Fibrillar Pathology Research Articles

Pathological conformations involving the amino terminus of tau occur early in Alzheimer's disease and are differentially detected by monoclonal antibodies

Conformational changes involving the amino terminus of the tau protein are among the earliest alterations associated with tau pathology in Alzheimer's disease and other tauopathies. This region of tau contains a phosphatase-activating domain (PAD) that is aberrantly exposed in pathological forms of the protein, an event that is associated with disruptions in anterograde fast axonal transport. We utilized four antibodies that recognize the amino terminus of tau, TNT1, TNT2 (a novel antibody), Tau12, and Tau13, to further study this important region. Using scanning alanine mutations in recombinant tau proteins, we refined the epitopes of each antibody. We examined the antibodies' relative abilities to specifically label pathological tau in non-denaturing and denaturing assays to gain insight into some of the mechanistic details of PAD exposure. We then determined the pattern of tau pathology labeled by each antibody in human hippocampal sections at various disease stages in order to characterize PAD exposure in the context of disease progression. The characteristics of reactivity for the antibodies fell into two groups. TNT1 and TNT2 recognized epitopes within amino acids 7–12 and specifically identified recombinant tau aggregates and pathological tau from Alzheimer's disease brains in a conformation-dependent manner. These antibodies labeled early pre-tangle pathology from neurons in early Braak stages and colocalized with thiazine red, a marker of fibrillar pathology, in classic neurofibrillary tangles. However, late tangles were negative for TNT1 and TNT2 indicating a loss of the epitope in later stages of tangle evolution. In contrast, Tau12 and Tau13 both identified discontinuous epitopes in the amino terminus and were unable to differentiate between normal and pathological tau in biochemical and tissue immunohistological assays. Despite the close proximity of these epitopes, the antibodies demonstrated remarkably different abilities to identify pathological changes in tau indicating that detection of conformational alterations involving PAD exposure is not achieved by all N-terminal tau antibodies and that a relatively discrete region of the N-terminus (i.e., amino acids 7–12, the TNT1 and TNT2 epitope) is central to the differences between normal and pathological tau. The appearance of PAD in early tau pathology and its disappearance in late-stage tangles suggest that toxic forms of tau are associated with the earliest forms of tau deposits. Collectively, these findings demonstrate that the TNT antibodies are useful markers for early conformational display of PAD and provide information regarding conformational changes that have potential implications in the toxic mechanisms of tau pathology.

100 Years of Alzheimer’s Disease Research: Are We Any Closer to a Cure?

Alzheimer’s disease (AD), the most common form of senile dementia, is characterized by a progressive disruption of memory, cognition, functional ability, mood and behavior. At the 100th anniversary of Alois Alzheimer’s (1864–1915) first description of AD, a tremendous amount of scientific insight into this devastating neurological affliction has been obtained; however, many gaps in our knowledge still remain. Our increased life expectancy and the demographics of our aging population cast significant healthcare concerns over the future management of AD. There are currently no curative or preventive treatments for this leading cause of cognitive failure, and pharmacological strategies directed at AD symptoms, and specifically targeted at the progressive and inflammatory nature of this brain degeneration, have met with disappointing results. In the second century of AD research, further objective studies, alternative pharmacological strategies, and the development of more efficacious drugs are clearly required to better address this complex and expanding healthcare problem. Alois Alzheimer first described the clinical and neuropathological findings of a novel neuropsychiatric disorder in a female patient who died of a progressive, atypical, senile psychosis, in 1906 at a meeting of European psychiatrists in Tubingen, Germany [1]. By the time he died, 9 years later, Alzheimer had become the first clinician–neuropathologist to correlate senile plaque (‘miliary foci’) and neurofibrillary tangle (‘fibrillar pathology’) densities with the degree of age-related senile dementia he observed in several AD patients. It is not as well known that Alzheimer also associated a peculiar cerebral vascular vessel involvement (‘focal lesions in the endothelium’) and angiogenesis (‘new vessel formation’) with the ‘steadily progressive disease process’ that now bears his name [1–5]. Over 100 years have passed since these original descriptions of AD; however, it was only in the last 40–50 years that several fundamental discoveries on the basic neuropathology and molecular neurobiology of senile plaques, neurofibrillary tangles and cerebrovascular changes have revolutionized our understanding of AD [2–34]. In part due to the concerns of the healthcare of our aging population, this complex neurological disorder now stands at the forefront of contemporary medical research. The ‘amyloid-β (Aβ) peptide cascade hypothesis’, that is, that Aβ peptide generation, speciation and aggregation, and Aβ peptide’s role as a trigger for brain-specific oxidative stress and inflammatory processes, lies at the very heart of the AD process, has advanced to the extent that the neurobiology of β-amyloid precursor protein (βAPP), and βAPP-derived peptides such as Aβ42, now represent one of the most thoroughly studied aspects of all of brain research [2–10]. However, despite these massive investigative efforts, the defining role of βAPP and Aβ42 peptides in the initiation and progression of AD remains largely unresolved, and the contributory role of neurofibrillary tangle formation to the AD process has received considerably less research attention [3–5]. Several relatively recent findings have not only expanded our insight into the pathogenic mechanisms of the AD process, but have had an impact on the direction of drug research and development and treatment strategies for targeting AD-specific pathways in the brain [3–8,11–19]. These include:

