Proper protein folding is vital for biological function. Unfortunately, many proteins, especially membrane proteins, tend to misfold either because of errors during synthesis, mutations, changes in the balance of protein folding/chaperone systems, or because of environmental changes. Misfolded proteins must be eliminated otherwise they could interfere with cellular function, eventually causing disease. Furthermore, accumulation of misfolded proteins in organisms can shorten lifespan. Therefore, understanding how misfolded proteins are removed from cells has important implications both in terms of our basic understanding of the biology of the process as well as for devising therapeutic methods to treat diseases linked to protein misfolding. A particular challenge is to understand how misfolded proteins are eliminated from the endoplasmic reticulum (ER), the site where most membrane and secretory proteins are made. An appreciation of the importance of protein degradation from the ER, called ER-associated degradation (ERAD), is underscored by defects that sometimes occur in the system, which can lead to activation of ER stress, prolonged activation of which leads to cell death and tissue malfunction. Unfortunately, at present there are few, if any, reporters that can be used to directly monitor and/or visualize ERAD in living organisms. In this application we propose to fill this void by generating transgenic mice that could be used for studying ERAD in vivo. Accordingly, in Aim 1 we propose to generate a transgenic mouse model that expresses a fluorescent-based reporter that can be used to biochemically and visually measure ERAD activity in neurons (driven by the Thy1.2 promoter). The ERAD reporter we have chosen is CD3? tagged with the photoswitchable fluorescent protein Dendra2. We enumerate the many advantages why this reporter is particularly well suited for measuring ERAD activity in neurons. Once generated the mice will be characterized to ensure that the reporter can be reliably used to measure defects in ERAD. Upon validation we will cross the reporter mice in Aim 2 with wild type (WT) and a P497S UBQLN2 transgenic mouse model of ALS, which we recently generated. The P497S line develops cognitive deficits and motor neuron disease, whereas UBQLN2 WT mice do not show similar disease. Immunoblots indicate a build- up of ubiquitinated proteins in the brain and spinal cord of the P497S animals compared to WT and non- transgenic animals. In addition, ER stress markers (PDI and phosphorylated eIF2?) are elevated in spinal cord of end-stage P497S animals. The increase in ER stress is consistent with expression of the mutant P497S protein interfering with ERAD, which is a known function of UBQLN2 protein. The animal cross will allow us to evaluate if this is the case, and the time course of any interference. The generation and validation Thy1.2 expressing CD3?-Dendra2 mice as proposed here should provide an extremely powerful tool for evaluating interference in ERAD caused by UBQLN2 and other mutant proteins linked to neurodegenerative diseases.
Disturbances in protein homeostasis in the endoplasmic reticulum, the factory where many proteins vital for cellular signaling are made, leads to ER stress, which is frequently, activated in many diseases, particularly neurodegenerative diseases. However, currently there is no convenient system to measure defects in the system (called ER-associated degradation or ERAD) that clears misfolded proteins from cells. Here we propose to fill this void by generating a convenient photoswitchable and fluorescent system that will enable visualization and biochemical studies of ERAD in neurons of mice. Once generated this system could have wide utility to study and interrogate defects in protein homeostasis in the ER, especially of mutant proteins involved in neurodegenerative disease.