Lysosomal cathepsins and their regulation in aging and neurodegeneration

Lysosomes and lysosomal hydrolases, including the cathepsins, have been shown to change their properties with aging brain a long time ago, although their function was not really understood. The first biochemical and clinical studies were followed by a major expansion in the last 20 years with the development of animal disease models and new approaches leading to a major advancement of understanding of the role of physiological and degenerative processes in the brain at the molecular level. This includes the understanding of the major role of autophagy and the cathepsins in a number of diseases, including its critical role in the neuronal ceroid lipofuscinosis. Similarly, cathepsins and some other lysosomal proteases were shown to have important roles in processing and/or degradation of several important neuronal proteins, thereby having either neuroprotective or harmful roles. In this review, we discuss lysosomal cathepsins and their regulation with the focus on cysteine cathepsins and their endogenous inhibitors, as well as their role in several neurodegenerative diseases.     

Activation of microglia in the brains of humans with heart disease and hypercholesterolemic rabbits

 Activated microglial cells are concentrated in senile plaques characteristic of Alzheimer’s disease. Such accumulations of activated microglia may contribute towards neurodegeneration via production of cytokines and free radicals. Studies suggesting a link between Alzheimer’s disease and heart disease led us to study microglia immunohistochemically, using monoclonal antibody LN-3, in age-matched nondemented humans with and without heart disease. Using a qualitative staging system for assessing morphological changes occurring in microglia, we found higher microglial activation in the brains of subjects with heart disease than in those without it. Lectin histochemical examination of brains from rabbits maintained on a high-cholesterol diet also revealed increased microglial activation and leukocyte infiltration. Collectively our observations from humans and rabbits suggest that hypercholesterolemia and heart disease accelerate brain aging, and that the formation of senile plaques may be the end result of progressive microglial activation that occurs with aging. Received: 8 October 1996 / Accepted: 4 November 1996     

Gene expression changes in aging retinal microglia: relationship to microglial support functions and regulation of activation

Microglia, the resident immune cells of the central nervous system (CNS), are thought to contribute to the pathogenesis of age-related neurodegenerative disorders. It has been hypothesized that microglia undergo age-related changes in gene expression patterns that give rise to pathogenic phenotypes. We compared the gene expression profiles in microglia isolated ex vivo from the retinas of mice ranging from early adulthood to late senescence. We discovered that microglial gene expression demonstrated progressive change with increasing age, and involved genes that regulate microglial supportive functions and immune activation. Molecular pathways involving immune function and regulation, angiogenesis, and neurotrophin signaling demonstrated age-related change. In particular, expression levels of complement genes, C3 and CFB, previously associated with age-related macular degeneration (AMD), increased with aging, suggesting that senescent microglia may contribute to complement dysregulation during disease pathogenesis. Taken together, senescent microglia demonstrate age-related gene expression changes capable of altering their constitutive support functions and regulation of their activation status in ways relating to neuroinflammation and neurodegeneration in the CNS.     

Neurodegeneration in the Niemann-Pick C mouse: glial involvement

A mouse model of Niemann-Pick type C disease has been found that exhibits neuropathology similar to the human condition. There is an age-related neurodegeneration in several brain regions and a lack of myelin in the corpus callosum in these mice. The purpose of the present study was to examine the Niemann-Pick mouse and determine whether: (1) microglia and astrocytes exhibit ultrastructural pathology similar to that found in neurons; (2) nerve fiber number is reduced when the myelin sheath is absent; and (3) the lysosomal hydrolase, cathepsin-D, is involved in the neurodegenerative process. Using light and electron microscopic methods, and immunocytochemistry, Niemann-Pick and control animals were examined at several ages. Cathepsin-D content was semi-quantitatively measured in neurons and glial cells in brain regions known to exhibit neurodegeneration, as was the density of glial fibrillary acidic protein-labeled astrocytes. The Niemann-Pick mouse exhibited: (1) an age-related increase in inclusion bodies in microglia and astrocytes, similar to that observed within neurons; (2) an almost complete absence of myelin in the corpus callosum by 7-8 weeks of age, along with a 30% reduction in the number of corpus callosum axons; (3) a mild age-related increase in cathepsin-D content within nerve cells in many brain regions. However, the cathepsin-D elevation was greatest in microglial cells; (4) an age-related increase in the number of microglial cells containing intense cathepsin-D immunoreactivity in both the thalamus and cerebellum. Both of these brain regions have been shown previously to exhibit an age-related loss of neurons; and (5) an increase in the number of reactive astrocytes immunostained for glial fibrillary acidic protein, especially in the thalamus and cerebellum.These data indicate that glial cells are a major target for pathology in the Niemann-Pick mouse. The lack of myelin within the corpus callosum may be related to the loss of nerve fibers in this structure. The increase in cathepsin-D-laden microglial cells, in brain regions previously shown to undergo neurodegeneration, is consistent with a role for microglia in the phagocytosis of dead neurons and in actively contributing to the neurodegenerative process. The activation of astrocytes in regions that undergo neurodegeneration is also consistent with a role for these glial cells in the neurodegenerative process.     

