X-linked adrenoleukodystrophy

Adrenoleukodystrophy

Abstract:

X-linked adrenoleukodystrophy (X-ALD) is a severe neurological disease which is caused by a mutation in the ABCD1 (ALD) gene which usually results in the loss of a functional protein. This results in the build up of very long chain fatty acids (VLCFAs), demyelination in the central and peripheral nervous system and impaired function of the adrenal glands. In this study by Pujol et al., the authors tested to see if the overlap between the proteins ALD and ALDR (ALD-related) and overexpression of ALDR can prevent the disease. They found that by overexpressing ALDR in mice that have a mutation in ALD, the typical phenotype of the disease can be prevented to a certain degree. This article was well written and easy to understand. A good number of controls were used and the authors were accurate in concluding that ALDR is a promising therapeutic target. Although the overexpression of ALDR was shown to compensate for the mutation in ALD, it was not proven if this treatment had any other side effects. In the future, it would be beneficial to study if the mice that were subjected to this experiment were normal in functions that were unrelated to the disease.

Overview:

X-Adrenoleukodystrophy is a severe neurological disorder in which patients show demyelination in the nervous system and impaired function of adrenals. People who have the disease show an increase in the level of very long chain fatty acids (VLCFAs) in tissues from the nervous system and adrenal cortex, as well as in plasma. There are two main types of this disease: severe childhood cerebral form (CCALD) and the adult adrenomyeloneuropathy (AMN). ALD is caused by mutations in the ALD (ABCD1) gene that inactive the protein it encodes, the ALD protein (ALDP). This protein belongs to an ATP-binding cassette (ABC) subfamily D. The ALDP is a transmembrane protein and is thought to be involved in the transport of VLCFA or into the peroxisome for oxidation (Takahashi, 2007).

Functional gene redundancy is a common theme seen in vertebrate history. Genetic redundancy means that two or more genes are performing the same function. This is thought to increase the defence of an individual against deleterious mutations by “backing-up” functionally important genes (Thomas, 1993). Evidence for overlapping function has been found in various types of important genes. In a human genetic disease, a gene from the same family that has overlapping functions with the mutated gene may be used to compensate for the loss of function. The ABCD2, (ALDR) gene is the closest known homologue to the ALD gene. There is 88% similarity between their sequences and 66% similarity between their encoded proteins (Lombard, 1996). Based on this information, ABCD2 can be used to compensate for a mutation in ABCD1.

To test if mice could have the same disease, they created a knockout in the ABCD1 gene in mice. These mice showed an increase in the level of VLCFAs in target organs. They did not show an observable phenotype until at least 6 months, despite the rise in VLCFAs. The mice developed a late onset phenotype that is similar to AMN (Pujol et al., 2002).

To test the possible redundancy of ALD and ALDR transporters in vivo, Pujol et al. took on a dual strategy. One way they tested this was by creating transgenic mice which overexpressed the ALDR gene. The second way was to generate mice that carry an inactivation of the ALDR gene. The authors then performed several experiments on these mice.

The first experiment was done to test the generation of the different genotypes of the mice that were used in this study. The first genotype they tested was the generation of ALDR-/-, inactivation of the ALDR gene. This leads to an absence of the ALDR protein, and was observed through the use of a western blot. The ALDR-/- mice were fertile and they showed no physical or behavioural abnormalities until at least 6 months. To create the double mutant ALD-/ALDR-/-, the authors crossed ALDR-/- and ALD- mice. They used a ubiquitous promoter to direct the overexpression expression of ALDR in various body tissues. This expression was found in all the tissues that were tested, therefore there was successful overexpression of ALDR. The transgenic mice are termed as tg ALDR. To create a mouse that overproduced ALDR but had the disease (ALD-/tg ALDR) the authors crossed the transgenic mice with ALD- mice. As a control, the authors also crossed the transgenic mice with the ALD+ (wild type) mice to produce ALD+/tg ALDR. These mice were all grown to maturity for the other experiments.

