Molecular Biology Neurodegenerative Disease

Question: Discuss about theMolecular Biologyfor Neurodegenerative Disease. Answer: Introduction In this research topic the role of autophagy in the neurodegenerative disease is investigated taking the mouse model C57BL/6j which must contain the GFP-LC3 transgene and also expresses GFPLC3. In this study an outline of the rationale for using mouse model C57BL/6j is performed. The light chain 3 (LC3) is a microtubule- associated protein which is widely used for the monitoring autophagy. This is an approach of detection of the conversion procedure of LC3 conversion from LC3-I to LC3-II by the process of analysis of the immunoblot. The reason behind this that the amount LC3-II is evidently interrelated with autophagosomes number. Many pathological and physiological processes involves the implication of the autophagy. However, autophagy degrades the LC3-II and makes an interpretation immunoblotting result of the LC3-II problematic (Black et al. 2014). Additionally, it sometimes happens that the quantity of the LC3 at a certain time point is not able to indicate the autophagic flux. C onsequently it becomes very important to measure the amount of the LC3-II that is delivered to the lysosomes and also a comparison should be made on the LC3-II levels in the conditions like the presence and absence of the lysosomal protease inhibitors. There is another problem exist with this method that is the LC3-II have a tendency to be more sensitive to be detected by the process of immunoblotting in comparison to the LC3-I (Lau et al. 20113). Among the all markers of the autolysosomes and autophagosomes the GFP-LC3 is the best known technique. In this method the green fluorescence tagged with the LC3 protein is utilized for the detection. The reason behind this that the techniques that are involving other markers can provide important snapshot of the autophagic compartments within the cells but they have some limitation in the capability to measure the autophagic activity rates. The autophagic activity is also known as the autophagic flux. One of the approach that is used for the elimination of this problem is the comparison of the markers GFP-LC3 or LC3-II in the condition of presence and absence of the lysosomal inhibitors (Sreelatha et al. 2013). The rationale of this experiment is that a stimulus will increases LC3 by the process of inducing autophagy and this will subsequently have increased effect when there is a disruption of the degradation by the inhibitors. On the other the blocking of this pathway causes accumu lation of the LC3 proteins. This is the best way for the assay of the autophagic flux (Weikel et al.2013). The process of the identification of the autophagic flux is very important to evaluate and distinguish between the induction and the suppression of the autophagy. The proposed autophagic flux assay is the western blot analysis for the detection of the GFP fragmentswhich are free and are resulting from the GFP-LC3 degradation inside the autolysosome. Though the accurate dynamics of the GFP-LC3 during the process of the autophagy is not very transparent and also the representation of this assay in mammalian cells is very inadequate. It is found here that the lysosomal acidity is a very essential regulating factor of the step wise degradation of the GFP-LC3. In this process the free GFP fragments are generated first but at the time of the lysosomal acidity is in a moderate condition the GFP fragments starts to accumulate. This moderate type of the condition of the lysosomal acidity can be achieved during the time of rapamycin treatment. It was found that the GFP fragments concentration is increased whenthere is the presence of the autophagy inhibitors like the E64D plus pepstatin A and chloroquine. The amount of the GFP fragments is dependent on the concentrations of these inhibitors (Yue et al. 2013). Early studies reveal that RAB7 plays a very important role and it is related to the fusion of the autophagosomes and with lysosomes. It has been confirmed that RAB7 perform the autophagic flux assays. By the process of the blocking off the lysosomal acidification. This the way in which chloroquine stabilizes the MAP1LC3B-II and also allows for the estimation of the autophagic flux. The therapeutic approaches that would you done to make the autophagic flux efficient in the disease mice or to reduce the efficiency of the autophagic flux. The autophagic flux is only produced due to the moderate condition of the lysosomal acidity. This lysosomal acidity is responsible for the autophagic flux. To block the autophagic flux the molecule RAB7 must be eliminated because the elimination of the RAB7 there occurs an accumulation of the chloroquine which stabilizes MAP1LC3B-II that is responsible for the increase in the autophagic flux (Zhang et al. 2014). Reference Black, C., Henrie, B., Pei, S. and Boudina, S., 2014. Despite Impaired Autophagic Flux Cardiac and Mitochondrial Functions are Preserved in Aged Mice Lacking Insulin Receptors in the Whole Heart. Circulation, 130(Suppl 2), pp.A18841-A18841. Lau, A., Zheng, Y., Tao, S., Wang, H., Whitman, S.A., White, E. and Zhang, D.D., 2013. Arsenic inhibits autophagic flux, activating the Nrf2-Keap1 pathway in a p62-dependent manner. Molecular and cellular biology, 33(12), pp.2436-2446. Sreelatha, A., Bennett, T.L., Zheng, H., Jiang, Q.X., Orth, K. and Starai, V.J., 2013. Vibrio effector protein, VopQ, forms a lysosomal gated channel that disrupts host ion homeostasis and autophagic flux. Proceedings of the National Academy of Sciences, 110(28), pp.11559-11564. Weikel, K.A., Ido, Y. and Ruderman, N., 2013. Decreased autophagic flux as a mechanism of glucose-and fatty acid-induced dysfunction in human aortic endothelial cells. The FASEB Journal, 27(1 Supplement), pp.565-6. Yue, W., Hama, A., Tonelli, G., Bauvy, C., Nicolas, V., Tharinger, H., Codogno, P. and Mehrpour, M., 2013. Inhibition of the autophagic flux by salinomycin in breast cancer stem-like/progenitor cells interferes with their maintenance. Autophagy, 9(5), pp.714-729. Zhang, X., Chen, S., Song, L., Tang, Y., Shen, Y., Jia, L. and Le, W., 2014. MTOR-independent, autophagic enhancer trehalose prolongs motor neuron survival and ameliorates the autophagic flux defect in a mouse model of amyotrophic lateral sclerosis. Autophagy, 10(4), pp.588-602.