Role of Rab5 in synaptic vesicle recycling

During synaptic transmission, NT-filled synaptic vesicles are released by Ca2+-triggered exocytosis at the active zone. Following exocytosis, SV membrane is immediately re-internalized and synaptic vesicles (SVs) are regenerated by a local recycling mechanism within the presynaptic terminal. It is debated whether an endosomal compartment is involved in this recycling process. In contrast, it is well known from cultured mammalian cells, that endocytic vesicles fuse to the early sorting endosome. The early endosome is a major sorting station of the cell where cargo is send into the degradative pathway to late endosome and lysosome or towards recycling. Each trafficking step is mediated by a certain protein of the Rab family. Rab proteins are small GTPases belonging to the Ras superfamily. They accumulate at their target compartments and have thereby been used as markers for the different endocytic organelles in cultured mammalian cells. Rab5 controls trafficking from the PM to the early endosome and has thereby been used as marker for this compartment. A second marker is based on the specific binding of the FYVE zinc finger protein domain to the lipid PI(3)P that is specifically generated at the early endosomal membrane. This study used the Drosophila NMJ as a model system to investigate the SV recycling process. In particular, three questions were addressed: First, is an endosomal compartment present at the synapse? Second, do SVs recycle through an endosome? Third, is Rab5 involved in SV recycling? We used GFP fusions of Rab5 and 2xFYVE to visualize endosomal compartments at the presynaptic terminal of Drosophila third instar larval NMJs. Furthermore, the endosomes are located within the pool of recycling SVs, labeled with the styryl-dye FM5-95. Using the temperature-sensitive mutation in Dynamin, shibirets, we showed that SV recycling involves trafficking through an intermediate endosomal compartment. In cultured mammalian cells, interfering with Rab5 function by expressing the dominant negative version, Rab5SN causes the fragmentation of the endosome and the accumulation of endocytic vesicles. In contrast, when Rab5 is overexpressed enlarged endosomal compartments were observed. In Drosophila, the endosomal compartment was disrupted when loss of function and dominant negative mutants of Rab5 were expressed. In addition, at the ultrastructural we observed an accumulation of endocytic vesicles in Rab5S43N expressing terminals and enlarged endosomes when Rab5 was overexpressed. Furthermore, interfering with Rab5 function using the dominant negative Rab5S43N caused a decrease in the SV recycling kinetics as shown by FM1-43 experiments. In contrast, overexpression of Rab5 or GFP-Rab5 caused an increase in the FM1-43 internalization rate. Finally, standard electrophysiological techniques were used to measure synaptic function. We found that the Rab5-mediated endosomal SV recycling pathway generates vesicles with a higher fusion efficacy during Ca2+-triggered release, compared to SVs recycled when Rab5 function was impaired. We therefore suggest a model in which the endosome serves as organelle to control the SV fusion efficacy and thereby the synaptic strength. Since changes in the synaptic strength are occuring during learning and memory processes, controlling endosomal SV recycling might be a new molecular mechanism involved in learning and memory.

Recycling urban waste as possible use for rooftop vegetable garden

Urban authorities in Europe are confronted with increasing demands by urban dwellers for allotment gardens, but vacant urban soil tends to be scarce and/or polluted by past industrial activities. A possible solution for local authorities could therefore be to promote rooftop gardening. However little technical information exists on certain forms of rooftop urban agriculture, called Z-Farming. In 2012, a pilot experiment was run in Paris (France). Simple and cheap systems of rooftop gardening were tested on a rooftop using as crop substrates only local urban organic waste so as to contribute to the urban metabolism. Production levels and heavy metal contents in cropping substrates and edible vegetables were measured. Available results show (i) high levels of crop production with limited inputs compared to land professional gardening, (ii) low levels of heavy metal pollutants in the edible parts of the crops, especially for Cd and Pb with respect to EU norms for vegetables and (iii) positive influence on yields on organizing the substrate in layers and enhancing the biological activity through earthworm inoculation. These encouraging results allow us to consider that rooftop gardening is feasible and seem to have a great potential to improve urban resiliency. It will nevertheless be necessary to identify more precisely the types of roof that can be used and to assess more fully the generic result of the low level of pollution, as well as the global sustainability of these cropping systems.