DHA partially counteracts cognitive decline in the elderly 
. Moreover, omega-3 essential fatty acid-rich diets are associated with a trend in reduced risk for MCI and with MCI conversion to AD, whereas DHA has been shown to be beneficial in transgenic AD models 
. The 15-lipoxygenase-1- (15-LOX-1) DHA-derived NPD1 displays neuroprotective bioactivity in brain and retinal cells against various insults, including oxidative injury, ischemia-reperfusion and inflammation 
. Both AD brain 
and the 3xTg-AD mouse exhibit reductions in DHA and NPD1 (). In this study we further characterized the anti-inflammatory and anti-apoptotic activity of NPD1 in co-cultures of HNG cells stressed with Aβ42 oligomer, and studied the NPD1-mediated modulation of α- and β-secretase activity that resulted in reduced shedding of Aβ42.
AD is marked by synaptic damage, neuronal atrophy and cell death in the hippocampus and entorhinal cortex 
. Neurotoxicity induced by Aβ42 aggregates appears to drive microglial-mediated neuroinflammatory responses and apoptosis 
. Oxidative stress, calcium overload, mitochondrial dysfunction and membrane impairments, along with activation of caspases and cell death are associated with Aβ42 up-regulation 
. We found that NPD1 induces HNG cell survival after Aβ42-oligomer-mediated stress and reduced Aβ42-triggered apoptosis. NPD1 attenuated caspase-3 activation and decreased compacted nuclei and fragmented DNA 
(). These observations are in agreement with the NPD1-mediated up-regulation of anti-apoptotic Bcl-2, Bcl-xl and Bfl-1 expression and the decrease in the pro-apoptotic expression of Bax, Bad and Bik 
Neuroinflammatory neurodegeneration associated with Aβ42 is an important contributory event to AD neuropathology 
. In these experiments primary HNG cells were used, as human primary neurons do not survive well in the absence of glial cells 
(). While we cannot exclude the possibility that glial cells are providing some neuroprotective ‘shielding’, both neuronal and glial cells release cytokines when exposed to Aβ42 that, in turn, activate more microglia and astrocytes that reinforce pathogenic signaling. NPD1 is anti-inflammatory and promotes inflammatory resolution 
. In HNG cell models of Aβ42 toxicity, microarray analysis and Western blot analysis revealed down-regulation of pro-inflammatory genes (COX-2, TNF-α and B94), suggesting NPD1's anti-inflammatory bioactivity targets, in part, this gene family 
. These effects are persistent, as shown by time-course Western blot analysis in which protein expression was examined up to 12 h after treatment by Aβ42 and NPD1.
Although counteracting Aβ42-induced neurotoxicity is a promising strategy for AD treatment, curbing excessive Aβ42 release during neurodegeneration is also desirable. DHA could lower Aβ42 load in the CNS by stimulating non-amyloidogenic βAPP processing, reducing PS1 expression, or by increasing the expression of the sortilin receptor, SorLA/LR11 
. In contrast to a previous report by Green et al. 
that suggested that Aβ peptide reductions in whole brain homogenates of 3xTg AD after dietary supplementation of DHA were the result of decreases in the steady state levels of PS1, our experiments in primary HNG cells showed no effects of NPD1 on PS1 levels, but a significant increase in ADAM10 coupled to a decrease in BACE1 (). These later observations were further confirmed by both activity assays ( and
) and siRNA knockdown (). NPD1 reduces Aβ42 levels released from HNG cells over-expressing APPsw
in a dose-dependent manner. Our examination of other βAPP fragments revealed after NPD1 addition, a reduction in the β-secretase products sAPPβsw
and CTFβ occurred, along with an increase in α-secretase products sAPPα and CTFα, while levels of βAPP expression remained unchanged in response to NPD1. Hence these abundance- and activity-based assays indicate a shift by NPD1 in βAPP processing from the amyloidogenic to non-amyloidogenic pathway. Previously sAPPα has been found to promote NPD1 biosynthesis from DHA 
, while in the present study NPD1 works to stimulate sAPPα secretion, creating positive feedback and neurotrophic reinforcement. Secreted sAPPα's beneficial effects include enhanced learning, memory and neurotrophic properties 
. NPD1 further down-regulated the β-secretase BACE1 and activated ADAM10, a putative α-secretase. Our ADAM10 siRNA knockdown and BACE1 over-expression-activity experiments confirmed that ADAM10 and BACE1 are required in NPD1's regulation of βAPP. NPD1 therefore appears to function favorably in both of these competing βAPP processing events.
