Stomatin Inhibits Pannexin-1-Mediated Whole-Cell Currents by Interacting with Its Carboxyl Terminal

The pannexin-1 (Panx1) channel (often referred to as the Panx1 hemichannel) is a large-conductance channel in the plasma membrane of many mammalian cells. While opening of the channel is potentially detrimental to the cell, little is known about how it is regulated under physiological conditions. Here we show that stomatin inhibited Panx1 channel activity. In transfected HEK-293 cells, stomatin reduced Panx1-mediated whole-cell currents without altering either the total or membrane surface Panx1 protein expression. Stomatin coimmunoprecipitated with full-length Panx1 as well as a Panx1 fragment containing the fourth membrane-spanning domain and the cytosolic carboxyl terminal. The inhibitory effect of stomatin on Panx1-mediated whole-cell currents was abolished by truncating Panx1 at a site in the cytosolic carboxyl terminal. In primary culture of mouse astrocytes, inhibition of endogenous stomatin expression by small interfering RNA enhanced Panx1-mediated outward whole-cell currents. These observations suggest that stomatin may play important roles in astrocytes and other cells by interacting with Panx1 carboxyl terminal to limit channel opening

Glial Pathology in Globoid Cell Leukodystrophy

The goal of this dissertation was to examine the pathogenic potential of microglia in globoid cell leukodystrophy (GLD). We determined that psychosine, the pathogenic lipid toxin that accumulates in GLD, induced microglial transformation to globoid cells in vitro. Psychosine also induced matrix metalloproteinase (MMP)-3 in cultured glia. This mirrored the elevated expression of MMP-3 in the central nervous system (CNS) of twitcher mice, a murine model of GLD. Pharmacological inhibition or genetic ablation of MMP-3 blocked psychosine-induced globoid cell formation in vitro. These findings indicate that MMP-3 mediated psychosine-induced globoid cell formation. These findings were the first to relate the putative functions of MMPs in the CNS pathogenesis of a leukodystrophy. We next determined the effects of the extracellular matrix (ECM) in GLD pathology. We determined that tenascin-c (TnC), an ECM molecule, was aberrantly expressed in the brain of murine and human GLD. We determined that TnC enhances both psychosine-induced MMP-3 expression and globoid cell formation in microglia. Additionally, TnC altered the phenotype of microglia and globoid cells from a protective toward a harmful phenotype toward oligodendrocytes in co-cultures. These findings suggest that TnC modulates cellular behaviors that may contribute to the GLD pathogenesis. Overall, results of this thesis determined that microglial responses to psychosine are a primary event of the pathogenesis in GLD. Overall, the findings of this dissertation have provided a new level to our understanding of GLD pathogenesis and provided new potential therapeutic targets to treat GLD

MMP-3 Mediates Psychosine-Induced Globoid Cell Formation: Implications for Leukodystrophy Pathology

Globoid cell leukodystrophy (GLD) or Krabbe disease, is a fatal demyelinating disease attributed to mutations in the galactocerebrosidase (GALC) gene. Loss of function mutations in GALC result in accumulation of the glycolipid intermediate, galactosylsphingosine (psychosine). Due to the cytotoxicity of psychosine, it has been hypothesized that accumulated psychosine underlie the pathophysiology of GLD. However, the cellular mechanisms of GLD pathophysiology remain unclear. Globoid cells, multinucleated microglia/macrophages in the central nervous system (CNS), are a defining characteristic of GLD. Here we report that exposure of primary glial cultures to psychosine induces the expression and the production of matrix metalloproteinase (MMP)-3 that mediated a morphological transformation of microglia into a multinucleated globoid cell type. Additionally, psychosine-induced globoid cell formation from microglia was prevented by either genetic ablation or chemical inhibition of MMP-3. These effects are microglia-specific as peripheral macrophages exposed to psychosine did not become activated or express increased levels of MMP-3. In the brain from twitcher mice, a murine model of human GLD, elevated MMP-3 expression relative to wild-type littermates was contemporaneous with disease onset and further increased with disease progression. Further, bone marrow transplantation (BMT), currently the only therapeutically beneficial treatment for GLD, did not mitigate the elevated expression of MMP-3 in twitcher mice. Hence, elevated expression of MMP-3 in GLD may promote microglial responses to psychosine that may represent an important pathophysiological process in this disease and its treatment

Human ESC-Derived MSCs Outperform Bone Marrow MSCs in the Treatment of an EAE Model of Multiple Sclerosis

Current therapies for multiple sclerosis (MS) are largely palliative, not curative. Mesenchymal stem cells (MSCs) harbor regenerative and immunosuppressive functions, indicating a potential therapy for MS, yet the variability and low potency of MSCs from adult sources hinder their therapeutic potential. MSCs derived from human embryonic stem cells (hES-MSCs) may be better suited for clinical treatment of MS because of their unlimited and stable supply. Here, we show that hES-MSCs significantly reduce clinical symptoms and prevent neuronal demyelination in a mouse experimental autoimmune encephalitis (EAE) model of MS, and that the EAE disease-modifying effect of hES-MSCs is significantly greater than that of human bone-marrow-derived MSCs (BM-MSCs). Our evidence also suggests that increased IL-6 expression by BM-MSCs contributes to the reduced anti-EAE therapeutic activity of these cells. A distinct ability to extravasate and migrate into inflamed CNS tissues may also be associated with the robust therapeutic effects of hES-MSCs on EAE

