Donna M. Barten

Dynamics of β-Amyloid Reductions in Brain, Cerebrospinal Fluid, and Plasma of β-Amyloid Precursor Protein Transgenic Mice Treated with a γ-Secretase Inhibitor

γ-Secretase inhibitors are one promising approach to the development of a therapeutic for Alzheimers disease (AD). γ-Secretase inhibitors reduce brain β-amyloid peptide (Aβ), which is believed to be a major contributor in the etiology of AD. Transgenic mice overexpressing the human β-amyloid precursor protein (APP) are valuable models to examine the dynamics of Aβ changes with γ-secretase inhibitors in plaque-free and plaque-bearing animals. BMS-299897 2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoic acid, a γ-secretase inhibitor, showed dose- and time dependent reductions of Aβ in brain, cerebrospinal fluid (CSF), and plasma in young transgenic mice, with a significant correlation between brain and CSF Aβ levels. Because CSF and brain interstitial fluid are distinct compartments in composition and location, this correlation could not be assumed. In contrast, aged transgenic mice with large accumulations of Aβ in plaques showed reductions in CSF Aβ in the absence of measurable changes in plaque Aβ in the brain after up to 2 weeks of treatment. Hence, CSF Aβ levels were a valuable measure of γ-secretase activity in the central nervous system in either the presence or absence of plaques. Transgenic mice were also used to examine potential side effects due to Notch inhibition. BMS-299897 was 15-fold more effective at preventing the cleavage of APP than of Notch in vitro. No changes in the maturation of CD8+ thymocytes or of intestinal goblet cells were observed in mice treated with BMS-299897, showing that it is possible for γ-secretase inhibitors to reduce brain Aβ without causing Notch-mediated toxicity.

Discovery and Evaluation of BMS-708163, a Potent, Selective and Orally Bioavailable γ-Secretase Inhibitor

During the course of our research efforts to develop a potent and selective γ-secretase inhibitor for the treatment of Alzheimers disease, we investigated a series of carboxamide-substituted sulfonamides. Optimization based on potency, Notch/amyloid-β precursor protein selectivity, and brain efficacy after oral dosing led to the discovery of 4 (BMS-708163). Compound 4 is a potent inhibitor of γ-secretase (Aβ40 IC50 = 0.30 nM), demonstrating a 193-fold selectivity against Notch. Oral administration of 4 significantly reduced Aβ40 levels for sustained periods in brain, plasma, and cerebrospinal fluid in rats and dogs.

γ-Secretase Inhibitors for Alzheimer’s Disease

The amyloid hypothesis, which states that β-amyloid (Aβ) aggregates cause the onset and progression of Alzheimer’s disease (AD), is a leading proposal to explain AD aetiology. Based on this hypothesis, compounds that inhibit γ-secretase, one of the enzymes responsible for forming Aβ, are potential therapeutics for AD. Preclinical studies clearly establish that γ-secretase inhibitors can reduce brain Aβ in rodent models. The initial investigation of the effects of a γ-secretase inhibitor on Aβ-induced cognitive deficits in transgenic mice showed that modest Aβ reductions (15–30%) are sufficient to reverse Aβ-induced cognitive deficits in Tg2576 mice. Extending these studies to other γ-secretase inhibitors and other models with Aβ-induced cognitive deficits will be important. Unfortunately, γ-secretase inhibitors also cause abnormalities in the gastrointestinal tract, thymus and spleen in rodents. These changes likely result from inhibition of Notch cleavage, a transmembrane receptor involved in regulating cell-fate decisions. Two recent studies in rodents suggest that Aβ reduction using γ-secretase inhibitors can be partially separated from Notch inhibition. Given the uncertain Aβ reduction target and the potential for mechanism-based toxicity, biomarkers for efficacy and toxicity would be helpful in clinical trials. The first report of γ-secretase inhibitors in clinical trials was recently published. In this study, LY-450139 reduced plasma Aβ, but not cerebrospinal fluid Aβ. Taken together, the results of studies to date suggest that γ-secretase inhibitors have the potential to address a large unmet medical need if the technical challenges can be overcome.

