Branislav Kovacech

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.

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.

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.

Transition of tau protein from disordered to misordered in Alzheimer's disease.

The neuronal protein tau, a member of the class of intrinsically disordered proteins, is characterized by the absence of any firm 3-D structure and high solubility when free in solution. The tau protein forms insoluble fibrils in the brain of people suffering from Alzheimer’s disease (AD) and other tauopathies and plays a key role in the neurodegenerative process. Posttranslational events leading to the transition of tau from a disordered highly soluble protein to the insoluble aggregate seem to be associated with hyperphosphorylation, truncation or a combination of both. These modifications are assumed to change the native disorder of tau into a preaggregation state, which then directly initiates the fibrillization process. Conformation-specific anti-tau antibody DC11 detects pathological truncated tau forms present in AD brains but not in the normal human brain. A truncated tau protein selected from this pool of DC11-positive molecules was sufficient to initiate and drive the complete tau cascade of neurofibrillary pathology in a rat model of tauopathy. Thus, DC11 antibody recognizes the AD-specific conformation of tau not found in the normal human brain. We propose the term ‘misdisordered’ for this distinct conformational state of tau, which is the first step in the transition of tau from a soluble disordered protein to its insoluble, misordered aggregated form.

The self-perpetuating tau truncation circle

Pathological truncations of human brain proteins represent the common feature of many neurodegenerative disorders including AD (Alzheimers disease), Parkinsons disease and Huntingtons disease. Protein truncations significantly change the structure and function of these proteins and thus can engender their pathological metamorphosis. We have shown previously that truncated forms of tau protein are contained in the core of the paired helical filaments that represent the main constituent of neurofibrillary pathology. Recently, we have identified truncated tau species of a different molecular signature. We have found that tau truncation is not produced by a random process, but rather by highly specific proteolytic cleavage and/or non-enzymatic fragmentation. In order to characterize the pathophysiology of AD-specific truncated tau species, we have used a transgenic rat model for AD expressing human truncated tau. Expression of the tau protein induces the formation of novel truncated tau species that originate from both transgenic human tau and endogenous rat tau proteins. Moreover, these truncated tau proteins are found exclusively in the misfolded fraction of tau, suggesting that they actively participate in the tau misfolding process. These findings corroborate further the idea that the appearance of truncated tau species starts a self-perpetuating cycle of further tau protein truncation leading to and accelerating tau misfolding and formation of neurofibrillary pathology.

Hyperphosphorylated truncated protein tau induces caspase-3 independent apoptosis-like pathway in the Alzheimer's disease cellular model.

Neurofibrillary degeneration and neuronal loss represent key pathological hallmarks of Alzheimers disease (AD). It has been demonstrated that the decrease of total neuronal numbers correlates with the presence of neurofibrillary degeneration in AD brain. In order to unravel the mechanism leading to the cell death in AD, we developed a stably transfected human neuroblastoma cellular model with doxycycline-regulatable expression of AD truncated tau protein (AT tau, 151-391 4R). Cells expressing the longest tau isoform (Tau 40) were used as a control. We found that more than 80% of the total amount of AT tau and Tau 40 were phosphorylated. Strikingly, both AT tau and Tau 40 reduced the metabolic activity of the cells in a time-dependent manner (p < 0.0001) suggesting that tau overexpression slows down cell proliferation. However, AT tau showed significantly higher toxicity than Tau 40 (p < 0.0001), which indicates that truncation leads to a toxic gain of function. The analysis of the type of the cell death revealed the characteristic features of apoptosis such as cell shrinkage, nuclear, and DNA fragmentation. However, we did not find either the activation of executive caspase (caspase-3) or the caspase cleavage products (PARP and fodrin). These results show that posttranslationally modified truncated tau protein induces caspase-3-independent apoptosis-like programmed cell death, a phenomenon we term tauoptosis.

