Knockdown of polypyrimidine tract binding protein facilitates motor function recovery after spinal cord injury.

After spinal cord injury (SCI), a fibroblast- and microglia-mediated fibrotic scar is formed in the lesion core, and a glial scar is formed around the fibrotic scar as a result of the activation and proliferation of astrocytes. Simultaneously, a large number of neurons are lost in the injured area. Regulating the dense glial scar and replenishing neurons in the injured area are essential for SCI repair. Polypyrimidine tract binding protein (PTB), known as an RNA-binding protein, plays a key role in neurogenesis. Here, we utilized short hairpin RNAs (shRNAs) and antisense oligonucleotides (ASOs) to knock down PTB expression. We found that reactive spinal astrocytes from mice were directly reprogrammed into motoneuron-like cells by PTB downregulation in vitro. In a mouse model of compression-induced SCI, adeno-associated viral shRNA-mediated PTB knockdown replenished motoneuron-like cells around the injured area. Basso Mouse Scale scores and forced swim, inclined plate, cold allodynia, and hot plate tests showed that PTB knockdown promoted motor function recovery in mice but did not improve sensory perception after SCI. Furthermore, ASO-mediated PTB knockdown improved motor function restoration by not only replenishing motoneuron-like cells around the injured area but also by modestly reducing the density of the glial scar without disrupting its overall structure. Together, these findings suggest that PTB knockdown may be a promising therapeutic strategy to promote motor function recovery during spinal cord repair.

Modulation of natural killer cell function by alpha-adrenoreceptor-coupled signalling.

OBJECTIVE: Our previous work has shown that α-adrenoreceptor (α-AR)-coupled signaling modulates T lymphocyte function. Here, we investigate the expression of α1- and α2-ARs in natural killer (NK) cells and roles of the two subtypes of α-ARs and their coupled signals in modulation of NK cell function. METHODS: NK cells were purified by Ficoll-Isopaque one-step gradient centrifugation and in discontinuous Percoll density gradients from splenic cells of rats. The mRNA expressions of α1-ARs and α2-ARs in NK cells were measured by reverse transcription-polymerase chain reaction (RT-PCR). Flow cytometry was employed to detect the cytotoxicity of NK cells. RESULTS: NK cells expressed both α1-AR and α2-AR mRNAs. Phenylephrine, a selective α1-AR agonist, increased the cytotoxicity of NK cells. This effect of phenylephrine was reduced by corynanthine, a selective α1-AR antagonist, and was blocked by PLC inhibitor U-73122, but not by PKA inhibitor H-89. Clonidine, a selective α2-AR agonist, also enhanced the cytotoxicity of NK cells. This action of clonidine was blocked by α2-AR antagonist yohimbine or by PKA inhibitor H-89, but not by PLC inhibitor U-73122. CONCLUSIONS: NK cells express α1- and α2-ARs. Activation of the either subtype of α-ARs augments NK cell function. This action of α1-ARs is transduced by PLC, while α2-AR effect is mediated by PKA signaling.

Expression of alpha-AR subtypes in T lymphocytes and role of the alpha-ARs in mediating modulation of T cell function.

OBJECTIVES: Previous work in our laboratory has shown that alpha-adrenoreceptors (alpha-ARs) and beta-ARs exist on lymphocytes from functional profile, and that the receptors mediate the regulation of lymphocyte function by catecholamines. In the present study, we directly examined the expression of alpha-AR subtypes, alpha(1)-AR and alpha(2)-AR mRNAs, in T lymphocytes and explored the roles of the alpha-AR subtypes and intracellular signal transduction mechanisms linked to the receptors in mediating the modulation of T lymphocyte function. METHODS: T lymphocytes from mesenteric lymph nodes of rats were purified by using a nylon wool column. Reverse transcription polymerase chain reaction was used to detect the expression of alpha(1)-AR and alpha(2)-AR mRNAs in the freshly isolated T cells and the mitogen concanavalin A (Con A)-activated lymphocytes. Colorimetric methylthiazoletetrazolium assay was employed to measure lymphocyte proliferation induced by Con A. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) levels in the Con A-stimulated lymphocyte culture supernatants were examined by enzyme-linked immunosorbent assay. RESULTS: T cells expressed both alpha(1)-AR and alpha(2)-AR mRNAs. The expression of both alpha(1)-AR and alpha(2)-AR mRNAs was significantly higher in the Con A-activated lymphocytes than in the resting lymphocytes. Phenylephrine, a selective alpha(1)-AR agonist, had no evident effect on lymphocyte proliferation nor on IFN-gamma and IL-4 production induced by Con A. However, the selective alpha(2)-AR agonist clonidine attenuated Con A-induced lymphocyte proliferation as well as IFN-gamma and IL-4 production. The inhibited lymphocyte proliferation and IFN-gamma and IL-4 production by clonidine were blocked by yohimbine, an alpha(2)-AR antagonist. Either phospholipase C inhibitor U-73122 or protein kinase C inhibitor chelerythrine partially prevented the suppressive effect of clonidine on Con A-stimulated lymphocyte proliferation and IL-4 production. CONCLUSIONS: T lymphocytes express both alpha(1)-ARs and alpha(2)-ARs, but only the alpha(2)-ARs participate in the suppressive modulation of lymphocyte proliferation and cytokine production in vitro. The inhibitory effect of alpha(2)-AR stimulation on lymphocyte function is partially mediated via the phospholipase C-protein kinase C pathway.