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Background Major prevention trials for Alzheimer’s disease (AD) are now focusing on multidomain lifestyle interventions. However, the exact combination of behavioral factors related to AD pathology remains unclear. In 2 cohorts of cognitively unimpaired individuals at risk of AD, we examined which combinations of personality traits, neuropsychiatric symptoms, and cognitive lifestyle (years of education or lifetime cognitive activity) related to the pathological hallmarks of AD, amyloid-β, and tau deposits. Methods A total of 115 older adults with a parental or multiple-sibling family history of sporadic AD (PREVENT-AD [PRe-symptomatic EValuation of Experimental or Novel Treatments for AD] cohort) underwent amyloid and tau positron emission tomography and answered several questionnaires related to behavioral attributes. Separately, we studied 117 mutation carriers from the DIAN (Dominant Inherited Alzheimer Network) study group cohort with amyloid positron emission tomography and behavioral data. Using partial least squares analysis, we identified latent variables relating amyloid or tau pathology with combinations of personality traits, neuropsychiatric symptoms, and cognitive lifestyle. Results In PREVENT-AD, lower neuroticism, neuropsychiatric burden, and higher education were associated with less amyloid deposition (p = .014). Lower neuroticism and neuropsychiatric features, along with higher measures of openness and extraversion, were related to less tau deposition (p = .006). In DIAN, lower neuropsychiatric burden and higher education were also associated with less amyloid (p = .005). The combination of these factors accounted for up to 14% of AD pathology. Conclusions In the preclinical phase of both sporadic and autosomal dominant AD, multiple behavioral features were associated with AD pathology. These results may suggest potential pathways by which multidomain interventions might help delay AD onset or progression.

The APOE ε4 allele was originally reported to contribute to risk of Alzheimer's disease (AD) in women, yet male and female AD patient-derived data are routinely pooled. Histopathological hallmarks of AD include neurofibrillary tangles centered on hyperphosphorylated Tau and plaques composed of the β-amyloid (Aβ) peptide that is derived by sequential secretase-mediated cleavage of the Amyloid Protein Precursor (APP). We chose to examine profiles of Aβ(1-40), Aβ(1-42), and N-truncated (i.e., p3-related) fragments in the plaque-associated fraction of autopsied cortical and corresponding hippocampal samples from donors with a diagnosis of early-onset (EOAD) and late-onset (LOAD) AD. Levels of Aβ(1-40), Aβ(1-42), and the p3 fragment-enriched pool were increased in EOAD and LOAD samples, and correlated well within -but not between- regions. Counterintuitively, these increases were similar regardless of the AD donor's APOE ε4 status. Focusing on the donor's sex and APOE ε4 status as nominal variables (i.e., omitting diagnosis from the stratification) revealed that increases in Aβ peptides were specific to female carriers of the ε4 allele and correlated with the proportional expression of BACE1/β-secretase and ADAM10/α-secretase in the cortex and with nicastrin (γ-secretase) expression in the hippocampus. These data preliminarily support the possibility that AD follows distinct amyloidogenic processes in males and females, and that the APOE ε4 allele exerts a major influence on the disease process, particularly in women. This knowledge could significantly impact the (re)interpretation of unsuccessful outcomes of clinical interventions targeting either Aβ peptides directly or the secretases implicated in APP processing.

Introduction Because currently known Alzheimer's disease (AD) single-nucleotide polymorphisms only account for a small fraction of the genetic variance in this disease, there is a need to identify new variants associated with AD. Methods Our team performed a genome-wide association study in the Quebec Founder Population isolate to identify novel protective or risk genetic factors for late-onset sporadic AD and examined the impact of these variants on gene expression and AD pathology. Results The rs10984186 variant is associated with an increased risk of developing AD and with a higher CDK5RAP2 mRNA prevalence in the hippocampus. On the other hand, the rs4837766 variant, which is among the best cis-expression quantitative trait loci in the CDK5RAP2 gene, is associated with lower mild cognitive impairment/AD risk and conversion rate. Discussion The rs10984186 risk and rs4837766 protective polymorphic variants of the CDK5RAP2 gene might act as potent genetic modifiers for AD risk and/or conversion by modulating the expression of this gene.

Imaging biomarkers are frequently proposed as endpoints for clinical trials targeting brain amyloidosis in Alzheimer's disease (AD); however, the specific impact of amyloid-β (Aβ) aggregation on biomarker abnormalities remains elusive in AD. Using the McGill-R-Thy1-APP transgenic rat as a model of selective Aβ pathology, we characterized the longitudinal progression of abnormalities in biomarkers commonly used in AD research. Middle-aged (9–11 months) transgenic animals (both male and female) displayed mild spatial memory impairments and disrupted cingulate network connectivity measured by resting-state fMRI, even in the absence of hypometabolism (measured with PET [18F]FDG) or detectable fibrillary amyloidosis (measured with PET [18F]NAV4694). At more advanced ages (16–19 months), cognitive deficits progressed in conjunction with resting connectivity abnormalities; furthermore, hypometabolism, Aβ plaque accumulation, reduction of CSF Aβ1-42concentrations, and hippocampal atrophy (structural MRI) were detectable at this stage. The present results emphasize the early impact of Aβ on brain connectivity and support a framework in which persistent Aβ aggregation itself is sufficient to impose memory circuits dysfunction, which propagates to adjacent brain networks at later stages.SIGNIFICANCE STATEMENTThe present study proposes a “back translation” of the Alzheimer pathological cascade concept from human to animals. We used the same set of Alzheimer imaging biomarkers typically used in large human cohorts and assessed their progression over time in a transgenic rat model, which allows for a finer spatial resolution not attainable with mice. Using this translational platform, we demonstrated that amyloid-β pathology recapitulates an Alzheimer-like profile of biomarker abnormalities even in the absence of other hallmarks of the disease such as neurofibrillary tangles and widespread neuronal losses.

