Constitutive autophagy: vital role in clearance of unfavorable proteins in neurons.

Investigations pursued during the last decade on neurodegenerative diseases have revealed a common mechanism underlying the development of such diseases: conformational disorder of certain proteins leads to the formation of misfolded protein oligomers, which subsequently develop into large protein aggregates. These aggregates entangle other denatured proteins and lipids to form disease-specific inclusion bodies. The failure of the ubiquitin-proteasome system to shred the protein aggregates has led investigators to focus their attention to autophagy, a bulk degradative system coupled with lysosomes, which is involved in non-selective shredding of large amounts of cytoplasmic components. Research in this field has demonstrated the accumulation of autophagic vacuoles and intracytoplasmic protein aggregates in patients with various neurodegenerative diseases. Although autophagy fails to degrade large protein aggregates once they are formed in the cytoplasm, drug-induced activation of autophagy is effective in preventing aggregate deposition, indicating that autophagy significantly contributes to the clearance of aggregate-prone proteins. The pivotal role of autophagy in the clearance of aggregate-prone proteins has been confirmed by a deductive approach using a brain-specific autophagy-ablated mouse model. In this review, we discuss the consequences of autophagy deficiency in neurons.

Retardation of chemical hypoxia-induced necrotic cell death by Bcl-2 and ICE inhibitors: possible involvement of common mediators in apoptotic and necrotic signal transductions.

Inhibition of the respiratory chain reaction by cyanide, rotenone or antimycin A (chemical hypoxia) induces necrotic cell death characterized by apparently intact chromatin, remarkable mitochondrial swelling with loss of crista structure, and loss of plasma membrane integrity. The treatments induce no apoptotic cell death, as defined by fragmented nuclei with condensed chromatin, fragmented or condensed cytoplasm. The anti-apoptotic proteins Bcl-2 and Bcl-xL effectively retard the chemical hypoxia-induced necrotic cell death. The necrotic cell death is also retarded by inhibitors of ICE(-like) proteases, including interleukin-1beta converting enzyme (ICE), which are common mediators of apoptosis. These results indicate that Bcl-2/Bcl-xL and ICE(-like) proteases modulate apoptotic and at least some forms of necrotic cell death. Both cell death pathways appear to involve some common mediators; however necrotic or apoptotic cell death signals might be transduced through multiple pathways, because Bcl-2/ Bcl-xL or inhibitors of ICE(-like) proteases are relatively less potent in blocking necrotic cell death than in preventing apoptosis.

Bcl-2 blocks loss of mitochondrial membrane potential while ICE inhibitors act at a different step during inhibition of death induced by respiratory chain inhibitors.

Bcl-2, Bcl-xL, CrmA and tetrapeptide ICE inhibitor reduce the extent of necrotic cell death induced by cyanide, which primarily damages mitochondria. Although none of them affects the drastic decrease in ATP levels induced by cyanide, Bcl-2 and Bcl-xL but not CrmA or ICE inhibitor inhibit the cyanide-induced decrease in mitochondrial membrane potential. A similar blocking effect is observed on necrotic cell death induced by other respiration inhibitors, rotenone and antimycin A, and on apoptotic cell death induced by etoposide or calcium ionophore. These results indicate that Bc1-2 and Bcl-xL protect mitochondria against the loss of function during both apoptosis and at least some forms of necrotic cell death. The ICE family proteases act at a different step other than the loss of mitochondrial membrane potential.

Cysteine proteinases in GH4C1 cells, a rat pituitary tumor cell line, are secreted by the constitutive and regulated secretory pathways.

