Suzana Petanceska

Ovariectomy and 17β-estradiol modulate the levels of Alzheimer’s amyloid β peptides in brain

Objective: To test whether female gonadal hormone status and estrogen modulate the metabolism of Aβ peptides in vivo. Background: AD is a neurodegenerative disorder characterized by accumulation of aggregated forms of the 40- and 42-amino acid Aβ peptides (Aβ40 and Aβ42). Estrogen replacement therapy in postmenopausal women is associated with decreased risk for AD or delay in disease onset or both. The mechanism by which estrogen exerts this neuroprotective effect is elusive. 17β-estradiol (E2) was shown to reduce the release of Aβ peptides by primary neuronal cultures of murine and human origin. Methods: For this purpose, four experimental sets of guinea pigs were used: intact animals, ovariectomized animals (ovx), and ovariectomized animals that received E2 at two different doses (ovx+low-dose E2 and ovx+high-dose E2). Brain Aβ40 and Aβ42 levels were assessed using Aβ40 and Aβ42-specific ELISA assays. Results: Prolonged ovariectomy resulted in uterine atrophy and decreased serum E2 levels and was associated with a pronounced increase in brain Aβ levels. Total brain Aβ in the ovx animals was increased by 1.5-fold on average as compared to intact controls. E2 treatment of ovariectomized animals led to uterine hypertrophy and a dose-dependent increase in serum E2 levels. In addition, both doses of E2 significantly reversed the ovariectomy-induced increase in brain Aβ levels. The high-dose E2 treatment did not lead to a further decrease in brain Aβ beyond that observed with the low-dose E2 treatment. Conclusions: Our results infer that cessation of ovarian estrogen production in postmenopausal women might facilitate Aβ deposition by increasing the local concentrations of Aβ40 and Aβ42 peptides in brain. In addition, our finding that E2 treatment is associated with diminution of brain Aβ levels suggests that modulation of Aβ metabolism may be one of the ways by which estrogen replacement therapy prevents or delays the onset of AD or both in postmenopausal women.

App Gene Dosage Modulates Endosomal Abnormalities of Alzheimer's Disease in a Segmental Trisomy 16 Mouse Model of Down Syndrome

Altered neuronal endocytosis is the earliest known pathology in sporadic Alzheimers disease (AD) and Down syndrome (DS) brain and has been linked to increased Aβ production. Here, we show that a genetic model of DS (trisomy 21), the segmental trisomy 16 mouse Ts65Dn, develops enlarged neuronal early endosomes, increased immunoreactivity for markers of endosome fusion (rab5, early endosomal antigen 1, and rabaptin5), and endosome recycling (rab4) similar to those in AD and DS individuals. These abnormalities are most prominent in neurons of the basal forebrain, which later develop aging-related atrophy and degenerative changes, as in AD and DS. We also show that App, one of the triplicated genes in Ts65Dn mice and human DS, is critical to the development of these endocytic abnormalities. Selectively deleting one copy of App or a small portion of the chromosome 16 segment containing App from Ts65Dn mice eliminated the endosomal phenotype. Overexpressing App at high levels in mice did not alter early endosomes, implying that one or more additional genes on the triplicated segment of chromosome 16 are also required for the Ts65Dn endosomal phenotype. These results identify an essential role for App gene triplication in causing AD-related endosomal abnormalities and further establish the pathogenic significance of endosomal dysfunction in 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.

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.

Carboxyl-terminal fragments of alzheimer β-amlyloid precursor protein accumulate in restricted and unpredicted intracellular compartments in presenilin 1-deficient cells

Absence of functional presenilin 1 (PS1) protein leads to loss of γ-secretase cleavage of the amyloid precursor protein (βAPP), resulting in a dramatic reduction in amyloid β peptide (Aβ) production and accumulation of α- or β-secretase-cleaved COOH-terminal fragments of βAPP (α- or β-CTFs). The major COOH-terminal fragment (CTF) in brain was identified as βAPP-CTF-(11–98), which is consistent with the observation that cultured neurons generate primarily Aβ-(11–40). In PS1−/− murine neurons and fibroblasts expressing the loss-of-function PS1D385A mutant, CTFs accumulated in the endoplasmic reticulum, Golgi, and lysosomes, but not late endosomes. There were some subtle differences in the subcellular distribution of CTFs in PS1−/− neurons as compared with PS1D385A mutant fibroblasts. However, there was no obvious redistribution of full-length βAPP or of markers of other organelles in either mutant. Blockade of endoplasmic reticulum-to-Golgi trafficking indicated that in PS1−/− neurons (as in normal cells) trafficking of βAPP to the Golgi compartment is necessary before α- and β-secretase cleavages occur. Thus, although we cannot exclude a specific role for PS1 in trafficking of CTFs, these data argue against a major role in general protein trafficking. These results are more compatible with a role for PS1 either as the actual γ-secretase catalytic activity or in other functions indirectly related to γ-secretase catalysis (e.g. an activator of γ-secretase, a substrate adaptor for γ-secretase, or delivery of γ-secretase to βAPP-containing compartments).

