Clara Theunis

Efficacy and Safety of A Liposome-Based Vaccine against Protein Tau, Assessed in Tau.P301L Mice That Model Tauopathy

Progressive aggregation of protein Tau into oligomers and fibrils correlates with cognitive decline and synaptic dysfunction, leading to neurodegeneration in vulnerable brain regions in Alzheimers disease. The unmet need of effective therapy for Alzheimers disease, combined with problematic pharmacological approaches, led the field to explore immunotherapy, first against amyloid peptides and recently against protein Tau. Here we adapted the liposome-based amyloid vaccine that proved safe and efficacious, and incorporated a synthetic phosphorylated peptide to mimic the important phospho-epitope of protein Tau at residues pS396/pS404. We demonstrate that the liposome-based vaccine elicited, rapidly and robustly, specific antisera in wild-type mice and in Tau.P301L mice. Long-term vaccination proved to be safe, because it improved the clinical condition and reduced indices of tauopathy in the brain of the Tau.P301L mice, while no signs of neuro-inflammation or other adverse neurological effects were observed. The data corroborate the hypothesis that liposomes carrying phosphorylated peptides of protein Tau have considerable potential as safe and effective treatment against tauopathies, including Alzheimers disease.

Neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) slows down Alzheimer's disease-like pathology in amyloid precursor protein-transgenic mice.

Pituitary adenylate cyclase‐activating polypeptide (PACAP) has neuroprotective and neurotrophic properties and is a potent α‐secretase activator. As PACAP peptides and their specific receptor PAC1 are localized in central nervous system areas affected by Alzheimers disease (AD), this study aims to examine the role of the natural peptide PACAP as a valuable approach in AD therapy. We investigated the effect of PACAP in the brain of an AD transgenic mouse model. The long‐term intranasal daily PACAP application stimulated the nonamyloidogenic processing of amyloid precursor protein (APP) and increased expression of the brain‐derived neurotrophic factor and of the antiapoptotic Bcl‐2 protein. In addition, it caused a strong reduction of the amyloid β‐peptide (Aβ) transporter receptor for advanced glycation end products (RAGE) mRNA level. PACAP, by activation of the somatostatin‐neprilysin cascade, also enhanced expression of the Aβ‐degrading enzyme neprilysin in the mouse brain. Furthermore, daily PAC1‐receptor activation via PACAP resulted in an increased mRNA level of both the PAC1 receptor and its ligand PACAP. Our behavioral studies showed that long‐term PACAP treatment of APP[V717I]‐transgenic mice improved cognitive function in animals. Thus, nasal application of PACAP was effective, and our results indicate that PACAP could be of therapeutic value in treating AD.—Rat, D., Schmitt, U., Tippmann, F., Dewachter, I., Theunis, C., Wieczerzak, E, Postina, R., van Leuven, F., Fahrenholz, F., Kojro, E. Neuropeptide pituitary adenylate cyclase‐activating polypeptide (PACAP) slows down Alzheimers disease‐like pathology in amyloid precursor protein‐transgenic mice. FASEB J. 25, 3208‐3218 (2011). www.fasebj.org

Increasing Brain Protein O-GlcNAc-ylation Mitigates Breathing Defects and Mortality of Tau.P301L Mice

The microtubule associated protein tau causes primary and secondary tauopathies by unknown molecular mechanisms. Post-translational O-GlcNAc-ylation of brain proteins was demonstrated here to be beneficial for Tau.P301L mice by pharmacological inhibition of O-GlcNAc-ase. Chronic treatment of ageing Tau.P301L mice mitigated their loss in body-weight and improved their motor deficits, while the survival was 3-fold higher at the pre-fixed study endpoint at age 9.5 months. Moreover, O-GlcNAc-ase inhibition significantly improved the breathing parameters of Tau.P301L mice, which underpinned pharmacologically the close correlation of mortality and upper-airway defects. O-GlcNAc-ylation of brain proteins increased rapidly and stably by systemic inhibition of O-GlcNAc-ase. Conversely, biochemical evidence for protein Tau.P301L to become O-GlcNAc-ylated was not obtained, nor was its phosphorylation consistently or markedly affected. We conclude that increasing O-GlcNAc-ylation of brain proteins improved the clinical condition and prolonged the survival of ageing Tau.P301L mice, but not by direct biochemical action on protein tau. The pharmacological effect is proposed to be located downstream in the pathological cascade initiated by protein Tau.P301L, opening novel venues for our understanding, and eventually treating the neurodegeneration mediated by protein tau.