3-Hydroxy-3-methyglutaryl coenzyme A reductase (HMGCR) is a cholesterol-regulating gene with statin relevance. rs3846662 being involved in regulation of HMGCR alternative splicing, we explored its impact on HMGCR messenger RNA (mRNA) and protein levels in the brain and the associations between those levels and levels of Alzheimer's disease pathological markers. We used brain samples derived from a cohort of 33 non-demented controls and 90 Alzheimer's disease autopsied-confirmed cases. HMGCR mRNA levels were determined in the frontal cortex (n = 114) and cerebellum (n = 110) using Taqman-qPCR, and HMGCR protein levels were determined in the frontal cortex (n = 117) using a commercial enzyme immunoassay. While densities of neurofibrillary tangles and senile plaques were determined in the frontal cortex (n = 74), total tau, phosphorylated Tau, and beta-amyloid 1-42 levels were determined in the frontal cortex (n = 94) and cerebellum (n = 91) using commercial enzyme immunoassays. Despite an increase in full-length HMGCR mRNA ratio in the frontal cortex of women carrying the AA genotype, there were no associations between rs3846662 and HMGCR mRNA or protein levels. An increased Δ13 HMGCR mRNA ratio was associated with increased levels of HMGCR proteins and neurofibrillary tangles in the frontal cortex but with reduced beta-amyloid 1-42 levels in the cerebellum, suggesting a brain cell type- or a disease progression-dependent association.

One important aspect in Alzheimer's disease pathology is the presence of chronic inflammation. Considering its role as a key receptor in the microglial innate immune system, TLR4 was shown to regulate the binding and phagocytosis of amyloid plaques by microglia in several mouse models of amyloidosis, as well as the production of pro-inflammatory cytokines. To our knowledge, TLR4 and its association with cytokines have not been thoroughly examined in the brains of subjects affected with Alzheimer's disease. Using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in postmortem human brains, we observed increased expression for the TLR4 and TNF genes (p = 0.001 and p = 0.025, respectively), as well as a trend for higher IL6 gene expression in the frontal cortex of AD subjects when compared to age-matched controls. Similarly, using a mouse model of hippocampal deafferentation without amyloidosis, (i.e., the entorhinal cortex lesioned mouse), we observed significant increases in the expression of both the Tlr4 (p = 0.0367 and p = 0.0193 compared to sham-lesioned mice or to the contralateral side, respectively) and Il1b (p = 0.0055 and p = 0.0066 compared to sham-lesioned mice or to the contralateral side, respectively) genes in the deafferentation phase, but not during the ensuing reinnervation process. In conclusion, we suggest that the modulation of cytokines by TLR4 is differentially regulated whether by the presence of amyloid plaques or by the ongoing deafferentation process.

Background: Both subjective cognitive decline (SCD) and a family history of Alzheimer's disease (AD) portend risk of brain abnormalities and progression to dementia. Posterior default mode network (pDMN) connectivity is altered early in the course of AD. It is unclear whether SCD predicts similar outcomes in cognitively normal individuals with a family history of AD. Methods: We studied 124 asymptomatic individuals with a family history of AD (age 64 ± 5 years). Participants were categorized as having SCD if they reported that their memory was becoming worse (SCD+). We used extensive neuropsychological assessment to investigate five different cognitive domain performances at baseline (n = 124) and 1 year later (n = 59). We assessed interconnectivity among three a priori defined ROIs: pDMN, anterior ventral DMN, medial temporal memory system (MTMS), and the connectivity of each with the rest of brain. Results: Sixty-eight (55%) participants reported SCD. Baseline cognitive performance was comparable between groups (all false discovery rate-adjusted p values >.05). At follow-up, immediate and delayed memory improved across groups, but the improvement in immediate memory was reduced in SCD+ compared with SCD− (all false discovery rate–adjusted p values −, SCD+ subjects showed increased pDMN–MTMS connectivity (false discovery rate–adjusted p

Background: Hypercholesterolemia is a major risk factor for the late-onset form of Alzheimer disease (AD). Loss-of-function (LOF) mutations of PCSK9 and PCSK9 inhibitors lower low-density lipoprotein cholesterol (LDL-C) and have been associated with a reduced risk of cardiovascular disease. The aim of this study was to examine the effect of PCSK9 LOF variants on risk and age of onset of AD. Methods: A total of 878 participants (410 controls and 468 AD cases) from the Quebec Founder Population were included in the study. Results: Fifty-four (6.2%) participants carried the R46L mutation, whereas 226 (26.2%) participants carried the InsLEU mutation. There was no protective or no deleterious effect of carrying PCSK9 LOF mutations on AD prevalence nor on age of onset, even when stratified by apolipoprotein E epsilon 4 genotype or by gender. Conclusion: Our data indicate that carrying PCSK9 LOF mutations has a neutral effect on neurocognitive health and the prevalence of AD.

