Amelioration of tau related pathology with a novel anti-prion protein monoclonal antibody in an AD mouse model
Boutajangout, Allal; Zhang, Wei; Abdali, Wed; Kim, Justin Sung Tae; Prelli, Frances; Wisniewski, Thomas
Passive Immunization With a Novel Monoclonal Anti-PrP Antibody TW1 in an Alzheimer's Mouse Model With Tau Pathology
Boutajangout, Allal; Zhang, Wei; Kim, Justin; Abdali, Wed Ali; Prelli, Frances; Wisniewski, Thomas
Neurofibrillary tangles (NFTs) are a major pathologic hallmark of Alzheimer's disease (AD). Several studies have shown that amyloid Î² oligomers (AÎ²o) and tau oligomers mediate their toxicity, in part, via binding to cellular prion protein (PrPC) and that some anti-PrP antibodies can block this interaction. We have generated a novel monoclonal anti-PrP antibody (TW1) and assessed the efficacy of passive immunization with it in a mouse model of AD with extensive tau pathology: hTau/PS1 transgenic (Tg) mice. These mice were injected intraperitoneally once a week with TW1 starting at 5 months of age. Behavior was assessed at 8 months of age and brain tissue was subsequently harvested for analysis of treatment efficacy at 9 months. Mice treated with TW1 did not show any significant difference in sensorimotor testing including traverse beam, rotarod, and locomotor activity compared to controls. Significant cognitive benefits were observed with the novel object recognition test (ORT) in the immunized mice (two-tailed, t-test p = 0.0019). Immunized mice also showed cognitive benefits on the closed field symmetrical maze (day 1 two-tailed t-test p = 0.0001; day 2 two-tailed t-test p = 0.0015; day 3 two-tailed t-test p = 0.0002). Reduction of tau pathology was observed with PHF-1 immunohistochemistry in the piriform cortex by 60% (two-tailed t-test p = 0.01) and in the dentate gyrus by 50% (two-tailed t-test p = 0.02) in animals treated with TW1 compared to controls. There were no significant differences in astrogliosis or microgliosis observed between treated and control mice. As assessed by Western blots using PHF-1, the TW1 therapy reduced phosphorylated tau pathology (two-tailed t-test p = 0.03) and improved the ratio of pathological soluble tau to tubulin (PHF1/tubulin; two-tailed t-test p = 0.0006). Reduction of tau pathology also was observed using the CP13 antibody (two-tailed t-test p = 0.0007). These results indicate that passive immunization with the TW1 antibody can significantly decrease tau pathology as assessed by immunohistochemical and biochemical methods, resulting in improved cognitive function in a tau transgenic mouse model of AD.
Disease modifying therapy by targeting generic protein secondary structure of pathological oligomers at any stages of Alzheimer's disease models [Meeting Abstract]
Goni, F; Herline, K; Marta-Ariza, M; Prelli, F; Wisniewski, T
Background: We have previously demonstrated the feasibility of eliciting a unique antibody response independent of the primary and tertiary structure of proteins/peptides and specifically recognizing generic secondary beta-sheet structure dominant on misfolded proteins of oligomeric form; a hallmark of toxic prion-like "infectious" pathology in neurodegenerative diseases and specifically in the dual Abeta and tau Alzheimer's disease (AD) (Goni et al 2013 J. Neuroinflammation). As a putative antigen we have used a small peptide with a non-self primary structure, only beta-sheet secondary structure and no tertiary structure do to its size. The highly polymerized peptide (pBri) preserved a stable 90% beta-sheet secondary structure without any other outstanding epitope that could be recognized by an immune system. Young animals of AD models APP/PS1, 3xTg and SwDI as well as wild type animals, all developed polyclonal antibodies IgM and IgG that recognized oligomers of Abeta and tau and significantly ameliorated pathology. CD-1 animals were used to produced monoclonal antibodies (mAbs) that proved unequivocally the recognition was not to a sequence dependent epitope but to a generic beta-sheet secondary structure of pathologic oligomers but not fibrils or native proteins (Goni et al 2017 Scientific reports). The mAbs were characterized as specific anti-beta-sheet secondary structure monoclonal antibodies (AbetaComAbs). Either the stable IgM or the engineered IgG AbetaComAbs could reverse pathology and produce cognitive rescue in 3xTg animals (Goni et al 2018, Alz Res Therapy; Herline et al 2018 Alz Res Therapy).
