Here we have demonstrated that DHA decreased Aβ40 and Aβ42 peptide secretion from aging brain cells and that this was accompanied by biosynthesis of NPD1. In turn, NPD1 inhibited Aβ42-induced apoptosis. Our observations on the occurrence of apoptosis in both glial and neuronal cell populations in our stressed HN cell model are in concordance with the findings of others on apoptosis in glial and neuronal cells in AD brain (42
). The neuropathologic features of the CA1 region for the control and AD samples used in this study, including age, sex, postmortem interval, and senile plaque and neurofibrillary tangle densities, are summarized in Table ; additional neuropathologic parameters for these same tissues (tissue pH, clinical dementia rating, etc.) were presented in a recent publication (33
). Concerning the brain samples used for lipidomic analysis, while the senile plaque densities in the CA1 region for control tissues used (control samples 1–4; Table ) averaged 0.25 lesions/mm2
, the senile plaque densities in the CA1 for all AD tissues used in the lipidomic study (AD samples 1–4; Table ) averaged greater than 6.7 lesions/mm2
Aβ42 is a weak inducer of NPD1 and potentiates DHA ability to stimulate the synthesis of this lipid (Figure H). The neuroprotective sAPPα peptide was a potent agonist for NPD1 synthesis. Aβ42 induces proinflammatory and proapoptotic genes, whereas DHA, and, more strikingly, NPD1, enhanced antiinflammatory and antiapoptotic gene expression. Notably, Bfl-1(A1) gene expression was upregulated by NPD1 about 6-fold in HN cells. cPLA2 expression was increased, whereas 15-LOX, the candidate enzyme for NPD1 synthesis, was decreased in the CA1 region of hippocampi from patients with moderate to severe stages of AD. In this same brain region, NPD1 content was dramatically reduced.
The aging of HN cells in primary culture is accompanied by the release of Aβ40 and Aβ42 peptides into the cell culture medium, and for each Aβ peptide this secretion increases almost 8-fold over 8 weeks of culture (Figure , D and E). Importantly, we observed no significant change in neuronal and glial cell morphology in these cultures after 18 hours of Aβ42 (or NPD1) treatment, and there was no considerable cell death at the concentrations of Aβ42 used over the time course investigated (Figure ). This suggests that Aβ42 is setting in motion potential cell-damaging signals accompanied by the early onset of apoptosis, and changes in gene-expression patterns that may be in part emulating the neurodegenerative process characteristic of AD. Accumulation of secreted Aβ40 and Aβ42 peptides during HN cell aging has important implications in the development of Aβ-related neuropathology and resembles Aβ deposition observed during brain aging and in AD (39
). Intracellular processing of the transmembrane glycoprotein βAPP through a sequential β- and γ-secretase–catalyzed proteolysis generates Aβ peptides that are subsequently shuttled to the plasma membrane and secreted (26
). Interestingly, exposure of HN cells to the glial cell–derived, proinflammatory cytokine IL-1β significantly stimulated both Aβ40 and Aβ42 secretion as a function of HN cell aging. IL-1β directly stimulates γ-secretase–mediated cleavage of βAPP into Aβ peptides through a JNK-dependent MAPK pathway (34
). Conversely, in the present work, instillation of DHA into the HN cell culture medium suppressed both Aβ40 and Aβ42 peptide release. DHA attenuates increases in the levels of lipid peroxides and reactive oxygen species in the cerebral cortex and the hippocampus of Aβ-infused rats, suggesting that DHA elicits neuroprotection by blocking Aβ40/Aβ42 neurotoxicity (45
). This protection might be achieved by decreasing γ-secretase activation, by decreasing antioxidative defenses, or both (22
). Along those lines, recent studies indicate that DHA attenuates neuronal degeneration and rescues learning ability in rodent models of AD (22
). Moreover, the neuroprotective mediator NPD1 (2
) is formed, while DHA downregulates Aβ release in aging HN cells in culture (Figure ). Since NPD1 inhibits Aβ42-induced apoptosis in HN cells (Figure ), DHA protection in cells in culture and in in vivo models may involve NPD1 synthesis.
Striking changes in the expression of Bcl-2 family members correlate with Aβ42, DHA, or NPD1 exposure. Pro- and antiapoptotic proteins are modulators proximal to mitochondria and irreversible cell damage. Proapoptotic proteins Bik and Bax were enhanced by Aβ42, but not by DHA or NPD1, whereas Bcl-2, Bcl-xl, and Bfl-1(A1) were enhanced in the presence of DHA. NPD1, on the other hand, promoted a much larger increase in antiapoptotic Bcl-2 proteins. Bfl-1(A1) increased almost 6-fold. Antiapoptotic Bcl-2 family members such as Bfl-1(A1) play critical roles in the survival of aged and terminally differentiated cells and break the mechanistic link between inflammatory signaling and apoptosis (37
). In fact, NPD1 also induces the antiapoptotic Bcl-2 family proteins Bcl-2 and Bcl-xl in oxidatively challenged human retinal pigment epithelial cells (6
) and promotes cytoprotection. A further suggestion of the significance of NPD1 in AD is the observation that hippocampal CA1 from AD patients shows a dramatic reduction in NPD1. Whether decreased NPD1 levels in AD brain hippocampal CA1 are the result or the cause of the AD process remains to be clarified. All except 1 postmortem AD brains displayed moderate histopathologic changes (Table ). Since these tissues were sampled within 3 hours postmortem, the NPD1 pool size may reflect the capacity of the CA1 hippocampal region to activate synthesis of the mediator. In mouse brain undergoing ischemia/reperfusion, NPD1 increases during the initial 8 hours after 1 hour of ischemia (5
). In the postmortem brain, the differences found between age-matched controls and AD brains point to the relative inability of the AD CA1 region to accumulate NPD1.
In summary, the interplay of DHA-derived neuroprotective signaling aims to counteract proinflammatory, cell-damaging events triggered by multiple, converging cytokine and amyloid peptide factors in AD. Amyloid peptide–mediated oxidative stress, the activation of microglia associated with Aβ peptide deposition, and excessive production of microglial-derived cytokines such as IL-1β and TNF-α support progressive inflammatory episodes in AD (4
). These noxious stimuli further orchestrate pathogenic gene-expression programs in stressed brain cells, thereby linking a cascade of caspase-mediated cell death pathways with apoptosis and neuronal demise (3
). Neural mechanisms leading toward NPD1 generation from DHA thereby appear to redirect cellular fate toward successful brain cell aging. The Bcl-2 pro- and antiapoptotic gene families, sAPPα, and NPD1 lie along a cell fate–regulatory pathway whose component members are highly interactive, and have potential to function cooperatively in brain cell survival, acting through modulation of Aβ42-directed pathogenic events. Taken together, these data suggest that NPD1 induces an antiapoptotic, neuroprotective gene-expression program that regulates the secretion of Aβ peptides, resulting in the modulation of inflammatory signaling, neuronal survival, and the preservation of brain cell function. Agonists of NPD1 biosynthesis or NPD1 analogs may be useful for exploring new therapeutic strategies for AD and related neurodegenerative disease.