Hongkyun Kim - US grants

[unreadable] DESCRIPTION (provided by applicant): The dystrophin associated protein complex is a multimeric protein complex found in many different tissues, including muscle. Genetic defects in the dystrophin associated protein complex lead to muscular dystrophy in humans. The dystrophin associated protein complex plays several different roles in the plasma membrane. However, given that large percentages of patients with muscular dystrophy remain to be molecularly diagnosed, there is a possibility that some of the components may not have been identified yet. Furthermore, we do not know how the dystrophin complex is exactly assembled, processed and transported to the plasma membrane. The nematode C. elegans is an established genetic model organism, and possesses most of components of the dystrophin associated protein complex. In C. elegans, mutations in components of the dystrophin associated protein complex cause a unique locomotory phenotype that is not observed in any other class of uncoordinated or hyperactive mutants, and lead to muscle degeneration under certain conditions. We previously designed a genetic screen that identifies specifically mutants exhibiting the same locomotory phenotype as the dystrophin mutant, and identified several genes encoding known components of the dystrophin complex. Additionally, we identified a novel gene that encodes an acetylcholine/choline transporter. In a modified genetic screen we now have identified at least two additional novel genes. We have cloned one of the genes and are continuing to characterize the gene. We propose to expand the genetic screen to completion and identify mutants that exhibit the same locomotory phenotype as the dystrophin mutant. We will determine whether these mutants represent known genes or novel genes of the dystrophin associated complex. We will clone the novel genes by a combination of genetic mapping and transformation rescue. These novel genes may be unidentified components of the dystrophin complex, regulate assembly or trafficking of the complex, and mediate cellular functions. We will characterize the molecular functions of these novel genes using genetic, molecular and cell biology techniques. We will also identify and study the functional mammalian homologues. These findings will improve our understanding of the pathogenesis mechanism of muscular dystrophy in humans and may help to devise new therapeutic strategies. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Many types of cells in metazoan species operate under conditions of mechanical stress, and are evolved to cope with plasma membrane damage. A failure in either the prevention or repair of plasma membrane damage can cause disease, or can influence disease progression, as observed in several muscular dystrophies. A more detailed mechanistic understanding of membrane repair is required to find therapeutic interventions for a variety of diseases where membrane damage underlies the pathophysiology. The first step towards the detailed understanding of the repair mechanism is a comprehensive identification of the component molecules. We developed a method that uses the microscopic nematode C. elegans for identifying molecular and cellular components that mediate the repair of damaged sarcolemma (muscle plasma membrane). By taking advantage of the transparency of the C. elegans body that allows detection of fluorescence proteins in vivo, we developed a novel, simple assay that can easily evaluate the degree of sarcolemmal damage. With this assay, we found that C. elegans dystrophin mutants (a model of Duchenne muscular dystrophy) exhibit sarcolemmal leakage and damage, albeit weak. Based on this finding, we performed a genetic screen to isolate mutants that have defects in sarcolemmal repair and, as a result, aggravate sarcolemmal damage of dystrophin mutants. In this exploratory proposal, we seek to establish the screen as a valuable tool for identifying genes responsible for sarcolemmal repair. With several prominent mutants in hands, we specifically propose to clone causal genes for defects in sarcolemmal repair by a combination of genetic mapping and whole genome sequencing. Once we identify the responsible genes, we will characterize the cloned genes using well-established C. elegans genetic techniques. In parallel, we will determine the relationship between these identified genes to understand how they function together to protect and repair the sarcolemma. The successful completion of this project will lead to a better understanding of the molecular mechanism of sarcolemmal repair and, more importantly, will reveal potential druggable targets for blocking the progression of muscular dystrophies that are influenced by membrane repair. PUBLIC HEALTH RELEVANCE: Many forms of muscular dystrophy, including Duchenne muscular dystrophy, cause a disruption of the integrity of the muscle membrane. Hence, understanding how muscle membrane integrity is maintained and repaired has a therapeutic implication. The proposed C. elegans genetic study will identify and characterize genes responsible for muscle membrane repair.

