubation for 2 h at room temperature with 5% nonfat dry milk (Bio-Rad) in Tris-buffered saline/Tween 20 buffer. Films were developed using a standard photographic procedure, and quantitative analysis of detected bands was carried out by densitometer scanning.
KV3.4 Immunofluorescence
NGF-differentiated PC12 cells and hippocampal neurons, both during control condition and after 24 h of A1–42 exposure, were washed in cold PBS and fixed in 4% (w/v) paraformaldehyde in PBS for 30 min. After four washes in PBS, the cells were first preincubated in PBS containing 3% (w/v) bovine serum albumin (Sigma) and 0.05% (v/v) Triton X-100 (Bio-Rad) for 30 min and then with the primary anti N-terminal KV3.4 polyclonal antibody (1:2000) at 4°C overnight. Next, cells were washed in PBS and finally incubated with the secondary antibody Alexa-fluor 546 anti-rabbit IgG (Molecular Probes; dilution, 1:200) for 1 h at room temperature. Cell nuclei were stained with Hoechst (Sigma). After the final wash, cells were mounted and placed on coverslips with Vectashield (Vector Laboratories, Burlingame, CA). Slides were analyzed with a Nikon Ecilpse 400 upright microscope (Nikon Instruments, Florence, Italy) equipped with a charge-coupled device digital camera (Coolsnap-Pro) and Image Pro-Plus software (both from Media Cybernetics).
-Amyloid Peptide Treatment
A1–42 was prepared as 0.1 mg/0.1 ml stock solution in sterile PBS, incubated at 37°C for 24 h to enhance aggregation, and stored at –20°C. Stock solution was directly diluted in cell culture media to give the desired experimental concentrations (5 µM).
Statistical Analysis
Statistical comparisons between controls and treated experimental groups were performed by ANOVA followed by Newman test or Student's t test. Differences were considered to be statistically significant when p values were <0.05.
Up-Regulation of the Transcripts of KV3.4 Channel Subunits and of Its Accessory Subunit MIRP2 in NGF-Differentiated PC-12 Cells Exposed to A1–42 Fragment. RT-PCR performed with specific primers against KV1.4, KV3.1, KV3.2, KV3.3, KV3.4, KV4.2, KV4.3, and their related channel subunits, MIRP1 and MIRP2 (Table 1), showed that A1–42 treatment (5 µM for 24 h) induced a selective increase in KV3.4 and MIRP2 mRNA expression but a decrease in KV4.2 (Fig. 1). By contrast, A-treatment did not modify KV1.4, KV3.1, KV3.2, KV3.3, KV4.3, and MIRP1 mRNA (Fig. 1).
Fig. 1. Up-regulation of the transcripts of KV3.4 channels in NGF-differentiated PC-12 cells exposed to A1–42 fragment. A, RT-PCR analysis of KV3.4, KV1.4, and GAPDH mRNA in control and A1–42-treated NGF-differentiated PC-12 cells. B, RT-PCR analysis of KV3.1, KV3.2, and GAPDH mRNA in control and A1–42-treated NGF-differentiated PC-12 cells. C, RT-PCR analysis of KV3.3, KV4.2, KV4.3, MIRP1, MIRP2, and GAPDH mRNA in control and A1–42-treated NGF-differentiated PC-12 cells. GADPH mRNA was used as an internal control to normalize the values. D, densitometric analysis of the ratios between the intensity of the KV1.4, KV3.1, KV3.2, KV3.3, KV3.4, KV4.2, KV4.3, MIRP1, and MIRP2 immunoreactive bands and that of the band corresponding to GAPDH (used as internal control). Data are expressed as mean (±S.E.) values obtained in three separate experiments for NGF-differentiated PC-12 cells and NGF-differentiated PC-12 cells treated with A1–42.
