Raúl Mena

Staging the pathological assembly of truncated tau protein into paired helical filaments in Alzheimer's disease.

Abstract Tau protein, which is incorporated into the core of paired helical filaments (PHFs) in Alzheimer’s disease (AD), can be characterised immunochemically by C-terminal truncation at Glu-391 recognised by monoclonal antibody (mAb) 423, and acid-reversible occlusion of a generic tau epitope in the tandem repeat region recognised by mAb 7.51. PHFs are also characterised by the presence of binding sites for a fluorescent dye (thiazin red) which can be used to differentiate between amorphous and fibrillar states of tau and β-amyloid proteins in AD. We have used double labelling confocal microscopy to investigate the state of aggregation of the tau antigens associated with the core structure of the PHF at early stages of neurofibrillary pathology. We report that the early abnormal tau deposits in cells vulnerable to neurofibrillary degeneration are characterised by C-terminal truncation at Glu-391, acid-reversible occlusion of the mAb 7.51 epitope, and the absence of binding sites for thiazin red, consistent with the amorphous non-fibrillar structure demonstrated by immunoelectron microscopy. Transition to the fibrillar state in the PHF is associated with acid-reversible occlusion of both mAb 7.51 and 423 epitopes, and acquisition of binding sites for thiazin red. In neurites, the transition between the two states of aggregation shows distal to proximal polarity, with the fibrillar state found nearest the cell body. These findings demonstrate that the assembly of tau protein into the PHF occurs in at least two stages, an amorphous stage characterised by C-terminal truncation and occlusion of sites within the tandem repeat region, and a fibrillar stage characterised by acid-reversible occlusion of both epitopes via addition of intact tau molecules in the fuzzy coat of the PHF.

Earliest Stages of Tau Conformational Changes are Related to the Appearance of a Sequence of Specific Phospho-Dependent Tau Epitopes in Alzheimer's Disease1

Neurofibrillary tangles (NFT) and dystrophic neurites represent dense cytoplasmic accumulations of abnormal polymers in the brain of patients with Alzheimers disease (AD). These polymers are referred to as paired helical filaments (PHFs) whose main structural core is composed of tau protein. Tau processing has been associated with hyperphosphorylation and truncation that results in PHF assembly. Both molecular events appear to cause conformational change of tau molecules [11,17,32]. In this regard, in a previous work focused on the analysis of patterns of immunolabeling in pre-tangle cells, we found that regional changes precede the structural modifications in tau [32]. In the present study, we further analyzed the early stages of tau processing in pre-tangle cells by using a variety of immunological markers of specific N-terminus phosphorylation tau sites. We used AT100, TG-3, AT8, pT231, Alz-50, Tau-C3 and 423 antibodies that recognize different abnormal tau epitopes in AD brains. These antibodies were combined and analyzed using a confocal microscope. Our results indicate that the early stages of abnormal tau processing are characterized by a sequential appearance of specific phospho-dependent epitope. The cascade of appearance of the antibodies is: pT231 --> TG-3 --> AT8 -->AT100 --> Alz-50. In addition; truncation at Asp-421 of the C-terminus of tau protein, as detected by Tau-C3, is also an early molecular event in tau protein aggregation prior to PHF formation in AD.

Cleavage and conformational changes of tau protein follow phosphorylation during Alzheimer’s disease

Phosphorylation, cleavage and conformational changes in tau protein all play pivotal roles during Alzheimer’s disease (AD). In an effort to determine the chronological sequence of these changes, in this study, using confocal microscopy, we compared phosphorylation at several sites (Ser199/202/396/404/422‐Thr205 and the second repeat domain), cleavage of tau (D421) and the canonical conformational Alz‐50 epitope. While all of these posttranslational modifications are found in neurofibrillary tangles (NFTs) at all stages of the disease, we found significantly higher numbers of phospho‐tau positive NFTs when compared with cleaved tau (P = 0.006 in Braak III; P = 0.002 in Braak IV; P = 0.012 in Braak V) or compared with the Alz‐50 epitope (P < 0.05). Consistent with these findings, in a double transgenic mice model (Tet/GSK‐3β/VLW) overexpressing the enzyme glycogen synthase kinase‐3β (GSK‐3β) and tau with a triple FTDP‐17 mutation (VLW) with AD‐like neurodegeneration, phosphorylation at sites Ser199/202‐Thr205 was greater than truncated tau. Taken together, these data strongly support the notion that the conformational changes and truncation of tau occur after the phosphorylation of tau. We propose two probable pathways for the pathological processing of tau protein during AD, either phosphorylation and cleavage of tau followed by the Alz‐50 conformational change or phosphorylation followed by the conformational change and cleavage as the last step.

