Cathepsin D,but not cathepsin B,releases T cell stimulatory fragments from lysozyme that are functional in the context of multiple murine class II MHC molecules

In this study, the major endosomal/lysosomal proteases cathepsin D and cathepsin B were tested on their ability to release T cell stimulatory peptides from hen egg white lysozyme (HEL) in vitro. Whereas neither enzyme could cleave unreduced HEL under mild conditions, reduced HEL was readily cleaved by cathepsin D but not by cathepsin B. Instead, cathepsin B was found to be very active in the trimming of HEL peptides after their release by cathepsin D. Following high-performance liquid chromatography (HPLC) fractionation, cathepsin D-released HEL fragments were screened for recognition by HEL-specific T cells from three strains of mice, i.e. B10. A (H-2^a), C57BL/6 (H-2^b) and BALB/c (H-2^d). Peptides in a large number of different HPLC fractions triggered significant T cell responses in all three strains. Interestingly, the response profiles of T cells from the three different strains showed marked similarities. Also, several individual synthetic HEL sequences corresponding to selected cathepsin D-released fragments were recognized by murine T cells in the context of all three major histocompatibility complex (MHC) haplotypes tested. Our data suggest that cathepsin D rather than cathepsin B may play a central role in the initial release of HEL fragments during endosomal/lysosomal processing. The relatively long HEL fragments released by cathepsin D, containing about 20—30 amino acid residues, are significantly more promiscuous in murine class II MHC binding than the shorter synthetic HEL sequences previously employed by others for the delineation of HEL epitopes. Extensive documentation of HEL epitopes in previous investigations indicate that this promiscuity cannot be explained by simply assuming that longer peptides contain additional epitopes. Rather, an increased peptide length by itself appears to promote promiscuous MHC binding.     

Participation of autophagy in storage of lysosomes in neurons from mouse models of neuronal ceroid-lipofuscinoses (Batten disease)

In cathepsin D-deficient (CD-/-) and cathepsins B and L double-deficient (CB-/-CL-/-) mice, abnormal vacuolar structures accumulate in neurons of the brains. Many of these structures resemble autophagosomes in which part of the cytoplasm is retained but their precise nature and biogenesis remain unknown. We show here how autophagy contributes to the accumulation of these vacuolar structures in neurons deficient in cathepsin D or both cathepsins B and L by demonstrating an increased conversion of the molecular form of MAP1-LC3 for autophagosome formation from the cytosolic form (LC3-I) to the membrane-bound form (LC3-II). In both CD-/- and CB-/-CL-/- mouse brains, the membrane-bound LC3-II form predominated whereas MAP1-LC3 signals accumulated in granular structures located in neuronal perikarya and axons of these mutant brains and were localized to the membranes of autophagosomes, evidenced by immunofluorescence microscopy and freeze-fracture-replica immunoelectron microscopy. Moreover, as in CD-/- neurons, autofluorescence and subunit c of mitochondrial ATP synthase accumulated in CB-/-CL-/- neurons. This suggests that not only CD-/- but also CB-/-CL-/- mice could be useful animal models for neuronal ceroid-lipofuscinosis/Batten disease. These data strongly argue for a major involvement of autophagy in the pathogenesis of Batten disease/lysosomal storage disorders.     

