RESUMO
Familial Dysautonomia (FD) is an autosomal recessive disorder caused by a splice site mutation in the gene ELP1, which disproportionally affects neurons. While classically characterized by deficits in sensory and autonomic neurons, neuronal defects in the central nervous system have also been described. Although ELP1 expression remains high in the normal developing and adult cerebellum, its role in cerebellar development is unknown. To explore the role of Elp1 in the cerebellum, we knocked out Elp1 in cerebellar granule cell progenitors (GCPs) and examined the outcome on animal behavior and cellular composition. We found that GCP-specific conditional knockout of Elp1 (Elp1cKO) resulted in ataxia by 8 weeks of age. Cellular characterization showed that the animals had smaller cerebella with fewer granule cells. This defect was already apparent as early as 7 days after birth, when Elp1cKO animals also had fewer mitotic GCPs and shorter Purkinje dendrites. Through molecular characterization, we found that loss of Elp1 was associated with an increase in apoptotic cell death and cell stress pathways in GCPs. Our study demonstrates the importance of ELP1 in the developing cerebellum, and suggests that loss of Elp1 in the GC lineage may also play a role in the progressive ataxia phenotypes of FD patients.
Assuntos
Cerebelo , Disautonomia Familiar , Camundongos Knockout , Fenótipo , Animais , Disautonomia Familiar/genética , Disautonomia Familiar/patologia , Cerebelo/metabolismo , Cerebelo/patologia , Camundongos , Modelos Animais de Doenças , Ataxia/genética , Ataxia/patologia , Ataxia/metabolismo , Células-Tronco Neurais/metabolismo , Apoptose/fisiologia , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Familial Dysautonomia (FD) is a rare disease caused by ELP1 exon 20 skipping. Here we clarify the role of RNA Polymerase II (RNAPII) and chromatin on this splicing event. A slow RNAPII mutant and chromatin-modifying chemicals that reduce the rate of RNAPII elongation induce exon skipping whereas chemicals that create a more relaxed chromatin exon inclusion. In the brain of a mouse transgenic for the human FD-ELP1 we observed on this gene an age-dependent decrease in the RNAPII density profile that was most pronounced on the alternative exon, a robust increase in the repressive marks H3K27me3 and H3K9me3 and a decrease of H3K27Ac, together with a progressive reduction in ELP1 exon 20 inclusion level. In HEK 293T cells, selective drug-induced demethylation of H3K27 increased RNAPII elongation on ELP1 and SMN2, promoted the inclusion of the corresponding alternative exons, and, by RNA-sequencing analysis, induced changes in several alternative splicing events. These data suggest a co-transcriptional model of splicing regulation in which age-dependent changes in H3K27me3/Ac modify the rate of RNAPII elongation and affect processing of ELP1 alternative exon 20.
Assuntos
Processamento Alternativo , Cromatina , Disautonomia Familiar , Éxons , RNA Polimerase II , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Disautonomia Familiar/genética , Disautonomia Familiar/metabolismo , Humanos , Éxons/genética , Animais , Cromatina/metabolismo , Cromatina/genética , Camundongos , Células HEK293 , Histonas/metabolismo , Camundongos Transgênicos , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Cinética , Splicing de RNA , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
Familial dysautonomia (FD) is a rare recessive neurodevelopmental disease caused by a splice mutation in the Elongator acetyltransferase complex subunit 1 (ELP1) gene. This mutation results in a tissue-specific reduction of ELP1 protein, with the lowest levels in the central and peripheral nervous systems (CNS and PNS, respectively). FD patients exhibit complex neurological phenotypes due to the loss of sensory and autonomic neurons. Disease symptoms include decreased pain and temperature perception, impaired or absent myotatic reflexes, proprioceptive ataxia, and progressive retinal degeneration. While the involvement of the PNS in FD pathogenesis has been clearly recognized, the underlying mechanisms responsible for the preferential neuronal loss remain unknown. In this study, we aimed to elucidate the molecular mechanisms underlying FD by conducting a comprehensive transcriptome analysis of neuronal tissues from the phenotypic mouse model TgFD9; Elp1Δ20/flox. This mouse recapitulates the same tissue-specific ELP1 mis-splicing observed in patients while modeling many of the disease manifestations. Comparison of FD and control transcriptomes from dorsal root ganglion (DRG), trigeminal ganglion (TG), medulla (MED), cortex, and spinal cord (SC) showed significantly more differentially expressed genes (DEGs) in the PNS than the CNS. We then identified genes that were tightly co-expressed and functionally dependent on the level of full-length ELP1 transcript. These genes, defined as ELP1 dose-responsive genes, were combined with the DEGs to generate tissue-specific dysregulated FD signature genes and networks. Within the PNS networks, we observed direct connections between Elp1 and genes involved in tRNA synthesis and genes related to amine metabolism and synaptic signaling. Importantly, transcriptomic dysregulation in PNS tissues exhibited enrichment for neuronal subtype markers associated with peptidergic nociceptors and myelinated sensory neurons, which are known to be affected in FD. In summary, this study has identified critical tissue-specific gene networks underlying the etiology of FD and provides new insights into the molecular basis of the disease.
