Elevated expression of wildtype RhoC promotes ErbB2- and Pik3ca-induced mammary tumor formation.

Copy number gains in genes coding for Rho activating exchange factors as well as losses affecting genes coding for RhoGAP proteins are common in breast cancer (BC), suggesting that elevated Rho signaling may play an important role. Extra copies and overexpression of RHOC also occur, although a role for RhoC overexpression in driving tumor formation has not been assessed in vivo. To this end, we report on the development of a Rosa26 (R26)-targeted Cre-conditional RhoC overexpression mouse (R26RhoC). This mouse was crossed to two models for ERBB2/NEU+ breast cancer: one based on expression of an oncogenic ErbB2/Neu cDNA downstream of the endogenous ErbB2 promoter (FloxNeoNeuNT), the other, a metastatic model that is based on high-level expression from MMTV regulatory elements (NIC). RhoC overexpression dramatically enhanced mammary tumor formation in FloxNeoNeuNT mice but showed a more subtle effect in the NIC line, which forms multiple mammary tumors after a very short latency. RhoC overexpression also enhanced mammary tumor formation in an activated Pik3ca model for breast cancer (Pik3caH1047R). The transforming effect of RhoC was associated with epithelial/mesenchymal transition (EMT) in ErbB2/NeuNT and Pik3caH1047R systems. Thus, our study reveals the importance of elevated wildtype Rho protein expression as a driver of breast tumor formation and highlights the significance of Copy Number Abberations that affect Rho signalling.

Mechanistic role of RND3-regulated IL33/ST2 signaling on cardiomyocyte senescence.

Hyperinflammatory responses are pivotal in the cardiomyocyte senescence pathophysiology, with IL33 serving as a crucial pro-inflammatory mediator. Our previous findings highlighted RND3's suppressive effect on IL33 expression. This study aims to explore the role of RND3 in IL33/ST2 signaling activation and in cardiomyocyte senescence. Intramyocardial injection of exogenous IL33 reduces the ejection fraction and fractional shortening of rats, inducing the appearance of senescence-associated secretory phenotype (SASP) in myocardial tissues. Recombinant IL33 treatment of AC16 cardiomyocytes significantly upregulated expression of SASP factors like IL1α, IL6, and MCP1, and increased the p-p65/p65 ratio and proportions of SA-ß-gal and γH2AX-positive cells. NF-κB inhibitor pyrrolidinedithiocarbamate ammonium (PDTC) and ST2 antibody astegolimab treatments mitigated above effects. RND3 gene knockout H9C2 cardiomyocytes using CRISPR/Cas9 technology upregulated IL33, ST2L, IL1α, IL6, and MCP1 levels, decreased sST2 levels, and increased SA-ß-gal and γH2AX-positive cells. A highly possibility of binding between RND3 and IL33 proteins was showed by molecular docking and co-immunoprecipitation, and loss of RND3 attenuated ubiquitination mediated degradation of IL33; what's more, a panel of ubiquitination regulatory genes closely related to RND3 were screened using qPCR array. In contrast, RND3 overexpression in rats by injection of AAV9-CMV-RND3 particles inhibited IL33, ST2L, IL1α, IL6, and MCP1 expression in cardiac tissues, decreased serum IL33 levels, and increased sST2 levels. These results suggest that RND3 expression in cardiomyocytes modulates cell senescence by inhibiting the IL33/ST2/NF-κB signaling pathway, underscoring its potential as a therapeutic target in cardiovascular senescence.

The amyloid precursor protein and its derived fragments concomitantly contribute to the alterations of mitochondrial transport machinery in Alzheimer's disease.

