- Email: [email protected]
THE FIRST TAU VACCINE FOR THERAPY OF ALZHEIMER'S DISEASE AND FTLD: FROM TAU STRUCTURE TO HUMAN CLINICAL TRIALS
Podium Presentations: Wednesday, July 27, 2016
manner; and (2) the success of tau immunotherapy could depend on the site targeted which does not have to be any phosphorylation site. O4-04-04
HYPERPHOSPHORYLATION DETERMINES BOTH THE SPREAD AND THE MORPHOLOGY OF TAU PATHOLOGY
Wen Hu, Xinhua Zhang, Shutao Xie, Yunn Chyn Tung, Fei Liu, Khalid Iqbal, New York State Institute for Basic Research in Developmental Disabilities, New York, NY, USA. Contact e-mail: [email protected] Background: Neurofibrillary pathology of abnormally hyper-
phosphorylated tau (P-tau) is a hallmark of Alzheimer’s disease (AD) and other tauopathies. Tau pathology can be experimentally induced and propagated. However, what induces the prion-like transmission-character to tau and produces morphologically distinct tau lesions remains elusive. We sought to investigate the role of hyperphosphorylation in the spread of tau pathology in hTau transgenic mice. Methods: We first isolated oligomeric, hyperphosphorylated tau from AD brain (AD P-tau) using an established protocol by us previously, and dephosphorylated AD P-tau in vitro with protein phosphatase 2A (PP2A). We then injected AD P-tau or PP2A-treated AD P-tau, using recombinant Tau441 and saline as controls, bilaterally into hippocampi (0.12 mg tau each) of 3 month-old hTau mice which express all six isoforms of wildtype human tau, and characterized the induced tau pathology by immunostaining for phospho-tau and Thioflavin-S staining at 6, 9 and 11 months post-injection. Results: We found that intra-hippocampal injection with AD P-tau, but not non-phosphorylated tau or saline, produced numerous P-tau tangles and neuropil threads both locally and in cerebral cortex lateral to injection site and upstream to the hippocampus. Dephosphorylation of AD P-tau with protein phosphatase-2A dramatically reduced (by w75%) and switched tau pathology from tangles to argyrophilic grainlike morphology. Conclusions: Abnormal hyperphosphorylation of tau determines the spread and the morphology of tau lesions and the propagation of tau pathology takes place both locally and in axonally connected areas, highlighting tau hyperphosphorylation as a potential therapeutic target. O4-04-05
HSC70/DNAJC5 COMPLEXES CONTROL THE RELEASE OF EXTRACELLULAR NEURODEGENERATIVE PROTEINS
Sarah Fontaine1, Dali Zheng1, Jonathan J. Sabbagh1, Mackenzie D. Martin1, Dale Chaput1, Justin H. Trotter2, Stanley M. Stevens, Jr,1, Chad A. Dickey1, 1University of South Florida, Tampa, FL, USA; 2Stanford University, Stanford, CA, USA. Contact e-mail: [email protected] Background: Extracellular tau, a-synuclein, and TDP43 have
been detected in a number of neurodegenerative diseases including Alzheimer’s disease. The presence of these extracellular proteins is hypothesized to be important for the pathophysiological neuron-neuron spread and accumulation of misfolded proteins. However, details of how these proteins are released from neurons has yet to be established. We hypothesized that the Hsc70 machinery, known to regulate a number of pathways important for the proteostasis of neurodegenerative disease proteins, may be involved in this phenomenon as well. Methods: Extracellular proteins were analyzed with a combination of mass spectroscopy, Western blot and dot blot from immortalized neuronal and non-neuronal cells, primary neurons, and organotypic slices. Intracellular protein levels
P341
and subcellular distribution were determined using Western blot and immunocytochemistry. mRNA levels were quantified with RT-qPCR. Results: Overexpression of DnaJC5 facilitated non-canonical exocytosis of wt and mutant tau, TDP43, and asynuclein in both neuronal and non-neuronal cells. Knocking down components of this exocytic pathway reduced extracellular tau release. Further, this release mechanism was dependent on the Hsc70/DnaJC5 complex: extracellular tau release was inhibited when DnaJC5 was knocked out or unable to bind Hsc70, or when levels of Hsc70 were reduced, or when the chaperone was inactive. Conclusions: These results describe, for the first time, a mechanism for the release of extracellular tau from neuronal cells dependent on the Hsc70 chaperone machinery. Here we show that in concert with DnaJC5, Hsc70 complexes regulate the extracellular release of tau, a-synuclein, and TDP-43. This extracellular release mechanism may be a universal phenomenon contributing to the propagation of proteinopathies such as AD, Parkinson’s disease, and ALS. Finally, these results illuminate a new Hsc70-driven pathway for therapeutic intervention for the treatment of these diseases. O4-04-06
