Neuroimaging outcomes in clinical trials in Alzheimer's disease.

The first disease modifying drugs targeting beta amyloid that were tested in phase II and III clinical trials have been disappointing. We believe that failures descended from a leaky drug development pipeline where insufficient attention has been devoted to valid animal models and valid imaging markers of disease progression. In the future, valid animal models will need to take into greater consideration the natural and molecular history of AD, where both beta amyloid and tau play a key role. Valid imaging markers of disease progression will need to be identified in humans and translated into animal versions. Future testing of putative disease modifying drugs in valid animal models with valid imaging markers of disease progression will allow to maximize the predictability of their effect in phase II and III clinical trials.

Biochemical staging of synucleinopathy and amyloid deposition in dementia with Lewy bodies.

The primary feature of dementia with Lewy bodies (DLB) is the aggregation of alpha-synuclein into characteristic lesions: Lewy bodies (LBs) and Lewy neurites. However, in most of DLB cases, LBs are associated with neurofibrillary tangles and amyloid plaques (both Alzheimer disease [AD]-related lesions). We wanted to determine if this overlap of lesions is statistical, as a result of the late onset of both diseases, or results from a specific physiopathological synergy between synucleinopathy and either tauopathy or amyloid pathology. All patients with DLB from our prospective and multidisciplinary study were analyzed. These cases were compared with cases with pure AD and patients with Parkinson disease and controls. All cases were analyzed thoroughly at the neuropathologic and biochemical levels with a biochemical staging of aggregated alpha-synuclein, tau, and Abeta species. All sporadic cases of DLB were associated with abundant deposits of Abeta x-42 that were similar in quality and quantity to those of AD. Amyloid precursor protein (APP) dysfunction is a risk factor for AD as demonstrated by pathogenic mutations and Abeta accumulation. The constant and abundant Abeta x-42 deposition in sporadic DLB suggests that synucleinopathy is also promoted by APP dysfunction. Therefore, we conclude that APP is a therapeutic target for both AD and DLB.

[From physiopathology to treatment of Alzheimer's disease]. / De la physiopathologie au traitement de la maladie d'Alzheimer.

The natural and molecular history of familial or sporadic Alzheimer's disease (AD) shows that APP (amyloid protein precursor) dysfunction is a consensual central etiological factor in Alzheimer's disease (AD). This is demonstrated by 1) genetic defects involving APP gene or APP dysfunction (such as PS1 or PS2), leading to the formation of neocortical amyloid plaques in familial AD; 2) transgenic mice with these mutated genes that develop plaques; 3) both sporadic and familial AD develop plaques. But two alternatives to explain the physiopathology can be proposed: a gain of toxic function of AB peptide (reflected by the amyloid cascade hypothesis) or a loss of function of APP, a ubiquitous and well conserved protein with numerous possible neurotrophic activities. On the other hand, AD is also characterized by another inescapable degenerating process: tauopathy, an intraneuronal aggregation of tau proteins into neurofibrillary tangles. Remarkably enough, progression of tauopathy in neocortical areas fully explains the progressive clinical deficits of AD, from memory loss to aphasia, apraxia, agnosia. Also one has to bare in mind that most demented patients and most dementing neurodegenerative disorders have a tauopathy. From that, it is concluded that APP an Tau are solid therapeutic targets. But if we know that APP and Tau dysfunctions interact to boost neurodegeneration in AD, we still do no know what are the intraneuronal signaling pathways to activate or to inhibit to stop the degenerating process. There are many hypotheses and many possible approaches: the inhibition of toxicity of plaque, of AB protofibrils, or of AB oligomers inside or outside the neuron, using vaccination or ligands (Alzhemed). On the other hand, modulation of secretases that cleave APP by inhibiting those involved in the amyloidogenic pathway or by stimulating those of the non-amyloidogenic pathway, is a major route of research. Also modulation of kinases or phosphatases possibly involved in the aggregation of tau is also investigated. Because animal models are not perfectly relevant, at the end of the long and costly pathway of drug discovery, therapeutic trials are the only way to test these different hypotheses.

