Microglia and astroglia prevent oxidative stress-induced neuronal cell death: implications for aceruloplasminemia

We partially characterized the transferrin-independent iron uptake (Tf-IU) of neuronal and glial cells in the previous report. In the present study, we further examined a mechanism of which glial cells protect neuronal cells against iron stress using neuron-microglia (N-MG) and neuron-astrocyte (N-AS) co-cultures. When each solely purified cell was treated with iron citrate, cell death occurred in N and MG. However, AS proliferated under the same condition. Both N-MG and N-AS co-cultures were effective in resistance to excessive iron. The total and specific Tf-IU activities of N-MG co-cultures similar to those of N did not increase in a density-dependent manner. Contrarily, the total activity of AS was extremely high and the specific activity was extremely low as a result of proliferation. Regarding of effect of co-cultures on H(2)O(2)-induced cell death, N-MG co-cultures were less effective, but N-AS co-cultures were more effective in protecting N from the oxidative stress. These results suggest that N-MG co-cultures suppress the Tf-IU and N-AS co-cultures stimulate AS proliferation to protect neuronal cells. Brain cells from aceruloplasminemia with mutations in the ceruloplasmin gene take up iron by Tf-IU. Therefore, the different mechanisms of neuronal cell protection by MG and AS may explain the pathophysiological observations in the brains of patient with aceruloplasminemia.     

Free radicals and neuronal cell death

Production of oxygen free radicals is a natural consequence of aerobic metabolism and they are constantly generated in vivo by chemical reactions and metabolic processes. Antioxidant defence systems scavenge and minimise the formation of oxygen-radical-derived biochemical products, however, these defences are not completely effective even under normal physiological conditions. In pathologic situations, oxygen free radicals can be generated in excess of a cell's antioxidant capacity resulting in severe damage to cellular constituents including proteins, DNA and lipids. The inherent biochemical and physiological charateristics of the brain, including high lipid concentrations and energy requirements, make it particularly susceptible to free radical mediated insult. Increasing evidence indicates that many neurological disorders may have components of free radical and oxidative stress induce injury.     

Ubiquitinated inclusions and neuronal cell death

Ubiquitinated inclusions and selective neuronal cell death are considered the pathological hallmarks of Parkinson's disease and other neurodegenerative diseases. Recent genetic, pathological and biochemical evidence suggests that dysfunction of ubiquitin-dependent protein degradation by the proteasome might be a contributing, if not initiating factor in the pathogenesis of these diseases. In neuronal cell culture models inhibition of the proteasome leads to cell death and formation of fibrillar ubiquitin and alpha-synuclein-positive inclusions, thus modeling some aspects of Lewy body diseases. The processes of inclusion formation and neuronal cell death share some common mechanisms, but can also be dissociated at a certain level.     

Extracellular proteases and neuronal cell death.

Neuronal cell death occurs during development of the central nervous system as well as in pathological situations such as acute injury and progressive degenerative diseases. For instance, granule cells in the developing cerebellum and neuronal precursor cells in the cortex undergo programmed cell death, or apoptosis. There is currently strong debate conceming the mechanism of death in many degenerative events such as ischemia, blunt head trauma, excitotoxicity and neurodegenerative diseases, i.e. Alzheimer's disease. Neurons can die a necrotic death when the initial insult is too great; apoptosis requires "planning." For example, the cell death seen in the core of an ischemic infarct is necrotic, while in the surrounding penumbra region the death is probably apoptotic. Regardless of the degenerative pathway, damaged or dead neurons are a hallmark of many diseases including Alzheimer's, Parkinson's, glaucoma, ischemia and multiple sclerosis. Molecules such as cytokines, chemokines, reactive nitrogen/oxygen species, and proteases play an important role in promoting and/or mediating neurodegeneration. Proteases have been implicated in both physiological and pathological events, suggesting their intervention in key points when things go awry. In this review we will summarize recent findings linking extracellular proteases with neuronal cell death in both human diseases and their animal models.     

Nerve growth factor and neuronal cell death

The regulation of neuronal cell death by the neuronotrophic factor, nerve growth factor (NGF), has been described during neural development and following injury to the nervous system. Also, reduced NGF activity has been reported for the aged NGF-responsive neurons of the sympathetic nervous system and cholinergic regions of the central nervous system (CNS) in aged rodents and man. Although there is some knowledge of the molecular structure of the NGF and its receptor, less is known as to the mechanism of action of NGF. Here, a possible role for NGF in the regulation of oxidant--antioxidant balance is discussed as part of a molecular explanation for the known effects of NGF on neuronal survival during development, after injury, and in the aged CNS.     

