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

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

Therapeutic manipulation of IKBKAP mis-splicing with a small molecule to cure familial dysautonomia.

Approximately half of genetic disease-associated mutations cause aberrant splicing. However, a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Here, we analyze the mechanism whereby IKBKAP-familial dysautonomia (FD) exon 20 inclusion is specifically promoted by a small molecule splice modulator, RECTAS, even though IKBKAP-FD exon 20 has a suboptimal 5' splice site due to the IVS20 + 6 T > C mutation. Knockdown experiments reveal that exon 20 inclusion is suppressed in the absence of serine/arginine-rich splicing factor 6 (SRSF6) binding to an intronic splicing enhancer in intron 20. We show that RECTAS directly interacts with CDC-like kinases (CLKs) and enhances SRSF6 phosphorylation. Consistently, exon 20 splicing is bidirectionally manipulated by targeting cellular CLK activity with RECTAS versus CLK inhibitors. The therapeutic potential of RECTAS is validated in multiple FD disease models. Our study indicates that small synthetic molecules affecting phosphorylation state of SRSFs is available as a new therapeutic modality for mechanism-oriented precision medicine of splicing diseases.

Carbidopa for Afferent Baroreflex Failure in Familial Dysautonomia: A Double-Blind Randomized Crossover Clinical Trial.

Afferent lesions of the arterial baroreflex occur in familial dysautonomia. This leads to excessive blood pressure variability with falls and frequent surges that damage the organs. These hypertensive surges are the result of excess peripheral catecholamine release and have no adequate treatment. Carbidopa is a selective DOPA-decarboxylase inhibitor that suppresses catecholamines production outside the brain. To learn whether carbidopa can inhibit catecholamine-induced hypertensive surges in patients with severe afferent baroreflex failure, we conducted a double-blind randomized crossover trial in which patients with familial dysautonomia received high dose carbidopa (600 mg/day), low-dose carbidopa (300 mg/day), or matching placebo in 3 4-week treatment periods. Among the 22 patients enrolled (13 females/8 males), the median age was 26 (range, 12-59 years). At enrollment, patients had hypertensive peaks to 164/116 (range, 144/92 to 213/150 mm Hg). Twenty-four hour urinary norepinephrine excretion, a marker of peripheral catecholamine release, was significantly suppressed on both high dose and low dose carbidopa, compared with placebo (P=0.0075). The 2 co-primary end points of the trial were met. The SD of systolic BP variability was reduced at both carbidopa doses (low dose: 17±4; high dose: 18±5 mm Hg) compared with placebo (23±7 mm Hg; P=0.0013), and there was a significant reduction in the systolic BP peaks on active treatment (P=0.0015). High- and low-dose carbidopa were similarly effective and well tolerated. This study provides class Ib evidence that carbidopa can reduce blood pressure variability in patients with congenital afferent baroreflex failure. Similar beneficial effects are observed in patients with acquired baroreflex lesions.

Combinatorial treatment increases IKAP levels in human cells generated from Familial Dysautonomia patients.

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.

Intranasal dexmedetomidine for adrenergic crisis in familial dysautonomia.

PURPOSE: To report the use of intranasal dexmedetomidine, an α2-adrenergic agonist for the acute treatment of refractory adrenergic crisis in patients with familial dysautonomia. METHODS: Case series. RESULTS: Three patients with genetically confirmed familial dysautonomia (case 1: 20-year-old male; case 2: 43-year-old male; case 3: 26-year-old female) received intranasal dexmedetomidine 2 mcg/kg, half of the dose in each nostril, for the acute treatment of adrenergic crisis. Within 8-17 min of administering the intranasal dose, the adrenergic crisis symptoms abated, and blood pressure and heart rate returned to pre-crises values. Adrenergic crises eventually resumed, and all three patients required hospitalization for investigation of the cause of the crises. CONCLUSIONS: Intranasal dexmedetomidine is a feasible and safe acute treatment for adrenergic crisis in patients with familial dysautonomia. Further controlled studies are required to confirm the safety and efficacy in this population.