Genome-wide association studies have identified several cholesterol metabolism-related genes as top risk factors for late-onset Alzheimer's disease (LOAD). We hypothesized that specific genetic variants could act as disease-modifying factors by altering the expression of those genes. Targeted association studies were conducted with available genomic, transcriptomic, proteomic, and histopathological data from 3 independent cohorts: the Alzheimer's Disease Neuroimaging Initiative (ADNI), the Quebec Founder Population (QFP), and the United Kingdom Brain Expression Consortium (UKBEC). First, a total of 273 polymorphisms located in 17 cholesterol metabolism-related loci were screened for associations with cerebrospinal fluid LOAD biomarkers beta amyloid, phosphorylated tau, and tau (from the ADNI) and with amyloid plaque and tangle densities (from the QFP). Top polymorphisms were then contrasted with gene expression levels measured in 134 autopsied healthy brains (from the UKBEC). In the end, only SREBF2 polymorphism rs2269657 showed significant dual associations with LOAD pathological biomarkers and gene expression levels. Furthermore, SREBF2 expression levels measured in LOAD frontal cortices inversely correlated with age at death; suggesting a possible influence on survival rate.

The mevalonate pathway has been described to play a key role in Alzheimer's disease (AD) physiopathology. Farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are nonsterol isoprenoids derived from mevalonate, which serve as precursors to numerous human metabolites. They facilitate protein prenylation; hFPP and hGGPP synthases act as gateway enzymes to the prenylation of the small guanosine triphosphate (GTP)ase proteins such as RhoA and cdc42 that have been shown to facilitate phospho-tau (p-Tau, i.e., protein tau phosphorylated) production in the brain. In this study, a significant positive correlation was observed between the synthases mRNA prevalence and disease status (FPPS, p0.001, n = 123; GGPPS, p0.001, n = 122). The levels of mRNA for hFPPS and hGGPPS were found to significantly correlate with the amount of p-Tau protein levels (p0.05, n = 34) and neurofibrillary tangle density (p 0.05, n = 39) in the frontal cortex. Interestingly, high levels of hFPPS and hGGPPS mRNA prevalence are associated with earlier age of onset in AD (p0.05, n = 58). Together, these results suggest that accumulation of p-Tau in the AD brain is related, at least in part, to increased levels of neuronal isoprenoids.

Several retrospective epidemiological studies report that utilization of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) inhibitors called statins at mid-life can reduce the risk of developing sporadic Alzheimer's disease (AD) by as much as 70%. Conversely, the administration of these inhibitors in clinically diagnosed subjects with AD confers little or no benefits over time. Here, we investigated the association between AD and HMGCR rs3846662, a polymorphism known to be involved in the regulation of HMGCR exon 13 skipping, in a founder population and in two distinct mixed North American populations of converting mild cognitively impaired (MCI) subjects (Alzheimer's disease Cooperative study (ADCS) and Alzheimer's disease Neuroimaging Initiative (ADNI) cohorts). Targeting more specifically women, the G allele negative (G-) AD subjects exhibit delayed age of onset of AD (P=0.017) and significantly reduced risk of AD (OR: 0.521; P=0.0028), matching the effect size reported by the apolipoprotein E type 2 variant. Stratification for APOE4 in a large sample of MCI patients from the ADCS cohort revealed a significant protective effect of G negative carriers on AD conversion 3 years after MCI diagnosis (odds ratio (OR): 0.554; P=0.041). Conversion rate among APOE4 carriers with the HMGCR's G negative allele was markedly reduced (from 76% to 27%) to levels similar to APOE4 non-carriers (27.14%), which strongly indicate protection. Conversion data from the independent ADNI cohort also showed significantly reduced MCI or AD conversion among APOE4 carriers with the protective A allele (P=0.005). In conclusion, HMGCR rs3846662 acts as a potent genetic modifier for AD risk, age of onset and conversion.

BACKGROUND Genetic heterogeneity in amnestic mild cognitively impaired (aMCI) subjects could lead to variations in progression rates and response to cholinomimetic agents. Together with the apolipoprotein E4 (APOE-ɛ4) gene, butyrylcholinesterase (BCHE) has become recently one of the few Alzheimer's disease (AD) susceptibility genes with distinct pharmacogenomic properties. OBJECTIVE To validate candidate genes (APOE/BCHE) which display associations with age of onset of AD and donepezil efficacy in aMCI subjects. METHODS Using the Petersen et al. (2005) study on vitamin E and donepezil efficacy in aMCI, we contrasted the effects of BCHE and APOE variants on donepezil drug response using the Alzheimer's Disease Assessment Score-Cognition (ADAS-Cog) scale. Independently, we assessed the effects of APOE/BCHE genotypes on age of onset and cortical choline acetyltransferase activity in autopsy-confirmed AD and age-matched control subjects. RESULTS Statistical analyses revealed a significant earlier age of onset in AD for APOE-ɛ4, BCHE-K*, and APOE-ɛ4/BCHE-K* carriers. Among the carriers of APOE-ɛ4 and BCHE-K*, the benefit of donepezil was evident at the end of the three-year follow-up. The responder's pharmacogenomic profile is consistent with reduced brain cholinergic activity measured in APOE-ɛ4 and BCHE-K* positive subjects. CONCLUSIONS APOE-ɛ4 and BCHE-K* positive subjects display an earlier age of onset of AD, an accelerated cognitive decline and a greater cognitive benefits to donepezil therapy. These results clearly emphasize the necessity of monitoring potential pharmacogenomic effects in this population of subjects, and suggest enrichment strategies for secondary prevention trials involving prodromal AD subjects.

