Leptin regulates amyloid ß production via the γ-secretase complex.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, affecting an estimated 5.3million people in the United States. While many factors likely contribute to AD progression, it is widely accepted that AD is driven by the accumulation of ß-amyloid (Aß), a small, fibrillogenic peptide generated by the sequential proteolysis of the amyloid precursor protein by the ß- and γ-secretases. Though the underlying causes of Aß accumulation in sporadic AD are myriad, it is clear that lifestyle and overall health play a significant role. The adipocyte-derived hormone leptin has varied systemic affects, including neuropeptide release and neuroprotection. A recent study by Lieb et al. (2009) showed that individuals with low plasma leptin levels are at greater risk of developing AD, through unknown mechanisms. In this report, we show that plasma leptin is a strong negative predictor of Aß levels in the mouse brain, supporting a protective role for the hormone in AD onset. We also show that the inhibition of Aß accumulation is due to the downregulation of transcription of the γ-secretase components. On the other hand, ß-secretase expression is either unchanged (BACE1) or increased (BACE2). Finally, we show that only presenilin 1 (PS1) is negatively correlated with plasma leptin at the protein level (p<0.0001). These data are intriguing and may highlight a role for leptin in regulating the onset of amyloid pathology and AD.

Pittsburgh compound B and the postmortem diagnosis of Alzheimer disease.

OBJECTIVE: Deposition of the amyloid-ß (Aß) peptide in neuritic plaques is a requirement for the diagnosis of Alzheimer disease (AD). Although the continued development of in vivo imaging agents such as Pittsburgh compound B (PiB) is promising, the diagnosis of AD is still challenging. This can be partially attributed to our lack of a detailed understanding of the interrelationship between the various pools and species of Aß and other common indices of AD pathology. We hypothesized that recent advances in our ability to accurately measure Aß postmortem (for example, using PiB), could form the basis of a simple means to deliver an accurate AD diagnosis. METHODS: We conducted a comprehensive analysis of the amount of Aß40 and Aß42 in increasingly insoluble fractions, oligomeric Aß, and fibrillar Aß (as defined by PiB binding), as well as plaques (diffuse and neuritic), and neurofibrillary tangles in autopsy specimens from age-matched, cognitively normal controls (n = 23) and AD (n = 22) cases, across multiple brain regions. RESULTS: Both PiB binding and the amount of sodium dodecyl sulfate (SDS)-soluble Aß were able to predict disease status; however, SDS-soluble Aß was a better measure. Oligomeric Aß was not a predictor of disease status. PiB binding was strongly related to plaque count, although diffuse plaques were a stronger correlate than neuritic plaques. INTERPRETATION: Although postmortem PiB binding was somewhat useful in distinguishing AD from control cases, SDS-soluble Aß measured by standard immunoassay was substantially better. These findings have important implications for the development of imaging-based biomarkers of AD.

Aß aggregation profiles and shifts in APP processing favor amyloidogenesis in canines.

The aged canine is a higher animal model that naturally accumulates ß-amyloid (Aß) and shows age-related cognitive decline. However, profiles of Aß accumulation in different species (40 vs. 42), its assembly states, and Aß precursor protein (APP) processing as a function of age remain unexplored. In this study, we show that Aß increases progressively with age as detected in extracellular plaques and biochemically extractable Aß40 and Aß42 species. Soluble oligomeric forms of the peptide, with specific increases in an Aß oligomer migrating at 56 kDa, also increase with age. Changes in APP processing could potentially explain why Aß accumulates, and we show age-related shifts toward decreased total APP protein and nonamyloidogenic (α-secretase) processing coupled with increased amyloidogenic (ß-secretase) cleavage of APP. Importantly, we describe Aß pathology in the cingulate and temporal cortex and provide a description of oligomeric Aß 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 Aß pathogenesis.

RNA oxidation adducts 8-OHG and 8-OHA change with Aß42 levels in late-stage Alzheimer's disease.

