Schwann cell-specific ablation of Beclin 1 impairs peripheral myelination and leads to a progressive motor and sensory neuropathy
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ABSTRACT: The core component of the class III phosphatidylinositol 3-kinase complex, Beclin 1, takes part in different protein networks, thus switching its role from inducing autophagy to regulating autophagosomal maturation and endosomal trafficking. While assessed in neurons, astrocytes and microglia, its role was far less investigated in myelinating glia, including Schwann cells (SCs), responsible for peripheral nerve myelination. Remarkably, the dysregulation in endosomal trafficking is emerging as a pathophysiological mechanism underlying peripheral neuropathies, such as demyelinating Charcot-Marie-Tooth diseases. By knocking out Beclin 1 in SCs we here generated a novel mouse model (Becn1 cKO), developing a severe and progressive neuropathy, accompanied by involuntary tremors, body weight loss and premature death. Ultrastructural analysis revealed abated myelination and SCs displaying enlarged cytoplasm with progressive accumulation of intracellular vesicles. Transcriptomic and histological analysis from sciatic nerves of 10-day and 2-month-old mice revealed pro-mitotic gene deregulation and increased SCs proliferation at both stages with axonal loss and increased immune infiltration in adults, well reflecting the progressive motor and sensory functional impairment that characterizes Becn1 cKO mice, compared to controls. Our study establishes a further step in understanding key mechanisms in SC development and points to Beclin 1 and its regulated pathways as targets for demyelinating CMT forms. A novel conditional gene knockout was generated to investigate the role that Beclin 1 exerts in Schwann cells and in myelination. The newly generated mouse line resulted in a model for severe and progressive peripheral neuropathy. Together with defective myelination, we described radial sorting defects, motor and sensory neuron impairments, immune infiltrate in adults and a huge transcripts deregulation. The emerged deregulated signalling pathways highlighted how Beclin 1 is able to coordinate integrated signaling pathways essential for Schwann cell maturation and nerve development and homeostasis.
Project description:Elucidating the genes regulated during cell-cell communication remains fascinating considering the importance of cell recognition and downstream signaling through development and in many diseases. In the peripheral nervous system, the interaction between Schwann cells (SCs) and axons is crucial as it allows their survival and induces SCs to differentiate and engage a process of myelination. To get further insight the molecular mechanisms resulting from this cell interaction, comparative gene analysis between SCs and SCs co-cultured with DRG neurons were performed and led us to identify a set of 32 genes regulated in SCs in the early stage of neuron-SC contact. Expression of several candidates were analyzed by QPCR during development and we demonstrate using a blocking antibody approach in an in vitro myelination assay that one candidate was not only upregulated in response to axonal contact but also controls peripheral myelination. Three biological replicates each in dye swap for 22k slides and two biological replicates each in dye swap for NeuroDev2
Project description:Gene expression analysis of 2-month-old Ctrl and Tfam-SCKO mice. At this age mitochondrial function is disrupted in the Schwann cells of Tfam-SCKO mice ,but their nerves display only very limited pathology. Mitochondrial dysfunction is a common cause of peripheral neuropathy. Much effort has been devoted to examining the role played by neuronal/axonal mitochondria, but how mitochondrial deficits in peripheral nerve glia (Schwann cells, SCs) contribute to peripheral nerve diseases remains unclear. Here, we investigate a mouse model of peripheral neuropathy secondary to SC mitochondrial dysfunction (Tfam-SCKOs). We show that disruption of SC mitochondria activates a maladaptive integrated stress response through actions of heme-regulated inhibitor kinase (HRI), and causes a shift in lipid metabolism away from fatty acid synthesis toward oxidation. These alterations in SC lipid metabolism result in depletion of important myelin lipid components as well as in accumulation of acylcarnitines, an intermediate of fatty acid b-oxidation. Importantly, we show that acylcarnitines are released from SCs and induce axonal degeneration. A maladaptive integrated stress response as well as altered SC lipid metabolism are thus underlying pathological mechanisms in mitochondria-related peripheral neuropathies. Total RNA samples were prepared by isolating and pooling RNA from three different 2-month-old MPZ-Tfam KO and Ctrl mice. 2 replicates per genotype were used in this experiment and they were prepared entirely independently.
Project description:Elucidating the genes regulated during cell-cell communication remains fascinating considering the importance of cell recognition and downstream signaling through development and in many diseases. In the peripheral nervous system, the interaction between Schwann cells (SCs) and axons is crucial as it allows their survival and induces SCs to differentiate and engage a process of myelination. To get further insight the molecular mechanisms resulting from this cell interaction, comparative gene analysis between SCs and SCs co-cultured with DRG neurons were performed and led us to identify a set of 32 genes regulated in SCs in the early stage of neuron-SC contact. Expression of several candidates were analyzed by QPCR during development and we demonstrate using a blocking antibody approach in an in vitro myelination assay that one candidate was not only upregulated in response to axonal contact but also controls peripheral myelination.
