Neuroprotective properties of Bcl-w in Alzheimer ... - Wiley Online Library

Journal of Neurochemistry, 2004, 89, 1233–1240

doi:10.1111/j.1471-4159.2004.02416.x

Neuroprotective properties of Bcl-w in Alzheimer disease Xiongwei Zhu,* Yang Wang, Osamu Ogawa,* Hyoung-gon Lee,* Arun K. Raina,* Sandra L. Siedlak,* Peggy L. R. Harris,* Hisashi Fujioka,* Shun Shimohama, Massimo Tabaton,§ Craig S. Atwood,* Robert B. Petersen,* George Perry* and Mark A. Smith* *Institute of Pathology, Department of Biostatistics, Case Western Reserve University, Cleveland, Ohio, USA Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto, Japan §University of Genova, Genova, Italy

Abstract While there is a host of pro-apoptotic stimuli that target neurons in Alzheimer disease (AD), given the chronicity of the disease and the survival of many neurons, those neurons must either avoid or, at minimum, delay apoptotic death signaling. In this study, we investigated Bcl-w, a novel member of the Bcl-2 family that promotes cell survival. In AD, we found increased levels of Bcl-w associated with neurofibrillary pathology and punctate intracytoplasmic structures whereas, in marked contrast, there are only low diffuse levels of Bcl-w in the neuronal cytoplasm of agematched control cases. Immunoblot analysis confirmed that Bcl-w levels were significantly increased in AD. By electron microscopy, we determined that the increased Bcl-w

expression in AD was ultrastructurally localized to mitochondria and neurofibrillary pathology. To investigate the cause and consequence of Bcl-w up-regulation in neurons, we found that fibrillized amyloid-b led to increased Bcl-w protein levels in M17 human neuroblastoma cells, and that overexpression of Bcl-w significantly protected neurons against staurosporine- and amyloid-b-induced apoptosis. Taken together, these series of results suggest that Bcl-w may play an important protective role in neurons in the diseased brain and that this aspect could be therapeutically harnessed to afford neuroprotection. Keywords: Alzheimer disease, amyloid-b, apoptosis Bcl-w, neuroprotection, signal transduction. J. Neurochem. (2004) 89, 1233–1240.

Alzheimer disease (AD), the most prevalent dementing neurodegenerative disease, is characterized by a progressive deterioration of cognitive function and memory in association with widespread neuronal cell loss and dysfunction (Smith 1998). The causes and mechanisms underlying such neuronal death are extremely controversial and none more so than whether apoptosis plays a role. On the one hand, neurons in AD face a wide assortment of apoptotic stimuli including oxidative stress (Zhu et al. 2003), amyloid-b (Yankner 1996) and metabolic compromise (Vander Heiden et al. 2000), which likely contribute to the expression or activation of apoptotic markers such as Par-4 and caspase family members (Guo et al. 1998; LeBlanc et al. 1999; Selznick et al. 1999; Raina et al. 2001). On the other hand, the absence of the hallmark end stage signs of apoptosis, such as nuclear chromatin condensation, apoptotic bodies and other histopathological and morphological manifestations of apoptosis in AD, cast doubt on whether such apoptotic stimuli actually lead to cell death in AD (Perry et al. 1998a,b, 2001; Stadelmann et al. 1998). Indeed, the temporal dichotomy between the acute nature of apoptosis

versus the chronic duration of AD suggests that, despite initiation, the apoptotic process in AD does not proceed to the point of cell death by classic mechanisms (Perry et al. 1998a; Raina et al. 2001). To this end, we speculated that survival factors that prevent propagation of the apoptotic signaling pathways would lead to termination, or at least delay, of the classic apoptotic cascade in neurons in AD. Apoptosis is regulated by members of the Bcl-2 family such as Bcl-2, Bcl-XL, Bad, Bax and Bak, which promote or prevent completion of the apoptotic cascade (Davies 1995; Farrow

Received October 30, 2003; revised manuscript received January 23, 2004; accepted January 27, 2004. Address correspondence and reprint requests to Xiongwei Zhu Ph.D., Institute of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, Ohio 44106, USA. E-mail: [email protected] Abbreviations used: Ab, amyloid-b peptide; AD, Alzheimer disease; DAB, 3-3¢-diaminobenzidene tetrahydrochloride; fAb, fibrillar Ab1)42; LDH, lactate dehydrogenase; NFT, neurofibrillary tangles; NGS, normal goat serum; PBS, phosphate-buffered saline; PHF, paired helical filament; STS, staurosporine; TAK1, TGF-b activating kinase 1; TBS, Trisbuffered saline.

