Adrenoleukodystrophy is a rare, X-linked, recessive genetic disorder with a single enzyme deficiency that is characterized by the accumulation of saturated very-long-chain fatty acids due to a peroxisomal disorder (5). Peroxisomes are simple, membrane bound vesicles and are also known as microbodies. These organelles are multifunctional and contain more than fifty enzymes. One of their functions is the B-oxidation of very-long- chain fatty acids, which are usually twenty-four to twenty-six carbons long and rare fatty acids twenty-six to thirty-eight carbon chains that are found in the brain (13,16). The peroxisome also function as the site for synthesis and of H₂O₂ (hydrogen peroxide). The peroxisomal enzyme catalase reduces the toxic H₂O₂ to H₂O.

ALD involves mainly the nervous system white matter, adrenal cortex and testes. This disease causes an abnormal accumulation of very-long-chain fatty acids in tissues and body fluids due to incomplete degradation of the fatty acids that are found in the blood plasma. These fatty acids accumulate in the brain, the fibroblasts, the testes, and the adrenal glands causing the breakdown or loss of the myelin sheath surrounding the nerve cells in the brain. This will be explained later in the paper. There is also progressive dysfunction of the adrenal glands, and these are located on top of each kidney. The white matter of the brain appears white because the axons are covered and insulated by the myelin sheath. The myelin sheath is made by the oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system. It consists of a variety of lipids including the lipid substance sphingomyelin. Spingomyelin is an excellent component for insulation that prevents almost all flow of ions. The function of the myelin sheath is to insulate and conduct nerve impulses away from the cell body and its presence speeds up the conduction of the impulse tremendously. The myelin sheath is a very complex substance, made up of at least ten distinct chemicals. Each of the leuko- dystrophies affects one, and only one, of these substances that affects the myelin sheath in some way. For example, metachromatic leukodystrophy is caused by a deficiency of arylsulfatase A enzyme. Due to an inability to hydrolyze sulfatides, they accumulate in the CNS and are toxic to the myelin sheath. The result is demyelination of the white matter of the brain, spinal cord, and the peripheral nerves. There is infant, childhood, and adult onset. Another example is globoid cell leukodystrophy. This is caused by a deficiency of galactocerebrosidase, which allows an accumulation of psychosine as a toxic metabolite (10). Otherwise the leukodystophies are distinct diseases and one type does not predispose to, or increase the risk of another type of leukodystrophy (20).

With X-linked ALD, the defect only involves a single gene that encodes for a specific protein known as the ALD protein (ALDP). ALDP is essential in tissues such as myelinated tracts and the adrenal gland, which have a high lipid turnover (6). This can explain why the fatty acids accumulate in the brain, the adrenal glands, and the testes. The myelin sheath degenerates possibly because the myelin sheath has a high lipid turnover in those areas. The very-long-chain fatty acids are metabolized into shorter fatty acids, in the peroxisome, and then used for the biosynthesis of complex lipids that can be used for the myelin sheath. The very-long-chain fatty acids are in high concentration and readily available to the oligodendrocytes and the Schwann cells. The incorporation of the VLCFA in the components of the multimellar myelin membrane must destabilize the ALD myelin (6). In the human CNS the ALD protein is expressed in all glial cell types, which are supporting cells of the central nervous system. The glial cells that we are concerned with are astrocytes, microglial cells, and oligodendrocytes. There is some localization of the ALD protein that is restricted to oligodendrocytes located in the corpus callosum, internal capsules and the anterior commissure, which are all located in the brain. These myelinated regions are the ones that are first affected by the disease. The ALD protein is transiently expressed in rodent developing neurons and is almost completely absent from mature neurons which appear to be spared in ALD. The central nervous system myelin is 70% lipid, which is comprised of cholesterol, galactolipids and phospholipids. In cultured oligodendrocyte processes the peroxisomes are abundant where the myelin lipids are being incorporated into the myelin membrane during the ensheathment of axons (6).

As previously mentioned, ALD is an X-linked disorder. ALD affects 1 in 20,000 boys (6, 11, 16). For X-linked disorders, females are carriers of this recessive gene. Carriers tend to have no disability because it is a recessive gene. Women who are carriers for ALD, occasionally do develop mild symptoms such as ataxia, urinary problems, and excessive muscle tone, called hypertonia. Symptoms can appear around the third decade of life appearing as a spinal disease and the progression is very slow. These symptoms are often mistaken for multiple sclerosis (10). One-half of her daughters will be carriers, while the other half will be normal. One-half of her sons will suffer from the illness, while the other half will be normal. If an affected male has children, all of his sons will be normal and his daughters will be carriers. Unless there is a family history most people do not know that they are carriers.

