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Analysing intrinsic and extrinsic factors involved in an impaired differentiation of induced pluripotent stem cell derived-oligodendrocytes in Multiple Sclerosis patients (#473)
L. Starost1, 2, M. Herold3, L. Ottoboni4, M. Ehrlich1, 2, M. Stehling2, H. R. Schöler2, L. Klotz3, G. Martino4, T. Kuhlmann1
1 University Hospital Münster, Institute of Neuropathology, Münster, Germany
Multiple Sclerosis (MS), a demyelinating and inflammatory disease, affects approximately 2.5 million people worldwide. The destruction of the myelin sheath results in axonal degeneration and loss of function. Although remyelination exists in MS patients, it is restricted during disease progression.
The proliferation and migration of oligodendroglial precursor cells (OPCs) followed by the differentiation and maturation of OPCs into oligodendrocytes is crucial for successful remyelination. Even though OPCs are still detectable in chronic MS lesions, the reconstitution of the myelin sheath fails due to an impaired differentiation of OPCs into mature oligodendrocytes.
Since the underlying process is not entirely understood, it needs to be clarified.
To address the question whether this differentiation block is mediated by intrinsic oligodendroglial factors or caused by the extrinsic inflammatory milieu, we generated induced pluripotent stem cell (iPSC)-derived oligodendrocytes (iOL) from MS patients and sex-matched healthy control individuals. By comparing these iOL with respect to proliferation, migration, differentiation, myelination and stress response, we examined possible phenotypic differences. To see whether the inflammatory environment potentially influences iOL differentiation, we applied the supernatant of peripheral blood derived mononuclear cells (PBMCs) to differentiating iOL of healthy controls.
On the one hand, comparison of iOL from MS patients and controls revealed a similar capability to proliferate, migrate and differentiate and resulted in the same cell death rate after induction of oxidative stress. Moreover, to see whether iOL could give rise to functional oligodendrocytes, we used an in vitro myelination assay by seeding the cells on nanofibers showing that iOL from MS patients and controls were able to form processes along nanofibers.
On the other hand, we found a significantly impaired differentiation of iOL after application of the supernatant of activated PBMCs during differentiation. The application of supernatants of activated single immune cell types indicated that this effect was predominantly caused by T cells.
In conclusion, our data suggest that the oligodendroglial differentiation block observed in MS is predominantly caused by extrinsic inflammatory mediators and is not due to intrinsic oligodendroglial factors. These findings contribute to a better understanding of MS pathogenesis and may help to develop new treatment options to promote remyelination in MS.
Keywords: Induced pluripotent stem cells (iPSCs), Oligodendrocytes
Is TRPA1 involved in fatty acid dysregulation-induced myelin disorders? (#474)
V. Giacco1, N. B. Hamilton1
1 Kin'g College London, Wolfson Centre for Age-Related Diseases, London, United Kingdom
Myelin is the most essential component for speeding up action potential propagation across long distance neurons. Oligodendrocytes are specialized in wrapping axons to create these myelin sheets whose defects occur in a wide range of degenerative diseases, such as Multiple Sclerosis and Leukodystrophies. It is known that myelin damage can be receptor-mediated and recently oligodendrocytes have been shown to express Ca2+-permeable TRPA1 channels, whose activation can result in myelin damage in ischaemia. Moreover, accumulation of fatty acids and their metabolites induce several receptor-signalling mechanisms that are associated with demyelination. As TRPA1 can be activated by long chain polyunsaturated fatty acids (VLCFA) and their derivatives, our aim is to investigate a possible role of TRPA1 activation in fatty acid associated myelin damage.
To answer this question, we are using i) patch-clamp experiments from freshly isolated brain slices to record oligodendrocyte currents during application of VLCFA and their derivatives; ii) Ca2+ imaging to determine the role of Ca2+ changes in response to VLCFA; and iii) organotypic brain slices to assess the chronic effect of VLCFA on myelination.
Keywords: Ion channels, Myelin diseases, Oligodendrocytes
Atypical myelin physiology and dynamics in a cortistatin-deficient environment (#495)
C. P. Falo1, J. Castillo-González1, I. Forte-Lago1, A. Stucchi1, F. O'Valle2, M. Caro1, E. González-Rey1
1 CSIC, 1Institute of Parasitology and Biomedicine / Cell Biology and Immunology, Granada, Spain
Multiple sclerosis (MS) is a chronic autoimmune inflammatory pathology that leads to myelin depletion and progressive axonal degeneration. Natural remyelination happens in a healthy central nervous system, and it partially works in the early stages of MS. However, once the disease becomes chronic, remyelination capability is lost. Current research is focused on characterizing the factors and pathways involved in remyelination in order to understand and prevent its failure. Between them, molecular and cellular mechanisms involved in the oligodendrocyte (OL) dynamics (including the proliferation and migration of these glial cells to the damage location in the CNS and the myelin sheet synthesis) and myelin debris clearance during MS, have received special attention. Therefore, novel therapeutic goals are focused not only on developing therapies with an immunomodulatory effect but also in promoting neuroprotection and neuroregeneration by encouraging the remyelination process. Cortistatin (CST) is a neuropeptide with anti-inflammatory and immunomodulatory functions distributed in the nervous and immune systems. In spite of our previous work, in which we demonstrated the immunomodulatory and therapeutic effect of CST in the experimental autoimmune encephalomyelitis murine model of MS, its therapeutic and endogenous role in the dynamics of glial cells during de-and remyelination is unknown. In order to investigate the role of CST ignoring the effects of infiltrating peripheral immune cells, we used the preclinical model of acute cuprizona (CPZ) intoxication. Our results showed that CST seemed to modulate the OL lineage through the course of the disease, and that lack of CST was associated with aberrant myelin patterns as observed by transmission electron microscopy in corpus callosum samples of CPZ mice. This atypical myelin belonging to CST-deficient mice was related to an unsuccessful phagocytosis rate by microglia. According to this, in vitro OL cell culture revealed a role of CST in the cell dynamics, during both proliferation and differentiation processes. Besides, co-culture of dorsal root ganglia neurons and OL demonstrated the functionality of mature OL is also modulated by this neuropeptide. Together, these results bring to light the significance of knowing the factors, as CST, that play a key role in the crosstalk between the nervous and immune system, acting as potential therapeutic agents in immune neurodegenerative sickness.
Funding: Spanish Ministry of Economy and Competitiveness-SAF2014-58354-R and SAF2017-85602-R
Keywords: CNS myelin, Neuromodulation, Oligodendrocytes
A conditional mouse model and in vitro system to study Gba1 in myelinating glia: novel contribution for Gaucher Disease and Parkinson’s Disease (#519)
I. Gregorio1, M. Chrisam1, D. Bizzotto1, E. Moro1, M. Cescon1
1 Università degli Studi di Padova, Department of Molecular Medicine, Padova, Italy
Glucocerebrosidase (GBA1) is a lysosomal enzyme that cleaves the β-glycosidic linkage of the molecule glucocerebroside, an intermediate in glycolipid metabolism. Mutations affecting the functionality of this gene cause two diseases impinging on the nervous system: Gaucher Disease (GD) and Parkinson’s Disease (PD). GD is a lysosomal storage disease caused by the pathological accumulation of glucocerebroside, mainly affecting blood cells, spleen, bones and both peripheral and central nervous system to different extents. PD is characterized by long-term neurodegeneration and mutations of GBA1 are numerically the most important risk factor for this disease. For both GD and PD, the neurodegenerative and neuropathic symptoms are often linked to neuronal degeneration, but the contribution of myelin was so far almost neglected, despite its fundamental role in neuronal and axonal support. Thus, we are exploiting both in vivo and in vitro systems to study for the first time the role of myelinating glial GBA1 and how this is involved in the onset of GD and PD.