2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 1233–1240

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et al. 1995; Yang et al. 1995). Bad, Bax and Bak belong to the pro-apoptotic subfamily which promotes apoptosis by translocating into the mitochondrial membrane and facilitating cytochrome c release (Eskes et al. 1998, 2000; Shimizu et al. 1999; Shimizu and Tsujimoto 2000; Wei et al. 2001; Zornig et al. 2001). In contrast, Bcl-2 and Bcl-XL belong to the prosurvival subfamily which prevents apoptotic death in neurons and several neuron-like cell lines by forming heterodimers with pro-apoptotic subfamily members and abolishing their activity (Lindsten et al. 2000; Zornig et al. 2001). Thus, the ratio between the levels of pro-apoptotic versus pro-survival factors determines the fate of cells subjected to stimuli that initiate apoptotic pathways. While many of these factors have been detected in AD brain, whether they lead to apoptosis or instead protection against apoptosis is controversial and has led to often markedly conflicting interpretations (Satou et al. 1995; O’Barr et al. 1996; Nagy and Esiri 1997; Kitamura et al. 1998; Tortosa et al. 1998). Bcl-w, a recently described regulator of apoptosis, is a Bcl-2 family member that promotes cell survival. Bcl-w is present in almost all myeloid cell lines and in a wide range of tissues at low levels, with the highest level of expression in the brain, colon and salivary gland (Gibson et al. 1996). It is of interest to note that during brain development, the level of Bcl-w increases, with the highest neuronal expression located to specific regions of the mature brain including the hippocampus, where it mediates a functional role (Hamner et al. 1999). Further, Bcl-w is increased during ischemia and in seizureinduced brain injury, and it is postulated to act as an endogenous neuroprotector (Minami et al. 2000; Yan et al. 2000a; Henshall et al. 2001). In this study, we assessed the potential role of Bcl-w in the pathogenesis of AD.

Materials and methods Brain tissue Hippocampal, frontal cortical and cerebellar brain tissue obtained post-mortem was fixed in methacarn (methanol : chloroform : acetic acid, 6 : 3 : 1) or 10% buffered formalin, embedded in paraffin, and 6 lm-thick consecutive sections prepared on silane-coated (Sigma, St. Louis, MO, USA) slides for immunocytochemistry. Cases used in this study included AD (n ¼ 23; ages ¼ 60–91 years; post-mortem interval ¼ 1–23 h), younger control (n ¼ 5; ages ¼ 3–56 years; post-mortem interval ¼ 2–23 h) and age-matched control (n ¼ 16; ages ¼ 65–91 years; post-mortem interval ¼ 3–24 h) cases [based on clinical and pathological criteria established by CERAD and an NIA consensus panel (Khachaturian 1985; Mirra et al. 1991)]. Immunocytochemical procedures Immunocytochemistry was performed by the peroxidase antiperoxidase protocol essentially as described previously (Zhu et al. 2000). Briefly, following immersion in xylene, hydration through graded ethanol solutions and elimination of endogenous peroxidase activity by incubation in 3% hydrogen peroxide for 30 min, sections

