Multicenter assessment of CSF-phosphorylated tau for the prediction of conversion of MCI.

BACKGROUND: The measurement of hyperphosphorylated tau (p-tau) in CSF has been proposed as a biomarker candidate for the prediction of Alzheimer disease (AD) in patients with mild cognitive impairment (MCI). However, a standard quantitative criterion of p-tau has not been evaluated. OBJECTIVE: To assess in a multicenter study the predictive accuracy of an a priori defined criterion of tau phosphorylated at threonine 231 (p-tau(231)) for the prediction of conversion from MCI to AD during a short-term observation interval. METHODS: The study included 43 MCI converters, 45 stable MCI (average follow-up interval = 1.5 years), and 57 healthy controls (at baseline only). Subjects were recruited at four international expert sites in a retrospective study design. Cox regression models stratified according to center were used to predict conversion status. Bootstrapped 95% CIs of classification accuracy were computed. RESULTS: Levels of p-tau(231) were a significant predictor of conversion (B = 0.026, p = 0.001), independent of age, gender, Mini-Mental State Examination, and ApoE genotype. For an a priori-defined cutoff point (27.32 pg/mL), sensitivity ranged between 66.7 and 100% and specificity between 66.7 and 77.8% among centers. The bootstrapped mean percentage of correctly classified cases was 79.95% (95% CI = 79.9 to 80.00%). Post hoc defined cutoff values yielded a mean bootstrapped classification accuracy of 80.45% (95% CI = 80.24 to 80.76%). CONCLUSIONS: An a priori defined cutoff value of p-tau(231) yields relatively stable results across centers, suggesting a good feasibility of a standard criterion of p-tau(231) for the prediction of Alzheimer disease.

Imaging and CSF studies in the preclinical diagnosis of Alzheimer's disease.

It is widely believed that the path to early and effective treatment for Alzheimer's disease (AD) requires the development of early diagnostic markers that are both sensitive and specific. To this aim, using longitudinal study designs, we and others have examined magnetic resonance imaging (MRI), 2-fluoro-2-deoxy-d-glucose-positron emission tomography (FDG/PET), and cerebrospinal fluid (CSF) biomarkers in cognitively normal elderly (NL) subjects and in patients with mild cognitive impairment (MCI). Such investigations have led to the often replicated findings that structural evidence of hippocampal atrophy as determined by MRI, as well as metabolic evidence from FDG-PET scan of hippocampal damage, predicts the conversion from MCI to AD. In this article we present a growing body of evidence of even earlier diagnosis. Brain pathology can be detected in NL subjects and used to predict future transition to MCI. This prediction is enabled by examinations revealing reduced glucose metabolism in the hippocampal formation (hippocampus and entorhinal cortex [EC]) as well as by the rate of medial temporal lobe atrophy as determined by MRI. However, neither regional atrophy nor glucose metabolism reductions are specific for AD. These measures provide secondary not primary evidence for AD. Consequently, we will also summarize recent efforts to improve the diagnostic specificity by combining imaging with CSF biomarkers and most recently by evaluating amyloid imaging using PET. We conclude that the combined use of conventional imaging, that is MRI or FDG-PET, with selected CSF biomarkers incrementally contributes to the early and specific diagnosis of AD. Moreover, selected combinations of imaging and CSF biomarkers measures are of importance in monitoring the course of AD and thus relevant to evaluating clinical trials.

Longitudinal CSF and MRI biomarkers improve the diagnosis of mild cognitive impairment.

The diagnosis of Alzheimer's disease (AD) in patients with mild cognitive impairment (MCI) is limited because it is based on non-specific behavioral and neuroimaging findings. The lesions of Alzheimer's disease: amyloid beta (Abeta) deposits, tau pathology and cellular oxidative damage, affect the hippocampus in the earlier stages causing memory impairment. In a 2-year longitudinal study of MCI patients and normal controls, we examined the hypothesis that cerebrospinal fluid (CSF) markers for these pathological features improve the diagnostic accuracy over memory and magnetic resonance imaging (MRI)-hippocampal volume evaluations. Relative to control, MCI patients showed decreased memory and hippocampal volumes and elevated CSF levels of hyperphosphorylated tau and isoprostane. These two CSF measures consistently improved the diagnostic accuracy over the memory measures and the isoprostane measure incremented the accuracy of the hippocampal volume achieving overall diagnostic accuracies of about 90%. Among MCI patients, over 2 years, longitudinal hippocampal volume losses were closely associated with increasing hyperphosphorylated tau and decreasing amyloid beta-42 levels. These results demonstrate that CSF biomarkers for AD contribute to the characterization of MCI.