Mitochondria dysfunctions and mitophagy failure have been associated with several Alzheimer's disease (AD) related molecular actors including amyloid beta (Aß) and recently the amyloid precursor protein-C terminal fragments (APP-CTFs). The efficacy of the mitophagy process in neurons relies on regulated mitochondrial transport along axons involving a complex molecular machinery. The contribution of the amyloid precursor protein (APP) and its derived fragments to the mitochondrial transport machinery alterations in AD have not been investigated before. We report herein a change of the expression of mitochondrial transport proteins (SNPH and Miro1), motor adapters (TRANK1 and TRAK2), and components of the dynein and kinesin motors (i.e., IC1,2 and Kif5 (A, B, C) isoforms) by endogenous APP and by overexpression of APP carrying the familial Swedish mutation (APPswe). We show that APP-CTFs and Aß concomitantly regulate the expression of a set of transport proteins as demonstrated in APPswe cells treated with ß- and γ-secretase inhibitors and in cells Knock-down for presenilin 1 and 2. We further report the impact of APP-CTFs on the expression of transport proteins in AAV-injected C99 mice brains. Our data also indicate that both Aß oligomers (Aßo) and APP-CTFs impair the colocalization of mitochondria and transport proteins. This has been demonstrated in differentiated SH-SY5Y naive cells treated with Aßo and in differentiated SH-SY5Y and murine primary neurons expressing APPswe and treated with the γ-secretase inhibitor. Importantly, we uncover that the expression of a set of transport proteins is modulated in a disease-dependent manner in 3xTgAD mice and in human sporadic AD brains. This study highlights molecular mechanisms underlying mitochondrial transport defects in AD that likely contribute to mitophagy failure and disease progression.

A computational analysis of the role of integrins and Rho-GTPases in the emergence and disruption of apical-basal polarization in renal epithelial cells.

Apical-basal polarization in renal epithelial cells is crucial to renal function and an important trigger for tubule formation in kidney development. Loss of polarity can induce epithelial-to-mesenchymal transition (EMT), which can lead to kidney pathologies. Understanding the relative and combined roles of the involved proteins and their interactions that govern epithelial polarity may provide insights for controlling the process of polarization via chemical or mechanical manipulations in an in vitro or in vivo setting. Here, we developed a computational framework that integrates several known interactions between integrins, Rho-GTPases Rho, Rac and Cdc42, and polarity complexes Par and Scribble, to study their mutual roles in the emergence of polarization. The modeled protein interactions were shown to induce the emergence of polarized distributions of Rho-GTPases, which in turn led to the accumulation of apical and basal polarity complexes Par and Scribble at their respective poles, effectively recapitulating polarization. Our multiparametric sensitivity analysis suggested that polarization depends foremost on the mutual inhibition between Rac and Rho. Next, we used the computational framework to investigate the role of integrins and GTPases in the generation and disruption of polarization. We found that a minimum concentration of integrins is required to catalyze the process of polarization. Furthermore, loss of polarization was found to be only inducible via complete degradation of the Rho-GTPases Rho and Cdc42, suggesting that polarization is fairly stable once it is established. Comparison of our computational predictions against data from in vitro experiments in which we induced EMT in renal epithelial cells while quantifying the relative Rho-GTPase levels, displayed that EMT coincides with a large reduction in the Rho-GTPase Rho. Collectively, these results demonstrate the essential roles of integrins and Rho-GTPases in the establishment and disruption of apical-basal polarity and thereby provide handles for the in vitro or in vivo regulation of polarity.

Molecular dissection of PI3Kß synergistic activation by receptor tyrosine kinases, GßGγ, and Rho-family GTPases.

Phosphoinositide 3-kinase (PI3K) beta (PI3Kß) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kß prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kß localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kß when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kß membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kß prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GßGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kß to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GßGγ or pY/Rac1(GTP), PI3Kß activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kß is synergistically activated by pY/GßGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.

RND3 modulates microglial polarization and alleviates neuroinflammation in Parkinson's disease by suppressing NLRP3 inflammasome activation.

Neuroinflammation mediated by microglia plays an important role in the etiology of Parkinson's disease (PD). Rho family GTPase 3 (RND3) exerts anti-inflammatory effects and may act as a potential new inducer of neuroprotective phenotypes in microglia. However, whether RND3 can be used to regulate microglia activation or reduce neuroinflammation in PD remains elusive. The study investigated the microglia modulating effects and potential anti-inflammatory effects of RND3 in vivo and in vitro, using animal models of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD and cell models of BV-2 cells stimulated by LPS plus IFN-γ with or without RND3-overexpression. The results showed that RND3 was highly expressed in the MPTP-induced PD mouse model and BV-2 cells treated with LPS and IFN-γ. In vivo experiments confirmed that RND3 overexpression could modulate microglia phenotype and ameliorate MPTP-induced neuroinflammation through inhibiting activation of the NLRP3 inflammasome in substantia nigra pars compacta (SNpc). In vitro study showed that RND3 overexpression could attenuate the production of pro-inflammatory factors in BV2 cells stimulated by LPS and IFN-γ. Mechanistically, RND3 reduced the activation of the NLRP3 inflammasome upon LPS and IFN-γ stimulation. Taken together, these findings suggest that RND3 modulates microglial polarization and alleviates neuroinflammation in Parkinson's disease by suppressing NLRP3 inflammasome activation.

