We show here that CD40 mRNA and protein are expressed by neuronal cells, and are increased in differentiated versus undifferentiated N2a and PC12 cells as measured by RT±PCR, western blotting and immuno¯uorescence staining. Additionally, immunohistochemistry reveals that neurons from adult mouse and human brain also express CD40 in situ. CD40 ligation results in a time-dependent increase in p44/42 MAPK activation in neuronal cells. Furthermore, ligation of CD40 opposes JNK phosphorylation and activity induced by NGF-b removal from differentiated PC12 cells or serum withdrawal from primary cultured neurons. Importantly, CD40 ligation also protects neuronal cells from NGF-b or serum withdrawal-induced injury and affects neuronal differentiation. Finally, adult mice de®cient for the CD40 receptor demonstrate neuronal dysfunction as evidenced by decreased neuro®lament isoforms, reduced Bcl-x L :Bax ratio, neuronal morphological change, increased DNA fragmentation, and gross brain abnormality. These changes occur with age, and are clearly evident at 16 months. Taken together, these data demonstrate a role of CD40 in neuronal development, maintenance and protection in vitro and in vivo.
Reactive microglia have been suggested to play a role in the Alzheimer's disease (AD) process, and previous studies have shown that expression of CD45, a membrane-bound proteintyrosine phosphatase (PTP), is elevated in microglia in AD brain compared with controls. To investigate the possible role of CD45 in microglial responsiveness to -amyloid (A) peptides, we first co-treated primary cultured microglia with a tyrosine phosphatase inhibitor [potassium bisperoxo (1,10-phenanthroline) oxovanadate (phen), 5 M] and freshly solubilized A peptides (1000 nM). Data show synergistic induction of microglial activation as evidenced by tumor necrosis factor ␣ (TNF-␣) production and nitric oxide (NO) release, both of which we show to be dependent on activation of p44/42 mitogen-activated protein kinase (MAPK). Furthermore, co-treatment with phen and A peptides results in microglia-induced neuronal cell injury. Stimulation of microglial CD45 by anti-CD45 antibody markedly inhibits these effects via inhibition of p44/42 MAPK, suggesting that CD45 is a negative regulator of microglial activation. Accordingly, primary cultured microglia from CD45-deficient mice demonstrate hyper-responsiveness to A, as evidenced by TNF-␣ release, NO production, and neuronal injury after stimulation with A peptides. As a validation of these findings in vivo, brains from a transgenic mouse model of AD [transgenic Swedish APPoverexpressing (Tg APP sw ) mice] deficient for CD45 demonstrate markedly increased production of TNF-␣ compared with Tg APP sw mice. Taken together, these results suggest that therapeutic agents that stimulate the CD45 PTP signaling pathway may be effective in suppressing microglial activation associated with AD. Key words: Alzheimer's disease; -amyloid; microlgia; neurons; mitogen-activated protein kinase; CD45; protein-tyrosine phosphatase; tyrosine phospatase inhibitor; TNF-␣; nitric oxideIt has been suggested that activated microglia play a key role in the inflammatory processes of neurodegenerative diseases such as Alzheimer's disease (AD), because reactive microglia secrete cytokines, including tumor necrosis factor ␣ (TNF-␣) and interleukin-1, which promote neurodegeneration (Meda et al., 1995;Rogers et al., 1996;Barger and Harmon, 1997). However, current antiinflammatory therapeutics directed against AD, such as nonsteroidal anti-inflammatory drugs (NSAIDs), only partially suppress microglial activation (Mackenzie and Munoz, 1998) and, therefore, may not provide the greatest therapeutic benefits for AD. This suggestion is supported by clinical evidence, in which elderly persons using NSAIDs demonstrate only an ϳ20% reduction in risk for AD (Beard et al., 1998), and AD patients using NSAIDs have only partial amelioration of disease symptoms (Rich et al., 1995). Thus, a more viable therapeutic strategy may be combination of NSAIDs with specific inhibitors of microglial activation.Most of our knowledge concerning the molecular mediators of microglial activation comes from studies involving peripheral lymphocytes....
Alzheimer's disease (AD) is pathologically characterized by deposition of amyloid-beta peptides (Abeta) as senile plaques and by the occurrence of neurofibrillary tangles (NFTs) composed primarily of hyperphosphorylated tau protein. Activation of cyclin-dependent kinase 5 (Cdk5) via its potent activator p25 has recently been shown to promote phosphorylation of tau at AD-specific phosphoepitopes, and increased cleavage of p35 to p25 has been demonstrated in AD patients, suggesting that Cdk5 may represent a pathogenic tau protein kinase. We were interested in the potential effect of soluble forms of Abeta on Cdk5-mediated AD-like tau phosphorylation, insofar as previous studies of human biopsies and aged canine and primate brains have shown that dystrophic neurites appear before the formation of neuritic plaques. We transfected N2a cells with a p35 vector (N2a/p35 cells) and, after differentiation, challenged these cells with Abeta(1-42) peptide in soluble form (sAbeta(1-42)). Results show that sAbeta(1-42) at relatively low levels (1-5 microM) dose-dependently increases tau phosphorylation at AD-specific phosphoepitopes in differentiated N2a/p35 cells compared with controls, an effect that is blocked by antisense oligonucleotides against p35. sAbeta(1-42)-induced tau phosphorylation is concomitant with an increase in both p25 to p35 ratio and Cdk5 activity (but not protein levels). Additionally, blockade of L-type calcium channels or inhibition of calpain completely abolishes this effect. Taken together, these data indicate that sAbeta is a potent activator of the p25/Cdk5 pathway, resulting in promotion of AD-like tau phosphorylation in vitro.
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