Identifying therapeutic targets for Alzheimer's disease: a cross-species genetic screen for molecules that lower tau levels Jiyoen Kim1,2, Ismael Al-Ramahi1,2, Maria de Haro1,2, Lorena Garaicoechea1,2, Hyun-HwanJeong1,2, Jean Pierre Revelli1,2, Maxime Rousseaux1,2, Qikai Xu3, Stephen J. Elledge3, Juan Botas1, Huda Y. Zoghbi1 1Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital; 2Dept. Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA; 3Dept. Genetics, Harvard Medical School, Boston, MA, USA
Tau is a central causative agent in the pathogenesis of Alzheimer's Disease (AD) and various dementias. Data from several studies indicate that the brain is particularly vulnerable to toxicity resulting from increased expression of aggregation-prone proteins. Conversely, reduction of tau levels has proven effective in AD mouse models. Thus, lowering tau levels might mitigate disease progression and provide an attractive target for therapy in AD and other dementias. However, to date, very few suppressors of tau levels have been identified. To identify novel regulators of tau, we employed a cross-species high-throughput screen. We used a pooled shRNA-retrovirus library targeting 7,532 “druggable” human genes in genetically engineered Daoy cells overexpressing a tau: EGFP. Cells with shRNAs that decreased tau levels were separated by flow cytometry and the bar-coded shRNAs identified by deep sequencing. In parallel, we conducted a siRNA screen in Drosophila targeting 4,000 fly homologs of the human genes tested. Our combined primary screens and in vitro validation in HEK293T cells led to the discovery of 91 potential tau modifiers. For in vivo validation in the mouse, we performed postnatal day zero intracerebroventricular (ICV) injection of AAV-shRNA to knockdown 19 prioritized targets and measured tau protein levels. Among these, gene suppression of 12 hits, including an ubiquitin specific protease (USP7), reduced tau protein levels by more than 20 percent. Interestingly, Usp7 interacts with tau and promote its deubiquitination, presenting a potential mechanism by which tau is stabilized. These results establish our cross-species high-throughput screen as a powerful approach to identify new targets involved in the regulation and processing of tau. Understanding the molecular mechanisms that underlie tau regulation will provide a foundation for identifying new potential therapeutics for AD as well as candidate risk genes.
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