# Structural Biochemistry/Cell Signaling Pathways/Necrosis
## Introduction

Necrosis is a type of cell death, distinct for apoptosis and autophagy, which has commonly thought to be an uncontrolled form of death. However, recent research has shown that the process and initial start may actually be regulated. Necrosis is typified by distinguishable signs such as generation of reactive oxygen species and ATP depletion. Necrosis is considered a more inflammatory form of cell death that might contribute to antiviral immunity. Additionally, it has been found that inhibition of proteins that are involved in apoptosis or autophagy lead to necrosis.

## Necrosis Characteristics

There are three types of mammalian cell death that can be distinguished by criteria of the morphological kind. While apoptosis is characterized by plasma membrane blebbing, among other criterion, and autophagy by accumulation of autophagic vacuoles, necrosis has been commonly defined negatively in that it is death lacking the aforementioned characteristics. Necrosis is associated with the loss of cells in many pathologies and is linked to local inflammation. This is through immune system alerting factors that are released. Furthermore, necrotic cells are cleared through a macropinocytotic mechanism whereby only parts of the cell are phagocytosed.

## Classification

Necrosis is the combine of cell changes after localized cellular death through a process called autolysis. Necrosis consists of five distinctive morphological patterns. The five are listed below:
* Coagulative necrosis- characterized by the formation of a gelatinous (gel-like) substance in dead tissues.
* Liquefactive necrosis- also known as colliquative necrosis and is characterized by the digestion of dead cells to form a viscous liquid mass.
* Caseous necrosis- combination of coagulative and liquefavtive necroses that is usually caused by mycobacteria.
* Fat Necrosis- specialized necrosis of fat tissue.
* Fibrinoid necrosis- special form of necrosis that caused by immune-mediated vascular damage containing antigen and antibodies.
Besides these five distinctive morphological patterns, there are also other clinical classifications of necrosis such as gangrene, gummatous, and haemorrhagic. Often time, spider bites may also lead to necrosis.

## Evidence of a Programmed Course

Experimental evidence in different species with similar necrosis with early plasma membrane rupture with little signs of apoptosis or autophagy has shown several traits leading to a sequence of events that are specific to necrotic cell death. These include mitochondrial dysfunction through production of reactive oxygen species (ROS) and swelling, depletion of ATP, lack of Ca$^{2+}$ homeostasis, perinuclear organelle clustering, protease activation such as calpains and cathepsins, lysosomal rupture, and plasma membrane rupture. This is further confirmed by the existence of a similar pathway in plant cells. Although research remains to be done to completely elucidate the molecular and chronological components of this pathway, it is possible that necrotic cell death is part of an organized programmed cascade to self-destruction.

## Evidence of a Programmed Occurrence

While there are necrotic processes that are poorly defined and cannot be regulated, such as that from harsh external conditions as freeze-thawing and detergents, there are programmed necrotic happenstances in its occurrence. Evidence for this includes developmental necrosis such as that limiting the growth of bones and that of intestinal epithelial cells. Additionally, certain plasma membrane receptors, if triggered by attachment of their physiological ligands, can initiate necrosis, suggesting that there are signal transduction pathways connected to the induction of necrosis specifically. In addition, certain types of genetic and epigenetic factors can increase susceptibility to necrotic death, as seen in mouse brain ischemia. Furthermore, the inhibition of specific enzymes can prevent necrosis, indicating that certain enzymes play a vital role in the necrotic pathway. Also telling is that inhibition of caspases can change the type of cell death from that of apoptosis or autophagy to necrotic cell death.

## Evidence of a Necrosis as a Default Pathway

The idea that necrotic cell death is the default pathway for cell death is supported by observations that inhibition of apoptosis as well as autophagy leads to necrosis in a variety of cell models. In a model using baby mouse kidney epithelial cells, suppression of both mitochondrial membrane permeabilization and transfection with Akt protein kinase changes the type of cell death from apoptosis to autophagy to necrosis. Simultaneous inhibition is required.
It has also been seen that inhibition of caspases induces necrosis, as shown in mice interdigital cells. By adding Z-VAD-fmk, a caspase inhibitor, the cells from the limb anlage undergo a non-apoptotic, non-autophagic cell death. What is notable is that this necrosis undergoes that same spatial and temporal pattern as what apoptosis what produce, and leads to the same formation of normal digits. Here, necrosis is a substitute for a lack of apoptosis. Furthermore, caspase inhibition can lead to sensitization of the cells for necrosis, leading to a reduced dosage of tumor-necrosis factor-α needed for necrosis. This indicates that elements of the apoptotic signaling cascase might inhibit necrosis.
Other signs include the depletion of ATP favoring necrotic cell death (due to reducing optimal activation of caspases), presence of nitric oxide (inhibiting caspases), and inactivation of atg1 gene for autophagy. As such, it would appear that necrosis can occur as a part of signal transduction cascades or upon inhibition of other cell death pathways.

### Cell Death Evolution Hypothesis

The occurrence of necrosis upon the inhibition of other pathways indicates its potential emergence early on in evolution as primordial. Apoptosis and autophagy may have been added later on. One hypothesis is that a primordial necrotic pathway existed. Addition of apoptosis or autophagy came later and was recruited for optimal dismantling of cells. However, this pathway would appear after the point of no return of cell death has already been reached. Autonomization of that pathway would arrive later, where two mechanisms are available for cell death, either apoptosis or necrosis. Both happen independently and the point of no return is not shared. Exclusivity of one mechanism, such as apoptosis, would come later in certain species, where necrosis is no longer a viable cell death pathway and cell death relies exclusively on apoptosis, such as in C. elegans.

