Research Paper
- 1. The Absence of Functional Peroxisomes within the Grey Matter of Multiple Sclerosis Patients Virginia Western Community College Mitchell Shelton 12-19-2014
- 2. Shelton 1 Eukaryotic organisms are highly advanced. Our bodies contain many systems that work together to promote one common goal: life. This is a phenomenal feat of evolution, as our bodies rarely malfunction or break down. However, in some instances, the systems of the body can accidently become faulty and work against each other, causing an extremely hostile environment inside of the body. For example, in multiple sclerosis, instead of the immune system attacking foreign bodies, or antigens, it attacks the body’s cells, particularly those that make up the central nervous system (Bright, Natarajan, Muthian, Barak, & Evans, 2003). Multiple sclerosis is categorized by the inflammation of the brain and demyelination of the nerve fibers—this leads to an interruption of communication within the body (Schmierer, et al., 2010). Specifically, the immune system is “re-wired” to attack nerve fibers, as well as the fatty substance that surrounds the fibers, termed myelin (What is MS?, n.d.). When the insulation around the nerve fibers (myelin) or the fibers themselves, for that matter, are destroyed, the brain is unable to process, interpret, or carry information to the rest of the body. In the brain, there are regions of matter termed grey matter that contain the majority of the brain’s nerve cell bodies, and subsequently the nerve fibers, and these areas appear in large patches throughout the brain’s lobes; as a patient progresses through various stages of multiple sclerosis, his or her regions of grey matter become filled with lesions. These lesions are brought about by the reduction of the nerve cell fibers and their insulating myelin covers, which effectively hinders normal brain function and central nervous system communications (Mandal MD, 2014). Neural cells (neurons) are highly specialized differentiated cells, and are responsible for the electrical impulses that carry information to and from the brain to the body (Alberts, et al.,
- 3. Shelton 2 2014). As eukaryotic cells, neurons contain a membrane bound nucleus and many organelles; that being said, they contain a highly useful organelle that is still being studied for its multiple functions today—the peroxisome. Peroxisomes are spherical organelles that perform multiple metabolic functions in the cell, such as the oxidation of carboxylates and fatty acids and the metabolism of oxygen (Hulshagen, et al., 2008). Peroxisomes are essential for normal brain development due to the massive amount of reactions that take place as a neuron “fires” an electrical signal—without peroxisomes, the cell body could not break down toxins or metabolize molecules (Bottelbergs, et al., 2010). Earlier this year, researchers of multiple sclerosis discovered that neurons in the grey matter of patients lacked functional peroxisomes. This began many experimental efforts to understand the implications of these missing organelles (Gray, et al., 2014). One research group was interested in not only why neural peroxisomes were absent, but also as to if their absence had any consequences on the cells (in addition to the brain itself). Gray, et al., examined the grey matter of many multiple sclerosis patients, and discovered that there was an abnormally low amount of functional peroxisomes; that being said, the group set out to prove that the grey matter of multiple sclerosis patients contain an absence of functional peroxisomes and to show how this promotes disease progression (Gray, et al., 2014). The researchers’ main objective was to determine whether there was a lower amount of peroxisomes in the grey matter of multiple sclerosis patients in contrast to normal grey matter (Gray, et al., 2014). This hypothesis should be supported, as research in the past few years has supported this accusation in mice, showing that without functional peroxisomes in the central nervous system, neural cells are broken down and the myelin sheaths surrounding neural fibers are disintegrated (Hulshagen, et al., 2008). To begin the project, the research group gathered
- 4. Shelton 3 frozen brain samples of grey matter from multiple sclerosis patients, in addition to samples of normal grey matter (Gray, et al., 2014). The frontal and parietal lobes of the brain were targeted in the selection of samples, due to their large amount of grey matter and their uses in brain and central nervous system communications (Trapp, et al., 1998). The samples were taken from multiple disease progression states and stained with antibodies to myelin and peroxisomal- membrane proteins; this staining will show the presence of myelin surrounding the neural fibers and the presence peroxisomal membrane proteins. Peroxisomal membrane protein 70 (PMP70) is an ATP binding transporter, which is responsible for the import of the fatty acids and molecules that it is to break down (Bottelbergs, et al., 2012). Since this protein is likely responsible for peroxisome biogenesis, the researchers decided to focus on this