Differential Smad2/3 linker phosphorylation is a crosstalk mechanism of Rho/ROCK and canonical TGF-ß3 signaling in tenogenic differentiation.

The transforming growth factor (TGF)-ß3 is a well-known inducer for tenogenic differentiation, signaling via the Smad2/3 pathway. Furthermore, other factors like extracellular matrix or mechanical force can induce tenogenic differentiation and possibly alter the response to TGF-ß3 by signaling via the Rho/ROCK pathway. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-ß3/Smad signaling in tenogenic differentiation, with the Smad2/3 molecule hypothesized as a possible interface. Cultured as monolayers or on collagen I matrices, mesenchymal stromal cells (MSC) were treated with the ROCK inhibitor Y-27632 (10 µM), TGF-ß3 (10 ng/ml) or both combined. Control cells were cultured accordingly, without Y-27632 and/or without TGF-ß3. At different time points, MSC were analyzed by real-time RT-PCR, immunofluorescence, and Western blot. Cultivation of MSC on collagen matrices and ROCK inhibition supported tenogenic differentiation and fostered the effect of TGF-ß3. The phosphorylation of the linker region of Smad2 was reduced by cultivation on collagen matrices, but not by ROCK inhibition. The latter, however, led to increased phosphorylation of the linker region of Smad3. In conclusion, collagen matrices and the Rho/ROCK signaling pathway influence the TGF-ß3/Smad2/3 pathway by regulating different phosphorylation sites of the Smad linker region.

Spaced training activates Miro/Milton-dependent mitochondrial dynamics in neuronal axons to sustain long-term memory.

Neurons have differential and fluctuating energy needs across distinct cellular compartments, shaped by brain electrochemical activity associated with cognition. In vitro studies show that mitochondria transport from soma to axons is key to maintaining neuronal energy homeostasis. Nevertheless, whether the spatial distribution of neuronal mitochondria is dynamically adjusted in vivo in an experience-dependent manner remains unknown. In Drosophila, associative long-term memory (LTM) formation is initiated by an early and persistent upregulation of mitochondrial pyruvate flux in the axonal compartment of neurons in the mushroom body (MB). Through behavior experiments, super-resolution analysis of mitochondria morphology in the neuronal soma and in vivo mitochondrial fluorescence recovery after photobleaching (FRAP) measurements in the axons, we show that LTM induction, contrary to shorter-lived memories, is sustained by the departure of some mitochondria from MB neuronal soma and increased mitochondrial dynamics in the axonal compartment. Accordingly, impairing mitochondrial dynamics abolished the increased pyruvate consumption, specifically after spaced training and in the MB axonal compartment, thereby preventing LTM formation. Our results thus promote reorganization of the mitochondrial network in neurons as an integral step in elaborating high-order cognitive processes.

SUMOylation of annexin A6 retards cell migration and tumor growth by suppressing RHOU/AKT1-involved EMT in hepatocellular carcinoma.

BACKGROUND: The protein annexin A6 (AnxA6) is involved in numerous membrane-related biological processes including cell migration and invasion by interacting with other proteins. The dysfunction of AnxA6, including protein expression abundance change and imbalance of post-translational modification, is tightly related to multiple cancers. Herein we focus on the biological function of AnxA6 SUMOylation in hepatocellular carcinoma (HCC) progression. METHODS: The modification sites of AnxA6 SUMOylation were identified by LC-MS/MS and amino acid site mutation. AnxA6 expression was assessed by immunohistochemistry and immunofluorescence. HCC cells were induced into the epithelial-mesenchymal transition (EMT)-featured cells by 100 ng/mL 12-O-tetradecanoylphorbol-13-acetate exposure. The ability of cell migration was evaluated under AnxA6 overexpression by transwell assay. The SUMO1 modified AnxA6 proteins were enriched from total cellular proteins by immunoprecipitation with anti-SUMO1 antibody, then the SUMOylated AnxA6 was detected by Western blot using anti-AnxA6 antibody. The nude mouse xenograft and orthotopic hepatoma models were established to determine HCC growth and tumorigenicity in vivo. The HCC patient's overall survival versus AnxA6 expression level was evaluated by the Kaplan-Meier method. RESULTS: Lys579 is a major SUMO1 modification site of AnxA6 in HCC cells, and SUMOylation protects AnxA6 from degradation via the ubiquitin-proteasome pathway. Compared to the wild-type AnxA6, its SUMO site mutant AnxA6K579R leads to disassociation of the binding of AnxA6 with RHOU, subsequently RHOU-mediated p-AKT1ser473 is upregulated to facilitate cell migration and EMT progression in HCC. Moreover, the SENP1 deSUMOylates AnxA6, and AnxA6 expression is negatively correlated with SENP1 protein expression level in HCC tissues, and a high gene expression ratio of ANXA6/SENP1 indicates a poor overall survival of patients. CONCLUSIONS: AnxA6 deSUMOylation contributes to HCC progression and EMT phenotype, and the combination of AnxA6 and SENP1 is a better tumor biomarker for diagnosis of HCC grade malignancy and prognosis.