Characterization of mammalian neurofilament subunits by circular dichroism.

Critical steps in the disassembly and reassembly of neurofilaments, the intermediate filaments of neurons, have been investigated. Bovine neurofilament subunits (Mr 210 000, 160 000 and 70 000) were purified by urea-polyacrylamide gel electrophoresis and renatured by dialysis against several non-denaturing buffers. The quality of the protein renaturation was measured by circular dichroism. The spectra of renatured neurofilament subunits were interpreted in terms of secondary structure and this showed that the solubilization of proteins in guanidine-HCl buffers is more suitable than in urea buffer for a good recovery of a filamentous structure. Furthermore, it is shown that (i) the three neurofilament subunits exhibit specific CD spectra, with shapes reminiscent of those obtained for the alpha/beta class of proteins and that (ii) there is good correlation between CD spectra, the state of renaturation and the ability of the proteins to assemble into filamentous structures. We conclude that CD studies of neurofilament proteins should help in understanding the numerous variables affecting the disassembly and reassembly of neurofilaments.

Proteasome inhibition and Tau proteolysis: an unexpected regulation.

Increasing evidence suggests that an inhibition of the proteasome, as demonstrated in Parkinson's disease, might be involved in Alzheimer's disease. In this disease and other Tauopathies, Tau proteins are hyperphosphorylated and aggregated within degenerating neurons. In this state, Tau is also ubiquitinated, suggesting that the proteasome might be involved in Tau proteolysis. Thus, to investigate if proteasome inhibition leads to accumulation, hyperphosphorylation and aggregation of Tau, we used neuroblastoma cells overexpressing Tau proteins. Surprisingly, we showed that the inhibition of the proteasome led to a bidirectional degradation of Tau. Following this result, the cellular mechanisms that may degrade Tau were investigated.

Normal and pathological Tau proteins as factors for microtubule assembly.

Tau proteins are microtubule-associated proteins. They regulate the dynamics of the microtubule network, especially involved in the axonal transport and neuronal plasticity. Tau proteins belong to a family of developmentally regulated isoforms generated by alternative splicing and phosphorylation. This generates several Tau variants that interact with tubulin and other proteins. Therefore, Tau proteins are influenced by many physiological regulations. Tau proteins are also powerful markers of the neuronal physiological state. Their degree of phosphorylation is a good marker of cell integrity. It is heavily disturbed in numerous neurodegenerative disorders, leading to a collapse of the microtubule network and the presence of intraneuronal lesions resulting from Tau aggregation. However, different biochemical and immunological patterns of pathological Tau proteins found among neurodegenerative disorders are useful markers for the understanding of the role of Tau protein isoforms and the diagnosis of these pathological conditions.

Specific pathological Tau protein variants characterize Pick's disease.

Pick's disease (PiD) is characterized by a pan-laminar frontotemporal cortical atrophy, widespread degeneration of the white matter, chromatolytic neurons, and Pick bodies (PB). Microtubule-associated Tau proteins are the main cytoskeletal components modified during the neurodegenerative changes. In the present study, pathological alterations of Tau proteins were investigated in the brains of five PiD cases at both neuropathological and biochemical levels, using the monoclonal antibody AD2 which recognizes a phosphorylation-dependent Tau epitope and strongly labeled PB. A large number of cortical and subcortical regions were studied on frozen materials. Tau proteins were analyzed on mono- and two-dimensional gel electrophoreses using a quantitative western blot approach. In all specimens, a 55 and 64 kDa Tau doublet was observed in limbic, frontal, and temporal cortices as well as in striatum and substantia nigra. In contract, Alzheimer's disease (AD) brains are characterized by the presence of the 55, 64, and 69 kDa Tau triplet whereas the 64 and 69 kDa doublet is more typical of the progressive supranuclear palsy and corticobasal degeneration. Thus, the 55 and 64 kDa doublet appears to be specific to PiD, less acidic than AD Tau proteins, and well correlated with the presence of PB.

