Necrosis

Homeostasis depends on the balance between cell proliferation and cell death. This balance can be disrupted by environmental agents that cause damage to the tissue and organs. The process of cell death is tightly regulated and occurs in various ways. 

Necrosis (accidental cell death) is the pathological process which occurs when cells are exposed to either physical or chemical insult such as high temperature, UV radiation, ionising radiation, anti-cancer drugs and oxidative stress.

 

 

The term necrosis was derived from the Greek word 'necros' - meaning dead - and refers to accidental cell death. Necrosis is not regulated by homeostatic mechanism and is a passive process as it does not require new protein biosynthesis and requires minimal energy. 

Necrotic cell death occurs in response to changes in physiological conditions including hypoxia (low oxygen), ischemia (restricted blood supply), hypoglycaemia (low blood sugar), toxin exposure, extreme temperature changes, nutrient deprivation and other environmental pressures.

The cell swells up and bursts thereby releasing its contents and inducing an inflammatory response.

 

Cellular events of necrosis

Simple model organisms such as Drosophila melanogaster and Caenorhabditis elegans have been used to study molecular events underlying necrosis. 

Changes in cell cycle

Necrotic changes may take place in two different phases of the cell cycle, namely interphase and mitosis.

Interphase cell death: This type of cell death is triggered following severe injury occuring prior to cell division.

Mitotic cell death: If the cells enter mitosis prematurely due to the failure of cell-cycle check points necrosis may be triggered. Cytotoxic drugs and ionising radiation are common causes of mitotic cell death. 

 

Depletion of ATP

ATP levels are an important determinant in distinguishing the mode of cell death. Generally, cellular stress from either nutrient starvation or reduced oxygen supply results in the depletion of ATP. Sufficient ATP depletion may trigger necrosis. This is a condition experienced in tumour cells.

 

Mediators of necrosis

Several mediators are involved in the process of necrosis.

Calcium-mediated necrosis

Intracellular Ca2+ is an important signalling molecule that elicits numerous cell responses including necrosis. In some cases, even extracellular ligands are able to induce Ca2+-dependent necrosis. 

In viable cells, both plasma membrane and intracellular membranes are impermeable to calcium uptake and the concentration of calcium varies in a cell. Under physiological conditions, the Ca2+ concentration is approximately 0.1uM in the cytoplasm and 0.2mM extracellularly.

The endoplasmic reticulum (ER) acts as a storage house for calcium. When Ca2+ from ER is released, or extracellular Ca2+ enters the plasma membrane, necrosis can be triggered due to the activation of either Ca2+-dependent proteases or mitochondrial Ca2+ overload (Figure 2). 

 

Fig.2. Calcium-mediated necrosis: Intracellular calcium increases in response to activation of glutamate receptors on the channels of both plasma membrane and ER membrane. This activates Ca2+-dependent proteases and the citric acid (aka tricarboxylic acid or TCA) cycle and reactive oxygen species (ROS) production. ROS activates mPT which is dependent on CypD. mPT then inhibits ATP production and hence necrosis. 



Reactive oxygen species mediated necrosis

Reactive oxygen species (ROS) are constantly generated in cells in an aerobic environment. At the same time, excessive production of ROS results in oxidative stress, damage to organelles and other intracellular molecules and finally leading to necrosis.

Reactive oxygen species is composed of a number of molecules which are derived from oxygen. One such molecule is the unpaired electron commonly known as a free radical. Major species include hydrogen peroxide, superoxide and nitric oxide. Mitochondria, the power house of energy, are the source of ROS that initiate necrosis. Increased mitochondrial ROS leads to oxidation of purines, cleavage of DNA strands and DNA-protein cross-links. This damage stabilises and activates p53 and poly(A)ribopolymerase (PARP). p53 activation results in either apoptosis or cell-cycle arrest while PARP activation leads to necrosis. Inhibition of PARP by inhibitors or its knock down prevents necrosis induced by hydrogen peroxide. 

 ROS are able to modify lipids as the double bonds present in polyunsaturated fatty acids are the major targets of ROS. Oxidation of lipds results in the loss of integrity of plasma membrane and also intracellular membranes. Loss of integrity of lysosomes and ER leads to release of proteases or influx of Ca2+resulting in necrosis. 

 Another target of ROS are proteins with sulhydral links or amino acids with sulfur as they break the disulfide bonds or sulfhydral links thereby changing the function of modified proteins. This contributes necrosis through modification of ca channels. The calcium channels of both plasma membrane and ER are affected by ROS mediated protein modification. 

 

Immune-induced necrosis

Death receptor-mediated necrosis is another necrotic pathway which involves the ROS. This pathway is also known as the extrinsic pathway and is initiated by the binding of extracellular ligands to the death receptors present on the plasma membrane.

The well-known and most studied death pathways are those mediated by the tumour necrosis factor receptor (TNFR) superfamily. Upon binding of TNF-alpha ligand, the TNFR1 receptor in the plasma membrane forms trimers and recruits numerous death domain-containing proteins to create the death-inducing complex (DISC).

One of the DISC components - FADD - is involved in necrosis. RIP1 is also involved in death receptor-induced necrosis. In the presence of caspase inhibitors, FADD and RIP1 both induce necrosis (Figure 3). The mechanism underlying the necrosis by FADD and RIP1 is still poorly understood. But one possible mechanism is by the generation of mitochondrial ROS. 

