Proteogenomics of the human hippocampus: The road ahead.

The hippocampus is one of the most essential components of the human brain and plays an important role in learning and memory. The hippocampus has drawn great attention from scientists and clinicians due to its clinical importance in diseases such as Alzheimer's disease (AD), non-AD dementia, and epilepsy. Understanding the function of the hippocampus and related disease mechanisms requires comprehensive knowledge of the orchestration of the genome, epigenome, transcriptome, proteome, and post-translational modifications (PTMs) of proteins. The past decade has seen remarkable advances in the high-throughput sequencing techniques that are collectively called next generation sequencing (NGS). NGS enables the precise analysis of gene expression profiles in cells and tissues, allowing powerful and more feasible integration of expression data from the gene level to the protein level, even allowing "-omic" level assessment of PTMs. In addition, improved bioinformatics algorithms coupled with NGS technology are finally opening a new era for scientists to discover previously unidentified and elusive proteins. In the present review, we will focus mainly on the proteomics of the human hippocampus with an emphasis on the integrated analysis of genomics, epigenomics, transcriptomics, and proteomics. Finally, we will discuss our perspectives on the potential and future of proteomics in the field of hippocampal biology. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.

The uniqueness of biobanks for neurological and psychiatric diseases: potentials and pitfalls.

OBJECTIVES: Central nervous system (CNS) biobanks are facing difficult and specific challenges due to the sensitive issue of collecting specimens of the CNS, and especially the brain. At present, there is no global network/central database to serve researchers, clinicians and pharma companies, or to supply the special specimens and the accompanying data in sufficient numbers and detail, respectively. The main challenge/objective is to standardize and harmonize all the facets involved in CNS biobanking in order to maximize efficient sample collection. METHODS: Since the number of CNS biospecimens stored in existing biobanks is relatively limited and the accompanying data are not always readily available and hard to identify, we propose using optimal procedures for handling and storage of these specimens, and the global standardization of the cliniconeuropathological diagnostic criteria. RESULTS: One of the prominent achievements of the current global activity in brain tissue biobanks (BTB-banks) is the development of an inventory of international standards, available specimens and concomitant data, and national registries. CONCLUSIONS: Taking into consideration the huge variety of the specimens stored in different repositories and the enormous differences in medicolegal systems and ethics regulations in different countries, we strongly recommend that healthcare systems and institutions who host BTB-banks make efforts to secure adequate funding for the infrastructure and daily activities. BTB-banks will refine standard operating procedures and their internal guides of best practices/codes of conduct. This in turn will enable the BTB-banks to share the collected specimens and data with the largest possible number of researchers, aiming at maximal scientific spin-off and advance of public health research.

Large-scale analysis of posttranslational modifications in the hippocampus of patients with Alzheimer's disease using pI shift and label-free quantification without enrichment.

Posttranslational modifications modulate protein function in cells. Global analysis of multiple posttranslational modifications can provide insight into physiology and disease, but presents formidable challenges. In the present study, we used a technique that does not require target enrichment to analyze alterations in the phosphorylation and ubiquitination of proteins from patients with Alzheimer's disease (AD). Guided by our previous findings, we applied three strategies to further our understanding of the dysregulation of posttranslationally modified proteins. We first identified phosphorylation sites by determining peptide pI shifts using OFFGEL. Second, using tandem mass spectrometry, we determined the ubiquitination status of the proteins using an assay for a trypsin digestion remnant of ubiquitination (Gly-Gly). Third, for large-scale discovery, we quantified the global differences in protein expression. Of the proteins expressed in AD tissue at levels of 2.0 or greater compared with controls, 60 were phosphorylated and 56 were ubiquitinated. Of the proteins expressed at levels of 0.5 or lower compared with controls, 81 were phosphorylated and 56 were ubiquitinated. Approximately 98 % of the phosphopeptides exhibited a pI shift. We identified 112 new phosphorylation sites (51.38 %), and 92 new ubiquitination sites (96.84 %). Taken together, our findings suggest that analysis of the alterations in posttranslationally modified proteins may contribute to understanding the pathogenesis of AD and other diseases.

Chromosome 11-centric human proteome analysis of human brain hippocampus tissue.

