Dual spatio-temporal regulation of axon growth and microtubule dynamics by RhoA signaling pathways.

RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only an inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localized in growth cones of undifferentiated neurites, whereas in developing axons it displayed a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevented axon initiation but had no effect on elongation, whereas formin inhibition reduced axon extension without significantly altering initial outgrowth. In addition, RhoA-mDia signaling promoted axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK signaling, which restrained growth cone microtubule assembly and protrusion.

RhoA/rho kinase pathway activation in age-associated endothelial cell dysfunction and thrombosis.

The small GTPase RhoA and the downstream Rho kinase (ROCK) regulate several cell functions and pathological processes in the vascular system that contribute to the age-dependent risk of cardiovascular disease, including endothelial dysfunction, excessive permeability, inflammation, impaired angiogenesis, abnormal vasoconstriction, decreased nitric oxide production and apoptosis. Frailty is a loss of physiological reserve and adaptive capacity with advanced age and is accompanied by a pro-inflammatory and pro-oxidative state that promotes vascular dysfunction and thrombosis. This review summarises the role of the RhoA/Rho kinase signalling pathway in endothelial dysfunction, the acquisition of the pro-thrombotic state and vascular ageing. We also discuss the possible role of RhoA/Rho kinase signalling as a promising therapeutic target for the prevention and treatment of age-related cardiovascular disease.

Angioimmunoblastic T-cell lymphoma and correlated neoplasms with T-cell follicular helper phenotype: from molecular mechanisms to therapeutic advances.

Angioimmunoblastic T-cell lymphoma (AITL) is the second most frequent subtype of mature T-cell lymphoma (MTCL) in the Western world. It derives from the monoclonal proliferation of T-follicular helper (TFH) cells and is characterized by an exacerbated inflammatory response and immune dysregulation, with predisposition to autoimmunity phenomena and recurrent infections. Its genesis is based on a multistep integrative model, where age-related and initiator mutations involve epigenetic regulatory genes, such as TET-2 and DNMT3A. Subsequently, driver-mutations, such as RhoA G17V and IDH-2 R172K/S promote the expansion of clonal TFH-cells ("second-hit"), that finally begin to secrete cytokines and chemokines, such as IL-6, IL-21, CXCL-13 and VEGF, modulating a network of complex relationships between TFH-cells and a defective tumor microenvironment (TME), characterized by expansion of follicular dendritic cells (FDC), vessels and EBV-positive immunoblasts. This unique pathogenesis leads to peculiar clinical manifestations, generating the so-called "immunodysplastic syndrome", typical of AITL. Its differential diagnosis is broad, involving viral infections, collagenosis and adverse drug reactions, which led many authors to use the term "many-faced lymphoma" when referring to AITL. Although great advances in its biological knowledge have been obtained in the last two decades, its treatment is still an unmet medical need, with highly reserved clinical outcomes. Outside the setting of clinical trials, AITL patients are still treated with multidrug therapy based on anthracyclines (CHOP-like), followed by up-front consolidation with autologous stem cell transplantation (ASCT). In this setting, the estimated 5-year overall survival (OS) is around 30-40%. New drugs, such as hypomethylating agents (HMAs) and histone deacetylase inhibitors (HDAi), have been used for relapsed/refractory (R/R) disease with promising results. Such agents have their use based on a biological rationale, have significant potential to improve the outcomes of patients with AITL and may represent a paradigm shift in the therapeutic approach to this lymphoma in the near future.

Tumor mutation burden involving epigenetic regulatory genes and the RhoA GTPase predicts overall survival in nodal mature T-cell lymphomas.

Nodal mature T-cell lymphomas (nMTCL) comprises a heterogeneous group of rare malignancies with aggressive biological behavior and poor prognosis. Epigenetic phenomena, including mutations in genes that control DNA methylation and histone deacetylation, in addition to inactivating mutations in the RhoA GTPase, play a central role in its pathogenesis and constitute potential new targets for therapeutic intervention. Tumor mutational burden (TMB) reflects the process of clonal evolution, predicts response to anti-cancer therapies and has emerged as a prognostic biomarker in several solid neoplasms; however, its potential prognostic impact remains unknown in nMTCL. In this study, we conducted Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) diagnostic tumor samples using a target-panel to search for recurrent mutations involving the IDH-1/IDH-2, TET-2, DNMT3A and RhoA genes in 59 cases of nMTCL. For the first time, we demonstrated that high-TMB, defined by the presence of ≥ two mutations involving the aforementioned genes, was associated with decreased overall survival in nMTCL patients treated with CHOP-like regimens. Additionally, high-TMB was correlated with bulky disease, lower overall response rate, and higher mortality. Future studies using larger cohorts may validate our preliminary results that indicate TMB as a potential molecular biomarker associated with adverse prognosis in nMTCL.

