b-Site APP cleaving enzyme up-regulation ... - Wiley Online Library

Journal of Neurochemistry, 2005, 92, 628–636

doi:10.1111/j.1471-4159.2004.02895.x

b-Site APP cleaving enzyme up-regulation induced by 4-hydroxynonenal is mediated by stress-activated protein kinases pathways Elena Tamagno,* Maurizio Parola,* Paola Bardini,* Alessandra Piccini, Roberta Borghi, Michela Guglielmotto,* Gianni Santoro,* Annalisa Davit,* Oliviero Danni,* M. A. Smith,à G. Perryà and Massimo Tabaton *Department of Experimental Medicine and Oncology, General Pathology Section, University of Turin, Turin, Italy Department of Neuroscience, Ophthalmology and Genetics, University of Genoa, Genoa, Italy àCase Western Reserve University, Cleveland, Ohio, USA

Abstract 4-Hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, up-regulates expression of the b-site APP cleaving enzyme (BACE-1), an aspartyl protease responsible for the b-secretase cleavage of amyloid precursor protein (AbPP), and results in increased levels of amyloid b (Ab) peptide. The mechanisms underlying this remain unclear but are of fundamental importance because prevention of BACE-1 upregulation is viewed as an important therapeutic strategy. In this study, we exposed NT2 neurons to a range of HNE concentrations (0.5–5 lM) that elicited an up-regulation of BACE1 expression, a significant increase in intracellular and secreted levels of Ab peptides as well as apoptosis involving poly-ADP ribose polymerase cleavage and activation of caspase 3. To delineate the molecular events involved in HNE-mediated BACE-1 activation, we investigated the

involvement of stress-activated protein kinases (SAPK), signal transducers and activators of transcription (STAT) and serine–threonine kinase B/phosphatidylinositol phosphate 3 kinase (Akt/PtdIns3K). Using specific pharmacological inhibitors, our results show that activation of c-Jun N-terminal kinases and p38MAPK., but not STAT or Akt/PtdIns3K, pathways mediate the HNE-dependent up-regulation of BACE-1 expression. Therefore, HNE, an oxidative stress mediator detected in vivo in the brains of Alzheimer’s disease patients, may play a pathogenetic role in Alzheimer’s disease by selectively activating SAPK pathways and BACE-1 that regulate the proteolytic processing of AbPP. Keywords: Alzheimer’s disease, b-site APP cleaving enzyme, cell signaling, 4-hydroxynonenal, oxidative stress, stress-activated protein kinase. J. Neurochem. (2005) 92, 628–636.

Alzheimer’s disease (AD) is characterized by the selective loss of synapses and neurons and the presence of amyloid plaques primarily composed of aggregated amyloid b peptide (Ab) 40–42 amino acids in length (Iversen et al. 1995; McDonald et al. 1998). The accumulation and polymerization of Ab are considered to be pathologically important in AD (Selkoe 2000), and this is supported by the effect of gene mutations causing familial early-onset AD and by the impairment exerted by Ab on synaptic transmission and neuronal viability (Giovanni et al. 2000; Eckert et al. 2003). Another key player in AD pathology is oxidative stress, which, like Ab, is an early event in disease pathogenesis (Nunomura et al. 2001) and affects membrane damage, cytoskeletal alterations and cell death (Perry et al. 2000a,b).

Received July 29, 2004; revised manuscript received September 22, 2004; accepted September 28, 2004. Address correspondence and reprint requests to Elena Tamagno, Department of Experimental Medicine and Oncology, General Pathology Section, University of Turin, Corso Raffaello 30, 10125, Turin, Italy. E-mail:[email protected] Abbreviations used: Ab, amyloid b; AD, Alzheimer’s disease; AbPP, amyloid precursor protein; Akt, serine–threonine kinase B; BACE-1, b-site APP cleaving enzyme; DAPI, 4¢,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; ELISA, enzyme-linked immunosorbent assay; HNE, 4-hydroxynonenal; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; PARP, poly ADP ribose polymerase; PBS, phosphate-buffered saline; PtdIns3K, phosphoinositide 3 kinase; SAPK, stress-activated protein kinases; STAT, signal transducers and activators of transcription.

