Cysteine-Independent Inhibition of Alzheimer's Disease-like Paired Helical Filament Assembly by Leuco-Methylthioninium (LMT).

Alzheimer's disease is a tauopathy characterized by pathological fibrillization of tau protein to form the paired helical filaments (PHFs), which constitute neurofibrillary tangles. The methylthioninium (MT) moiety reverses the proteolytic stability of the PHF core and is in clinical development for treatment of Alzheimer's disease in a stable reduced form as leuco-MT. It has been hypothesized that MT acts via oxidation of cysteine residues, which is incompatible with activity in the predominantly reducing environment of living cells. We have shown recently that the PHF-core tau unit assembles spontaneously in vitro to form PHF-like filaments. Here we describe studies using circular dichroism, SDS-PAGE, transmission electron microscopy and site-directed mutagenesis to elucidate the mechanism of action of the MT moiety. We show that MT inhibitory activity is optimal in reducing conditions, that the active moiety is the reduced leuco-MT form of the molecule and that its mechanism of action is cysteine independent.

Common fibrillar spines of amyloid-ß and human islet amyloid polypeptide revealed by microelectron diffraction and structure-based inhibitors.

Amyloid-ß (Aß) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues and are associated with Alzheimer's disease (AD) and type II diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aß and hIAPP. Using the cryoEM method microelectron diffraction, we determined the atomic structures of 11-residue segments from both Aß and hIAPP, termed Aß(24-34) WT and hIAPP(19-29) S20G, with 64% sequence similarity. We observed a high degree of structural similarity between their backbone atoms (0.96-Å root mean square deviation). Moreover, fibrils of these segments induced amyloid formation through self- and cross-seeding. Furthermore, inhibitors designed for one segment showed cross-efficacy for full-length Aß and hIAPP and reduced cytotoxicity of both proteins, although by apparently blocking different cytotoxic mechanisms. The similarity of the atomic structures of Aß(24-34) WT and hIAPP(19-29) S20G offers a molecular model for cross-seeding between Aß and hIAPP.

mTor mediates tau localization and secretion: Implication for Alzheimer's disease.

Abnormally hyperphosphorylated tau aggregates form paired helical filaments (PHFs) in neurofibrillary tangles, a key hallmark of Alzheimer's disease (AD) and other tauopathies. The cerebrospinal fluid (CSF) levels of soluble total tau and phospho-tau from clinically diagnosed AD patients are significantly higher compared with controls. Data from both in vitro and in vivo AD models have implied that an aberrant increase of mammalian target of rapamycin (mTor) signaling may be a causative factor for the formation of abnormally hyperphosphorylated tau. In the present study, we showed that in post-mortem human AD brain, tau was localized within different organelles (autophagic vacuoles, endoplasmic reticulum, Golgi complexes, and mitochondria). In human SH-SY5Y neuroblastoma cells stably carrying different genetic variants of mTor, we found a common link between the synthesis and distribution of intracellular tau. mTor overexpression or the lack of its expression was responsible for the altered balance of phosphorylated (p-)/-non phosphorylated (Np-) tau in the cytoplasm and different cellular compartments, which might facilitate tau deposition. Up-regulated mTor activity resulted in a significant increase in the amount of cytosolic tau as well as its re-localization to exocytotic vesicles that were not associated with exosomes. These results have implicated that mTor is involved in regulating tau distribution in subcellular organelles and in the initiation of tau secretion from cells to extracellular space.

Extracellular monomeric tau protein is sufficient to initiate the spread of tau protein pathology.

Understanding the formation and propagation of aggregates of the Alzheimer disease-associated Tau protein in vivo is vital for the development of therapeutics for this devastating disorder. Using our recently developed live-cell aggregation sensor in neuron-like cells, we demonstrate that different variants of exogenous monomeric Tau, namely full-length Tau (hTau40) and the Tau-derived construct K18 comprising the repeat domain, initially accumulate in endosomal compartments, where they form fibrillar seeds that subsequently induce the aggregation of endogenous Tau. Using superresolution imaging, we confirm that fibrils consisting of endogenous and exogenous Tau are released from cells and demonstrate their potential to spread Tau pathology. Our data indicate a greater pathological risk and potential toxicity than hitherto suspected for extracellular soluble Tau.