Protein Tau: Prime Cause of Synaptic and Neuronal Degeneration in Alzheimer's Disease

The microtubule-associated protein Tau (MAPT) is a major component of the pathogenesis of a wide variety of brain-damaging disorders, known as tauopathies. These include Alzheimers disease (AD), denoted as secondary tauopathy because of the obligatory combination with amyloid pathology. In all tauopathies, protein Tau becomes aberrantly phosphorylated, adopts abnormal conformations, and aggregates into fibrils that eventually accumulate as threads in neuropil and as tangles in soma. The argyrophilic neurofibrillary threads and tangles, together denoted as NFT, provide the postmortem pathological diagnosis for all tauopathies. In AD, neurofibrillary threads and tangles (NFTs) are codiagnostic with amyloid depositions but their separated and combined contributions to clinical symptoms remain elusive. Importantly, NFTs are now considered a late event and not directly responsible for early synaptic dysfunctions. Conversely, the biochemical and pathological timeline is not exactly known in human tauopathy, but experimental models point to smaller Tau-aggregates, termed oligomers or multimers, as synaptotoxic in early stages. The challenge is to molecularly define these Tau-isoforms that cause early cognitive and synaptic impairments. Here, we discuss relevant studies and data obtained in our mono- and bigenic validated preclinical models, with the perspective of Tau as a therapeutic target.

Phosphorylation of protein Tau by GSK3β prolongs survival of bigenic Tau.P301L × GSK3β mice by delaying brainstem tauopathy

Tau.P301L transgenic mice suffer precocious mortality between ages 8 and 11 months, resulting from upper airway defects caused by tauopathy in autonomic brainstem circuits that control breathing (Dutschmann et al., 2010). In individual mice, the clinical phenotype evolves progressively and rapidly (3-6weeks) from clasping, over general motor impairment to severe reduction in body-weight into the terminal phase that announces imminent death (<3days). Surprisingly, co-expression of GSK3β with Tau.P301L significantly prolonged survival of bigenic biGT mice (Terwel et al., 2008), which we here assign to delayed development of brainstem tauopathy. Eventually, brainstem tauopathy became as prominent in old biGT mice in the specified brainstem nuclei as in the parental Tau.P301L mice, resulting in similar clinical deterioration and terminal phase preceding death, although at later age. Biochemically, in both genotypes the pathway to neurofibrillary tangles and neuropil threads was similar: phosphorylation of protein Tau and formation of soluble oligomers and insoluble aggregates, ending in the typical tangles and threads of tauopathy. The extra GSK3β activity led to expected increased phosphorylation of protein Tau, particularly at residues S262 and S396, which we must conclude to delay the aggregation of protein Tau in the brainstem of aging biGT mice. The unexpected, paradoxical alleviation of the brainstem problems in biGT mice allowed them to grow older and thereby develop more severe tauopathy in forebrain than Tau.P301L mice, which succumb at younger age.

