- Email: [email protected]
Specific targeting of tau oligomeric seeds by passive immunization
P330
Oral Sessions: O2-08: Basic Science: Protective and Pathological Protein-Protein Interactions in Alzheimer’s Disease
Pengfei Li2, Ying Jin2, Christopher Atkins2, Chad Dickey2, 1University of Kentucky, Lexington, Kentucky, United States; 2University of South Florida, Tampa, Florida, United States. Contact e-mail: [email protected] Background: Tauopathies are a group of over 15 diseases that share a common pathogenic hallmark: the deposition of the microtubule associated protein tau in the brain. The mechanism leading to this phenomenon is still unclear, thereby diminishing treatment options. Another co-pathology in tauopathies is the appearance of ubiquitin aggregates; this was described over 20 years ago, but it has been widely overlooked. In this study, we sought to determine the cause of this co-pathology and assess whether it was linked to tau pathogenesis. Methods: We used the doxycycline-regulated rTg4510 tau transgenic mouse as well as Braak stage 6 brains of Alzheimer’s disease individuals to determine changes in the levels and localization of ubiquitin with Western blot analyses, immunofluorescent imaging, and subcellular fractionation assays. We also performed live-cell imaging in an inducible cell culture model of tau over-expression. Results: Here we show that tau increased the levels of ubiquitinated proteins in the brain, which triggered activation of the Unfolded Protein Response (UPR). This suggested that tau interfered with protein quality control in the endoplasmic reticulum (ER). Consistent with this, ubiquitin was found to associate with the ER in human AD brains and rTg4510 brains, but this was not always co-localized with tau. Increased levels of phosphorylated PERK accompanied the increased levels of ubiquitinated proteins, a marker that indicates UPR activation. Importantly, depleting soluble tau levels in cells and brain could reverse UPR activation. Tau accumulation facilitated its deleterious interaction with ER membrane and associated proteins that are essential for ER-associated degradation (ERAD), including VCP and Hrd1. Based on this, the effects of tau accumulation on ERAD efficiency were evaluated using the CD3v‘ reporter, an ERAD substrate. Indeed, CD3v‘ accumulated in cell culture and mouse models of tauopathy, as well as in AD brains. Conclusions: These data suggest that soluble tau impairs ERAD, and the result is activation of the UPR. Importantly, the reversibility of this process suggests that tau-based therapeutics could significantly delay this type disease progression. O2-08-02
SPECIFIC TARGETING OF TAU OLIGOMERIC SEEDS BY PASSIVE IMMUNIZATION
Diana Castillo-Carranza, Marcos Guerrero-Munoz, Urmi Sengupta, Shashirekha Krishnamurthy, Julia Gerson, Kelly Dineley, George Jackson, Rakez Kayed, University of Texas Medical Branch, Galveston, Texas, United States. Contact e-mail: [email protected] Background: Tau oligomers represent the most toxic form of tau aggregates and the primary seeds responsible for the spread of tau pathology. In support of this idea, we recently demonstrated that oligomeric tau causes neurotoxicity in vivo and that increased oligomeric tau species are present in post mortem brain samples from Alzheimer’s disease (AD) and PSP patients. Moreover, we observed that brain-derived tau oligomers propagate abnormal tau conformation of endogenous murine tau after prolonged incubation. Methods: We have developed a novel anti tau oligomer specific mouse monoclonal antibody (TOMA). Herein, we investigated the specific modulation of tau oligomers in animal models. 30 mg of TOMA injected, intravenously, reversed the phenotypes in aged tau transgenic animals, the 8 months old P301Land 12 months old htau mice within 6 days after receiving the TOMA injection. Moreover, we investigated the effects biweekly 15 mg of TOMA injections in protecting wild-type and h-tau animals from the effects of intracerebral injection of brain-derived tau oligomers. Two groups of 3 months mice, one receive 15 mg of TOMA the other received 15 mg of no-specific IgG, 4 hours before being injected with pure brain-derived tau oligomers, as previously described (Lasagna et al. Sci Rep. 2012;2:700). Results: TOMA halts the spread of tau pathology and reverses phenotypes associated with AD and tauopathies. A single intravenous dose of TOMA was sufficient to reverse both locomotor and memory deficits in aged mice. Phenotypic rescue coincided with rapid reduction of tau oligomers. Long term administration of TOMA was effective as prevention therapy, preventing tau accumulation and preserving memory and locomoter activities. Conclusions: These results support the critical role of oligomeric
