Though Krabbe Disease was identified as a progressive leukodystrophy as early as 1916, it was not until 1970 that Suzuki and Suzuki identified β-galactocerebrocidase as the defective enzyme behind the disease pathology. Suzuki and Suzuki proposed a change in the paradigm of thought regarding Krabbe Disease from focusing on the loss of sulfatide to the high concentrations of galactocerebroside in globoid cells, Schwann cells, and oligodendrocyte cells. Based on previous knowledge that β-galactocerebrocidase breaks down galactocerebroside, these authors conducted activity assays on human post-morem brain tissue isolated from both pediatric and adult patients suffering from a variety of neurodegenerative diseases (including Krabbe disease); β-galactocerebrocidase activity was also measured in a set of control tissues (Suzuki and Suzuki 1970). Out of all the samples studied, only Krabbe disease tissue samples were deficient in β-galactocerebrocidase activity (Figure 1).
Suzuki and Suzuki confirmed that the deficiency in β-galactocerebrocidase activity was not due to a change in tissue pH or an endogenous inhibitor, and then noted that a lack in β-galactocerebrocidase activity would explain the pathology of Krabbe Disease—the authors proposed that this enzyme deficiency would lead to an accumulation of galactocerebroside, which would permit globoid cell infiltration. The authors believed that the globoid cells eventually overwhelmed the oligodendrocytes and Schwann cells, causing cell death. Without these myelin-producing cells present, newly synthesized neurons would not by myelinated, resulting in loss of white matter and neuronal death (Suzuki and Suzki 1970).
Deficiencies and defects in β-galactocerebrocidase stem from mutations to the GALC gene on the long arm of chromosome 14 (14q31; Debs et al., 2012). There are a huge variety of potential GALC mutations, the most common which being a 30 kb deletion from intron 10 through the protein’s C-terminus due to a nonsense mutation caused by a single nucleotide polymporphism in the gene sequence from cytidine to thymidine at position 502. The T1637C single nucleotide polymorphism is the second most frequently observed mutation, with about 35-45% of all alleles and causes a significant decrease in enzymatic activity (Hill et. al., 2013).
Yet another frequently observed mutation occurs at residue 380 (R380W and R380L are the most common mutations at this residue in early infantile Krabbe diseases; Hill et al., 2013). As can can now be determined through crystal structures that model the catalytic cycle of β-galactocerebrocidase (Figure 2), this residue directly interacts with the galactosyl-6-hydroxyl group via hydrogen bonding that is transferred to the myelin precursor (Hill et al., 2013). Catalytic modeling of β-galactocerebrocidase also identified E182 and E258 as critical to catalytic activity—E258 serves as the catalytic nucleophile and forms a covalent bond with the galactose substrate, and E182 utilizes acid –base catalysis to activate water for nucleophilic attack of the enzyme bound intermediate (Debs et al., 2012).
While Suzuki and Suzuki certainly identified much of the basic pathology of Krabbe Disease, insight into the molecular basis of this neurodegenerative process was significantly expanded with the identification of psychosine as a toxic metabolite that is also broken down by β-galactocerebrocidase. Miyatake and Suzuki had proposed that psychosine, a well-known cytotoxic metabolite, could be playing a role in Krabbe Disease pathology by inducing Schwann cell and oligodendrocyte death and subsequent loss of myelination—this was famously called the “psychosine hypothesis (Miyatake and Suzuki, 1972), and it was supported by the identification of high concentrations of psychosine in brain tissue from patients with Krabbe Disease post-mortem (Svernnerholm et al., 1980).
In 1980, Duchen et al. provided a means for studying the molecular basis of Krabbe Disease in vivo by developing an autosomal recessive mutation in the Twitcher mouse. The mouse experiences macrophages resembling globoid cells and an increase in myelination components followed by complete demyelination, thus greatly resembling Krabbe Disease pathology. The mouse’s condition was fatal within three months (Duchen et al., 1980).
Shortly thereafter in 1984, Igisu and Suzuki utilized the mutant Twitcher mice by killing them at several time points early within the disease course (as early as seven days) and measuring the levels of psychosine in their brain tissue—the authors demonstrated rapid and significant accumulation of psychosine in the brain even before abnormalities in myelination were observed (Igisu and Suzuki, 1984). The authors corroborated these results with time point evaluations of psychosine in dogs with globoid leukodystrophy and also re-affirmed elevated levels of psychosine in the post-mortem brain tissue of Krabbe Disease patients as compared to healthy patients and patients with other neurodegenerative diseases such as Tay-Sachs. These results clearly implicated psychosine as the toxic metabolite of Krabbe Disease (Igisu and Suzuki, 1984).
Since 1984, studies on psychosine’s many mechanisms of cytotoxicity and its relation to Krabbe Disease have been rampant. In 2006, Giri et al. demonstrated that one molecular mechanism by which psychosine may cause neuronal death occurs through reaction oxygen species generation. The authors showed that Psychosine activates secretory phospholipase A2, which stimulates the overexpression of lycophosphatidylcholine (LPC) and arachadonic acid (AA). Metabolism of AA results in the generation of reactive oxygen species, which can accumulate and trigger oligodendrocyte death. Small molecule inhibition of secretory phospholipase A2 blocks AA and LPC release, decreases reactive oxygen species, and promotes cell survival in vitro and in vivo, thus identifying a potential avenue for therapy development (Giri et al., 2006). Interestingly, activation secretory phospholipase A2 by psychosine has also been shown to directly inhibit oligodendrocyte differentiation and induce oligodendrocyte death (Won et al., 2013). Psychosine is also proposed to contribute many early and late infantile onset Krabbe patients’ inability to thrive due to an in vivo study using the Twitcher mice that indicated that psychosine accumulation decreases IGF-1 mRNA levels, which decreases somatic growth (Contreras et al., 2010).
Last year, psychosine was linked to one of the main pathologic features of Krabbe Disease: dying-back degeneration of neurons, or the disintegration of the axonal skeleton and membrane followed by demyelination. In this landmark in vivo and in vitro study, psychosine was shown to activate PP1, PP2, and GSK3β—these enzymes hyperphosphorylate kinesin light chains (KLCs) which subsequently block fast axonal transport. Inhibition of fast axonal transport has the known physiological consequence of dying-back degeneration (Castelvetri et al., 2013). Psychosine was also demonstrated to be required and sufficient to rapidly induce axonal swelling and neruonal death (the two other main aspects of Krabbe Disease pathology) in vivo (Figure 3; Castelvetri et al., 2011):
Most recently, Smith et al. demonstrated that a well-known pathologic characteristic of Krabbe disease, fibrillized deposits of proteins including α-synuclein, is psychosine-mediated (2014). Studies are underway to explore the potential use of neural stem cells to create a population of neurons within the brain of Twitcher mice that are resistant to pscyhosine accumulation as a therapeutic intervention for Krabbe Disease (Taylor et al., 2006).
Click on the following links to learn more about Krabbe Disease!