Human ESC-Derived MSCs Outperform Bone Marrow MSCs in the Treatment of an EAE Model of Multiple Sclerosis

Current therapies for multiple sclerosis (MS) are largely palliative, not curative. Mesenchymal stem cells (MSCs) harbor regenerative and immunosuppressive functions, indicating a potential therapy for MS, yet the variability and low potency of MSCs from adult sources hinder their therapeutic potential. MSCs derived from human embryonic stem cells (hES-MSCs) may be better suited for clinical treatment of MS because of their unlimited and stable supply. Here, we show that hES-MSCs significantly reduce clinical symptoms and prevent neuronal demyelination in a mouse experimental autoimmune encephalitis (EAE) model of MS, and that the EAE disease-modifying effect of hES-MSCs is significantly greater than that of human bone-marrow-derived MSCs (BM-MSCs). Our evidence also suggests that increased IL-6 expression by BM-MSCs contributes to the reduced anti-EAE therapeutic activity of these cells. A distinct ability to extravasate and migrate into inflamed CNS tissues may also be associated with the robust therapeutic effects of hES-MSCs on EAE

Stomatin inhibited Panx1 channel-mediated outward whole-cell currents in transfected HEK-293 cells.

<p>HEK-293 cells were transfected independently with Panx1, stomatin, Panx1 plus stomatin, and empty vectors (Control). Whole-cell currents in response to a voltage ramp (–60 to +90 mV over 10 sec) were recorded from the transfected cells either in the absence or presence of the hemichannels blockers carbenoxolone (CBX 25 µM) or ¹⁰Panx1 (100 µM). <b>A.</b> Averaged current traces in response to a voltage ramp (–60 to +90 mV over 10 sec). Cells expressing Panx1 showed large voltage-dependent outward currents compared with the Control, which were blocked by either CBX or ¹⁰Panx1 and reduced by stomatin. Note that the Control current trace is almost invisible due to overlap with other traces. <b>B.</b> Statistical comparisons of the whole-cell current density at indicated membrane voltages. The asterisk (*) indicates a significant difference compared with the Control whereas the pound sign (#) indicates a significant difference compared with the Panx1 group (<i>p</i><0.01, one-way ANOVA with Bonferroni posthoc tests).</p

Inhibition of stomatin expression enhanced Panx1-dependent outward whole-cell currents in astrocytes.

<p>Cells were transfected with either stomatin siRNA or scrambled siRNA. <b>A.</b> Effects of scrambled and stomatin siRNA on stomatin mRNA level as determined by real-time PCR (from 3 independent experiments). The asterisk (*) indicates a significant difference compared with scrambled siRNA (<i>p</i><0.01, paired <i>t</i>-test). <b>B.</b> Averaged current traces in response to a voltage ramp (–60 to +90 mV over 10 sec) showing the effects of siRNA and the specific Panx1 channel inhibitor ¹⁰Panx1. <b>C & D.</b> Comparisons of the peak outward (at +90 mV) and inward (at –60 mV) currents among the different groups. The asterisk (*) indicates a significant difference (<i>p</i><0.01) compared with scrambled siRNA whereas the pound sign (#) indicates a significant difference (<i>p</i><0.05) compared with stomatin siRNA (one-way ANOVA with Bonferroni posthoc test).</p

Stomatin did not inhibit Panx1-dependent ethidium uptake in transfected HEK-293 cells.

<p>HEK-293 cells were transfected with Panx1, stomatin, Panx1 plus stomatin, or empty vectors (Control). <b>A.</b> Transfected cells were incubated with either phosphate-buffered saline (1 mM K⁺) or 150-mM [K⁺] solution containing ethidium bromide (20 µM) for 5 min. Fluorescence intensity of the cells (identified by GFP fluorescence) at 5 min of the incubation was background subtracted and compared among the different groups. The asterisk (*) indicates significant difference compared with the Control while the pound sign (#) indicate significant difference compared with the Panx1 group at 1 mM [K⁺]_o (<i>p</i><0.01, one-way ANOVA with Bonferroni posthoc tests). The numbers inside the columns indicate the number of cells analyzed. <b>B.</b> Transfected cells were voltage-clamped to +80 mV (5 sec) and –60 mV (1.5 sec) alternatively for a total duration of 2 min immediately following the addition of ethidium (20 µM) to the bath solution. The brief sojourns to –60 mV were necessary for maintaining the whole-cell configuration. Fluorescence intensities of the cells at the beginning and end of the voltage pulses were imaged. The red horizontal lines in the graph indicate the means. Fluorescence intensities are shown in arbitrary units in the plots of both A and B.</p

Stomatin did not alter total or cell surface expression of Panx1.

<p>The amount of total and surface Panx1 protein levels were determined with transfected HEK-293 cells expressing either Myc-tagged Panx1 (Panx1::Myc) alone or Panx1::Myc plus HA-tagged stomatin (Stomatin::HA). The bar graphs show the densities of the Panx1::Myc total and surface protein bands (normalized by actin) based on 5 independent experiments.</p