7alpha-Hydroperoxycholesterol causes CNS neuronal cell death

Brain cholesterol, which is synthesized in the central nervous system and also partly taken up from lipoproteins via the blood-brain barrier, is a major component of neuronal membranes. Oxidation of cholesterol leads to the formation of oxysterols, which have been shown to act cytotoxic. The influence of 7alpha-hydroperoxycholesterol, was investigated using the human neuroblastoma cell line SH-SY5Y. 7alpha-Hydroperoxycholesterol caused neuronal cell death; this neurotoxic effect was dose-dependent, within 48 h 10 microM led to 50%, 50 microM to 92% loss of cell viability, which was detected by cell morphology and Trypan blue exclusion. DNA-fragmentation or caspase-3 activity were not detectable, LDH release occurred rapidly and reactive oxygen species (ROS) were generated. Therefore we infer that 7alpha-hydroperoxycholesterol, apart from its role in atherosclerosis, leads to necrosis of neuronal cells.     

Internucleosomal DNA cleavage and neuronal cell survival/death

Serum-free PC12 cell cultures have been used to study the mechanisms of neuronal death after neurotrophic factor deprivation. We previously reported that PC12 cells undergo "apoptotic" internucleosomal DNA cleavage after withdrawal of trophic support. Here, we have used a sensitive method to detect PC12 cell DNA fragmentation within three hrs of serum removal and have exploited this assay to examine several aspects regarding the mechanisms of neuronal survival/death. Major advantages of this assay are that it permits acute experiments to be performed well before other manifest signs of cell death and under conditions that cannot be applied chronically. We find that this apopotic DNA fragmentation is distinct from the random DNA degradation that occurs during necrotic death. Major observations include the following: (a) There is a good correlation between the ability of trophic substances to promote PC12 cell survival and to inhibit early DNA fragmentation. (b) Phorbol ester, an activator of PKC, acutely suppresses DNA fragmentation, but does not promote long-term survival or inhibition of endonuclease activity when applied chronically due to its downregulation of PKC. (c) Cells undergoing apoptosis within 3 h of serum withdrawal have a "commitment point" of only 1.0-1.5 h beyond which they can no longer be rescued by NGF. (d) Aurin, a non-carboxylic analog of the endonuclease inhibitor ATA, also inhibits DNA fragmentation and promotes short-term survival of PC12 cells. (e) Macromolecular synthesis is not required for DNA fragmentation or for NGF to prevent this event. (f) Extracellular Ca2+ is not required for internucleosomal DNA cleavage caused by serum withdrawal or for suppression of this by NGF. (g) DNA fragmentation can also be detected in cultures of rat sympathetic neurons as early as 10 h after removal of NGF. As in PC12 cell cultures, this precedes morphological signs of cell death.     

Salinomycin induces calpain and cytochrome c-mediated neuronal cell death

Salinomycin is a polyether antibiotic with properties of an ionophore, which is commonly used as cocciodiostatic drug and has been shown to be highly effective in the elimination of cancer stem cells (CSCs) both in vitro and in vivo. One important caveat for the potential clinical application of salinomycin is its marked neural and muscular toxicity. In the present study we show that salinomycin in concentrations effective against CSCs exerts profound toxicity towards both dorsal root ganglia as well as Schwann cells. This toxic effect is mediated by elevated cytosolic Na⁺ concentrations, which in turn cause an increase of cytosolic Ca²⁺ by means of Na⁺/Ca²⁺ exchangers (NCXs) in the plasma membrane as well as the mitochondria. Elevated Ca²⁺ then leads to calpain activation, which triggers caspase-dependent apoptosis involving caspases 12, 9 and 3. In addition, cytochrome c released from depolarized mitochondria directly activates caspase 9. Combined inhibition of calpain and the mitochondrial NCXs resulted in significantly decreased cytotoxicity and was comparable to caspase 3 inhibition. These findings improve our understanding of mechanisms involved in the pathogenesis of peripheral neuropathy and are important to devise strategies for the prevention of neurotoxic side effects induced by salinomycin.     