? DESCRIPTION (provided by applicant): Alcohol abuse is a major health issue with enormous socio-economic costs. Muscle is one of the major tissues that are negatively impacted by alcohol abuse. While binge drinking causes acute muscle damage such as rhabdomyolysis (muscle fiber dissolution), chronic alcohol abusers suffer from alcoholic muscle disease, such as cardiomyopathy or myopathy. Previous studies in the mammalian muscle showed that habitual alcohol consumption impairs mitochondria function. Along with the unique role of mitochondria in apoptotic cell death, the alcohol-induced impairment in mitochondrial function may explain partially muscle weakness and the atrophy-like symptoms in chronic alcoholic muscle disease. Recent studies have demonstrated that alcohol influences mitochondrial fusion and fission dynamics, which in turn modulates mitochondrial bioenergetics, autophagy and other signaling pathways. However, it is also recognized that chronic alcohol intake causes an array of adverse changes in cellular events and signaling pathways, including reduced mTOR (mechanistic target of rapamycin) signaling and cellular stress responses. One important question that has arisen from these studies is how these altered events and pathways are related to the dynamics of mitochondrial fusion and fission in the context of alcohol toxicity. Are these events and pathways sequential or causal to altered mitochondrial dynamics? Do the cellular events and pathways influence mitochondrial dynamics or vice versa if they occur independently of each other? Understanding their relationship will provide the basis for the identification of effective therapeutic targets. The nematode C. elegans is an excellent model organism that has widely been used to dissect complex cellular events and pathways, such as stress response and apoptotic cell death. We found that the C. elegans muscle exhibited changes in mitochondrial tubular networks upon ethanol exposure. In addition, we observed that chronic alcohol exposure reduced muscle size compared to controls, implicating the involvement of the mTOR signaling pathway that controls cell size and mitochondria biogenesis. Finally, chronic alcohol exposure to C. elegans resulted in the mitochondrial unfolded protein response (UPRmt). The induction of UPRmt by chronic alcohol exposure indicates that alcohol causes mitochondrial protein misfolding. Together, our data showed that alcohol preferentially targets mitochondrial function in muscle. In this exploratory proposal, we will define the inter-relationship between alcohol-induced mitochondrial dynamics and other cellular events or pathways. First, we will define the molecular components responsible for alcohol-mediated muscle mitochondrial fission. Second, we will determine the relationship between UPRmt and mTOR signaling and the alcohol-induced change in mitochondrial dynamics. The successful completion of this project will lead to a better understanding of how alcohol-induced mitochondrial fission interacts with other signaling pathways implicated in alcoholic muscle diseases. Hence, our study will reveal effective therapeutic targets for ameliorating alcohol-induced muscle disease.

Environmental and pathophysiological challenges, such as oxidative stress, infection, and excessive neural activation, disrupt the functions of excitable cells and thus threaten the health and survival of animals. These challenges lead to the accumulation of misfolded and aggregated proteins in the endoplasmic reticulum (ER), a condition referred to as ER stress. ER stress is closely associated with cellular calcium ions; excessive cytosolic calcium ions disrupt ER calcium homeostasis and result in ER stress, while ER stress causes calcium leak from the ER and in turn calcium accumulation in the cytoplasm and mitochondria. Excessive calcium accumulation can lead to the activation of apoptotic factors and calcium-dependent proteases. Thus, it is critical that excitable cells have a protective mechanism that antagonizes calcium increases. The BK SLO-1 channel is a calcium-activated potassium channel that has been described as an ?emergency brake? that responds to excessive depolarization and accumulation of intracellular calcium ions. A critical factor that influences BK channel function is its density at the plasma membrane. Indeed, changes in BK channel density affect disease course or symptoms in an increasing number of disease conditions and drug use disorders, including autism, Alzheimer's disease, and alcohol abuse. Despite the importance of BK channel density, three key processes that determine BK channel density, channel trafficking, recycling, and degradation, are poorly understood. By implementing a C. elegans forward genetic screen designed to identify genes responsible for BK channel trafficking and recycling, we have identified three novel genes that influence the trafficking of BK channels. In this proposal, we will molecularly characterize these genes and how these genes contribute to BK channel trafficking in the basal and stress conditions. Our proposed study will improve our understanding of the pathological mechanisms for diseases that are associated with BK channel dysfunction, and provide therapeutic opportunities to selectively modulate BK channel density by altering the activities of proteins that mediate channel trafficking and recycling.