Optogenetic "low-theta" pacing of the septohippocampal circuit is sufficient for spatial goal finding and is influenced by behavioral state and cognitive demand.

Hippocampal theta oscillations show prominent changes in frequency and amplitude depending on behavioral state or cognitive demands. How these dynamic changes in theta oscillations contribute to the spatial and temporal organization of hippocampal cells, and ultimately behavior, remain unclear. We used low-theta frequency optogenetic stimulation to pace coordination of cellular and network activity between the medial septum (MS) and hippocampus during baseline and MS stimulation while rats were at rest or performing a spatial accuracy task with a visible or hidden goal zone. Hippocampal receptivity to pan-neuronal septal stimulation at low-theta frequency was primarily determined by speed and secondarily by task demands. Competition between artificial and endogenous field potentials at theta frequency attenuated hippocampal phase preference relative to local theta, but the spike-timing activity of hippocampal pyramidal cells was effectively driven by artificial septal output, particularly during the hidden goal task. Notwithstanding temporal reorganization by artificial theta stimulation, place field properties were unchanged and alterations to spatial behavior were limited to goal zone approximation. Our results indicate that even a low-theta frequency timing signal in the septohippocampal circuit is sufficient for spatial goal finding behavior. The results also advance a mechanistic understanding of how endogenous or artificial somatodendritic timing signals relate to displacement computations during navigation and spatial memory.

COL1A1 oligodeoxynucleotides decoy: biochemical and morphologic effects in an acute wound repair model.

Type I collagen is an integral component of granulation tissue and scar, that is highly dependent on TGFß1, a member of a pro-fibrotic family of cytokines, for its promotion and deposition. Blocking COL1A1 gene transcription obstructs type I collagen synthesis, hindering the progress of granulation tissue deposition and fibrosis. Local injections of a double stranded oligodeoxynucleotide (dsODN) decoy, containing the TGFß1 regulatory element that is located in the distal promoter of the COL1A1 gene, were investigated in a rat polyvinyl alcohol (PVA) sponge granulation tissue implant model. The effects on the granulation tissue deposition by dsODN decoy therapy were evaluated by the synthesis of types I and III collagens as well as ED-A (cellular) fibronectin. Fluorescently labeled dsODN was used to identify the distribution of the decoy molecules in the sponge implant relative to the observed histological effects. Morphological alterations in cells and changes in the organization of connective tissue were documented and evaluated. Collagen levels were reduced by half in implants treated with 10 nM dsODN decoy compared to scrambled dsODN-treated implants. Histologically, dsODN decoy treated implants had an increased cellular density without a corresponding increase in deposited connective tissue. Polarized light birefringence pattern of Sirius red-stained sections showed less collagen fibers accumulating between fibroblasts. The highest concentration of fluorescently labeled dsODN was identified within the interior margin of sponge implants, correlating to increased cellular density and an altered birefringence patterns. In conclusion, 10 nM dsODN decoy therapy reduced collagen deposition and altered the organization of granulation tissue, supporting its potential as a localized anti-fibrotic therapy for limiting fibrotic conditions.

Tissue stretch decreases soluble TGF-beta1 and type-1 procollagen in mouse subcutaneous connective tissue: evidence from ex vivo and in vivo models.

Transforming growth factor beta 1 (TGF-beta1) plays a key role in connective tissue remodeling, scarring, and fibrosis. The effects of mechanical forces on TGF-beta1 and collagen deposition are not well understood. We tested the hypothesis that brief (10 min) static tissue stretch attenuates TGF-beta1-mediated new collagen deposition in response to injury. We used two different models: (1) an ex vivo model in which excised mouse subcutaneous tissue (N = 44 animals) was kept in organ culture for 4 days and either stretched (20% strain for 10 min 1 day after excision) or not stretched; culture media was assayed by ELISA for TGF-beta1; (2) an in vivo model in which mice (N = 22 animals) underwent unilateral subcutaneous microsurgical injury on the back, then were randomized to stretch (20-30% strain for 10 min twice a day for 7 days) or no stretch; subcutaneous tissues of the back were immunohistochemically stained for Type-1 procollagen. In the ex vivo model, TGF-beta1 protein was lower in stretched versus non-stretched tissue (repeated measures ANOVA, P < 0.01). In the in vivo model, microinjury resulted in a significant increase in Type-1 procollagen in the absence of stretch (P < 0.001), but not in the presence of stretch (P = 0.21). Thus, brief tissue stretch attenuated the increase in both soluble TGF-beta1 (ex vivo) and Type-1 procollagen (in vivo) following tissue injury. These results have potential relevance to the mechanisms of treatments applying brief mechanical stretch to tissues (e.g., physical therapy, respiratory therapy, mechanical ventilation, massage, yoga, acupuncture).