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2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 628–636

BACE-1 and signaling pathways

Notably, oxidative stress and Ab are inexorably linked because Ab aggregation induces oxidative stress in vitro and in vivo (Harkany et al. 2000) and oxidants increase the production of Ab (Misonou et al. 2000; Paola et al. 2000). This toxic loop likely plays a crucial role in sporadic AD because oxidative stress is an age-dependent phenomenon (Floyd and Hensley 2002) and aging is the biggest risk factor for AD. Ab derives from a type I integral membrane amyloid precursor protein (AbPP), by two consecutive proteolytic cleavages operated by b and c secretase (Selkoe 2001). b-Site APP cleaving enzyme (BACE-1) is a 501 amino acid aspartyl protease widely expressed in brain, that fulfills most of the requirements expected for a candidate b secretase (Hussain et al. 1999; Sinha et al. 1999; Vassar et al. 1999; Yan et al. 1999; Lin et al. 2000). We have previously shown that oxidants including the aldehydic end product of lipid peroxidation, 4-hydroxynonenal (HNE), up-regulate BACE1 expression and activity in differentiated neuronal NT2 cells (Tamagno et al. 2002) and this finding was subsequently extended to other oxidants (Kao et al. 2004). The cellular signaling pathways that mediate this event are unknown, however, members of the stress-activated protein kinases (SAPK) family, such as c-Jun N-terminal kinase (JNKs) and p38MAPK, which are markedly up-regulated in AD (Zhu et al. 2000, 2001a,b,c, 2003b) and are activated by a variety of stress signals, including oxidative stress (Wang et al. 1998; Le Niculescu et al. 1999), have been proposed to be important signaling components linking extracellular stimuli to cellular responses. Recently, it has also been shown that interferon c up-regulates the expression and stimulates the activity of BACE-1 in astrocytes, through induction of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway (Hong et al. 2003). Because it is known that this pathway is activated in response to the generation of oxidative stress (Madamanchi et al. 2001), it may also be potentially involved in the activation of BACE-1 in neuronal cells. Also, the serine/threonine kinase Akt/ protein kinase B is induced via a phosphatidylinositol 3-kinase (PtdIns3K)-dependent signaling pathway under conditions of oxidative stress, including HNE, and therefore might also be involved in HNE-dependent BACE-1 activation (Nakashima et al. 2003). In this study, we investigated the role of the above signaling pathways (JNK, p38, JAK/STAT and Akt) on amyloid processing and BACE-1 expression induced by HNE in NT2 neuronal cells.

Materials and methods Antibodies and reagents The following antibodies were used: polyclonal antibody against BACE-1, recognizing residues 455–501 of the molecule (Oncogene Research Products, La Jolla, CA, USA); monoclonal antibody 6E10,

