Clinical Development of Aducanumab, an Anti-Aß Human Monoclonal Antibody Being Investigated for the Treatment of Early Alzheimer's Disease.

The amyloid hypothesis has been the dominant framework for Alzheimer's disease (AD) research, including the development of anti-AD therapies. However, none of the phase III clinical trials conducted to date that targeted amyloid ß (Aß) production, aggregation, or clearance demonstrated a statistically significant treatment effect in patients with AD. This includes the approach of using monoclonal antibodies that recognize various Aß epitopes and display different binding selectivity. While some monoclonal antibodies have failed in phase III trials, several are still in development. Aducanumab (BIIB037) is a human antibody that selectively targets aggregated forms of Aß, including soluble oligomers and insoluble fibrils. In PRIME (NCT01677572), an ongoing phase Ib trial (N=196 patients dosed), aducanumab was shown to reduce Aß plaques and slow decline in clinical measures in patients with prodromal or mild AD, with acceptable safety and tolerability. The main safety finding was amyloid-related imaging abnormalities (ARIA), a side effect associated with removal of Aß, which was dose-dependent and occurred more often in ApoE ε4 carriers than non-carriers. ENGAGE (NCT02477800) and EMERGE (NCT02484547), the ongoing phase III trials of aducanumab in early AD, have been designed based on the outcomes of PRIME and on lessons from past clinical trials in patients with AD. Those study design features include patient selection with confirmed Aß pathology, ensuring sufficient target engagement, and conducting clinical trials in patients at earlier symptomatic stages of AD.

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.

Tangle and neuron numbers, but not amyloid load, predict cognitive status in Alzheimer's disease.

OBJECTIVE: To examine the relationship between stereologic estimates of AD-related pathology and severity of cognitive deficits in brain aging. BACKGROUND: Previous studies reported substantial contributions of neurofibrillary tangles (NFT), amyloid deposits, and neuronal loss to the development of dementia. However, the prediction of cognitive status based on nonstereologic quantification of these measures has led to conflicting results. Such studies have measured densities, rather than absolute numbers, and most do not take into account the potential interaction between the above pathologic hallmarks in a global multivariate analysis. METHODS: Clinicopathologic study in 22 elderly cases. Cognitive status assessed prospectively using the Mini-Mental State Examination (MMSE); stereologic assessment of NFT, unaffected neurons, and total amyloid volume in the CA1 field of the hippocampus, entorhinal cortex, and area 9. Statistical analysis was performed using both univariate and multivariate linear regression models. RESULTS: High total NFT counts but not amyloid volume were strongly associated with a lower number of unaffected neurons in all areas studied. A high proportion of variability in MMSE scores was explained by NFT and neuronal counts in the CA1 field (83% and 85.4%), entorhinal cortex (87.8% and 83.7%), and area 9 (87% and 79%); amyloid volume in the entorhinal cortex, but not in the CA1 field and area 9, accounted for 58.5% of MMSE variability. Multivariate analyses showed that total NFT counts in the entorhinal cortex and area 9 as well as neuron numbers in the CA1 field were the best predictors of MMSE score. CONCLUSIONS: These new stereologic data indicate that neuronal pathology in hippocampal formation and frontal cortex closely reflects the progression of cognitive deficits in brain aging and AD. They also demonstrate that amyloid volume has no additional predictive value, in terms of clinicopathologic correlations, beyond its interaction with NFT.

Stereologic analysis of neurofibrillary tangle formation in prefrontal cortex area 9 in aging and Alzheimer's disease.

Alzheimer's disease (AD) is characterized neuropathologically by several features including extensive neuronal death in the cerebral cortex. In fact, while neuropathological changes restricted to the hippocampal formation are a consistent reflection of age-related memory impairment, overt dementia is present only in cases with neocortical involvement. Several quantitative studies have reported a substantial loss of neurons from these regions and a parallel increase in the number of neurofibrillary tangles (NFT). However, accurate quantitative data on the dynamics of NFT formation are lacking. In the present study, we performed a stereologic analysis of the proportions of intracellular and extracellular (ghost) NFT, and unaffected neurons in the deep part of layer III (layer IIIc) and the superficial part of layer V (layer Va) of Brodmann's prefrontal cortex area 9. Elderly cognitively unimpaired cases were compared with cases with different degrees of cognitive dysfunction. The data revealed differential rates of formation of intracellular and extracellular NFT between the two layers, and confirmed the presence of a severe disease-associated, but not age-related, neuronal loss. It was also shown that a susbtantial number of pyramidal cells may persist either unaffected or in a transitional stage of NFT formation in both neocortical layers. These results suggest that a considerable number of neurons containing an intracellular NFT exists in the neocortex until late in the course of AD. Whereas it is not possible to assess whether such transitional neurons are fully functional, these affected neurons might respond positively to therapeutic strategies aimed at protecting the cells that are prone to neurofibrillary degeneration in AD.