THE FIRST TAU VACCINE FOR THERAPY OF ALZHEIMER’S DISEASE AND FTLD: FROM TAU STRUCTURE TO HUMAN CLINICAL TRIALS
Michal Novak, Rostislav Skrabana, Norbert Zilka, AXON Neuroscience SE, Bratislava, Slovakia. Contact e-mail: [email protected] Background: Previously, we have identified structural determinants
on disease modified protein tau that have essential regulatory function for formation of neurofibrillary pathology in Alzheimer’s disease (AD). The identified tau determinants, which are recognized by monoclonal antibody DC8E8, served as a lead for the design of an active vaccine AADvac1 that entered the Phase II clinical trial on human AD patients. In the present study we address the questions about the sequence and structure requirements of DC8E8based immunotherapy. Methods: The kinetics of DC8E8 interaction with tau were studied by surface plasmon resonance (SPR), conformation of tau epitope and DC8E8 combining site was revealed by X-ray crystallography, and observed tau binding mode was verified by alanine mutagenesis of DC8E8 paratope residues. Results: Monoclonal antibody DC8E8 is unique among the anti-tau antibodies as it interacts with three, respectively four homologous epitopes on a single tau molecule. SPR affinity studies uncovered finely tuned specificity of DC8E8 to individual components of its quadruple epitope on the tau molecule. Further, we solved several X-ray structures of the DC8E8 Fab fragment and its complex with a panel of tau peptides up to 1.69 A resolution. Antibody recognition is mediated by a unique paratope binding pocket embracing the conserved sequence motif HXPGGG on the tau molecule, which adopts a tight type I beta-turn. The binding mode of DC8E8 effectively blocks the beta-structure forming hexapeptides adjacent to the epitope, which explains the potent DC8E8 inhibitory effect on tau aggregation. Finally, alanine scanning mutagenesis experimentally confirmed the importance of specific DC8E8 paratope residues that are indispensable for binding therapeutic tau epitopes. Conclusions: Present results of structural and functional analysis shed light on the mechanism of action of active and passive immunotherapy based on the monoclonal antibody DC8E8 and its therapeutic tau epitope encompassed in the tau vaccine AADvac1. These findings further substantiate the potency of active and passive vaccine to protect the brain from tau neurodegeneration.
manner; and (2) the success of tau immunotherapy could depend on the site targeted which does not have to be any phosphorylation site. O4-04-04
HYPERPHOSPHORYLATION DETERMINES BOTH THE SPREAD AND THE MORPHOLOGY OF TAU PATHOLOGY
Wen Hu, Xinhua Zhang, Shutao Xie, Yunn Chyn Tung, Fei Liu, Khalid Iqbal, New York State Institute for Basic Research in Developmental Disabilities, New York, NY, USA. Contact e-mail: [email protected] Background: Neurofibrillary pathology of abnormally hyper-
phosphorylated tau (P-tau) is a hallmark of Alzheimer’s disease (AD) and other tauopathies. Tau pathology can be experimentally induced and propagated. However, what induces the prion-like transmission-character to tau and produces morphologically distinct tau lesions remains elusive. We sought to investigate the role of hyperphosphorylation in the spread of tau pathology in hTau transgenic mice. Methods: We first isolated oligomeric, hyperphosphorylated tau from AD brain (AD P-tau) using an established protocol by us previously, and dephosphorylated AD P-tau in vitro with protein phosphatase 2A (PP2A). We then injected AD P-tau or PP2A-treated AD P-tau, using recombinant Tau441 and saline as controls, bilaterally into hippocampi (0.12 mg tau each) of 3 month-old hTau mice which express all six isoforms of wildtype human tau, and characterized the induced tau pathology by immunostaining for phospho-tau and Thioflavin-S staining at 6, 9 and 11 months post-injection. Results: We found that intra-hippocampal injection with AD P-tau, but not non-phosphorylated tau or saline, produced numerous P-tau tangles and neuropil threads both locally and in cerebral cortex lateral to injection site and upstream to the hippocampus. Dephosphorylation of AD P-tau with protein phosphatase-2A dramatically reduced (by w75%) and switched tau pathology from tangles to argyrophilic grainlike morphology. Conclusions: Abnormal hyperphosphorylation of tau determines the spread and the morphology of tau lesions and the propagation of tau pathology takes place both locally and in axonally connected areas, highlighting tau hyperphosphorylation as a potential therapeutic target. O4-04-05
HSC70/DNAJC5 COMPLEXES CONTROL THE RELEASE OF EXTRACELLULAR NEURODEGENERATIVE PROTEINS