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specific for residues 6–10 of Ab (Signet Pathology System Inc., Dedham, MA, USA); polyclonal antibody specific for 22 amino acids of C-terminus of AbPP (Zymed Laboratories Inc., San Francisco, CA, USA); monoclonal antibody 22C11, that recognizes AbPP N-terminus (Boehringer-Mannheim, Germany); monoclonal antibody pSTAT1 and polyclonal antibodies JNKs, STAT1, PARP and caspase 3 were from Santa Cruz Biotecnology (La Jolla, CA, USA); polyclonal antibodies pSAPK/JNKs, pp38MAPK, p38MAPK were from New England Biolabs (Hertfordshire, UK); monoclonal antibody anti-b-actin was from Sigma Chemical Co (St. Louis, MO, USA). AG490 (inhibitor of JAK family tyrosine kinase), SB203580 (inhibitor of p38MAPK) and 4-hydroxynonenal were purchased from Calbiochem (La Jolla, CA, USA). JNKs inhibitor SP600125 was from Biomol Research Laboratories (Plymouth, PA, USA). All reagents used for cell culture and differentiation were from Sigma. Cell culture and differentiation NT2 undifferentiated cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM/F12), supplemented with 5% fetal bovine serum and 1% antibiotic mixture comprising penicillin–streptomycin–amphotericin, in a humified atmosphere at 37C with 5% CO2. For differentiation, 2 · 106 cells were plated in 75 cm2 culture flasks (Costar) and exposed to 10 lM retinoic acid for 5 weeks. Growth medium was changed three times a week. Cells were then replated and, 48 h after, mitotic inhibitors cytosine arabinoside (1 lM), fluorodeoxyuridine (10 lM) and uridine (10 lM) were added for 2 weeks to inhibit the division of non-neuronal cells. Experiments were performed 4–5 weeks after cessation of retinoic acid treatment. Treatment conditions NT2 neuronal cells were left for 16 h in serum-free DMEM and then incubated for 1 and 24 h with HNE 0.5–1–5 lM or for 5 h with H2O2 20 lM or with anisomycin 10 lM. Inhibitors of JNKs and p38MAPK (SP600125 20 lM and SB203580 10 lM, respectively) were added to culture medium 15 min before exposure of cells to HNE concentrations. Inhibitor of JAK (AG490 50 lM) was added 16 h before treatment with pro-oxidant conditions. Cychloheximide 10 lM and actinomycin D 1 lM were added 30 min before HNE. Enzyme-linked immunosorbent assay The levels of Abx-40 and Abx-42 were measured by sandwich enzyme-linked immunosorbent assay (ELISA) method following the manufacturer’s instructions (IBL, Gumna, Japan). Three 75 cm2 flasks of differentiated NT2 cells for each condition were collected to prepare cell lysates. Media were concentrated 20-fold. Samples were then analyzed following the manufacturer’s instructions (IBL, Gumna, Japan). The measurement range of this assay is given between 7 and 1000 pg/mg of proteins, for both Ab species. The Ab concentration was detected using a Benchmark Microplate Reader and evaluated by MICROPLATE MANAGER v. 5.1 software (Bio-Rad, Hercules, CA, USA). Values were expressed as variations in percentage from basal condition for each sample and normalized to total protein concentration. ELISA analysis of all samples was performed in two different experiments. Immunoblot analysis NT2 neuronal cells, treated under appropriate experimental conditions, were quickly placed on ice and washed with ice-cold