Overexpression of KV3.4 Channel and of Its Subunit MIRP2 Induced by A1–42 in NGF-Differentiated PC-12 Cells and in Hippocampal Neurons. Immunoblot analysis performed with a KV3.4-specific antibody on protein extracts from NGF-differentiated PC-12 cells and hippocampal neurons revealed two bands, one at 75 kDa and the other at 110 kDa, that seem to correspond to native subunit monomer and to glycosylated subunit monomers (McMahon et al., 2004), respectively (Fig. 2). Densitometric analysis showed that the bands at 75 and 110 kDa were both significantly more intense in hippocampal neurons and NGF-differentiated PC-12 cells treated with A1–42 fragment than in the controls (Fig. 2, C and D). In contrast, A1–42 treatment failed to modify KV3.3 protein expression in these neurons (Fig. 2, A and B). In addition, A1–42 exposure induced an increase in MIRP2 protein expression (Fig. 2).
Fig. 2. Overexpression of KV3.4 channels in NGF-differentiated PC-12 cells and in hippocampal neurons induced by A1–42. A, effect of A1–42 on KV3.4, KV3.3, and MIRP2 protein expression in NGF-treated PC-12 cell. B, densitometric analysis of each experimental group, normalized for the corresponding actin value (used as internal control), is expressed as a percentage of controls. Data are expressed as percentage (±S.E.) values obtained in three separate experiments for NGF-differentiated PC-12 cells and NGF-differentiated PC-12 cells treated with A1–42. C, effect of A1–42 on KV3.4 and MIRP2 protein expression in hippocampal neurons. D, densitometric analysis of each experimental group, normalized for the corresponding actin value (used as internal control), is expressed as a percentage of controls. Data are expressed as percentage (±S.E.) values obtained in three separate experiments in hippocampal neurons.*, p < 0.05 versus control values (Student's t test).
The KV3.4 Inhibitor BDS-I Prevents A1–42 Induced Increase of IA Currents. Patch-clamp experiments, performed in hippocampal neurons, revealed that A1–42 treatment (5 µM for 24 h) caused an increase in IA current amplitude, previously isolated with the voltage subtraction protocol described under Materials and Methods (Fig. 3). IA currents, obtained upon subtraction of IDR from IK currents, were blocked by the extracellular application of the KV3 subfamily inhibitor BDS-I (50 nM) (Diochot et al., 1998; Yeung et al., 2005) (Fig. 3). The percentage of the BDS-I inhibition was higher in A1–42-treated hippocampal neurons than in control neurons (Fig. 3D).
Fig. 3. The KV3.4 inhibitor BDS-I prevents A1–42-induced increase of IA currents. A, total outward K+ currents recorded from hippocampal neurons exposed to vehicle or 50 nM BDS-I for 5 min (top) or to A1–42 (24 h) and A1–42+BDS-I (bottom). B, IDR currents recorded from hippocampal neurons exposed to vehicle or 50 nM BDS-I for 5 min (top) or to A1–42 (24 h) and A1–42+BDS-I (bottom). C, IA currents obtained in each cell upon subtraction of IDR from IK currents. D, IA current quantification expressed as a percentage of inhibition compared with control in the absence or presence of A1–42 in hippocampal neurons exposed to 50 nM BDS-I. The values are expressed as mean (±S.E.M.) of current densities recorded from 6 to 40 cells in each experimental group, as indicated. *, p < 0.05 versus control values (Student's t test).