Human chromosome 11 is the third gene-rich chromosome having 1304 protein-coding genes. According to the GeneCards, this chromosome contains 240 genes related to diseases, as it is well known as a disease-rich chromosome. Although there are many protein-coding genes, the proteomic identification ratio is rather low. As a model study, human hippocampal tissues from patients suffering from Alzheimer's disease and epilepsy were prepared to evaluate the gene-centric statistics related to the gene expression and disorders of chromosome 11. A total of 8828 protein coding genes from brain tissues were extensively off-gel fractionated and profiled by a high resolution mass spectrometer with collision induced dissociation and electron transfer dissociation. Five-hundred twenty-three of the proteins from brain tissues were determined to belong to chromosome 11, representing 37% of the proteins reported in the Global Proteome Machine Database. We extracted gene clusters from a specific biological process or molecular function in gene ontology, among which the olfactory receptor genes showed the largest cluster on chromosome 11. Analysis of the proteome data set from the hippocampus provides a significant network associated with genes and proteins and leads to new insights into the biological and genetic mechanisms of chromosome 11-specific diseases such as Alzheimer's disease.

GAB2 alleles modify Alzheimer's risk in APOE epsilon4 carriers.

The apolipoprotein E (APOE) epsilon4 allele is the best established genetic risk factor for late-onset Alzheimer's disease (LOAD). We conducted genome-wide surveys of 502,627 single-nucleotide polymorphisms (SNPs) to characterize and confirm other LOAD susceptibility genes. In epsilon4 carriers from neuropathologically verified discovery, neuropathologically verified replication, and clinically characterized replication cohorts of 1411 cases and controls, LOAD was associated with six SNPs from the GRB-associated binding protein 2 (GAB2) gene and a common haplotype encompassing the entire GAB2 gene. SNP rs2373115 (p = 9 x 10(-11)) was associated with an odds ratio of 4.06 (confidence interval 2.81-14.69), which interacts with APOE epsilon4 to further modify risk. GAB2 was overexpressed in pathologically vulnerable neurons; the Gab2 protein was detected in neurons, tangle-bearing neurons, and dystrophic neuritis; and interference with GAB2 gene expression increased tau phosphorylation. Our findings suggest that GAB2 modifies LOAD risk in APOE epsilon4 carriers and influences Alzheimer's neuropathology.

A meta-analysis of two-dimensional electrophoresis pattern of the Parkinson's disease-related protein DJ-1.

MOTIVATION: The two-dimensional electrophoresis (2-DE) pattern of proteins is thought to be specifically related to the physiological or pathological condition at the moment of sample preparation. On this ground, most proteomic studies move to identify specific hallmarks for a number of different conditions. However, the information arising from these investigations is often incomplete due to inherent limitations of the technique, to extensive protein post-translational modifications and sometimes to the paucity of available samples. The meta-analysis of proteomic data can provide valuable information pertinent to various biological processes that otherwise remains hidden. RESULTS: Here, we show a meta-analysis of the PD protein DJ-1 in heterogeneous 2-DE experiments. The protein was shown to segregate into specific clusters associated with defined conditions. Interestingly, the DJ-1 pool from neural tissues displayed a specific and characteristic molecular weight and isoelectric point pattern. Moreover, changes in this pattern have been related to neurodegenerative processes and aging. These results were experimentally validated on human brain specimens from control subjects and PD patients. AVAILABILITY: ImageJ is a public domain image processing program developed by the National Institutes of Health and is freely available at http://rsbweb.nih.gov/ij. All the ImageJ macros used in this study are available as supplementary material and upon request at info@biodigitalvalley.com. XLSTAT can be purchased online at http://www.xlstat.com/en/home/ at a current cost of approximately 300 EUR.

[Changes of nuclear factor and inflammatory chemotactic factors in brain of patients with Alzheimer's disease].

