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Project

Development and validation of active and passive immunotherapy targeted at protein Tau in transgenic mouse models for Alzheimer's disease

Over 30 million patients are affected worldwide by Alzheimers disease (AD), the most frequent and prominent type of dementia. Paralleling the graying of our society, resulting from improved hygiene and advanced medical care, the prevalence of dementia and in particular AD is expectedto quadruple by 2050. The dependence of a dementing person on the care of others for almost all daily tasks, poses a high financial and emotional burden on spouses, family and on society at large. More than 100 years after the original description by Professor Alois Alzheimer, many questions still remain unanswered about the molecular mechanisms behind thisdisease. Ongoing research and development of urgently needed early diagnosis and effective treatment will hopefully lead to cure or prevention,in the not too far future.
 
Besides marked neuroinflammation, AD is characterized by aggregation of mis-folded amyloid peptides andprotein Tau. These give rise to respectively the extracellular amyloid or senile plaques and to the intracellular neurofibrillary tangles and neuropil threads. While the exact synergy between amyloid and Tau pathology remains vague, available genetic, pathological and experimental observations soundly corroborate tauopathy as an essential parameter, and not a mere side-effect in AD. The LEGTEGG research group incorporates both types of pathology in their working hypothesis. The cascade of events that eventually leads to memory impairment and cognitive decline inAD, is initiated by an excess of amyloid peptides. These activate different kinases, with GSK3 as most important mediator between amyloid and Tau pathology. Higher than normal kinase activity results in abnormal phosphorylation of protein Tau, which induces aggregation and tauopathy. Combined, they trigger the execution of synaptic and neuronal malfunction,leading to neurodegeneration and dementia. Besides the pathological malfunctions, the normal physiological functions of amyloid peptides, if any, and of protein Tau in the neuronal mechanisms of learning and memory,remain to be elucidated.  
 
Aggregates of proteinTau occur in soma and neuronal processes and correlate well with the severity of cognitive decline in ageing patients. However, in mice with tauopathy, memory defects appear more early, before the formation of intracellular aggregates in brain, indicating that not the large fibrils are responsible but a cause of the underlying tauopathy. Smaller molecular intermediates in the aggregation cascade, situated between single molecules and large fibrils, must be the actual neurotoxic agents that we denoted TauP*, because their exact biochemical nature remains unknown. Their identification and the understanding of their formation would be the major step forward to untangle the disease and define new therapeutic targets. 
 
The aim of this PhD was on one hand to characterize the molecular nature of the elusive TauP* species and the causes and effects of Tau pathology, and on the other hand to develop and validate immunotherapy to counteract tauopathy. For both goals we relied on the different transgenic mouse models for tauopathy and AD developed at the LEGTEGG research group.  
 
We studied the molecularcharacteristics of tauopathy to define TauP* first in Tau.P301L mice that model tauopathy. We compared them to two bigenic mouse strains, namedbiAT and biGT that are models for Alzheimers disease and frontotemporal dementia, respectively. They express the same Tau.P301L transgenes butin combination with APP.V717I in biAT and with GSK3β in biGT mice.Our observations define important correlations between biochemical and pathological parameters in forebrain and brainstem. The forebrain, with hippocampus and cortex is most essential for learning and memory, while the brainstem controls involuntary physiological actions, like heartbeatand breathing. 
 
Tauopathy in the brainstem causes upper airway defects and early death of Tau.P301L mice, and was now demonstrated to be very similar in the brainstem of terminal biGT mice, but at much older age. Biochemical analysis identified phosphorylation of S262 and S396 in protein Tau by GSK3β as the origin of this delayed Tau aggregation in the brainstem of biGT mice. This explained the longer life-expectancy of the biGT mice, which at the same time allows for more important, even dramatic forebrain tauopathy that evidently increases withage. In biAT mice, similar reduced brainstem Tau pathology could be proposed to delay death in older surviving biAT mice, in contrast to the epileptic problems that are surmised to cause early mortality. Note that also AD patients are susceptible to epileptic seizures, meaning that the biAT mice recapitulate AD not only by the combined amyloid and Tau pathology. 
 
The biochemical analysis of protein Tau in the different transgenic models confirmed that oligomeric and conformationalchanged forms are the likely candidates for TauP*. ELISA with the MC1 antibody detected the conformational abnormal forms of protein Tau, whileELISA with the TN5-20 antibody, both for capture and detection, measured oligomers and small aggregates of protein Tau. Both types of ELISA yielded very similar results in mice of both genotypes, at different age and with different clinical conditions. Moreover the ELISA data fitted closely the detection of the 68 kDa Tau isoform in western blotting as marker of tauopathy, and as candidate for TauP*. Because no major qualitative differences in phosphorylation were noted in protein Tau in forebrain or brainstem between pre-symptomatic, clinically ill and terminal Tau.P301L and biGT mice, the molecular generation or origin of TauP* remains elusive. Several posttranslational modifications of Tau were considered, although O-GluNAc-ylation was excluded in a parallel project. Phosphorylation of TauP* was considered as main important, in first instance analysed for the quantitative aspect, i.e. the overall level of phosphorylation, but also the precise combinations of phospho-epitopes should be evaluated, which unfortunately are "in-numeral" on this naturally unfolded protein Tau, with 45 serine, 35 threonine and 5 tyrosine residues exposedto the plethora of neuronal kinases. Further biochemical elucidation isongoing, including analysis with the newly developed antibodies that are conformation and/or phosphorylation specific. 
 
To counteract the fundamental pathological processes that cause AD, differenttherapeutic approaches were primarily and historically concentrated on amyloid peptides, conform the amyloid cascade hypothesis. Many of the approaches failed in clinical trials, showing inadequate efficacy or majorside-effects. Consequently, many other treatment strategies that targetother pathological markers or events are currently evaluated in pre-clinical studies in transgenic mouse models. 
 
The second aim of this PhD project was to develop, evaluate and validate (i) activeimmunotherapy with liposome-based vaccines carrying synthetic pathological phospho-Tau peptides and (ii) passive immunotherapy with newly generated monoclonal antibodies. By concentrating on protein Tau not only treatment of AD but also of the many primary tauopathies can become possible. Both strategies were found effective in decreasing phosphorylated soluble Tau in respectively Tau.P301L and biGT mouse models. Moreover active immunotherapy in Tau.P301L mice prevented, at least partially, the formation of higher order fibrillar aggregates in the forebrain of these mice. Beside the effectiveness, we were concerned to demonstrate the safety of both strategies, but mostly that of active vaccination against protein Tau. The liposomal vaccines were proven to be safe even after prolonged and multiple administration, as corroborated by the improved condition, but more essentially by the absence of any signs of neuroinflammation and without detectable T-cell responses. 
 
The combined results of experimental active and passive immunotherapy in the LEGTEGG mouse models for tauopathy allow us to be cautiously optimistic for their promise for the treatment of primary tauopathies and Alzheimer's disease, either active on their own or as an important element in a combination of therapeutic treatments.  
Date:1 Oct 2008 →  13 Sep 2013
Keywords:Post-translational modification, Therapy applications, Diagnostic applications, Transgenic mice, Tauopathy, Neurofibrillary tangles, Tau, Alzheimer's disease
Disciplines:Biochemistry and metabolism, Medical biochemistry and metabolism, Systems biology, Neurosciences, Biological and physiological psychology, Cognitive science and intelligent systems, Developmental psychology and ageing, Immunology
Project type:PhD project