1) When laminin stimulates neurite outgrowth, is gene expression in the PC12 affected? If so which gene or genes are activated by laminin? Reference your answer.
In the study, “Laminin stimulates protein tyrosine dephosphorylation in PC12 cells,” conducted by Weeks et al. in 1999, the laminin signal transduction mechanisms were explored and the effect of laminin on the gene expression in neuronal cells was observed after the cells were given laminin treatment. “The results demonstrated that laminin-mediated neurite outgrowth involves protein tyrosine dephosphorylation and suggested that the mechanism may have specificity to neuronal cells” (Weeks et al. 1999) According to an experiment they had previously conducted in 1995, the authors stated that “while neurite-promoting laminin domains and neuronal cell laminin receptors are known, little is known about the genes induced by laminin during neurite outgrowth.” (Weeks et al. 1995)
However, as a result of the laminin treatment, increased levels of steady-state mRNA and protein expression of mitochondrial proteins such as cytochrome and chargerin II were observed. Furthermore, an increase in the transcription and replication of the mitochondrial genome during the differentiation of neuroblastoma cells was also observed. Moreover, the neuronal mitochondria were also seen to localize at the synapse which increased the synaptic cytochrome-c activity. This showed a correlation with the increase in the current frequency of the action potentials at the neuron terminal. Therefore, it was concluded that laminin does encourage rapid stimulation of mitochondrial protein synthesis in neuronal cells. Past studies have suggested that mitochondrial abnormalities may be responsible for numerous neurodegenerative diseases. (Weeks et al. 1995). And in the experiment conducted in 1999, Laminin has shown to activate the c-fos and c-jun oncogenes in PC12 cells and the expression of cytochrome b and an ATP synthase in the neural x glial hybridoma NG108-15 cells. (Weeks et al. 1999).
2) PC12 cells are neural crest derived. Compare and contrast the role of laminin and fibronectin in neural crest cell migration into the periphery of the body plan. Reference your answer.
Laminin and Fibronectin are two large glycoproteins. They are responsible for the organization of the matrix and the cells into an orderly structure. Fibronectin helps mark the path on which certain migrating cells are to travel. These fibronectin “roads” direct the germ cells and lead them to the gonads. Similarly, they also direct the heart cells to the embryo’s midline.
An experiment conducted by Ravasio et al, demonstrated that the cells migrate more quickly on fibronectin (FN) than on Laminin (LN) or Type I collagen substrates. This conclusion was made when the migrating cells were given the option to migrate on FN strips, LN strips, and glass. The results showed that the cells gave preference to FN among the three options.
Migration on the cell binding part of FN was inhibited by the antibodies, but this inhibition could be reversed by adding exogenous FN subsequently. The migration on FN was deemed as random and it showed little to no persistence; however, for cells with high densities, directionality was observed since there was more frequent contact with the FN strip. For the migration of cells on FN-containing matrices, the in vitro rate was 50 microns/h, which was similar to the migration rate of the cells on the narrow strips of FN-containing extracellular matrix in migratory pathways, in vivo. These results showed that FN is vital for the migration of neural crest cells. Furthermore, it showed that high cell densities of neural crest cells in the transient, narrow migratory pathways found in the embryo are also important for effective directional migration. So their major findings were: (a) avian trunk neural crest cells preferentially utilize FN over LN or collagen for motility; (b) motility are inhibited by anti-FN cell-binding fragment antibodies in both simple and complex matrices; (c) in vitro manipulation of crest cells can produce cells capable of utilizing LN for adhesion but not for migration; (d) directional migration on FN requires an unusually high cell density; (e) rates of directional migration on purified FN were half those found in vivo, and rates equal to those found in vivo require other as yet undefined factors in the extracellular matrix (Ravasio et al. 1983). By contrast in another study, migration was significantly greater on laminin-1 than on the other substrate molecules in avian. In other words, the preferential migration of neural crest cells along basal laminae can be accounted for by the ability of laminin-1 to promote migration with great efficiency (Desban and Duband 1997). In a third study, in laminin alpha5 mutant mice, neural crest migratory streams appear expanded in width compared to wild type. Conversely, neural folds exposed to laminin alpha5 in vitro show a reduction by half in the number of migratory neural crest cells (Coles et al. 2006).
3) Recently, it has been suggested that gut bacterial peptides may cause Parkinson’s disease. I hypothesize that these peptides cause altered gene expression in neurons. Describe an experiment to test this hypothesis. Provide an experimental design, hypothetical results that support the a) hypothesis and b) the null hypothesis, describe both of these results, what technique was used and your conclusions.
Parkinson’s disease (also known as Parkinsonism) is a neurodegenerative, movement disorder that results from the loss of selective dopaminergic neurons in the the specific brain area. The specific brain area consists of the basal ganglia and the substantia nigra. If a bacterial peptide is suspected to cause the loss of dopaminergic neurons, an experiment in cultured dopaminergic neurons must be designed. Moreover, the neuron culture must be treated with purified bacterial protein. Then a protein or a gene must be chosen that is related to an apoptotic pathway such as caspase-3, caspase-9.
In the first step of the experiment, the neuronal cells will be closely monitored to check if they are dying because of the bacterial protein treatment. This can be determined by photomicrography and the color change of the media (which would contain a pH indicator) the cells are in. If they are dying, it means that the treatment is inducing the inflammatory cytokines that will increase the apoptotic gene expression. The experiment would entail taking gut cell line, treating it with bacterial protein, collecting the supernatant, and then putting the supernatant secreted into a cultured neuron. If the neuron cells start to die, check for the expression level of apoptotic proteins. If in the experiment the neurons show more expression of the apoptotic genes, then conduct at least four more trials using different cell batches of similarly cultured dopaminergic neurons. For example, more expression of alpha synuclein protein can be because of an elevated level of cytokines in the brain, which in turn could have induced more expression of the alpha synuclein protein which would lead to oxidative stress and ultimately the apoptotic death of the cell. To get the results, a western blot can be performed to check the expression of the proteins in the experiments. If cell death is observed, the Western Blot will be positive, and the null hypothesis that the bacterial peptide does not cause the loss of dopaminergic neurons will be rejected. The results will be significant to accept the alternative hypothesis i.e. the bacterial peptide does cause the loss of dopaminergic neurons.
I think that the experiment will be similar to the one we conducted with Vitamin C and PC12 cells, but since we’re testing the expression of a protein, simple photomicrography won’t suffice, so we’ll get results by conducting a Western Blot to determine which hypothesis is accepted.