Atorvastatin therapy lowers circulating cholesterol but not free radical activity in advance of identifiable clinical benefit in the treatment of mild-to-moderate AD.

Cholesterol-induced production of amyloid beta (Abeta) as a putative neurotoxin in Alzheimers disease (AD), along with epidemiological evidence, suggests that statin drugs may provide benefit in treatment of the disorder. We tested the effect of once daily atorvastatin calcium (80 mg; two 40 mg tablets) on cognitive and/or behavioral decline in patients with mild-to-moderate AD. The study was designed as a pilot intention-to-treat, proof-of-concept, double-blind, placebo-controlled, randomized (1:1) trial with a 1-year exposure to study medication employing last-observation-carried-forward (LOCF) ANCOVA as the primary statistical method of assessment. Alternate statistical methods were employed to further explore the effect of atorvastatin treatment on progression of deterioration. Of the 98 individuals with mild-to-moderate AD (Mini-Mental State Examination score of 12-28) providing Informed Consent, 71 were eligible for randomization, 67 were randomized and 63 completed the 3-month visit and were statistically evaluable. The primary outcome measures were change in the Alzheimer Disease Assessment Scale-Cognitive (ADAS-cog) performance and the Clinical Global Impression of Change (CGIC). Secondary outcome measures included the MMSE, Geriatric Depression Scale (GDS), the Neuropsychiatric Inventory (NPI) and the ADCS Activities of Daily Living inventory (ADCS-ADL). Tertiary outcome measures included levels of total circulating cholesterol, LDL and VLDL, and circulating activity of the free radical scavenger enzymes superoxide dismutase (SOD) and glutathione peroxidase (GpX). Atorvastatin reduced circulating cholesterol levels and produced a positive signal on each of the clinical outcome measures compared to placebo, but did not elicit a difference in circulating SOD or GpX activities. The observed beneficial clinical effect reached significance for the GDS (p = 0.040) and the ADAS-cog at 6 months (p = 0.003), was all but significant for the ADAS-cog (p = 0.055) at 12 months, and was of marginal significance for the CGIC (p = 0.073) and NPI (p = 0.071) at 12 months when employing the primary statistical approach (ANCOVA with LOCF). Application of repeated measures ANCOVA statistics revealed the difference was significant for the CGIC and marginally significant for the ADAS-cog, but not significant for the other clinical indices. This evaluation indicated significant time-by-treatment interactions (altered progression) for the ADAS-cog and MMSE, but not the CGIC. Application of random intercept regression analysis revealed a significant difference for the CGIC, ADAS-cog and MMSE. Regression analysis also indicated that atorvastatin produced change in the slope of deterioration on the MMSE. Accordingly, atorvastatin therapy may be an effective treatment and may slow the progression of AD among mild-to-moderately affected patients.

Mutant Presenilin 1 Increases the Levels of Alzheimer Amyloid β-Peptide Aβ42 in Late Compartments of the Constitutive Secretory Pathway

Abstract: Mutations in the presenilin 1 (PS1) gene are associated with autosomal dominant, early‐onset, familial Alzheimers disease and result in increased release of the hyperaggregatable 42‐amino acid form of the amyloid β‐peptide (Aβ42). To determine which subcellular compartments are potential source(s) of released Aβ42, we compared the levels and spatial segregation of intracellular Aβ40 and Aβ42 peptides between N2a neuroblastoma cells doubly transfected with the “Swedish” familial Alzheimers disease‐linked amyloid precursor protein variant and either wild‐type PS1 (PS1wt) or familial Alzheimers disease‐linked Δ9 mutant PS1 (PS1Δ9). As expected, PS1Δ9‐expressing cells had dramatically higher levels of intracellular Aβ42 than did cells expressing PS1wt. However, the highest levels of Aβ42 colocalized not with endoplasmic reticulum or Golgi markers but with rab8, a marker for trans‐Golgi network (TGN)‐to‐plasma membrane (PM) transport vesicles. We show that PS1 mutants are capable of causing accumulation of Aβ42 in late compartments of the secretory pathway, generating there a readily releasable source of Aβ42. Our findings indicate that PS1 “bioactivity” localizes to the vicinity of the TGN and/or PM and reconcile the apparent discrepancy between the preponderant concentration of PS1 protein in proximal compartments of the secretory pathway and the recent findings that PS1 “bioactivity” can control γ‐secretase‐like processing of another trans‐membrane substrate, Notch, at or near the PM.

