Abnormal distribution of cathepsins in the brain of patients with Alzheimer's disease

Formalin-fixed paraffin-embedded hippocampal sections of brains with early-onset and late-onset Alzheimer's disease were studied immunohistochemically with antisera against cathepsin D and cathepsin B. In addition to the staining of neuronal perikarya, some of the senile plaques visualized by Bielshowsky silver staining and some of reactive astrocytes were positively stained with the antisera against cathepsin D and cathepsin B in brains with Alzheimer's disease. Abnormal localization of cathepsin D and cathepsin B immunoreactivity in neuronal perikarya was observed in brains with early-onset Alzheimer's disease. These findings demonstrate that the distribution of lysosomal proteases was altered in brains with Alzheimer's disease, suggesting the primary and/or secondary involvement of the lysosomal proteases in the pathological process of Alzheimer's disease.     

Autosomal dominant adult neuronal ceroid lipofuscinosis: a novel form of NCL with granular osmiophilic deposits without palmitoyl protein thioesterase 1 deficiency

We describe the neuropathological and biochemical autopsy findings in 3 patients with autosomal dominant adult neuronal ceroid lipofuscinosis (ANCL, Parry type; MIM 162350), from a family with 6 affected individuals in 3 generations. Throughout the brain of these patients, there was abundant intraneuronal lysosomal storage of autofluorescent lipopigment granules. Striking loss of neurons in the substantia nigra was found. In contrast, little neuronal cell loss occurred in other cerebral areas, despite massive neuronal inclusions. Visceral storage was present in gut, liver, cardiomyocytes, skeletal muscle, and in the skin eccrine glands. The storage material showed highly variable immunoreactivity with antiserum against subunit c of mitochondrial ATP synthase, but uniform strong immunoreactivity for saposin D (sphingolipid activating protein D). Protein electrophoresis of isolated storage material revealed a major protein band of about 14 kDa, recognized in Western blotting by saposin D antiserum (but not subunit c of mitochondrial ATPase (SCMAS) antiserum). Electron microscopy showed ample intraneuronal granular osmiophilic deposits (GRODs), as occurs in CLN1 and congenital ovine NCL. These forms of NCL are caused by the deficiencies of palmitoyl protein thioesterase 1 and cathepsin D, respectively. However, activities of these enzymes were within normal range in our patients. Thus we propose that a gene distinct from the cathepsin D and CLN1-CLN8 genes is responsible for this autosomal dominant form of ANCL.     

Regionally selective changes in brain lysosomes occur in the transition from young adulthood to middle age in rats

The possibility that brain aging in rats exhibits regional variations of the type found in humans was studied using lysosomal chemistry as a marker. Age-related (two vs 12months; male Sprague-Dawley) differences in cathepsin D immunostaining were pronounced in the superficial layers of entorhinal cortex and in hippocampal field CA1, but not in neocortex and field CA3. Three changes were recorded: an increase in the intraneuronal area occupied by labeled lysosomes; clumping of immunopositive material within neurons; more intense cytoplasmic staining. Western blot analyses indicated that the increases involved the active forms of cathepsin D rather than their proenzyme. Shrinkage of cathepsin-D-positive neuronal cell bodies was observed in entorhinal cortex but not in neocortical sampling zones. Age-related lysosomal changes as seen with cathepsin B immunocytochemistry were considerably more subtle than those obtained with cathepsin D antibodies. In contrast, a set of glial and/or vascular elements located in a distal dendritic field of the middle-aged hippocampus was much more immunoreactive for cathepsin B than cathepsin D. The areas exhibiting sizeable changes in the present study are reported to be particularly vulnerable to aging in humans.The results thus suggest that aspects of brain aging common to mammals help shape neurosenescence patterns in humans.     

