THE BIOCHEMISTRY OF NEURONAL NECROSIS: ROGUE BIOLOGY? | |
При сильных стрессовых воздействиях клетки подвергаются некрозу – острой, не апоптозной форме клеточной гибели. Некроз является тяжелым повреждением, нарушающим в числе прочих и функционирование нервной системы. Недавние исследования выявили молекулярные механизмы некроза и доказали, что механизмы некротической клеточной гибели эволюционно высококонсервативны и почти одинаковы от нематоды до человека. Однако в отличие от механизмов апоптоза, механизмы некроза не эволюционировали специфическим образом. При экстремальных условиях нормальная активность клеток дестабилизируется с тяжелыми для нее последствиями. Дан обзор механизмов некроза и обсуждаются события, которые превращают эти механизмы в катастрофические для выживаемости клеток.
 (Рис.1.) | Necrotic cell death. Electron micrographs of a normal cell (a) and a cell undergoing necrotic cell death (b). Extensive distortion of the cytoplasm and the plasma membrane is evident. The scanning electron micrograph shown in c illustrates the marked lesions that appear on the surface of the plasma membrane at late stages of necrotic cell death. Adapted, with permission, from Ref. 181 © (1997) MMK Holdings Inc. in association with Purdue University Cytometry Laboratories  (Рис.2.) | Neurodegenerative disease in Caenorhabditis elegans. A nematode neuron undergoing necrosis as a result of degenerin ion channel hyperactivation. In panel a, the degenerating cell (orange arrow) seems swollen to several times its normal diameter, whereas the nucleus (white arrow) is distended and has a distorted morphology. Panel b shows the peculiar electron dense membranous circumvolutions (arrowheads) that accompany cellular destruction, under the electron microscope. Modified, with permission, from Ref. 61 © (1997) Society for Neuroscience  (Рис.3.) | Calcium homeostasis mechanisms. Intracellular calcium concentration is tightly regulated within narrow limits. Under pathological conditions however, regulatory mechanisms are overwhelmed and intracellular calcium concentration ([Ca2+]i) increases through calcium influx from extracellular pools through various channels (voltage-, ligand- or concentration-gated channels) and, under extreme circumstances, through the sodium/calcium exchanger (NCX). Under normal conditions, NCX is the main pathway for calcium efflux, but it can also contribute to Ca2+ influx (reverse mode exchange) especially during strong depolarization, and with increased intracellular sodium182. Calcium concentration can also increase through release from endoplasmic reticulum stores, through the ryanodine (RyR), and inositol-1,4,5-trisphosphate receptors (Ins(1,4,5)P3R). Counterbalancing mechanisms fight to halt calcium concentration increase in the cytoplasm. The plasma membrane calcium pump (PMCA), NCX and sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) function to restore normal calcium levels. Increased intracellular calcium concentration drives calcium overload at mitochondria, through PMCA, and relaxed specificity channels (Uniporter). In turn, calcium overload triggers secondary release of calcium from mitochondrial stores, through the mitochondrial NCX (MNCX) and mitochondrial pores opened during MITOCHONDRIAL PERMEABILITY TRANSITION (MPT)136, 183-185. Calcium-binding proteins in the cytoplasm and in the endoplasmic reticulum offer additional calcium buffering capacity. MMCA, mitochondrial membrane Ca2+ ATPase  (Рис.4.) | Proteases effecting necrosis. Necrotic insults, either directly or indirectly, trigger the activation of proteolytic activities that participate in dismantling the cell. Calcium-activated calpain proteases together with lysosomal cathepsins liberated in the cytoplasm and caspases contribute to necrosis. Destructive proteolytic events are shown with bold arrows. ROS, reactive oxygen species.  (Рис.5.) | Deadly proteolytic cascades in the nematode. Execution of necrotic cell death in the nematode requires the activity of both calpain and cathepsin proteases. Two specific calpain proteases TRA-3 and CLP-1 function redundantly upstream of aspartyl proteases ASP-3 and ASP-4 to mediated necrotic death79. Such an arrangement is consistent with a function of calpains in the activation of non-specific acidic proteases such as cathepsin D, in accordance with the calpain–cathepsin hypothesis122 DATABASES | | | | | | | | | | | | | | | | FURTHER INFORMATION |
Некроз – это форма не апоптозной клеточной гибели, вносящий определенный вклад в некоторые патологические состояния у человека. Несмотря на значительное влияние некроза на здоровье человека, его молекулярные механизмы оставались плохо изученными. Это отчасти обусловливает широкое распространение идей о том, что некроз является хаотическим уничтожением клеток, а также отсутствие подходящих генетических моделей.
