Conceived and designed the experiments: SS CWC. Performed the experiments: SS GA TS. Analyzed the data: SS NB. Contributed reagents/materials/analysis tools: DP CWC. Wrote the paper: SS NB CWC.
The authors have declared that no competing interests exist.
Lifestyle interventions such as diet, exercise, and cognitive training represent a quietly emerging revolution in the modern approach to counteracting age-related declines in brain health. Previous studies in our laboratory have shown that long-term dietary supplementation with antioxidants and mitochondrial cofactors (AOX) or behavioral enrichment with social, cognitive, and exercise components (ENR), can effectively improve cognitive performance and reduce brain pathology of aged canines, including oxidative damage and Aβ accumulation. In this study, we build on and extend our previous findings by investigating if the interventions reduce caspase activation and ceramide accumulation in the aged frontal cortex, since caspase activation and ceramide accumulation are common convergence points for oxidative damage and Aβ, among other factors associated with the aged and AD brain. Aged beagles were placed into one of four treatment groups: CON – control environment/control diet, AOX– control environment/antioxidant diet, ENR – enriched environment/control diet, AOX/ENR– enriched environment/antioxidant diet for 2.8 years. Following behavioral testing, brains were removed and frontal cortices were analyzed to monitor levels of active caspase 3, active caspase 9 and their respective cleavage products such as tau and semaphorin7a, and ceramides. Our results show that levels of activated caspase-3 were reduced by ENR and AOX interventions with the largest reduction occurring with combined AOX/ENR group. Further, reductions in caspase-3 correlated with reduced errors in a reversal learning task, which depends on frontal cortex function. In addition, animals treated with an AOX arm showed reduced numbers of cells expressing active caspase 9 or its cleavage product semaphorin 7A, while ENR (but not AOX) reduced ceramide levels. Overall, these data demonstrate that lifestyle interventions curtail activation of pro-degenerative pathways to improve cellular health and are the first to show that lifestyle interventions can regulate caspase pathways in a higher animal model of aging.
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
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
Mitochondrial dysfunction, oxidative damage and Aβ accumulation are thought to be primary factors contributing to declining function in aging and Alzheimer's disease (AD)
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
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.
To evaluate if behavioral enrichment (ENR), dietary enrichment (AOX), or the combined intervention (ENR/AOX) affected the extent of caspase 3 activation in the frontal cortex, cells immune-positive for activated caspase 3 were counted. An antibody specific to activated caspase 3 was used that detects the large (17 kDa) fragment but that does not recognize either the full-length procaspase-3 or other cleaved caspases. One-way ANOVA identified a significant intervention effect (F3,20 = 8.3; p<0.01) and post-hoc analysis revealed fewer cells positive for activated caspase 3 following AOX (p<0.01), ENR (p<0.01) and ENR/AOX (p<0.001) treatment relative to aged controls. The greatest reduction occurred with the combined treatment, which reduced counts of activated caspase-3 positive cells by 80% relative to untreated control values (
We next evaluated if the extent of caspase 3 activation related to cognitive performance, with the prediction that better cognition would be present in those animals where fewer cells contained activated caspase 3. To determine whether caspase activation was correlated with cognitive function, we used behavioral data from our previously published findings
We selected a task that engages frontal lobe function (black/white discrimination learning and reversal) and that was conducted at the end of the 2.8 yr intervention period
We next evaluated levels of casapase-3 cleavage products to confirm that reduced immunoreactivity for activated caspase 3 with the interventions also reduced downstream proteolytic consequences. Two well-characterized cleavage targets of caspase 3 are the cytoskeletal proteins beta-actin and tau. Specifically, activated caspase-3 cleaves beta-actin between Asp244 and Gly245, generating a 32 kDa N-terminal fragment of actin known as Fractin, while tau is cleaved at the D421 site (tau421). Using an activity test followed by dot-blot analysis, we confirmed that caspase 3 cleaves tau to produce tau421 in canine frontal cortex homogenates (data not shown), similar to the effect of caspase 3 on tau in human and rodent brain tissue.