MRI and CSF studies in the early diagnosis of Alzheimer's disease.

The main goal of our studies has been to use MRI, FDG-PET, and CSF biomarkers to identify in cognitively normal elderly (NL) subjects and in patients with mild cognitive impairment (MCI), the earliest clinically detectable evidence for brain changes due to Alzheimer's disease (AD). A second goal has been to describe the cross-sectional and longitudinal interrelationships amongst anatomical, CSF and cognition measures in these patient groups. It is now well known that MRI-determined hippocampal atrophy predicts the conversion from MCI to AD. In our summarized studies, we show that the conversion of NL subjects to MCI can also be predicted by reduced entorhinal cortex (EC) glucose metabolism, and by the rate of medial temporal lobe atrophy as determined by a semi-automated regional boundary shift analysis (BSA-R). However, whilst atrophy rates are predictive under research conditions, they are not specific for AD and cannot be used as primary evidence for AD. Consequently, we will also review our effort to improve the diagnostic specificity by evaluating the use of CSF biomarkers and to evaluate their performance in combination with neuroimaging. Neuropathology studies of normal ageing and MCI identify the hippocampal formation as an early locus of neuronal damage, tau protein pathology, elevated isoprostane levels, and deposition of amyloid beta 1-42 (Abeta42). Many CSF studies of MCI and AD report elevated T-tau levels (a marker of neuronal damage) and reduced Abeta42 levels (possibly due to increased plaque sequestration). However, CSF T-tau and Abeta42 level elevations may not be specific to AD. Elevated isoprostane levels are also reported in AD and MCI but these too are not specific for AD. Importantly, it has been recently observed that CSF levels of P-tau, tau hyperphosphorylated at threonine 231 (P-tau231) are uniquely elevated in AD and elevations found in MCI are useful in predicting the conversion to AD. In our current MCI studies, we are examining the hypothesis that elevations in P-tau231 are accurate and specific indicators of AD-related changes in brain and cognition. In cross-section and longitudinally, our results show that evaluations of the P-tau231 level are highly correlated with reductions in the MRI hippocampal volume and by using CSF and MRI measures together one improves the separation of NL and MCI. The data suggests that by combining MRI and CSF measures, an early (sensitive) and more specific diagnosis of AD is at hand. Numerous studies show that neither T-tau nor P-tauX (X refers to all hyper-phosphorylation site assays) levels are sensitive to the longitudinal progression of AD. The explanation for the failure to observe longitudinal changes is not known. One possibility is that brain-derived proteins are diluted in the CSF compartment. We recently used MRI to estimate ventricular CSF volume and demonstrated that an MRI-based adjustment for CSF volume dilution enables detection of a diagnostically useful longitudinal P-tau231 elevation. Curiously, our most recent data show that the CSF isoprostane level does show significant longitudinal elevations in MCI in the absence of dilution correction. In summary, we conclude that the combined use of MRI and CSF incrementally contributes to the early diagnosis of AD and to monitor the course of AD. The interim results also suggest that a panel of CSF biomarkers can provide measures both sensitive to longitudinal change as well as measures that lend specificity to the AD diagnosis.

Longitudinal cerebrospinal fluid tau load increases in mild cognitive impairment.

Cross-sectional cerebrospinal fluid (CSF) levels of tau and amyloid (A) beta (beta) are of diagnostic importance for Alzheimer's disease (AD) and mild cognitive impairment (MCI). However, most longitudinal studies of tau fail to demonstrate progression. Because predominantly brain-derived proteins such as tau, have higher ventricle to lumbar ratios, we hypothesized that adjusting for the ventricular enlargement of AD would correct for the dilution of tau, and improve detection of longitudinal change. Abeta which is not exclusively brain derived, shows a ratio <1, and no benefit was expected from adjustment. In a 1 year longitudinal study of eight MCI and ten controls, we examined CSF levels of hyperphosphorylated (P) tau231, Abeta40, and Abeta42. In cross-section, MCI patients showed elevated Ptau231 and Abeta40 levels, and greater ventricular volumes. Longitudinally, only after adjusting for the ventricular volume and only for Ptau231, were increases seen in MCI. Further studies are warranted on mechanisms of tau clearance and on using imaging to interpret CSF studies.

