Truncated tau protein is the characteristic feature of human sporadic Alzheimer's disease. We have identified truncated tau proteins conformationally different from normal healthy tau. Subpopulations of these structurally different tau species promoted abnormal microtubule assembly in vitro suggesting toxic gain of function. To validate pathological activity in vivo we expressed active form of human truncated tau protein as transgene, in the rat brain. Its neuronal expression led to the development of the neurofibrillary degeneration of Alzheimer's type. Furthermore, biochemical analysis of neurofibrillary changes revealed that massive sarcosyl insoluble tau complexes consisted of human Alzheimer's tau and endogenous rat tau in ratio 1:1 including characteristic Alzheimer's disease (AD)-specific proteins (A68). This work represents first insight into the possible causative role of truncated tau in AD neurofibrillary degeneration in vivo.

No abstract

BackgroundAbnormal misfolded tau protein is a driving force of neurofibrillary degeneration in Alzheimer’s disease. It has been shown that tau oligomers play a crucial role in the formation of intracellular neurofibrillary tangles. They are intermediates between soluble tau monomers and insoluble tau filaments and are suspected contributors to disease pathogenesis. Oligomeric tau can be released into the extracellular space and spread throughout the brain. This finding opens the question of whether brain macrophages or blood monocytes have the potential to phagocytose extracellular oligomeric tau.MethodsWe have used stable rat primary microglial cells, rat peripheral monocytes-derived macrophages, BV2 microglial and TIB67 macrophage immortalized cell lines that were challenged by tau oligomers prepared by an in vitro aggregation reaction. The efficiency of cells to phagocytose oligomeric protein was evaluated with confocal microscopy. The ability to degrade tau protein was analyzed by immunoblotting.ResultsConfocal microscopy analyses showed that macrophages were significantly more efficient in phagocytosing oligomerized tau proteins than microglial cells. In contrast to macrophages, microglia are able to degrade the internalized oligomeric tau only after stimulation with lipopolysaccharide (LPS).ConclusionsOur data suggests that microglia may not be the principal phagocytic cells able to target extracellular oligomeric tau. We found that peripheral macrophages display a high potency for elimination of oligomeric tau and therefore could play an important role in the modulation of neurofibrillary pathology in Alzheimer’s disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0161-z) contains supplementary material, which is available to authorized users.

Recent findings showed that vascular dysfunction is an integral part of Alzheimer's disease pathology. Increased microvascular permeability is mainly associated with cerebrovascular amyloid-beta deposits. In contrast, little is known about the relationship between functional impairment of the blood-brain barrier and misfolded tau. In the present study, we examined whether human truncated tau is able to impair the blood-brain barrier in an in vitro model. We have found that truncated tau induced a very strong polarity-dependent effect in the rat blood-brain barrier model. When the tau was added to the upper compartment of the model containing endothelial cells (apical treatment), no effect was observed. However, the application of tau to the lower compartment (basolateral treatment), consisting of astrocyte-microglia culture, triggered significant decrease of transendothelial electrical resistance and increase of endothelial permeability for mannitol. Further, we found that truncated tau showed cytotoxic effects on astrocyte-microglia culture manifested by increased extracellular adenylate kinase levels. Molecular analysis of underlying mechanisms of tau-induced blood-brain barrier damage revealed the contribution of pro-inflammatory cytokine tumor necrosis factor-alpha and chemokine MCP-1 released from activated microglial cells. This study for the first time uncovers a novel toxic gain of function of misfolded tau that could contribute to the cerebral microvascular damage in human tauopathies.

Neuroinflammation plays a key role in the pathogenesis of Alzheimer’s disease and related tauopathies. We have previously shown that expression of nonmutated human truncated τ (151-391, 4R), derived from sporadic Alzheimer's disease, induced neurofibrillary degeneration accompanied by microglial and astroglial activation in the brain of transgenic rats. The aim of the current study was to determine the molecular mechanism underlying innate immune response induced by misfolded truncated τ. We found that purified recombinant truncated τ induced morphological transformation of microglia from resting into the reactive phenotype. Simultaneously, truncated τ caused the release of NO, proinflammatory cytokines (IL-1β, IL-6, TNF-α), and tissue inhibitor of metalloproteinase-1 from the mixed glial cultures. Notably, when the pure microglial culture was activated with truncated τ, it displayed significantly higher levels of the proinflammatory cytokines, suggesting a key role of microglia in the τ-mediated inflammatory response. Molecular analysis showed that truncated τ increased the mRNA levels of three MAPKs (JNK, ERK1, p38β) and transcription factors AP-1 and NF-κB that ultimately resulted in enhanced mRNA expression of IL-1β, IL-6, TNF-α, and NO. Our results showed for the first time, to our knowledge, that misfolded truncated protein τ is able to induce innate immune response via a MAPK pathway. Consequently, we suggest that misfolded truncated protein τ represents a viable target for immunotherapy of Alzheimer’s disease.

