Microglial secreted cathepsin B induces neuronal apoptosis

Activated microglia release a number of substances that can influence neuronal signalling and survival. Here we report that microglia stimulated with the peptide chromogranin A (CGA), secreted the cysteine protease, cathepsin B. Conditioned medium from CGA exposed microglia was neurotoxic to the HT22 hippocampal cell line and to primary cultures of cerebellar granule neurones. In both neuronal cell types, the neurotoxicity could be significantly attenuated with z-FA-fmk or by depletion of microglial conditioned medium with cathepsin B antibody. Conditioned medium from activated microglia or cathepsin B alone induced neuronal apoptosis and caspase 3 activation. Our data indicate that CGA-activated microglia can trigger neuronal apoptosis and that this may be mediated through the secretion of cathepsin B. Since cathepsins may also play a role in the amyloidogenic processing of amyloid precursor protein, these results may have significance for tissue damage and neuronal loss in the neuropathology of Alzheimer's disease.     

Intra- versus extracellular effects of microglia-derived cysteine proteases in a conditioned medium transfer model

Activated microglia release inflammatory mediators that display either beneficial or harmful effects on neuronal survival and signaling. In the present study we demonstrate that exposure to lipopolysaccharide leads to an increase in the lysosomal cysteine proteases, cathepsin B, K, S, and X, in culture supernatants of the microglia cell line BV-2. In addition, we observed an up-regulation of cathepsins in the cytoplasmic fraction in response to stimulation with lipopolysaccharide. Conditioned medium from these cells was toxic to the neuroblastoma cell line Neuro2a. Experiments with membrane-permeable and membrane-impermeable cysteine protease inhibitors suggested that blocking extracellular cathepsins had no effect on microglia-mediated neuron death in this medium transfer model. However, intracellular cathepsins seem to trigger the release of neurotoxic factors. In lipopolysaccharide-stimulated BV-2 cells, inhibition of intracellular cathepsins significantly diminished microglial activation characterized by reduced expression of different proinflammatory cytokines, thereby reducing the neurotoxic effects of the medium. This hitherto unknown intracellular effect of cysteine proteases in activated microglia might connect chronic neuroinflammation with neurodegeneration.     

Neurotoxicity of microglial cathepsin D revealed by secretome analysis

Microglia-driven inflammatory responses have both neuroprotective and neurotoxic effects in the CNS. The excessive and chronic activation of microglia, however, may shift the balance towards neurotoxic effects. In this regard, proteins secreted from activated microglia likely play a key role in the neurotoxic effects. To characterize secreted proteins of activated microglia, conditioned media obtained from BV-2 mouse microglia cells were analyzed by two-dimensional gel electrophoresis or liquid chromatography coupled with tandem mass spectrometry. Among many proteins identified in the secretome of activated microglia, an aspartic endoprotease cathepsin D has been found to mediate microglial neurotoxicity based on the following results: (i) the expression of cathepsin D protein was markedly increased in lipopolysaccharide/interferon-γ-stimulated microglia compared with resting microglia as determined by western blot analysis of conditioned media; (ii) knockdown of cathepsin D expression in microglia using short hairpin RNA diminished the neurotoxicity in the coculture of microglia and neuroblastoma cells and (iii) recombinant procathepsin D protein exerted cytotoxic effects toward cultured neurons. In conclusion, cathepsin D appears to play a central role in the microglial neurotoxicity, and could be a potential biomarker or drug target for the diagnosis and treatment of neurodegenerative diseases that are associated with excessive microglial activation and subsequent neurotoxic inflammation.     