were incubated for 4 min at room temperature in 70% formic acid and then incubated for 30 min at room temperature in 10% normal goat serum (NGS) in Tris-buffered saline (TBS; 50 mM Tris-HCl, 150 mM NaCl, pH 7.6) to reduce non-specific binding. After rinsing briefly with 1% NGS/TBS, the sections were sequentially incubated overnight at 4C with either (i) immunoaffinity purified rabbit polyclonal antibody to Bcl-w (StressGen Biotechnologies Corporation, Victoria, British Columbia, Canada), (ii) immunoaffinity purified rabbit polyclonal antibody to Bcl-w (Chemicon International, Temecula, CA, USA) or (iii) mouse monoclonal AT8 antibody (Innogenetics, Ghent, Belgium) to phosphorylated tau protein. The sections were then incubated in either goat anti-rabbit (ICN, Costa Mesa, CA, USA) or goat anti-mouse (ICN) antisera, followed by species-specific peroxidase anti-peroxidase complex (Sternberger Monoclonals Inc., Lutherville, MA, USA and ICN, Costa Mesa, CA, USA). 3–3¢-Diaminobenzidine tetrahydrochrolide (DAB) was used as a chromagen. For some experiments, sections were double-labeled with two different antibodies. Rabbit antisera were localized using the peroxidase–anti-peroxidase method with DAB as the chromogen. Monoclonal antibodies were localized using the alkaline phosphatase anti-alkaline phosphatase method with Fast Blue as the chromogen (Zhu et al. 2000). Double-stained slides were used for quantification. Briefly, five adjacent fields (20 · 1.6) of CA1 region from the hippocampus of each case were examined on a Zeiss Axiophot-microscope for neurons stained with either Bcl-w or AT8, or both. Positively-stained neurons were counted. Absorption experiments were performed to verify the specificity of antibody binding. Briefly, the purified Bcl-w immunizing peptide (StressGen Biotechnologies Corporation), as well as an irrelevant peptide [TGF-b activating kinase 1 (TAK1) peptide (100 lg/mL) (StressGen Biotechnologies Corporation)], was coupled to CNBractivated 4B beads (Sigma, St Louis, MO, USA) according to the manufacturer’s instructions. The immunostaining protocol was repeated using absorbed antibody produced by an overnight incubation at 4C of primary antibody with beads coupled to the Bcl-w immunizing peptide followed by centrifugation. In parallel, as a control against artifactual absorption, absorption of the Bcl-w antibody with beads coupled to an irrelevant peptide (i.e. TAK1) and absorption of an irrelevant antibody (i.e. TAK1 antibody) with beads coupled to Bcl-w peptide was performed. Immunoblotting and immunodotting Samples from gray matter of temporal cortex of AD (n ¼ 6) and control cases (n ¼ 6) were homogenized in 10 volumes of TBS containing 0.02% sodium azide, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 lg/mL aprotinin and 1 lg/ mL antipain (lysis buffer). Brain homogenates were centrifuged for 10 min at 16 000 g and the supernatant fluids were then transferred to new tubes. Protein concentration was determined by the bicinchoninic acid assay (BCA Kit) method (Pierce, Rockford, IL, USA) and an equal amount of protein from each sample was loaded. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto Immobilon-P (Millipore, Bedford, MA, USA) by standard procedures as previously described (Zhu et al. 2000).


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Blots were incubated sequentially with blocking agent (10% nonfat milk in 0.1% Tween-20 in TBS), rabbit anti-Bcl-w antibody (1 : 200, StressGen Biotechnologies Corporation) and affinitypurified goat anti-rabbit immunoglobulin peroxidase conjugate preabsorbed to eliminate human cross-reactivity. Blots were developed by the ECL technique (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) according to the manufacturer’s instructions. Parallel blots were probed with antibodies against actin or HO-2 (1 : 500, StressGen Biotechnologies Corporation), which are constitutively expressed in neuronal cells, as a control to demonstrate equivalent loading. Blots were scanned at high resolution and the immunoreactive bands were quantitated with KS300 image analysis software (Zeiss, Thornwood, NY, USA) as follows. The optical density was determined for the entire band in each case by outlining the immunoreactive area and subtracting the background density of the surrounding unlabeled blot area with similar size. The values for the AD and control cases were averaged and the Student’s t-test used to determine whether the differences were significant (p < 0Æ05). Dot blots were prepared by applying 5 lg of s protein (0.5–1.0 mg/mL), immunizing peptide of Bcl-w (1 mg/mL), 5 lg of insoluble PHF or a control fraction directly onto Immobilon (Millipore) membrane and then air-dried. Human s from a normal human brain and PHF-enriched fractions from AD brain were prepared by previously described methods (Takeda et al. 2000). The membrane was incubated sequentially with blocking agent (10% non-fat milk in TBS-Tween), rabbit anti-Bcl-w and goat anti-rabbit peroxidase conjugate. AT8 antibody that detects PHF-s and 5E2 antibody that detects total s were used as a positive control, and rabbit anti-Ras was used as a negative control. Dot blots were developed using ECL technique (Santa Cruz Biotechnologies). Electron microscopy Brain tissue was obtained at autopsy and fixed in 2% paraformaldehyde/0.5% glutaraldehyde and sectioned at 60 lm using a Vibratome. Sections were rinsed with phosphate-buffered saline (PBS), incubated in 10% NGS for 1 h, followed by incubation with antibody against Bcl-w for 16 h at 4C. After rinsing with 1% NGS, goat-anti-rabbit conjugated to 17 nm colloidal gold was applied. After incubating for 4 h, sections were rinsed overnight in PBS, followed by fixation in 2.5% glutaraldehyde for 1 h and rinsed with PBS. After incubation in 1% osmium tetraoxide for 1 h, sections were dehydrated with acetone and embedded in Spurr embedding media (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections were stained with uranyl acetate and lead citrate, and examined in a Zeiss CEM 902 electron microscope. The primary antibody was omitted on one section as a negative control.