RhoA/ROCK-1 Signaling Pathway and Oxidative Stress in Coronary Artery Disease Patients

Abstract Introduction: Coronary artery disease (CAD) is an ischemic condition that occurs as a result of partial or complete interruption of blood flow by narrowing or complete blockage of the vessels supplying the heart, which are called coronary arteries. Our objective in this study is to investigate the RhoA/Rho-associated kinase (ROCK)-1 signaling pathway and oxidative stress in CAD patients. Methods: A total of 81 individuals aged between 40-70 years - including 45 patients (15 females and 30 males) who were admitted to the Artvin State Hospital Cardiovascular Surgery Clinic and were diagnosed with CAD and 36 healthy volunteers (15 females and 21 males) - participated in this study. Serum samples were tested for total cholesterol, triglyceride, low-density lipoprotein, high-density lipoprotein, malondialdehyde (MDA), superoxide dismutase (SOD), RhoA, and ROCK-1 values. Results: Serum RhoA, MDA levels, and ROCK-1 activity in the CAD group were found to be statistically significantly higher than in the control group (P<0.001). Concordantly, serum SOD activity was found to be statistically significantly lower in the CAD group than in the control group (P<0.001). Conclusion: Inhibition of the activity of RhoA/ROCK-1 pathway would be beneficial in treating cardiovascular diseases since this pathway plays an important role in the development of these diseases.

RhoA/ROCK-1 Signaling Pathway and Oxidative Stress in Coronary Artery Disease Patients.

INTRODUCTION: Coronary artery disease (CAD) is an ischemic condition that occurs as a result of partial or complete interruption of blood flow by narrowing or complete blockage of the vessels supplying the heart, which are called coronary arteries. Our objective in this study is to investigate the RhoA/Rho-associated kinase (ROCK)-1 signaling pathway and oxidative stress in CAD patients. METHODS: A total of 81 individuals aged between 40-70 years - including 45 patients (15 females and 30 males) who were admitted to the Artvin State Hospital Cardiovascular Surgery Clinic and were diagnosed with CAD and 36 healthy volunteers (15 females and 21 males) - participated in this study. Serum samples were tested for total cholesterol, triglyceride, low-density lipoprotein, high-density lipoprotein, malondialdehyde (MDA), superoxide dismutase (SOD), RhoA, and ROCK-1 values. RESULTS: Serum RhoA, MDA levels, and ROCK-1 activity in the CAD group were found to be statistically significantly higher than in the control group (P<0.001). Concordantly, serum SOD activity was found to be statistically significantly lower in the CAD group than in the control group (P<0.001). CONCLUSION: Inhibition of the activity of RhoA/ROCK-1 pathway would be beneficial in treating cardiovascular diseases since this pathway plays an important role in the development of these diseases.

Gßγ mediates activation of Rho guanine nucleotide exchange factor ARHGEF17 that promotes metastatic lung cancer progression.

Metastatic lung cancer is a major cause of death worldwide. Dissemination of cancer cells can be facilitated by various agonists within the tumor microenvironment, including by lysophosphatidic acid (LPA). We postulate that Rho guanine nucleotide exchange factors (RhoGEFs), which integrate signaling cues driving cell migration, are critical effectors in metastatic cancer. Specifically, we addressed the hypothetical role of ARHGEF17, a RhoGEF, as a potential effector of Gßγ in metastatic lung cancer cells responding to LPA. Here, we show that ARHGEF17, originally identified as a tumor endothelial marker, is involved in tumor growth and metastatic dissemination of lung cancer cells in an immunocompetent murine model. Gene expression-based analysis of lung cancer datasets showed that increased levels of ARHGEF17 correlated with reduced survival of patients with advanced-stage tumors. Cellular assays also revealed that this RhoGEF participates in the invasive and migratory responses elicited by Gi protein-coupled LPA receptors via the Gßγ subunit complex. We demonstrate that this signaling heterodimer promoted ARHGEF17 recruitment to the cell periphery and actin fibers. Moreover, Gßγ allosterically activates ARHGEF17 by the removal of inhibitory intramolecular restrictions. Taken together, our results indicate that ARHGEF17 may be a valid potential target in the treatment of metastatic lung cancer.