Cysteine Cathepsins in Neurological Disorders

Increased proteolytic activity is a hallmark of several pathological processes, including neurodegeneration. Increased expression and activity of cathepsins, lysosomal cysteine proteases, during degeneration of the central nervous system is frequently reported. Recent studies reveal that a disturbed balance of their enzymatic activities is the first insult in brain aging and age-related diseases. Leakage of cathepsins from lysosomes, due to their membrane permeability, and activation of pro-apoptotic factors additionally contribute to neurodegeneration. Furthermore, in inflammation-induced neurodegeneration the cathepsins expressed in activated microglia play a pivotal role in neuronal death. The proteolytic activity of cysteine cathepsins is controlled by endogenous protein inhibitors—the cystatins—which evidently fail to perform their function in neurodegenerative processes. Exogenous synthetic inhibitors, which may augment their inhibitory potential, are considered as possible therapeutic tools for the treatment of neurological disorders.     

Dysregulation of macrophage-secreted cathepsin B contributes to HIV-1-linked neuronal apoptosis

Chronic HIV infection leads to the development of cognitive impairments, designated as HIV-associated neurocognitive disorders (HAND). The secretion of soluble neurotoxic factors by HIV-infected macrophages plays a central role in the neuronal dysfunction and cell death associated with HAND. One potentially neurotoxic protein secreted by HIV-1 infected macrophages is cathepsin B. To explore the potential role of cathepsin B in neuronal cell death after HIV infection, we cultured HIV-1(ADA) infected human monocyte-derived macrophages (MDM) and assayed them for expression and activity of cathepsin B and its inhibitors, cystatins B and C. The neurotoxic activity of the secreted cathepsin B was determined by incubating cells from the neuronal cell line SK-N-SH with MDM conditioned media (MCM) from HIV-1 infected cultures. We found that HIV-1 infected MDM secreted significantly higher levels of cathepsin B than did uninfected cells. Moreover, the activity of secreted cathepsin B was significantly increased in HIV-infected MDM at the peak of viral production. Incubation of neuronal cells with supernatants from HIV-infected MDM resulted in a significant increase in the numbers of apoptotic neurons, and this increase was reversed by the addition of either the cathepsin B inhibitor CA-074 or a monoclonal antibody to cathepsin B. In situ proximity ligation assays indicated that the increased neurotoxic activity of the cathepsin B secreted by HIV-infected MDM resulted from decreased interactions between the enzyme and its inhibitors, cystatins B and C. Furthermore, preliminary in vivo studies of human post-mortem brain tissue suggested an upregulation of cathepsin B immunoreactivity in the hippocampus and basal ganglia in individuals with HAND. Our results demonstrate that HIV-1 infection upregulates cathepsin B in macrophages, increases cathepsin B activity, and reduces cystatin-cathepsin interactions, contributing to neuronal apoptosis. These findings provide new evidence for the role of cathepsin B in neuronal cell death induced by HIV-infected macrophages.     

Cysteine Cathepsins in the secretory vesicle produce active peptides: Cathepsin L generates peptide neurotransmitters and cathepsin B produces beta-amyloid of Alzheimer's disease

Recent new findings indicate significant biological roles of cysteine cathepsin proteases in secretory vesicles for production of biologically active peptides. Notably, cathepsin L in secretory vesicles functions as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell-cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in secretory vesicles has been recently identified as a β-secretase for production of neurotoxic β- amyloid (Aβ) peptides that accumulate in Alzheimer's disease (AD), participating as a notable factor in the severe memory loss in AD. These secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrast with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin functions. Features of the secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Cysteine Cathepsins in the secretory vesicle produce active peptides: Cathepsin L generates peptide neurotransmitters and cathepsin B produces beta-amyloid of Alzheimer's disease

Recent new findings indicate significant biological roles of cysteine cathepsin proteases in secretory vesicles for production of biologically active peptides. Notably, cathepsin L in secretory vesicles functions as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell-cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in secretory vesicles has been recently identified as a β-secretase for production of neurotoxic β- amyloid (Aβ) peptides that accumulate in Alzheimer's disease (AD), participating as a notable factor in the severe memory loss in AD. These secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrast with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin functions. Features of the secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Regulation of inflammatory response in neural cells in vitro by thiadiazolidinones derivatives through peroxisome proliferator-activated receptor gamma activation