Assuntos
Disautonomia Familiar , Humanos , Camundongos , Animais , Disautonomia Familiar/genética , Disautonomia Familiar/metabolismo , Disautonomia Familiar/patologia , Proteínas de Transporte/metabolismo , Sistema Nervoso Periférico/metabolismo , Células Receptoras Sensoriais/metabolismo , Perfilação da Expressão Gênica , Expressão GênicaRESUMO
BACKGROUND: Children with familial dysautonomia (FD) are smaller and grow more slowly than the general population. It is unknown whether this abnormal growth is due to comorbidities that patients with FD live with, or if it is a direct effect of the disease-causing homozygous ELP-1 mutations. Here, we created growth curves for weight, height, and body mass index (BMI) in male and female children with FD to monitor the nutritional status of patients with FD. METHODS: We used the New York University (NYU) FD Registry which includes data from 680 individuals with FD who were followed longitudinally since birth. We generated sex-specific FD growth charts for three age ranges (birth to 36 months, 2 to 20 years, and 2 to 40 years) and compared them to the general population. We generated Kaplan-Meier curves to test the hypothesis that FD patients with low BMI had shorter survival than the rest of the cohort. RESULTS: Growth charts generated from 591 individuals with FD show that these patients grow more slowly, reach less height, and gain less weight than the general population. The impact of FD on height was more pronounced in girls than in boys. However, both groups showed markedly low weights, which resulted in low BMI. Low weight, but not height, is already evident at birth. In a subpopulation of FD patients, we found that treatment with growth hormone or spinal fusion surgery helped patients achieve the expected growth characteristic of FD patients, but these treatments did not lead FD patients to achieve the growth pattern of the general population. Contrary to our hypothesis, low BMI had no impact on patient survival. CONCLUSIONS: Pediatric patients with FD have lower height, weight, and BMI compared to the general pediatric population, but this does not appear to affect survival. Growth curves specific to the FD population are an important tool to monitor growth and nutritional status in pediatric patients with FD when the general population growth curves are of limited use.
Assuntos
Disautonomia Familiar , Recém-Nascido , Humanos , Criança , Masculino , Feminino , Pré-Escolar , Índice de Massa Corporal , Peso Corporal , Disautonomia Familiar/genética , Estado Nutricional , Magreza , Redução de Peso , EstaturaRESUMO
Familial dysautonomia (FD) is a rare neurodevelopmental and neurodegenerative disease caused by a splicing mutation in the Elongator Acetyltransferase Complex Subunit 1 (ELP1) gene. The reduction in ELP1 mRNA and protein leads to the death of retinal ganglion cells (RGCs) and visual impairment in all FD patients. Currently patient symptoms are managed, but there is no treatment for the disease. We sought to test the hypothesis that restoring levels of Elp1 would thwart the death of RGCs in FD. To this end, we tested the effectiveness of two therapeutic strategies for rescuing RGCs. Here we provide proof-of-concept data that gene replacement therapy and small molecule splicing modifiers effectively reduce the death of RGCs in mouse models for FD and provide pre-clinical foundational data for translation to FD patients.
