Norbert Zilka

Truncated tau from sporadic Alzheimer's disease suffices to drive neurofibrillary degeneration in vivo

Truncated tau protein is the characteristic feature of human sporadic Alzheimers 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 Alzheimers type. Furthermore, biochemical analysis of neurofibrillary changes revealed that massive sarcosyl insoluble tau complexes consisted of human Alzheimers tau and endogenous rat tau in ratio 1:1 including characteristic Alzheimers disease (AD)‐specific proteins (A68). This work represents first insight into the possible causative role of truncated tau in AD neurofibrillary degeneration in vivo.

First-in-man tau vaccine targeting structural determinants essential for pathological tau–tau interaction reduces tau oligomerisation and neurofibrillary degeneration in an Alzheimer’s disease model

IntroductionWe have identified structural determinants on tau protein that are essential for pathological tau–tau interaction in Alzheimer’s disease (AD). These regulatory domains, revealed by monoclonal antibody DC8E8, represent a novel target for tau-directed therapy. In order to validate this target, we have developed an active vaccine, AADvac1.MethodsA tau peptide encompassing the epitope revealed by DC8E8 was selected for the development of an active vaccine targeting structural determinants on mis-disordered tau protein that are essential for pathological tau–tau interaction. The efficacy of the vaccine was tested in a transgenic rat model of human tauopathies. Toxicology and safety pharmacology studies were conducted under good laboratory practice conditions in multiple rodent and nonrodent species.ResultsWe have administered the tau peptide vaccine to a rat model of AD to investigate whether the vaccine can improve its clinical, histopathological and biochemical AD phenotype. Our results show that vaccination induced a robust protective humoral immune response, with antibodies discriminating between pathological and physiological tau. Active immunotherapy reduced the levels of tau oligomers and the extent of neurofibrillary pathology in the brains of transgenic rats. Strikingly, immunotherapy has reduced AD-type hyperphosphorylation of tau by approximately 95%. Also, the tau peptide vaccine improved the clinical phenotype of transgenic animals. Toxicology and safety pharmacology studies showed an excellent safety and tolerability profile of the AADvac1 vaccine.ConclusionsActive immunisation targeting crucial domains of Alzheimer tau eliminated tau aggregation and neurofibrillary pathology. Most importantly, the AD type of tau hyperphosphorylation was abolished by vaccination across a wide range of AD phospho-epitopes. Our results demonstrate that active immunisation led to elimination of all major hallmarks of neurofibrillary pathology, which was reflected by a profound improvement in the clinical presentation of transgenic rats. This makes the investigated tau peptide vaccine a highly promising candidate therapeutic for the disease-modifying treatment of AD. The tested vaccine displayed a highly favourable safety profile in preclinical toxicity studies, which opens up the possibility of using it for AD prophylaxis in the future. The vaccine has already entered phase I clinical trial under the name AADvac1.Trial registrationCurrent Controlled Trials NCT01850238. Registered 7 May 2013.

Human misfolded truncated tau protein promotes activation of microglia and leukocyte infiltration in the transgenic rat model of tauopathy

It has been hypothesized that misfolded tau protein could be a mediator of the inflammatory response in human tauopathies. Here we show that neurodegenerative lesions caused by human truncated tau promote inflammatory response manifested by upregulation of immune-molecules (CD11a,b, CD18, CD4, CD45 and CD68) and morphological activation of microglial cells in a rat model of tauopathy. In parallel, the innate immune brain response promotes activation of MHC class II positive blood-borne leukocytes and their influx into the brain parenchyma. These findings have important consequences for the rationale drug development of effective inflammation-based therapeutic strategies for human tauopathies.

Safety and immunogenicity of the tau vaccine AADvac1 in patients with Alzheimer's disease: a randomised, double-blind, placebo-controlled, phase 1 trial