Novel Phospho-Tau Monoclonal Antibody Generated Using a Liposomal Vaccine, with Enhanced Recognition of a Conformational Tauopathy Epitope

The microtubule-associated protein Tau is an intrinsically unfolded, very soluble neuronal protein. Under still unknown circumstances, Tau protein forms soluble oligomers and insoluble aggregates that are closely linked to the cause and progression of various brain pathologies, including Alzheimer’s disease. Previously we reported the development of liposome-based vaccines and their efficacy and safety in preclinical mouse models for tauopathy. Here we report the use of a liposomal vaccine for the generation of a monoclonal antibody with particular characteristics that makes it a valuable tool for fundamental studies as well as a candidate antibody for diagnostic and therapeutic applications. The specificity and affinity of antibody ACI-5400 were characterized by a panel of methods: (i) measuring the selectivity for a specific phospho-Tau epitope known to be associated with tauopathy, (ii) performing a combination of peptide and protein binding assays, (iii) staining of brain sections from mouse preclinical tauopathy models and from human subjects representing six different tauopathies, and (iv) evaluating the selective binding to pathological epitopes on extracts from tauopathy brains in non-denaturing sandwich assays. We conclude that the ACI-5400 antibody binds to protein Tau phosphorylated at S396 and favors a conformation that is typically present in the brain of tauopathy patients, including Alzheimer’s disease.

Epileptogenicity and precocious mortality of bigenic biat mice that express amyloid and tau

Background:Across the three clinical profiles caused by frontotemporal lobar degeneration (FTLD) (behavioural variant (bvFTD), semantic dementia (SD) and progressive non-fluent aphasia (PNFA)), pathology may be characterized at a molecular level by either tau or TDP-43 proteinopathies. In sporadic cases, correlations between profile and proteinopathy are weak, though among patients with SD around 90% have TDP-43 positive pathology in post mortem series. There is emerging evidence that inflammation underpins TDP-43 proteinopathy: in one study, antibodies against progranulin were found in up to 43% of patients with systemic autoimmune conditions, compared with <1% of controls. Another study found a history of autoimmunity in significantly more cases of definite (familial) or highly probable (SD) TDP-43 pathology than controls. Might autoimmune disease and TDP-43 proteinopathy therefore be linked by the presence of antibodies affecting the activity of proand anti-inflammatory cytokines such as PGRN and TNFa? If so, we may expect to see evidence of FTLD-TDP in patients with autoimmunity. Since TDP43 correlates strongly with an SD phenotype, subclinical pathology may be marked by subtle semantic deficits. Methods: Participants with rheumatoid arthritis (RA) or psoriatic arthritis (PsA) and a group of matched controls with osteoarthritis (OA) completed three tests of semantic memory: Graded Naming Test (GNT), Pyramids and Palm Trees (PPT) and verbal fluency, a cognitive screening questionnaire and the MMSE. Plasma samples were obtained to measure levels of PGRN and TNFa. Results: We present the semantic memory performance in individuals with and without an autoimmune condition. The putative inflammatory mechanism is examined based on correlations between aggregated semantic test performance and levels of PGRN and TNFa. Conclusions: The differential prevalence of subclinical semantic deficits in patients with a common autoimmune disease and a similar condition without an autoimmune mechanism contributes to the debate concerning a possible autoimmune, inflammatory mechanism underpinning FTLD-TDP.

A liposomal anti-phospho-tau vaccine demonstrates higher immunogenicity and specificity than a classical peptide vaccine approach

However, the relationship between these two proteins and neuronal loss lacks amechanistic explanation. Furthermore, evidence from animal models suggests that amyloid beta toxicity is mediated by tau [1-6]. We hypothesize that tau oligomers formation plays curial role in driving AD pathogenesis. Thus, tau oligomers represent an ideal therapeutic target for the treatment of AD. In order to study the removal of toxic tau assemblies in an animal model of AD (Tg2576), we generated a tau oligomer specific antibody (TOMA). This antibody does not recognize the functional monomeric tau or oligomers from other amyloidogenic proteins. Methods: Here we used the Tg2576 mouse model which overexpress the human APP with the Swedish double mutations (K670N, M671L) under the control of a hamster prion protein promoter .14-month old Tg2576 mice, received a single iv injection of 30 mg of the TOMA antibody. Control group received 30 mg of non-specific IgG. Cognitive function was assessed by novel object recognition test, 15 days after injection. In addition, western blot, ELISA and Immunostaining were performed to evaluate the response to treatment. Results: Our results indicate that single iv-injection of the TOMA antibody, reduce endogenous tau oligomers and improve cognition in the Tg2576 mouse. Interestingly, removal of tau oligomers by immunotherapy decreases beta-amyloid-56* and increases deposition of plaques in immunized mice. Conclusions: Our results support the findings that tau oligomers mediate beta-amyloid toxicity in vivo. Moreover, removal of tau oligomers by immunotherapy may induce beta-amyloid aggregates to assembly into inert and perhaps protective plaques. Thus, targeting tau oligomers by immunotherapymay represent a novel strategy for the treatment of AD and other neurodegenerative tauopathies.