Cholesterol, Copper and Aβ in Controls, MCI, AD and the AD Cholesterol- Lowering Treatment Trial (ADCLT)

Cholesterol clearly plays an influential role in promoting the production of amyloid beta (Abeta) and possibly the progression of Alzheimers Disease (AD). The AD Cholesterol-Lowering Treatment trial (ADCLT; 1 year duration) tested atorvastatin and found significant benefit on measures of cognition and depressive symptoms in treated patients (N = 32) compared to placebo (N = 31). We assessed the circulating levels of Abeta(1-40), Abeta(1-42), ceruloplasmin (copper chaperone), apolipoprotein E and HDL-cholesterol in blood collected at each clinical visit during the ADCLT. We also determined the circulating cholesterol, ceruloplasmin, and Abeta levels in AD and MCI (mild cognitive impairment) patients, and controls (two groups stratified by function; high and low) participating in our Brain Bank Program. Each Brain Bank individual was clinically assessed for performance on the Mini-Mental Status Exam (MMSE), Rey auditory verbal learning test (AVLT), Clock draw, and UPSIT (smell identification test). Among individuals of equal age and education, scores on the MMSE were significantly reduced in AD compared to both MCI and controls, as were scores on the UPSIT. Ability on delayed verbal recall was significantly reduced in AD compared to MCI, and in MCI compared to both control groups. Performance on the Clock draw was similar for AD and MCI patients, but was significantly reduced when comparing MCI to control. Both cholesterol and ceruloplasmin levels were significantly increased in low-function controls compared to the high-function control group, but were not different from levels identified in the MCI and AD patients. Significantly increased levels of Abeta(1-40) occurred in low- compared to high-function controls, with a further significant increase in MCI compared to low-function controls. Circulating Abeta(1-40) levels were decreased in AD compared to MCI. Levels of Abeta(1-42) were not significantly different between the groups. The slight gradual increase in circulating Abeta(1-40) and Abeta(1-42) levels produced by atorvastatin treatment in the ADCLT were not significant compared placebo. There was a trend for significant reduction in circulating ceruloplasmin levels after a year of atorvastatin therapy compared to levels observed at screen. The levels of HDL-cholesterol remained stable in the atorvastatin treated AD patients for 9 months and then decreased significantly compared to the placebo group at the 1-year time-point. The combined data support a role for cholesterol in AD and a possible influence of increasing circulating copper levels. The deterioration of function in controls and transition to MCI may be associated with concomitant incremental increases in circulating Abeta(1-40) levels. Increased cholesterol and ceruloplasmin levels may be associated with slight deterioration in function among controls as a precursor to impairment considered MCI. The clinical benefit of atorvastatin therapy is clearly not associated with decreased circulating Abeta or increased HDL-cholesterol, but a positive influence of reduced copper (ceruloplasmin) levels may be a consideration.

Aging, gender and APOE isotype modulate metabolism of Alzheimer's Aβ peptides and F2‐isoprostanes in the absence of detectable amyloid deposits

Aging and apolipoprotein E (APOE) isoform are among the most consistent risks for the development of Alzheimers disease (AD). Metabolic factors that modulate risk have been elusive, though oxidative reactions and their by‐products have been implicated in human AD and in transgenic mice with overt histological amyloidosis. We investigated the relationship between the levels of endogenous murine amyloid β (Aβ) peptides and the levels of a marker of oxidation in mice that never develop histological amyloidosis [i.e. APOE knockout (KO) mice with or without transgenic human APOEɛ3 or human APOEɛ4 alleles]. Aging‐, gender‐, and APOE‐genotype‐dependent changes were observed for endogenous mouse brain Aβ40 and Aβ42 peptides. Levels of the oxidized lipid F2‐isoprostane (F2‐isoPs) in the brains of the same animals as those used for the Aβ analyses revealed aging‐ and gender‐dependent changes in APOE KO and in human APOEɛ4 transgenic KO mice. Human APOEɛ3 transgenic KO mice did not exhibit aging‐ or gender‐dependent increases in F2‐isoPs. In general, the changes in the levels of brain F2‐isoPs in mice according to age, gender, and APOE genotype mirrored the changes in brain Aβ levels, which, in turn, paralleled known trends in the risk for human AD. These data indicate that there exists an aging‐dependent, APOE‐genotype‐sensitive rise in murine brain Aβ levels despite the apparent inability of the peptide to form histologically detectable amyloid. Human APOEɛ3, but not human APOEɛ4, can apparently prevent the aging‐dependent rise in murine brain Aβ levels, consistent with the relative risk for AD associated with these genotypes. The fidelity of the brain Aβ/F2‐isoP relationship across multiple relevant variables supports the hypothesis that oxidized lipids play a role in AD pathogenesis, as has been suggested by recent evidence that F2‐isoPs can stimulate Aβ generation and aggregation.