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phosphate-buffered saline (PBS). Cell lysates and nuclear extracts were obtained as previously described by the method of Andrew and Faller (1991). Lysates and nuclear extracts were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 9.3% (JNKs, p38MAPK., STAT and Akt phosphorylated and not phosphorylated forms) or 7.5% (BACE-1 and full-length AbPP) acrylamide gels using the mini-PROTEAN II electrophoresis cell (Bio-Rad) according to Laemmli (1970). Proteins were transferred onto nitrocellulose membranes (Hybond-C extra Amersham Life Science, Arlington Heights, IL, USA). For C-terminal fragment extraction, cell lysates were immunoprecipitated with the polyclonal antibody anti-Cterminus of AbPP conjugated with protein A–Sepharose beads for 12 h at 4C. Immunoprecipitated proteins were separated in 10–16% Tris–tricine gels, transferred onto nitrocellulose membranes, and detected with the monoclonal antibody 6E10. Aliquots of cell lysates used for immunoprecipitation were separated by 10% gels and revealed with anti-actin. Unspecific binding was blocked with 50 g/L non-fat dry milk in 50 mM Tris–HCl, pH 7.4, containing 200 mM NaCl and 0.5 mM Tween-20 (TBS-Tween). The blots were incubated with different primary antibodies, followed by incubation with peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulins in TBS-Tween containing 20 g/L-non-fat dry milk. Reactions were developed with an enhanced chemiluminescence system according to the manufacturer’s protocol (Amersham-Pharmacia Biotech Italia, Cologno Monzese, Italy). Reactivities of BACE-1 were normalized to corresponding actin levels and then expressed as arbitrary units of optical density, means ± SD of three independent experiments. Real-time PCR For the quantitative SYBR Green real-time PCR, 40 ng of cDNA was used per reaction. Each 25 lL SYBR Green reaction consisted of 1 lL of cDNA (40 ng/lL), 12.5 lL of 2X SYBR Green PCR Master Mix (Applied Biosystems, Monza, Italy), and 2.5 lL of 1 lM BACE-1 forward and reverse primers or 1 lL of 5 lM GADPH forward and reverse primers. Optimization was performed for each gene-specific primer prior to the experiment to confirm that 100 nM primer concentrations for BACE-1 and 200 nM primers did not produce unspecific primer–dimer amplification signal in template-free control tubes. Primer sequences were designed using PRIMER EXPRESS software (Applied Biosystems) and are BACE-1 forward, 5¢-TGGAGGGCTTCTACGTTGTCTT-3¢ and reverse 5¢-CCTGAACTCATCGTGCACATG-3¢; GAPDH forward, 5¢-GTCGGAGTCAACGGATTTGG-3¢ and reverse, 5¢-GGGTGGAATCATATTGGAACATG-3¢. Quantitative PCR was performed on an ABI PRISM 7700 sequence detector PCR instrument (Applied Biosystems) by using three-stage three-step program parameters provided by the manufacturer exactly as follows: 2 min at 50C, 10 min at 95C and then 40 cycles of 15 s at 95C, 40 s at 56C and 40 s at 72C. The specificity of the produced amplification product was confirmed by examination of dissociation reaction plots. A distinct single peak indicated that a single DNA sequence was amplified during PCR. In addition, end products were visualized on ethidium bromide-stained 2.5% agarose gels. The appearance of a single band of the expected molecular size confirmed the specificity of the PCR. Each sample was tested in triplicate and threshold cycle (Ct) values were averaged from each reaction. The results were

obtained with the comparative Ct method using the arithmetic formula 2–DDCt, according to User Bulletin 2 (ABI PRISM 7700 Sequence Detection System, User’s Manual, Applied Biosystems). Samples obtained from at least three independent experiments were used to calculate the means and SD. Morphological detection of apoptosis The occurrence of apoptosis was evaluated by 4¢,6-diamidino2-phenylindole (DAPI) staining. To identify apoptotic nuclei, cells were grown on glass coverslips, washed in PBS, fixed and permeabilized with 95% ethanol for 5 min, and then stained with DAPI solution for 30 min at 37C. After rinsing in PBS, coverslips were mounted on glass slides and observed by fluorescence microscopy (Leitz Dialux 20 Microscope) (Kaltschmidt et al. 1999). All the experiments were repeated three times and the number of stained cells was counted in 10 randomly selected fields. Statistical analysis Statistical analysis was performed when appropriate, by means of Student’s t-test or ANOVA followed by the Bonferroni post test (Mattwes and Farewell 1988).

Results

HNE increases Ab production We previously showed that HNE up-regulates BACE-1 expression in neuronally differentiated NT2 cells and that this induction is followed by an increased production of C-terminal fragments, the transmembrane polypeptides produced by b-secretase cleavage, without affecting AbPP synthesis (Tamagno et al. 2002). To further evaluate the functional significance of increased BACE-1 after HNE treatment, we quantitatively analyzed the intra- and extracellular levels of Abx-40 and Abx-42 by ELISA technique. The data reported in Fig. 1 indicate that the oxidative stressdependent induction of the amyloidogenic processing of AbPP mediated by BACE-1 leads to a significant increase of 70–80% and 60–140% in the steady-state concentrations of intracellular Abx-40 and Abx-42, respectively (Fig. 1a,c). Secreted levels of Abx-40 were not affected by HNE treatment (Fig. 1b), whereas Abx-42 was increased by 50% (Fig. 1d) compared with control cells. HNE activates different signaling pathways We studied signaling pathways potentially involved in the oxidative stress-dependent increase in BACE-1 activity. To determine whether HNE treatment led to STAT1, Akt, JNKs and/or p38MAPK activation, nuclear extracts obtained from cells treated with HNE at 1 and 5 lM for 15–30 min were subjected to western blot analysis using anti-phospho-STAT, phospho-Akt, phospho-JNKs and phosphop38MAPK, respectively (Fig. 2a–d). Membranes were stripped and reprobed with antibodies recognizing total STAT1, Akt, JNKs and p38MAPK to verify equal protein loading in the samples. HNE



