Eliot J. Davidowitz

Extracellular Tau Oligomers Produce An Immediate Impairment of LTP and Memory

Non-fibrillar soluble oligomeric forms of amyloid-β peptide (oAβ) and tau proteins are likely to play a major role in Alzheimer’s disease (AD). The prevailing hypothesis on the disease etiopathogenesis is that oAβ initiates tau pathology that slowly spreads throughout the medial temporal cortex and neocortices independently of Aβ, eventually leading to memory loss. Here we show that a brief exposure to extracellular recombinant human tau oligomers (oTau), but not monomers, produces an impairment of long-term potentiation (LTP) and memory, independent of the presence of high oAβ levels. The impairment is immediate as it raises as soon as 20 min after exposure to the oligomers. These effects are reproduced either by oTau extracted from AD human specimens, or naturally produced in mice overexpressing human tau. Finally, we found that oTau could also act in combination with oAβ to produce these effects, as sub-toxic doses of the two peptides combined lead to LTP and memory impairment. These findings provide a novel view of the effects of tau and Aβ on memory loss, offering new therapeutic opportunities in the therapy of AD and other neurodegenerative diseases associated with Aβ and tau pathology.

Trimeric Tau Is Toxic to Human Neuronal Cells at Low Nanomolar Concentrations

In Alzheimers disease (AD), tau aggregates into fibrils and higher order neurofibrillary tangles, a key histopathological feature of AD. However, soluble oligomeric tau species may play a more critical role in AD progression since these tau species correlate better with neuronal loss and cognitive dysfunction. Recent studies show that extracellular oligomeric tau can inhibit memory formation and synaptic function and also transmit pathology to neighboring neurons. However, the specific forms of oligomeric tau involved in toxicity are still unknown. Here, we used two splice variants of recombinant human tau and generated monomeric, dimeric, and trimeric fractions of each isoform. The composition of each fraction was verified chromatographically and also by atomic force microscopy. The toxicity of each fraction toward both human neuroblastoma cells and cholinergic-like neurons was assessed. Trimeric, but not monomeric or dimeric, tau oligomers of both splice variants were neurotoxic at low nanomolar concentrations. Further characterization of tau oligomer species with disease-specific modifications and morphologies is necessary to identify the best targets for the development of biomarker and therapeutic development for AD and related tauopathies.

Pathogenic chaperone-like RNA induces congophilic aggregates and facilitates neurodegeneration in Drosophila

Abstract Protein aggregation is a hallmark of many neurodegenerative diseases. RNA chaperones have been suggested to play a role in protein misfolding and aggregation. Noncoding, highly structured RNA recently has been demonstrated to facilitate transformation of recombinant and cellular prion protein into proteinase K-resistant, congophilic, insoluble aggregates and to generate cytotoxic oligomers in vitro. Transgenic Drosophila melanogaster strains were developed to express highly structured RNA under control of a heat shock promoter. Expression of a specific construct strongly perturbed fly behavior, caused significant decline in learning and memory retention of adult males, and was coincident with the formation of intracellular congophilic aggregates in the brain and other tissues of adult and larval stages. Additionally, neuronal cell pathology of adult flies was similar to that observed in human Parkinsons and Alzheimers disease. This novel model demonstrates that expression of a specific highly structured RNA alone is sufficient to trigger neurodegeneration, possibly through chaperone-like facilitation of protein misfolding and aggregation.

Isolation and characterization of antibody fragments selective for toxic oligomeric tau

Oligomeric tau species are important in the onset and progression of Alzheimers disease (AD), as they are neurotoxic and can propagate tau-tangle pathology. Therefore, reagents that selectively recognize different key morphologies of tau are needed to help define the role of tau in AD and related diseases. We utilized a biopanning protocol that combines the binding diversity of phage-displayed antibody libraries with the powerful imaging capability of atomic force microscopy to isolate single-chain antibody fragments (scFvs) that selectively bind toxic oligomeric tau. We isolated 3 different antibody fragments that bind oligomeric but not monomeric or fibrillar tau. The scFvs differentiate brain tissue homogenates of both 3×TG and tau-AD mice from wild-type mice, detecting oligomeric tau at much earlier ages than when neurofibrillary tangles are typically detected. The scFvs also distinguish human postmortem AD brain tissue from cognitively normal postmortem human brain tissue, demonstrating the potential of this approach for developing biomarkers for early detection and progression of AD.