In most neurodegenerative disorders, including multiple sclerosis, Parkinson disease, and Alzheimer disease, a massive neuronal cell death occurs as a consequence of an uncontrolled inflammatory response, where activated astrocytes and microglia and their cytotoxic agents play a crucial pathological role. Current treatments for these diseases are not effective. In the present study we investigate the effect of thiadiazolidinone derivatives, which have been recently suggested to play a role in neurodegenerative disorders. We have found that thiadiazolidinones are potent neuroprotector compounds. Thiadiazolidinones inhibited inflammatory activation of cultured brain astrocytes and microglia by diminishing lipopolysaccharide-induced interleukin 6, tumor necrosis factor alpha, inducible nitric-oxide synthase, and inducible cyclooxygenase type 2 expression. In addition, thiadiazolidinones inhibited tumor necrosis factor-alpha and nitric oxide production and, concomitantly, protected cortical neurons from cell death induced by the cell-free supernatant from activated microglia. The neuroprotective effects of thiadiazolidinones are completely inhibited by the peroxisome proliferator-activated receptor gamma antagonist GW9662. In contrast the glycogen synthase kinase 3beta inhibitor LiCl did not show any effect. These findings suggest that thiadiazolidinones potently attenuate lipopolysaccharide-induced neuroinflammation and reduces neuronal death by a mechanism dependent of peroxisome proliferator-activated receptor gamma activation.     

Microglial functions and proteases

There is accumulating evidence that intracellular and extracellular proteases of microglia contribute to various events in the central nervous system (CNS) through both nonspecific and limited proteolysis. Cathepsin E and cathepsin S, endosomal/lysosomal proteases, have been shown to play important roles in the major histocompatibility complex (MHC) class II-mediated antigen presentation of microglia by processing of exogenous antigens and degradation of the invariant chain associated with MHC class II molecules, respectively. Some members of cathepsins are also involved in neuronal death after secreted from microglia and clearance of phagocytosed amyloid-β peptides. Tissue-type plasminogen activator, a serine protease, secreted from microglia participates in neuronal death, enhancement of N-methyl-d-aspartate receptor-mediated neuronal responses, and activation of microglia via either proteolytic or nonproteolytic activity. Calpain, a calcium-dependent cysteine protease, has been shown to play a pivotal role in the pathogenesis of multiple sclerosis by degrading myelin proteins extracellulary. Furthermore, matrix metalloproteases secreted from microglia also receive great attention as mediators of inflammation and tissue degradation through processing of pro-inflammatory cytokines and damage to the blood-brain barrier. The growing knowledge about proteolytic events mediated by microglial proteases will not only contribute to better understanding of microglial functions in the CNS but also may aid in the development of protease inhibitors as novel neuroprotective agents.     

Inflammatory responses are not sufficient to cause delayed neuronal death in ATP-induced acute brain injury

Background

Brain inflammation is accompanied by brain injury. However, it is controversial whether inflammatory responses are harmful or beneficial to neurons. Because many studies have been performed using cultured microglia and neurons, it has not been possible to assess the influence of multiple cell types and diverse factors that dynamically and continuously change in vivo. Furthermore, behavior of microglia and other inflammatory cells could have been overlooked since most studies have focused on neuronal death. Therefore, it is essential to analyze the precise roles of microglia and brain inflammation in the injured brain, and determine their contribution to neuronal damage in vivo from the onset of injury.

Methods and Findings

Acute neuronal damage was induced by stereotaxic injection of ATP into the substantia nigra pars compacta (SNpc) and the cortex of the rat brain. Inflammatory responses and their effects on neuronal damage were investigated by immunohistochemistry, electron microscopy, quantitative RT-PCR, and stereological counting, etc. ATP acutely caused death of microglia as well as neurons in a similar area within 3 h. We defined as the core region the area where both TH⁺ and Iba-1⁺ cells acutely died, and as the penumbra the area surrounding the core where Iba-1⁺ cells showed activated morphology. In the penumbra region, morphologically activated microglia arranged around the injury sites. Monocytes filled the damaged core after neurons and microglia died. Interestingly, neither activated microglia nor monocytes expressed iNOS, a major neurotoxic inflammatory mediator. Monocytes rather expressed CD68, a marker of phagocytic activity. Importantly, the total number of dopaminergic neurons in the SNpc at 3 h (∼80% of that in the contralateral side) did not decrease further at 7 d. Similarly, in the cortex, ATP-induced neuron-damage area detected at 3 h did not increase for up to 7 d.

