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.

Overexpression of MBNL1 fetal isoforms and modified splicing of Tau in the DM1 brain: two individual consequences of CUG trinucleotide repeats.

Neurofibrillary degeneration is often observed in the brain of patients with type 1 myotonic dystrophy (DM1). It consists principally of the aggregation of Tau isoforms that lack exon 2/3 encoded sequences, and is the consequence of the modified splicing of Tau pre-mRNA. In experimental models of DM1, the splicing of several transcripts is modified due to the loss of Muscleblind-like 1 (MBNL1) function. In the present study, we demonstrate that the MBNL1 protein is also present in the human brain, and consists of several isoforms, as shown by RT-PCR and sequencing. In comparison with controls, we show that the adult DM1 brain exhibits modifications in the splicing of MBNL1, with the preferential expression of long MBNL1 isoforms--a splicing pattern similar to that seen in the fetal human brain. In cultured HeLa cells, the presence of long CUG repeats, such as those found in the DM1 mutation, leads to similar changes in the splicing pattern of MBNL1, and the localization of MBNL1 in nuclear RNA foci. Long CUG repeats also reproduce the repression of Tau exon 2/3 inclusion, as in the human disease, suggesting that their effect on MBNL1 expression may lead to changes in Tau splicing. However, while an overall reduction in the expression of MBNL1 mimics the effect of the DM1 mutation, none of the MBNL1 isoforms tested so far modulates the endogenous splicing of Tau. The modified splicing of Tau thus results from a possibly CUG-mediated loss of function of MBNL1, but not from changes in the MBNL1 expression pattern.

[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.

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.

Similar brain tau pathology in DM2/PROMM and DM1/Steinert disease.

Neurofibrillary degeneration (NFD) occurs in the brains of patients with myotonic dystrophy (DM) type 1. The authors report a similar tau pathology in the CNS of a patient with DM2 and compare it to that of patients with DM1. A reduced expression of tau exon 2 and exon 3 epitopes is observed in both DM1 and DM2. This suggests a similar physiopathologic process that may contribute to common neurologic features in patients with DM.

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.

Tau aggregates are abnormally phosphorylated in inclusion body myositis and have an immunoelectrophoretic profile distinct from other tauopathies.

Sporadic inclusion body myositis (s-IBM) is the most frequent progressive acquired inflammatory myopathy in people older than 50 years. Abnormal aggregates of 'Alzheimer's proteins', including tau proteins, have been previously demonstrated in s-IBM. In the present study, we have investigated by immunohistochemistry and immunoblotting analysis the presence of tau proteins in muscle biopsy samples from patients with s-IBM and other myopathies with rimmed vacuoles, using newly developed antibodies raised against tau protein epitopes found in Alzheimer's disease brain. Tau immunoreactivity was shown in rimmed vacuoles or inclusions, preferentially with antibodies directed against phosphorylated carboxy-terminal epitopes of tau proteins. Cytoplasmic reactivity was also demonstrated in atrophic, nonvacuolated fibres, as well as in non-necrotic fibres invaded by inflammatory cells. Abnormally phosphorylated tau aggregates were also found in other compartments of the muscle fibre in s-IBM and other myopathies. Tau immunoblotting showed an electrophorectic profile of a doublet within the range of 60-62 kDa isovariants, which was different from tauopathies of the central nervous system. Finally, the unique pattern of immunoreactivity of s-IBM samples towards anti-tau antibodies is a new clue to a possible distinct subclass of peripheral tauopathy, different from the tauopathies of the central nervous system.

Coexistence of reactive plasticity and neurodegeneration in Alzheimer diseased brains.

Alzheimer's disease (AD) is a pathological process characterized by neuron degeneration and, as recently suggested, brain plasticity. In this work, we compared the reactive plasticity in AD brains associated to O-glycosydically linked glycans, recognized by lectins from Amaranthus leucocarpus (ALL) and Macrobrachium rosenbergii (MRL), and the tau neuritic degeneration. The neuritic degenerative process was evaluated by the quantification of aggregated neuritic structures. Lesions were determined using antibodies against hyperphosphorylated-tau (AD2), amyloid-beta, and synaptophysin. In these conditions, we classified and quantified three pathological structures associated to the neuritic degenerative process: 1) Amyloid-beta deposits (AbetaDs), 2) Classic neuritic plaques (NPs), and 3) Dystrophic neurites clusters (DNCs) lacking amyloid-beta deposits. Reactive plasticity structures were constituted by meganeuritic clusters (MCs) and peri-neuronal sprouting in neurons of the CA4 region of the hippocampus, immunoreactive to synaptophysin (exclusively in AD brains) and GAP-43. Besides, MCs were associated to sialylated O-glycosydically linked glycans as determined by positive labeling with ALL and MRL. Considering that these lectins are specific for the synaptic sprouting process in AD, our results suggest the co-occurrence of of several areas of reactive plasticity and neuron degeneration in AD.

Endothelin-converting enzyme-1 is expressed in human cerebral cortex and protects against Alzheimer's disease.