 

Fig. 3. Immune-induced necrosis: Tumour necrosis factor initiates the signal transduction pathway to increase mitochondrial ROS production, leading to DNA damage or lysosomal membrane permeabilization. Hydrogen peroxide also increases the mitochondrial ROS production. 



DNA damage-induced necrosis

Cells switch between oxidative phosphorylation and glycolysis for generating energy under the circumstances where oxygen supply is limited, or when mitochondrial respiration is inhibited. In the case of highly proliferating cells such as cancer cells and lymphocytes, protein and lipid biosynthesis maintains ATP production via aerobic glycolysis.

One efficient way to block glycolysis is by activating PARP. PARP binds to DNA double-strand breaks and catalyzes poly(ADP) ribosylation of a number of proteins by converting NAD into NAM and ADP-ribose. PARP depletes the cytosolic NAD pool without hindering the mitochondrial NAD pool as cytosolic and mitochondrial NAD pools do not exchange across the mitochondrial inner membrane.

This depletion results in the further inhibition of glucose catabolism and hence prevents glucose-dependent ATP production. Whilst activation of PARP induces necrosis, NAD rescues cells from PARP-mediated necrosis (Fig. 5).

 

Fig. 4. DNA damage-induced necrosis: In response to DNA damage, the nuclear enzyme PARP is activated and catalyzes the degradation of NAD into poly(ADP)-ribose polymers and nicotinic acid mononucleotide (NAM). The NAD consumption depletes the cellular NAD pool and blocks the cells ability to degrade glucose to support ATP production resulting in necrosis.



Role of proteases in necrosis

Multiple proteases are involved in the process of necrosis. A number of proteases are essential for necrosis. Among them, calpains and cathepsins are the major ones. The evidence for the involvement of these proteases came from genetic studies carried out in C.elegans.

Calpains: These belong to a family of Ca2+-dependent cysteine proteases. Based on the requirement of Ca2+ they are categorized into two sub-categories: u-calpains and m-calpains. u-calpains are activated in response to micromolar, and m-calpains  in response to millimolar concentrations of Ca2+.

Both share a common regulatory subunit of 30kDa and a distinct catalytic subunit of 80kDa. Calpains are latent in cells, but in response to increased levels of Ca2+, they translocate to intracellular membrane and are later activated by autocatalytic hydrolysis. This activation contributes to necrotic cell death through cleavage of the Na+/Ca2+ exchanger in the plasma membrane, leading to secondary intracellular Ca2+ overload and ultimately necrotic cell death. Calpains can also act indirectly through activation of cathepsins by causing lysosome membrame permeable and thereby releasing the contents of lysosome leading to necrotic cell death. 

Cathepsins: Cathepsins are another group of proteases involved in necrosis. They are basically grouped into three types depending on their substrate residues: aspartyl, cysteine and serine. They are generally found within lysosomes in healthy cells and help breakdown phagocytosed molecules and are active in the acidic lysosomal environment.

When there is loss of lysosomal integrity, cathepsins leak out of lysosomes and digest molecules which are not exposed to proteases eventually resulting in cell necrosis. One of the molecules that regulates lysosome membrane permeability is sphingosine which is a metabolite of sphingolipid

Caspases: Caspases form another group of proteases that are involved in necrotic cell death. They are subcategorized into three classes: initiator caspases, executioner caspases and inflammatory caspases. The initiator and executioner caspase activation is involved in apoptosis but not in necrosis.  

Significance of necrosis

Necrosis plays a major role in several physiological, pathological and pharamacological conditions. Some of them are described below. 

Microbial and viral infections: There is a dynamic interplay between the pathogen and host immune sytem during the process of infection. The pathogens have the ability to suppress as well as induce host cell death. Recent reports show that HIV1 kills CD4(+) T lymphocytes by necrosis. 

Cancer therapy: Anti-cancer drugs are still considered effective because of their ability to induce selective cell death. Researchers are finding chemotherapeutic drugs that induces cell death by activating necrosis. Some of the agents that activate necrosis include photodynamic treatment, DNA alkylating agents, apoptolidin and honokil. 

Neurodegenerative disease: Cell death plays a significant role in the development of the central nervous system as well as the brain. Growth factor deprivation and persistent excitation are the two strategies which are responsible for cell death in the central nervous system. In the former case, there is excessive elimination of neurons at the completion of neural develoment, whereas in the latter case there is death of the mature neurons. 

Excitotoxicity: Excitotoxicity is a condition that results from the excess release of neurotransmitters. In addition, the cell membrane interacts with certain excitatory amino acids such as N-methyl-D-aspartate, Kainate etc. These excitotoxins have the ability to either induce intracellular Ca2+ by the release of ER Ca2+, or induce the transport of extracellular Ca2+ through plasma membrane transporters. This plays an essential role in neurological disorders like trauma, Alzheimer's and Huntington's disease. 

References

1. Walker, N.I., Harmon, B.V., Gobe, G.C. and Kerr, J.F. 1988. Pattens of cell death. Methods Achiev. Exp. pathol. 13, 18-54.

2. Kroemer, G. and Martin, S.J. 2005. Caspase-independent cell death. nat. Med. 11, 725-730.

3. Majno, G. and Joris, I. 1995. Apoptosis, oncosis and necrosis. An overview of cell death. Am. J. Pathol. 146: 3-15.

4. Wang, K.K. 2000. Calpain and caspase: Can you tell the difference? Trends Neurosci. 23: 20-26.

5. Weinrauch, Y. and Zychlinsky, A. 1999. The induction of apoptosis by bacterial pathogens. Annu. rev. Microbiol. 53: 155-187.

 

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