spectroscopy by W. M. Keck Foundation Biotechnology Resource Laboratory (Yale University, New Haven, CT). The peptide was identified as a single peak upon HPLC and showed no chemical modification. A 1 mM stock solution of Ab1)42 in deionized water was allowed to fibrillize for 7 days at 37C. fAb was stored at ) 80C until use. Determination of cell death A lactate dehydrogenase (LDH) release assay (Roche Molecular Biochemicals, Indianapolis, IN, USA), combined with immunoblot analysis of cleaved caspase 3 and cleaved PARP were used to determine cell death and apoptosis, respectively. For immunoblot, after each treatment, both detached and attached cells were spun down, harvested together and washed with PBS, then lysed with lysis buffer (Cell Signaling Technology, Beverly, MA, USA). Antibody against cleaved caspase 3 (Cell Signaling Technology) only recognizes cleaved casapse 3 at 17 kDa, while antibody against human PARP (Pharmingen) recognizes both full length and cleaved PARP. The LDH concentration in the media was determined according to manufacturer’s instructions. Transfection and selection Plasmids containing the cDNAs encoding Bcl-w were kind gifts from Dr Suzanne Cory (Gibson et al. 1996). M17 cells were grown in OPTI-MEMI supplemented with 5% DCS and 1% penicillin/ strptomycin. After serum-starving overnight, they were transfected with 5 lg of mammalian expression vectors containing Bcl-w using Superfect transfection reagent (Qiagen, Valencia, CA, USA). Stablytransfected cells were selected in 600 lg/mL G418 (Life Technologies) for 4 weeks. Pools of 50 or more colonies were used to avoid bias of single cell colonies. Protein expression of Bcl-w was confirmed by immunoblot and RT-PCR. RT-PCR Total RNA was extracted from cells using RNeasy Midi kit (Qiagen). cDNAs were prepared with random hexamers (Firststrand cDNA synthesis kit; Amersham Pharmacia Biotech, Piscataway, NJ, USA) and used as templates for subsequent PCR. For Bcl-w cDNA amplification, samples were heated at 94C for 5 min and then subjected to thermocycling (30 cycles of 1 min at 94C, 1 min at 55C and 1 min at 72C) using the specific primers 5¢-TTATGTCTGTGGAGCTGGCC-3¢ and 5¢-TCTCCAGGTAGGCCACCATC-3¢ (Kitamura et al. 2000). The PCR products were separated on a 2% agarose gel in 1 · Tris borate-EDTA buffer and visualized with UV light after ethidium bromide staining.

Results Cells The human neuroblastoma cell line M17 was maintained in serumfree Opti-MEM media (Life Technologies, Gaithersburg, MD, USA) supplemented with 5% donor calf serum and 1% penicillin/ streptomycin with fungizone (Life Technologies). Cells were serum-starved overnight before Ab treatment. Preparation of fibrillar Ab1)42 (fAb) Human amyloid-b peptide (Ab1)42) was synthesized and purified by HPLC, and characterized by amino acid analysis and mass

Bcl-w was increased and associated with pathological alterations in AD In situ localization of Bcl-w protein was performed on hippocampal and cortical sections from patients with AD and from cognitively normal age-matched individuals, using two polyclonal antibodies against Bcl-w from different sources, and identical results were found. In 14 out of 16 age-matched control cases and all younger control cases, Bcl-w was found