A1–42-Induced Apoptosis Is Accompanied by an Up-Regulation in KV3.4 Channel Subunits in NGF-Differentiated PC-12 Cells and in Hippocampal Neurons. Immunocytochemical analysis, performed with the selective anti-KV3.4 antibodies, revealed a plasma membrane localization and a punctated staining pattern mostly confined throughout the neuropil of NGF-differentiated PC-12 cells and hippocampal neurons (Figs. 4, A and C, and 5A). Similarly to what was observed in Western blot analysis, A1–42 exposure increased KV3.4 immunoreactivity signal (Figs. 4, D, F, and G, and 5, B and C). It is interesting that the anti-KV3.4 antibody revealed a pronounced perikaryal staining that was intensely confined to the somatic plasma membrane (Figs. 4, D, F, and G, and 5, B and C). In addition, double-labeling experiments of KV3.4 channel subunits with the fluorescent-DNA binding dye Hoechst 33258 revealed that cells showing KV3.4 channels immunoreactivity after amyloid peptide exposure also displayed an abnormal nuclear morphology (Figs. 4, B and E, and 5, A-a and C-c), thus suggesting that neurons overexpressing this K+ channel undergo apoptosis. This result was confirmed by Western blot analysis performed with a caspase-3-specific antibody on protein extracts from NGF-differentiated PC-12 cells. In fact, A1–42 exposure induced an increase of caspase-3 expression (Fig. 4H). Likewise, 24-h A1–42 exposure induced the activation of caspase-3 in hippocampal neurons (Fig. 5D).
Fig. 4. Effect of A1–42 on the distribution of KV3.4 channel subunits and on the apoptotic process in NGF-differentiated PC-12 cells. Double immunofluorescence images of NGF-differentiated PC-12 cells displaying both KV3.4 protein expression and Hoechst-33258 staining in control (A and B, respectively) and in cells exposed to A1–42 for 24 h (D and E, respectively). Arrows point to normal (B) or abnormal nuclei (E) showing pyknosis, fragmentation, or a decreased nuclear size. High-magnification images of representative KV3.4 staining pattern in control (C) and A1–42-treated NGF-differentiated PC-12 cell (F and G). Arrows point to the KV3.4 immunoreactivity signal along cellular processes of control cells (A and C) or around the soma plasma membrane in A1–42-exposed cells (D, F, and G). H, effect of A1–42 on caspase-3 expression in NGF-differentiated PC-12 cells. Staurosporine was used as positive control. Scale bar, 50 µm in A, B, D, and E; 20 µm in C, F, and G.
Fig. 5. Effect of A1–42 on the distribution of KV3.4 channel subunits and on the apoptotic process in hippocampal neurons. Confocal double immunofluorescence images of a single representative hippocampal neuron displaying both KV3.4 protein expression and nuclear staining in control (A and a, respectively) and in cells exposed to A1–42 for 24 h (B–C and c, respectively). B and C refer to the same cell registered with differing focus points. Note the finely punctated KV3.4 labeling in control cells (A, arrows) or the intense perikaryal KV3.4 immunoreactivity in A1–42-exposed cells (C, arrows). D, effect of A1–42 on caspase-3 expression in hippocampal neuron. Scale bar, 20 µm in A, B, and C.
Prevention of A1–42-Induced Apoptosis by BDS-I in NGF-Differentiated PC-12 Cells and in Hippocampal Neurons. BDS-I (50 nM), the KV3 subfamily inhibitor, was able to significantly prevent the abnormal nuclear morphology induced by the exposure of NGF-differentiated PC-12 cells (Fig. 6, A and B) and of hippocampal neurons (Fig. 6, C and D) to A1–42 (5 µM for 24 h), as detected by the Hoechst 32258 dye staining.
Fig. 6. Prevention of A1–42-induced apoptosis by the inhibition of KV3.4 channel activity in NGF-differentiated PC-12 cells and in hippocampal neurons. A, assessment of nuclear morphology with Hoechst-33258 in NGF-differentiated PC-12 cells exposed for 24 h either to 5 µM A1–42, to A1–42+BDS-I, or to only BDS-I. B, quantification of the results obtained in four separate experiments in which at least 10 microscopic fields were analyzed (approximately 1000 cells per group). *, p < 0.05 versus controls; **, p < 0.05 versus A1–42 groups (Student's and ANOVA test). C, assessment of nuclear morphology evaluated with Hoechst 33258 in primary rat hippocampal neurons in different experimental conditions. D, nuclear morphology quantification assessment with Hoechst 33258 in primary rat hippocampal neurons in different experimental conditions. The mean (±S.E.) values were obtained in four separate experiments in which at least 10 microscopic fields were analyzed (approximately 1000 cells per group). *, p < 0.05 versus control groups **, p < 0.05 versus A1–42 groups (Student's t test and ANOVA).