tau in the disease progression and validate tau oligomers as a potential drug target. Moreover, these results may impact a variety of fields as the concept of prion-like induction and spreading of pathogenic proteins recently has been proposed for many neurodegenerative diseases. Antibodies and drugs that target amyloid and tau oligomers may be valuable for the treatment of a variety of neurodegenerative disease and preventing spread of pathology. O2-08-03
SELECTION AND CHARACTERIZATION OF TAUBINDING D-ENANTIOMERIC PEPTIDES FOR THERAPEUTIC APPLICATIONS IN NEURODEGENERATIVE DISEASES
Susanne Funke1, Christina Dammers1, Deniz Yolcu1, Laura Kukuk2, Stephan Rudolph1, Dieter Willbold1, 1Forschungszentrum J€ulich, J€ulich, Germany; 2Heinrich-Heine-Universit€at, D€usseldorf, Germany. Contact e-mail: [email protected] Background: A variety of neurodegenerative disorders, including Alzheimer’s disease, are associated with amyloid fibrils and neurofibrillaryt tangles composed of the tau protein. Inhibitors of pathological amyloid fibril formation could be useful in the development of therapeutics, provided that the inhibitors were specific enough to avoid interference with physiological processes. Methods: Employing mirror-image phage display with a large peptide library (> 1 billion different peptides), we have identified tau-fibril binding peptides consisting of D-enantiomeric amino acids. Dpeptides are known to be extremely protease resistant and less immunogenic than the respective L-enantiomers. Selections were performed using fibrils of the D-enantiomeric hexapeptide VQIVYK, representing residues 306311 of the tau protein, as a target. VQIVYK has been shown to be important for fibril formation of the full-length protein and itself forms fibrils with biophysical properties similar to full-length tau fibrils. Results: Here, we report on D-enantiomeric peptides which bind to VQIVYK as well as to full length tau fibrils and modulate the aggregation thereof. Binding of the peptides and their influence on tau aggregation was investigated using a variety of biochemical and biophysical methods. Conclusions: We present D-enantiomeric peptides which bind to tau protein fibrils and influence aggregation thereof. The peptides might therefore be interesting for therapeutic and diagnostic applications in Alzheimer’s disease research. O2-08-04
EXTRACELLULAR VERSUS INTRACELLULAR ANTI-TAU IMMUNOTHERAPY EFFECTS IN RTG4510 TAU TRANSGENIC MICE
Naruhiko Sahara, Yona Levites, Golda Sinyavskiy, Awilda Rosario, Pedro Cruz, Todd Golde, University of Florida, Gainesville, Florida, United States. Contact e-mail: [email protected] Background: In order to rapidly and cost-effectively evaluate potential modifiers of Alzheimer’s disease (AD) pathology in mouse models, we have developed a "somatic brain transgenics" paradigm, established by delivery of gene constructs packaged into adeno-associated viral vectors and injected into the cerebral ventricles of P0 mice. The mechanisms underlying the abnormal phosphorylation and accumulation of tau in AD remain unclear, but one of the possibilities is that it might be due to conformational changes in tau in the diseased brain. Anti-tau immunotherapy has recently emerged as a promising approach to target tau, but many mechanistic questions regarding the optimal form of anti-tau immunotherapy remain. We hypothesize that anti-tau immunotherapy may be optimized by targeting both intracellular and extracellular pools of tau and that specific binding of hyperphosphorylated tau by single chain variable fragments (scFv) or by intracellularly expressed intrabodies will prevent its toxicity and formation of neurofibrillary tangles. Methods: We cloned scFvs from two monoclonal antibodies (PHF and CP13) that recognize different forms of phosphorylated tau and generated stable intracellular anti-Ptau scFvs (intrabodies), as well as scFvs under IgG kappa-leader, directed into the secretory pathway. Then we expressed these intrabodies and scFvs in the brains of newborn transgenic rTg4510 mice. Results: Analysis of three month old brains revealed that attenuation of tau pathology relative to PBS-injected controls was achieved by the treatment, but the effect varied depending on the epitope and nature of tested intrabodies and scFvs.
