Gregory R. Thatcher

Effects of aromatase inhibition versus gonadectomy on hippocampal complex amyloid pathology in triple transgenic mice

Alzheimers disease (AD) is the most common form of dementia among the elderly with women exhibiting a higher risk than men for the disease. Due to these gender differences, there is great interest in the role that estrogens play in cognitive impairment and the onset of the classic amyloid and tau lesions in AD. Human and rodent studies indicate a strong association between low brain aromatase, sex hormone levels, and beta amyloid deposition. Therefore, the effects of depleting both circulating and brain estrogen levels, through gonadectomy and/or treatment with the aromatase inhibitor, anastrozole, upon hippocampal AD-like pathology in male and female 3xTgAD mice were evaluated. Liquid chromatography-mass spectrometry revealed anastrozole serum levels of 10.19 ng/mL and for the first time brain levels were detected at 4.7 pg/mL. Densitometric analysis of the hippocampus revealed that anastrozole significantly increased Aβ- but not APP/Aβ-immunoreactivity in intact 3xTgAD females compared to controls (p<0.001). Moreover, anastrozole significantly increased the number of Aβ- compared to APP/Aβ-positive hippocampal CA1 neurons in intact and gonadectomized female mice. Concurrently, anastrozole significantly reduced the APP/Aβ plaque load in 9 month old female 3xTgAD mice. These data suggest that anastrozole treatment differentially affects select amyloid species which in turn may play a role in the extraneuronal to intraneuronal deposition of this peptide.

Novel analogues of chlormethiazole are neuroprotective in four cellular models of neurodegeneration by a mechanism with variable dependence on GABA A receptor potentiation

Chlormethiazole (CMZ), a clinical sedative/anxiolytic agent, did not reach clinical efficacy in stroke trials despite neuroprotection demonstrated in numerous animal models. Using CMZ as a lead compound, neuroprotective methiazole (MZ) analogues were developed, and neuroprotection and GABAA receptor dependence were studied.

NO/cGMP/pCREB re-activation reverses cognition deficits and attenuates amyloid-β neuropathology in transgenic models of Alzheimer’s disease

Results and Conclusion The hypothesis that activation of CREB through NO/ cGMP signaling might modify the amyloid-b (Ab) neuropathology, linked to AD pathogenesis, was demonstrated in both APP/PS1 and 3xTg transgenic mouse models of AD using small molecules, termed nomethiazoles, also designed to provide neuroprotection and attenuate pro-inflammatory cytokine release. Functional restoration of long-term potentiation was shown in hippocampal slices from AD transgenic mice in accord with observation of restoration of cognitive function in vivo, and was dependent upon soluble guanylyl cyclase (sGC) activation. Levels of pCREB and BDNF were significantly elevated, whereas TNFa, Ab, oligomeric Ab142, and also tau protein were significantly lowered after drug treatment. In the absence of neuronal loss in animal models of AD, neuroprotection was demonstrated in rat primary neurons after oxygen-glucose deprivation or application of oligomeric Ab. The lead nomethiazole was also studied in a novel transgenic mouse model, E4FAD, which incorporates familial AD mutations, but also has targeted replacement of mouse apolipoproteinE with human ApoE4, the major genetic risk factor for sporadic and age-related AD.

Abstract 3738: Three-dimensional treatment-resistant breast cancer spheroids as a predictive model of in vivo response to endocrine therapy

1 out of every 8 U.S. women will develop invasive breast cancer during her lifetime, making it the second most common form of cancer affecting women. Breast cancer is also a leading cause of cancer related deaths for women in the U.S., second only to lung cancer. Activation of estrogen receptor alpha (ERα) is the primary proliferative mechanism of breast cancer cells, making it a logical target for therapy. ER ligands with antiestrogenic activity, such as the selective estrogen receptor modulator (SERM), tamoxifen, and selective estrogen receptor degrader (SERD), fulvestrant, have proven clinically successful as treatments for breast cancer; however, resistance in up to 50% of patients provides a therapeutic challenge. Once resistant, breast cancer cells become endocrine-independent, because of this, there is an urgent need for both novel therapy and improved models of resistant breast cancer. Our lab has created a panel of various endocrine-independent cell lines to mimic SERM and SERD resistance. Along with traditional 2D cell culturing, 3D spheroids have also been utilized to gain a better understanding of resistance. Importantly, the response to therapeutic agents, of cell lines in 2D versus 3D cultures is not identical. We observe that 3D cultures better replicate observations in mouse xenograft models, demonstrating that elements of the spheroid microenvironment, such as cell-cell interactions and the presence of extracellular matrix (EM), mimic aspects of the tumor microenvironment in vivo. Cells cultured as spheroids are therefore a suitable in vitro model for drug discovery, predictive of response in preclinical animal models, in contrast to 2D monolayer cell cultures. Citation Format: Carlo I. Rosales, Jiong Zhao, Lauren M. Gutgesell, Rui Xiong, Debra A. Tonetti, Gregory R. Thatcher. Three-dimensional treatment-resistant breast cancer spheroids as a predictive model of in vivo response to endocrine therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3738.

Discovery of neuroprotective soluble guanylate cyclase modulators (GCMs) aciting via NO/GC/cGMP/CREB pathway

NO/cGMP signaling is essential for normal brain function, including learning and memory, and mediation of long-term potentiation (LTP). NO/cGMP signaling is coupled to cholinergic, glutamatergic, and dopaminergic systems and plays key roles in motor function associated with Parkinson’s disease pathogenesis and L-DOPA therapy. In Alzheimer’s Disease (AD), early synaptic failure, has been linked to dysfunction of gene expression programs mediated via the transcription factor cAMP-response element binding protein (CREB), activation of which is tightly regulated by NO/cGMP. Since several neurodegenerative disorders continue to be dogmatically linked to the chemical toxicity of NO, activation of soluble guanylyl cyclase (sGC) represents a potential therapeutic approach in both AD and PD; whereas negative modulation of sGC may be of use in some stages of PD. Such small molecule sGC modulators (GCMs) can be assayed in cell cultures by measuring levels of phosphorylated CREB (pCREB). GCMs that allosterically potentiate NO activation of sGC have proven successful in clinical trials for peripheral indications, however, there are no reports directed at therapeutic activity in the CNS. The aim of this study is to develop GCMs for use in CNS disorders, including AD and PD. A library of GCMs was synthesized, including positive allosteric modulators (PAMs) and negative modulators, as assessed by increasing levels of pCREB in SH-SY5Y human neuroblastoma cell cultures. Molecules were designed by classical bioisosteric replacement, aiming for desirable physiochemical properties and chemical diversity. GCM PAMs were assayed for cGMP elevation and reversal of neurotoxicity induced by 6-hydroxydopamine in dopaminergic neuronal cells. One GCM PAM, active in cell culture, was further tested for the reversal of memory deficits in mice treated with scopolamine, and drug levels in brain and plasma measured. The ligand binding site for sGC stimulators and GCM PAMs on the sGC protein remains to be definitively defined. Photoaffinity probes were designed that retained the activity of the parent GCM PAM in cell cultures in order to elucidate the binding mode to sGC and to indicate other protein partners for such compounds.