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(d) Fig. 1 Intracellular and secreted Abx-40 and Abx-42 levels in NT2 neurons. Incubation of NT2 cells with a range of HNE concentrations (0.5–5 lM) for 1 h results in a significant increase of intracellular (60–140%) and secreted (50%) Abx-42; only intracellular Abx-40 showed a significant increase (70–80%). Values are means ± SD of three experiments performed in duplicate. (w) significantly different from control (p < 0.05).

at 1 and 5 lM activated STAT1 at 15 min (Fig. 2a) but did not affect Akt signaling (Fig. 2b). Notably, the same HNE concentrations induced SAPK activation as shown in Fig. 2(c,d); in particular, JNK was rapidly phosphorylated after 15 min (Fig. 2c) and p38MAPK was still activated after 30 min (Fig. 2d). Activation of STAT1 did not affect HNE-induced BACE-1 protein levels To investigate the role of STAT1 on the up-regulation of BACE-1 induced by HNE, we pre-treated NT2 neurons with AG490, an inhibitor of JAK 2, that selectively inhibits STAT1 activity. Although, as expected, AG490 completely abolished HNE-elicited STAT1 activation (Fig. 3a), HNEinduced BACE-1 protein levels were not affected (Fig. 3b). The Akt signaling pathway was not investigated because HNE treatment in our experimental conditions did not affect this pathway (Fig. 2b). Activation of JNKs and p38MAPK is nedeed for HNE induced BACE-1 protein levels To investigate whether BACE-1 induction mediated by HNE treatment occurs through activation of JNKs and p38MAPK pathways, we pre-treated cells with the pharmacological inhibitors SP600125 (20 lM) and SB203580 (10 lM), respectively, 15 min before exposure to HNE. Figures 4(a) and (5a) show levels of pJNKs and pp38MAPK before and

Fig. 2 Effect of HNE on STAT, Akt, JNKs and p38MAPK activation in NT2 neurons. Western blot analysis shows phosphorylation of STAT1 (a), Akt (b), JNKs (c) and p38MAPK (d) obtained from cells starved for 16 h and then treated with HNE (1 and 5 lM) for the indicated times. Blots are representative of three separate experiments. Equal protein loading was evaluated by stripping membranes and reprobing them with appropriated antibodies such as against STAT1, c-Akt, JNKs and p38MAPK.

after treatment with the inhibitors, as evaluated by western blot analysis to confirm the complete abrogation of activation of JNK and p38 pathways, respectively. Importantly, inhibition of JNK and p38 kinases was followed by a complete prevention of HNE-mediated BACE-1 induction, indicating that both pathways mediate the response to HNE (Figs 4b and 5b). The behavior of BACE-1 expression parallels its protein level Expression of BACE-1, evaluated using real-time PCR, is reported in Fig. 6. Quantitative analysis of BACE-1 mRNA confirms the results obtained by western blot technique. Neuronal cells exposed to HNE, 0.5 lM for 1 h, increased BACE-1 mRNA levels by 2-fold, whereas higher (1 and 5 lM) HNE concentrations increased BACE-1 expression by 4-fold compared with control cells. BACE-1 transcription was not prevented by an inhibitor of the STAT1 cascade (data not shown), whereas the role of JNKs and p38MAPK pathways is confirmed by the reduction



