Abstract:The small GTPase Rab7 is a key regulator of endosomal maturation in eukaryotic cells. Mutations in rab7 are thought to cause the dominant neuropathy Charcot-Marie-Tooth 2B (CMT2B) by a gain-of-function mechanism. Here we show that loss of rab7, but not overexpression of rab7 CMT2B mutants, causes adult-onset neurodegeneration in a Drosophila model. All CMT2B mutant proteins retain 10–50% function based on quantitative imaging, electrophysiology, and rescue experiments in sensory and motor neurons in vivo. Cons… Show more
“…S4), confirming previous studies in different animal models ( Drosophila , zebrafish) showing that CMT2B mutants could behave both as inhibitory and as active proteins, depending on the kinetic requirements of the processes controlled by RAB7 (see, also, the Introduction). 17–19
10.1080/15548627.2017.1388475-F0005Figure 5.Analysis of autophagy in CMT2B fibroblasts. ( A ) Normal dermal human fibroblasts and CMT2B patient derived fibroblasts were incubated with full medium (FM) or starvation medium (ST) for 30 min, or incubated with 400 nM bafilomycin A 1 (BAF) for 3 h. Immunoblot of LC3B, HA-RAB7 proteins and MAPK1.…”
Section: Resultsmentioning
confidence: 99%
“…14 However, given that K off for GTP is also increased compared to RAB7 WT , 14–16 thus determining early release of GTP, and that binding of GTP is not correctly regulated, 16 these mutants although being mainly GTP-bound and interacting more strongly with a number of effector proteins, 14 , 16 could also display reduced efficiency in the activation of downstream specific effectors and pathways. In fact, studies on disease animal models established that in Drosophila , the presence of CMT2B mutant proteins induces sensory defects and dosage-dependent neurodegeneration due to partial loss of function, 17 , 18 while in zebrafish axon growth and guidance defects are due to gain-of-function mechanisms. 19 Therefore, depending on the kinetic requirements of the processes controlled by RAB7, these mutants could behave as inhibitory or active.…”
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 RAB7AWT, expression of these mutants caused a reduced autophagic flux, similar to what happens in cells expressing the dominant negative RAB7AT22N mutant. Consistently, both basal and starvation-induced autophagy were strongly inhibited in skin fibroblasts from a CMT2B patient carrying the RAB7AV162M mutation, suggesting that alteration of the autophagic flux could be responsible for neurodegeneration.
“…S4), confirming previous studies in different animal models ( Drosophila , zebrafish) showing that CMT2B mutants could behave both as inhibitory and as active proteins, depending on the kinetic requirements of the processes controlled by RAB7 (see, also, the Introduction). 17–19
10.1080/15548627.2017.1388475-F0005Figure 5.Analysis of autophagy in CMT2B fibroblasts. ( A ) Normal dermal human fibroblasts and CMT2B patient derived fibroblasts were incubated with full medium (FM) or starvation medium (ST) for 30 min, or incubated with 400 nM bafilomycin A 1 (BAF) for 3 h. Immunoblot of LC3B, HA-RAB7 proteins and MAPK1.…”
Section: Resultsmentioning
confidence: 99%
“…14 However, given that K off for GTP is also increased compared to RAB7 WT , 14–16 thus determining early release of GTP, and that binding of GTP is not correctly regulated, 16 these mutants although being mainly GTP-bound and interacting more strongly with a number of effector proteins, 14 , 16 could also display reduced efficiency in the activation of downstream specific effectors and pathways. In fact, studies on disease animal models established that in Drosophila , the presence of CMT2B mutant proteins induces sensory defects and dosage-dependent neurodegeneration due to partial loss of function, 17 , 18 while in zebrafish axon growth and guidance defects are due to gain-of-function mechanisms. 19 Therefore, depending on the kinetic requirements of the processes controlled by RAB7, these mutants could behave as inhibitory or active.…”
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 RAB7AWT, expression of these mutants caused a reduced autophagic flux, similar to what happens in cells expressing the dominant negative RAB7AT22N mutant. Consistently, both basal and starvation-induced autophagy were strongly inhibited in skin fibroblasts from a CMT2B patient carrying the RAB7AV162M mutation, suggesting that alteration of the autophagic flux could be responsible for neurodegeneration.
