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Tocotrienols induce IKBKAP expression: A possible therapy for familial dysautonomia
Abstract
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.
... The incomplete penetrance of the IVS20 + 6 T> C mutation that allows for the production of some fulllength transcript in cell lines and tissues from FD patients (5), prompted the screening of chemical libraries for compounds capable of increasing the production of the correctly spliced ELP1 transcript, either by increasing the expression of the gene or by correcting the mis-splicing of the transcript. Chemical compounds with therapeutic potential identified to date include: tocotrienols (5), epigallocatechin gallate (6), kinetin (7), phosphatidylserine (8), genistein (9), cardiac glycosides (10) and rectifier of aberrant splicing (RECTAS) (11). ...
... The incomplete penetrance of the IVS20 + 6 T> C mutation that allows for the production of some fulllength transcript in cell lines and tissues from FD patients (5), prompted the screening of chemical libraries for compounds capable of increasing the production of the correctly spliced ELP1 transcript, either by increasing the expression of the gene or by correcting the mis-splicing of the transcript. Chemical compounds with therapeutic potential identified to date include: tocotrienols (5), epigallocatechin gallate (6), kinetin (7), phosphatidylserine (8), genistein (9), cardiac glycosides (10) and rectifier of aberrant splicing (RECTAS) (11). Clinical studies evaluating the impact of tocotrienol ingestion on FD patients have revealed its ability to increase production of the exon 20-containing ELP1 transcript in peripheral blood mononuclear cells (PBMCs), improve cardiac response to exercise, reduce the number of hypertensive/dysautonomic crises, decrease the number of pneumonias and increase stability in walking (12)(13)(14)(15). ...
... The previously noted ability of several edible plantderived molecules to increase the production of the correctly spliced, exon 20-containing transcript from the IVS20 + 6 T> C-bearing ELP1 allele, which in a homozygous state causes FD, by either increasing the rate of transcription of this gene (5) or by altering the splicing process (6,9), prompted the continued search for plant-derived compounds capable of increasing the production of the exon 20-containing transcript from the mutant allele. For this analysis, RNA isolated from two FD patient-derived fibroblast cell lines treated with approximately 1100 plant extracts and purified natural compounds for 48 h was subjected to RT-PCR analysis using primers located 5 and 3 of exon 20 of the ELP1 transcript and which amplify both the exon 20-containing and exon 20-lacking transcripts. ...
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 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 RBM24 and RBM38 proteins., two multifunctional RNA binding proteins. Transfection-mediated expression of either of these 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 PBMCs facilitates the inclusion of exon 20 into the ELP1 transcript. Increased levels of the ELP1 and RBM38 transcripts and the alternative splicing of the 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.
... HSAN-III is the first HSAN for which promising interventions are on the horizon. It is speculated that therapeutic agents could be developed to raise the wild-type protein expression of the mutated gene, thus slowing progression and improving symptoms [90]. Tocotrienol, epigallocatechin gallate, phosphatidylserine, and kinetin have been shown to modify genetic expression in vitro [80,[90][91][92][93]. ...
... It is speculated that therapeutic agents could be developed to raise the wild-type protein expression of the mutated gene, thus slowing progression and improving symptoms [90]. Tocotrienol, epigallocatechin gallate, phosphatidylserine, and kinetin have been shown to modify genetic expression in vitro [80,[90][91][92][93]. The initial tocotrienol study reported increased IKBKAP/ ELP1 expression [90]; however, this was not replicated in subsequent studies [91,94]. ...
... Tocotrienol, epigallocatechin gallate, phosphatidylserine, and kinetin have been shown to modify genetic expression in vitro [80,[90][91][92][93]. The initial tocotrienol study reported increased IKBKAP/ ELP1 expression [90]; however, this was not replicated in subsequent studies [91,94]. Phosphatidylserine is an overthe-counter compound that was also shown to increase IKBKAP/ELP1 expression. ...
Purpose of Review
Hereditary sensory and autonomic neuropathies (HSANs) are a clinically heterogeneous group of inherited neuropathies featuring prominent sensory and autonomic involvement. Classification of HSAN is based on mode of inheritance, genetic mutation, and phenotype. In this review, we discuss the recent additions to this classification and the important updates on management with a special focus on the recently investigated disease-modifying agents.
Recent Findings
In this past decade, three more HSAN types were added to the classification creating even more diversity in the genotype–phenotype. Clinical trials are underway for disease-modifying and symptomatic therapeutics, targeting mainly HSAN type III.
Summary
Obtaining genetic testing leads to accurate diagnosis and guides focused management in the setting of such a diverse and continuously growing phenotype. It also increases the wealth of knowledge on HSAN pathophysiologies which paves the way toward development of targeted genetic treatments in the era of precision medicine.
... Kinetin, a plant growth regulator, was clinically tested on healthy clinically asymptomatic individual heterozygous for the FD mutation and was demonstrated to increase IKBKAP mRNA level in leucocytes (Gold-von Simson et al. 2009). In another study, by Anderson SL et al. (Anderson et al. 2003b) cultured FD cells were treated with either 2.5 or 25 μg/ml of α; β; γ, and δ-tocotrienol, (a member of the vitamin E family with a strong antioxidant effect) and resulted in the increased transcription of IKBKAP gene and consequently produced more IKAP protein. This group also reported in open-label non-comparative clinical study a reduction in the frequency of crises in FD patients taking 100 mg of tocotrienol per day for 3-4 months (Rubin et al. 2008). ...
... It was shown, that tocotrienol could increase IKBKAP WT and MT transcript levels with a corresponding substantive elevation in IKAP protein in FD fibroblasts in vitro (Anderson et al. 2003b). Tocotrienols, members of the Vitamin E family, induce expression of the IKBKAP-encoded transcript in the FD-derived fibroblasts cell culture (Anderson et al. 2003b). ...
... It was shown, that tocotrienol could increase IKBKAP WT and MT transcript levels with a corresponding substantive elevation in IKAP protein in FD fibroblasts in vitro (Anderson et al. 2003b). Tocotrienols, members of the Vitamin E family, induce expression of the IKBKAP-encoded transcript in the FD-derived fibroblasts cell culture (Anderson et al. 2003b). In addition, Anderson et al. has reported a reduction in the frequency of crises in 19 out of 23 FD patients followed by 100 mg daily tocotrienol treatment for the period of 3-4 months (Rubin et al. 2008) and that administration of tocotrienol and green tea to one patient was reported to have a positive effect on postoperative clinical conditions compared to previous surgical experiences (Cook-Sather et al. 2012). ...
Familial dysautonomia (FD) is an autosomal recessive congenital neuropathy, primarily presented in Ashkenazi Jews. The most common mutation in FD patients results from a single base pair substitution of an intronic splice site in the IKBKAP gene which disrupts normal mRNA splicing and leads to tissue-specific reduction of IKBKAP protein (IKAP). To date, treatment of FD patients remains preventative, symptomatic and supportive. Based on previous in vitro evidence that tocotrienols, members of the vitamin E family, upregulate transcription of the IKBKAP gene, we aimed to investigate whether a similar effects was observed in vivo. In the current study, we assessed the effects of tocotrienol treatment on FD patients' symptoms and IKBKAP expression in white blood cells. The initial daily doses of 50 or 100 mg tocotrienol, doubled after 3 months, was administered to 32 FD patients. Twenty-eight FD patients completed the 6-month study. The first 3 months of tocotrienol treatment was associated with a significant increase in IKBKAP expression level in FD patients' blood. Despite doubling the dose after the initial 3 months of treatment, IKBKAP expression level returned to baseline by the end of the 6-month treatment. Clinical improvement was noted in the reported clinical questionnaire (with regard to dizziness, bloching, sweating, number of pneumonia, cough episodes, and walking stability), however, no significant effect was observed in any clinical measurements (weight, height, oxygen saturation, blood pressure, tear production, histamine test, vibration threshold test, nerve conduction, and heart rate variability) following Tocotrienol treatment. In conclusion, tocotrienol treatment appears significantly beneficial by clinical evaluation for some FD patients in a few clinical parameters; however it was not significant by clinical measurements. This open-label study shows the complexity of effect of tocotrienol treatment on FD patients' clinical outcomes and on IKBKAP expression level compared to in vitro results. A longitudinal study with an increased sample size is required in the future to better understand tocotrienol affect on FD patients.
... The observed production of a small amount of the exon-20-containing IKBKAP transcript in cell lines and tissues from individuals with FD [16] prompted a search for therapeutic modalities capable of facilitating the production of the exon-20-containing IKBKAP transcript in those affected with this disorder. In 2003, we reported that tocotrienols, members of the vitamin E family [16], and epigallocatechin gallate (EGCG), a flavonoid present in plants, elevate the production of the exon-20-containing transcript in FD-derived cells [17]. ...
... The observed production of a small amount of the exon-20-containing IKBKAP transcript in cell lines and tissues from individuals with FD [16] prompted a search for therapeutic modalities capable of facilitating the production of the exon-20-containing IKBKAP transcript in those affected with this disorder. In 2003, we reported that tocotrienols, members of the vitamin E family [16], and epigallocatechin gallate (EGCG), a flavonoid present in plants, elevate the production of the exon-20-containing transcript in FD-derived cells [17]. Tocotrienols mediate their effect by increasing the rate of transcription of the IKBKAP gene [16] and EGCG alters the splicing process, resulting in the inclusion of exon 20 in the IKBKAP-derived transcript [17]. ...
... In 2003, we reported that tocotrienols, members of the vitamin E family [16], and epigallocatechin gallate (EGCG), a flavonoid present in plants, elevate the production of the exon-20-containing transcript in FD-derived cells [17]. Tocotrienols mediate their effect by increasing the rate of transcription of the IKBKAP gene [16] and EGCG alters the splicing process, resulting in the inclusion of exon 20 in the IKBKAP-derived transcript [17]. Others have reported the ability of both kinetin, a plant cytokinin which alters the splicing process [18][19][20][21], and phosphatidylserine, through its effect on IKB-KAP gene expression [21], to increase the production of the full-length transcript from the IVS20 +6T?Cbearing allele. ...
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 IKAP 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. This article is protected by copyright. All rights reserved.
... The observed production of a small, but detectable, amount of the exon-20-containing IKBKAP transcript in cell lines and tissues from individuals with FD [16] prompted a search for therapeutic modalities capable of facilitating the production of the exon-20-containing IKBKAP transcript in those affected. In 2003, we reported that the tocotrienols, members of the vitamin E family, elevate the production of the exon-20-containing transcript in FD-derived cells by increasing the rate of transcription of the IKBKAP gene [16]. ...
... The observed production of a small, but detectable, amount of the exon-20-containing IKBKAP transcript in cell lines and tissues from individuals with FD [16] prompted a search for therapeutic modalities capable of facilitating the production of the exon-20-containing IKBKAP transcript in those affected. In 2003, we reported that the tocotrienols, members of the vitamin E family, elevate the production of the exon-20-containing transcript in FD-derived cells by increasing the rate of transcription of the IKBKAP gene [16]. Clinical analysis of the impact of the tocotrienol administration in individuals with FD revealed the tocotrienol-mediated elevated production of the full-length IKBKAP transcript in peripheral blood cells, improvement of cardiac function, a reduced incidence of dysautonomic crises, and increased eye moisture [4,[17][18][19], http://emedicine.medscape.com/article/1200921]. ...
... The previous identification of commonly consumed compounds that facilitate the production of the wild-type IKBKAP transcript in cells derived from individuals with FD [16,20] encouraged us to evaluate the impact of several hundred compounds, including genistein, on the production of the wildtype transcript. Genistein, a compound found in soy, has been previously reported to have protein tyrosine kinase inhibitor activity [32,33] and neuro-and cardio-protective properties [34][35][36][37][38][39][40][41][42]. ...
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.
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.
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.
... The ELP1 cDNA segment between nucleotides 2194 and 2593 (numbering corresponding to the NM_003640.5 transcript) was amplified by the polymerase chain reaction using 5′-CAGGTGTCGCTTTTTCATCA-3′ and 5′-CATTTCCAAGAAACACCTTAGGG-3′ primers (Anderson et al., 2003) ...