Identification of polypeptides in neurofibrillary tangles and total homogenates of brains with Alzheimer's disease by tandem mass spectrometry.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly. AD brains are characterized by the presence of neurofibrillary tangles (NFTs) and neuritic plaques. NFTs are constituted of paired helical filaments, which are structurally composed by assembled hyperphosphorylated and truncated tau polypeptides. To date, the integral constituents of NFTs remain unknown mainly due to the high insolubility of NFTs. The aim of this study was to identify by tandem mass spectrometry, the polypeptides contained in both isolated NFTs by laser capture microdissection and total homogenates, using tissue sections from paraformaldehyde-fixed AD brains. In the first case, we isolated 2,000 NFTs from tissue samples of hippocampus from each of the three Mexican AD brains used in our study. These were previously stained with anti-hyperphosphorylated tau AT-100 antibodies. After the removal of paraformaldehyde and delipidation with organic solvents, we tested three solubilization methods. We identified 102 polypeptides from total homogenates and 41 from isolated NFTs. We selected UCH-L1, transferrin, and GAPDH polypeptides to be studied by immunofluorescence and confocal microscopy. Only UCH-L1 and GAPDH colocalized with hyperphosphorylated tau in NFTs.

Immunoelectron microscopic and biochemical studies of caspase-cleaved tau in a mouse model of tauopathy.

Neurofibrillary tangles (NFTs) have been implicated in mediating neuronal death and disease progression in human tauopathies; however, mounting in vivo data suggest that NFTs may not be the primary initiators of neurotoxicity. Caspase activity has been implicated in processes associated with the development of tauopathy, but the position that caspase activation holds in neurodegenerative cascades remains uncertain. Using multiphoton real-time imaging microscopy, de Calignon et al recently demonstrated that caspase activation precedes and leads to tangle formation within 24 hours in the rTg4510 mouse model of tauopathy. Here, we used immunoelectron microscopy to determine whether caspase-cleaved tau was present in NFTs of rTg4510 mice. Using a caspase-cleaved tau-specific antibody (TauC3), we found very little immunogold labeling in NFTs in the brains of rTg4510 mice. By immunohistochemistry, the number of TauC3-positive neurons was far less than the numbers of neurons stained with the MC1 antibody, which recognizes abnormal conformations of tau. Biochemically, caspase-cleaved tau was barely detectable in fractions of rTg4510 mouse brain extracts. Our data suggest that caspase activation might be one of multiple routes through which NFT formation occurs, rather than an obligatory initiation step in pathologic tau production in rTg4510 mice.

Grape derived polyphenols attenuate tau neuropathology in a mouse model of Alzheimer's disease.

Aggregation of microtubule-associated protein tau into insoluble intracellular neurofibrillary tangles is a characteristic hallmark of Alzheimer's disease (AD) and other neurodegenerative diseases, including progressive supranuclear palsy, argyrophilic grain disease, corticobasal degeneration, frontotemporal dementias with Parkinsonism linked to chromosome 17, and Pick's disease. Tau is abnormally hyperphosphorylated in AD and aberrant tau phosphorylation contributes to the neuropathology of AD and other tauopathies. Anti-aggregation and anti-phosphorylation are main approaches for tau-based therapy. In this study, we report that a select grape-seed polyphenol extract (GSPE) could potently interfere with the assembly of tau peptides into neurotoxic aggregates. Moreover, oral administration of GSPE significantly attenuated the development of AD type tau neuropathology in the brain of TMHT mouse model of AD through mechanisms associated with attenuation of extracellular signal-receptor kinase 1/2 signaling in the brain.

Pathogenic missense MAPT mutations differentially modulate tau aggregation propensity at nucleation and extension steps.

