Enhanced expression of the central survival of motor neuron (SMN) protein during the pathogenesis of osteoarthritis

The identification of new components implicated in the pathogenesis of osteoarthritis (OA) might improve our understanding of the disease process. Here, we investigated the levels of the survival of motor neuron (SMN) expression in OA cartilage considering the fundamental role of the SMN protein in cell survival and its involvement in other stress-associated pathologies. We report that SMN expression is up-regulated in human OA compared with normal cartilage, showing a strong correlation with the disease severity, a result confirmed in vivo in an experimental model of the disease. We further show that the prominent inflammatory cytokines (IL-1β, TNF-α) are critical inducers of SMN expression. This is in marked contrast with the reported impaired levels of SMN in spinal muscular atrophy, a single inherited neuromuscular disorder characterized by mutations in the smn gene whereas OA is a complex disease with multiple aetiologies. While the precise functions of SMN during OA remain to be elucidated, the conclusions of this study shed light on a novel pathophysiological pathway involved in the progression of OA, potentially offering new targets for therapy

A combination of mutations enhances the neurotropism of AAV-2

AbstractThere is strong interest in developing practical strategies for gene delivery to the central nervous system (CNS). Direct delivery into the brain or spinal cord is highly invasive as well as inefficient or hazardous using most current vector systems. Our objective was to generate innocuous gene vehicles that would be effectively taken up by axons and then home to the neuron cell bodies. Vectors derived from Adeno-Associated Virus (AAV), a harmless human parvovirus, offer strong starting candidates for deriving such vehicles. Enhancing the axonal uptake of AAV, and conferring more efficient retrograde transport capabilities upon the virus, should produce near ideal gene transfer vehicles for the CNS. To enhance retrograde transport of the virus, peptides mimicking binding domains for cytoplasmic dynein were inserted in the capsid by directed mutagenesis. In separate clones, peptides derived from an NMDA receptor antagonist were also introduced to provide a specific affinity for this receptor. When combined, these two functionally distinct classes of mutation enabled efficient gene transfer into neurons under conditions not permissive for standard AAV-2 vectors prepared under the same conditions. These results hold strong promise for the development of safe, convenient vehicles to target genes and other sequences to neurons, enabling new and novel approaches for the treatment of multiple neurological disorders

Characterization of an Envelope Mutant of HIV-1 That Interferes with Viral Infectivity

AbstractA mutant human immunodeficiency virus (HIV-1) provirus encoding an envelope (Env) protein with a truncated transmembrane protein cytoplasmic domain was defective for replication. Coexpression of the mutant with a wild-type (wt) HIV-1 provirus potently inhibited the production of infectious virus. The maximum inhibitory effect was reached when the ratio of mutant to wt proviral DNA was 2:1. This transdominant defect in infectivity conferred by the mutant Env did not appear to involve the late steps of virus replication, since the synthesis, precursor processing, and intracellular transport of the Env proteins were not blocked; nor did it prevent the incorporation of the envelope proteins into virions or the subsequent release of the virus. Although the mutant Env protein still retained syncytia-forming ability, the truncated protein was unable to mediate cell-to-cell transmission of the virus. Moreover, coexpression with the mutant effectively inhibited the ability of the wt Env to mediate cell-to-cell transmission. The mutant Env protein formed a complex with the wt protein when they were coexpressed, producing heterooligomeric structures which appeared to be severely defective in an early, post-CD4 binding step of the virus life cycle despite the inclusion of wt Env in the complexes

Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9

Objective. Human osteoarthritis (OA) is characterized by a pathologic shift in articular cartilage homeostasis toward the progressive loss of extracellular matrix (ECM). The purpose of this study was to investigate the ability of rAAV-mediated SOX9 overexpression to restore major ECM components in human OA articular cartilage. Methods. We monitored the synthesis and content of proteoglycans and type II collagen in 3-dimensional cultures of human normal and OA articular chondrocytes and in explant cultures of human normal and OA articular cartilage following direct application of a recombinant adeno-associated virus (rAAV) SOX9 vector in vitro and in situ. We also analyzed the effects of this treatment on cell proliferation in these systems. Results. Following SOX9 gene transfer, expression levels of proteoglycans and type II collagen increased over time in normal and OA articular chondrocytes in vitro. In situ, overexpression of SOX9 in normal and OA articular cartilage stimulated proteoglycan and type II collagen synthesis in a dose-dependent manner. These effects were not associated with changes in chondrocyte proliferation. Notably, expression of the 2 principal matrix components could be restored in OA articular cartilage to levels similar to those in normal cartilage. Conclusion. These data support the concept of using direct, rAAV-mediated transfer of chondrogenic genes to articular cartilage for the treatment of OA in humans

The tyrosine kinase Pyk2 mediates lipopolysaccharide-induced IL-8 expression in human endothelial cells

Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the role of a nonreceptor proline-rich tyrosine kinase, Pyk2, in LPS-induced IL-8 (CXCL8) production in endothelial cells. First, we observed a marked activation of Pyk2 in response to LPS. Furthermore, inhibition of Pyk2 activity in these cells by transduction with the catalytically inactive Pyk2 mutant, transfection with Pyk2-specific small interfering RNA, or treatment with Tyrphostin A9 significantly blocked LPS-induced IL-8 production. The supernatants of LPS-stimulated cells exhibiting attenuated Pyk2 activity blocked transendothelial neutrophil migration in comparison to the supernatants of LPS-treated controls, thus confirming the inhibition of functional IL-8 production. Investigations into the molecular mechanism of this pathway revealed that LPS activates Pyk2 leading to IL-8 production through the TLR4. In addition, we identified the p38 MAPK pathway to be a critical step downstream of Pyk2 during LPS-induced IL-8 production. Taken together, these results demonstrate a novel role for Pyk2 in LPS-induced IL-8 production in endothelial cells

Tob1 is a constitutively expressed repressor of liver regeneration

How proliferative and inhibitory signals integrate to control liver regeneration remains poorly understood. A screen for antiproliferative factors repressed after liver injury identified transducer of ErbB2.1 (Tob1), a member of the PC3/BTG1 family of mito-inhibitory molecules as a target for further evaluation. Tob1 protein decreases after 2/3 hepatectomy in mice secondary to posttranscriptional mechanisms. Deletion of Tob1 increases hepatocyte proliferation and accelerates restoration of liver mass after hepatectomy. Down-regulation of Tob1 is required for normal liver regeneration, and Tob1 controls hepatocyte proliferation in a dose-dependent fashion. Tob1 associates directly with both Caf1 and cyclindependent kinase (Cdk) 1 and modulates Cdk1 kinase activity. In addition, Tob1 has significant effects on the transcription of critical cell cycle components, including E2F target genes and genes involved in p53 signaling. We provide direct evidence that levels of an inhibitory factor control the rate of liver regeneration, and we identify Tob1 as a crucial check point molecule that modulates the expression and activity of cell cycle proteins

Improved Tissue Repair in Articular Cartilage Defects in Vivo by rAAV-Mediated Overexpression of Human Fibroblast Growth Factor 2

Therapeutic gene transfer into articular cartilage is a potential means to stimulate reparative activities in tissue lesions. We previously demonstrated that direct application of recombinant adeno-associated virus (rAAV) vectors to articular chondrocytes in their native matrix in situ as well as sites of tissue damage allowed for efficient and sustained reporter gene expression. Here we test the hypothesis that rAAV-mediated overexpression of fibroblast growth factor 2 (FGF-2), one candidate for enhancing the repair of cartilage lesions, would lead to the production of a biologically active factor that would facilitate the healing of articular cartilage defects. In vitro, FGF-2 production from an rAAV-delivered transgene was sufficient to stimulate chondrocyte proliferation over a prolonged period of time. In vivo, application of the therapeutic vector significantly improved the overall repair, filling, architecture, and cell morphology of osteochondral defects in rabbit knee joints. Differences in matrix synthesis were also observed, although not to the point of statistical significance. This process may further benefit from cosupplementation with other factors. These results provide a basis for rAAV application to sites of articular cartilage damage to deliver agents that promote tissue repair

Tob1 is a constitutively expressed repressor of liver regeneration

How proliferative and inhibitory signals integrate to control liver regeneration remains poorly understood. A screen for antiproliferative factors repressed after liver injury identified transducer of ErbB2.1 (Tob1), a member of the PC3/BTG1 family of mito-inhibitory molecules as a target for further evaluation. Tob1 protein decreases after 2/3 hepatectomy in mice secondary to posttranscriptional mechanisms. Deletion of Tob1 increases hepatocyte proliferation and accelerates restoration of liver mass after hepatectomy. Down-regulation of Tob1 is required for normal liver regeneration, and Tob1 controls hepatocyte proliferation in a dose-dependent fashion. Tob1 associates directly with both Caf1 and cyclin-dependent kinase (Cdk) 1 and modulates Cdk1 kinase activity. In addition, Tob1 has significant effects on the transcription of critical cell cycle components, including E2F target genes and genes involved in p53 signaling. We provide direct evidence that levels of an inhibitory factor control the rate of liver regeneration, and we identify Tob1 as a crucial check point molecule that modulates the expression and activity of cell cycle proteins

The V1a Vasopressin Receptor Is Necessary and Sufficient for Normal Social Recognition: A Gene Replacement Study

SummaryVasopressin modulates many social and nonsocial behaviors, including emotionality. We have previously reported that male mice with a null mutation in the V1a receptor (V1aR) exhibit a profound impairment in social recognition and changes in anxiety-like behavior. Using site-specific injections of a V1aR-specific antagonist, we demonstrate that the lateral septum, but not the medial amygdala, is critical for social recognition. Reexpressing V1aR in the lateral septum of V1aR knockout mice (V1aRKO) using a viral vector resulted in a complete rescue of social recognition. Furthermore, overexpression of the V1aR in the lateral septum of wild-type (wt) mice resulted in a potentiation of social recognition behavior and a mild increase in anxiety-related behavior. These results demonstrate that the V1aR in the lateral septum plays a critical role in the neural processing of social stimuli required for complex social behavior