OBJECTIVES: To investigate the changes of nuclear factor (NF-)κBp65 and inflammatory chemotactic factors including monocyte chemoattractant protein 1 (MCP-1/CCL-2), macrophage inflammatory protein 1α (MIP-1α/CCL-3), glial fibrillary acidic protein (GFAP) in brains of the patients with Alzheimer's disease (AD) and reveal the correlation of these factors. METHODS: Ten patients with AD and 8 age-matched control subjects were selected in the study. Immunohistochemistry was performed to determine the protein expression of NF-κBp65, MCP-1, MIP-1α and GFAP. Double-immunohistochemistry was used to detect the expression of GFAP and ß-amyloid peptide 1-42 (Aß(1-42)) in the hippocampus, temporal and frontal cortices. RESULTS: As compared to age-matched controls (the numbers of the positively stained neuronal cells: 0.31 ± 0.20, 0.25 ± 0.20 and 0.25 ± 0.20, respectively), the immunoreactivities of NF-κBp65 in the hippocampus and the temporal and frontal cortices (numbers of the positively stained cells: 3.6 ± 1.5, 2.2 ± 1.2 and 2.2 ± 1.2, respectively) were significantly increased in AD brains. The levels of MCP-1 and MIP-1α in the hippocampus, and the temporal and frontal cortices (numbers of the positively stained neuronal cells: 8.0 ± 1.3, 8.8 ± 1.0, 9.3 ± 1.4, respectively;and 8.1 ± 1.5, 12.5 ± 1.1, 6.4 ± 1.1, respectively) with AD were significantly higher than those of controls (the numbers of the positive neuronal cells: 4.5 ± 0.9, 4.5 ± 0.6, 4.0 ± 1.8, respectively; and 5.0 ± 1.9, 6.3 ± 2.2, 3.8 ± 1.5, respectively). An increased number of glial cells stained with GFAP were observed to extensively distribute around the senile plaques in AD brains. There were significant correlations between NF-κBp65 and these inflammatory chemotactic factors in AD brains. CONCLUSION: Correlative expressions of NF and inflammatory chemotactic factors were found in the brains of AD patients, through a mechanism that may involve the inflammatory response induced by Aß in the processing of AD.

Changes in readthrough acetylcholinesterase expression modulate amyloid-beta pathology.

Alzheimer's disease has long been known to involve cholinergic deficits, but the linkage between cholinergic gene expression and the Alzheimer's disease amyloid pathology has remained incompletely understood. One known link involves synaptic acetylcholinesterase (AChE-S), shown to accelerate amyloid fibrils formation. Here, we report that the 'Readthrough' AChE-R splice variant, which differs from AChE-S in its 26 C-terminal residues, inversely exerts neuroprotective effects from amyloid beta (Abeta) induced toxicity. In vitro, highly purified AChE-R dose-dependently suppressed the formation of insoluble Abeta oligomers and fibrils and abolished Abeta toxicity to cultured cells, competing with the prevalent AChE-S protein which facilitates these processes. In vivo, double transgenic APPsw/AChE-R mice showed lower plaque burden, fewer reactive astrocytes and less dendritic damage than single APPsw mice, inverse to reported acceleration of these features in double APPsw/AChE-S mice. In hippocampi from Alzheimer's disease patients (n = 10), dentate gyrus neurons showed significantly elevated AChE-R mRNA and reduced AChE-S mRNA. However, immunoblot analyses revealed drastic reductions in the levels of intact AChE-R protein, suggesting that its selective loss in the Alzheimer's disease brain exacerbates the Abeta-induced damages and revealing a previously unforeseen linkage between cholinergic and amyloidogenic events.

P2X(7) receptor blockade prevents ATP excitotoxicity in oligodendrocytes and ameliorates experimental autoimmune encephalomyelitis.

Oligodendrocyte death and demyelination are hallmarks of multiple sclerosis (MS). Here we show that ATP signaling can trigger oligodendrocyte excitotoxicity via activation of calcium-permeable P2X(7) purinergic receptors expressed by these cells. Sustained activation of P2X(7) receptors in vivo causes lesions that are reminiscent of the major features of MS plaques, i.e., demyelination, oligodendrocyte death, and axonal damage. In addition, treatment with P2X(7) antagonists of chronic experimental autoimmune encephalomyelitis (EAE), a model of MS, reduces demyelination and ameliorates the associated neurological symptoms. Together, these results indicate that ATP can kill oligodendrocytes via P2X(7) activation and that this cell death process contributes to EAE. Importantly, P2X(7) expression is elevated in normal-appearing axon tracts in MS patients, suggesting that signaling through this receptor in oligodendrocytes may be enhanced in this disease. Thus, P2X(7) receptor antagonists may be beneficial for the treatment of MS.

Heparan sulfate accumulation with Abeta deposits in Alzheimer's disease and Tg2576 mice is contributed by glial cells.

