Tauopathies are a class of neurodegenerative disorders characterized by the pathological accumulation of microtubule-associated protein tau in the human brain. Tau aggregate accumulation is observed in many diseases, such as Alzheimer's disease and Frontotemporal Lobar Degeneration. In the early stage of disease, the tau pathology is often restricted to discrete and stereotyped regions of the brain. With disease progression, however, the pathological changes typically spread through the nervous system according to specific anatomical patterns. Emerging evidence demonstrates that tau aggregates are capable of transcellular spread to co-cultured cells, consistent with the notion that aggregated tau can serve as an agent of disease propagation. However, the mechanisms by which tau aggregates enter cells and subsequently access the cytoplasm to induce misfolding of native tau protein remain unknown. The experiments outlined in this proposal are designed to add to the limited understanding of these processes and to inform therapeutic advances that may reduce the burden of neurodegenerative disease.
In Aim 1, the role of heparan sulfate proteoglycans (HSPGs) as a cellular mediator of tau aggregate binding and uptake will be tested. Pharmacological HSPG inhibitors such as heparin, heparinase III, sodium chlorate, soluble glycans and heparan mimetics will be evaluated for reduced cellular binding and internalization of tau aggregates. Genetic approaches exploiting mutant cell lines deficient in glycosaminoglycan synthesis as well as shRNA knockdown technology will then be employed to further determine the role of HSPGs on tau aggregate internalization. Quantification of MTBR aggregate binding and uptake will be assayed using flow cytometry and automated analysis microscopy.
Aim 2 consists of testing if exogenously derived tau aggregates, once internalized via macropinocytosis, can escape the vesicular lumen to contact the cytoplasm. Neuroblastoma cell lines will be exposed to recombinant tau fibrils and subsequently fractionated via ultracentrifugation. The resulting fractions will be probed for the presence of tau aggregates using biochemical approaches. These studies will help delineate the cellular environment in which tau aggregates convert natively folded tau into an aggregated, fibrillar form.
Recent evidence suggests that transfer of misfolded tau protein from cell to cell may underlie the progression of many neurodegenerative diseases, such as Alzheimer's disease. A clear understanding of how the misfolded tau enters cells and subsequently corrupts the normal tau protein does not yet exist. This research project will allow us to mechanistically understand these processes so that disease progression may be targeted by new therapies for neurodegeneration.
|Holmes, Brandon B; Diamond, Marc I (2014) Prion-like properties of Tau protein: the importance of extracellular Tau as a therapeutic target. J Biol Chem 289:19855-61|
|Holmes, Brandon B; Furman, Jennifer L; Mahan, Thomas E et al. (2014) Proteopathic tau seeding predicts tauopathy in vivo. Proc Natl Acad Sci U S A 111:E4376-85|
|Zheng, Zhiqiang; Li, Aimin; Holmes, Brandon B et al. (2013) An N-terminal nuclear export signal regulates trafficking and aggregation of Huntingtin (Htt) protein exon 1. J Biol Chem 288:6063-71|
|Holmes, Brandon B; DeVos, Sarah L; Kfoury, Najla et al. (2013) Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds. Proc Natl Acad Sci U S A 110:E3138-47|