Oral Sessions: O2-08: Basic Science: Protective and Pathological Protein-Protein Interactions in Alzheimer’s Disease
Pengfei Li2, Ying Jin2, Christopher Atkins2, Chad Dickey2, 1University of Kentucky, Lexington, Kentucky, United States; 2University of South Florida, Tampa, Florida, United States. Contact e-mail: [email protected] Background: Tauopathies are a group of over 15 diseases that share a common pathogenic hallmark: the deposition of the microtubule associated protein tau in the brain. The mechanism leading to this phenomenon is still unclear, thereby diminishing treatment options. Another co-pathology in tauopathies is the appearance of ubiquitin aggregates; this was described over 20 years ago, but it has been widely overlooked. In this study, we sought to determine the cause of this co-pathology and assess whether it was linked to tau pathogenesis. Methods: We used the doxycycline-regulated rTg4510 tau transgenic mouse as well as Braak stage 6 brains of Alzheimer’s disease individuals to determine changes in the levels and localization of ubiquitin with Western blot analyses, immunofluorescent imaging, and subcellular fractionation assays. We also performed live-cell imaging in an inducible cell culture model of tau over-expression. Results: Here we show that tau increased the levels of ubiquitinated proteins in the brain, which triggered activation of the Unfolded Protein Response (UPR). This suggested that tau interfered with protein quality control in the endoplasmic reticulum (ER). Consistent with this, ubiquitin was found to associate with the ER in human AD brains and rTg4510 brains, but this was not always co-localized with tau. Increased levels of phosphorylated PERK accompanied the increased levels of ubiquitinated proteins, a marker that indicates UPR activation. Importantly, depleting soluble tau levels in cells and brain could reverse UPR activation. Tau accumulation facilitated its deleterious interaction with ER membrane and associated proteins that are essential for ER-associated degradation (ERAD), including VCP and Hrd1. Based on this, the effects of tau accumulation on ERAD efficiency were evaluated using the CD3v‘ reporter, an ERAD substrate. Indeed, CD3v‘ accumulated in cell culture and mouse models of tauopathy, as well as in AD brains. Conclusions: These data suggest that soluble tau impairs ERAD, and the result is activation of the UPR. Importantly, the reversibility of this process suggests that tau-based therapeutics could significantly delay this type disease progression. O2-08-02
SPECIFIC TARGETING OF TAU OLIGOMERIC SEEDS BY PASSIVE IMMUNIZATION
Diana Castillo-Carranza, Marcos Guerrero-Munoz, Urmi Sengupta, Shashirekha Krishnamurthy, Julia Gerson, Kelly Dineley, George Jackson, Rakez Kayed, University of Texas Medical Branch, Galveston, Texas, United States. Contact e-mail: [email protected] Background: Tau oligomers represent the most toxic form of tau aggregates and the primary seeds responsible for the spread of tau pathology. In support of this idea, we recently demonstrated that oligomeric tau causes neurotoxicity in vivo and that increased oligomeric tau species are present in post mortem brain samples from Alzheimer’s disease (AD) and PSP patients. Moreover, we observed that brain-derived tau oligomers propagate abnormal tau conformation of endogenous murine tau after prolonged incubation. Methods: We have developed a novel anti tau oligomer specific mouse monoclonal antibody (TOMA). Herein, we investigated the specific modulation of tau oligomers in animal models. 30 mg of TOMA injected, intravenously, reversed the phenotypes in aged tau transgenic animals, the 8 months old P301Land 12 months old htau mice within 6 days after receiving the TOMA injection. Moreover, we investigated the effects biweekly 15 mg of TOMA injections in protecting wild-type and h-tau animals from the effects of intracerebral injection of brain-derived tau oligomers. Two groups of 3 months mice, one receive 15 mg of TOMA the other received 15 mg of no-specific IgG, 4 hours before being injected with pure brain-derived tau oligomers, as previously described (Lasagna et al. Sci Rep. 2012;2:700). Results: TOMA halts the spread of tau pathology and reverses phenotypes associated with AD and tauopathies. A single intravenous dose of TOMA was sufficient to reverse both locomotor and memory deficits in aged mice. Phenotypic rescue coincided with rapid reduction of tau oligomers. Long term administration of TOMA was effective as prevention therapy, preventing tau accumulation and preserving memory and locomoter activities. Conclusions: These results support the critical role of oligomeric