Amyloid beta-peptide (Abeta) plaques, one of the major neuropathological lesions in Alzheimer's disease (AD), can be broadly subdivided into two morphological categories: neuritic and diffuse. Heparan sulfate (HS) and HS proteoglycans (HSPGs) are codeposits of multiple amyloidoses, including AD. Although HS has been considered a limiting factor in the initiation of amyloid deposition, the pathological implications of HS in Abeta deposits of AD remain unclear. In this study, immunohistochemistry combined with fluorescence and confocal microscopy was employed to gain deeper insight into the accumulation of HS with Abeta plaques in sporadic and familial AD. Here we demonstrate that HS preferentially accumulated around the Abeta40 dense cores of neuritic plaques, but was largely absent from diffuse Abeta42 plaques, suggesting that Abeta42 deposition may occur independently of HS. A codeposition pattern of HS with Abeta deposits in Tg2576 mice was also examined. We identified the membrane-bound HSPGs, glypican-1 (GPC1) and syndecan-3 (SDC3), in glial cells associated with Abeta deposits, proximal to sites of HS accumulation. In mouse primary glial cultures, we observed increased levels of GPC1 and SDC3 following Abeta stimulation. These results suggest that HS codeposits with Abeta40 in neuritic plaques and is mainly derived from glial cells.

Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue.

BACKGROUND: Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Quantitative real-time PCR (RT qPCR) is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB. RESULTS: The present study investigated the expression stability of 8 candidate reference genes, (ubiquitin C [UBC], tyrosine-3-monooxygenase [YWHAZ], RNA polymerase II polypeptide [RP II], hydroxymethylbilane synthase [HMBS], TATA box binding protein [TBP], beta-2-microglobulin [B2M], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and succinate dehydrogenase complex-subunit A, [SDHA]) in cerebellum and medial temporal gyrus of 6 AD, 6 PD, 6 DLB subjects, along with 5 matched controls using RT qPCR (TaqMan(R) Gene Expression Assays). Gene expression stability was analysed using geNorm to rank the candidate genes in order of decreasing stability in each disease group. The optimal number of genes recommended for accurate data normalization in each disease state was determined by pairwise variation analysis. CONCLUSION: This study identified validated sets of mRNAs which would be appropriate for the normalization of RT qPCR data when studying gene expression in brain tissue of AD, PD, DLB and control subjects.

Sorl1 as an Alzheimer's disease predisposition gene?

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressively disabling impairments in memory, cognition, and non-cognitive behavioural symptoms. Sporadic AD is multifactorial and genetically complex. While several monogenic mutations cause early-onset AD and gene alleles have been suggested as AD susceptibility factors, the only extensively validated susceptibility gene for late-onset AD is the apolipoprotein E (APOE) epsilon4 allele. Alleles of the APOE gene do not account for all of the genetic load calculated to be responsible for AD predisposition. Recently, polymorphisms across the neuronal sortilin-related receptor (SORL1) gene were shown to be significantly associated with AD in several cohorts. Here we present the results of our large case-control whole-genome scan at over 500,000 polymorphisms which presents weak evidence for association and potentially narrows the association interval.

The DeltaK280 mutation in MAP tau favors exon 10 skipping in vivo.

Tau mutations in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) are associated with changes in alternative splicing of exon 10. The DeltaK280 mutation in exon 10 is exceptional because in vitro observations suggest a dramatic effect on microtubule binding, enhanced self-aggregation, as well as a decrease of the 4R/3R ratio by the ablation of an exon splicing enhancer element. Using immunohistochemistry, Western blotting, and electron microscopy on brain material with the DeltaK280 mutation, we investigated which of these effects is most dominant in vivo. The brain showed abundant Pick bodies in several brain regions, which stained positive with 3-repeat-specific but not with 4-repeat-specific tau antibodies. Western blots of sarkosyl-insoluble tau showed exclusively three repeat (3R0N and 3R1N) tau in most regions, although some 4R1N could be detected in the frontal cortex. In addition, the sarkosyl-soluble tau fraction showed a significantly higher amount of 3-repeat tau. Because quantitative analysis of 4R and 3R mRNA transcripts showed a 4R/3R ratio of only 0.3, association between increased transcription and protein expression was observed. These observations confirm the postulated hypothesis that the DeltaK280 mutation abolishes a splice enhancer element, which overrules the decreased microtubule binding and enhanced self-aggregation.

Effects of formalin fixation, paraffin embedding, and time of storage on DNA preservation in brain tissue: a BrainNet Europe study.

