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Non-amyloid beta (Aβ) component of Alzheimer's disease (AD) amyloid (NAC) coexists with Aβ protein in senile plaques. After exposure to NAC fibrils, cortical neurons of rat brain primary culture became apoptotic, while astrocytes were activated with extension of their processes. NAC fibrils decreased the activity of reducing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in cortical neurons more markedly (IC50 = 5.6 µm) than in astrocytes (IC50≈ 50 µm). The neuron-specific toxicity of NAC fibrils was indicated also by an increased release of lactate dehydrogenase from the cells. Neuronal apoptosis was suppressed by pre-treatment with the antioxidants, propyl gallate (PG) and N-t-butyl-phenylnitrone (BPN), or overexpression of human Bcl-2. Exposure to NAC fibrils enhanced generation of reactive oxygen species (ROS) in neurons and less efficiently in astrocytes, as demonstrated by oxidation of 2′,7′-dichlorofluorescin. The site of ROS generation was shown to be mitochondria by oxidation of chloromethyl-tetramethyl rosamine. Exposure to NAC fibrils increased also the nuclear translocation of nuclear factor kappa B (NF-κB) and enhanced its DNA-binding activity, which was inhibited by PG and BPN more efficiently in neurons than in astrocytes. These results suggest that NAC fibrils increase mitochondrial ROS generation and activate NF-κB, thereby causing a differential change in gene expression between neurons and astrocytes in the AD brain.
Amyloid deposition in senile plaque cores is one of the histopathological changes characteristic of Alzheimer's disease (AD). The AD amyloid consists of Aβ protein and many minor substances, including the non-Aβ component of AD amyloid (NAC; Uéda et al. 1993). Immunohistochemical studies have shown that NAC is located more abundantly in the central portion than the peripheral portion of senile plaque cores, whereas Aβ is distributed homogeneously throughout cores (Masliah et al. 1996). NAC has been demonstrated to bind to Aβ and stimulate its aggregation invitro (Yoshimoto et al. 1995). NAC is a hydrophobic peptide that displays an amyloidogenic β-sheet structure (Iwai et al. 1995a), and consists of at least 35 amino acids derived from a precursor protein, termed NACP (Uéda et al. 1993). NACP was later identified as α-synuclein, a pre-synaptic protein of the synuclein family (Iwai et al. 1995b). This family comprises α-, β- and γ-synucleins, among whichonly α-synuclein has the NAC domain. The mechanism of NAC production from α-synuclein remains to be elucidated.
Oxidative stress has been claimed to play a central role in the pathogenesis of AD (Smith et al. 2000), and a number of sources of ROS have been suggested, including activated glial cells (Colton and Gilbert 1987), accumulated metals (Smith et al. 1997), and mutated mitochondria (Davis et al. 1997). The Aβ protein is another potential source of ROS; Aβ itself generates ROS in vitro (Hensley et al. 1994) and also induces intracellular production of ROS (Behl et al. 1994). The idea of ROS involvement in the neurotoxicity of Aβ protein is supported by the protective action of Bcl-2 (Sailléet al. 1999) which decreases the formation of ROS and suppresses oxidative stress in neuronal cell lines (Kane et al. 1993).
Oxidative stress is known to activate the transcription factor nuclear factor kappa B (NF-κB) in a neuronal cell line (Behl et al. 1994). This factor is ubiquitously expressed in both neurons and glial cells in the brain (O'Neill and Kaltschmidt 1997), and its activation has been demonstrated in neurons and astrocytes around the senile plaques in AD brains (Kaltschmidt et al. 1997). The activation was also observed in cultured neurons, astrocytes (Akama et al. 1998), and microglia (Bonaiuto et al. 1997) after exposure to Aβ protein.
Recently, the neurotoxicity of NAC and α-synuclein was reported (El-Agnaf et al. 1998; Liu and Schubert 1998); however, the molecular mechanism(s) involved have yet to be elucidated. Here we report that NAC as well as Aβ fibrils induce mitochondrial ROS generation and lead to apoptosis of neurons, whereas both fibrils activate astrocytes in rat brain primary cultures. It is of interest that, although ROS could be a potent activator of NF-κB in both neurons and astrocytes, the ultimate cellular responses are different between the two cell types.
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Amyloidogenic peptides, such as Aβ protein and prion protein, which form a β-pleated sheet structure, are generally cytotoxic to neurons (Yankner et al. 1990; Forloni et al. 1993; Lorenzo and Yankner 1994). Similarly, NAC peptide prone to form a β-pleated sheet structure (Uéda et al. 1993) is cytotoxic. The neurotoxicity of NAC was first reported by Liu and Schubert (1998) using rat brain tumor B12 cells, and by El-Agnaf et al. (1998) using human neuroblastoma SH-SY5Y cells. Bodles et al. (2001) showed that amino acid residues 8–16 of NAC are crucial for the cytotoxic action. We previously reported that NAC fibrils induce cell death in differentiated PC12 cells partly by necrosis and partly by apoptosis (Tanaka et al. 2001). The present study clearly showed induction of apoptosis by NAC fibrils in cortical neurons of rat brain.
We then examined the effects of NAC fibrils on astrocytes. Glial cell cultures comprised type-1 and type-2 astrocytes that can be distinguished by morphology and phenotypic expression of A2B5 antigen (Raff 1989). Exposure to NAC fibrils induced extension of processes or activation of type-2 astrocytes, in sharp contrast to induction of apoptosis in neurons. The neuron-specific cytotoxicity was also demonstrated by an increased release of LDH from neurons, but not from astrocytes. It is of interest that this difference between neuronal and glial cells closely resembles the situation in the AD brain; neurons are degenerated, whereas astrocytes and microglias are markedly activated in and around senile plaques (Pike et al. 1995).