BACKGROUND Neurofibrillary pathology composed of tau protein is a main correlate of cognitive impairment in patients with Alzheimers disease. Immunotherapy targeting pathological tau proteins is therefore a promising strategy for disease-modifying treatment of Alzheimers disease. We have developed an active vaccine, AADvac1, against pathological tau proteins and assessed it in a phase 1 trial. METHODS We did a first-in-man, phase 1, 12 week, randomised, double-blind, placebo-controlled study of AADvac1 with a 12 week open-label extension in patients aged 50-85 years with mild-to-moderate Alzheimers disease at four centres in Austria. We randomly assigned patients with a computer-generated sequence in a 4:1 ratio overall to receive AADvac1 or placebo. They received three subcutaneous doses of AADvac1 or placebo from masked vaccine kits at monthly intervals, and then entered the open-label phase, in which all patients were allocated to AADvac1 treatment and received another three doses at monthly intervals. Patients, carers, and all involved with the trial were masked to treatment allocation. The primary endpoint was all-cause treatment-emergent adverse events, with separate analyses for injection site reactions and other adverse events. We include all patients who received at least one dose of AADvac1 in the safety assessment. Patients who had a positive IgG titre against the tau peptide component of AADvac1 at least once during the study were classified as responders. The first-in-man study is registered with EU Clinical Trials Register, number EudraCT 2012-003916-29, and ClinicalTrials.gov, number NCT01850238; the follow-up study, which is ongoing, is registered with EU Clinical Trials Register, number EudraCT 2013-004499-36, and ClinicalTrials.gov, number NCT02031198. FINDINGS This study was done between June 9, 2013, and March 26, 2015. 30 patients were randomly assigned in the double-blind phase: 24 patients to the AADvac1 group and six to the placebo group. A total of 30 patients received AADvac1. Two patients withdrew because of serious adverse events. The most common adverse events were injection site reactions after administration (reported in 16 [53%] vaccinated patients [92 individual events]). No cases of meningoencephalitis or vasogenic oedema occurred after administration. One patient with pre-existing microhaemorrhages had newly occurring microhaemorrhages. Of 30 patients given AADvac1, 29 developed an IgG immune response. A geometric mean IgG antibody titre of 1:31415 was achieved. Baseline values of CD3+ CD4+ lymphocytes correlated with achieved antibody titres. INTERPRETATION AADvac1 had a favourable safety profile and excellent immunogenicity in this first-in-man study. Further trials are needed to corroborate the safety assessment and to establish proof of clinical efficacy of AADvac1. FUNDING AXON Neuroscience SE.

Who fans the flames of Alzheimer's disease brains? Misfolded tau on the crossroad of neurodegenerative and inflammatory pathways

Neurodegeneration, induced by misfolded tau protein, and neuroinflammation, driven by glial cells, represent the salient features of Alzheimers disease (AD) and related human tauopathies. While tau neurodegeneration significantly correlates with disease progression, brain inflammation seems to be an important factor in regulating the resistance or susceptibility to AD neurodegeneration. Previously, it has been shown that there is a reciprocal relationship between the local inflammatory response and neurofibrillary lesions. Numerous independent studies have reported that inflammatory responses may contribute to the development of tau pathology and thus accelerate the course of disease. It has been shown that various cytokines can significantly affect the functional and structural properties of intracellular tau. Notwithstanding, anti-inflammatory approaches have not unequivocally demonstrated that inhibition of the brain immune response can lead to reduction of neurofibrillary lesions. On the other hand, our recent data show that misfolded tau could represent a trigger for microglial activation, suggesting the dual role of misfolded tau in the Alzheimers disease inflammatory cascade. On the basis of current knowledge, we can conclude that misfolded tau is located at the crossroad of the neurodegenerative and neuroinflammatory pathways. Thus disease-modified tau represents an important target for potential therapeutic strategies for patients with Alzheimers disease.

Neurodegeneration caused by expression of human truncated tau leads to progressive neurobehavioural impairment in transgenic rats

Human truncated tau protein is an active constituent of the neurofibrillary degeneration in sporadic Alzheimers disease. We have shown that modified tau protein, when expressed as a transgene in rats, induced AD characteristic tau cascade consisting of tau hyperphosphorylation, formation of argyrophilic tangles and sarcosyl-insoluble tau complexes. These pathological changes led to the functional impairment characterized by a variety of neurobehavioural symptoms. In the present study we have focused on the behavioural alterations induced by transgenic expression of human truncated tau. Transgenic rats underwent a battery of behavioural tests involving cognitive- and sensorimotor-dependent tasks accompanied with neurological assessment at the age of 4.5, 6 and 9 months. Behavioural examination of these rats showed altered spatial navigation in Morris water maze resulting in less time spent in target quadrant (p<0.05) and fewer crossings over previous platform position (p<0.05) during probe trial. Spontaneous locomotor activity and anxiety in open field was not influenced by transgene expression. However beam walking test revealed that transgenic rats developed progressive sensorimotor disturbances related to the age of tested animals. The disturbances were most pronounced at the age of 9 months (p<0.01). Neurological alterations indicating impaired reflex responses were other added features of behavioural phenotype of this novel transgenic rat. These results allow us to suggest that neurodegeneration, caused by the non-mutated human truncated tau derived from sporadic human AD, result in the neuronal dysfunction consequently leading to the progressive neurobehavioural impairment.