Frontal cortex sections were immunolabeled with an antibody specific to fractin and cell counts were assessed in CON, AOX, ENR and AOX/ENR groups. One-way ANOVA revealed a significant treatment effect (F3,20 = 3.30, p<0.05), with modest reductions in fractin-positive cells in the AOX and ENR groups and a significant reduction to 40% of untreated controls in the ENR/AOX combined treatment group (p<0.05) (
Because caspase 3 is primarily activated by two initiator pathways driven by caspases 8 and 9, we next investigated if either of these caspases may be a mechanism by which the dietary and behavioral interventions reduce caspase 3 activation. Immunohistochemistry using an antibody specific to activated caspase 8 revealed the presence of this activated caspase in frontal cortical sections, however the incidence of immune-positive cells was rare. Activated caspase-3 was detected in all groups, suggesting that the effects of the dietary and behavioral interventions on caspase 3 activation are not mediated via caspase 8. On the other hand, the interventions reduced caspase 9 activation, based on counts of cells immunopositive for active caspase 9 and semaphorin 7A, a specific cleavage product of active caspase 9. One-way ANOVA revealed a significant treatment effect on cells positive for active caspase 9 (F3,18 = 4.85, p = 0.016,
The sphingolipid ceramide can disrupt the mitochondrial respiratory chain, activate caspases and has been implicated in signaling pathways that impair learning and memory. In particular, increases in the levels of ceramide species in brain and plasma were linked to cognitive impairment/decline in AD/MCI
CON | AOX | ENR | AOX/ENR | |
|
0.06±0.001 | 0.006±0.002 | 0.004±0.001 | 0.010±0.005 |
|
0.20±0.02 | 0.24±0.07 | 0.170±0.02 | 0.196±0.02 |
|
6.05±0.55 | 5.00±0.90 | 3.701±0.39 | 5.951±0.53 |
|
0.12±0.02 | 0.14±0.03 | 0.126±0.02 | 0.129±0.19 |
Data expressed as ±SEM. CON: control environment/control diet; AOX: control environment/antioxidant diet; ENR: behavioral enrichment/control diet; AOX/ENR: behavioral enrichment/antioxidant diet.
While caspase activation has traditionally been associated with apoptosis, recent findings reveal that activated caspase 3 can be present in the brain with no evidence of cell death. To determine if caspase 3 activation in the aged dog frontal cortex is accompanied by apoptosis, we used TUNEL-labeling to detect the DNA fragmentation characteristic of apoptotic cells. Interestingly, while extensive TUNEL-positive labeling was apparent in the positive control tissue (from a patient who had died from AD), no TUNEL-positive cells were identified in any of the four treatment groups of aged dogs, suggesting that activation of caspase 3 is not sufficient to trigger cell death in the aged canine brain.
Behavioral lifestyle interventions represent a quietly emerging revolution in the modern approach to counteracting age-related declines in brain health and cognitive function. In this study, we investigated if activation of caspases and ceramide accumulation are reduced by long term dietary and behavioral interventions. Caspase activation and ceramide accumulation are common convergence points for oxidative damage and AB, among other factors associated with the aged and AD brain. While activated caspases and ceramides are present in the AD brain and are known risk factors for cognitive decline and neurodegeneration, no studies to date have assessed if these are targeted by ENR, AOX or combined intervention using higher animal models of human brain aging.
Our data reveal a high abundance of cells with activated caspase 3 in the frontal cortex of the aged dog brain, present primarily in neurons. In parallel, caspase cleavage products, such as cleaved cytoskeletal proteins actin and tau are present. A sustained change in lifestyle consisting of behavioral enrichment, dietary supplementation with antioxidants and mitochondrial cofactors, or the combined intervention dramatically reduced the abundance of cells expressing activated caspase 3, with the combined intervention having the greatest effect, reducing numbers of immune-positive cells by 80% relative to untreated levels. The reduction in active caspase 3 correlated strongly with reduced error scores on discrimination learning, suggesting that reduction in caspase 3 activation is an important target of the interventions to promote cognitive stability with age. In parallel with fewer cells expressing active caspase-3, there were fewer cells expressing caspase cleavage products, including fractin and cleaved tau, which may underlie the benefits of reduced caspase 3 activation. For example, the truncation state of tau influences many of its normal and pathologic characteristics, including its ability to bind to and stabilize microtubules
Caspase 3 is traditionally known as one of the primary effector caspases driving apoptosis. However, our finding that activated caspase 3 is not accompanied by TUNEL labeling strongly suggests that caspase activation in the canine brain is associated with non-apoptotic processes. This is supported by our previous finding that neuron loss in the aged canine brain is minimal
While activation of caspase 3 is regulated primarily by the initiator caspases 8 and 9, our data suggest that only caspase 9 activation is targeted by the interventions. Active caspase 8 was present but sparse in the aged frontal cortex, and was detected in all treatment groups. In contrast, many cells were immunopositive for active caspase 9, with animals treated with an AOX arm (eg AOX, or AOX/ENR) showing approximately 50% fewer cells expressing active caspase 9 or its cleavage product semaphorin 7A. Because caspase 9 is selectively released by stressed mitochondria these findings suggest that mitochondrial health is improved by the interventions, consistent with our previous finding that the antioxidant diet reduced mitochondrial ROS production and improved NADH respiration in the canine
Like caspase 3, the bioactive lipid group of ceramides has recently been identified as potential causes of cognitive decline and onset of AD. Levels of ceramides have been shown to be elevated in the cerebral cortex during normal aging and in Alzheimer's disease
Taken together, our findings reveal that life style interventions can engage a range of molecular mechanisms to improve brain health and cognitive function. Along with our previous findings that behavioral and dietary interventions improve mitochondrial NADH respiration, reduce generation of mitochondrial reactive oxygen species (ROS)
Twenty-four beagles ranging in age at the start of the study from 8.05 to 12.35 years (mean = 10.69 years, SE = 0.25, 12 males/12 females) were obtained from the colony at the Lovelace Respiratory Research Institute. Animals were born and maintained in the same environment and all had documented dates of birth and comprehensive medical histories.