Kinetin (N⁶-furfuryladenine), a plant growth substance of the cytokinin family, has been shown to modulate aging and various age-related conditions in animal models. Here we report the synthesis of kinetin isosteres with the purine ring replaced by other bicyclic heterocycles, and the biological evaluation of their activity in several in vitro models related to neurodegenerative diseases. Our findings indicate that kinetin isosteres protect Friedreich́s ataxia patient-derived fibroblasts against glutathione depletion, protect neuron-like SH-SY5Y cells from glutamate-induced oxidative damage, and correct aberrant splicing of the ELP1 gene in fibroblasts derived from a familial dysautonomia patient. Although the mechanism of action of kinetin derivatives remains unclear, our data suggest that the cytoprotective activity of some purine isosteres is mediated by their ability to reduce oxidative stress. Further, the studies of permeation across artificial membrane and model gut and blood-brain barriers indicate that the compounds are orally available and can reach central nervous system. Overall, our data demonstrate that isosteric replacement of the kinetin purine scaffold is a fruitful strategy for improving known biological activities of kinetin and discovering novel therapeutic opportunities.
... 27 Tocotrienols increase, in vitro, transcription of IKBKAP gene, mutated in the neurodegenerative genetic disorder Familial dysautonomia, leading to an increase in the correctly spliced transcript and normal protein. 28 Moreover, tocotrienols possess in vitro antifibrotic properties, 29 and, recently, Yang et al. elucidated the mechanisms by which d-Tocotrienol reduces inflammation through the inhibition of TNF-a-induced activation of NF-jB and the LPSstimulated IL-6 30 expression. Finally, tocotrienols are able to modulate the sensitivity to ferroptosis by inhibiting lipid peroxidation. ...
Friedreich’s ataxia is an autosomal recessive disorder characterized by impaired mitochondrial function, resulting in oxidative stress. In this study, we aimed at evaluating whether tocotrienol, a phytonutrient that diffuses easily in tissues with saturated fatty layers, could complement the current treatment with idebenone, a quinone analogue with antioxidant properties. Five young Friedreich’s ataxia patients received a low-dose tocotrienol supplementation (5 mg/kg/day), while not discontinuing idebenone treatment. Several oxidative stress markers and biological parameters related to oxidative stress were evaluated at the time of initiation of treatment and 2 and 12 months post-treatment. Some oxidative stress-related parameters and some inflammation indices were altered in Friedreich’s ataxia patients taking idebenone alone and tended to be normal values following tocotrienol supplementation; likewise, a cardiac magnetic resonance study showed some improvement following one-year tocotrienol treatment. The pathway by which tocotrienol affects the Nrf2 modulation of hepcidin gene expression, a peptide involved in iron handling and in inflammatory responses, is viewed in the light of the disruption of the iron intracellular distribution and of the Nrf2 anergy characterizing Friedreich’s ataxia. This research provides a suitable model to analyze the efficacy of therapeutic strategies able to counteract the excess free radicals in Friedreich’s ataxia, and paves the way to long-term clinical studies.
Impact statement
Oxidative stress is involved in the pathogenesis of Friedreich's ataxia (FRDA), a genetic disorder causing neurodegeneration due to the dramatic reduction in the expression of frataxin. To date, no cure is available for FRDA patients. In some countries, FRDA patients assume idebenone in order to counteract the effects of frataxin deficiency. We demonstrate that idebenone treatment alone is not able to abrogate oxidative stress in FRDA patients, whereas the combined treatment with tocotrienols might be more efficient and perhaps produce clinical improvement. In fact, a decrease in oxidative stress and inflammation markers can be seen after two months and is more pronounced after one year of treatment. This is, in our opinion, valuable information for clinicians, since idebenone is the treatment of choice for FRDA patients in some countries.
... In our pursuit to find a way to improve the therapeutic effect in FD, we considered different agents with the potential to affect IKBKAP. Several potential therapies for FD have been investigated including PS [30- 32,34], kinetin [36,37], tocotrienols [47,48], and the green tea component epigallocatechin gallate [49]. Other therapy strategies are based on the FD mutation acting by altering gene splicing in the nerve system in a tissue-specific manner [25,27,[50][51][52]. ...
Familial Dysautonomia (FD) is an autosomal recessive congenital neuropathy that results from a point mutation at the 5’ splice site of intron 20 in the IKBKAP gene. This mutation decreases production of the IKAP protein, and treatments that increase the level of the full-length IKBKAP transcript are likely to be of therapeutic value. We previously found that phosphatidylserine (PS), an FDA-approved food supplement, elevates IKAP levels in cells generated from FD patients. Here we demonstrate that combined treatment of cells generated from FD patients with PS and kinetin or PS and the histone deacetylase inhibitor trichostatin A (TSA) resulted in an additive elevation of IKAP compared to each drug alone. This indicates that the compounds influence different pathways. We also found that pridopidine enhances production of IKAP in cells generated from FD patients. Pridopidine has an additive effect on IKAP levels when used in combination with kinetin or TSA, but not with PS; suggesting that PS and pridopidine influence IKBKAP levels through the same mechanism. Indeed, we demonstrate that the effect of PS and pridopidine is through sigma-1 receptor-mediated activation of the BDNF signaling pathway. A combination treatment with any of these drugs with different mechanisms has potential to benefit FD patients.
... In our pursuit to find a way to improve the therapeutic effect in FD, we considered different agents with the potential to affect IKBKAP. Several potential therapies for FD have been investigated including PS [30][31][32]34 , kinetin 40,41 , tocotrienols 47,48 , and the green tea component epigallocatechin gallate . CC-BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
Familial Dysautonomia (FD) is an autosomal recessive congenital neuropathy that results from a point mutation at the 5′ splice site of intron 20 in the IKBKAP gene. This mutation decreases production of the IKAP protein, and treatments that increase the level of the full-length IKBKAP transcript are likely to be of therapeutic value. We previously found that phosphatidylserine (PS), an FDA-approved food supplement, elevates IKAP levels in cells generated from FD patients. Here we demonstrate that combined treatment of cells generated from FD patients with PS and kinetin or PS and the histone deacetylase inhibitor trichostatin A (TSA) resulted in an additive elevation of IKAP compared to each drug alone. This indicates that the compounds influence different pathways. We also found that pridopidine enhances production of IKAP in cells generated from FD patients. Pridopidine has an additive effect on IKAP levels when used in combination with kinetin or TSA, but not with PS; suggesting that PS and pridopidine influence IKBKAP levels through the same mechanism. Indeed, we demonstrate that the effect of PS and pridopidine is through sigma-1 receptor-mediated activation of the BDNF signaling pathway. A combination treatment with any of these drugs with different mechanisms has potential to benefit FD patients.
... It has been recently demonstrated that small molecules (such as resveratrol analogues) are able to modulate the expression of splicing factors with a subsequent rescue of multiple aspects related to cellular senescence including increased telomere length, re-entry into the cell cycle and restarted proliferation [33]. In this context, it has been reported that T3 are capable to correct aberrant splicing of IkappaB kinase complex-associated protein (IKAP) in cells derived from patients with familial dysautonomia [2] and to modulate the expression of a specific set of miRNAs in HeLa cells involved in the alternative splicing of pro-apoptotic proteins, such as the X-box binding protein 1 (XBP-1) [10]. Hence, alteration of splicing factor levels may be an additional mechanism by which T3 can reverse cellular senescence. ...
Tocotrienols (T3) have been shown to represent a very important part of the vitamin E family since they have opened new opportunities to prevent or treat a multitude of age-related chronic diseases. The beneficial effects of T3 include the amelioration of lipid profile, the promotion of Nrf2 mediated cytoprotective activity and the suppression of inflammation. All these effects may be the consequence of the ability of T3 to target multiple pathways. We here propose that these effects may be the result of a single target of T3, namely senescent cells. Indeed, T3 may act by a direct suppression of the senescence-associated secretory phenotype (SASP) produced by senescent cells, mediated by inhibition of NF-kB and mTOR, or may potentially remove the origin of the SASP trough senolysis (selective death of senescent cells). Further studies addressed to investigate the impact of T3 on cellular senescence “in vitro” as well as in experimental models of age-related diseases “in vivo” are clearly encouraged.
... The use of mouse models to study compounds that alter the splicing process in human cells or transcriptionally upregulate genes of interest are further fraught with challenges, as the splicing process in humans and mice is very different 83,84 and transcriptional regulation and promoter sequences have evolutionarily diverged in these species. 85,86 Despite these potential pitfalls, investigators have endeavored to create a suitable FD mouse model in which to characterize the in vivo effects of chemical compounds demonstrated to increase the production of the exon 20-containing ELP1 transcript in cell lines 61,63 and induced pluripotent stem cell-derived lineages 87 generated from individuals homozygous for the FD-causing IVS20+6T>C mutation. Attempts to generate an FD mouse model bearing the major FD-causing mutation in the conserved intron 20 donor splice site of the mouse ELP1 homolog, Ikbkap (current nomenclature as per the Mouse Genomic Nomenclature Committee), which would most closely model the human FD-causing mutation, failed. ...
Berish Y Rubin, Sylvia L Anderson Department of Biological Sciences, Fordham University, Bronx, NY, USA Abstract: The successful completion of the Human Genome Project led to the discovery of the molecular basis of thousands of genetic disorders. The identification of the mutations that cause familial dysautonomia (FD), an autosomal recessive disorder that impacts sensory and autonomic neurons, was aided by the release of the human DNA sequence. The identification and characterization of the genetic cause of FD have changed the natural history of this disease. Genetic testing programs, which were established shortly after the disease-causing mutations were identified, have almost completely eliminated the birth of children with this disorder. Characterization of the principal disease-causing mutation has led to the development of therapeutic modalities that ameliorate its effect, while the development of mouse models that recapitulate the impact of the mutation has allowed for the in-depth characterization of its impact on neuronal development and survival. The intense research focus on this disorder, while clearly benefiting the FD patient population, also serves as a model for the positive impact focused research efforts can have on the future of other genetic diseases. Here, we present the research advances and scientific breakthroughs that have changed and will continue to change the natural history of this centuries-old genetic disease. Keywords: HSAN, splicing, MAO, tocotrienol, EGCG, Ashkenazi
... Therefore, a rational approach to reduce carfilzomib toxicity while maintaining splicing profile improvement and proteasome blocking could be to combine this drug with EGCG or with tocotrienols, Table 2 Validated mRNA targets of dysregulated miRNAs associated with the ubiquitin/proteasome machinery. Target miRNA Target miRNA Target miRNA Target miRNA BIRC6 H members of the vitamin E family, that have been shown to upregulate IKBKAP transcription and to provide clinical benefit to FD patients (Anderson et al., 2003b;Cheishvili et al., 2016). Proteasome inhibitors are pharmacological agents mainly used to kill tumor cells but also able to induce toxicity toward healthy cells. ...
FD is a rare neurodegenerative disorder caused by a mutation of the IKBKAP gene, which induces low expression levels of the Elongator subunit IKAP/hELP1 protein. A rational strategy for FD treatment could be to identify drugs increasing IKAP/hELP1 expression levels by blocking protein degradation pathways such as the 26S proteasome. Proteasome inhibitors are promising molecules emerging in cancer treatment and could thus constitute an enticing pharmaceutical strategy for FD treatment. Therefore, we tested three proteasome inhibitors on FD human olfactory ecto-mesenchymal stem cells (hOE-MSCs): two approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA), bortezomib and carfilzomib, as well as epoxomicin. Although all 3 inhibitors demonstrated activity in correcting IKBKAP mRNA aberrant splicing, carfilzomib was superior in enhancing IKAP/hELP1 quantity. Moreover, we observed a synergistic effect of suboptimal doses of carfilzomib on kinetin in improving IKBKAP isoforms ratio and IKAP/hELP1 expression levels allowing to counterbalance carfilzomib toxicity. Finally, we identified several dysregulated miRNAs after carfilzomib treatment that target proteasome-associated mRNAs and determined that IKAP/hELP1 deficiency in FD pathology is correlated to an overactivity of the 26S proteasome. Altogether, these results reinforce the rationale for using chemical compounds inhibiting the 26S proteasome as an innovative option for FD and a promising therapeutic pathway for many other neurodegenerative diseases.