Accumulation of Aspartic Acid421- and Glutamic Acid391-Cleaved Tau in Neurofibrillary Tangles Correlates With Progression in Alzheimer Disease

Truncations of tau protein at aspartic acid421 (D421) and glutamic acid391 (E391) residues are associated with neurofibrillary tangles (NFTs) in the brains of Alzheimer disease (AD) patients. Using immunohistochemistry with antibodies to D421- and E391-truncated tau (Tau-C3 and MN423, respectively), we correlated the presence of NFTs composed of these truncated tau proteins with clinical and neuropathologic parameters in 17 AD and 23 non-AD control brains. The densities of NFTs composed of D421- or E391-truncated tau correlated with clinical dementia index and Braak staging in AD. Glutamic acid391 tau truncation was prominent in the entorhinal cortex, whereas D421 truncation was prominent in the subiculum, suggesting that NFTs composed of either D421- or E391-truncated tau may be formed mutually exclusively in these areas. Both truncations were associated with the prevalence of the apolipoprotein E ϵ4 allele. By double labeling, intact tau in NFTs was commonly associated with D421-cleaved tau but not with E391-truncated tau; D421-cleaved tau was never associated with E391-truncated tau. These results indicate that tau is not randomly proteolyzed at different domains, and that proteolysis occurs sequentially from the C-terminus to inner regions of tau in AD progression. Identification of NFTs composed of tau at different stages of truncation may facilitate assessment of neurofibrillary pathology in AD.

Monitoring pathological assembly of tau and β-amyloid proteins in Alzheimer's disease

This double-labelling confocal microscopy study of the neuropathology of Alzheimers disease (AD) reports the use of a fluorescent dye, thiazin red, which has staining properties similar to thioflavin-S. Thiazin red fluorescence can be visualised selectively in the red channel, and we have used this property to compare it with the labelling seen using monoclonal antibody (mAb) 423, which detects tau protein C-terminally truncated at Glu-391, and mAb 4G8, which detects β-amyloid protein. Thiazin red is shown to recognized the typical histopathological deposits associated with both proteins. However, not all deposits containing these proteins are stained. Specifically, diffuse β-amyloid plaques and severely degraded extracellular tangles are unlabelled. Likewise a characteristic mAb 423-reactive granular plaque-like structure, typically present in cases with abundant extracellular tangels, is unlabelled by thiazin red. Such plaques can be shown to be continuous with the basal dendrites of degraded tanglebearing pyramidal cells. These findings suggest that paired helical filaments (PHFs) continue to undergo degradation in the extracellular space, which is associated with loss of thiazin red binding sites, but preservation of mAb 423 immunoreactivity. This epitope appears to be characteristic of a stable core element of the PHF which is highly resistant to proteolysis. Compounds such as thiazin red with high affinity for β-pleated protein structures can be used to monitor the state of pathological assembly of amyloidogenic protein species found in AD.

Regional conformational change involving phosphorylation of tau protein at the Thr231, precedes the structural change detected by Alz-50 antibody in Alzheimer's disease

Neurofibrillary tangles (NFTs) are the neuropathological hallmarks in Alzheimers disease (AD). Densities of NFTs correlate with the dementia status. NFTs reflect the intracellular accumulation of abnormal paired helical filaments (PHFs) composed of the microtubule-associated protein tau. Hyperphosphorylation and truncation have been proposed as key events leading to the genesis of PHFs. A recent hypothesis involving conformational changes has been emerging. These structural modifications of the tau protein were detected by monoclonal antibodies (mAbs) recognizing discontinuous epitopes along the tau molecule such as Alz-50, Tau-66 and MC1. A new mAb, TG-3, detects an early pathology in AD. The epitope of mAb TG-3 maps to phosphorylated Thr231 when the tau molecule is conformationally altered. In the present study, we used confocal microscopy to analyze the state of tau molecule adopting the TG-3 conformation during tangle formation. We also compared mAb TG-3 immunoreactivity with that of mAb Alz-50. Immunoelectronmicroscopy was also performed. N- and C- termini markers evidenced that the tau molecule is intact when it adopts the TG-3 conformation. In addition to NFT, mAb TG-3 also recognized NFT-not bearing-neurons suggesting an early processing of tau prior to NFT formation. Ultrastructural analysis evidenced the presence of TG-3 and Alz-50 immunoreactive products on organelles including mitochondria and endoplasmic reticulum. Nuclear heterochromatin was densely immunolabelled. These results together with the fact that TG-3 immunoreactivity is related to intact tau suggest that the conformation recognized by TG-3 is early staged in the neuronal pathology of AD. In addition, we document that the earliest changes in tau occur closely associated with organelles and heterochromatin.

Biochemical and histochemical analysis of 71 kDa dystrophin isoform (Dp71f) in rat brain.

Dp71 is a member of the dystrophin family and the most abundant dmd gene product in the brain. In the present study, we focused on a short dystrophin transcript named Dp71f, which is alternatively spliced when exon 78 is absent The topographic localization of this protein in the encephalon has not been properly described yet, nor its cellular or subcellular localization, and even less its functions. Dp71f was found to be a cytoplasmic 70 kDa protein and localized in all encephalon regions studied. Double labeling using specific markers for various cell types confirmed Dp71f distribution in the cytoplasm of all cell types studied. Labeling was more conspicuous near the nucleus and diminished towards the periphery of cells. In some cases, we observed cells that were positive for actin and Dp71f in regions corresponding to lamellipodia-like structures. Dp71f and Dp71d isoforms were differently distributed. Our study is the first specific and unambiguous description of the topography and cellular localization patterns of Dp71f in brain, suggesting that Dp71f is a ubiquitous protein.

