Abstract

Recent evidence indicates that U1-70K and other U1 small nuclear ribonucleoproteins are Sarkosyl-insoluble and associate with Tau neurofibrillary tangles selectively in Alzheimer disease (AD). Currently, the mechanisms underlying the conversion of soluble nuclear U1 small nuclear ribonucleoproteins into insoluble cytoplasmic aggregates remain elusive. Based on the biochemical and subcellular distribution properties of U1-70K in AD, we hypothesized that aggregated U1-70K itself or other biopolymers (e.g. proteins or nucleic acids) interact with and sequester natively folded soluble U1-70K into insoluble aggregates. Here, we demonstrate that total homogenates from AD brain induce soluble U1-70K from control brain or recombinant U1-70K to become Sarkosyl-insoluble. This effect was not dependent on RNA and did not correlate with detergent-insoluble Tau levels as AD homogenates with reduced levels of these components were still capable of inducing U1-70K aggregation. In contrast, proteinase K-treated AD homogenates and Sarkosyl-soluble AD fractions were unable to induce U1-70K aggregation, indicating that aggregated proteins in AD brain are responsible for inducing soluble U1-70K aggregation. It was determined that the C terminus of U1-70K, which harbors two disordered low complexity (LC) domains, is necessary for U1-70K aggregation. Moreover, both LC1 and LC2 domains were sufficient for aggregation. Finally, protein cross-linking and mass spectrometry studies demonstrated that a U1-70K fragment harboring the LC1 domain directly interacts with aggregated U1-70K in AD brain. Our results support a hypothesis that aberrant forms of U1-70K in AD can directly sequester soluble forms of U1-70K into insoluble aggregates.

Highlights

  • The mechanisms underlying U1-70K aggregation in Alzheimer disease (AD) are unknown

  • AD brain homogenates can induce the aggregation of soluble U1-70K, and disordered low complexity domains are necessary for U1-70K aggregation

  • Induced Aggregation of recombinant U1-70K (rU1-70K) by AD Homogenate Is Protein-dependent—We previously reported that U1 small nuclear RNA (snRNA) co-aggregates with U1-70K in AD brain [8, 35] suggesting that RNA may play an important role in rU1-70K aggregation in AD homogenates

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Summary

Background

The mechanisms underlying U1-70K aggregation in AD are unknown. Results: AD brain homogenates can induce the aggregation of soluble U1-70K, and disordered low complexity domains are necessary for U1-70K aggregation. Most detectable U1-70K was found in the Sarkosyl-insoluble fraction of AD brain tissue following biochemical fractionation, despite the fact that most neurons and glia harbor nonfilamentous nuclear U1-70K Based on these observations, we hypothesized that following tissue homogenization, aggregated U1-70K itself or other biopolymers (i.e. proteins or nucleic acids) sequester natively folded soluble U1-70K into detergent-insoluble aggregates. Using protein cross-linking and mass spectrometry, we show that a rU1-70K fragment harboring the LC1 domain directly interacts with aggregated U1-70K in AD brain These results support the hypothesis that aggregated U1-70K in AD brain can interact with and sequester normal soluble forms of U1-70K through the intrinsically disordered LC1 domain, providing important insight into the mechanisms underlying U1-70K protein aggregation events in AD

EXPERIMENTAL PROCEDURES
RESULTS
B Total Homogenate kDa HEK
DISCUSSION
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