New schwann cell markers identified by gene profiling analysis after axonal contact
ABSTRACT: 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:The myelination of axons in peripheral nerves requires precisely coordinated proliferation and differentiation of Schwann cells (SCs). We found that the activity of the mechanistic target of rapamycin complex 1 (mTORC1), a key signaling hub for the regulation of cellular growth and proliferation, is progressively extinguished as SCs differentiate during nerve development. To study the effects of different levels of sustained mTORC1 hyperactivity in the SC lineage, we disrupted negative regulators of mTORC1, including TSC2 or TSC1, in developing SCs of mutant mice. Surprisingly, the phenotypes ranged from arrested myelination in nerve development to focal hypermyelination in adulthood, depending on the level and timing of mTORC1 hyperactivity. For example, mice lacking TSC2 in developing SCs displayed hyperproliferation of undifferentiated SCs incompatible with normal myelination. However, these defects and myelination could be rescued by pharmacological mTORC1 inhibition. The subsequent reconstitution of SC mTORC1 hyperactivity in adult animals resulted in focal hypermyelination. Together our data suggest a model in which high mTORC1 activity promotes proliferation of immature SCs and antagonizes SC differentiation during nerve development. Down-regulation of mTORC1 activity is required for terminal SC differentiation and subsequent initiation of myelination. In distinction to this developmental role, excessive SC mTORC1 activity stimulates myelin growth, even overgrowth, in adulthood. Thus, our work delineates two distinct functions of mTORC1 in the SC lineage essential for proper nerve development and myelination. Moreover, our studies show that SCs retain their plasticity to myelinate and remodel myelin via mTORC1 throughout life.
Project description:Proper function of the nervous system depends on myelination. In peripheral nerves, Schwann cells (SCs) myelinate axons and the miRNA biogenesis pathway is required for developmental myelination and myelin maintenance. However, regulatory roles of this pathway at different stages of myelination are only partially understood. We addressed the requirement of the core miRNA biogenesis pathway components Dgcr8, Drosha, and Dicer in developing and adult SCs using mouse mutants with a comparative genetics and transcriptomics approach. We found that the microprocessor components Dgcr8 and Drosha are crucial for axonal radial sorting and to establish correct SC numbers upon myelination. Transcriptome analyses revealed a requirement of the microprocessor to prevent aberrantly increased expression of injury-response genes. Those genes are predicted targets of abundant miRNAs in sciatic nerves (SNs) during developmental myelination. In agreement, Dgcr8 and Dicer are required for proper maintenance of the myelinated SC state, where abundant miRNAs in adult SNs are predicted to target injury-response genes. We conclude that the miRNA biogenesis pathway in SCs is crucial for preventing inappropriate activity of injury-response genes in developing and adult SCs.
Project description:The formation of myelin by Schwann cells (SCs) occurs via a series of orchestrated molecular events. We previously used global expression profiling to examine peripheral nerve myelination and identified the NAD(+)-dependent deacetylase Sir-two-homolog 2 (Sirt2) as a protein likely to be involved in myelination. Here, we show that Sirt2 expression in SCs is correlated with that of structural myelin components during both developmental myelination and remyelination after nerve injury. Transgenic mice lacking or overexpressing Sirt2 specifically in SCs show delays in myelin formation. In SCs, we found that Sirt2 deacetylates Par-3, a master regulator of cell polarity. The deacetylation of Par-3 by Sirt2 decreases the activity of the polarity complex signaling component aPKC, thereby regulating myelin formation. These results demonstrate that Sirt2 controls an essential polarity pathway in SCs during myelin assembly and provide insights into the association between intracellular metabolism and SC plasticity.