Secretory granules of GH4C1 cells, a rat pituitary tumor cell line, are known to be induced by the treatment of estradiol (E2), insulin, and epidermal growth factor (EGF). We examined changes in the localization of cathepsins B, H, and L, lysosomal cysteine proteinases, in GH4C1 cells before and after hormonal treatment. Northern blotting and immunofluorescence microscopy showed that both mRNAs and intracellular protein concentrations of these enzymes were increased in the hormone-induced cells. By immunoelectron microscopy, immunogold particles indicating cathepsins B, H, and L were localized not only in lysosomes but also in some secretory granules. To further examine the molecular forms of these proteinases in secretory granules, radiolabeling and immunoprecipitation methods were applied to the media of the cells incubated with or without secretagogues (100 nM 12-O-tetradecanoylphorbol-13-acetate and 50 microM forskolin); the proforms of cathepsins B, H, and L were secreted from the cells by the constitutive pathway, whereas the mature forms of cathepsins B and H, and the proform and mature form of cathepsin L were secreted by the regulated pathway. These results suggest that in hormone-induced GH4C1 cells, cathepsins B, H, and L are sorted from the Golgi complex not only into lysosomes but also into secretory granules, in which proforms of cathepsins B and H, and a part of procathepsin L are processed into mature forms.

Effects of sex steroids on secretory granule formation in gonadotropes of castrated male rats with respect to granin expression.

Pituitary gonadotropes show sex-related differences in their ultrastructure. Typical gonadotropes of male rats exhibit both large granules, which contain chromogranin A (CgA), and small granules, which contain secretogranin II (SgII). In contrast, typical female rat gonadotropes show only a very few large granules among the numerous small granules. To clarify the nature of the biogenesis of these secretory granules and the effects of sex steroids, the ultrastructural and immunocytochemical changes in gonadotropes were examined in castrated male rats supplied with a testosterone or estradiol implant. In castrated rats, pituitary expression and plasma levels of LH increased drastically, but the pituitary content of CgA decreased. The majority of gonadotropes then showed features of "castration cells" containing many small secretory granules. A testosterone implant to castrated rats remarkably suppressed the expression and circulating levels of LH and increased the CgA content in the pituitary to near-normal levels. In this situation, immunocytochemical studies demonstrated that gonadotropes again exhibited large and small secretory granules with the respective localization of CgA and SgII. On the contrary, in castrated rats supplied with an estradiol implant, the expression and content of CgA in the pituitary were remarkably suppressed, and large secretory granules disappeared from gonadotropes. These results suggest that the expression of CgA in gonadotropes is regulated differently by male and female sex steroids. These different effects of androgen and estrogen on the expression level of CgA are closely associated with the sex-related differences in the ultrastructure of secretory granules within gonadotropes.

Regulation of a novel pathway for cell death by lysosomal aspartic and cysteine proteinases.

PC12 cells undergo apoptosis when cultured under conditions of serum deprivation. In this situation, the activity of caspase-3-like proteinases was elevated, and the survival rate could be maintained by treatment with acetyl-DEVD-cho, a specific inhibitor of caspase-3. In a culture of PC12 cells treated with acetyl-DEVD-cho, where caspase-3-like proteinases are not activated, CA074, a specific inhibitor of cathepsin B induced active death of the cells. Cathepsin B antisense oligonucleotides showed a similar effect to CA074 on the induction of active cell death. By double staining of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling and activated caspase-3, the dying cells treated with CA074 were positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling staining but negative for activated caspase-3. Ultrastructurally, the cells were relatively large and had nuclei with chromatin condensation. The initiation of cell death by CA074 or the cathepsin B antisense were inhibited by the addition of pepstatin A, a lysosomal aspartic proteinase inhibitor, or by cathepsin D antisense. To examine whether this cell death pathway was present in cell types other than PC12 cells, we analysed dorsal root ganglion neurons obtained from rat embryos on the 15th gestational day, a time when they require nerve growth factor for survival and differentiation in culture. When cultured in the absence of nerve growth factor, the neurons survived in the presence of acetyl-DEVD-cho or acetyl-YVAD-cho. Under these conditions, CA074 reduced the survival rate of the neurons, which was subsequently restored by the further addition of pepstain A. These results suggest that a novel pathway for initiating cell death exists which is regulated by lysosomal cathepsins, and in which cathepsin D acts as a death factor. We speculate that this death-inducing activity is normally suppressed by cathepsin B.

Cysteine proteinases in rat parathyroid cells with special reference to their correlation with parathyroid hormone (PTH) in storage granules.