The small yeast GTPase Rho5 requires specific mitochondrial outer membrane proteins for translocation under oxidative stress and interacts with the VDAC Por1.

Yeast Rho5 is a small GTPase which mediates the response to nutrient and oxidative stress, and triggers mitophagy and apoptosis. We here studied the rapid translocation of a GFP-tagged Rho5 to mitochondria under such stress conditions by live-cell fluorescence microscopy in the background of strains lacking different mitochondrial outer membrane proteins (MOMP). Fun14, Msp1 and Alo1 were found to be required for efficient recruitment of the GTPase, whereas translocation of Dck1 and Lmo1, the subunits of its dimeric GDP/GTP exchange factor (GEF), remained unaffected. An influence of the voltage-dependent anion channel (VDAC) Por1 on the association of GFP-Rho5 with mitochondria under oxidative stress conditions appeared to be strain-dependent. However, epistasis analyses and bimolecular fluorescence complementation (BiFC) studies indicate a genetic and physical interaction. All four strains lacking a single MOMP were investigated for their effect on mitophagy.

Armcx1 Reduces Neurological Damage Via a Mitochondrial Transport Pathway Involving Miro1 After Traumatic Brain Injury.

Armcx1 is a member of the ARMadillo repeat-Containing protein on the X chromosome (ARMCX) family, which is recognized to have evolutionary conserved roles in regulating mitochondrial transport and dynamics. Previous research has shown that Armcx1 is expressed at higher levels in mice after axotomy and in adult retinal ganglion cells after crush injury, and this protein increases neuronal survival and axonal regeneration. However, its role in traumatic brain injury (TBI) is unclear. Therefore, the aim of this study was to assess the expression of Armcx1 after TBI and to explore possible related mechanisms by which Armcx1 is involved in TBI. We used C57BL/6 male mice to model TBI and evaluated the role of Armcx1 in TBI by transfecting mice with Armcx1 small interfering RNA (siRNA) to inhibit Armcx1 expression 24 h before TBI modeling. Western blotting, immunofluorescence, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, Nissl staining, transmission electron microscopy, adenosine triphosphate (ATP) level measurement, neuronal apoptosis analysis, neurological function scoring and the Morris water maze were performed. The results demonstrated that Armcx1 protein expression was elevated after TBI and that the Armcx1 protein was localized in neurons and astroglial cells in cortical tissue surrounding the injury site. In addition, inhibition of Armcx1 expression further led to impaired mitochondrial transport, abnormal morphology, reduced ATP levels, aggravation of neuronal apoptosis and neurological dysfunction, and decrease Miro1 expression. In conclusion, our findings indicate that Armcx1 may exert neuroprotective effects by ameliorating neurological injury after TBI through a mitochondrial transport pathway involving Miro1.

Exploration of the Mechanisms Underlying Yu's Enema Formula in Treating Ulcerative Colitis by Blocking the RhoA/ROCK Pathway based on Network Pharmacology, High-performance Liquid Chromatography Analysis, and Experimental Verification.