Comparative biochemistry of tau in progressive supranuclear palsy, corticobasal degeneration, FTDP-17 and Pick's disease.

Neurodegenerative disorders referred to as tauopathies have cellular hyperphosphorylated tau protein aggregates in the absence of amyloid deposits. Comparative biochemistry of tau aggregates shows that they differ in both phosphorylation and content of tau isoforms. The six tau isoforms found in human brain contain either three (3R) or four microtubule-binding domains (4R). In Alzheimer's disease, all six tau isoforms are abnormally phosphorylated and aggregate into paired helical filaments. They are detected by immunoblotting as a major tau triplet (tau55, 64 and 69). In corticobasal degeneration and progressive supranuclear palsy, only 4R-tau isoforms aggregate into twisted and straight filaments respectively. They appear as a major tau doublet (tau64 and 69). Finally, in Pick's disease, only 3R-tau isoforms aggregate into random coiled filaments. They are characterized by another major tau doublet (tau55 and 64). These differences in tau isoforms may be related to either the degeneration of particular cell populations in a given disorder or aberrant cell trafficking of particular tau isoforms. Finally, recent findings provide a direct link between a genetic defect in tau and its abnormal aggregation into filaments in fronto-temporal dementia with Parkinsonism linked to chromosome 17, demonstrating that tau aggregation is sufficient for nerve cell degeneration. Thus, tau mutations and polymorphisms may also be instrumental in many neurodegenerative disorders.

Rapid tau protein dephosphorylation and differential rephosphorylation during cardiac arrest-induced cerebral ischemia and reperfusion.

The effects of cerebral ischemia/reperfusion on phosphorylation of microtubule-associated tau proteins were assessed in a canine model of cardiac arrest. As tau proteins are phosphorylated by kinases involved in different transduction signal pathways, their phosphorylation state is an excellent marker of neuronal homeostasis and microtubule dynamics. Canine brain tau proteins were characterized by immunoblotting using phosphorylation-dependent antibodies and antisera raised against different amino- and carboxy-terminal tau sequences. The present study reports a complete dephosphorylation of tau proteins during ischemia, which is shown by a higher electrophoretic mobility and the almost (if not total) disappearance of phosphorylation-dependent monoclonal antibody labeling. After 2-hour restoration of spontaneous circulation, a decrease in the electrophoretic mobility was observed, and after 24 hours of reperfusion, a full restoration of the phosphorylation was visualized using phosphorylation-dependent monoclonal antibodies directed against Ser/Thr-Pro sites. However, one particular phosphorylation site involved in tau binding to microtubules, located on Ser262/356, was never fully significantly rephosphorylated, suggesting that microtubule metabolism was still affected after 24 hours of reperfusion. Thus, the sequential and differential recovery of tau phosphorylation after ischemia followed by reperfusion is a useful marker with which to monitor neuronal integrity after brain ischemia.

Abnormal tau species are produced during Alzheimer's disease neurodegenerating process.

Tau proteins were detected in human brain using two polyclonal antibodies: anti-paired helical filaments and anti-human native tau proteins. Both antisera detected identically the normal set of tau proteins in control brains. Moreover they detected two abnormal tau variants of 64 and 69 kDa exclusively in brain areas showing neurofibrillary tangles and senile plaques. Tau 64 and 69 were abnormally phosphorylated as revealed by the decrease in their molecular mass observed after alkaline phosphatase treatment. Therefore, tau 64 and 69 are specific markers of the neurofibrillary degeneration of the Alzheimer type and might be useful tools for studying the first pathological events that lead to neuronal death.

Alzheimer's disease: microtubule-associated proteins 2 (MAP 2) are not components of paired helical filaments.