Project description:Isolated Schwann cells (SCs) respond to cAMP elevation by adopting a differentiated post-mitotic state that exhibits high levels of Krox-20, a transcriptional enhancer of myelination, and mature SC markers such as the myelin lipid galactocerebroside (O1). To address how cAMP controls myelination, we performed a series of cell culture experiments which compared the differentiating responses of isolated and axon-related SCs to cAMP analogs and ascorbate, a known inducer of axon ensheathment, basal lamina formation and myelination. In axon-related SCs, cAMP induced the expression of Krox-20 and O1 without a concomitant increase in the expression of myelin basic protein (MBP) and without promoting axon ensheathment, collagen synthesis or basal lamina assembly. When cAMP was provided together with ascorbate, a dramatic enhancement of MBP expression occurred, indicating that cAMP primes SCs to form myelin only under conditions supportive of basal lamina formation. Experiments using a combination of cell permeable cAMP analogs and type-selective adenylyl cyclase (AC) agonists and antagonists revealed that selective transmembrane AC (tmAC) activation with forskolin was not sufficient for full SC differentiation and that the attainment of an O1 positive state also relied on the activity of the soluble AC (sAC), a bicarbonate sensor that is insensitive to forskolin and GPCR activation. Pharmacological and immunological evidence indicated that SCs expressed sAC and that sAC activity was required for morphological differentiation and the expression of myelin markers such as O1 and protein zero. To conclude, our data indicates that cAMP did not directly drive myelination but rather the transition into an O1 positive state, which is perhaps the most critical cAMP-dependent rate limiting step for the onset of myelination. The temporally restricted role of cAMP in inducing differentiation independently of basal lamina formation provides a clear example of the uncoupling of signals controlling differentiation and myelination in SCs.
Project description:OBJECTIVE:Polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE), which is a piezoelectric, biocompatible polymer, holds promise as a scaffold in combination with Schwann cells (SCs) for spinal cord repair. Piezoelectric materials can generate electrical activity in response to mechanical deformation, which could potentially stimulate spinal cord axon regeneration. Our goal in this study was to investigate PVDF-TrFE scaffolds consisting of aligned fibers in supporting SC growth and SC-supported neurite extension and myelination in vitro. APPROACH:Aligned fibers of PVDF-TrFE were fabricated using the electrospinning technique. SCs and dorsal root ganglion (DRG) explants were co-cultured to evaluate SC-supported neurite extension and myelination on PVDF-TrFE scaffolds. MAIN RESULTS:PVDF-TrFE scaffolds supported SC growth and neurite extension, which was further enhanced by coating the scaffolds with Matrigel. SCs were oriented and neurites extended along the length of the aligned fibers. SCs in co-culture with DRGs on PVDF-TrFE scaffolds promoted longer neurite extension as compared to scaffolds without SCs. In addition to promoting neurite extension, SCs also formed myelin around DRG neurites on PVDF-TrFE scaffolds. SIGNIFICANCE:This study demonstrated PVDF-TrFE scaffolds containing aligned fibers supported SC-neurite extension and myelination. The combination of SCs and PVDF-TrFE scaffolds may be a promising tissue engineering strategy for spinal cord repair.
Project description:Myelination is a biosynthetically demanding process in which mTORC1, the gatekeeper of anabolism, occupies a privileged regulatory position. We have shown previously that loss of mTORC1 function in Schwann cells (SCs) hampers myelination. Here, we genetically disrupted key inhibitory components upstream of mTORC1, TSC1 or PTEN, in mouse SC development, adult homeostasis, and nerve injury. Surprisingly, the resulting mTORC1 hyperactivity led to markedly delayed onset of both developmental myelination and remyelination after injury. However, if mTORC1 was hyperactivated after myelination onset, radial hypermyelination was observed. At early developmental stages, physiologically high PI3K-Akt-mTORC1 signaling suppresses expression of Krox20 (Egr2), the master regulator of PNS myelination. This effect is mediated by S6K and contributes to control mechanisms that keep SCs in a not-fully differentiated state to ensure proper timing of myelination initiation. An ensuing decline in mTORC1 activity is crucial to allow myelination to start, while remaining mTORC1 activity drives myelin growth.
