A recurring theme in neurobiology is the role of a set of molecules that support proliferation, differentiation and survival of neurons. These molecules, collectively referred to as neurotrophins, are essential for the development and maintenance of the vertebrate nervous system, mediating their signal into the cell by specific interaction with tyrosine kinase receptors of the TRK (Tyrosine Kinase Receptor) family. TRK
family is composed of related transmembrane tyrosine receptor kinases that specifically bind neurotrophins: TRKA binds NGF (Nerve Growth Factor); TRKB binds BDNF (Brain Derived Neurotrophic Factor), NT3 (Neurotrophin-3) and NT4/5 (Neurotrophin-4/5); and TRKC binds NT3. Out of these, TRKA forms a high-affinity binding site for NGF, the best characterized member of the Neurotrophin family, which is involved in a variety of signaling events such as cell differentiation and survival; growth cessation; and apoptosis of diverse peripheral and central neurons. Of the five domains comprising its extracellular portion, the immunoglobulin-like domain proximal to the membrane (TRKA-d5 domain) is necessary and sufficient for NGF binding and subsequent signaling. Thus, NGF-TRKA complex serves as an important messenger that delivers the NGF signal from axon terminals to cell bodies of the sympathetic and some sensory neurons (Ref.1 & 2).
NGF induces the dimerization and autophosphorylation of tyrosine residues on TRKA receptor which provides docking sites for signal transduction molecules, associated with the activation of MAPK (Mitogen-Activated Protein Kinase), PI3K (Phosphatidylinositiol-3-Kinase), PLC-Gamma
(Phospholipase-C-Gamma), SHP2 (SH2-containing Protein Tyrosine Phosphatase-2),and SNT (Suc-Associated Neurotrophic Factor-Induced Tyrosine Phosphorylated Target) signaling pathways. The activation of these pathways regulates the expression of NGF-inducible genes and contributes to NGF-induced neurite outgrowth. Activation of TRKA results in rapid association of TRKA with PI3K, PLC-Gamma (Phospholipase-C-Gamma), and the phosphotyrosine phosphatase SHP1 (SH2-Containing Protein Tyrosine Phosphatase-1) and the adaptor proteins. TRKA tyrosine phosphorylates the adaptor proteins GAB1 (GRB2-Associated Binding Protein-1) and SHC (SH2 Containing Protein), resulting in their association with GRB2 (Growth Factor Receptor-Bound Protein-2)-SOS, a complex that enhances the rate of GDP-GTP exchange on Ras, leading to Ras activation. Activated Ras recruits and activates Raf, followed by the activation of MEK1/2 (MAPK/ERK Kinases. MEKs in turn, phosphorylate the MAPKs: ERK1, or ERK2 (Extracellular Signal Regulated Kinases). Phosphorylated ERK1/2 then participate in at least two cascades. ERK1/2 may translocate into the nucleus, where they phosphorylate the transcription factor Elk, or they may phosphorylate the RSK
(Ribosomal-S6-Kinase). Phosphorylation of RSK
leads to its nuclear translocation and consequent phosphorylation of CREB
(c-AMP Response Element Binding Protein). Phosphorylated CREB
binds to the transcriptional coactivator protein CBP (CREB Binding Protein) and the SRF-Elk complex creating an extended transcriptional factor complex leading to c-Fos transcription. Crk and SNT also modulate MAPK activity in some neuronal cells. The small G-protein Rap1
that lies downstream of Crk, links to the MAPK cascade by physically binding to and activating Raf, whereas, SNT contributes to Ras activation. The roles of PLC-Gamma include the regulation of intracellular Ca2+ and PKC-Delta (Protein Kinase-C-Delta), which is catalyzed by the formation of DAG (Diacylglycerol) and IP3 (Inositol Triphosphate) from PIP2 (Phosphatidylinositol-4,5-Bisphosphate) (Ref.2, 3, 4 & 5).
and CDC42, members of the Rho family GTPase proteins, actively participate in neurite outgrowth and differentiation. Activated TRKA gives rise to Actin Polymerization, by activating the Rac
-->RhoA-->ROCK (Rho-Associated Coiled-Coil-Containing Protein Kinase) pathway. Actin polymerization and formation of filopodia or lamellipodia are important events leading to neurite differentiation. Another type of Rho family GTPase, RhoG, transduces the Ras signal to Rac1 and CDC42 activation in neuronal cells. Neurite outgrowth requires not only GTPase cycle of Rac1 and CDC42 activities but also their appropriate localization to the sites where neurites are formed and extend, and RhoG may function to appropriately promote the GTPase cycle of Rac1 and CDC42 and localize them to the protrusion sites. Trio (Triple Functional Domain-PTPRF Interacting) is an upstream regulator of RhoG for the NGF-induced neurite outgrowth. Trio-->RhoG-->Rac1 and Trio-->RhoG-->CDC42 pathways form a central signaling route for neurite outgrowth (Ref.6).