Evidence suggests that the genes involved in brain lipid homeostasis are of particular relevance for Alzheimer's disease (AD) etiology. Among these genes, that encoding paraoxonase 1 (PON1) has gained newfound interest from a public health perspective, as recent studies have suggested that PON1 L55M and Q192R genetic variants might affect individual susceptibility to environmental events, such as exposure to cholinesterase inhibitors. Cholinesterase inhibitor therapy being the treatment of choice for patients with mild to moderate AD, we sought to answer two main questions: (i) are these genetic variants associated with increased AD risk, earlier age of onset/death, or shorter AD duration; and (ii) do they affect the neuropathological hallmarks of AD? This genetic study used a large cohort of clinical and autopsy-confirmed AD cases and age-matched, cognitively intact controls from the Douglas Hospital Brain Bank, Quebec, Canada (n = 1066). The evidence presented here suggests multiple gender-specific effects of PON1 polymorphisms on AD etiopathology. The L55M Met allele exerts an AD risk-enhancing effect only in men (P < 0.001), whereas both men and women carrying the M55M/Q192Q genotype exhibit increased survival (2.5 years, P < 0.05) and later age of onset (1.5 years, P < 0.05). These genetic variants are also individually and significantly associated, sometimes in opposite directions for both genders, with beta-amyloid levels (P < 0.001), senile plaque accumulation (P < 0.001) and choline acetyltransferase activity (P < 0.05) in, respectively, two of two, five of six, and three of six brain areas. These results suggest an involvement of the PON1 gene in AD etiopathology and responses to treatment.

Emerging evidences indicate a role for lipoprotein lipase (LPL) in degenerative states. Genetic variations in the LPL gene were previously associated to lipid imbalance and coronary artery disease (CAD) risk and severity, a condition that shares pathological features with common Alzheimer's disease (AD). To evaluate whether these genetic variations associate with the risk and pathophysiology of common AD, autopsy-confirmed patients (242 controls, 153 AD) were genotyped for a PvuII single nucleotide polymorphism (SNP; rs285; referred to as the P+ allele) of LPL. Brain LPL mRNA levels, cholesterol levels, amyloid concentration, senile plaques and neurofibrillary tangles density counts were measured and contrasted with specific LPL genotypes. When adjusted for age and sex, homozygosity for the P+ allele resulted in an odds ratio of 2.3 for the risk of developing AD. More importantly, we report that the presence of the P+ allele of LPL significantly affects its mRNA expression level (n = 51; P = 0.026), brain tissue cholesterol levels (n = 55; P = 0.0013), neurofibrillary tangles (n = 52; P = 0.025) and senile plaque (n = 52; P = 0.022) densities. These results indicate that a common polymorphism in the lipoprotein lipase gene modulates the risk level for sporadic AD in the eastern Canadian population but more importantly, indirectly modulates the pathophysiology of the brain in autopsy-confirmed cases.

Increasing evidence suggests that cholesterol plays a central role in the pathophysiology of Alzheimer's disease (AD). Caveolin is a cholesterol-binding membrane protein involved in cellular cholesterol transport. We investigated the changes in the protein amount of hippocampal caveolin of autopsy-confirmed AD and aged-matched control subjects. Our results demonstrate that caveolin protein levels in the hippocampus and caveolin mRNA in the frontal cortex are up-regulated in AD by approximately two-fold, compared to control brains. These results suggest a relationship between caveolin-1 expression levels and a dysregulation of cholesterol homeostasis at the plasma membrane of brain cells. In support of this hypothesis, a significant increase in caveolin protein levels has also been observed in hippocampal tissue from ApoE-deficient (knockout) and aged wild-type mice; two situations associated with modifications of transbilayer distribution of cholesterol in brain synaptic plasma membranes. These results indicate that caveolin over-expression is linked to alterations of cholesterol distribution in the plasma membrane of brain cells and are consistent with the notion of a deterioration of cholesterol homeostasis in AD.

were 1.05 for cases and 0.85 for controls (p

The discovery that the apolipoprotein E (apoE) e4 allele is genetically linked to both sporadic and familiallate-onset Alzheimer’s disease (AD) raises the possibility that a dysfunction of the lipid transport systemcould seriously affect lipid homeostasis in the brain of AD subjects. The presence of the e4 allele has beenassociated with lower levels of apoE in both serum and brain tissues of normal and AD subjects. In anattempt to reverse the apoE deficit in AD, we identified and characterized several apoE inducer agentsusing a low-throughput in vitro screening assay. The most promising of these compounds is calledprobucol. Administration of probucol, an old cholesterol-lowering drug, in a pilot trial in mild-to-moderate sporadic AD led to a significant increase in cerebrospinal fluid (CSF) apoE levels and adecrease in CSF in both phosphorylated tau 181 and beta-amyloid 1e42 concentrations without sig-nificant modifications of lipid hydroperoxide levels. 2014 Elsevier Inc. All rights reserved.

