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      Alterations of autophagy in the peripheral neuropathy Charcot-Marie-Tooth type 2B

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          ABSTRACT

          Charcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by 5 mutations in the RAB7A gene, a ubiquitously expressed GTPase controlling late endocytic trafficking. In neurons, RAB7A also controls neuronal-specific processes such as NTF (neurotrophin) trafficking and signaling, neurite outgrowth and neuronal migration. Given the involvement of macroautophagy/autophagy in several neurodegenerative diseases and considering that RAB7A is fundamental for autophagosome maturation, we investigated whether CMT2B-causing mutants affect the ability of this gene to regulate autophagy. In HeLa cells, we observed a reduced localization of all CMT2B-causing RAB7A mutants on autophagic compartments. Furthermore, compared to expression of RAB7A WT, expression of these mutants caused a reduced autophagic flux, similar to what happens in cells expressing the dominant negative RAB7A T22N mutant. Consistently, both basal and starvation-induced autophagy were strongly inhibited in skin fibroblasts from a CMT2B patient carrying the RAB7A V162M mutation, suggesting that alteration of the autophagic flux could be responsible for neurodegeneration.

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          Most cited references 59

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          Compromised autophagy and neurodegenerative diseases.

          Most neurodegenerative diseases that afflict humans are associated with the intracytoplasmic deposition of aggregate-prone proteins in neurons and with mitochondrial dysfunction. Autophagy is a powerful process for removing such proteins and for maintaining mitochondrial homeostasis. Over recent years, evidence has accumulated to demonstrate that upregulation of autophagy may protect against neurodegeneration. However, autophagy dysfunction has also been implicated in the pathogenesis of various diseases. This Review summarizes the progress that has been made in our understanding of how perturbations in autophagy are linked with neurodegenerative diseases and the potential therapeutic strategies resulting from the modulation of this process.
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            Role for Rab7 in maturation of late autophagic vacuoles.

            The small GTP binding protein Rab7 has a role in the late endocytic pathway and lysosome biogenesis. The role of mammalian Rab7 in autophagy is, however, unknown. We have addressed this by inhibiting Rab7 function with RNA interference and overexpression of dominant negative Rab7. We show here that Rab7 was needed for the formation of preferably perinuclear, large aggregates, where the autophagosome marker LC3 colocalised with Rab7 and late endosomal and lysosomal markers. By electron microscopy we showed that these large aggregates corresponded to autophagic vacuoles surrounding late endosomal or lysosomal vesicles. Our experiments with quantitative electron microscopy showed that Rab7 was not needed for the initial maturation of early autophagosomes to late autophagic vacuoles, but that it participated in the final maturation of late autophagic vacuoles. Finally, we showed that the recruitment of Rab7 to autophagic vacuoles was retarded in cells deficient in the lysosomal membrane proteins Lamp1 and Lamp2, which we have recently shown to accumulate late autophagic vacuoles during starvation. In conclusion, our results showed a role for Rab7 in the final maturation of late autophagic vacuoles.
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              Rab7: a key to lysosome biogenesis.

              The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)-tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein-1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and the trans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment.
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                Author and article information

                Journal
                Autophagy
                Autophagy
                KAUP
                kaup20
                Autophagy
                Taylor & Francis
                1554-8627
                1554-8635
                2018
                4 May 2018
                4 May 2018
                : 14
                : 6
                : 930-941
                Affiliations
                [a ]Consiglio Nazionale delle Ricerche, Istituto di Fisiologia Clinica and Istituto Toscano Tumori-Core Research Laboratory, Signal Transduction Unit, AOU Senese , Siena, Italy
                [b ]Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento , Lecce, Italy
                [c ]Department of Neurosciences, University of Naples “Federico II” , Naples, Italy
                [d ]Salvatore Maugeri Foundation, Institute of Telese Terme , Benevento, Italy
                [e ]Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico II” , Naples, Italy
                [f ]Department of Biosciences, Division of Biochemistry and Biotechnology, University of Helsinki , Helsinki, Finland
                Author notes
                Mario Chiariello mario.chiariello@ 123456cnr.it , Consiglio Nazionale delle Ricerche, Istituto di Fisiologia Clinica and Istituto Toscano Tumori-Core Research Laboratory, Signal Transduction Unit, AOU Senese, Siena, Italy
                Cecilia Bucci cecilia.bucci@ 123456unisalento.it , Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Provinciale Monteroni 165, 73100 Lecce, Italy.

                Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/KAUP.

                Article
                1388475
                10.1080/15548627.2017.1388475
                6103410
                29130394
                644e5e21-1970-4ac3-9de4-a8158cbffd18
                © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License ( http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

                Page count
                Figures: 6, Tables: 0, References: 69, Pages: 12
                Product
                Funding
                Funded by: Italian Association for Cancer Research (AIRC) 10.13039/501100005010
                Award ID: IG2016 N.19068
                Funded by: Telethon Italy 10.13039/501100002426
                Award ID: GGP16037
                Italian Association for Cancer Research (AIRC), Telethon Italy
                Categories
                Research Paper - Basic Science

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