CSF tau protein phosphorylated at threonine 231 correlates with cognitive decline in MCI subjects.

In this longitudinal study of 77 patients with mild cognitive impairment (MCI), the authors analyzed whether levels of tau protein phosphorylated at threonine 231 (p-tau(231)) in CSF correlate with progression of cognitive decline. High CSF p-tau(231) levels at baseline, but not total tau protein levels, correlated with cognitive decline and conversion from MCI to AD. Independently, old age and APOE-epsilon 4 carrier status were predictive as well. Our data indicate that an increased p-tau(231) level is a potential risk factor for cognitive decline in patients with MCI.

Detection of tau phosphorylated at threonine 231 in cerebrospinal fluid of Alzheimer's disease patients.

A new sandwich ELISA is described which shows specificity for tau phosphorylated at threonine 231 and preferentially reacts with Alzheimer's disease (AD) brain extracts relative to other dementias. This assay was used to analyze 58 antemortem cerebrospinal fluid samples. Twenty-three of 27 AD samples (85% sensitivity) yielded signals greater than the cutoff, while only one of 31 non-AD samples (97% specificity) were greater. This indicates that detection of phosphotau in cerebrospinal fluid with this sandwich ELISA could prove useful in the diagnosis of AD.

Tau as a nucleolar protein in human nonneural cells in vitro and in vivo.

The Tau-1 monoclonal antibody was localized to the nucleolus of interphase cells and the nucleolar organizing regions (NORs) of acrocentric chromosomes in cultured human cells. Putative nucleolar and NOR tau was found in HeLa cells and lymphoblasts as well as in nontransformed fibroblasts and lymphocytes. To confirm the presence of tau in the nuclei of these nonneural cells, immunoblotting analysis was performed on isolated nuclei from lymphoblasts. Several tau bands were noted on the blot of the nuclear extract suggesting the presence of multiple tau isoforms. Tau-1 immunostaining demonstrated variable staining intensities between individual acrocentric chromosomes in all cells tested. In cultured peripheral lymphocytes, these staining patterns were the same from one chromosome spread to the next within an individual. This consistency of Tau-1 staining and its variability among NORs was reminiscent of staining patterns obtained using the silver-NOR procedure. Comparisons of Tau-1 immunostaining with silver staining of chromosome spreads from human lymphocytes demonstrated that Tau-1 did not immunostain all of the NORs that were silver stained. The intensity of Tau-1 fluorescence in nucleoli was further shown to be increased in phytohemagglutinin-stimulated lymphocytes, indicating an upregulation of nuclear tau when cells reentered the cell cycle. These results contribute to a growing body of evidence defining tau as a multifunctional protein that may be involved in ribosomal biogenesis and/or rRNA transcription in the nucleus of all cells as well as microtubule-stabilizing functions in the neuronal cytoplasm.

Functional implications for the microtubule-associated protein tau: localization in oligodendrocytes.

We present evidence that the microtubule-associated protein tau is present in oligodendrocytes (OLGs), the central nervous system cells that make myelin. By showing that tau is distributed in a pattern similar to that of myelin basic protein, our results suggest a possible involvement of tau in some aspect of myelination. Tau protein has been identified in OLGs in situ and in vitro. In interfascicular OLGs, tau localization, revealed by monoclonal antibody Tau-5, was confined to the cell somata. However, in cultured ovine OLGs with an exuberant network of processes, tau was detected in cell somata, cellular processes, and membrane expansions at the tips of these processes. Moreover, in such cultures, tau appeared localized adjacent to or coincident with myelin basic protein in membrane expansions along and at the ends of the cellular processes. The presence of tau mRNA was documented using fluorescence in situ hybridization. The distribution of the tau mRNA was similar to that of the tau protein. Western blot analysis of cultured OLGs showed the presence of many tau isoforms. Together, these results demonstrate that tau is a genuine oligodendrocyte protein and pave the way for determining its functional role in these cells.

Dissociation of centrosome replication events from cycles of DNA synthesis and mitotic division in hydroxyurea-arrested Chinese hamster ovary cells.