The H2 allele of apolipoprotein (apo) C-I is associated with Alzheimer's disease (AD). However, this association is potentially confounded by the linkage disequilibrium of H2 with the epsilon2 and epsilon4 alleles of apoE and of H1 with the epsilon3 allele. To establish plausibility for a direct role for apoC-I in AD, we compared apoC-I and apoE protein and mRNA levels in postmortem specimens of frontal cortex and hippocampus from AD patients with levels in nondemented controls. In H2-allelic individuals (usually also epsilon4 carriers), apoC-I mRNA levels were strikingly lower with AD (by 65%, P0.05), but apoC-I protein levels in AD were significantly higher (by 34%, P0.05). The opposite direction of the apoC-I mRNA and apoC-I protein level changes in AD in the epsilon4/H2 genotype may reflect decreased clearance of CNS lipoproteins associated with apoE4. In H1/H1 (usually epsilon3/epsilon3) individuals, both apoC-I protein and mRNA were lower in AD. ApoC-I protein levels in hippocampus were nearly twice those in frontal cortex. Immunohistochemistry of hippocampus revealed colocalization of apoC-I protein with the astrocytic marker GFAP. In addition, cultured human astrocytes expressed the mRNA for apoC-I. This study confirms apoC-I expression in the CNS and identifies astrocytes as the source of apoC-I. In addition, it has revealed differences in apoC-I expression based on site, genotype, and disease status that may reflect a role for apoC-I in the pathogenesis of AD.

Both apolipoprotein E (apoE) and amyloid peptides are associated with Alzheimer's disease (AD). Using primary hippocampal neurons, we demonstrate that apoE is capable of reducing potentially toxic extracellular amyloid peptides, likely through a receptor mediated mechanism. We hypothesize that isoform-specific differences in apoE-mediated amyloid clearance and intracellular accumulation may be responsible, at least in part, for the increased number of amyloid plaques observed in apoE ϵ4 allele AD individuals.

Apolipoprotein E (apoE) is recognized as a key actor in brain remodeling. It has been shown to increase after peripheral and central injury, to modulate reparative capacity in neurodegenerative conditions like Alzheimer's disease (AD) and to be associated with a number of other neurodegenerative diseases. This particular function of apoE has been postulated to underlie the robust association with risk and age at onset of AD. ApoE associations studies with Parkinson's disease (PD), the second most prevalent neurodegenerative disease, have generated contradictory results but associations with age at onset and dementia in PD stand out. We investigate here whether apoE is involved in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced degeneration that models PD-like deafferentation of the striatum in the mouse and participates in compensatory reinnervation mechanisms. We examined the modifications in gene expression and protein levels of apoE and its key receptors, the low density lipoprotein receptor (LDLR) and the LDLR-related protein (LRP), as well as the reactive astrocyte marker glial fibrillary acidic protein (GFAP) in different brain structures throughout the degenerative and reactive regenerative period. In the striatum, upregulations of GFAP, apoE and LRP mRNAs at 1 day post-treatment were associated with marked decreases in dopamine (DA) levels, loss in tyrosine hydroxylase protein content, as well as to a compensatory increase in dopaminergic metabolism. Subsequent return to near control levels coincided with indications of reinnervation in the striatum: all consistent with a role of apoE during the degenerative process and regenerative period. We also found that this cascade was activated in the hippocampus and more so than in the striatum, with a particular contribution of LDLR expression. The hippocampal activation did not correlate with substantial neurochemical reductions but appears to reflect local subtle alteration of DA metabolism and the regulation of plasticity-related event in this structure. This study provides first evidence of an activation of the apoE/apoE receptors cascade in a mouse model of PD, specifically in the MPTP-induced deafferentation of the striatum. Results are also quite consistent with the postulated role of apoE in brain repair but, raise the issue of possible lesion- and region-specific alterations in gene expression.

Validating the expression stability of reference genes is crucial for reliable normalization of real-time quantitative PCR (qPCR) data, but relatively few studies have investigated this issue in brain human tissues. The present study thus aimed at identifying in human post-mortem brain tissues a set of suitable endogenous reference genes (ERG) for the expression analysis of potential candidate genes associated with Alzheimer's disease (AD). The mRNA levels of ten common ERGs (ACTB, GAPDH, GPS1, GUSB, M-RIP, PGK1, POL2RF, PPIA, UBE2D2, and YES1) were determined in the frontal cortex of autopsy-confirmed AD and non-demented control cases ( n = 20) using SYBR Green technology. Then, these levels were ranked according to their expression stability using three software applications: geNorm, NormFinder and BestKeeper. Whereas PPIA and UBE2D2 were among the ERGs with the most reliable expression, ACTB was the worst. Subsequently, using PPIA and UBE2D2 as ERGs for normalization, the mRNA levels of paraoxonase 1 (PON1) and paraoxonase 2 (PON2) were quantified in the frontal cortex of AD and control cases ( n = 80) and analyzed using the REST 2009 program. Our results indicate that both paraoxonases are expressed in the human frontal cortex and that PON2 but not PON1 mRNA levels are up-regulated in AD relative to non-demented controls. However, re-analysis of the results by ANCOVA indicated that the significance of the difference between AD and control groups depended upon the ERG used for normalization. The use of a computational method allowing the inclusion of possible confounding factors is thus recommended for the analysis of data.

