A functional DyneinMicrotubule Network Is Required for NGF signaling through the Rap1MAPK pathway

Neurons are known to be the receptors and conveyors of signals throughout our body.One of the processes that have gotten much attention is the mechanism which governs the transport of neurotrophin to the neurons. NTs have been proven to be essential to the survival and proper function of the neurons by the works of many studies such as that of Campenot and MacInnis (2004) which have identified that the absence of NTs results to destruction of the cell body.Establishing the importance of NTs on neurons was one thing. finding out how it is transported to the cell body was another. Shedding light on the problem are the works of Sofroniew, Howe and Mobley (2001) and Ginty and Segal (2002) which both found evidence for the retrograde transport hypothesis. According to the hypothesis, the binding of the nerve growth factor (NGF) to the receptor kinase A (TrkA) activates the latter at the axon (AX) terminal. The NGF -TrkA then enters the cell body thru clathrin-mediated and independent pathways. However, much still has to be learned about the retrograde transport process.In 2003, the role of dynein-based transport of NT signals with the use of intact microtubules was identified by Delcroix and his colleagues. The said study provided the impetus for further research on the trafficking and signaling mechanism of the NGF leading to the conception and completion of this research paper that it is being reviewed.In this paper, it is hypothesized that there are certain steps that govern the transport of TrkA/Rap1 signaling endosome. The transport process begins when NGF binds to TrkA at the cell surface to activate the receptor. Then, the NGF-TrkA complex passes thru clathrin-mediated or nonclathrin- mediated pathways. The transfer and docking process of the NGF-TrkA is facilitated by Rap1, a small guanosine triphosphatase, which transduces signaling in early endosomes thereby ensuring that the p44/p42 mitogenactivated protein kinases (MAPK1/2) signaling pathway is activated. It will be shown that dynein- and microtubule-based transport is involved and necessary for the trafficking, signaling and accumulation of NGF, Trka and Rap 1 and MAPK1/2.

3.0 Major Findings
When NGF treatment was initiated, the microtubules were visually observed (microtubules and Rap1 were stained). Normal cells with the complex network of microtubules intact were found to have Rap1 in its perinuclear region. However, diffusing and disintegrating the microtubules resulted to the absence of Rap1 in the perinuclear region. The next was to test whether the microtubules had any bearing on the traffic of TrkA. Nocodazole was introduced in the experimental cells to diffuse the microtubules. When the cells were treated with NGF, visual observation of immunostained TrkA revealed that it had not accumulated in the perinuclear region and remained dispersed. Cells which were not treated with nocodazole had TrkA accumulation in the said region. Although this was the case, it was found that the disruption of microtubules did not affect the magnitude or the duration of TrkA activation induced by NGF.
Nocodazole was also used to determine whether disruption of the microtubule network suppresses Rap1 and MAPK1/2 signaling. Results confirmed that Rap1 activation was suppressed. MAPK1/2 activation was unaffected in the initial phase but was markedly reduced at the 30 min phase of NGF treatment. In addition, the researchers also found that the dispersion of microtubules caused by nocodazole partially suppressed the phosphatidylinositide 3-kinase (PI3K)/Akt pathway.
To determine the role of dynein in retrograde transport, the 50kD subunit of dynein-dynactin complex was over expressed. It was previously found Burkhardt, Echeverri , Nilsson and Vallee (1997)