The Second-Generation PGI2 Analogue Treprostinil Fails to Chemoprevent Tumors in a Murine Lung Adenocarcinoma Model.

Prostacyclin (prostaglandin I2, PGI2) overproduction in FVB/N mice prevents the formation of carcinogen and tobacco smoke-induced adenomas, and administration of the oral prostacyclin analogue iloprost to wild-type mice also prevented carcinogen-induced mouse lung adenoma formation. Former smokers taking oral iloprost showed improved bronchial dysplasia histology compared with placebo. Next-generation oral prostacyclin analogues, like treprostinil, were developed for the treatment of pulmonary arterial hypertension (PAH). On the basis of our prior studies with iloprost, we performed preclinical studies examining the ability of treprostinil to chemoprevent urethane-induced murine lung adenocarcinoma. We determined the MTD in chow (prior studies had delivered treprostinil by gavage), and this dose produced serum levels in the experimental animals similar to those found in PAH patients treated with treprostinil. We then examined the chemopreventive efficacy of treprostinil exposure initiated both before (1 week) and after (6 weeks) urethane exposure to better model chemoprevention studies conducted in former smokers. Neither of these dosing strategies prevented murine lung cancer; however, we did detect changes in pulmonary inflammatory cell infiltrate and expression of CXCR4 (a chemokine receptor previously shown to increase in response to treprostinil exposure) in tumor-bearing, treprostinil-treated animals, indicating that the drug was bioavailable. One potential explanation stems from iloprost and treprostinil differentially activating cell surface prostaglandin receptors and intracellular peroxisome proliferator-activated receptors. When murine lung tumor cells were treated with treprostinil, their proliferation rate increased; in contrast, iloprost had no effect on proliferation. Future investigations comparing these two agents will provide insight into iloprost's chemopreventive mechanisms. Cancer Prev Res; 10(11); 671-9. ©2017 AACR.

Amyloid-beta antibody treatment leads to rapid normalization of plaque-induced neuritic alterations.

The accumulation of amyloid-beta into insoluble plaques is a characteristic feature of Alzheimer's disease. Neuronal morphology is distorted by plaques: rather than being essentially straight, they are substantially more curved than those in control tissue, their trajectories become altered, and they are frequently distended or swollen, presumably affecting synaptic transmission. Clearance of plaques by administration of antibodies to amyloid-beta is a promising therapeutic approach to the treatment of Alzheimer's disease, leading to stabilization of dementia by an unknown cellular mechanism. The effect of plaque clearance on plaque-induced neuronal alterations has not been studied previously. Here we show that both plaques and neuritic lesions are reversible in a strikingly short period of time after administration of a single dose of amyloid-beta antibody. Amyloid clearance and recovery of normal neuronal geometries were observed as early as 4 d and lasted at least 32 d after a single treatment. These results demonstrate that, once plaques are cleared, neuronal morphology is self-correcting and that passive antibody treatment has the potential to reverse neuronal damage caused by Alzheimer's disease and, hence, directly impact cognitive decline. Moreover, the rapid normalization of neuritic dystrophy suggests an unexpected degree of plasticity in the adult nervous system.

Cortical synaptic integration in vivo is disrupted by amyloid-beta plaques.

The accumulation of amyloid-beta protein into plaques is a characteristic feature of Alzheimer's disease. However, the contribution of amyloid-beta plaques to neuronal dysfunction is unknown. We compared intracellular recordings from neocortical pyramidal neurons in vivo in APP-Sw (Tg2576 transgenic mice overexpressing amyloid precursor protein with the Swedish mutation) transgenic mice to age-matched nontransgenic cohorts at ages either before or after deposition of cortical plaques. We show that the evoked synaptic response of neurons to transcallosal stimuli is severely impaired in cortex containing substantial plaque accumulation, with an average 2.5-fold greater rate of response failure and twofold reduction in response precision compared with age-matched nontransgenic controls. This effect correlated with the presence of amyloid-beta plaques and alterations in neuronal process geometry. Responses of neurons in younger APP-Sw animals, before plaque accumulation, were similar to those in nontransgenic controls. In all cases, spontaneous membrane potential dynamics were similar, suggesting that overall levels of synaptic innervation were not affected by plaques. Our results show that plaques disrupt the synchrony of convergent inputs, reducing the ability of neurons to successfully integrate and propagate information.