Elevation of Cystatin C in Susceptible Neurons in Alzheimer’s Disease

A common polymorphism in the cystatin C gene is associated with increased risk of developing Alzheimer's disease (AD). To explore possible neuropathological consequences of this genetic association, we examined expression of cystatin C in brains from 22 AD and 11 control patients by immunohistochemistry. In the temporal cortex of all AD brains, there was strong cystatin C immunostaining of neurons and activated glia, whereas staining was absent or minimal in 7 of the 11 control brains. Neuronal staining of cystatin C in AD brains was primarily limited to pyramidal neurons in cortical layers III and V, which are the neurons most susceptible to cell death in AD. The increase in cystatin C staining in AD was independent of cystatin C genotype. Immunostaining of cystatin C within neurons showed a punctate distribution, which co-localized with the endosomal/lysosomal proteinase, cathepsin B. A primarily glial source for cystatin C was suggested by parallel studies using in situ hybridization of mouse brain. In human AD brain, there was little co-localization of cystatin C with parenchymal Abeta deposits, although a small fraction of cerebral blood vessels and neurofibrillary tangles were cystatin C-positive. The regional distribution of cystatin C neuronal immunostaining also duplicated the pattern of neuronal susceptibility in AD brains: the strongest staining was found in the entorhinal cortex, in the hippocampus, and in the temporal cortex; fewer pyramidal neurons were stained in frontal, parietal, and occipital lobes. These neuropathological observations reinforce the association between cystatin C and AD, and support a model of cystatin C involvement in the process of neuronal death in AD.     

Neuronal and microglial cathepsins in aging and age-related diseases

It has been long believed that cathepsins compensate for each other because of their overlapping substrate specificities. However, there is increasing evidence that disturbance of the normal balance of their enzymatic activities is the first insult in brain aging and age-related diseases. The imbalance of cathepsins may further cause age-related neuropathological changes such as accumulation of autophagic vacuoles and the formation of ceroid-lipofuscin leading to neuronal dysfunction and damage. Leakage of cathepsins due to the fragility of lysosomal membranes during aging also contributes to neurodegeneration. Furthermore, the deficiency of cathepsin D has been recently revealed to provoke a novel type of lysosomal storage disease associated with massive neurodegeneration. In these animals, microglia are activated to initiate inflammatory and cytotoxic responses by binding and phagocytosis of storage neurons. Activated microglia also release some members of cathepsins to induce neuronal death by degrading extracellular matrix proteins. Thus the microglial activation possibly through sensing neuronal storage may also be an important causative factor for neurodegeneration in lysosomal storage diseases and age-related diseases such as Alzheimer's disease. This review describes the pathological roles of neuronal and microglial cathepsins in brain aging and age-related diseases.     

Up-regulation of the lysosomal system in experimental models of neuronal injury: implications for Alzheimer's disease

Previous studies established that the populations of neurons that frequently degenerate in Alzheimer's disease exhibit robust up-regulation of the lysosomal system. In this study, we investigated alterations of the lysosomal system during different forms of experimental injury in rat hippocampal neurons in culture, utilizing a combination of immunocytochemical and biochemical methods. Using triple-label immnocytochemistry for activated caspase-3, fragmentation of DNA and the microtubule-associated protein-2, we characterized treatment paradigms as models of the apoptotic (staurosporine, camptothecin), the oncotic (high-dose menadione, glutamate), and the mixed apoptotic and oncotic (low-dose menadione) pathways of neuronal death. Slowly developing apoptotic or slowly developing mixed apoptotic and oncotic forms of neuronal injury were associated with substantial increases in the number and size of cathepsin D-positive vesicles (late endosomes and mature lysosomes) as determined by immunocytochemistry, and elevated levels of cathepsin D by western blotting. In agreement with our previous findings in Alzheimer's disease, where lysosomal system activation was not restricted to overtly degenerating neurons, up-regulation of this system was also detected quite early during the course of experimental neuronal injury, preceding the development of dystrophic neurites, nuclear segmentation or fragmentation of DNA. These findings implicate lysosomal system activation, both in Alzheimer's disease and in experimental models of neuronal injury, as an important event associated with early stages of neurodegeneration.     

