Transcriptome analysis of familial dysautonomia reveals tissue-specific gene expression disruption in the peripheral nervous system
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ABSTRACT: Familial Dysautonomia (FD) is a rare recessive neurodevelopmental disease caused by a splice site mutation in the Elongator acetyltransferase complex subunit 1 (ELP1) leading to tissue-specific skipping of exon 20 and reduction of the ELP1 protein, distinctly in the central nervous system (CNS) and peripheral nervous system (PNS). Here we performed a transcriptome-wide study to dissect the molecular mechanisms underlying FD in specific neuronal tissues from the FD phenotypic mouse which expresses human ELP1, including the dorsal root ganglion (DRG), trigeminal ganglion (TG), medulla (MED), cortex, and spinal cord (SC). We focused our analyses on differentially expressed genes (DEGs) representing the most dominant transcriptomic alterations; and on genes in co-expression modules that are highly correlated with full-length ELP1 expression (ELP1 dose-responsive genes). We identified higher number of DEGs (342) in the PNS (DRG, TG) as compared to the CNS (MED, SC, Cortex) (143). ELP1 dose-responsive genes are only found in DRG, TG, and MED, not in Cortex or SC, tissues. Gene Ontology analyses of both DEGs and ELP1-dose-responsive genes highlight the regulation of neurotransmitters. The transcriptome-wide signals were highly convergent between PNS tissues (DRG and TG) but not among CNS tissues. Those convergent genes were enriched for known protein-protein interactions and cell type-specific markers defining myelinated neurons and peptidergic nociceptors. Our findings support the involvement of specific neuronal subtypes underlying the PNS phenotypes in FD. Our study comprehensively investigates transcriptome-wide alterations in FD neuronal tissues and identifies the functional dysregulations in the peripheral nervous system contributing to disease.
Project description:Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a splice mutation in Elongator complex protein 1 (ELP1, also known as IKBKAP) which leads to variable skipping of exon 20 and to a drastic reduction of ELP1 levels in the nervous system. Clinically, many of the debilitating aspects of the disease are related to a progressive loss of proprioception, which 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. 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 of mutant ELP1 splicing promoted by the small molecule kinetin 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 levels of full length ELP1 mRNA and protein in multiple tissues including 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.
Project description:The peripheral nervous system (PNS) is essential for proper body function. A high percentage of the world’s population suffers nerve degeneration or peripheral nerve damage. Despite this, there are major gaps in the knowledge of human PNS development and degeneration and therefore, there are no available treatments. Familial Dysautonomia (FD) is a devastating disorder caused by a homozygous point mutation in the gene ELP1. FD specifically affects the development and causes degeneration of the PNS. We previously employed patient-derived induced pluripotent stem cells (iPSCs) to show that peripheral sensory neurons (SNs) recapitulate the developmental and neurodegenerative defects observed in FD. Here, we conducted a chemical screen to identify compounds that rescue the SN differentiation inefficiency in FD. We identified that genipin restores neural crest and SN development in patient derived iPSCs and in two mouse models of FD. Additionally, genipin prevented FD degeneration in SNs derived from patients with FD, suggesting that it could be used to ameliorate neurodegeneration. Moreover, genipin crosslinked the extracellular matrix (ECM), increased the stiffness of the ECM, reorganized the actin cytoskeleton, and promoted transcription of YAP-dependent genes. Finally, genipin enhanced axon regeneration in healthy sensory and sympathetic neurons (part of the PNS), and in prefrontal cortical neurons (part of the central nervous system), in in vitro axotomy models. Our results suggest that genipin has the potential to treat FD-related neurodevelopmental and neurodegenerative phenotypes, and to enhance neuronal regeneration of healthy neurons after injury. Moreover, this suggests that the ECM can be targeted to treat FD.
Project description:Elongator is a highly conserved protein complex required for transcriptional elongation, intracellular transport and translation. Elongator complex protein 1 (ELP1) is the scaffolding protein of Elongator and is essential for its assembly and stability. Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by a mutation in ELP1 that lead to a tissue-specific reduction of ELP1 protein. Our work to generate a phenotypic mouse model for FD led 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 the formation and development of the nervous system. 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. Overall, this study highlights the crucial role of ELP1 during early embryonic neuronal development and reveals gene networks and biological pathways that are regulated by Elongator.
Project description:Familial dysautonomia (FD) results from mutation in IKBKAP/ELP1, a gene encoding the scaffolding protein for the Elongator complex. This highly conserved complex is required for the methoxy-carbonyl-methyl (mcm5) modification of uridines located in the wobble position of tRNA molecules (U34). In FD, the peripheral nervous system is particularly devastated by loss of IKBKAP. Here we investigate protein expression within the dorsal root ganglia (DRG) of a mouse model of FD. In this model, Ikbkap is selectively deleted in the neural crest lineage using a Wnt1-Cre transgene and floxed Ikbkap. DRG were collected and pooled from seven Ikbkap conditional knockout (Wnt1-Cre;IkbkapLoxP/LoxP) and seven control E17.5 mice and analyzed via UHPLC-MS/MS.