Prevention of Protein and Transcript Expression of KV3.4 Channel Subunits and of Apoptotic Nuclear Features by the Inhibition of NF-B Nuclear Translocation in NGF-Differentiated PC-12 Cells. RT-PCR (Fig. 7, A and B) and Western blot analysis (Fig. 7, C and D) performed in the presence and in the absence of SN-50 (5 µM)—a cell membrane-permeable peptide capable of inhibiting the nuclear translocation of NF-B complexes (Lin et al., 1995)—showed that this agent largely prevented the increase in KV3.4 mRNA and protein expression, respectively, caused by A1–42 exposure (5 µM, 24 h). Furthermore SN-50, the NF-B inhibitor, was able to prevent A1–42-induced increase of IA current (Fig. 7E).
Fig. 7. The inhibition of NF-B nuclear translocation prevents protein and transcript expression of KV3.4 channel subunits and apoptotic nuclear feature. RT-PCR analysis of KV3.4 in NGF-differentiated PC-12 cells in basal conditions (control group) or exposed for 24 h to either 5 µM A1–42, A1–42 + SN-50, or SN-50 alone. B, densitometric analysis of the ratios between the intensity of the KV3.4 in the same experimental conditions. Data are expressed as mean (±S.E.) values obtained in three separate experiments in NGF-differentiated PC-12 cells or in cells treated with A1–42 in the presence or absence of SN-50, respectively, or in cells exposed only to SN-50. C, effect on KV3.4 protein expression in NGF-treated PC-12 cells under the same experimental conditions. D, densitometric analysis of each experimental group, normalized for the corresponding actin value (used as internal control), is expressed as a percentage of controls. Data are expressed as the percentage mean (±S.E.) values obtained in three separate experiments in NGF-differentiated PC-12 cells or in cells treated with A1–42 in the presence or absence of SN-50, respectively, or in cells exposed only to SN-50. E, IA superimposed traces recorded from NGF-differentiated PC-12 cells untreated and/or exposed for 24 h to 5 µM A1–42 in the presence or absence of SN-50, respectively. *, p < 0.05 versus control groups; **, p < 0.05 versus A1–42 groups (Student's t test and ANOVA).
In this study, we provide evidence that hippocampal neurons and NGF-differentiated PC-12 cells exposed to A1–42 treatment display a selective up-regulation of KV3.4 channel subunits through the activation of the transcriptional factor NF-B. In addition, this transcriptional event is associated with a current increase carried by this channel. It is interesting that the action of A1–42 was not limited to the poreforming KV3.4 subunit. Indeed, it also induced an overexpression of MIRP2, a neuronal -subunit coassembling with KV3.4 subunit and playing a crucial role in the control of its biophysical properties and pharmacological profile (Abbott et al., 2001, 2006). Such MIRP2 overexpression might profoundly interfere with the function of pore-forming KV3.4 -subunits, suggesting that the neurotoxic A peptide might influence the IA current by modulating simultaneously the pore-forming -subunit and its accessory component expression.
