Increased Expression of Golli Myelin Basic Proteins Enhances Calcium Influx into Oligodendroglial Cells

Paez, Pablo M.; Spreuer, Vilma; Handley, Vance; Feng, Ji‐Ming; Campagnoni, Celia W.; Campagnoni, Anthony T.

doi:10.1523/jneurosci.2381-07.2007

Cited by 61 publications

(102 citation statements)

References 56 publications

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“…This cell line stains positive for both the NG2 and A2B5 antibodies, which are markers for oligodendroglial progenitor cells (OPCs), and also lacks expression of classic MBP and proteolipid protein mRNAs [45]. This cell line has previously been used to examine the effects of apotransferrin over-expression on OLG differentiation, elaboration of neurite cell processes in co-culture with neurons, and more recently, the effects of golli and classic MBP over-expression on calcium homeostasis and cell migration [7,38,[46][47][48]. Another interaction of classic MBP that has been studied in N19 cells is that with SH3-domaincontaining proteins such as Fyn, where we have demonstrated that disruption and/or pseudophosphorylation of the SH3-ligand in MBP has significant effects on cell morphology, and on the trafficking of the protein [35].…”

Section: Classic 185-and 215-kda Mbp Isoforms Localize To Membrane-mentioning

confidence: 99%

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

et al. 2012

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The 18.5-kDa classic myelin basic protein (MBP) is an intrinsically disordered protein arising from the Golli (Genes of Oligodendrocyte Lineage) gene complex and is responsible for compaction of the myelin sheath in the central nervous system. This MBP splice isoform also has a plethora of post-translational modifications including phosphorylation, deimination, methylation, and deamidation, that reduce its overall net charge and alter its protein and lipid associations within oligodendrocytes (OLGs). It was originally thought that MBP was simply a structural component of myelin; however, additional investigations have demonstrated that MBP is multifunctional, having numerous protein-protein interactions with Ca 2+ -calmodulin, actin, tubulin, and proteins with SH3-domains, and it can tether these proteins to a lipid membrane in vitro. Here, we have examined cytoskeletal interactions of classic 18.5-kDa MBP, in vivo, using early developmental N19-OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). We show that MBP redistributes to distinct 'membrane-ruffled' regions of the plasma membrane where it co-localizes with actin and tubulin, and with the SH3-domaincontaining proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. Moreover, using phospho-specific antibody staining, we show an increase in phosphorylated Thr98 MBP (human sequence numbering) in membrane-ruffled OLGs. CIHR Author ManuscriptCIHR Author Manuscript CIHR Author ManuscriptPreviously, Thr98 phosphorylation of MBP has been shown to affect its conformation, interactions with other proteins, and tethering of other proteins to the membrane in vitro. Here, MBP and actin were also co-localized in new focal adhesion contacts induced by IGF-1 stimulation in cells grown on laminin-2. This study supports a role for classic MBP isoforms in cytoskeletal and other protein-protein interactions during membrane and cytoskeletal remodeling in OLGs.

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Section: Classic 185-and 215-kda Mbp Isoforms Localize To Membrane-mentioning

confidence: 99%

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

et al. 2012

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“…This cell line stains positive for both the NG2 and A2B5 antigens, which are markers for oligodendroglial progenitor cells (OPCs), and lacks expression of classic MBP and proteolipid protein mRNAs [35]. The N19-OLG line has been successfully employed in other studies, including specifically examining the effect of Golli and classic 18.5-kDa MBP over-expression on calcium homeostasis [13,20,40,41], and on the co-localization of 18.5-kDa MBP with cytoskeletal proteins and SH3-domain-containing proteins [21,22].…”

Section: Resultsmentioning

confidence: 99%

The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

Smith

Seymour

Boggs

et al. 2012

Biochemical and Biophysical Research Communications

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The predominant 18.5-kDa classic myelin basic protein (MBP) is mainly responsible for compaction of the myelin sheath in the central nervous system, but is multifunctional, having numerous interactions with Ca 2+ -calmodulin, actin, tubulin, and SH3-domains, and can tether these proteins to a lipid membrane in vitro. The full-length 21.5-kDa MBP isoform has an additional 26 residues encoded by exon-II of the classic gene, which causes it to be trafficked to the nucleus of oligodendrocytes (OLGs). We have performed site-directed mutagenesis of selected residues within this segment in red fluorescent protein (RFP)-tagged constructs, which were then transfected into the immortalized N19-OLG cell line to view protein localization using epifluorescence microscopy. We found that 21.5-kDa MBP contains two nontraditional PYnuclear-localization signals, and that arginine and lysine residues within these motifs were involved in subcellular trafficking of this protein to the nucleus, where it may have functional roles during myelinogenesis.

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“…The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15,5 , and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g. 6,10 ). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13,12 .…”

Section: Introductionmentioning

confidence: 99%

“…The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1,10 . In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.…”

mentioning

confidence: 99%

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

Smith

Voyer-Grant

Harauz

2012

JoVE

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The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8,16 . If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14 . Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4,14, 3,11,7 .In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15,5 , following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15,5 , and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g. 6,10 ). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13,12 . Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9 . We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2 . The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1,10 . In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques. Video LinkThe video component of this article can be found at https://www.jove.com/video/3422/ Protocol 1. Thawing and Culturing Cells 1. Obtain stock of frozen immortalized N19-oligodendroglial cells from long term liquid nitrogen stores. 2. Immerse vial containing cells at 37 °C water bath until the cell suspension is completely thawed. 3. Add 7 mL of DMEM (Dulbecco's Modified Eagle Medium) with high-glucose ...

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Increased Expression of Golli Myelin Basic Proteins Enhances Calcium Influx into Oligodendroglial Cells

Cited by 61 publications

References 56 publications

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

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