Stereologic assessment of the total cortical volume occupied by amyloid deposits and its relationship with cognitive status in aging and Alzheimer's disease.

Although the presence of amyloid deposits is required to establish the neuropathologic diagnosis of Alzheimer's disease, from a clinical point of view, a direct contribution of these cerebral lesions to cognitive deficits is still controversial. The development and standardization of quantitative and accurate biochemical and neuropathologic methods may be critical to improve the postmortem diagnosis and clinicopathologic correlations. Here, we used a point counting method, based on the Cavalieri principle, to estimate the volume occupied by amyloid deposits in a discrete region of the prefrontal cortex and in the hippocampal formation, in brains from patients with cognitive status ranging from normal to severely demented. We demonstrate that the assessment of the total volume occupied by the amyloid deposits in the entorhinal cortex and subiculum can be considered an effective predictor of dementia severity. We also reveal the existence of a high degree of regional and interindividual heterogeneity in amyloid distribution and relative volume. Our data suggest that even though a correlation was observed between the stereologic point counting method and a non-stereologic random field thresholding approach, in most cases non-stereologic methods may not provide adequate samples of the tissue and may lead to unreliable estimates of amyloid burden due to the inhomogeneous distribution of amyloid in the cerebral cortex and the large variability among brains.

Clinical validity of A beta-protein deposition staging in brain aging and Alzheimer disease.

Braak's neurofibrillary tangle (NFT) pathology staging system of Alzheimer disease (AD) correlates generally with clinical data. Recently, Braak's group proposed an Abeta-protein staging based on the progression of amyloid deposition in the medial temporal lobe. To examine its clinical validity and evaluate whether it adds predictive power to NFT-based staging, we performed a study comparing both neuropathological classifications with clinical dementia rating scale (CDR) scores in a large autopsy series. The 2 neuropathological staging systems were strongly correlated. Their association with clinical severity was highly significant. However, the strength of the relationship was greater for NFT-based staging. It accounted for 26.5% of the variability in clinical severity, Abeta-protein-based staging for 13.0%, and age for 4.4%. Compared to NFT-based staging, the Abeta-protein-based system was less able to distinguish mild cognitive changes from dementia and showed marked overlap among the various stages of cognitive decline. In a multivariate model, NFT and age together accounted for 27.2% of the clinical variability and the addition of Abeta-protein deposition staging could only explain an extra 2.9%. Our data support the close relationship between NFT progression and amyloid formation within the medial temporal lobe proposed by Braak's group but demonstrate the limited value of Abeta-protein deposition staging in terms of clinicopathological correlations.

Practical approaches to stereology in the setting of aging- and disease-related brain banks.

The unbiased sampling techniques of stereology have been developed to avoid the inaccuracies of using 'representative' sections for morphometric studies. In order to carry out a stereologically valid study, the region of interest must be fully available for sampling and its boundaries or its constituents must also be distinctly identifiable. However, in the setting of a brain bank in which only one half of the brain specimen is fixed for morphology, a logistic problem arises in satisfying the needs of the diagnostic neuropathologist with that of the stereologically oriented morphologist. We present a dissection approach in which the region for analysis must be used for unbiased sampling and also be available for paraffin-embedded neuropathologic work-up. Following fixation, a block consisting of the entire region of interest is removed intact and using a multibladed knife the block is subsectioned in the coronal plane at regular intervals. Alternate blocks are chosen for either paraffin embedding and destined to neuropathologic evaluation or are processed for stereology. The stereology blocks can be either cryoprotected or placed in phosphate buffer and are serially sectioned on a cryostat or a Vibratome. Preliminary analyses using this approach have provided reliable estimates of the total number of different neuronal populations and disease-related lesions in a variety of human and non-human banked specimens. In addition, this approach has definite advantages in that it provides rigorous quantitative estimates of neuropathologic changes that can be correlated to clinical data and does not compromise the routine neuropathological diagnostic procedure of the materials.

Tau protein isoforms, phosphorylation and role in neurodegenerative disorders.

Tau proteins belong to the family of microtubule-associated proteins. They are mainly expressed in neurons where they play an important role in the assembly of tubulin monomers into microtubules to constitute the neuronal microtubules network. Microtubules are involved in maintaining the cell shape and serve as tracks for axonal transport. Tau proteins also establish some links between microtubules and other cytoskeletal elements or proteins. Tau proteins are translated from a single gene located on chromosome 17. Their expression is developmentally regulated by an alternative splicing mechanism and six different isoforms exist in the human adult brain. Tau proteins are the major constituents of intraneuronal and glial fibrillar lesions described in Alzheimer's disease and numerous neurodegenerative disorders referred to as 'tauopathies'. Molecular analysis has revealed that an abnormal phosphorylation might be one of the important events in the process leading to their aggregation. Moreover, a specific set of pathological tau proteins exhibiting a typical biochemical pattern, and a different regional and laminar distribution could characterize each of these disorders. Finally, a direct correlation has been established between the progressive involvement of the neocortical areas and the increasing severity of dementia, suggesting that pathological tau proteins are reliable marker of the neurodegenerative process. The recent discovery of tau gene mutations in frontotemporal dementia with parkinsonism linked to chromosome 17 has reinforced the predominant role attributed to tau proteins in the pathogenesis of neurodegenerative disorders, and underlined the fact that distinct sets of tau isoforms expressed in different neuronal populations could lead to different pathologies.