BACKGROUND: The traditional Chinese medicine formula, Yu's Enema Formula (YEF), has demonstrated potential in the treatment of Ulcerative Colitis (UC). OBJECTIVE: This study aimed to unveil the anti-UC mechanisms of YEF. METHODS: Utilizing public databases, we obtained YEF and UC-related targets. GO and KEGG analyses were conducted via clusterProfiler and Reactome. The STRING database facilitated the construction of the PPI network, and hub targets were selected using cytoHubba. We used R software for differential expression and correlation analyses, and molecular docking was performed with PyMOL and AutoDock. HPLC analysis identified the compounds in YEF. For in vivo validation, a UC rat model was employed. RESULTS AND DISCUSSION: 495 YEF-UC overlapping targets were identified. GO and KEGG analyses indicated enrichment in exogenous stimuli response, peptide response, positive MAPK cascade regulation, interleukin- related signaling, and the TLR4 cascade. Hub targets included CTNNB1, JUN, MAPK1, MAPK3, SRC, STAT3, TLR4, TP53, and RELA, which were often interconnected. Molecular docking revealed quercetin's strong binding affinity with CTNNB1, MAPK1, MAPK3, SRC, STAT3, TLR4, and TP53, consistent with HPLC analysis. In vivo experiments suggested that YEF has the potential to alleviate UC symptoms and protect the intestinal mucosal barrier by inhibiting the RhoA/ROCK pathway. CONCLUSION: YEF may safeguard the intestinal mucosal barrier in UC by targeting CTNNB1, MAPK1, MAPK3, SRC, STAT3, TLR4, and TP53, while blocking the RhoA/ROCK pathway.

Identification of New Markers of Angiogenic Sprouting Using Transcriptomics: New Role for RND3.

BACKGROUND: New blood vessel formation requires endothelial cells to transition from a quiescent to an invasive phenotype. Transcriptional changes are vital for this switch, but a comprehensive genome-wide approach focused exclusively on endothelial cell sprout initiation has not been reported. METHODS: Using a model of human endothelial cell sprout initiation, we developed a protocol to physically separate cells that initiate the process of new blood vessel formation (invading cells) from noninvading cells. We used this model to perform multiple transcriptomics analyses from independent donors to monitor endothelial gene expression changes. RESULTS: Single-cell population analyses, single-cell cluster analyses, and bulk RNA sequencing revealed common transcriptomic changes associated with invading cells. We also found that collagenase digestion used to isolate single cells upregulated the Fos proto-oncogene transcription factor. Exclusion of Fos proto-oncogene expressing cells revealed a gene signature consistent with activation of signal transduction, morphogenesis, and immune responses. Many of the genes were previously shown to regulate angiogenesis and included multiple tip cell markers. Upregulation of SNAI1 (snail family transcriptional repressor 1), PTGS2 (prostaglandin synthase 2), and JUNB (JunB proto-oncogene) protein expression was confirmed in invading cells, and silencing JunB and SNAI1 significantly reduced invasion responses. Separate studies investigated rounding 3, also known as RhoE, which has not yet been implicated in angiogenesis. Silencing rounding 3 reduced endothelial invasion distance as well as filopodia length, fitting with a pathfinding role for rounding 3 via regulation of filopodial extensions. Analysis of in vivo retinal angiogenesis in Rnd3 heterozygous mice confirmed a decrease in filopodial length compared with wild-type littermates. CONCLUSIONS: Validation of multiple genes, including rounding 3, revealed a functional role for this gene signature early in the angiogenic process. This study expands the list of genes associated with the acquisition of a tip cell phenotype during endothelial cell sprout initiation.

Functional Conservation of the Small GTPase Rho5/Rac1-A Tale of Yeast and Men.

Small GTPases are molecular switches that participate in many essential cellular processes. Amongst them, human Rac1 was first described for its role in regulating actin cytoskeleton dynamics and cell migration, with a close relation to carcinogenesis. More recently, the role of Rac1 in regulating the production of reactive oxygen species (ROS), both as a subunit of NADPH oxidase complexes and through its association with mitochondrial functions, has drawn attention. Malfunctions in this context affect cellular plasticity and apoptosis, related to neurodegenerative diseases and diabetes. Some of these features of Rac1 are conserved in its yeast homologue Rho5. Here, we review the structural and functional similarities and differences between these two evolutionary distant proteins and propose yeast as a useful model and a device for high-throughput screens for specific drugs.

Assessing the mechanism of fast-cycling cancer-associated mutations of Rac1 small Rho GTPase.