Novel phospho-tau-specific liposome-based vaccines to treat tau pathology

Clara Theunis, Natalia Crespo-Biel, Maria Pihlgren, Peter Borghgraef, Herman Devijver, Valerie Gafner, David Hickman, Nathalie Chuard, Maria Pilar Lopez Deber, Pedro Reis, Anna Lucia Buccarello, Oskar Adolfsson, Sigrun Roeber, Hans Kretzschmar, Andrea Pfeifer, Andreas Muhs, Fred Van Leuven, LEGTEGG Katholieke Universiteit Leuven, Leuven, Belgium; 2 ACImmune, Lausanne, Switzerland; LEGTEGGKULeuven, Leuven, Belgium; LEGTEGG-KULeuven, Leuven, Belgium; AC Immune SA, Lausanne, Switzerland; Center for Neuropathology, M€unchen, Germany; 7 AC Immune, Lausanne, Switzerland; 8 Experimental Genetics Group LEGTEGG KULeuven, Leuven, Belgium.

Novel phospho-Tau specific monoclonal antibodies for the treatment of Tau-mediated pathology

Background: Research over the last decade has highlighted the role that metals may play in the pathogenesis of Alzheimer’s disease (AD). To date, the primary end point for these studies has been s-amyloid. Recent literature supports the notion that metals may also interact with tau and be important for the formation of neurofibrillary tangles (NFTs). In this study we surveyed metal levels in the rTg(tauP301L)4510 mouse model and utilised clioquinol (CQ) to assess the effect of metal modulation on tangle pathology. Methods: Metal levels were assessed by ICPMS in cohorts of mice at either w2 months (n 1⁄4 6 WT; n 1⁄4 6 Tg) or w5 months (n 1⁄4 12 WT; n 1⁄4 11 Tg) of age. The effect of oral clioquinol (6 months of treatment, 30mg/kg) was assessed in a separate cohort of Tg (n 1⁄4 25) and WT mice (n 1⁄4 30). Drug treatment began at w6 months of age and prior to culling animals were assessed in the open field, rotarod, y-maze and water maze. Young rTg4510 mice, prior to the formation of any pathology, had significantly altered brain metal content, including elevations in zinc (25%, p 1⁄4 0.01) and copper (31%, p 1⁄4 0.02) and a trend to elevated iron (20%, p 1⁄4 0.08). In contrast, aged rTg4510 mice had significantly decreased levels of zinc (18%, p 1⁄4 0.01) and iron levels were also decreased (20%, p 1⁄4 0.06). Behavioural analysis of older mice did not reveal any effect of CQ on performance in the water maze, Y-maze or open field. In the rotarod, however, the Tg animals spent a significantly shorter amount of time on the rotarod (3.5 sec, Tg; 35 sec, WT; p < 0.0001), and clioquinol treatment of the Tg animals significantly improved their performance (10.2 sec, Tg+clioquinol; p 1⁄4 0.01 compared to Tg). Further metal analyses and histological studies are currently being finalised and will be discussed. Conclusions: We have demonstrated that there is a dysregulation in metal ion homeostasis in the rTg4510 model of tauopathy, and that there are also age-dependent alterations in metal levels that occur coincident with the evolution of neurofibrillary pathology. These data support a potential interaction between tau/NFTs and various metal ions and provide further insight into novel therapeutic approaches for the treatment of tauopathies.