Lorenzo M. Refolo

Mild hypercholesterolemia is an early risk factor for the development of Alzheimer amyloid pathology.

Background: Epidemiologic and experimental data suggest that cholesterol may play a role in the pathogenesis of AD. Modulation of cholesterolemia in transgenic animal models of AD strongly alters amyloid pathology. Objective: To determine whether a relationship exists between amyloid deposition and total cholesterolemia (TC) in the human brain. Methods: The authors reviewed autopsy cases of patients older than 40 years and correlated cholesterolemia and presence or absence of amyloid deposition (amyloid positive vs amyloid negative subjects) and cholesterolemia and amyloid load. Amyloid load in human brains was measured by immunohistochemistry and image analysis. To remove the effect of apoE isoforms on cholesterol levels, cases were genotyped and duplicate analyses were performed on apoE3/3 subjects. Results: Cholesterolemia correlates with presence of amyloid deposition in the youngest subjects (40 to 55 years) with early amyloid deposition (diffuse type of senile plaques) (p = 0.000 for all apoE isoforms; p = 0.009 for apoE3/3 subjects). In this group, increases in cholesterolemia from 181 to 200 almost tripled the odds for developing amyloid, independent of apoE isoform. A logistic regression model showed consistent results (McFadden ρ2 = 0.445). The difference in mean TC between subjects with and without amyloid disappeared as the age of the sample increased (>55 years: p = 0.491), possibly reflecting the effect of cardiovascular deaths among other possibilities. TC and amyloid load were not linearly correlated, indicating that there are additional factors involved in amyloid accumulation. Conclusions: Serum hypercholesterolemia may be an early risk factor for the development of AD amyloid pathology.

Melatonin increases survival and inhibits oxidative and amyloid pathology in a transgenic model of Alzheimer's disease

Increased levels of a 40–42 amino‐acid peptide called the amyloid β protein (Aβ) and evidence of oxidative damage are early neuropathological markers of Alzheimers disease (AD). Previous investigations have demonstrated that melatonin is decreased during the aging process and that patients with AD have more profound reductions of this hormone. It has also been recently shown that melatonin protects neuronal cells from Aβ‐mediated oxidative damage and inhibits the formation of amyloid fibrils in vitro. However, a direct relationship between melatonin and the biochemical pathology of AD had not been demonstrated. We used a transgenic mouse model of Alzheimers amyloidosis and monitored over time the effects of administering melatonin on brain levels of Aβ, abnormal protein nitration, and survival of the mice. We report here that administration of melatonin partially inhibited the expected time‐dependent elevation of β‐amyloid, reduced abnormal nitration of proteins, and increased survival in the treated transgenic mice. These findings may bear relevance to the pathogenesis and therapy of AD.

Statin therapy for Alzheimer's disease: will it work?

Disease-modifying therapies are being developed for Alzheimer’s disease (AD). These are expected to slow the clinical progression of the disease or delay its onset. Cerebral accumulation of amyloid β (Aβ) peptides is an early and perhaps necessary event for establishing AD pathology. Consequently therapies aimed at attenuating brain amyloidosis are expected to be disease modifying. Based on the epidemiological evidence pointing to a link between cholesterol metabolism and AD and the numerous laboratory studies implicating cholesterol in the process of Aβ production and accumulation, it is now believed that cholesterol-lowering therapies will be of value as disease modifying agents. Several epidemiological studies revealed that statin use for the treatment of coronary arterial disease is associated with a decreased prevalence or a decreased risk of developing AD. These observations require both preclinical and clinical validation. The former involves testing statins in one or more animal models of AD in order to establish which disease features are affected by statin treatment, the relative efficacy with which different statins modify these features and the mechanism(s) by which statins affect AD phenotypes. The latter requires prospective, randomized, placebo controlled trials to evaluate the effect of statin treatment on cognitive and AD biomarker outcomes. We have initiated a study aimed at determining the effects of atorvastatin (LipitorR), a statin with the largest US market share, on brain Aβ deposition in the PSAPP transgenic mouse model of Alzheimer’s amyloidosis. Our results indicate that Lipitor treatment markedly attenuates Aβ deposition in this animal model.