While research supports amyloid-ß (Aß) as the etiologic agent of Alzheimer's disease (AD), the mechanism of action remains unclear. Evidence indicates that adducts of RNA caused by oxidation also represent an early phenomenon in AD. It is currently unknown what type of influence these two observations have on each other, if any. We quantified five RNA adducts by gas chromatography/mass spectroscopy across five brain regions from AD cases and age-matched controls. We then used a reductive directed analysis to compare the RNA adducts to common indices of AD neuropathology and various pools of Aß. Using data from four disease-affected brain regions (Brodmann's Area 9, hippocampus, inferior parietal lobule, and the superior and middle temporal gyri), we found that the RNA adduct 8-hydroxyguanine (8-OHG) decreased, while 8-hydroxyadenine (8-OHA) increased in AD. The cerebellum, which is generally spared in AD, did not show disease related changes, and no RNA adducts correlated with the number of plaques or tangles. Multiple regression analysis revealed that SDS-soluble Aß(42) was the best predictor of changes in 8-OHG, while formic acid-soluble Aß(42) was the best predictor of changes in 8-OHA. This study indicates that although there is a connection between AD related neuropathology and RNA oxidation, this relationship is not straightforward.

Purified high molecular weight synthetic Aß(1-42) and biological Aß oligomers are equipotent in rapidly inducing MTT formazan exocytosis.

Synthetic soluble Aß oligomers are often used as a surrogate for biologic material in a number of model systems. We compared the activity of Aß oligomers (synthetic and cell culture media derived) on the human SH-SY5Y neuroblastoma and C2C12 mouse myoblast cell lines in a novel, modified MTT assay. Separating oligomers from monomeric peptide by size exclusion chromatography produced effects at peptide concentrations approaching physiologic levels (10-100 nM). Purified oligomers, but not monomers or fibrils, elicited an increase of a detergent-insoluble form of MTT formazan within 2h as opposed to a control toxin (H(2)O(2)). This effect was comparable for biological and synthetic peptide in both cell types. Monomeric Aß attenuated the effect of soluble oligomers. This study suggests that the activities of biological and synthetic oligomers are indistinguishable during early stages of Aß oligomer-cell interaction.

Changes in cognition and amyloid-ß processing with long term cholesterol reduction using atorvastatin in aged dogs.

Human studies suggest either a protective role or no benefit of statins against the development of Alzheimer's disease (AD). We tested the hypothesis that statin-mediated cholesterol reduction in aged dogs, which have cognitive impairments and amyloid-ß (Aß) pathology, would improve cognition and reduce neuropathology. In a study of 12 animals, we treated dogs with 80 mg/day of atorvastatin for 14.5 months. We did not observe improvements in discrimination learning; however, there were transient impairments in reversal learning, suggesting frontal dysfunction. Spatial memory function did not change with treatment. Peripheral levels of cholesterol, LDLs, triglycerides, and HDL were significantly reduced in treated dogs. Aß in cerebrospinal fluid and brain remained unaffected. However, ß-secretase-1 (BACE1) protein levels and activity decreased and correlated with reduced brain cholesterol. Finally, lipidomic analysis revealed a significant decrease in the ratio of omega-6 to omega-3 essential fatty in temporal cortex of treated aged dogs. Aged beagles are a unique model that may provide novel insights and translational data that can predict outcomes of statin use in human clinical trials. Treatment with atorvastatin may be beneficial for brain aging by reducing BACE1 protein and omega6:omega3 ratio, however, the potential adverse cognitive outcomes reported here should be more carefully explored given their relevance to human clinical outcomes.

Neuron specific toxicity of oligomeric amyloid-ß: role for JUN-kinase and oxidative stress.

Recent studies have demonstrated a potential role for oligomeric forms of amyloid-ß (Aß) in the pathogenesis of Alzheimer's disease (AD), although it remains unclear which aspects of AD may be mediated by oligomeric Aß. In the present study, we found that primary cultures of rat cortical neurons exhibit a dose-dependent increase in cell death following Aß oligomer administration, while primary cultures of astrocytes exhibited no overt toxicity with even the highest concentrations of oligomer treatment. Neither cell type exhibited toxicity when treated by equal concentrations of monomeric Aß. The neuron death induced by oligomer treatment was associated with an increase in reactive oxygen species (ROS), altered expression of mitochondrial fission and fusion proteins, and JUN kinase activation. Pharmacological inhibition of JUN kinase ameliorated oligomeric Aß toxicity in neurons. These data indicate that oligomeric Aß is sufficient to selectively induce toxicity in neurons, but not astrocytes, with neuron death occurring in a JUN kinase-dependent manner. Additionally, these observations implicate a role for oligomeric Aß as a contributor to neuronal oxidative stress and mitochondrial disturbances in AD.

