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Netrin-1 (NTN1) is a protein that in humans is encoded by the NTN1 gene.

In an embryo, NTN1 is produced in the floor plate of the neural tube, where it guides the growth of commissural axons. In conjunction with other chemoattractants, such as Sonic Hedgehog (Shh), NTN1 signals for axons to cross the floor plate. NTN1 is also able to prevent the crossing of some neurons, such as trochlear motor axons. Different combinations of extracellular signals can result in a variety of axonal growth patterns, allowing the complex network of axons in the CNS to be vigilantly constructed during development.

Recent research has shown that axonal response to NTN1 may change during neural development; for example, early in development, retinal axons are attracted to NTN1, and then later are repelled by the same signal. This response is due to a downregulation of ‘deleted in colorectal cancer’ (DCC) receptors on the axonal growth cone.

Outside of neural development, NTN1 is also involved in angiogenesis, specifically during embryogenesis, as well as morphogenesis of organs such as the pancreas,  lung, and mammary gland.

Netrin
During embryonic development of the brain, axons extend away from their anchored cell bodies to their terminal sites to form synapses. Neurons often have to cross the midline of the brain, from one cerebral hemisphere to the next. Growth cones at the lengthening end of axons mediate the physical movement through the mesenchyme. The growth cone is populated with receptors that respond to extracellular signals. Netrin is one such protein class that act as signals for axon guidance functioning through chemotropism. Netrins are produced primarily at the midline of the brain, and are therefore involved in crossing of commissural neurons between cerebral hemispheres.

Netrins can act both as chemoattractants and chemorepelents, depending on the receptor it binds. Binding of netrin to DCC receptors attracts the axon towards the target site, which is producing a higher concentration of netrin. However, if netrin binds to ‘uncoordinated locomotion-5’ (UNC-5) receptors on the axonal growth cone, the axon is repelled from that region. Axon guidance functions through concentration gradients; higher concentrations of netrin have a greater effect on axon migration. Netrin concentrations can act over a distance as large as a few millimeters.

Axon guidance


NTN1 is involved with the process of axon guidance, inducing or inhibiting axon migration towards a target site.

Axon movement is mediated by altering the growth of filopodia on the growth cone. Binding of NTN1 to DCC stimulates growth of filopodia, while binding with UNC-5 results in depolymerization of filopodia, repelling the axon from areas of higher NTN1 concentration. Signaling pathway complexes induce rearrangement of axon cytoskeleton, allowing the axon to turn as it extends through the mesenchyme. Further combinations of DCC or UNC-5 with other receptors and signaling compounds can result in a variety of reactions upon exposure to NTN1.

NTN1 mediated axon movement involves activation of protein kinases. NTN1 binds protein tyrosine kinase 2 (PTK2) and Src-family kinases (SFK), inducing phosphorylation of DCC receptors. The resultant activation of DCC triggers a variety of signaling pathways, including a MAPK cascade that stimulates axon growth and turning. NTN1 also activates phospholipase C (PLC); one crucial response of this interaction is subsequent opening of Ca2+ channels. The resultant influx of Ca2+, as well as the release of intracellular stores of Ca2+, prompts local actin polymerization, and axons grows towards NTN1 concentrated areas.

NTN1 stimulates phosphorylation of UNC-5 receptors, which results in a Rho GTPase signaling pathway that induces repulsion of axons from NTN1 concentrated areas.

Axon response to NTN1 is dependent upon its internal state. For example, axon sensitivity to NTN1 is dependent on protein concentrations inside the axon growth cone. Activation of MAPK-dependent protein synthesis is necessary for resensitization of the axon growth cone to NTN1. Furthermore, the ratio of cAMP to cGMP determines whether the axon will be attracted to, or repulsed by, NTN1. This may be because cAMP is involved in the expression of receptors on the axon growth cone.

Angiogenesis
NTN1 induces angiogenesis by a similar mechanism in which it induces axon migration. It has been proposed that a feed-forward mechanism based on protein kinases with nitric oxide (NO) mediate and amplify the effect of NTN1. By measuring NO- levels and endothelial proliferation in response to knocking out several siRNA kinases and receptors, the researchers suggested a mechanism similar to the irreversible differentiation of cells during haematopoiesis. In the endothelial cell, NTN1 binds to the DCC receptor which then activates ERK1/2 protein kinases, which subsequently phosphorylates eNOS to yield NO. NO stimulates endothelial cell migration and proliferation, and it functions in a positive feedback loop by activating the ERK1/2 protein. Thus NO acts as a feed forward mechanism to continue its synthesis after a basal NTN1 initiation step.