Cerebral accumulation of beta-amyloid peptide (A beta) is a central event in the pathogenesis of Alzheimer's disease (AD). Endothelin-converting enzyme-1 (ECE-1) is a candidate A beta-degrading enzyme in brain, but its involvement in AD pathogenesis was never assessed. We first performed brain immunocytochemistry, using a monoclonal anti-ECE-1 antibody, and observed neuronal ECE-1 expression in various cortical regions of nondemented subjects. In the hippocampus, ECE-1 immunoreactivity showed a stereotypical pattern inversely correlated with susceptibility to A beta deposition, further suggesting a physiological role in A beta clearance. In order to undertake a genetic association study, we identified a functional genetic variant (ECE1B C-338A) located in a regulatory region of the ECE1 gene. We showed that the A allele is associated with increased transcriptional activity in promoter-reporter gene assays and with increased ECE-1 mRNA expression in human neocortex. In a case-control study involving 401 patients with late-onset AD and 461 aged controls, we found that homozygous carriers of the A allele had a reduced risk of AD (OR=0.47, 95% CI 0.25-0.88). This finding was strengthened by the analysis of two other genetic variants of the ECE1 gene, which showed that the genetic association is extended over at least 13 kilobases of the gene sequence. Our results suggest that ECE-1 expression in brain may be critical for cortical A beta clearance and offer new potential targets for therapeutic interventions in AD.

The proprotein convertase PC2 is involved in the maturation of prosomatostatin to somatostatin-14 but not in the somatostatin deficit in Alzheimer's disease.

A somatostatin deficit occurs in the cerebral cortex of Alzheimer's disease patients without a major loss in somatostatin-containing neurons. This deficit could be related to a reduction in the rate of proteolytic processing of peptide precursors. Since the two proprotein convertases (PC)1 and PC2 are responsible for the processing of neuropeptide precursors directed to the regulated secretory pathway, we examined whether they are involved first in the proteolytic processing of prosomatostatin in mouse and human brain and secondly in somatostatin defect associated with Alzheimer's disease. By size exclusion chromatography, the cleavage of prosomatostatin to somatostatin-14 is almost totally abolished in the cortex of PC2 null mice, while the proportions of prosomatostatin and somatostatin-28 are increased. By immunohistochemistry, PC1 and PC2 were localized in many neuronal elements in human frontal and temporal cortex. The convertases levels were quantified by Western blot, as well as the protein 7B2 which is required for the production of active PC2. No significant change in PC1 levels was observed in Alzheimer's disease. In contrast, a marked decrease in the ratio of the PC2 precursor to the total enzymatic pool was observed in the frontal cortex of Alzheimer patients. This decrease coincides with an increase in the binding protein 7B2. However, the content and enzymatic activity of the PC2 mature form were similar in Alzheimer patients and controls. Therefore, the cortical somatostatin defect is not due to convertase alteration occuring during Alzheimer's disease. Further studies will be needed to assess the mechanisms involved in somatostatin deficiency in Alzheimer's disease.

Association of ATP synthase alpha-chain with neurofibrillary degeneration in Alzheimer's disease.

Amyloid deposits and neurofibrillary tangles (NFT) are the two hallmarks that characterize Alzheimer's disease (AD). In order to find the molecular partners of these degenerating processes, we have developed antibodies against insoluble AD brain lesions. One clone, named AD46, detects only NFT. Biochemical and histochemistry analyses demonstrate that the labeled protein accumulating in the cytosol of Alzheimer degenerating neurons is the alpha-chain of the ATP synthase. The cytosolic accumulation of the alpha-chain of ATP synthase is observed even at early stages of neurofibrillary degenerating process. It is specifically observed in degenerating neurons, either alone or tightly associated with aggregates of tau proteins, suggesting that it is a new molecular event related to neurodegeneration. Overall, our results strongly suggest the implication of the alpha-chain of ATP synthase in neurofibrillary degeneration of AD that is illustrated by the cytosolic accumulation of this mitochondrial protein, which belongs to the mitochondrial respiratory system. This regulatory subunit of the respiratory complex V of mitochondria is thus a potential target for therapeutic and diagnostic strategies.

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.

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.

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.

Mice transgenic for the human myotonic dystrophy region with expanded CTG repeats display muscular and brain abnormalities.

The autosomal dominant mutation causing myotonic dystrophy (DM1) is a CTG repeat expansion in the 3'-UTR of the DM protein kinase (DMPK) gene. This multisystemic disorder includes myotonia, progressive weakness and wasting of skeletal muscle and extramuscular symptoms such as cataracts, testicular atrophy, endocrine and cognitive dysfunction. The mechanisms underlying its pathogenesis are complex. Recent reports have revealed that DMPK gene haploinsufficiency may account for cardiac conduction defects whereas cataracts may be due to haploinsufficiency of the neighboring gene, the DM-associated homeobox protein (DMAHP or SIX5) gene. Furthermore, mice expressing the CUG expansion in an unrelated mRNA develop myotonia and myopathy, consistent with an RNA gain of function. We demonstrated that transgenic mice carrying the CTG expansion in its human DM1 context (>45 kb) and producing abnormal DMPK mRNA with at least 300 CUG repeats, displayed clinical, histological, molecular and electrophysiological abnormalities in skeletal muscle consistent with those observed in DM1 patients. Like DM1 patients, these transgenic mice show abnormal tau expression in the brain. These results provide further evidence for the RNA trans-dominant effect of the CUG expansion, not only in muscle, but also in brain.