protein in order to quantify peroxisomal distribution in multiple sclerosis grey matter and control grey matter (Gray, et al., 2014). The labelling of the brain sections was performed using an enzyme-linked immunosorbent assay (ELISA); the primary antibodies were added and allowed to incubate overnight in favorable conditions, and the following morning the unbound primary antibodies were washed off and secondary antibodies were added and allowed to bind to the primary antibodies (Gray, et al., 2014). Additionally, the samples were coated with several series of buffers and other chemicals to enhance the areas of the grey matter that were either lesional or non-lesional. After staining, the regions of grey matter could be randomly selected by a computer system and the lesional and non-lesional areas could be quantified (Gray, et al., 2014). In control brains, where lesions are not present, the peroxisomes of neurons were increasingly abundant and had an even distribution throughout the tissue. Additionally, control brains showed a normal amount of myelin surrounding the nerve fibers, which was expected due to the lack of lesions or
- 5. Shelton 4 presence of a neurodegenerative disease. Conversely, the grey matter regions of brains that have multiple sclerosis showed an extremely low number of PMP70 expression in neural peroxisomes. Since these brains contained lesions brought on by the progression of multiple sclerosis, demyelination was constant and showed a decreased extension of nerve fibers throughout the tissue (Gray, et al., 2014). In order to test to see if peroxisomes and the PMP70 protein were being coded for in mRNA, the research group produced complementary DNA (cDNA) from the neural cells’ transcriptomes. Using random sections of the grey matter of normal brains and multiple sclerosis affected brains, mRNA was extracted and isolated by means of cell lysing, and the mRNA was prepared with DNase to remove any DNA fragments before the production of cDNA (Gray, et al., 2014). The researchers used the general procedure of cDNA production: the lysing of cells, the applying of a poly-T primer tail, and the sequencing of the strands using reverse transcriptase and DNA polymerase (Alberts, et al., 2014). The coding regions of the DNA were revealed and those for peroxisome production were isolated for further testing. The researchers quantified gene expression using the method presented above and took the mean of both the control group and the multiple sclerosis grey matter group (Gray, et al., 2014). The production of cDNA showed that in control brains, the genes that code for peroxisomes were producing normal amounts of the mRNA, and therefore, a normal number of peroxisomes; additionally, the mRNAs for PMP70 were very much apparent and were being produced in normal amounts. In contrast to the control brains, the multiple sclerosis brain sections were analyzed for the presence of peroxisomes and PMP70. The quantitative analysis of the cDNA showed that the grey matter of multiple sclerosis patients was severely lacking functional peroxisomes, and subsequently, PMP70 (Gray, et al., 2014).
- 6. Shelton 5 The researchers decided to perform another experiment so that the functions of peroxisomes could be compared in the grey matter of multiple sclerosis brains and the grey matter of control brains. Peroxisomes are essential for breaking down very long chain fatty acids (VLCFA); if the organelles did not break down the large fatty acids, then the cell body would become overwhelmed and likely die (Hulshagen, et al., 2008). Brain tissue that contains the organelles can therefore be easily compared against those that do not. The researchers choose nine control brains and nine multiple sclerosis affected brains, and isolated the fatty acids by extraction and evaporation. In order to be able to isolate the VLCFAs, transesterification was used, which is the process of producing fatty acid methyl esters from normal fatty acids (Gray, et al., 2014). Now that the VLCFAs could be used in laboratory techniques, the researchers quantified total VLCFAs by using stable-isotope dilution capillary gas chromatography—mass spectrometry (Gray, et al., 2014). Using capillary gas chromatography columns, the researchers could isolate their target VLCFAs from other acids and proteins that normally are identical to the VLCFAs (it is similar to protein chromatography columns, such as those used in GFP isolation). Finally, in order to produce a definite count of VLCFAs in brain matter, mass spectrometry was used to detect the concentration of VLCFAs in the grey matter of multiple sclerosis brains and in the control brains (Struys, et al., 1998). The experiment presented data that showed a high concentration of VLCFAs present in multiple sclerosis grey matter, making the connection that there was a distinct decrease in peroxisomes, since they were not present to break down these large molecules. On the converse, control brains showed a very miniscule concentration of VLCFAs in the grey matter, due to the normal amount of peroxisomes that were present in the neural cell bodies (Gray, et al., 2014).