Mechanisms of neuronal cell death in Huntington's disease

Huntington's disease (HD) is a genetically dominant neurodegenerative condition caused by an unique mutation in the disease gene huntingtin. Although the Huntington protein (Htt) is ubiquitously expressed, expansion of the polyglutamine tract in Htt leads to the progressive loss of specific neuronal subpopulations in HD brains. In this article, we will summarize the current understanding on mechanisms of how mutant Htt can elicit cytotoxicity, as well as how the selective sets of neuronal cell death occur in HD brains.     

Diversity in the mechanisms of neuronal cell death

Neurons may die as a normal physiological process during development or as a pathological process in diseases. The best-understood mechanism of neuronal cell death is apoptosis, which is regulated by an evolutionarily conserved cellular pathway that consists of the caspase family, the Bcl-2 family, and the adaptor protein Apaf-1. Apoptosis, however, may not be the only cellular mechanism that regulates neuronal cell death. Neuronal cell death may exhibit morphological features of autophagy or necrosis, which differ from that of the canonical apoptosis. This review evaluates the evidence supporting the existence of alternative mechanisms of neuronal cell death and proposes the possible existence of an evolutionarily conserved pathway of necrosis.     

Neurogenesis and cell cycle-reactivated neuronal death during pathogenic tau aggregation

The aim of the present study was to investigate the relation between neurogenesis, cell cycle reactivation and neuronal death during tau pathology in a novel tau transgenic mouse line THY-Tau22 with two frontotemporal dementia with parkinsonism linked to chromosome-17 mutations in a human tau isoform. This mouse displays all Alzheimer disease features of neurodegeneration and a broad timely resolution of tau pathology with hyperphosphorylation of tau at younger age (up to 6 months) and abnormal tau phosphorylation and tau aggregation in aged mice (by 10 months). Here, we present a follow-up of cell cycle markers with aging in control and transgenic mice from different ages. We show that there is an increased neurogenesis during tau hyperphosphorylation and cell cycle events during abnormal tau phosphorylation and tau aggregation preceding neuronal death and neurodegeneration. However, besides phosphorylation, other mechanisms including tau mutations and changes in tau expression and/or splicing may be also involved in these mechanisms of cell cycle reactivation. Altogether, these data suggest that cell cycle events in THY-Tau22 are resulting from neurogenesis in young animals and cell death in older ones. It suggests that neuronal cell death in such models is much more complex than believed.     

Microglia: phagocyte and glia cell

Microglia are the resident immune cells of the brain, and are located within the brain parenchyme behind the blood-brain barrier. They originate from mesodermal hemapoietic precursors and are slowly turned over and replenished by proliferation in the adult central nervous system. In the healthy brain resting, ramified microglia function as supportive glia cells, and their activation status is regulated by neurons through soluble mediators and cell-cell contact. However, in response to brain pathology microglia become activated: acquisition of innate immune cell functions render microglia competent to react towards brain injury through tissue repair or induction of immune responses. In certain pathological conditions, however, microglia activation may sustain a chronic inflammation of the brain, leading to neuronal dysfunction and cell death. This might be mediated by the microglial release of extracellular toxic reactive oxygen and nitrogen species. Nevertheless, in the future microglia may potentially be harnessed for therapeutical purposes.     

Metalloproteins and neuronal death

Neurodegenerative diseases include Alzheimer's and Parkinson's disease that are very common and other diseases that are notorious but occur less often such as Creutzfeldt-Jakob disease. In each case a protein is closely linked to the pathology of these diseases. These proteins include alpha-synuclein, the prion protein and Aβ. Despite first being discovered because of aggregates of these amyloidogenic proteins found in the brains of patients, these proteins all exist in the healthy brain where their normal function involves binding of metals. Recognition of these proteins as metalloproteins implies that the diseases they are associated with are possibly diseases with altered metal metabolism at their heart. This review considers the evidence that cell death in these diseases involves not just the aggregated proteins but also the metals they bind.     