Hyperdynamic Microtubules, Cognitive Deficits, and Pathology Are Improved in Tau Transgenic Mice with Low Doses of the Microtubule-Stabilizing Agent BMS-241027

Tau is a microtubule (MT)-stabilizing protein that is altered in Alzheimers disease (AD) and other tauopathies. It is hypothesized that the hyperphosphorylated, conformationally altered, and multimeric forms of tau lead to a disruption of MT stability; however, direct evidence is lacking in vivo. In this study, an in vivo stable isotope-mass spectrometric technique was used to measure the turnover, or dynamicity, of MTs in brains of living animals. We demonstrated an age-dependent increase in MT dynamics in two different tau transgenic mouse models, 3xTg and rTg4510. MT hyperdynamicity was dependent on tau expression, since a reduction of transgene expression with doxycycline reversed the MT changes. Treatment of rTg4510 mice with the epothilone, BMS-241027, also restored MT dynamics to baseline levels. In addition, MT stabilization with BMS-241027 had beneficial effects on Morris water maze deficits, tau pathology, and neurodegeneration. Interestingly, pathological and functional benefits of BMS-241027 were observed at doses that only partially reversed MT hyperdynamicity. Together, these data suggest that tau-mediated loss of MT stability may contribute to disease progression and that very low doses of BMS-241027 may be useful in the treatment of AD and other tauopathies.

Tau Transgenic Mice as Models for Cerebrospinal Fluid Tau Biomarkers

Levels of tau in cerebrospinal fluid (CSF) are elevated in Alzheimers disease (AD) patients. It is believed this elevation is related to the tau pathology and neurodegeneration observed in AD, but not all tauopathies have increased CSF tau. There has been little pre-clinical work to investigate mechanisms of increased CSF tau due to the difficulty in collecting CSF samples from mice, the most commonly used pre-clinical models. We developed methods to collect CSF from mice without contamination from tau in brain tissue, which is approximately 50,000 fold more abundant in brain than CSF. Using these methods, we measured CSF tau from 3xTg, Tg4510, and Tau Alone transgenic mice. All three lines of mice showed age-dependent increases in CSF tau. They varied in phenotype from undetectable to severe tau pathology and neurodegeneration, suggesting that degenerating neurons are unlikely to be the only source of pathologic CSF tau. Overall, CSF tau levels mirrored expression levels and changes of tau in the brain, but they did not always correlate exactly. CSF tau was often more sensitive to changes in brain transgene expression and pathology. In addition, we also developed ELISA assays specific to different regions of the tau protein. We used these assays to provide evidence that CSF tau exists as fragments, with little intact C-terminus and partial loss of the N-terminus. Taken together, these assays and mouse models may be used to facilitate a deeper understanding of CSF tau in neurodegenerative disease.

The Amyloid-β Rise and γ-Secretase Inhibitor Potency Depend on the Level of Substrate Expression

The amyloid-β (Aβ) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-β precursor protein (APP) through consecutive proteolytic cleavages by β-site APP-cleaving enzyme and γ-secretase. Unexpectedly γ-secretase inhibitors can increase the secretion of Aβ peptides under some circumstances. This “Aβ rise” phenomenon, the same inhibitor causing an increase in Aβ at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Aβ rise depends on the β-secretase-derived C-terminal fragment of APP (βCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Aβ, known as “p3,” formed by α-secretase cleavage, did not exhibit a rise. In addition to the Aβ rise, low βCTF or C99 expression decreased γ-secretase inhibitor potency. This “potency shift” may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, γ-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Aβ under conditions of low substrate expression. The Aβ rise was also observed in rat brain after dosing with the γ-secretase inhibitor BMS-299897. The Aβ rise and potency shift are therefore relevant factors in the development of γ-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Aβ rise, including the “incomplete processing” and endocytic models, are discussed.

Passive Immunization with Phospho-Tau Antibodies Reduces Tau Pathology and Functional Deficits in Two Distinct Mouse Tauopathy Models

In Alzheimer’s disease (AD), an extensive accumulation of extracellular amyloid plaques and intraneuronal tau tangles, along with neuronal loss, is evident in distinct brain regions. Staging of tau pathology by postmortem analysis of AD subjects suggests a sequence of initiation and subsequent spread of neurofibrillary tau tangles along defined brain anatomical pathways. Further, the severity of cognitive deficits correlates with the degree and extent of tau pathology. In this study, we demonstrate that phospho-tau (p-tau) antibodies, PHF6 and PHF13, can prevent the induction of tau pathology in primary neuron cultures. The impact of passive immunotherapy on the formation and spread of tau pathology, as well as functional deficits, was subsequently evaluated with these antibodies in two distinct transgenic mouse tauopathy models. The rTg4510 transgenic mouse is characterized by inducible over-expression of P301L mutant tau, and exhibits robust age-dependent brain tau pathology. Systemic treatment with PHF6 and PHF13 from 3 to 6 months of age led to a significant decline in brain and CSF p-tau levels. In a second model, injection of preformed tau fibrils (PFFs) comprised of recombinant tau protein encompassing the microtubule-repeat domains into the cortex and hippocampus of young P301S mutant tau over-expressing mice (PS19) led to robust tau pathology on the ipsilateral side with evidence of spread to distant sites, including the contralateral hippocampus and bilateral entorhinal cortex 4 weeks post-injection. Systemic treatment with PHF13 led to a significant decline in the spread of tau pathology in this model. The reduction in tau species after p-tau antibody treatment was associated with an improvement in novel-object recognition memory test in both models. These studies provide evidence supporting the use of tau immunotherapy as a potential treatment option for AD and other tauopathies.

