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Netrins are a class of involved in axon guidance. They are named after the Sanskrit word "netr", which means "one who guides". Netrins are genetically conserved across nematode worms, Drosophila, Xenopus laevis, Mus musculus, and Human. Structurally, netrin resembles the extracellular matrix protein laminin.
Netrins are chemotropism; a growing axon will either move towards or away from a higher concentration of netrin. Though the detailed mechanism of axon guidance is not fully understood, it is known that netrin attraction is mediated through UNC-40/DCC cell surface receptors and repulsion is mediated through UNC-5 receptors. Netrins also act as growth factors, encouraging cell growth activities in target cells. Mice deficient in netrin fail to form the hippocampal comissure or the corpus callosum.
A proposed model for netrin activity in the spinal column of developing human embryos is that netrins are released by the floor plate and then are picked up by receptor proteins embedded in the growth cones of axons belonging to neurons in the developing spinal column. The bodies of these neurons remain stationary while the axons follow a path defined by netrins, eventually connecting to neurons inside the embryonic brain by developing synapses. Research supports that new axons tend to follow previously traced pathways rather than being guided by netrins or related chemotropic factors.
There is a high degree of conservation in the secondary structure of netrins, which has several domains which are homologous with laminin at the amino terminal end. The C-terminal domain is where most of the variation is found between species and contains different amino acids which allow interaction with specific proteins in extracellular matrix or on the cell surface. The differences in terms of structure and function have led to the identifications of several different types of netrins including netrin-1, netrin-3, and netrins-G.
Netrin-3 is different from other netrins. While expressed during development of the peripheral nervous system in the motor, sensory and sympathetic neurons, it is very limited in the central nervous system. Studies with netrin-3 have noticed a reduced ability to bind with DCC when compared with netrin-1. This suggests that it mainly operates through other receptors.
Netrins-G are secreted but remain bound to the extracellular surface of the cell membrane through Glycophosphatidylinositol (GPI). They are expressed predominantly in the central nervous system in places such as the thalamus and Mitral cell of the olfactory bulb. They do not bind to DCC or UNC-5 and instead bind to ligand NGL-1, which results in an intracellular transduction cascade. The two versions, netrin-G1 and netrin-G2, are found only in vertebrates. It is believed that they evolved independently of other netrins in order to facilitate the construction of the brain.
Recently, scientists have characterized many of the cellular mechanisms by which netrin-1 binding to DCC motivates axonal attraction through at least three independent signaling pathways. In all three pathways netrin-1 is observed to cause the homodimerization of DCC that begins the chemoattraction cascade. In the first pathway, the focal adhesion kinase (FAK) is bound to DCC and both undergo tyrosine phosphorylation upon netrin-1 binding that induces the recruitment and phosphorylation of Src and Fyn, which is hypothesized to lead to an increase in second messengers Rac1 and Cdc42 thereby promoting growth cone extension. In a second possible pathway, phosphatidylinositol transfer protein α (PITP) binds to phosphorylated DCC which induces phospholipase C Phospholipase C to increase the ratio of cAMP to cGMP. This increase of cAMP relative to cGMP activates L-type Ca2+ channels as well as transient receptor potential channels (TRPC's) causing an influx of extracellular Ca2+. Evidence suggests that this increased calcium is responsible for the activation of Rho GTPases, Cdc42 Rac1 and the nuclear transcription factor NFAT which can all initiate growth cone extension. Additional studies have also shown that netrin-induced signaling between DCC downstream targets NcK, and Wiskott–Aldrich syndrome protein WASP trigger Rac1 and Cdc42 and subsequently axonal growth.
It is currently hypothesized that long range chemorepulsion involves initiation of the Arachidonic acid pathway upon netrin-1 interaction with the DCC/UNC-5 complex. This pathway increases the intracellular levels of 12-HPETE (12-Hydroperoxy-5, 8, 10, 14-Eicosatetraenoic Acid), which induces cGMP signaling and subsequently causes a decrease in the cAMP/cGMP ratio. Reducing this ratio inhibits calcium conductance through the L-type calcium channels (LCC) and ultimately results in growth cone repulsion though a possible activation of RHOA. A similar RhoA-mediated mechanism is proposed for short range chemorepulsion whereby netrin-1 binding to UNC-5 homodimers alone induces tyrosine phosphorylation requiring FAK and Src, which as a result activates RhoA. An additional mechanism proposes that binding of the tyrosine phosphatase Shp2 to the netrin-1/UNC-5 complex may also trigger chemorepulsion through RhoA.