Objective(s): To demonstrate that small engineered peptides with similar beta-sheet dominant structure but different primary sequences would elicit similar anti-beta-sheet responses and ameliorate pathology on 3xTg animals. To demonstrate that old 3xTg animals with already flourished pathology would develop the same type of anti-beta-sheet polyclonal IgM and IgG response and compared to AbetaComAbs infused animals produced comparable cognitive rescue and reduced oligomeric pathology. Finally, to demonstrate the specific reaction to the secondary structure but not the primary/tertiary structure does not produce antibody dependent cerebral microhemorrhages in an old SwDI CAA model of AD.
Method(s): Groups of 8 to 12 male and female 16 months old 3xTg AD and SwDI animals were inoculated i.p. weekly for 9 weeks with either 100 mug of the stable IgMk GW-23B7 or the derived TWF9 IgG2ak AbetaComAbs in 100 muL of sterile saline or with 100 muL of vehicle alone. Other groups of young 3xTg were inoculated with several tailored made mutations of the original pBri; whereas groups of 17 months old 3xTg were inoculated with the highly polymerized original pBri. The pBri immunizations and the AbetaComAbs were repeated in aged 16 month old SwDI animals prone to develop cerebral amyloid angiopathy due to the nature of the model, exacerbated by eventual reaction to Abeta amyloid epitopes existing in vessels of the brain. All animals were subjected to radial arm maze, locomotor tests or Barnes maze after the treatment and before sacrifice. Histochemical and biochemical analysis were performed on brains from treated and control animals.
Result(s): No adverse reactions were demonstrated during any one of the different treatments. Old animals inoculated with the pBri produced a fare anti-beta-sheet response; however, lower than in young animals. There was a significant cognitive rescue compared to controls and a significant reduction of both Abeta and tau oligomers. The mutated peptides were not as efficient to elicit anti-beta-sheet polyclonal response but still produce a significant amelioration on cognitive decline close to the results obtained by the original pBri. All SwDI animals either pBri immunized or AbetaComAbs infused -stable IgMk or IgG2aK- did not show any signs of vascular complication. In all cases the main biochemical improvement was the significant decrease on the number of oligomers of both Abeta and tau as assessed by specific blots of solubilized brain extractions, ELISAs and MSD measurements. Immunohistochemically the extracellular Abeta in plaques was significantly decreased whereas the intracellular PHF-tau remained fairly the same.
Conclusion(s): The development of a specific antibody response -polyclonal or monoclonal- to strictly the secondary structure of a protein or peptide frozen in a stable beta-sheet state is successfully achieved by the immunization with a highly polymerized, beta-sheet only, immunogen with no other visible epitope. That specific response is completely independent of the primary structure of pathological conformers; thus, it interferes only with oligomeric pathologic conformers that show the dominant beta-sheet secondary structure. Either treatment can produce , in various degrees, amelioration of AD pathology or evident cognitive rescue depending on the time of start of treatment. The mechanism is likely related to reductions of the levels of soluble oligomeric forms of Abeta and Tau; the species most closely linked to cognitive deficits in AD patients and the prionlike propagation. These results are extremely encouraging for the further testing of potential combinations of immunizations and AbetaComAbs in clinical trials with disease modifying potential and minimal risk of autoimmune complications
Immunotherapy to improve cognition and reduce pathological species in an Alzheimer's disease mouse model
Herline, Krystal; Prelli, Frances; Mehta, Pankaj; MacMurray, Claire; Goñi, Fernando; Wisniewski, Thomas
BACKGROUND:Alzheimer's disease (AD) is characterized by physiologically endogenous proteins amyloid beta (AÎ²) and tau undergoing a conformational change and accumulating as soluble oligomers and insoluble aggregates. Tau and AÎ² soluble oligomers, which contain extensive Î²-sheet secondary structure, are thought to be the most toxic forms. The objective of this study was to determine the ability of TWF9, an anti-Î²-sheet conformation antibody (aÎ²ComAb), to selectively recognize pathological AÎ² and phosphorylated tau in AD human tissue compared with cognitively normal age-matched controls and to improve the performance of old 3xTg-AD mice with advanced pathology in behavioral testing after acute treatment with TWF9. METHODS:In this study, we used immunohistochemistry, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) to characterize TWF9 specificity. We further assessed cognitive performance in old (18-22Â months) 3xTg-AD mice using both a Barnes maze and novel object recognition after intraperitoneal administration of TWF9 (4Â mg/kg) biweekly for 2 weeks before the start of behavioral testing. Injections continued for the duration of the behavioral testing, which lasted 2 weeks. RESULTS:Histological analysis of TWF9 in formalin-fixed paraffin-embedded human control and AD (ABC score: A3B3C3) brain tissue revealed preferential cytoplasmic immunoreactivity in neurons in the AD tissue compared with controls (pâ€‰<â€‰0.05). Furthermore, ELISA using oligomeric and monomeric AÎ² showed a preferential affinity for oligomeric AÎ². Immunoprecipitation studies showed that TWF9 extracted both phosphorylated tau (pâ€‰<â€‰0.01) and AÎ² (pâ€‰<â€‰0.01) from fresh frozen brain tissues. Results show that treated old 3xTg-AD mice have an enhanced novel object recognition memory (pâ€‰<â€‰0.01) and Barnes maze performance (pâ€‰=â€‰0.05) compared with control animals. Overall plaque burden, neurofibrillary tangles, microgliosis, and astrocytosis remained unchanged. Soluble phosphorylated tau was significantly reduced in TWF9-treated mice (pâ€‰<â€‰0.05), and there was a trend for a reduction in soluble AÎ² levels in the brain homogenates of female 3xTg-AD mice (pâ€‰=â€‰0.06). CONCLUSIONS:This study shows that acute treatment with an aÎ²ComAb can effectively improve performance in behavioral testing without reduction of amyloid plaque burden, and that peripherally administered IgG can affect levels of pathological species in the brain.
