Preserving Mitochondrial Structure and Motility Promotes Recovery of White Matter After Ischemia.

Stroke significantly affects white matter in the brain by impairing axon function, which results in clinical deficits. Axonal mitochondria are highly dynamic and are transported via microtubules in the anterograde or retrograde direction, depending upon axonal energy demands. Recently, we reported that mitochondrial division inhibitor 1 (Mdivi-1) promotes axon function recovery by preventing mitochondrial fission only when applied during ischemia. Application of Mdivi-1 after injury failed to protect axon function. Interestingly, L-NIO, which is a NOS3 inhibitor, confers post-ischemic protection to axon function by attenuating mitochondrial fission and preserving mitochondrial motility via conserving levels of the microtubular adaptor protein Miro-2. We propose that preventing mitochondrial fission protects axon function during injury, but that restoration of mitochondrial motility is more important to promote axon function recovery after injury. Thus, Miro-2 may be a therapeutic molecular target for recovery following a stroke.

Mediating effects of aryl-hydrocarbon receptor and RhoA in altering brain vascular integrity: the therapeutic potential of statins.

We have demonstrated previously that focal adhesion kinase (FAK)/RhoA alteration by the aryl-hydrocarbon receptor (AhR) agonist 3-methylcholanthrene (3MC) is involved in the antimigratory effects of 3MC in human umbilical vascular endothelial cells. Here, we identified that signaling properties and molecular mechanisms of RhoA/ß-catenin were both implicated in alterations to blood-brain barrier integrity. The mechanisms of action were the down-regulation of integrin, the extracellular matrix, and adherens junction stability. PTEN phosphorylation by 3MC-mediated AhR/RhoA activation increased the proteasomal degradation of ß-catenin through PKCδ/pGSK3ß-mediated ß-catenin phosphorylation; the crucial roles of AhR/RhoA in this process were verified by using gain- or loss-of-function experiments. The decrease in ß-catenin led to decreased expression of fibronectin and α5ß1 integrin. Additionally, protein interactions among FAK, VE-cadherin, vinculin, and ß-actin were simultaneously decreased, resulting in adherens junction instability. Novel functional TCF/LEF1 binding sites in the promoter regions of fibronectin and α5/ß1 integrin were identified by electrophoretic mobility shift and chromatin immunoprecipitation assays. The results indicate that the binding activities of ß-catenin decreased in mouse cerebrovascular endothelial cells treated with 3MC. In addition, simvastatin and pravastatin treatment reversed 3MC-mediated alterations in mouse cerebrovascular endothelial cells by RhoA inactivation, and the in vitro findings were substantiated by an in vivo blood-brain barrier assay. Thus, endothelial barrier dysfunction due to 3MC occurs through AhR/RhoA-mediated ß-catenin down-regulation, which is reversed by simvastatin treatment in vivo.

The calcium-sensing receptor-dependent regulation of cell-cell adhesion and keratinocyte differentiation requires Rho and filamin A.

Extracellular Ca(2+) (Ca(2+)(o)) functioning through the calcium-sensing receptor (CaR) induces E-cadherin-mediated cell-cell adhesion and cellular signals mediating cell differentiation in epidermal keratinocytes. Previous studies indicate that CaR regulates cell-cell adhesion through Fyn/Src tyrosine kinases. In this study, we investigate whether Rho GTPase is a part of the CaR-mediated signaling cascade regulating cell adhesion and differentiation. Suppressing endogenous Rho A expression by small interfering RNA (siRNA)-mediated gene silencing blocked the Ca(2+)(o)-induced association of Fyn with E-cadherin and suppressed the Ca(2+)(o)-induced tyrosine phosphorylation of ß-, γ-, and p120-catenin and formation of intercellular adherens junctions. Rho A silencing also decreased the Ca(2+)(o)-stimulated expression of terminal differentiation markers. Elevating the Ca(2+)(o) level induced interactions among CaR, Rho A, E-cadherin, and the scaffolding protein filamin A at the cell membrane. Inactivation of CaR expression by adenoviral expression of a CaR antisense complementary DNA inhibited Ca(2+)(o)-induced activation of endogenous Rho. Ca(2+)(o) activation of Rho required a direct interaction between CaR and filamin A. Interference of CaR-filamin interaction inhibited Ca(2+)(o)-induced Rho activation and the formation of cell-cell junctions. These results indicate that Rho is a downstream mediator of CaR in the regulation of Ca(2+)(o)-induced E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation.