Destructive proteolysis by cysteine proteases in antigen presentation of ovalbumin

Most native antigens require digestion by acidic proteases in order to be recognized in the context of major histocompatibility complex class II by T helper cells (Th). We have studied the roles of three different acidic proteases, cathepsin D, cathepsin B and cathepsin L, in the processing of ovalbumin (OVA) for presentation in the context of I-A^d. We report that digestion of OVA in vitro with the aspartyl protease cathepsin D generates the epitope OVA_322–336, which is recognized by I-A^d-restricted OVA-specific Th in the presence of paraformaldehyde-fixed antigen-presenting cells (APC). In contrast, digestion of OVA with the cysteine proteases cathepsin B and L not only failed to generate an epitope, but also destroyed OVA_322–336. In the presence of fixed APC expressing I-A^d, OVA_322–336 was protected from destructive proteolysis by cathepsin L. These results illustrate the dependence of epitope selection on the intracellular proteolytic environment in APC, and suggest that mechanisms must exist for protection of epitopes from destructive proteolysis in the processing compartments.     

Identification and characterization of an increased glycoprotein in aging: age-associated translocation of cathepsin D

We found that 14 N-glycosylated proteins were accumulated in the rat cerebral cortex cytosolic fraction in the aging process by a comparative study with two-dimensional gel electrophoresis and concanavalin A staining. All proteins had high mannose and/or hybrid-type N-glycans, as indicated by the fact that they were sensitive to endoglycosidase H digestion. Three of these cytosolic glycoproteins were identified as cathepsin D, a lysosomal protease, by tryptic digestion and nano liquid chromatography electrospray ionization quadrupole time of flight mass spectrometry. The increase of cytosolic cathepsin D during aging was not due to lysosomal membrane disruption, as shown by the fact that the activities of beta-hexosaminidase and beta-glucuronidase, other lysosomal enzymes, did not increase in the cytosolic fraction. Although the total amount of cathepsin D increased during aging, the amount of cathepsin D in the microsomal fraction did not change, indicating a selective increase of cytosolic cathepsin D. This phenomenon was also observed in the hippocampus, cerebellum, kidney, liver, and spleen. Based on these results, we propose that cytosolic cathepsin D is a new biomarker of aging.     

Aluminum-induced neurofibrillary degeneration affects a subset of neurons in rabbit cerebral cortex, basal forebrain and upper brainstem

Neurofibrillary tangles in Alzheimer's disease show a predilection for cortical pyramidal and subcortical projection neurons. The antigenic composition, neuronal specificity and distribution of aluminum-induced neurofibrillary degeneration were examined in regions of rabbit brain analogous to those that develop neurofibrillary tangles in Alzheimer's disease. Neurofibrillary degeneration was induced by intraventricular instillation of aluminum chloride. In aluminum-treated rabbits, intensely immunoreactive filamentous aggregates were seen in affected neuronal perikarya after staining with an antiphosphorylated neurofilament antibody (SMI 31), while in controls immunoreactivity was confined to axon-like elements. Monoclonal antibodies against Microtubule-associated protein 2 and tau, which stain human neurofibrillary tangles, did not stain aluminum-induced neurofibrillary degeneration. Pyramidal neurons exhibiting neurofibrillary degeneration formed a discrete linear pattern in layers III and V of cortex. Cortical somatostatin and nicotinamide adenine dinucleotide phosphate diaphorase-reactive neurons identified in double-stained sections were unaffected. Large perikarya in the vicinity of the globus pallidus, some of which contained acetylcholinesterase, were frequently SMI 31-immunoreactive. Among the cell groups affected in the upper brainstem were the nucleus raphe dorsalis and locus coeruleus. These findings show that aluminum-induced neurofibrillary degeneration differs antigenically from neurofibrillary tangles in Alzheimer's disease. Nevertheless, many neuronal subsets that are particularly susceptible to Alzheimer's disease, including cortical pyramidal neurons, basal forebrain cholinergic neurons and upper brainstem catecholaminergic neurons, are also affected by aluminum-induced neurofibrillary degeneration.     