To further understand the roles of storage granules in parathyroid cells, we examined by immunocytochemistry the localization of cathepsins B and H and of PTH in rat parathyroid gland. In semi-thin sections, small and large granular immunodeposits for cathepsins B and H appeared in the cells, whereas those for PTH were detected throughout the cells, especially in perinuclear regions. By electron microscopy, immunogold particles indicating cathepsins B and H labeled lysosomes and storage granules, whereas those showing PTH were localized in storage granules, small secretory granules, and the trans-Golgi network. Small vesicles labeled by immunogold particles showing these proteinases often appeared close to the storage granules. By double immunostaining, immunogold particles indicating these proteinases were co-localized with those for PTH in storage granules. By EDTA treatment, immunoreactivity for cathepsins B and H and for PTH was notably reduced in the cells, but immunoreactivity for the proteinases was still seen in lysosomes. These results suggest that storage granules in the rat parathyroid cells fuse with small vesicles containing cathepsins B and H, which may participate in regulating the intracellular PTH levels by degrading PTH in the granules.

Coexistence of renin and cathepsin B in secretory granules of granular duct cells in male mouse submandibular gland.

Cathepsin B, a representative lysosomal cysteine proteinase, has been demonstrated to coexist with renin in secretary granules of rat pituitary LH/FSH cells and renal juxtaglomerular cells. We investigated immunocytochemically the localization of cathepsins B, H, and L in the submandibular gland of male mice, in which active renin is also produced. By light microscopy, granular immunodeposits for cathepsin B were detected in epithelial cells of the gland, particularly in granular duct cells and interstitial cells. Immunoreactivity for cathepsins H and L was mainly found in interstitial cells, although that for cathepsin H was weakly seen in acinar cells. By electron microscopy, immunogold particles indicating cathepsin B intensely labeled small granules near the Golgi complex of granular duct cells and weakly labeled large secretory granules, whereas those showing renin labeled both granules. Double immunostaining co-localized immunogold particles showing renin and cathepsin B in small perinuclear granules near the Golgi complex. Some immunopositive granules seemed to be closely associated with the Golgi elements. These results indicate that the co-localization of renin and cathepsin B is also seen in secretory granules of granular duct cells in the mouse submandibular gland, as seen in rat juxtaglomerular and LH/FSH cells. This suggests that cathepsin B is one of the possible candidates for the renin-processing enzyme.

Immunocytochemical localization of cathepsin B in rat anterior pituitary endocrine cells, with special reference to its co-localization with renin and prorenin in gonadotrophs.

We examined by immunocytochemistry the localization of cathepsin B in endocrine cells of rat anterior pituitary lobe, using a monospecific antibody to cathepsin B. By light microscopy, granular immunodeposits for cathepsin B were detected in most endocrine cells of anterior pituitary lobe. Cells immunoreactive for luteinizing hormone (LH) were diffusely immunostained by anti-cathepsin B. By electron microscopy, immunogold particles for cathepsin B were localized in lysosomes of thyrotrophs, somatotrophs, and mammotrophs. In mammotrophs, immunogold particles for cathepsin B were also detected in crinophagic bodies. Double immunostaining co-localized immunogold particles for LH and cathepsin B in secretory granules of gonadotrophs. Immunocytochemistry was also applied to demonstrate localization of renin and prorenin in LH-producing gonadotrophs; immunogold particles for renin were co-localized with those for LH, cathepsin B, or prorenin in their secretory granules. Immunogold particles for prorenin were also co-localized with those for LH or cathepsin B in secretory granules, but prorenin-positive granules appeared less frequently than renin-positive granules. These results suggest that cathepsin B not only plays a role in the protein degradation in lysosomes of anterior pituitary endocrine cells but also participates in the activation of renin in gonadotrophs, as has been demonstrated in secretory granules of juxtaglomerular cells.

Immunocytochemical localization of prorenin, renin, and cathepsins B, H, and L in juxtaglomerular cells of rat kidney.