Arhgap21 Deficiency Results in Increase of Osteoblastic Lineage Cells in the Murine Bone Marrow Microenvironment.

ARHGAP21 is a member of the RhoGAP family of proteins involved in cell growth, differentiation, and adhesion. We have previously shown that the heterozygous Arhgap21 knockout mouse model (Arhgap21+/-) presents several alterations in the hematopoietic compartment, including increased frequency of hematopoietic stem and progenitor cells (HSPC) with impaired adhesion in vitro, increased mobilization to peripheral blood, and decreased engraftment after bone marrow transplantation. Although these HSPC functions strongly depend on their interactions with the components of the bone marrow (BM) niche, the role of ARHGAP21 in the marrow microenvironment has not yet been explored. In this study, we investigated the composition and function of the BM microenvironment in Arhgap21+/- mice. The BM of Arhgap21+/- mice presented a significant increase in the frequency of phenotypic osteoblastic lineage cells, with no differences in the frequencies of multipotent stromal cells or endothelial cells when compared to the BM of wild type mice. Arhgap21+/- BM cells had increased capacity of generating osteogenic colony-forming units (CFU-OB) in vitro and higher levels of osteocalcin were detected in the Arhgap21+/- BM supernatant. Increased expression of Col1a1, Ocn and decreased expression of Trap1 were observed after osteogenic differentiation of Arhgap21+/- BM cells. In addition, Arhgap21+/- mice recipients of normal BM cells showed decreased leucocyte numbers during transplantation recovery. Our data suggest participation of ARHGAP21 in the balanced composition of the BM microenvironment through the regulation of osteogenic differentiation.

Effects of RhoA and RhoC upon the sensitivity of prostate cancer cells to glutamine deprivation.

RhoA and RhoC contribute to the regulation of glutamine metabolism, which is a crucial determinant of cell growth in some types of cancer. Here we investigated the participation of RhoA and RhoC in the response of prostate cancer cells to glutamine deprivation. We found that RhoA and RhoC activities were up- or downregulated by glutamine reduction in PC3 and LNCaP cell lines, which was concomitant to a reduction in cell number and proliferation. Stable overexpression of wild type RhoA or RhoC did not alter the sensitivity to glutamine deprivation. However, PC3 cells expressing dominant negative RhoAN19 or RhoCN19 mutants were more resistant to glutamine deprivation. Our results indicate that RhoA and RhoC activities could affect cancer treatments targeting the glutamine pathway.

RhoA/ROCKs signaling is increased by treatment with TKI-258 and leads to increased apoptosis in SCC-4 oral squamous cell carcinoma cell line.

BACKGROUND: This study evaluated the effect of treatment with TKI-258 on apoptosis, involving Rho GTPases and their effectors in SCC-4 cells of oral squamous cell carcinoma. METHODS: Markers of cell death and apoptosis were analyzed in control and TKI-258-treated SCC-4 cells by flow cytometry. The involvement of Rho GTPases and effectors in the induction of apoptosis by TKI-258 was evaluated by quantification of cleaved PARP. Also, gene expression analysis of those proteins was performed. RESULTS: The treatment with TKI-258 led to a significant increase in cell death (7-AAD) and apoptosis (annexin V and cleaved PARP). When Rho GTPases were stimulated with LPA and inhibited with toxin A Clostridium difficile, the percentage of apoptotic cells increased and decreased, respectively. A similar effect was found when the treatment was with TKI-258 combined with LPA and toxin A. Treatment with TKI-258 significantly increased RhoA gene expression, while RhoB, RhoC, Rac1, and Cdc42 decreased significantly. ROCKs inhibitors (Y-27632 and HA-1077) reduced apoptosis compared with control. TKI-258 combined with Y-27632 or HA-1077 led to an increase in apoptosis compared with inhibitors only. Treatment with TKI-258 led to an increase in ROCK1 and ROCK2 gene expression, and a decrease in PAK1 and PAK2 gene expression. CONCLUSIONS: TKI-258 stimulates apoptosis in SCC-4 cells of oral squamous cell carcinoma. Possibly, RhoA GTPase and their effectors ROCKs participate in the signaling pathway inhibited by TKI-258. CLINICAL RELEVANCE: Therapies with multi-target inhibitors, such as TKI-258, may be promising alternatives for the clinical treatment of oral squamous cell carcinoma.