Hyperhomocysteinemic Alzheimer's mouse model of amyloidosis shows increased brain amyloid β peptide levels

Recent epidemiological and clinical data suggest that elevated serum homocysteine levels may increase the risk of developing Alzheimers disease (AD), but the underlying mechanisms are unknown. We tested the hypothesis that high serum homocysteine concentration may increase amyloid beta-peptide (Abeta) levels in the brain and could therefore accelerate AD neuropathology. For this purpose, we mated a hyperhomocysteinemic CBS(tm1Unc) mouse carrying a heterozygous dominant mutation in cystathionine-beta-synthase (CBS*) with the APP*/PS1* mouse model of brain amyloidosis. The APP*/PS1*/CBS* mice showed significant elevations of serum homocysteine levels compared to the double transgenic APP*/PS1* model of amyloidosis. Results showed that female (but not male) APP*/PS1*/CBS* mice exhibited significant elevations of Abeta40 and Abeta42 levels in the brain. Correlations between homocysteine levels in serum and brain Abeta levels were statistically significant. No increases in beta secretase activity or evidence of neuronal cell loss in the hyperhomocysteinemic mice were found. The causes of neuronal dysfunction and degeneration in AD are not fully understood, but increased production of Abeta seems to be of major importance. By unveiling a link between homocysteine and Abeta levels, these findings advance our understanding on the mechanisms involved in hyperhomocysteinemia as a risk factor for AD.

Atorvastatin-induced activation of Alzheimer's α secretase is resistant to standard inhibitors of protein phosphorylation-regulated ectodomain shedding

Studies of metabolism of the Alzheimer amyloid precursor protein (APP) have focused much recent attention on the biology of juxta‐ and intra‐membranous proteases. Release or ‘shedding’ of the large APP ectodomain can occur via one of two competing pathways, the α‐ and β‐secretase pathways, that are distinguished both by subcellular site of proteolysis and by site of cleavage within APP. The α‐secretase pathway cleaves within the amyloidogenic Aβ domain of APP, precluding the formation of toxic amyloid aggregates. The relative utilization of the α‐ and β‐secretase pathways is controlled by the activation of certain protein phosphorylation signal transduction pathways including protein kinase C (PKC) and extracellular signal regulated protein kinase [ERK/mitogen‐activated protein kinase (MAP kinase)], although the relevant substrates for phosphorylation remain obscure. Because of their apparent ability to decrease the risk for Alzheimer disease, the effects of statins (HMG CoA reductase inhibitors) on APP metabolism were studied. Statin treatment induced an APP processing phenocopy of PKC or ERK activation, raising the possibility that statin effects on APP processing might involve protein phosphorylation. In cultured neuroblastoma cells transfected with human Swedish mutant APP, atorvastatin stimulated the release of α‐secretase‐released, soluble APP (sAPPα). However, statin‐induced stimulation of sAPPα release was not antagonized by inhibitors of either PKC or ERK, or by the co‐expression of a dominant negative isoform of ERK (dnERK), indicating that PKC and ERK do not play key roles in mediating the effect of atorvastatin on sAPPα secretion. These results suggest that statins may regulate α‐secretase activity either by altering the biophysical properties of plasma membranes or by modulating the function of as‐yet unidentified protein kinases that respond to either cholesterol or to some intermediate in the cholesterol metabolic pathway. A ‘phospho‐proteomic’ analysis of N2a cells with and without statin treatment was performed, revealing changes in the phosphorylation state of several protein kinases plausibly related to APP processing. A systematic evaluation of the possible role of these protein kinases in statin‐regulated APP ectodomain shedding is underway.