Pharmacodynamics of Selective Inhibition of γ -Secretase by Avagacestat

A hallmark of Alzheimer’s disease (AD) pathology is the accumulation of brain amyloid β-peptide (Aβ), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aβ and offer a potential new approach to treat AD. As γ-secretase also cleaves Notch proteins, GSIs can have undesirable effects due to interference with Notch signaling. Avagacestat (BMS-708163) is a GSI developed for selective inhibition of APP over Notch cleavage. Avagacestat inhibition of APP and Notch cleavage was evaluated in cell culture by measuring levels of Aβ and human Notch proteins. In rats, dogs, and humans, selectivity was evaluated by measuring plasma blood concentrations in relation to effects on cerebrospinal fluid (CSF) Aβ levels and Notch-related toxicities. Measurements of Notch-related toxicity included goblet cell metaplasia in the gut, marginal-zone depletion in the spleen, reductions in B cells, and changes in expression of the Notch-regulated hairy and enhancer of split homolog-1 from blood cells. In rats and dogs, acute administration of avagacestat robustly reduced CSF Aβ40 and Aβ42 levels similarly. Chronic administration in rats and dogs, and 28-day, single- and multiple-ascending–dose administration in healthy human subjects caused similar exposure-dependent reductions in CSF Aβ40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aβ levels without causing Notch-related toxicities. Our results demonstrate the selectivity of avagacestat for APP over Notch cleavage, supporting further evaluation of avagacestat for AD therapy.

P-Glycoprotein Efflux and Other Factors Limit Brain Amyloid β Reduction by β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitors in Mice

Alzheimers disease (AD) is a progressive neurodegenerative disease. Amyloid β (Aβ) peptides are hypothesized to cause the initiation and progression of AD based on pathologic data from AD patients, genetic analysis of mutations that cause early onset forms of AD, and preclinical studies. Based on this hypothesis, β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) inhibitors are an attractive therapeutic approach for AD because cleavage of the APP by BACE1 is required to form Aβ. In this study, three potent BACE1 inhibitors are characterized. All three inhibitors decrease Aβ formation in cultured cells with IC50 values less than 10 nM. Analysis of APP C-terminal fragments by immunoblotting and Aβ peptides by mass spectrometry showed that these inhibitors decreased Aβ by inhibiting BACE1. An assay for Aβ1–40 in mice was developed and used to show that these BACE1 inhibitors decreased plasma Aβ1–40, but not brain Aβ1–40, in wild-type mice. Because these BACE1 inhibitors were substrates for P-glycoprotein (P-gp), a member of the ATP-binding cassette superfamily of efflux transporters, these inhibitors were administered to P-gp knockout (KO) mice. These studies showed that all three BACE1 inhibitors decreased brain Aβ1–40 in P-gp KO mice, demonstrating that P-gp is a major limitation for development of BACE1 inhibitors to test the amyloid hypothesis. A comparison of plasma Aβ1–40 and brain Aβ1–40 dose responses for these three compounds revealed differences in relative ED50 values, indicating that factors other than P-gp can also contribute to poor brain activity by BACE1 inhibitors.

Tau Overexpression Impacts a Neuroinflammation Gene Expression Network Perturbed in Alzheimer’s Disease

Filamentous inclusions of the microtubule-associated protein, tau, define a variety of neurodegenerative diseases known as tauopathies, including Alzheimer’s disease (AD). To better understand the role of tau-mediated effects on pathophysiology and global central nervous system function, we extensively characterized gene expression, pathology and behavior of the rTg4510 mouse model, which overexpresses a mutant form of human tau that causes Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We found that the most predominantly altered gene expression pathways in rTg4510 mice were in inflammatory processes. These results closely matched the causal immune function and microglial gene-regulatory network recently identified in AD. We identified additional gene expression changes by laser microdissecting specific regions of the hippocampus, which highlighted alterations in neuronal network activity. Expression of inflammatory genes and markers of neuronal activity changed as a function of age in rTg4510 mice and coincided with behavioral deficits. Inflammatory changes were tau-dependent, as they were reversed by suppression of the tau transgene. Our results suggest that the alterations in microglial phenotypes that appear to contribute to the pathogenesis of Alzheimer’s disease may be driven by tau dysfunction, in addition to the direct effects of beta-amyloid.