Anti-Î²-sheet conformation monoclonal antibody reduces tau and AÎ² oligomer pathology in an Alzheimer's disease model
Goñi, Fernando; MartÃ¡-Ariza, Mitchell; Herline, Krystal; Peyser, Daniel; Boutajangout, Allal; Mehta, Pankaj; Drummond, Eleanor; Prelli, Frances; Wisniewski, Thomas
BACKGROUND:Oligomeric forms of amyloid-Î² (AÎ²) and tau are increasing being recognized as key toxins in the pathogenesis of Alzheimer's disease (AD). METHODS:We developed a novel monoclonal antibody (mAb), GW-23B7, that recognizes Î²-sheet secondary structure on pathological oligomers of neurodegenerative diseases. RESULTS:The pentameric immunoglobulin M kappa chain (IgMÎºp) we developed specifically distinguishes intra- and extracellular pathology in human AD brains. Purified GW-23B7 showed a dissociation constant in the nanomolar range for oligomeric AÎ² and did not bind monomeric AÎ². In enzyme-linked immunosorbent assays, it recognized oligomeric forms of both AÎ² and hyperphosphorylated tau. Aged triple-transgenic AD mice with both AÎ² and tau pathology infused intraperitoneally for 2Â months showed IgMÎºp in the soluble brain homogenate, peaking at 24Â h postinoculation. Treated mice exhibited significant cognitive rescue on radial arm maze testing compared with vehicle control-infused mice. Immunohistochemically, treatment resulted in a significant decrease of extracellular pathology. Biochemically, treatment resulted in significant reductions of oligomeric forms of AÎ² and tau. CONCLUSIONS:These results suggest that GW-23B7, an anti-Î²-sheet conformational mAb humanized for clinical trials, may be an effective therapeutic agent for human AD.
Potential Novel Approaches to Understand the Pathogenesis and Treat Alzheimer's Disease
Drummond, Eleanor; Goñi, Fernando; Liu, Shan; Prelli, Frances; Scholtzova, Henrieta; Wisniewski, Thomas
There is growing genetic and proteomic data highlighting the complexity of Alzheimer's disease (AD) pathogenesis. Greater use of unbiased "omics" approaches is being increasingly recognized as essential for the future development of effective AD research, that need to better reflect the multiple distinct pathway abnormalities that can drive AD pathology. The track record of success in AD clinical trials thus far has been very poor. In part, this high failure rate has been related to the premature translation of highly successful results in animal models that mirror only limited aspects of AD pathology to humans. We highlight our recent efforts to increase use of human tissue to gain a better understanding of the AD pathogenesis subtype variety and to develop several distinct therapeutic approaches tailored to address this diversity. These therapeutic approaches include the blocking of the AÎ²/apoE interaction, stimulation of innate immunity, and the simultaneous blocking of AÎ²/tau oligomer toxicity. We believe that future successful therapeutic approaches will need to be combined to better reflect the complexity of the abnormal pathways triggered in AD pathogenesis.