Distribution of cathepsin D immunoreactivity in the central nervous system of rat and selected brain regions of man

The regional distribution and cellular localization of cathepsin D immunoreactivity was demonstrated at the light microscopic level in the CNS of rat and man by use of unlabelled immunoenzyme technique. A wide but uneven distribution was substantiated for the rat brain. Furthermore, we present evidence that antiserum produced against rat liver enzyme is capable of recognizing cathepsin D in human brain.     

Immunohistochemical localization of glial cell line-derived neurotrophic factor in the human central nervous system

Glial cell line-derived neurotrophic factor, initially purified from the rat glial cell line B49, has the ability to promote the survival and differentiation of various types of neurons in the central and peripheral nervous systems. In the present study, to evaluate the physiological role of glial cell line-derived neurotrophic factor in the central nervous system, we investigated the cellular and regional distribution of glial cell line-derived neurotrophic factor immunoreactivity in autopsied control human brains and spinal cords using a polyclonal glial cell line-derived neurotrophic factor-specific antibody. On western blot analysis, the antibody reacted with recombinant human glial cell line-derived neurotrophic factor, and recognized a single band at a molecular weight of approximately 34,000 in human brain homogenates. Glial cell line-derived neurotrophic factor immunoreactivity was observed mainly in the neuronal somata, dendrites and axons. In the telencephalon, diencephalon and brainstem, the cell bodies and proximal processes of several neuronal subtypes were immunostained with punctate dots. Furthermore, immunopositive nerve fibers were also observed, and numerous axons were intensely immunolabeled in the internal segment of the globus pallidus and the pars reticulata of the substantia nigra. In the cerebellum, the most conspicuous immunostaining was found in the Purkinje cells, in which the somata and dendrites were strongly immunolabeled. Intense immunoreactivity was also detected in the posterior horn of the spinal cord. In addition to the neuronal elements, immunopositive glial cell bodies and processes were observed in various regions.

Our results suggest that glial cell line-derived neurotrophic factor is widely localized, but can be found selectively in certain neuronal subpopulations of the human central nervous system. Glial cell line-derived neurotrophic factor may regulate the maintenance of neuronal functions under normal circumstances.     

Endosomal sorting related protein CHMP2B is localized in Lewy bodies and glial cytoplasmic inclusions in α-synucleinopathy

Charged multivesicular body protein 2B (CHMP2B) is a component of the endosomal sorting complex required for transport-III, which is involved in the degradation of proteins in the endocytic and autophagic pathways. Mutations in the CHMP2B gene cause frontotemporal dementia and amyotrophic lateral sclerosis characterized by accumulation of ubiquitinated protein aggregates. Recent studies have shown that autophagosomal proteins are present in α-synuclein aggregates in neurons and glial cells in Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). We therefore immunohistochemically examined the brains of various neurodegenerative diseases using CHMP2B-specific antibody. CHMP2B immunoreactivity was present in intracytoplasmic and axonal Lewy bodies in PD and DLB as well as in neuronal and glial cytoplasmic inclusions in MSA. No CHMP2B immunoreactivity was found in a variety of other neuronal and glial inclusions in TDP-43 proteinopathy and tauopathy. These findings suggest that endosomal and autophagic pathway is associated with degradation or formation of α-synuclein aggregates in α-synucleinopathy.     

Expression of cathepsins B, H, K, L, and S during human fetal lung development.

Cathepsins are involved in lysosomal protein degradation, proenzyme activation, antigen processing, and hormone maturation. They are secreted by tumor cells and macrophages and catalyze the remodeling of extracellular matrix proteins. To gain insight into the expression pattern of cathepsins during fetal lung development, the expression of cathepsins B, H, K, L, and S at protein and mRNA levels were evaluated by using immunohistochemistry and in situ hybridization. Early expression of cathepsins B, H, and K was found in epithelial cells of the branching presumptive bronchi (<12th week of gestation). The most intense cathepsin K-specific immunoreactivity was found in developing airways with a lumen. Cathepsin K was found in epithelial cells only, whereas in contrast, cathepsins B and H were detected both in epithelial and interstitial cells. During fetal maturation, interstitial cells displayed cathepsin L immunoreactivity and, in the saccular phase (>26th week of gestation), both cathepsin L and S immunoreactivities. A continuous decline in the proportion of cathepsin H-positive interstitial CD68-positive cells was observed. These discrete temporal and spatial variations in cathepsin expression during organogenesis of the human lung indicate different physiological roles for the individual enzymes in different cell types and developmental stages.     