Increased RNA editing in EAAT2 pre-mRNA from amyotrophic lateral sclerosis patients: involvement of a cryptic polyadenylation site

Microglial cells play critical roles in the immune and inflammatory responses of the brain. Under pathological conditions, the activation of microglia helps to restore brain homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. As such, regulators of microglial activation have been considered as potential therapeutic candidates to reduce the risk of neurodegeneration associated with neurodegenerative diseases, including Alzheimer's and, Parkinson's diseases. Indirubin-3'-oxime, a potent inhibitor of cyclin-dependent kinases and glycogen synthase kinase-3β, has been shown to have neuroprotective potential. The specific aim of this study was to examine the efficacy of indirubin-3'-oxime in the repression of microglial activation. Indirubin-3'-oxime was shown to effectively inhibit lipopolysaccharide (LPS)-induced nitric oxide release from cultured rat brain microglia. This compound reduced the LPS-stimulated productions of tumor necrosis factor-α, interleukin-1β, prostaglandin E(2), and intracellular reactive oxygen species and also effectively reduced LPS-elicited NF-κB activation. In organotypic hippocampal slice cultures, indirubin-3'-oxime blocked LPS-related hippocampal cell death. These results suggest that indirubin-3'-oxime provides neuroprotection by reducing the productions of various neurotoxic molecules in activated microglia.     

小胶质细胞及其在阿尔茨海默病中的作用

阿尔茨海默病(AD)是一种常见的老年神经变性疾病,病因十分复杂。目前多数学者认为：β-淀粉样蛋白沉积使得神经胶质细胞活化引起脑内慢性炎症反应可能是AD发病的核心病理机制之一。在AD炎症过程中,渉及到诸多细胞如小胶质细胞、星形胶质细胞及神经元参与,小胶质细胞则是其最主要的炎症细胞,小胶质细胞被β-淀粉样蛋白（Aβ）激活,产生大量致炎性细胞因子和神经元毒性介质,从而诱发脑内炎症反应,导致神经元损伤、死亡。Aβ的持续存在,小胶质细胞被持续激活,形成炎症发生和持续的恶性循环,最后导致AD的发生发展。     

Microglial clearance function in health and disease

Microglial cells are of hematopoietic origin, populate the CNS during early development and form the brain's innate immune cell type. Besides their well-known role in immune defense, microglia have an active and homeostatic function in the normal CNS based on high motility of their ramified processes and endocytic clearance of apoptotic vesicular material. During development microglia contribute to the reorganization of neuronal connections, however microglia have also pivotal roles during acute and chronic neurodegeneration. Microglia become attracted to site of injury by nucleotides released from damaged neurons. Scavenger receptors expressed on microglia bind to debris and microglial phagocytic receptors signal via immunoreceptor tyrosine-based activation motif (ITAM)--containing adaptor proteins to promote phagocytosis of extracellular material. Insufficient clearance by microglia appears to be prevalent in neurodegenerative diseases such as Alzheimer's disease.     

Increased microglial activation and protein nitration in white matter of the aging monkey

Activated microglia are important pathological features of a variety of neurological diseases, including the normal aging process of the brain. Here, we quantified the level of microglial activation in the aging rhesus monkey using antibodies to HLA-DR and inducible nitric oxide synthase (iNOS). We observed that 3 out of 5 white matter areas but only 1 of 4 cortical gray matter regions examined showed significant increases in two measures of activated microglia with age, indicating that diffuse white matter microglial activation without significant gray matter involvement occurs with age. Substantial levels of iNOS and 3-nitrotyrosine, a marker for peroxynitrite, increased diffusely throughout subcortical white matter with age, suggesting a potential role of nitric oxide in age-related white matter injury. In addition, we found that the density of activated microglia in the subcortical white matter of the cingulate gyrus and the corpus callosum was significantly elevated with cognitive impairment in elderly monkeys. This study suggests that microglial activation increases in white matter with age and that these increases may reflect the role of activated microglia in the general pathogenesis of normal brain aging.     

