Reversing Neuronal Cell Damage Through Mitophagy Induction

Familial Dysautonomia (FD) is caused by the single point mutation of the major signaling scaffolding elongator protein IKAP/ELP1. This disease disrupts the autonomic nervous system, leading to decreased sensory perception, poor reflexes, inhibition of vascular and cardiac functions, orthostatic hypotension, and recurrent severe autonomic crises. Patients with FD experience increased incidence of severe adverse reaction and increased neuropathy after anesthesia. Neuropathy is associated with mitochondrial damage and increased cellular reactive oxidative species (ROS). Mitophagy is a cellular process that recycles damaged or dysfunctional mitochondria. We hypothesize that increasing mitophagy or reducing ROS levels, will lead to greater neuronal cell viability. We will employ the dopaminergic LUHMES cell line and apply a mitophagy stimulator known as CCCP. We expect the cellular viability to be increased because CCCP will cull the defective mitochondrial pool as measured by MTT assay and Western blot for mitophagy markers (PINK1) and apoptosis markers (Caspase-3). Future directions of this experimental plan will include the addition of the anesthetic Ketamine. We hypothesize that a greater level of neuronal cell death will occur after Ketamine application. To counter this death, pre-treatment with CCCP or a ROS inhibitor will delay or prevent Ketamine induced cellular apoptosis. We envision patient pre-treatment with a ROS inhibitor or mitophagy stimulator may counteract anesthetic neuronal damage. Implications of this study could elucidate new methods in preventing anesthesia-associated nerve damage in FD patients as well as diabetic- and cancer-induced neuropathic conditions.

I’m Sofie Strompf and this is my OSCAR URSP Spring 2024 project. My research focuses on the role of mitophagy in anesthesia and neuropathy. The goal of this research is to, in the far future, allow us to pretreat patients who are predisposed to experiencing neuropathy following anesthesia with a mitophagy-inducing drug, to prevent or decrease the likelihood of their developing neuropathy. My mentor is Dr. Ali Andalibi, and I want to give special thanks to Drs. Marissa Howard, Lance Liotta, Jessica Roman, Alessandra Luchini, and Karen Lee, as well as Purva Gade, Matthew Gadziala, and Thomas Philipson for their mentorship. This research was funded in part by the George Mason University Office of Student Creative Activities and Research Undergraduate Research Scholars Program.
So, Familial Dysautonomia is a rare genetic neurodegenerative condition caused by the single point mutation of the major signaling scaffolding elongator protein IKAP/ELP1. This disease disrupts the autonomic nervous system, leading to autonomic distress and recurrent severe autonomic crises. These patients also experience increased incidence of severe adverse reaction and increased neuropathy after anesthesia. Anesthesia is known to induce mitochondrial stress. Neuropathy is associated with mitochondrial damage and increased cellular reactive oxidative species (ROS). This implies a correlation between the increased incidence of neuropathy following anesthesia in patients with FD and the known increase in mitochondrial stress during anesthesia. Mitophagy is a cellular process that recycles damaged or dysfunctional mitochondria. From this knowledge, our hypothesis is that increasing mitophagy or reducing ROS levels will lead to greater neuronal cell viability. Exploration of this hypothesis may elucidate new methods in preventing anesthesia-associated nerve damage in Familial Dysautonomia patients as well as diabetic- and cancer-induced neuropathic conditions. To be clear, this research is not only applicable to patients with Familial Dysautonomia””it is applicable to all patients experiencing neuropathy, especially following anesthesia. Familial Dysautonomia patients are a helpful control group for this experiment, as it is extremely likely that they will experience neuropathy directly related to induction of anesthetic; this occurs at far higher rates in this population than any other patient group, as far as is currently known.
This project is a continuation of my Fall 2023 OSCAR URSP Project. Last semester, I specifically focused on learning skills and techniques that I need to work in biological research and gained valuable certifications that I need to work in a laboratory setting safely.
This semester, I utilized my knowledge of laboratory skills to complete experiments. I utilized skills in cell passaging, cell viability, luminescence imaging, drug dosing protocols, and western blots to do so. The experiments mostly utilized the IOMM-Lee cell line, a malignant meningioma cell line derived from the human brain. In my most successful, complete experiment, the cells were maintained in DMEM-FBS 10% media. They were then plated with control, control-DMSO, varying concentrations of heregulin beta 1″”a tumor growth polypeptide””and CCCP, a known mitophagy stimulator. Half of the wells were imaged for cell viability; half were lysed. Cell lysate was employed in a drug dosing procedure which introduced phosphorylated PINK1 with Actin as a control. A Western blot was utilized to measure P-PINK1 and Actin after 24 hours. This procedure was the first of a series of planned procedures measuring mitophagy markers. This semester, I also had the incredible opportunity to present my current research at the National Conference on Undergraduate Research, or NCUR, in Long Beach, California.
In the future, continuing to use the IOMM-Lee cell line and also introducing the dopaminergic LUHMES cell line, we will continue to measure mitochondrial dysfunction following application of CCCP. We will continue to measure mitophagy markers, including PINK1 and Parkin, and the apoptosis marker Caspase-3, utilizing MTT assay and Western blot. We will also attempt to measure markers of cellular stress associated with induction of anesthesia in order to compare this preliminary analysis of mitochondrial stress with data derived from future experimental processes. Future directions of this experimental plan will possibly include the addition of anesthetics to further explore our hypothesis. We expect that a greater level of neuronal cell death will occur after anesthetic application. To explore new methods in prevention and treatment of anesthesia-associated nerve damage, we will attempt to counter this expected increased incidence of cell death. We will pre-treatment with CCCP or a ROS inhibitor will delay or prevent anesthetic induced cellular apoptosis. We envision patient pre-treatment with a ROS inhibitor or mitophagy stimulator, which may counteract anesthetic neuronal damage.
Obviously, getting anywhere near pre-treatment of patients will happen very, very far in the future. So, in the near future, I plan to continue my experiments and present advances in my research at next year‘s NCUR conference and OSCAR celebration. I plan to continue to work in the Institute for Advanced Biomedical Research, in the Center for Applied Proteomics and Molecular Medicine, where I have been completing my research so far. I have applied for additional funding through outside sources and am hoping that I will be able to not only continue my research next year or through the remainder of my undergraduate career, but that I will be able to carry this research into my graduate career. This has been an incredible experience, and I look forward to continuing my research in the years to come.