On the other hand, A1–42 exposure selectively modulated the expression and the activity of KV3.4 channel subunits because the amyloid peptide did not modify the mRNA expression of the other KV channels, KV1.4, KV3.1, KV3.2, KV3.3, and KV4.3 subunits. In agreement with biochemical and electrophysiological results, the immunocytochemical analysis confirmed that the A1–42-induced up-regulation of KV3.4 channel subunit was associated with an altered subcellular distribution of this protein. In fact, A1–42 treatment induced a more intense staining for KV3.4 subunits; after A1–42 treatment, KV3.4 immunoreactivity was mainly confined to the somatic plasma membrane, compared with that of control cells in which the fluorescent signal was mostly distributed through the neuropil. This different KV3.4 distribution induced by the neurotoxic peptide could be caused by the altered integrity of the cytoskeletal network prompted by oligomeric or fibrillar forms of A (Michaelis et al., 2005). These findings are in accordance with other experiments carried out with rhodaminate-phalloidin, a selective actin fluorescence staining, which revealed that A1–42 causes a cytoskeletal rearrangement (Pannaccione et al., 2005). In addition, hippocampal neurons and NGF-differentiated PC-12 cells expressing increased KV3.4 immunoreactivity showed an apoptotic nuclear process, as revealed by caspase-3 activation (Amoroso et al., 2002; Tamagno et al., 2006) and by Hoechst 33258-monitored abnormal nuclear morphology, thus suggesting a possible link between the enhanced expression and function of this K+ channel and the neurotoxic consequences prompted by A exposure. That the exposure to A fragments may alter the properties of K+ currents in mammalian neurons (Jalonen et al., 1997; Colom et al., 1998; Yu et al., 1998; Jhamandas et al., 2001; Ramsden et al., 2001; Pannaccione et al., 2005) and therefore may cause an increase in cell death as a result of a decrease in cytoplasmic K+ concentrations (Bortner et al., 1997; Hughes and Cidlowski, 1999) has already been reported. Nevertheless, the molecular identification of the K+ channel involved has been a matter of debate. In particular, Pan et al. (2004) showed that a single intracerebroventricular injection of A25–35 in rat up-regulates KV1.4 and KV2.1 expression in the hippocampus and KV4.2 in the cortex. Likewise, an upregulation of KV4.2 channel subunit, carrying IA current, has also been observed in rat cerebellar granule cells exposed to neurotoxic A peptides (Plant et al., 2006). By contrast, in the present study, the expression of KV4.2 mRNA was down-regulated by neurotoxic A1–42. It is noteworthy that the expression of KV4.2 gene can be differentially regulated in different cell types or by following several injury models (Tsaur et al., 1992; Jia and Takimoto, 2003).
The hypothesis that KV3.4 channels are involved in the A1–42 neurotoxic effect was further supported by the results showing that BDS-I, a KV3.4 blocker (Diochot et al., 1998), exerted a potent neuroprotective action in hippocampal neurons and NGF-differentiated PC-12 cells exposed to A1–42 peptide. On the other hand, the possibility that the neuroprotective action of BDS-I is also caused by a blockade of the other HKV3 subfamily members KV3.1, KV3.2, and KV3.3 (Yeung et al., 2005) does not seem a likely explanation because their expression is unmodified after A peptide exposure.
Regarding the NF-B family of transcriptional factors, their involvement in AD pathogenesis has long been corroborated by insightful evidence. In fact, an increased NF-B activity has been detected in neurons and astrocytes in the immediate vicinity of amyloid plaques in brain sections from patients with AD (Ferrer et al., 1998); furthermore, a significant NF-B activation in primary neuronal cells exposed to A peptides has also been documented (Barger and Mattson, 1996). In addition, data obtained previously in our laboratory (Pannaccione et al., 2005) have revealed that the highly specific and cell membrane-permeable NF-B inhibitor SN-50 (Lin et al., 1995) exerts a significant neuroprotective effect upon A1–42 exposure (Pannaccione et al., 2005). In the present study, SN-50 largely prevented the increase in KV3.4 protein expression and activity triggered by A1–42 exposure, suggesting that NF-B mediates the transcriptional regulation of KV3.4 occurring during A exposure. Further evidence that the increased expression of KV3.4 channels was caused by the stimulation of protein transcription and synthesis rather than by an inhibition of its catabolism is that KV3.4 mRNA also increased. In addition, other experiments have revealed that the protein synthesis inhibitor cycloheximide and the transcriptional inhibitor actinomycin D fully prevent A-induced K+ currents (Pannaccione et al., 2005). It is interesting that cDNA array analysis has also demonstrated that the KV3.4 gene is up-regulated in the brain extracts of transgenic mice with the amyloid precursor protein 695 Swedish mutation and in the cerebral cortex of patients in the early and later stages of AD (Angulo et al., 2004).