... Therefore, the attempt to correct IKBKAP gene aberrant splicing in FD patients has been made and several chemical compounds were identified to improve exon 20 inclusion. For example, tocotrienols were shown to upregulate both full-length and exon 20-lacking mRNAs (Anderson et al. 2003b). Epigallocatechin gallate (EGCG) was found to reduce the level of hnRNP A2/B1 proteins and to correct aberrant splicing (Anderson et al. 2003a). ...
Pre-mRNA splicing is an essential step for gene expression in higher eukaryotes. Alternative splicing contributes to diversity of the expressed proteins from the limited number of genes. Disruption of splicing regulation often results in hereditary and sporadic diseases called as 'RNA diseases'. Modulation of splicing by small chemical compounds and nucleic acids has been tried to target aberrant splicing in those diseases. Several RNA diseases and splicing-target therapeutic approaches will be briefly introduced in this review. Accumulating knowledge about molecular mechanism of aberrant splicing and their correction by chemical compounds is important not only for RNA biologists, but also for clinicians who desire therapies for those diseases.
... Familial dysautonomia is a brutal neurological disease for which there is no cure. The discovery that certain vitamins (Anderson et al., 2003b), botanicals (Anderson et al., 2003a; Slaugenhaupt et al., 2004), or supplements (Keren et al., 2011) that increase IKBKAP mRNA levels, lead many patients to believe that these may have physiological benefits. With the expansion of the health food industry, many of these compounds can be purchased easily. ...
... Several potential therapeutics have been tested for treatment of FD, such as kinetin (29,30), RECTAS (17) and tocotrienols (31,32); however, their therapeutic effects on FD patients remain to be seen. Phosphatidylserine (PS), an FDA-approved food supplement, was also tested for FD with promising results, both in cell lines derived from FD patients (11) and in an FD mouse model (33) and in a small-scale clinical trial in FD patients (34). ...
Familial dysautonomia (FD) is a genetic disorder manifested due to abnormal development and progressive degeneration of the
sensory and autonomic nervous system. FD is caused by a point mutation in the IKBKAP gene encoding the IKAP protein, resulting in decreased protein levels. A promising potential treatment for FD is phosphatidylserine
(PS); however, the manner by which PS elevates IKAP levels has yet to be identified. Analysis of ChIP-seq results of the IKBKAP promoter region revealed binding of the transcription factors CREB and ELK1, which are regulated by the mitogen-activated
protein kinase (MAPK)/extracellular-regulated kinase (ERK) signaling pathway. We show that PS treatment enhanced ERK phosphorylation
in cells derived from FD patients. ERK activation resulted in elevated IKBKAP transcription and IKAP protein levels, whereas pretreatment with the MAPK inhibitor U0126 blocked elevation of the IKAP protein
level. Overexpression of either ELK1 or CREB activated the IKBKAP promoter, whereas downregulation of these transcription factors resulted in a decrease of the IKAP protein. Additionally,
we show that PS improves cell migration, known to be enhanced by MAPK/ERK activation and abrogated in FD cells. In conclusion,
our results demonstrate that PS activates the MAPK/ERK signaling pathway, resulting in activation of transcription factors
that bind the promoter region of IKBKAP and thus enhancing its transcription. Therefore, compounds that activate the MAPK/ERK signaling pathway could constitute
potential treatments for FD.
... To date, several compounds have been proposed as potential therapeutics for FD. Phosphatidylserine, was reported to modify IKBKAP splicing in mice, and Tocotrienols were reported to increase total IKBKAP transcript production in human FD cell lines [58,59]. Kinetin has been shown to be one of the most potent drugs to correct IKBKAP alternative splicing and upregulate IKAP expression in FD cells [25,26] although its therapeutic potential is uncertain due to possible side effects and toxicity in FD patients [60]. ...
A splicing mutation in the IKBKAP gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC) line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD) analysis. We found that IKBKAP downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC) while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects IKBKAP alternative splicing) promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD.
... 80 Since FD is caused by a tissue specific splicing defect, attempts to find compounds that may correct IKBKAP splicing and relieve some of the symptoms have been undertaken. [81][82][83][84] Several small molecules have been identified, but only Kinetin has shown positive clinical effects. 85 Thus, FD is the only RNA modification disease for which treatment options may exist through restoration of RNA modification levels. ...
Chemical RNA modifications are present in all kingdoms of life and many of these post-transcriptional modifications are conserved throughout evolution. However, most of the research has been performed on single cell organisms, whereas little is known about how RNA modifications contribute to the development of metazoans. In recent years, the identification of RNA modification genes in genome wide association studies (GWAS) has sparked new interest in previously neglected genes. In this review, we summarize recent findings that connect RNA modification defects and phenotypes in higher eukaryotes. Furthermore, we discuss the implications of aberrant tRNA modification in various human diseases including metabolic defects, mitochondrial dysfunctions, neurological disorders, and cancer. As the molecular mechanisms of these diseases are being elucidated, we will gain first insights into the functions of RNA modifications in higher eukaryotes and finally understand their roles during development.
... Patients are homozygous for a mutation at the sixth nucleotide of the 5′ splice site of intron 20 (IVS20 + 6T > C) in the IKBKAP gene. 78,79 Tocotrienols were found to upregulate both full-length and exon 20-lacking transcripts, 80 and epigallocatechin gallate (EGCG) reduced the level of hnRNP A2/B1 and corrected aberrant splicing without increasing exon 20-lacking transcripts. 81 Using a lymphoblast cell line from an FD patient, 1040 compounds were screened to find the plant cytokinin kinetin to correct splicing of the mutant IKBKAP gene and to increase IKAP protein expression. ...
Alternative splicing is a critical step where a limited number of human genes generate a complex and diverse proteome. Various diseases, including inherited diseases with abnormalities in the “genome code,” have been found to result in an aberrant mis-spliced “transcript code” with correlation to the resulting phenotype. Chemical compound-based and nucleic acid-based strategies are trying to target this mis-spliced “transcript code”. We will briefly mention about how to obtain splicing-modifying-compounds by high-throughput screening and overview of what is known about compounds that modify splicing pathways. The main focus will be on RNA-binding protein kinase inhibitors. In the main text, we will refer to diseases where splicing-modifying-compounds have been intensively investigated, with comparison to nucleic acid-based strategies. The information on their involvement in mis-splicing as well as nonsplicing events will be helpful in finding better compounds with less off-target effects for future implications in mis-splicing therapy.
... Anderson and colleagues have shown that tocotrienol is able to regulate directly the expression of IKBKAP whose alteration is responsible for a neurodegenerative disease, familial dysautonomia [24,25]. ...
Friedreich's ataxia (FRDA) is caused by deficient expression of the mitochondrial protein frataxin involved in the formation of iron-sulphur complexes and by consequent oxidative stress. We analysed low-dose tocotrienol supplementation effects on the expression of the three splice variant isoforms (FXN-1, FXN-2, and FXN-3) in mononuclear blood cells of FRDA patients and healthy subjects. In FRDA patients, tocotrienol leads to a specific and significant increase of FXN-3 expression while not affecting FXN-1 and FXN-2 expression. Since no structural and functional details were available for FNX-2 and FXN-3, 3D models were built. FXN-1, the canonical isoform, was then docked on the human iron-sulphur complex, and functional interactions were computed; when FXN-1 was replaced by FXN-2 or FNX-3, we found that the interactions were maintained, thus suggesting a possible biological role for both isoforms in human cells. Finally, in order to evaluate whether tocotrienol enhancement of FXN-3 was mediated by an increase in peroxisome proliferator-activated receptor- γ (PPARG), PPARG expression was evaluated. At a low dose of tocotrienol, the increase of FXN-3 expression appeared to be independent of PPARG expression. Our data show that it is possible to modulate the mRNA expression of the minor frataxin isoforms and that they may have a functional role.
... We listed 460 non-redundant NDD genes from Biobase Knowledge Library (BKL) (http://www.biobase-international. com/) out of 848 readily available NDD annotations by matching their functional description with any one of the neurodegenerative diseases common to literatures (such as Alzheimer disease, Parkinson disease, Huntington disease, Adrenoleukodystrophy, Creutzfeldt-Jakob disease, Friedreich ataxia, Leigh syndrome, Neuronal Ceroid lipofuscinosis, Myoclonic epilepsy, Pick disease, Spinocerebellar ataxia, Supranuclear palsy, Charcot-Marie-tooth disease, Wolfram syndrome, Alexander disease, Amyotrophic lateral sclerosis, Canavan disease, Familial dysautonomia, Leukoencephalopathy, Metachromatic leukodystrophy, Multiple sclerosis, Myotonic dystrophy, Prion diseases, Rett syndrome, Schizophrenia, Spastic paraplegia, Spinal muscular atrophy, Multiple system atrophy and Tay-sachs disease) [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34]. However, some of the afore-mentioned diseases may belong to neuropathy or lysosomal storage disease groups [35,36] and were excluded from our gene set. ...
Comparative analyses between human disease and non-disease genes are of great interest in understanding human disease gene evolution. However, the progression of neurodegenerative diseases (NDD) involving amyloid formation in specific brain regions is still unknown. Therefore, in this study, we mainly focused our analysis on the evolutionary features of human NDD genes with respect to non-disease genes. Here, we observed that human NDD genes are evolutionarily conserved relative to non-disease genes. To elucidate the conserved nature of NDD genes, we incorporated the evolutionary attributes like gene expression level, number of regulatory miRNAs, protein connectivity, intrinsic disorder content and relative aggregation propensity in our analysis. Our studies demonstrate that NDD genes have higher gene expression levels in favor of their lower evolutionary rates. Additionally, we observed that NDD genes have higher number of different regulatory miRNAs target sites and also have higher interaction partners than the non-disease genes. Moreover, miRNA targeted genes are known to have higher disorder content. In contrast, our analysis exclusively established that NDD genes have lower disorder content. In favor of our analysis, we found that NDD gene encoded proteins are enriched with multi interface hubs (party hubs) with lower disorder contents. Since, proteins with higher disorder content need to adapt special structure to reduce their aggregation propensity, NDD proteins found to have elevated relative aggregation propensity (RAP) in support of their lower disorder content. Finally, our categorical regression analysis confirmed the underlined relative dominance of protein connectivity, 3'UTR length, RAP, nature of hubs (singlish/multi interface) and disorder content for such evolutionary rates variation between human NDD genes and non-disease genes.
... It is important to note that the cellular systems have been proved successful for testing a plethora of different treatments aimed at restoring splicing with supplements such as kinetin (52,53), something that cannot be tested in our mouse models. Our mice, however, are the only in vivo models of FD and not only are useful for the elucidation of the pathophysiology of the disease, but are also invaluable for testing the efficacy of current and future therapies that aim at increasing IKAP protein levels through transcriptional activation, such as tocotrienols and phosphatidylserine (54)(55)(56)(57). Our finding that Ikbkap D20/flox mice display a severe reduction in nociceptive (trkA/CGRP positive) neuronal numbers at late gestation is consistent with the severe impairment in pain and temperature perception observed in some FD patients in infancy (1). ...
Hereditary sensory and autonomic neuropathies (HSANs) encompass a group of genetically inherited disorders characterized by
sensory and autonomic dysfunctions. Familial dysautonomia (FD), also known as HSAN type III, is an autosomal recessive disorder
that affects 1/3600 live births in the Ashkenazi Jewish population. The disease is caused by abnormal development and progressive
degeneration of the sensory and autonomic nervous systems and is inevitably fatal, with only 50% of patients reaching the
age of 40. FD is caused by a mutation in intron 20 of the Ikbkap gene that results in severe reduction in the expression of
its encoded protein, inhibitor of kappaB kinase complex-associated protein (IKAP). Although the mutation that causes FD was
identified in 2001, so far there is no appropriate animal model that recapitulates the disorder. Here, we report the generation
and characterization of the first mouse models for FD that recapitulate the molecular and pathological features of the disease.