Oligodeoxynucleotide decoy therapy blocks type 1 procollagen transcription and the prolyl hydroxylase beta subunit translation.

Persistent transforming growth factor-beta1 (TGF-beta1) exposure to lungs increases type 1 collagen synthesis and deposition resulting in excess fibrosis which leads to morbidity and possibly death. We now report using human embryonic lung fibroblasts in the presence of TGF-beta1, a novel double-stranded (ds) DNA decoy with phosphorothioate (PT) linkages, containing the TGF-beta cis-element found in the distal promoter region of the COL1A1 gene which silences COL1A1 gene expression. In a cell-free protein translation system, we have previously reported that collagen synthesis was inhibited by disulfide isomerase, the prolyl-4-hydroxylase (P-4-H) beta subunit. By comparative proteomics dsdecoy therapy increased the levels of disulfide isomerase, the P-4-H beta subunit. These findings taken together support the notion that the dsdecoy inhibits type 1 collagen synthesis at both the transcriptional and translational levels.

Direct cysteine sulfenylation drives activation of the Src kinase.

The Src kinase controls aspects of cell biology and its activity is regulated by intramolecular structural changes induced by protein interactions and tyrosine phosphorylation. Recent studies indicate that Src is additionally regulated by redox-dependent mechanisms, involving oxidative modification(s) of cysteines within the Src protein, although the nature and molecular-level impact of Src cysteine oxidation are unknown. Using a combination of biochemical and cell-based studies, we establish the critical importance of two Src cysteine residues, Cys-185 and Cys-277, as targets for H2O2-mediated sulfenylation (Cys-SOH) in redox-dependent kinase activation in response to NADPH oxidase-dependent signaling. Molecular dynamics and metadynamics simulations reveal the structural impact of sulfenylation of these cysteines, indicating that Cys-277-SOH enables solvent exposure of Tyr-416 to promote its (auto)phosphorylation, and that Cys-185-SOH destabilizes pTyr-527 binding to the SH2 domain. These redox-dependent Src activation mechanisms offer opportunities for development of Src-selective inhibitors in treatment of diseases where Src is aberrantly activated.

Modulation of pituitary adenylate cyclase-activating polypeptide (PACAP) expression in explant-cultured guinea pig cardiac neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) expression was quantified in explant-cultured guinea pig cardiac ganglia neurons. In explant culture, both the percentage of PACAP-immunoreactive neurons and pro-PACAP transcript levels increased significantly. Treatment with neurturin or glial-derived neurotrophic factor significantly suppressed the percentage of PACAP-IR neurons, but not pro-PACAP transcript levels.

Mechanisms of enhanced basal tone of brain parenchymal arterioles during early postischemic reperfusion: role of ET-1-induced peroxynitrite generation.

The contributions of vasoconstrictors (endothelin-1 (ET-1), peroxynitrite) and endothelium-dependent vasodilatory mechanisms to basal tone were investigated in parenchymal arterioles (PAs) after early postischemic reperfusion. Transient middle cerebral artery occlusion (tMCAO) was induced for 2 hours with 30 minutes reperfusion in male Wistar rats and compared with ischemia alone (permanent MCAO (pMCAO); 2.5 hours) or sham controls. Changes in lumen diameter of isolated and pressurized PAs were compared. Quantitative PCR was used to measure endothelin type B (ETB) receptors. Constriction to intravascular pressure ('basal tone') was not affected by tMCAO or pMCAO. However, constriction to inhibitors of endothelial cell, small- (SK) and intermediate- (IK) conductance, Ca(2+)-sensitive K(+) channels (apamin and TRAM-34, respectively) were significantly enhanced in PAs from tMCAO compared with pMCAO or sham. Addition of the ETB agonist sarafotoxin caused constriction in PAs from tMCAO but not from sham animals (21 ± 4% versus 3 ± 3% at 1 nmol/L; P<0.01) that was inhibited by the peroxynitrite scavenger FeTMPyP (5,10,15,20-tetrakis (N-methyl-4'-pyridyl) porphinato iron (III) chloride) (100 µmol/L). Expression of ETB receptors was not found on PA smooth muscle, suggesting that constriction to sarafotoxin after tMCAO was due to peroxynitrite and not ETB receptor expression. The maintenance of basal tone in PAs after tMCAO may restrict flow to the ischemic region and contribute to infarct expansion.