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Fig. 3 Effect of pharmacological inhibitor of JAK-2 (AG490) on STAT1 phosphorylation and BACE-1 protein levels induced by HNE in NT2 neurons. (a) Western blot analysis of phosphorylation of STAT1 in NT2 cells starved for 16 h, then treated or not with AG490, and finally exposed for 1 h to HNE (1 and 5 lM). Blots are representative of three separate experiments. Equal protein loading was evaluated by stripping membranes and reprobing them with appropriated antibody such as against STAT1. (b) Western blot analysis of BACE-1 protein levels in NT2 cells starved for 16 h, then treated or not with AG490 and exposed for 1 h to HNE (1 and 5 lM). The blots are representative of three separate experiments. Optical density values of BACE-1 protein levels, normalized against b actin, are means ± SD of three independent experiments. (w) significantly different from control (p < 0.05). (q) significantly different from control (p < 0.02).

of BACE-1 mRNA to baseline levels after pre-treating NT2 neurons with the specific inhibitors (Fig. 6a). Because BACE-1 transcripts and protein levels increase 1 h after the exposure of cells to various concentrations of HNE, we suspect stabilization of BACE-1 mRNA and protein, rather than an actual transcriptional effect. To test this, we pre-treated NT2 neuronal cells with actinomycin, a transcription inhibitor, and with cycloheximide, a translation inhibitor. Data show that pre-treatment with both compounds was able to rescue BACE1 induction (Fig. 6b,c), suggesting that HNE induces increased BACE-1 expression at the transcriptional level. Other kinase inducers increased BACE-1 protein levels To further confirm the role of SAPK pathway in the oxidative stress-dependent BACE-1 induction, we tested other known inducers of JNKs and p38MAPK, such as hydrogen peroxide and anisomycin. As shown, treatment of cells with H2O2 (20 lM) or anisomycin (10 lM) for 5 h induces an increase in BACE-1 protein levels of 100 and 300%, respectively (Fig. 7a,b). Inhibition of JNKs and p38MAPK completely abolished this BACE-1 induction, confirming that both JNK and p38MAPK pathways mediate this response.

Fig. 4 Effect of pharmacological inhibitors of JNK isoforms (SP600125) on JNK phosphorylation and BACE-1 protein levels induced by HNE in NT2 neurons. (a) Western blot analysis of phosphorylation of JNKs in NT2 cells starved for 16 h, then pre-treated or not with SP600125 and finally exposed for 1 h to HNE (1 and 5 lM). Blots are representative of three separate experiments. Equal protein loading has been evaluated by stripping membranes and reprobing them with appropriated antibody such as against JNKs. (b) Western blot analysis of BACE-1 protein levels in NT2 cells starved for 16 h, then treated or not with SP600125 and exposed for 1 h to HNE (1 and 5 lM). The blots are representative of three separate experiments. Optical density values of BACE-1 protein levels, normalized against b-actin, are means ± SD of three independent experiments. (w) significantly different from control (p < 0.05). (q) significantly different from control (p < 0.02).

Apoptotic cell death, induced by HNE, is prevented by SAPK inhibitors Activation of SAPKs is associated with induction of apoptosis (Tamagno et al. 2003b; Zhu et al. 2003b). Hence, we evaluated apoptotic cell death induced by HNE by means of DAPI staining. HNE at a concentration of 5 lM was able to induce apoptosis in 30% nuclei in differentiated NT2 cells after 24 h in comparison with control cells. Pretreatment of cells with specific inhibitors of JNKs and p38MAPK significantly decreased the percentage of apoptotic nuclei (Fig. 8a). The induction of classic apoptotic cell death was confirmed by data obtained monitoring caspase 3 and PARP protein levels. Caspase 3 activation was evaluated by quantifying the 20 kDa active form in NT2 neuron lysates by western blot analysis. A significant increase (150%) in active isoform levels was detected after treatment with 5 lM HNE (Fig. 8b). Furthermore, cleavage of PARP (an endogenous substrate of caspase 3) to the 86 kDa C-terminal