Apolipoprotein E is synthesized and secreted by astrocytes in the hippocampus following lesions of the entorhinal cortex. It was proposed that apolipoprotein E, by analogy to its role in cholesterol transport in circulation, could be involved in the salvage and reutilization of non-esterified cholesterol released during terminal breakdown. The salvaged cholesterol could then be transported to neurons by apolipoprotein E-complexes and taken up via the apolipoprotein E/apolipoprotein B (low-density lipoprotein) receptor. To test this hypothesis, we have examined low-density lipoprotein receptor binding in brain sections of rats undergoing hippocampal reinnervation. The number of neuronal cells labelled by fluorescent Dil-low-density lipoprotein as well as the density of [125I]low-density lipoprotein binding sites in the dentate gyrus were found to increase in parallel with the extent of cholinergic reinnervation occurring in the deafferented hippocampus. In contrast, hippocampal cholesterol synthesis fell by more than 60% at eight days post-lesion, but eventually returned to control levels at 30 days post-lesion. The transient loss of cholesterol synthesis coincided with a peak in hippocampal apolipoprotein E expression. A concomitant accumulation of sudanophilic lipids (cholesterol esters and phospholipids) was detected in the outer molecular layer of the dentate gyrus and in the hilar region. The present findings suggest that non-esterified cholesterol released during terminal breakdown is esterified, transported via the apolipoprotein E transport system to neurons undergoing reinnervation, and take-up through the low-density lipoprotein receptor pathway where it is presumably used as a precursor molecule for the synthesis of new synapses and terminals.

Entorhinal cortex lesioning (ECL) produces a loss of more than 80% of the synapses in the outer molecular layer of the hippocampus. However, the loss of synapses is transient. Beginning a few days after denervation, new synapses are formed, virtually replacing the lost inputs within 2 months. Synaptic remodelling induced by ECL is associated with specific modifications of neurotransmitters, hormones and growth factors. Particularly, protein kinase C (PKC) plays important functional roles in receptor-mediated transmembrane signal transduction. PKC is also involved in various aspects of synaptic plasticity, such as cellular growth and differentiation. To investigate further the potential roles of PKC in synaptic plasticity observed in the ECL model, [ 3 H]phorbol 12,13-dibutyrate ([ 3 H]PDBu) binding, a putative marker of PKC, was examined at different times post-lesion. [ 3 H]PDBu binding sites transiently decreased bilaterally at 2 and 8 days post-lesion (20%) in different laminae and sub-fields of the rostral hippocampus but returned to control values at 14 and 30 days post-lesion. In caudal portion of the hippocampus, [ 3 H]PDBu binding was also decreased at 2 days post-lesion but only on the contralateral side. Interestingly, [ 3 H]PDBu binding sites in the cortex increased by up to 30% in the contralateral side while no significant change was observed in the ipsilateral side at any time post-lesion. It is known that PKC can be regulated by different systems following alterations of neuronal and glial activity. We suggest that these could be involved in the response of PKC and [ 3 H]PDBu binding sites following ECL. Moreover, PKC seemed to be modified in different brain areas in addition to the hippocampal formation in this model. This can be associated to a rather general reorganization observed following losses of neuronal inputs from the entorhinal cortex and the subsequent reinnervation process.

Currently data from 119 mitochondrial polymorphisms were extracted from GWAS data from 500 Alzheimer’s disease patients and 300 individuals diagnosed as cognitively normal. Polymorphism data for all mitochondrial genes will be combined into a composite score from the entire mitochondria to assess mitochondrial genetic load. This genetic load score will be included in a logistic regression to assess potential associations between mitochondrial gene variants and the development or progression of Alzheimer’s disease. Conclusions: Genetic variation within the mitochondria electron transport chain may impact the development or progression of Alzheimer’s disease.

Apolipoproteins, lipid carrier molecules, are instrumental in the coordination of the metabolism of lipid following peripheral and central nervous system (CNS) injuries. Apolipoprotein E (ApoE) in particular is unique among apolipoproteins in that it has a special relevance to nervous tissue. It was shown to coordinate the mobilization and redistribution of cholesterol and phospholipids for the repair, growth, and maintenance of myelin and neuronal membranes, during development or after injury, in the peripheral nervous system. In the brain, ApoE plays a pivotal role in the mobilization and redistribution of cholesterol and phospholipids during membrane remodeling associated with synaptic plasticity [1]. The low concentrations of other key plasma apolipoproteins such asApoA1, C1, and B in the brain further emphasize the critical role of ApoE in this particular tissue [2].

The insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is a multifunctional membrane glycoprotein, which binds different classes of ligands including IGF-II and M6P-bearing lysosomal enzymes. Besides participating in the process of endocytosis this receptor functions in the trafficking of lysosomal enzymes from the trans-Glogi network (TGN) or the cell surface to lysosomes. In Alzheimer's disease (AD) brain, marked overexpression of certain lysosomal enzymes in vulnerable neuronal populations and their association to beta-amyloid (Abeta) containing neuritic plaques has been correlated to altered metabolic functions. In the present study, we measured the levels of IGF-II/M6P receptor and characterized its distribution profile in selected regions of AD and age-matched normal postmortem brains. Western blot analysis revealed no significant alteration in the levels of IGF-II/M6P receptor either in the hippocampus, frontal cortex or cerebellum between AD and age-matched control brains. However, a significant gene dose effect of apolipoprotein E (APOE) epsilon4 allele on IGF-II/M6P receptor levels was evident in the hippocampus of the AD brain. At the cellular level, immunoreactive IGF-II/M6P receptors were localized in the neurons of the frontal cortex, hippocampus and cerebellum of control brains. In AD brains, the labeling of the neurons was less intense in the frontal cortex and hippocampus than in the age-matched control brains. Additionally, IGF-II/M6P receptor immunoreactivity was observed in association with a subpopulation of Abeta-containing neuritic plaques as well as tau-positive neurofibrillary tangles both in the frontal cortex and the hippocampus. Reactive glial cells localized adjacent to the plaques also occasionally exhibited IGF-II/M6P receptor immunoreactivity. These results, when analyzed in context of the established role of the IGF-II/M6P receptor in the regulation of the intracellular trafficking of lysosomal enzymes, suggest that alterations in IGF-II/M6P receptor levels/distribution are possibly associated with altered functioning of the lysosomal enzymes and/or loss of neurons observed in AD brains, especially in patients carrying APOE epsilon4 alleles.