In vivo imaging of amyloid-beta deposits in mouse brain with multiphoton microscopy.

With the advent of transgenic mouse models expressing cortical amyloid pathology, the potential to study its progression in an intact brain has been realized. Multiphoton microscopy provides a non-destructive means of imaging with micron resolution up to 500 microm deep into the cortex. We detail a surgical procedure and discuss a multiphoton imaging approach that allows for labeling and chronic visualization of amyloid-beta deposits through a cranial window. The ability to monitor these hallmarks of Alzheimer's disease enables studies aimed at evaluating the efficacy of treatment and prevention strategies.

Fluorescence resonance energy transfer determinations using multiphoton fluorescence lifetime imaging microscopy to characterize amyloid-beta plaques.

We describe the implementation of a commercial fluorescence lifetime imaging microscopy (FLIM) instrument used in conjunction with a commercial laser scanning multiphoton microscope. The femtosecond-pulsed near-infrared laser is an ideal excitation source for time-domain fluorescence lifetime measurements. With synchronization from the x-y scanners, fluorescence lifetimes can be acquired on a pixel-by-pixel basis, with high spatial resolution. Multiexponential curve fits for each pixel result in two-dimensional fluorescence resonance energy transfer (FRET) measurements that allow the determination of both proximity of fluorescent FRET pairs, as well as the fraction of FRET pairs close enough for FRET to occur. Experiments are described that characterize this system, as well as commonly used reagents valuable for FRET determinations in biological systems. Constructs of CFP and YFP were generated to demonstrate FRET between this pair of green fluorescent protein (GFP) color variants. The lifetime characteristics of the FRET pair fluorescein and rhodamine, commonly used for immunohistochemistry, were also examined. Finally, these fluorophores were used to demonstrate spatially resolved FRET with senile plaques obtained from transgenic mouse brain. Together these results demonstrate that FLIM allows sensitive measurements of protein-protein interactions on a spatial scale less than 10 nm using commercially available components.

A limited role for microglia in antibody mediated plaque clearance in APP mice.

Amyloid-beta (Abeta) accumulation in senile plaques is a hallmark of Alzheimer's disease (AD). Immunotherapy is a leading approach for amyloid clearance, despite the early termination of the Elan clinical trial with active immunization due to a few cases of meningoencephalitis. The mechanisms of immunotherapy-mediated amyloid clearance and this deleterious side effect are largely unknown. While clearance of Abeta probably results in part from microglia-mediated inflammation, it can be microglia independent. Therefore, establishing the role of microglia in Abeta clearance is important for the treatment of AD. We analyzed the effects of direct microglia activation and inhibition on antibody-mediated Abeta clearance. Robust microglia activation with interferon-gamma led to modest Abeta clearance alone but did not potentiate antibody-mediated clearance. Microglia elimination/inactivation with immunotoxin or minocycline only partially limited antibody-induced Abeta clearance suggesting that although there is a role for microglia in Abeta clearance, it does not account for the majority of the effect observed after anti-Abeta antibody treatment.

Four-dimensional multiphoton imaging of brain entry, amyloid binding, and clearance of an amyloid-beta ligand in transgenic mice.

The lack of a specific biomarker makes preclinical diagnosis of Alzheimer's disease (AD) impossible, and it precludes assessment of therapies aimed at preventing or reversing the course of the disease. The development of a tool that enables direct, quantitative detection of the amyloid-beta deposits found in the disease would provide an excellent biomarker. This article demonstrates the real-time biodistribution kinetics of an imaging agent in transgenic mouse models of AD. Using multiphoton microscopy, Pittsburgh compound B (PIB) was imaged with sub-microm resolution in the brains of living transgenic mice during peripheral administration. PIB entered the brain quickly and labeled amyloid deposits within minutes. The nonspecific binding was cleared rapidly, whereas specific labeling was prolonged. WT mice showed rapid brain entry and clearance of PIB without any binding. These results demonstrate that the compound PIB has the properties required for a good amyloid-imaging agent in humans with or at risk for AD.