Signaling in neurons. We demonstrate that beclin 1 recruits the retromer to ALK5 and regulates its recycling, and that loss of beclin 1 results in neuronal death.targeting shRNA (bec shRNA). Infection of primary forebrain neurons resulted in efficient knockdown of beclin 1 by 75 (Fig. 1a, b). To assess neuronal survival upon beclin 1 knockdown, we followed the experimental design outlined in Fig. 1c. Infected cells were maintained in culture for 2 or 3 weeks post infection (P.I), then fixed and stained for MAP2, a marker of neuronal dendrites. At 2 weeks P.I, there was no significant change in MAP2+ neurons (Fig. 1d). However, by 3 weeks postinfection, beclin 1 knockdown resulted in a significant decrease in the number of surviving MAP2+ neurons (Fig. 1d, e), supporting previous findings that neurons with decreased levels of beclin 1 degenerate in vivo.Beclin 1 localizes to the endosomal system in neuronsResultsBeclin 1 is required for neuronal survival in vitroGiven that decreased beclin 1 levels lead to neuronal death in vivo [6, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/15501003 7], we wanted to establish an in vitro system to study how beclin 1 maintains neuronal survival. To do this, we generated cultures of primary forebrain neurons from wild type CF1 mice and manipulated beclin 1 levels using a lentivirus encoding either a control scrambled (ctrl shRNA) or beclin 1-In order to determine where beclin 1 may function in neurons, we first looked at localization of beclin 1 in primary neurons. Beclin 1 has been reported to localize primarily to the trans-Golgi network [27] and the endoplasmic reticulum under basal conditions [28, 29] and to nascent autophagosomes during autophagy initiation [30]. However, work from our lab that demonstrates beclin 1 recruits the retromer to phagosomes [5], and others have only recently suggested beclin 1 may function more broadly in the endosomal system [7]. We first confirmed the specificity of the antibody in COS7 cells transfected with FLAG-beclin 1. We stained with antibodies against beclin 1 and FLAG and found a large degree of overlap between the signals (Additional file 1: Figure S2A). We also stained untransfected cells we had infected with our ctrl or bec shRNA. Knockdown of beclin 1 resulted in a significant decrease in staining with the beclin antibody (Additional file 1: Figure S2B, C). Together these results corroborate the specificity of the beclin 1 antibody. To determine the Alectinib localization of beclin 1, we looked for colocalization between beclin 1 and several subcellular markers. In primary hippocampal neurons, beclin 1 was distributed throughout the soma and dendrites where it colocalized with Rab5-GFP, a marker of early endosomes (Fig. 2a, mean colocalization coefficient: 0.49 ?0.03), and Rab7-GFP, a marker of late endosomes (Fig. 2b, mean colocalization coefficient: 0.50 ?0.03). Surprisingly, beclin 1 colocalized with these markers to a greater degree than the trans-Golgi marker, Golgin 97 (Fig. 2c, mean colocalization coefficient: 0.13 ?0.01). This is similar to what we observed in the unpolarized cell line, COS7. In these cells, beclin 1 was found primarily in a perinuclear region (Additional file 1: Figure S3A, B) where it colocalized with Rab5-GFP (Additional file 1: Figure S3C, colocalization coefficient 0.71 ?0.06), Rab7-GFP (Additional file 1: Figure S3D, colocalization coefficient (0.77 ?0.08), and to a lesser extent, Golgin 97 (Additional file 1: Figure S3E, colocalization coefficientO'Brien et al. Molecular Neurodegenerat.