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Fig. 5 Effect of pharmacological inhibitors of p38MAPK (SB203580) on p38MAPK phosphorylation and BACE-1 protein levels induced by HNE in NT2 neurons. (a) Western blot analysis of phosphorylation of p38MAPK in NT2 cells starved for 16 h, then pre-treated or not with SB203580 and finally exposed for 1 h to HNE (1 and 5 lM). Blots are representative of three separate experiments. Equal protein loading was evaluated by stripping membranes and reprobing them with appropriated antibody such as that against p38MAPK. (b) Western blot analysis of BACE-1 protein levels in NT2 cells starved for 16 h, then treated or not with SB203580 and exposed for 1 h to HNE (1 and 5 lM). The blots are representative of three separate experiments. Optical density values of BACE-1 protein levels, normalized against b-actin, are means ± SD of three independent experiments. (w) significantly different from control (p < 0.05). (q) significantly different from control (p < 0.02).

fragment resulted also increased ( 100%) after treatment with 5 lM HNE (Fig. 8c). Pre-treatment of differentiated NT2 cells with SAPK inhibitors significantly prevented activation of caspase 3 and cleavage of PARP (Fig. 8b,c). Discussion

Ab accumulation and oxidative stress are linked and are both viewed as central to the pathogenesis of AD (Perry et al. 1998; Selkoe 2000). HNE, a major product of lipid peroxidation that occurs early in AD brain (Sayre et al. 1997), is proportional to the extent of neuronal lesions (Takeda et al. 2000; Wang et al. 2003), mediates the expression, protein levels and activity of BACE-1 and, consequently, Ab levels (Tamagno et al. 2002, 2003a). Here, we report the novel finding that exposure of NT2 neurons to HNE is followed by a significant increase of intra- and extracellular levels of Ab40 and Ab42 species and that Ab overproduction paralleled HNE-mediated BACE-1 overexpression, establishing a direct relationship between oxidative stress and the amyloidogenic processing of AbPP. These

Fig. 6 Effect of pharmacological inhibitors of JNK and p38MAPK pathways on BACE-1 over-expression induced by HNE. (a) Real-time PCR analysis of BACE-1 mRNA expression in NT2 cells, starved for 16 h, then pre-treated or not with SP600125 or SB203580, and exposed for 1 h to HNE (1 and 5 lM). Results are means ± SD of three independent experiments. (b) Western blot analysis of BACE-1 protein levels in NT2 cells starved 16 h, then treated or not with cycloheximide and exposed for 1 h to HNE 5 lM. The blots are representative of three separate experiments. Optical density values of BACE-1 protein levels, normalized against b actin, are means ± SD of three independent experiments. (c) Real-time PCR analysis of BACE-1 mRNA expression in NT2 cells, starved for 16 h, then pretreated or not with actinomycin D and exposed for 1 h to HNE 5 lM. (w) significantly different from control (p < 0.05). (q) significantly different from control (p < 0.02). ( ) significantly different from HNE without, respectively, inhibitor compounds (p < 0.02).

in vitro findings are in agreement with the increase in BACE-1 protein levels and activity detected in brains of patients affected by sporadic forms of AD (Fukumoto et al. 2002; Holsinger et al. 2002; Yang et al. 2003). The cellular signaling pathways that mediate this event are unknown. Recently, Christensen et al. (2004) showed that the transcription factor Sp1 plays an important role in regulating BACE-1 gene expression, providing the first clue on the molecular mechanisms by which human BACE-1 gene expression is regulated. Here, we show that SAPK signaling pathways modulate the expression of BACE-1 induced by HNE. Indeed, HNEdependent activation of JNKs and p38MAPK signaling pathways is needed for the overexpression of BACE-1, because specific pharmacological inhibition of these two pathways results in the abrogation of up-regulation of