Downregulation of GluA2 AMPA receptor subunits reduces the dendritic arborization of developing spinal motoneurons.

AMPA receptors lacking the GluA2 subunit allow a significant influx of Ca(2+) ions. Although Ca(2+)-permeable AMPA receptors are a familiar feature at early stages of development, the functional significance of these receptors during the maturation of the nervous system remains to be established. Chicken lumbar motoneurons express Ca(2+)-permeable AMPA receptors at E6 but the Ca(2+) permeability of AMPA receptors decreases ∼3-fold by E11. Considering that activity-dependent changes in intracellular Ca(2+) regulates dendritic outgrowth, in this study we investigated whether downregulation of GluA2 expression during a critical period of development alters the dendritic arborization of spinal motoneurons in ovo. We use an avian replication-competent retroviral vector RCASBP (B) carrying the marker red fluorescent protein (RFP) and a GluA2 RNAi construct to downregulate GluA2 expression. Chicken embryos were infected at E2 with one of the following constructs: RCASBP(B)-RFP, RCASBP(B)-RFP-scrambled RNAi, or RCASBP(B)-RFP-GluA2 RNAi. Infection of chicken embryos at E2 resulted in widespread expression of RFP throughout the spinal cord with ≥60% of Islet1/2-positive motoneurons infected, resulting in a significant reduction in GluA2 protein expression. Downregulation of GluA2 expression had no effect on the dendritic arborization of E6 motoneurons. However, downregulation of GluA2 expression caused a significant reduction in the dendritic arborization of E11 motoneurons. Neither motoneuron survival nor maturation of network activity was affected by changes in GluA2 expression. These findings demonstrate that increased GluA2 expression and changes in the Ca(2+) permeability of AMPA receptors regulate the dendritic arborization of spinal cord motoneurons during a critical period of development.

Functional effects of the hypertrophic cardiomyopathy R403Q mutation are different in an alpha- or beta-myosin heavy chain backbone.

The R403Q mutation in the beta-myosin heavy chain (MHC) was the first mutation to be linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle. The initial studies with R403Q myosin, isolated from biopsies of patients, showed a large decrease in myosin motor function, leading to the hypothesis that hypertrophy was a compensatory response. The introduction of the mouse model for FHC (the mouse expresses predominantly alpha-MHC as opposed to the beta-isoform in larger mammals) created a new paradigm for FHC based on finding enhanced motor function for R403Q alpha-MHC. To help resolve these conflicting mechanisms, we used a transgenic mouse model in which the endogenous alpha-MHC was largely replaced with transgenically encoded beta-MHC. A His(6) tag was cloned at the N terminus of the alpha-and beta-MHC to facilitate protein isolation by Ni(2+)-chelating chromatography. Characterization of the R403Q alpha-MHC by the in vitro motility assay showed a 30-40% increase in actin filament velocity compared with wild type, consistent with published studies. In contrast, the R403Q mutation in a beta-MHC backbone showed no enhancement in velocity. Cleavage of the His-tagged myosin by chymotrypsin made it possible to isolate homogeneous myosin subfragment 1 (S1), uncontaminated by endogenous myosin. We find that the actin-activated MgATPase activity for R403Q alpha-S1 is approximately 30% higher than for wild type, whereas the enzymatic activity for R403Q beta-S1 is reduced by approximately 10%. Thus, the functional consequences of the mutation are fundamentally changed depending upon the context of the cardiac MHC isoform.

Phenotypic expression of human hepatoma cells in culture.

Hepatomas thrive in a hypoxic environment resulting in the induction of a cluster of hypoxia related genes. The protein phenotypic expression include hypoxia inducible factor-alpha, prolyl-4-hydroxylase, vascular endothelear growth factor and erythropoietin. The present study was undertaken to determine if human hepatoma cells when cultured for 72 h in the presence of serum under normoxia would maintain their cancerous phenotypic expression of certain hypoxia inducible genes. Our positive results affords an in vitro model system to test hypoxia inhibitors on the expression and the intracellular compartmentalization or the secretion of these hypoxia-inducible proteins.

