FUS/TLS in Stress Response - Implications for Amyotrophic Lateral Sclerosis: A Dissertation
Authors
Sama, Reddy Ranjith KumarFaculty Advisor
Daryl A. Bosco, PhDAcademic Program
Cell BiologyUMass Chan Affiliations
NeurologyDocument Type
Doctoral DissertationPublication Date
2014-03-28Keywords
Dissertations, UMMSAmyotrophic Lateral Sclerosis
DNA Damage
DNA-Binding Proteins
Mutation
RNA-Binding Protein FUS
p38 Mitogen-Activated Protein Kinases
Amyotrophic Lateral Sclerosis
DNA Damage
DNA-Binding Proteins
Mutation
RNA-Binding Protein FUS
p38 Mitogen-Activated Protein Kinases
Biochemistry, Biophysics, and Structural Biology
Cellular and Molecular Physiology
Molecular Genetics
Nervous System Diseases
Metadata
Show full item recordAbstract
Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease is a fatal neurodegenerative disease. ALS is typically adult onset and is characterized by rapidly progressive loss of both upper and lower motor neurons that leads to death usually within 3-5 years. About 90% of all the cases are sporadic with no family history while the remaining 10% are familial cases with mutations in several genes including SOD1, FUS/TLS, TDP43 and C9ORF72. FUS/TLS (Fused in Sarcoma/Translocated in Liposarcoma or FUS) is an RNA/DNA binding protein that is involved in multiple cellular functions including DNA damage repair, transcription, mRNA splicing, RNA transport and stress response. More than 40 mutations have now been identified in FUS that account for about 5% of all the familial cases of ALS. However, the exact mechanism by which FUS causes ALS is unknown. While significant progress has been made in understanding the disease mechanism and identifying therapeutic strategies, several questions still remain largely unknown. The work presented here aims at understanding the normal functions of FUS as well as the pathogenic mechanisms by which it leads to disease. Several studies showed the association of mutant-FUS with structures made up of RNA and proteins, called stress granules that form under various stress conditions. However, little is known about the role of endogenous FUS under stress conditions. I have shown that under hyperosmolar conditions, the predominantly nuclear FUS translocates into the cytoplasm and incorporates into stress granules. The response is specific to hyperosmolar stress because FUS remains nuclear under other stress conditions tested, such as oxidative stress, ER stress and heat shock. The response of FUS is rapid, and cells with reduced FUS levels are susceptible to the hyperosmolar stress, indicating a pro-survival role for FUS. In addition to investigating the functions of endogenous wild-type (WT) FUS, the work presented also focuses on identifying the pathogenic mechanism(s) of FUS variants. Using various biochemical techniques, I have shown that ALS-causing FUS variants are misfolded compared to the WT protein. Furthermore, in a squid axoplasm based vesicle motility assay, the FUS variants inhibit fast axonal transport (FAT) in a p38 MAPK dependent manner, indicating a role for the kinase in mutant-FUS mediated disease pathogenesis. Analysis of human ALS patient samples indicates higher levels of total and phospho p38, supporting the notion that aberrant regulation of p38 MAPK is involved in ALS. The results presented in this dissertation 1) support a novel prosurvival role for FUS under hyperosmolar stress conditions and, 2) demonstrate that protein misfolding and aberrant kinase activation contribute to ALS pathogenesis by FUS variants.DOI
10.13028/M2QG74Permanent Link to this Item
http://hdl.handle.net/20.500.14038/32062Rights
Copyright is held by the author, with all rights reserved.ae974a485f413a2113503eed53cd6c53
10.13028/M2QG74
Scopus Count
Related items
Showing items related by title, author, creator and subject.
-
Dynamic Regulation at the Neuronal Plasma Membrane: Novel Endocytic Mechanisms Control Anesthetic-Activated Potassium Channels and Amphetamine-Sensitive Dopamine Transporters: A DissertationGabriel, Luke R. (2013-06-13)Endocytic trafficking dynamically regulates neuronal plasma membrane protein presentation and activity, and plays a central role in excitability and plasticity. Over the course of my dissertation research I investigated endocytic mechanisms regulating two neuronal membrane proteins: the anesthetic-activated potassium leak channel, KCNK3, as well as the psychostimulant-sensitive dopamine transporter (DAT). My results indicate that KCNK3 internalizes in response to Protein Kinase C (PKC) activation, using a novel pathway that requires the phosphoserine binding protein, 14-3-3β, and demonstrates for the first time regulated KCNK3 channel trafficking in neurons. Additionally, PKC-mediated KCNK3 trafficking requires a non-canonical endocytic motif, which is shared exclusively between KCNK3 and sodium-dependent neurotransmitter transporters, such as DAT. DAT trafficking studies in intact ex vivo adult striatal slices indicate that DAT endocytic trafficking has both dynamin-dependent and –independent components. Moreover, DAT segregates into two populations at the neuronal plasma membrane: trafficking-competent and -incompetent. Taken together, these results demonstrate that novel, non-classical endocytic mechanisms dynamically control the plasma membrane presentation of these two important neuronal proteins.
-
Selective interaction of JNK protein kinase isoforms with transcription factorsGupta, Shashi; Barrett, Tamera; Whitmarsh, Alan J.; Cavanagh, Julie; Sluss, Hayla Karen; Derijard, Benoit; Davis, Roger J. (1996-06-03)The JNK protein kinase is a member of the MAP kinase group that is activated in response to dual phosphorylation on threonine and tyrosine. Ten JNK isoforms were identified in human brain by molecular cloning. These protein kinases correspond to alternatively spliced isoforms derived from the JNK1, JNK2 and JNK3 genes. The protein kinase activity of these JNK isoforms was measured using the transcription factors ATF2, Elk-1 and members of the Jun family as substrates. Treatment of cells with interleukin-1 (IL-1) caused activation of the JNK isoforms. This activation was blocked by expression of the MAP kinase phosphatase MKP-1. Comparison of the binding activity of the JNK isoforms demonstrated that the JNK proteins differ in their interaction with ATF2, Elk-1 and Jun transcription factors. Individual members of the JNK group may therefore selectively target specific transcription factors in vivo.
-
Role of the Raf/mitogen-activated protein kinase pathway in p21ras desensitizationKlarlund, Jes K.; Cherniack, Andrew D.; McMahon, Martin; Czech, Michael P. (1996-07-12)Desensitization of p21(ras) after stimulation of cells by growth factors and phorbol 12-myristate 13-acetate (PMA) correlates with hyperphosphorylation of the guanine nucleotide exchange factor Son-of-sevenless (Sos) and its dissociation from the adaptor protein Grb2 (Cherniack, A., Klarlund, J. K., Conway, B. R., and Czech, M. P. (1995) J. Biol. Chem. 270, 1485-1488). To test the role of the Raf/mitogen-activated protein (MAP) kinase pathway, we utilized cells expressing a chimera composed of the catalytic domain of p74Raf-1 and the hormone binding domain of the estradiol receptor (DeltaRaf-1:ER). Estradiol markedly stimulated DeltaRaf-1:ER and the downstream MEK and MAP kinases in these cells as well as Sos phosphorylation. However, the dissociation of Grb2 from Sos observed in response to PMA was not apparent upon DeltaRaf-1:ER activation. Furthermore, stimulation of DeltaRaf-1:ER did not impair GTP loading of p21(ras) in response to platelet-derived growth factor or epidermal growth factor. We conclude that activation of the Raf/MAP kinase pathway alone in these cells is insufficient to cause disassembly of Sos from Grb2 or to interrupt the ability of Sos to catalyze activation of p21(ras).