Detection of Proteins in Normal and Alzheimer's Disease Cerebrospinal Fluid with a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay

Abstract— Alzheimer's disease is a progressive degenerative dementia characterized by the abundant presence of neurofibrillary tangles in neurons. This study was designed to test whether the microtubule-associated protein, a major component of neurofibrillary tangles, could be detected in CSF. Additionally, we investigated whether CSF levels were abnormal in Alzheimer's disease as compared with a large group of control patients. We developed a sensitive sandwich enzyme-linked immunosorbent assay using AT120, a monoclonal antibody directed to human, as a capturing antibody. With this technique, the detection limit for was less than 5 pg/ml of CSF. Using ATS, which recognizes abnormally phosphorylated ser-ines 199–202 in, the detection limit was below 20 pg/ml of CSF. However, with AT8, we found no immunoreactiv-ity in CSF, suggesting that only a small fraction of CSF contains the abnormally phosphorylated AT8 epitope. Our results indicate that CSF levels are significantly increased in Alzheimer's disease. Also, CSF levels in a large group of patients with a diversity of neurological diseases showed overlap with CSF levels in Alzheimer's disease.     

An in vitro model for the study of microglia-induced neurodegeneration: involvement of nitric oxide and tumor necrosis factor-alpha

The precise function of activated microglia and their secretory products remains controversial. In order to assess the role of microglial secretion products, we established an in vitro model of an inflammatory reaction in the brain by co-culturing microglial and neuronal cell lines. Upon stimulation with interferon-gamma and lipopolysaccharides, the microglial cells adopted an activated phenotype and secreted tumor necrosis factor-alpha (TNF-alpha), prostaglandin E(2) and nitric oxide (NO). Neuronal degeneration was quantified by measuring the concentrations of microtubule associated protein tau and neuron specific enolase, which are also used as diagnostic tool in Alzheimer's disease, in supernatants. In activated contact co-cultures, the levels of these neuronal markers were significantly raised compared to non-activated co-cultures. NO-synthase inhibitors significantly diminished the rise of tau in activated co-cultures, while indomethacin, superoxide dismutase, or a neutralizing TNF-alpha antibody did not. When a chemical NO-donor or TNF-alpha were added to pure neuronal cultures, cell viability was significantly reduced. TNF-alpha increased neuronal sensitivity towards NO. There were indications that a part of the cells died by apoptosis. This model demonstrates a neurotoxic role for NO in microglia-induced neurodegeneration and provides a valuable in vitro tool for the study of microglia-neuron interactions during inflammation in the brain.     

Affinity Purification of Human τ Proteins and the Construction of a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay for Human τ Detection

Immunoaffinity chromatography with a monoclonal antibody produced against bovine tau protein was used to purify tau proteins from human brain. Fifty grams of brain tissue yielded approximately 2 mg of pure tau proteins. The affinity-purified human tau was used to produce a high-titered rabbit anti-human tau serum. The monoclonal anti-tau antibody and the polyclonal rabbit anti-tau serum were then used to construct a sandwich enzyme-linked immunosorbent assay for detection of human tau proteins, with a sensitivity of 1 ng/ml.     

Tau Monoclonal Antibody Generation Based on Humanized Yeast Models: IMPACT ON TAU OLIGOMERIZATION AND DIAGNOSTICS*

