Dysregulation of the unfolded protein response in the pathogenesis of non-alcoholic steatohepatitis - Project Summary/Abstract Non-alcoholic steatohepa��s (NASH) disease will supplant hepa��s C as the leading indica�on for liver transplanta�on this decade and has no FDA-approved therapy. To understand the root cause of NASH, we performed single-cell RNA sequencing (scSeq) in a mouse model of NASH at �mepoints before and during disease progression. We find that even before NASH occurs, a protec�ve program called the unfolded protein response (UPR) is lost in HFD livers. Strikingly, this UPR loss is most profound in the precise region of the liver where NASH is known to originate (“Zone 3”) in human livers. We subsequently confirmed that the UPR is also lost during human NASH. In this proposal, we seek to understand the mechanism of UPR loss (Aim 1) in human NASH, and to more clearly delineate whether and how the Zone 3 UPR protects against NASH (Aim 2). In Aim 1 we propose using precision cut liver slicing (PCLS), single-nuclei RNA sequencing, and spa�al immunofluorescence in human specimens to understand the rela�onship between lipid, oxida�ve stress, and the UPR. In Aim 2, we propose rescuing the UPR and key signaling molecules in our mouse model of NASH (long-term) and in human PCLS specimens (short-term) to understand how the UPR protects against NASH. These thema�cally related but independent aims will help establish mechanisms of Zone 3 loss of the UPR, and how the UPR protects against NASH, in turn clarifying the precise UPR targets that may be therapeu�cally tractable for trea�ng human NASH disease. This proposal supports my long-term goal to understand how func�onal adapta�on across the liver Zones leads to a vulnerability to specific liver diseases, of which the Zone-3 vulnerability of NASH is an exemplar. I have amassed significant exper�se in measuring Zone-specific gene expression using scSeq and immunofluorescence coupled with microscopy. This career development grant will enable me to expand this exper�se into specific tools which let me interrogate func�on in specific liver zones. The goal and keystone of the career development por�on of this proposal is to learn precision cut liver slicing and ex vivo culture, which would allow me to perturb zone-specific mechanisms chemically and gene�cally in human liver �ssue. We have established a collabora�on with a leading liver PCLS expert who has kindly invited me to train in his laboratory to learn this valuable method. In mice, I will learn how to use adenoviral vectors (AAVs) as a zone-specific tool to study rescue of UPR genes lost in NASH. And finally, this award will allow me solidify exper�se in scSeq analysis by learning advanced network analysis tools, which will in turn allow me to analyze scSeq data in a zone-specific manner. The environment to conduct this research is superb. Dr. Goessling, the primary mentor, is a renowned liver disease modeler, has exper�se in scSeq, and retains an excellent track record of developing mentees into independently funded scien�sts. The broader Harvard and MGH communi�es offer ample opportunity for rich collabora�on, as evidenced by support of this applica�on from Dr. Omer Yilmaz at MIT, an expert in modeling murine hypernutri�on, Dr. David Cohen at Brigham and Women’s, an expert in NASH and metabolism, and Dr. Alex Shalek at MIT, an expert in scSeq, among others. My mentorship team and the broader ins�tu�onal environment offers ample resources and ins�tu�onal support to successfully carry out the proposed aims.