Conclusions

Different cellular components (microglia, astrocytes, monocytes, and neutrophils) and different factors (proinflammatory and neurotrophic) could be produced in inflammatory processes depending on the nature of the injury. The results in this study suggest that the inflammatory responses of microglia and monocytes in response to ATP-induced acute injury could not be neurotoxic.     

Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit

Microglia cells are the brain counterpart of macrophages and function as the first defense in the brain. Although they are neuroprotective in the young brain, microglia cells may be primed to react abnormally to stimuli in the aged brain and to become neurotoxic and destructive during neurodegeneration. Aging-induced immune senescence occurs in the brain as age-associated microglia senescence, which renders microglia to function abnormally and may eventually promote neurodegeneration. Microglia senescence is manifested by both morphological changes and alterations in immunophenotypic expression and inflammatory profile. These changes are likely caused by microinvironmental factors, but intrinsic factors cannot yet be completely excluded. Microglia senescence appears to underlie the switching of microglia from neuroprotective in the young brain to neurotoxic in the aged brain. The hypothesis of microglia senescence during aging offers a novel perspective on their roles in aging-related neurodegeneration. In Parkinson's disease and Alzheimer's disease, over-activation of microglia may play an active role in the pathogenesis because microglia senescence primes them to be neurotoxic during the development of the diseases.     

The executioners sing a new song: killer caspases activate microglia

Activation of microglia and inflammation-mediated neurotoxicity are suggested to have key roles in the pathogenesis of several neurodegenerative disorders. We recently published an article in Nature revealing an unexpected role for executioner caspases in the microglia activation process. We showed that caspases 8 and 3/7, commonly known to have executioner roles for apoptosis, can promote microglia activation in the absence of death. We found these caspases to be activated in microglia of PD and AD subjects. Inhibition of this signaling pathway hindered microglia activation and importantly reduced neurotoxicity in cell and animal models of disease. Here we review evidence suggesting that microglia can have a key role in the pathology of neurodegenerative disorders. We discuss possible underlying mechanisms regulating their activation and neurotoxic effect. We focus on the provocative hypothesis that caspase inhibition can be neuroprotective by targeting the microglia rather than the neurons themselves.     

小胶质细胞在脑中的生理功能

小胶质细胞是脑中的巨噬细胞,也是脑实质中唯一的一种免疫细胞,因而被看作是中枢神经系统抵御病原入侵的第一道防线。在其他非感染病理状态下,如脑损伤及神经退行性疾病等,小胶质细胞也发挥着保护和毒性损伤的双重作用。相比较其病理功能,人们对小胶质细胞的生理功能长期以来很少关注。然而,近几年关于小胶质细胞生理功能的研究在多个方面都有突破。这些研究结果揭示,小胶质细胞在发育的神经系统中起着调控神经元存活和修饰突触的作用,并且在成熟的健康脑中具有探测和调控神经元活动的功能。将着重对近几年关于小胶质细胞生理功能的相关研究做一综述。     

Cysteine cathepsins B,X and K expression in peri-arteriolar glioblastoma stem cell niches