To examine the correlation of localization of prorenin, renin, and cathepsins B, H, and L, immunocytochemistry was applied to rat renal tissue, using a sequence-specific anti-body (anti-prorenin) that recognizes the COOH terminus of the rat renin prosegment. In serial semi-thin sections, immunodeposits for prorenin, renin, and cathepsins B, H, and L were localized in the same juxtaglomerular (JG) cells. Immunodeposits for renin were detected throughout the cytoplasm of the cells, whereas those for prorenin were detected in the perinuclear region. Immunoreactivity for cathepsin B was stronger than that for cathepsins H and L. By electron microscopy, prorenin was localized in small (immature) granules but not in large mature granules, whereas renin was localized mainly in mature granules. In serial thin sections, prorenin, renin, and cathepsin B were colocalized in the same immature granules containing heterogeneously dense material (intermediate granules). By double immunostaining, co-localization of renin with cathepsins B, H, or L was demonstrated in mature granules. The results suggest the possibility that processing of prorenin to renin occurs in immature granules of rat JG cells, and cathepsin B detected in JG cells may be a major candidate for the maturation of renin.

Lysosomal cysteine proteinases in rat epididymis.

To examine the precise localization of lysosomal cysteine proteinases, cathepsins B, H, and L in rat epididymal epithelial cells, immunohistochemistry and enzyme assay were applied to the epididymal tissue. Granular immunodeposits for cathepsins B and H were detected in epididymal epithelial cells, whereas faint or no immunoreactivity for cathepsin L was found. Moreover, immunoreactivity for cathepsin B appeared mainly in principal cells and was more intense in the head of the epididymis than in the tail, whereas that for cathepsin H appeared in both principal and clear cells and was more intense in the tail than the head. By enzyme assay, activities of cathepsins B and H showed a similar distribution to that of the immunoreactivity. The cathepsin L-specific activity was distributed evenly in each part of the epididymis and was also detected in epididymal fluids obtained from the body and tail parts. By immunoblotting, proforms of cathepsins B, H, and L were present in the seminal fluid. The results suggest that cathepsins B and H are involved in the intracellular degradation system of epididymal epithelial cells, and proforms of cathepsins B, H, and L may be secreted into the epididymal duct lumen.

Immunocytochemical localization of cathepsins B, H, L, and T4 in follicular cells of rat thyroid gland.

To localize cathepsins B, H, and L in follicular cells of rat thyroid gland, we applied immunocytochemistry to the thyroid tissue using their respective monospecific antibodies. On serial semi-thin sections, cathepsins B, H, and L were localized in granules of various sizes located throughout the cytoplasm, whereas T4 was detected in larger granules located in the apical and supranuclear regions. By electron microscopy, cathepsins B, H, and L were localized in large less-dense granules (so-called colloid droplets) and in dense bodies of various sizes, whereas T4 was localized more intensely in large less-dense granules than in smaller dense bodies. By double immunostaining using an immunogold method, cathepsins H and B or L were co-localized in the same cytoplasmic granules. Moreover, immunoblotting demonstrated that proteins similar to cathepsins B, H, and L in the liver are present in the thyroid gland. These results suggest that cathepsins B, H, and L participate not only in degradation of thyroglobulin but in maturation of thyroid hormones, although it remains unknown whether all of them participate in the maturation process.

Immunocytochemical localization of cathepsins B and H in corticotrophs and melanotrophs of rat pituitary gland.

We examined by immunocytochemistry the localization of cathepsins B and H in corticotrophs and melanotrophs in anterior and intermediate lobes of rat pituitary gland, using monospecific antibodies to cathepsins B and H. In serial semithin sections, immunodeposits for cathepsin H were detected throughout the cytoplasm of cells immunoreactive for ACTH and alpha-MSH in anterior and intermediate pituitary. Granular immunodeposits for cathepsin B were demonstrated in anterior and intermediate cells. Double immunostaining colocalized immunogold particles for cathepsin H and ACTH or alpha-MSH in secretory granules of corticotrophs or melanotrophs, whereas those for cathepsin B were detected only in their lysosomes. Enzyme assay demonstrated cathepsin B activity in both anterior and intermediate pituitary tissue, but did not detect cathepsin H activity in the intermediate pituitary. Western blotting, however, revealed the presence of cathepsin H and cystatin beta in intermediate pituitary. These results suggest that cathepsin B plays a role in protein degradation in lysosomes of corticotrophs and melanotrophs. Moreover, the presence of cathepsin H in secretory granules of the cells may indicate that the enzyme participates in the activation of secretory products.

