Charcot-Marie-Tooth 2B mutations in rab7 cause dosage-dependent neurodegeneration due to partial loss of function.

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. Consequently, expression of CMT2B mutants at levels between 0.5 and 10-fold their endogenous levels fully rescues the neuropathy-like phenotypes of the rab7 mutant. Live imaging reveals that CMT2B proteins are inefficiently recruited to endosomes, but do not impair endosomal maturation. These findings are not consistent with a gain-of-function mechanism. Instead, they indicate a dosage-dependent sensitivity of neurons to rab7-dependent degradation. Our results suggest a therapeutic approach opposite to the currently proposed reduction of mutant protein function. DOI: http://dx.doi.org/10.7554/eLife.01064.001.

Membrane trafficking in neuronal maintenance and degeneration.

Defects in membrane trafficking and degradation are hallmarks of most, and maybe all, neurodegenerative disorders. Such defects typically result in the accumulation of undegraded proteins due to aberrant endosomal sorting, lysosomal degradation, or autophagy. The genetic or environmental cause of a specific disease may directly affect these membrane trafficking processes. Alternatively, changes in intracellular sorting and degradation can occur as cellular responses of degenerating neurons to unrelated primary defects such as insoluble protein aggregates or other neurotoxic insults. Importantly, altered membrane trafficking may contribute to the pathogenesis or indeed protect the neuron. The observation of dramatic changes to membrane trafficking thus comes with the challenging need to distinguish pathological from protective alterations. Here, we will review our current knowledge about the protective and destructive roles of membrane trafficking in neuronal maintenance and degeneration. In particular, we will first focus on the question of what type of membrane trafficking keeps healthy neurons alive in the first place. Next, we will discuss what alterations of membrane trafficking are known to occur in Alzheimer's disease and other tauopathies, Parkinson's disease, polyQ diseases, peripheral neuropathies, and lysosomal storage disorders. Combining the maintenance and degeneration viewpoints may yield insight into how to distinguish when membrane trafficking functions protectively or contributes to degeneration.

Similarities of Drosophila rab GTPases based on expression profiling: completion and analysis of the rab-Gal4 kit.

We recently generated rab-Gal4 lines for 25 of 29 predicted Drosophila rab GTPases. These lines provide tools for the expression of reporters, mutant rab variants or other genes, under control of the regulatory elements of individual rab loci. Here, we report the generation and characterization of the remaining four rab-Gal4 lines. Based on the completed 'rab-Gal4 kit' we performed a comparative analysis of the cellular and subcellular expression of all rab GTPases. This analysis includes the cellular expression patterns in characterized neuronal and non-neuronal cells and tissues, the subcellular localization of wild type, constitutively active and dominant negative rab GTPases and colocalization with known intracellular compartment markers. Our comparative analysis identifies all Rab GTPases that are expressed in the same cells and localize to the same intracellular compartments. Remarkably, similarities based on these criteria are typically not predicted by primary sequence homology. Hence, our findings provide an alternative basis to assess potential roles and redundancies based on expression in developing and adult cell types, compartment identity and subcellular localization.

Systematic discovery of Rab GTPases with synaptic functions in Drosophila.

BACKGROUND: Neurons require highly specialized intracellular membrane trafficking, especially at synapses. Rab GTPases are considered master regulators of membrane trafficking in all cells, and only very few Rabs have known neuron-specific functions. Here, we present the first systematic characterization of neuronal expression, subcellular localization, and function of Rab GTPases in an organism with a brain. RESULTS: We report the surprising discovery that half of all Drosophila Rabs function specifically or predominantly in distinct subsets of neurons in the brain. Furthermore, functional profiling of the GTP/GDP-bound states reveals that these neuronal Rabs are almost exclusively active at synapses and the majority of these synaptic Rabs specifically mark synaptic recycling endosomal compartments. Our profiling strategy is based on Gal4 knockins in large genomic fragments that are additionally designed to generate mutants by ends-out homologous recombination. We generated 36 large genomic targeting vectors and transgenic rab-Gal4 fly strains for 25 rab genes. Proof-of-principle knockout of the synaptic rab27 reveals a sleep phenotype that matches its cell-specific expression. CONCLUSIONS: Our findings suggest that up to half of all Drosophila Rabs exert specialized synaptic functions. The tools presented here allow systematic functional studies of these Rabs and provide a method that is applicable to any large gene family in Drosophila.

The response of high and low polyamine-producing cell lines to aluminum and calcium stress.

The diamine putrescine (Put) has been shown to accumulate in tree leaves in response to high Al and low Ca in the soil, leading to the suggestion that this response may provide a physiological advantage to leaf cells under conditions of Al stress. The increase in Put is reversed by Ca supplementation in the soil. Using two cell lines of poplar (Populus nigra x maximowiczii), one with constitutively high Put (resulting from transgenic expression of a mouse ornithine decarboxylase--called HP cells) and the other with low Put (control cells), we investigated the effects of reduced Ca (0.2-0.8 mM vs. 4 mM) and treatment with 0.1 mM Al on several biochemical parameters of cells. We found that in the presence of reduced Ca concentration, the HP cells were at a disadvantage as compared to control cells in that they showed greater reduction in mitochondrial activity and a reduction in the yield of cell mass. Upon addition of Al to the medium, the HP cells, however, showed a reversal of low-Ca effects. We conclude that due to increased ROS production in the HP cells, their tolerance to low Ca is compromised. Contrary to the expectation of deleterious effects, the HP cells showed an apparent advantage in the presence of Al in the medium, which could have come from reduced uptake of Al, enhanced extrusion of Al following its accumulation, and perhaps a reduction in Put catabolism as a result of a reduction in its biosynthesis.