Regulation of store-operated and voltage-operated Ca2+ channels in the proliferation and death of oligodendrocyte precursor cells by golli proteins

OPCs (oligodendrocyte precursor cells) express golli proteins which, through regulation of Ca2+ influx, appear to be important in OPC process extension/retraction and migration. The aim of the present study was to examine further the role of golli in regulating OPC development. The effects of golli ablation and overexpression were examined in primary cultures of OPCs prepared from golli-KO (knockout) and JOE (golli J37-overexpressing) mice. In OPCs lacking golli, or overexpressing golli, differentiation induced by growth factor withdrawal was impaired. Proliferation analysis in the presence of PDGF (platelet-derived growth factor), revealed that golli enhanced the mitogen-stimulated proliferation of OPCs through activation of SOCCs (store-operated Ca2+ channels). PDGF treatment induced a biphasic increase in OPC intracellular Ca2+, and golli specifically increased Ca2+ influx during the second SOCC-dependent phase that followed the initial release of Ca2+ from intracellular stores. This store-operated Ca2+ uptake appeared to be essential for cell division, since specific SOCC antagonists completely blocked the effects of PDGF and golli on OPC proliferation. Additionally, in OPCs overexpressing golli, increased cell death was observed after mitogen withdrawal. This phenomenon could be prevented by exposure to VOCC (voltage-operated Ca2+ channel) blockers, indicating that the effect of golli on cell death involved increased Ca2+ influx through VOCCs. The results showed a clear effect of golli on OPC development and support a role for golli in modulating multiple Ca2+-regulatory events through VOCCs and SOCCs. Our results also suggest that PDGF engagement of its receptor resulting in OPC proliferation proceeds through activation of SOCCs

Targeted overexpression of a golli–myelin basic protein isoform to oligodendrocytes results in aberrant oligodendrocyte maturation and myelination

Recently, several in vitro studies have shown that the golli–myelin basic proteins regulate Ca2+ homoeostasis in OPCs (oligodendrocyte precursor cells) and immature OLs (oligodendrocytes), and that a number of the functions of these cells are affected by cellular levels of the golli proteins. To determine the influence of golli in vivo on OL development and myelination, a transgenic mouse was generated in which the golli isoform J37 was overexpressed specifically within OLs and OPCs. The mouse, called JOE (J37-overexpressing), is severely hypomyelinated between birth and postnatal day 50. During this time, it exhibits severe intention tremors that gradually abate at later ages. After postnatal day 50, ultrastructural studies and Northern and Western blot analyses indicate that myelin accumulates in the brain, but never reaches normal levels. Several factors appear to underlie the extensive hypomyelination. In vitro and in vivo experiments indicate that golli overexpression causes a significant delay in OL maturation, with accumulation of significantly greater numbers of pre-myelinating OLs that fail to myelinate axons during the normal myelinating period. Immunohistochemical studies with cell death and myelin markers indicate that JOE OLs undergo a heightened and extended period of cell death and are unable to effectively myelinate until 2 months after birth. The results indicate that increased levels of golli in OPC/OLs delays myelination, causing significant cell death of OLs particularly in white matter tracts. The results provide in vivo evidence for a significant role of the golli proteins in the regulation of maturation of OLs and normal myelination

Modulation of canonical transient receptor potential channel 1 in the proliferation of oligodendrocyte precursor cells by the golli products of the myelin basic protein gene

Golli proteins, products of the myelin basic protein gene, function as a new type of modulator of intracellular Ca(++) levels in oligodendrocyte progenitor cells (OPCs). Because of this, they affect a number of Ca(++) dependent functions such as OPC migration and process extension. To examine further the Ca(++) channels regulated by golli, we studied the store operated Ca(++) channels (SOCCs) in OPCs and acute brain slice preparations from golli-KO and golli-overexpressing mice. Our results showed that pharmacologically-induced Ca(++) release from intracellular stores evoked a significant extracellular Ca(++) entry after store depletion in OPCs. They also indicated that under these pharmacological conditions golli promoted activation of Ca(++) influx by SOCCs in cultured OPCs as well as in tissue slices. The Canonical TRP (Transient Receptor Potential) family of Ca(++) channels (TRPCs) has been postulated to be SOCC subunits in oligodendrocytes. Using a siRNA knock down approach, we provided direct evidence that TRPC1 is involved in store-operated Ca(++) influx in OPCs, and that it is modulated by golli. Furthermore, our data indicated that golli is probably associated with TRPC1 at OPC processes. Additionally, we found that TRPC1 expression is essential for the effects of golli on OPC proliferation. In summary, our data indicate a key role for golli proteins in the regulation of TRPC mediated Ca(++) influx, a finding that has profound consequences for the regulation of multiple biological processes in OPCs. More important, we have shown that extracellular Ca(++) uptake through TRPC1 is an essential component in the mechanism of OPC proliferation

Impact of simulated microgravity on oligodendrocyte development: implications for central nervous system repair.

