Exclusion of integrins from CNS axons is regulated by Arf6 activation and the AIS.

Integrins are adhesion and survival molecules involved in axon growth during CNS development, as well as axon regeneration after injury in the peripheral nervous system (PNS). Adult CNS axons do not regenerate after injury, partly due to a low intrinsic growth capacity. We have previously studied the role of integrins in axon growth in PNS axons; in the present study, we investigate whether integrin mechanisms involved in PNS regeneration may be altered or lacking from mature CNS axons by studying maturing CNS neurons in vitro. In rat cortical neurons, we find that integrins are present in axons during initial growth but later become restricted to the somato-dendritic domain. We investigated how this occurs and whether it can be altered to enhance axonal growth potential. We find a developmental change in integrin trafficking; transport becomes predominantly retrograde throughout axons, but not dendrites, as neurons mature. The directionality of transport is controlled through the activation state of ARF6, with developmental upregulation of the ARF6 GEF ARNO enhancing retrograde transport. Lowering ARF6 activity in mature neurons restores anterograde integrin flow, allows transport into axons, and increases axon growth. In addition, we found that the axon initial segment is partly responsible for exclusion of integrins and removal of this structure allows integrins into axons. Changing posttranslational modifications of tubulin with taxol also allows integrins into the proximal axon. The experiments suggest that the developmental loss of regenerative ability in CNS axons is due to exclusion of growth-related molecules due to changes in trafficking.The authors thank Dr. Matthew N. Rasband for kindly providing the adenoviruses for ankG silencing experiment and Dr. Juan Bonifacino for AP-1 constructs. We also thank Menghon Cheah for his assistance. We acknowledge funding from the Medical Research Council, the Christopher and Dana Reeve Foundation, EU Framework 7 Project Plasticise, the European Research Council, the John and Lucille van Geest Foundation, and the NIHR Cambridge Biomedical Research Centre.This is the final version of the article. It first appeared from the Society for Neuroscience via http://dx.doi.org/10.1523/JNEUROSCI.2850-14.201

TREM2 shedding by cleavage at the H157-S158 bond is accelerated for the Alzheimer’s disease-associated H157Y variant

We have characterised the proteolytic cleavage events responsible for the shedding of Triggering Receptor Expressed on Myeloid cells 2 (TREM2) from primary cultures of human macrophages, murine microglia and TREM2-expressing human embryonic kidney (HEK293) cells. In all cell types, a soluble 17 kDa N-terminal cleavage fragment was shed into the conditioned media in a constitutive process that is inhibited by G1254023X and metalloprotease inhibitors and siRNA targeting ADAM10. Inhibitors of serine proteases and matrix metalloproteinases 2/9, and ADAM17 siRNA did not block TREM2 shedding. Peptidomimetic protease inhibitors highlighted a possible cleavage site and mass spectrometry confirmed that shedding occurred predominantly at the H157-S158 peptide bond for both wild type and H157Y human TREM2 and for the wild type murine orthologue. Crucially, we also show that the Alzheimer diseaseassociated H157Y TREM2 variant was shed more rapidly than wild type from HEK293 cells, possibly by a novel, batimastat- and ADAM10-siRNA-independent, sheddase activity. These insights offer new therapeutic targets for modulating the innate immune response in Alzheimer’s and other neurological diseases.Funding from the Wellcome Trust and the Canadian Institutes of Health Research contributed to the support of this study