APP(DeltaNL695) expression in murine tissue downregulates CNBP expression.

The cellular nucleic acid binding protein (CNBP) is a ubiquitously expressed protein involved in regulation of transcription and translation. CNBP, and its encoding gene ZNF9, have been shown to be involved in type 2 myotonic dystrophy. Both Alzheimer's disease (AD) and sporadic inclusion body myositis (sIBM) are age-related degenerative diseases associated with the accumulation of beta-amyloid. Overexpression of amyloid precursor protein (APP) in mice has been used to generate models of both diseases. We show here that overexpression of APP in skeletal muscle from a mouse model of sIBM reduces the expression of CNBP significantly. We examined CNBP expression in a brain-specific APP-overexpressing strain, and a whole body APP knock-in strain, and found that there was a reduction in CNBP expression in tissue expressing APP(Swe). We conclude that expression of APP(Swe) in murine tissue induces a decrease in CNBP expression. This effect does not appear to be due to alterations in CNBP transcription. APP(Swe) expression may provide a tool for the study of CNBP regulation and clues to the roles of both proteins in disease.

Effects of nonsteroidal anti-inflammatory drugs on amyloid-beta pathology in mouse skeletal muscle.

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.

NOX activity is increased in mild cognitive impairment.

This study was undertaken to investigate the profile of NADPH oxidase (NOX) in the clinical progression of Alzheimer's disease (AD). Specifically, NOX activity and expression of the regulatory subunit p47phox and the catalytic subunit gp91phox was evaluated in affected (superior and middle temporal gyri) and unaffected (cerebellum) brain regions from a longitudinally followed group of patients. This group included both control and late-stage AD subjects, and also subjects with preclinical AD and with amnestic mild cognitive impairment (MCI) to evaluate the profile of NOX in the earliest stages of dementia. Data show significant elevations in NOX activity and expression in the temporal gyri of MCI patients as compared with controls, but not in preclinical or late-stage AD samples, and not in the cerebellum. Immunohistochemical evaluations of NOX expression indicate that whereas microglia express high levels of gp91phox, moderate levels of gp91phox also are expressed in neurons. Finally, in vitro experiments showed that NOX inhibition blunted the ability of oligomeric amyloid beta peptides to injure cultured neurons. Collectively, these data show that NOX expression and activity are upregulated specifically in a vulnerable brain region of MCI patients, and suggest that increases in NOX-associated redox pathways in neurons might participate in the early pathogenesis of AD.

Cognitive decline in Alzheimer's disease is associated with selective changes in calcineurin/NFAT signaling.

Upon activation by calcineurin, the nuclear factor of activated T-cells (NFAT) translocates to the nucleus and guides the transcription of numerous molecules involved in inflammation and Ca(2+) dysregulation, both of which are prominent features of Alzheimer's disease (AD). However, NFAT signaling in AD remains relatively uninvestigated. Using isolated cytosolic and nuclear fractions prepared from rapid-autopsy postmortem human brain tissue, we show that NFATs 1 and 3 shifted to nuclear compartments in the hippocampus at different stages of neuropathology and cognitive decline, whereas NFAT2 remained unchanged. NFAT1 exhibited greater association with isolated nuclear fractions in subjects with mild cognitive impairment (MCI), whereas NFAT3 showed a strong nuclear bias in subjects with severe dementia and AD. Similar to NFAT1, calcineurin-Aalpha also exhibited a nuclear bias in the early stages of cognitive decline. But, unlike NFAT1 and similar to NFAT3, the nuclear bias for calcineurin became more pronounced as cognition worsened. Changes in calcineurin/NFAT3 were directly correlated to soluble amyloid-beta (Abeta((1-42))) levels in postmortem hippocampus, and oligomeric Abeta, in particular, robustly stimulated NFAT activation in primary rat astrocyte cultures. Oligomeric Abeta also caused a significant reduction in excitatory amino acid transporter 2 (EAAT2) protein levels in astrocyte cultures, which was blocked by NFAT inhibition. Moreover, inhibition of astrocytic NFAT activity in mixed cultures ameliorated Abeta-dependent elevations in glutamate and neuronal death. The results suggest that NFAT signaling is selectively altered in AD and may play an important role in driving Abeta-mediated neurodegeneration.

Effects of short-term Western diet on cerebral oxidative stress and diabetes related factors in APP x PS1 knock-in mice.

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.