Protrudin functions from the endoplasmic reticulum to support axon regeneration in the adult CNS

Adult mammalian central nervous system axons have intrinsically poor regenerative capacity, so axonal injury has permanent consequences. One approach to enhancing regeneration is to increase the axonal supply of growth molecules and organelles. We achieved this by expressing the adaptor molecule Protrudin which is normally found at low levels in non-regenerative neurons. Elevated Protrudin expression enabled robust central nervous system regeneration both in vitro in primary cortical neurons and in vivo in the injured adult optic nerve. Protrudin overexpression facilitated the accumulation of endoplasmic reticulum, integrins and Rab11 endosomes in the distal axon, whilst removing Protrudin’s endoplasmic reticulum localization, kinesin-binding or phosphoinositide-binding properties abrogated the regenerative effects. These results demonstrate that Protrudin promotes regeneration by functioning as a scaffold to link axonal organelles, motors and membranes, establishing important roles for these cellular components in mediating regeneration in the adult central nervous system.


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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Dr Veselina Petrova (vp351@cam.ac.uk)

21/09/2020
Images of immunostained cells were taken with a confocal microscope (Leica DMI 4000 B) using LAS-AF software (Leica Application Suite, Version 2.7.3.9723). For Protrudin localisation and intensity measurements as well as for ER localisation at the growth cone, a z-stack of images was obtained through each cell by taking an image at every 0.5 um thickness and an average intensity z-projection was created in Fiji ImageJ software where fluorescence intensity was measure against background (Schindelin et al., 2012). In vitro axotomy videos and data analysis was also performed using LAS-AF software. Images of optic nerves and retinas were obtained using the ZEN Digital Imaging Suite. Live-cell imaging was performed using spinning disk confocal microscopy, using an Olympus IX70 microscope with a Hamamatsu EM-CCD Image-EM camera and a PerkinElmer Ultra-VIEW scanner. Videos were taken using Meta-Morph software (Version 7.6.1.0). Alliance (Version 16.05) software was used for detection of Western blot signal. BCA assays were analysed using Gen5.1 software (Version 5.1).
Data analysis was performed using GraphPad Prism software (Version 8.0). Sholl analysis was performed in SPSS software (Version 26).
The RNA-sequencing datasets from peripheral DRG neurons in development and after injury, from retinal ganglion cells during development or from cultured rat primary cortical neurons used for analysis in this study have previously been deposited in NCBI Gene Expression Omnibus (accession numbers: GSE66128, GSE90654 nature research | reporting summary

October 2018
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Complete experiments were repeated a minimum of three times under controlled conditions and variation was tested between individual experiments. No significant variation was found between different series of the same experiments. Control and experimental groups were tested under identical conditions. Different viral treatments were assigned to animals randomly. Cell culture samples were assigned randomly, with control and experimental groups analysed in identical conditions to minimise potential covariates. For example, a typical laser axotomy experiment would run across four to five days (DIV13-17), with four experimental groups analysed. We took steps to ensure that no particular group was analysed on the same day. In this way samples would be analysed on a different day for each replicate of the experiments.
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