BGP-15 prevents the death of neurons in a mouse model of familial dysautonomia.

Hereditary sensory and autonomic neuropathy type III, or familial dysautonomia [FD; Online Mendelian Inheritance in Man (OMIM) 223900], affects the development and long-term viability of neurons in the peripheral nervous system (PNS) and retina. FD is caused by a point mutation in the gene IKBKAP/ELP1 that results in a tissue-specific reduction of the IKAP/ELP1 protein, a subunit of the Elongator complex. Hallmarks of the disease include vasomotor and cardiovascular instability and diminished pain and temperature sensation caused by reductions in sensory and autonomic neurons. It has been suggested but not demonstrated that mitochondrial function may be abnormal in FD. We previously generated an Ikbkap/Elp1 conditional-knockout mouse model that recapitulates the selective death of sensory (dorsal root ganglia) and autonomic neurons observed in FD. We now show that in these mice neuronal mitochondria have abnormal membrane potentials, produce elevated levels of reactive oxygen species, are fragmented, and do not aggregate normally at axonal branch points. The small hydroxylamine compound BGP-15 improved mitochondrial function, protecting neurons from dying in vitro and in vivo, and promoted cardiac innervation in vivo. Given that impairment of mitochondrial function is a common pathological component of neurodegenerative diseases such as amyotrophic lateral sclerosis and Alzheimer's, Parkinson's, and Huntington's diseases, our findings identify a therapeutic approach that may have efficacy in multiple degenerative conditions.

Dexmedetomidine for refractory adrenergic crisis in familial dysautonomia.

OBJECTIVE: Adrenergic crises are a cardinal feature of familial dysautonomia (FD). Traditionally, adrenergic crises have been treated with the sympatholytic agent clonidine or with benzodiazepines, which can cause excessive sedation and respiratory depression. Dexmedetomidine is a centrally-acting α 2-adrenergic agonist with greater selectivity and shorter half-life than clonidine. We evaluated the preliminary effectiveness and safety of intravenous dexmedetomidine in the treatment of refractory adrenergic crisis in patients with FD. METHODS: Retrospective chart review of patients with genetically confirmed FD who received intravenous dexmedetomidine for refractory adrenergic crises. The primary outcome was preliminary effectiveness of dexmedetomidine defined as change in blood pressure (BP) and heart rate (HR) 1 h after the initiation of dexmedetomidine. Secondary outcomes included incidence of adverse events related to dexmedetomidine, hospital and intensive care unit (ICU) length of stay, and hemodynamic parameters 12 h after dexmedetomidine cessation. RESULTS: Nine patients over 14 admissions were included in the final analysis. At 1 h after the initiation of dexmedetomidine, systolic BP decreased from 160 ± 7 to 122 ± 7 mmHg (p = 0.0005), diastolic BP decreased from 103 ± 6 to 65 ± 8 (p = 0.0003), and HR decreased from 112 ± 4 to 100 ± 5 bpm (p = 0.0047). The median total adverse events during dexmedetomidine infusion was 1 per admission. Median hospital length of stay was 9 days [interquartile range (IQR) 3-11 days] and median ICU length of stay was 7 days (IQR 3-11 days). CONCLUSIONS: Intravenous dexmedetomidine is safe in patients with FD and appears to be effective to treat refractory adrenergic crisis. Dexmedetomidine may be considered in FD patients who do not respond to conventional clonidine and benzodiazepine pharmacotherapy.

Familial dysautonomia: History, genotype, phenotype and translational research.