In addition to inducing angiogenesis, NTN1 expression by neurons after a stroke may help to prevent apoptosis of nearby endothelial cells. Researchers found in cell cultures under conditions of serum starvation, human umbilical vein and artery endothelial cells had a high propensity to undergo apoptosis, but when given a dosage of NTN1 this propensity was greatly reduced. NTN1 appeared to block the UNC-5B apoptotic pathway, which works by inhibiting DAP kinase phosphorylation. When NTN1 was expressed, the DAP kinase (DAPK1/2/3) phosphorylation levels were much higher, likely because NTN1 inhibited the apoptotic cascade by binding to UNC-5B receptors.

Moreover, further evidence for NTN1 inhibition of the UNC-5B apoptotic pathway was provided by a study using zebrafish (Danio rerio). NTN1 expression was silenced, which led to vascular defects; however, these defects were rescued with the inhibition of the UNC-5B and DAP kinase.

Potential application to stroke therapy
Immediately after a stroke, levels of DCC, UNC-5 and NTN1 decrease. Neurons surrounding the newly damaged area become hypersensitive to stimulation which can result in further tissue damage. However, it is thought that functional ability of remaining neurons and their connections are more important to functional recovery post-stroke than the number of neurons remaining. Intact axons take over the synaptic connections previously made by neurons now damaged by stroke. Tissue recovery after a stroke involves angiogenesis, recruitment of progenitors to form new neurons, and axon sprouting in the areas surrounding tissue damage.

NTN1 is  involved in axon guidance and angiogenesis, and may possibly be involved in attraction of progenitors to the site of tissue damage; these functions are important for neural plasticity in recovering tissue post-stroke.

After a stroke expression of UNC-5 receptors increase while DCC and NTN1 levels remain low. Increased UNC-5 receptors without NTN1 present signals cells to die; this occurs after a stroke. Also, it is possible that the upregulation of UNC-5 directs axon branches of surrounding neurons away from the damaged tissue when in the presence of NTN1. Later during recovery, axon growth cones can be signaled, directing them to take over the previous synaptic connections of now damaged neurons. The neuronal growth that does occur after a stroke seems to be implicated with NTN1 expression. Growth and guidance of axon branches to take over pre-existing connections from damaged neurons allows for motor recovery.

From a study using the brains of Wistar rats (Rattus norvegicus), it was found that NTN1 expression after a middle artery occlusion appeared most strongly at the edges of the ischemic areas, and peaked around two weeks after the stroke. By immunohistological staining the cells, it was discovered that NTN1 expression overlapped the neurons but failed to overlap the glial cells, suggesting that the damaged neurons adjacent the stroke release NTN1 to induce axon migration from surrounding areas.

Another study found similar results when the same method for inducing stroke in the rat brain was used, demonstrating that NTN1 expression began as soon as one day following the stroke and peaked after about one week. Vectors were used to induce overexpression of NTN1 following the stroke. Increased numbers of migrating neuroblasts towards the ischemic area were found in rats with overexpression of NTN1. A significant gain in microvessel density in the adjacent cortex was observed and accounted to the high concentration of NTN1. This increase in adjacent blood vessels may precede neuroblast migration because any new neurons that are formed will need new microvessels to supply nutrients for their growth.

In the same study, these findings were coupled with several behavioral improvements, including; significantly increased motor activity; decreased time resting while exploring an open area over three days; increased the rats time spent in the center of the open field, suggesting decreased anxiety. Other behavioral improvements included a reduction in the time spent favoring the healthy limb and more time spent using the limb affected by the stroke during exploratory activity, indicating a significantly increased ability of motor coordination in the rats with an overexpression of NTN1. This suggests that the expression of NTN1 plays a primary role in the recovery of motor activities after stroke. Repeated coupling of behavioral and neurobiological data will provide a strong argument for taking NTN1 therapy to the next stage in clinical research for therapeutic uses on humans after stroke.

From the data, it appears that an overexpression of NTN1 in the affected tissue results in an increased recovery. Future studies could focus on comparing gene expression and molecular function in damaged neurons versus undamaged neurons. The end goal would be to come to better understand the function of NTN1 in stroke recovery, and verify that increasing NTN1 concentrations at the site of damage is a recommendable treatment (either by injection or increasing NTN1 production within the cells).