- 7. Shelton 6 The conclusion of the experiment came with large amounts of supporting data to link peroxisome deficiency in multiple sclerosis grey matter and concur that peroxisomes are needed for correct central nervous system functions. The results came from the staining & gas chromatography, ELISA, and cDNA production. In figure 1, part A shows normal grey matter in control brains, while part B shows grey matter of multiple sclerosis patients. The neurons were stained with a dye that shows areas that contain PMP70, and are clearly shown to be prominent throughout the neurons in part A, while severely lacking in part B; this data suggests that neurons lack PMP70, and therefore peroxisomes altogether. The black bar represents 100µm (Gray, et al., 2014). In figure 2, multiple sclerosis grey matter is compared with the grey matter of control brains. The control grey matter showed a higher mean count of PMP70, showing that the neurons were responding normally and performing normal functions. The multiple sclerosis grey matter showed a lower mean count of PMP70, which confirms the absence of peroxisomes (Gray, et al., 2014). Finally, in figure 3, control grey matter is compared with grey matter of multiple sclerosis patients. The data in the graph was found by gas Figure 1 (Gray, et al., 2014) Figure 2 (Gray, et al, 2014)
- 8. Shelton 7 chromatography and shows the amount of VLCFAs present inside of the neural cells of the grey matter. The control grey matter showed an average of 0.8µmol/mg of VLCFAs; consequently, the density of VLCFAs in multiple sclerosis grey matter was a high 1.2µmol/mg. In conclusion, without peroxisomes present, VLCFAs are able to accumulate inside neural cells, which causes the cells to become overwhelmed—they likely die due to lysing (Gray, et al., 2014). The experiment provided sufficient data to conclude that the multiple sclerosis grey matter contained a substantially lower amount of PMP70, and therefore a decrease in the amount of peroxisomes in the neural cells. Additionally, the experiment showed a larger concentration of VLCFAs inside the grey matter of multiple sclerosis affected neural cell bodies, showing that peroxisomes are absent in the tissue; peroxisomes are needed to breakdown the extremely long fatty acid chains. The hypothesis proposed by the study was supported, as the experiment concluded that the grey matter of patients affected by multiple sclerosis contained a significantly lower level of gene expression for peroxisomes, as well as a reduction in the number of peroxisomal proteins, such as PMP70 (Gray, et al., 2014). The experiment was successful in respect to how it supported the idea that peroxisomes are less abundant in the grey matter of patients affected by multiple sclerosis. Interestingly, the data also revealed a new piece to the puzzle of multiple sclerosis disease progression. A quantitative analysis of affected grey matter shows that as the disease progresses, the levels of Figure 3 (Gray, et al., 2014)
- 9. Shelton 8 PMP70 actually decrease. As shown in figure 4, the mean counts of PMP70 were higher in the earlier stages of the disease. During the duration of the disease, the mean counts of PMP70 actually decreased, showing that as time goes on, peroxisomes become less and less abundant in the grey matter of affected patients. This data supports the findings of the main experiment, as it shows the reduction of neural peroxisomes as multiple sclerosis progresses (Gray, et al., 2014). This preceding data has the potential to stem into an entirely different study, as researchers could now focus on what happens if PMP70 levels in grey matter did not decrease as multiple sclerosis progresses. Consequently, researchers could try experiments on mice, due to the fact that as their central nervous system loses peroxisomes, their neural bodies lose myelin and the neural fibers are broken down—which is exactly what happens in humans when they lose neural peroxisomes (Hulshagen, et al., 2008). This promotes the study of possible pharmaceuticals, such as a medicine that prevents the malfunctioning of peroxisomes by binding to PMP70 receptors; if a drug helps to prevent the breakdown of neural peroxisomes in mice, then it is very possible that this could also be used in humans affected by multiple sclerosis. This study showed that the grey matter of multiple sclerosis patients contain an extremely low number of functioning peroxisomes, which is shown to be a possible connection in the progression of the disease. The study sheds light onto these usually overlooked organelles, and how without them, we can be faced with extreme disorders, and even death. The course text book, Essential Cell Biology, 4th Edition, sheds light on the importance of these organelles in Figure 4 (Gray, et al., 2014)
- 10. Shelton 9 chapter 15, page 498, by commenting on what peroxisomes do, what happens when they are absent, and a disease that is relevant when discussing the absence of peroxisomes in a cell. If we can begin to understand these extremely complex organelles, we can possibly discover the answers to several questions that arise in the fields of cell biology and medicine.
