Neuritic plaques surround amyloid deposits and are made up of swollen, dystrophic neural processes that contain aggregated phosphorylated tau. Neurofibrillary tangles are made up of phosphorylated tau that has abnormally been aggregated in Alzheimer’s disease. Tau normally is microtubule adaptor protein involved in the highways of neurons that allow for trafficking.Significant preliminary data (published and unpublished), as well as reports from other groups, indicate that tau aggregate binding to heparan sulfate proteoglycans (HSPGs) on the cell surface mediates cell uptake and intracellular seeding by pathogenic aggregates. However, the precise mechanisms that could mediate progression or propagation of tau pathology in the brain have not been explicitly tested. Nor do we know the cellular mechanisms that facilitate tau uptake into the cell after initial binding to the cell surface. This proposal seeks to answer these questions.
It is unknown why neurodegenerative diseases such as Alzheimer’s disease are progressive. It is clear that neurodegeneration in AD is caused by accumulation of the protein tau in highly ordered assemblies, or aggregates, inside neurons. Our work and that of many labs around the world suggests that progression of AD is due to these aggregates escaping one cell and moving to others, where they are taken up. Once inside the second cell, the aggregate acts like a pathological crystal, or “seed,” interacting with normal protein and converting it to an abnormal, aggregated form. This idea can explain why neurodegeneration starts in a defined area of the brain and spreads inexorably through it. In 2013, we originally determined the mechanism by which tau binds the cell surface to enter and corrupt normal protein on the cell interior. This involves tau aggregate binding to specific cell surface “receptor” proteins, which have to go through a defined cellular processing pathway to become functional. Multiple processing enzymes modify the receptors in a stepwise fashion. Once tau aggregates bind these surface receptors, they trigger their own uptake. This process is not well understood, and appears to involve multiple signaling pathways. Each step in this pathway might represent a “pressure point” to stop the progression of AD. This proposal extends our prior work in this regard. Whereas our first study tested 23 genes in a defined pathway important for uptake of tau, we now have individually tested virtually every gene in the human genome. We have identified approximately 900 new candidate genes that must be validated in further tests, but promise to improve our therapeutic options for AD. We also will use mouse models to definitively test the candidate gene that we identified in the prior years.
Trans-cellular propagation of tau pathology has been implicated in the progression of Alzheimer’s disease and other tauopathies. We previously have determined the mechanism by which tau aggregates bind the cell surface to trigger uptake via macropinocytosis. This involves direct binding of tau to heparan sulfate proteoglycans (HSPGs) on the cell surface. HSPGs are glycolipid-anchored and transmembrane core proteins that are extensively glycosylated and sulfated by a defined set of cellular enzymes. In prior published and unpublished work, we have determined that disruption of EXT1, a gene that plays a proximal role in the extension of sugar chains on HSPG core proteins, strongly inhibits tau aggregate uptake, seeding and transcellular propagation in vitro and in vivo. We hypothesize that individual HSPG synthetic genes required for tau uptake will represent viable drug targets.
Aim 1: We will individually test each of 24 genes associated with HSPG synthesis using Cas9/CRISPR-mediated gene knockout in HEK293T cells. We will confirm hits in HEK293T cells and primary cultured neurons.
Aim 2: We will test candidate genes in an in vivo model of spreading tau pathology by AAV-shRNA knockdown. If we are successful, a limited number of candidates will represent important new drug targets to block Alzheimer’s disease progression.