Недавние исследования обнаружили некоторые общие закономерности в некротической гибели клеток, указывающие на ограниченный репертуар биохимических каскадов в клеточной деструкции. Эти механизмы, в отличие от большинства апоптозных путей, не эволюционировали. Они представляют нормальные физиологические процессы, которые становятся деструктивными при неблагоприятных условиях.
Определяющими детерминантами некроза являются механизмы ионного гомеостаза. Самые разные воздействия, инициирующие некроз, приводят к нарушениям ионного баланса, выходящего за пределы критического уровня и, таким образом, запускается клеточная гибель. Увеличение внутриклеточной концентрации кальция и окисление могут индуцировать и усиливать клеточную некротическую гибель.
Как и при апоптозе, в некротической гибели задействован механизм белковой деградации. Calpain и cathepsin протеазы играют ключевую роль в «демонтаже» клеток во время некротической гибели. Кроме того, каспазы (caspases) – главные «убийцы» при апоптозе – и протеасомы (proteasome) также участвуют в некрозе.
Несколько экспериментальных моделей некротической гибели клеток были разработаны на простых организмах, легко поддающихся генетическим манипуляциям – Caenorhabditis elegans и Drosophila melanogaster. Эти организмы пригодны для анализа биохимических механизмов некроза и дают возможность получить данные, необходимые для разработки эффективных защитных мер против некротической гибели.
(Box.1) | Excitatory neurotransmitters such as glutamate are released from synapses on depolarization after the arrival of an action potential. The release process is carefully controlled and build-up of excessive neurotransmitter at the synapse is prevented by the action of dedicated transporters that clear the synaptic cleft. However, many deleterious conditions can converge to induce unrestrained glutamate release at synapses, initiating a cascade of events that leads to death of the postsynaptic cell51. For example, catastrophic depolarization occurs during hypoxia or hypoglycaemia, which compromise energy production and therefore the ability of the cell to maintain a membrane potential168, 169. Overstimulation of neurons during seizure has the same effect on glutamate release170, 171. Glutamate binds to and opens specific ionotropic receptor channels on postsynaptic neurons (AMPA, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; NMDA, N-methyl-D-aspartate). Gating of these channels provokes an influx of calcium ions inside the cell either directly (through glutamate receptors that conduct both calcium and sodium) or indirectly (through the secondary activation of voltage-gated calcium channels172). The sharp increase of intracellular calcium concentration is a principal death-signalling event that is involved in both necrosis and apoptosis. The contribution of each type of death to excitotoxicity correlates with the severity and the abruptness of the increase in intracellular calcium concentration82. More profound changes initiate necrosis, whereas relatively mild increases preferentially induce apoptosis15, 57. During stroke, the area immediately affected by restricted blood flow is usually the focal point of necrosis44. However, both necrotic and apoptotic cell death occur within the surrounding tissue, which suffers less from oxygen and nutrient deprivation48. EAAT2, excitatory amino acid transporter 2.