Genetic background modifies neurodegeneration and neuroinflammation driven by misfolded human tau protein in rat model of tauopathy: implication for immunomodulatory approach to Alzheimer's disease

BackgroundNumerous epidemiological studies demonstrate that genetic background modifies the onset and the progression of Alzheimers disease and related neurodegenerative disorders. The efficacious influence of genetic background on the disease pathway of amyloid beta has been meticulously described in rodent models. Since the impact of genetic modifiers on the neurodegenerative and neuroinflammatory cascade induced by misfolded tau protein is yet to be elucidated, we have addressed the issue by using transgenic lines expressing the same human truncated tau protein in either spontaneously hypertensive rat (SHR) or Wistar-Kyoto (WKY) genetic background.MethodsBrains of WKY and SHR transgenic rats in the terminal stage of phenotype and their age-matched non-transgenic littermates were examined by means of immunohistochemistry and unbiased stereology. Basic measures of tau-induced neurodegeneration (load of neurofibrillary tangles) and neuroinflammation (number of Iba1-positive microglia, their activated morphology, and numbers of microglia immunoreactive for MHCII and astrocytes immunoreactive for GFAP) were quantified with an optical fractionator in brain areas affected by neurofibrillary pathology (pons, medulla oblongata). The stereological data were evaluated using two-way ANOVA and Students t-test.ResultsTau neurodegeneration (neurofibrillary tangles (NFTs), axonopathy) and neuroinflammation (microgliosis, astrocytosis) appeared in both WKY and SHR transgenic rats. Although identical levels of transgene expression in both lines were present, terminally-staged WKY transgenic rats displayed significantly lower final NFT loads than their SHR transgenic counterparts. Interestingly, microglial responses showed a striking difference between transgenic lines. Only 1.6% of microglia in SHR transgenic rats expressed MHCII in spite of having a robust phagocytic phenotype, whereas in WKY transgenic rats, 23.2% of microglia expressed MHCII despite displaying a considerably lower extent of transformation into phagocytic phenotype.ConclusionsThese results show that the immune response represents a pivotal and genetically variable modifying factor that is able to influence vulnerability to neurodegeneration. Therefore, targeted immunomodulation could represent a prospective therapeutic approach to Alzheimers disease.

Microglia display modest phagocytic capacity for extracellular tau oligomers

BackgroundAbnormal misfolded tau protein is a driving force of neurofibrillary degeneration in Alzheimers 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 Alzheimers disease.

Validation of a quantitative cerebrospinal fluid alpha-synuclein assay in a European-wide interlaboratory study

Decreased levels of alpha-synuclein (aSyn) in cerebrospinal fluid (CSF) in Parkinsons disease and related synucleinopathies have been reported, however, not consistently in all cross-sectional studies. To test the performance of one recently released human-specific enzyme-linked immunosorbent assay (ELISA) for the quantification of aSyn in CSF, we carried out a round robin trial with 18 participating laboratories trained in CSF ELISA analyses within the BIOMARKAPD project in the EU Joint Program - Neurodegenerative Disease Research. CSF samples (homogeneous aliquots from pools) and ELISA kits (one lot) were provided centrally and data reported back to one laboratory for data analysis. Our study showed that although factors such as preanalytical sample handling and lot-to-lot variability were minimized by our study design, we identified high variation in absolute values of CSF aSyn even when the same samples and same lots of assays were applied. We further demonstrate that although absolute concentrations differ between laboratories the quantitative results are comparable. With further standardization this assay may become an attractive tool for comparing aSyn measurements in diverse settings. Recommendations for further validation experiments and improvement of the interlaboratory results obtained are given.

CSF phospho-tau correlates with behavioural decline and brain insoluble phospho-tau levels in a rat model of tauopathy

The aim of the present study was to identify the relationship between progressive neurobehavioural decline and phospho-tau levels (p-tau181) in the cerebrospinal fluid (CSF) and the brain in transgenic rats expressing human truncated tau protein. Behavioural analyses, as quantified using the NeuroScale scoring method, revealed that the transgenic rats fell into two main groups based on the baseline behavioural functioning: (1) mild neurobehavioural impairment (MNI, score 3.3–26) and (2) severe neurobehavioural impairment (SNI, score 36–44). SNI transgenic rats showed a significant increase in brain sarkosyl insoluble p-tau181 when compared to their MNI counterparts. In order to determine whether CSF phospho-tau reflects the behavioural decline and increase in sarkosyl insoluble tau in the brain, p-tau181 was measured in the CSF in a longitudinal study. The study showed a significant increase in CSF p-tau181 during the progression of the disease from MNI to SNI. Moreover, increased levels of p-tau181 in CSF correlated with an increase in the sarkosyl insoluble p-tau181 levels in the brain. The increase in the CSF level of p-tau181 during progressive behavioural decline suggests that it may represent a useful surrogate biomarker for preclinical drug development and a potential surrogate endpoint for clinical trials of disease-modifying therapy for Alzheimer’s disease and related human tauopathies.