Mutations in the MAPT gene encoding tau protein lead to neurofibrillary lesion formation, neurodegeneration, and cognitive decline associated with frontotemporal lobar degeneration. While some pathogenic mutations affect MAPT introns, resulting in abnormal splicing patterns, the majority occur in the tau coding sequence leading to single amino acid changes in tau primary structure. Depending on their location within the polypeptide chain, tau missense mutations have been reported to augment aggregation propensity. To determine the mechanisms underlying mutation-associated changes in aggregation behavior, the fibrillization of recombinant pathogenic mutants R5L, G272V, P301L, V337M, and R406W prepared in a full-length four-repeat human tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Results indicated that the mutants differ from each other and from wild-type tau in their aggregation propensity. G272V and P301L mutations increased the rates of both filament nucleation and extension reactions, whereas R5L and V337M increased only the nucleation phase. R406W did not differ from wild-type in any kinetic parameter. The results show that missense mutations can directly promote tau filament formation at different stages of the aggregation pathway.

Tauopathy with paired helical filaments in an aged chimpanzee.

An enigmatic feature of age-related neurodegenerative diseases is that they seldom, if ever, are fully manifested in nonhuman species under natural conditions. The neurodegenerative tauopathies are typified by the intracellular aggregation of hyperphosphorylated microtubule-associated protein tau (MAPT) and the dysfunction and death of affected neurons. We document the first case of tauopathy with paired helical filaments in an aged chimpanzee (Pan troglodytes). Pathologic forms of tau in neuronal somata, neuropil threads, and plaque-like clusters of neurites were histologically identified throughout the neocortex and, to a lesser degree, in allocortical and subcortical structures. Ultrastructurally, the neurofibrillary tangles consisted of tau-immunoreactive paired helical filaments with a diameter and helical periodicity indistinguishable from those seen in Alzheimer's disease. A moderate degree of Abeta deposition was present in the cerebral vasculature and, less frequently, in senile plaques. Sequencing of the exons and flanking intronic regions in the genomic MAPT locus disclosed no mutations that are associated with the known human hereditary tauopathies, nor any polymorphisms of obvious functional significance. Although the lesion profile in this chimpanzee differed somewhat from that in Alzheimer's disease, the copresence of paired helical filaments and Abeta-amyloidosis indicates that the molecular mechanisms for the pathogenesis of the two canonical Alzheimer lesions--neurofibrillary tangles and senile plaques--are present in aged chimpanzees.

Role of calpain and caspase in beta-amyloid-induced cell death in rat primary septal cultured neurons.

The invariant characteristic features associated with Alzheimer's disease (AD) brain include the presence of extracellular neuritic plaques composed of amyloid beta (Abeta) peptide, intracellular neurofibrillary tangles containing hyper-phosphorylated tau protein and the loss of basal forebrain cholinergic neurons. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that in vivo accumulation of Abeta(1-42) may initiate the process of neurodegeneration observed in AD brains. However, the cause of degeneration of the basal forebrain cholinergic neurons and their association to Abeta peptides or phosphorylated tau protein have not been clearly established. In the present study, using rat primary septal cultures, we have shown that Abeta(1-42), in a time (1-48 h) and concentration (0.01-20 microM)-dependent manner, induce toxicity in cultured neurons. Subsequently, we have demonstrated that Abeta toxicity is mediated via activation of cysteine proteases, i.e., calpain and caspase, and proteolytic breakdown of their downstream substrates tau, microtubule-associated protein-2 and alpha II-spectrin. Additionally, Abeta-treatment was found to induce phosphorylation of tau protein along with decreased levels of phospho-Akt and phospho-Ser(9)glycogen synthase kinase-3beta. Exposure to specific inhibitors of caspase or calpain can partially protect cultured neurons against Abeta-induced toxicity but their effects are not found to be additive. These results, taken together, suggest that Abeta peptide can induce toxicity in rat septal cultured neurons by activating multiple intracellular signaling molecules. Additionally, evidence that inhibitors of caspase and calpains can partially protect the cultured basal forebrain neurons raised the possibility that their inhibitors could be of therapeutic relevance in the treatment of AD pathology.

Antioxidant compounds have potent anti-fibrillogenic and fibril-destabilizing effects for alpha-synuclein fibrils in vitro.

