Maï Panchal

Enrichment of cholesterol in microdissected Alzheimer's disease senile plaques as assessed by mass spectrometry

Extensive knowledge of the protein components of the senile plaques, one of the hallmark lesions of Alzheimers disease, has been acquired over the years, but their lipid composition remains poorly known. Evidence suggests that cholesterol contributes to the pathogenesis of Alzheimers disease. However, its presence within senile plaques has never been ascertained with analytic methods. Senile plaques were microdissected from sections of the isocortex in three Braak VI Alzheimers disease cases and compared with a similar number of samples from the adjoining neuropil, free of amyloid-β peptide (Aβ) deposit. Two cases were apoε4/apoε3, and one case was apoε3/apoε3. A known quantity of 13C-labeled cholesterol was added to the samples as a standard. After hexane extraction, cholesterol content was analyzed by liquid chromatography coupled with electrospray ionization mass spectrometry. The mean concentration of free cholesterol was 4.25 ± 0.1 attomoles/µm3 in the senile plaques and 2.2 ± 0.49 attomoles/µm3 in the neuropil (t = 4.41, P < 0.0009). The quantity of free cholesterol per senile plaque (67 ± 16 femtomol) is similar to the published quantity of Aβ peptide. The highly significant increase in the cholesterol concentration, associated with the increased risk of Alzheimers disease linked to the apoε4 allele, suggests new pathogenetic mechanisms.

Cholesterol changes in Alzheimer's disease: methods of analysis and impact on the formation of enlarged endosomes

An increasing number of results implicating cholesterol metabolism in the pathophysiology of Alzheimers disease (AD) suggest cholesterol as a target for treatment. Research in genetics, pathology, epidemiology, biochemistry, and cell biology, as well as in animal models, suggests that cholesterol, its transporter in the brain, apolipoprotein E, amyloid precursor protein, and amyloid-beta all interact in AD pathogenesis. Surprisingly, key questions remain unanswered due to the lack of sensitive and specific methods for assessing cholesterol levels in the brain at subcellular resolution. The aims of this review are not only to discuss the various methods for measuring cholesterol and its metabolites and to catalog results obtained from AD patients but also to discuss some new data linking high plasma membrane cholesterol with modifications of the endocytic compartments. These studies are particularly relevant to AD pathology, since enlarged endosomes are believed to be the first morphological change observed in AD brains, in both sporadic cases and Down syndrome.

Ceramides and sphingomyelinases in senile plaques.

The senile plaque is a hallmark lesion of Alzheimer disease (AD). We compared, without a priori, the lipidome of the senile plaques and of the adjacent plaque-free neuropil. The analysis by liquid chromatography coupled with electrospray ionization mass spectrometry revealed that laser microdissected senile plaques were enriched in saturated ceramides Cer(d18:1/18:0) and Cer(d18:1/20:0) by 33 and 78% respectively with respect to the surrounding neuropil. This accumulation of ceramides was not explained by their affinity for Aβ deposits: no interaction between ceramide-liposomes and Aβ fibrils was observed in vitro by surface plasmon resonance and fluorescent ceramide-liposomes showed no affinity for the senile plaques in AD brain tissue. Accumulation of ceramides could be, at least partially, the result of a local production by acid and neutral sphingomyelinases that we found to be present in the corona of the senile plaques.

Home is where the future is: The BrightFocus Foundation consensus panel on dementia care

A national consensus panel was convened to develop recommendations on future directions for home‐based dementia care (HBDC).

Lipidomic characterization of postmortem amyloid plaques isolated by laser capture microdissection

Background: Recent evidence suggests a role for APP processing in the regulation of apoE and cholesterol endocytosis. We have previously demonstrated that the entire cholesterol biosynthesis pathway is upregulated in Tg2576 mouse brain. Tg2576 mice over-express the APPswe mutation and accumulate the BACE1 cleavage products, C99 and Ab. Methods: It is well known that impairment of cholesterol endocytosis increases cholesterol biosynthesis gene expression. We therefore determined whether apoE endocytosis was dysregulated in Tg2576. Results: We found that extracellular cholesterol levels and apoE protein levels were significantly increased in young Tg2576 brain, implying that apoE lipoprotein particles are not being endocytosed and degraded in vivo. By directly examining the effect of APPswe overexpression on lipoprotein endocytosis in vitro we found that primary neurons from Tg2576 showed a dramatic deficit in lipoprotein endocytosis relative to wild type (WT) neurons. Given that Tg2576 cultures massively accumulate both C99 and Ab we wished to determine which cleavage product mediated the inhibitory effects on endocytosis. To directly test this, we treated WT neurons with a g-secretase inhibitor to increase intracellular C99, but reduce Ab levels. Inhibition of g-secretase led to a dose dependant inhibition of lipoprotein endocytosis. This inhibition could be blocked when co-dosed with a b-secretase inhibitor. Importantly, Ab was inhibited > 95 % by the g-secretase inhibitor alone, implying that it was the accumulation of C99 that inhibits lipoprotein endocytosis. Conclusions: These experimental results suggest a role for the amyloidogenic processing of APP in regulating apoE lipoprotein endocytosis and provide further evidence in support of the growing association between Alzheimer’s disease and cholesterol metabolism. Moreover they further establish a biological link between APP, g-secretase and apoE, the major genetic determinants of Alzheimer’s disease.