Fig. 7 Effect of pharmacological inhibitors of JNKs (SP600125) or p38MAPK (SB203580) on BACE-1 protein levels induced by H2O2 or anysomicin in NT2 neurons. (a) Western blot analysis of BACE-1 protein levels in NT2 cells starved for 16 h, then treated or not with SP600125 or SB203580 and exposed for 5 h to H2O2 (20 lM). The blots are representative of three separate experiments. Optical density values of BACE-1 protein levels, normalized against b actin, are means ± SD of three independent experiments. (b) Western blot analysis of BACE-1 protein levels in NT2 cells starved for 16 h, then treated or not with SP600125 or SB203580 and exposed for 5 h to anysomicin (10 lM). The blots are representative of three separate experiments. Optical density values of BACE-1 protein levels, normalized against b actin, are means ± SD of three independent experiments. (q) significantly different from control (p < 0.02).

BACE-1 expression. Contrasting findings in astrocytes stimulated with interferon-c (Hong et al. 2003), the STAT/ JAK pathway does not contribute to BACE-1 expression in our experimental model. Notably, activation of SAPK family members has been described in AD brain tissue and correlate with oxidative stress. JNK is not only activated but also redistributed (Zhu et al. 2001b), from nuclei to the cytoplasm, concomitant with the progression of the neurofibrillary pathology. Moreover, JNK is strongly activated in mutant AbPP transgenic mice with extensive oxidative damage, but not in mutant AbPP transgenic mice with little oxidative damage (Smith et al. 1998). The activation of p38 signaling in AD is also well documented. p38MAPK is shown to be active and present in Ab deposits of AbPP transgenic mice (Savage et al. 2002). Levels of p38MAPK are increased significantly after ischemic injury in APP over-expressing mice (Koistinaho et al. 2002), indicating a role in oxidative stress-induced cell damage. The essentially identical staining patterns for phospho-JNK and phospho-p38 in AD cases suggest that JNK and p38 are activated by the same signal

Fig. 8 Effect of pharmacological inhibitors of JNKs (SP600125) or p38MAPK (SB203580) on induction of apoptotic cell death by HNE. (a) Nuclear NT2 neurons morphology evaluated in terms of DAPI staining 24 h after treatment with HNE 5 lM. Inhibitors SP600125 and SB203580 were added 15 min before treatment with HNE. Apoptotic nuclei are indicated by arrows. (b) Western blot analysis of active caspase 3 protein levels in NT2 cells starved for 16 h, then treated or not with SP600125 and SB203580 and exposed to HNE 1 and 5 lM. The blots are representative of three separate experiments. (c) Western blot analysis of PARP protein levels in NT2 cells starved for 16 h, then treated or not with SP600125 and SB203580 and exposed to HNE 1 and 5 lM. The blots are representative of three separate experiments.

(Zhu et al. 2001a). Moreover, the abnormal involvement of these pathways is confirmed by the phosphorylation of JKK1 and MKK-6, the upstream activators of the JNKs and p38MAPK (Zhu et al. 2001c, 2003a). Alterations in gene expression and enzyme activity induced by oxidative stress are mediated through the interplay of multiple signaling pathways. In neuronal cells, potentially deleterious stimuli such as free radicals provoke an intracellular stress response that either leads to apoptosis or defensiveprotective adaptations. SAPK and their downstream effectors are the major molecules involved in this bipartite response,



which can lead to either neurodegeneration or neuroprotection depending on the cellular and environmental conditions as well as the cooperation with other signaling pathways (Mielke et al. 2000). In this scenario, BACE-1 over-expression mediated by oxidative stress may shift the signaling equilibrium toward cell damage and apoptosis. According to this view, neuronal apoptotic cell death can depend on a significant increase in BACE-1 activity, and Ab may be the active player of neuronal damage induced by oxidative stress through activation of SAPK cascade. The relationship among oxidative stress, SAPK pathways, BACE, and Ab outlined in this study supports the hypothesis that the overproduction of Ab, dependent on the overexpression of BACE-1 induced by oxidative stress, contributes to the pathogenesis of the common sporadic late-onset form of AD, in which multiples coexisting factors are suspected to determine Ab accumulation. Acknowledgement This study was supported by Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica.

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