In developing mammary glands, the growing tips of the ductal network consist of two layers made up of luminal epithelial cells and cap cells. The luminal cells secrete netrin 1, which binds to the receptor neogenin (a homologue of DCC) on the cap cells. This allows for adhesion between the two cell layers, which is necessary for the proper morphogenesis of the terminal end buds (TEBs) in the mammary glands. Loss of the gene coding for either netrin 1 or neogenin leads to the improper formation of the (TEBs), suggesting that rather than acting as a guidance molecule as in neuronal systems, netrin 1 serves as an adhesive in mammary tissue.
During the morphogenesis of the embryonic lung, epithelial cells express netrin 1 and netrin 4. These netrins surround endoderm buds in the basement membrane, preventing distal tip cells from expressing DCC and UNC5B. This allows for normal development of the lung and halts potentially dangerous over-branching and budding from occurring.
In pancreatic development, netrin 1 is expressed in epithelial ductal cells and localizes to the basal membrane. Netrin 1 associates with several elements in the extracellular matrix, including Type-IV collagen, fibronectin, and integral proteins α6β4 and α3β1. These elements in the extracellular matrix are responsible for epithelial cell adhesion and migration, suggesting that netrin 1 is associated with the guidance of epithelial cells in the embryonic pancreas.
Netrin has been implicated as a vital molecule for the proliferation of vascular networks. Multiple studies have found different effects of netrin on these branching vessels. The endothelial tip cells in vascular tissue display similar properties to the growth cone found in neuronal tissue. Studies have discovered that these same endothelial tip cells also express UNC5B, which netrin 1 can bind to, inhibiting angiogenesis. In contrast, several studies show that netrin-1 actually promotes blood vessel branching. In conjunction with this research, it has been found that netrin 4 is responsible for growth in the Lymphatic system. Overall, these studies show that regulating effects of netrin is dependent on the type of vascular tissue. Recently, netrin has been implicated in angiogenesis in the placenta, making it vital to the survival of the fetus. This finding has implications in the future treatment of vascular disease in the placenta.
In adults, netrin has been implicated in the regulation of stem cell movement and inflammation. Netrin 1 has been found to inhibit leukocyte migration to inflamed areas in the body. This provides evidence that the up regulation of netrin protects injured tissue from excess inflammation. Also, the migration of adult Progenitor cell and adult spinal cord progenitor cells to the spine is netrin 1 dependent. Little is known of the mechanism controlling the inhibition or attraction of these stem cells.
Because netrin-1 has been found to be upregulated in tumors, recent research has attempted to identify netrin-1 as a biomarker for the onset of cancer in the human body. It was found that netrin can be found at above-normal levels in the blood plasma of patients who are positive for renal, liver, prostate, meningioma of brain, pituitary adenoma, glioblastoma and breast cancer. Netrin-3 appears to be specifically expressed in Neublastoma (a paediatric tumour) and in small cell lung cancer (SCLC) where it correlates with a bad patient prognosis.
Another important line of current research targets netrin as a treatment for various diseases, including cancer, myocardial infarction, and Alzheimer's disease. In avian and mouse model organisms suffering from neuroblastoma, interfering with the netrin-1 autocrine loop in malignant tumors leads to cell death. This could lead to possible alternative therapies resulting from future trials. Similar treatments regarding the down-regulation of netrin-1 are also being investigated for metastatic breast and colorectal cancers. Recent studies also suggest that netrin is involved in a cardioprotective role by releasing Nitrous oxide gas. In mice, netrin has also been associated with the regulation of Beta amyloid, which is responsible for amyloid plaques in Alzheimer's disease.
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