(Box.2) | Tight regulation of intracellular pH and subcellular organelle proton concentration is paramount for the normal function and survival of the cell. For example, fibroblasts in cultures go into cell quiescence (G0 phase of cell cycle) with a pH change as little as 0.2 units173. Under such conditions, gene transcription stops, DNA synthesis ceases, rates of metabolism and protein synthesis decrease, and the cell does not grow or divide until the pH is brought back to normal levels. Given that most molecules cannot function outside a certain pH range, cells have a battery of specialized homeostatic mechanisms to keep their internal pH (pHi) at a constant level (usually around 7.2, in the cytoplasm). These mechanisms can be divided into active, which require the use of ATP, and passive, which do not require ATP. Cells passively regulate their pH with an overabundance of buffer molecules174. Buffers sequester excess protons under conditions of low pH, and release protons if the cytosolic pH becomes too high. The most important and abundant cytosolic buffer is phosphoric acid (H3PO4), which has a pKa of 7.2, helping to maintain a pH of 7.2. Amino acids are also important pH buffers. Lysine, arginine, and histidine have basic side chains with amino groups, and aspartate and glutamate have acidic carboxyl groups on their side chains. Free amino acids have additional amino and carboxyl groups, which can add to their buffering capabilities. Normal cellular metabolism also creates buffer molecules. These include acetic, lactic and citric acids, and the production of carbon dioxide (CO2). Passive pH regulatory mechanisms are augmented by energy-dependent processes that maintain cytoplasmic and organelle pH at its optimum value. Many ion exchangers regulate intracellular pH, by letting one charged ion into the cell while releasing another charged ion. These 'antiporters' draw energy from electrochemical gradients across the plasma or organelle membranes to drive the electro-neutral exchange of protons for Na+ or K+ ions175. Dedicated pumps such as the vacuolar H+ ATPase also regulate cellular pH at the expense of ATP by forcing protons out of the cytoplasm to the extracellular space or into subcellular organelles176. The vacuolar ATPase acidifies lysosomes, generating a low pH that is necessary for the optimum activity of lysosomal hydrolytic enzymes such as cathepsin proteases. NHX, sodium/proton exchanger; pHL, lysosomal pH.
(Box.3.) | The calpain-cathepsin hypothesis This hypothesis was formulated on the basis of observations in mammalian systems122, 177 and encompasses two central players as key mediators of cellular destruction during necrosis: calpains and cathepsins. Calpains become activated when calcium concentration is elevated. Increases in intracellular calcium concentration occur either directly or indirectly in response to many diverse necrosis-initiating stimuli, and have been implicated as principal death-inducing signals in various organisms. Cathepsin proteases are liberated in the cytoplasm after activated calpains compromise the integrity of lysosomal membranes. Lysosomes contain over 80 types of hydrolytic enzymes, including cathepsins. Although, the mechanism of calpain-mediated rupture of lysosomes is unclear, spilling of hydrolytic enzymes from lysosomes into the cytoplasm owing to injury or rupture of lysosomal membranes has been implicated in necrotic cell death after ischaemic injury to both heart and brain178. In the cytoplasm, these enzymes degrade cellular structures and interfere with normal metabolism - death is unavoidable. This process is reminiscent of autophagy, and supports de Duve's original categorization of lysosomes as the cell's 'suicide bag'179. The mechanism by which overactivation of autophagy causes cell demise is not clear. A probable scenario is that cell death is triggered by severe energy depletion following destruction of mitochondria121, 180. Genes that encode proteins involved in cellular calcium homeostasis, as well as genes for lysosomal and calpain proteases, have been detected in genetic screens as suppressors of neurodegeneration in C. elegans, a result that is consistent with observations in cultured mammalian neurons78
(Box.1) | Excitatory neurotransmitters such as glutamate are released from synapses on depolarization after the arrival of an action potential. The release process is carefully controlled and build-up of excessive neurotransmitter at the synapse is prevented by the action of dedicated transporters that clear the synaptic cleft. However, many deleterious conditions can converge to induce unrestrained glutamate release at synapses, initiating a cascade of events that leads to death of the postsynaptic cell51. For example, catastrophic depolarization occurs during hypoxia or hypoglycaemia, which compromise energy production and therefore the ability of the cell to maintain a membrane potential168, 169. Overstimulation of neurons during seizure has the same effect on glutamate release170, 171. Glutamate binds to and opens specific ionotropic receptor channels on postsynaptic neurons (AMPA, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; NMDA, N-methyl-D-aspartate). Gating of these channels provokes an influx of calcium ions inside the cell either directly (through glutamate receptors that conduct both calcium and sodium) or indirectly (through the secondary activation of voltage-gated calcium channels172). The sharp increase of intracellular calcium concentration is a principal death-signalling event that is involved in both necrosis and apoptosis. The contribution of each type of death to excitotoxicity correlates with the severity and the abruptness of the increase in intracellular calcium concentration82. More profound changes initiate necrosis, whereas relatively mild increases preferentially induce apoptosis15, 57. During stroke, the area immediately affected by restricted blood flow is usually the focal point of necrosis44. However, both necrotic and apoptotic cell death occur within the surrounding tissue, which suffers less from oxygen and nutrient deprivation48. EAAT2, excitatory amino acid transporter 2.
