Abstract
Charcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by five mutations in the RAB7A gene. Autophagy and late endocytic trafficking were already characterized in CMT2B. Indeed, impairment of autophagy and an increase in lysosomal degradative activity were found in cells expressing the mutant proteins. Recently, we described a novel RAB7 mutation associated with predominantly motor CMT2 and impaired EGFR trafficking. With the aim to analyze the autophagy process and lysosomal activity in CMT2B fibroblasts carrying the p.K126R RAB7 novel mutation and to investigate further the causes of the different phenotype, we have performed Western blot, immunofluorescence and cytometric analyses monitoring autophagic markers and endocytic proteins. Moreover, we investigated lipophagy by analyzing accumulation of lipid droplets and their co-localization with endolysosomal degradative compartments. We found that cells expressing the RAB7K126R mutant protein were characterized by impairment of autophagy and lipophagy processes and by a moderate increase in lysosomal activity compared to the previously studied cells carrying the RAB7V162M mutation. Thus, we concluded that EGFR trafficking alterations and a moderate increase in lysosomal activity with concomitant impairment of autophagy could induce the specific predominantly motor phenotype observed in K126R patients.
Highlights
Charcot-Marie-Tooth (CMT) is a rare inherited disorder affecting the peripheral nervous system
In skin patient’s fibroblasts carrying the K126R mutation, we found a strong accumulation of Epidermal Growth Factor Receptor (EGFR) caused by inhibition of EGFR degradation, in contrast to what has been observed for fibroblasts carrying the V162M classical CMT type 2B (CMT2B) mutation [22,33]
Quantification of autophagic flux by measuring the ratio of LC3B-II expression levels between bafilomycin A1 and full medium of the same sample revealed a strong inhibition in CMT2B cells carrying V162M or K126R mutations compared to control cells (Figure 1C)
Summary
Charcot-Marie-Tooth (CMT) is a rare inherited disorder affecting the peripheral nervous system. RAB7A, hereafter referred to as RAB7, is a well-characterized ubiquitous GTPase with multiple important roles in cellular physiology, in particular in the regulation of late endocytic traffic from endosomes to lysosomes and of lysosomal biogenesis [8,9,10]. RAB7 is important for other processes in which the late endocytic route is involved, such as phagosome and autophagosome maturation, interaction and fusion between phagosomes and autophagosomes with lysosomes, and phagolysosome and autolysosome biogenesis [11,12,13]. It regulates the shape of the nascent autophagosome isolation membrane around damaged mitochondria during mitophagy and the breakdown of lipid droplets (LDs) during lipophagy [14,15]. RAB7 has been demonstrated to play specific roles in neurons where it regulates neurotrophin trafficking and signaling, neurite outgrowth and neuronal migration during development [19,20,21]
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