Cyclooxygenase and 5-lipoxygenase inhibitors protect against mononuclear phagocyte neurotoxicity

Neuroinflammation and oxidative stress are believed to be contributing factors to neurodegeneration in normal aging, as well as in age-related neurological disorders. Reactive microglia are found in increased numbers in aging brain and are prominently associated with lesions in such age-related degenerative conditions as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). In vitro, stimulated microglia or microglial-like cells secrete neurotoxic materials and are generators of free radicals through their respiratory burst system. Agents that suppress microglial activation are therefore candidates for neuroprotection. We have developed quantitative in vitro assays for measuring neurotoxicity of microglia or other mononuclear phagocytes. Neuronal like SH-SY5Y cells are cultured in supernatants from activated cells of the human monocytic THP-1 line and their survival is followed. Respiratory burst is directly measured on the activated cells. We tested inhibitors of the cyclooxygenase (COX) or the 5-lipoxygenase (5-LOX) pathways as possible neuroprotective agents. The COX pathway generates inflammatory prostaglandins, while the 5-LOX pathway generates inflammatory leukotrienes. We found that inhibitors of both these pathways suppressed neurotoxicity in a dose-dependent fashion. They included the COX-1 inhibitor indomethacin; the COX-2 inhibitor NS-398; the mixed COX-1/COX-2 inhibitor ibuprofen; the nitric oxide (NO) derivatives of indomethacin, ibuprofen and flurbiprofen; the 5-LOX inhibitor REV 5901; and the 5-LOX activating protein (FLAP) inhibitor MK-886. The FLAP inhibitor also reduced respiratory burst activity in a more potent manner than indomethacin. Combinations of COX and 5-LOX inhibitors were more effective than single inhibitors. The data suggest that both COX inhibitors and 5-LOX inhibitors may be neuroprotective in vivo by suppressing toxic actions of microglia/macrophages, and that combinations of the two might have greater therapeutic potential than single inhibitors of either class.     

免疫应答异常与中枢神经系统退变性疾病

免疫功能异常参与了中枢神经系统退变性疾病的发生。中枢免疫功能异常主要为小胶质细胞异常激活。激活的小胶质细胞可形成活性中间代谢产物、一氧化氮、促炎因子等细胞毒性物质。发病率最高的两种中枢神经系统退变性疾病阿尔茨海默病（Alzheimer′s disease,AD)和帕鑫森病（Parkinson′s disease,PD)的发生都与免疫功能异常密切相关。由于免疫功能异常特别是小胶质细胞激活普遍存在于中枢神经系统退变性疾病过程中，调节小胶质细胞功能的药物可能会具有神经保护作用，延迟甚至阻止神经元变性。     

Activation of microglia by retroviral infection correlates with transient clearance of prions from the brain but does not change incubation time

Prion diseases are fatal transmissible diseases, where conversion of the endogenous prion protein (PrP^C) into a misfolded isoform (PrP^Sc) leads to neurodegeneration. Microglia, the immune cells of the brain, are activated in neurodegenerative disorders including prion diseases; however, their impact on prion disease pathophysiology is unclear with both beneficial PrP^Sc‐clearing and detrimental potentially neurotoxic effects. Moreover, monocytes entering the brain from the periphery during disease course might add to disease pathophysiology. Here, the degree of microglia activation in the brain of prion infected mice with and without an additional intraperitoneal retrovirus infection was studied. Peripheral murine retrovirus infection leads to activation of parenchymal microglia without recruitment of monocytes. This activation correlated with transient clearance or delay in accumulation of infectious prions specifically from the brain at early time points in the diseases course. Microglia expression profiling showed upregulation of genes involved in protein degradation coinciding with prion clearance. This enforces a concept where microglia act beneficial in prion disease if adequately activated. Once microglia activation has ceased, prion disease reemerges leading to disease kinetics undistinguishable from the situation in prion‐only infected mice. This might be caused by the loss of microglial homeostatic function at clinical prion disease.     

