The retromer complex component VPS35 prevents activation of the BACE1 and Aβ production and thus plays an essential role in limiting Alzheimer’s disease neuropathology.

SummaryPear (Pyrus pyrifolia L.) has an S-RNase-based gametophytic self-incompatibility (SI) mechanism, and S-RNase has also been implicated in the rejection of self-pollen and genetically identical pollen. However, RNA degradation might be only the beginning of the SI response, not the end. Recent in vitro studies suggest that S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia, and it seems that a relationship exists between self S-RNase, actin depolymerization and DNA degradation. To further uncover the SI response in pear, the relationship between self S-RNase and tip-localized reactive oxygen species (ROS) was evaluated. Our results show that S-RNase specifically disrupted tip-localized ROS of incompatible pollen tubes via arrest of ROS formation in mitochondria and cell walls. The mitochondrial ROS disruption was related to mitochondrial alteration, whereas cell wall ROS disruption was related to a decrease in NADPH. Tip-localized ROS disruption not only decreased the Ca 2+ current and depolymerized the actin cytoskeleton, but it also induced nuclear DNA degradation. These results indicate that tip-localized ROS disruption occurs in Pyrus pyrifolia SI. Importantly, we demonstrated nuclear DNA degradation in the incompatible pollen tube after pollination in vivo. This result validates our in vitro system in vivo. Journal of Cell Science Results S-RNase disrupts tip-localized ROS in incompatible pollen tubesTo assess the effect of ROS on pollen tube growth, the pollen was grown in the basal medium for 1 hour at 25°C, then the NADPH oxidase inhibitor diphenylene iodonium chloride (DPI) and ROS scavenger 10,15,21H,23H-porphin (TMPP) were added to the medium. As expected, DPI and TMPP obviously inhibited pollen tube growth (Fig. 1A), which indicated that ROS are necessary for pear pollen tube growth. Furthermore, to evaluate the effect of S-RNase on tip-localized ROS, pollen tubes were stained with the ROS fluorescence probe, 5-(and 6-)chloromethyl-2Ј,7Ј-dichlorodihydrofluorescein diacetate (CM-H 2 DCFDA). Two types of pollen tubes were stained with CM-H 2 DCFDA (Fig. 1B): pollen tubes with strongest fluorescence in the tip (from subapical domain to apex) were regarded as normal, whereas pollen tubes with uniform fluorescence suggested that ROS in the tip-localized pollen tube were disrupted. The sample was stained with CM-H 2 DCFDA at 30 minutes after S-RNase, DPI or TMPP challenge to count the pollen tube with strongest fluorescence in tip (Fig. 1C). In the control, the proportion of pollen tubes with strongest fluorescence in the tip relative to the total number of tubes was 52.7±2.1% (means ± s.e.; n>100), and with the DPI or TMPP treatments, only 32.4±3.4% (n>100) and 35.9± 4.4% (n>100), respectively. In the compatible treatment, similarly to the control, 52.0±1.4% (n>100) of pollen tubes had strongest fluorescence in the tip. However, in the incompatible treatment, this was only 28.8±1.7% (n>100), nearly half the control value.Nitroblue tetrazo...

SummaryVPS35, a major component of the retromer, plays an important role in the selective endosome-to-Golgi retrieval of membrane proteins. Dysfunction of retromer is a risk factor for neurodegenerative disorders, but its function in developing mouse brain remains poorly understood. Here we provide evidence for VPS35 promoting dendritic growth and maturation, and axonal protein transport in developing mouse hippocampal neurons. Embryonic hippocampal CA1 neurons suppressing Vps35 expression by in utero electroporation of its micro RNAs displayed shortened apical dendrites, reduced dendritic spines, and swollen commissural axons in the neonatal stage, those deficits reflecting a defective protein transport/trafficking in developing mouse neurons. Further mechanistic studies showed that Vps35 depletion in neurons resulted in an impaired retrograde trafficking of BACE1 (β1-secretase) and altered BACE1 distribution. Suppression of BACE1 expression in CA1 neurons partially rescued both dendritic and axonal deficits induced by Vps35-deficiency. These results thus demonstrate that BACE1 acts as a critical cargo of retromer in vitro and in vivo, and suggest that VPS35 plays an essential role in regulating apical dendritic maturation and in preventing axonal spheroid formation in developing hippocampal neurons.

Neurite extension is essential for wiring the nervous system during development. Although several factors are known to regulate neurite outgrowth, the underlying mechanisms remain unclear. Here, we provide evidence for a role of phosphatidylinositol transfer protein-alpha (PlTPalpha) in neurite extension in response to netrin-1, an extracellular guidance cue. PlTPalpha interacts with the netrin receptor DCC (deleted in colorectal cancer) and neogenin. Netrin-1 stimulates PlTPalpha binding to DCC and to phosphatidylinositol (5) phosphate [Pl(5)P], increases its lipid-transfer activity and elevates hydrolysis of phosphatidylinositol bisphosphate (PlP2). In addition, the stimulated PIP2 hydrolysis requires PlTPalpha. Furthermore, cortical explants of PlTPalpha mutant mice are defective in extending neurites in response to netrin-1. Commissural neurons from chicken embryos expressing a dominant-negative PlTPalpha mutant show reduced axon outgrowth. Morpholino-mediated knockdown of PlTPalpha expression in zebrafish embryos leads to dose-dependent defects in motor-neuron axons and reduced numbers of spinal-cord neurons. Taken together, these results identify a crucial role for PlTPalpha in netrin-1-induced neurite outgrowth, revealing a signalling mechanism for DCC/neogenin and PlTPalpha regulation.