## Necrosis Specific Molecular Processes

Studies have shown that there may be certain molecules that are specifically required for necrosis. Some potential ones are described below. However, unlike apoptosis where genes necessary for its course have been identified, in necrosis, genes identified are not as specific.

### Receptor-Induced Necrosis and RIP1

In the L929 mouse fibrosarcoma cell line with TNFα, necrosis is induced through a signal transduction pathway involving the TNF receptor 1 and the recruitment of Fas-associated death domain to that receptor. This is accompanied by a rapid burst in ROS production by the mitochondria. Here, necrosis is blocked by rotenone, an inhibitor of the respiratory chain complex I, and lipophilic antioxidants. Additionally, knocking down expression of RIP1, a kinase, prevents these mitochondrial manifestations. RIP1 knockout cells are refractory to the propagation of necrosis, indicating the role of RIP1 in necrotic signaling.
In certain types of cell death signaling involving activation of caspase-8, RIP1 is cleaved by caspase-8, inactivating any pro-necrotic activity. By inhibiting caspase-8, cell death follows a necrotic-like pattern which is dependent on RIP1 expression. RIP1 has been implicated in other necrotic signaling such as that induced by DNA alkylation. The mechanism for this, however, is not completely elucidated, and more research is needed. However, it appears that RIP1 is a necrosis contributor as an upstream signal.

### RIP1-RIP3 Pro-Necrotic Complex

Since RIP1 activates signaling pathways other than necrosis, it suggests that there are additional mechanisms existing to regulate its pro-necrotic function. RNA interference screens have identified a kinase known as RIP3, which showed that RIP3 over-expression prompts apoptotic and necrotic cell death. This kinase serves as an inducer of programmed necrosis. The assembly of the pro-necrotic RIP1-RIP3 complex is during the TNF-induced programmed necrosis. The complex is mediated through the RHIM (RIP homotypic interaction motif), which is a representation of an emerging protein-protein interaction motif. Evidence favor RIP1 over RIP3 as the upstream kinase activator in necrotic signaling cascade. RIP1/RIP3-dependent programmed necrosis may be important in other inflammatory diseases as well as cancer.

### Cyclophilin D

Cyclophilin D (CypD) is a mitochondrial matrix protein which interacts with certain proteins that result in the removal of the transmembrane potential of the inner mitochondria. The strongest stimulus is Ca$^{2+}$, but ROS condtions or ATP depletion can also favor this permeability. However, knockout of the CypD gene gives resistance to necrotic cell death in many cell types. Furthermore, cyclosporin A, which targets CypD, has been shown to reduce the loss of cells induced by necrotic stimuli such as TNFα.
However, CypD is not the cause of permeabilization mediated by the Bax/Bak genes, suggesting two different mechanisms of MMP, one necrotic and the other apoptotic. Cyclosporin A, strangely though, has been shown to inhibit apoptotic death in some situations, indicating some cross over between the two pathways. As involved in necrosis as CypD may be, it is not involved in all necrosis cases and is not specific to necrosis either.

### Non-Caspase Proteases

Calpains, cysteine proteases that are Ca$^{2+}$-dependent, and lysosomal cathepsins have been shown to be the likely executors of cell death through cleavage of the plasma membrane sodium/calcium exchanger, resulting in the inactivation of homeostasis. In C. elegans cases where there is a deg-3(d) allele, lysosomal proteases appear to be important as lysosomes fuse together and eventually result in necrosis, possible due to calpain activation. It would appear that altered ion homeostasis results in calpain activation and eventually cell death by necrosis.

## Therapeutic Manipulation

To therapeutically inhibit necrosis may be desirable. One way to do this would be to block surface receptors that are linked to necrosis. Studies have shown that inhibition of PARP, RIP1, CypD, calpains, and cathepsins lead to necrosis inhibition in in vivo settings. On the other side, we may want to initiate necrosis in cells such as tumors that are resistant to cell death. Switching from necrotic cell death to the non-inflammatory apoptosis may also be a desirable deviation. By activating caspases, necrosis may be attenuated and inflammation decreased.
The most likely desirable outcome is the prevention of cell death completely. Knowing that several cell death pathways exist and are inter-related, inhibition of all of the pathways would prevent cell death. Combined inhibition of caspases, apoptotic proteins and necrotic factors would truly prevent cell death.

## References

* Golstein, P., & Kroemer, G. (2007). Cell death by necrosis: towards a molecular definition. Trends in biochemical sciences, 32(1), 37–43. doi:10.1016/j.tibs.2006.11.001
* Golstein, P. and Kroemer, G. (2005) Redundant cell death mechanisms as relics and backups. Cell Death Differ. 12 (Suppl. 2), 1490–1496
* Xu, K. et al. (2001) Necrotic cell death in C. elegans requires the function of calreticulin and regulators of Ca2+ release from the endoplasmic reticulum. Neuron 31, 957–971
* http://en.wikipedia.org/wiki/Necrosis
* Moquin, David, and Francis Ka-Ming Chan. The Molecular Regulation of Programmed Necrotic Cell Injury. Worcestor: Cell Press, n.d. PDF.