Small-molecule lead identification for inhibition of tau oligomer formation

Background: There is a need for alternative approaches for the development of disease-modifying therapeutics for Alzheimer’s disease (AD) as all phase III drug development programs to-date based on the amyloid hypothesis have failed to meet their clinical endpoints. Research advances indicate that pre-tangle aggregates of tau cause transmission of pathology and symptoms related to memory formation validating tau oligomers as a target for drug discovery. A highly differentiated HTS assay (proximity-based) was developed to discover inhibitors of tau oligomer formation and transferred to MHTSC to screen a drug-like library of 100,000 compounds. Methods: Cytotoxicity studies were performed using SH-SY5Y neuroblastoma cells on a high throughput platform. Medicinal chemistry analysis was performed that enabled clustering of hits into chemical series that demonstrated a preliminary SAR. HTS hit triage was performed including confirmation of activity in primary assays with dose response, mass spec evaluation of structure and purity, elimination of reactive species, and removal of known/suspected promiscuous inhibitors or compounds that may contribute to tau aggregation at high concentrations. Lastly, lead series were prioritized based on an analysis of novelty, freedom-to-operate, and the potential for intellectual property on composition of matter. Results: For neurocytotoxicity, 338 active hits and 3 toxic control compounds were evaluated. Cell growth was inhibited at less than 50 uM by fifty compounds and the controls. Only 14 compounds had IC50 values<10 uM.Medicinal chemistry analysis was performed on the 576 most active inhibitors from the HTS assay. Cytotoxic compounds were eliminated and initial SAR was developed with compounds clustered by structure type. The clustering revealed that there are 3 large series (>10 compounds), 6 medium series (4 5 compounds), 8 small sized series (2 3 compounds), and 22 singletons. Conclusions: The preliminary analysis of the structures clearly demonstrates that certain structural classes are active in this assay. It also indicates that hit optimization is possible since a range of potency is observed within structural classes. The HTS has achieved the important goal of finding viable compounds as starting points for medicinal chemical optimization to generate leads.

SMALL MOLECULE TAU OLIGOMERIZATION INHIBITORS

used for chronic efficacy studies. Results: CNP520 is selective for BACE-1 over BACE-2 and highly selective over pepsin, cathepsin D & E, and renin. Low nanomolar inhibition of Ab and sAPPb release was observed in cell assays using wt-APP cells. The free fraction of CNP520 in the rat brain, and the concentration of CNP520 in the CSF, was comparable to unbound blood concentrations, indicating excellent brain penetration. Oral dosing of CNP520 reduced Ab in the rat brain by more than 80%. A single CNP520 dose in dogs reduced CSF Ab for 72 hours, in agreement with long terminal half-lives (9.5-23 hours) in animals. CNP520 did not induce any hair depigmentation when dosed to mice for 8 weeks at a dose for > 90% Ab reduction. No hypopigmentation was observed in chronic studies in transgenic mice, and during long-term toxicology studies. CNP520 was dosed into APP23 mice 6 months and showed dose-dependent reduction of Triton TX-100 soluble and insoluble Ab. At the high dose, the levels of deposited Ab40/42 were indistinguishable from baseline. Conclusions: Preclinical data predict that more than 80% Ab reduction can be reached in humans at steady state. CNP520 stopped further amyloid-b deposition in APP transgenic mice, indicating that the compound may be able to show long term efficacy against Ab deposition in humans.

Characterization of tool compounds targeting tau oligomers

of disease-modifying therapeutics. However, a major impediment to progress may lie in fundamental differences between humans and animal model species, largely rodents. Disruption of hippocampal insulin signaling has recently been described in the brains of AD patients and animal models of disease, which could contribute to cognitive impairment in this disease. Therefore, drugs that can restore normal insulin function in the central nervous system have been recently suggested as a promising novel approach to treat AD. Methods:Here, we evaluated the neuroprotective effects of liraglutide in cynomolgus macaques (Macaca fascicularis) that received intracerebroventricular injections of Ab oligomers (AbOs). Liraglutide is an anti-diabetic agent that activates pathways common to insulin signaling through stimulation of glucagon-like peptide 1 (GLP-1) receptors. Nine female cynomolgus macaques were used. Three of them were sham-operated and served as controls. We performed intracerebroventricular (i.c.v.) injections of AbOs into six cynomolgus macaques. Two of these monkeys had been pre-treated with daily i.p. injections of liraglutide. Liraglutide administration continued daily until the last injection of oligomers. Brain sections were used for immunohistochemystry to evaluate the levels of synaptic markers. Results:We found that AbOs induced a decrease in synapse number in the primate brain and reduced the levels of NMDA (GluN1 and GluN2B subunits), AMPA (GluA1 and GluA2 subunits) and insulin receptors. Liraglutide attenuated the impact of AbOs on synapses and on plasticity related receptors. Conclusions: These results establish the protective actions of liraglutide in the primate brain and indicates that a primate model of AD may be valuable not only for studying mechanisms responsible for AbOs toxicity, but also for exploring and evaluating new preventive therapeutic strategies for AD.