Rho-GTPases proteins function as molecular switches alternating from an active to an inactive state upon Guanosine triphosphate (GTP) binding and hydrolysis to Guanosine diphosphate (GDP). Among them, Rac subfamily regulates cell dynamics, being overexpressed in distinct cancer types. Notably, these proteins are object of frequent cancer-associated mutations at Pro29 (P29S, P29L, and P29Q). To assess the impact of these mutations on Rac1 structure and function, we performed extensive all-atom molecular dynamics simulations on wild-type (wt) and oncogenic isoforms of this protein in GDP- and GTP-bound states. Our results unprecedentedly elucidate that P29Q/S-induced structural and dynamical perturbations of Rac1 core domain weaken the binding of the catalytic site Mg2+ ion, and reduce the GDP residence time within protein, enhancing the GDP/GTP exchange rate and Rac1 activity. This broadens our knowledge of the role of cancer-associated mutations on small GTPases mechanism supplying valuable information for future drug discovery efforts targeting specific Rac1 isoforms.

Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin.

Neurotransmission at synapses is mediated by the fusion and subsequent endocytosis of synaptic vesicle membranes. Actin has been suggested to be required for presynaptic endocytosis but the mechanisms that control actin polymerization and its mode of action within presynaptic nerve terminals remain poorly understood. We combine optical recordings of presynaptic membrane dynamics and ultrastructural analysis with genetic and pharmacological manipulations to demonstrate that presynaptic endocytosis is controlled by actin regulatory diaphanous-related formins mDia1/3 and Rho family GTPase signaling in mouse hippocampal neurons. We show that impaired presynaptic actin assembly in the near absence of mDia1/3 and reduced RhoA activity is partly compensated by hyperactivation of Rac1. Inhibition of Rac1 signaling further aggravates impaired presynaptic endocytosis elicited by loss of mDia1/3. Our data suggest that interdependent mDia1/3-Rho and Rac1 signaling pathways cooperatively act to facilitate synaptic vesicle endocytosis by controlling presynaptic F-actin.

Dbl family RhoGEFs in cancer: different roles and targeting strategies.

Small Ras homologous guanosine triphosphatase (Rho GTPase) family proteins are highly associated with tumorigenesis and development. As intrinsic exchange activity regulators of Rho GTPases, Rho guanine nucleotide exchange factors (RhoGEFs) have been demonstrated to be closely involved in tumor development and received increasing attention. They mainly contain two families: the diffuse B-cell lymphoma (Dbl) family and the dedicator of cytokinesis (Dock) family. More and more emphasis has been paid to the Dbl family members for their abnormally high expression in various cancers and their correlation to poor prognosis. In this review, the common and distinctive structures of Dbl family members are discussed, and their roles in cancer are summarized with a focus on Ect2, Tiam1/2, P-Rex1/2, Vav1/2/3, Trio, KALRN, and LARG. Significantly, the strategies targeting Dbl family RhoGEFs are highlighted as novel therapeutic opportunities for cancer.

ICAM-1 Deletion Using CRISPR/Cas9 Protects the Brain from Traumatic Brain Injury-Induced Inflammatory Leukocyte Adhesion and Transmigration Cascades by Attenuating the Paxillin/FAK-Dependent Rho GTPase Pathway.

Intercellular adhesion molecule-1 (ICAM-1) is identified as an initiator of neuroinflammatory responses that lead to neurodegeneration and cognitive and sensory-motor deficits in several pathophysiological conditions including traumatic brain injury (TBI). However, the underlying mechanisms of ICAM-1-mediated leukocyte adhesion and transmigration and its link with neuroinflammation and functional deficits following TBI remain elusive. Here, we hypothesize that blocking of ICAM-1 attenuates the transmigration of leukocytes to the brain and promotes functional recovery after TBI. The experimental TBI was induced in vivo by fluid percussion injury (25 psi) in male and female wild-type and ICAM-1-/- mice and in vitro by stretch injury (3 psi) in human brain microvascular endothelial cells (hBMVECs). We treated hBMVECs and animals with ICAM-1 CRISPR/Cas9 and conducted several biochemical analyses and demonstrated that CRISPR/Cas9-mediated ICAM-1 deletion mitigates blood-brain barrier (BBB) damage and leukocyte transmigration to the brain by attenuating the paxillin/focal adhesion kinase (FAK)-dependent Rho GTPase pathway. For analyzing functional outcomes, we used a cohort of behavioral tests that included sensorimotor functions, psychological stress analyses, and spatial memory and learning following TBI. In conclusion, this study could establish the significance of deletion or blocking of ICAM-1 in transforming into a novel preventive approach against the pathophysiology of TBI.

