In neuronal ceroid lipofuscinosis (NCL), cerebral and retinal atrophy has been reported coincident with abnormal inclusion bodies found in astrocytes and neurons of the brain and pigmented epithelium of the retina. In addition to undegraded proteins, these structures contain lysosomal acid hydrolases, dolichyl oligosaccharides normally found in rough endoplasmic reticulum (RER), and the c-subunit of the mitochondrial ATP synthase. From our recent studies, it is likely that autophagy is responsible for the delivery of these cellular components to the inclusion bodies. Autophagy is a normal process whereby cellular components (ie, mitochondria) are sequestered within RER-derived vacuoles and ultimately degraded following fusion of these vacuoles with lysosomes. Therefore, we propose the following Primary Hypothesis: The accumulation of inclusion bodies within NCL cells is coincident with alterations in autophage-mediated protein degradation. To test our hypothesis, we will examine the autophagic response in astroglial and neuronal cells isolated from normal and NCL English setters. The rates of protein degradation in astroastroglial and neuronal cells from normal and NCL English setters will be measured under conditions whereby autophage is enhanced or inhibited (AIM #1). Cultured cells whose proteins have been previously labeled with 14C-valine will be incubated in media with or without amino acids and the release of acid-soluble radioactivity quantified over time. The rates of degradation will be correlated to the occurrence of the NCL inclusion bodies identified morphologically. The abnormal deposition of the inclusion bodies may be due to either an increase in autophagy or a decrease in intralysosomal proteolysis or both. Therefore, we will measure the rate of formation of autophagic vacuoles and the rate of intralysosomal degradation (AIM #2). The increase in the fractional volume represented by autophagosomes will be quantified morphometrically in respect to time of incubation with amino acid depleted medium. Relative rates of intralysosomal degradation will be estimated by measuring the release of 14C-valine from autophagolysosomes which had been isolated from cells previously incubated in amino acid depleted medium. Finally, we will compare the characteristics of autophagic vacuoles to those of inclusion bodies (AIM #3). Specifically, we will determine whether or not the inclusion bodies are acidic and contain lysosomal proteins (eg, cathepsin D, lysosomal membrane proteins, and superoxide dismutase) normally found in autophagolysosomes. Alternatively, we will evaluate whether or not the c-subunit of the ATP synthase, normally found in mitochondria and NCL inclusion bodies, is present in autophagosomes and autophagolysosomes. This information will allow us to evaluate the mode of entry (eg, autophagy) of the c-subunit into these inclusion bodies. The studies we propose will provide new insights into the mechanisms and regulation of protein degradation in normal and diseased astroglial and neuronal cells.
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