Early axonal loss accompanied by impaired endocytosis, abnormal axonal transport, and decreased microtubule stability occur in the model of Krabbe's disease
Introduction
Krabbe's disease (KD) is an autosomal recessive lysosomal storage disorder caused by mutations in β-galactosylceramidase (GALC), an enzyme responsible for the catabolism of myelin galactolipids. The loss-of-function of GALC leads to accumulation of its substrates, galactocerebroside and psychosine (Wenger et al., 1997). Galactocerebroside builds up inside macrophages and microglia giving rise to the globoid cells (Duchen et al., 1980, Jesionek-Kupnicka et al., 1997). Psychosine is considered the main pathological cause of KD (Igisu & Suzuki, 1984), as it disrupts lipid rafts in myelinating glia, culminating in apoptosis of oligodendrocytes and Schwann cells (White et al., 2009, Zaka and Wenger, 2004). As a consequence, demyelination affecting both the central nervous system (CNS) and the peripheral nervous system (PNS) is the principal hallmark of KD (Krabbe, 1916, Sabatelli et al., 2002, Sourander and Olsson, 1968). The Twitcher mouse, a model of KD, mimics the most common form of the human disease, which has infantile onset. This naturally occurring model presents a premature stop codon (W339X) in the GALC gene that abolishes enzymatic activity (Lee et al., 2006).
Despite the view that axonopathy in KD is secondary to demyelination, recent evidence supports that axonal defects, not directly caused by loss of myelin, may be a common feature in leukodystrophies, including metachromatic leukodystrophy, Pelizaeus–Merzbacher disease and KD (Castelvetri et al., 2011, Griffiths et al., 1998, Mar and Noetzel, 2010). In KD patients, axonal degeneration and neuronal degeneration have been reported sporadically in autopsy material (Sourander & Olsson, 1968). Whether neural degeneration or a dying-back process with Wallerian degeneration takes place in KD nerves has been under discussion for many years (Joosten et al., 1974, Sourander and Olsson, 1968). Recently, psychosine was shown to accumulate not only in myelinating glia, but also in KD neurons (Castelvetri et al., 2011), further supporting a myelin-independent neuronal pathology. In Twitcher mice, axonal swellings and transections occur in sciatic nerves before the onset of myelin loss and progress with age, synchronously with demyelination (Castelvetri et al., 2011, Smith et al., 2011). In these animals, a dying-back neuropathy has been proposed, since apoptosis of motor neurons develops later, while in the sciatic nerve, distal axonal defects are already evident before myelin loss occurs (Castelvetri et al., 2011). Despite the presence of axonopathy, there is still conflicting evidence as to axonal loss in Twitcher nerves (Jacobs et al., 1982, Kobayashi et al., 1988, Tanaka et al., 1988).
The characterization of axonal pathology in KD is still limited, and its understanding is of great interest since therapies devised for this disorder, including enzyme replacement therapy, gene therapy and cell transplantation aim mainly at targeting myelinating cells whereas neuroprotective strategies have been neglected. Currently, the recommended therapy for KD is hematopoietic stem cell transplantation which slows disease progression when performed presymptomatically (Escolar et al., 2005), but fails overtime to address the complexity of the disorder and to counteract neurologic damage. Here we aimed at clarifying the occurrence of axonal loss in KD and at further characterizing the underlying molecular defects leading to axonopathy in this disorder.
Section snippets
Animals
Twitcher mice and WT littermates of either sex were obtained from heterozygous breeding pairs (Jackson Laboratory) and genotyped as described (Sakai et al., 1996). All mice were handled according to the European legislation. Accessible wet rodent's chow was provided to Twitcher mice after weaning.
Sciatic nerve crush
The sciatic nerve of 3–4 week-old WT (n = 4) and Twitcher mice (n = 6), was crushed twice during 15 s using a hemostat (Fine Science Tools). The crush site was maintained constant and identified with a
In Twitcher nerves, a decrease in axon number precedes demyelination
Given the conflicting evidence related to axonal loss in Twitcher nerves, and to establish whether besides axonopathy, a decreased axon number occur prior to demyelination, we performed a comprehensive evaluation of nerve pathology in this animal model. In peripheral nerves from Twitcher mice, demyelination initiates around P15 (Smith et al., 2011) with a clear decrease in myelin thickness starting only around P20 (Tanaka et al., 1988). Our analysis showed that sciatic nerves from P9 Twitcher
Discussion
In KD, axonal damage and loss are generally seen as a consequence of the progressive demyelination. Our work shows that however, before the onset of demyelination, the number of both myelinated and unmyelinated axons is already decreased in Twitcher mice. Neuronal defects, resulting from the combined or synergistic contribution of psychosine-induced lipid raft clustering, impaired endocytosis, decreased microtubule stability and defective axonal transport, may originate from abnormal signaling
Acknowledgments
This work was funded by the European Leukodystrophy Association to M.M.S. and P.B. (ELA 2010-042C5) and by FEDER funds through the Operational Competitiveness Programme — COMPETE and by National Funds through FCT — Fundação para a Ciência e a Tecnologia under projects PTDC/SAU-ORG/112406/2009 (to P.B.) and PTDC/SAU-GMG/111761/2009 (to M.M.S.). G.H.M. is currently supported by the National Institutes of Health (NIH) through NIMH 1R03MH098689 and NIH-NINDS 1R01NS079655 grants, by the National MPS
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These authors contributed equally to this work.