Evan B. Stubbs

Forced-exercise delays neuropathic pain in experimental diabetes: effects on voltage-activated calcium channels.

J. Neurochem. (2011) 118, 224–236.

Lovastatin attenuates nerve injury in an animal model of Guillain–Barré syndrome

Statins, widely used as clinically effective inhibitors of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, exhibit anti‐inflammatory properties that may be of therapeutic benefit for the management of some neurological disorders. In this study, a short‐term course of lovastatin treatment is shown to markedly inhibit the development of experimental autoimmune neuritis (EAN) in the absence of hepatotoxic or myotoxic complications. Independent of cholesterol reduction, lovastatin treatment prevented EAN‐induced peripheral nerve conduction deficits and morphologic nerve injury. Co‐administration with mevalonate neutralized the prophylactic effects of lovastatin. When administered therapeutically, lovastatin significantly shortened the disease course. Autoreactive immunity, measured in vitro by myelin‐stimulated proliferation of splenocytes, was significantly diminished by in vivo lovastatin treatment. Th1‐dominant immune responses, measured by cytokine profiling, however, were not affected by lovastatin. Sciatic nerves of lovastatin‐treated immunized rats showed markedly reduced levels of cellular infiltrates. Treating peripheral nerve endothelial monolayers with lovastatin significantly inhibited the in vitro migration of autoreactive splenocytes. Together, these data demonstrate that a short‐term course of lovastatin attenuates the development and progression of EAN in Lewis rats by limiting the proliferation and migration of autoreactive leukocytes.

Increased RhoA and RhoB Protein Accumulation in Cultured Human Trabecular Meshwork Cells by Lovastatin

PURPOSE This study aimed to determine the effect of lovastatin on Rho G-protein expression and activation in human trabecular meshwork (TM) cells. METHODS Confluent cultures of low-passage (primary) or transformed (GTM3) human TM cells were incubated overnight with vehicle (0.01% ethanol) or activated lovastatin (10 microM). Changes in Rho mRNA, protein content, and activation were quantified by qRT-PCR, immunoblotting, and ELISA, respectively. F-actin organization was determined using Alexa Fluor 488-conjugated phalloidin. RESULTS Low-passage or transformed TM cells treated with lovastatin exhibited marked increases in RhoA and RhoB mRNA and protein content. Actinomycin D prevented lovastatin-dependent increases in RhoB, but not RhoA, protein accumulation. In contrast, cycloheximide prevented lovastatin from increasing both RhoA and RhoB. Supplementation with mevalonate or geranylgeranyl pyrophosphate prevented, whereas inhibition of geranylgeranyl transferase mimicked, the effects of lovastatin on RhoA and RhoB accumulation. The effect of lovastatin was dose dependent, with newly synthesized protein accumulating in the cytosol. The amount of functionally active (GTP-bound) RhoA in cell lysates was significantly reduced by lovastatin. Lovastatin altered the morphology of TM cells by disrupting F-actin organization. CONCLUSIONS Lovastatin enhances the accumulation of RhoA and RhoB in human TM cells, in part, by limiting geranylgeranyl isoprenylation of these G-proteins. We propose that post-translational geranylgeranylation serves as a regulator of both RhoA and RhoB protein expression and processing in human TM cells. Increased accumulation of unprenylated forms of RhoA and RhoB may disrupt Rho-dependent regulation of TM cell cytoskeletal organization.

Geranylgeranylation Facilitates Proteasomal Degradation of Rho G-Proteins in Human Trabecular Meshwork Cells

PURPOSE To determine the role of posttranslational isoprenylation in regulating Rho G-protein activation and stability in human trabecular meshwork (TM) cells. METHODS Transformed human TM cells (GTM3) were incubated for 24 hours in the presence of activated lovastatin (10 μM) to enhance the endogenous synthesis of latent Rho proteins. Medium was replaced, cycloheximide (CHX) was added to inhibit synthesis of new proteins, and lovastatin-pretreated cells were subsequently incubated (0-24 hours) in the absence (control) or presence of farnesyl pyrophosphate (10 μM) or geranylgeranyl pyrophosphate (10 μM). Relative changes in the content of total and GTP-bound Rho G-proteins were quantified by Western immunoblot and GTP-binding ELISA, respectively. Changes in filamentous actin stress fiber organization were visualized with AlexaFluor488-conjugated phalloidin. RESULTS GTM3 cells cultured in the presence of lovastatin exhibited a loss of actin stress fiber organization concomitant with a marked accumulation of cytosolic inactive (GDP-bound) Rho G-proteins. Addition of geranylgeranyl pyrophosphate to the culture medium restored actin stress fiber organization while selectively facilitating the subcellular redistribution of accumulated Rho proteins from cytosol to membrane and increasing RhoA activation. Geranylgeranyl pyrophosphate selectively enhanced the degradation of newly synthesized Rho proteins. Epoxomicin, a potent and selective inhibitor of the 20S proteasome, prevented geranylgeranyl-enhanced degradation of Rho proteins. CONCLUSIONS Posttranslational geranylgeranylation selectively alters the lifecycle of newly synthesized Rho proteins by facilitating their membrane translocation, functional activation, and turnover. Geranylgeranylation represents a novel mechanism by which active Rho proteins are targeted to the proteasome for degradation in human TM cells.