The aggregation of alpha-synuclein (alphaS) in the brain has been implicated as a critical step in the development of Lewy body diseases (LBD) and multiple system atrophy (MSA). Various antioxidants not only inhibit the formation of beta-amyloid fibrils (fAbeta), but also destabilize preformed fAb in vitro. Using fluorescence spectroscopy with thioflavin S and electron microscopy, here we examined the effects of the antioxidants nordihydroguaiaretic acid, curcumin, rosmarinic acid, ferulic acid, wine-related polyphenols [tannic acid, myricetin, kaempferol (+)-catechin and (-)-epicatechin], docosahexaenoic acid, eicosapentaenoic acid, rifampicin and tetracycline on the formation of alphaS fibrils (falphaS) and on preformed falphaS. All molecules, except for docosahexaenoic acid and eicosapentaenoic acid, dose-dependently inhibited the formation of falphaS. Moreover, these molecules dose-dependently destabilized preformed falphaS. The overall activity of the molecules examined was in the order of: tannic acid=nordihydroguaiaretic acid=curcumin=rosmarinic acid=myricetin>kaempferol=ferulic acid>(+)-catechin=(-)-epicatechin>rifampicin=tetracycline. These compounds with anti-fibrillogenic as well as antioxidant activities could be key molecules for the development of preventives and therapeutics for LBD and MSA as well as Alzheimer's disease.

Nuclear pore complex proteins in Alzheimer disease.

Ultrastructural studies of neurofibrillary tangles in Alzheimer disease (AD) have demonstrated a close relationship between nuclear pores and the cytoplasmic paired helical filaments comprising the tangles, as well as nuclear irregularity in many tangle-bearing neurons; nuclear pore aggregation has been observed in nearby neurons. These observations prompted examination of the nuclear pore complex (NPC) and proteins critical to nucleocytoplasmic transport in neurons with and without tangles in AD and control cases. Light microscopic study of hippocampus and neocortex in AD and controls revealed that all nuclei were labeled by antibodies to NPC proteins, including the central transporter nucleoporin Nup62. Nucleoporin and tau label revealed significantly more nuclear irregularity in AD, often associated with neurofibrillary tangles. Double label of Nup62 with apoptotic markers (TUNEL and active caspase-3) and a cell-cycle protein (cyclin B1) revealed no clear relationship of nuclear irregularity to apoptosis or cell-cycle protein expression. However, cytoplasmic accumulation of nuclear transport factor 2 (NTF2), a protein that transports cargo from the cytoplasm into the nucleus, was observed in a subset of hippocampal neurons with and without tangles in AD but not control cases. Further investigation of the NPC and nucleocytoplasmic transport in AD is warranted.

The role of electron microscopy in neuropathology.

The electron microscope has been an essential instrument for elucidating the morphology of cells and tissue. Fine structural investigation of nervous tissue has disclosed numerous new findings, which were once invisible. In this communication I present my personal perspective on the role of electron microscopy in neuropathology through the presentation of selected electron microscopic projects engaged in our institution. These include brain edema, structural analysis of the myelin in the central nervous system, endodermal cysts in the central nervous system, aberrant synaptic development, toxoplasmosis in AIDS, and Hirano bodies.

Anthraquinones inhibit tau aggregation and dissolve Alzheimer's paired helical filaments in vitro and in cells.

The abnormal aggregation of tau protein into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease. Aggregation takes place in the cytoplasm and could therefore be cytotoxic for neurons. To find inhibitors of PHF aggregation we screened a library of 200,000 compounds. The hits found in the PHF inhibition assay were also tested for their ability to dissolve preformed PHFs. The results were obtained using a thioflavin S fluorescence assay for the detection and quantification of tau aggregation in solution, a tryptophan fluorescence assay using tryptophan-containing mutants of tau, and confirmed by a pelleting assay and electron microscopy of the products. Here we demonstrate the feasibility of the approach with several compounds from the family of anthraquinones, including emodin, daunorubicin, adriamycin, and others. They were able to inhibit PHF formation with IC50 values of 1-5 microm and to disassemble preformed PHFs at DC50 values of 2-4 microm. The compounds had a similar activity for PHFs made from different tau isoforms and constructs. The compounds did not interfere with the stabilization of microtubules by tau. Tau-inducible neuroblastoma cells showed the formation of tau aggregates and concomitant cytotoxicity, which could be prevented by inhibitors. Thus, small molecule inhibitors could provide a basis for the development of tools for the treatment of tau pathology in AD and other tauopathies.