Glioblastoma (GBM) is the most lethal brain tumor also due to malignant and therapy-resistant GBM stem cells (GSCs) that are localized in protecting hypoxic GSC niches. Some members of the cysteine cathepsin family of proteases have been found to be upregulated in GBM. Cathepsin K gene expression is highly elevated in GBM tissue versus normal brain and it has been suggested to regulate GSC migration out of the niches. Here, we investigated the cellular distribution of cathepsins B, X and K in GBM tissue and whether these cathepsins are co-localized in GSC niches. Therefore, we determined expression of these cathepsins in serial paraffin sections of 14 human GBM samples and serial cryostat sections of two samples using immunohistochemistry and metabolic mapping of cathepsin activity using selective fluorogenic substrates. We detected cathepsins B, X and K in peri-arteriolar GSC niches in 9 out of 16 GBM samples, which were defined by co-expression of the GSC marker CD133, the niche marker stromal-derived factor-1α (SDF-1α) and smooth muscle actin as a marker for arterioles. The expression of cathepsin B and X was detected in stromal cells and cancer cells throughout the GBM sections, whereas cathepsin K expression was more restricted to arteriole-rich regions in the GBM sections. Metabolic mapping showed that cathepsin B, but not cathepsin K is active in GSC niches. On the basis of these findings, it is concluded that cathepsins B, X and K have distinct functions in GBM and that cathepsin K is the most likely GSC niche-related cathepsin of the three cathepsins investigated.     

Cysteine cathepsins in extracellular matrix remodeling: Extracellular matrix degradation and beyond

Cysteine cathepsins have been for a long time considered to execute mainly nonspecific bulk proteolysis in the endolysosomal system. However, this view has been changing profoundly over the last decade as cathepsins were found in the cytoplasm, nucleus and in the extracellular milieu. Cathepsins are currently gaining increased attention largely because of their extracellular roles associated with disease development and progression. While kept under tight control under physiological conditions, their dysregulated and elevated activity in the extracellular milieu are distinctive hallmarks of numerous diseases such as various cancers, inflammatory disorders, rheumatoid arthritis, bone disorders and heart diseases. In this review, we discuss cysteine cathepsins with a major focus on their extracellular roles and extracellular proteolytic targets beyond degradation of the extracellular matrix. We further highlight the perspectives of cathepsin research and novel avenues in cathepsin-based diagnostic and therapeutic applications.     

Cathepsin expression during skeletal development.

Cysteine proteinases, cathepsins B, H, K, L and S, have been implicated in several proteolytic processes during development, growth, remodeling and aging, as well as in a variety of pathological processes. For systematic analysis of cathepsin gene expression we have produced cDNA clones for mouse and human cysteine cathepsins. Northern analysis of a panel of total RNAs isolated from 16-19 different human and mouse tissues revealed the presence of mRNAs for cathepsin B, H, K, L and S in most tissues, but each with a distinct profile. Of the different cathepsin mRNAs, those for cathepsin K were clearly the highest in bone and cartilage. However, relatively high mRNA levels for the other cathepsins were also present in these tissues. To better understand the roles of different cathepsins during endochondral ossification in mouse long bones, cathepsin mRNAs were localized by in situ hybridization. Cathepsin K mRNAs were predominantly seen in multinucleated chondroclastic and osteoclastic cells at the osteochondral junction and on the surface of bone spicules. The other cathepsin mRNAs were also seen in osteoclasts, and in hypertrophic and proliferating chondrocytes. These observations were confirmed by immunohistochemistry and suggest that all cysteine cathepsins are involved in matrix degradation during endochondral ossification.     

Involvement of Kv1.3 and p38 MAPK signaling in HIV-1 glycoprotein 120-induced microglia neurotoxicity

Inflammatory responses mediated by activated microglia play a pivotal role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorders. Studies on identification of specific targets to control microglia activation and resultant neurotoxic activity are imperative. Increasing evidence indicate that voltage-gated K⁺ (K_v) channels are involved in the regulation of microglia functionality. In this study, we investigated K_v1.3 channels in the regulation of neurotoxic activity mediated by HIV-1 glycoprotein 120 (gp120)-stimulated rat microglia. Our results showed treatment of microglia with gp120 increased the expression levels of K_v1.3 mRNA and protein. In parallel, whole-cell patch-clamp studies revealed that gp120 enhanced microglia K_v1.3 current, which was blocked by margatoxin, a K_v1.3 blocker. The association of gp120 enhancement of K_v1.3 current with microglia neurotoxicity was demonstrated by experimental results that blocking microglia K_v1.3 attenuated gp120-associated microglia production of neurotoxins and neurotoxicity. Knockdown of K_v1.3 gene by transfection of microglia with K_v1.3-siRNA abrogated gp120-associated microglia neurotoxic activity. Further investigation unraveled an involvement of p38 MAPK in gp120 enhancement of microglia K_v1.3 expression and resultant neurotoxic activity. These results suggest not only a role K_v1.3 may have in gp120-associated microglia neurotoxic activity, but also a potential target for the development of therapeutic strategies.     