TRKA-activated PI3K activity primarily regulates survival signaling through anti-apoptotic responses in neurons. PI3K enzymes are normally present in cytosol and can be activated directly by recruitment to activated TRKA receptor, or indirectly through activated Ras. PI3K catalyzes the production of 3-phosphoinositides, including PIP3 (Phosphatidylinositol-3,4,5-Trisphosphate), which bind to and activate PDK-1 (Phosphoinositol Dependent Kinase-1). PDK-1 regulates neuronal survival by simultaneous activation of two pathways: PKC pathway (by activating PKC-Zeta), and by the activation of Akt pathways. PDK-1 associates with and phosphorylates the serine-threonine kinase Akt. Akt signaling through various downstream signaling intermediates culminates in neuronal cell survival and differentiation. Akt suppresses neuronal cell death by various mechanisms, which include: phosphorylation and inactivation of GSK3Beta (Glycogen Synthase Kinase-3-Beta), FKHRL1 (Forkhead Transcription Factor) and of cell death proteins BAD (BCL2 Associated Death Promoter), and Caspase9; and the fifth one involves an indirect suppression of the levels of p53, the tumor suppressor and neuronal death sensor protein. Akt
Pathway directly or indirectly also affects transcription factors CREB and NF-KappaB
(Nuclear Factor-KappaB), which are involved in regulating cell survival. The phosphorylation of CREB and IKK
stimulates the transcription of pro-survival factors, whereas the phosphorylation of BAD, FKHRL1 and Caspase9 inhibits the pro-apoptotic pathway. BAD and CREB are also the targets of RSK that might act synergistically with Akt
to activate the survival pathway (Ref.2, 4 & 5). Sometimes CREB phosphorylation requires activation and endocytosis of TRKA located at the axon terminals. Within distal axons, activated TRKA are internalized into vesicles. These vesicles give rise to signaling endosomes that are transported from distal axons back to the cell bodies. The binding of NGF to TRKA receptors at the axon terminal results in local activation of ERK1/2 and ERK5 within the axon terminal and also activation of ERK5 in the cell body. Components of the ERK5 cascade are transported in endosomes together with TRKA. The activated ERK5 moves from the cell body to the nucleus, where it mediates CREB phosphorylation and gene expression. The ERK5 pathway is required for CREB phosphorylation following neurotrophin stimulation of distal axons, whereas both ERK1/2 and ERK5 contribute to CREB
activation following neurotrophin stimulation of cell bodies. SHP2 and SNT also appear to play roles in neuritogenesis. Activation of these signaling molecules culminates in downstream events involved in the promotion and maintenance of neuronal survival and differentiation (Ref.3 & 7).
In the nervous system, a critical balance of cell survival and death is being continually maintained. While NGF stimulation of TRKA leads to the sustained activation of the cell survival pathways leading to neurite outgrowth, withdrawal of NGF from TRKA culminates in apoptosis in sympathetic neurons. The removal of NGF results in a decrease in MAPK and PI3K activities, followed by a series of early metabolic changes including the increased production of ROS (Reactive Oxygen Species), decreased glucose uptake and decreased RNA and protein synthesis. NGF withdrawal induces JNK (c-Jun N-terminal Kinase) activation and the phosphorylation of c-Jun, which in turn induces the expression of DP5, a member of the BCL2 family containing a BH3-domain only, and p53 in NGF-deprived neurons. It is the synthesis of DP5-related proteins that is vital to the execution of the cell death programme. DP5 and p53 are required to help BAX (BCL2 Associated X-protein) move from its location in the cytosol to the mitochondria, causing mitochondrial damage, which results in the release of CytoC (Cytochrome-C). APAF1 (Apoptotic Protease Activating Factor-1) forms a complex with mitochondrial-released CytoC and Caspase9 to mediate the activation of Procaspase9. Activated Caspase9 in turn cleaves and activates Caspase3, resulting in apoptosis. Thus, a lack of NGF signaling also induces a non-nuclear competence-to-die pathway that facilitates the formation of the CytoC-APAF1-Caspase9 complex, resulting in Caspase9 activation (Ref.8 & 9).
Overproduction of cells followed by massive cell death is a common phenomenon in the mammalian nervous system. Neuronal cell death is associated with the pathogenesis of several neurodegenerative diseases such as Alzheimers disease, Parkinsons disease, amyotrophic lateral sclerosis, and stroke. The NGF-TRKA complex plays a role in the repair, regeneration, and protection of neurons, and as such could serve as a therapeutic agent in the neurodegenerative conditions. The NGF-TRKA pathway has also been suggested to play a role in other physiological systems and tissues such as the immune system (Ref.2 & 10).