Assuntos
Disautonomia Familiar , Doenças Neurodegenerativas , Camundongos , Animais , Humanos , Células Ganglionares da Retina/metabolismo , Disautonomia Familiar/genética , Disautonomia Familiar/terapia , Disautonomia Familiar/metabolismo , Doenças Neurodegenerativas/metabolismo , Splicing de RNA , Terapia Genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Familial dysautonomia (FD) is a rare neurodegenerative disease caused by a splicing mutation in elongator acetyltransferase complex subunit 1 (ELP1). This mutation leads to the skipping of exon 20 and a tissue-specific reduction of ELP1, mainly in the central and peripheral nervous systems. FD is a complex neurological disorder accompanied by severe gait ataxia and retinal degeneration. There is currently no effective treatment to restore ELP1 production in individuals with FD, and the disease is ultimately fatal. After identifying kinetin as a small molecule able to correct the ELP1 splicing defect, we worked on its optimization to generate novel splicing modulator compounds (SMCs) that can be used in individuals with FD. Here, we optimize the potency, efficacy, and bio-distribution of second-generation kinetin derivatives to develop an oral treatment for FD that can efficiently pass the blood-brain barrier and correct the ELP1 splicing defect in the nervous system. We demonstrate that the novel compound PTC258 efficiently restores correct ELP1 splicing in mouse tissues, including brain, and most importantly, prevents the progressive neuronal degeneration that is characteristic of FD. Postnatal oral administration of PTC258 to the phenotypic mouse model TgFD9;Elp1Δ20/flox increases full-length ELP1 transcript in a dose-dependent manner and leads to a 2-fold increase in functional ELP1 in the brain. Remarkably, PTC258 treatment improves survival, gait ataxia, and retinal degeneration in the phenotypic FD mice. Our findings highlight the great therapeutic potential of this novel class of small molecules as an oral treatment for FD.
Assuntos
Disautonomia Familiar , Doenças Neurodegenerativas , Degeneração Retiniana , Camundongos , Animais , Disautonomia Familiar/genética , Cinetina , Marcha Atáxica , Administração OralRESUMO
Familial dysautonomia (FD) is a rare genetic neurologic disorder caused by impaired neuronal development and progressive degeneration of both the peripheral and central nervous systems. FD is monogenic, with >99.4% of patients sharing an identical point mutation in the elongator acetyltransferase complex subunit 1 (ELP1) gene, providing a relatively simple genetic background in which to identify modifiable factors that influence pathology. Gastrointestinal symptoms and metabolic deficits are common among FD patients, which supports the hypothesis that the gut microbiome and metabolome are altered and dysfunctional compared to healthy individuals. Here we show significant differences in gut microbiome composition (16 S rRNA gene sequencing of stool samples) and NMR-based stool and serum metabolomes between a cohort of FD patients (~14% of patients worldwide) and their cohabitating, healthy relatives. We show that key observations in human subjects are recapitulated in a neuron-specific Elp1-deficient mouse model, and that cohousing mutant and littermate control mice ameliorates gut microbiome dysbiosis, improves deficits in gut transit, and reduces disease severity. Our results provide evidence that neurologic deficits in FD alter the structure and function of the gut microbiome, which shifts overall host metabolism to perpetuate further neurodegeneration.
Assuntos
Disautonomia Familiar , Microbioma Gastrointestinal , Humanos , Camundongos , Animais , Disautonomia Familiar/genética , Disbiose/metabolismo , Neurônios/metabolismo , Sistema Nervoso Central/metabolismoRESUMO
Familial dysautonomia (FD), a rare neurodevelopmental and neurodegenerative disorder affects the sympathetic and sensory nervous system. Although almost all patients harbor a mutation in ELP1, it remains unresolved exactly how function of sympathetic neurons (symNs) is affected; knowledge critical for understanding debilitating disease hallmarks, including cardiovascular instability or dysautonomic crises, that result from dysregulated sympathetic activity. Here, we employ the human pluripotent stem cell (hPSC) system to understand symN disease mechanisms and test candidate drugs. FD symNs are intrinsically hyperactive in vitro, in cardiomyocyte co-cultures, and in animal models. We report reduced norepinephrine transporter expression, decreased intracellular norepinephrine (NE), decreased NE re-uptake, and excessive extracellular NE in FD symNs. SymN hyperactivity is not a direct ELP1 mutation result, but may connect to NET via RAB proteins. We found that candidate drugs lowered hyperactivity independent of ELP1 modulation. Our findings may have implications for other symN disorders and may allow future drug testing and discovery.