We generated a conditional mouse model crossing transgenic mice carrying the Cre Recombinase under the control of the myelin specific Cnp1 promoter (Cnp1-Cre) with a line in which the loxP sequences flank the region coding for the catalytic site of Gba1 (Gba1floxed/floxed). Recombination was checked by genomic PCR performed on myelinated tissues, and enzyme inactivation was confirmed by Western blot analysis and enzymatic activity assay.
In parallel, to better understand the molecular alterations occurring in oligodendrocytes upon GBA1 inhibition, we performed in vitro analysis using undifferentiated and differentiated oli-neu cells treated with Conduritol B epoxide (CBE) an irreversible GBA1 inhibitor.
Our results on mice show motor and non-motor behavioural deficits in Cnp1Cre::Gba1floxed/floxed compared to controls, already at 3 mo of age.
On oli-neu cultures, enzymatic activity assay confirmed the enzyme inhibition and the accumulation of glucocerebroside upon CBE treatment, especially on differentiated cells. Moreover, perturbed lysosomal function as well as alterations in the expression of myelin proteins were observed, highlighting the role of Gba1 in lysosomal homeostasis and CNS myelination.
Our results for the first time point to the impact of myelinating glia contribution to the onset of GD and PD, since Gba1 is expressed by these cells types, and its absence in oligodendrocytes affects the lysosomal machinery and myelin proteins expression.
Keywords: Neurodegenerative diseases, Oligodendrocytes, Cellular and Developmental Neuroscience
CRISPR/Cas9-mediated gene editing strategy to modulate Plp1 expression for Pelizaeus Merzbacher disease caused by Plp1 duplication (#527)
G. - B. Cho1, H. S. Bae1, H. Shin1, J. M. Lee1, J. Y. Lee1
1 Toolgen, R&D Therapeutics, Seoul, Republic of Korea
Pelizaeus Merzbacher disease (PMD) is a leukodystrophy caused by mutations in gene encodes for proteolipid protein 1 (PLP1). PLP1 is the most abundant myelin protein in the central nervous system (CNS) and alterations in PLP1 expression in the CNS can cause hypomyelination and axonal damage as exhibited by PMD patients. Majority of PMD patients harbor duplication of PLP1 gene which results in over-produced PLP1 protein. On the other hand, deletion mutations of PLP1 gene which prevents PLP1 protein production can also cause PMD. Therefore, tight maintenance of normal physiological PLP1 expression is crucial. In this regard, the rationale therapeutic approach for PMD caused by PLP1 duplication is to modulate PLP1 gene expression to normal range. To modulate PLP1 gene expression we utilized CRISPR/Cas9 derived from Streptococcus pyogenes (SpCas9) and Campylobacter jejuni (CjCas9; smallest Cas9 characterized by far) to target regulatory regions of PLP1. Specifically, we aim to target TATA-box of promoter region or intronic enhancer region by CRISPR/Cas9. Targeted deletion of these regulatory regions in oligodendrocyte-like cell line and primary oligodendrocytes resulted in modulation of Plp1 expression as measured by qRT-PCR or western blot analysis under differentiation culture condition. Furthermore AAV-mediated delivery of these CRISPR/Cas9 targeting regulatory regions of Plp1 reduced its expression in vivo. Taken together these gene editing strategy to modulate Plp1 expression warrants further proof-of-principle study to evaluate therapeutic efficacy in animal models of Plp1 duplication.
Keywords: Inherited disorders of myelination of the central nervous system, Regulation of gene expression, CRISPR-Cas9 gene editing
Therapeutic genome editing for Charcot-Marie-Tooth 1A (#536)
J. Y. Lee1, J. - S. Lee2, D. W. Song1, H. S. Bae1, H. S. Yu1, K. J. Lee1, S. Kim1, Y. B. Hong2, B. - O. Choi2, J. M. Lee1
1 ToolGen, R&D Therapeutics, Seoul, Republic of Korea
Charcot-Marie-Tooth 1A (CMT1A) is the most common inherited neuropathy without a known therapy, which is caused by a 1.4 Mb duplication on human chromosome 17, which includes the gene encoding the peripheral myelin protein of 22 kDa (PMP22). Overexpressed PMP22 protein from its gene duplication is thought to cause demyelination and subsequently axonal degeneration in the peripheral nervous system (PNS). Here, we targeted TATA-box of human PMP22 promoter to normalize overexpressed PMP22 level in C22 mice, a mouse model of CMT1A harboring multi copies of human PMP22. Direct local intraneural delivery of CRISPR/Cas9 designed to target TATA-box of PMP22 before the onset of disease, downregulates gene expression of PMP22 and preserves both myelin and axons. Notably, the same approach was effective in partial rescue of demyelination even after the onset of disease. Collectively, our data present a proof-of-concept that CRISPR/Cas9-mediated targeting of TATA-box can be utilized to treat CMT1A.
This work was supported by grants from the Korean Health Technology R&D Project, Ministry of Health & Welfare (HI14C3484 and HI16C0426) and by NRF grants funded by the Korean government (2016R1A5A2007009, 2017R1A2B2004699, 2018R1A4A1024506, 2017M3A9B4061404, and 2018M3A9H3020844) and by the National Institute of Health (RO1 NS094388).
Keywords: Neuropathy, Regulation of gene expression, CRISPR-Cas9 gene editing
Brain hypoxia in demyelination and remyelination (#554)
A. M. Rondelli1, K. R. Kranc2, S. R. Walmsley3, A. Williams1
1 MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
In the normal mammalian brain, interstitial tissue oxygen levels are low (~1 to 8 %). The brain is very sensitive to reductions in tissue oxygen levels, and during mild hypoxia develops adaptive mechanisms (via the transcription factor HIF1α and its target genes) that help sustain physiological functions, with changes in metabolism, angiogenesis, cell proliferation and cell death. In both human and animal models of multiple sclerosis (MS), neuropathological and imaging studies show the presence of tissue hypoxia and evidence of tissue adaptation to this hypoxia. It is within this hypoxic environment that remyelination of MS lesions occurs, but we do not understand whether this worsens tissue repair or whether the tissue adaptations aid repair, similarly to the effects of hypoxia pre-conditioning in other organs/diseases.
To test this hypothesis, we have used two experimental paradigms: 1) exposure to hypoxia during remyelination and 2) exposure to hypoxia prior to demyelination, as pre-conditioning. We have used stereotactic injection of Lysophosphatidylcholine (LPC) into the mouse corpus callosum as a focal model of demyelination, exposed mice to mild hypoxia (10% normobaric oxygen) and assessed remyelination efficiency at 10 and 15 days post lesion by electron microscopy.