Curcumin has potent anti-amyloidogenic effects for Alzheimer's beta-amyloid fibrils in vitro.

Inhibition of the accumulation of amyloid beta-peptide (Abeta) and the formation of beta-amyloid fibrils (fAbeta) from Abeta, as well as the destabilization of preformed fAbeta in the central nervous system, would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). We reported previously that nordihydroguaiaretic acid (NDGA) and wine-related polyphenols inhibit fAbeta formation from Abeta(1-40) and Abeta(1-42) and destabilize preformed fAbeta(1-40) and fAbeta(1-42) dose-dependently in vitro. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of curcumin (Cur) and rosmarinic acid (RA) on the formation, extension, and destabilization of fAbeta(1-40) and fAbeta(1-42) at pH 7.5 at 37 degrees C in vitro. We next compared the anti-amyloidogenic activities of Cur and RA with NDGA. Cur and RA dose-dependently inhibited fAbeta formation from Abeta(1-40) and Abeta(1-42), as well as their extension. In addition, they dose-dependently destabilized preformed fAbetas. The overall activities of Cur, RA, and NDGA were similar. The effective concentrations (EC(50)) of Cur, RA, and NDGA for the formation, extension, and destabilization of fAbetas were in the order of 0.1-1 microM. Although the mechanism by which Cur and RA inhibit fAbeta formation from Abeta and destabilize preformed fAbeta in vitro remains unclear, they could be a key molecule for the development of therapeutics for AD.

Alzheimer beta-amyloid homodimers facilitate A beta fibrillization and the generation of conformational antibodies.

We reported previously that stabilized beta-amyloid peptide dimers were derived from mutant amyloid precursor protein with a single cysteine in the ectodomain juxtamembrane position. In vivo studies revealed that two forms of SDS-stable A beta homodimers exist, species ending at A beta 40 and A beta 42. The phenomenon of the transformation of the initially deposited 42-residue beta-amyloid peptide into the amyloid fibrils of Alzheimer's disease plaques remains to be explained in physical terms, i.e. energetically and structurally. We therefore performed spectroscopic analyses revealing that engineered dimeric peptides ending at residue 42 displayed a much more pronounced beta-structural transition than corresponding monomers. Specifically, the single chemically induced dimerization of A beta peptides significantly increased the beta-sheet content by a factor of 2. The C-terminal residues Ile-41 and Ala-42 of dimeric forms further increased the beta-sheet content by roughly one-third. In contrast to A beta 42, the beta-sheet content of the alpha- and gamma-secretase-generated p3 fragments did not necessarily correlate with the tendency to form fibrils, although p3/17-42 had a pronounced thread forming character with fibril lengths of up to 2.5 microM. Electron microscopic images show that forms of p3/17-42 generated smaller granular particles than forms ending at residue 40. We discuss these findings in terms of A beta 1-42 dimers representing paranuclei, which self-aggregate into ribbon-like ordered fibrils by elongation. Based on A beta 42 dimer-specific titers of a polyclonal antiserum we propose that the A beta homodimer represents a nidus for plaque formation and a well defined novel therapeutic target.

Toward a unified scheme for the aggregation of tau into Alzheimer paired helical filaments.

Alzheimer's disease is characterized by aggregates of tau protein. Attempts to study the conditions for aggregation in vitro have led to different experimental systems, some of which appear mutually exclusive (e.g., oxidative vs reductive conditions, induction by polyanions vs fatty acids). We show here that different approaches and pathways can be viewed in a common framework, and that apparent differences can be explained by variations in the kinetics of subreactions. A unified view of PHF aggregation should help to analyze the causes of PHF aggregation and devise methods to prevent it.

Amyloid fibril formation by a synthetic peptide from a region of human acetylcholinesterase that is homologous to the Alzheimer's amyloid-beta peptide.