Sarah Fontaine1, Dali Zheng1, Jonathan J. Sabbagh1, Mackenzie D. Martin1, Dale Chaput1, Justin H. Trotter2, Stanley M. Stevens, Jr,1, Chad A. Dickey1, 1University of South Florida, Tampa, FL, USA; 2Stanford University, Stanford, CA, USA. Contact e-mail: [email protected] Background: Extracellular tau, a-synuclein, and TDP43 have
been detected in a number of neurodegenerative diseases including Alzheimer’s disease. The presence of these extracellular proteins is hypothesized to be important for the pathophysiological neuron-neuron spread and accumulation of misfolded proteins. However, details of how these proteins are released from neurons has yet to be established. We hypothesized that the Hsc70 machinery, known to regulate a number of pathways important for the proteostasis of neurodegenerative disease proteins, may be involved in this phenomenon as well. Methods: Extracellular proteins were analyzed with a combination of mass spectroscopy, Western blot and dot blot from immortalized neuronal and non-neuronal cells, primary neurons, and organotypic slices. Intracellular protein levels
P341
and subcellular distribution were determined using Western blot and immunocytochemistry. mRNA levels were quantified with RT-qPCR. Results: Overexpression of DnaJC5 facilitated non-canonical exocytosis of wt and mutant tau, TDP43, and asynuclein in both neuronal and non-neuronal cells. Knocking down components of this exocytic pathway reduced extracellular tau release. Further, this release mechanism was dependent on the Hsc70/DnaJC5 complex: extracellular tau release was inhibited when DnaJC5 was knocked out or unable to bind Hsc70, or when levels of Hsc70 were reduced, or when the chaperone was inactive. Conclusions: These results describe, for the first time, a mechanism for the release of extracellular tau from neuronal cells dependent on the Hsc70 chaperone machinery. Here we show that in concert with DnaJC5, Hsc70 complexes regulate the extracellular release of tau, a-synuclein, and TDP-43. This extracellular release mechanism may be a universal phenomenon contributing to the propagation of proteinopathies such as AD, Parkinson’s disease, and ALS. Finally, these results illuminate a new Hsc70-driven pathway for therapeutic intervention for the treatment of these diseases. O4-04-06
THE FIRST TAU VACCINE FOR THERAPY OF ALZHEIMER’S DISEASE AND FTLD: FROM TAU STRUCTURE TO HUMAN CLINICAL TRIALS
Michal Novak, Rostislav Skrabana, Norbert Zilka, AXON Neuroscience SE, Bratislava, Slovakia. Contact e-mail: [email protected] Background: Previously, we have identified structural determinants
on disease modified protein tau that have essential regulatory function for formation of neurofibrillary pathology in Alzheimer’s disease (AD). The identified tau determinants, which are recognized by monoclonal antibody DC8E8, served as a lead for the design of an active vaccine AADvac1 that entered the Phase II clinical trial on human AD patients. In the present study we address the questions about the sequence and structure requirements of DC8E8based immunotherapy. Methods: The kinetics of DC8E8 interaction with tau were studied by surface plasmon resonance (SPR), conformation of tau epitope and DC8E8 combining site was revealed by X-ray crystallography, and observed tau binding mode was verified by alanine mutagenesis of DC8E8 paratope residues. Results: Monoclonal antibody DC8E8 is unique among the anti-tau antibodies as it interacts with three, respectively four homologous epitopes on a single tau molecule. SPR affinity studies uncovered finely tuned specificity of DC8E8 to individual components of its quadruple epitope on the tau molecule. Further, we solved several X-ray structures of the DC8E8 Fab fragment and its complex with a panel of tau peptides up to 1.69 A resolution. Antibody recognition is mediated by a unique paratope binding pocket embracing the conserved sequence motif HXPGGG on the tau molecule, which adopts a tight type I beta-turn. The binding mode of DC8E8 effectively blocks the beta-structure forming hexapeptides adjacent to the epitope, which explains the potent DC8E8 inhibitory effect on tau aggregation. Finally, alanine scanning mutagenesis experimentally confirmed the importance of specific DC8E8 paratope residues that are indispensable for binding therapeutic tau epitopes. Conclusions: Present results of structural and functional analysis shed light on the mechanism of action of active and passive immunotherapy based on the monoclonal antibody DC8E8 and its therapeutic tau epitope encompassed in the tau vaccine AADvac1. These findings further substantiate the potency of active and passive vaccine to protect the brain from tau neurodegeneration.