Epidemiological studies have shown that use of non-steroidal anti-inflammatory drugs (NSAIDs) by the elderly is associated with a decreased relative risk and a delayed onset of Alzheimer's disease (AD). In contrast, the apolipoprotein E (apoE) gene has proven to be a risk factor for AD with apoE epsilon 4 AD patients having been found to show lower levels of brain apoE. In the present study, treatment of primary rat mixed glial cell cultures with the common NSAIDs, indomethacin and aspirin, induced significant increases in extracellular apoE protein levels. Similarly, treatment of primary rat astrocyte cell cultures with aspirin and a cyclooxygenase (COX)-2-selective aspirin derivative also stimulated significant increases in apoE protein. However, astrocyte and mixed glial apoE protein levels were significantly reduced following exposure to COX-2-specific indomethacin amides and an inactive indomethacin derivative. ApoE protein modulation was observed at physiological and subphysiological concentrations well below the COX inhibition IC50 values of the NSAIDs used, suggestive of a COX-independent mechanism. In contrast to these results, indomethacin and aspirin treatment failed to induce any significant changes in apoE mRNA levels. The failure of NSAIDs to significantly alter apoE expression may have been indicative of a nontranscriptional mechanism of apoE protein induction. Consequently, NSAID-induced increases in apoE protein may enhance apoE-mediated immunosuppression and compensatory synaptic plasticity, potentially resulting in decreased AD risk and delay of disease onset.

Several recent epidemiological studies have proposed that cholesterol-lowering drug Statin may provide protection against Alzheimer's disease (AD). Probucol is a non-Statin cholesterol-lowering drug and a potent inducer of apolipoprotein E (apoE) production in peripheral circulation. A recent clinical study using Probucol in elderly AD subjects revealed a concomitant stabilisation of cognitive symptoms and significant increases in apoE levels in the cerebral spinal fluid in these patients. To gain insight into the mechanisms underlying these effects, we treated a cohort of aged male rats (26-month-old) with oral dose of Probucol for 30 days. Specifically, we examined the effects of Probucol on apoE production and its receptors (low density lipoprotein receptor [LDLr] and low density lipoprotein receptor-related protein [LRP]), astroglial marker of cell damage (glial fibrillary acidic protein [GFAP]), markers of neuronal synaptic plasticity and integrity (synaptosomal associated protein of 25 kDa [SNAP-25] and synaptophysin) as well as cholesterol biosynthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase [HMGCoAr]) in the hippocampus. We report that Probucol induces the production of apoE and one of its main receptors, LRP, increases HMGCoAr (rate-limiting enzyme in cholesterol synthesis), substantially attenuates age-related increases in glial activation, and induces production of synaptic marker SNAP-25, a molecule commonly associated with synaptogenesis and dendritic remodeling. These findings suggest that Probucol could promote neural and synaptic plasticity to counteract the synaptic deterioration associated with brain aging through an apoE/LRP-mediated system. Consistent with the beneficial effects of other cholesterol-lowering drugs such as the Statin, Probucol could also offers additional benefits based on apoE neurobiology.

ApoER2 is one of the major receptors for ApoE in the brain, and has been shown to be involved not only in lipoprotein endocytosis, as other members of the LDL receptor family of receptors, but also in various cellular functions such as signalling and cellular guidance. By using a model of synaptic plasticity in mice lacking none, one or two alleles of the apoER2 gene, we investigated the implication of such a receptor deficiency on the remodelling process. Our results indicate that animals lacking apoER2 express higher levels of brain APP, as well as both key amyloid peptides, while apoE levels are slightly lower. Following entorhinal cortex lesioning, apoE levels increase in the deafferented hippocampus, while a delay in the increase of APP was observed. Hippocampal amyloid levels are also increased in response to the lesion, and highly potentiated by the complete absence of apoER2 gene. The results suggest a significant role for apoER2 in signalling various proteins in response to massive deafferentation and may participate in maintaining efficient synaptic plasticity and dendritic remodelling.

