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Metabolic Disease: Krabbe Disease
Discovered in 1916 by Danish neurologist Knud Haraldsen Krabbe whom observed globoid cells in the brain, Krabbe disease (KD), or globoid cell leukodystrophy, is a metabolic disorder in which the body does not produce sufficient amounts of galactosylceramidase (GALC). This enzyme is necessary to make myelin. KD is a genetically inherited lysosomal storage disorder that is rare and only effects about 1 in 100,000 people but can be extremely deadly. Myelin surrounds nerve fibers and insulates and protects them. When nerve cells aren’t sufficiently myelinated, nerve cells in the brain will die resulting in other nerves in other parts of the body to not work properly. This disease is mostly seen in infants typically starting around six months of age (early-onset form of the disease) with death ensuing typically by age two. However, Krabbe disease can also develop later in life (late-onset form of the disease) but the earlier symptoms start to occur the quicker the disease will progress. Symptoms of the disease include but are not limited to: fevers, loss of ability to support the head, poor coordination, seizures, muscle spasms, deafness and or blindness, decrease in mental function, and troubles eating. The gene for Krabbe disease occurs by receiving the mutated gene from both parents resulting in the shortage of galactosylceramidase. Currently, there is no cure for this disease and the only treatments that exist for it are ones that are in place to ease some of the symptoms. However, as far as diagnosis there are both genetic and enzymatic assays that are available and are useful for prenatal diagnosis of the disease. The assays are able to measure the GALC activity level to determine if the individual possesses the phenotype indicative of Krabbe disease (Graziano and Cardile 2014).
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In 1970, Japanese scientist Suzuki was able to identify the genetic deficiency of the GALC enzyme which caused Krabbe disease (Suzuki and Suzuki). This eventually led to GALC cDNA to be cloned following the screening of human testes and brain cDNA by using a primer from a GALC gene fragment that was obtained from human urine and brain matter (Graziano and Cardile 2014). The GALC gene has a length of 3795 base pairs including 2007 in the coding region and is located on chromosome 14. At least 128 mutations at this location have been reported to be causes of Krabbe disease. In an infant of European decent, the most common mutation includes the deletion of exons 11-17 which eliminates the coding region for the 30-kDa subunit and part of the coding region of the 50 to 52-kDa subunit that make up this protein. An infant of Japanese descent often has a slightly different mutation which includes a 12 base pair deletion and a 3 base pair insertion in the gene which results in the deletion of 5 amino acids and the insertion of 2 other amino acids which therefore impacts the overall quaternary structure of GALC (Graziano and Cardile 2014).
The mature GALC protein has a total of 669 amino acids and is made up of two subunits of varying weight with up to six possible glycosylation sites. Genetic defects on lysosomal enzymes such as GALC are known to impact the viability of cells and are often called lysosomal storage diseases due to non-catabolized products being stored. What causes this accumulation is a defect in a protein that possesses enzymatic functions involved in hydrolyzing, activating, or transporting, or it can be an enzyme involved in the processing of other lysosomal proteins (Graziano and Cardile 2014). In the case of the GALC enzyme, it acts hydrolytically upon glycolipid substrates with a terminal galactose. The structure of this hydrolase was recently determined to have three domains that all contribute to the substrate binding pocket and it was found that mutations can occur on many locations throughout the protein that cause disease (Deane et al 2011). GALC essentially catabolizes myelin lipid during myelin turnover and has a primary substrate called galactosylceramide (Gal-cer) which is a sphingolipid that is located in the myelin sheath. However, this enzyme can also act on other substrates such as galactosylsphingosine, monogalactosyldiglyceride, and a precursor of seminolipid (Graziano and Cardile 2014). In order for the enzyme to become activated, it requires the presence of a protein called saposin A. A mutation in this activator protein can result in similar symptoms as Krabbe disease but technically someone possessing this mutation would not be diagnosed with KD.
Substrates of galactosylceramidase are processed by the lysosome in a properly functioning nervous system and the recycled components are used by the remyelination pathway. Krabbe disease is characterized by a decreased amount of this enzyme and mutations to the gene that produce it result in an impaired ability to degrade lipids during myelin turnover. Specifically, GALC is unable to degrade the lipids galactosylceramide which is its primary substrate and monoglycosylsphingosine (psy) which is a toxic metabolite (Graziano and Cardile 2014) This inevitably results in an impaired ability to perform remyelination due to the build-up of psy which inhibits and kills the cells that perform remyelination. While psy accumulates, which is believed to be the cause of Krabbe disease, myelin turnover is still proceeding.
Figure 1 displays the pathway in which galactosylceramide, along with its substrates and compounds that are associated with it. As can be seen by the image sphingosine is converted to ceramide via acylation and is then galactosylated by UDPgalactose: ceramide galactosylceramidase (CGT) to form Gal-cer (Graziano and Cardile 2014). CGT can also act directly on sphinogosine to produce psychosine (psy) which, along with Gal-cer are degraded by GALC in a normal functioning body. Since they are unable to be degraded, they accumulate in the brain and cause detrimental effects.
Galactosylceramide is an abundant glycosphingolipid found in all nervous tissues amounting in up to 12% of the white matters mass. They are a major component of oligodendrocytes in the central nervous system as well as Schwann cells in the peripheral nervous system. During myelin turnover, Gal-cer is broken down by GALC into galactose and ceramide which are reused in a different remyelination pathway. Since Gal-cer is not broken down, it triggers macrophages to enter the brain which phagocytose the lipid and change form into multinucleated globoid cells (Graziano and Cardile 2014). However, the formation of globoid cells doesn’t explain the loss of myelin forming cells since they have not been proven to be cytotoxic. Psy, on the other hand, has been found to have such properties. It is a basic molecule formed by the galactosylation of sphingosine by ceramide galactosyltransferase that has unusual binding properties not seen in most lipids. Typically having a concentration in the brain of less than 10 pmol/mg, it is able to accumulate to a much greater mass in patients with Krabbe disease. In a study done by Zaka and Wenger (2004), they were able to implant psy into cells in vivo and observe the cytotoxic effects of the build-up of the lipid. More specifically, psy is now known to indirectly cause the release of pro-inflammatory cytokines: tumor necrosis factor- (TNF-) and interleukin-6, which are apoptotic cells. As seen in Figure 2, a number of other factors are triggered by the accumulation of psy but it appears that increased inflammation in the brain caused by the release of these cytokines and it seems to be the primary biochemical mechanism for the myelin forming cell death. Additional inflammation in the brain is also induced by astrocytosis and microgliosis due to psy collections. Other side effects include the AMP-activated protein kinase (AMPK) being decreased which causes inflammation of astrocytes without killing oligodendrocytes (Graziano and Cardile 2014).
Figure from Fletcher et al. (2009)
There appear to be multiple hypotheses as to how psy accumulation inhibits the remyelination of nerve cells. The most agreed upon mechanism of action is that psy accumulates in the lipid rafts of the membrane structure of the central nervous system and peripheral nervous system in patients with KD. This leads to an overall weakening of the lipid raft structure and eventually induces large clots of actin filaments which inhibits a protein kinase C (PKC) that acts as a signaling molecule. This interference in cell signaling appears to be the reason that accumulation of psy eventually leads to neurodegeneration (White et al 2009). A visual of this phenomenon is shown in figure 3 in which it shows inflammation as being one of the observable side effects of psy accumulation. Due to the spatial separation of molecules on the membrane from accumulation of psy, cell signaling is disrupted which eventually leads to demyelination and neurodegeneration. When neuron cells become demyelinated, this results in the symptoms that define Krabbe disease and eventually death ensues.
Figure from White et al (2009)
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