A mutation in the cathepsin D gene (CTSD) in American Bulldogs with neuronal ceroid lipofuscinosis

We obtained DNA, brains, and eyes from American Bulldogs with neurodegeneration due to neuronal ceroid lipofuscinosis (NCL). The diagnosis of NCL was confirmed by detection of autofluorescent cytoplasmic inclusions within neurons throughout the brains, in retinal ganglion cells, and along outer limiting membranes of the retinas. Electron microscopy revealed that the inclusions had coarsely granular matrices surrounding well-delineated spherical structures and that the inclusions near the retinal outer limiting membranes were within photoreceptor cells, mostly cones. Affected American Bulldogs were homozygous for the A allele of a G to A transition in the cathepsin D gene (CTSD), which predicts the conversion of methionine-199 to an isoleucine. Only the G allele was detected in DNA samples from 131 randomly selected dogs representing 108 breeds other than American Bulldog; however, the A allele had a frequency of 0.28 among 123 genotyped American Bulldogs. Transmission analysis in a 99 dog pedigree of American Bulldogs indicated a probability of less than 10(-7) that alleles from any mutation unlinked to CTSD would be concordant with the pedigree and phenotypes of the dogs. Brain samples from affected dogs had 36% of the cathepsin D-specific enzymatic activity found in control dog brains; whereas, specific enzymatic activities of 15 other lysosomal enzymes were unchanged or increased. Compared to previously described NCLs in mice and sheep that completely lack cathepsin D activity, the clinical course of NCL in the American Bulldogs was less severe and more closely resembled that of many human NCLs.     

Clinicopathologic significance of cystatin C expression in gliomas

Cathepsin B, one of the lysosomal cysteine proteases, has been related to tumor invasiveness. Cystatin C is the strongest inhibitor of cathepsin B. Knowledge of its participation in the progression of gliomas is limited. We investigated the expression of cystatin C and its association with the clinicopathologic features of 57 gliomas. Cystatin C and cathepsin B expressions were evaluated by immunohistochemical methods and by semiquantitative real-time polymerase chain reaction analysis for the corresponding messenger RNA. Disease-free survival was analyzed by the Kaplan-Meier method. Tumors with low cystatin C protein expression and high cathepsin B protein expression were significantly more likely to be of high grade, and this pattern was significantly correlated with high Ki-67 LI and tumor recurrence. Depressed expression of cystatin C messenger RNA in glioblastomas compared with low-grade astrocytomas was demonstrated. Multivariate analysis demonstrated high tumor grade, high Ki-67 labeling index, high cathepsin B expression, and low cystatin C expression correlated significantly with shorter disease-free survival. These results suggest that gliomas in patients with an unfavorable clinical outcome are characterized by depressed expression of cystatin C. Evaluation of cystatin C expression in gliomas provides useful clinical information, especially as a prognostic indicator.     

The distribution of cathepsin B in human tissues

Cathepsin B is a lysosomal enzyme of importance in many physiological and pathological processes. Its distribution in human tissues was studied by an indirect immunoperoxidase method. Cathepsin B was demonstrated in macrophages, hepatocytes, renal tubules, gastrointestinal epithelium and fibroblasts, confirming previous studies. It was demonstrated for the first time by immunohistology in several other tissues, especially stratified squamous epithelium, transitional epithelium, salivary glands, pancreas, central and peripheral neuronal cell bodies, trophoblast and all endocrine organs. Widespread distribution of cathepsin B has been postulated several times but this is the fullest evidence that the enzyme indeed occurs in many organs. In pathology cathepsin B has so far been thought to be involved in demyelination, emphysema, rheumatoid arthritis and neoplastic infiltration.     

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.     

Accumulation of inactive cathepsin D in old rats

From the 2nd to the 29th month of life seven different rat organs (liver, heart, kidney, lung, spleen, skeletal muscle, intestinal mucosa) show a continuous increase in the concentration of protein reacting with rabbit antiserum against rat liver cathepsin D. However, the activity of aspartate proteinases, probably mainly cathepsin D, does not show the same increase. This result is interpreted as accumulation of enzymatically inactive but immunologically reactive cathepsin D. This phenomenon, which is well known for some cytosolic enzymes in senescent animals, is first reported to occur for a lysosomal proteinase as well.     