“…However, in some cases, dominant-negative mutations may not completely inactivate the protein but, rather, slow its activation. This is the case in Drosophila, where Rab7 T22N overexpression can partially rescue Rab7-null mutants, showing that Rab7 T22N retains some of its wild-type function (20). Therefore, we sought to examine whether the mCh-Rab13 T22N mutant used here retains wildtype function by performing pulldown assays using the Rabbinding domain of the Rab13 effector MICAL-L1 (21).…”
Rab GTPases are critical regulators of membrane trafficking. The canonical view is that Rabs are soluble in their inactive GDP-bound form, and only upon activation and conversion to their GTP-bound state are they anchored to membranes through membrane insertion of a C-terminal prenyl group. Here we demonstrate that C-terminal prenylation is not required for Rab13 to associate with and traffic on vesicles. Instead, inactive Rab13 appears to associate with vesicles via protein-protein interactions. Only following activation does Rab13 associate with the plasma membrane, presumably with insertion of the C-terminal prenyl group into the membrane.Rab GTPases control all aspects of membrane trafficking, ranging from vesicle formation and transport to tethering and fusion with the target membrane (1). The understanding of Rab regulation is thus critical for a full understanding of membrane trafficking. Furthermore, disturbances in Rab function are observed in several diseases. Notably, alterations in the levels or activity of Rab5, Rab35, Rab8, and Rab13 have been implicated in phenotypes associated with cancer (2-5).As for all small GTPases, Rabs cycle between an active GTPbound form and an inactive GDP-bound form. They are activated by guanine nucleotide exchange factors (GEFs) 3 that facilitate the exchange of GDP for GTP, and they are inactivated by GTPase-activating proteins, which enhance Rab-mediated hydrolysis of GTP to GDP. Furthermore, Rabs are posttranslationally modified by prenylation; that is, the covalent attachment of either a Cys-15 (farnesyl) or Cys-20 (geranylgeranyl) isoprenoid to cysteine residues at the carboxyl terminus (6). When the Rab is active, it associates with membranes with insertion of the prenyl group into the lipid bilayer, whereas effector molecules bind to the switch region (7). This allows the Rab to coordinate the function of the effector protein to the membrane compartment where it is anchored. When the Rab is inactive, it loses affinity for the effector molecules and is extracted from membrane by GDP dissociation inhibitors (GDIs) (8, 9). GDIs bind the hydrophobic prenyl group to prevent its reassociation with the membrane and keep the Rab soluble in the cytosol. Consistently, deletion or mutation of the C-terminal cysteine residues results in complete cytosolic localization of both Rab4 and Rab5 (10).Rabs are thought to associate with membranes only upon activation (11). However, it seems that at least some Rabs can associate with the membrane in their inactive GDP-bound form. For example, inactive Rab35 exists on the plasma membrane, whereas inactive Rab11 can be found on cytoplasmic vesicles (12, 13). Here we show that inactive Rab13, in the absence of prenylation, traffics on vesicles derived from recycling and late endosomal compartments. It appears that Rab13 associates with these vesicles as part of a protein complex.
Experimental ProceduresAntibodies, DNA Constructs, and shRNA-Mouse monoclonal antibodies used were as follows: Rab8 and flotillin (BD Biosciences), Rab9 (A...
“…In the autosomal dominant neuropathy Charcot–Marie–Tooth disease type 2B, mutations in the late endosomal Rab7 GTPase interfere with receptor trafficking, resulting in alteration in intracellular signalling events 23. Specifically, mutant Rab7 delays trafficking of epidermal growth factor receptor (EGFR) to the lysosome, thereby slowing down the process of receptor degradation, causing enhanced EGFR signalling, and increased p38 and Erk1/2 activation 24.…”
We speculate that in neuronal cells, impairment of fusion of the late endosome to the vacuole would attenuate the degradation of plasma membrane receptors, thereby underlying the progressive neuronal phenotype in our patients. The VPS11 p.Cys846Gly mutation should be added to the AJ carrier screening panel.
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