The frequency of the epsilon4 allele of apolipoprotein E (apoE) is increased in late-onset and sporadic forms of Alzheimer's disease (AD). ApoE also binds to beta-amyloid (A beta) and both proteins are found in AD plaques. To further investigate the potential interaction of apoE and A beta in the pathogenesis of AD, we have determined the binding, internalization, and degradation of human apoE isoforms in the presence and absence of A beta peptides to rat primary hippocampal neurons. We demonstrate that the lipophilic A beta peptides, in particular A beta(1-42), A beta(1-40), and A beta(25-35), increase significantly apoE-liposome binding to hippocampal neurons. For each A beta peptide, the increase was significantly greater for the apoE4 isoform than for the apoE3 isoform. The most effective of the A beta peptides to increase apoE binding, A beta(25-35), was further shown to increase significantly the internalization of both apoE3- and apoE4-liposomes, without affecting apoE degradation. Conversely, A beta(1-40) uptake by hippocampal neurons was shown to be increased in the presence of apoE-liposomes, more so in the presence of the apoE4 than the apoE3 isoform. These results provide evidence that A beta peptides interact directly with apoE lipoproteins, which may then be transported together into neuronal cells through apoE receptors.

Apolipoprotein (apo) E is likely involved in redistributing cholesterol and phospholipids during compensatory synaptogenesis in the injured CNS. Three common isoforms of apoE exist in human (E2, E3, and E4). The apoE4 allele frequency is markedly increased in both late-onset sporadic and familial Alzheimer's disease (AD). ApoE concentration in the brain of AD subjects follows a gradient : ApoE levels decrease as a function of E2 > E3 >> E4. It has been proposed that the poor reinnervation capacity reported in AD may be caused by impairment of the apoE/low-density lipoprotein (LDL) receptor activity. To understand further the role of this particular axis in lipid homeostasis in the CNS, we have characterized binding, internalization, and degradation of human 125 I-LDL to primary cultures of rat astrocytes. Specific binding was saturable, with a K D of 1.8 nM and a B max of 0.14 pmol/mg of proteins. Excess unlabeled human LDL or very LDL (VLDL) displaced 70% of total binding. Studies at 37°C confirmed that astrocytes bind, internalize, and degrade 125 I-LDL by a specific, saturable mechanism. Reconstituted apoE (E2, E3, and E4)-liposomes were labeled with 125 I and incubated with primary cultures of rat astrocytes and hippocampal neurons to examine specific binding. Human LDL and VLDL displaced binding and internalization of all apoE isoforms similarly in both astrocytes and neurons. 125 I-ApoE2 binding was significantly lower than that of the other 125 I-apoE isoforms in both cell types, 125 I-ApoE4 binding was similar to that of 125 I-apoE3 in both astrocytes and neurons. On the other hand, 125 I-apoE3 binding was significantly higher in neurons than in astrocytes. These isoform-specific alterations in apoE-lipoprotein pathway could explain some of the differences reported in the pathophysiology of AD subjects carrying different apoE alleles.

Apolipoprotein E (apoE) is a lipid-binding, 37 kDa, 299 amino acid glycoprotein involved in cholesterol and phospholipid transport and metabolism. ApoE mediates the uptake of lipid complexes through interaction with the apoB/ApoE (LDL) receptor and other receptors1. The LDL receptor pathway consists of cell surface binding of apoE-containing lipoproteins followed by internalization and degradation of the lipoprotein by a lysosomal pathway2. In the peripheral nervous system, apoE has been proposed to be involved in the transport of cholesterol in repair, growth and maintenance of membranes during development or following injury3,4. In the central nervous system, ApoE released from astrocytes in response to injury such as entorhinal cortex lesions in the rat plays a pivotal role in the redistribution of cholesterol and phospholipids during synaptic remodeling and compensatory synaptogenesis5,6.

Injured neurons in the CNS are known to synthesize high levels of proliferation related oncogene products and heat shock proteins without dividing. Statin is a cell cycle regulated nuclear phosphoprotein, selectively associated with the non-proliferative state in a wide variety of cell types. In the present study, neuronal statin was examined following lethal or sublethal neuronal injuries in the hippocampus of Alzheimer's disease patients, in rats receiving kainate lesions to the dorsal hippocampus and in entorhinal cortex lesioned rats. Immunolabelling of nuclear statin showed that statin immunoreactivity increased preferentially in CA1 pyramidal neurons of the hippocampus in Alzheimer's disease. In kainate lesioned rats, statin immunoreactivity was markedly induced in the CA3 hippocampal region in association with neuronal loss. Entorhinal cortex lesioned rats showed a transient induction of statin between 2 and 6 days post lesion in CA1 neurons. However, cell counts in entorhinal cortex lesioned rats remained unaltered in the CA1 and granule cell layers during the entire 30 day time course, indicating that increased statin levels are not secondary to neuronal degeneration and are not necessarily accompanied by irreversible neuronal death. It is concluded that, in addition to proliferation related gene products, neuronal injury induces an increase in levels of statin, a nuclear marker of cell cycle arrest. Furthermore, statin may be a potentially useful marker of injurious neuronal stress, even under conditions that do not necessarily lead to irreversible cell death.

Evidence is given for altered gene expression of gamma-actin in the hippocampus in response to entorhinal cortex lesion (ECL). Time course analysis reveals a progressive repression of gamma-actin expression between 4 and 14 days post-lesion, coinciding with the early and middle phases of the hippocampal reinnervation process. RNA prevalence returns to near control values at 30 days post-lesion. Corticosterone administration, which is known to impair the reinnervation process in ECL rats, prevents the lesion-induced reduction in gamma-actin expression and blocks the induction of alpha 1-tubulin in the deafferented hippocampus. The timing of response of gamma-actin to ECL and its modulation by glucocorticoid administration support suggestions that gamma-actin has an important role to play in neuronal cytoarchitecture remodelling during hippocampal reinnervation.