Characterization of Alzheimer paired helical filaments by electron microscopy

We show how electron microscopy can be used to answer several critical issues in neurodegenerative disorders that course with the formation of aberrant filamentous structures. Thus, electron microscopy is a useful technique to study in vitro assembly of pathogenic proteins, to map the regions involved in filament formation, as well as to detect by immunoelectron microscopy which proteins bind to the filaments. Furthermore, electron microscopy is the main technique used to discover if an animal model develops fibrillar pathology and if those filaments are similar to those found in human patients. This review focuses on Alzheimer's disease and related tauopathies, although similar studies have been done with other neurodegenerative disorders as, for example, Huntington's disease.

C-terminal inhibition of tau assembly in vitro and in Alzheimer's disease.

Alzheimer's disease (AD) is, in part, defined by the polymerization of tau into paired helical and straight filaments (PHF/SFs) which together comprise the fibrillar pathology in degenerating brain regions. Much of the tau in these filaments is modified by phosphorylation. Additionally, a subset also appears to be proteolytically truncated, resulting in the removal of its C terminus. Antibodies that recognize tau phosphorylated at S(396/404 )or truncated at E(391) do not stain control brains but do stain brain sections very early in the disease process. We modeled these phosphorylation and truncation events by creating pseudo-phosphorylation and deletion mutants derived from a full-length recombinant human tau protein isoform (ht40) that contains N-terminal exons 2 and 3 and all four microtubule-binding repeats. In vitro assembly experiments demonstrate that both modifications greatly enhance the rates of tau filament formation and that truncation increases the mass of polymer formed, as well. Removal of as few as 12 or as many as 121 amino acids from the C terminus of tau greatly increases the rate and extent of tau polymerization. However, deletion of an additional 7 amino acids, (314)DLSKVTS(320), from the third microtubule-binding repeat results in the loss of tau's ability to form filaments in vitro. These results suggest that only part of the microtubule-binding domain (repeats 1, 2 and a small portion of 3) is crucial for tau polymerization. Moreover, the C terminus of tau clearly inhibits the assembly process; this inhibition can be partially reversed by site-specific phosphorylation and completely removed by truncation events at various sites from S(320) to the end of the molecule.