- 11. Shelton 10 Works Cited Alberts, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J., Raff, M., . . . Walter, P. (2014). Essential Cell Biology, Fourth Edition. New York: Garland Science, Taylor & Francis Group, LLC. Bottelbergs, A., Verheijden, S., Hulshagen, L., Gutmann, D. H., Goebbels, S., Nave, K.-A., . . . Baes, M. (2010). Axonal Integrity in the Absence of Functional Peroxisomes from Projection Neurons and Astrocytes. GLIA, 1532-1543. Bottelbergs, A., Verheijden, S., Van Veldhoven, P. P., Just, W., Devos, R., & Baes, M. (2012). Peroxisome deficiency but not the defect in ether lipid synthesis causes activation of the innate immune system and axonal loss in the central nervous system. Journal of Neuroinflammation, 61-89. Bright, J. J., Natarajan, C., Muthian, G., Barak, Y., & Evans, R. M. (2003). Peroxisome Proliferator-Activated Receptor-γ-Deficient Heterozygous Mice Develop an Exacerbated Neural Antigen-Induced Th1 Response and Experimental Allergic Encephalomyelitis. The Journal of Immunology, 5743-5750. Gray, E., Rice, C., Hares, K., Redondo, J., Kemp, K., Williams, M., . . . Wilkins, A. (2014). Reductions in Neuronal Peroxisomes in Multiple Sclerosis Grey Matter. Multiple Sclerosis Journal, 651-659. Hulshagen, L., Krysko, O., Bottelbergs, A., Huyghe, S., Klein, R., Van Veldhoven, P. P., . . . Baes, M. (2008). Absence of Functional Peroxisomes from Mouse CNS Causes Dysmyelination and Axon Degeneration. The Journal of Neuroscience, 4015-4027. Mandal MD, A. (2014, October 29). What is Grey Matter? Retrieved from News Medical: http://www.news-medical.net/health/Grey-Matter-What-is-Grey-Matter.aspx
- 12. Shelton 11 Schmierer, K., Parkes, H. G., So, P.-W., An, S. F., Brandner, S., Ordidge, R. J., . . . Miller, D. H. (2010). High Field (9.4 Tesla) Magnetic Resonance Imaging of Cortical Grey Matter Lesions in Multiple Sclerosis. Brain, 858-867. Struys, E. A., W, J. E., ten Brink, H. J., Verhoeven, N. M., van der Knaap, M. S., & Jakobs, C. (1998). An accurate stable isotope dilution gasy chromatographic--mass spectrometric approach to the diagnosis of guanidinoacetate methyltransferase deficiency. Journal of Pharmaceutical and Biomedical Analysis, 659-665. Trapp, B. D., Peterson, J., Ranschoff, R. M., Rudick, R., Mork, S., & Bo, L. (1998). Axonal Transection in the Lesions of Multiple Sclerosis. The New England Journal of Medicine, 278-290. What is MS? (n.d.). Retrieved from National Multiple Sclerosis Society: http://www.nationalmssociety.org/What-is-MS