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 Alzheimer's disease, has been acquired over the years, but their lipid composition remains poorly known. Evidence suggests that cholesterol contributes to the pathogenesis of Alzheimer's 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 Alzheimer's disease cases and compared with a similar number of samples from the adjoining neuropil, free of amyloid-beta peptide (A beta) deposit. Two cases were apo epsilon 4/apo epsilon 3, and one case was apo epsilon 3/apoepsilon3. A known quantity of (13)C-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/microm(3) in the senile plaques and 2.2 +/- 0.49 attomoles/microm(3) 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 A beta peptide. The highly significant increase in the cholesterol concentration, associated with the increased risk of Alzheimer's disease linked to the apo epsilon 4 allele, suggests new pathogenetic mechanisms.

Cholesterol in the senile plaque: often mentioned, never seen.

The lipid components of the senile plaque (SP) remain largely unknown. Senile plaques were said to be enriched in cholesterol in a few studies using the cholesterol probe filipin and a histoenzymatic method based upon cholesterol oxidase activity. We provide data that strongly suggest that these results are false-positive: the SPs were still stained in the absence of the enzyme cholesterol oxidase; filipin still labeled the plaques after lipid extraction. On the other hand, resorufin, the highly fluorescent end-product of the histoenzymatic method, bound with high affinity to the SPs and neurofibrillary tangles in a cholesterol-independent manner, and might serve as a new marker of amyloid. In conclusion, the probable cholesterol enrichment of the SPs has never been proven so far, and might necessitate non-histological methods to be ascertained.

Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies.

Camelids produce antibodies made of homodimeric heavy chains, and the antigen-binding region being composed of a single domain called VHH. These VHHs are much smaller than complete IgG. They are also more thermostable and more soluble in water; they should, therefore, diffuse more readily in the tissues. VHHs, expressed in bacteria, are easier to produce than conventional monoclonal antibodies. Because of these special characteristics, these antibody fragments could have interesting developments in immunohistochemistry and in the development of biomarkers. To test the possibility of their use in immunohistochemistry (IHC), we selected the glial fibrillary acidic protein (GFAP), a well-known marker of astrocytes. One alpaca (Lama pacos) was immunized against GFAP. Lymphocytes were isolated; the DNA was extracted; the VHH-coding sequences were selectively amplified. Three VHHs with a high affinity for GFAP and their corresponding mRNA were selected by ribosome display. Large quantities of the recombinant VHHs coupled with different tags were harvested from transfected bacteria. One of them was shown to immunolabel strongly and specifically to GFAP of human astrocytes in tissue sections. The quality of the IHC was comparable or, in some aspects, superior to the quality obtained with conventional IgG. The VHH was shown to diffuse on a longer distance than conventional monoclonal antibodies in fixed cortical tissue: a property that may be useful in immunolabeling of thick sections.

[The lesions of Alzheimer's disease: which therapeutic perspectives?]. / "La maladie d'Alzheimer, Les lésions de la maladie d'Alzheimer. Perspectives thérapeutiques?

The brain lesions associated with Alzheimer's disease are caused by extracellular accumulation of Abeta peptide and intracellular accumulation of tau protein. Abeta peptide makes the core of the senile plaque (the "focal deposit"); it is also present in the extracellular "diffuse deposits" and in the vessel walls. Neurofibrillary tangles, and neuropil threads are composed of hyperphosphorylated tau that also accumulates in the processes of the corona of the senile plaque. The Abeta deposits first involve the neocortex, while the tau pathology is initially found in the hippocampal region. Abeta deposits first occur in the neocortex, while intracellular tau accumulation mainly affect the hippocampal region. Abeta peptide deposits are initially found in all the neocortical areas, then involve the hippocampus and the subcortical nuclei. Tau lesions successively involve the hippocampal regions, multi- and uni-modal areas and finally the primary cortices in stereotyped stages. Mutations of APP, the precursor of Abeta peptide, cause autosomal dominant familial Alzheimer disease, suggesting that a cascade of reactions link Abeta overproduction, tau pathology and the clinical phenotype. Transgenic mice bearing the mutated human APP gene (APP mice) develop A deposits. Systemic injection of Abeta peptide prevents the deposition of Abeta peptide. However, a clinical trial had to be interrupted when meningoencephalitis occurred in a significant proportion of treated patients. Post mortem studies showed a relative scarcity of Abeta deposits. Forthcoming immunotherapy studies should soon show whether the prevention of Abeta deposition interrupts disease progression.