A region near the C-terminus of human acetylcholinesterase (AChE) is weakly homologous with the N-terminus of the Alzheimer's disease amyloid-beta peptide. We report that a 14-amino acid synthetic polypeptide whose sequence corresponds to residues 586-599 of the human synaptic or T form of AChE assembles into amyloid fibrils under physiological conditions. The fibrils have all the classical characteristics of amyloid: they have a diameter of 6-7 nm and bind both Congo red and thioflavin-T. Furthermore, the kinetics of assembly indicate that fibril formation proceeds via a two-step nucleation-dependent polymerization pathway, and a transition in the peptide conformation from random coil to beta-sheet is observed during fibril formation using far-UV circular dichroism spectroscopy. We also show that the peptide in aggregated fibrillar form has a toxic effect upon PC-12 cells in vitro. AChE normally resides mainly on cholinergic neuronal membranes, but is abnormally localized to senile plaques in Alzheimer's disease. Recently, an in vitro interaction between AChE and A beta, the principal constituent of the amyloid fibrils in senile plaques, has been documented. The presence of a fibrillogenic region within AChE may be relevant to the interaction of AChE with amyloid fibrils formed by Abeta.

Amyloid cored plaques in Tg2576 transgenic mice are characterized by giant plaques, slightly activated microglia, and the lack of paired helical filament-typed, dystrophic neurites.

We examined the brains of Tg2576 transgenic mice carrying human amyloid precursor protein with the Swedish mutation and Alzheimer's disease (AD) by means of immunohistochemistry and electron microscopy to clarify the characteristics of amyloid-associated pathology in the transgenic mice. In 12- to 29-month-old Tg2576 mice, congophilic cored plaques in the neocortex and hippocampus were labeled by all of the Abeta1-, Abeta40- and 42-specific antibodies, as seen in the classical plaques in AD. However, large-sized (>50 micro m in core diameter) plaques were seen more frequently in the older mice (18-29 months) than in those with AD (approximately 20% vs 2% in total cored plaques), and Tg2576 mice contained giant plaques (>75 micro m in core diameter), which were almost never seen in the brain of those with AD. Neither thread-like structures nor peripheral coronas were observed in the cored plaques of the transgenic mice in the silver impregnations. Immunohistochemically, plaque-accompanied microglia showed a slight enlargement of the cytoplasm with consistent labeling of Mac-1 and macrosialin (murine CD68), and with partial labeling of Ia antigen and macrophage-colony stimulating factor receptor. Ultrastructurally, the microglia surrounding the extracellular amyloid fibrils in the large, cored plaques showed some organella with phagocytic activity, such as secondary lysosomal, dense bodies, but intracellular amyloid fibrils were not evident. Dystrophic neurites in the plaques of the transgenic mice contained many dense multilaminar bodies, but no paired helical filaments. Our results suggest that giant cored plaques without coronas or paired helical filament-typed, dystrophic neurites are characteristic in Tg2576 mice, and that plaque-associated microglia in transgenic mice are activated to be in phagocytic function but not sufficient enough to digest extracellularly deposited amyloid fibrils.

[Neuropathology of the cerebral vessels of centenarians]. / Neuropathologie des vaisseaux cérébraux des centenaires.

Neuropathological study of brain and brain vessels was performed in two series of 12 and 20 centenarians, focusing on the prevalence of small vessel lesions, infarction, Alzheimer's changes and mental status. These are discussed as a function of vascular risk factors. In the first series (12 cases), there was no correlation between the severity of small vessel lesions: hyalinosis (12/12), mineralisation (10/12), amyloid angiopathy (9/12), vascular risk factors (high blood pressure or diabetes), Alzheimer's lesions. However, there was a tendency for an association between amyloid angiopathy and high density of neurofibrillary tangles. In the second series (20 cases), small infarcts and lacunes were found in 9/20 cases, neurofibrillary tangles and diffuse deposits of A beta peptide were constant, senile plaques were very frequent (19/20). Five patients were demented (one vascular dementia, one Alzheimer dementia, and 3 mixed dementias). These data indicate that: 1) Lesions of the walls of small cerebral vessels do not seem linked to the vascular risk factors observed at the end of the life of centenarians. 2) Cerebral infarcts and lacunes are frequent in these patients, and are responsible, at least in part, for a high proportion of the cognitive dysfunctions. The study of larger series is needed for a better understanding of relationships between vascular and degenerative lesions in the oldest old.