Localization of prosaposin in rat cochlea

Prosaposin, the precursor of the sphingolipid hydrolase activator proteins called saposins A, B, C, and D, is abundant in the nervous system and muscles. Besides its role as the precursor of saposins, prosaposin is reported to function as a neurotrophic factor, initiating neural differentiation and preventing neuronal cell death in vivo and in vitro. In this study, we examined the localization and synthesis of prosaposin in the rat cochlea. Intense prosaposin immunoreactivity was observed in the organ of Corti, stria vascularis, and spiral ganglion. In an immuno-electron microscopic study, prosaposin immunoreactivity was found mainly in lysosomal granules of the cells in these regions. In the lysosome, prosaposin does not always colocalize with cathepsin D, but was localized mainly in the dark area of the lysosome. Prosaposin mRNA was observed in these same regions. Our results suggest that prosaposin plays a role in homeostasis in the peripheral auditory system.     

Effect of colchicine on the activity of cathepsin B and D in human liver cirrhosis

The activity of cathepsin B and D in human liver biopsy specimens from cirrhotic patients before and after one year of colchicine treatment was studied. The hydroxyproline content as a marker of the amount of collagen in tissue specimens was also determined. The hydroxyproline content in the liver samples was two or threefold that of the control group. After colchicine it remained unchanged or in some patients its values were decreased. Cathepsin B activity was higher in cirrhotic liver samples as compared with the controls, whereas the increased activity of cathepsin D was not significant. The ratio of cathepsin B and D activity to hepatic hydroxyproline content was significantly reduced in cirrhotic livers. Colchicine treatment was followed by an increase in the levels of the enzymes investigated as well as by a significant rise in the ratio of cathepsin B and D activity to hepatic hydroxyproline content.     

Microinjection of cathepsin d induces caspase-dependent apoptosis in fibroblasts

Recent reports have indicated that enzymes such as cathepsins D and B are translocated from lysosomal compartments to the cytosol early during apoptosis. We have previously noted that a translocation of cathepsins D and B occur before cytochrome c release and caspase activation in cardiomyocytes and human fibroblasts during oxidative stress-induced apoptosis. In the present report, we use a microinjection technique to investigate if cytosolic location of the cathepsins D and B are important for induction of apoptosis. We found that microinjection of cathepsin D into the cytosol of human fibroblasts caused apoptosis, which was detected as changes in distribution of cytochrome c, cell shrinkage, activation of caspases, chromatin condensation, and formation of pycnotic nuclei. No apoptosis was, however, induced by microinjection of cathepsin B. Moreover, apoptosis was prevented in fibroblasts pretreated with a caspase-3-like inhibitor, and also when microinjected with cathepsin D mixed with the cathepsin D inhibitor, pepstatin A. These results show that cytosolic cathepsin D can act as a proapoptotic mediator upstream of cytochrome c release and caspase activation in human fibroblasts.     

A comparative immunohistochemical study of calcineurin and S-100 protein in mammalian and avian brains

Summary The cellular and topographic localization of calcineurin and S-100 protein was examined immunohistochemically in the mammalian and avian brain. Calcineurin immunoreactivity in both the avian and mammalian brain was located only in neuronal cells. S-100 protein was localized mainly in the glial and Schwann cells within the mammalian brain. However, in the avian brain, neuronal cells in certain regions such as the paleostriatum primitivum and the cerebellum, as well as other non-neuronal cells, exhibited S-100 protein immunoreactivity. A distinct difference was demonstrated in the macroscopic topographic distribution patterns of S-100 protein immunoreactivity between the mammalian and avian brains, while the patterns of calcineurin distribution were essentially identical. In addition, we provided calcineurin- and S-100 protein-immunocytochemical results for the turtle, frog and fish brain.     