FHOD3 promotes carcinogenesis by regulating RhoA/ROCK1/LIMK1 signaling pathway in medulloblastoma.

PURPOSE: Medulloblastoma (MB) is a malignant brain disease in young children. The overall survival of MB patients is disappointing due to absence of effective therapeutics and this could be attributed to the lack of molecular mechanism underlying MB. FHOD3 was an important gene during cardio-genesis and was reported to promote cell migration in cancer. However, its role in MB is not clear to date. METHODS: RT-qPCR and IHC analysis were used to determine expression of FHOD3. Survival curve was drawn by K-M analysis. FHOD3 was knocked down by RNAi technology. The effects of FHOD3 on medulloblastoma cells were determined by CCK-8 assay, colony formation assay, transwell assay and FACs analysis. RESULTS: FHOD3 expression increased by 1.5 fold in tumor tissues compared to the control and IHC analysis further confirmed strong expression of FHOD3 in medulloblastoma tissues. Then higher FHOD3 expression was associated with shorter survival time in MB patients (13.0 months versus 43.8 months). In medulloblastoma cells such as Daoy and D283med, FHOD3 also displayed abundant expression. When FHOD3 was knocked down, the ability of cell proliferation and colony formation was reduced over greatly. The capability of cell migration and invasion was also inhibited significantly. However, cell apoptotic rate increased significantly reversely. Mechanistically, the phosphorylation level of RhoA, ROCK1, and LIMK1 was decreased when FHOD3 was knocked down but increased reversely when FHOD3 was over-expressed in Daoy cells. CONCLUSIONS: FHOD3 was associated with overall survival time in medulloblastoma patients and was essential to cell proliferation, growth and survival in medulloblastoma and might regulates activation of RhoA/ROCK1/LIMK1 signaling pathway.

ZO-2 Is a Master Regulator of Gene Expression, Cell Proliferation, Cytoarchitecture, and Cell Size.

ZO-2 is a cytoplasmic protein of tight junctions (TJs). Here, we describe ZO-2 involvement in the formation of the apical junctional complex during early development and in TJ biogenesis in epithelial cultured cells. ZO-2 acts as a scaffold for the polymerization of claudins at TJs and plays a unique role in the blood-testis barrier, as well as at TJs of the human liver and the inner ear. ZO-2 movement between the cytoplasm and nucleus is regulated by nuclear localization and exportation signals and post-translation modifications, while ZO-2 arrival at the cell border is triggered by activation of calcium sensing receptors and corresponding downstream signaling. Depending on its location, ZO-2 associates with junctional proteins and the actomyosin cytoskeleton or a variety of nuclear proteins, playing a role as a transcriptional repressor that leads to inhibition of cell proliferation and transformation. ZO-2 regulates cell architecture through modulation of Rho proteins and its absence induces hypertrophy due to inactivation of the Hippo pathway and activation of mTOR and S6K. The interaction of ZO-2 with viral oncoproteins and kinases and its silencing in diverse carcinomas reinforce the view of ZO-2 as a tumor regulator protein.

Neurotoxicity of the pesticide rotenone on neuronal polarization: a mechanistic approach.

Neurons are the most extensive and polarized cells that display a unique single long axon and multiple dendrites, which are compartments exhibiting structural and functional differences. Polarity occurs early in neuronal development and it is maintained by complex subcellular mechanisms throughout cell life. A well-defined and controlled spatio-temporal program of cellular and molecular events strictly regulates the formation of the axon and dendrites from a non-polarized cell. This event is critical for an adequate neuronal wiring and therefore for the normal functioning of the nervous system. Neuronal polarity is very sensitive to the harmful effects of different factors present in the environment. In this regard, rotenone is a crystalline, colorless and odorless isoflavone used as insecticide, piscicide and broad spectrum pesticide commonly used earlier in agriculture. In the present review we will summarize the toxicity mechanism caused by this pesticide in different neuronal cell types, focusing on a particular biological mechanism whereby rotenone could impair neuronal polarization in cultured hippocampal neurons. Recent advances suggest that the inhibition of axonogenesis produced by rotenone could be related with its effect on microtubule dynamics, the actin cytoskeleton and their regulatory pathways, particularly affecting the small RhoGTPase RhoA. Unveiling the mechanism by which rotenone produces neurotoxicity will be instrumental to understand the cellular mechanisms involved in neurodegenerative diseases influenced by this environmental pollutant, which may lead to research focused on the design of new therapeutic strategies.

