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The aged canine is a higher animal model that naturally accumulates β-amyloid (Aβ) and shows age-related cognitive decline. However, profiles of Aβ accumulation in different species (40 vs. 42), its assembly states, and Aβ precursor protein (APP) processing as a function of age remain unexplored. In this study, we show that Aβ increases progressively with age as detected in extracellular plaques and biochemically extractable Aβ40 and Aβ42 species. Soluble oligomeric forms of the peptide, with specific increases in an Aβ oligomer migrating at 56kDa, also increase with age. Changes in APP processing could potentially explain why Aβ accumulates, and we show age-related shifts towards decreased total APP protein and non-amyloidogenic (α-secretase) processing coupled with increased amyloidogenic (β-secretase) cleavage of APP. Importantly, we describe Aβ pathology in the cingulate and temporal cortex and provide a description of oligomeric Aβ across the canine lifespan. Our findings are in line with observations in the human brain, suggesting that canines are a valuable higher animal model for the study of Aβ pathogenesis.

Using APPNLh/APPNLh x PS-1P246L/PS-1P246L human double knock-in (APP/PS-1) mice, we examined whether phosphatidylserine (PtdSer) asymmetry is significantly altered in brain of this familial Alzheimer disease mouse model in an age-dependent manner as a result of oxidative stress, toxic Aβ(1-42) oligomer production, and/or apoptosis. Annexin V (AV) and NBD-PS fluorescence in synaptosomes of wild-type (WT) and APP/PS-1 mice were used to determine PtdSer exposure with age, while Mg2+ ATPase activity was determined to correlate PtdSer asymmetry changes with PtdSer translocase, flippase, activity. AV and NBD-PS results demonstrated significant PtdSer exposure beginning at 9 months compared to 1-month-old WT controls for both assays, a trend that was exacerbated in synaptosomes of APP/PS-1 mice. Decreasing Mg2+ ATPase activity confirms that the age-related loss of PtdSer asymmetry is likely due to loss of flippase activity, more prominent in APP/PS-1 brain. Two-site sandwich ELISA on SDS- and FA-soluble APP/PS-1 brain fractions were conducted to correlate Aβ(1–40) and Aβ(1–42) levels with age-related trends determined from the AV, NBD-PS, and Mg2+ ATPase assays. ELISA revealed a significant increase in both SDS- and FA-soluble Aβ(1–40) and Aβ(1–42) with age, consistent with PtdSer and flippase assay trends. Lastly, because PtdSer exposure is affected by pro-apoptotic caspase-3, levels of both latent and active forms were measured. Western blotting results demonstrated an increase in both active fragments of caspase-3 with age, while levels of pro-caspase-3 decrease. These results are discussed with relevance to loss of lipid asymmetry and consequent neurotoxicity in brain of subjects with Alzheimer disease.

Region specific declines in the cerebral glucose metabolism are an early and progressive feature of Alzheimer's disease (AD). Such declines occur pre-symptomatically and offer a potential point of intervention in developing AD therapeutics. Medium chain triglycerides (MCTs), which are rapidly converted to ketone bodies, were tested for their ability to provide an alternate energy source to neurons suffering from compromised glucose metabolism. The present study determined the short-term effects of ketosis in aged dogs, a natural model of amyloidosis. The animals were administered a 2 g/kg/day dose of MCTs for 2 months. Mitochondrial function and oxidative damage assays were then conducted on the frontal and parietal lobes. Amyloid-beta (Abeta), amyloid precursor protein (APP) processing and beta-site APP cleaving enzyme (BACE1) assays were conducted on the frontal, parietal and occipital lobes. Aged dogs receiving MCTs, as compared to age-matched controls, showed dramatically improved mitochondrial function, as evidenced by increased active respiration rates. This effect was most prominent in the parietal lobe. The improved mitochondrial function may have been due to a decrease in oxidative damage, which was limited to the mitochondrial fraction. Steady-state APP levels were also decreased in the parietal lobe after short-term MCT administration. Finally, there was a trend towards a decrease in total Abeta levels in the parietal lobe. BACE1 levels remained unchanged. Combined, these findings suggest that short-term MCT administration improves energy metabolism and decreases APP levels in the aged dog brain.