tau in the disease progression and validate tau oligomers as a potential drug target. Moreover, these results may impact a variety of fields as the concept of prion-like induction and spreading of pathogenic proteins recently has been proposed for many neurodegenerative diseases. Antibodies and drugs that target amyloid and tau oligomers may be valuable for the treatment of a variety of neurodegenerative disease and preventing spread of pathology. O2-08-03
SELECTION AND CHARACTERIZATION OF TAUBINDING D-ENANTIOMERIC PEPTIDES FOR THERAPEUTIC APPLICATIONS IN NEURODEGENERATIVE DISEASES
Susanne Funke1, Christina Dammers1, Deniz Yolcu1, Laura Kukuk2, Stephan Rudolph1, Dieter Willbold1, 1Forschungszentrum J€ulich, J€ulich, Germany; 2Heinrich-Heine-Universit€at, D€usseldorf, Germany. Contact e-mail: [email protected] Background: A variety of neurodegenerative disorders, including Alzheimer’s disease, are associated with amyloid fibrils and neurofibrillaryt tangles composed of the tau protein. Inhibitors of pathological amyloid fibril formation could be useful in the development of therapeutics, provided that the inhibitors were specific enough to avoid interference with physiological processes. Methods: Employing mirror-image phage display with a large peptide library (> 1 billion different peptides), we have identified tau-fibril binding peptides consisting of D-enantiomeric amino acids. Dpeptides are known to be extremely protease resistant and less immunogenic than the respective L-enantiomers. Selections were performed using fibrils of the D-enantiomeric hexapeptide VQIVYK, representing residues 306311 of the tau protein, as a target. VQIVYK has been shown to be important for fibril formation of the full-length protein and itself forms fibrils with biophysical properties similar to full-length tau fibrils. Results: Here, we report on D-enantiomeric peptides which bind to VQIVYK as well as to full length tau fibrils and modulate the aggregation thereof. Binding of the peptides and their influence on tau aggregation was investigated using a variety of biochemical and biophysical methods. Conclusions: We present D-enantiomeric peptides which bind to tau protein fibrils and influence aggregation thereof. The peptides might therefore be interesting for therapeutic and diagnostic applications in Alzheimer’s disease research. O2-08-04
EXTRACELLULAR VERSUS INTRACELLULAR ANTI-TAU IMMUNOTHERAPY EFFECTS IN RTG4510 TAU TRANSGENIC MICE
Naruhiko Sahara, Yona Levites, Golda Sinyavskiy, Awilda Rosario, Pedro Cruz, Todd Golde, University of Florida, Gainesville, Florida, United States. Contact e-mail: [email protected] Background: In order to rapidly and cost-effectively evaluate potential modifiers of Alzheimer’s disease (AD) pathology in mouse models, we have developed a "somatic brain transgenics" paradigm, established by delivery of gene constructs packaged into adeno-associated viral vectors and injected into the cerebral ventricles of P0 mice. The mechanisms underlying the abnormal phosphorylation and accumulation of tau in AD remain unclear, but one of the possibilities is that it might be due to conformational changes in tau in the diseased brain. Anti-tau immunotherapy has recently emerged as a promising approach to target tau, but many mechanistic questions regarding the optimal form of anti-tau immunotherapy remain. We hypothesize that anti-tau immunotherapy may be optimized by targeting both intracellular and extracellular pools of tau and that specific binding of hyperphosphorylated tau by single chain variable fragments (scFv) or by intracellularly expressed intrabodies will prevent its toxicity and formation of neurofibrillary tangles. Methods: We cloned scFvs from two monoclonal antibodies (PHF and CP13) that recognize different forms of phosphorylated tau and generated stable intracellular anti-Ptau scFvs (intrabodies), as well as scFvs under IgG kappa-leader, directed into the secretory pathway. Then we expressed these intrabodies and scFvs in the brains of newborn transgenic rTg4510 mice. Results: Analysis of three month old brains revealed that attenuation of tau pathology relative to PBS-injected controls was achieved by the treatment, but the effect varied depending on the epitope and nature of tested intrabodies and scFvs.