Tony Yuen, Ph.D. - US grants

PROJECT SUMMARY Alzheimer?s disease (AD) stands out as notable in not having a cure among many diseases that affect elderly men and women??in essence, creating an urgent need for a new therapy. It is also unclear why menopausal women have a preponderance of AD, and, while declining estrogen bas been implicated, there is clear clinical correlation with rising levels of follicle stimulating hormone (FSH). We have identified FSH as a target for several aging disorders??osteoporosis, obesity, hypercholesteremia??and now, AD. Inhibiting the action of FSH using blocking antibodies reduces body fat, increases bone mass, lowers serum cholesterol, and from our newest and most exciting results, prevents AD in two mouse models. We have designed a novel humanized monoclonal antibody, Hu6, that binds to a small epitope within the receptor?binding domain of FSH?, thus blocking its action on the FSH receptor (FSHR). Our aspirational goal is to use this lead therapeutic for the therapy and prevention of all four disorders??or, at the very least, AD. Selected from a pool of 30 newly synthesized humanized antibodies, Hu6 displays high?affinity binding to FSH (KD ~7 nM) and thus prevents its action on hippocampal FSHRs to improve cognition in AD mice. These observations, together with its optimal pharmacokinetic profile, lay the groundwork for Hu6 to enter early stage development. In Specific Aim 1, we propose to scale up production of research?grade Hu6; create an optimal formulation; test its physicochemical properties; study the structure of the FSH:Hu6 complex; and manufacture a master cell bank for cGMP?grade Hu6. In Specific Aim 2, we will perform pharmacokinetic studies in Tg32 mice ?humanized? for antibody clearance, and in African green vervet monkeys; determine minimum effective dose(s) in preventing and/or treating AD in 3xTg mice; examine efficacy and safety in young and aged 3xTg mice; and document safety in vervet monkeys. The work will be conducted using Good Laboratory Practice (GLP) standards established at Mount Sinai, Emory, Wake Forest and San Antonio. We have also created cross?functional research teams that will be supported by a distinguished panel of advisors, comprising science and medicine experts, business leaders, and entrepreneurs in biotechnology. Definitive information on dosage, route and frequency, together with early proof of safety should propel us into late stage development and first?in?human studies.

PROJECT SUMMARY Alzheimer?s disease (AD) stands out as notable in two respects??in not having a cure and in affecting women more than men. While declining estrogen has been thought to underpin post?menopausal AD, there is a clear clinical correlation of AD with rising levels of follicle?stimulating hormone (FSH). Most notably, there is a ?spike? in cognitive decline in women in the early years of the menopausal transition, when serum estrogen is normal and FSH levels begin to rise. Collaborative studies between the Mount Sinai and Emory groups have identified FSH as a potential driver for AD?and suggest that rising FSH levels may contribute to the disproportionate increase of AD in aging women. Notably, we find that FSH receptors (FSHRs) are expressed in both mouse and human brain, and that the injection of recombinant FSH or ovariectomy (that elevates serum FSH) aggravates AD pathology and cognitive decline in 3xTg mice. Inhibiting the action of FSH in 3xTg or APP/PS1 mice by an FSH?blocking antibody or downregulating Fshr expression in the hippocampus prevents onset of the AD phenotype. The Emory group also provides strong preliminary evidence that FSH upregulates C/EBP?, which activates asparagine endopeptidase (AEP), a ??secretase that cleaves amyloid precursor protein (APP) and Tau??resulting in neuritic plaques and neurofibrillary tangles, respectively. The goal of the transdisciplinary collaboration between the disciplines of endocrinology and neuroscience is to fully understand the mechanism of FSH action on AD?vulnerable brain regions. Thus, in Specific Aim 1, we will map the distribution and cellular localization of the FSHR and its signaling partners CEBPB and LGMN in human and mouse brain using single?transcript technologies. In Specific Aim 2, we will examine the function of the brain FSHR in driving AD pathology and cognitive decline. For this, we will downregulate or overexpress the Fshr in specific brain areas of 3xTg mice by stereotaxically injecting AAV expressing siFshr or Fshr. We will also study the effect of high FSH in 3xTg mice rendered haploinsufficient in Cebpb, and delineate the transcriptomic architecture of FSH?treated human neuronal cells by RNA?seq. In Specific Aim 3, we will determine whether deleting the Fshr or inhibiting FSH action by our murine FSH blocking antibody, Hf2, injected over the lifespan of 3xTg mice can prevent the onset of cognitive decline. To contemporaneously replicate our data, the Emory group will study the effect of treating established cognitive impairment with Hf2 in 18?month?old APP knock?in (KI) mice. In all, our proof?of?concept studies??conducted using our Good Laboratory Practices (GLP) Platform??should not only establish a role for high FSH in driving AD, but also provide a framework for the future testing of our humanized FSH?blocking antibody, Hu6, in aging women.

The record of safety and efficacy of the four FDA?approved drugs for erectile dysfunction, namely tadalafil, vardenafil, sildenafil and avanafil, is predicated on their ability to potently inhibit the cellular enzyme, phosphodiesterase 5A (PDE5A). PDE5A hydrolyzes cyclic guanosine monophosphate (cGMP), so that PDE5A inhibitors stimulate the nitric oxide?cGMP?protein kinase G (PKG) pathway. In 1991, we documented for the first time that nitric oxide regulates the osteoclast, the cell that resorbs bone (PMID: 1849281). Multiple studies have since established robust effects of modulating this pathway on both components of bone remodeling ? bone resorption by osteoclasts and bone formation by osteoblasts. Prompted by observations that erectile dysfunction and osteoporosis track together in older men, in men with diabetes, and in men receiving androgen?deprivation therapy for prostate cancer, we sought to investigate the action of tadalafil and vardenafil on bone. The overarching hypothesis was that PDE5A inhibitors could be repurposed for the co?therapy of erectile dysfunction and osteoporosis in men and, even perhaps, solely for osteoporosis in women. We found in mouse studies that tadalafil and vardenafil increased bone mass, importantly in female mice, by stimulating bone formation and inhibiting bone resorption (Kim et al, PNAS, In press). Despite net bone gain, the anabolic action of the drugs was antagonized by a unique sympathetic relay signature originating from central PDE5A?positive neurons in the locus coeruleus, raphe pallidus and hypothalamic paraventricular nucleus. Noting that most osteoporosis drugs are either anti?resorptive or anabolic, any dual?acting agent will have unique value particularly with oral use. Therefore, towards the potential for repurposing PDE5A inhibitors for osteoporosis, our current goal is to understand precisely how the drugs work on bone and to evaluate preclinical efficacy in models of bone loss. In Specific Aim 1, using global and cell?selective knock out mice, we will determine whether the drugs inhibit PDE5A to activate the NO?cGMP?PKG2 pathway in bone, and if so, which cell is the primary target. In Specific Aim 2, we will comprehensively map the distribution of PDE5A in brain at the single transcript level by RNAscope, and interrogate PDE5A?positive nodes through AAV?mediated Pde5a overexpression or knock down. In Specific Aim 3, we will study the ability of tadalafil, vardenafil, sildenafil and/or avanafil to trigger bone gain in 1?year?old aging mice; to prevent hypogonadal bone loss in rats and mice; and to restore bone that is already lost 28 weeks following ovariectomy in rats. Together, our mechanistic and efficacy studies should provide a firm foundation for future clinical trials towards repurposing PDE5A inhibitors for the prevention and treatment of osteoporosis.