We have shown that these demyelinated lesions show signs of tissue hypoxia and activation of the hypoxia pathway even at 15 days post lesion, when remyelination is occurring. Exposure to hypoxia during remyelination resulted in a significant decrease in the number of remyelinated axons, with no effect on the total number of axons compared to mice undergoing remyelination in normoxia. We will next assess whether hypoxia preconditioning affects subsequent remyelination.
These results suggest that tissue hypoxia during the process of remyelination is detrimental for myelin repair providing possible targets for intervention to improve remyelination.
Keywords: CNS myelin, Myelin diseases, Remyelination
G protein coupled receptor 37 (GPR37) inhibits remyelination (#567)
H. - J. Yang1, 2, A. Vainshtein1, Y. Eshed-Eisenbach1, E. Peles1
1 Weizmann Institute of Science, Department of Molecular Cell Biology, Rehovot, Israel
GPR37 has been recognized as a negative regulator of oligodendrocyte differentiation and myelination during development. However, its role during remyelination under pathological condition is yet to be determined. To understand the function of GPR37 in remyelination, we have compared between wild type and mice lacking GPR37 after the induction of immune (EAE), focal toxin (lysolecithin), and systemic toxin (cuprizone) mediated demyelination. Lumbar spinal cords and corpus callosum were used for analysis of EAE and two toxin models, respectively.
We found that the absence of GPR37 led to enhanced remyelination in all three demyelinated models. In EAE, clinical score of Gpr37 null mice was significantly lower than wild type animals from day 16 until day 30 post induction of demyelination. We observed a 1.7-fold increase in the number of remyelinated axons in lumbar spinal cords of Gpr37 null mice compared to wild type animals. In the focal demyelination lysolecithin model, we noted that while the lesioned area was similar between genotypes at day 7, a week later there was a marked increase in the PLP positive remyelinated area in Gpr37 null mice compared to wild type animals. In contrast to the comparable demyelination observed in Gpr37 null and wild type mice in the lysolecithin models, cuprizone induce much faster and severe demyelination in the corpus callosum of the mutant, suggesting a protective role for GPR37. Nevertheless, similar to the other demyelination models, we noted a significant increase in the presence of remyelinated axons in Gpr37 null mice compare to wild type animals. Our study suggests that GPR37 not only negatively regulate developmental myelination but also remyelination in the adult brain.
Keywords: CNS myelin, Oligodendrocytes, Remyelination
Dynamic myelinogenesis is required for spatial learning and memory in mice (#568)
F. Wang1, Y. S. Ren1, F. Mei1
1 Third Military Medical University, Histology and Embryology, Chongqing, China
Active myelinogenesis persists throughout lifetime in CNS, but its dynamics and functional significance for spatial learning and memory remains unclear. By lineage-specific tracing of newly-generated oligodendrocytes (OLs), we demonstrated that myelinogenesis in mouse brains is gradually declining in an age-dependent manner. To address the functional significance of the active myelinogenesis, we inhibited oligodendroglial differentiation and myelination in adult mice by inducing genetically deletion of the transcriptional factor Olig2 in OPCs. The inhibited myelinogenesis results in impaired memory and cognitive functions in adult mice. Together, our data indicates that active myelinogenesis is required for maintaining neural plasticity.
This work was supported by the National Natural Science Foundation of China (31771120, 31471043).
Keywords: Ageing, CNS myelin, Oligodendrocytes
Cell-autonomous requirement of TDP-43, an ALS/FTD signature protein, for oligodendrocyte survival and myelination (#570)
S. - C. Ling1, J. Wang1, W. Y. Ho1, K. Lim1, J. Feng1, G. Tucker-Kellogg2, K. - A. Nave3
1 National University of Singapore, Physiology, Singapore, Singapore
TDP-43 aggregates in neurons and glia are the defining pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), raising the possibility of glial damage in the disease pathogenesis. However, the normal physiological functions of TDP-43 in glia are largely unknown. To address how TDP-43 may be required for oligodendroglial functions, we selectively deleted TDP-43 in mature oligodendrocytes in mice. Although mice with TDP-43 deleted in oligodendrocytes are born in an expected Mendelian ratio, they develop progressive neurological phenotypes leading to early lethality accompanied by a progressive reduction in myelination. The progressive myelin reduction is likely due to a combination of the cell-autonomous RIPK1-mediated necroptosis of mature oligodendrocytes and the TDP-43-dependent reduction in the expression of myelin genes. Strikingly, enhanced proliferation of NG2-positive oligodendrocyte precursor cells within the white matter, but not the grey matter, was able to replenish the loss of mature oligodendrocytes, indicating an intrinsic regeneration difference between the grey and white matter oligodendrocytes. By contrast, there was no loss of spinal cord motor neurons and no sign of denervation at the neuromuscular synapses. Taken together, our data demonstrate that TDP-43 is indispensable for oligodendrocyte survival and myelination, and loss of TDP-43 in oligodendrocytes exerts no apparent toxicity on motor neurons.
This work was supported by grants to S.-C. L. from the Swee Liew-Wadsworth Endowment fund, National University of Singapore (NUS), National Medical Research Council (NMRC/OFIRG/0001/2016 and NMRC/OFIRG/0042/2017) and Ministry of Education (MOE2016-T2-1-024), Singapore. S.-C. Ling.
Wang, J., Ho, H.W., Lim, K., Feng, J., Tucker-Kellogg, G., Nave., K.-A., Ling, S.-C.*, “Cell-autonomous requirement of TDP-43, an ALS/FTD signature protein, for oligodendrocyte survival and myelination”, Proc Natl Acad Sci U S A., 2018, 115(46):E10941-10950
Graphic summary of a cell-autonomous role for TDP-43 in mature oligodendrocytes.
TDP-43 depletion in mature oligodendrocytes leads to RIPK1-mediated cell-autonomous degeneration. Enhanced proliferation of OPCs compensate for the loss of mature oligodendrocyte in the white, but not the grey, matter. In parallel, TDP-43 regulates the expression of myelin-related genes, such as Plp1 and Mbp and Plp1, that is essential for myelin. Deletion of TDP-43 down-regulates myelin-related genes leading to loss of myelination capacity.
Keywords: ALS, Neurodegenerative diseases, Oligodendrocytes
Hypoxia-inducible factor 1 alpha promotes peripheral nerve myelination (#574)
Y. Ujiie-Kobayashi1, S. Wakatsuki1, T. Araki1
1 National Center of Neurology and Psychiatry, Department of Peripheral Nervous System Research, National Institute of Neuroscience, Kodaira, Japan
Schwann cells (SCs) generate myelin sheath in the peripheral nervous system (PNS), and their differentiation plays an important role in PNS myelination. Mature SCs are formed via a stepwise differentiation during development from neural crest cell-derived SC precursors to immature SCs and pro-myelinating SCs, followed by myelinating/non-myelinating SCs. Characteristic expression of genes, including several transcriptional factors, have been described in each developmental stage. However, underlying mechanisms of SC differentiation remain unclear. Recent studies have shown that hypoxia-inducible factor-1 alpha (HIF-1α) plays a role in myelination in the central nervous system. Here, we investigated the role of HIF-1α in PNS myelination. We found that HIF-1α can be stabilized in nuclei of SCs cultured under hypoxia (1% O2). Culturing in hypoxia or overexpression of HIF-1α bearing mutation to be resistant to proteasomal degradation resulted in upregulation of myelin related gene expressions in SCs. By immunohistochemistry, HIF-1α was localized in S100β-positive SCs in murine sciatic nerve during development. HIF-1α expression in protein level was higher during development than in adulthood in mice, while HIF-1α mRNA expression was almost constant. Moreover, HIF-1α stabilizing drug that inhibits prolyl hydroxylation was able to upregulate myelin protein expression and promoted myelination in culture. Transient hypoxic incubation also facilitated in vitro myelination. These finding suggest that HIF-1α induces SCs differentiation and promotes PNS myelination during development.