There is a large amount of tissue stored in brain collections and brain banks, but little is known about whether formalin-fixed tissues and paraffin blocks stored for years in brain banks are suitable for the retrospective genetic studies. The study was carried out in order to: (i) compare DNA preservation in frozen, formalin-fixed and paraffin-embedded tissues stored for different periods; (ii) study point mutations and triplet expansions in frozen, formalin-fixed and paraffin-embedded material stored for variable periods, and using different fixative solutions; (iii) compare different methods to optimize DNA extraction and DNA amplification from suboptimally preserved brain tissue. DNA preservation is suitable for genetic studies in samples stored at -80 degrees C for several years. Formalin-fixed, paraffin-embedded tissue was inferior to frozen tissue, but did yield adequate results in many cases depending on the type of fixative solution and time of fixation before embedding. Prolonged fixation in formalin rarely yielded useful DNA. Similar results were obtained in samples from prion diseases. The best results were obtained by using the Qiagen kits (QIAmp DNA Micro) in frozen material, paraffin blocks and formalin-fixed tissue. Genomiphi and TaKaRa Ex Taq methods were also assayed in paraffin blocks and in formalin-fixed samples with limited success.

CCL5 and CCR5 genotypes modify clinical, radiological and pathological features of multiple sclerosis.

Chemokines mediate selective recruitment of leukocyte subsets into the CNS during inflammatory episodes. We hypothesised that functional polymorphisms in CCR5 and CCL5 influence perivascular leukocyte infiltration, inflammation, axonal loss, and remyelination, and disease course. Therefore, we determined genotypes at four possibly functional polymorphisms in CCR5 and CCL5 for 637 patients and 92 brain donors with multiple sclerosis (MS). For a subset of 192 patients, MRI data were available. We found that low-producer allele CCL5-403*G was associated with reduced risk of severe axonal loss, whereas high-producer allele CCL5-403*A was associated with a worse clinical disease course measured by the MS Functional Composite Score and MS Severity Score. Low-producer allele CCR5+303*G was associated with reduced T2 hyperintense and T1 hypointense lesion volumes on MRI, and high-producer allele CCR5+303*A with early age at onset. Furthermore, low-producer allele CCR5Delta32 was associated with reduced T2 lesion volume, lower black hole ratio on MRI, and with a higher percentage of lesions with signs of remyelination, histopathologically. In summary, our multifaceted study supports the notion that polymorphisms in CCL5 and CCR5 modify the course of MS.

Comparison analysis of gene expression patterns between sporadic Alzheimer's and Parkinson's disease.

Sporadic Alzheimer's (AD) and Parkinson's disease (PD) are late-onset neurodegenerative diseases with tremendous impact on lives of affected individuals. There is a great probability of developing concurrent Parkinsonism in AD and vice-versa than would be predicted by independent prevalence of each disease. We hypothesize that in sporadic AD as well as PD a combination of environmental effects and gene expression may affect specific brain areas leading to neurodegeneration. We profiled gene expression of AD compared to PD and age matched controls post-mortem in the hippocampus, the gyrus-frontalis-medius (Gfm) and the cerebellum using Gene-Chip microarray (Affymetrix) and quantitative-real-time-RT-PCR. Twelve genes altered in similar manner in AD and PD, while four genes showed differential expression profiles between AD and PD in different brain regions (cannabinoid-receptor-2, Histone-cluster-1-H3e, nicotinic-cholinergic-receptor-alpha6 and beta-site-APP-cleaving enzyme-1). Knowledge of selective gene expression profile can lead to better understanding of disease pathology and development of specific diagnosis and effective therapy.

Large-scale expression study of human mesial temporal lobe epilepsy: evidence for dysregulation of the neurotransmission and complement systems in the entorhinal cortex.

Human mesial temporal lobe epilepsies (MTLE) are the most frequent form of partial epilepsies and display frequent pharmacoresistance. The molecular alterations underlying human MTLE remain poorly understood. A two-step transcriptional analysis consisting in cDNA microarray experiments followed by quantitative RT-PCR validations was performed. Because the entorhinal cortex (EC) plays an important role in the pathophysiology of the MTLE and usually discloses no detectable or little cell loss, resected EC and each corresponding lateral temporal neocortex (LTC) of MTLE patients were used as the source of disease-associated and control RNAs, respectively. Six genes encoding (i) a serotonin receptor (HTR2A) and a neuropeptide Y receptor type 1 (NPY1R), (ii) a protein (FHL2) associating with the KCNE1 (minK) potassium channel subunit and with presenilin-2 and (iii) three immune system-related proteins (C3, HLA-DR-gamma and CD99), were found consistently downregulated or upregulated in the EC of MTLE patients as compared with non-epileptic autopsy controls. Quantitative western blot analyses confirmed decreased expression of NPY1R in all eight MTLE patients tested. Immunohistochemistry experiments revealed the existence of a perivascular infiltration of C3 positive leucocytes and/or detected membrane attack complexes on a subset of neurons, within the EC of nine out of eleven MTLE patients. To summarize, a large-scale microarray expression study on the EC of MTLE patients led to the identification of six candidate genes for human MTLE pathophysiology. Altered expression of NPY1R and C3 was also demonstrated at the protein level. Overall, our data indicate that local dysregulation of the neurotransmission and complement systems in the EC is a frequent event in human MTLE.