Familial dysautonomia (FD) is a rare neurological disorder caused by a splice mutation in the IKBKAP gene. The mutation arose in the 1500s within the small Jewish founder population in Eastern Europe and became prevalent during the period of rapid population expansion within the Pale of Settlement. The carrier rate is 1:32 in Jews descending from this region. The mutation results in a tissue-specific deficiency in IKAP, a protein involved in the development and survival of neurons. Patients homozygous for the mutations are born with multiple lesions affecting mostly sensory (afferent) fibers, which leads to widespread organ dysfunction and increased mortality. Neurodegenerative features of the disease include progressive optic atrophy and worsening gait ataxia. Here we review the progress made in the last decade to better understand the genotype and phenotype. We also discuss the challenges of conducting controlled clinical trials in this rare medically fragile population. Meanwhile, the search for better treatments as well as a neuroprotective agent is ongoing.

Phosphatidylserine Ameliorates Neurodegenerative Symptoms and Enhances Axonal Transport in a Mouse Model of Familial Dysautonomia.

Familial Dysautonomia (FD) is a neurodegenerative disease in which aberrant tissue-specific splicing of IKBKAP exon 20 leads to reduction of IKAP protein levels in neuronal tissues. Here we generated a conditional knockout (CKO) mouse in which exon 20 of IKBKAP is deleted in the nervous system. The CKO FD mice exhibit developmental delays, sensory abnormalities, and less organized dorsal root ganglia (DRGs) with attenuated axons compared to wild-type mice. Furthermore, the CKO FD DRGs show elevated HDAC6 levels, reduced acetylated α-tubulin, unstable microtubules, and impairment of axonal retrograde transport of nerve growth factor (NGF). These abnormalities in DRG properties underlie neuronal degeneration and FD symptoms. Phosphatidylserine treatment decreased HDAC6 levels and thus increased acetylation of α-tubulin. Further PS treatment resulted in recovery of axonal outgrowth and enhanced retrograde axonal transport by decreasing histone deacetylase 6 (HDAC6) levels and thus increasing acetylation of α-tubulin levels. Thus, we have identified the molecular pathway that leads to neurodegeneration in FD and have demonstrated that phosphatidylserine treatment has the potential to slow progression of neurodegeneration.

Tocotrienol Treatment in Familial Dysautonomia: Open-Label Pilot Study.

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.

Phosphatidylserine enhances IKBKAP transcription by activating the MAPK/ERK signaling pathway.

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.

Rectifier of aberrant mRNA splicing recovers tRNA modification in familial dysautonomia.

Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by missplicing of exon 20, resulting from an intronic mutation in the inhibitor of kappa light polypeptide gene enhancer in B cells, kinase complex-associated protein (IKBKAP) gene encoding IKK complex-associated protein (IKAP)/elongator protein 1 (ELP1). A newly established splicing reporter assay allowed us to visualize pathogenic splicing in cells and to screen small chemicals for the ability to correct the aberrant splicing of IKBKAP. Using this splicing reporter, we screened our chemical libraries and identified a compound, rectifier of aberrant splicing (RECTAS), that rectifies the aberrant IKBKAP splicing in cells from patients with FD. Here, we found that the levels of modified uridine at the wobble position in cytoplasmic tRNAs are reduced in cells from patients with FD and that treatment with RECTAS increases the expression of IKAP and recovers the tRNA modifications. These findings suggest that the missplicing of IKBKAP results in reduced tRNA modifications in patients with FD and that RECTAS is a promising therapeutic drug candidate for FD.

Modulation of alternative splicing with chemical compounds in new therapeutics for human diseases.

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.

Current treatments in familial dysautonomia.