7Li Nuclear Magnetic Resonance Study for the Determination of Li+ Properties in Neuroblastoma SH‐SY5Y Cells

Abstract: Lithium has been used clinically in the treatment of manic depression. However, its pharmacologic mode of action remains unclear. Characteristics of Li+ interactions in red blood cells (RBCs) have been identified. We investigated Li+ interactions on human neuroblastoma SH‐SY5Y cells by developing a novel 7Li NMR method that provided a clear estimation of the intra‐ and extracellular amounts of Li+ in the presence of the shift reagent thulium‐1,4,7,10‐tetrazacyclododecane‐N,N′,N″,N‴‐tetramethylene phosphonate (HTmDOTP4−). The first‐order rate constants of Li+ influx and efflux for perfused, agarose‐embedded SH‐SY5Y cells in the presence of 3 mM HTmDOTP4− were 0.055 ± 0.006 (n = 4) and −0.025 ± 0.006 min−1 (n = 3), respectively. Significant increases in the rate constants of Li+ influx and efflux in the presence of 0.05 mM veratridine indicated the presence of Na+ channel‐mediated Li+ transport in SH‐SY5Y cells. 7Li NMR relaxation measurements showed that Li+ is immobilized more in human neuroblastoma SH‐SY5Y cells than in human RBCs.

Cdc42 GTPases facilitate TNF‐α‐mediated secretion of CCL2 from peripheral nerve microvascular endoneurial endothelial cells

Trafficking of autoreactive leukocytes across the blood‐nerve barrier and into peripheral nerves is an early pathological hallmark of Guillain‐Barré syndrome (GBS). Tumor necrosis factor‐α (TNF‐α), a proinflammatory cytokine, promotes transendothelial migration by upregulating endothelial expression of inflammatory mediators, including CCL2, a chemokine implicated in GBS. We sought to determine the mechanism by which TNF‐α induces expression and secretion of CCL2 from peripheral nerve microvascular endoneurial endothelial cells (PNMECs). Expression of CCL2 mRNA and protein in quiescent PNMEC cultures was minimal. In contrast, cultures treated with TNF‐α exhibited increased CCL2 mRNA and protein content, as well as protein secretion. Simvastatin significantly attenuated TNF‐α‐induced CCL2 secretion without affecting CCL2 mRNA or protein expression. Co‐incubation with geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate, prevented the effect of simvastatin. By comparison, inhibiting protein isoprenylation with GGTI‐298, but not FTI‐277, mimicked the effect of simvastatin and significantly attenuated transendothelial migration in vitro. Inhibition of the monomeric GTPase Cdc42, but not Rac1 or RhoA‐C, attenuated TNF‐α‐mediated CCL2 secretion. TNF‐α‐mediated trafficking of autoreactive leukocytes into peripheral nerves during GBS may proceed by a mechanism that involves Cdc42‐facilitated secretion of CCL2.

Prenylation of Rho G-Proteins: a Novel Mechanism Regulating Gene Expression and Protein Stability in Human Trabecular Meshwork Cells

Endogenous prenylation with sesquiterpene or diterpene isoprenoids facilitates membrane localization and functional activation of small monomeric GTP-binding proteins. A direct effect of isoprenoids on regulation of gene expression and protein stability has also been proposed. In this study, we determined the role of sesquiterpene or diterpene isoprenoids on the regulation of Rho G-protein expression, activation, and stability in human trabecular meshwork (TM) cells. In both primary and transformed human TM cells, limiting endogenous isoprenoid synthesis with lovastatin, a potent HMG-CoA reductase inhibitor, elicited marked increases in RhoA and RhoB mRNA and protein content. The effect of lovastatin was dose-dependent with newly synthesized inactive protein accumulating in the cytosol. Supplementation with geranylgeranyl pyrophosphate (GGPP) prevented, while inhibition of geranylgeranyl transferase-I mimicked, the effects of lovastatin on RhoA and RhoB protein content. Similarly, lovastatin-dependent increases in RhoA and RhoB mRNA expression were mimicked by geranylgeranyl transferase-I inhibition. Interestingly, GGPP supplementation selectively promoted the degradation of newly synthesized Rho proteins which was mediated, in part, through the 20S proteasome. Functionally, GGPP supplementation prevented lovastatin-dependent decreases in actin stress fiber organization while selectively facilitating the subcellular redistribution of accumulated Rho proteins from the cytosol to the membrane and increasing RhoA activation. Post-translational prenylation with geranylgeranyl diterpenes selectively facilitates the expression, membrane translocation, functional activation, and turnover of newly synthesized Rho proteins. Geranylgeranyl prenylation represents a novel mechanism by which active Rho proteins are targeted to the 20S proteasome for degradation in human TM cells.