We also evaluated cytotoxicity of NAC fibrils by the MTT reduction assay. Two mechanisms have been suggested for suppression of MTT-reducing activity by Aβ protein. One is inhibition of mitochondrial succinate dehydrogenase (Kaneko et al. 1995) that is a component of complex II of the electron transport system located in the inner mitochondrial membrane. The suppression of MTT-reducing activity by NAC fibrils in cortical neurons might reflect their deleterious effects on mitochondria. The other mechanism is acceleration of MTT formazan exocytosis, as reported for rat brain tumor B12 cells (Liu and Schubert 1997) and astrocytes (Abe and Saito 1998; Kerokoski et al. 2001). In the present study, an accelerated formation of needle-like MTT formazan crystals by NAC fibrils was observed on the cell surface of both neurons and astrocytes (data not shown), suggesting that the second mechanism is, in part, responsible.
The neuroprotective effects of antioxidants suggest a role of oxidative stress in NAC-induced neurotoxicity, whereas their negative effects on the morphological changes of astrocytes preclude the role of ROS on NAC-induced astrocytic activation. However, intracellular oxidation of DCFH in astrocytes as well as neurons indicated intracellular generation of ROS in both cell types. Oxidative stress in the AD brain has been suggested by several lines of indirect evidence, such as increased protein oxidation (Smith et al. 1991), increased lipid peroxidation (Lovell et al. 1995), and the presence of advanced glycation end products in senile plaques (Vitek et al. 1994). While DCFH–DA monitors ROS generation in all cell compartments (Royall and Ischiropoulos 1993), rosamine monitors it more specifically in mitochondria. The increased staining of amyloid fibril-treated cells with rosamine suggests the generation of ROS to be mitochondrial in origin. These results are in agreement with a generally accepted view that mitochondria are the primary source ofROS (Loschen et al. 1974). In this context, Hsu et al. (2000) reported that α-synuclein overexpression led to morphological changes and dysfunction of mitochondria accompanied by an increased level of ROS.
Overexpression of Bcl-2 suppressed ROS generation as well as apoptosis in neuronal cell lines (Kane et al. 1993). The regulation of ROS generation proved not to be the principal mechanism of its anti-apoptotic function, but a secondary effect of preserving mitochondrial membrane functions (Jacobson and Raff 1995; Shimizu et al. 1995). Bcl-2 is localized in the outer membrane of mitochondria and blocks the opening of pores or channels through which apoptotic factors such as cytochrome c, apoptosis-inducing factor (AIF) and Smac/DIABLO are released (Hengartner 2000). Our results indicating the inhibition of NAC- or Aβ-induced apoptosis by Bcl-2 overexpression are expected to result from preservation of the mitochondrial membrane integrity and down-regulation of ROS.
The transcription factor NF-κB was activated in both cortical neurons and astrocytes exposed to NAC or Aβ fibrils. This finding may explain why NF-κB was activated in neurons and astrocytes in the close vicinity of diffuse and primitive plaques in the AD brain (Kaltschmidt et al. 1997). Furthermore, it is likely that activation of NF-κB, in astrocytes and microglias, augments neurodegenerative changes in the AD brain by up-regulating expression of proinflammatory or cytotoxic factors such as IL-1β, IL-6 (Bales et al. 1998) and NO synthase (Akama et al. 1998). On the other hand, NF-κB is known to induce expression of anti-apoptotic factors such as c-IAP1 and c-IAP2 in damaged cells (Wang et al. 1998), suggesting a possibility that activation of NF-κB in neurons may help protection against the cytotoxic action of amyloid. This idea is supported by the finding that pretreatment with IκB (NF-κB-binding inhibitor) antisense oligonucleotide promoted NF-κB activation and inhibited the neurotoxic action of Aβ protein (Barger et al. 1995).
The mechanism how NAC fibrils activate NF-κB has not been clarified. Our present study suggested that mitochondrial ROS is responsible for not only NAC cytotoxicity but also NAC-induced NF-κB activation. A role of mitochondrial ROS in NF-κB activation was suggested in TNF-induced gene transcription (Schulze-Osthoff et al. 1993). We observed a gel shift with the nuclear extract of NAC-treated neurons, implying that mitochondrial ROS is involved in the release of the NF-κB p50/p65 complex from IκB, its nuclear translocation, and the binding to target sequences of DNA.
Recently, Culvenor et al. (1999) reported no immunoreactivity of NAC in senile plaques and argued against the presence of NAC thereabout. Hashimoto et al. (2000), who originally reported the immunoreactivity of NAC in senile plaques (Uéda et al. 1993; Masliah et al. 1996), ascribed the discrepancy to a difference in antibodies and/or methods of tissue preparation. In support of this view,we have experienced a marked difference in the avidityamong antibodies against NAC.
Our preliminary experiment suggested that NAC accelerates Aβ amyloid formation invitro and enhances its neurotoxicity in cultures (unpublished data). These findings suggest a possibility that NAC interacts with Aβ protein to form composite amyloid fibrils and such fibrils may contribute, partly or principally, to neuronal cell injury in the AD brain. In order to assess this possibility, we need knowledge how α-synuclein is processed to NAC in the brain. In view of the presence of α-synuclein in extracellular spaces, e.g. the cerebrospinal fluid (Borghi et al. 2000) and extracellular Lewy bodies (Togo et al. 2001), it seems plausible that α-synuclein is released from damaged neurites and extracellularly proteolysed to NAC. Thus, the elucidation of α-synuclein processing will shed light on the molecular mechanisms of amyloid formation and neurodegeneration in the AD brain, thereby giving a clue to preventive and therapeutic interventions.