Clinical implications of the involvement of tPA in neuronal cell death

 Tissue plasminogen activator (tPA), the serine protease that converts inactive plasminogen to the protease plasmin, was recently shown to mediate neurodegeneration in the mouse hippocampus. Mice deficient in tissue plasminogen activator (tPA) display a dramatic resistance to a paradigm of excitotoxic neuronal death that involves intrahippocampal injection of the excitotoxin. This model is thought to reproduce the mechanism of neuronal death observed during acute (such as ischemic stroke) and degenerative (such as amyotrophic lateral sclerosis) diseases of the nervous system. The requirement for the proteolytic activity of tPA to mediate neuronal death is acute in the adult mouse. Serine protease inhibitors, specific for tPA or the tPA/plasmin proteolytic cascade, are effective in conferring extensive neuroprotection following the excitotoxic injection. These findings suggest possible new ways for interfering with the neuronal death observed in the hippocampus as a result of excitotoxicity. In addition, tPA is produced in the hippocampus primarily by microglial cells, which become activated in response to the neuronal injury. Blocking microglial activation has been shown in other injury paradigms to protect against neuronal death, therefore suggesting another way to retard neurodegeneration in the CNS. Furthermore, after the insult has been inflicted and in the presence of a compromised blood-brain barrier macrophages (cells deriving from the same lineage as microglia) migrate into the brain, where they are thought to contribute to the neuronal cell loss by secreting neurotoxic molecules. If these macrophages/microglia expressed, however, a tPA inhibitor, rather than the possibly neurotoxic tPA, they might be able to protect the neurons from dying. Received: 1 October 1996 / Accepted: 27 January 1997     

A2E accumulation influences retinal microglial activation and complement regulation

Age-related macular degeneration is an outer retinal disease that involves aging and immune dysfunction. In the aging retina, microglia aggregate in the outer retina and acquire intracellular autofluorescent lipofuscin deposits. In this study, we investigated whether accumulation of A2E, a key bisretinoid constituent of ocular lipofuscin, alters the physiology of retinal microglia in pathologically relevant ways. Our findings show that sublethal accumulations of intracellular A2E in cultured retinal microglia increased microglial activation and decreased microglial neuroprotection of photoreceptors. Increased A2E accumulation also lowered microglial expression of chemokine receptors and suppressed microglial chemotaxis, suggesting that lipofuscin accumulation may potentiate subretinal microglial accumulation. Significantly, A2E accumulation altered microglial complement regulation by increasing complement factor B and decreasing complement factor H expression, favoring increased complement activation and deposition in the outer retina. Taken together, our findings highlight the role of microglia in the local control of complement activation in the retina and present the age-related accumulation of ocular lipofuscin in subretinal microglia as a cellular mechanism capable of driving outer retinal immune dysregulation in age-related macular degeneration pathogenesis.     

Activated microglia proliferate at neurites of mutant huntingtin-expressing neurons

In Huntington's disease (HD), mutated huntingtin (mhtt) causes striatal neurodegeneration which is paralleled by elevated microglia cell numbers. In vitro corticostriatal slice and primary neuronal culture models, in which neuronal expression of mhtt fragments drives HD-like neurotoxicity, were employed to examine wild type microglia during both the initiation and progression of neuronal pathology. As neuronal pathology progressed, microglia initially localized in the vicinity of neurons expressing mhtt fragments increased in number, demonstrated morphological evidence of activation, and expressed the proliferation marker, Ki67. These microglia were positioned along irregular neurites, but did not localize with mhtt inclusions nor exacerbate mhtt fragment-induced neurotoxicity. Prior to neuronal pathology, microglia upregulated ionized calcium binding adaptor molecule 1 (Iba1), signaling a functional shift. With neurodegeneration, interleukin-6 and complement component 1q were increased. The results suggest a stimulatory, proliferative signal for microglia present at the onset of mhtt fragment-induced neurodegeneration. Thus, microglia effect a localized inflammatory response to neuronal mhtt expression that may serve to direct microglial removal of dysfunctional neurites or aberrant synapses, as is required for reparative actions in vivo.     

Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism

Inflammation, a common denominator among the diverse list of neurodegenerative diseases, has recently been implicated as a critical mechanism responsible for the progressive nature of neurodegeneration. Microglia are the resident innate immune cells in the central nervous system and produce a barrage of factors (IL-1, TNFalpha, NO, PGE2, superoxide) that are toxic to neurons. Evidence supports that the unregulated activation of microglia in response to environmental toxins, endogenous proteins, and neuronal death results in the production of toxic factors that propagate neuronal injury. In the following review, we discuss the common thread of microglial activation across numerous neurodegenerative diseases, define current perceptions of how microglia are damaging neurons, and explain how the microglial response to neuronal damage results in a self-propelling cycle of neuron death.     