Truncated tau expression levels determine life span of a rat model of tauopathy without causing neuronal loss or correlating with terminal neurofibrillary tangle load

We have previously demonstrated in a transgenic rat model of tauopathy that human misfolded truncated tau derived from Alzheimer’s disease suffices to drive neurofibrillary degeneration in vivo. We employed this model to investigate the impact of truncated tau expression levels on life span, neuronal loss and the final load of neurofibrillary tangles (NFTs) in transgenic rats. Two independent transgenic lines (SHR72, SHR318), that display different expression levels of truncated tau, were utilized in this study. We found that transgene expression levels in the brain of SHR72 rats were 44% higher than in SHR318 rats and that truncated tau protein levels determined the survival rate of transgenic rats. The line with higher expression levels of truncated tau (SHR72) showed decreased median survival (222.5 days) when compared with the line with lower expression (SHR318; 294.5 days). Interestingly, NFT loads (total NFT/total neurons) were very similar in terminal stages of disease in both transgenic lines (SHR72 – 10.9%; SHR318 – 11.6%), despite significantly different expression levels of truncated tau. Moreover, mean neuron numbers in the hippocampus (CA1–3) and brain stem (gigantocellular reticular nucleus) in the two transgenic rat strains in the terminal stages of disease were similar, and did not differ significantly from those observed in age‐matched non‐transgenic controls. These findings suggest that the expression levels of misfolded truncated tau determine the life span in a transgenic rat model of tauopathy without causing neuronal loss or correlating with terminal NFT load.

Rat tau proteome consists of six tau isoforms: implication for animal models of human tauopathies

Human brain encompasses six tau isoforms, containing either three (3R) or four (4R) repeat domains, all of which participate in the pathogenesis of human tauopathies. To investigate the role of tau protein in the disease, transgenic rat models have been created. However, unlike humans, it has been suggested that rat brain expresses only three 4R tau isoforms. Because of the significance of the number of tau isoforms for faithful reproducibility of neurofibrillary pathology in transgenic rat models, we reopened this issue. Surprisingly, our results showed that adult rat brain contains six tau isoforms like humans. Protein expression of 4R tau isoforms was ninefold higher than 3R isoforms. Furthermore, the protein levels of tau isoforms with none, one or two N‐terminal inserts were 30%, 35%, and 35% of total tau, respectively. Moreover, amount and ratio of tau isoforms were developmentally regulated. The levels of 4R tau isoforms progressively increased from early postnatal period until adulthood, whereas the expression of 3R tau isoforms reached maximum at P10 and then gradually declined. Our results show that rat brain encompasses full tau proteome similar to humans. These findings support the use of rat as an animal model in human tauopathies research.

First transgenic rat model developing progressive cortical neurofibrillary tangles.

Neurofibrillary degeneration induced by misfolded protein tau is considered to be one of the key pathological hallmarks of Alzheimers disease (AD). In the present study, we have introduced a novel transgenic rat model expressing a human truncated tau that encompasses 3 microtubule binding domains (3R) and a proline-rich region (3R tau151-391). The transgenic rats developed progressive age-dependent neurofibrillary degeneration in the cortical brain areas. Neurofibrillary tangles (NFTs) satisfied several key histological criteria used to identify neurofibrillary degeneration in human Alzheimers disease including argyrophilia, Congo red birefringence, and Thioflavin S reactivity. Neurofibrillary tangles were also identified with antibodies used to detect pathologic tau in the human brain, including DC11, recognizing an abnormal tau conformation and antibodies that are specific for hyperphosphorylated forms of tau protein. Moreover, neurofibrillary degeneration was characterized by extensive formation of sarkosyl insoluble tau protein complexes consisting of rat endogenous and truncated tau species. Interestingly, the transgenic rats did not show neuronal loss either in the cortex or in the hippocampus. We suggest that novel transgenic rat model for human tauopathy represents a valuable tool in preclinical drug discovery targeting neurofibrillary degeneration of Alzheimers type.

Chaperone-like Antibodies Targeting Misfolded Tau Protein: New Vistas in the Immunotherapy of Neurodegenerative Foldopathies

Neurodegenerative foldopathies are characterized by aberrant folding of proteins leading to the intracellular and extracellular accumulation of insoluble misfolded proteins. One of the most prominent protein folding disorders is Alzheimers disease (AD). In AD, there were identified two major driving forces behind neurodegeneration, misfolded proteins tau and amyloid-beta. Tau belongs to a family of intrinsically disordered proteins that are characterized by the absence of a rigid three-dimensional structure in their natural environment. However, in disease condition, tau truncation and hyperphosphorylation could lead to tau transformation from intrinsically disordered protein into highly ordered soluble and insoluble misfolded structures. Increased understanding of the molecular mechanism underlying pathological transformation of tau protein has opened up the possibility of targeting misfolded tau for therapeutic purposes. Pharmacological research has identified several therapeutic approaches targeting directly or indirectly tau cascade. Novel promising field of AD treatment represent monoclonal antibodies with chaperon like activities that will be able to neutralize the toxic gain of function of misfolded tau and thus increase its degradation. We suggest that chaperon like antibodies targeting disease modified tau may hold promise for the successful treatment of AD and related foldopathies.