Important for therapeutic interventions is also our finding that a slight increase in IKAP levels is enough to ameliorate
the phenotype and increase the life span. Understanding the mechanisms underlying FD will provide insights for potential new
therapeutic interventions not only for FD, but also for other peripheral neuropathies.
... Availability of human peripheral sensory neurons (PSNs), for example, would permit the study of how the truncated protein product of the IKBKAP gene, the gene mutated in the vast majority of FD patients (reviewed in [2]), leads to specific degeneration of this type of neuron in this disease. In addition, normal human sensory neurons could be useful for screening of new and improved drugs for treating FD and other peripheral neuropathies [3,4]. It is even possible that such neurons could eventually be used for cell-replacement therapy in patients who have significant sensory neuron loss. ...
Human embryonic stem cells (hESCs) have been directed to differentiate into neuronal cells using many cell-culture techniques. Central nervous system cells with clinical importance have been produced from hESCs. To date, however, there have been no definitive reports of generation of peripheral neurons from hESCs. We used a modification of the method of Sasai and colleagues for mouse and primate embryonic stem cells to elicit neuronal differentiation from hESCs. When hESCs are cocultured with the mouse stromal line PA6 for 3 weeks, neurons are induced that coexpress (a) peripherin and Brn3a, and (b) peripherin and tyrosine hydroxylase, combinations characteristic of peripheral sensory and sympathetic neurons, respectively. In vivo, peripheral sensory and sympathetic neurons develop from the neural crest (NC). Analysis of expression of mRNAs identified in other species as NC markers reveals that the PA6 cells induce NC-like cells before neuronal differentiation takes place. Several NC markers, including SNAIL, dHAND, and Sox9, are increased at 1 week of coculture relative to naive cells. Furthermore, the expression of several NC marker genes known to be downregulated upon in vivo differentiation of NC derivatives, was observed to be present at lower levels at 3 weeks of PA6-hESC coculture than at 1 week. Our report is the first on the expression of molecular markers of NC-like cells in primates, in general, and in humans, specifically. Our results suggest that this system can be used for studying molecular and cellular events in the almost inaccessible human NC, as well as for producing normal human peripheral neurons for developing therapies for diseases such as familial dysautonomia.
... Characterization of alternatively spliced transcripts that are specifically present or absent in a specific condition provides another opportunity for therapeutic intervention of diabetic retinopathy. Tocotrienol and EGCG are known to target splice sites and to modulate the abundance of alternatively spliced transcripts [60,61]. Exploration of the ability of these agents to inhibit diabetic retinopathy will be an interesting avenue of further research. ...
To define gene expression changes associated with diabetic retinopathy in a mouse model using next generation sequencing, and to utilize transcriptome signatures to assess molecular pathways by which pharmacological agents inhibit diabetic retinopathy.
We applied a high throughput RNA sequencing (RNA-seq) strategy using Illumina GAIIx to characterize the entire retinal transcriptome from nondiabetic and from streptozotocin-treated mice 32 weeks after induction of diabetes. Some of the diabetic mice were treated with inhibitors of receptor for advanced glycation endproducts (RAGE) and p38 mitogen activated protein (MAP) kinase, which have previously been shown to inhibit diabetic retinopathy in rodent models. The transcripts and alternatively spliced variants were determined in all experimental groups.
Next generation sequencing-based RNA-seq profiles provided comprehensive signatures of transcripts that are altered in early stages of diabetic retinopathy. These transcripts encoded proteins involved in distinct yet physiologically relevant disease-associated pathways such as inflammation, microvasculature formation, apoptosis, glucose metabolism, Wnt signaling, xenobiotic metabolism, and photoreceptor biology. Significant upregulation of crystallin transcripts was observed in diabetic animals, and the diabetes-induced upregulation of these transcripts was inhibited in diabetic animals treated with inhibitors of either RAGE or p38 MAP kinase. These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin.
Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects. These therapies also changed the abundance of various alternatively spliced versions of signaling transcripts, suggesting a possible role of alternative splicing in disease etiology. Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.
... Moreover, the number of paxillin-positive cells was reduced in FD-iPSCs-derived neural crest progenitors, referring to the aberration of cell spreading and migration [54]. They have reported that plant hormone kinetin could reduce the levels of the mutant IKBKAP splice in FD cells545556. Epigallocatechin gallate and tocotrinal were exposed to FD-iPSCs-derived neural crest precursors and showed dramatic reduction of the mutant IKBKAP splice form; however, the hormone did not show a significant increase in the expression of neurogenic markers or improve the migration behavior [54]. ...
Studies of human brain development are critical as research on neurological disorders have been progressively advanced. However, understanding the process of neurogenesis through analysis of the early embryo is complicated and limited by a number of factors, including the complexity of the embryos, availability, and ethical constrains. The emerging of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) has shed light of a new approach to study both early development and disease pathology. The cells behave as precursors of all embryonic lineages; thus, they allow tracing the history from the root to individual branches of the cell lineage tree. Systems for neural differentiation of hESCs and iPSCs have provided an experimental model that can be used to augment in vitro studies of in vivo brain development. Interestingly, iPSCs derived from patients, containing donor genetic background, have offered a breakthrough approach to study human genetics of neurodegenerative diseases. This paper summarizes the recent reports of the development of iPSCs from patients who suffer from neurological diseases and evaluates the feasibility of iPSCs as a disease model. The benefits and obstacles of iPSC technology are highlighted in order to raising the cautions of misinterpretation prior to further clinical translations.
... In FD-iPSC lines, ASCL1 + neurons differentiated from neural crest precursors revealed a significantly delayed time course of development, which appears consistent with the symptomatology in the human disease. To investigate the potential for pharmaceutical rescue and putative therapeutic targets, Studer and colleagues screened a small set of candidate compounds identified in earlier studies using FD lymphoblast cell lines (Anderson et al., 2003bAnderson et al., , 2003a Slaugenhaupt et al., 2004). Interestingly, the plant hormone kinetin was able to rescue several FD-iPSC phenotypes, including splicing and impaired production of autonomic neurons. ...
The remarkable advances in cellular reprogramming have made it possible to generate a renewable source of human neurons from fibroblasts obtained from skin samples of neonates and adults. As a result, we can now investigate the etiology of neurological diseases at the cellular level using neuronal populations derived from patients, which harbor the same genetic mutations thought to be relevant to the risk for pathology. Therapeutic implications include the ability to establish new humanized disease models for understanding mechanisms, conduct high-throughput screening for novel biogenic compounds to reverse or prevent the disease phenotype, identify and engineer genetic rescue of causal mutations, and develop patient-specific cellular replacement strategies. Although this field offers enormous potential for understanding and treating neurological disease, there are still many issues that must be addressed before we can fully exploit this technology. Here we summarize several recent studies presented at a symposium at the 2011 annual meeting of the Society for Neuroscience, which highlight innovative approaches to cellular reprogramming and how this revolutionary technique is being refined to model neurodevelopmental and neurodegenerative diseases, such as autism spectrum disorders, schizophrenia, familial dysautonomia, and Alzheimer's disease.
... To date, three other compounds have been proposed as potential therapeutics for FD. Epigallocatechin gallate, an antioxidant present in green tea, was reported to modify IKBKAP splicing, and tocotrienols were reported to increase total IKBKAP transcript production in FD cell lines (39,40). However, two recent studies have failed to show that either of these two compounds has a significant effect on IKBKAP levels (41,42). ...
Recent studies emphasize the importance of mRNA splicing in human genetic disease, as 20-30% of all disease-causing mutations are predicted to result in mRNA splicing defects. The plasticity of the mRNA splicing reaction has made these mutations attractive candidates for the development of therapeutics. Familial dysautonomia (FD) is a severe neurodegenerative disorder, and all patients have an intronic IVS20+6T>C splice site mutation in the IKBKAP gene, which results in tissue-specific skipping of exon 20 and a corresponding reduction in ikappaB kinase complex associated protein (IKAP) levels. We created transgenic mouse lines using a human IKBKAP bacterial artificial chromosome (BAC) into which we inserted the IKBKAP splice mutation (FD BAC) and have shown that the transgenic mice exhibit the same tissue-specific aberrant splicing patterns as seen in FD patients. We have previously demonstrated that the plant cytokinin kinetin can significantly improve the production of wild-type IKBKAP transcripts in FD lymphoblast cell lines by improving exon inclusion. In this study, we tested the ability of kinetin to alter IKBKAP splicing in the transgenic mice carrying the FD BAC and show that it corrects IKBKAP splicing in all major tissues assayed, including the brain. The amount of wild-type IKBKAP mRNA and IKAP protein was significantly higher in the kinetin-treated mice. These exciting results prove that treatment of FD, as well as other mechanistically related splicing disorders, with kinetin holds great promise as a potential therapeutic aimed at increasing normal protein levels, which may, in turn, slow disease progression.
... Our own published work, as well as similar efforts in SMA [17], has focused on testing the iPS-based disease model against a small set of candidate compounds. We were able to test most of the key compounds previously proposed to be of potential value for treating FD such as the vitamin E derivative tocotrienols [83], the polyphenol epigallocatechin gallate [84] and the plant hormone kinetin [50]. We only observed robust rescue of FD-iPS cell phenotypes such as splicing and the generation of autonomic neurons with kinetin but not any of the other compounds. ...
Induced pluripotent stem (iPS) cells have considerable promise as a novel tool for modelling human disease and for drug discovery. While the generation of disease-specific iPS cells has become routine, realizing the potential of iPS cells in disease modelling poses challenges at multiple fronts. Such challenges include selecting a suitable disease target, directing the fate of iPS cells into symptom-relevant cell populations, identifying disease-related phenotypes and showing reversibility of such phenotypes using genetic or pharmacological approaches. Finally, the system needs to be scalable for use in modern drug discovery. Here, we will discuss these points in the context of modelling familial dysautonomia (FD, Riley-Day syndrome, hereditary sensory and autonomic neuropathy III (HSAN-III)), a rare genetic disorder in the peripheral nervous system. We have demonstrated three disease-specific phenotypes in FD-iPS-derived cells that can be partially rescued by treating cells with the plant hormone kinetin. Here, we will discuss how to use FD-iPS cells further in high throughput drug discovery assays, in modelling disease severity and in performing mechanistic studies aimed at understanding disease pathogenesis. FD is a rare disease but represents an important testing ground for exploring the potential of iPS cell technology in modelling and treating human disease.
... A significant increase in wild-type IKAP mRNA levels and a shift towards a higher level of exon 20 inclusion was observed with an optimal concentration of 100 mM of kinetin (Figure 7A), with no toxicity observed. In contrast, tocotrienol, a member of the vitamin E family that was reported to induce IKAP expression in FD cells [36,37,38], did not affect IKAP mRNA expression in our system (Figure 7B); this observation is similar to that of another publication [39]. These results indicate that the FD cells used in this study are sensitive to kinetin treatment but not to tocotrienol. ...
Familial Dysautonomia (FD) is an autosomal recessive congenital neuropathy that results from abnormal development and progressive degeneration of the sensory and autonomic nervous system. The mutation observed in almost all FD patients is a point mutation at position 6 of intron 20 of the IKBKAP gene; this gene encodes the IκB kinase complex-associated protein (IKAP). The mutation results in a tissue-specific splicing defect: Exon 20 is skipped, leading to reduced IKAP protein expression. Here we show that phosphatidylserine (PS), an FDA-approved food supplement, increased IKAP mRNA levels in cells derived from FD patients. Long-term treatment with PS led to a significant increase in IKAP protein levels in these cells. A conjugate of PS and an omega-3 fatty acid also increased IKAP mRNA levels. Furthermore, PS treatment released FD cells from cell cycle arrest and up-regulated a significant number of genes involved in cell cycle regulation. Our results suggest that PS has potential for use as a therapeutic agent for FD. Understanding its mechanism of action may reveal the mechanism underlying the FD disease.