Project description:The important contribution of glia to mechanisms of injury and repair of the nervous system is increasingly recognized. In stark contrast to the central nervous system (CNS), the peripheral nervous system (PNS) has a remarkable capacity for regeneration after injury. Schwann cells are recognized as key contributors to PNS regeneration but the molecular underpinnings of the Schwann cell response to injury remain incompletely understood. To gain insight into the acute SC injury response, we provide an RNAseq database of Schwann cells purified acutely from the naïve and injured rodent sciatic nerve at 3, 5, and 7 days post injury. Bioinformatic analysis provides validation of cell purity and dataset integrity as well as identification of discrete modules of genes that follow distinct patterns of regulation in the first days after injury and their corresponding molecular pathways. Our dataset provides a helpful resource for further deciphering the SC injury response and provides a depth of transcriptional data that can complement the findings of recent single cell sequencing approaches. In addition, as more data becomes available on the response of CNS glia to injury, we anticipate that this dataset will provide a valuable platform for understanding key differences in the PNS and CNS glial responses to injury and for designing approaches to ameliorate CNS regeneration.
Project description:Neural crest-derived neural stem cells (NCSCs) from the embryonic PNS can be reprogrammed in neurosphere culture (NS) to rNCSCs that produce CNS progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord (SCSCs). Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3- and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed towards a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSCs. These findings show that embryonic NCSCs acquire a full CNS identity in neurosphere culture. In contrast, MSCs are generated from adult pNCSCs and BMP NCSCs, which reveals that postmigratory NCSCs are a source for MSCs up to the adult stage. Affymetrix Mouse 430_2 arrays were used to compare the gene expression profiles of embryonic DRG-derived reprogrammed NCSCs and adult NCSCs from mouse palate.
Project description:Neural crest-derived neural stem cells (NCSCs) from the embryonic PNS can be reprogrammed in neurosphere culture (NS) to rNCSCs that produce CNS progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord (SCSCs). Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3- and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed towards a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSCs. These findings show that embryonic NCSCs acquire a full CNS identity in neurosphere culture. In contrast, MSCs are generated from adult pNCSCs and BMP NCSCs, which reveals that postmigratory NCSCs are a source for MSCs up to the adult stage. Affymetrix Mouse 430_2 arrays were used to compare the gene expression profiles of E12.5 mouse spinal cord-derived neurospheres (SCSCs) and E12.5 DRG-derived neurospheres, cultured in the absence (rNCSCs) or in the presence of BMP4 (BMP NCSCs).
Project description:Glial cells are present throughout the entire nervous system and paly a crucial role in regulating physiological and pathological functions, such as infections, acute injuries and chronic neurodegenerative disorders. The glial cells mainly include astrocytes, microglia, and oligodendrocytes in the central nervous system (CNS), and satellite glial cells (SGCs) in the peripheral nervous system (PNS). Although the glial subtypes and functional heterogeneity is relatively well understood in mice by recent studies using single-cell or single-nucleus RNA-sequencing, no evidence yet has elucidate the transcriptomic profiles of glia cells in PNS and CNS. Here, we used high-throughput single-nucleus RNA-sequencing to map the cellular and functional heterogeneity of SGCs in human dorsal root ganglion (DRG), and astrocytes, microglia, and oligodendrocytes in human spinal cord. In addition, we compared the human findings with previous single-nucleus transcriptomic profiles of glial cells from mouse DRG and spinal cord. This work will comprehensively profile glial cells heterogeneity and will provide a powerful resource for probing the cellular basis of human physiological and pathological conditions related to glial cells.
Project description:Familial Dysautonomia (FD; OMIM #223900) is both a developmental and progressive autosomal recessive neurodegenerative disorder that results from a nervous-system reduction in the IKAP/ELP1 protein due to a mutation in a splice acceptor site of the IKBKAP/ELP1 gene. The function of this gene in the nervous system is unresolved. To obviate the embryonic lethality of mice completely null for Ikbkap, we generated conditional knock out (CKO) mouse models for FD that recapitulate hallmarks of the human disease. To derive insight into potential intracellular functions for Ikbkap, we conducted a genome-wide transcriptome analysis of both the peripheral and central nervous systems from Ikbkap CKO mice, and identify over 100 shared misregulated genes that reveal roles for IKAP in several metabolic and signaling pathways in addition to synaptic transmission. Importantly, our data are the first to demonstrate that in the absence of IKAP, neurons undergo intracellular stress that is marked by transcriptional elevations in ATF5, p53, and several CREB target genes, as well as an increase in reactive oxygen species. These data will aid in the identification of common upstream and downstream targets for therapeutics for preventing the progressive demise of neurons in FD and potentially other neuropathies.
Project description: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.