A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the phrenic motor neuron (PhMN) pool that innervates the diaphragm. While initial trauma is uncontrollable, a valuable opportunity exists in the hours to days following SCI for preventing PhMN loss and consequent respiratory dysfunction that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. GLT1, mainly expressed by astrocytes, is responsible for the vast majority of functional uptake of extracellular glutamate in the CNS, particularly in spinal cord. We found that, in bacterial artificial chromosome-GLT1-enhanced green fluorescent protein reporter mice following unilateral midcervical (C4) contusion SCI, numbers of GLT1-expressing astrocytes in ventral horn and total intraspinal GLT1 protein expression were reduced soon after injury and the decrease persisted for Ն6 weeks. We used intraspinal delivery of adeno-associated virus type 8 (AAV8)-Gfa2 vector to rat cervical spinal cord ventral horn for targeting focal astrocyte GLT1 overexpression in areas of PhMN loss. Intraspinal delivery of AAV8-Gfa2-GLT1 resulted in transduction primarily of GFAP ϩ astrocytes that persisted for Ն6 weeks postinjury, as well as increased intraspinal GLT1 protein expression. Surprisingly, we found that astrocyte-targeted GLT1 overexpression increased lesion size, PhMN loss, phrenic nerve axonal degeneration, and diaphragm neuromuscular junction denervation, and resulted in reduced functional diaphragm innervation as assessed by phrenic nerve-diaphragm compound muscle action potential recordings. These results demonstrate that GLT1 overexpression via intraspinal AAV-Gfa2-GLT1 delivery exacerbates neuronal damage and increases respiratory impairment following cervical SCI.

Approximately half of traumatic spinal cord injury (SCI) cases affect cervical regions, resulting in chronic respiratory compromise. The majority of these injuries affect midcervical levels, the location of phrenic motor neurons (PMNs) that innervate the diaphragm. A valuable opportunity exists following SCI for preventing PMN loss that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxicity due to dysregulation of extracellular glutamate homeostasis. Astrocytes express glutamate transporter 1 (GLT1), which is responsible for the majority of CNS glutamate clearance. Given our observations of GLT1 dysfunction post-SCI, we evaluated intraspinal transplantation of Glial-Restricted Precursors (GRPs)--a class of lineage-restricted astrocyte progenitors--into ventral horn following cervical hemicontusion as a novel strategy for reconstituting GLT1 function, preventing excitotoxicity and protecting PMNs in the acutely injured spinal cord. We find that unmodified transplants express low levels of GLT1 in the injured spinal cord. To enhance their therapeutic properties, we engineered GRPs with AAV8 to overexpress GLT1 only in astrocytes using the GFA2 promoter, resulting in significantly increased GLT1 protein expression and functional glutamate uptake following astrocyte differentiation in vitro and after transplantation into C4 hemicontusion. Compared to medium-only control and unmodified GRPs, GLT1-overexpressing transplants reduced lesion size, diaphragm denervation and diaphragm dysfunction. Our findings demonstrate transplantation-based replacement of astrocyte GLT1 is a promising approach for SCI.

Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSAs) following cervical contusion SCI as a novel strategy for reconstituting GLT1 expression and for protecting diaphragmatic respiratory neural circuitry. Transplant-derived cells showed robust long-term survival post-injection and efficiently differentiated into astrocytes in injured spinal cord of both immunesuppressed mice and rats. However, the majority of transplant-derived astrocytes did not express high levels of GLT1, particularly at early times post-injection. To enhance their ability to modulate extracellular glutamate levels, we engineered hIPSAs with lentivirus to constitutively express GLT1. Overexpression significantly increased GLT1 protein and functional GLT1-mediated glutamate uptake levels in hIPSAs both in vitro and in vivo post-transplantation. Compared to human fibroblast control and unmodified hIPSA transplantation, GLT1-overexpressing hIPSAs reduced (1) lesion size within the injured cervical spinal cord, (2) morphological denervation by respiratory phrenic motor neurons at the diaphragm neuromuscular junction, and (3) functional diaphragm denervation as measured by recording of spontaneous EMGs and evoked compound muscle action potentials. Our findings demonstrate that hiPSA transplantation is a therapeutically-powerful approach for SCI.

No abstract

Phytochemicals are among the natural chemopreventive agents with most potential for delaying, blocking or reversing the initiation and promotional events of carcinogenesis. They therefore offer cancer treatment strategies to reduce cancer related death. One such promising chemopreventive agent which has attracted considerable attention is sulforaphane (SFN), which exhibits anti-cancer, anti-diabetic, and anti-microbial properties. The present study was undertaken to assess effect of SFN alone and in combination with a chemotherapeutic agent, gemcitabine, on the proliferative potential of MCF-7 cells by cell viability assay and authenticated the results by nuclear morphological examination.

Bladder dysfunction is associated with the overexpression of the intermediate filament (IF) proteins desmin and vimentin in obstructed bladder smooth muscle (BSM). However, the mechanisms by which these proteins contribute to BSM dysfunction are not known. Previous studies have shown that desmin and vimentin directly participate in signal transduction. In this study, we hypothesized that BSM dysfunction associated with overexpression of desmin or vimentin is mediated via c‐Jun N‐terminal kinase (JNK). We employed a model of murine BSM tissue in which increased expression of desmin or vimentin was induced by adenoviral transduction to examine the sufficiency of increased IF protein expression to reduce BSM contraction. Murine BSM strips overexpressing desmin or vimentin generated less force in response to KCl and carbachol relative to the levels in control murine BSM strips, an effect associated with increased JNK2 phosphorylation and reduced myosin light chain (MLC20) phosphorylation. Furthermore, desmin and vimentin overexpressions did not alter BSM contractility and MLC20 phosphorylation in strips isolated from JNK2 knockout mice. Pharmacological JNK2 inhibition produced results qualitatively similar to those caused by JNK2 knockout. These findings suggest that inhibition of JNK2 may improve diminished BSM contractility associated with obstructive bladder disease.

No abstract

Caveolins (CAVs) are structural proteins of caveolae that function as signaling platforms to regulate smooth muscle contraction. Loss of CAV protein expression is associated with impaired contraction in obstruction-induced bladder smooth muscle (BSM) hypertrophy. In this study, microarray analysis of bladder RNA revealed down-regulation of CAV1, CAV2, and CAV3 gene transcription in BSM from models of obstructive bladder disease in mice and humans. We identified and characterized regulatory regions responsible for CAV1, CAV2, and CAV3 gene expression in mice with obstruction-induced BSM hypertrophy, and in men with benign prostatic hyperplasia. DNA affinity chromatography and chromatin immunoprecipitation assays revealed a greater increase in binding of GATA-binding factor 6 (GATA-6) and NF-kB to their cognate binding motifs on CAV1, CAV2, and CAV3 promoters in obstructed BSM relative to that observed in control BSM. Knockout of NF-kB subunits, shRNA-mediated knockdown of GATA-6, or pharmacologic inhibition of GATA-6 and NF-kB in BSM increased CAV1, CAV2, and CAV3 transcription and promoter activity. Conversely, overexpression of GATA-6 decreased CAV2 and CAV3 transcription and promoter activity. Collectively, these data provide new insight into the mechanisms by which CAV gene expression is repressed in hypertrophied BSM in obstructive bladder disease.