Early axonal loss accompanied by impaired endocytosis, abnormal axonal transport, and decreased microtubule stability occur in the model of Krabbe's disease

Highlights

• In Twitcher nerves, a decrease in axon number precedes demyelination.

• Twitcher nerves are regeneration-competent and capable of remyelination.

• Activation of Erk1/2 and Akt pathways is impaired in Twitcher neurons.

• Endocytosis and transport of vesicles are defective in Twitcher neurons.

• Twitcher neurons have decreased microtubule stability.

Abstract

In Krabbe's disease (KD), a leukodystrophy caused by β-galactosylceramidase deficiency, demyelination and a myelin-independent axonopathy contributes to the severe neuropathology. Beyond axonopathy, we show that in Twitcher mice, a model of KD, a decreased number of axons both in the PNS and in the CNS, and of neurons in dorsal root ganglia (DRG), occurred before the onset of demyelination. Despite the early axonal loss, and although in vitro Twitcher neurites degenerated over time, Twitcher DRG neurons displayed an initial neurite overgrowth and, following sciatic nerve injury, Twitcher axons were regeneration-competent, at a time point where axonopathy was already ongoing. Psychosine, the toxic substrate that accumulates in KD, induced lipid raft clustering. At the mechanistic level, TrkA recruitment to lipid rafts was dysregulated in Twitcher neurons, and defective activation of the ERK1/2 and AKT pathways was identified. Besides defective recruitment of signaling molecules to lipid rafts, the early steps of endocytosis and the transport of endocytic and synaptic vesicles were impaired in Twitcher DRG neurons. Defects in axonal transport, specifically in the retrograde component, correlated with decreased levels of dynein, abnormal levels of post-translational tubulin modifications and decreased microtubule stability. The identification of the axonal defects that precede demyelination in KD, together with the finding that Twitcher axons are regeneration-competent when axonopathy is already installed, opens new windows of action to effectively correct the neuropathology that characterizes this disorder.

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.