We have confirmed that MPNSP supplementation can modulate markers of brain energy metabolism and alter other indicators of cognition and pathophysiology in a mouse model of AD. MPNSP supplementation prevented or reversed cognitive decline, with the modality affected dependent upon sex in this model. In post hoc analyses, MPNSP supplementation increased WT FDG uptake in several regions, including retrosplenial cingulate, posterior cingulate, and subiculum, but showed few effects in the 3×Tg mice, who maintained significant FDG decreases in most ROIs. In contrast, analysis of regional CO activity in the 3×Tg demonstrated several key regions in which the enzyme activity was corrected to WT levels. Following on the behavioral analysis, a breakdown by sex revealed striking differences in brain regional effects, particularly in regions in and closely connected to the hippocampal formation (e.g., entorhinal cortex).
Deposition of fibrillar plaques and AT8-positive tau did not appear to be ameliorated by the supplementation, possibly a function of the timing of intervention: evidence of intracellular and extracellular Aβ in the 3×Tg mice has been reported at 4 and 6 months, respectively [39
]. Memory impairment has been reported at 4 months [39
] and synaptic dysfunction at 6 months [20
]. Our earliest intervention began at approximately 7 months, after these processes should be underway; that said, biochemical measurements of the levels of human Aβ40
in 3×Tg mice showed supplementation-induced changes, with a strong trend toward differences dependent upon sex. A significant Aβ42
-lowering effect was observed in the female subgroup, which had much higher Aβ levels overall as reported in this model previously [40
]. Our analyses indicate that our sample may have been underpowered for detection of several of the tested effects; alternatively, an earlier start time or higher dosing may be required to maximize the benefit of broad-based nutritional supplementation.
Our earlier regional FDG analyses of mouse models of AD, including this model [25
], indicated consistent declines in brain regional FDG uptake, some similar to those seen in human AD [27
]. We were surprised to find that the supplementation did not correct this aberrant FDG uptake, which is an extremely plastic marker of brain activity, while it did correct mitochondrial CO function, also a plastic marker but much less so, in many of the same regions. One possible explanation for this dichotomy may be that aspects of the transgene-induced changes have direct and perhaps irreversible effects on mechanisms of FDG uptake, while the mitochondrial changes may be more amenable to our treatment, which includes compounds thought to be mitochondrial enhancers [41
Concerning cognition, we were able to detect robust differences between the WT and 3×Tg groups for both spatial working and short-term memory on the DMP task, and spatial reference memory on the MWM, establishing an excellent paradigm for the evaluation of interventions in the 3×Tg AD model. Importantly, MPNSP supplementation generated significant changes in short-term spatial memory as demonstrated in the DMP task, but not for spatial reference memory on the MWM. These divergent effects may represent a specificity of memory type enhancement through MPNSP supplementation, aiding more in short-term working memory functional recovery rather than reference memory. Indeed, these effects are in line with the brain region-specific recovery of CO activity in supplemented male 3×Tg entorhinal cortex, an area that may be more important for short-term working memory processing than long-term reference memory, as shown in patients with MCI using MRI voxel-based morphometry [42
]. Although behavioral analysis often revealed only marginal effects of the supplementation, we could determine that subject sex contributed substantially to variability and, when analyzed accordingly, differences between the sexes were indeed significant. Further, in the subsequent imaging and pathology analyses, including sex as a covariate illuminated other sex-specific changes. Specifically, in entorhinal cortex, medial mammillary nuclei, and in the CA3 and dentate gyrus of the hippocampus, a pattern emerged whereby in these regions supplementation corrected CO activity to WT levels in 3×Tg males only, and decreased levels in supplemented 3×Tg females only. Given the prominent role of these nuclei in the behavioral tasks tested, these regional effects may reflect the sex-specific performance differences we observed. It follows then, that these network differences may reflect differences between male and female correction and/or compensation of functional deficits; i.e., 3×Tg females activate different pathways than males in order to perform the task, or suffer enhanced regional vulnerability (or differences in recovery) which forces them to do so. That the entorhinal cortex and hippocampus appeared central to this sex-specific network change in mitochondrial activity was no surprise; previous research from our group has indicated that these regions are particularly sensitive to sex steroid-induced alterations in female rodents [43
]. In fact, estrogen and progesterone have been shown to modulate AD pathology and hippocampal-dependent behavioral function in 3×Tg mice [44
], and female 3×Tg mice display larger deficits in hippocampal-dependent behavior than matched males [45
]. The CO biomarker identified changes in areas (including the hippocampal formation) relevant to the apparent preservation of cognitive function that resulted from supplementation in 3×Tg males. This lends support to further examination of CO as an endpoint in preclinical interventional studies, and our concomitant work in humans [30
] indicates that CO may be a useful biomarker early in the disease process and a quantifiable endpoint for the effects of consequential intervention.
As many components of the supplement diet used in this study may possess anti-inflammatory properties, we also examined whether the diet could alter the activation of glial cells. We found that in female 3×Tg mice the supplementation significantly increased the expression levels of ARG1, a marker for alternative activation of microglia. This result suggested that the supplementation might have potential to shift a subpopulation of microglia from classical to alternative activation phenotype. Alternatively-activated microglia have an enhanced ability to phagocytize Aβ [38
]. However, ARG1 expression could also be increased with severity of AD as indicated by previous data obtained in AD brains and in 70 week-old Tg2576 mice, an animal model for amyloid pathology in AD [36
]. Increases in ARG1 have also been observed in the lipopolysaccharide-induced inflammatory response in the rTG2510 transgenic mice, an animal model over-expressing P301 L tau [46
This study has demonstrated that broad-based nutritional supplementation can positively impact certain parameters of cognition, mitochondrial function, and pathophysiology in a transgenic mouse model of AD. There were no indications that the supplementation carried unwanted side effects; indeed, these supplements have a long history of safe use in humans, including in formal clinical trials [3
]. Further research is needed to establish the optimal time for such supplementation to begin and its optimal dose (in both mice and humans). The youngest mice in this study at initiation were approximately 7 months old, and cognitive deficits, Aβ accumulation, and bioenergetic deficits have been previously reported in this model prior to that age; nonetheless, MPNSP supplementation induced several potentially beneficial effects. And, while not directly addressed in this study, it is possible that supplementation may be more effective as a preventative therapy, possibly via mechanisms bolstering brain reserve (i.e., increasing metabolic capacity via effects on mitochondrial function) or keeping levels of pathological species of tau or Aβ below critical thresholds. Given the relative safety and low cost of such intervention relative to current traditional pharmaceutical treatments with similar efficacy, broad-based supplementation such as the MPNSP or comprehensive diet plans of similar design hold great promise in the prevention and potential treatment of AD.