Assuntos
Disautonomia Familiar , Animais , Humanos , Disautonomia Familiar/genética , Disautonomia Familiar/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/genética , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Neurônios/metabolismo , Norepinefrina/metabolismo , MutaçãoRESUMO
Familial dysautonomia (FD) is a currently untreatable, neurodegenerative disease caused by a splicing mutation (c.2204+6T>C) that causes skipping of exon 20 of the elongator complex protein 1 (ELP1) pre-mRNA. Here, we used adeno-associated virus serotype 9 (AAV9-U1-FD) to deliver an exon-specific U1 (ExSpeU1) small nuclear RNA, designed to cause inclusion of ELP1 exon 20 only in those cells expressing the target pre-mRNA, in a phenotypic mouse model of FD. Postnatal systemic and intracerebral ventricular treatment in these mice increased the inclusion of ELP1 exon 20. This also augmented the production of functional protein in several tissues including brain, dorsal root, and trigeminal ganglia. Crucially, the treatment rescued most of the FD mouse mortality before one month of age (89% vs 52%). There were notable improvements in ataxic gait as well as renal (serum creatinine) and cardiac (ejection fraction) functions. RNA-seq analyses of dorsal root ganglia from treated mice and human cells overexpressing FD-ExSpeU1 revealed only minimal global changes in gene expression and splicing. Overall then, our data prove that AAV9-U1-FD is highly specific and will likely be a safe and effective therapeutic strategy for this debilitating disease.
Assuntos
Disautonomia Familiar , Doenças Neurodegenerativas , Animais , Modelos Animais de Doenças , Disautonomia Familiar/genética , Éxons/genética , Humanos , Camundongos , Doenças Neurodegenerativas/genética , Precursores de RNA/genética , Splicing de RNA/genética , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismoRESUMO
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.
Assuntos
Disautonomia Familiar , Rosmarinus , Fatores de Elongação da Transcrição/metabolismo , Abietanos/farmacologia , Acetiltransferases , Adulto , Proteínas de Transporte/genética , Disautonomia Familiar/tratamento farmacológico , Disautonomia Familiar/genética , Disautonomia Familiar/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Rosmarinus/genética , Rosmarinus/metabolismo , Sirtuína 2/metabolismo , Fatores de Elongação da Transcrição/genéticaRESUMO
Familial dysautonomia (FD) is a sensory and autonomic neuropathy caused by mutations in elongator complex protein 1 (ELP1). FD patients have small trigeminal nerves and impaired facial pain and temperature perception. These signals are relayed by nociceptive neurons in the trigeminal ganglion, a structure that is composed of both neural crest- and placode-derived cells. Mice lacking Elp1 in neural crest derivatives ('Elp1 CKO') are born with small trigeminal ganglia, suggesting Elp1 is important for trigeminal ganglion development, yet the function of Elp1 in this context is unknown. We demonstrate that Elp1, expressed in both neural crest- and placode-derived neurons, is not required for initial trigeminal ganglion formation. However, Elp1 CKO trigeminal neurons exhibit abnormal axon outgrowth and deficient target innervation. Developing nociceptors expressing the receptor TrkA undergo early apoptosis in Elp1 CKO, while TrkB- and TrkC-expressing neurons are spared, indicating Elp1 supports the target innervation and survival of trigeminal nociceptors. Furthermore, we demonstrate that specific TrkA deficits in the Elp1 CKO trigeminal ganglion reflect the neural crest lineage of most TrkA neurons versus the placodal lineage of most TrkB and TrkC neurons. Altogether, these findings explain defects in cranial gangliogenesis that may lead to loss of facial pain and temperature sensation in FD.