Memory decline in human aging and dementia is linked to dysfunction of the cholinergic system. Aging dogs demonstrate cognitive impairments and neuropathology that models human aging and dementia. This paper reviews recent evidence suggesting cholinergic involvement in canine cognitive aging based on studies with the anti-cholinergic drug, scopolamine, and a novel acetylcholinesterase inhibitor, phenserine. In particular, we examine: (1) the cognitive specificity of scopolamine's impairment in dogs, (2) the effect of age on scopolamine impairment and (3) the effect of phenserine on cognitive performance in dogs. Our findings indicate that working memory performance is disrupted by scopolamine at doses that do not disrupt non-cognitive behavior or long-term, semantic-like, memory, as indicated by performance of previously learned discriminations. This pattern of deficits is also seen in human and canine aging. We demonstrate that aged dogs are more sensitive to the impairing effects of scopolamine than young dogs, suggesting a decrease in cholinergic tone with increasing age. Dogs receiving phenserine demonstrate improved learning and memory compared to placebo controls. Our findings suggest that cholinergic decline could result in memory impairment, but that the memory impairment may be secondary to deficits in attention and/or encoding of new information. Together, these results suggest that the canine cholinergic system declines with age and that the aged dog is a unique model for screening therapeutics and for examining the relationship between amyloid pathology and cholinergic dysfunction in age-dependent cognitive decline.

β-Amyloid (Aβ), a small, fibrillogenic peptide, is known to play an important role in the pathogenesis of Alzheimer's disease (AD) in the brain. In addition, Aβ accumulates in skeletal muscle cells in individuals with sporadic inclusion body myositis (sIBM), an age-related muscle disease. Because of the socioeconomic burden associated with age-related diseases, particularly AD, there has been considerable emphasis on studying potential therapeutic strategies. The high-fat, low carbohydrate ketogenic diet has been used extensively to treat refractory childhood epilepsy and has been studied as a potential treatment for other neurological diseases, including Parkinson's disease and AD. In this study, we fed young APP/PS1 knock-in mice, which have a whole body knock-in of AD-related genes, a ketogenic diet and determined the effect on Aβ levels in the brain and skeletal muscle, as well motor performance and oxidative stress. Aβ and its precursor, the β-C-terminal fragment of amyloid precursor protein (CTFβ), were unchanged overall in both the brain and quadriceps after 1 month on the ketogenic diet, and there was no effect on nitrotyrosine, a product of oxidative stress. The ketogenic diet improved performance on the Rota-rod apparatus (p=0.007), however. These data indicate that the ketogenic diet may have some efficacy in the treatment of both neurologic and muscle diseases though the underlying mechanisms do not involve amelioration of Aβ pathology.

Sporadic inclusion body myositis (sIBM) is a common age-related inflammatory myopathy characterized by the presence of intracellular inclusions that contain the amyloid-beta (Abeta) peptide, a derivative of the amyloid precursor protein (APP). Abeta is believed to cause Alzheimer's disease (AD), suggesting that a link may exist between the two diseases. If AD and sIBM are linked, then treatments that lower Abeta in brain may prove useful for sIBM. To test this hypothesis, transgenic mice that overexpress APP in skeletal muscle were treated for 6 months with a variety of nonsteroidal anti-inflammatory drugs (NSAIDs; naproxen, ibuprofen, carprofen or R-flurbiprofen), a subset of which reduce Abeta in brain and cultured cells. Only ibuprofen lowered Abeta in muscle, and this was not accompanied by corresponding improvements in phenotype. These results indicate that the effects of NSAIDs in the brain may be different from other tissues and that Abeta alone cannot account for skeletal muscle dysfunction in these mice.