Project description:DNA methylation is a key epigenetic regulator of mammalian embryogenesis and somatic cell differentiation. Using high-resolution genome-scale maps of methylation patterns, we show that the formation of myelin in the peripheral nervous system, proceeds with progressive DNA demethylation, which coincides with an upregulation of critical genes of the myelination process. More importantly, we found that, in addition to expression of DNA methyltransferases and demethylases, the levels of S-adenosylmethionine (SAMe), the principal biological methyl donor, could also play a critical role in regulating DNA methylation during myelination and in the pathogenesis of diabetic neuropathy. In summary, this study provides compelling evidence that SAMe levels need to be tightly controlled to prevent aberrant DNA methylation patterns, and together with recently published studies on the influence of SAMe on histone methylation in cancer and embryonic stem cell differentiation show that in diverse biological processes, the methylome, and consequently gene expression patterns, are critically dependent on levels of SAMe. Axonal myelination by Schwann cells in the peripheral nervous system is essential for rapid saltatory impulse conduction, and malformation or destruction of myelin sheaths can lead to severe motor and sensory disabilities (peripheral neuropathies). Using high-resolution genome-scale methylome maps, we found that DNA methylation could play a critical role in the generation of myelinated Schwann cells. This process was accompanied by a global DNA demethylation at most genomic elements. Notably, demethylation at gene-regulatory regions was associated with activation of critical myelination-specific genes. Furthermore, we found an aberrant DNA methylation pattern in a mouse model of diabetic neuropathy, which could be involved in the pathogenesis of the disease. Importantly, we found that these methylation patterns in both situations could be regulated by levels of S-adenosylmethionine (SAMe), the principal biological methyl donor. Together with recent studies on the influence of SAMe on histone methylation in diverse biological processes, we conclude that the methylation landscape of cells could be critically dependent on levels of SAMe. These provide a mechanistic link between metabolism and gene regulatory networks in normal and pathological situations. Sciatic nerves from post natal day 90 GNMT (wt and KO) mice of either sex were dissected and pooled together, 2 replicates per sample group.
Project description:DNA methylation is a key epigenetic regulator of mammalian embryogenesis and somatic cell differentiation. Using high-resolution genome-scale maps of methylation patterns, we show that the formation of myelin in the peripheral nervous system, proceeds with progressive DNA demethylation, which coincides with an upregulation of critical genes of the myelination process. More importantly, we found that, in addition to expression of DNA methyltransferases and demethylases, the levels of S-adenosylmethionine (SAMe), the principal biological methyl donor, could also play a critical role in regulating DNA methylation during myelination and in the pathogenesis of diabetic neuropathy. In summary, this study provides compelling evidence that SAMe levels need to be tightly controlled to prevent aberrant DNA methylation patterns, and together with recently published studies on the influence of SAMe on histone methylation in cancer and embryonic stem cell differentiation show that in diverse biological processes, the methylome, and consequently gene expression patterns, are critically dependent on levels of SAMe. Axonal myelination by Schwann cells in the peripheral nervous system is essential for rapid saltatory impulse conduction, and malformation or destruction of myelin sheaths can lead to severe motor and sensory disabilities (peripheral neuropathies). Using high-resolution genome-scale methylome maps, we found that DNA methylation could play a critical role in the generation of myelinated Schwann cells. This process was accompanied by a global DNA demethylation at most genomic elements. Notably, demethylation at gene-regulatory regions was associated with activation of critical myelination-specific genes. Furthermore, we found an aberrant DNA methylation pattern in a mouse model of diabetic neuropathy, which could be involved in the pathogenesis of the disease. Importantly, we found that these methylation patterns in both situations could be regulated by levels of S-adenosylmethionine (SAMe), the principal biological methyl donor. Together with recent studies on the influence of SAMe on histone methylation in diverse biological processes, we conclude that the methylation landscape of cells could be critically dependent on levels of SAMe. These provide a mechanistic link between metabolism and gene regulatory networks in normal and pathological situations. Sciatic nerves from C57Bl6J mice of either sex, were dissected and pooled together at different developmental stages, 3 replicates per sample group.