Keywords: PNS myelin, Schwann cells, Molecular Neuroscience
Requirement of TDP-43 in myelin-competent glia (#584)
S. Bachofner1, J. A. Pereira1, C. Fimiani1, J. Keller1, J. Gerber1, U. Suter1
1 Swiss Federal Institute of Technology, ETH Zurich, Institute of Molecular Health Sciences, Zurich, Switzerland
TAR DNA binding protein 43 (TDP-43) is an RNA binding protein involved in RNA splicing and stability. Typically found in the nucleus, it is known to interact with consecutive UG-repeats mostly found in distal intronic regions. Binding to these sites precludes integration of intronic sequences, designated cryptic exons, into mature mRNAs. The majority of such cryptic splicing events lead to frame shifts and/or premature termination codons, thereby reducing mRNA levels or diminishing translation to functional proteins. Cryptic splicing events are poorly conserved between different species and variable between cell types. Whether cells are mainly dependent on the cryptic splicing silencing function, or on the other functions attributed to TDP-43, remains to be systematically evaluated.
TDP-43 has been mostly studied in the context of disease models and neurons, since it is frequently found in aggregates of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Mutations in TDP-43 have also been associated with these severe human disorders. Beyond neurons, oligodendrocytes have also been implicated in TDP-43 FTLD pathology. TDP-43 (TBPH) knockdown in Drosophila glia cells results in age-dependent motor deficits, premature lethality, and defects in motor neuron wrapping. These data argue for a vital function of TDP-43 in glia cells. Furthermore, mice harboring TDP-43 deficient oligodendrocytes display progressive motor deficits and premature lethality, at least in part associated with the death of differentiated oligodendrocytes.
Despite the current published knowledge, there are still numerous questions awaiting experimental resolution. Does TDP-43 affect myelinating glia beyond triggering cell death, by interfering with differentiation, myelination, and the maintenance of the adult myelinated state? What is the impact of TDP-43 deletion on the transcriptome of myelin-competent glia, including on the retention of unwanted cryptic exons? Is there a detrimental impact on axons and neurons upon TDP-43 deletion in myelinating-competent glia?
To address such questions, we conditionally excised mouse TDP-43 in myelination-competent glia. We confirmed cryptic splicing events in several described targets, supporting the successful TDP-43 functional loss from recombined cells. KO animals progressively develop behavioral deficits, along with vacuolation of myelinated fibers.
Keywords: CNS myelin, Development, PNS myelin
In vivo study on adenosine A1 receptor functions in oligodendrocyte precursor cells (#593)
Q. Guo1, Q. Liu1, L. Caudal1, A. Scheller1, W. Huang1, F. Kirchhoff1
1 University of Saarland, Center for Integrative Physiology and Molecular Medicine (CIPMM), Molecular Physiology, Homburg, Saarland, Germany
Adenosine A1 receptor (A1AR) signaling exerts important functions in the central nervous system (CNS). Among all neural cell types, A1AR mRNA levels are highest in oligodendrocyte precursor cells (OPCs). However, in vivo data are still missing regarding the functions of A1ARs in oligodendrocyte (OL) lineage cells, largely due to the lack of cell specific transgenic animal models. In this study, we generated NG2-CreERT2xA1ARfl/fl conditional knockout (cKO) mice to specifically delete A1ARs from OPCs upon tamoxifen administration. We induced Cre DNA recombinase activity at postnatal day 7 (P7) or P30 and analyzed these mice at P35 (P7:P35) or P90 (P30:P90), respectively. Immunostainings with lineage markers PDGFRα, APC CC1 and GSTπ as well as BrdU incorporation revealed no alterations of differentiation and proliferation in the corpus callosum (CC) and dorsal cortex of A1AR cKO mice. To study the functions of OPC-specific A1ARs during remyelination, we induced demyelination in mice at P30:P60 by cuprizone treatment for three weeks. One week after withdrawal of cuprizone, we observed enhanced remyelination in the CC of A1AR cKO mice compared to control mice. We also observed increased myelin basic protein (MBP) expression and more mature CC1+ OLs, approximately 30% and 10% more in comparison to control mice, respectively. However, Ki67 immunostaining suggested that the proliferation of OPCs was not altered in the A1AR cKO mice during remyelination. Taken together, our results suggest that A1ARs do not significantly affect the proliferation and differentiation of OPCs during development, but negatively regulate remyelination after cuprizone-induced demyelination.
Keywords: NG2 cells, Remyelination
The epigenetic role of vitamin C in Schwann cell myelination (#609)
T. C. Huff1, D. W. Sant1, V. Camarena1, S. Mustafi1, P. V. Monje2, G. Wang1
1 University of Miami Miller School of Medicine, Hussman Institute for Human Genetics, Miami, Florida, United States of America
Peripheral neuropathies are among the most common morbidities in the United States, the most notable being Charcot-Marie-Tooth disease type 1 (CMT1) and diabetic peripheral neuropathy (DPN). Both diseases are characterized by heterogeneous sensory deficits, neuropathic pain, and pathological Schwann cell demyelination. Many molecular agents have been implicated in Schwann cell myelination and development. Among these signals critical for myelination is vitamin C, the dietary micronutrient classically implicated in the prevention of scurvy. Vitamin C is vital cofactor for the ten-eleven translocation (TET) enzyme family which promotes active DNA demethylation. This finding implicates vitamin C as a vital mediator of the epigenome. Although the importance of vitamin C for Schwann cell myelination in vitro has been known for decades, its epigenetic function in this process has been largely unexplored. We hypothesized that vitamin C promotes Schwann cell myelination via DNA demethylation of genes associated with the myelin program. We found that vitamin C deficiency in mice resulted in hypomyelination of peripheral nerves throughout development. Using RNA and hydroxymethylated DNA Immunoprecipitation (hMeDIP) sequencing, we discovered that vitamin C treatment of Schwann cells upregulated numerous genes of the myelin program while downregulating negative myelin regulators and precursor markers. Upregulation of myelin genes such as Krox20, PRX, and MBP occurred in tandem with changes in DNA demethylation at these loci. Finally, we show that the expression of these genes throughout development and remyelination after injury is dysregulated in vitamin C deficiency. Overall, these results suggest that vitamin C-mediated DNA demethylation may be critical for peripheral myelination and may be a potential therapeutic target for peripheral neuropathies.
This work is supported by NIH grants (R01NS089525, R21CA191668), a Craig H. Neilson Foundation grant (M1501061), and by a Lois Pope Life Fellowship. We would like to thank Dr. Rong Wen for his technical support on semithin sectioning and Dr. Marcia Boulina of the Analytical Imaging Core Facility for her support with microscopy.