Schweinfurthin A selectively inhibits proliferation and Rho signaling in glioma and neurofibromatosis type 1 tumor cells in a NF1-GRD-dependent manner.

Neurofibromatosis type 1 (NF1) is the most common genetic disease affecting the nervous system. Patients typically develop many tumors over their lifetime, leading to increased morbidity and mortality. The NF1 gene, mutated in NF1, is also commonly mutated in sporadic glioblastoma multiforme (GBM). Because both NF1 and GBM are currently incurable, new therapeutic approaches are clearly needed. Natural products represent an opportunity to develop new therapies, as they have been evolutionarily selected to play targeted roles in organisms. Schweinfurthin A is a prenylated stilbene natural product that has previously shown specific inhibitory activity against brain and hematopoietic tumor lines. We show that patient-derived GBM and NF1 malignant peripheral nerve sheath tumor (MPNST) lines, as well as tumor lines derived from the Nf1-/+;Trp53-/+ (NPcis) mouse model of astrocytoma and MPNST are highly sensitive to inhibition by schweinfurthin A and its synthetic analogs. In contrast, primary mouse astrocytes are resistant to the growth inhibitory effects of schweinfurthin A, suggesting that schweinfurthin A may act specifically on tumor cells. Stable transfection of the GTPase-activating protein related domain of Nf1 into Nf1-/-;Trp53-/- astrocytoma cells confers resistance to schweinfurthin A. In addition, the profound effect of schweinfurthin A on dynamic reorganization of the actin cytoskeleton led us to discover that schweinfurthin A inhibits growth factor-stimulated Rho signaling. In summary, we have identified a class of small molecules that specifically inhibit growth of cells from both central and peripheral nervous system tumors and seem to act on NF1-deficient cells through cytoskeletal reorganization correlating to changes in Rho signaling.

Rho GTPases as therapeutic targets for the treatment of inflammatory diseases.

Diseases related to inflammation are a major cause of morbidity and mortality throughout the world and affect the functions of several tissues. The pathophysiology of these diseases involves release of many pro-inflammatory cytokines, such as TNF and IL-1, in addition to anti-inflammatory molecules. Recent studies have demonstrated that neuroimmune interactions are important in the initiation and progress of inflammatory processes. TNF, IL-1 and neuropeptides such as substance P and neurotensin stimulate the release of chemokines, in particular IL-8, a potent neutrophil chemoattractant. Expression of IL-8 is regulated mainly by the transcription factors NF-kappaB, activating protein-1 and CCAAT/enhancer-binding proteins. Recent exciting results indicate that the Rho family of small GTP-binding proteins plays an important role in the expression of NF-kappaB-dependent genes and migration of leukocytes. These results suggest that these proteins may represent a potential therapeutic target to treat several inflammatory states.

Erectile physiological and pathophysiological pathways involved in erectile dysfunction.