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at background low levels in the cytoplasm of neurons (Fig. 1a). However, conversely, Bcl-w was markedly increased in large pyramidal neurons in the hippocampus of all individuals with AD, i.e. in those neurons vulnerable to degeneration during the disease (Figs 1b–e). Most strikingly, in AD, Bcl-w was increased in association with intracellular neurofibrillary tangles (NFT), neuritic senile plaques and neuropil threads, i.e. the classic neuropathology of the AD brain (Fig. 1b), as well as with punctate structures (Fig. 1c and at higher magnification in Fig. 1d). Extracellular NFTs are also occasionally stained. Indeed, there was an obvious overlap of the immunostaining profiles of Bcl-w and s-positive neurofibrillary pathology in AD cases as assessed by staining of adjacent sections (result not shown) and double staining (result not shown) using AT8, an antibody which only recognizes s protein when serine 202 and threonine 205 are phosphorylated. Quantification revealed that neurons are either Bcl-w positive or both Bcl-w and AT8 positive, with no neurons being only stained by AT8. As often found in normal aging, a few pyramidal neurons contained NFTs and in these cells, Bcl-w is increased and also localized to tangles. Importantly, in the cerebellum, an area that is unaffected by AD, there was no difference in the staining pattern between AD and control cases (result not shown). Extending the light level studies, electron microscopy showed that Bcl-w was localized to the paired helical filaments in NFT (Fig. 2a, see figure legend) and to dystrophic neurites (Fig. 2b). Additionally, in neurons containing neurofibrillary pathology, Bcl-w was also found localized to mitochondrial structures (Fig. 2c). In the control section, in which the primary antibody was omitted, no gold particles were detected (data not shown).

Fig. 2 Immunogold labeling of Bcl-w in AD hippocampus is detected in the paired helical filaments of neurofibrillary tangles and in the dystrophic neurites: (a) longitudinal section; (b) cross section. Additionally, Bcl-w is also detected in mitochondrial membrane (c). Scale bar: a ¼ 1 lm; b ¼ 0.5 lm, c ¼ 0.1 lm.

To confirm the specificity of Bcl-w immunocytochemistry, several control experiments were performed in parallel. Absorption of the Bcl-w antibody with the immunizing peptide of Bcl-w, conjugated with Sepharose Cl-4B beads, almost completely abolished immunostaining (compare Figs 1f and 1g). No effect was observed by the absorption of: (i) antibody to TAK1 with Bcl-w peptide (free or conjugated); (ii) antibody to Bcl-w with sepharose Cl-4B beads conjugated to TAK1 peptide; or (iii) antibody to Bcl-w with amyloid b protein precursor (results not shown). To exclude the possibility that the Bcl-w antibody was crossreacting with s protein or PHF-s, no immunoreactivity was found on dot blots of enriched human s protein from normal patients or of purified PHF-s from AD brain (result not shown). Furthermore, there was no significant sequence homology between Bcl-w and tau protein (Meg Align, DNAStar).

Fig. 1 Immunocytochemical localization of Bcl-w reveals diffuse and low levels of Bcl-w in hippocampal neurons from control cases (a) but, by marked contrast, Bcl-w is increased in AD and localized to neurofibrillary tangles and dystrophic neurites (b) as well as punctate neuronal staining (c). A representative neuron containing granular structure (d) and a representative neuron with increased Bcl-w but without pathology at a higher magnification (e) are shown. Neuronal immunostaining in the hippocampus with Bcl-w antibody in AD (f) is completely abolished by absorption with immunizing peptide (g) but not by irrelevant peptide (not shown). *Indicates landmark blood vessel in adjacent sections in f–h. Scale bars: a,b,c,f,g ¼ 50 lm, d,e ¼ 100 lm.



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Fig. 3 (a) A representative result of immunoblots of cortical gray matter, homogenized in lysis buffer and probed with antisera against Bcl-w, shows a strong band around the expected molecular weight of 21 kDa in Alzheimer disease (AD) and weaker in control (C) samples. (b) Quantification of the Bcl-w reactive band, normalized to actin levels, shows that Bcl-w is significantly increased in AD (*p < 0.05).