Project description:Myelination requires extensive plasma membrane rearrangements, implying that molecules controlling membrane dynamics play prominent roles. The large GTPase dynamin 2 (DNM2) is a well-known regulator of membrane remodeling, membrane fission, and vesicular trafficking. Here, we genetically ablated Dnm2 in Schwann cells (SCs) and in oligodendrocytes of mice. Dnm2 deletion in developing SCs resulted in severely impaired axonal sorting and myelination onset. Induced Dnm2 deletion in adult SCs caused a rapidly-developing peripheral neuropathy with abundant demyelination. In both experimental settings, mutant SCs underwent prominent cell death, at least partially due to cytokinesis failure. Strikingly, when Dnm2 was deleted in adult SCs, non-recombined SCs still expressing DNM2 were able to remyelinate fast and efficiently, accompanied by neuropathy remission. These findings reveal a remarkable self-healing capability of peripheral nerves that are affected by SC loss. In the central nervous system, however, we found no major defects upon Dnm2 deletion in oligodendrocytes.
Project description:Shiverer-immunodeficient (Shi-id) mice demonstrate defective myelination in the central nervous system (CNS) and significant ataxia by 2 to 3 weeks of life. Expanded, banked human neural stem cells (HuCNS-SCs) were transplanted into three sites in the brains of neonatal or juvenile Shi-id mice, which were asymptomatic or showed advanced hypomyelination, respectively. In both groups of mice, HuCNS-SCs engrafted and underwent preferential differentiation into oligodendrocytes. These oligodendrocytes generated compact myelin with normalized nodal organization, ultrastructure, and axon conduction velocities. Myelination was equivalent in neonatal and juvenile mice by quantitative histopathology and high-field ex vivo magnetic resonance imaging, which, through fractional anisotropy, revealed CNS myelination 5 to 7 weeks after HuCNS-SC transplantation. Transplanted HuCNS-SCs generated functional myelin in the CNS, even in animals with severe symptomatic hypomyelination, suggesting that this strategy may be useful for treating dysmyelinating diseases.
Project description:Signaling through cyclic AMP (cAMP) has been implicated in the regulation of Schwann cell (SC) proliferation and differentiation. In quiescent SCs, elevation of cAMP promotes the expression of proteins associated with myelination such as Krox-20 and P0, and downregulation of markers associated with the non-myelinating SC phenotype. We have previously shown that the motor protein myosin II is required for the establishment of normal SC-axon interactions, differentiation and myelination, however, the mechanisms behind these effects are unknown. Here we report that the levels and activity of myosin light chain kinase (MLCK), an enzyme that regulates MLC phosphorylation in non-muscle cells, are dramatically downregulated in SCs after cAMP treatment, in a similar pattern to that of c-Jun, a known inhibitor of myelination. Knockdown of MLCK in SCs mimics the effect of cAMP elevation, inducing plasma membrane expansion and expression of Krox-20 and myelin proteins. Despite activation of myelin gene transcription these cells fail to make compact myelin when placed in contact with axons. Our data indicate that myosin II activity is differentially regulated at various stages during myelination and that in the absence of MLCK the processes of SC differentiation and compact myelin assembly are uncoupled.
Project description:Our ultimate goal of in vitro derivation of Schwann cells (SCs) from adult bone marrow stromal cells (BMSCs) is such that they may be used autologously to assist post-traumatic nerve regeneration. Existing protocols for derivation of SC-like cells from BMSCs fall short in the stability of the acquired phenotype and the functional capacity to myelinate axons. Our experiments indicated that neuro-ectodermal progenitor cells among the human hBMSCs could be selectively expanded and then induced to differentiate into SC-like cells. Co-culture of the SC-like cells with embryonic dorsal root ganglion neurons facilitated contact-mediated signaling that accomplished the switch to fate-committed SCs. Microarray analysis and in vitro myelination provided evidence that the human BMSC-derived SCs were functionally mature. This was reinforced by repair and myelination phenotypes observable in vivo with the derived SCs seeded into a nerve guide as an implant across a critical gap in a rat model of sciatic nerve injury.