Transforming growth factor-β2 induces synthesis and secretion of endothelin-1 in human trabecular meshwork cells.

PURPOSE Analysis of aqueous humor from patients with primary open-angle glaucoma (POAG) revealed marked increases in the content of endothelin-1 (ET-1) and transforming growth factor-beta (TGF-β). We determined the consequences of TGF-β signaling on ET-1 expression and secretion by human trabecular meshwork (TM) cells. METHODS Primary or transformed (NTM5 and GTM3) human TM cells conditioned in serum-free media were incubated in the absence or presence of TGF-β1 or -β2. Relative changes in preproendothelin (ppET)-1 mRNA content and secreted ET-1 peptide were quantified by real-time PCR and ELISA, respectively. In some experiments, TGF-β or ET-1 receptor antagonists, or Rho G-protein inhibitors, were evaluated for effects on TGF-β signaling. Filamentous actin organization was visualized by phalloidin. RESULTS Primary or transformed human TM cells cultured in the presence of TGF-β1 or -β2 exhibit a marked (>8-fold) increase in ppET-1 mRNA content compared to vehicle controls. Coincubation with SB-505124, an inhibitor of TGFβRI/ALK-5 signaling, prevented TGF-β-mediated ppET-1 mRNA expression. In contrast, coincubation with ET(A) (BQ-123) or ET(B) (BQ-788) receptor antagonists had no effect on TGF-β-mediated ppET-1 mRNA expression. TGF-β1 and -β2 each elicited a robust (>7-fold) secretion of ET-1 while enhancing stress fiber organization. Inhibition of Rho signaling attenuated TGF-β-mediated increases in ppET-1 mRNA content, ET-1 secretion, and stress fiber organization. CONCLUSIONS TGF-β, signaling through the TGFβRI/ALK-5 receptor, elicits marked increases in ET-1 mRNA content and ET-1 secretion from cultured primary or transformed human TM cells. Elevated levels of TGF-β2 present in AH of POAG patients may elevate intraocular pressure, in part, by eliciting aberrant Rho G-protein dependent cell contraction, and increasing ET-1 synthesis and secretion, in human TM cells.

Tumour Necrosis Factor α Enhances CCL2 and ICAM-1 Expression in Peripheral Nerve Microvascular Endoneurial Endothelial Cells

Recruitment and trafficking of autoreactive leucocytes across the BNB (blood–nerve barrier) is an early pathological insult in GBS (Guillain-Barré syndrome), an aggressive autoimmune disorder of the PNS (peripheral nervous system). Whereas the aetiology and pathogenesis of GBS remain unclear, pro-inflammatory cytokines, including TNFα (tumour necrosis factor α), are reported to be elevated early in the course of GBS and may initiate nerve injury by activating the BNB. Previously, we reported that disrupting leucocyte trafficking in vivo therapeutically attenuates the course of an established animal model of GBS. Here, PNMECs (peripheral nerve microvascular endothelial cells) that form the BNB were harvested from rat sciatic nerves, immortalized by SV40 (simian virus 40) large T antigen transduction and subsequently challenged with TNFα. Relative changes in CCL2 (chemokine ligand 2) and ICAM-1 (intercellular adhesion molecule 1) expression were determined. We report that TNFα elicits marked dose- and time-dependent increases in CCL2 and ICAM-1 mRNA and protein content and promotes secretion of functional CCL2 from immortalized and primary PNMEC cultures. TNFα-mediated secretion of CCL2 promotes, in vitro, the transendothelial migration of CCR2-expressing THP-1 monocytes. Increased CCL2 and ICAM-1 expression in response to TNFα may facilitate recruitment and trafficking of autoreactive leucocytes across the BNB in autoimmune disorders, including GBS.

Anti-tubulin antibodies in a sensorimotor neuropathy patient alter tubulin polymerization.

Abstract The effect of anti-tubulin antibodies present in the serum of a patient with a progressive sensorimotor neuropathy on microtubule assembly was examined. The patient’s serum was reactive on immunoblots with a single band of proteins of 55-kDa from homogenates of neural tissues. Tubulin was identified as the quantitatively major component of these 55-kDa proteins. Polymerization of tubulin in vitro was significantly enhanced by the patient’s serum. A monoclonal antibody to nerve-specific class III β-tubulin precisely duplicated the immunoreactive profile of the patient’s serum, while an antibody to class (I + II) β-tubulins also reacted with tubulins in non-neural tissues. The results indicate for the first time that human antisera reactive with nerve specific β-tubulin can alter tubulin polymerization-depolymerization dynamics.