Targeting Apolipoprotein E/Amyloid beta Binding by Peptoid CPO_Abeta17-21 P Ameliorates Alzheimer's Disease Related Pathology and Cognitive Decline
Liu, Shan; Park, Shinae; Allington, Grant; Prelli, Frances; Sun, Yanjie; Marta-Ariza, Mitchell; Scholtzova, Henrieta; Biswas, Goutam; Brown, Bernard; Verghese, Philip B; Mehta, Pankaj D; Kwon, Yong-Uk; Wisniewski, Thomas
Inheritance of the apolipoprotein E4 (apoE4) genotype has been identified as the major genetic risk factor for late onset Alzheimer's disease (AD). Studies have shown that apoE, apoE4 in particular, binds to amyloid-beta (Abeta) peptides at residues 12-28 of Abeta and this binding modulates Abeta accumulation and disease progression. We have previously shown in several AD transgenic mice lines that blocking the apoE/Abeta interaction with Abeta12-28 P reduced Abeta and tau-related pathology, leading to cognitive improvements in treated AD mice. Recently, we have designed a small peptoid library derived from the Abeta12-28 P sequence to screen for new apoE/Abeta binding inhibitors with higher efficacy and safety. Peptoids are better drug candidates than peptides due to their inherently more favorable pharmacokinetic properties. One of the lead peptoid compounds, CPO_Abeta17-21 P, diminished the apoE/Abeta interaction and attenuated the apoE4 pro-fibrillogenic effects on Abeta aggregation in vitro as well as apoE4 potentiation of Abeta cytotoxicity. CPO_Abeta17-21 P reduced Abeta-related pathology coupled with cognitive improvements in an AD APP/PS1 transgenic mouse model. Our study suggests the non-toxic, non-fibrillogenic peptoid CPO_Abeta17-21 P has significant promise as a new AD therapeutic agent which targets the Abeta related apoE pathway, with improved efficacy and pharmacokinetic properties.
Chemical Fluorescent Probe for Detection of Abeta Oligomers
Teoh, Chai Lean; Su, Dongdong; Sahu, Srikanta; Yun, Seong-Wook; Drummond, Eleanor; Prelli, Frances; Lim, Sulgi; Cho, Sunhee; Ham, Sihyun; Wisniewski, Thomas; Chang, Young-Tae
Aggregation of amyloid beta-peptide (Abeta) is implicated in the pathology of Alzheimer's disease (AD), with the soluble, Abeta oligomeric species thought to be the critical pathological species. Identification and characterization of intermediate species formed during the aggregation process is crucial to the understanding of the mechanisms by which oligomeric species mediate neuronal toxicity and following disease progression. Probing these species proved to be extremely challenging, as evident by the lack of reliable sensors, due to their heterogeneous and transient nature. We describe here an oligomer-specific fluorescent chemical probe, BoDipy-Oligomer (BD-Oligo), developed through the use of the diversity-oriented fluorescent library approach (DOFLA) and high-content, imaging-based screening. This probe enables dynamic oligomer monitoring during fibrillogenesis in vitro and shows in vivo Abeta oligomers staining possibility in the AD mice model.
Characterization of a Novel Monoclonal Antibody Targeting Pathological Proteins in Alzheimer's Disease [Meeting Abstract]
Herline, Krystal; Goni, Fernando; Drummond, Eleanor; Marta-Ariza, Mitchell; Prelli, Frances; Wisniewski, Thomas
Styryl-based and tricyclic compounds as potential anti-prion agents
Chung, Erika; Prelli, Frances; Dealler, Stephen; Lee, Woo Sirl; Chang, Young-Tae; Wisniewski, Thomas
Prion diseases currently have no effective therapy. These illnesses affect both animal and human populations, and are characterized by the conformational change of a normal self protein PrP(C) (C for cellular) to a pathological and infectious conformer, PrP(Sc) (Sc for scrapie). We used a well characterized tissue culture model of prion infection, where mouse neuroblastoma cells (N2a) were infected with 22L PrP(Sc), to screen compounds for anti-prion activity. In a prior study we designed a library of styryl based, potential imaging compounds which were selected for high affinity binding to Alzheimer's disease beta-amyloid plaques and good blood-brain barrier permeability. In the current study we screened this library for activity in the N2a/22L tissue culture system. We also tested the anti-prion activity of two clinically used drugs, trimipramine and fluphenazine, in the N2a/22L system. These were selected based on their structural similarity to quinacrine, which was previously reported to have anti-prion activity. All the compounds were also screened for toxicity in tissue culture and their ability to disaggregate amyloid fibrils composed of PrP and beta-amyloid synthetic peptides in vitro. Two of the imaging agents, 23I and 59, were found to be both effective at inhibiting prion infection in N2a/22L tissue culture and to be non-toxic. These two compounds, as well as trimipramine and fluphenazine were evaluated in vivo using wild-type CD-1 mice infected peripherally with 139A PrP(Sc). All four agents significantly prolonged the asymptomatic incubation period of prion infection (p<0.0001 log-rank test), as well as significantly reducing the degree of spongiform change, astrocytosis and PrP(Sc) levels in the brains of treated mice. These four compounds can be considered, with further development, as candidates for prion therapy