AMPA receptors modulate the reorganization of F-actin in Bergmann glia cells through the activation of RhoA.

Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid glutamate receptors have been shown to modulate the morphology of the lamelar processes of Bergmann glia cells in the molecular layer of the cerebellum. Here we suggest that reorganization of F-actin may underlay the changes in the morphology of the lamelar processes. Using the fluorescent staining of F-actin with Phalloidin and the quantification of RhoA activation through immunoprecipitation or pull-down assays, we show that RhoA is activated after stimulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors and leads to the reorganization of the actin cytoskeleton of Bergmann fibers. This reorganization of the actin cytoskeleton is reflected in the form of an increase in the intensity of the F-actin staining as well as in the loss of the number of Bergmann fibers stained with Phalloidin. Moreover, using a pharmacological approach, we show that activation of RhoA and the change in the intensity of the F-actin staining depends on the activation of PI3-K, focal adhesion kinase, and protein kinase C, whereas changes in the number of Bergmann fibers depend on external calcium in a RhoA independent manner. Our findings show that glutamate may induce a form of structural plasticity in Bergmann glia cells through the reorganization of the actin cytoskeleton. This may have implications in the way the synaptic transmission is processed in the cerebellum.

Inhibiting 6-phosphogluconate dehydrogenase enhances chemotherapy efficacy in cervical cancer via AMPK-independent inhibition of RhoA and Rac1.

BACKGROUND: The oxidative pentose phosphate pathway (PPP) is essential for cancer metabolism and growth. However, the contribution of 6-phosphogluconate dehydrogenase (6PGD), a key enzyme of PPP, to cervical cancer development remains largely unknown. METHODS: mRNA and protein levels of 6PGD were analyzed in cervical cancer cells and tissues derived from patients and compared to normal counterparts. Using cell culture system and xenograft mouse model, the functions of 6PGD in cervical cancer are determined and its molecular mechanism is analyzed. 6PGD inhibitor physcion and siRNA knockdown were used. RESULTS: In this work, we demonstrate that 6PGD is aberrantly upregulated and activated in cervical cancer cells and patient tissues compared to normal counterparts. Using different approaches and preclinical models, we show that 6PGD inhibition decreases growth and migration, and enhances chemosensitivity in cervical cancer. Mechanistically, inhibition of 6PGD activates AMP-activated protein kinase (AMPK) and decreases RhoA and Rac1 activities. AMPK depletion significantly reduces the effects of 6PGD inhibition in decreasing RhoA and Rac1 activities, growth and migration in cervical cancer cells. CONCLUSIONS: Our work is the first to demonstrate the aberrant expression of 6PGD and its predominant roles in cervical cancer cell growth and migration, via a AMPK-dependent activation. Our findings suggest 6PGD as a potential therapeutic target to enhance chemosensitivity in cervical cancer.

Focal adhesion kinase, RhoA, and p38 mitogen-activated protein kinase modulates apoptosis mediated by angiotensin II AT2 receptors.

Apoptosis plays an important role in cellular processes such as development, differentiation, and homeostasis. Although the participation of angiotensin II (Ang II) AT2 receptors (AT 2 R) in cellular apoptosis is well accepted, the signaling pathway involved in this process is not well established. We evaluated the participation of signaling proteins focal adhesion kinase (FAK), RhoA, and p38 mitogen-activated protein kinase (p38MAPK) in apoptosis induced by Ang II via AT 2 R overexpressed in HeLa cells. Following a short stimulation time (120 to 240 minutes) with Ang II, HeLa-AT 2 cells showed nuclear condensation, stress fibers disassembly and membrane blebbing. FAK, classically involved in cytoskeleton reorganization, has been postulated as an early marker of cellular apoptosis. Thus, we evaluated FAK cleavage, detected at early stimulation times (15 to 30 minutes). Apoptosis was confirmed by increased caspase-3 cleavage and enzymatic activity of caspase-3/7. Participation of RhoA was evaluated. HeLa-AT 2 cells overexpressing RhoA wild-type (WT) or their mutants, RhoA V14 (constitutively active form) or RhoA N19 (dominant-negative form) were used to explore RhoA participation. HeLa-AT 2 cells expressing the constitutively active variant RhoA V14 showed enhanced apoptotic features at earlier times as compared with cells expressing the WT variant. RhoA N19 expression prevented nuclear condensation/caspase activation. Inhibition of p38MAPK caused an increase in nuclear condensation and caspase-3/7 activation, suggesting a protective role of p38MAPK. Our results clearly demonstrated that stimulation of AT 2 R induce apoptosis with participation of FAK and RhoA while p38MAPK seems to play a prosurvival role.