The peripheral nervous system (PNS) is essential for proper body function. A high percentage of the population suffer nerve degeneration or peripheral damage. For example, over 40% of patients with diabetes or undergoing chemotherapy develop peripheral neuropathies. Despite this, there are major gaps in the knowledge of human PNS development and therefore, there are no available treatments. Familial Dysautonomia (FD) is a devastating disorder that specifically affects the PNS making it an ideal model to study PNS dysfunction. FD is caused by a homozygous point mutation in ELP1 leading to developmental and degenerative defects in the sensory and autonomic lineages. We previously employed human pluripotent stem cells (hPSCs) to show that peripheral sensory neurons (SNs) are not generated efficiently and degenerate over time in FD. Here, we conducted a chemical screen to identify compounds able to rescue this SN differentiation inefficiency. We identified that genipin, a compound prescribed in Traditional Chinese Medicine for neurodegenerative disorders, restores neural crest and SN development in FD, both in the hPSC model and in a FD mouse model. Additionally, genipin prevented FD neuronal degeneration, suggesting that it could be offered to patients suffering from PNS neurodegenerative disorders. We found that genipin crosslinks the extracellular matrix, increases the stiffness of the ECM, reorganizes the actin cytoskeleton, and promotes transcription of YAP-dependent genes. Finally, we show that genipin enhances axon regeneration in an in vitro axotomy model in healthy sensory and sympathetic neurons (part of the PNS) and in prefrontal cortical neurons (part of the central nervous system, CNS). Our results suggest genipin can be used as a promising drug candidate for treatment of neurodevelopmental and neurodegenerative diseases, and as a enhancer of neuronal regeneration.
One sentence summary
Genipin rescues the developmental and degenerative phenotypes of the peripheral neuropathy familial dysautonomia and enhances neuron regeneration after injury.
Drug repurposing is the development of existing drugs for new uses: given that 9 in 10 drugs that enter drug development are never marketed and therefore represent wasted effort, it is an attractive as well as inherently more efficient process. Three repurposed drugs can be brought to market for the same cost as one new chemical entity; and they can also be identified more quickly, an important benefit for patients whose diseases are progressing faster than therapeutic innovation. But repurposing also requires a fresh look at configuring pharmaceutical R&D, considering clinical, regulatory and patent issues much earlier than would otherwise be the case; a holistic gedanken experiment almost needs to be undertaken at the very start of any repurposing development. In addition to new ways of thinking, the discovery of repurposing opportunities can take advantage of artificial intelligence techniques to match the perfect new use for an existing drug. And while repurposing of medicines has been in the mind of every doctor since Hypocrates, modern clinical practice will simply have to adapt to new repurposing techniques in an age where the number of known diseases is increasing much faster than the healthcare dollars available.
Pre-mRNA splicing is a critical step for protein gene expression in higher eukaryotes. Another mode of splicing, termed alternative splicing, contributes to diversity of the expressed proteins from the limited number of genes in chromosomes. Those steps are highly regulated and must be accurate. Therefore, disruption of splicing regulation often results in hereditary and sporadic diseases called as “RNA diseases” in human. Modulation of splicing by small chemical compounds and nucleic acids has been targeting aberrant splicing in those diseases. In this chapter, I will introduce several RNA diseases and splicing-target therapeutic approaches with chemical compounds. Unveiling molecular mechanism and correction of aberrant splicing by small chemical compounds are important for both RNA biologists and clinicians who desire therapies for those diseases.
The 5' splice site mutation (IVS20+6T>C) of the inhibitor of kappa light polypeptide gene enhancer in B cells, kinase complex-associated protein (IKBKAP) gene in familial dysautonomia (FD) is at the sixth intronic nucleotide of the 5' splice site. It is known to weaken U1 snRNP recognition and result in aberrantly spliced mRNA product in neuronal tissue, but normally spliced mRNA in other tissues. Aberrantly spliced IKBKAP mRNA abrogates IKK complex-associated protein (IKAP)/elongator protein 1 (ELP1) expression and results in a defect of neuronal cell development in FD. To elucidate the tissue-dependent regulatory mechanism, we screened an expression library of major RNA-binding proteins (RBP)s with our mammalian dual-color splicing reporter system, and identified RBM24 as a regulator. RBM24 functioned as a cryptic intronic splicing enhancer binding to an element (IVS20+13~29) downstream of the intronic 5' splice site mutation in the IKBKAP gene, and promoted U1 snRNP recognition only to the mutated 5' splice site, but not the wild-type 5' splice site. Our results show that tissue-specific expression of RBM24 can explain the neuron-specific aberrant splicing of IKBKAP exon 20 in familial dysautonomia, and that ectopic expression of RBM24 in neuronal tissue could be a novel therapeutic target of the disease.
Transfer RNAs (tRNAs) are essential components of the protein translation machinery. In order to become fully active, they need to be heavily modified post-transcriptionally. Such modifications affect the structure, stability and functionality of tRNAs; however, their exact roles at the molecular level remain largely elusive. Here we focus on the biological functions of tRNA modifications associated to human diseases and how such information can be used for biomedical applications. We put an emphasis on mitochondrial-linked dysfunctions, metabolic disorders, neurological defects and cancer. We also present methods and approaches currently used in the clinic to detect and monitor different human pathologies involving tRNA modifications or tRNA modification enzymes, and, additionally, we propose novel tRNA modification-based strategies that could be used for diagnosis, prognosis or treatment of human diseases.
The brain and neural cells are highly susceptible to oxidative damage because they (1) contain high amounts of polyunsaturated (20:4 and 22:6) fatty acids that are susceptible to lipid peroxidation, (2) have high oxygen consumption, (3) are relatively deficient in certain antioxidant enzymes, and (4) are rich in iron concentration. Thus, antioxidant defenses are critically important to protect the brain and neural tissues from oxidative damage. Numerous pathophysiological conditions have indeed been associated with increased levels of oxidative stress indices [1]. Neuroprotection by antioxidants has therefore drawn much interest. Much of the available research on the role of antioxidant nutrients in neurological function and disease has focused on vitamin E. Vitamin E is essential for normal neurological function [2,3]. It represents a family of major lipid-soluble, chain-breaking antioxidants in the body protecting the integrity of membranes by inhibiting lipid peroxidation. Mostly based on symptoms of primary vitamin E deficiency, research has demonstrated that vitamin E has a central role in maintaining neurological structure and function [3]. Most of the vitamin E-sensitive neurological disorders are associated with elevated levels of oxidative damage markers. During the last decade, this led to the popular hypothesis stating that the neuroprotective effects of vitamin E are mediated by its antioxidant property [4].
Familial dysautonomia (FD, Riley-Day syndrome, hereditary sensory and autonomic neuropathy III) is a recessive disorder with a carrier frequency of 1 in 30 in Ashkenazi Jews. Clinical features include gastrointestinal dysfunction, abnormal responses to hypoxia and hypercarbia, gastroesophageal reflux, vomiting crises, alacrima, and postural hypotension. FD is caused by. IKBKAP gene mutations; all FD patients have at least one copy of a mutation resulting in variable, tissue-specific . IKBKAP expression, with lowest levels in the central and peripheral nervous systems. As FD is associated with neuronal degeneration, a therapeutic option may be to enhance correct mRNA splicing and increase functional protein.
Familial dysautonomia (FD), also known as Riley-Day syndrome or recessive hereditary sensory and autonomic neuropathy type III (HSAN III), is caused by a single-base noncoding mutation in intron 20 (IVS20+6T > C) of the IKBKAP/ELP1 gene. This mutation results in variable skipping of exon 20 in IKBKAP/ELP1 transcripts, which leads to tissue-specific reduction of ELP1 (IKBKAP) protein, particularly in the nervous system. FD is a devastating disorder with a high mortality rate due primarily to autonomic dysfunction. The identification of the gene and the disease-causing mutation has promised the development of potential treatments that directly target mRNA splicing to increase normal mRNA and protein. FD is a developmental disease with diagnostic symptoms present at birth, whereas patients show progressive neurodegeneration throughout life. Drugs that can increase ELP1 protein may slow this degeneration and improve the quality of life in aging patients. Since these compounds target the mRNA splicing mechanism and not a specific gene, it is likely that they will prove useful in other disorders with similar splice-site mutations. Given that 20-30% of human mutations are predicted to alter mRNA splicing, direct modification of splicing efficiency poses an important target for the design of therapeutics in the future. Copyright © 2010 Prous Science, S.A.U. or its licensors All rights reserved.
The adherence of monocytes to the vascular endothelial cells is an important early event in atherogenesis. Monocyte adherence to endothelial cells is induced by oxidized low density lipoprotein (LDL) and mediated by multiple cell adhesion molecules including vascular cell-adhesion molecule 1 (VCAM-1). Enhanced endothelial expression of these molecules by oxidized LDL has been shown to be a critical step in foam cell formation and the development of atherosclerosis. Recent studies have demonstrated that vitamin E inhibited the expression of mRNA and protein for VCAM-1 by endothelial cells in response to stimulation with oxidized LDL or inflammatory cytokines. Compared to α- tocopherol, α-tocotrienol displayed a more profound inhibitory effect on adhesion molecule expression and monocytic cell adherence. Among the isomers of tocotrienol, δ-tocotrienol was a potent and effective agent for the reduction of cellular VCAM-1 expression and monocytic cell adherence. The inhibitory effects of vitamin E analogs on the adhesiveness of endothelial cells correlated with their intracellular concentrations. Anti-atherogenic effects of tocotrienols may be derived from their properties of cholesterol-lowering effect, protein kinase C inhibition, and modulation of cyclooxygenase cascade. Although recent human studies have raised some doubts on the efficacy of vitamin E in the prevention of progression of atherosclerotic lesions, the experimental studies using tocotrienol strongly support its positive effect on the reduction of risk of atherogenesis. Further studies will be necessary for clarifying the anti-atherogenic effect of tocotrienol and its bioavailability after oral administration.
Cellular reprogramming allows for the de novo generation of human neurons and glial cells from patients with neurological and psychiatric disorders. Crucially, this technology preserves the genome of the donor individual and thus provides a unique opportunity for systematic investigation of genetic influences on neuronal pathophysiology. Although direct reprogramming of adult somatic cells to neurons is now possible, the majority of recent studies have used induced pluripotent stem cells (iPSCs) derived from patient fibroblasts to generate neural progenitors that can be differentiated to specific neural cell types. Investigations of monogenic diseases have established proof-of-principle for many aspects of cellular disease modeling, including targeted differentiation of neuronal populations and rescue of phenotypes in patient iPSC lines. Refinement of protocols to allow for efficient generation of iPSC lines from large patient cohorts may reveal common functional pathology and genetic interactions in diseases with a polygenic basis.We review several recent studies that illustrate the utility of iPSC-based cellular models of neurodevelopmental and neurodegenerative disorders to identify novel phenotypes and therapeutic approaches.
Hereditary sensory and autonomic neuropathies (HSN/HSAN) are clinically and genetically heterogeneous disorders of the peripheral nervous system that predominantly affect the sensory and autonomic neurons. Hallmark features comprise not only prominent sensory signs and symptoms and ulcerative mutilations but also variable autonomic and motor disturbances. Autosomal dominant and autosomal recessive inheritance has been reported. Molecular genetics studies have identified disease-causing mutations in 11 genes. Some of the affected proteins have nerve-specific roles but underlying mechanisms have also been shown to involve sphingolipid metabolism, vesicular transport, structural integrity, and transcription regulation. Genetic and functional studies have substantially improved the understanding of the pathogenesis of the HSN/HSAN and will help to find preventive and causative therapies in the future.
