Carnosol, a diterpene present in rosemary, increases ELP1 levels in familial dysautonomia patient-derived cells and healthy adults: a possible therapy for FD.

Recent research on familial dysautonomia (FD) has focused on the development of therapeutics that facilitate the production of the correctly spliced, exon 20-containing, transcript in cells and individuals bearing the splice-altering, FD-causing mutation in the elongator acetyltransferase complex subunit I (ELP1) gene. We report here the ability of carnosol, a diterpene present in plant species of the Lamiaceae family, including rosemary, to enhance the cellular presence of the correctly spliced ELP1 transcript in FD patient-derived fibroblasts by upregulating transcription of the ELP1 gene and correcting the aberrant splicing of the ELP1 transcript. Carnosol treatment also elevates the level of the RNA binding motif protein 24 (RBM24) and RNA binding motif protein 38 (RBM38) proteins, two multifunctional RNA-binding proteins. Transfection-mediated expression of either of these RNA binding motif (RBMs) facilitates the inclusion of exon 20 sequence into the transcript generated from a minigene-bearing ELP1 genomic sequence containing the FD-causing mutation. Suppression of the carnosol-mediated induction of either of these RBMs, using targeting siRNAs, limited the carnosol-mediated inclusion of the ELP1 exon 20 sequence. Carnosol treatment of FD patient peripheral blood mononuclear cells facilitates the inclusion of exon 20 into the ELP1 transcript. The increased levels of the ELP1 and RBM38 transcripts and the alternative splicing of the sirtuin 2 (SIRT2) transcript, a sentinel for exon 20 inclusion in the FD-derived ELP1 transcript, are observed in RNA isolated from whole blood of healthy adults following the ingestion of carnosol-containing rosemary extract. These findings and the excellent safety profile of rosemary together justify an expedited clinical study of the impact of carnosol on the FD patient population.

Pollen dispersal patterns differ among sites for a wind-pollinated species and an insect-pollinated species.

PREMISE: Pollen dispersal, the main component of overall plant gene flow, generally decreases with increasing distance from the pollen source, but the pattern of this relationship may differ among sites. Although site-based differences in pollen dispersal may lead to over- or underestimation of gene flow, no studies have investigated pollen dispersal patterns among differing urban site types, despite the incongruent range of habitats in urban areas. METHODS: We used paternity assignment to assess pollen dispersal patterns in a wind-pollinated species (waterhemp; Amaranthus tuberculatus) and in an insect-pollinated species (tomato; Solanum lycopersicum) in experimental arrays at four disparate sites (two roof-level sites, two ground-level sites) in the New York (New York, USA) metropolitan area. RESULTS: The number of seeds or fruits, a proxy for the number of flowers pollinated, decreased with increasing distance from the pollen donors at all sites for both species. However, the mean number of Amaranthus tuberculatusseeds produced at a given distance differed two-fold among sites, while the slope of the relationship between Solanum lycopersicumfruit production and distance differed by a factor of four among sites. CONCLUSIONS: Pollen dispersal patterns may differ substantially among sites, both in the amount of pollen dispersed at a given distance and in the proportional decrease in pollen dispersal with increasing distance, and these effects may act independently. Accordingly, the capacity of plant species to adapt to climate change and other selection pressures may be different from predictions based on pollen dispersal patterns at a single location.

Cardiac glycosides correct aberrant splicing of IKBKAP-encoded mRNA in familial dysautonomia derived cells by suppressing expression of SRSF3.

The ability to modulate the production of the wild-type transcript in cells bearing the splice-altering familial dysautonomia (FD) causing mutation in the IKBKAP gene prompted a study of the impact of a panel of pharmaceuticals on the splicing of this transcript, which revealed the ability of the cardiac glycoside digoxin to increase the production of the wild-type, exon-20-containing, IKBKAP-encoded transcript and the full-length IκB-kinase-complex-associated protein in FD-derived cells. Characterization of the cis elements and trans factors involved in the digoxin-mediated effect on splicing reveals that this response is dependent on an SRSF3 binding site(s) located in the intron 5' of the alternatively spliced exon and that digoxin mediates its effect by suppressing the level of the SRSF3 protein. Characterization of the digoxin-mediated effect on the RNA splicing process was facilitated by the identification of several RNA splicing events in which digoxin treatment mediates the enhanced inclusion of exonic sequence. Moreover, we demonstrate the ability of digoxin to impact the splicing process in neuronal cells, a cell type profoundly impacted by FD. This study represents the first demonstration that digoxin possesses splice-altering capabilities that are capable of reversing the impact of the FD-causing mutation. These findings support the clinical evaluation of the impact of digoxin on the FD patient population.

Nutraceutical-mediated restoration of wild-type levels of IKBKAP-encoded IKAP protein in familial dysautonomia-derived cells.

SCOPE: The reported ability to modulate the production of the wild-type transcript in cells bearing the splice-altering familial dysautonomia (FD)-causing mutation in the IKBKAP gene prompted an evaluation of the impact of commonly consumed nutraceuticals on the splicing of this transcript. METHODS AND RESULTS: Screening efforts revealed the ability of the isoflavones, genistein, and daidzein, to impact splicing and increase the production of the wild-type, exon-20-containing, transcript, and the full-length IKBKAP-encoded IΚB kinase complex associated protein(IKAP) in FD-derived cells. Genistein was also found to impact splicing in neuronal cells, a cell type profoundly impacted by FD. The simultaneous exposure of FD-derived cells to genistein and epigallocatechin gallate (EGCG) resulted in the almost exclusive production of the exon-20-containing transcript and the production of wild-type amounts of IKAP protein. CONCLUSION: This study represents the first demonstration that the isoflavones, genistein and daidzein, possess splice-altering capabilities and that simultaneous treatment with genistein and EGCG reverses the splice-altering impact of the FD-causing mutation. These findings support the clinical evaluation of the therapeutic impact of the combined administration of these two commonly consumed nutraceuticals on this patient population and suggest a broader evaluation of the impact of these nutraceuticals on the in vivo RNA splicing process.

Tocotrienols induce IKBKAP expression: a possible therapy for familial dysautonomia.

Familial dysautonomia (FD), a neurodegenerative genetic disorder primarily affecting individuals of Ashkenazi Jewish descent, is caused by mutations in the IKBKAP gene which encodes the IkappaB kinase complex-associated protein (IKAP). The more common or major mutation causes aberrant splicing, resulting in a truncated form of IKAP. Tissues from individuals homozygous for the major mutation contain both mutant and wild-type IKAP transcripts. The apparent leaky nature of this mutation prompted a search for agents capable of elevating the level of expression of the wild-type IKAP transcript. We report the ability of tocotrienols, members of the vitamin E family, to increase transcription of IKAP mRNA in FD-derived cells, with corresponding increases in the correctly spliced transcript and normal protein. These findings suggest that in vivo supplementation with tocotrienols may elevate IKBKAP gene expression and in turn increase the amount of functional IKAP protein produced in FD patients.