Characterization of the Formation of Functional Solid Condensates of the Neuropeptides CRH and Neuropeptide Y in the Context of Anxiety and Depression

Abstract

Structural biology is undergoing a revolution with advancements in cryo-electron microscopy (Cryo-EM) and artificial intelligence (AI), allowing for the unprecedented precision study of high molecular weight proteins and large protein complexes. However, these technologies do not yet fully address intrinsically disordered proteins (IDPs) and their intrinsically disordered regions (IDRs), especially in the context of the formation of solid condensates and liquid-liquid phase separation (LLPS). IDPs are dynamic and respond to the environment in diverse ways, making them challenging to study. Solid condensates formed by IDPs have a complex history and play a fundamental role in various pathologies, such as Alzheimer's, Parkinson's, type 2 diabetes, and cancers. For many years, amyloid aggregates were viewed merely as aberrant states of protein aggregation, but recent studies show that they also have important native functions due to their ability to transform soluble proteins into insoluble ones, their high structural stability, and resistance to proteolysis. Additionally, amyloid aggregates play a role in the hypothesis of the origin of life, being simple and stable structures that can interact with and shape biomembranes. A functional example of amyloid-type solid condensates is the storage and release of neuropeptides via secretory granules. Neuropeptides can reach extremely high concentrations within cellular vesicles and are released in response to stress triggers. This packing process exploits the transition to the amyloid form, which must be reversible to allow the dispersion of neuropeptides into the bloodstream. However, the molecular and physicochemical requirements for neuropeptide disaggregation are still poorly understood. Neuropeptides, such as CRH and Neuropeptide Y, play crucial roles in the physiology of stress and are clinically relevant for mood disorders related to stress. Antagonists of the CRH receptor, such as pexacerfont, are being investigated to treat anxiety disorders, while high levels of CRH are associated with conditions like depression and Alzheimer's. Conversely, Neuropeptide Y acts as an endogenous anxiolytic, promoting stress resilience and dampening the fear response. This PhD project aims to provide a detailed description of the process of forming functional amyloids by neuropeptides, focusing on the neuropeptides CRH and Neuropeptide Y. The work will involve the production of these neuropeptides through heterologous expression and/or solid-phase peptide synthesis, and the physicochemical characterization of the amyloid condensate formation process under biologically relevant conditions. Techniques such as circular dichroism, static fluorescence, various microscopies, and solid-state nuclear magnetic resonance will be used. This study will significantly impact the understanding of how cells control the formation and release of functional neuropeptides, as well as contribute to the pharmacological management and therapeutic applications of these neuropeptides in treating anxiety and depression. The project timeline includes completing program coursework, producing and characterizing neuropeptides, and a BEPE internship to deepen knowledge in Cryo-EM techniques. The expected results will provide a solid foundation for new therapeutic and pharmacological approaches in the context of stress-related mood disorders.