Search

Metabotropic glutamate receptors (mGluRs) are important modulators of excitatory transmission, and have been implicated in anxiety and stress-related behaviors. Previously, we showed that group III mGluR agonists could depress excitatory synaptic transmission in the bed nucleus of the stria terminalis (BNST), an integral component of the anxiety circuitry. Here, we provide converging evidence indicating that this effect is mediated primarily by mGluR8, is exerted presynaptically, and is modulated by noradrenergic signaling and stress. The effects of the group III mGluR agonist L-AP4 on excitatory transmission are not potentiated by the mGluR4-selective allosteric potentiator PHCCC, but are mimicked by the mGluR8-selective agonist DCPG. Consistent with these results, mGluR8-like immunoreactivity is seen in the BNST, and the actions of L-AP4 on excitatory transmission are absent in slices from mGluR8 knockout (KO) mice. Application of DCPG is associated with an increase in paired-pulse evoked glutamate synaptic currents, and a decrease in spontaneous glutamate synaptic current frequency, consistent with a primarily presynaptic action. mGluR8-mediated suppression of excitatory transmission is disrupted ex vivo by activation of α1 adrenergic receptors (α1 ARs). BNST mGluR8 function is also disrupted by both acute and chronic in vivo exposure to restraint stress, and in brain slices from α2A AR KO mice. These studies show that mGluR8 is an important regulator of excitatory transmission in the BNST, and suggest that this receptor is selectively disrupted by noradrenergic signaling and by both acute and chronic stress.

Retinal neovascularization is central to the pathogenesis of proliferative diabetic retinopathy, the leading cause of blindness among the middle-aged population. Angiostatin, a proteolytic fragment of plasminogen is one of the most promising inhibitors of angiogenesis currently in clinical trials. Here we show that recombinant angiostatin can inhibit retinal neovascularization in a mouse model of proliferative retinopathy. Because proliferative diabetic retinopathy is a recurrent disease, effective therapy will need to be sustained. Recombinant adeno-associated viruses permit long-term expression of transfected genes; however, they can only accommodate a small insert sequence. Thus, we engineered and tested a shortened recombinant angiostatin derivative containing a signal sequence to permit secretion. Recombinant protein was purified from the medium of transfected HEK293 cells and injected subcutaneously into treated animals. The retinal vasculature was analyzed in retinal flat mounts and using immunohistochemically stained sections. Both methods demonstrate that this short, secreted form of angiostatin is effective in reducing the development of blood vessels in a nontumor environment and has therapeutic potential for neovascular retinopathies such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion and, possibly, age-related macular degeneration.

Membrane-enclosed organelles, a defining characteristic of eukaryotic cells, are lost during differentiation of specific cell types such as reticulocytes (an intermediate in differentiation of erythrocytes), central fibre cells of the eye lens, and keratinocytes. The degradation of these organelles must be tightly regulated with respect to both the time of activation and the specificity of membrane degradation. The expression of 15-lipoxygenase (15-LOX) peaks in reticulocytes immediately before organelle degradation. Here we show that 15-LOX integrates into the membranes of various organelles, allowing release of proteins from the organelle lumen and access of proteases to both lumenal and integral membrane proteins. In addition, by sparing the plasma membrane, 15-LOX shows the required specificity for organellar membranes. Thus, the action of 15-LOX provides a mechanism by which the natural degradation process can be explained. This conclusion is supported by our finding that lipoxygenase expression in the eye lens is restricted to the region at which organelle degradation occurs.