Alix is required during development for normal growth of the mouse brain

www.nature.com/scientificreports OPEN received: 24 October 2016 accepted: 14 February 2017 Published: 21 March 2017 Alix is required during development for normal growth of the mouse brain Marine H. Laporte1,2, Christine Chatellard1,2, Victoria Vauchez1,2, Fiona J. Hemming1,2, Jean-Christophe Deloulme1,2, Frédérique Vossier1,2, Béatrice Blot1,2, Sandrine Fraboulet1,2 & Rémy Sadoul1,2 Alix (ALG-2 interacting protein X) drives deformation and fission of endosomal and cell surface membranes and thereby intervenes in diverse biological processes including cell proliferation and apoptosis. Using embryonic fibroblasts of Alix knock-out mice, we recently demonstrated that Alix is required for clathrin-independent endocytosis. Here we show that mice lacking Alix suffer from severe reduction in the volume of the brain which affects equally all regions examined. The cerebral cortex of adult animals shows normal layering but is reduced in both medio-lateral length and thickness. Alix controls brain size by regulating its expansion during two distinct developmental stages. Indeed, embryonic surface expansion of the Alix ko cortex is reduced because of the loss of neural progenitors during a transient phase of apoptosis occurring between E11.5 and E12.5. Subsequent development of the Alix ko cortex occurs normally until birth, when Alix is again required for the post-natal radial expansion of the cortex through its capacity to allow proper neurite outgrowth. The need of Alix for both survival of neural progenitor cells and neurite outgrowth is correlated with its role in clathrinindependent endocytosis in neural progenitors and at growth cones. Thus Alix-dependent, clathrin independent endocytosis is essential for controlling brain size. The size of adult brains is the resultant of a delicate balance between neural progenitor proliferation, differentiation and neurite outgrowth, which occur during both embryogenesis and post-natally. A multitude of cell surface receptors and their downstream signaling harmoniously orchestrate these processes to allow proper brain development. The endolysosomal system is crucial for this orchestration, not only by regulating cell surface expression and degradation of the receptors, but also by organising signaling hubs inside endosomes. Alg-2 interacting protein X (Alix/PDCD6IP) is a cytosolic protein1 acting at the plasma membrane to allow clathrin-independent endocytosis (CIE) of receptors through its interaction with endophilins2,3 and at endosomes to regulate caspase activation through binding to proteins of the endosomal sorting complexes required for transport (ESCRT)4,5. Alix is ubiquitously expressed1 and is also involved in virus egress, cytokinesis, cell spreading and membrane repair6 which all rely on ESCRT-mediated membrane deformation and fission. In order to better appreciate the role of the protein in vivo, we have made an Alix knock out (ko) mouse line and report here that the mice are viable and appear normal except for severe reduction in size of testes and brain as well as hydrocephaly. We focused our present study of the Alix ko mice on deciphering the mechanisms leading to microcephaly. During early development, proliferation of neuroepithelial cells surrounding the ventricle allows expansion of the telencephalon giving rise to most of the brain including the cerebral cortex7. Neuroepithelial cells mature into radial glial cells (RGCs), which continue to proliferate. At the onset of neurogenesis around E11.5, RGCs start to give birth to deep-layer neurons or intermediate progenitors, which further divide to produce more neurons8,9. Newly generated neurons migrate radially along RGC processes towards the pial surface and settle to build the cortical neuron layers10,11. Subsequently, neurons elaborate axon and dendrites, the growth of which accounts for most of the post-natal lateral expansion of the cortex12. 1 Institut National de la Santé et de la Recherche Médicale (INSERM), U1216, F-38042 Grenoble, France. 2Université Grenoble Alpes, Institut des Neurosciences, F-38042 Grenoble, France. Correspondence and requests for materials should be addressed to S.F. (email: ) or R.S. (email: ) Scientific Reports | 7:44767 | DOI: 10.1038/srep44767 1 www.nature.com/scientificreports/ Figure 1. Strategy used for making Alix ko mice. (a) Constructs used for the recombination of alix. Alixfloxed mice were crossed with Actin-cre mice or Nestin-cre mice. (b) Cropped images of Western blot demonstrate the lack of Alix expression in all organs of Alix ko mice (upper panel) and a strong reduction in the brain of Nestin;Alixfl/fl mice (lower panel). Here, we demonstrate that one aspect of the microcephaly observed in Alix ko mice is partly due to a massive but transient wave of apoptosis of a fraction of neural progenitors in the E11.5 and E12.5 telencephalon correlating with a reduced lateral expansion of the tissue and loss of deep-layer neurons. Subsequent developmental steps appear normal giving rise to newborn (P0) cortices with reduced volumes but normal cortical layers except for thinner layer VI. However, post-natal radial expansion is altered giving rise to an adult cortex with all layers significantly thinner. This alteration correlates with a reduction in post-natal neurite extension detected both in vivo and in vitro. Thus, Alix plays a major role in determining the size of the brain by controlling neural progenitor survival at the start of neurogenesis and later on, by regulating post-natal dendrite development. Results Alix ko mice suffer from severe microcephaly. Alix floxed animals were crossed with mice expressing Cre under an actin promoter to knock out alix ubiquitously (Fig. 1a,b, see Supplementary Fig. S7 for full-length Western blot). Homozygous Alix ko animals were viable even though there was a significant reduction in the proportion of Alix ko born from crossing Alix heterozygotes (15% vs the expected 25% Mendelian ratio), thus the lack of alix leads to a lethal phenotype with varying penetrance. Mendelian ratios were, however, normal in E12.5 litters demonstrating that lethality must occur beyond this developmental stage (Supplementary Fig. S1a). Alix ko mice had a normal life span and the body weight of embryos, pups and adults was not different between ko and wild type (wt) littermates (Supplementary Fig. S1b). The size of adult animals was equivalent in wt and Alix ko animals (Supplementary Fig. S1c). No gross organ abnormalities were detected, except for testes which were severely reduced in size, but had an apparently normal organization (Supplementary Fig. S1d,e). The only other obvious difference in the ko was the significant reduction in their brain size (Fig. 2), already obvious at P0 (Fig. 2a) and reflected by a decreased brain weight at all ages studied (Fig. 2b). Coronal sections of 3-month old adult brains, showed no gross abnormalities in overall brain architecture despite a 20 to 30% decrease i (...truncated)