The Absence of Functional Peroxisomes within the Grey Matter of Multiple Sclerosis Patients

Virginia Western Community College

Mitchell Shelton12-19-2014

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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.,

2014). As eukaryotic cells, neurons contain a membrane bound nucleus and many organelles;

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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

frozen brain samples of grey matter from multiple sclerosis patients, in addition to samples of

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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

presence of a neurodegenerative disease. Conversely, the grey matter regions of brains that have

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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).

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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).

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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

Figure 1 (Gray, et al., 2014)

Figure 2 (Gray, et al, 2014)

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graph was found by gas 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).

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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

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

Figure 4 (Gray, et al., 2014)

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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

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.

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