Mechanism of Neuromuscular Dysfunction in Krabbe Disease

Abstract

The atrophy of skeletal muscles in patients with Krabbe disease is a major debilitating manifestation that worsens their quality of life and limits the clinical efficacy of current therapies. The pathogenic mechanism triggering muscle wasting is unknown. This study examined structural, functional, and metabolic changes conducive to muscle degeneration in Krabbe disease using the murine (twitcher mouse) and canine [globoid cell leukodystrophy (GLD) dog] models. Muscle degeneration, denervation, neuromuscular [neuromuscular junction (NMJ)] abnormalities, and axonal death were investigated using the reporter transgenic twitcher-Thy1.1-yellow fluorescent protein mouse. We found that mutant muscles had significant numbers of smaller-sized muscle fibers, without signs of regeneration. Muscle growth was slow and weak in twitcher mice, with decreased maximum force. The NMJ had significant levels of activated caspase-3 but limited denervation. Mutant NMJ showed reduced surface areas and lower volumes of presynaptic terminals, with depressed nerve control, increased miniature endplate potential (MEPP) amplitude, decreased MEPP frequency, and increased rise and decay rate constants. Twitcher and GLD dog muscles had significant capacity to store psychosine, the neurotoxin that accumulates in Krabbe disease. Mechanistically, muscle defects involved the inactivation of the Akt pathway and activation of the proteasome pathway. Our work indicates that muscular dysfunction in Krabbe disease is compounded by a pathogenic mechanism involving at least the failure of NMJ function, activation of proteosome degradation, and a reduction of the Akt pathway. Akt, which is key for muscle function, may constitute a novel target to complement in therapies for Krabbe disease.

Axonal pathology in Krabbe's disease: The cytoskeleton as an emerging therapeutic target

“…In fact, impaired protein kinase C activity (Yamada et al, ; White et al, ) and increased basal location of TrkA in lipid rafts were found in twitcher cells with defective activation of the downstream ERK1/2 and AKT pathways (Teixeira et al, ). It is notable that the defect in ERK and AKT activation was not restricted to KD neurons; it has also been described in mouse oligodendrocyte progenitor cells exposed to psychosine (Zaka et al, ), and, more recently, the twitcher muscle was also shown to have inactivation of the AKT pathway (Cantuti‐Castelvetri et al, ). In twitcher dorsal root ganglion neurons expressing Rab5‐GFP, a marker of early endosomes retained in Trk‐containing endosomes, not only was a decreased density of Rab5‐containing (Rab5 + ) vesicles observed but also a decreased percentage of Rab5 + vesicles containing TrkA was found, further suggesting that the endocytic pathway, specifically the formation of Trk‐signaling endosomes, was impaired (Teixeira et al, ).…”

“…As previously discussed, independent studies have shown that the AKT‐GSK3β pathway is dysregulated in KD, with neurons (Teixeira et al, ), oligodendrocyte precursors (Zaka et al, ), and muscle (Cantuti‐Castelvetri et al, ) presenting decreased AKT activation and/or the resulting increased GSK3β activity. These findings raise the possibility that inhibiting GSK3β could be considered a relevant therapeutic strategy in KD.…”

“…These findings raise the possibility that inhibiting GSK3β could be considered a relevant therapeutic strategy in KD. In support of this, in vitro pretreatment with TDZD‐8, a specific GSK3β inhibitor, was shown to prevent the decreased dephosphorylation of KLC mediated by psychosine (Cantuti‐Castelvetri et al, ). In twitcher mice, in vivo treatment with another specific GSK3β inhibitor, myristoylated L803, was able to increase the phosphorylation of KLC significantly.…”

Perspective on innovative therapies for globoid cell leukodystrophy

“…Defective autophagosome–lysosome fusion resulting in accumulation of toxic metabolites and cell death has been proposed for several neurodegenerative LSD (Settembre et al, ) but has never been reported for GLD. These pathways may indeed be implicated in the decline of cellular function that ultimately results in the appearance of clinical symptoms and are worth investigating in appropriate GLD models. g)Psychosine contributes to muscle pathology acting at multiple levels (decreasing Akt signaling and increasing ubiquitination of muscle proteins and their degradation), ultimately resulting in presynaptic and postsynaptic defects in the neuromuscular junction (Cantuti‐Castelvetri et al, ).…”

“…Also, they might be beneficial to slow the progression of the disease, until enzymatic activity is effectively restored and the storage defects have been corrected. In this perspective, pharmacological modulation of the ERK1/2, AKT, and/or GSK3β pathways, which are affected (inhibited or hyperactivated) as a consequence of psychosine accumulation and contribute to neuronal (Cantuti Castelvetri et al, ; Teixeira et al, ), oligodendroglial (Zaka et al, ), and muscular (Cantuti‐Castelvetri et al, ) dysfunction in Twitcher mice may constitute a promising target to complement therapies for Krabbe's disease.…”