Assuntos
Disautonomia Familiar , Animais , Disautonomia Familiar/genética , Disautonomia Familiar/metabolismo , Dor Facial/metabolismo , Camundongos , Crista Neural/metabolismo , Neurônios/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Gânglio TrigeminalRESUMO
Cardiovascular instability and a blunted respiratory drive in hypoxic conditions are hallmark features of the genetic sensory and autonomic neuropathy, familial dysautonomia (FD). FD results from a mutation in the gene ELP1, the encoded protein of which is a scaffolding subunit of the six-subunit Elongator complex. In mice, we and others have shown that Elp1 is essential for the normal development of neural crest-derived dorsal root ganglia sensory neurons. Whether Elp1 is also required for development of ectodermal placode-derived visceral sensory receptors, which are required for normal baroreception and chemosensory responses, has not been investigated. Using mouse models for FD, we here show that the entire circuitry underlying baroreception and chemoreception is impaired due to a requirement for Elp1 in the visceral sensory neuron ganglia, as well as for normal peripheral target innervation, and in their central nervous system synaptic partners in the medulla. Thus, Elp1 is required in both placode- and neural crest-derived sensory neurons, and its reduction aborts the normal development of neuronal circuitry essential for autonomic homeostasis and interoception. This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas de Transporte , Disautonomia Familiar , Animais , Proteínas de Transporte/metabolismo , Sistema Nervoso Central/metabolismo , Disautonomia Familiar/genética , Gânglios Espinais/metabolismo , Humanos , Camundongos , Crista Neural/metabolismoRESUMO
Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by a mutation in the Elongator complex protein 1 (ELP1) gene that leads to a tissue-specific reduction of ELP1 protein. Our work to generate a phenotypic mouse model for FD headed to the discovery that homozygous deletion of the mouse Elp1 gene leads to embryonic lethality prior to mid-gestation. Given that FD is caused by a reduction, not loss, of ELP1, we generated two new mouse models by introducing different copy numbers of the human FD ELP1 transgene into the Elp1 knockout mouse (Elp1-/-) and observed that human ELP1 expression rescues embryonic development in a dose-dependent manner. We then conducted a comprehensive transcriptome analysis in mouse embryos to identify genes and pathways whose expression correlates with the amount of ELP1. We found that ELP1 is essential for the expression of genes responsible for nervous system development. Further, gene length analysis of the differentially expressed genes showed that the loss of Elp1 mainly impacts the expression of long genes and that by gradually restoring Elongator, their expression is progressively rescued. Finally, through evaluation of co-expression modules, we identified gene sets with unique expression patterns that depended on ELP1 expression.
Assuntos
Proteínas de Transporte , Disautonomia Familiar , Animais , Proteínas de Transporte/genética , Modelos Animais de Doenças , Disautonomia Familiar/genética , Disautonomia Familiar/metabolismo , Expressão Gênica , Homozigoto , Humanos , Camundongos , Deleção de SequênciaRESUMO
PURPOSE: Patients with the autosomal recessive disorder of familial dysautonomia typically exhibit exacerbated adverse side effects to many common drugs. We aimed to catalog these adverse effects - with a focus on common drugs that are frequently administered to FD patients and compare their incidences to those within the general population. METHODS: We used data of 595 FD patients from an international database with information on drugs received and adverse effects. To investigate the molecular causes of reported differences in drug responses in FD patients, we used expression microarrays to compare the mRNA expression profiles in peripheral blood leukocytes of FD patients (n = 12) and healthy individuals (n = 10). RESULTS: Several drug classes, including cholinergics, anti-cholinergics, anti-convulsants, methylxanthines, SSRIs, and antibiotics caused either unreported symptoms or elevated rates of adverse events in FD patients. FD patients experienced different or more frequent adverse side effects than the general population in 31/123 drugs. These side effects included blood cell dyscrasias, amenorrhea, gastrointestinal bleeding, and bronchospasm. New findings include enhanced reaction of FD patients to H2 antagonist agents and to serotonin receptor agonists. We also detected eight genes differentially expressed between FD patients and healthy individuals that may underlie the differential drug responses of FD patients. CONCLUSION: We provide evidence that suggests the use of several common drugs should be discontinued or reduced in FD patients.
Assuntos
Disautonomia Familiar , Preparações Farmacêuticas , Proteínas de Transporte , Disautonomia Familiar/epidemiologia , Disautonomia Familiar/genética , Feminino , Humanos , Fatores de Elongação da TranscriçãoRESUMO
Familial Dysautonomia (FD) is an autosomal recessive congenital neuropathy affecting the development and function of the peripheral nervous system. FD causing gene is IKBKAP, encoding IkappaB kinase complex-associated protein also named elongator complex like protein 1 (IKAP/ELP1). The most common mutation (IVS20 + 6 T > C) causes an exon 20 skipping, leading to a truncated protein. We report the generation of two induced pluripotent stem cell lines from an FD patient with a homozygous mutation in ELP1 and his heterozygous healthy family relative. Both lines highly express pluripotency markers, can differentiate into the three germ layers, retain the disease-causing mutation and display normal karyotypes.