Cholesterol, oxidative stress, and Alzheimer’s disease: expanding the horizons of pathogenesis

Recent epidemiological, clinical, and experimental data suggest that cholesterol may play a role in Alzheimers disease (AD). We have recently shown that cholesterolemia has a profound effect in the development and modulation of amyloid pathology in a transgenic model of AD. This review summarizes recent advancements in our understanding of the potential role of cholesterol and the amyloid beta protein in initiating the generation of free radicals and points out their role in a chain of events that causes damage of essential macromolecules in the central nervous system and culminates in neuronal dysfunction and loss. Experimental data links cholesterol and oxidative stress with some neurodegenerative aspects of AD.

Changes in apolipoprotein E expression in response to dietary and pharmacological modulation of cholesterol.

Apolipoprotein E (ApoE) influences the risk of late onset Alzheimer’s disease (AD) in an isoform-dependent manner, such that the presence of the apoE ε4 allele increases the risk of AD while the presence of the apoE ε2 allele appears to be protective. Although a number of ApoE functions are isoform dependent and may underlie the “risk factor” activity of AD, its ability to bind amyloid β peptides and influence their clearance and/or deposition has gained strong experimental support. Evidence suggests that in addition to genotype, increased ApoE transcription can contribute to AD risk. There is growing evidence in support of the hypothesis that disrupted cholesterol metabolism is an early risk factor for AD. Studies in animal models have shown that chronic changes in cholesterol metabolism associate with changes in brain Aβ accumulation, a process instrumental for establishing AD pathology. ApoE mediates cholesterol homeostasis in the body and is a major lipid carrier in brain. As such, its expression in the periphery and in brain changes in response to changes in cholesterol metabolism. Here, we used a transgenic mouse model of Alzheimer’s amyloidosis to examine whether the diet-induced or pharmacologically induced changes in plasma cholesterol that result in altered brain amyloidosis also affect ApoE content in liver and in brain. We found that chronic changes in total cholesterol in plasma lead to changes in ApoE mRNA levels in brain. We also found that cholesterol loading of primary glial cells increases cellular and secreted ApoE levels and that long-term treatment of astrocytes and microglia with statins leads to a decrease in the cellular and/or secreted ApoE. These observations suggest that disrupted cholesterol metabolism may increase the risk of developing AD in part due to the effect of cholesterol on brain ApoE expression.