Therapies for bleomycin induced lung fibrosis through regulation of TGF-beta1 induced collagen gene expression.

This review describes normal and abnormal wound healing, the latter characterized by excessive fibrosis and scarring, which for lung can result in morbidity and sometimes mortality. The cells, the extracellular matrix (ECM) proteins, and the growth factors regulating the synthesis, degradation, and deposition of the ECM proteins will be discussed. Therapeutics with particular emphasis given to gene therapies and their effects on specific signaling pathways are described. Bleomycin (BM), a potent antineoplastic antibiotic increases TGF-beta1 transcription, TGF-beta1 gene expression, and TGF-beta protein. Like TGF-beta1, BM acts through the same distal promoter cis-element of the COL1A1 gene causing increased COL1 synthesis and lung fibrosis. Lung fibroblasts exist as subpopulations with one subset predominantly responding to fibrogenic stimuli which could be a specific cell therapeutic target for the onset and development of pulmonary fibrosis.

Tissue fibrosis and carcinogenesis: divergent or successive pathways dictate multiple molecular therapeutic targets for oligo decoy therapies.

The extracellular matrix (ECM) is composed of several families of macromolecular components: fibrous proteins such as collagens, type I collagen (COL1), type III collagen (COL3), fibronectin, elastin, and glycoconjugates such as proteoglycans and matrix glycoproteins. Their receptors on the cell membrane, most of which in the case of the ECM belong to the integrins, which are heterodimeric proteins composed of alpha and beta chains. COL1 is the major fibrous collagen of bone, tendon, and skin; while COL3 is the more pliable collagen of organs like liver. Focus will not only be given to the regulation of synthesis of several fibrogenic parameters but also modulation of their degradation during growth factor-induced tissue fibrosis and cancer development. Evidence will be provided that certain tissues, which undergo fibrosis, also become cancerous. Why does there exist a divergency between tissues, which undergo frank fibrosis as an endpoint, and those tissues that undergo fibrosis and subsequently are susceptible to carcinogenicity; resulting from the etiological factor(s) causing the initial injury? For example, why does a polyvinyl alcohol (PVA) sponge implant become encapsulated and filled with fibrous tissue then fibrosis tissue growth stops? Why does the subcutaneous injection of a fibrogenic growth factor cause a benign growth and incisional wounding results in fibrosis and ultimately scarring? There are many examples of tissues, which undergo fibrosis as a prerequisite to carcinogenesis. Is there a cause-effect relationship? If you block tissue fibrosis in these precancerous tissues, would you block cancer formation? What are the molecular targets for blocking fibrosis and ultimately carcinogenesis? How can oligo decoys may be used to attenuate carcinogenesis and which oligo decoys specifically attenuate fibrogenesis as a prelude to carcinogenesis? What are other molecular targets for oligo decoy therapy in carcinogenesis?

Fibroblast spreading induced by connective tissue stretch involves intracellular redistribution of alpha- and beta-actin.

Mechanical stretching of connective tissue occurs with normal movement and postural changes, as well as treatments including physical therapy, massage and acupuncture. Connective tissue fibroblasts were recently shown to respond actively to short-term mechanical stretch (minutes to hours) with reversible cytoskeletal remodeling, characterized by extensive cell spreading and lamellipodia formation. In this study, we have examined the effect of tissue stretch on the distribution of alpha- and beta-actin in subcutaneous tissue fibroblasts ex vivo. Normal fibroblasts uniformly exhibited alpha-smooth muscle actin (alpha-SMA) immunoreactivity. Unlike cultured fibroblasts and smooth muscle cells, alpha-SMA in these fibroblasts was not in F-actin form (indicated by lack of phalloidin co-localization) nor was it organized into distinct stress fibers. The lack of stress fibers and fibronexus was confirmed by electron microscopy, indicating that these cells were not myofibroblasts. In unstretched tissue, the pattern of alpha-actin was diffuse and granular. With tissue stretch (30 min), alpha-actin formed a star-shaped pattern centered on the nucleus, while beta-actin extended throughout the cytoplasm including lamellipodia and cell cortex. This dual response pattern of alpha- and beta-actin may be an important component of cellular mechanotransduction mechanisms relevant to physiologic and therapeutic mechanical forces applied to connective tissue.