Characterization of liver lysosomal enzyme activity in hepatocytes, Kupffer and endothelial cells during aging: effect of dietary restriction

The specific activity of 4 lysosomal enzymes was studied in homogenate, hepatocytes, Kupffer and endothelial cells isolated from the livers of female Sprague-Dawley rats aged 3.5, 12 and 24 months. Cells were obtained by enzymatic digestion and centrifugal elutriation. Cell viability was not affected by age or diet. In hepatocytes, the activities of all enzymes (acid phosphatase, beta-galactosidase, arylsulfatase B and cathepsin D) increased with age in rats fed ad libitum (A) but were not altered significantly by dietary restriction. The activities of all enzymes except acid phosphatase were systematically higher at 3.5 months of age in Kupffer and endothelial cells than in hepatocytes. Acid phosphatase, arylsulfatase B and cathepsin D activities increased with age in both Kupffer and endothelial cells. Beta galactosidase was decreased significantly with age in Kupffer cells but was elevated in endothelial cells. Rats exposed to dietary restriction (R) showed higher activities of beta-galactosidase, arylsulfatase B and cathepsin D when compared to corresponding A animals with the exception of the younger age group. No clear cut pattern was observed in acid phosphatase activity. Thus, the activities of liver lysosomal enzymes increase with age but the pattern of change differs with respect to enzyme and cell populations. The heightened enzyme activity in Kupffer and endothelial cells from R rats may reflect a more efficient phagocytic capacity in these animals.     

Lysis and killing of bacteria by lysosomal proteinases.

The bacteriolytic and bactericidal effects of the human proteinases cathepsin B, cathepsin D, cathepsin G, and elastase were investigated. Cathepsin G and elastase were 5 to 10% as active as egg white lysozyme in the lysis of Micrococcus lysodeikticus. All four enzymes slowly lysed the lysozyme-resistant Staphylococcus aureus. The gram-negative Acinetobacter 199A was rendered sensitive to lysozyme by all of the proteinases. Only elastase caused marked proteolysis of the outer membrane, which would permit access by lysozyme to the underlying peptidoglycan. When the surface layer of regularly arranged a protein was removed, however, the outer membrane proteins became susceptible to the other proteinases. Cathepsin G, elastase, and cathepsin D were bactericidal to Acinetobacter 199A. The bactericidal activity of cathepsin D was shown to be dependent on enzymatic activity, unlike that of cathepsin G, which was related to its cationic nature.     

Direct comparison of the sensitivity of enzyme histochemical and immunohistochemical methods: cathepsin B expression in human colorectal mucosa

Immunohistochemical localization of the proteinase cathepsin B has been compared directly with localization of cathepsin B activity with a catalytic (enzyme) histochemical method. The 2 approaches demonstrate principally different aspects of an enzyme. The immunohistochemical method localizes the enzyme protein whether it is active or not whereas the catalytic method visualizes the functionally active enzyme only. Sensitivity of both approaches to localize low amounts of enzyme protein or activity has never been compared. In the present study, we show that cathepsin B protein has a wider distribution pattern than cathepsin B activity in human colorectal mucosa, which means that inactive cathepsin B protein is present. With respect to sensitivity of the methods, it is shown that cathepsin B protein could only be demonstrated properly when strong signal amplification was applied by using Nanogold with silver enhancement, whereas activity could be demonstrated with a simple and direct fluorogenic histochemical assay. It is concluded that catalytic histochemical methods are relatively simple methods for the localization of activity of enzymes in tissues and cells and that their sensitivity is high in comparison with immunohistochemical methods.     