Relatively little is known about the mechanisms used by somatic cells to regulate the replication of the centrosome complex. Centrosome doubling was studied in CHO cells by electron microscopy and immunofluorescence microscopy using human autoimmune anticentrosome antiserum, and by Northern blotting using the cDNA encoding portion of the centrosome autoantigen pericentriolar material (PCM)-1. Centrosome doubling could be dissociated from cycles of DNA synthesis and mitotic division by arresting cells at the G1/S boundary of the cell cycle using either hydroxyurea or aphidicolin. Immunofluorescence micros-copy using SPJ human autoimmune anticentrosome antiserum demonstrated that arrested cells were able to undergo numerous rounds of centrosome replication in the absence of cycles of DNA synthesis and mitosis. Northern blot analysis demonstrated that the synthesis and degradation of the mRNA encoding PCM-1 occurred in a cell cycle-dependent fashion in CHO cells with peak levels of PCM-1 mRNA being present in G1 and S phase cells before mRNA amounts dropped to undetectable levels in G2 and M phases. Conversely, cells arrested at the G1/S boundary of the cell cycle maintained PCM-1 mRNA at artificially elevated levels, providing a possible molecular mechanism for explaining the multiple rounds of centrosome replication that occurred in CHO cells during prolonged hydroxyurea-induced arrest. The capacity to replicate centrosomes could be abolished in hydroxyurea-arrested CHO cells by culturing the cells in dialyzed serum. However, the ability to replicate centrosomes and to synthesize PCM-1 mRNA could be re-initiated by adding EGF to the dialyzed serum. This experimental system should be useful for investigating the positive and negative molecular mechanisms used by somatic cells to regulate the replication of centrosomes and for studying and the methods used by somatic cells for coordinating centrosome duplication with other cell cycle progression events.

Presence of tau in isolated nuclei from human brain.

Previous studies have demonstrated that the microtubule-associated protein (MAP) tau is present in the axonal and somatodendritic compartment of neurons. In cultured primate cell lines, tau has been found localized to the NOR regions of the acrocentric chromosomes in mitotic cells and the dense fibrillar regions of nucleoli in interphase cells. We report here the presence of nuclear tau in nuclei isolated from fresh, frozen human frontal cortex. Using several monoclonal antibodies against tau, Tau-1, Tau 46.1, and 5E2, we have established by both indirect immunofluorescence and Western blotting that tau is an integral component of nuclei isolated from Alzheimer's disease (AD) and pathologically normal control brains. Brain nuclear tau, like nuclear tau in primate cells, is insoluble in SDS and must first be extracted with formic acid prior to analysis by Western blot. Immunoblot analysis of isolated brain nuclei displays the characteristic ladder of tau proteins and demonstrates that all isoforms of tau are present. It is unclear whether levels of nuclear tau can be correlated to pathologic events in AD, but its insoluble nature along with reports of intranuclear PHFs warrant further studies of nuclear tau as a molecular candidate in the genesis of AD.

A novel tau transcript in cultured human neuroblastoma cells expressing nuclear tau.

We previously reported the presence of the microtubule-associated protein, tau in the nuclei of primate cells in culture. The present study confirms the existence of nuclear tau in two human neuroblastoma cells lines by indirect immunofluorescence and Western blot using mAbs to tau. Northern blot analysis of poly A+ mRNA detects a novel 2-kb tau transcript coexpressed with the 6-kb message in cultured human cells and human frontal cortex. PCR and cDNA sequencing demonstrate that the 2-kb message contains the entire tau coding region. Furthermore, actinomycin D transcription inhibition experiments indicate that the 2-kb message is not derived from the 6-kb message, but instead arises from the original tau transcript. One of the human neuroblastoma cell lines examined contains both nuclear and cytoplasmic tau as assayed by both Western blot and indirect immunofluorescence. Northern blot analysis of this cell line indicates that copious amounts of the 2-kb message are present while little of the 6-kb transcript is obvious. Immunofluorescence analysis of this cell line demonstrates that the cytoplasmic tau is not localized to microtubules. Together, these results indicate that the 2-kb tau message in humans may specify tau for non-microtubule functions in both the cytoplasm and the nucleus. We hypothesize that this is accomplished via a message targeting mechanism mediated by the untranslated regions of the tau messages.

Actin-binding protein is a component of bovine erythrocytes.

Actin-binding protein (ABP) is a well-characterized polypeptide capable of crosslinking filamentous actin. To date, this polypeptide has been shown to exist in a number of tissues and cultured cell lines. This report shows that by using a panel of three monoclonal antibodies for immunoblotting and immunofluorescence analysis, that ABP is present in bovine erythrocytes. Moreover, the data obtained suggest that this protein is a component of the erythrocyte membrane skeleton. Additionally, bovine erythrocyte ABP is shown to possess both an apparent molecular weight and an isoelectric point identical to that of bovine smooth muscle filamin, implying that these two polypeptides are identical.

