Netrin

Netrin 1 knockout disrupts thalamocortical projections topography. From Powell et al., 2008.^[1]

Netrins are a class of proteins involved in axon guidance. They are named after the Sanskrit word "netr", which means "one who guides." Netrins are genetically conserved across nematode worms^[2], fruitflies, frogs, and mice. Structurally, netrin resembles laminin.

Netrin 1 Overview of Netrin-1 signalling in axonal guidance. Chemoattraction is mediated through netrin-1 binding with the DCC receptor. Chemrepulsion is mediated by the DCC/UNC-5 association. Image was courtesy of QIAGEN.

Netrins are chemotropic; 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 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. It seems that once a pathway has been traced by an axon, new axons tend to follow it rather than being guided by netrins or related chemotropic factors.^[3]

Discovery

Netrin was first described in the nematode Caenorhabditis elegans in 1990, and named UNC-6, according to standard C. elegans naming protocol.^[4] The first mammalian homologue of UNC-6 was discovered in 1994, where it was discovered to be a vital guidance cue for rodent commissural axons in the spinal cord.^[5] As of 2009, there have been five types of Netrins identified in mammals. Netrins 1, 3, and 4 are secreted proteins, whereas G1 and G2 are membrane bound proteins tethered by Glycophosphatidylinositol tails. All Netrins discovered in invertebrates thus far are secreted.^[6]

Overview of Netrins

The netrin family is composed mostly of secreted proteins which serve as bifunctional signals: attracting some neurons while repelling others during the development of brain. Expressed in the midline of all animals possessing bilateral symmetry, they can act as long or short range signals during neurogenesis. In order to carry out their functions, netrins interact with specific receptors: DCC or UNC-5 depending on whether they are trying to attraction or repel neurons respectively. There is a high degree of conservation in the second structure of netrins, which has several domains which are homologous domains with laminin at the amino terminal end. The C-terminal domain is where most of the variation is found between species and contain different amino acids which allow interaction with specific proteins in extracellular matrix or on 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.

Key Netrins

Netrin-1 is found in the floor plate and neuroepithelial cells of the ventral region of the spinal cord, as well as other locations in the nervous system including the somatic mesoderm, pancreas and cardiac muscle.^[7] Its main role is in axonal guidance, neuronal migration and morphogenesis of different branching structures. Mice with mutations in the netrin-1 gene were observed to be lacking in forebrain and spinal cord commissural axons. Netrin-3 is different from other netrins. While expressed during development of the periphery nervous system in the motor, sensory and sympathetic neurons, it is very limited in the central nervous system.^[8] Studies with netrin-3 have noticed a reduced ability to bind with DCC receptors when compared with netrin-1. This suggests that it mainly operates through other receptors. Netrins-G are non-secreted proteins which are bound to the cell membrane through Glycophosphatidylinositol (GPI). They are expressed predominantly in the central nervous system in places such as the thalamus and mitral cells of the olfactory bulb.^[9] They do not bind to DCC or UNC-5. Instead, they bind to ligand NGL-1, which results in an intracellular transduction cascade. The two versions, netrins-G1 and netrins-G2, are found only vertebrates. It is believed that they evolved independently of other netrins in order to facilitate the construction of the brain.

Netrin Receptors

DCC and UNC-5 proteins carefully mediate netrin-1 responses. The UNC-5 protein is mainly involved in signaling repulsion. DCC, which is mainly involved in attraction, can also serve as a co-factor in repulsion signaling when far away from the source of netrin-1. DCC is highly expressed in the central nervous system and associated with the basal lamina of epithelia cells. In the absence of netrin-1, these receptors are known to induce apoptosis.

