Abstract
Identification of fluorescent dyes that label the filamentous protein aggregates characteristic of neurodegenerative disease, such as β-amyloid and tau in Alzheimer's disease, in a live cell culture system has previously been a major hurdle. Here we show that pentameric formyl thiophene acetic acid (pFTAA) fulfills this function in living neurons cultured from adult P301S tau transgenic mice. Injection of pFTAA into 5-month-old P301S tau mice detected cortical and DRG neurons immunoreactive for AT100, an antibody that identifies solely filamentous tau, or MC1, an antibody that identifies a conformational change in tau that is commensurate with neurofibrillary tangle formation in Alzheimer's disease brains. In fixed cultures of dorsal root ganglion (DRG) neurons, pFTAA binding, which also identified AT100 or MC1+ve neurons, followed a single, saturable binding curve with a half saturation constant of 0.14 μM, the first reported measurement of a binding affinity of a beta-sheet reactive dye to primary neurons harboring filamentous tau. Treatment with formic acid, which solubilizes filamentous tau, extracted pFTAA, and prevented the re-binding of pFTAA and MC1 without perturbing expression of soluble tau, detected using an anti-human tau (HT7) antibody. In live cultures, pFTAA only identified DRG neurons that, after fixation, were AT100/MC1+ve, confirming that these forms of tau pre-exist in live neurons. The utility of pFTAA to discriminate between living neurons containing filamentous tau from other neurons is demonstrated by showing that more pFTAA+ve neurons die than pFTAA-ve neurons over 25 days. Since pFTAA identifies fibrillar tau and other misfolded proteins in living neurons in culture and in animal models of several neurodegenerative diseases, as well as in human brains, it will have considerable application in sorting out disease mechanisms and in identifying disease-modifying drugs that will ultimately help establish the mechanisms of neurodegeneration in human neurodegenerative diseases.
Highlights
Tau is a soluble microtubule-associated protein with dynamic phospho-epitopes that is expressed throughout the CNS and PNS (Cleveland et al, 1977; Binder et al, 1985)
We first examined whether dorsal root ganglion (DRG) neurons in P301S tau transgenic mice—like CNS neurons (Klingstedt et al, 2013)—were labeled after injection of pentameric formyl thiophene acetic acid (pFTAA) in vivo, and whether pFTAA-labeled neurons co-label with antibodies that detect insoluble tau aggregates and fibrils
A subset of neurons in the DRG were labeled with pFTAA and, as in the brain, this labeling coincided with immunolabeling with HT7 or the MC1 antibody (Figure 1D), which detects a conformational change in tau that is associated with neurofibrillary tangles in human Alzheimer’s disease (AD) brains (Weaver et al, 2000) and is only evident in DRG neurons from P301S tau mice when they are AT100+ve (Mellone et al, 2013)
Summary
Tau is a soluble microtubule-associated protein with dynamic phospho-epitopes that is expressed throughout the CNS and PNS (Cleveland et al, 1977; Binder et al, 1985). The driving force behind tau aggregation is not known but familial mutations in the MAPT gene such as P301S can increase aggregation propensity by reducing tau binding to microtubules, and possibly by introducing a new phosphorylation site (Hong et al, 1998). Individuals affected by this mutation have an early to midlife age of onset and an aggressive disease progression (Bugiani et al, 1999; Sperfeld et al, 1999; Yasuda et al, 2000; Lossos et al, 2003)
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