As a whole, our data suggest that A1–42 fragment exposure causes the up-regulation not only of KV3.4 but also of its accessory subunit MIRP2. This increased expression is associated with an enhanced activity of the K+ current carried by this channel. Furthermore, the changes resulting from this channel activation may contribute to a reduction in [K+]i, which in turn triggers the apoptotic process, as already demonstrated (Yu, 2003).
In conclusion, the relationship between KV3.4 and apoptotic cell death is demonstrated in the current study by the fact that the blockade of this channel was able to prevent the apoptotic process triggered by A1–42 fragment. These results reveal new and additional therapeutic strategies for AD using selective voltage-dependent potassium channels as molecular targets.
Acknowledgements
We thank Vincenzo Grillo for technical help with animal housing and Dr. Paola Merolla for editorial revision.
ABBREVIATIONS: KV channels, voltage-gated K+ channels; A, -amyloid peptide; A1–42, -amyloid peptide 1–42; AD, Alzheimer's disease; BDS-I, blood depressing substance-I; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IA, fast inactivating K+ currents; MIRP, MinK-related peptide; NGF, nerve growth factor; NF-B, nuclear factor B; PBS, phosphate-buffered saline; RT-PCR, reverse transcription polymerase chain reaction; ANOVA, analysis of variance; bp, base pair(s); SN-50, NF-B cell-permeable inhibitory peptide (AAVALLPAVLLALLAPVQRKRQKLMP).
【参考文献】
Abbott GW, Butler MH, Bendahhou S, Dalakas MC, Ptacek LJ, and Goldstein SA (2001) MiRP2 forms potassium channels in skeletal muscle with Kv3.4 and is associated with periodic paralysis. Cell 104: 217–231.
Abbott GW, Butler MH, and Goldstein SA (2006) Phosphorylation and protonation of neighboring MiRP2 sites: function and pathophysiology of MiRP2-Kv3.4 potassium channels in periodic paralysis. FASEB J 20: 293–301.[Abstract/Free Full Text]
Amoroso S, D'Alessio A, Sirabella R, Di Renzo G, and Annunziato L (2002) Ca2+-independent caspase-3 but not Ca2+-dependent caspase-2 activation induced by oxidative stress leads to SH-SY5Y human neuroblastoma cell apoptosis. J Neurosci Res 68: 454–562.
Angulo E, Noe V, Casado V, Mallol J, Gomez-Isla T, Lluis C, Ferrer I, Ciudad CJ, and Franco R (2004) Up-regulation of the Kv3.4 potassium channel subunit in early stages of Alzheimer's disease. J Neurochem 91: 547–557.
Barger SW and Mattson MP (1996) Induction of neuroprotective kB-dependent transcription by secreted forms of the Alzheimer's -amyloid precursor. Brain Res Mol Brain Res 40: 116–126.
Birnbaum SG, Varga AW, Yuan LL, Anderson AE, Sweatt JD, and Schrader LA (2004) Structure and function of Kv4-family transient potassium channels. Physiol Rev 84: 803–833.[Abstract/Free Full Text]
Bortner CD, Hughes FM, and Cidlowski JA Jr (1997) A primary role for K+ and Na+ efflux in the activation of apoptosis. J Biol Chem 272: 32436–32442.[Abstract/Free Full Text]
Colom LV, Diaz ME, Beers DR, Neely A, Xie WJ, and Appel SH (1998) Role of potassium channels in amyloid-induced cell death. J Neurochem 70: 1925–1934.
