AAV-mediated Interrogation of the Brain FSH Receptor

PROJECT SUMMARY The biology underpinning the preponderance of Alzheimer’s disease (AD) in postmenopausal women has until recently remained unclear. While declining estrogen levels have been implicated, there is a clear correlation, in post–menopausal women, between cognitive decline and high levels of the pituitary glycoprotein, follicle–stimulating hormone (FSH). We have discovered FSH as a target for several aging disorders––namely, osteoporosis, obesity, and AD. Inhibiting FSH action, either genetically in Fshr–deficient mice or using FSH– blocking antibodies reduces body fat, increases bone mass, and from our newest and most exciting results, prevents AD in mice [Nature, 2022, PMID: 35236988]. The deleterious effect of FSH on cognition is mediated through abundant FSH receptors (FSHRs) in several AD–vulnerable brain regions, including the granular cell layer of the dentate gyrus, pyramidal layer, and cortical layer V. Activation of molecules downstream of brain FSHRs, namely C/EBPb and arginine endopeptidase, in AD–prone 3xTg mice results in neuropathology and memory loss, while Fshr downregulation in hippocampal neurons attenuates the FSH–induced AD phenotype. Here, we seek to develop, validate, and study newly engineered adeno–associated viruses (AAVs) that carry a cell–specific enhancer to enable gene knockdown in Fshr–positive neurons. We hypothesize that high– efficiency, AAV–mediated Fshr knockdown in Fshr–positive, AD–vulnerable neurons in the granular layer of the dentate gyrus, pyramidal layer, and cortical layer V will reduce AD–like neuropathology and memory loss. The R61 Pilot Phase will consist of studies focused on creating and validating two enhancer–AAV constructs–– containing either a neuron–specific enhancer, mscRE4, or Fshr distal enhancers consisting of evolutionarily conserved regions (ECRs)––packaged in one of 8 existing brain–permeant AAV capsids. As a readout of enhancer–driven gene expression, we will study the expression of YFP or mCherry, respectively, in Fshr–positive cells. Once we establish a novel enhancer–AAV toolkit, the R33 Implementation Phase will focus on studying the extent of Fshr knockdown by the AAV–enhancers in AD–vulnerable neurons. Importantly, we will ask the question whether Fshr downregulation in these neurons translates into an attenuation of FSH–induced neuropathology and memory loss. Our studies represent the first attempt at using an innovative AAV–based strategy to interrogate a brain–resident pituitary hormone receptor in relation to its role in the pathogenesis of AD. Carrying different cargos, our new toolkit could be deployed to probe additional pathogenic mechanisms of AD, thus laying the groundwork for multiple therapeutic options.