Paradoxical phosphorylation of the serine 199 on tau proteins from young individuals.

The microtubule-associated tau proteins are abnormally aggregated in many tauopathies. Phosphorylation modulates the functions of tau. The serine 199 residue of tau is abnormally phosphorylated at early and late stages of Alzheimer's disease. The presence of the phosphorylated Ser199 was investigated in autopsy-derived and biopsy-derived brain tissue samples from non-demented individuals. A paradoxical expression was found in the hippocampus of the youngest ones, in granule cells of the dentate gyrus and in pyramidal cells of the Ammon's horn, which are particularly prone to neurodegeneration in several tauopathies. The rate of positive cells decreased with age. These data emphasize the importance of the phosphorylation of the Ser199 residue of tau in ageing and susceptibility to neurodegeneration.

Dysregulation of human brain microtubule-associated tau mRNA maturation in myotonic dystrophy type 1.

Intraneuronal aggregates of hyperphosphorylated tau proteins, referred to as pathological tau, are found in brain areas of demented patients affected by numerous different neurodegenerative disorders. We previously described a particular biochemical profile of pathological tau proteins in myotonic dystrophy type 1 (DM1). This multisystemic disorder is characterized by an unstable CTG repeat expansion in the 3'-untranslated region of the DM protein kinase gene. In the human central nervous system, tau proteins consist of six isoforms that differ by the presence or absence of the alternatively spliced exons 2, 3 and 10. Here we show that the pattern of tau isoforms aggregated in DM1 brain lesions is characteristic. It consists mainly of the aggregation of the shortest human tau isoform. A disruption in normal tau isoform expression consisting of a reduced expression of tau isoforms containing the exon 2 was observed at both the mRNA and protein levels. Large expanded CTG repeats were detected and showed marked somatic heterogeneity between DM1 cases and in cortical brains regions analysed. Our data suggest a relationship between the CTG repeat expansion and the alteration of tau expression showing that DM1 is a peculiar tauopathy.

Differential phosphorylation of tau proteins during kitten brain development and Alzheimer's disease.

Differential distribution and phosphorylation of tau proteins were studied in developing kitten brain by using several antibodies, and was compared to phosphorylation in Alzheimer's disease. Several antibodies demonstrated the presence of phosphorylated tau proteins during kitten brain development and identified pathological structures in human brain tissue. Antibody AD2, recognized tau in kittens and adult cats, but reacted in Alzheimer's tissue only with a pathological tau form. Antibody AT8 was prominent in developing kitten neurons and was found in axons and dendrites. After the first postnatal month this phosphorylation type disappeared from axons. Furthermore, dephosphorylation of kitten tau with alkaline phosphatase abolished immunoreactivity of AT8, but not that of AD2, pointing to a protection of the AD2 epitope in cats. Tau proteins during early cat brain development are phosphorylated at several sites that are also phosphorylated in paired helical filaments during Alzheimer's disease. In either event, phosphorylation of tau may play a crucial role to modulate microtubule dynamics, contributing to increased microtubule instability and promoting growth of processes during neuronal development or changing dynamic properties of the cytoskeleton and contributing to the formation of pathological structures in neurodegenerative diseases.

Hyperphosphorylation of tau is mediated by ERK activation during anticancer drug-induced apoptosis in neuroblastoma cells.

Phosphorylated tau protein is the major component of paired helical filaments in Alzheimer disease (AD). We have previously shown that abnormal tau phosphorylation was induced in neuroblastoma SK-N-SH cells by the anticancer drug, paclitaxel, during apoptosis [Guise et al., 1999: Apoptosis 4:47-58]. In the present study, we first demonstrated a shift from fetal tau to hyperphosphorylated tau after incubation with paclitaxel, that showed some similarities with the hyperphosphorylated tau in AD, by using several tau antibodies, N-Term, Tau-1 and AT-8. Tau phosphorylation occurred independently of caspase-3 activation. We next showed that a sustained activation of ERK (extracellular signal-regulated kinase) induced both tau phosphorylation and apoptosis during paclitaxel treatment (1 microM). The inhibition of ERK activation by using the pharmacological MEK1/2 inhibitor, PD98059 (50 microM), or an antisense strategy, reduced tau phosphorylation and neuronal apoptosis (P < 0.001), indicating a link between ERK activation, tau phosphorylation and apoptosis. Doxorubicin (0.2 microM), an anticancer drug whose mechanism of action is independent of microtubules, also induced ERK activation, tau phosphorylation and apoptosis. Moreover, doxorubicin induced some morphological features of neurodegeneration such as loss of neurites and disorganization of the cytoskeleton in apoptotic neuroblastoma cells. Altogether, our results suggest that tau phosphorylation plays a significant role in apoptosis enhancing disruption of microtubules that in turn leads to formation of apoptotic bodies, suggesting that neurodegeneration and apoptosis are related.