Modulating CCR2 and CCL2 at the blood-brain barrier: relevance for multiple sclerosis pathogenesis.

Chemokines and chemokine receptors play a key role in the transmigration of leucocytes across the blood-brain barrier (BBB). CCR2 is the major receptor for CCL2, a potent monocyte and T cell chemoattractant. CCR2 and CCL2 have been consistently associated with a pathogenic role in experimental autoimmune encephalomyelitis, using knockout and transgenic mice, neutralizing antibodies, peptide antagonists and DNA vaccination. However, the significance of CCL2 and CCR2 in multiple sclerosis is enigmatic, because CCL2 levels are consistently decreased in the CSF of patients with this disease and other chronic neuroinflammatory conditions, despite abundant expression within lesional multiple sclerosis tissues. This study used an in vitro BBB model to test the hypothesis that CCL2 is removed from the extracellular fluid by CCR2-positive migrating cells as they cross the BBB, resulting in decreased CSF CCL2 levels. We showed that CCR2-positive T cells and monocytes migrated selectively across the in vitro BBB, and that CCL2 on the abluminal (tissue) side was consumed by migrating T cells and monocytes. Next, we used a new anti-CCR2 antibody to show that CCR2-positive mononuclear inflammatory cells could be readily detected in appropriate positive control tissues, but that CCR2+ cells were very infrequently found in multiple sclerosis lesions. We then showed that CCR2 receptor density on T cells and monocytes was specifically downregulated upon in vitro BBB transmigration in response to CCL2, but not irrelevant chemokines. These findings document a novel strategy for analysing chemokine receptor function in inflammatory CNS disease, and support the hypothesis that CCL2 is consumed by migrating inflammatory cells, which downregulate CCR2, as they cross the BBB.

Interlaboratory comparison of assessments of Alzheimer disease-related lesions: a study of the BrainNet Europe Consortium.

This interlaboratory study evaluated the reproducibility of the assessments of neuritic plaques and neurofibrillary tangles (NFTs)--the hallmark lesions of Alzheimer disease--and compared the staining between the BrainNet Europe centers. To reduce the topography-related inconsistencies in assessments, we used a 2-mm tissue microarray (TMA) technique. The TMA block included 42 core samples taken from 21 paraffin blocks. The assessments were done on Bielschowsky and Gallyas silver stains using an immunohistochemical (IHC) method with antibodies directed to beta-amyloid (IHC/Abeta) and hyperphosphorylated tau (IHC/HPtau). The staining quality and the assessments differed between the participants, being most diverse with Bielschowsky (good/acceptable stain in 53% of centers) followed by Gallyas (good/acceptable stain in 57%) and IHC/Abeta (good/acceptable stain in 71%). The most uniform staining quality and assessment was obtained with the IHC/HPtau method (good/acceptable stain in 94% of centers). The neuropathologic diagnostic protocol (Consortium to Establish a Registry for Alzheimer Disease, Braak and Braak, and the National Institute of Aging and Reagan [NIA-Reagan] Institute) that was used significantly influenced the agreement, being highest with NIA-Reagan (54%) recommendations. This agreement was improved by visualization of NFTs using the IHC/HPtau method. Therefore, the IHC/HPtau methodology to visualize NFTs and neuropil threads should be considered as a method of choice in a future diagnostic protocol for Alzheimer disease.

Altered beta-secretase enzyme kinetics and levels of both BACE1 and BACE2 in the Alzheimer's disease brain.

beta-Secretase is the rate limiting enzymatic activity in the production of amyloid-beta peptide, the primary component of senile plaque pathology in Alzheimer's disease (AD). This study performed the first comparative analysis of beta-secretase enzyme kinetics in AD and control brain tissue. Results found V(max) values for beta-secretase to be significantly increased, and K(m) values unchanged in AD temporal cortex compared to matched control temporal cortex. The increased V(max) in AD cases, did not correlate with levels of BACE1, and decreased BACE1 and BACE2 levels correlated with the severity of neurofibrillary pathology (I-VI), and synaptic loss in AD. These results indicate that increased V(max) for beta-secretase is a feature of AD pathogenesis and this increase does not correlate directly with levels of BACE1, the principal beta-secretase in brain.