Amyloid precursor protein compartmentalization restricts β-amyloid production

Alzheimers disease (AD) is defined by deposits of the 42-residue amyloid-beta peptide (Abeta42) in the brain. Abeta42 is a minor metabolite of the amyloid precursor protein (APP), but its relative levels are increased by mutations on APP and presenilins 1 and 2 linked to familial AD. beta-secretase (BACE-1), an aspartyl protease, cleaves approx 10% of the APP in neuronal cells on the N-terminal side of Abeta to produce the C-terminal fragment (CTFbeta), which is cleaved by gamma-secretase to produce mostly Abeta of 40 residues (90%) and approx10% Abeta42. A third enzyme, alpha-secretase, cleaves APP after Abeta16 to secrete sAPPalpha and CTFalpha, the major metabolites of APP. Moreover, previous studies have demonstrated that phorbol esters stimulate processing of APP by alpha-secretase. Because alpha-secretase and BACE-1 cleave APP within the secretory pathway, it is likely that the two enzymes compete for the APP substrate. This type of competition can explain the failure to saturate the minor BACE-1 pathway by overexpressing APP in the cell. In this study, we demonstrate that inhibition of constitutive alpha-secretase processing in a human neuroblastoma cell line does not increase the yield of Abeta, suggesting that the APP substrate targeted for alpha-secretase processing is not diverted to the BACE-1 pathway. However, when phorbol ester-induced alpha-secretase was similarly inhibited, we detected an increase in BACE-1 processing and AB yield. We explain these results compartmentalization of BACE-1 and alpha-secretase with processing depending on sorting of APP to the two compartments. The simplest explanation for the detection of competition between the two pathways upon phorbol ester stimulation is the partial failure of this compartmentalization by phorbol ester-induced release of secretory vesicles.Alzheimer’s disease (AD) is defined by deposits of the 42-residue amyloid-β peptide (Aβ42) in the brain. Aβ42 is a minor metabolite of the amyloid precursor protein (APP), but its relative levels are increased by mutations on APP and presenilins 1 and 2 linked to familial AD. β-secretase (BACE-1), an aspartyl protease, cleaves approx 10% of the APP in neuronal cells on the N-terminal side of Aβ to produce the C-terminal fragment (CTFβ), which is cleaved by γ-secretase to produce mostly Aβ of 40 residues (90%) and approx10% Aβ42. A third enzyme, α-secretase, cleaves APP after Aβ16 to secrete sAPPα and CTFα, the major metabolites of APP. Moreover, previous studies have demonstrated that phorbol esters stimulate processing of APP by α-secretase. Because α-secretase and BACE-1 cleave APP within the secretory pathway, it is likely that the two enzymes compete for the APP substrate. This type of competition can explain the failure to saturate the minor BACE-1 pathway by overexpressing APP in the cell. In this study, we demonstrate that inhibition of constitutive α-secretase processing in a human neuroblastoma cell line does not increase the yield of Aβ, suggesting that the APP substrate targeted for α-secretase processing is not diverted to the BACE-1 pathway. However, when phorbol ester-induced α-secretase was similarly inhibited, we detected an increase in BACE-1 processing and AB yield. We explain these results compartmentalization of BACE-1 and α-secretase with processing depending on sorting of APP to the two compartments. The simplest explanation for the detection of competition between the two pathways upon phorbol ester stimulation is the partial failure of this compartmentalization by phorbol ester-induced release of secretory vesicles.

Common Alzheimer’s Disease Research Ontology: National Institute on Aging and Alzheimer’s Association Collaborative Project

Alzheimers disease is recognized as a public health crisis worldwide. As public and private funding agencies around the world enhance and expand their support of Alzheimers disease research, there is an urgent need to coordinate funding strategies and leverage resources to maximize the impact on public health and avoid duplication of effort and inefficiency. Such coordination requires a comprehensive assessment of the current landscape of Alzheimers disease research in the United States and internationally. To this end, the National Institute on Aging at the National Institutes of Health and the Alzheimers Association developed the Common Alzheimers Disease Research Ontology (CADRO) as a dynamic portfolio analysis tool that can be used by funding agencies worldwide for strategic planning and coordination.

Apolipoprotein E4 as a target for developing new therapeutics for Alzheimer’s disease

The identification of factors that influence the onset or progression of the sporadic form of Alzheimer’s disease (AD) is a key step toward understanding its mechanism(s) and developing successful rational therapies. The apoE genotype has been identified as a powerful risk factor for AD that may account for as much as 50% of the sporadic form of the disease. As the major risk factor for late-onset AD, apolipoprotein E4 (apoE4) should be considered a good target for AD drug discovery. However, despite knowing for over a decade that apoE4 is detrimental to the disease process, we still remain uncertain about the molecular mechanisms subserving the risk-factor activity of apoE4. This, coupled with the fact that we know relatively little about the function(s) of apoE in brain, has presented a barrier to developing apoE-based therapeutics for AD. Progress has been made in understanding the neurobiology of apoE; a number of potentially overlapping functions have been ascribed, which include lipid transport, neuronal repair, dendritic growth, maintenance of synaptic plasticity, and anti-inflammatory activities. Until the gaps are filled in our understanding of the pathogenic function(s) of apoE4, therapeutic strategies targeting this protein will lag behind the development of other AD therapies. Putative pathological functions, or risk-factor activities, of apoE4 include its role in β-amyloid deposition, neurofibrillary tangle formation, synaptic loss, lipid dysfunction, neuroinflammation, and oxidative stress.