Project description:DNA methylation is a key epigenetic regulator of mammalian embryogenesis and somatic cell differentiation. Using high-resolution genome-scale maps of methylation patterns, we show that the formation of myelin in the peripheral nervous system, proceeds with progressive DNA demethylation, which coincides with an upregulation of critical genes of the myelination process. More importantly, we found that, in addition to expression of DNA methyltransferases and demethylases, the levels of S-adenosylmethionine (SAMe), the principal biological methyl donor, could also play a critical role in regulating DNA methylation during myelination and in the pathogenesis of diabetic neuropathy. In summary, this study provides compelling evidence that SAMe levels need to be tightly controlled to prevent aberrant DNA methylation patterns, and together with recently published studies on the influence of SAMe on histone methylation in cancer and embryonic stem cell differentiation show that in diverse biological processes, the methylome, and consequently gene expression patterns, are critically dependent on levels of SAMe. DNA methylome maps of developmental Schwann cell myelination, GNMT-KO and diabetic mice were generated by Reduced-Representation Bisulfite Sequencing, with 2-3 replicates per sample group.
Project description:Axonal myelination is essential for neuronal function and health. In peripheral nerves, deficient myelination is responsible for the morbidity of various forms of inherited or acquired neuropathies, including Charcot-Marie-Tooth disease and diabetic neuropathy. Decades of research have uncovered a complex transcriptional and post-transcriptional program that co-ordinates the formation and maintenance of the myelin sheath. In contrast, much less is known about the functional role of post-translational modification (PTM) of proteins in this remarkable biogenic process. Neddylation, a PTM that involves the conjugation of the ubiquitin-like protein Nedd8 to protein targets, has recently emerged as a central and versatile regulator of many cellular processes, including gene transcription, metabolism, and cellular differentiation. In this study, we show that genetic and pharmacological inhibition of neddylation in vivo in developing Schwann cells lead to striking nerve defects that exhibit the classical hallmarks of a severe neuropathy, including gait abnormalities, muscle weakness, and hindlimb clasping, ultimately leading to early death. The mutant mice lack peripheral myelin and develop secondary axonal loss, and we demonstrate, at the mechanistic level, that neddylation regulates multiple critical myelination-related pathways. Together, our findings identify neddylation as a central regulatory hub of control of peripheral myelination and delineate the potential pathogenetic mechanisms in inherited human PNS disorders, characterized by mutations in genes related to the neddylation pathway.
Project description:Mutations in myelin protein zero (MPZ) are generally associated with Charcot-Marie-Tooth type 1B (CMT1B) disease, one of the most common forms of demyelinating neuropathy. Pathogenesis of some MPZ mutants, such as S63del and R98C, involves protein misfolding and retention in the endoplasmic reticulum (ER) of myelinating Schwann cells. To cope with proteotoxic ER-stress, Schwann cells mount an unfolded protein response (UPR) characterized by activation of the PERK, ATF6 and IRE1/XBP1 pathways. Previous studies have reported that targeting PERK pathway can mitigate the neuropathy in CMT1B mice. To unravel the role of the XBP1 pathway in normal myelination and in CMT1B, we generated mouse models of in which XBP1 is deleted specifically in Schwann cells. We observed that whereas XBP1 is dispensable for normal developmental myelination, myelin maintenance and remyelination after injury, the absence of XBP1 dramatically worsens the hypomyelination and the electrophysiological and locomotor parameters in young and adult CMT1B neuropathic animals. RNAseq analysis suggested that XBP1 exerts its adaptive function in large part via the induction of genes involved in misfolded protein degradation. Accordingly, the exacerbation of the neuropathy was accompanied by upregulation of ER-stress pathways and of IRE1-mediated RIDD signaling, suggesting that the activation of XBP1 plays a critical role in limiting mutant proteins toxicity, which cannot be compensated by other stress responses. Schwann cell specific overexpression of spliced XBP1 partially re-established Schwann cell proteostasis and attenuated CMT1B severity in vivo in both the S63del and R98C mouse models. In addition, the pharmacologic selective activation of XBP1 signaling ameliorated myelination in S63del dorsal root ganglia explants.
Project description:DNA methylation is a key epigenetic regulator of mammalian embryogenesis and somatic cell differentiation. Using high-resolution genome-scale maps of methylation patterns, we show that the formation of myelin in the peripheral nervous system, proceeds with progressive DNA demethylation, which coincides with an upregulation of critical genes of the myelination process. More importantly, we found that, in addition to expression of DNA methyltransferases and demethylases, the levels of S-adenosylmethionine (SAMe), the principal biological methyl donor, could also play a critical role in regulating DNA methylation during myelination and in the pathogenesis of diabetic neuropathy. In summary, this study provides compelling evidence that SAMe levels need to be tightly controlled to prevent aberrant DNA methylation patterns, and together with recently published studies on the influence of SAMe on histone methylation in cancer and embryonic stem cell differentiation show that in diverse biological processes, the methylome, and consequently gene expression patterns, are critically dependent on levels of SAMe. DNA methylome maps of mouse Schwann cells in which GNMT was silenced by lentiviral vectors, cultured in normal medium or low methionine medium.