Research in the past decade shows that the commonly used non-desmethyl vitamin Es (e.g., alpha-tocopherol and alpha-tocotrienol) do not share the beneficial effects of desmethyl vitamin Es (e.g., gamma and delta isomers of toco- pherols and tocotrienols). Research also shows that high lev- els of alpha-tocopherol may attenuate the bioavailability and functional activity of other vitamin E isomers. In general, desmethyl tocotrienols are much more bioactive than desmethyl tocopherols, especially in cancer inhibition. This paper delineates the role of desmethyl tocopherols and desmethyl tocotrienols in biological studies and in human health. A new perspective is presented for applications of delta-tocotrienol, gamma-tocotrienol, delta-tocopherol and gamma-tocopherol that are consistent with the emerging sci- ence of vitamin E. The paper concludes that formulated vita- min E should be "appropriate spectrum" and not merely "full spectrum" based on 35-40 mg of daily consumption (DC) of vitamin E in foods. Formulated "appropriate spectrum" vita- min E should more closely reflect the composition of our diet, and is therefore well suited for maintenance; (1X DC); pre- vention (10X DC) and treatment (50-200 mg/day desmethyl tocotrienols) formulations may require higher doses. Diversity brings out the best in unique cultures. This diversity can be appreciated not only in human societies but also in the plant kingdom. For example, there are in excess of 600 naturally occurring carotenoids in plants yet only a handful, namely beta-carotene, lycopene and lutein, are actively being researched. Similarly, there are more than twelve vitamin Es found in nature but only alpha-toco- pherol is primarily being studied. This paper calls into ques- tion the suitability of unqualified use of large doses of alpha-tocopherol as well as the unqualified use of the "full spectrum" vitamin E. The advent of "appropriate spectrum" vitamin E for human health is a derivative concept1 from punctuated research development. This review addresses recent research developments to delineate the functional roles of desmethyl tocotrienols and desmethyl tocopherols apart from common alpha-tocopherol.
The common familial dysautonomia (FD) mutation causes a splicing defect that leads to production of both wild-type (WT) and mutant (MU) IKBKAP mRNA. Because drugs may alter splicing, seven drugs, fludrocortisone, midodrine, diazepam, albuterol, clonidine, caffeine, and dopamine were screened. Since only fludrocortisone negatively altered gene expression, we assessed fludrocortisone’s efficacy in treating postural hypotension, and its effect on survival and secondary long-term FD problems. For 341 FD patients we obtained demographic data and clinical information from the last Center evaluation (most current or prior to death) including mean blood pressures (supine, 1 min erect and 5 min erect) and history regarding syncope and presyncope symptoms. For 175 fludrocortisonetreated patients, data from the evaluation prior to start of fludrocortisone and from the last Center evaluation were compared. The fludrocortisone-treated patient cohort was compared to the nontreated patient cohort with respect to overall survival and event-free survival for crisis frequency, worsening gait, frequent fractures, spine curvature, renal insufficiency, and pacemaker insertion. Overall survivals of patients on fludrocortisone alone, on fludrocortisone and midodrine, and on neither drug were compared. Cumulative survival was significantly higher in fludrocortisone-treated patients than in non-treated patients during the first decade. In subsequent decades, the addition of midodrine improved cumulative survival. Fludrocortisone significantly increased mean blood pressures and decreased dizziness and leg cramping, but not headaches or syncope. Fludrocortisone was associated with more long-term problems, which may reflect more symptomatic status associated with longer survival. Our data suggest that fludrocortisone has clinical efficacy despite negative in vitro observations on gene expression.
Vitamin E is a generic term that refers to a family of eight natural compounds, and is further divided into two subgroups called tocopherols and tocotrienols. Tocopherols are found in a wide variety of dietary fats, whereas tocotrienols are relatively rare and found naturally in high concentrations in only a few sources, such as palm and rice bran oil. Tocopherols and tocotrienols have the same basic chemical structure characterized by a long aliphatic side chain attached to a chroman ring. The difference between the tocopherols and tocotrienols is that the phytyl chain in tocopherols is saturated whereas the phytyl chain in tocotrienols is unsaturated. In addition, the chroman ring can have different methylation patterns in both tocopherols and tocotrienols resulting in further subdivision of these subgroups into alpha (α), beta (β), gamma (γ), and delta (δ) isoforms. Although, tocopherols and tocotrienols are chemically very similar and both display powerful antioxidant activities, it has become increasing evident that their biological activities are quite distinctive. Recently, a large body of research has demonstrated that tocotrienols display more potent antioxidant, anticancer, anti-cholesterolemic, antiatherosclerotic, antihypertensive, anti-inflammatory, immune-modulatory, and neuroprotective activity than do tocopherols. In addition, studies are rapidly identifying and characterizing the mechanism of action(s) mediating these effects of tocotrienols, and thereby providing the necessary information required for understanding the potential health benefits that may be obtained from enhanced tocotrienol dietary intake or supplementation. This review summarizes the beneficial effects that tocotrienol supplementation may provide in the prevention and treatment of human disease.
Familial dysautonomia (FD) is caused by an intronic splice mutation in the IKBKAP gene that leads to partial skipping of exon 20 and tissue-specific reduction in I-κ-B kinase complex-associated protein/elongation protein 1 (IKAP/ELP-1) expression. Kinetin (6-furfurylaminopurine) has been shown to improve splicing and increase WT IKBKAP mRNA and IKAP protein expression in FD cell lines and carriers. To determine whether oral kinetin treatment could alter mRNA splicing in FD subjects and was tolerable, we administered kinetin to eight FD individuals homozygous for the splice mutation. Subjects received 23.5 mg/Kg/d for 28 d. An increase in WT IKBKAP mRNA expression in leukocytes was noted after 8 d in six of eight individuals; after 28 d, the mean increase compared with baseline was significant (p = 0.002). We have demonstrated that kinetin is tolerable in this medically fragile population. Not only did kinetin produce the desired effect on splicing in FD patients but also that effect seems to improve with time despite lack of dose change. This is the first report of a drug that produces in vivo mRNA splicing changes in individuals with FD and supports future long-term trials to determine whether kinetin will prove therapeutic in FD patients.
Dysfunction and loss of retinal pigment epithelium (RPE) leads to degeneration of photoreceptors in age-related macular degeneration and subtypes of retinitis pigmentosa. Human embryonic stem cells (hESCs) may serve as an unlimited source of RPE cells for transplantation in these blinding conditions. Here we show the directed differentiation of hESCs toward an RPE fate under defined culture conditions. We demonstrate that nicotinamide promotes the differentiation of hESCs to neural and subsequently to RPE fate. In the presence of nicotinamide, factors from the TGF-beta superfamily, which presumably pattern RPE development during embryogenesis, further direct RPE differentiation. The hESC-derived pigmented cells exhibit the morphology, marker expression, and function of authentic RPE and rescue retinal structure and function after transplantation to an animal model of retinal degeneration caused by RPE dysfunction. These results are an important step toward the future use of hESCs to replenish RPE in blinding diseases.
The isolation of human induced pluripotent stem cells (iPSCs)1-3 offers a novel strategy for modeling human disease. Recent studies have reported the derivation and differentiation of disease-specific human iPSCs4-7. However, a key challenge in the field is the demonstration of disease-related phenotypes and the ability to model pathogenesis and treatment of disease in iPSCs. Familial dysautonomia (FD) is a rare but fatal peripheral neuropathy caused by a point mutation in IKBKAP8 involved in transcriptional elongation9. The disease is characterized by the depletion of autonomic and sensory neurons. The specificity to the peripheral nervous system and the mechanism of neuron loss in FD are poorly understood due to the lack of an appropriate model system.
Here we report the derivation of patient specific FD-iPSCs and the directed differentiation into cells of all three germ layers including peripheral neurons. Gene expression analysis in purified FD-iPSC derived lineages demonstrates tissue specific mis-splicing of IKBKAP in vitro. Patient-specific neural crest precursors express particularly low levels of normal IKBKAP transcript suggesting a mechanism for disease specificity. FD pathogenesis is further characterized by transcriptome analysis and cell based assays revealing marked defects in neurogenic differentiation and migration behavior. Finally, we use FD-iPSCs for validating the potency of candidate drugs in reversing aberrant splicing and ameliorating neuronal differentiation and migration. Our study illustrates the promise of iPSC technology for gaining novel insights into human disease pathogenesis and treatment.
A cDNA clone complementary to an interferon (IFN)-induced mRNA was isolated and used to characterize the regulation of expression of its RNA by the IFNs and to identify the protein its RNA encodes. This cDNA hybridizes to IFN-induced 3.1- and 2.3-kilobase mRNAs that are synthesized in response to both IFN-alpha and IFN-gamma. IFN-gamma induces the sustained accumulation of these mRNAs while IFN-alpha induces their transient accumulation. Cycloheximide (50 micrograms/ml) failed to inhibit the induction of these mRNAs by either IFN-alpha or IFN-gamma, suggesting that their induction does not require de novo protein synthesis. DNA sequence analysis of this cDNA reveals that it encodes a protein of Mr 53,168 that has sequence homology with and the biological activity of a tryptophanyl-tRNA synthetase, an enzymatic activity that has been demonstrated to play a role in and be modulated by the growth of cells. Elevated levels of this enzyme may be involved in the cell growth inhibitory activity of the IFNs.
A factor with tumor necrosis factor (TNF) activity produced by the LuKII human lymphoblastoid cell line [designated TNF(LuKII)] was purified sequentially by using controlled-pore glass, lentil lectin-Sepharose, and procion red agarose chromatography, yielding TNF with a specific activity of 1.5 X 10(7) units per mg of protein and an isoelectric point of approximately equal to 6.7. Purified TNF(LuKII) fractionated by NaDodSO4/PAGE under reducing as well as nonreducing conditions was found to contain seven protein bands of Mr 80,000, 70,000, 43,000, 25,000, 23,000, 21,000, and 19,000. The proteins of Mr 80,000 and 70,000 could not be dissociated into lower molecular weight components. Peptide mapping analysis and immunoblotting analysis revealed that the seven protein bands in the purified TNF(LuKII) preparations are related. After fractionation of TNF(LuKII) by NaDodSO4/PAGE under reducing conditions, TNF activity was recovered from the regions of Mr 70,000 and 19,000-25,000. Purified human TNF(LuKII) (i) produces hemorrhagic necrosis of Meth A mouse sarcoma in the standard in vivo mouse TNF assay; (ii) has the same pattern of reactivity as mouse TNF (cytotoxic/cytostatic/no effect) on a panel of human cancer cell lines; and (iii) has its anticellular effect potentiated by interferon, also a feature of mouse TNF.
N-myc gene copy numbers and transcription rates are similar in N (neuroblastic, tumorigenic) and S (non-neuronal, non-tumorigenic) neuroblastoma cells with chromosomally integrated amplified N-myc genes. However, N cells show significantly higher N-myc mRNA levels than S cells. Therefore, post-transcriptional control of N-myc gene expression must differ between these cell types. Since no differences in N-myc mRNA half-life were found between N and S cells from two cell lines, steady-state levels of N-myc pre-mRNA processing intermediates were analysed. Results suggest that the differences in N-myc expression arise primarily at the nuclear post-transcriptional level. The neuronal-specific RNA-binding Hu proteins are present in cytoplasmic and nuclear fractions of N cells and one of them, HuD, binds specifically to both exonic and intronic N-myc RNA sequences. In sense and antisense HuD-transfected N cells, there are coordinate changes in HuD and N-myc expression levels. Thus, we propose that HuD plays a role in the nuclear processing/stability of N-myc pre-mRNA in N-type neuroblastoma cells.
HT4 hippocampal neuronal cells were studied to compare the efficacy of tocopherols and tocotrienol to protect against glutamate-induced death. Tocotrienols were more effective than alpha-tocopherol in preventing glutamate-induced death. Uptake of tocotrienols from the culture medium was more efficient compared with that of alpha-tocopherol. Vitamin E molecules have potent antioxidant properties. Results show that at low concentrations, tocotrienols may have protected cells by an antioxidant-independent mechanism. Examination of signal transduction pathways revealed that protein tyrosine phosphorylation processes played a central role in the execution of death. Activation of pp60(c-Src) kinase and phosphorylation of ERK were observed in response to glutamate treatment. Nanomolar amounts of alpha-tocotrienol, but not alpha-tocopherol, blocked glutamate-induced death by suppressing glutamate-induced early activation of c-Src kinase. Overexpression of kinase-active c-Src sensitized cells to glutamate-induced death. Tocotrienol treatment prevented death of Src-overexpressing cells treated with glutamate. alpha-Tocotrienol did not influence activity of recombinant c-Src kinase suggesting that its mechanism of action may include regulation of SH domains. This study provides first evidence describing the molecular basis of tocotrienol action. At a concentration 4-10-fold lower than levels detected in plasma of supplemented humans, tocotrienol regulated unique signal transduction processes that were not sensitive to comparable concentrations of tocopherol.