Axonal Guidance

Located at the end of developing axons during embryogenesis are growth cones responsible for the elongation of the axon during migration. Elongation occurs in response to both trophic and tropic factors present in the surrounding environment. Netrins are one such tropic factor secreted by axonal target cells that function as a crucial axonal guidance protein in both vertebrate and invertebrate organisms. Studies in multiple organisms including, mice, rats, chicks, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and the zebrafish Danio rerio have indicated that secreted netrins are bifunctional, meaning that they can act as either attractants or repellants in directing axonal extension. In addition, many studies have characterized netrins as both short and long range cues, acting in the immediate or distant vicinity of their source cell (the axonal target cell).^[10]

Studies of central nervous system (CNS) development in chick and rodent models have identified the netrin-1 protein as a particularly important vertebrate axonal guidance cue. Most significantly, it was observed that the specialized cells of the floor plate located at the ventral midline of the embryonic brain secrete netrin-1, resulting in a protein gradient. This gradient is most concentrated at the ventral midline and becomes increasing diffuse as you move dorsally. Additional research in netrin deficient mice found that when netrin associates with the Deleted in Colorectal Cancer (DDC) receptor on the axonal growth cone an attractant response is initiated. This was further supported by an observed absence of ventral commissure (i.e. corpus callosum) development in mice lacking either netrin-1 or DDC. Similar results were observed in experiments with the netrin-1 homolog UNC-6 discovered in C. elegans. The same early expression and formation of a protein concentration gradient emanating from the ventral midline is observed in epidermal cells of the developing worm. Evidence suggests that this gradient is essential for the long-range function of UNC-6 in guiding the initial circumferential migration of axons to the ventral midline and further that the UNC-40 receptor mediates the attractive response. As additional axons reach the midline, the temporal and spatial expression of UNC-6 becomes increasing restricted, indicating that after a more general dorsal-ventral guidance of axons, UNC-6 is further involved in directing axons to more specific locations.^[11], ^[12]

In current years, scientists have been able to elucidate many of cellular mechanisms by which netrin-1 binding to DCC motivates axonal attraction. It has been shown that the association between netrin-1 and the DDC receptor causes a depolarization of the neuronal plasma membrane through the release of calcium ions Ca2+ from its internal stores. Depolarization activates L-type Ca2+ channels as well as transient receptor potential channels (TRPC’s) causing an influx of extracellular Ca2+, which has been evidenced to induce activation of the nuclear transcription factor NFAT responsible for axon growth. Additional studies have shown that a high internal concentration ratio of cAMP to cGMP is necessary for the Ca2+ mediated chemoattractive response to netrin-1. Using, Xenopus spinal neurons in vitro it has been observed that a low concentration of cAMP to cGMP inhibits the Ca2+ influx through the L-type Ca2+ channels thereby preventing axonal growth, and possibly inducing axonal repulsion according to some sources. A simultaneous chemoattractive mechanism involves the remodeling of the actin microfilaments that comprise the growth cone. Recent evidence has indicated that increased signaling between DCC downstream targets such as NcK, and Wiskott–Aldrich syndrome protein WASP as well as others leads to an increase in the second messengers including Rac and Cdc42, which are known to trigger cytoskeletal remodeling. Additionally is has been suggested that the Rho family of GTPases stimulated by the above mentioned increase in the intracellular Ca2+ concentration contributes significantly to the assembly and disassembly of actin filaments of the growth cone in response to netrin-1 binding.

Interestingly, both DDC in vertebrates and UNC-40 in C. elegans have shown to initiate a repulsive rather than attractive response when associated with the netrin receptor UNC-5. In the same ventral midline gradient discussed above, netrin-1 acts as a chemorepellant for axons of the trochlear motor neurons, thus directing their growth dorsally. Also, antibody inhibition of DCC receptors in embryonic Xenopus spinal cord inhibited both attraction and repulsion in vitro. Likewise multiple defects were observed in C. elegans unc-40 mutants; however errors in migration patterns were more profoundly affected by mutations in the unc-5 gene, indicating that UNC-6 binding to UNC-5 alone can repel axonal growth. In both vertebrate and invertebrate systems short range chemorepulsion in which the concentration of netrins is high seems to primarily occur via the UNC-5 receptor, while long range repulsive effects at more diffuse concentrations require the coordination of DCC or UNC-40 and UNC-5.