Assuntos
Disautonomia Familiar , Células-Tronco Pluripotentes Induzidas , Proteínas de Transporte/genética , Disautonomia Familiar/genética , Heterozigoto , Humanos , MutaçãoRESUMO
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.
Assuntos
Processamento Alternativo/genética , Disautonomia Familiar/genética , Mutação , Fatores de Elongação da Transcrição/genética , Processamento Alternativo/efeitos dos fármacos , Animais , Células Cultivadas , Modelos Animais de Doenças , Disautonomia Familiar/tratamento farmacológico , Disautonomia Familiar/metabolismo , Elementos Facilitadores Genéticos/genética , Éxons/genética , Células HeLa , Humanos , Íntrons/genética , Camundongos Transgênicos , Estrutura Molecular , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , Sítios de Splice de RNA/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Elongação da Transcrição/metabolismoRESUMO
PURPOSE: Familial dysautonomia (FD) is associated with a high prevalence of bone fractures, but the impacts of the disease on bone mass and quality are unclear. The purpose of this study was to evaluate tissue through whole-bone scale bone quality in a mouse model of FD. METHODS: Femurs from mature adult Tuba1a-Cre; Elp1LoxP/LoxP conditional knockouts (CKO) (F = 7, M = 4) and controls (F = 5, M = 6) were evaluated for whole-bone flexural material properties, trabecular microarchitecture and cortical geometry, and areal bone mineral density (BMD). Adjacent maps spanning the thickness of femur midshaft cortical bone assessed tissue-scale modulus (nanoindentation), bone mineralization, mineral maturity, and collagen secondary structure (Raman spectroscopy). RESULTS: Consistent with prior studies on this mouse model, the Elp1 CKO mouse model recapitulated several key hallmarks of human FD, with one difference being the male mice tended to have a more severe phenotype than females. Deletion of Elp1 in neurons (using the neuronal-specific Tuba1a-cre) led to a significantly reduced whole-bone toughness but not strength or modulus. Elp1 CKO female mice had reduced trabecular microarchitecture (BV/TV, Tb.Th, Conn.D.) but not cortical geometry. The mutant mice also had a small but significant reduction in cortical bone nanoindentation modulus. While bone tissue mineralization and mineral maturity were not impaired, FD mice may have altered collagen secondary structure. Changes in collagen secondary structure were inversely correlated with bone toughness. BMD from dual-energy x-ray absorptiometry (DXA) was unchanged with FD. CONCLUSION: The deletion of Elp1 in neurons is sufficient to generate a mouse line which demonstrates loss of whole-bone toughness, consistent with the poor bone quality suspected in the clinical setting. The Elp1 CKO model, as with human FD, impacts the nervous system, gut, kidney function, mobility, gait, and posture. The bone quality phenotype of Elp1 CKO mice, which includes altered microarchitecture and tissue-scale material properties, is complex and likely influenced by these multisystemic changes. This mouse model may provide a useful platform to not only investigate the mechanisms responsible for bone fragility in FD, but also a powerful model system with which to evaluate potential therapeutic interventions for bone fragility in FD patients.
Assuntos
Osso e Ossos/fisiopatologia , Disautonomia Familiar , Peptídeos e Proteínas de Sinalização Intracelular/genética , Absorciometria de Fóton , Animais , Densidade Óssea , Modelos Animais de Doenças , Disautonomia Familiar/genética , Disautonomia Familiar/fisiopatologia , Feminino , Humanos , Masculino , Camundongos , Camundongos KnockoutRESUMO
Stuve-Wiedemann syndrome (SWS; MIM 601559) is a rare autosomal recessive disease caused by mutations in the leukemia inhibitor factor receptor gene (LIFR). Common clinical and radiological findings are often observed, and high neonatal mortality occurs due to respiratory distress and hyperthermic episodes. Despite initially considered as a lethal disorder during the newborn period, in recent years, several SWS childhood survivors have been reported. We report a detailed clinical and radiological characterization of four unrelated childhood SWS molecularly confirmed patients and review 22 previously reported childhood surviving cases. We contribute to the definition of the childhood survival phenotype of SWS, emphasizing the evolving phenotype, characterized by skeletal abnormalities with typical radiological findings, distinctive dysmorphic features, and dysautonomia. Based on the typical features and clinical course, early diagnosis is possible and crucial to plan appropriate management and prevent potential complications. Genetic confirmation is advisable in order to improve genetic counseling to the patients and their families.