Diochot S, Schweitz H, Beress L, and Lazdunski M (1998) Sea anemone peptides with a specific blocking activity against the fast inactivating potassium channel Kv3.4. J Biol Chem 273: 6744–6749.[Abstract/Free Full Text]
Ferrer I, Marti E, Lopez E, and Tortosa A (1998) NF-kB immunoreactivity is observed in association with A4 diffuse plaques in patients with Alzheimer's disease. Neuropathol Appl Neurobiol 24: 271–277.
Greene LA and Tischler AS (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A 73: 2424–2428.[Abstract/Free Full Text]
Heinemann SH, Rettig J, Graack HR, and Pongs O (1996) Functional characterization of Kv channel beta-subunits from rat brain. J Physiol 493 (Pt 3): 625–633.[Abstract/Free Full Text]
Hughes FM Jr and Cidlowski JA (1999) Potassium is a critical regulator of apoptotic enzymes in vitro and in vivo. Adv Enzyme Regul 39: 157–171.
Jalonen TO, Charniga CJ, and Wielt DB (1997) beta-Amyloid peptide-induced morphological changes coincide with increased K+ and Cl– channel activity in rat cortical astrocytes. Brain Res 746: 85–97.
Jhamandas JH, Cho C, Jassar B, Harris K, MacTavish D, and Easaw J (2001) Cellular mechanisms for amyloid beta-protein activation of rat cholinergic basal forebrain neurons. J Neurophysiol 86: 1312–1320.[Abstract/Free Full Text]
Jia Y and Takimoto K (2003) GATA and FOG2 transcription factors differentially regulate the promoter for Kv4.2 K+ channel gene in cardiac myocytes and PC12 cells. Cardiovasc Res 60: 278–287.[Abstract/Free Full Text]
Lauritzen I, Zanzouri M, Honore E, Duprat F, Ehrengruber MU, Lazdunski M, and Patel AJ (2003) K+-dependent cerebellar granule neuron apoptosis. Role of task leak K+ channels. J Biol Chem 278: 32068–32076.[Abstract/Free Full Text]
Lin YZ, Yao SY, Veach RA, Torgerson TR, and Hawiger J (1995) Inhibition of nuclear translocation of transcription factor NF-B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J Biol Chem 270: 14255–14258.[Abstract/Free Full Text]
McMahon A, Fowler SC, Perney TM, Akemann W, Knopfel T, and Joho RH (2004) Allele-dependent changes of olivocerebellar circuit properties in the absence of the voltage-gated potassium channels Kv3.1 and Kv3.3. Eur J Neurosci 19: 3317–3327.
Michaelis ML, Ansar S, Chen Y, Reiff ER, Seyb KI, Himes RH, Audus KL, and Georg GI (2005) -Amyloid-induced neurodegeneration and protection by structurally diverse microtubule-stabilizing agents. J Pharmacol Exp Ther 312: 659–668.[Abstract/Free Full Text]
Pan Y, Xu X, Tong X, and Wang X (2004) Messenger RNA and protein expression analysis of voltage-gated potassium channels in the brain of Abeta(25–35)-treated rats. J Neurosci Res 77: 94–99.
Pannaccione A, Secondo A, Scorziello A, Calì G, Tagliatatela M, and Annunziato L (2005) NF-kB activation by reactive oxygen species mediates voltage-gated K+ current enhancement by neurotoxic -amyloid peptides in NGF-differentiated PC-12 cells and hippocampal neurons. J Neurochem 94: 572–586.
Pike CJ, Balazs R, and Cotman CW (1996) Attenuation of beta-amyloid neurotoxicity in vitro by potassium-induced depolarization. J Neurochem 67: 1774–1777.
Plant LD, Webster NJ, Boyle JP, Ramsden M, Freir DB, Peers C, and Pearson HA (2006) Amyloid beta peptide as a physiological modulator of neuronal `A'-type K+ current. Neurobiol Aging 27: 1673–1683.
