Protein degradation in the mouse visual system. I. Degradation of axonally transported and retinal proteins.
The analysis of proteolysis in the nervous system is complicated by the heterogeneity of cell types, extensive reutilization of liberated amino acids, and artifacts that may arise when the integrity of the tissue is disrupted during experimentation. For these reasons, changes in proteolytic activity that are observed during brain development and in neuropathological states may often be difficult to interpret. To minimize these problems, we have developed a technique that permits protein degradation to be investigated specifically within axons of the mouse retinal ganglion cells (RGC). In the present study, the method has been used to examine the degradation of proteins conveyed in the slow phases of axoplasmic transport. When adult C57Bl/6J mice were injected intravitreally with L-[3H]proline, labeled proteins within the primary optic pathway (optic nerve and tract) after 5 days were almost exclusively the slow phase axonal proteins. The rate of degradation of these proteins was then determined within the excised, but otherwise intact, optic pathway by measuring the release of acid soluble radioactivity at 37 degrees C in vitro. At physiological pH, the amino acids released by proteolysis were extensively reutilized. Unless amino acid reutilization was prevented, protein degradative rates were artifactually lowered 3-fold. At least two proteolytic systems within RGC axons actively degraded the slowly transported axonal proteins. A 'neutral' system, stimulated by exogenous calcium ions, was optimally active within the physiological pH range (pH 7.0--7.8). The rate of protein degradation at pH 7.4 was uniform along the RGC axon. An 'acidic' system was optimally active with the incubation was carried out at pH 3.8. This proteolytic activity was calcium-independent and exhibited a proximodistal gradient within the RCG axon with higher activity proximally. Similar proteolytic activities were present in isolated intact retinas but in different proportions. The half-lives of axonal and retinal proteins were comparable to CNS protein half-lives estimated in vivo by methods that take amino acid reutilization into account. These and other recent findings demonstrate the utility of this neuron-specific approach in characterizing proteolytic processes within one cell type that may otherwise be obscured by proteolytic events in other cells when brain tissue is analyzed by conventional methods.
D000818:Animals; D001370:Axonal Transport; D001369:Axons / Q000187:drug effects / Q000378:metabolism*; D002701:Chloramphenicol / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009900:Optic Nerve / Q000187:drug effects / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012160:Retina / Q000187:drug effects / Q000378:metabolism*
D011506:Proteins; D002701:Chloramphenicol; D003513:Cycloheximide; D010447:Peptide Hydrolases