Adrenoleukodystrophy is the most frequently occurring leukodystrophy in children and adults (6). ALD is categorized into various subtypes. In most childhood forms the boys are usually diagnosed between the ages of four to ten years old (6, 9, 10,). Some problems are learning disabilities, perceptual problems, attention deficit disorder, short- and long-term memory loss, and various personality and behavioral changes. Other symptoms suffered from childhood ALD is fatigue, seizures, intermittent vomiting, deafness, blindness and progressive dementia. Patients usually die within one to ten years after the onset of symptoms of adrenoleukodystrophy (24).

Adult-onset ALD is called adrenomyeloneuropathy (AMN). This subtype is more mild than the childhood form. The symptoms do not appear until the male is between the ages of twenty-one and thirty-five. The symptoms may include leg stiffness, progressive paralysis of the lower extremities, and ataxia. Some other characteristics are dementia and short-term memory loss. Although adult-onset ALD progresses more slowly than the childhood form it can also result in the deterioration of brain function and death several decades later (10). Patients with the Addision-only form has adrenal dysfunction but they have normal neurological functions (13, 23).

Neonatal adrenoleukodystrophy affects both male and female newborns probably because their immune systems are weak and underdeveloped. Symptoms may include seizures, adrenal dysfunction, enlarged liver, hypotonia, retinal degeneration, and facial abnormalities. This form progresses very quickly and usually results in death within a year. Considering these few subtypes mentioned, patients with the same gene mutation will not necessarily have the exact same phenotypes (8).

There is a gene on chromosome X that encodes a peroxisomal membrane protein that is a member of the ATP-binding-cassette (ABC) transporter family. When this gene is mutated, the outcome is adrenoleukodystrophy. The oxidation of very-long-chain fatty acids normally occurs in the peroxisomes and is initiated by VLCFA-acyl-coenzyme A synthetase. This activates VLCFA into acyl-CoA derivatives. Initially, the gene associated with ALD was thought to code for VLCFA-acyl-CoA synthetase, but it was later found that the ALD gene encoded an ABC transporter, called ALD-related protein. This transporter appears to be needed for the transfer of VLCFA or VLCFA-CoA into the peroxisomes. The VLCFA or VLCFA-CoA are then metabolized by enzymes in the peroxisomes into shorter fatty acid chains that are used for the biosynthesis of complex lipids and acylated proteins in the cytoplasm (17,21). The fibroblasts of ALD patients have been shown to have the ability to oxidize VLCFA-CoA but not VLCFA (6). This means that the VLCFA’s need to be converted to their CoA derivatives before entering the peroxisome for B-oxidation.

In 1981 the ALD gene was mapped to chromosome X, segment q, at position 28 (Xq28) (Fig.1). The gene that codes for the ALD protein (ALDP) was isolated in 1993. The ALDP gene is comprised of ten exons, is approximately twenty to twenty-one kilobases of the genomic sequence and encodes a predicted mRNA with a open reading frame of 2625 bases which encodes for a protein of 745-834 amino acids. The primary nucleotide sequence for ALDP suggests that ALDP is a member of a superfamily of transmembrane transporters called the ATP binding cassette (ABC) transporter proteins (1, 6, 11, 23).

Figure 1.

The peroxisomal membrane has many ABC transporters. The ALD protein and other peroxisomal transporters are hemitransporters that need to dimerize in order to activate an import function. There are a total of four hemi-transporters, that are homologous to one another, in the peroxisomal membrane. They are the ALD protein, the ALD-related protein, the peroxisomal membrane protein 70 (PMP70), and the PMP70- related protein (P70R) (2, 6, 11, 14, 20). Functional ABC transporters contain four domains. Two of these domains are nucleotide-binding folds located in the hydrophilic region. The other two domains are membrane-spanning regions, each containing six transmembrane alpha helices (6, 11, 21). In the central nervous system ALDP is detected mostly in the glial cells, especially the oligodendrocytes. This pattern of expression correlates with the demyelinating nature of the disease.