Ramsden M, Plant LD, Webster NJ, Vaughan PF, Henderson Z, and Pearson HA (2001) Differential effects of unaggregated and aggregated amyloid beta protein (1–40) on K+ channel currents in primary cultures of rat cerebellar granule and cortical neurones. J Neurochem 79: 699–712.
Rudy B, Chow A, Lau D, Amarillo Y, Ozaita A, Saganich M, Moreno H, Nadal MS, Hernandez-Pineda R, Hernandez-Cruz A, et al. (1999) Contributions of Kv3 channels to neuronal excitability. Ann N Y Acad Sci 868: 304–343.
Rudy B and McBain CJ (2001) Kv3 channels: voltage-gated channels designed for high frequency repetitive firing. Trends Neurosci 24: 517–526.
Scorziello A, Pellegrini C, Forte L, Tortiglione A, Gioielli A, Iossa S, Amoroso S, Tufano R, Di Renzo G, and Annunziato L (2001) Differential vulnerability of cortical and cerebellar neurons in primary culture to oxygen glucose deprivation followed by reoxygenation. J Neurosci Res 63: 20–26.
Secondo A, Sirabella R, Formisano L, D'Alessio A, Castaldo P, Amoroso S, Ingleton P, Di Renzo G, and Annunziato L (2003) Involvement of PI3'-K, mitogen-activated protein kinase and protein kinase B in the up-regulation of the expression of nNOSalpha and nNOSbeta splicing variants induced by PRL-receptor activation in GH3 cells. J Neurochem 84: 1367–1377.
Suh YH and Checler F (2002) Amyloid precursor protein, presenilins, and alpha-synuclein: molecular pathogenesis and pharmacological applications in Alzheimer's disease. Pharmacol Rev 54: 469–525.[Abstract/Free Full Text]
Tamagno E, Bardini P, Guglielmotto M, Danni O, and Tabaton M (2006) The various aggregation states of beta-amyloid 1–42 mediate different effects on oxidative stress, neurodegeneration, and BACE-1 expression. Free Radic Biol Med 41: 202–212.
Tsaur ML, Sheng M, Lowenstein DH, Jan YN, and Jan LY (1992) Differential expression of K+ channel mRNAs in the rat brain and down-regulation in the hippocampus following seizures. Neuron 8: 1055–1067.
Yeung SY, Thompson D, Wang Z, Fedida D, and Robertson B (2005) Modulation of Kv3 subfamily potassium currents by the sea anemone toxin BDS: significance for CNS and biophysical studies. J Neurosci 25: 8735–8745.[Abstract/Free Full Text]
Yu SP (2003) Regulation and critical role of potassium homeostasis in apoptosis. Prog Neurobiol 70: 363–386.
Yu SP, Farhangrazi ZS, Ying HS, Yeh CH, and Choi DW (1998) Enhancement of outward potassium current may participate in beta-amyloid peptide-induced cortical neuronal death. Neurobiol Dis 5: 81–88.
Yu SP, Yeh C, Strasser U, Tian M, and Choi DW (1999b) NMDA receptor-mediated K+ efflux and neuronal apoptosis. Science 284: 336–339.[Abstract/Free Full Text]
Yu SP, Yeh CH, Gottron F, Wang X, Grabb MC, and Choi DW (1999a) Role of the outward delayed rectifier K+ current in ceramide-induced caspase activation and apoptosis in cultured cortical neurons. J Neurochem 73: 933–941.
Yu SP, Yeh CH, Sensi SL, Gwag BJ, Canzoniero LM, Farhangrazi ZS, Ying HS, Tian M, Dugan LL, and Choi DW (1997) Mediation of neuronal apoptosis by enhancement of outward potassium current. Science 278: 114–117.[Abstract/Free Full Text]
作者单位：Division of Pharmacology, Department of Neuroscience, School of Medicine, Federico II University of Naples, Naples, Italy