The extent of neurofibrillary pathology in perforant pathway neurons is the key determinant of dementia in the very old

Abstract Neurofibrillary pathology as found in Alzheimer’s disease (AD) is also found in the normal elderly, suggesting that these changes may be part of the aging process. In this study, we assessed the densities and distribution of structures recognized by the monoclonal antibody (mAb) to phosphorylated tau (AT8) in the hippocampal formation and medial temporal isocortex of 19 centenarians. Of these, 4 cases were demented and 15 non-demented. AT8 immunoreactivity correlated with the global deterioration scale (GDS). The density of both intraneuronal neurofibrillary tangles (I-NFTs) and neuritic clusters (NCs) significantly correlated with the GDS in the layer II of the entorhinal cortex (r = 0.66, P = 0.005 and r = 0.611, P = 0.01, respectively). Density of I-NFTs in the subiculum (r = 0.491; P = 0.034) also correlated significantly. No other area was found to be statistically significant. Importantly, no correlation was found when demented and non-demented centenarian cases were analyzed separately, suggesting that the difference marks a fundamental shift between AD and non-demented individuals. This assertion is supported by the significantly higher densities of I-NFTs and NCs in the transentorhinal (P = 0.043 and P = 0.011, respectively) and layer II of the entorhinal cortex (P = 0.02 and P = 0.007, respectively), and I-NFTs in the subiculum (P < 0.001) and CA1 (P = 0.011) in the demented group when compared with the non-demented cases. Granular diffuse deposits, an early stage parameter of the neurofibrillary pathology involving accumulation of non-fibrillar abnormally phosphorylated tau protein did not correlate with the GDS or between the two groups studied. This study, combining morphometric and confocal analyses, not only provides further evidence that, in the brains of patients with AD, the perforant pathway is highly sensitive to tau pathology but also that involvement is distinct from the changes of normal aging, even of the oldest old.

Whole Transcriptome RNA-Seq Analysis of Breast Cancer Recurrence Risk Using Formalin-Fixed Paraffin-Embedded Tumor Tissue

RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue form the basis for widely used clinical diagnostic tests; however, RT-PCR is practically constrained in the number of transcripts that can be interrogated. We have developed and optimized RNA-Seq library chemistry as well as bioinformatics and biostatistical methods for whole transcriptome profiling from FFPE tissue. The chemistry accommodates low RNA inputs and sample multiplexing. These methods both enable rediscovery of RNA biomarkers for disease recurrence risk that were previously identified by RT-PCR analysis of a cohort of 136 patients, and also identify a high percentage of recurrence risk markers that were previously discovered using DNA microarrays in a separate cohort of patients, evidence that this RNA-Seq technology has sufficient precision and sensitivity for biomarker discovery. More than two thousand RNAs are strongly associated with breast cancer recurrence risk in the 136 patient cohort (FDR <10%). Many of these are intronic RNAs for which corresponding exons are not also associated with disease recurrence. A number of the RNAs associated with recurrence risk belong to novel RNA networks. It will be important to test the validity of these novel associations in whole transcriptome RNA-Seq screens of other breast cancer cohorts.

Accumulation of C-terminally truncated tau protein associated with vulnerability of the perforant pathway in early stages of neurofibrillary pathology in Alzheimer's disease.

Neurofibrillary pathology is a characteristic hallmark of Alzheimers disease that is closely correlated with cognitive decline. We have analysed the density and distribution of neurofibrillary tangles (NFTs) that are immunoreactive with the monoclonal antibody (mAb) 423 in a prospectively analysed population of Alzheimers disease (AD) cases and age-matched controls. NFTs were examined in allocortical and isocortical areas and correlated with Braak pathological stage and clinical severity of dementia. The mAb 423 was used as it recognises a C-terminally truncated tau fragment that is a major constituent of NFTs. Our results show that extracellular NFTs and, to a lesser extent, intracellular NFTs, correlated significantly with both Braak stages and the clinical index of severity. Furthermore, a differential distribution of the two types of tangles indicates that layer II of the entorhinal cortex and the transentorhinal area are particularly vulnerable to neurofibrillary degeneration. These areas serve as a point of connection between isocortex and hippocampus. Our findings, therefore, suggest that the perforant pathway may be substantially affected by the accumulation of truncated tau protein in AD and that this represents a neuropathological predictor for the clinical severity of dementia. When neurofibrillary pathology was examined by combined labelling with mAbs 423 and Alz-50 and the dye thiazin red, we were able to demonstrate various stages of tau aggregation. The different stages may represent a sequence of conformational changes that tau proteins undergo during tangle formation in the allocortex during the early development of dementia in AD.