Aging is characterized by cognitive decline, synaptic dysfunction, and the accumulation of brain pathology. Lifestyle interventions such as diet, exercise, and cognitive training have emerged as effective strategies to prevent cognitive decline and reduce brain pathology 
. However, how these lifestyle interventions improve the health of the aging brain remains unclear.
For several years now, our group has used the aged canine to study the specific effects of lifestyle interventions on brain aging and cognitive function. Like aged humans, the aged canine brain naturally shows cognitive decline, increased oxidative damage, mitochondrial dysfunction, selective neuron loss, and beta-amyloid (Aβ) accumulation 
. We have demonstrated that long-term (2.8 years) behavioral enrichment (with social, cognitive, and exercise stimulation), dietary supplementation (with antioxidants and mitochondrial cofactors), or the combination of the behavioral and dietary interventions slow age-related cognitive decline and reduce brain pathology in aged canines 
. The combination treatment was particular effective in improving cognitive performance 
, suggesting that the effects of the behavioral and dietary treatment approaches are additive.
Mitochondrial dysfunction, oxidative damage and Aβ accumulation are thought to be primary factors contributing to declining function in aging and Alzheimer's disease (AD) 
. We have evaluated these mechanisms as potential targets of the behavioral and dietary interventions, and foundprominent effects on improving mitochondrial function and reducing oxidative stress. Notably, the interventions improved mitochondrial NADH respiration, reduced generation of mitochondrial reactive oxygen species (ROS) 
lowered levels of protein carbonyls, and bolstered antioxidant defense mechanisms in the brain 
. The reduction in oxidative damage correlated with cognitive improvement 
, suggesting that accumulating oxidative stress is likely to be a central feature underlying cognitive decline in aging. In parallel with reducing oxidative damage, the behavioral and dietary interventions modestly reduced the accumulation of Aβ in the aged canine brain, particularly with the combined intervention 
. Surprisingly, while Aβ load was reduced in entorhinal, cingulate and parietal cortices, improvements in cognitive performance did not correlate with Aβ load 
, suggesting that effects on Aβ are likely not a central mechanism underlying the cognitive benefits of the interventions. However, because Aβ is well established to compromise neuronal health and synaptic function 
, accumulation of Aβ in the aged canine brain is likely not benign, and it must be considered that AB may trigger downstream mechanisms that contribute to declines in brain health and cognitive function with age.
It is likely that the combined effects of oxidative stress, impaired mitochondrial function and AB accumulation can propagate harmful cascades that converge on common downstream mechanisms that ultimately cause neuronal damage and dysfunction. Two downstream targets that have recently come into increasing focus for their roles in compromising synaptic function and cognition are activated caspase 3 and the bioactive lipid ceramide. While caspases are best known for their role in apoptosis, recent evidence implicates caspase 3 in non-apoptotic processes, including impairing synaptic plasticity 
, spine atrophy and degeneration 
, and memory deficits 
in the absence of neuron loss. In addition, caspase 3 mediates some of the harmful effects of certain pathologies such as Aβ, as inhibition of long term potentiation (LTP) by Aβ1–42 is dependent on caspase 3 activation 
. Like caspase 3, ceramides have recently been identified as potential causes of cognitive decline and onset of AD. Ceramide levels are elevated in the brain even at the earliest clinical stages of AD 
, and there is evidence that ceramide in CSF and serum may be a useful biomarker predicting cognitive decline 
. These signaling molecules are important in an array of physiological processes and are generated in response to inflammatory cytokines and oxidative stress either by hydrolysis of sphingomyelin or by de novo synthesis 
. One effect of ceramide is to suppress the mitochondrial respiratory chain, resulting in increased production of ROS and oxidative stress 
. Oxidative stress, in turn, can activate caspase 3 
, and stimulate ceramide generation 
revealing that oxidative stress, ceramides, and caspase 3 are linked in a self-perpetuating feed-forward cycle. Taken together, this literature suggests that oxidative damage, mitochondrial dysfunction and AB might ultimately converge on caspase 3 to impair synaptic and cognitive function.
In this study, we build on our previous findings that long-term behavioral enrichment, dietary supplementation, or the combined therapies improve cognitive function, improve mitochondrial health and function, reduce oxidative damage, and decrease AB in the aged canine brain. Using this same set of dog tissue, here we investigate if caspase activation and ceramide accumulation are reduced in the aged frontal cortex, serving as potential readout targets of intervention efficacy. In particular, we focus on caspase 3, based on its recently identified role in driving synaptic dysfunction in mouse models of AD. We assess if dietary, behavioral, or the combined interventions reduce the extent of caspase 3 activation in the aged canine frontal cortex, and assess potential roles of caspases 8 and 9 in mediating the effects of the interventions on caspase 3. In parallel, we assess levels of several species of ceramides, candidate risk factors for triggering pathological cascades and cognitive impairment in the aging brain.