Gene deletion of cystatin C aggravates brain damage following focal ischemia but mitigates the neuronal injury after global ischemia in the mouse

Cystatin C is distributed in all human tissues and fluids with a particular abundance in the cerebrospinal fluid. Cystatin C is a strong endogenous inhibitor of lysosomal cysteine proteases, such as cathepsin B, L, H and S, that are involved in various biological processes such as degradation of cellular proteins and regulation of enzymes, as well as in pathological processes. Pharmacological inhibition of cathepsins has been shown to reduce neuronal damage after brain ischemia, suggesting that cystatin C is an endogenous neuroprotectant. Cystatin C has also amyloidogenic properties and is co-localized with beta-amyloid in degenerated neurons in Alzheimer's disease, suggesting a role in neuronal degeneration. To test the hypothesis that endogenous cystatin C is neuroprotective during brain ischemia, global and focal brain ischemia was induced in mice with the cystatin C gene knocked out. Following focal ischemia, larger brain infarcts were found in cystatin C knockout mice, probably due to a reduced inhibition of the cathepsins during ischemia. In contrast, brain damage after global ischemia was diminished in cystatin C knockout mice, suggesting that cystatin C has an aggravating effect on selective neuronal damage after global ischemia.     

New cathepsin d inhibitors with hydroxyethylamine isosteres: preparation and characterization

The lysosomal aspartyl protease, cathepsin D, has been suggested to play a role in the metastatic potential of several types of cancer. Cathepsin D is secreted by malignant cells, and is believed to be involved in the breakdown of the extracellular matrix. High levels of active cathepsin D have been found in colon cancer, prostate cancer, uterine cancer and ovarian cancer. Also cathepsin D has recently been associated with the development of Alzheimer's disease. Hydroxyethyl isosteres with cyclic tertiary amine have proven to be clinically useful as inhibitors of aspartyl proteases similar to cathepsin D in activity, such as the HIV-1 aspartyl protease. In the present study twenty-eight compounds containing (hydroxyethyl)amine isosteres with cyclic tertiary amines have been synthesized. These compounds show significant activity as cathepsin D inhibitors, many with IC(50) values in the nanomolar range. For example, the compounds that contain hydroxyethylamines where the amine is formed from N-piperazine-2-carboxylic acid methyl ester, 4y-bb, show IC(50) values ranging from 2.5 to 15 nM.     

Immunohistochemical identification of thrombospondin in normal human brain and in Alzheimer''s disease.

Thrombospondin is part of a family of adhesive glycoproteins and is involved in a number of physiologic processes such as angiogenesis and neurite outgrowth. Immunohistochemical localization of thrombospondin in normal human brains was investigated in the hippocampus and inferior temporal cortex. Two antibodies (one polyclonal and one monoclonal) against thrombospondin-labeled microvessels, glial cells, and a subpopulation of pyramidal neurons. The distribution of thrombospondin staining in patients with Alzheimer's disease was found to be comparable to control subjects. However, in patients with Alzheimer's disease a subset of pyramidal neurons that may be vulnerable in Alzheimer's disease exhibited decreased staining. This decrease in the intensity of labeling might constitute a marker for a neuronal population prone to early degeneration. In addition, thrombospondin staining was demonstrated in senile plaques in Alzheimer's disease. These results suggest that thrombospondin may be involved in the process of neuronal degeneration and senile plaque formation.     

