Effects of Conditional Knockout of Pdia3 during Development of the Mouse Hippocampus: Assessment of Behavioral, Histological and Molecular Alterations

• Grant number: 24/00317-9
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Effective date (Start): March 01, 2024
• Effective date (End): February 28, 2027
• Field of knowledge: Biological Sciences - Biology
• Principal Investigator: Danilo Bilches Medinas
• Grantee: Nayrob Pereira
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/06287-6 - Role of protein disulfide isomerase A3 in learning and memory, AP.JP

Abstract

The redox folding through disulfide bond formation constitutes an essential step in biogenesis of membrane and secreted proteins trafficking through the endoplasmic reticulum (ER) that confers structural stability while allowing conformational transitions for cellular signaling. This process is tightly regulated to maintain protein homeostasis (or proteostasis) of the ER, being catalyzed by oxidoreductases of the protein disulfide isomerases (PDIs) family. Around 20 members of the PDIs family have been described, with different substrates specificity and tissue distribution. Alteration of PDIs activity can perturb proteostasis of the ER, leading to accumulation of misfolded and aggregated proteins in the organelle, a condition referred to as ER stress. Such condition has been associated to pathogenic events underlying myriad neurological disorders, such as synaptic dysfunction, neuroinflammation, and apoptosis. There is clear genetic and biochemical evidence transversally linking protein disulfide isomerase A3 (PDIA3) to pathologies of the nervous systems, but its roles in neuronal populations comprising different neural networks affected in these conditions remain unknown. The ER is a key organelle contributing to neuronal morphogenesis and synaptic transmission where PDIA3 catalyzes redox folding of glycosylated proteins interacting with the lectin-like chaperones calnexin (CNX) and calreticulin (CRT). The elucidation of molecular and cellular mechanisms governed by PDIA3 in neurons of different regions of the nervous system can unravel fundamental aspects of neuronal function and selective vulnerability in pathological conditions. In this thesis project, we aim to define the contribution of PDIA3 to learning and memory, cognitive processes compromised in disorders associated to PDIA3, by performing loss of function manipulation during development of the mouse hippocampus. We hypothesize that PDIA3 promotes learning and memory by catalyzing the redox folding of essential proteins involved in formation of neural circuits and synaptic activity of neurons. Using a battery of behavioral tests, we will determine whether Pdia3 ablation at late stages of embryonic development of mice affect different types of learning and memory in the adulthood. Then, we will perform systematic histological examination of hippocampal tissue to investigate morphological abnormalities of neuronal subpopulations, alterations of synaptic content, and ER stress responses underlying possible cognitive phenotypes. In parallel, we will perform primary hippocampal culture to monitor the impact of Pdia3 ablation on neuronal maturation. In addition, we will conduct unbiased proteomic analysis of proteins aggregates and synaptosomes fractions of hippocampal tissue for determination of molecular alterations resulting from PDIA3 deficiency. Using this experimental strategy, we will generate critical knowledge about the participation of PDIA3 in the formation and function of neuronal connections in the hippocampus supporting learning and memory that can be disrupted under pathological conditions.