The three homologues of the ALD protein are closely related to the ALD protein. The ALD-related protein has a 66% amino-acid identity to the ALD protein and they both have a 250-residue C-terminal domain that encompasses the ATP-binding folds in common. There is also a highly conserved region, showing (a 92% similarity), consisting of 290-amino acids that encompass the transmembrane segments, TM2-TM6. PMP70 protein which has a 38% amino-acid identity and the P70R protein which has a 27% identity and are more distantly related. ALDR is presently known to be the closest homologue to the ALD gene (6, 11).

In humans the ABC transporter family also includes the cystic fibrosis trans- membrane conductance regulator, the multidrug resistance gene product, and theTAP1 and TAP2 peptide transporters encoded in the major histocompatibility complex (MHC) cluster (5).

After the mutated gene that is responsible for adrenoleukodystrophy was discovered, two closely related hemi-transporter genes were cloned from labeled yeast, Pxa1p and Pxa2p. Studies of these genes and their mutations have provided many clues about the function of the ALD protein. In humans, long-chain-fatty acids (LCFA) and medium-chain-fatty acids (MCFA) are oxidized in the mitochondria after being converted into acyl-carnitine derivatives. In yeast LCFA are activated to CoA derivatives within the cytosol by a LCFA-CoA synthetase and then imported into the peroxisome through the Pxa1p and the Pxa2p heterodimers. The MCFA are first imported into the peroxisome by an unknown mechanism and then activated into CoA esters by an MCFA-CoA synthetase (6). In mammals the B-oxidation of fats occurs both in the mitochondria and the peroxisomes. Evidence shows that VLCFA’s are converted to their VLCFA-CoA derivatives by very-long-chain acyl-coenzyme A synthetase (VLCS) on the cytoplasmic side of the peroxisome (figure 2) (6, 16, 17, 22). The VLCFA-CoA’s are imported onto the peroxisome and B-oxidation occurs. Contradicting this evidence a recent study, using immunofluorescence, determined that the C-terminus, which is the ATP-binding site, of fibroblast human VLCS (hVLCS) faces the peroxisomal matrix . The ALD protein, on the other hand, has its C-terminal hydrophilic domain, which is the ATP-binding site, known as the nuclear binding fold, localized on the cytoplasmic side of the peroxisomal membrane (4, 5, 6, 17). This means that the ALD protein might actually import VLCFA rather than their VLCFA-COA derivatives, as previously thought (17).

Whether the ALD protein forms homodimers or heterodimers, or both, with the ALD-related protein or with the PMP70 and P70R is still unknown. The precise function of ALDP remains unknown as well (19). Since there are four hemi- transporters that are a part of the peroxisomal membrane there is the possibility that different types of dimers can form and each will regulate the import of various lengths of fatty acid chains or of other substrates. Different combinations of transporters are sometimes found in the peroxisomal membrane of various cell types. For example, the ALD protein and the ALD-related protein are more prominent in the oligodenrocytes. The ALD-related protein and PMP70 are both abundant in neurons. With this in mind the two transporters could have similar, if not identical functions as homodimers or hetero- dimers (6). B-oxidation of very-long-chain fatty acids, particularly hexacosanoic acid (C26:0), is impaired in ALD and adrenomyeloneuropathy patients (21). To support this idea, it has been observed that the overexpression of PMP70 or the ALD-related protein can partially or completely correct the B-oxidation defect that is observed in patients with adrenoleukodystrophy (6, 17).

X-linked adrenoleukodystrophy is not caused by mutations in the VLCS gene, but investigating the properties of VLCS is crucial to determining the mechanism of this disease. In this experiment they cloned the gene encoding hVLCS and produced a polyclonal antibody that is specific for this protein. They examined the fibroblasts of four controls (normal) and four ALD patients. All four ALD patients lacked detectable ALDP and their hVLCS immunofluorescence was indistinguishable from the controls. This suggests that the hVLCS activity of X-ALD peroxisomes is not caused by the absence of hVLCS protein (17).

The ALD protein is a transmembrane protein that spans the membrane six times. Three regions have been conserved between yeast and humans. The first region of the transmembrane protein (TM1) might control the correct membrane insertion of the ABC transporter or its targeting to peroxisomes. In the cytoplasmic domain between TM4 and TM5 there is a hydrophilic sequence with a Glu-Ala-Ala, which determines the various substrates in prokaryotic and yeast ABC transporters (5, 6, 14, 21).