Streptozotocin-induced diabetes mellitus affects lysosomal enzymes in rat liver

It has been previously shown that dextran sulfate administered to diabetic rats accumulates in the liver and kidney, and this could be due to a malfunction of the lysosomal digestive pathway. The aim of the present study was to evaluate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in the livers of diabetic rats. Diabetes mellitus was induced by streptozotocin in 26 male Wistar rats (12 weeks old), while 26 age-matched controls received only vehicle. The livers were removed on either the 10^th or the 30^th day of the disease, weighed, and used to evaluate the activity, expression, and localization of lysosomal enzymes. A 50-60% decrease in the specific activities of cysteine proteases, especially cathepsin B, was observed in streptozotocin-induced diabetes mellitus. Expression (mRNA) of cathepsins B and L was also decreased on the 10^th, but not on the 30^th day. Sulfatase decreased 30% on the 30^th day, while glycosidases did not vary (or presented a transitory and slight decrease). There were no apparent changes in liver morphology, and immunohistochemistry revealed the presence of cathepsin B in hepatocyte granules. The decrease in sulfatase could be responsible for the dextran sulfate build-up in the diabetic liver, since the action of sulfatase precedes glycosidases in the digestive pathway of sulfated polysaccharides. Our findings suggest that the decreased activities of cathepsins resulted from decreased expression of their genes, and not from general lysosomal failure, because the levels of glycosidases were normal in the diabetic liver.     

Massive accumulation of modified tau and severe depletion of normal tau characterize the cerebral cortex and white matter of Alzheimer''s disease. Demonstration using the hydrated autoclaving method.

Using the hydrated autoclaving method, a new immunohistochemical procedure to enhance tau immunoreactivity in formalin-fixed brain tissue, the authors recently reported that tau protein is detected in neuronal cell bodies and proximal dendrites, gray matter neuropil, axons, and glial cells in normal human hippocampus and neocortex. In the this study, the authors performed a comparative study of the distribution of normal and modified forms of tau in Alzheimer's disease (AD) and control brains. In the cerebral cortex and white matter of AD brains, a massive accumulation of modified tau and/or severe depletion of normal tau were documented in all the tau compartments. In mild AD cases, gray matter neuropil, axons, and glial cells were less severely involved than neuronal perikarya. In the controls, neuronal perikarya were often involved by modified tau accumulation, but the other compartments showed normal distribution. These observations suggest that modifications of tau which lead to neurofibrillary lesions in AD may begin in neuronal perikarya and extend to the other tau compartments in advanced stages of the disease.     

Cell type-specific distribution of cathepsin B and D immunoreactivity within the rabbit retina

The cellular localization of cathepsin B and D immunoreactivity was demonstrated at the light microscopic level in the retina of adult rabbits by use of the peroxidase-antiperoxidase technique. Antisera were raised against rat liver enzymes. Whereas cathepsin D immunoreactivity was confined to Müller (glial) cells, cathepsin B was demonstrated in some, but not all, neuronal cell types. It is proposed that the two enzymes might carry different functions within the neuronal versus glial compartment.     

Cholesterol accumulation is associated with lysosomal dysfunction and autophagic stress in Npc1 -/- mouse brain

Niemann-Pick type C (NPC) disease is an autosomal recessive disorder caused by mutations of NPC1 and NPC2 genes. Progressive neurodegeneration that accompanies NPC is fatal, but the underlying mechanisms are still poorly understood. In the present study, we characterized the association of autophagic-lysosomal dysfunction with cholesterol accumulation in Npc1(-/-) mice during postnatal development. Brain levels of lysosomal cathepsin D were significantly higher in mutant than in wild-type mice. Increases in cathepsin D occurred first in neurons and later in astrocytes and microglia and were both spatially and temporally associated with intracellular cholesterol accumulation and neurodegeneration. Furthermore, levels of ubiquitinated proteins were higher in endosomal/lysosomal fractions of brains from Npc1(-/-) mice than from wild-type mice. Immunoblotting results showed that levels of LC3-II were significantly higher in brains of mutant than wild-type mice. Combined LC3 immunofluorescence and filipin staining showed that LC3 accumulated within filipin-labeled cholesterol clusters inside Purkinje cells. Electron microscopic examination revealed the existence of autophagic vacuole-like structures and multivesicles in brains from Npc1(-/-) mice. These results provide strong evidence that cholesterol accumulation-induced changes in autophagy-lysosome function are closely associated with neurodegeneration in NPC.