Keywords: Myelin diseases, PNS myelin, Schwann cells
The role of oligodendrocyte cholesteryl esters in Alzheimer's disease (#613)
Y. Zhang1, T. Gao1, H. Li1
1 University College London, The Wolfson Institute for Biomedical Research, London, United Kingdom
Myelin is the fatty multi-layered membranous sheath surrounding axons in the nervous system of jawed vertebrates. In the central nervous system, myelin is generated by oligodendrocytes. Myelin is a vital evolutionary acquisition that facilitates extremely rapid and efficient conduction of action potentials along axons. An essential component of myelin is cholesterol, which provides stability for the myelin structure. Cholesterol is known to play critical roles in the function of cell membrane and several metabolic pathways. Myelin houses most (70-80%). of the brain cholesterol, which is mainly synthesized de novo. In the brain, excess cellular cholesterol can be converted to cholesteryl esters by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) and stored as lipid droplets in cytoplasm. Recent evidence suggests that the balance between free and esterified cholesterol may be an important factor in AD pathology and ACAT1 has been proposed as a therapeutic target for Alzheimer’s disease (AD). Given that most brain cholesterol resides in myelin, we aim to reveal the role of oligodendrocyte cholesterol esters in age-related neurodegenerative diseases in this project. We evaluated ACAT1 expression and the distribution of cholesteryl esters in the developing, aging and AD mouse brain by immunostaining and histochemical methods. By cross-breeding, we generated an oligodendrocyte-specific Sox10-iCreERT2;Acat1fl/fl mouse line, which was then bred to J20, an AD mouse line, to tailor-make an AD model for subsequent experiments. We are investigating if knocking out Acat1 specifically in oligodendrocytes can rescue AD phenotypes in this mouse model.
Keywords: Alzheimer’s disease, Metabolism, Oligodendrocytes
Autotaxin, a regulator of oligodendrocyte differentiation (#620)
E. Suárez-Pozos1, F. S. Afshari1, K. M. Gorse1, W. H. Moolenaar2, J. L. Dupree1, B. Fuss1
1 Virginia Commonwealth University School of Medicine , Department of Anatomy and Neurobiology, Richmond , Virginia, United States of America
Autotaxin (ATX), also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) or phosphodiesterase-Iα (PD-Iα)/ATX, is a secreted glycoprotein primarily known for its enzymatic lysophospholipase D (lysoPLD) activity, which generates the lipid signaling molecule lysophosphatidic acid (LPA). LPA, in turn, exerts its functions through activation of a family of G protein-coupled receptors (GPCRs), the so-called LPA receptors. In our own studies, we identified ATX as a protein that is released by cells of the oligodendrocyte (OLG) lineage and functions via two mechanisms to drive OLG differentiation. Initially, we uncovered that ATX, via its C-terminally located modulator of oligodendrocyte remodeling and focal adhesion organization (MORFO) domain, promotes the establishment of a complex and expanded process network by post-migratory, premyelinating OLGs. More recently, we focused on ATX’s lysoPLD activity and found that the ATX-LPA axis promotes the expression of genes well-known to be associated with the earlier stages of OLG differentiation via, at least in part, the modulation of histone deacetylation. Studies undertaken in the developing zebrafish substantiated a critical role of ATX in regulating OLG differentiation during development. Interestingly, in the major demyelinating disease in humans, Multiple Sclerosis, there is evidence for reduced levels of ATX within the central nervous system (CNS) parenchyma. Here, we show that in a model of toxin-induced demyelination ATX levels are also reduced within affected CNS areas. In addition, we present data that support a role of OLG-derived ATX in regulating OLG differentiation not only during development but also after toxin-induced demyelination.
This work was supported by grants from the National Institute of Health (NIH/NINDS) and the National Multiple Sclerosis Society (NMSS).
Keywords: CNS myelin, Oligodendrocytes, Remyelination
Teneurin-4 is a positive regulator of CNS myelination through oligodendrocyte process formation (#668)
N. Suzuki1, 2, Y. Yamada2
1 Tokyo Medical and Dental University, Tokyo, Japan
Myelination by oligodendrocytes enables the rapid propagation of action potential and is essential for proper functioning of the CNS. Therefore, defects in CNS myelination cause various types of neural and mental disorders. However, the molecular mechanisms of oligodendrocyte development and CNS myelination have not been fully understood. In this study, we identified a mouse mutation caused by a transgene insertion that resulted in tremors and hypomyelination in the CNS, particularly in the spinal cord, but not in the PNS. Interestingly, myelination of small diameter axons and the number of oligodendrocytes were dramatically reduced in the mutant mice. We subsequently identified the transgene insertion site into the teneurin-4 (Ten-4) gene encoding a type II transmembrane protein, whose function was unknown. We found that Ten-4 was highly expressed in the spinal cord of wild-type mice and was induced during normal oligodendrocyte differentiation. In the mutant mice, however, the expression of Ten-4 was absent and no transgene expression was observed in the CNS, indicating that the deficiency of Ten-4 expression was responsible for the defects of myelination by oligodendrocytes. Primary oligodendrocytes from the mutant mice failed to form well-branched cellular processes in culture. In the oligodendrocyte progenitor cell line CG-4, suppression of Ten-4 expression by shRNA inhibited process formation. Further, the deficiency of Ten-4 attenuated the activation of the focal adhesion kinase, which is a regulator of myelination of small diameter axon in the CNS. These findings indicate that Ten-4 is a key regulator of oligodendrocyte process formation and is required for CNS myelination. This mutant mouse model will facilitate a better understanding of oligodendrocyte biology, as well as of the development of diagnostic and therapeutic reagents for dysmyelinating diseases.