Next, several experiments were conducted to test if ALDR expression levels can prevent the development of several characteristics of the disease. The first characteristic they analyzed was the level of VLCFAs in tissues from the main organs and serum (body fluid) targeted by the disease. In 8-month-old mice the authors found that ALD inactivation leads to an increase in the levels of VLCFAs in the spinal cord, the adrenal gland and in the peripheral nerves. The ALDR-/- mice showed no change in VLCFA levels in the central of peripheral nervous system, but they did show accumulation in the adrenal gland. The double mutants showed even higher levels of VLCFAs than the ALD- mice. The ALD-/tg ALDR mice showed normal levels of VLCFAs in all of the organs analyzed and the ALD+/tg ALDR mice showed lower than normal levels of VLCFAs.

The next characteristic the authors studied was the histology of the adrenal glands. The adrenals of 18-month of ALD- mice showed abnormal development of connective tissues and intralysosomal needle-like cholesterol inclusions. Overexpression of ALDR in ALD- mice prevented this. The double mutants showed lipid droplets across cell layers and the cholesterol inclusions were spread through the cell and more numerous.

The third characteristic of the disease they studied was the activity of the peripheral nervous system (PNS). The authors did this by measuring two parameters: the compound muscle action potential (CMAP), representative of fast, motor conduction fibers and the sensitive nerve conduction velocities (SNCV). At 12 months, ALD- mice showed normal CMAP and SNCV waves. In double mutants, the waves were slower than normal. ALDR-/- mice showed normal waves at this age. At 20 months, ALD mutants showed further slowing of the two waves. ALDR-/- mice did not show any abnormalities and the double mutants did not show differences from the ALD- mice. Overexpression of ALDR protein prevented the slowing of the two waves in ALD- mice. The ALD+/tg ALDR mice did not show any difference from the wild type mice.

The fourth characteristic studied was the histology of the spinal cord. In a previous study, myelin and axonal degeneration was found in the spinal cord of ALD- animals. In this study, the authors wanted to determine whether myelin disturbances happen before or after axonal pathology. They did this by studying the mice at 12 and 22 months. At 22 months, ALD- mice showed signs of immune system activation. Myelin debris was found as well as signs of axonal damage, but the axonal lesions were not found in the same regions as the myelin lesions. A similar pathology was found in ALDR-/- mice and double mutants exhibited a more severe pathology. All of these features are prevented by overexpression of ALDR protein in ALD- mice. After observing the 12-month-old mice, they saw signs of axonal damage before myelin damage in both ALD- and ALDR-/- mice.

The final characteristic the authors studied was the neurological phenotype. They found that at 20 months of age, the ALD- mice showed impaired coordination and exploratory abilities through different tests. When ALDR was overexpressed in ALD- mice, this behaviour was prevented. The double mutants showed worsened performance at a younger age. The ALD+/tg ALDR mice showed similar behaviour to the wild type mice.

The results from these experiments show that overexpression of ALDR can correct VLCFA levels in the mouse observed mouse tissues and can prevent the development of different characteristics of X-linked adrenoleukodystrophy. This shows that there is functional redundancy between the ALD and ALDR gene in vivo (Pujol et al., 2004). Overexpression of ALDR is then a plausible therapeutic target strategy for treatment of the disease ALD. As overexpression of ALDR is necessary for the mice to exhibit a normal phenotype, Pujol et al. concluded that it is most likely that ALDR is less efficient than ALD. The authors showed that ALDR inactivation did not have a large effect on the mice. Double mutants showed a more severe version and earlier appearance of several characteristics of ALD. This raises the question of whether VLCFA accumulation causes the neurological phenotype or if it is caused by unknown substrates of ALD or ALDR (Pujol et al., 2004). In the adrenal gland, the ALDR-/- mice showed accumulation of VLCFAs, as did the double mutants. This may suggest that ALDR plays a more important role in the adrenal gland than elsewhere in the body (Pujol et al., 2004). As the double mutants showed a distinct and worsened phenotype than the single mutants, these mice could be used as a model to understand the mechanisms of interactions between ALDP and ALDPR in ALD individuals.

Critique:

This was a well structured and well-written article. It was easy to read and to understand what each experiment involved. The results were presented in a way that was easy to understand and find. The tables and figures were easy to understand, the only issue was the placement of some of the tables and figures. It is easier to understand tables or figures if they are visible when the results are being discussed in the article. The authors did not always do this.