All studies were conducted in compliance with approved IACUC protocols, consistent with the National Research Council's Guide for the care and use of laboratory animals.
All dogs underwent extensive baseline cognitive testing as has been described previously
The behavioral enrichment protocol has been described previously
Both the control and supplemented test foods were formulated to meet the nutrient profile for the American Association of Feed Control Officials (AAFCO) recommendations for adult dogs (AAFCO 1999) and has been described previously
Dogs were ex-sanguinated under anesthesia (5% isoflurane), by cardiac puncture and within 15 min the brain was removed from the skull. The brain was sectioned midsagitally, with the entire left hemisphere being immediately placed in 4% paraformaldehyde for 48–72 h at 4°C then transferred to phosphate buffered saline with 0.05% sodium azide at 4°C for long term storage. The left hemisphere was sent to NeuroScience Associates for sectioning. NeuroScience Associates treated individual canine hemispheres with 20% glycerol and 2% dimethylsulfoxide to prevent freeze-artifacts and subsequently embedded two hemispheres (i.e. two animals) per block in a gelatin matrix using MultiBrain Technology™ (NeuroScience Associates, Knoxville, TN;
Standard immunohistochemical methods were used and have been published elsewhere
Immunofluorescence was used to evaluate colocalization of cleaved caspase 3 (1∶100) with either NeuN (1∶500) or GFAP (1∶1000). The primary antibody/antigen complex was detected using secondary antibodies conjugated to either Alexa 488 or Alexa 568 (Invitrogen), and was visualized using a Zeiss LSM510 META confocal system configured with a Zeiss Axiovert 200 M motorized inverted microscope. Multilabeled fluorescent samples were imaged acquiring each fluorescent channel sequentially to avoid signal bleed-over. The Alexa 488 component was obtained using the 488 nm line of an argon ion laser for excitation and a band pass 505–530 nm emission filter. The Alexa 564 component was obtained using the 543 nm line of a green helium/neon laser for excitation and a band pass 585–620 nm emission filter.
Nitrocellulose membranes were dotted with 1 µl of frontal cortical crude extracts (4 ug/ul) from 4 aged beagles. All extracts were suspended in 100 µl of Laemmli buffer prior to absorption on the membrane. The membrane was air dried for 30 minutes and blocked with TBS-5% BSA and incubated with caspase cleaved tau antibody (1∶1000, overnight at 4°C). After three washes with TBS-0.05% Tween 20, the immune complexes were revealed with HRP-labeled goat anti-mouse secondary antibody (Sigma) (1/10,000 dilution, 1 hour at room temperature) followed by ECL detection. In addition, to detect caspase 3-induced cleavage of tau in crude frontal cortex homogenates, an activity test was performed. Specifically, wells were coated with 1 µl of active caspase-3 protease (Chemicon) and incubated with frontal cortex homogenates (1 µl, 4 ug/ul) from 4 aged beagles at 37°C for 1 hour. A dot blot assay to detect cleaved tau was then performed on the samples as described above.
Lipid extractions analysis was conducted as previously described
Apoptotic cells can be detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Using slide-mounted tissue sections, DNA fragmentation was detected using a TUNEL labeling apoptosis detection kit (Chemicon) according to the manufacturer's instruction. As a positive control, a brain section from a patient who had died from AD was included.
To quantify immunohistochemical staining, numbers of labeled cells were counted in at least three different sites from each frontal cortex section, using a light microscope at 20× magnification. For each section, values from the multiple sites were averaged to generate a single mean value per dog. Average and standard error of the mean (n = 5–6 per group) were then generated for each treatment group. Dot blots were quantified by using Image J software and the intensity of the dots are expressed as optical density (OD). Statistical analysis on immunopositive cell counts, dot blot intensity, and ceramide levels consisted of one-way ANOVA across the 4 treatment groups to detect a main effect of intervention, followed by post-hoc bonferroni t-test (p<0.05) if a significant main effect was detected.