Pathological tau phenotypes. The weight of mutations, polymorphisms, and differential neuronal vulnerabilities.

In tauopathies, comparative biochemistry of tau aggregates shows that they differ in both phosphorylation and content of tau isoforms. Six tau isoforms are found in human brain that contain either three (3R) or four microtubule-binding domains (4R). In Alzheimer's disease, all six of the tau isoforms are phosphorylated and aggregate into paired helical filaments. They are detected by immunoblotting as a major tau triplet (tau 55, 64, and 69). In corticobasal degeneration and progressive supranuclear palsy, only phosphorylated 4R-tau isoforms aggregate and appear as a major tau doublet (tau 64 and 69). In Pick's disease, only phosphorylated 3R-tau isoforms aggregate into filaments and are characterized by another major tau doublet (tau 55 and 64). Finally, recent findings provide a direct link between a genetic defect in tau and its abnormal aggregation into filaments in frontotemporal dementia with parkinsonism linked to chromosome 17. In the present study, the question of a relationship between tau isoforms and cell morphology is raised. To answer this question, stably transfected human neuroblastoma SY5Y cell lines with either 3R- or 4R-tau isoforms are established. Cell morphology and tau phosphorylation were modified, suggesting that cells undergo profound changes in their metabolism and viability.

Phosphorylated serine422 on tau proteins is a pathological epitope found in several diseases with neurofibrillary degeneration.

Neuronal inclusions with bundles of abnormal filaments made of tau polymers are found in numerous diseases with neurofibrillary degeneration. Tau proteins are the basic components of paired helical filaments (PHF) in Alzheimer's disease (AD), and are abnormally phosphorylated. A disease-specific phosphorylation site at serine422 was demonstrated on PHF, but not on tau proteins from biopsy-derived brain samples. In the present study, we report the characterization of a polyclonal antibody (988) against the serine422 phosphorylation site. By using biochemical and immunohistochemical methods, we confirmed that it is not found on tau proteins from biopsy- or autopsy-derived control samples, and we investigated the presence of this epitope on tau proteins in several neurodegenerative disorders, including AD, Down syndrome (DS), Guamanian amyotrophic lateral sclerosis/Parkinsonism-dementia complex (ALS/PDC), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), postencephalitic parkinsonism (PEP) and Pick's disease (PiD). By Western blotting, antibody 988 labeled the characteristic tau triplet (tau 55, 64, 69) in AD, DS, Guamanian ALS/PDC and PEP. PSP and CBD exhibited their typical tau doublet (tau 64, 69), whereas the doublet tau 55 and 64 was detected in PiD. In all of these neurodegenerative disorders, antibody 988 clearly labeled NFT and dystrophic neurites, as well as Pick bodies in PiD cases, whereas no staining was observed in control cases. These data indicate that phosphorylation of serine422 on tau proteins is a common feature among neurodegenerative disorders and is therefore not specific of AD. Moreover, phosphorylation of this epitope permits the distinction between normal tau proteins and pathological tau proteins.

Alzheimer-specific epitope of AT100 in transfected cell lines with tau: toward an efficient cell model of tau abnormal phosphorylation.

Intraneuronal aggregation of specific hyperphosphorylated tau isoforms in subsets of neurons may explain many neurodegenerative processes. Only some antibodies including AP422 and AT100 are specific to the abnormal phosphorylation of tau proteins in these processes. AT100-immunoreactivity was never observed in cell models with the exception of Sf9 cells. In the present study, we developed a way to induce AT100-immunoreactivity in different cell types including COS and SY5Y cells after tau cDNA transfection and treatment by okadaic acid. This represents a useful model to study abnormal tau phosphorylation in situ.

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.

Isoelectric point differentiates PHF-tau from biopsy-derived human brain tau proteins.

In the present study, Tau proteins were detected by two monoclonal antibodies AD2 and Tau-1 raised against PHF-tau and normal Tau proteins respectively using single- and two-dimensional immunoblotting. We demonstrate here the presence of a Tau triplet in brain homogenates from patients with Alzheimer's disease (AD) processed human brain biopsies from controls. However PHF-tau proteins have a slight but significantly higher mol. wt and a much more acidic isoelectric point. Therefore, Tau proteins are more phosphorylated in AD.