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Exposure to fibrillized Ab up-regulates Bcl-w protein expression Amyloid-b (Ab), a critical protein in the pathophysiology of AD, has been demonstrated to be neurotoxic in cell cultures by an apoptotic mechanism. Therefore, given the elevated levels of Bcl-w in susceptible neurons in AD and the neuroprotective role that Bcl-w may play in vivo, we explored the role that Bcl-w may play in response to Ab stress. Cultured M17 human neuroblastoma cells were exposed to fibrillized Ab1)42 (fAb) and Bcl-w expression was analyzed by immunoblot experiments. After 24 h of treatment with fAb peptides, expression levels of Bcl-w protein were examined. A 24-h exposure of M17 cells to various concentrations of fAb1)42 (0.1–10 lM) led to significantly increased expression of Bcl-w protein compared with vehicle-treated controls (Fig. 4). Overexpression of Bcl-w protects against neuronal apoptosis To investigate the role of Bcl-w in Ab neurotoxicity and apoptotic cell death in susceptible neurons in AD, we generated M17 cell lines overexpressing Bcl-w by transfection with the plasmid pcDNA-Bcl-w and subsequent selection of stable transfectants. M17 cells stably transfected with Bcl-w construct expressed higher levels of Bcl-w mRNA expression (Fig. 5a) compared with cells transfected with the empty vector (mock).

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Fig. 4 Exposure to fibrillized Ab1)42 increases Bcl-w protein expression in M17 human neuroblastoma cells. (a) A representative immunoblot analysis. (b) Quantification of the intensity of Bcl-w bands, normalized with constitutively expressed HO-2 levels and setting the intensity of controls as 100%. The experiments were repeated four times with comparable results (*p < 0.05).

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Immunoblot analysis of cortical brain homogenates revealed a major band at around 21 000 Da corresponding to the known molecular weight of Bcl-w (Gibson et al. 1996). In accord with our immunocytochemical findings, this band was much less intense in the control brain homogenates when compared with that from AD cases (Fig. 3a). Statistical analysis, normalized to actin level, showed an increase greater than twofold in the expression of Bcl-w in AD compared with age-matched control cases (p < 0.05) (Fig. 3b).

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Fig. 5 (a) Characterization of the Bcl-w transfectants by RT-PCR confirms the overexpression of Bcl-w in Bcl-w-transfected M17 cells. (b) Bcl-w-transfected cells are resistant to staurosporine-induced cell death. (c, d) Bcl-w-transfected cells are resistant to fAb-induced cell death. (c) LDH release assay; (d) Immunoblot with apoptosis markers. The arrow points to the cleaved PARP. Experiments were repeated three times with comparable results (*p < 0.05).

The effect of Bcl-w overexpression on the induction of neuronal death was first investigated by exposing cells to apoptosis-inducing agent, staurosporine (STS). Exposure to


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100 nM STS for 24 h led to more than 90% cell detachment and death in non-transfected and mock-transfected cells but only around 15% detachment in Bcl-w-transfected cells. Activation of caspases, reflected by the cleavage of caspase 3 and its substrate, PARP, were determined in cell lysates prepared from STS-exposed M17 cell cultures (Fig. 5b). Exposure of mock-transfected M17 cells to 100 nM STS led to a significant increase in cleaved caspase 3 and PARP products (Fig. 5b). In contrast, cleavage products in Bclw-transfected M17 cells exposed to STS were significantly decreased compared with mock-transfected cells (Fig. 5b). The effect of Bcl-w overexpression on fAb-induced neuronal death was also investigated. Cell death after exposure to 1 lM and 10 lM fAb was quantified as LDH release. At a sub-lethal concentration of 1 lM fAb, no significant cell death was induced in mock- or Bcl-wtransfected cells. However, a 48-h exposure of 10 lM fAb caused significant cell death in mock-transfected cells (Fig. 5c). In contrast, exposure to 10 lM fAb failed to induce a significant toxicity in cells overexpressing Bcl-w (Fig. 5c). To investigate the activation of caspases, the levels of cleaved caspase 3 and its substrate, PARP, were determined in cell lysates prepared from fAb-exposed M17 cell cultures (Fig. 5d). Cell lysates from vehicle-treated cultures showed a negligible cleavage rate of both caspase 3 and PARP (not shown). Exposure of mock-transfected M17 cells to 10 lM Ab led to a significant increase in cleaved caspase 3 and PARP products. In contrast, cleavage products in Bcl-w-transfected M17 cells exposed to 10 lM Ab were not significantly higher than the baseline activity of vehicletreated controls. Discussion

