The 42-aa-long β-amyloid protein—Aβ_1-42—is thought to play a central role in the pathogenesis of Alzheimer's disease (AD) (Walsh and Selkoe, 2007). Data from AD brain (Shankar et al., 2008), transgenic APP (amyloid precursor protein)-overexpressing mice (Lesné et al., 2006), and neuronal cultures treated with synthetic Aβ peptides (Lambert et al., 1998) indicate that self-association of Aβ_1-42monomers into soluble oligomers is required for neurotoxicity. The function of monomeric Aβ_1-42is unknown. The evidence that Aβ_1-42is present in the brain and CSF of normal individuals suggests that the peptide is physiologically active (Shoji, 2002). Here we show that synthetic Aβ_1-42monomers support the survival of developing neurons under conditions of trophic deprivation and protect mature neurons against excitotoxic death, a process that contributes to the overall neurodegeneration associated with AD. The neuroprotective action of Aβ_1-42monomers was mediated by the activation of the PI-3-K (phosphatidylinositol-3-kinase) pathway, and involved the stimulation of IGF-1 (insulin-like growth factor-1) receptors and/or other receptors of the insulin superfamily. Interestingly, monomers of Aβ_1-42carrying the Arctic mutation (E22G) associated with familiar AD (Nilsberth et al., 2001) were not neuroprotective. We suggest that pathological aggregation of Aβ_1-42may also cause neurodegeneration by depriving neurons of the protective activity of Aβ_1-42monomers. This “loss-of-function” hypothesis of neuronal death should be taken into consideration when designing therapies aimed at reducing Aβ burden.

Cultured neurons exposed to synthetic β-amyloid (Aβ) fragments reenter the cell cycle and initiate a pathway of DNA replication that involves the repair enzyme DNA polymerase-β (DNA pol-β) before undergoing apoptotic death. In this study, by performing coimmunoprecipitation experiments on cross-linked nucleoprotein fragments from Aβ-treated neurons, we demonstrate that DNA pol-β coimmunoprecipitates with cell division cycle 45 (Cdc45) and with DNA primase in short nucleoprotein fragments. This indicates that DNA pol-β is loaded into neuronal DNA replication forks after Aβ treatment. In response to Aβ the canonical DNA-synthesizing enzyme DNA pol-δ also was loaded into neuronal replication forks, but at later times than DNA pol-β. Methoxyamine, an inhibitor of the apurinic/apyrimidinic endonuclease that allows for the recruitment of DNA pol-β during the process of base excision repair (BER), failed to affect coimmunoprecipitation between DNA pol-β and Cdc45, indicating that DNA pol-β loading to the replication forks is independent of DNA breaks. However, methoxyamine reduced DNA replication and ensuing apoptosis in neurons exposed to Aβ, suggesting that an efficient BER process allows DNA replication to proceed up to the threshold for death.These data demonstrate that DNA pol-β is an essential component of the DNA replication machinery in Aβ-treated neurons and additionally support the hypothesis of a close association of cell cycle events with neuronal death in Alzheimer's disease (AD). Accordingly, by investigating the neuronal expression of DNA pol-β, along with phosphorylated retinoblastoma protein and neurofibrillary changes in AD brain, we show an early involvement of DNA pol-β in the pathogenesis of AD.

We have shown that cortical neurons challenged with toxic concentrations of β-amyloid peptide (βAP) enter the S phase of the cell cycle before apoptotic death. Searching for a signaling molecule that lies at the border between cell proliferation and apoptotic death, we focused on the disialoganglioside GD3. Exposure of rat cultured cortical neurons to 25 μmβAP(25–35) induced a substantial increase in the intracellular levels of GD3 after 4 hr, a time that precedes neuronal entry into S phase. GD3 levels decreased but still remained higher than in the control cultures after 16 hr of exposure to βAP(25–35). Confocal microscopy analysis showed that the GD3 synthesized in response to βAP colocalized with nuclear chromatin. The increase in GD3 was associated with a reduction of sphingomyelin (the main source of the ganglioside precursor ceramide) and with the induction of α-2,8-sialyltransferase (GD3 synthase), the enzyme that forms GD3 from the monosialoganglioside GM3. A causal relationship between GD3, cell-cycle activation, and apoptosis was demonstrated by treating the cultures with antisense oligonucleotides directed against GD3 synthase. This treatment, which reduced βAP(25–35)-stimulated GD3 formation by ∼50%, abolished the neuronal entry into the S phase and was protective against βAP(25–35)-induced apoptosis.

L-acetylcarnitine (LAC), a drug utilized for the treatment of neuropathic pain in humans, has been shown to induce analgesia in rodents by up-regulating the expression of metabotropic glutamate receptor 2 (mGlu2) in dorsal root ganglia (DRG). We now report that LAC-induced upregulation of mGlu2 expression in DRG cultures involves transcriptional activation mediated by nuclear factor-kappaB (NF-κB). A single application of LAC (250 μM) to DRG cultures induced a transient increase in mGlu2 mRNA, which was observable after 1 hour and was no longer detectable after 1 to 4 days. In contrast, LAC treatment had no effect on mGlu3 mRNA expression. Pharmacological inhibition of NF-κB binding to DNA by caffeic acid phenethyl ester (CAPE) (2.5 μg/ml for 30 minutes) reduced the constitutive expression of mGlu2 and mGlu3 mRNA after 1–4 days and reduced the constitutive expression of mGlu2/3 protein at 4 days. This evidence combined with the expression of p65/RelA and c-Rel in DRG neurons indicated that expression of mGlu2 and mGlu3 is endogenously regulated by the NF-κB family of transcription factors. Consistent with this idea, the transient increase in mGlu2 mRNA induced by LAC after 1 hour was completely suppressed by CAPE. Furthermore, LAC induced an increase in the acetylation of p65/RelA, a process that enhances the transcriptional activity of p65/RelA. These results are consistent with the hypothesis that LAC selectively induces the expression of mGlu2 by acting as a donor of acetyl groups, thus enhancing the activity of the NF-κB family of transcription factors. Accordingly, we show that carnitine, which has no effect on pain thresholds, had no effect on p65/RelA acetylation and did not enhance mGlu2 expression. Taken together, these results demonstrate that expression of mGlu2 and mGlu3 mRNA is regulated by the NF-κB transcriptional machinery, and that agents that increase acetylation and activation of NF-κB transcription factors might induce analgesia via upregulation of mGlu2 in DRG neurons.

An ectopic reentrance into the cell cycle with ensuing DNA replication is required for neuronal apoptosis induced by β‐amyloid. Here, we investigate the repertoire of DNA polymerases expressed in β‐amyloid‐treated neurons, and their specific role in DNA synthesis and apoptosis. We show that exposure of cultured cortical neurons to β‐amyloid induces the expression of DNA polymerase‐β, proliferating cell nuclear antigen, and the p49 and p58 subunits of DNA primase. Induction requires the activity of cyclin‐dependent kinases. The knockdown of the p49 primase subunit prevents β‐amyloid‐induced neuronal DNA synthesis and apoptosis. Similar effects are observed by knocking down DNA polymerase‐β or by using dideoxycytidine, a preferential inhibitor of this enzyme. Thus, the reparative enzyme DNA polymerase‐β unexpectedly mediates a large component of de novo DNA synthesis and apoptotic death in neurons exposed to βamyloid. These data indicate that DNA polymerases become death signals when erratically expressed by differentiated neurons.