Detection of soluble oligomeric tau with morphology-specific antibody fragments as a potential diagnostic method for Alzheimer's disease

Sequenom I-plex platform. CSF was obtained through lumbar puncture and analysed using multiplex xMAP Luminex platform. Statistical analyses were performed using mixed models, corrected for age, sex, APOE e 4 status and study site. Adjustment for multiple testing was set at a false discovery rate adjusted P 0.05). Conclusions: Our findings suggest that polymorphisms in the MS4A gene influence CSFA b 142 concentrations. This supports results from GWAS studies that this gene is a genetic risk factor for AD.

Characterization of a novel monoclonal antibody specific for a tau autoproteolytic cut site that differentiates between Alzheimer's disease and non-Alzheimer's specimens

Background: Tau pathology associates with several neurodegenerative diseases including Alzheimer’s disease (AD) collectively known as tauopathies. The role of inflammation in neurodegenerative diseases remains controversial yet provides opportunity for therapeutic intervention. Byproducts of certain activation profiles reduce disease pathology whereas others promote it. Inhibiting one pathway along the inflammatory milieu may promote an alternative cascade. Arginase 1 (Arg1) and nitric oxide synthases (NOS) increase during certain disease states and have been implicated in AD pathogenesis. Arg1 and NOS compete for L-arginine to produce either ornithine and polyamines or nitric oxide, respectively. Polyamines act as bivalent regulators of cellular function, promoting cell growth or cell death, depending on cell type and the microenvironment. Methods: We identified dysregulation of proteins associated with polyamine synthesis andmetabolism in rTg4510 tau transgenic mice compared to non-transgenic littermates. We postulate that increased Arg1 expression in the CNS impacts tau pathology. Four month-old rTg4510 tau transgenic mice receive an intracranial injection of recombinant adeno associated virus (rAAV) into the hippocampus of either rAAV-Arg1 or a control vector rAAV-GFP. Four months post injection brains were harvested for histology, immunohistochemistry, western blotting and ELISA. Results:We show that overexpression Arg1 for a duration of four months in the hippocampus of rTg4510 tau transgenic mice using rAAV-Arg1 reduced several phospho-tau epitopes (i.e. AT8, AT180, AT270, Ser262, Ser396) and tangle pathology indicated by Gallyas silver positive staining compared the control vector rAAVGFP (green fluorescent protein). Furthermore we found that Arg1 decreased several kinases associated with phospho-tau including phospho-GSK3 alpha 216/ beta 279 and CDK5 levels. Additionally, Arg1 overexpression decreased several cytokines and inflammatory markers including IL-1beta, TNF-alpha, IL-12, along with microglial activation measured by CD45 immunohistochemistry. Unbiased stereology showed no change in the number of neurons compared to the control vector however, Arg1 overexpression slightly but significantly mitigated hippocampal atrophy. Conclusions: These data suggests that Arg1 and the polyamine pathway may provide new and potential therapeutic targets for AD and tauopathies.

Tau autoproteolytic activity is induced by tau oligomerization

kinson’s disease (PD), Huntington’s disease (HD), transmissible spongiform encephalopathies (TSE) and amyotrophic lateral sclerosis (ALS). Recent evidence indicates that these diverse neurodegenerative diseases might have a common cause and pathological mechanism—the misfolding, aggregation and accumulation of proteins in the brain, resulting in neuronal apoptosis. Studies from different disciplines strongly support this hypothesis and indicate that a common therapy for these devastating disorders might be possible. We found that these proteins have a common mechanism that regulates their mRNA translation. Iron influx increases the translation of these proteins via an iron-responsive element (IRE) RNA stem loop in their 5’untranlated region, while iron regulatory protein 1 (IRP1) controls and down regulates the iron-dependent translation. Methods: Messenger RNA IREs were modeled with RNA Shape. Binding of IRPs to IREs was measured by coupling biotin to the 5’UTR of the specific mRNA, incubating the construct with IRPs, precipitating the complex with Avidin beads, and visualizing the precipitate by Western blot. In vitro and in vivo (mice and human) measurements of APP and SNCA levels were executed either by immunoprecipitation, Elisa or Western blot. Results: Posiphen increases binding of IRP1 to the IRE stem loop structure and further down regulates translation. We have data showing that Posiphen inhibits the translation of amyloid precursor protein (APP) and alpha synuclein (SNCA). We also have data showing that Posiphen inhibits their translation in a number of tissue culture cells, in normal, trisomic and transgenic mice, as well as in humans (MCI patients). At ICAD we want to present our newest data showing translational regulation via the 5’UTR IRE/IRP1 interaction for prions and superoxide dismutase (SOD) and further down regulation with Posiphen in vitro and in vivo. Conclusions: Our data shows that neurotoxic aggregating proteins, such as APP, SNCA, Prions and SODhave a regulatory element in their 5’UTR that is responsible for the amount of protein translated by the ribosome. This regulatory element consists of an IRE stem loop structure that when binding iron upregulates translation, whilewhen binding IRP1 down regulates translation. Posiphen further inhibits the translation of these mRNAs by the ribosomes, because it increases the affinity of IRP1 binding to the IRE.