Immunocytochemical localization of angiotensinogen and cathepsins B, H, and L in rat hepatocytes, with special reference to degradation of angiotensinogen in lysosomes after colchicine.

We examined the effects of bilateral nephrectomy and colchicine treatment on localization and content of angiotensinogen and cathepsins B, H, and L in rat liver using immunohistochemistry, radioimmunoassay, and enzyme assay. Angiotensinogen content increased in the liver of colchicine-treated rats, whereas a clear-cut increase was not detected in the liver of nephrectomized rats. This tendency was consistent with the immunocytochemical results; only perivenous hepatocytes in control and nephrectomized rats were diffusely immunostained by anti-angiotensinogen, whereas perivenous and periportal hepatocytes of colchicine-treated rats were strongly immunostained. Enzyme assay revealed no significant change in activities of cathepsins B, H, and L in liver extracts under these experimental conditions. Immunocytochemical localization of these cysteine proteinases in hepatocytes after colchicine treatment was more widespread in the cytoplasm than that in the control hepatocytes. By electron microscopy, angiotensinogen was localized in smaller vesicles and some larger vesicles (lysosomes) of hepatocytes after colchicine treatment. Double immunostaining demonstrated co-localization of cathepsins B, H, and L with angiotensinogen in lysosomes. These results suggest that cathepsins B, H, and L play a role in the degradation of excess angiotensinogen in hepatocytes of rats after colchicine treatment.

Cysteine proteinases in bronchoalveolar epithelial cells and lavage fluid of rat lung.

We examined the presence of cathepsins B, H, and L in bronchoalveolar epithelial cells, including alveolar macrophages, and in bronchoalveolar lavage fluid (BALF), using immunocytochemistry and immunoblotting. By light and electron microscopy, immunoreactivity for cathepsins B, H, and L was detected in lysosomes of ciliated and non-ciliated epithelial cells of bronchi and bronchioles, and in macrophages. Immunodeposits for cathepsin H only were demonstrated in lamellar bodies of Type II alveolar epithelial cells, suggesting the cosecretion of surfactants and cathepsin H from the cells into the alveolar space. By immunoblotting, cathepsins B and H were found to be present in BALF. To further investigate the origin of these enzymes in BALF, alveolar macrophages obtained from BALF were cultured for 6 hr in a serum-free medium. Immunoblotting revealed that protein bands corresponding to the pro-form and mature form of cathepsin B and the mature form of cathepsin H were present in the culture medium. From these results, the presence of cathepsins B and H in BALF can be explained by the fact that cathepsin B is secreted from alveolar macrophages and cathepsin H is secreted mainly with surfactants from Type II cells and also from macrophages.

Different distribution patterns of the two mannose 6-phosphate receptors in rat liver.

Two mannose 6-phosphate receptors, cation-dependent and -independent receptors (CDMPR and CIMPR), play an important role in the intracellular transport of lysosomal enzymes. To investigate functional differences between the two in vivo, their distribution was examined in the rat liver using immunohistochemical techniques. Positive signals corresponding to CIMPR were detected intensely in hepatocytes and weakly in sinusoidal Kupffer cells and interstitial cells in Glisson's capsule. In the liver acinus, hepatocytes in the perivenous region showed a more intense immunoreactivity than those in the periportal region. On the other hand, positive staining of CDMPR was detected at a high level in Kupffer cells, epithelial cells of interlobular bile ducts, and fibroblast-like cells, but the corresponding signal was rather weak in hepatocytes. In situ hybridization analysis also revealed a high level of expression of CIMPR mRNAs in hepatocytes and of CDMPR mRNA in Kupffer cells. By double immunostaining, OX6-positive antigen-presenting cells in Glisson's capsule were co-labeled with the CDMPR signal but were only faintly stained with anti-CIMPR. These different distribution patterns of the two MPRs suggest distinct functional properties of each receptor in liver tissue.