We have recently established a culture system to study the impact of simulated microgravity on oligodendrocyte progenitor cells (OPCs) development. We subjected mouse and human OPCs to a short exposure of simulated microgravity produced by a 3D-Clinostat robot. Our results demonstrate that rodent and human OPCs display enhanced and sustained proliferation when exposed to simulated microgravity as assessed by several parameters, including a decrease in the cell cycle time. Additionally, OPC migration was examined in vitro using time-lapse imaging of cultured OPCs. Our results indicated that OPCs migrate to a greater extent after stimulated microgravity than in normal conditions, and this enhanced motility was associated with OPC morphological changes. The lack of normal gravity resulted in a significant increase in the migration speed of mouse and human OPCs and we found that the average leading process in migrating bipolar OPCs was significantly longer in microgravity treated cells than in controls, demonstrating that during OPC migration the lack of gravity promotes leading process extension, an essential step in the process of OPC migration. Finally, we tested the effect of simulated microgravity on OPC differentiation. Our data showed that the expression of mature oligodendrocyte markers was significantly delayed in microgravity treated OPCs. Under conditions where OPCs were allowed to progress in the lineage, simulated microgravity decreased the proportion of cells that expressed mature markers, such as CC1 and MBP, with a concomitant increased number of cells that retained immature oligodendrocyte markers such as Sox2 and NG2. Development of methodologies aimed at enhancing the number of OPCs and their ability to progress on the oligodendrocyte lineage is of great value for treatment of demyelinating disorders. To our knowledge, this is the first report on the gravitational modulation of oligodendrocyte intrinsic plasticity to increase their progenies

Golli myelin basic proteins regulate oligodendroglial progenitor cell migration through voltage-gated Ca2+ influx

Migration of OL progenitor cells (OPCs) from proliferative zones to their final location in the brain is an essential step in nervous system development. Golli proteins, products of the myelin basic protein gene, can modulate voltage-gated Ca(++) uptake in OPCs during process extension and retraction. Given the importance of process extension/retraction on movement, the consequences of golli expression on OPC migration were examined in vivo and in vitro using time-lapse imaging of isolated OPCs and acute brain slice preparations from golli KO and golli overexpressing mice (JOE). The results indicated that golli stimulated migration, and this enhanced motility was associated with increases in the activity of voltage operated Ca(++) channels (VOCCs). Activation of VOCCs by high K(+) resulted in a significant increase in the migration speed of JOE OPCs vs control cells and golli-mediated modulation of OPC migration disappeared in the presence of VOCC antagonists. During migration, OPCs generated Ca(++) oscillations that were dependent on voltage-calcium influx and both the amplitude and frequency of these Ca(++) transients correlated positively with the rate of cell movement under a variety of pharmacological treatments. The Ca(++) transient amplitude and the rate of cell movement were significantly lower in KO cells and significantly higher in JOE cells suggesting that the presence of golli promotes OPC migration by increasing the size of voltage-mediated Ca(++) oscillations. These data define a new molecule that regulates Ca(++) homeostasis in OPCs, and are the first to demonstrate that voltage-gated Ca(++) channels can regulate an OPC function, such as migration

Simulated microgravity increases OPC migration.

<p>Mixed glial cultures of PLP-GFP labeled OPCs were imaged with a GFP filter at 6 min intervals for a period of 16 h. (<b>A</b>) Time-lapse series of OPCs from cultures treated during 24 h in 0G. Yellow arrowheads identify a migrating OPC. Each frame represents a single section of a time lapse video sequence. Time is denoted in hours in the bottom right corner. Scale bar = 50 µm. Cell migration speed and distances were analyzed off-line by tracing individual cells at different times, after which migratory values were statistically analyzed across the experimental conditions. (<b>B</b>) OPC migration speed was calculated from at least 60 cells in each experimental condition. (<b>C and D</b>) Total migration distance was followed for 12 h in 50 cells from each experimental condition and the percentage of migrating cells was calculated from the entire cell population. Values are expressed as mean ± SEM of at least four independent experiments. **p<0.01, ***p<0.001 versus control cells.</p

Simulated microgravity increases the number of DNA-synthesizing hOPCs.

<p>Cultures of hOPCs were treated during 24 and 48-four hour pulses of 10 µM bromo-deoxyuridine (BrdU) were done during the 0G treatment as well as in parallel untreated cultures. After each BrdU pulse, cells were fixed and immunostained with anti-BrdU. (<b>A</b>) Microphotographs showing BrdU positive cells at 24 and 48 h. green: BrdU immunostaining. Scale bar = 50 µm. (<b>B</b>) The percentage of BrdU positive cells in each experimental condition was compared with respective controls. Values are expressed as mean ± SEM of two independent experiments. ***p<0.001 versus control.</p

Like in mouse OPCs simulated microgravity shortens the cell cycle of human OPCs (hOPCs).

<p>hOPCs were incubated in a chamber with 5% CO2 at 37°C, which was placed on the stage of a spinning disc confocal inverted microscope. Cells were imaged at 6 min intervals for a period of 26 h. Estimated cell cycle times and percentage of mitotic hOPCs for control and 0G treated cultures are shown. Values are expressed as mean ± SEM of four independent experiments. *p<0.05, **p<0.01, versus respective control.</p

Cell cycle shortening in microgravity.

<p>Mixed glial cultures of PLP-GFP labeled OPCs were incubated in a chamber with 5% CO2 at 37°C, which was placed on the stage of a spinning disc confocal microscope. GFP-labeled OPC clones were imaged with a specific GFP filter at 6 min intervals for a period of 26 h. (<b>A</b>) Time-lapse series of OPC∼GFP clones from cultures treated during 24 h in 0G. Yellow arrowheads designate cytokinesis events. Tracking of cells between birth cytokinesis and division cytokinesis was noted with a yellow asterisk near the cell, which was generated from frame to frame. Each frame represents a single section of a time lapse video sequence. Time is denoted in hours in the bottom right corner. Scale bar = 50 µm. (<b>B and C</b>) Estimated cell cycle times and percentage of mitotic OPCs for each experimental condition. Values are expressed as mean ± SEM of four independent experiments. *p<0.05, **p<0.01, versus respective control.</p