(Box.2) | Tight regulation of intracellular pH and subcellular organelle proton concentration is paramount for the normal function and survival of the cell. For example, fibroblasts in cultures go into cell quiescence (G0 phase of cell cycle) with a pH change as little as 0.2 units173. Under such conditions, gene transcription stops, DNA synthesis ceases, rates of metabolism and protein synthesis decrease, and the cell does not grow or divide until the pH is brought back to normal levels. Given that most molecules cannot function outside a certain pH range, cells have a battery of specialized homeostatic mechanisms to keep their internal pH (pHi) at a constant level (usually around 7.2, in the cytoplasm). These mechanisms can be divided into active, which require the use of ATP, and passive, which do not require ATP. Cells passively regulate their pH with an overabundance of buffer molecules174. Buffers sequester excess protons under conditions of low pH, and release protons if the cytosolic pH becomes too high. The most important and abundant cytosolic buffer is phosphoric acid (H3PO4), which has a pKa of 7.2, helping to maintain a pH of 7.2. Amino acids are also important pH buffers. Lysine, arginine, and histidine have basic side chains with amino groups, and aspartate and glutamate have acidic carboxyl groups on their side chains. Free amino acids have additional amino and carboxyl groups, which can add to their buffering capabilities. Normal cellular metabolism also creates buffer molecules. These include acetic, lactic and citric acids, and the production of carbon dioxide (CO2). Passive pH regulatory mechanisms are augmented by energy-dependent processes that maintain cytoplasmic and organelle pH at its optimum value. Many ion exchangers regulate intracellular pH, by letting one charged ion into the cell while releasing another charged ion. These 'antiporters' draw energy from electrochemical gradients across the plasma or organelle membranes to drive the electro-neutral exchange of protons for Na+ or K+ ions175. Dedicated pumps such as the vacuolar H+ ATPase also regulate cellular pH at the expense of ATP by forcing protons out of the cytoplasm to the extracellular space or into subcellular organelles176. The vacuolar ATPase acidifies lysosomes, generating a low pH that is necessary for the optimum activity of lysosomal hydrolytic enzymes such as cathepsin proteases. NHX, sodium/proton exchanger; pHL, lysosomal pH.
(Box.3.) | The calpain-cathepsin hypothesis This hypothesis was formulated on the basis of observations in mammalian systems122, 177 and encompasses two central players as key mediators of cellular destruction during necrosis: calpains and cathepsins. Calpains become activated when calcium concentration is elevated. Increases in intracellular calcium concentration occur either directly or indirectly in response to many diverse necrosis-initiating stimuli, and have been implicated as principal death-inducing signals in various organisms. Cathepsin proteases are liberated in the cytoplasm after activated calpains compromise the integrity of lysosomal membranes. Lysosomes contain over 80 types of hydrolytic enzymes, including cathepsins. Although, the mechanism of calpain-mediated rupture of lysosomes is unclear, spilling of hydrolytic enzymes from lysosomes into the cytoplasm owing to injury or rupture of lysosomal membranes has been implicated in necrotic cell death after ischaemic injury to both heart and brain178. In the cytoplasm, these enzymes degrade cellular structures and interfere with normal metabolism - death is unavoidable. This process is reminiscent of autophagy, and supports de Duve's original categorization of lysosomes as the cell's 'suicide bag'179. The mechanism by which overactivation of autophagy causes cell demise is not clear. A probable scenario is that cell death is triggered by severe energy depletion following destruction of mitochondria121, 180. Genes that encode proteins involved in cellular calcium homeostasis, as well as genes for lysosomal and calpain proteases, have been detected in genetic screens as suppressors of neurodegeneration in C. elegans, a result that is consistent with observations in cultured mammalian neurons78