A link between Tau phosphorylation and aggregation has been shown in different models for Alzheimer disease, including yeast. We used human Tau purified from yeast models to generate new monoclonal antibodies, of which three were further characterized. The first antibody, ADx201, binds the Tau proline-rich region independently of the phosphorylation status, whereas the second, ADx215, detects an epitope formed by the Tau N terminus when Tau is not phosphorylated at Tyr¹⁸. For the third antibody, ADx210, the binding site could not be determined because its epitope is probably conformational. All three antibodies stained tangle-like structures in different brain sections of THY-Tau22 transgenic mice and Alzheimer patients, and ADx201 and ADx210 also detected neuritic plaques in the cortex of the patient brains. In hippocampal homogenates from THY-Tau22 mice and cortex homogenates obtained from Alzheimer patients, ADx215 consistently stained specific low order Tau oligomers in diseased brain, which in size correspond to Tau dimers. ADx201 and ADx210 additionally reacted to higher order Tau oligomers and presumed prefibrillar structures in the patient samples. Our data further suggest that formation of the low order Tau oligomers marks an early disease stage that is initiated by Tau phosphorylation at N-terminal sites. Formation of higher order oligomers appears to require additional phosphorylation in the C terminus of Tau. When used to assess Tau levels in human cerebrospinal fluid, the antibodies permitted us to discriminate patients with Alzheimer disease or other dementia like vascular dementia, indicative that these antibodies hold promising diagnostic potential.     

Identification of Novel ��-Synuclein Isoforms in Human Brain Tissue by using an Online NanoLC-ESI-FTICR-MS Method

Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are neurodegenerative diseases that are characterized by intra-neuronal inclusions of Lewy bodies in distinct brain regions. These inclusions consist mainly of aggregated α-synuclein (α-syn) protein. The present study used immunoprecipitation combined with nanoflow liquid chromatography (LC) coupled to high resolution electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) to determine known and novel isoforms of α-syn in brain tissue homogenates. N-terminally acetylated full-length α-syn (Ac-α-syn?????) and two N-terminally acetylated C-terminally truncated forms of α-syn (Ac-α-syn????? and Ac-α-syn?????) were found. The different forms of α-syn were further studied by Western blotting in brain tissue homogenates from the temporal cortex Brodmann area 36 (BA36) and the dorsolateral prefrontal cortex BA9 derived from controls, patients with DLB and PD with dementia (PDD). Quantification of α-syn in each brain tissue fraction was performed using a novel enzyme-linked immunosorbent assay (ELISA).     

Genetic distinction between contiguous urban and rural multimammate mice in Tanzania despite gene flow

Special conditions are required for genetic differentiation to arise at a local geographical scale in the face of gene flow. The Natal multimammate mouse, Mastomys natalensis, is the most widely distributed and abundant rodent in sub‐Saharan Africa. A notorious agricultural pest and a natural host for many zoonotic diseases, it can live in close proximity to humans and appears to compete with other rodents for the synanthropic niche. We surveyed its population genetic structure across a 180‐km transect in central Tanzania along which the landscape varied between agricultural land in a rural setting and natural woody vegetation, rivers, roads and a city (Morogoro). We sampled M. natalensis across 10 localities and genotyped 15 microsatellite loci from 515 individuals. Hierarchical STRUCTURE analyses show a K‐invariant pattern distinguishing Morogoro suburbs (located in the centre of the transect) from nine surrounding rural localities. Landscape connectivity analyses in Circuitscape and comparison of rainfall patterns suggest that neither geographical isolation nor natural breeding asynchrony could explain the genetic differentiation of the urban population. Using the isolation‐with‐migration model implemented in IMa2, we inferred that a split between suburban and rural populations would have occurred recently (<150 years ago) with higher urban effective population density consistent with an urban source to rural sink of effective migration. The observed genetic differentiation of urban multimammate mice is striking given the uninterrupted distribution of the animal throughout the landscape and the high estimates of effective migration (2N_eM = 3.0 and 29.7), suggesting a strong selection gradient across the urban boundary.     