Miro1 improves the exogenous engraftment efficiency and therapeutic potential of mitochondria transfer using Wharton's jelly mesenchymal stem cells.

Mitochondria are important for maintaining cellular energy metabolism and regulating cellular senescence. Mitochondrial DNA (mtDNA) encodes subunits of the OXPHOS complexes which are essential for cellular respiration and energy production. Meanwhile, mtDNA variants have been associated with the pathogenesis of neurodegenerative diseases, including MELAS, for which no effective treatment has been developed. To alleviate the pathological conditions involved in mitochondrial disorders, mitochondria transfer therapy has shown promise. Wharton's jelly mesenchymal stem cells (WJMSCs) have been identified as suitable mitochondria donors for mitochondria-defective cells, wherein mitochondrial functions can be rescued. Miro1 participates in mitochondria trafficking by anchoring mitochondria to microtubules. In this study, we identified Miro1 over-expression as a factor that could help to enhance the efficiency of mitochondrial delivery. More specifically, we reveal that Miro1 over-expressed WJMSCs significantly improved intercellular communications, cell proliferation rates, and mitochondrial membrane potential, while restoring mitochondrial bioenergetics in mitochondria-defective fibroblasts. Furthermore, Miro1 over-expressed WJMSCs decreased rates of induced apoptosis and ROS production in MELAS fibroblasts; although, Miro1 over-expression did not rescue mtDNA mutation ratios nor mitochondrial biogenesis. This study presents a potentially novel therapeutic strategy for treating mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), and other diseases associated with dysfunctional mitochondria, while the pathophysiological relevance of our results should be further verified by animal models and clinical studies.

Constraint-Induced Movement Therapy Promotes Myelin Remodeling and Motor Function by Mediating Sox2/Fyn Signals in Rats With Hemiplegic Cerebral Palsy.

OBJECTIVE: Hypoxic-ischemic brain injury in infants often leads to hemiplegic motor dysfunction. The mechanism of their motor dysfunction has been attributed to deficiencies of the transcription factor sex-determining region (SRY) box 2 (Sox2) or the non-receptor-type tyrosine kinase Fyn (involved in neuronal signal transduction), which causes a defect in myelin formation. Constraint-induced movement therapy (CIMT) following cerebral hypoxia-ischemia may stimulate myelin growth by regulating Sox2/Fyn, Ras homolog protein family A (RhoA), and rho-associated kinase 2 (ROCK2) expression levels. This study investigated how Sox2/Fyn regulates myelin remodeling following CIMT to improve motor function in rats with hemiplegic cerebral palsy (HCP). METHODS: To investigate the mechanism of Sox2 involvement in myelin growth and neural function in rats with HCP, Lentivirus (Lenti)-Sox2 adeno-associated virus and negative control-Lenti-Sox2 (LS) adeno-associated virus were injected into the lateral ventricle. The rats were divided into a control group and an HCP group with different interventions (CIMT, LS, or negative control-LS [NS] treatment), yielding the HCP, HCP plus CIMT (HCP + CIMT), HCP + LS, HCP + LS + CIMT, HCP + NS, and HCP + NS + CIMT groups. Front-limb suspension and RotaRod tests, Golgi-Cox staining, transmission electron microscopy, immunofluorescence staining, western blotting, and quantitative polymerase chain reaction experiments were used to analyze the motor function, dendrite/axon area, myelin ultrastructure, and levels of expression of oligodendrocytes and Sox2/Fyn/RhoA/ROCK2 in the motor cortex. RESULTS: The rats in the HCP + LS + CIMT group had better values for motor function, dendrite/axon area, myelin ultrastructure, oligodendrocytes, and Sox2/Fyn/RhoA/ROCK2 expression in the motor cortex than rats in the HCP and HCP + NS groups. The improvement of motor function and myelin remodeling, the expression of oligodendrocytes, and the expression of Sox2/Fyn/RhoA/ROCK2 in the HCP + LS group were similar to those in the HCP + CIMT group. CONCLUSION: CIMT might overcome RhoA/ROCK2 signaling by upregulating the transcription of Sox2 to Fyn in the brain to induce the maturation and differentiation of oligodendrocytes, thereby promoting myelin remodeling and improving motor function in rats with HCP. IMPACT: The pathway mediated by Sox2/Fyn could be a promising therapeutic target for HCP.