Keywords: CNS myelin, Oligodendrocytes
Molecular interaction between oligodendrocytes and axons through Teneurins for CNS myelination (#670)
C. Hayashi1, N. Suzuki1, N. Kikura1, Y. Hosoda1, Y. Mabuchi1, C. Akazawa1
1 Tokyo Medical and Dental University, Tokyo, Japan
Oligodendrocytes (OLs) form myelin in the CNS and accomplish the rapid propagation of action potential. At the initial stages of myelin formation is a complex sequence of the events as following; 1) adherence of oligodendrocyte precursor cells (OPCs) to targeted axons, 2) reception of the signals from axons, 3) extension of processes of differentiating OPCs into OLs to ensheathe axons, and 4) compaction with excluding uncompacted cytoplasm to form myelin sheath. However, molecular mechanisms of these events, particularly regarding the molecular interaction/signal between OL and axon, have not been well understood. In our previous study, we reported that Teneurin-4 (Ten-4), a type II transmembrane protein, is required for CNS myelination. Here we focused on Ten-4 function and aimed to elucidate the molecular mechanism of the OPCs/OLs interaction with axons at the initial stage of myelination. Our histological analysis of Ten-4 deficient (-/-) mice revealed that Ten-4 -/- OLs failed to form compacted myelin because of disorganization of cytoskeleton in their extending processes and to maintain their number between P3 and P7, when OPCs/OLs initially contact/interact with axons. We next explored the binding partners of Ten-4 using mass-spectrometry for identification of co-immunoprecipitated proteins with Ten-4. As a result, interestingly, all of Teneurin isoforms (Ten-1 to -4) were obtained as candidates. Since all of Ten isoforms and only Ten-4 are expressed on neuronal axons and OPCs/OLs, respectively, we hypothesized that Ten-4 on OPCs/OLs binds to Ten isoforms on axons and forms cell adhesion between them. To verify this, we assessed the cell-cell adhesion activity between Ten-4 and Ten isoforms and found that Ten-4-overexpressing cells formed larger cell aggregates with all of Ten isoforms-overexpressing cells, homophilically and heterophilically. In addition, OPCs/OLs attached to the recombinant extracellular domains of Ten-1 to -4 (rTen-1 to -4ECD). The attachment was inhibited in the presence of soluble rTen-4ECD or anti-Ten-4ECD antibody, suggesting that Ten-4 on OPCs/OLs interacted with Ten isoforms presumably on axons. To examine the biological effect of the bindings, we cultured OPCs/OLs on rTen-1 to -4ECD and found that OL process formation with cytoskeletal organization and cell survival were promoted. Further, the myelination assay using nanofibers revealed that rTen-1 to -4 promoted myelin formation by OLs. We finally demonstrated that Akt and Erk signaling is responsible for the Ten-4-Ten isoforms’ activity downstream of Ten-4. From these observations, we concluded that Ten-4 positively regulates the OL-axon interaction together with Ten isoforms and functions as a platform of the signaling pathways for CNS myelination.
Keywords: CNS myelin, Oligodendrocytes
Autophagy in oligodendrocytes (#674)
N. Ktena1, 2, V. Nikoletopoulou1, D. Karagogeos1, 2, M. Savvaki1, 2
1 Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology, Heraklion, Greece
(Macro)autophagy comprises a conserved lysosome-dependent catabolic pathway, facilitating degradation of cytoplasmic proteins and damaged organelles. Through its role in energy production and cellular homeostasis, autophagy is crucial during development as shown in many tissues and organisms, while its dysregulation has been linked to several disorders, including neurodegenerative diseases. Although a few studies implicate autophagy in CNS demyelinating pathologies, its role, particularly in oligodendrocytes, remains elusive.
In our study, we aim to shed light on the significance of autophagy in CNS myelin and oligodendrocytes. In a preliminary set of experiments, we have observed significant CNS hypomyelination in the Nestin-Cre; Atg5 fl/fl mouse line where autophagy is ablated in all CNS cells. We are currently examining the role of autophagy in both oligodendrocyte primary cultures as well as in vivo utilizing a new conditional mutant mouse line, in which autophagy is specifically ablated in the CNS myelinating glial cells after tamoxifen administration (Plp-CreERT2; Atg5 fl/fl). Using primary cultures, we showed that autophagy is active in mature oligodendrocytes and its pharmacological inhibition results in reduced maturation of these cells. More specifically, we showed that administration of SBI-0206965 resulted in a delay of oligodendrocyte differentiation over the three basic morphological categories that was restored by DIV8. At that time point, SBI-treated, myelin-producing oligodendrocytes showed a significantly altered morphology. The investigation of the underlying mechanism, as well as the in vivo effect of selective autophagy ablation in oligodendrocytes, is in progress.
This project has received funding from the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT), under grant agreement No 1676.
Keywords: CNS myelin, Oligodendrocytes
Single nuclei transcriptomics of human white matter oligodendroglia in multiple sclerosis (#687)
E. Agirre1, S. Jäkel2, A. M. Falcão1, D. van Bruggen1, I. Knuesel3, D. Malhotra3, K. W. Lee1, C. ffrench-Constant2, A. Williams2, G. Castelo-Branco1
1 Karolinska Institutet, Stockholm, Sweden
Oligodendrocytes myelinate and provide support to axons. Several diseases, such as multiple sclerosis (MS), are characterized by abnormal of defective myelination. Spontaneous remyelination occurs at initial stages of MS, promoted by endogenous oligodendrocyte precursor cells. However, this process progressively starts occurring with less efficiency, until it eventually fails.
In MS, demyelination in the central nervous system leads to neurodegeneration with a high variability between patients, which does not correlate with the extend of demyelination. One of the possible factors of such variability may be the oligodendrocyte heterogeneity. We previously found using single cell studies that oligodendrocytes are more heterogeneous than previously thought in mouse (Marques and Zeisel et al. 2016). However, the extent of human oligodendrocyte heterogeneity and its possible contribution to MS remains unclear.
In this study (Jäkel and Agirre et al. 2019) we performed single nuclei RNA-sequencing (snRNA-seq) from white matter (WM) areas of post mortem human brain both in control (Ctr) and MS patients. We applied canonical correlation analysis (CCA) to integrate control and disease nuclei from the different patients allowing us to define different oligodendroglia cell clusters. The sub-clusters of oligodendroglia in Ctr human WM, showed similarity to already described subclusters in mouse, but we were also able to define new markers for these cell states specific of human. Strikingly, some sub-clusters were under-represented in MS tissue, while others were more prevalent. Showing an altered pattern of oligodendroglia types between control and disease. These differences in mature oligodendrocyte sub-clusters may indicate different functional states of oligodendrocytes in MS lesions. Differential expression analysis between sub-clusters showed upregulation of myelin genes in the mature MS oligodendroglia sub-clusters, indicating that mature oligodendrocytes might increase transcriptional programs of myelination in the context of MS. Finally, our results of an altered oligodendroglial heterogeneity in MS show the power of single cell technologies to study and understand human pathology.
Keywords: Myelin diseases, Oligodendrocytes, Remyelination
L-prostaglandin D2 synthase regulates Schwann cells metabolism (#706)
A. Trimarco1, M. Cariello1, M. Audano3, A. Cestaro2, D. Caruso3, N. Mitro3, C. Taveggia1
1 San Raffaele Scientific Institute, Division of Neuroscience and INSPE, Axo-glial Interaction Unit, Milan, Italy
Myelin provides trophic support to neurons and electrical insulation for rapid impulse transmission. In the peripheral nervous system (PNS), myelin stability strictly depends on constant and optimal communication between axons and Schwann cells (SC). We previously showed that in the PNS, prostaglandin D2 (PGD2), one of the final products of the arachidonic acid metabolism, is synthesized by neuronal L-prostaglandin D2 synthase (L-PGDS). We also showed that loss of L-PGDS enzymatic activity causes hypomyelination early in development and myelin aberrations and degeneration in adulthood.
To identify the molecular constituents of myelin degeneration, we performed: 1) RNAseq analyses on rat SC–mouse neuronal cocultures presenting signs of demyelination; 2) metabolic flux experiments on these cocultures to determine changes in metabolites’ consumption and 3) lipidomic and metabolomic studies on sciatic nerves from aged L-PGDS null mice. In vitro assays revealed a set of genes specifically upregulated in SC, the majority of which are involved in arachidonic acid transport and glucose metabolism and are under the control of PPARγ nuclear receptor. Further, metabolic fluxes analyses revealed pyruvate accumulation and increased acetate metabolism. Finally, in vivo, we observed decreased levels of phospholipids containing arachidonic acid and of key metabolites of glycolysis and of Krebs cycle.