Interaction of the Receptor for Advanced Glycation End Products (RAGE) with Transthyretin Triggers Nuclear Transcription Factor kB (NF-kB) Activation

Mutated transthyretin (TTR) fibrils are associated with the pathology of familial amyloidotic polyneuropathy (FAP), in which extracellular amyloid deposits lead to degeneration of cells and tissues, in particular neurons of the peripheral nerve. Here we present evidence that the receptor for advanced glycation end products (RAGE), previously associated with Alzheimer's disease, acts as a selective cell surface acceptor site for both soluble and fibrillar TTR. Immunohistochemical studies demonstrating increased expression of RAGE in FAP tissues suggested the relevance of this receptor to TTR-induced fibrillar pathology. In vitro studies using soluble RAGE showed saturable specific interaction with soluble and fibrillar TTR with a Kd of ∼120 nm. However, no binding was observed when soluble TTR was combined with retinol-binding protein, which represents the form in which TTR normally circulates in plasma. Specific binding of TTR to RAGE-transfected Chinese hamster ovary cells (which was completely blocked by anti-RAGE) was observed, confirming that RAGE could mediate TTR binding to cellular surfaces. RAGE-dependent activation of nuclear transcription factor kB (NF-kB) by TTR fibrils was shown in PC-12 cells stably transfected to overexpress the receptor. Furthermore, FAP nerves showed up-regulation of p50, one of the NF-kB subunits, when compared with age-matched controls. From these observations we predict that, in vivo, the presence of TTR fibrils associated with cellular surfaces of FAP patients, by contributing to NF-kB activation, leads to the pathogenesis of neurodegeneration. Further insights into the consequences of the interaction of fibrillar TTR with RAGE may therefore provide a better understanding of neurodegeneration associated with FAP.

Defective ubiquitination of cerebral proteins in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the presence of neurofibrillary tangles (NFT), senile plaques, and cerebrovascular deposits of amyloid-beta. Ubiquitin has also been shown to be present in some of the inclusions characteristic of this disease. To obtain further insight into the role played by the ubiquitin pathway in AD, we investigated the capacity of postmortem samples of cerebral cortex from normal and AD patients to form high-molecular-weight ubiquitin-protein conjugates. Activity of the ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2) involved in the ubiquitin pathway was also determined. In normal samples, the amount of high-molecular-weight ubiquitin-protein conjugates (HMW-UbPC) in cytosol increased with incubation time, whereas, in samples of AD cases, these were almost undetectable. The addition of an adult rat fraction, enriched in ubiquitinating enzymes, restored the capacity of AD brain cytosolic fraction to form conjugates. The trypsin-like proteolytic activity of the 26S proteasome was found to be decreased in AD cytosol brain. Assay of the activity of E1 and E2 by thiol-ester formation revealed a significant decrease in AD samples. Moreover, Western blotting using a specific antibody against E1 showed a dramatic drop of this enzyme in the cytosolic fraction, whereas normal levels were found in the particulate fraction, suggesting a possible delocalization of the enzyme. Our results suggest that a failure in the ubiquitination enzymatic system in brain cytosol may contribute to fibrillar pathology in AD.

A New Molecular Link between the Fibrillar and Granulovacuolar Lesions of Alzheimer's Disease

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder involving select neurons of the hippocampus, neocortex, and other regions of the brain. Markers of end stage disease include fibrillar lesions, which accumulate hyperphosphorylated tau protein polymerized into filaments, and granulovacuolar lesions, which appear primarily within the hippocampus. The mechanism by which only select populations of neurons develop these lesions as well as the relationship between them is unknown. To address these questions, we have turned to AD tissue to search for enzymes specifically involved in tau hyperphosphorylation. Recently, we showed that the principal phosphotransferases associated with AD brain-derived tau filaments are members of the casein kinase-1 (CK1) family of protein kinases. Here we report the distribution of three CK1 isoforms (Ckialpha, Ckidelta, and Ckiepsilon) in AD and control brains using immunohistochemistry and Western analysis. In addition to colocalizing with elements of the fibrillar pathology, CK1 is found within the matrix of granulovacuolar degeneration bodies. Furthermore, levels of all CK1 isoforms are elevated in the CA1 region of AD hippocampus relative to controls, with one isoform, Ckidelta, being elevated >30-fold. We propose that overexpression of this protein kinase family plays a key role in the hyperphosphorylation of tau and in the formation of AD-related pathology.