Ovarian cysteine proteinases in the teleost Fundulus heteroclitus: molecular cloning and gene expression during vitellogenesis and oocyte maturation

The cysteine proteinases cathepsins B and L are members of the multigene family of lysosomal proteases that have been implicated in the processing of yolk proteins (YPs) in teleost oocytes. However, the full identification of the type of cathepsins expressed in fish ovarian follicles and embryos, as well as their regulatory mechanisms and specific function(s), are not yet elucidated. In this study, cDNAs encoding cathepsins B, L, F, K, S, Z, C, and H have been isolated from the teleost Fundulus heteroclitus, and the analysis of their deduced amino acid sequences revealed highly similar structural features to vertebrate orthologs, and confirmed in this species the existence of cathepsin L-like, cathepsin B-like, and cathepsin F-like subfamilies of cysteine proteinases. While all identified cathepsins were expressed in ovarian follicles, the corresponding mRNAs showed different temporal expression patterns. Thus, similar mRNA levels of cathepsins L, F, S, B, C, and Z were found throughout the oocyte growth or vitellogenesis period, whereas those for cathepsin H and K appeared to decrease as vitellogenesis advanced. During oocyte maturation, a transient accumulation of cathepsins L, S, H, and F mRNAs, approximately a 3-, 1.5-, 1.6-, and 6-fold increase, respectively, was detected in ovarian follicles within the 20-25 hr after hormone stimulation, coincident with the maximum proteolysis of the oocyte major YPs. The specific temporal pattern of expression of these genes may indicate a potential role of cathepsin L-like and cathepsin F proteases in the YP processing events occurring during fish oocyte maturation and/or early embryogenesis.     

Redundancy between Cysteine Cathepsins in Murine Experimental Autoimmune Encephalomyelitis

The cysteine cathepsins B, S, and L are functionally linked to antigen processing, and hence to autoimmune disorders such as multiple sclerosis. Stemming from several studies that demonstrate that mice can be protected from experimental autoimmune encephalomyelitis (EAE) through the pharmacologic inhibition of cysteine cathepsins, it has been suggested that targeting these enzymes in multiple sclerosis may be of therapeutic benefit. Utilizing mice deficient in cysteine cathepsins both individually and in combination, we found that the myelin-associated antigen myelin oligodendrocyte glycoprotein (MOG) was efficiently processed and presented by macrophages to CD4+ T cells in the individual absence of cathepsin B, S or L. Similarly, mice deficient in cathepsin B or S were susceptible to MOG-induced EAE and displayed clinical progression and immune infiltration into the CNS, similar to their wild-type counterparts. Owing to a previously described CD4+ T cell deficiency in mice deficient in cathepsin L, such mice were protected from EAE. When multiple cysteine cathepsins were simultaneously inhibited via genetic deletion of both cathepsins B and S, or by a cathepsin inhibitor (LHVS), MHC-II surface expression, MOG antigen presentation and EAE were attenuated or prevented. This study demonstrates the functional redundancy between cathepsin B, S and L in EAE, and suggests that the inhibition of multiple cysteine cathepsins may be needed to modulate autoimmune disorders such as multiple sclerosis.     

Functional roles of microglia in the central nervous system

Microglia, a type of perineuronal glial cells in the central nervous system, have been suggested to play various important roles in normal and pathologic brains. In this article, first, we described the association or roles of activated microglia in injury and various brain diseases, and subsequently, summarized microglia-derived physiologically active molecules which will affect the neuronal survival and neuronal growth, and glial function, and finally, discussed the molecular mechanism of microglial activation.