The identification, purification, and characterization of a pancreatic beta-cell form of the microtubule adenosine triphosphatase kinesin.

Microtubules have been implicated as being necessary for the secretion of insulin from beta-cells, although the mechanism by which cytoplasmic microtubules contribute to the release of insulin is unknown. Kinesin is a microtubule-dependent adenosine triphosphatase (ATPase) that is thought to be responsible for the intracellular transport of vesicles and organelles. In this manuscript, the purification and preliminary characterization of a beta-cell form of kinesin is described. A 120-kilodalton antikinesin-reactive polypeptide was identified on blots when cultured insulinoma tumor cell lines were subjected to immunoblot analysis using monoclonal antibodies specific for the heavy chain of mammalian kinesin. The beta-cell form of kinesin was isolated from solid rat insulinoma tumors by cosedimentation of the kinesin with microtubules from tissue homogenates in the presence of adenylyl-imidodiphosphate. The beta-cell kinesin was further purified by gel filtration chromatography, and then the pure enzyme was characterized using in vitro assays. Although beta-cell kinesin showed little ATPase activity alone, the enzyme exhibited considerable ATP hydrolysis activity in the presence of taxol-stabilized microtubules. Moreover, in motility assays beta-cell kinesin was able to translocate microtubules across microscope coverslips in the presence of Mg(2+)-ATP. In summary, we report the identity of a novel islet beta-cell form of the microtubule-dependent ATPase kinesin and suggest a possible contribution of the microtubule cytoskeleton in insulin secretion.

Structure and molecular organization of the centromere-kinetochore complex.

For over a century, the terms centromere and kinetochore have been used interchangeably to describe a complex locus on eukaryotic chromosomes that attaches chromosomes to spindle fibres and facilitates chromosome movement in mitosis and meiosis. This region has become the focus of research aimed at defining the mechanism of chromosome segregation. A variety of new molecular probes and vastly improved optical-imaging technology have provided much new information on the structure of this locus and raised new hopes that an understanding of its function may soon be at hand.

The centromere-kinetochore complex: a repeat subunit model.

The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species.

CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase.

We have identified a novel human centromere-associated protein by preparing monoclonal antibodies against a fraction of HeLa chromosome scaffold proteins enriched for centromere/kinetochore components. One monoclonal antibody (mAb177) specifically stains the centromere region of mitotic human chromosomes and binds to a novel, approximately 250-300 kd chromosome scaffold associated protein named CENP-E. In cells progressing through different parts of the cell cycle, the localization of CENP-E differed markedly from that observed for the previously identified centromere proteins CENP-A, CENP-B, CENP-C and CENP-D. In contrast to these antigens, no mAb177 staining is detected during interphase, and staining first appears at the centromere region of chromosomes during prometaphase. This association with chromosomes remains throughout metaphase but is redistributed to the midplate at or just after the onset of anaphase. By telophase, the staining is localized exclusively to the midbody. Microinjection of the mAb177 into metaphase cells blocks or significantly delays progression into anaphase, although the morphology of the spindle and the configuration of the metaphase chromosomes appear normal in these metaphase arrested cells. This demonstrates that CENP-E function is required for the transition from metaphase to anaphase.

Autoantibodies from a patient with scleroderma CREST recognized kinetochores of the higher plant Haemanthus.

Human autoantibodies from a patient with scleroderma CREST (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, telangiectasia) were used to immunostain kinetochores on chromosomes in endosperm of the seed of the monocot Haemanthus katherinae Bak. Kinetochores of mitotic chromosomes and prekinetochores of interphase cells were specifically stained using conventional indirect immunofluorescence procedures as well as a nonfading immunogold-silver-enhanced technique and analyzed by fluorescence and video microscopy. In interphase, prekinetochores were either single or double structures depending on the stage of the cell cycle but became quadruple (two distinct stained dots on each chromatid) in mid-to-late prophase. In favorable preparations of prometaphase chromosomes, multiple subunits could be resolved within each sister kinetochore suggesting a compound organization. Western blot analysis demonstrated common epitopes in centromeric peptides of HeLa and Haemanthus cell extracts. Although the molecular mass of individual polypeptides differed in the two species, the presence of shared epitopes indicates striking conservation of centromere/kinetochore components throughout evolution.