Christine P. Donahue

Stabilization of the Tau Exon 10 Stem Loop Alters Pre-mRNA Splicing

Neurofibrillary tangles containing filaments of the microtubule-associated protein tau are found in a variety of neurodegenerative diseases. Mutations in the tau gene itself cause frontotemporal dementia with parkinsonism, demonstrating the critical role of tau in pathogenesis. Many of these mutations in tau are silent, are found at the 5′-splice site of exon 10, and lead to increased inclusion of exon 10. These silent mutations are predicted to destabilize a stem loop structure at the exon 10 5′-splice site; however, the existence of this stem loop under physiological conditions and its role in splice regulation are controversial. Here we show that base changes that stabilize this stem loop in vitro substantially decrease exon 10 inclusion in a wild type tau minigene and rescue the increase in exon 10 splicing caused by a dementia-causing point mutation. Moreover, we probed the intracellular structure of the tau stem loop with antisense RNA and demonstrate that the stability of the stem loop dictates antisense effectiveness. Together these results validate the stem loop as a bona fide structure regulating tau exon 10 splicing.

Structural Basis for Stabilization of the Tau Pre-mRNA Splicing Regulatory Element by Novantrone (Mitoxantrone)

Some familial neurodegenerative diseases are associated with mutations that destabilize a putative stem-loop structure within an intronic region of the tau pre-messenger RNA (mRNA) and alter the production of tau protein isoforms by alternative splicing. Because stabilization of the stem loop reverses the splicing pattern associated with neurodegeneration, small molecules that stabilize this stem loop would provide new ways to dissect the mechanism of neurodegeneration and treat tauopathies. The anticancer drug mitoxantrone was recently identified in a high throughput screen to stabilize the tau pre-mRNA stem loop. Here we report the solution structure of the tau mRNA-mitoxantrone complex, validated by the structure-activity relationship of existing mitoxantrone analogs. The structure describes the molecular basis for their interaction with RNA and provides a rational basis to optimize the activity of this new class of RNA-binding molecules.

Distribution pattern of Notch3 mutations suggests a gain-of-function mechanism for CADASIL.

Mutations in Notch3 cause the syndrome CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). The mechanism by which these mutations result in a CADASIL phenotype has been widely speculated upon. A first step toward understanding a disease mechanism is to learn whether the mutations result in the loss of Notch3 function, in particular, its role in signaling or in the gain of a novel function. Notch3 genomic sequences were analyzed for sites of conservation across species. We present here a bioinformatic analysis of the Notch paralogs and orthologs that suggest that CADASIL mutations result in a gain of function. This finding diminishes the likelihood that a Notch3 signaling deficit is responsible for the phenotype and increases the likelihood that CADASIL joins the growing list of neurological diseases with protein deposits due to misfolding and aggregation.

Mitoxantrone Analogues as Ligands for a Stem–Loop Structure of Tau Pre-mRNA

A series of mitoxantrone (MTX) analogues have been designed, synthesized, and evaluated for binding to and stabilizing a stem-loop structure that serves as a splicing regulatory element in the pre-mRNA of tau, which is involved in Alzheimers and other neurodegenerative diseases. Several compounds showed significantly improved binding activity relative to the original screening hit mitoxantrone. These findings establish essential structure-activity relationships to further optimize the activity of this promising class of compounds.

Identification of Tau Stem Loop RNA Stabilizers

Alternative splicing of tau exon 10 produces tau isoforms with either 3 (3R) or 4 (4R) repeated microtubule-binding domains. Increased ratios of 4R to 3R tau expression, above the physiological 1:1, leads to neurofibrillary tangles and causes neurodegenerative disease. An RNA stem loop structure plays a significant role in determining the ratio, with decreasing stability correlating with an increase in 4R tau mRNA expression. Recent studies have shown that aminoglycosides are able to bind and stabilize the tau stem loop in vitro, suggesting that other druglike small molecules could be identified and that such molecules might lead to decreased exon 10 splicing in vivo. The authors have developed a fluorescent high-throughput fluorescent binding assay and screened a library of ∼110,000 compounds to identify candidate drugs that will bind the tau stem loop in vitro. In addition, they have developed a fluorescent-based RNA probe to assay the stabilizing effects of candiate drugs on the tau stem loop RNA. These assays should be applicable to the general problem of identifying small molecules that interact with mRNA secondary structures. (Journal of Biomolecular Screening 2007:789-799)