The mechanisms involved in the import of proteins into peroxisomes are highly conserved between yeast and humans. There was a study done on yeast and they found that there are at least seventeen peroxisome-biogenesis genes called peroxines (PEX), seven of which are conserved in humans (7). One of the main differences between mitochondria and peroxisomes is that peroxisomes do not contain DNA. There are many nuclear genes that encode the membrane and matrix proteins that have specific peroxisomal-targeting sequences (PTS). The interaction of PTS1 and PTS2 with the proteins, recognize receptors that shuttle these proteins to the import machinery within the peroxisomal membrane. PTS1 and PTS2 are encoded by the PEX genes. If there are mutations in some of the seven PEX genes that we know about in humans, they will cause peroxisomal disorders that will effect the nervous system (6, 15). This result gives us some possible insight into the cause of ALD. Peroxisomal proteins are synthesized on free polysomes and posttranslationally targeted to existing peroxisomes via peroxisomal targeting signals. The PTS1 signal has an absolute requirement of a basic residue, lysine, at position-2 from the carboxyl terminus in mammals. It is a carboxyl-terminal tripeptide signal with the structure (Ser/Ala/Cys)(Arg/Lys/His)(Leu/Met). The PTS1-receptor binds the proteins with the PTS1 signal in the cytosol and delivers them to the peroxisomal membrane by its interaction with the cytoplasmic SH3-domain-containing peroxisomal membrane protein PEX13p. The PTS2 signal is sometimes a cleaved amino-terminal signal Arg/Lys- Leu/Val/Ile- X₅-His/Gln-Leu/Ala (7). Catalase has the PTS2 signal. However, if there is a mutation and catalase cannot get into the peroxisome then there could be a build up of H₂O₂, which can be detrimental (15). This is an example of how important each signal is for the enzymes and their activities.

One of the most frequently used tests to determine if the patient has ALD is to test for excess very-long-chain fatty acids (VLCFA) in the blood plasma. A biochemical defect is the incapacity to form the coenzyme A derivatives of VLCFA.

Effective reduction of the concentration of very-long-chain fatty acids has been accomplished by the use of dietary restriction and "Lorenzo’s oil" (10). "Lorenzo’s oil" contains glycerol trioleate and trierucate (3, 13, 20). When cells are exposed to certain chemicals like oleic acid the number of peroxisomes in a cell can increase (18). Other therapies include immunosupression, interferon-B, thalidomide, and steroid replacement (9). All of these have failed to affect the disease progression (10). Bone marrow transplant is the only treatment that has been effective so far. The treatment works best the earlier the diagnosis. It is difficult to distinguish between the childhood and the adult forms during the asymptomatic stages. The mutation and the levels of VLCFA in the plasma are identical, but the onset of symptoms is different and there is no known explanation. There have been two cases in which asymptomatic ALD has been treated with a bone marrow transplant successfully. Whether the boys would have developed the childhood form or the adult form will remain unknown. Mutational analysis has failed to differentiate these two forms (10).

Two biochemical methods are being used to distinguish the difference between the adult and childhood form of adrenoleukodystrophy. It has been shown that monocytes have greater amounts of very-long-chain fatty acids in the childhood form than in the adult form. A possible reason why the adult form is less severe could be because apolipoprotein E serves as an immunodulator and inhibitor of tumor necrosis factor and could be the "modifying factor" that makes the adult form less severe. Using a specific medication that inhibits tumor necrosis factor has the potential of reducing the inflammatory response in the brain in the severely affected patients (10).

Magnetic resonance spectroscopy has shown that earlier diagnosis can be made by identifying abnormalities in changes in N-acetylaspartate, choline, and lipid concentrations. These markers could possibly differentiate between early and late onset and therapy could be more effective before the rapid progression of the disease begins (10).

Adrenoleukodystrophy has a very complex mechanism, which is still not fully understood. Unfortunately there is no way to determine ahead of time it the child will have the childhood or adult onset of the ALD disease until after the disease has set in. It is very difficult to differentiate between the two forms. Experiments have been done on yeast in order to determine whether the VLCFA or the VLCFA-CoA is imported to the cell for B-oxidation. The only treatment that has been found to be successful is a bone marrow transplant. Once the mechanism has been determined doctors may be able to find a more effective treatment for ALD. The severity of the disease proves that even though there is a deficiency of only one peroxisomal enzyme, it makes a difference between life and death.

Copyright © 2000 by Amethyst Schlecht and Koni Stone
Back to the Stanislaus Journal of Biochemistry 2000 Table of Contents