- Top of page
- Materials and methods
Accumulation of β-amyloid peptide (Aβ), which is a landmark of Alzheimer's disease, may alter astrocyte functions before any visible symptoms of the disease occur. Here, we examined the effects of Aβ on biosynthesis and release of diazepam-binding inhibitor (DBI), a polypeptide primarily expressed by astroglial cells in the CNS. Quantitative RT–PCR and specific radioimmunoassay demonstrated that aggregated Aβ25−35, at concentrations up to 10−4 m, induced a dose-dependent increase in DBI mRNA expression and DBI-related peptide release from cultured rat astrocytes. These effects were totally suppressed when aggregation of Aβ25−35 was prevented by Congo red. Measurement of the number of living cells revealed that Aβ25−35 induced a trophic rather than a toxic effect on astrocytes. Administration of cycloheximide blocked Aβ25−35-induced increase of DBI gene expression and endozepine accumulation in astrocytes, indicating that protein synthesis is required for DBI gene expression. Altogether, the present data suggest that Aβ-induced activation of endozepine biosynthesis and release may contribute to astrocyte proliferation associated with Alzheimer's disease.
The term endozepines designates a family of regulatory peptides that have been initially isolated from the rat brain on the basis of their ability to displace the binding of benzodiazepines from their receptors (Guidotti et al. 1983). All endozepines characterized so far derive from an 86-amino acid precursor polypeptide called diazepam-binding inhibitor (DBI), which generates, through proteolytic cleavage, several biologically active peptides including the triakontatetraneuropeptide DBI[17–50] (TTN) and the octadecaneuropeptide DBI[33–50] (ODN) (Ferrero et al. 1986; Slobodyansky et al. 1989).
Alzheimer's disease is a neurogenerative disorder characterized by the presence of senile plaques in the brain associated with intense astrogliosis. Senile plaques are primarily comprised of 39- to 43-amino acid peptides called beta-amyloid peptides (Aβ) (Kang et al. 1987). The Aβ25−35 fragment, which forms aggregates in vitro (Pike et al. 1993), is considered to bear the neurotoxic activity of Aβ (Yankner et al. 1990; Wei et al. 2000). During the prenatal period, a period which corresponds to intense astrogliosis (Kadhim et al. 1988; Rakic 1991), high concentrations of DBI mRNA and DBI-derived peptides occur in the rat brain (Malagon et al. 1993; Bürgi et al. 1999). These observations, together with the fact that endozepines increase [3H]thymidine incorporation in rat astrocytes (Gandolfo et al. 1999, 2000), suggest that endozepines may act as neurotrophic factors regulating proliferation and/or survival of astroglial cells.
It has been previously reported that the concentration of endozepines is higher in the cerebrospinal fluid of patients with Alzheimer's disease as compared to control subjects (Ferrarese et al. 1990). This observation prompted us to examine the effect of Aβ on endozepine production. The present study provides evidence that Aβ25−35 increases both transcription of the DBI gene and release of endozepines in cultured rat astrocytes, supporting the view that endozepines may play a role in glial cell proliferation in Alzheimer patients.
- Top of page
- Materials and methods
Although endozepines are very abundant and widely distributed in the central nervous system (Tonon et al. 1990; Alho et al. 1991), the role of these neuropeptides still remains enigmatic. A report has shown an increase in endozepine content in the cerebrospinal fluid of patients with Alzheimer's disease (Ferrarese et al. 1990). The present study demonstrates that β-amyloid peptides, the main constituents of senile plaques in Alzheimer's disease brain, activate the biosynthesis and release of endozepines in rat astrocytes.
We first observed that both Aβ1−40 and Aβ25−35 induce a concentration- and time-dependent stimulation of endozepine secretion by cultured astrocytes. Pre-incubation of Aβ1−40 and Aβ25−35 with Congo red, a chemical compound that prevents β-amyloid fibril formation (Lorenzo and Yankner 1994; Soto et al. 1996), suppressed the stimulatory effect of the peptides, indicating that only the aggregated forms of Aβ can activate endozepine release.
Prolonged incubation of astrocytes with Aβ25−35 induced a sustained increase of endozepine content in cells while the concentration of ODN-LI in culture medium gradually declined. It has been previously reported that cultured astrocytes release proteolytic enzymes which actively degrade various neuropeptides (Mentlein et al. 1990; Mentlein and Dahms 1994; Masmoudi et al. 2005). Therefore, the decrease in ODN-LI observed in the incubation medium after 12 h of culture is likely attributable to the accumulation of proteolytic enzymes in the medium, leading to degradation of DBI-related peptides and thus to the loss of immunoreactive material in the extracellular medium.