In Alzheimer's disease, the most characteristic neuropathological changes are the formation of neurofibrillary tangles (NFT) and neuritic plaques (NP) characterized by the presence of bundles of paired helical filaments (PHF) that accumulate in the degenerating neurites and neuronal cell bodies. Although the protein composition of the PHF is ill-defined, a number of microtubule-associated proteins have been implicated in these lesions. Here we report results with an antiserum monospecific for the microtubule-associated protein MAP 2 which does not cross-react with any other microtubular protein. Immunostaining with this antibody of sections from an Alzheimer's brain show a strong reactivity with NFT but no reactivity at the level of the NP. On the other hand, immunostaining of Alzheimer's brain sections with another antibody specific for the microtubule-associated protein tau shows strong staining of PHF on both NFT and NP. These findings confirm the presence of the tau proteins in the PHF and strongly suggest that MAP 2 may not be a main structural component of the PHF. Labelling of NFT with the anti-MAP 2 antiserum suggests a non-specific binding of MAP 2 to the PHF during the process of NFT formation.

Shift from fetal-type to Alzheimer-type phosphorylated Tau proteins in SKNSH-SY 5Y cells treated with okadaic acid.

Tau proteins are abnormally phosphorylated in Alzheimer's disease. Pathological Tau proteins named PHF-Tau 55, PHF-Tau 64, and PHF-Tau 69, are the main constituents of the paired helical filaments (PHF). When treating SKNSH-SY 5Y cells with okadaic acid (OA), Tau 55 protein was clearly induced whereas Tau 64 protein was only faintly induced. Here, we show that the absence of Tau 69 could be explained by the fact that adult isoforms containing N-terminal inserts are not detected. Phosphorylation is similar for untreated cellular Tau proteins and fetal Tau proteins, while OA cell treatment transformed fetal-type into Alzheimer-type phosphorylated proteins.

Different distribution of phosphorylated tau protein isoforms in Alzheimer's and Pick's diseases.

Tau proteins aggregate into different neuronal inclusions in several neurodegenerative disorders. In Alzheimer's disease (AD), hyperphosphorylated Tau from paired helical filaments (PHF) of neurofibrillary tangles, named PHF-Tau, have an electrophoretic profile with four main bands (Tau 55, 64, 69, 74 kDa). In Pick's disease, phosphorylated Tau from Pick bodies are made of two major components (Tau 55, 64 kDa) and a minor 69 kDa. Here we show, using specific antibodies against translated exon 2, 3 or 10 of Tau isoforms, that the set of Tau isoforms engaged in the most insoluble part of PHF in AD is made of Tau isoforms with exon 10 while they are lacking in phosphorylated Tau from Pick's disease. Our results suggest that specific sets of Tau isoforms distinguish between typical neuronal inclusions.

Phosphorylation of specific sets of tau isoforms reflects different neurofibrillary degeneration processes.

Tau proteins are the basic components of filaments that accumulate within neurons during neurofibrillary degeneration, a degenerating process with disease-specific phenotypes. This specificity is likely to be sustained by both phosphorylation state and isoform content of tau aggregates that form neuronal inclusions. In the present study, characterization of tau isoforms involved in neurofibrillary degeneration in Alzheimer's disease, Pick's disease, corticobasal degeneration and progressive supranuclear palsy was performed. Both analyses by immunoblotting using specific tau antibodies and cell transfection by tau isoform cDNAs allowed us to demonstrate the aggregation of (1) the six hyperphosphorylated tau isoforms in Alzheimer's disease, (2) tau isoforms without exon 10-encoding sequence in Pick's disease and (3) hyperphosphorylated exon 10-tau isoforms in corticobasal degeneration and progressive supranuclear palsy. Thus, neurofibrillary degeneration phenotypes are likely to be related to the phosphorylation of different combinations of tau isoforms (with and/or without exon 10-encoding sequence) in subpopulations of neurons.