Hippocampal Memory Recovery After Acute Stress: A Behavioral, Morphological and Molecular Study.

Several studies have shown that a single exposure to stress may improve or impair learning and memory processes, depending on the timing in which the stress event occurs with relation to the acquisition phase. However, to date there is no information about the molecular changes that occur at the synapse during the stress-induced memory modification and after a recovery period. In particular, there are no studies that have evaluated-at the same time-the temporality of stress and stress recovery period in hippocampal short-term memory and the effects on dendritic spine morphology, along with variations in N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits. The aim of our study was to take a multidimensional approach to investigate concomitant behavioral, morphological and molecular changes induced by a single restraint stress exposure (2.5 h) and a recovery period of 6 and 24 h in rats. We found that acute stress elicited a reduced preference to explore an object placed in a novel position (a hippocampal-dependent task). These changes were accompanied by increased activity of LIM kinase I (LIMK; an actin-remodeling protein) and increased levels of NR2A subunits of NMDA receptors. After 6 h of recovery from stress, rats showed similar preference to explore an object placed in a novel or familiar position, but density of immature spines increased in secondary CA1 apical dendrites, along with a transient rise in GluA2 AMPA receptor subunits. After 24 h of recovery from stress, the animals showed a preference to explore an object placed in a novel position, which was accompanied by a normalization of NMDA and AMPA receptor subunits to control values. Our data suggest that acute stress produces reversible molecular and behavioral changes 24 h after stress, allowing a full reestablishment of hippocampal-related memory. Further studies need to be conducted to deepen our understanding of these changes and their reciprocal interactions.Adaptive stress responses are a promising avenue to develop interventions aiming at restoring hippocampal function impaired by repetitive stress exposure.

Assessing the Roles of Rho GTPases in Cell DNA Repair by the Nucleotide Excision Repair Pathway.

Ultraviolet light crossing the ozone layer in the atmospheric barrier affects all forms of living beings on earth. In eukaryotic cells, the nucleotide excision repair (NER) pathway protects the DNA by removing cyclobutane pyrimidine dimers (CPDs) and 6-4-photoproduct (6-4-PP) lesions caused by ultraviolet (UV) light, allowing cells to proliferate. On the other hand, adhesion and invasion processes, primarily regulated by the typical Rho GTPases Rho, Rac, and Cdc42, are also affected by UV radiation effects. Studies focused on determining whether or not these GTPases might affect the NER pathway in different cell models are enlightening and should start with classical experimental methodologies. In this chapter we describe two methods (host cell reactivation assay, or HCR, and slot-blots for CPDs and 6-4-PPs) to assess the direct or indirect involvement of these three GTPases on the NER pathway.

Rotenone inhibits axonogenesis via an Lfc/RhoA/ROCK pathway in cultured hippocampal neurons.

Rotenone, a broad-spectrum insecticide, piscicide and pesticide, produces a complete and selective suppression of axonogenesis in cultured hippocampal neurons. This effect is associated with an inhibition of actin dynamics through activation of Ras homology member A (RhoA) activity. However, the upstream signaling mechanisms involved in rotenone-induced RhoA activation were unknown. We hypothesized that rotenone might inhibit axon growth by the activation of RhoA/ROCK pathway because of the changes in microtubule (MT) dynamics and the concomitant release of Lfc, a MT-associated Guanine Nucleotide Exchange Factor (GEF) for RhoA. In this study, we demonstrate that rotenone decreases MT stability in morphologically unpolarized neurons. Taxol (3 nM), a drug that stabilizes MT, attenuates the inhibitory effect of rotenone (0.1 µM) on axon formation. Radiometric Forster Resonance Energy Transfer, revealed that this effect is associated with inhibition of rotenone-induced RhoA and ROCK activation. Interestingly, silencing of Lfc, but not of the RhoA GEF ArhGEF1, prevents the inhibitory effect of rotenone on axon formation. Our results suggest that rotenone-induced MT de-stabilization releases Lfc from MT thereby promoting RhoA and ROCK activities and the consequent inhibition of axon growth. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.