Collectively, we posit that in the absence of PGD2 myelin degeneration could arise from increased levels of arachidonic acid in SC leading to an up-regulation in the expression of PPARγ target genes and an overall impairment in glial cells metabolism.
Keywords: Metabolism, PNS myelin, Schwann cells
Toward a comprehensive understanding of promyelinating drugs molecular mechanism of action for Central Nervous System remyelination. (#731)
A. Del Giovane1, M. Tiberi1, E. Nocita1, F. Basoli2, A. Rainer2, A. Ragnini-Wilson1
1 Università degli studi di Roma Tor Vergata, Biology, Roma, Steiermark, Italy
Myelin, oligodendrocyte losses and neurodegeneration are the hallmark of demyelinating diseases affecting the Central Nervous System (CNS). Pharmacological approaches to remyelination aim to restore neurological function by restoring the myelin sheath covering axons in order to impair neuronal loss and neurodegeneration.
A number of phenotypical screens, that made use of FDA-approved compound libraries and primary rat oligodendrocyte precursor cells (OPC), Epiblast-derived OPC or oligodendrocyte cell line expressing MyrF (Oli-neuM), have been performed in the recent years. They led to the identification of a handful number of biological active drugs (among which Clobetasol, Benztropine, Clotrimazole and Miconazole), that promote either Neuronal Precursor Cells (NPC) proliferation/migration and/or OPC differentiation and MBP expression. Those drugs were proven to promote remyelination in toxically-induced demyelination mouse model, Experimental Autoimmune Encephalomyelitis and/or in Neuromyelitis Optica murine model.
All together, these studies have shown that pharmacological approaches to CNS remyelination are feasible. On the other hand, their future clinical use remains strictly linked to the clarification of their mechanism of action (MOA), as their CNS remyelination potential is counterbalanced by their potential pharmacological activity on secondary targets at therapeutic concentration. We have performed one of the phenotypical screens that led to the identification of Clobetasol and other pro-myelinating drugs in Oli-neuM cells line. Here, we will present our current understanding of hit drugs MOA in Oli-neuM cells in an in vitro model of synthetic microfiber engagement.
Keywords: Cell determination and differentiation, CNS myelin, Oligodendrocytes
PINCH proteins regulate myelination of axons in the central nervous system (#752)
J. Paes de Faria1, 2, R. S. Silva1, 2, J. B. Relvas1, 2
1 Instituto de Biologia Molecular e Celular (IBMC), Porto, Portugal
In the central nervous system, myelin, which consists of the lipid-rich multilayered membrane sheath laid down along axons, is produced in a precise temporal sequence of events, by oligodendrocytes in response to different signals including those derived from the extracellular matrix (ECM). A major group of receptors for ECM molecules are integrins. Since their cytoplasmatic domains lack enzymatic activity, integrins require the recruitment of adaptor proteins such as ILK, PINCH, and parvin to form signaling hubs (focal adhesions) to enable the activation of intracellular pathways. Mammals display two PINCH proteins, PINCH1 and PINCH2, that bind in a mutually exclusive mode to ILK, and then to parvin to form the IPP complex. This complex serves as a molecular adaptor between integrin signaling and the actin cytoskeleton. Deletion of any of the IPP complex components results in disruption of the complex and has a dramatic effect on myelination. Binding of either PINCH confers the IPP complex specific interactors and activates potential distinct downstream targets. We genetically ablated PINCH1 and PINCH2 in oligodendrocytes at different stages of development, and we have examined in vivo progression of myelination in different white matter regions of the central nervous system. Our results showed that -with some degree of redundancy among them- the function of each PINCH is fundamental during the active phase of myelination. We also observed that PINCH2 protein acts as a negative regulator of myelination: we observed hypermyelinated axons in PINCH2-depleted CNS regions and hypomyelinated tracts upon in vivo overexpression of PINCH2. Overall, these findings reveal a critical role of PINCH in regulating myelin biogenesis and fine-tuning of different signaling pathways downstream of integrins during myelination.
Keywords: CNS myelin, Oligodendrocytes
The small GTPase RhoA regulates the onset of myelination and myelin production during PNS development (#753)
A. Seixas1, M. Morais1, J. A. Pereira2, S. Krause2, C. Brakebusch3, U. Suter2, J. B. Relvas1
1 i3S/IBMC, University of Porto, Porto, Portugal
In cells, control of actin cytoskeleton rearrangements mediated by classical Rho GTPase signaling is important for morphological plasticity governing many biological processes in development. Early postnatally, during development of the peripheral nervous system (PNS), Schwann cells (SCs) respond to extracellular signals by extending cytoplasmic protrusions into embryonic axon bundles to contact and segregate axons destined to be myelinated in a process called radial sorting. This morphological plasticity of SCs, important for the interaction with axons and wrapping to occur, is conveyed by signaling pathways regulating actin cytoskeleton rearrangement. Studies using constitutively active forms or chemical inhibition of its main effector ROCK suggest a critical regulatory role for RhoA signaling in SC biology and axon ensheathment. Conditional mutants for various extracellular matrix proteins, their receptors or downstream signaling molecules showed that inhibition of RhoA activation is required for radial sorting.
We sought to study the specific functions of the classical Rho GTPase RhoA in SC development and myelination in the PNS using a conditional RhoA mutant (RhoA-cKO, using Dhh-Cre and Cnp-Cre transgenic lines). Mutant postnatal sciatic nerves showed a decrease in the number of sorted and myelinated axons, suggesting an impairment in radial sorting and a delay in the onset of myelination. We observed no changes in survival, proliferation or differentiation of RhoA-cKO SCs. SCs from RhoA-cKO cultured in vitro extended less radial lamellipodia. Biochemical and FRET analyses suggest dysregulation of signaling molecules related to actin dynamics, including cofilin, RhoC and Rac1. Sciatic nerves from RhoA-cKO had thinner myelin sheaths at P15 and P25 but after developmental myelination resumed, at P90, were hypermyelinated, indicating that RhoA regulates myelin production in the PNS.
Past studies indicated that a switch in the levels of active Rho controls radial sorting. Here, we show that genetic ablation of RhoA specifically in SCs leads to defects in peripheral nerve development and that RhoA independently regulates myelination in the PNS.
Keywords: Cytoskeletal proteins, PNS myelin, Schwann cells
The adhesive properties of myelin basic protein C1/C8 charge isomers and their role in microglia plasticity. (#764)
L. V. Shanshiashvili1, 2, 3, M. V. Chikviladze1, M. M. Sepashvili1, 2, I. V. Kalandadze2, E. Zaalishvili2, J. J. Ramsden3, D. G. Mikeladze1, 2
1 Ilia State University, Institute of Chemical Biology, Tbilisi, Georgia
Myelin basic protein (MBP), a major protein of the myelin sheath, is encoded by the Golli-MBP gene in myelinating glia. Several isoforms of classic MBP are formed by alternative splicing of a single mRNA transcript. The major MBP isoform in the adult human and bovine CNS is 18.5 kDa protein that plays a structural role in maintaining myelin stability. MBP exhibits charge microheterogeneity as a result of post-translational modifications such as phosphorylation, deamidation, deimination, arginine methylation, and N-terminal acylation. The fractions of MBP isomers which are isolated by cation-exchange chromatography on a carboxymethyl cellulose-52 (CM-52) column have been named from C1 to C8. C1 is the least modified and most cationic, whereas C8 is the most modified and the least cationic. Among these modifications, deimination is the most significant that involves the conversion of MBP arginine into citrulline by the enzyme peptidylarginine deiminase. This modification reduces the cationicity of the protein. Citrullinated MBP is structurally less ordered and more susceptible to proteolytic attacks. Thus, the reduction in cationicity of citrullinated MBP impedes the membrane assembly and exposes an immunodominant epitope of the membrane-bound protein to proteases. It’s important that the degree of deimination limits MBP’s ability to maintain compact myelin and correlates with the severity of multiple sclerosis.