A critical step in the activation of NF-κB is the phosphorylation of IκBs by the IκB kinase (IKK) complex. IKKα and IKKβ are
the two catalytic subunits of the IKK complex and two additional molecules, IKKγ/NEMO and IKAP, have been described as further
integral members. We have analyzed the function of both proteins for IKK complex composition and NF-κB signaling. IKAP and
IKKγ belong to distinct cellular complexes. Quantitative association of IKKγ was observed with IKKα and IKKβ. In contrast
IKAP was complexed with several distinct polypeptides. Overexpression of either IKKγ or IKAP blocked tumor necrosis factor
α induction of an NF-κB-dependent reporter construct, but IKAP in addition affected several NF-κB-independent promoters. Whereas
specific down-regulation of IKKγ protein levels by antisense oligonucleotides significantly reduced cytokine-mediated activation
of the IKK complex and subsequent NF-κB activation, a similar reduction of IKAP protein levels had no effect on NF-κB signaling.
Using solely IKKα, IKKβ, and IKKγ, we could reconstitute a complex whose apparent molecular weight is comparable to that of
the endogenous IKK complex. We conclude that while IKKγ is a stoichiometric component of the IKK complex, obligatory for NF-κB
signaling, IKAP is not associated with IKKs and plays no specific role in cytokine-induced NF-κB activation.
Human Elongator complex was purified to virtual homogeneity from HeLa cell extracts. The purified factor can exist in two
forms: a six-subunit complex, holo-Elongator, which has histone acetyltransferase activity directed against histone H3 and
H4, and a three-subunit core form, which does not have histone acetyltransferase activity despite containing the catalytic
Elp3 subunit. Elongator is a component of early elongation complexes formed in HeLa nuclear extracts and can interact directly
with RNA polymerase II in solution. Several human homologues of the yeast Elongator subunits were identified as subunits of
the human Elongator complex, including StIP1 (STAT-interacting protein1) and IKAP (IKK complex-associatedprotein). Mutations in IKAP can result in the severe human disorder familial dysautonomia, raising the possibility that this
disease might be due to compromised Elongator function and therefore could be a transcription disorder.
We demonstrate here a novel role for the IκB kinase complex-associated protein (IKAP) in the regulation of activation of the
mammalian stress response via the c-Jun N-terminal kinase (JNK)-signaling pathway. We cloned IKAP as a JNK-associating protein
using the Ras recruitment yeast two-hybrid system. IKAP efficiently and specifically enhanced JNK activation induced by ectopic
expression of MEKK1 and ASK1, upstream activators of JNK. Importantly, IKAP also enhanced JNK activation induced by ultraviolet
light irradiation as well as treatments with tumor necrosis factor or epidermal growth factor. The JNK association site in
IKAP was mapped to the C-terminal part of IKAP. Interestingly, this region is deleted from IKAP expressed in the autonomous
nervous system of the patients affected by familial dysautonomia. Ectopic expression of this C-terminal fragment of IKAP was
sufficient to support JNK activation. Taken together, our data demonstrate a novel role for IKAP in the regulation of the
JNK-mediated stress signaling. Additionally, our results point to a role of JNK signaling in familial dysautonomia and, thus,
further support the involvement of JNK signaling in the development, survival, and degeneration of the sensory and autonomic
nervous system.
We characterized the 3272-26A→G mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, creating an alternative acceptor splice site in intron 17a, that competes with the normal one, although we predict from consensus values, with lower efficiency. We analyzed five Cystic Fibrosis (CF) Portuguese patients with the 3272-26A→G/F508del genotype. Besides clinical and haplotype characterization of those patients, we report here results from CFTR transcript analysis in nasal brushings from all five patients. RT-PCR analysis supports alternative splicing in all patients and carriers, but not in controls. By sequencing, we determined that the alternative transcript includes 25 nucleotides from intron 17a, which predictively cause frameshift and a premature stop codon. The use of this alternative splice site causes a reduction in the levels of normal transcripts from the allele with this mutation and, most probably, of normal protein as well. By immunocytochemistry of both epithelial primary cell cultures and slices from CF polyps, CFTR protein is detected at the cell membrane, with three different antibodies. Ussing chamber analysis of one nasal polyp shows a high sodium absorption, characteristic of CF. Altogether, the results suggest that the main defect caused by the 3272-26A→G mutation is a reduction in normal CFTR transcripts and protein and therefore this mutation should be included in class V, according to Zielenski and Tsui. Hum Mutat 14:133–144, 1999. © 1999 Wiley-Liss, Inc.
Tocotrienols exhibit antioxidant and cholesterol-biosynthesis-inhibitory activities and may be of value as antiatherosclerotic agents. The mechanism of their hypolipidemic action involves; posttranscriptional suppression of HMG-CoA reductase (HMGR) in a manner mimicking the; action of putative non-sterol feedback inhibitors. The in vitro cholesterol-biosynthesis-inhibitory and HMGR-suppressive activities in HepG2 cells of an expanded series of benzopyran and tetrahydronaphthalene isosteres and the hypocholesterolemic activity:of selected compounds assessed in orally dosed chickens are presented. Preliminary antioxidant data of these compounds have been obtained using cyclic voltammetry and Cu-induced:LDL oxidation assays. The farnesyl side chain and the methyl/hydroxy substitution pattern of gamma-tocotrienol deliver a high level of HMGR Suppression, unsurpassed by synthetic analogues of the present study. In orally dosed; chickens, 8-bromotocotrienol (40), 2-desmethyltocotrienol (4t), and the tetrahydronaphthalene derivative 35 exhibit a greater degree of LDL cholesterol lowering than the natural tocotrienols.
Glycogen storage disease type II (GSDII), an autosomal recessive myopathic disorder, results from deficiency of lysosomal acid alpha-glucosidase. We searched for mutations in an evolutionarily conserved region in 54 patients of differing phenotype. Four novel mutations (D645N, G448S, R672W, and R672Q) and a previously described mutation (C647W) were identified in five patients and their deleterious effect on enzyme expression demonstrated in vitro. Two novel frame-shifting insertions/deletions (delta nt766-785/insC and +insG@nt2243) were identified in two patients with exon 14 mutations. The remaining three patients were either homozygous for their mutations (D645N/D645 and C647W/C647W) or carried a previously described leaky splice site mutation (IVS1-13T-->G). For all patients "in vivo" enzyme activity was consistent with clinical phenotype. Agreement of genotype with phenotype and in vitro versus in vivo enzyme was seen in three patients (two infantile patients carrying C647W/C647W and D645N/+insG@nt2243 and an adult patient heteroallelic for G648S/IVS1-13T-->G). Relative discordance was found in a juvenile patient homozygous for the non-expressing R672Q and an adult patient heterozygous for the minimally expressing R672W and delta nt766-785/+insC. Possible explanations include differences in in vitro assays vs in vivo enzyme activity, tissue specific expression with diminished enzyme expression/stability in fibroblasts vs muscle, somatic mosaicism, and modifying genes.
The defective gene DYS, which is responsible for familial dysautonomia (FD) and has been mapped to a 0.5-cM region on chromosome 9q31, has eluded identification. We identified and characterized the RNAs encoded by this region of chromosome 9 in cell lines derived from individuals homozygous for the major FD haplotype, and we observed that the RNA encoding the IκB kinase complex–associated protein (IKAP) lacks exon 20 and, as a result of a frameshift, encodes a truncated protein. Sequence analysis reveals a T→C transition in the donor splice site of intron 20. In individuals bearing a minor FD haplotype, a missense mutation in exon 19 disrupts a consensus serine/threonine kinase phosphorylation site. This mutation results in defective phosphorylation of IKAP. These mutations were observed to be present in a random sample of Ashkenazi Jewish individuals, at approximately the predicted carrier frequency of FD. These findings demonstrate that mutations in the gene encoding IKAP are responsible for FD.
Tocotrienols are farnesylated benzopyran natural products that exhibit hypocholesterolemic activity in vitro and in vivo. The mechanism of their hypolipidemic action involves posttranscriptional suppression of HMG-CoA reductase by a process distinct from other known inhibitors of cholesterol biosynthesis. An efficient synthetic route to tocotrienols and their isolation from palm oil distillate using an improved procedure is presented. gamma-Tocotrienol exhibits a 30-fold greater activity toward cholesterol biosynthesis inhibition compared to alpha-tocotrienol in HepG2 cells in vitro. The synthetic (racemic) and natural (chiral) tocotrienols exhibit nearly identical cholesterol biosynthesis inhibition and HMG-CoA reductase suppression properties as demonstrated in vitro and in vivo.
d-Alpha-tocopherol (2R,4'R,8'R-Alpha-tocopherol) and d-alpha-tocotrienol are two vitamin E constituents having the same aromatic chromanol "head" but differing in their hydrocarbon "tail": tocopherol with a saturated and toctrienol with an unsaturated isoprenoid chain. d-Alpha-tocopherol has the highest vitamin E activity, while d-alpha-tocotrienol manifests only about 30% of this activity. Since vitamin E is considered to be physiologically the most important lipid-soluble chain-breaking antioxidant of membranes, we studied alpha-tocotrienol as compared to alpha-tocopherol under conditions which are important for their antioxidant function. d-Alpha-tocotrienol possesses 40-60 times higher antioxidant activity against (Fe2+ + ascorbate)- and (Fe2+ + NADPH)-induced lipid peroxidation in rat liver microsomal membranes and 6.5 times better protection of cytochrome P-450 against oxidative damage than d-alpha-tocopherol. To clarify the mechanisms responsible for the much higher antioxidant potency of d-alpha-tocotrienol compared to d-alpha-tocopherol, ESR studies were performed of recycling efficiency of the chromanols from their chromanoxyl radicals. 1H-NMR measurements of lipid molecular mobility in liposomes containing chromanols, and fluorescence measurements which reveal the uniformity of distribution (clusterizations) of chromanols in the lipid bilayer. From the results, we concluded that this higher antioxidant potency of d-alpha-tocotrienol is due to the combined effects of three properties exhibited by d-alpha-tocotrienol as compared to d-alpha-tocopherol: (i) its higher recycling efficiency from chromanoxyl radicals, (ii) its more uniform distribution in membrane bilayer, and (iii) its stronger disordering of membrane lipids which makes interaction of chromanols with lipid radicals more efficient. The data presented show that there is a considerable discrepancy between the relative in vitro antioxidant activity of d-alpha-tocopherol and d-alpha-tocotrienol with the conventional bioassays of their vitamin activity.
An autosomal recessive deficiency of acid alpha-glucosidase (GAA), type II glycogenosis, is genetically and clinically heterogeneous. The discovery of an enzyme-inactivating genomic deletion of exon 18 in three unrelated genetic compound patients--two infants and an adult--provided a rare opportunity to analyze the effect of the second mutation in patients who displayed dramatically different phenotypes. A deletion of Lys-903 in one patient and a substitution of Arg for Leu-299 in another resulted in the fatal infantile form. In the adult, a T-to-G base change at position -13 of intron 1 resulted in alternatively spliced transcripts with deletion of exon 2, the location of the start codon. The low level of active enzyme (12% of normal) generated from the leakage of normally spliced mRNA sustained the patient to adult life.
This chapter describes the antioxidant properties of α-tocopherol and α-tocotrienol. Tocopherols and tocotrienols are present in various components of the human diet. Tocopherols are found in polyunsaturated vegetable oils and in the germ of cereal seeds, whereas tocotrienols are found in the aleurone and subaleurone layers of cereal seeds and in palm oil. Although the tocopherols and tocotrienols are closely related chemically, they have widely varying degrees of biological effectiveness. The potency of α-tocotrienol evaluated by gestation-resorption assays are 32% of the potency of α-tocopherol. The chapter discusses the relative and the absolute antioxidant effectiveness in vitro of the individual tocopherols that make up vitamin E. It is recognized that differences in vivo in the antioxidant activity of tocopherols and tocotrienols may depend greatly on their pharmacokinetics. However, α-tocotrienol may have higher antioxidant activity in vivo under conditions of oxidative stress because of its more effective antioxidant potency in membranes.