Pathologically altered tau protein is a common denominator of neurodegenerative disorders including Alzheimer's disease (AD) and other tauopathies. Therefore, promising immunotherapeutic approaches target and eliminate extracellular pathogenic tau species, which are thought to be responsible for seeding and propagation of tau pathology. Tau isoforms in misfolded states can propagate disease pathology in a template-dependent manner, proposed to be mediated by the release and internalization of extracellular tau. Monoclonal antibody DC8E8, binding four highly homologous and independent epitopes in microtubule-binding domain (MTBD) of diseased tau, inhibits tau-tau interaction, discriminates between healthy and pathologically truncated tau and reduces tau pathology in animal model in vivo. Here, we show that DC8E8 antibody acts via extracellular mechanism and does not influence viability and physiological functions of neurons. Importantly, in vitro functional assays showed that DC8E8 recognises pathogenic tau proteins of different size and origin, and potently blocks their entry into neurons. Next, we examined the mechanisms by which mouse antibody DC8E8 and its humanized version AX004 effectively block the neuronal internalization of extracellular AD tau species. We determined a novel mode of action of a therapeutic candidate antibody, which potently inhibits neuronal internalization of AD tau species by masking of epitopes present in MTBD important for interaction with neuron surface Heparan Sulfate Proteoglycans (HSPGs). We show that interference of tau-heparane sulfate interaction with DC8E8 antibody via steric hindrance represents an efficient and important therapeutic approach halting tau propagation.

Combining proteomics and systems biology approaches, we demonstrate that neonatal microglial cells derived from two different CNS locations, cortex and spinal cord, and cultured in vitro displayed different phenotypes upon different physiological or pathological conditions. These cells also exhibited greater variability in terms of cellular and small extracellular vesicles (sEVs) protein content and levels. Bioinformatic data analysis showed that cortical microglia exerted anti-inflammatory and neurogenesis/tumorigenesis properties, while the spinal cord microglia were more inflammatory. Interestingly, while both sEVs microglia sources enhanced growth of DRGs processes, only the spinal cord-derived sEVs microglia under LPS stimulation significantly attenuated glioma proliferation. These results were confirmed using the neurite outgrowth assay on DRGs cells and glioma proliferation analysis in 3D spheroid cultures. Results from these in vitro assays suggest that the microglia localized at different CNS regions can ensure different biological functions. Together, this study indicates that neonatal microglia locations regulate their physiological and pathological functional fates and could affect the high prevalence of brain vs spinal cord gliomas in adults.

Neurofibrillary tau protein pathology is closely associated with the progression and phenotype of cognitive decline in Alzheimer’s disease and other tauopathies, and a high-priority target for disease-modifying therapies. Herein, we provide an overview of the development of AADvac1, an active immunotherapy against tau pathology, and tau epitopes that are potential targets for immunotherapy. The vaccine leads to the production of antibodies that target conformational epitopes in the microtubule-binding region of tau, with the aim to prevent tau aggregation and spreading of pathology, and promote tau clearance. The therapeutic potential of the vaccine was evaluated in transgenic rats and mice expressing truncated, non mutant tau protein, which faithfully replicate of human tau pathology. Treatment with AADvac1 resulted in reduction of neurofibrillary pathology and insoluble tau in their brains, and amelioration of their deleterious phenotype. The vaccine was highly immunogenic in humans, inducing production of IgG antibodies against the tau peptide in 29/30 treated elderly patients with mild-to-moderate Alzheimer’s. These antibodies were able to recognise insoluble tau proteins in Alzheimer patients’ brains. Treatment with AADvac1 proved to be remarkably safe, with injection site reactions being the only adverse event tied to treatment. AADvac1 is currently being investigated in a phase 2 study in Alzheimer’s disease, and a phase 1 study in non-fluent primary progressive aphasia, a neurodegenerative disorder with a high tau pathology component.