PURPOSE: The importance of signaling pathways in penile smooth muscles involved in normal erection and erectile dysfunction (ED) is discussed based on a review of the literature. MATERIALS AND METHODS: Erection is basically a spinal reflex that can be initiated by recruitment of penile afferents but also by visual, olfactory and imaginary stimuli. The generated nervous signals will influence the balance between the contractant and relaxant factors, which control the degree of contraction of penile smooth muscles and, thus, determine the functional state of the penis. The different steps involved in neurotransmission, impulse propagation and intracellular transduction of neural signals may be changed in different types of erectile dysfunction. RESULTS: Recent findings have suggested an important role for RhoA/Rho kinase in the regulation of cavernosal smooth muscle tone and that changes in this pathway may contribute to ED in various patient subgroups, eg diabetes and vascular disease. Neurogenic nitric oxide is still considered the most important factor for immediate relaxation of penile vessels and corpus cavernosum. However, endothelially generated nitric oxide seems essential for maintaining erection. Endothelial dysfunction can contribute to ED in several patient subgroups. In addition, in conditions associated with reduced function of nerves and endothelium, such as aging, hypertension, smoking, hypercholesterolemia and diabetes, circulatory and structural changes in the penile tissues can result in arterial insufficiency and defect muscle relaxation. CONCLUSIONS: Different types of ED often have overlapping pathophysiologies but may also have common pathways contributing to ED. Such pathways may be potential treatment targets.

RICS, a novel GTPase-activating protein for Cdc42 and Rac1, is involved in the beta-catenin-N-cadherin and N-methyl-D-aspartate receptor signaling.

Cadherin adhesion molecules are believed to be important for synaptic plasticity. beta-Catenin, which links cadherins and the actin cytoskeleton, is a modulator of cadherin adhesion and regulates synaptic structure and function. Here we show that beta-catenin interacts with a novel GTPase-activating protein, named RICS, that acts on Cdc42 and Rac1. The RICS-beta-catenin complex was found to be associated with N-cadherin, N-methyl-d-aspartate receptors, and postsynaptic density-95, and localized to the postsynaptic density. Furthermore, the GTPase-activating protein activity of RICS was inhibited by phosphorylation by Ca(2+)/calmodulin-dependent protein kinase II. These results suggest that RICS is involved in the synaptic adhesion- and N-methyl-d-aspartate-mediated organization of cytoskeletal networks and signal transduction. Thus, RICS may regulate dendritic spine morphology and strength by modulating Rho GTPases.

Lovastatin-induced cytoskeletal reorganization in lens epithelial cells: role of Rho GTPases.

PURPOSE: To understand the involvement of isoprenylated small guanosine triphosphatases (GTPases) in lovastatin-induced cataractogenesis, Rho- and Rac-mediated cell adhesion and actin cytoskeletal reorganization were investigated in lovastatin-treated lens epithelial cells. METHODS: The effects of lovastatin on F-actin reorganization (phalloidin staining), focal adhesion formation (paxillin or vinculin), cell-cell adhesions (cadherin and beta-catenin), and protein tyrosine phosphorylation were evaluated in human and porcine lens epithelial cells by immunocytochemical staining with specific antibodies. To explore the involvement of the Rho and Rac GTPases in lovastatin-mediated effects, changes in distribution of Rho and Rac GTPases were analyzed by Western blot analysis, and the effects of C3-exoenzyme on lovastatin-induced cytoskeletal changes were evaluated by immunocytochemical analysis. RESULTS: Lovastatin induced drastic changes in cell shape in both human and porcine lens epithelial cells, including a profound loss of actin stress fibers, focal adhesions, protein phosphotyrosine, and cell-cell adhesions. Lovastatin treatment also led to the accumulation of nonisoprenylated Rho and Rac GTPases in cytosolic fraction. Supplementation of culture media with geranylgeranyl pyrophosphate dramatically reversed the lovastatin-induced morphologic and cytoskeletal changes, whereas farnesyl pyrophosphate was ineffective. Treatment of cells with C3-exoenzyme (a Rho GTPase-specific inhibitor), however, abolished the geranylgeranyl-supplementation-induced recovery from the morphologic and cytoskeletal effects of lovastatin. CONCLUSIONS: This study demonstrates that inhibition of protein prenylation by lovastatin leads to disruption of actin cytoskeletal organization, and to loss of integrin-mediated focal adhesions and cadherin-mediated cell-cell adhesions in lens epithelial cells. Based on isoprenoid supplementation studies, it could be concluded that impairment of geranylgeranylated Rho and Rac GTPase function is most likely responsible for lovastatin-induced cytoskeletal changes in lens epithelial cells.

Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly.

Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is approximately 800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3-like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH(2)-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.