It has been previously reported that, in fetal bovine serum-containing medium, Aβ25−35 exerts a toxic effect on cultured cortical astrocytes (Brera et al. 2000). In order to determine whether the Aβ25−35-evoked increase in endozepine concentration in the conditioned medium could be ascribed to astrocyte death, we examined the effect of Aβ25−35 on cell viability. In fact, visualization of living cells by calcein-AM staining and direct cell counting both revealed that Aβ25−35 induces an increase in the number of astrocytes, indicating that Aβ does not affect the viability of cultured astrocytes, and may even stimulate cell proliferation. In agreement with this observation, it has already been shown that Aβ exhibits a modest mitogenic activity on C6 glioma cell line (Pena et al. 1995) and that endozepines, in very much the same way as IL-1β, stimulate proliferation of cultured astrocytes (Gandolfo et al. 1999, 2000; this study). The discrepancy between our data and those reported by Brera et al. (2000) can be accounted for by the different culture conditions used, i.e. the absence (this study) or the presence of serum in the culture medium. Consistent with this hypothesis, it has been previously shown that in the absence of serum, β-amyloid fragments, including Aβ25−35, do not affect survival of cultured hippocampal astrocytes (Meske et al. 1998).
A major histological feature observed in the brain of patients with Alzheimer's disease is the accumulation of activated astrocytes around senile plaques (Arelin et al. 2002; Nagele et al. 2004). It has been proposed that Aβ may activate the surrounding astrocytes to secrete proinflammatory factors, such as IL-1, IL-6 and prostaglandins, that elicit a cascade of cellular events leading to neurodegeneration (Landolfi et al. 1998; Hu and Van Eldik 1999). Indeed, it has been found that IL-1 levels are higher in the brain of Alzheimer patients than in controls (Griffin et al. 1989). In addition, in vitro experiments have shown that treatment of cultured rat cortical astrocytes with β-amyloid peptides induce up-regulation of IL-1β (Eriksson et al. 1998; Hu et al. 1998) and that IL-1β is involved in glia proliferation (Ait-Ikhlef et al. 1999; Parish et al. 2002). On the other hand, recent studies have shown that endozepines can modulate the immune response (Cosentino et al. 2000; Marino et al. 2003), suggesting that Aβ-induced stimulation of endozepine secretion may participate to the propagation of the inflammatory process and thus to the progression of Alzheimer's disease. In support of this hypothesis, it has been found that endozepines potentiate lipopolysaccharide-induced release of tumour necrosis factor α and IL-1β from macrophages (Taupin et al. 1993) and IL-6 from monocytes (Stepien et al. 1993). The observation that the endozepine ODN, as well as Aβ25−35, stimulates the release of IL-1β from cultured astrocytes provides additional evidence that endozepines may contribute to Aβ-evoked astrogliosis.
Besides their role in cell-to-cell signalling, endozepines also act as intracrine factors through activation of peripheral-type benzodiazepine receptors (PBR) located on the outer mitochondrial membrane (Papadopoulos 1993; Galiègue et al. 2003; Lacapère and Papadopoulos 2003). The observation that Aβ provokes an increase in DBI mRNA and accumulation of endozepines in cultured astrocytes thus suggests that the effect of Aβ on astrocyte activation can be ascribed, at least in part, to stimulation of PBR. Consistent with this notion, an increase in PBR ligand binding has been observed in the brain of Alzheimer patients (Diorio et al. 1991; Cagnin et al. 2001).
The mechanism by which Aβ stimulates the biosynthesis and release of endozepines in astrocytes is currently unknown. The present study has shown that the effect Aβ25−35 on DBI mRNA level and endozepine content is suppressed by cycloheximide, indicating that de novo protein synthesis is required for basal and Aβ-induced DBI gene. On the other hand, it has been previously reported that Aβ25−35 provokes an increase in intracellular calcium concentration in rat cortical astrocytes (Jalonen et al. 1997; Stix and Reiser 1998), suggesting that calcium mobilization may trigger DBI gene expression and/or endozepine secretion. Finally, it has been shown that Aβ25−35 induces the release of IL-1β from monocytes through activation of formyl peptide receptors (FPR) (Lorton et al. 2000). Whether FRP can mediate the effects of Aβ on DBI gene expression and endozepine release deserves further investigation.