Tissue Levels of Stefin A and Stefin B in Hepatocellular Carcinoma

Stefins have been reported to be associated with the progression and metastasis of various malignant tumors. However, the expressions of stefins in hepatocellular carcinoma (HCC) have not been well‐defined. In this study, the protein levels of stefin A and stefin B were assessed by immunohistochemical staining, and the mRNA levels were quantified by real‐time polymerase chain reaction in 85 primary HCC tissues, 85 surrounding non‐cancerous tissues, and 9 normal hepatic tissues. The immunohistochemical staining of cathepsin B and cathepsin D, and the ratio of cathepsins to stefins were assessed. The mRNA expressions of stefin A and stefin B in HCC tissues were significantly higher than surrounding noncancerous tissues and normal hepatic tissues, respectively. A significant positive relationship of stefin A and stefin B was found with node metastasis, tumor size, and Edmondson grade for HCC. Univariate and multivariate analyses revealed that Edmondson grade and stefin B expression were independent factors associated with the risk of lymph node metastasis in HCC. The ratios of cathepsin B to stefin A, cathepsin D to stefin A, cathepsin B to stefin B and cathepsin D to stefin B of the HCC group were significantly higher than that of the surrounding noncancerous group. A significant positive correlation between the ratio of cathepsins to stefins (cathepsin B/stefin A, cathepsin B/stefin B and cathepsin D/stefin B) and node metastasis was demonstrated. We concluded that high expressions of stefin A and stefin B may be an important factor contributing to the development and metastasis of HCC. Anat Rec, 299:428–438, 2016. © 2016 Wiley Periodicals, Inc.     

Effect of adjuvant arthritis on collagenase and certain lysosomal enzymes in relation to the catabolism of collagen

The activity of collagenase, cathepsin B1, cathepsin D and Hyaluronidase was determined in skin, bone, liver, kidney, spleen and serum of adjuvant induced arthritic rats during the acute and chronic phase of the disease. Collagenase was assayed directly in tissue extract by a solution method using radioactive labelled substrate. The activity of collagenase, cathepsin B1 and D was found to increase significantly at both phases of the disease. The activity of hyaluronidase decreased significantly in liver, kidney and spleen of arthritic rats, while in skin, bone and serum no significant change was observed. The results are discussed with respect to catabolism of collagen in adjuvant induced arthritis.Prednisolone andl-thyroxine were administered to arthritic rats and the activity of collagenase, cathepsin B1, cathepsin D and hyaluronidase was determined in the treated groups during the acute and chronic phase of the disease. Prednisolone was found to suppress the development of arthritis which, in turn, decreased the increased activity of collagenase and lysosomal enzymes cathepsin B1 and D in tissues and serum of arthritic rats.l-Thyroxine was found to slowly diminish the development of inflammation and its beneficial action was found in mesenchymal tissues and skin of arthritic rats but not in bone.Presented at the Symposium of the Society of Biological Chemists, India, held at New Dehli in October, 1978.     

Heterogeneous localizations of Trk B among individual periodontal Ruffini endings in the rat incisor

The present immunocytochemical study examined the localization of Trk B, a high affinity neurotrophin receptor, in the neural elements of the periodontal ligament of the rat incisor. In light microscopy, the immunoreactivity was demonstrated in dendritic profiles in the alveolar half of the periodontal ligament. Their location and morphological features indicated that they were periodontal Ruffini endings. Occasional rounded cells associated with periodontal Ruffini endings, which had immunonegative kidney-shaped nuclei, were immunoreactive; these were judged to be terminal Schwann cells. Immunoelectron microscopy revealed the heterogeneous localization of Trk B among individual Ruffini endings. Some terminal Schwann cells contained immunoreactive products for Trk B in the cytoplasm, while others did not. Similarly, a part of the Schwann sheaths covering the axon terminals showed Trk B immunoreactivity. Most axon terminals associated with periodontal Ruffini endings were immunopositive for Trk B, though a few of them were immunonegative. The ordinary Schwann cells did not contain Trk B immunoreactive products. These findings imply that Trk B is required for the maintenance of periodontal Ruffini endings. The different expression pattern of Trk B suggests that neuronal and glial elements comprising individual periodontal Ruffini endings are subject to heterogeneous conditions with regard to the requirement of Trk B.     