Modelling Alzheimer-specific abnormal Tau phosphorylation independently of GSK3beta and PKA kinase activities.

In Alzheimer's disease, neurofibrillary degeneration results from the aggregation of abnormally phosphorylated Tau proteins into paired helical filaments. These Tau variants displayed specific epitopes that are immunoreactive with anti-phospho-Tau antibodies such as AT100. As shown in in vitro experiments, glycogen synthase kinase 3 beta (GSK3beta) and protein kinase A (PKA) may be key kinases in these phosphorylation events. In the present study, Tau was microinjected into Xenopus oocytes. Surprisingly, in this system, AT100 was generated without any GSK3beta and PKA contribution during the progesterone or insulin-induced maturation process. Our results demonstrate that a non-modified physiological process in a cell model can generate the most specific Alzheimer epitope of Tau pathology.

Evidence for early vulnerability of the medial and inferior aspects of the temporal lobe in an 82-year-old patient with preclinical signs of dementia. Regional and laminar distribution of neurofibrillary tangles and senile plaques.

Detailed neuropathologic studies of neurofibrillary tangle and senile plaque distribution have shown that key elements of certain neocortical and hippocampal circuits are either compromised or lost in Alzheimer's disease. It has been suggested that a global corticocortical disconnection underlies dementia and leads to the dramatic disruption of integrated functions exhibited by patients with Alzheimer's disease. To investigate the distribution of lesions associated with the earliest indications of incipient dementia, we performed a quantitative neuropathologic evaluation of a non-demented 82-year-old patient demonstrating globally intact intellectual function but initial signs of impairment of specific cognitive functions before death. We observed densities of senile plaques comparable to those found in Alzheimer's disease throughout the cerebral cortex, whereas extensive neurofibrillary tangle formation was restricted to selective areas of the temporal lobe. The results of this systematic quantitative and comparative analysis of medial and inferior temporal lobe structures suggest a functional relationship between the degree of cognitive decline evidenced in the earliest stages of Alzheimer's disease and the anatomic progression of Alzheimer's disease-related pathologic changes along specific elements of the cortical circuitry.

General and dramatic glial reaction in Alzheimer brains.

We quantified glial fibrillary acidic protein (GFAP) using immunoblot techniques and anti-GFAP, in unfractionated homogenates of different brain regions from Alzheimer's disease (AD) patients. The amount of GFAP was significantly higher than in brains from controls and other neurodegenerative disorders (mean of 11 times), even in brain regions usually free of AD lesions such as caudate nucleus, thalamus, cerebellum, or brainstem. This dramatic increase of GFAP is not simply reflective of the astrocytic gliosis usually observed near the neurofibrillary tangles and senile plaques but more likely represents an astrocytic reaction in the whole brain corresponding to an overproduction or an accumulation of GFAP. The significance of such an increase is unknown, but it might be a key element in the pathogenesis of AD.

Specific tau variants in the brains of patients with myotonic dystrophy.

The mutation causing myotonic dystrophy (DM) is an unstable CTG trinucleotide repeat in a gene encoding for a protein with putative serine-threonine kinase activity. Several studies have reported the appearance of abnormally frequent neurofibrillary tangles (NFTs) in the cortex of patients with DM. Using immunologic probes against normal and pathologic hyperphosphorylated tau proteins, the basic components of NFTs, we performed a biochemical and immunohistochemical study of the brains of two DM cases. We compared the tau profiles with those found in Alzheimer's disease (AD) using mono- and two-dimensional immunoblotting. Patients were aged 53 and 61 years at death. In both cases, we observed few perikaryal and axonal inclusions in the hippocampus as well as the entorhinal and inferior temporal cortices. As in AD brain homogenates, pathologic tau proteins, named tau 55, 64, and 69, were exclusively immunodetected in the DM cases in the hippocampus, the entorhinal cortex, and in most of the temporal areas. Amounts of pathologic tau proteins were higher in the more severely affected case, but lower than in AD brain homogenates. Pathologic tau proteins were less acidic in DM than in AD. We found a very low amount of the tau 69 isoform in DM extracts, and in most of the cortical areas, tau 55 was overexpressed compared with AD homogenates. A link between the increase of kinase activity and the presence of pathologic tau proteins is discussed.

Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclear palsy.

BACKGROUND: Progressive supranuclear palsy (PSP) is characterized by a pure neurofibrillary tau pathology involving mainly basal ganglia and brainstem nuclei. In addition to a haplotype of the tau gene potentially favoring tau aggregation, lipoperoxidation has been shown to be associated with PSP tau pathology. OBJECTIVE: To analyze cdk5/p35 complex, a kinase that regulates neurite outgrowth, as a potential cellular mechanism underlying tau phosphorylation in brain tissues from PSP and control cases and comparatively in cerebral cortex from subjects with AD. METHODS: Cdk5/p35 protein levels and distribution were evaluated by immunoblotting and immunocytochemistry in brain regions from seven PSP, six AD, and seven control cases, with similar postmortem intervals. RESULTS: Total cdk5 protein levels were significantly increased by more than threefold in PSP tissue and were augmented in PSP neurons, codistributed with tau immunoreactivity. P35, the regulatory subunit of cdk5, was degraded by postmortem proteolysis to the same extent in PSP, AD, and control tissues. CONCLUSIONS: The proteolysis in vivo of p35, the regulatory subunit of the kinase, is not ascertainable because it is masked by its postmortem degradation. The study, however, indicates that in PSP, the alteration of cdk5 is different from that described in AD and suggests that the absence of amyloid beta protein deposition may account for the different pathways responsible for the same kinase activation.

Nonoverlapping but synergetic tau and APP pathologies in sporadic Alzheimer's disease.

OBJECTIVE: To determine the spatiotemporal mapping of tau pathologies and insoluble pools of Abeta in aging and sporadic AD, and their contribution to the physiopathologic, clinical, and neuropathologic features. METHODS: The authors studied 130 patients of various ages and different cognitive status, from nondemented controls (n = 60) to patients with severe definite AD (n = 70) who were followed prospectively. Insoluble Abeta 42 and 40 species were fully solubilized and quantified in the main neocortical areas, with a new procedure adapted to human brain tissue. Tau pathology staging was determined in 10 different brain areas, using Western blots. RESULTS: In AD, there is a constellation of amyloid phenotypes, extending from cases with exclusively aggregated Abeta 42 to cases with, in addition, large quantities of insoluble Abeta 40 species. Five other points were observed: 1) There was no spatial and temporal overlap in the distribution of these two insoluble Abeta species in cortical brain areas. 2) In contrast to solubilized Abeta 40 aggregates composed essentially of monomers and dimers, solubilized Abeta 42 was essentially observed as dimers and multimers. 3) Abeta 42 aggregates were observed at the early stages of tau pathology, whereas the insoluble Abeta 40 pool was found at the last stages. 4) During the progression of the disease, Abeta aggregates increase in quantity and heterogeneity, in close parallel to the extension of tau pathology. 5) There was no spatial overlap between Abeta aggregation that is widespread and heterogeneously distributed in cortical areas and tau pathology that is progressing sequentially, stereotypically, and hierarchically. CONCLUSIONS: These observations demonstrate that Abeta 42 aggregation, and not Abeta 40, is the marker that is close to Alzheimer etiology. It should be the main target for the early biological diagnosis of AD and modeling. Furthermore, the spatial mismatch between amyloid ss-precursor protein (APP) and tau pathologies in cortical brain areas demonstrates that neurodegeneration is not a direct consequence of extracellular Abeta neurotoxicity. Hence, there is a synergetic effect of APP dysfunction, revealed by Abeta aggregation, on the neuron-to-neuron propagation of tau pathology.