Glial and Mesodermal Guidance

Many studies have shown that Netrin-1, UNC-40, UNC-6, and UNC-5 are involved in the migration of glia during embryogenesis.^{[13] [14] [15]} During the migratory phase in Drosophila melanogaster, embryonic peripheral glia (ePG) express UNC-5. When the gene for UNC-5 is mutated, ePG do not migrate or stall.^[16] UNC-6 signaling in C. elegans, coupled with the UNC-40 receptor on neurons, promotes synaptogenesis and assembles the glial endfeet around the synapse.^[17]

Functions Outside of Neuronal Guidance

Although originally understood to be specifically involved in axonal guidance in the central nervous system, new research has linked netrin to cancer regulation, the development and formation of non-neural tissue, and the detection of cancer and other diseases.

Development and Regulation of Tissue

Netrin has been discovered to play a key role in the development and mature regulation of tissue outside the nervous system. Some of the non-neural tissues implicated include lung, placental, vasculature, pancreas, muscle and mammary gland tissue. Netrin contributes to tissue morphogenesis by controlling developing cell migration and cell adhesion in different organs. ^[18]

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 led 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. ^{[19] [20]}

During the morphogenesis of the embryonic lung, epithelial stalk 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.^[21]

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 collagin IV, fibronectin, and integral proteins α6β4 and α3β1. These elements in the ECM are responsible for epithelial cell adhesion and migration, therefore suggesting netrin 1 is correlated with the guidance of epithelial cells in the embryonic pancreas.^[22][23]

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 vascular system. 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. ^{[24] [25]}

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 neural progenitor cells and adult spinal cord progenitor cells to the spine is netrin 1 dependant. Little is known behind the mechanism regarding the inhibition or attraction of these stem cells. ^{[26] [27]}

Cancer regulation and disease markers

In various human cancers, it has been shown that netrin becomes over-expressed. In conjunction, it has been shown that certain receptors become down-regulated in this process. The netrin receptors DCC and UNC5H are responsible for apoptosis regulation. The absence of netrin 1 is responsible for apoptosis, while the presence of netrin 1 leads to an inhibition of the apoptosis pathway. This pathway is unique and independent of the mitochondrial and death receptor pathways that lead to controlled cell death. This has been observed in the human colon epithelium, where higher levels of natural cell death at the upper portion of the villi correlated with a smaller gradient of netrin-1. This correlated the role of netrin with tissue death and growth. Tumor suppressor p53 is responsible for the expression of the netrin-1, implicating that netrin may be the pathway through which p53 regulates the cell cycle. Because netrin is so influential in the regulation of cell death, the gene coding for netrin (NTN1) is considered to be an oncogene. ^[28]

Because netrin-1 has been found to be up regulated in tumors, recent research has been done to identify netrin-1 as a biomarker to indicate the onset of cancer in the human body. It was found that netrin can be found in excess in the blood plasma for patients who are positive for renal, liver, prostate, meningioma of brain, pituitary adenoma, glioblastoma and breast cancer. ^[29]

Continued research on Netrin

There are still many questions still unanswered regarding the netrin family of molecules. It is still unsure what role vertebrate homologues of UNC-5 play in chemo repulsion. Although much is known about the expression of netrin during development, little is still known about its regulation in later development in the brain. Netrin knockout mice show that there is much to learn about the many roles of netrin in axonal guidance. ^[30]

Another important line of research currently being done target's netrin as a treatment for various diseases, including cancer, myocardial infarction, and Alzheimer. In avian and mouse model organisms suffering from neuroblastoma, interefering with the netrin-1 autocrine loop in malignant tumors lead to cell death.^[31] This could lead to a possible alternative therapy with future trials. Similar treatments regarding the down-regulation of netrin-1 are also being looked into for metastatic breast and colorectal cancers.^[32] Recent studies also suggest that netrin is involved in a cardioprotective role by releasing NO gas. In mice, netrin has also been associated with the regulation of (Aβ) peptide, which is responsible for amyloid plaques in Alzheimer’s disease. ^[33]

References

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19. ^ Karen Lai Wing Sun, James P. Correia, and Timothy E. Kennedy Netrins: versatile extracellular cues with diverse functions Development 2011 138:2153-2169; doi:10.1242/dev.044529
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