P1-12 Le cholestérol et le peptide a myloïde dans la maladie d’Alzheimer

Introduction Le cholesterol pourrait interagir avec l’apolipoproteine E et le peptide Abeta dans le milieu extracellulaire et dans les depots amyloides des plaques seniles. Le taux de cholesterol membranaire modifie in vitro la secretion du peptide Abeta. Des donnees contradictoires ont ete publiees sur l’effet des statines (inhibitrices de la synthese du cholesterol) sur la prevalence de la maladie d’Alzheimer. La presence, la concentration, et le caractere esterifie du cholesterol n’ont jamais ete determines avec precision dans les plaques seniles. Methodes La concentration du cholesterol dans les plaques seniles a ete etudiee par chromatographie liquide suivie de spectrometrie de masse sur des plaques seniles microdissequees comparees a des volumes similaires de neuropile. Les plaques seniles ont ete reperees par immunohistochimie faisant appel a la phosphatase alcaline (pour eviter l’effet des peroxydases sur les lipides). Plus de 3000 plaques provenant de 3 cas (Braak VI) ont ete comparees a 3 000 echantillons de neuropile sans plaque, provenant des memes prelevements de la circonvolution temporale superieure, microdisseques dans les memes conditions. Un standard interne d’un isotope lourd de cholesterol a ete ajoute a l’echantillon pour permettre la quantification. Resultats La concentration de cholesterol libre etait de 4.25 +/-0.18 attomoles/μm3 dans les plaques seniles et de 2.2 +/-0.86 attomoles/μm3 (p<0,0009) dans le neuropile avoisinant. Le nombre de molecules de cholesterol par plaque senile n’etait pas statistiquement different du nombre de molecules de peptide Abeta. Conclusions Le rapport molaire 1/1 entre le cholesterol libre et le peptide Abeta au sein des plaques seniles suggere une interaction directe. Du point de vue quantitatif, il est surprenant de constater que le cholesterol libre est aussi abondant dans la plaque senile que le peptide Abeta lui-meme.

Cholesterol in amyloid plaques: Analysis by laser capture microdissection combined with mass spectrometry

total apoE. Moreover, the absolute amount of apoE3 per allele was similar between e3/3 and e3/4 mice, implying that the reduced levels of total apoE in e3/4 mice can be explained by the reduction in apoE4 levels. In culture medium from e3/4 human astrocytoma or e3/3, e4/4 and e3/4 primary astrocytes, apoE4 levels were consistently lower than apoE3. Secreted cholesterol levels were also lower from e4/4 astrocytes. Pulse-chase experiments showed an enhanced degradation and reduced half-life of newly synthesized apoE4 compared to apoE3. Taken together these data suggest that astrocytes preferentially degrade apoE4, leading to reduced apoE4 secretion and ultimately to reduced brain apoE levels. Next we examined the physiological relevance of these findings. Firstly, as apoE has been shown to enhance the degradation of Ab, we measured the endogenous levels of Ab in e4/4 and e3/3 mice. As previously reported for the human population, e4/4 mice had higher levels of Ab42 in their hippocampus and cortex. Next we examined the capacity of astrocytic e3/3 and e4/4 to promote neurite outgrowth. Interestingly, and unlike previous reports, we found that astrocytic lipoproteins prepared from e4/4 mice, was able to promote neurite outgrowth and increase synaptophysin expression, but with 10 fold less potency than particles from e3/3 mice. Conclusions: Taken together these data suggest that the low levels of astrocytic apoE/ cholesterol secreted by e4 carriers may directly contribute to the disease progression by reducing apoE’s capacity to promote synaptic repair and Ab clearance.