Immunohistochemical evidence of oxidative [corrected] stress in Alzheimer's disease.

Membrane and cytoskeletal structures are known targets of oxidative injury. Brains from patients with Alzheimer's disease have cytoskeletal abnormalities and platelet and possible neuronal membrane lesions. The authors have recently demonstrated that superoxide anion is a powerful inducer of heat-shock protein synthesis, and have also shown that in response to oxidative stress or hyperthermia, intracellular levels of antioxidant enzymes increase to several folds. Whether the aforementioned mechanisms play a role in Alzheimer's disease has been suggested but is not totally established. While exploring this possibility, tissue sections from five brains with Alzheimer's disease and five neuropathologically normal age-matched controls were immunostained with polyclonal antibodies against superoxide dismutase (CuZn- and Mn- forms) and catalase. A standard avidin-biotin-peroxidase method was used for antigen detection. A subgroup of neurofibrillary tangles (15-25%) and senile plaques (50%) showed immunoreactivity for both enzymes with a staining pattern similar (but not identical) to that usually observed with antibodies against ubiquitin. Senile plaques displayed a granular pattern of immunostaining. Amyloid cores in mature classical plaques remained unstained. In addition, occasional elements with features consistent with reactive glial cells were strongly immunostained. Tangle-free neurons in both diseased and control brains showed weak to absent intracytoplasmic immunoreactivity. The immunoreactivity was totally abolished by preincubation of the primary antibodies with the corresponding purified antigens. These findings support the hypothesis that oxidative stress may be involved in the pathogenesis of Alzheimer's disease.     

Autophagic cell death and its execution by lysosomal cathepsins

In the last decade, the molecular mechanisms of apoptosis, a major type of active cell death (type I cell death) have largely been clarified in mammalian cells. Particularly, the caspase family of proteinases has been shown to play crucial roles in the execution of apoptosis. Differing from apoptosis, type II cell death is known to be associated with autophagosomes/autolysosomes and appear in the developing nervous system (CLARKE, 1990). We have previously shown that delayed neuronal death occurring in the CA1 pyramidal layer of the gerbil hippocampus after brief forebrain ischemia is apoptotic in nature and autophagosomes/autolysosomes abundantly appear in the neurons before DNA fragmentation. To further understand the roles of autophagosomes/autolysosomes in active cell death, we examined the apoptosis of PC12 cells using morphological and biochemical techniques. PC12 cells are known to undergo apoptosis when cultured in the absence of serum. In such an environment, the mitochondrial pathway of apoptosis is activated; cytochrome c is released from mitochondria, and caspase-9/caspase-3 are activated. We have first examined morphological features of PC12 cells during the apoptotic process following serum deprivation, and found that autophagy is induced from the early stage of the process in the cells before typical nuclear changes. When autophagy is inhibited in the cells by 3-methyladenine, an autophagy inhibitor, they are largely protected from apoptosis. In relation to the induction of autophagy in PC12 cells following serum deprivation, immunoreactivity, protein amounts, and the proteolytic activity of lysosomal proteinases, particularly cathepsins B and D, are all greatly altered; those of cathepsin B drastically decrease in the cells from the early stage of serum-deprived cultures, whereas those of cathepsin D increase. Moreover, PC12 cells overexpressing cathepsin D undergo apoptosis more rapidly in serum-deprived cultures than wild-type cells, whereas those overexpressing cathepsin B increase the viability. These lines of evidence suggest that autophagy is involved in PC12 cell death following serum deprivation, this type of cell death being regulated by lysosomal proteinases, cathepsins B and D, downstream autophagy.