Molecular mechanisms of glutamate receptor-mediated excitotoxic neuronal cell death

Excitotoxicity is one of the most extensively studied processes of neuronal cell death, and plays an important role in many central nervous system (CNS) diseases, including CNS ischemia, trauma, and neurodegenerative disorders. First described by Olney, excitotoxicity was later characterized as an excessive synaptic release of glutamate, which in turn activates postsynaptic glutamate receptors. While almost every glutamate receptor subtype has been implicated in mediating excitotoxic cell death, it is generally accepted that the N-methyl-D-aspartate (NMDA) subtypes play a major role, mainly owing to their high calcium (Ca²⁺) permeability. However, other glutamate receptor subtypes such as 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionate (AMPA) or kainate receptors have also been attributed a critical role in mediating excitotoxic neuronal cell death. Although the molecular basis of glutamate toxicity is uncertain, there is general agreement that it is in large part Ca²⁺-dependent. The present review is aimed at summarizing the molecular mechanisms of NMDA receptor and AMPA/kainate receptor-mediated excitotoxic neuronal cell death.     

Effect of neurotrophic factors on neuronal stem cell death

Neural cell survival is an essential concern in the aging brain and many diseases of the central nervous system. Neural transplantation of the stem cells are already applied to clinical trials for many degenerative neurological diseases, including Huntington\'s disease, Parkinson\'s disease, and strokes. A critical problem of the neural transplantation is how to reduce their apoptosis and improve cell survival. Neurotrophic factors generally contribute as extrinsic cues to promote cell survival of specific neurons in the developing mammalian brains, but the survival factor for neural stem cell is poorly defined. To understand the mechanism controlling stem cell death and improve cell survival of the transplanted stem cells, we investigated the effect of plausible neurotrophic factors on stem cell survival. The neural stem cell, HiB5, when treated with PDGF prior to transplantation, survived better than cells without PDGF. The resulting survival rate was two fold for four weeks and up to three fold for twelve weeks. When transplanted into dorsal hippocampus, they migrated along hippocampal alveus and integrated into pyramidal cell layers and dentate granule cell layers in an inside out sequence, which is perhaps the endogenous pathway that is similar to that in embryonic neurogenesis. Promotion of the long term-survival and differentiation of the transplanted neural precursors by PDGF may facilitate regeneration in the aging adult brain and probably in the injury sites of the brain.     

Mitochondrial dynamics in the regulation of neuronal cell death

Mitochondria undergo continuous fission and fusion events in physiological situations. Fragmentation of mitochondria during cell death has been shown to play a key role in cell death progression, including release of the mitochondrial apoptotic proteins. Ultrastructural changes in mitochondria, such as cristae remodeling, is also involved in cell death initiation. Here, we emphasize the important role of mitochondrial fission/fusion machinery in neuronal cell death. Unlike many other cell types such as immortalized cell lines, neurons are distinct morphologically and functionally. We will discuss how this uniqueness presents special challenges in the cellular response to neurotoxic stresses, and how this affects the mitochondrial dynamics in the regulation of cell death in neurons.     

Role of nucleotide- and base-excision repair in genotoxin-induced neuronal cell death

Base-excision (BER) and nucleotide-excision (NER) repair play pivotal roles in protecting the genomes of dividing cells from damage by endogenous and exogenous agents (i.e. environmental genotoxins). However, their role in protecting the genome of post-mitotic neuronal cells from genotoxin-induced damage is less clear. The present study examines the role of the BER enzyme 3-alkyladenine DNA glycosylase (AAG) and the NER protein xeroderma pigmentosum group A (XPA) in protecting cerebellar neurons and astrocytes from chloroacetaldehyde (CAA) or the alkylating agent 3-methyllexitropsin (Me-Lex), which produce ethenobases or 3-methyladenine (3-MeA), respectively. Neuronal and astrocyte cell cultures prepared from the cerebellum of wild type (C57BL/6) mice or Aag(-/-) or Xpa(-/-) mice were treated with 0.1-50 microM CAA for 24h to 7 days and examined for cell viability, DNA fragmentation (TUNEL labeling), nuclear changes, and glutathione levels. Aag(-/-) neurons were more sensitive to the acute (>20 microM) and long-term (>5 microM) effects of CAA than comparably treated wild type neurons and this sensitivity correlated with the extent of DNA fragmentation and nuclear changes. Aag(-/-) neurons were also sensitive to Me-Lex at comparable concentrations of CAA. In contrast, Xpa(-/-) neurons were more sensitive than either wild type or Aag(-/-) neurons to CAA (>10 microM), but less sensitive than Aag(-/-) neurons to Me-Lex. Astrocytes from the cerebellum of wild type, Aag(-/-) or Xpa(-/-) mice were essentially insensitive to CAA at the concentrations tested. These studies demonstrate that BER and NER are required to protect neurons from genotoxin-induced cell death.