Tocotrienols exhibit antioxidant and cholesterol-biosynthesis-inhibitory activities and may be of value as antiatherosclerotic agents. The mechanism of their hypolipidemic action involves posttranscriptional suppression of HMG-CoA reductase (HMGR) in a manner mimicking the action of putative non-sterol feedback inhibitors. The in vitro cholesterol-biosynthesis-inhibitory and HMGR-suppressive activities in HepG2 cells of an expanded series of benzopyran and tetrahydronaphthalene isosteres and the hypocholesterolemic activity of selected compounds assessed in orally dosed chickens are presented. Preliminary antioxidant data of these compounds have been obtained using cyclic voltammetry and Cu-induced LDL oxidation assays. The farnesyl side chain and the methyl/hydroxy substitution pattern of gamma-tocotrienol deliver a high level of HMGR suppression, unsurpassed by synthetic analogues of the present study. In orally dosed chickens, 8-bromotocotrienol (4o), 2-desmethyltocotrienol (4t), and the tetrahydronaphthalene derivative 35 exhibit a greater degree of LDL cholesterol lowering than the natural tocotrienols.
The transcription factor NF-kappaB coordinates the activation of numerous genes in response to pathogens and pro-inflammatory cytokines, and is, therefore, vital in the development of acute and chronic inflammatory diseases. NF-kappaB is activated by phsophorylation of its inhibitory subunit, IkappaB-alpha, on serine residues 32 and 36 by cytokine-activated IKB kinases (IKKs); this phosphorylation precedes rapid degradation of IkappaB. IKK-alpha and IKK-beta isozymes are found in large complexes of relative molecular mass 700,000-900,000 (M(r) 70K-90K), but little is known about other components that organize and regulate these complexes. IKK-alpha was independently discovered as a NF-kappaB-inducing kinase (NIK)-associated protein in a yeast two-hybrid screen, and IKK-beta was also identified by homology screening. It is, however, unknown whether NIK is part of the IKK complex. Here we isolate large, interleukin-1-inducible IKK complexes that contain NIK, IKK-alpha, IKK-beta, IkappaB-alpha, NF-kappaB/RelA and a protein of M(r) 150K. This latter component is a new protein, termed IKK-complex-associated protein (IKAP), which can bind NIK and IKKs and assemble them into an active kinase complex. We show that IKAP is a scaffold protein and a regulator for three different kinases involved in pro-inflammatory cytokine signalling.
The form of RNA polymerase II (RNAPII) engaged in transcriptional elongation was isolated. Elongating RNAPII was associated with a novel multisubunit complex, termed elongator, whose stable interaction was dependent on a hyperphosphorylated state of the RNAPII carboxy-terminal domain (CTD). A free form of elongator was also isolated, demonstrating the discrete nature of the complex, and free elongator could bind directly to RNAPII. The gene encoding the largest subunit of elongator, ELP1, was cloned. Phenotypes of yeast elp1 delta cells demonstrated an involvement of elongator in transcriptional elongation as well as activation in vivo. Our data indicate that the transition from transcriptional initiation to elongation involves an exchange of the multiprotein mediator complex for elongator in a reaction coupled to CTD hyperphosphorylation.
We characterized the 3272-26A-->G mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, creating an alternative acceptor splice site in intron 17a, that competes with the normal one, although we predict from consensus values, with lower efficiency. We analyzed five Cystic Fibrosis (CF) Portuguese patients with the 3272-26A-->G/F508del genotype. Besides clinical and haplotype characterization of those patients, we report here results from CFTR transcript analysis in nasal brushings from all five patients. RT-PCR analysis supports alternative splicing in all patients and carriers, but not in controls. By sequencing, we determined that the alternative transcript includes 25 nucleotides from intron 17a, which predictively cause frameshift and a premature stop codon. The use of this alternative splice site causes a reduction in the levels of normal transcripts from the allele with this mutation and, most probably, of normal protein as well. By immunocytochemistry of both epithelial primary cell cultures and slices from CF polyps, CFTR protein is detected at the cell membrane, with three different antibodies. Ussing chamber analysis of one nasal polyp shows a high sodium absorption, characteristic of CF. Altogether, the results suggest that the main defect caused by the 3272-26A-->G mutation is a reduction in normal CFTR transcripts and protein and therefore this mutation should be included in class V, according to Zielenski and Tsui.
We report 2 patients with atypical glycogen storage disease type Ib without neutropenia or infectious complications. Neither patient was deficient in hepatic glucose-6-phosphatase activities in microsome-disrupted homogenates; both had mutations in the glucose-6-phosphate transporter gene, suggesting an allelic variant of glycogen storage disease type Ib.
The defective gene DYS, which is responsible for familial dysautonomia (FD) and has been mapped to a 0.5-cM region on chromosome 9q31, has eluded identification. We identified and characterized the RNAs encoded by this region of chromosome 9 in cell lines derived from individuals homozygous for the major FD haplotype, and we observed that the RNA encoding the IkappaB kinase complex-associated protein (IKAP) lacks exon 20 and, as a result of a frameshift, encodes a truncated protein. Sequence analysis reveals a T-->C transition in the donor splice site of intron 20. In individuals bearing a minor FD haplotype, a missense mutation in exon 19 disrupts a consensus serine/threonine kinase phosphorylation site. This mutation results in defective phosphorylation of IKAP. These mutations were observed to be present in a random sample of Ashkenazi Jewish individuals, at approximately the predicted carrier frequency of FD. These findings demonstrate that mutations in the gene encoding IKAP are responsible for FD.
Familial dysautonomia (FD; also known as "Riley-Day syndrome"), an Ashkenazi Jewish disorder, is the best known and most frequent of a group of congenital sensory neuropathies and is characterized by widespread sensory and variable autonomic dysfunction. Previously, we had mapped the FD gene, DYS, to a 0.5-cM region on chromosome 9q31 and had shown that the ethnic bias is due to a founder effect, with >99.5% of disease alleles sharing a common ancestral haplotype. To investigate the molecular basis of FD, we sequenced the minimal candidate region and cloned and characterized its five genes. One of these, IKBKAP, harbors two mutations that can cause FD. The major haplotype mutation is located in the donor splice site of intron 20. This mutation can result in skipping of exon 20 in the mRNA of patients with FD, although they continue to express varying levels of wild-type message in a tissue-specific manner. RNA isolated from lymphoblasts of patients is primarily wild-type, whereas only the deleted message is seen in RNA isolated from brain. The mutation associated with the minor haplotype in four patients is a missense (R696P) mutation in exon 19, which is predicted to disrupt a potential phosphorylation site. Our findings indicate that almost all cases of FD are caused by an unusual splice defect that displays tissue-specific expression; and they also provide the basis for rapid carrier screening in the Ashkenazi Jewish population.
Pure hereditary spastic paraplegia (SPG) type 4 is the most common form of autosomal dominant hereditary SPG, a neurodegenerative disease characterized primarily by hyperreflexia and progressive spasticity of the lower limbs. It is caused by mutations in the gene encoding spastin, a member of the AAA family of ATPases. We have screened the spastin gene for mutations in 15 families consistent with linkage to the spastin gene locus, SPG4, and have identified 11 mutations, 10 of which are novel. Five of the mutations identified are in noninvariant splice-junction sequences. Reverse transcription-PCR analysis of mRNA from patients shows that each of these five mutations results in aberrant splicing. One mutation was found to be "leaky," or partially penetrant; that is, the mutant allele produced both mutant (skipped exon) and wild-type (full-length) transcripts. This phenomenon was reproduced in in vitro splicing experiments, with a minigene splicing-vector construct only in the context of the endogenous splice junctions flanking the splice junctions of the skipped exon. In the absence of endogenous splice junctions, only mutant transcript was detected. The existence of at least one leaky mutation suggests that relatively small differences in the level of wild-type spastin expression can have significant functional consequences. This may account, at least in part, for the wide ranges in age at onset, symptom severity, and rate of symptom progression that have been reported to occur both among and within families with SPG linked to SPG4. In addition, these results suggest caution in the interpretation of data solely obtained with minigene constructs to study the effects of sequence variation on splicing. The lack of full genomic sequence context in these constructs can mask important functional consequences of the mutation.
The comparison of the gene expression profiles between two subpopulations of melanoma cells (1C8 and T1C3) derived from the tumor of one patient by cDNA array revealed differences in GAPDH and beta-actin gene levels. These two housekeeping genes were up-regulated in invasive T1C3 melanoma cells compared to noninvasive 1C8 cells. Since cDNA array results were not confirmed by conventional RT-PCR throughout the exponential phase of amplification, we performed duplex relative RT-PCR using ribosomal 18S RNA as internal standard including competimer technology. Statistical analyses provided significant evidence that invasive T1C3 melanoma cells exhibited a twofold higher mRNA level of both GAPDH and beta-actin than noninvasive 1C8 cells. This study demonstrates that the duplex relative RT-PCR procedure including ribosomal 18S RNA as internal standard and competimer technology is precise for RNA quantification and is tailored for cDNA array validation. Our data provide molecular evidence that cellular subpopulations of the same pathological origin are highly heterogeneous and extend the concept that the selection of an appropriate internal control for comparative mRNA analysis should be adapted to each model of human cancers.
Tocopherols and tocotrienols are metabolized by side chain degradation via initial omega-oxidation and subsequent beta-oxidation. omega-Oxidation is performed by cytochrome P450 (CYP) enzymes which are often regulated by their substrates themselves. Results presented here show that all forms of Vitamin E are able to activate gene expression via the pregnane X receptor (PXR), a nuclear receptor regulating a variety of drug metabolizing enzymes. In HepG2 cells transfected with the human PXR and the chloramphenicol acetyl transferase (CAT) gene linked to two PXR responsive elements, CAT activity was most strongly induced by alpha- and gamma-tocotrienol followed by rifampicin, delta-, alpha- and gamma-tocopherol. The inductive efficacy was concentration-dependent; its specificity was underscored by a lower response when cotransfection with PXR was omitted. Up-regulation of endogenous CYP3A4 and CYP3A5 mRNA was obtained by gamma-tocotrienol, the most potent activator of PXR, with the same efficacy as with rifampicin. This points to a potential interference of individual forms of Vitamin E with the metabolism and efficacy of drugs.
We recently identified a mutation in the I-kappa B kinase associated protein (IKBKAP) gene as the major cause of familial dysautonomia (FD), a recessive sensory and autonomic neuropathy. This alteration, located at base pair 6 of the intron 20 donor splice site, is present on >99.5% of FD chromosomes and results in tissue-specific skipping of exon 20. A second FD mutation, a missense change in exon 19 (R696P), was seen in only four patients heterozygous for the major mutation. Here, we have further characterized the consequences of the major mutation by examining the ratio of wild-type to mutant (WT:MU) IKBKAP transcript in EBV-transformed lymphoblast lines, primary fibroblasts, freshly collected blood samples, and postmortem tissues from patients with FD. We consistently found that WT IKBKAP transcripts were present, albeit to varying extents, in all cell lines, blood, and postmortem FD tissues. Further, a corresponding decrease in the level of WT protein is seen in FD cell lines and tissues. The WT:MU ratio in cultured lymphoblasts varied with growth phase but not with serum concentration or inclusion of antibiotics. Using both densitometry and real-time quantitative polymerase chain reaction, we found that relative WT:MU IKBKAP RNA levels were highest in cultured patient lymphoblasts and lowest in postmortem central and peripheral nervous tissues. These observations suggest that the relative inefficiency of WT IKBKAP mRNA production from the mutant alleles in the nervous system underlies the selective degeneration of sensory and autonomic neurons in FD.Therefore, exploration of methods to increase the WT:MU IKBKAP transcript ratio in the nervous system offers a promising approach for developing an effective therapy for patients with FD.
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- J L Biedler
- B A Spengler
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and two insertions/deletions in the acid alpha-glucosidase locus of patients of differing phenotype
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Central autonomic dysfunction with defective lacrimation
- C M Riley
- R L Day
- D Greely
- W S Langford
C.M. Riley, R.L. Day, D. Greely, W.S. Langford, Central
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Human neuroblastoma cell differentiation
- Biedler