The hypothesis of the article was not clearly defined, but it was easily implied after reading the introduction. The main hypothesis of this article was not novel. It had already been proven that the overexpression of ALDR can help prevent adrenoleukodystrophy; however, the authors did look at specific characteristics of the disease and they tested how overexpression of ALDR can help. In this way, this study was more thorough that those before it. The authors were also thorough when selecting controls for the experiments. They not only used ALD mutant mice and ALD-/tg ALDR mice, but also used double mutants, ALDR mutants and ALD+/tg ALDR. The use of these controls adds credibility to the results they found.

The lifetime of a mouse in the lab can be up to about two and a half years. In previous studies, it was found that mice do not develop any pathological symptoms until at least the age of 6 months (Pujol et al., 2002). In light of these facts, any experiments conducted on the lab mice should be done at multiple points between the ages of 6 months until about two and a half years. By doing this, you would be able to observe when the disease begins to develop and how it progresses over time. The authors did this in most of the experiments that were conducted except one. The experiment which measured the level of VLCFAs only used 8-month-old mice. In order to get accurate results as to how overexpression of ALDR affects the levels of VLCFAs over time, at least one more group of older mice should have been tested. By adding another group, the authors would have been able to observe the extent to which overexpression can prevent the accumulation of VLCFAs over time. It may be possible that the overexpression of ALDR may not have the same effect on the level of VLCFAs in young mice as it does in older mice. Also, by adding this extra group for testing, the authors would have been able to observe the different rates of accumulation of VLCFAs in the different mice that were used. Data from two points in time would be much more useful than one when comparing the different mice.

One other problem with this article was that it did not mention anywhere the environment or conditions in which the mice were raised. It did not mention what the mice were fed or how this was controlled, now did they mention the environment or treatment of the mice. It is important that the diet of the mice be mentioned because if the different mice are being fed separate and different diets, then this could skew the data that was collected. This is especially important for the experiment where the authors measured the levels of VLCFAs in the tissue and plasma. If the different mice are being fed diets which contain varying amounts of VLCFAs in them, then this would ruin the results. The authors may have assumed that whoever read this article would assume a controlled diet and environment, but this is important information and should have been included somewhere in the article to affirm the credibility of their results.

In the discussion of this article, the authors realized that there is a strong possibility that the way in which they overexpressed ALDR in mice may not be reproducible in humans. They only said that their data showed that is worth looking further into this possible treatment of ALD in humans. When the authors were discussing their results and what they meant, they never said that anything was 100 percent. They only discuss how their interpretation of the results was most probable. In this way, they did not over-interpret that data that they collected. The authors went further and also discussed some possible areas of future research.

There are still a lot of questions surrounding adrenoleukodystrophy and the possible treatment that is discussed in this article. It is well known that the disease is associated with high levels of VLCFAs, but it is still not known how this accumulation results in the physical characteristics of adrenoleukodystrophy, such as demyelination. This would be very helpful in understanding the disease and would be very helpful in developing better treatments for the disease. It is also not known exactly how the protein ALDR works. It would be extremely useful to understand how exactly VLCFAs pass through the protein and into the peroxisome. It is important to understand the mechanism or system before you start manipulating it.

In this study, the upregulation of ALDR was done through the generation of transgenic mice. The authors realize that this method may not be applicable in humans. This means that it may be necessary to find other ways to overexpress ALDR in humans. This will most likely be done through the use of drugs, which is currently being tested in mice. Another way that the expression of ALDR may be regulated is through diet. In current studies it has been shown that a decrease in cholesterol can increase the expression of ALDR (Weinhofer, 2002). There may be other ways to overexpress ALDR that have yet to be discovered, so this should also be researched further.

Based on the results presented in this article, there are several areas in which there should be further research. The first is the possible function of ALDR. When measuring the levels of VLCFAs, the authors discovered that ALDR-/- mice only showed accumulation of VLCFAs in the adrenal gland. This suggests that ALDR plays a more important role in the adrenals then in the brain. This should be researched further. Another aspect that should be studied further is any possible side effects that the overexpression of ALDR may have on an individual such as lifespan or any basic bodily functions.

This article had some flaws, but overall it was a well structured article and was easy to follow and understand. There is still a lot that is not know about adrenoleukodystrophy and how exactly overexpression of ALDR can prevent the progression of the disease. The authors, Pujol et al., presented strong results which justify further research of this system and how it can be applied to other organisms, specifically humans.

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