We have studied the association of C8 and C1 MBP isomers with the myelin lipids in a model membrane system using optical waveguide lightmode spectrometry. The analysis of association/dissociation kinetics to planar lipids under controlled hydrodynamic conditions has shown that MBP C8 isomer is less effectively adsorbed on the lipid membrane, than C1 isomer and packing densities for C1 MBP is higher than for C8 isomer.
On the other hand, we have studied the effects of C1 and C8 on the microglia plasticity. We have found that MBP charge isomers take part in microglia polarization.
These data indicate that citrullination changes the association/dissociation kinetics of MBP to planar lipids, as well as takes part in microglia plasticity.
This research was supported by the SRNSF Georgia RF17_534 grant.
Keywords: CNS myelin, Microglial cells, Myelin diseases
Elucidating the repertoire of RNA-binding proteins associating with Myelin Basic Protein mRNA during oxidative stress conditions in oligodendroglial cells (#790)
P. Hoch-Kraft1, C. Gonsior1, F. Butter2, J. Trotter1
1 Johannes Gutenberg-University Mainz, Institute for Developmental Biology and Neurobiology, Mainz, Rhineland-Palatinate, Germany
Myelin Basic Protein (MBP) is one of the most abundant myelin proteins and indispensable for the formation of a compacted myelin sheath around target axons in the CNS. MBP expression is strictly regulated on the post-transcriptional level and Mbp mRNA has been discovered to be transported to the myelin compartment where it can be locally translated dependent on neuronal signals, including neuronal activity. In white matter diseases like multiple sclerosis, differentiation of oligodendrocyte progenitor cells (OPC) into mature MBP+ oligodendrocytes (OL) is a prerequisite for remyelination. Also in neurodegenerative diseases like Alzheimer’s disease or Amyotrophic Lateral Sclerosis changes in white matter are suggested to contribute to pathology. A mutual hallmark is the presence of elevated intracellular stress levels by intrinsic (e.g. mutations, age) or extrinsic factors (e.g. lesion environment) that potentially also influence the OPC fate. Oxidative stress has been shown to prevent differentiation of OPC and we found an extensive decrease in MBP protein levels in response to stress preferentially during the immediate recovery phase. Activation of the integrated stress response leads to a global attenuation of CAP-dependent translation and the redistribution of stalled mRNAs into cytoplasmic RNA granules, including stress granules (SG). We found Mbp mRNA to be prominently sorted into oligodendroglial SG and a maturation-dependent prolonged persistence of SG in MBP+ OL during the recovery phase. We intended to identify the repertoire of RNA-binding proteins associating with the Mbp transcripts during oxidative stress, regulating the subsequent mRNA maintenance. We therefore have created stable Oli-neu cell lines expressing MS2-tagged isoforms of Mbp mRNA and the system served as a basis for RNA-centric affinity purification. We carried out a proteomic screen using SILAC to identify binding partners of MS2-labelled Mbp mRNA during oxidative stress. The obtained dataset includes known RNA-binding proteins as well as proteins previously not directly associated with RNA-binding or regulation and several candidates have been connected to CNS disease before. We currently analyse the function of selected candidates on Mbp mRNA localisation, stability and translation during acute stress situations and recovery from stress. We aim to better understand the regulation of MBP (and other myelin mRNAs) under cellular stress conditions and the consequences for OL maturation and myelination.
Keywords: Oligodendrocytes, Regulation of gene expression, In situ hybridization (RNA scope)
Activation of GABAB receptors promotes oligodendrocyte precursor cell differentiation and maturation (#799)
M. P. Serrano Regal1, 2, 3, L. Bayón2, I. Luengas2, N. Ibarra2, A. Pérez Sanmartín1, 2, 3, J. C. Chara1, 2, 3, V. Tepavcevic1, F. Pérez Cerdá1, 2, 3, C. Matute1, 2, 3, M. V. Sánchez Gómez1, 2, 3
1 Achucarro Basque Center for Neuroscience, Leioa, Spain
Differentiation of oligodendrocyte precursor cells (OPCs) is the most relevant event for myelination of axons in the central nervous system, and for remyelination in demyelinating diseases as multiple sclerosis. Myelination is a complex process mediated by neuron-glia interactions that involve several molecules such as growth factors and neurotransmitters like GABA. In this line, we previously reported that the expression and function of GABAA receptors in cultured oligodendrocytes (OLGs) is driven by axonal cues and that GABA signaling may play a relevant role in myelination and/or during axon-glia recognition (Arellano et al., 2016). Here, we show that GABA receptors, specifically GABAB subtypes, are functional regulators of OPC differentiation and myelination, both in vivo and in vitro models. First, we demonstrate in isolated cultures, cerebellar organotypic slices and rat brain sections, that OPCs express GABAB receptors (GABABRs) and maintain this expression during its maturity to OLGs. Additionally, OLGs express the two GABA-synthesizing enzymes, GAD65/67 and MAOB, and the GABA-transporters GAT-1 and GAT-3. Moreover, we have detected that purified OLGs content intracellular GABA that may be released to the supernatant. Second, we have observed that exogenous GABA increases the number of myelin segments and MBP expression in DRG-OPC cultures, indicating that GABA may regulate axonal myelination. Surprisingly, selective GABABR activation with baclofen in a purified rat OPC culture accelerates OPC differentiation, enhances process branching and promotes local translation of MBP in peripheral areas. Moreover, chronic exposure of baclofen to isolated OPCs and cerebellar organotypic cultures promotes the production of MAG and MBP, effects that are attenuated in the presence of the GABABR antagonist CGP55485. Finally, we examine baclofen effects on oligodendroglial development by daily administration of baclofen (4mg/kg/day) or CGP35348 (10mg/kg/day) in developing rats, from postnatal day 6 to 12. Preliminary experiments show that GABABR modulation in these critical ages provokes changes in OLG differentiation and myelination rates in the corpus callosum and cerebellum, and increases the conduction velocity of the fibers in the corpus callosum, as shown by electrophysiological recordings. In conclusion, these results strongly suggest that GABA signaling via GABABRs is relevant to OLG maturation during development.
Arellano RO, Sánchez-Gómez MV, Alberdi E, Canedo-Antelo M, Chara JC, Palomino A, Pérez-Samartín A and Matute C. (2016). Axon-to-glia interaction regulates GABAA receptor expression in oligodendrocytes. Mol Pharmacol, 89:63-74.
Keywords: CNS myelin, Neurotransmitter and hormone receptors, Oligodendrocytes