2012 — 2014 |
Kukar, Thomas L |
R00Activity Code Description: To support the second phase of a Career/Research Transition award program that provides 1 -3 years of independent research support (R00) contingent on securing an independent research position. Award recipients will be expected to compete successfully for independent R01 support from the NIH during the R00 research transition award period. |
Selective Modulation of Gamma-Secretase Processing Through Substrate Binding
Numerous lines of evidence support the hypothesis that targeting A[342 is an ideal therapeutic strategy to prevent and/or treat Alzheimer's disease (AD), the major cause of dementia among the elderly. Chemicals called y-secretase modulators (GSMs), are being developed as AD therapeutics because they are able to selectively decrease Ap42. The first GSMs to be discovered were non-steroidal antiinflammatory drugs (NSAIDs), which lower Ap42 without inhibition of APP processing. As a whole GSMs minimally alter total Abeta production and instead shift where gamma-secretase cleaves Abeta. The mechanism of GSMs is still unknown although different explanations for their activity have been proposed including: 1) allosteric binding to y-secretase 2) inhibition of the Rho-ROCK signaling pathway 3) conformational changes in presenilin or 4) decreased dimerization of APP. We have recently discovered using novel GSM photoaffinity probes that these drugs do not label the y-secretase enzyme but instead modulate cleavage by binding to the substrate, APP. We hypothesize that binding of APP by GSMs shifts the position of APP-CTF in the membrane resulting in altered gamma-secretase cleavage. This hypothesis will be tested through the following specific aims: 1) investigate how substrate targeting by GSMs produces a shift in the cleavage pattern of Ap using a combination of molecular biology and protein biochemistry 2) determine the specificity of GSMs for affecting APP proteolysis by y-secretase in comparison to other substrates and 3) incorporate unnatural amino acids to study proteolysis of APPCTF by y-secretase. These studies will provide additional insight into how NSAIDs and other GSMs shift A(3 cleavage and how they exert their protective effects in vivo. This work will also guide future efforts to design more potent GSMs which will be useful as chemical probes for understanding the biology of ysecretase and as potential therapeutics for Alzheimer's disease.
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0.958 |
2015 — 2019 |
Kukar, Thomas L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Defining the Role of Fus Phosphorylation in Neurodegeneration
? DESCRIPTION (provided by applicant): Fused in Sarcoma (FUS) is a ubiquitous multifunctional RNA-binding protein (RBP) located in the nucleus. The abnormal and pathogenic aggregation of FUS in the cytoplasm of neurons defines subtypes of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), termed FTLD-FUS or ALS-FUS. ALS- FUS cases are caused by mutations in the FUS gene. In these cases, the accumulation of FUS is thought to be driven by long-term increases in cytoplasmic FUS caused by mutations that decrease nuclear import through disruption of a conserved nuclear localization signal (NLS). However, it is unknown why FUS accumulates in FTLD-FUS cases. Moreover, it is unclear what causes cytoplasmic FUS in ALS to aggregate and become insoluble. We have discovered a novel mechanism that may explain both phenomena. We find that FUS can be phosphorylated and this event causes the cytoplasmic redistribution of FUS in multiple cells including human astrocytes and neurons. In particular, we find the DNA-damage, caused by chemical toxins, is a potent inducer of FUS phosphorylation. Furthermore, DNA-damage also causes cytoplasmic accumulation of EWS, TAF15, and TRN, which mimics a unique aspect of FTLD-FUS pathology. Preliminary evidence suggests that phosphorylation of FUS leads to increased amounts of FUS in the cytoplasm by disrupting a novel nuclear localization signal in the N-terminus. Consistent with this mechanism, we find that a FUS phospho-mimetic accumulates in the cytoplasm and forms aggregates. These aggregates co-label with markers of stress granules (SGs), RNA/protein granules that have been linked to the formation of inclusions in ALS-FUS and other forms of neurodegeneration. We theorize that cytoplasmic phosphorylated FUS can cause disease through a toxic gain of function by inducing granules that sequester RNA and RNA-binding proteins, impeding normal function. Importantly, we find that phosphorylated FUS occurs in the biochemically insoluble fraction of brains of human and mice with FUS inclusions. Finally, we find a large increase in ?-H2AX, a marker of DNA damage, in FTLD-FUS brains, supporting the idea that DNA damage and phosphorylation of FUS is a key component of disease pathogenesis. In this proposal we focus on the hypothesis that double-strand DNA damage induces phosphorylation of FUS by the DNA-dependent protein kinase (DNA-PK) causing FUS accumulation in the cytoplasm by impairing nuclear import. We will test this hypothesis by 1) Defining the kinase and types of DNA damage responsible for FUS phosphorylation, 2) Determining how phosphorylation of FUS causes cytoplasmic translocation and affects function, and 3) Determining the role of FUS phosphorylation in neurodegeneration. This research will provide insight into how FUS accumulation causes neurodegeneration and inform drug development strategies for ALS and FTLD. Our data suggest that methods to prevent FUS from forming pathogenic RNA/stress granules, potentially through modulation of the DNA- repair pathway or DNA-PK, may yield treatments for these devastating neurodegenerative diseases.
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0.958 |
2018 — 2021 |
Kukar, Thomas L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms of Progranulin in Neurodegeneration
Project Summary Frontotemporal degeneration (FTD) and Alzheimer?s disease (AD) are two of the most common causes of dementia, share overlapping pathologies, are huge health burdens, and are incurable. This proposal focuses on elucidating how loss of progranulin (PGRN), and its mature products the granulins, drive neurodegeneration and lysosomal dysfunction associated with FTD and AD. PGRN is a secreted protein composed of 7.5 tandem domains that are cleaved into 6kDa granulin proteins (GRNs), through a poorly defined pathway. Genetic variants and loss-of-function mutations in the progranulin gene (Grn), reduce the production of the progranulin (PGRN) protein and increase the risk of AD and cause FTD, respectively. Converging evidence suggest that decreased levels of PGRN/granulins induce lysosomal dysfunction leading to neuroinflammation and degeneration through an unknown mechanism. Based on our published work and new data, we propose that granulins are the functional unit of PGRN and are produced in the endo-lysosomal pathway. We find PGRN is trafficked to the lysosome and processed into stable granulins in multiple tissues and cells. Clinically, PGRN and granulins are equally decreased in iPSC-derived neurons and brain tissue from FTD-GRN carriers. Further, expression of the FTD-risk-factor TMEM106B reduces granulins. Finally, extracellular granulin can rescue lysosomal defects in Grn KO mouse fibroblasts, providing strong evidence that granulins facilitate lysosome function. Our findings fit into the larger narrative that lysosome-autophagy dysfunction is a critical pathogenic mechanism in FTD and AD. Our preliminary data lead us to propose the hypothesis that PGRN is trafficked to the lysosome and processed into mature, functional granulins that mediate lysosomal homeostasis and neuroprotection. Successful completion of the following specific aims will advance the neurodegeneration field by providing mechanism-based rationale for testing granulins as a novel therapy for FTD and AD. We will 1) delineate the molecular pathways that traffic PGRN to the lysosome, 2) determine the molecular mechanisms of granulin production and function in the lysosome, and 3) determine the in vivo role of PGRN and granulins in lysosome dysfunction and neurodegeneration. Completion of the proposed studies will enable us to critically evaluate the paradigm-shifting hypothesis that granulins are lysosomal, functional, and neuroprotective. In doing so, we will uncover why decreased levels of PGRN lead to FTD, AD, or NCL and a new approach to treat diseases caused by decreased PGRN.
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0.958 |