Neprilysin (NEP) is a zinc metallopeptidase that efficiently degrades the amyloid beta (Abeta) peptides believed to be involved in the etiology of Alzheimer disease (AD). The focus of this study was to develop a new and tractable therapeutic approach for treating AD using NEP gene therapy. We have introduced adeno-associated virus (AAV) expressing the mouse NEP gene into the hindlimb muscle of 6-month-old human amyloid precursor protein (hAPP) (3X-Tg-AD) mice, an age which correlates with early stage AD. Overexpression of NEP in muscle decreased brain soluble Abeta peptide levels by approximately 60% and decreased amyloid deposits by approximately 50%, with no apparent adverse effects. Expression of NEP on muscle did not affect the levels of a number of other physiological peptides known to be in vitro substrates. These findings demonstrate that peripheral expression of NEP and likely other peptidases represents an alternative to direct administration into brain and illustrates the potential for using NEP expression in muscle for the prevention and treatment of AD.

A chronic high fat Western diet (WD) promotes a variety of morbidity factors although experimental evidence for short-term WD mediating brain dysfunction remains to be elucidated. The amyloid precursor protein and presenilin-1 (APP x PS1) knock-in mouse model has been demonstrated to recapitulate some key features of Alzheimer's disease pathology, including amyloid-beta (Abeta) pathogenesis. In this study, we placed 1-month-old APP x PS1 mice and non-transgenic littermates on a WD for 4 weeks. The WD resulted in a significant elevation in protein oxidation and lipid peroxidation in the brain of APP x PS1 mice relative to non-transgenic littermates, which occurred in the absence of increased Abeta levels. Altered adipokine levels were also observed in APP x PS1 mice placed on a short-term WD, relative to non-transgenic littermates. Taken together, these data indicate that short-term WD is sufficient to selectively promote cerebral oxidative stress and metabolic disturbances in APP x PS1 knock-in mice, with increased oxidative stress preceding alterations in Abeta. These data have important implications for understanding how WD may potentially contribute to brain dysfunction and the development of neurodegenerative disorders such as Alzheimer's disease.

Visuospatial learning and memory impairments are an early marker for age-related cognitive decline and Alzheimer's disease. Similar to humans, aged dogs show visuospatial learning and memory deficits (). One hundred and nine beagle dogs ranging between 0.25 and 11.99 years were tested on a visuospatial delayed non-matching to position (DNMP) task to better characterize the progression of visuospatial deficits in the dog. Age predicted 48.2% of the variability in learning the DNMP, with dogs ranging from 1 to 11.99 years generally making more errors with increasing age. By contrast, puppies (1 year) likely were showing developmental deficits, possibly due to an immature prefrontal cortex. Mild visuospatial deficits were detected by 6 years, which precedes the typical onset of amyloid-beta (Abeta) accumulation in the dog brain by two years, and can serve as an early marker for cognitive decline in the dog. These findings suggest that (1) age-related changes in visuospatial function in the dog models that seen in humans, further validating the dog as a model for human aging and dementia; and (2) other mechanisms, such as oxidative stress, soluble Abeta oligomers or cholinergic deficits, are likely contributing to the early impairment.

For the past 15 years we have investigated the aged beagle dog as a model for human aging and dementia. We have shown that dogs develop cognitive deficits and neuropathology seen in human aging and dementia. These similarities increase the likelihood that the model will be able to accurately predict the efficacy of Alzheimer's disease (AD) treatments as well as detect therapeutics with limited or no efficacy. Better predictive validity of cognitive-enhancing therapeutics (CETs) could lead to enormous cost savings by reducing the number of failed human clinical trials and also may reduce the likelihood of negative outcomes such as those recently observed in the AN-1792 clinical trials. The current review assesses the pharmacological validity of the canine model of human aging and dementia. We tested the efficacy of (1) CP-118,954 and phenserine, two acetylcholinesterase inhibitors, (2) an ampakine, (3) selegiline hydrochloride, two drugs that have failed human AD trials, and (4) adrafinil, a putative CET. Our research demonstrates that dogs not only develop isomorphic changes in human cognition and brain pathology, but also accurately predict the efficacy of known AD treatments and the absence or limited efficacy of treatments that failed clinical trials. These findings collectively support the utilization of the dog model as a preclinical screen for identifying novel CETs for both age-associated memory disorder and dementia.