Variations in immunoreactivity for phenobarbital- and 3-methylcholanthrene-inducible cytochromes P-450, and NADPH-cytochrome P-450 reductase in rat liver over twenty-four hours.

To reveal distribution patterns of phenobarbital- and 3-methylcholanthrene-inducible cytochromes P-450 (PB and MC) and NADPH-cytochrome P-450 reductase (P-450red) within the liver acinus of untreated rats, and their variations over 24 h, hepatic samples were examined by immunohistochemistry and image-analyzer at evenly spaced six time points over 24 h. When examined in semi-thin sections obtained from Epon-embedded, freeze-dried, and paraformaldehyde vapor-phase fixed materials, the immunoreactivity for these enzymes showed different distribution patterns within the liver acinus. Immunodeposits for PB were predominantly distributed in perivenous hepatocytes, whereas those for MC and P-450red were slightly more intense in periportal hepatocytes at each time point. The immunoreactivity for PB and MC in both perivenous and periportal hepatocytes increased during the dark period, peaking early in the light period. These variations coincide well with our previous morphometric results (Uchiyama and Asari, 1984); the volume and surface densities of rough endoplasmic reticulum (rER) in hepatocytes increased during the dark period. On the other hand, weak fluctuation was demonstrated in the immunoreactivity for P-450red in hepatocytes of both zones. These results suggest that PB and MC are retained in rER rather than smooth endoplasmic reticulum (sER) of hepatocytes obtained from untreated rats. These enzymes in sER may be short in their half-life spans.

Bi-directional trafficking between the trans-Golgi network and the endosomal/lysosomal system.

Protein transport in the secretory and endocytic pathways of eukaryotic cells is mediated by vesicular transport intermediates. Their formation is a tightly controlled multistep process in which coat components are recruited onto specific membranes, and cargo, as well as targeting molecules, become segregated into nascent vesicles. At the trans-Golgi network, two transport systems deliver cargo molecules to the endosomal system. They can be distinguished with regard to coat components that select cargo molecules. AP-1 assembly proteins mediate transport of MPRs and furin, whereas AP-3 adaptors mediate transport of lysosomal membrane glycoproteins to the endosomal/lysosomal system. The molecular basis for protein-specific sorting lies within sorting signals that are present in the cytoplasmic tails of cargo proteins and allow specific interactions with individual coat components. In order to maintain cellular homeostasis, some proteins are retrieved from endosomal compartments and transported back to the trans-Golgi network. Distinct points for protein retrieval exist within the endosomal system, retrieval occurring from either early or late endosomes. Whereas significant progress has been made in recent years in identifying anterograde and retrograde transport pathways, the molecular mechanisms underlying protein sorting and retrieval are only poorly defined. Recently, however, novel vesicle coats (e.g. AP-4) and proteins that might be involved in sorting (e.g. PACS-1 and TIP47) have been described, and the interactions between assembly proteins and sorting signals are becoming increasingly well defined.

Variations in immunoreactivity of angiotensinogen and cathepsins B and H in rat hepatocytes over 24 hours.

To examine variations in immunoreactivity of angiotensinogen and cathepsins B and H in hepatocytes over 24 hr, rat liver was examined immunohistochemically. Immunoreactivity of angiotensinogen and cathepsins B and H in periportal and perivenous hepatocytes varied significantly over 24 hr, when analyzed by an image analyzer. In periportal and perivenous hepatocytes, immunoreactivity of angiotensinogen was highest at 0800 hr and lowest at 2000 hr or 0000 hr, whereas that of cathepsins B and H was maximal at 1600 hr and minimal at 0400 hr or 0800 hr. Proteolytic activities of cathepsins B and H in liver extracts varied in parallel to the variations in immunoreactivity of these enzymes. Localization of angiotensinogen in the liver acinus was inversely correlated to that of cathepsins B and H; angiotensinogen was predominantly localized in periportal hepatocytes, but cathepsins B and H were in perivenous hepatocytes at each time point examined. These results suggest that angiotensinogen in hepatocytes is actively synthesized and secreted early in the light period, whereas proteolytic activities in lysosomes of hepatocytes are augmented late in the light period.