PPARγ activation leads to anti-inflammatory, anti-amyloidogenic actions and anti-apoptotic bioactivity, as does NPD1. Some fatty acids are natural ligands for PPARγ, which have a predilection for binding polyunsaturated fatty acids 
. Our hypothesis that NPD1 is a PPARγ activator was confirmed by results from both human adipogenesis and cell-based-transactivation assay ( and
). NPD1 may activate PPARγ via direct binding or other interactive mechanisms 
. Analysis of βAPP-derived fragments revealed that PPARγ does play a role in the NPD1-mediated suppression of Aβ production. Over-expressing PPARγ or incubation with a PPARγ agonist led to reductions in Aβ, sAPPβ and CTFβ similar to that with NPD1 treatment, while a PPARγ antagonist abrogated these reductions. Activation of PPARγ signaling is further confirmed by the observation that PPARγ activity decreased BACE1 levels, and a PPARγ antagonist overturned this decrease. Thus, the anti-amyloidogenic bioactivity of NPD1 is associated with activation of the PPARγ and the subsequent BACE1 down-regulation. The difference between the bioactivity of NPD1 concentrations for anti-apoptotic and anti-amyloidogenic activities (50 nM vs. 500 nM) may be due to the different cell models used (i.e., Aβ-peptide stressed vs. βAPPsw
-over-expressing HNG cells) and/or related mechanisms.
Although Aβ-lowering effects of PPARγ have been reported, the molecular mechanism of this action remains unclear. Induction of βAPP ubiquitination, which leads to enhanced βAPP degradation and reduced Aβ peptide secretion, has been suggested 
. Alternatively, Aβ clearance might be involved, or regulation by PPARγ may be due to enhancement of insulin sensitivity and increases in brain insulin degrading enzyme 
. Our results suggest that decreases in BACE1 may be the cause for Aβ reduction 
. A reason for these conflicting reports may be that cell models and culture conditions used varied; in our study, we used HNG cells transiently over-expressing βAPPsw
while previous reports employed cell lines using stable βAPP expression. Similar to the model of Sastre et al. 
, our cells underwent increases in αβ overproduction. Excessive Aβ causes inflammatory responses in both neuronal and glial cells 
. Since inflammatory signaling plays a role in AD pathogenesis, we believe HNG cell cultures are a valuable model for Aβ42 -mediated cellular actions. The fact that comparable results of our study were obtained at a much lower drug concentration (0.5 µM of rosiglitazone vs. 10–30 µM in previous reports) () underscores the highly sensitive nature of HNG cells after βAPP transfection. It is still possible that PPARγ may repress BACE1 by antagonizing activities of other transcription factors that promote BACE1 expression, such as STAT1, NF-κB and AP1 
. It is noteworthy that BACE1 expression in HNG cells was increased after βAPP over-expression. The fact that PPARγ did not affect the levels of sAPPα and CTFα besides PPARγ antagonist being unable to reverse NPD1-elicited increase in these fragments, clearly show that PPARγ is not essential for NPD1's regulation on the non-amyloidogenic pathway. Further analysis of ADAM10 showed no change occurring in ADAM10 following PPARγ activation, nor did PPARγ antagonists affect NPD1-enhanced expression of mature ADAM10. Therefore, modulation by NPD1 of α-secretase and βAPP processing are independent of PPARγ. ADAM10 is synthesized as an inactive zymogene and is processed to its mature form by cleavage of the pro-domain by pro-protein convertases (PPCs), such as furin and PC7 
. Other evidence also demonstrated that protein kinase C (PKC) and mitogen-activated protein (MAP) kinase, particularly extracellular signal-regulated kinases (ERK1/2), are involved in regulation of α-secretase activity 
. No cross-talk between the PCs and PKC or MAP kinases has been reported. Since in our study only the mature ADAM10 was increased, it is likely that the PPCs are implicated in NPD1 actions.
PPARγ antagonist GW9662 also failed to reverse the anti-apoptotic effect of NPD1, indicating that PPARγ is not implicated in NPD1 anti-apoptotic bioactivity (). NPD1 attained this neuroprotection at a concentration of 50 nM, at which its PPARγ activity is far from physiologically relevant in the in vitro
system. Other mechanisms have been proposed to explain DHA's anti-apoptotic and anti-inflammatory effects, including maintenance of plasma membrane integrity, activation of Akt signaling 
, and conversion into other derivatives 
. These findings also provide clues for NPD1's potential targets. NPD1 inhibits NF-κB activation and COX-2 expression in brain ischemia-reperfusion 
, while Aβ peptide-induced apoptosis is associated with ERK and p38 MAPK-NF-κB mediated COX-2 up-regulation 
. Neuroprotection mediated by NPD1 may further involve components of signaling pathways upstream of NF-κB activation and DNA-binding 
Our results provide compelling evidence that NPD1 is endowed with strong anti-inflammatory, anti-amyloidogenic, and anti-apoptotic bioactivities in HNG cells upon exposure to Aβ42 oligomers, or in HNG cells over-expressing βAPPsw. These results suggest that NPD1's anti-amyloidogenic effects are mediated in part through activation of the PPARγ receptor, while NPD1's stimulation of non-amyloidogenic pathways is PPARγ-independent. Suggested sites of NPD1 actions are schematically presented in . NPD1 stimulation of ADAM10 coupled to suppression of BACE1-mediated Aβ42 secretion clearly warrants further study, as these dual secretase-mediated pathways may provide effective combinatorial or multi-target approaches in the clinical management of the AD process.