In this study, we demonstrated an increased level of Bcl-w protein in the susceptible neuronal populations of AD brain compared with age-matched controls. While the mechanism of activation of Bcl-w is poorly understood, Bcl-w translocates to the mitochondrial membrane following ischemia and inhibits cytochrome c release, presumably by forming heterodimers with Bax (Yan et al. 2000b). The increased Bcl-w protein with mitochondrial localization in susceptible neurons found in our study may therefore indicate an active role for Bcl-w in these cells in response to apoptotic stimuli found in AD. In this regard, we also demonstrated that exposure of cultured M17 human neuroblastoma cells to Ab increased the expression of Bcl-w, and that overexpression of Bcl-w protected neurons against apoptosis induced by Ab. It is therefore conceivable that the increased Bcl-w expression in response to Ab is a stress response that may help to protect neurons against cell death. Given that Ab plays a critical role in AD pathogenesis, the increased expression of Bcl-w in AD suggests that a similar stress response may also play a role in vivo and lead to neuronal survival. Strikingly, our studies

show, for the first time, that the pro-survival member, Bcl-w, is not only increased in AD but is also intimately associated with the lesions of the disease, i.e. neurofibrillary tangles, senile plaque neurites and neuropil threads. This is in marked contrast to other survival factors such as Bcl-2 and Bcl-XL, which, while up-regulated in AD, show no clear association with lesions. In fact, Bcl-2 is elevated in neurons without pathology and decreased in neurons with neurofibrillary pathology (Satou et al. 1995; O’Barr et al. 1996; Tortosa et al. 1998), which was suggested to make the neurons with neurofibrillary pathology particularly vulnerable, especially in light of the elevation of Bax, a pro-apoptotic regulator, in such neurons (MacGibbon et al. 1997; Su et al. 1997). However, this must be viewed in contrast with studies showing that activation of caspase 3 occurs at a much lower level (less than one in a thousand) (Stadelmann et al. 1999) and that neurons with neurofibrillary tangles survive for decades (Morsch et al. 1999), indicating that these neurons are probably mobilizing a protective mechanism. Although it is not clear whether Bcl-w protein associated with neurofibrillary pathology is functional or not, our study at least indicates that these neurons significantly up-regulate the prosurvival Bcl-w, part of which translocates to mitochondria membrane and may serve a neuroprotective role. Preliminary studies show that Bcl-w is also increased and localized to neurofibrillary pathology in other tauopathies, such as progressive superanuclear palsy (PSP) and Pick’s disease (results not shown), but not in Parkinson’s disease and Diffused Lewy Body disease (DLBD), suggesting that Bcl-w may play an important neuroprotective role in those neurofibrillary pathology-containing neurons. Indeed, it is likely that Bcl-w may, in part, serve as a compensatory protective mechanism for neurons assaulted by apoptogenic stimuli in AD and contribute to their survival. Such an assertion is further strengthened by the finding that Bcl-w plays an important role in determining neuronal cell survival after cerebral ischemia or induced seizure (Minami et al. 2000; Yan et al. 2000a; Henshall et al. 2001). In this regard, it is interesting to note that, compared with Bcl-XL, Bcl-w becomes more important in regulating sensory neuron survival with age (Middleton et al. 2001). While Bcl-w knockout mice showed no abnormalities in brain (Print et al. 1998), this does not exclude a role for Bcl-w in stressed or age-related conditions because the oldest mice examined were only 1 year old, corresponding to a human age of around 40 years, at which time point there are few AD-related alterations (Nunomura et al. 2000). It will be of interest in future studies to determine whether older Bcl-w knockout mice show AD-related changes. In conclusion, Bcl-w, a pro-survival, anti-apoptotic factor, is up-regulated in AD and intimately associated with neurofibrillary pathology. Exposure to fibrillized Ab leads to increased Bcl-w protein levels, and overexpression of Bcl-w results in the protection against Ab- and STS-induced



apoptosis. These findings indicate that Bcl-w may play an important protective role in neurons in the face of the various pro-apoptotic signals present in the brains of individuals with AD. How Bcl-w exerts its protective role in susceptible neurons certainly merits further investigation and may reveal novel neuroprotective strategies. Acknowledgements We thank Dr Suzanne Cory (Royal Melbourne Hospital) for providing Bcl-w constructs. This study was supported by grants from the National Institutes of Health (NS38648) and the Alzheimer Association (NIRG-02–3923).

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