A First Tetraplex Assay for the Simultaneous Quantification of Total α-Synuclein,Tau, β-Amyloid42 and DJ-1 in Human Cerebrospinal Fluid

The quantification of four distinct proteins (α-synuclein, β-amyloid_1-42, DJ-1, and total tau) in cerebrospinal fluid (CSF) has been proposed as a laboratory-based platform for the diagnosis of Parkinson’s disease (PD) and Alzheimer’s disease (AD). While there is some clinical utility in measuring these markers individually, their usage in routine clinical testing remains challenging, in part due to substantial overlap of concentrations between healthy controls and diseased subjects. In contrast, measurement of different analytes in a single sample from individual patients in parallel appears to considerably improve the accuracy of AD or PD diagnosis. Here, we report the development and initial characterization of a first, electrochemiluminescence-based multiplex immunoassay for the simultaneous quantification of all four proteins (‘tetraplex’) in as little as 50 μl of CSF. In analytical performance experiments, we assessed its sensitivity, spike-recovery rate, parallelism and dilution linearity as well as the intra- and inter-assay variability. Using our in-house calibrators, we recorded a lower limit of detection for α-synuclein, β-amyloid₄₂, DJ-1, and t-tau of 1.95, 1.24, 5.63, and 4.05 pg/ml, respectively. The corresponding, linear concentration range covered >3 orders of magnitude. In diluted CSF samples (up to 1:4), spike-recovery rates ranged from a low of 55% for β-amyloid₄₂ to a high of 98% for DJ-1. Hillslopes ranged from 1.03 to 1.30, and inter-assay variability demonstrated very high reproducibility. Our newly established tetraplex assay represents a significant technical advance for fluid-based biomarker studies in neurodegenerative disorders allowing the simultaneous measurement of four pivotal makers in single CSF specimens. It provides exceptional sensitivity, accuracy and speed.     

Truncated beta-amyloid peptide species in pre-clinical Alzheimer's disease as new targets for the vaccination approach

Vaccination against human beta-amyloid peptide (A beta) has been shown to remove the amyloid burden produced in transgenic mice overexpressing the mutated human amyloid precursor protein (APP) gene. For human beings, the efficiency of this therapeutic strategy has to take into account the specificities of human amyloid, especially at the early stages of 'sporadic' Alzheimer's disease (AD). A beta 40/42 were previously quantified in tissues from our well-established brain bank, including non-demented individuals with both mild amyloid and tau pathologies, hence corresponding to the earliest stages of Alzheimer pathology. Herein, we have adapted a proteomic method combined with western blotting and mass spectrometry for the characterization of insoluble A beta extracted in pure-formic acid. We demonstrated that amino-truncated A beta species represented more than 60% of all A beta species, not only in full blown AD, but also, and more interestingly, at the earliest stage of Alzheimer pathology. At this stage, A beta oligomers were exclusively made of A beta-42 species, most of them being amino-truncated. Thus, our results strongly suggest that amino-truncated A beta-42 species are instrumental in the amyloidosis process. In conclusion, a vaccine specifically targeting these pathological amino-truncated species of A beta-42 are likely to be doubly beneficial, by inducing the production of specific antibodies against pathological A beta products that are, in addition, involved in the early and basic mechanisms of amyloidosis in humans.     

CSF total tau, Aβ42 and phosphorylated tau protein as biomarkers for Alzheimer’s disease

With the arrival of effective symptomatic treatments and the promise of drugs that may delay progression, we now need to identify Alzheimer’s disease (AD) at an early stage of the disease. To diagnose AD earlier and more accurately, attention has been directed toward peripheral biochemical markers. This article reviews promising potential cerebrospinal fluid (CSF) biomarkers for AD focussing on their role in clinical diagnosis. In particular, two biochemical markers, CSF total tau (t-tau) protein and the 42 amino acid form of β-amyloid (Aβ42), perform satisfactorily enough to achieve a role in the clinical diagnostic settings of patients with dementia together with the cumulative information from basic clinical work-up, genetic screening, and brain imaging. These CSF markers are particularly useful to discriminate early or incipient AD from age-associated memory impairment, depression, and some secondary dementias. In order to discriminate AD from other primary dementia disorders, however, more accurate and specific markers are needed. Preliminary evidence strongly suggests that quantification of tau phosphorylated at specific sites in CSF improves early detection, differential diagnosis, and tracking of disease progression in AD.