Assuntos
Anormalidades Múltiplas/genética , Doenças do Desenvolvimento Ósseo/genética , Exostose Múltipla Hereditária/diagnóstico por imagem , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/genética , Osteocondrodisplasias/diagnóstico por imagem , Doenças do Desenvolvimento Ósseo/diagnóstico por imagem , Doenças Ósseas Metabólicas/genética , Pré-Escolar , Consanguinidade , Deficiências do Desenvolvimento/genética , Disautonomia Familiar/genética , Exostose Múltipla Hereditária/genética , Exostose Múltipla Hereditária/patologia , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/deficiência , Masculino , Hipotonia Muscular/genética , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Fenótipo , Roma (Grupo Étnico)/genética , SobreviventesRESUMO
Familial dysautonomia (FD) is the most prevalent form of hereditary sensory and autonomic neuropathy (HSAN). In FD, a germline mutation in the Elp1 gene leads to Elp1 protein decrease that causes sympathetic neuron death and sympathetic nervous system dysfunction (dysautonomia). Elp1 is best known as a scaffolding protein within the nuclear hetero-hexameric transcriptional Elongator protein complex, but how it functions in sympathetic neuron survival is very poorly understood. Here, we identified a cytoplasmic function for Elp1 in sympathetic neurons that was essential for retrograde nerve growth factor (NGF) signaling and neuron target tissue innervation and survival. Elp1 was found to bind to internalized TrkA receptors in an NGF-dependent manner, where it was essential for maintaining TrkA receptor phosphorylation (activation) by regulating PTPN6 (Shp1) phosphatase activity within the signaling complex. In the absence of Elp1, Shp1 was hyperactivated, leading to premature TrkA receptor dephosphorylation, which resulted in retrograde signaling failure and neuron death. Inhibiting Shp1 phosphatase activity in the absence of Elp1 rescued NGF-dependent retrograde signaling, and in an animal model of FD it rescued abnormal sympathetic target tissue innervation. These results suggest that regulation of retrograde NGF signaling in sympathetic neurons by Elp1 may explain sympathetic neuron loss and physiologic dysautonomia in patients with FD.
Assuntos
Disautonomia Familiar/metabolismo , Fator de Crescimento Neural/metabolismo , Neurônios/metabolismo , Transdução de Sinais , Sistema Nervoso Simpático/metabolismo , Animais , Disautonomia Familiar/genética , Disautonomia Familiar/patologia , Mutação em Linhagem Germinativa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Transgênicos , Fator de Crescimento Neural/genética , Neurônios/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptor trkA/genética , Receptor trkA/metabolismo , Sistema Nervoso Simpático/patologiaRESUMO
Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a splice mutation in Elongator complex protein 1 (ELP1, also known as IKBKAP); this mutation leads to variable skipping of exon 20 and to a drastic reduction of ELP1 in the nervous system. Clinically, many of the debilitating aspects of the disease are related to a progressive loss of proprioception; this loss leads to severe gait ataxia, spinal deformities, and respiratory insufficiency due to neuromuscular incoordination. There is currently no effective treatment for FD, and the disease is ultimately fatal. The development of a drug that targets the underlying molecular defect provides hope that the drastic peripheral neurodegeneration characteristic of FD can be halted. We demonstrate herein that the FD mouse TgFD9;IkbkapΔ20/flox recapitulates the proprioceptive impairment observed in individuals with FD, and we provide the in vivo evidence that postnatal correction, promoted by the small molecule kinetin, of the mutant ELP1 splicing can rescue neurological phenotypes in FD. Daily administration of kinetin starting at birth improves sensory-motor coordination and prevents the onset of spinal abnormalities by stopping the loss of proprioceptive neurons. These phenotypic improvements correlate with increased amounts of full-length ELP1 mRNA and protein in multiple tissues, including in the peripheral nervous system (PNS). Our results show that postnatal correction of the underlying ELP1 splicing defect can rescue devastating disease phenotypes and is therefore a viable therapeutic approach for persons with FD.