INTRODUCTION: Familial dysautonomia (FD) is a rare hereditary sensory and autonomic neuropathy (type III). The disease is caused by a point mutation in the IKBKAP gene that affects the splicing of the elongator-1 protein (ELP-1) (also known as IKAP). Patients have dramatic blood pressure instability due to baroreflex failure, chronic kidney disease, and impaired swallowing leading to recurrent aspiration pneumonia, which results in chronic lung disease. Diminished pain and temperature perception result in neuropathic joints and thermal injuries. Impaired proprioception leads to gait ataxia. Optic neuropathy and corneal opacities lead to progressive visual loss. AREAS COVERED: This article reviews current therapeutic strategies for the symptomatic treatment of FD, as well as the potential of new gene-modifying agents. EXPERT OPINION: Therapeutic focus on FD is centered on reducing the catecholamine surges caused by baroreflex failure. Managing neurogenic dysphagia with effective protection of the airway passages and prompt treatment of aspiration pneumonias is necessary to prevent respiratory failure. Sedative medications should be used cautiously due to the risk of respiratory depression. Non-invasive ventilation during sleep effectively manages apneas and prevents hypercapnia. Clinical trials of compounds that increase levels of IKAP (ELP-1) are underway and will determine whether they can reverse or slow disease progression.

Personalized drug discovery: HCA approach optimized for rare diseases at Tel Aviv University.

The Cell screening facility for personalized medicine (CSFPM) at Tel Aviv University in Israel is devoted to screening small molecules libraries for finding new drugs for rare diseases using human cell based models. The main strategy of the facility is based on smartly reducing the size of the compounds collection in similarity clusters and at the same time keeping high diversity of pharmacophores. This strategy allows parallel screening of several patient derived - cells in a personalized screening approach. The tested compounds are repositioned drugs derived from collections of phase III and FDA approved small molecules. In addition, the facility carries screenings using other chemical libraries and toxicological characterizations of nanomaterials.

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

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

Hyperdopaminergic crises in familial dysautonomia: a randomized trial of carbidopa.

OBJECTIVE: The purpose of this study was to determine whether carbidopa (Lodosyn), an inhibitor of dopa-decarboxylase that blocks the synthesis of dopamine outside the brain, is an effective antiemetic in patients with familial dysautonomia (FD) and hyperdopaminergic nausea/retching/vomiting attacks. METHODS: We enrolled 12 patients with FD in an open-label titration and treatment study to assess the safety of carbidopa. We then conducted a randomized, double-blind, placebo-controlled, crossover study to evaluate its antiemetic efficacy. RESULTS: Previous fundoplication surgery in each patient studied prevented vomiting, but all of the subjects experienced severe cyclical nausea and uncontrollable retching that was refractory to standard treatments. Carbidopa at an average daily dose of 480 mg (range 325-600 mg/day) was well tolerated. In the double-blind phase, patients experienced significantly less nausea and retching while on carbidopa than on placebo (p < 0.03 and p < 0.02, respectively). Twenty-four-hour urinary dopamine excretion was significantly lower while on carbidopa (147 ± 32 µg/gCr) than while on placebo (222 ± 41µg/gCr, p < 0.05). CONCLUSIONS: Carbidopa is a safe and effective antiemetic in patients with FD, likely by reducing the formation of dopamine outside the brain. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that carbidopa is effective in reducing nausea/retching/vomiting in patients with FD.

Large-scale screening using familial dysautonomia induced pluripotent stem cells identifies compounds that rescue IKBKAP expression.

Patient-specific induced pluripotent stem cells (iPSCs) represent a novel system for modeling human genetic disease and could provide a source of cells for large-scale drug-discovery screens. Here we demonstrate the feasibility of performing a primary screen in neural crest precursors derived from iPSCs that were generated from individuals with familial dysautonomia (FD), a rare, fatal genetic disorder affecting neural crest lineages. We tested 6,912 small-molecule compounds and characterized eight that rescued expression of IKBKAP, the gene responsible for FD. One of the hits, SKF-86466, was found to induce IKBKAP transcription through modulation of intracellular cAMP levels and PKA-dependent CREB phosphorylation. SKF-86466 also rescued IKAP protein expression and the disease-specific loss of autonomic neuronal marker expression. Our data implicate alpha-2 adrenergic receptor activity in regulating IKBKAP expression and demonstrate that small-molecule discovery using an iPSC-based disease model can identify candidate drugs for potential therapeutic intervention.