Dysfunction of the neurovascular unit in ischemic stroke and neurodegenerative diseases: An aging effect

Current understanding on the mechanisms of brain injury and neurodegeneration highlights an appreciation of multicellular interactions within the neurovascular unit (NVU), which include the evolution of blood-brain barrier (BBB) damage, neuronal cell death or degeneration, glial reaction, and immune cell infiltration. Aging is an important factor that influences the integrity of the NVU. The age-related physiological or pathological changes in the cellular components of the NVU have been shown to increase the vulnerability of the NVU to ischemia/reperfusion injury or neurodegeneration, and to result in deteriorated brain damage. This review describes the impacts of aging on each NVU component and discusses the mechanisms by which aging increases NVU sensitivity to stroke and neurodegenerative diseases. Prophylactic or therapeutic perspectives that may delay or diminish aging and thus prevent the incidence of these neurological disorders will also be reviewed.     

The microglial lysosomal system in Alzheimer’s disease: Guardian against proteinopathy

Microglia, the brain-resident immune cells, play an essential role in the upkeep of brain homeostasis. They actively adapt into specific activation states based on cues from the microenvironment. One of these encompasses the activated response microglia (ARMs) phenotype. It arises along a healthy aging process and in a range of neurodegenerative diseases, including Alzheimer’s disease (AD). As the phenotype is characterized by an increased lipid metabolism, phagocytosis rate, lysosomal protease content and secretion of neuroprotective agents, it leaves to reason that the phenotype is adapted in an attempt to restore homeostasis. This is important to the conundrum of inflammatory processes. Inflammation per se may not be deleterious; it is only when microglial reactions become chronic or the microglial subtype is made dysfunctional by (multiple) risk proteins with single-nucleotide polymorphisms that microglial involvement becomes deleterious instead of beneficial. Interestingly, the ARMs up- and downregulate many late-onset AD-associated risk factor genes, the products of which are particularly active in the endolysosomal system. Hence, in this review, we focus on how the endolysosomal system is placed at the crossroad of inflammation and microglial capacity to keep pace with degradation.     

Age-associated dysregulation of microglial activation is coupled with enhanced blood-brain barrier permeability and pathology in APP/PS1 mice

Aging adversely affects inflammatory processes in the brain, which has important implications in the context of disease progression. It has been proposed that microglia become dysfunctional with age and may lose their neuroprotective properties leading to chronic neurodegeneration. Here, we sought to characterize inflammatory changes in a mouse model of Alzheimer's disease and to delineate differences between normal aging and those associated with disease pathology. A proinflammatory profile, characterized by the upregulation of markers of classical activation, was evident in APPswe/PS1dE9 mice, associated with increased interferon-γ (IFNγ) concentration and dysregulation of mechanisms designed to limit the proinflammatory response. The data indicate that microglia are not less active with age but alter their phenotype; indeed, changes observed in the deactivation state appear to relate to aging rather than disease pathology. We hypothesize that disruption of the blood-brain barrier, in tandem with an enhanced chemokine profile, permits the infiltration of immune cells serving to reinforce classical activation of microglia through their enhanced responsiveness to IFNγ.     

Age-related changes in expression of metallothionein-III in rat brain

Metallothionein (MT)-III is a metal binding protein, called growth inhibitory factor, and is mainly expressed in the central nervous system. Since MT-III decreases in the brain of Alzheimer's disease (AD), a growing interest has been focused on its relationship to neurodegenerative diseases. To clarify age-related changes in the MT-III expression and its inducibility against oxidative stress, we analyzed the expression of MT-III and its mRNA in the brain of lipopolysaccharide (LPS)-treated aged rats. In the frontal cortex, basal expression of MT-III mRNA was significantly increased with aging, while it was observed no induction of MT-III mRNA against LPS administration in the aged rat brain. MT-III immunopositive cells were increased in the frontal, parietal and piriform cortices, hypothalamus and amygdaloid nucleus with aging. The LPS treatment induced MT-III expression in the brain of young-adult rats, but not in the aged rat brain. Furthermore, the MT-III induction with LPS treatment was mainly observed in oligodendrocyte and microglia. In the present study, we showed that inducibility of brain MT-III against oxidative stress may be reduced with aging. Since it has been reported that MT-III has neuroprotective roles as an antioxidant, present results suggest that MT-III is closely related to the neurodegeneration in the aged animals.