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Of neuronal markers [51], and in an AD model reduced amyloid pathology [6]. While these effects were attributed to increased autophagy, it is possible that enhanced protein sorting also contributed to this rescue. Our work presented here links beclin 1 with the TGF- pathway. A decrease in beclin 1 levels, as previously observed in AD [6], leads to impaired ALK5 recycling and deprives neurons of neuroprotective TGF- signaling. Future studies should determine the full extent of beclin 1-mediated protein sorting, and its contribution to neuronal survival. In particular, identification of the full receptor repertoire beclin 1 regulates may reveal additional trophic factor or other signaling pathways and provide a deeper insight into the consequences of altered beclin 1 levels. Knowledge of both the autophagy and protein sorting functions of beclin 1 should inform strategies to target beclin 1 in neurodegenerative disease.MethodsCell culture and primary neuron isolationSingle cell-suspensions of primary hippocampal and cortical neurons were isolated at E16.5 from CF1 pregnant mothers (Charles River) following the protocol described in Fath et al. [52]. Cells were seeded onto 24-well plates with or without coverslips coated with 0.1 mg/mL polyL-lysine in boric acid pH 8. Neurons were maintained in neurobasal medium + B27. Hippocampal neurons were used for microscopy (50,000 cells/well) Vandetanib only due to their limited numbers, while cortical neurons were used for western blotting (200,000 cells/well). COS7 and mouse fibroblast (MFB) F11 cells were cultured in DMEM media supplemented with 10 fetal bovine serum. For TGF- treatments in F11 cells, cells were washed 2?in PBS and incubated with 1 ng/mL TGF-1 (R D Systems) or TGF- 3 (National Institute for Biological Standards and Control #98/608) in serum-free media for 1 h.Plasmids and lentivirusesConclusions Neurodegenerative diseases such as Alzheimer's Disease are associated with alterations in multiple pathwaysThe lentivirus knockdown plasmids contain shRNA targeting mouse beclin 1 at nucleotides 405?23 [5] or a scrambled control shRNA in the pSIH-H1 vector from SBI System Biosciences. This plasmid also contains a copepod GFP (copGFP) to monitor infection efficiency. All lentiviruses were generated by the Stanford Neuroscience Gene Vector and Virus Core. Plasmids for VPS34, UVRAG, ATG7, and Atg14 shRNA were obtained from Santa Cruz Biotechnology Inc. Plasmids were providedO'Brien et al. Molecular Neurodegeneration (2015) 10:Page 12 ofas pools of three 19?5 bp target shRNA. The Rab5GFP and Rab7-GFP plasmids were generously provided by Dr. Craig Garner.Cell transfection and viral transductionPrimary cells were infected at 7 DIV at an MOI of 20 in neurobasal medium + B27. Virus was removed 16?0 h later and cells were analyzed 7 days post infection. F11 or COS7 cells were plated in media containing 8 g/mL polybrene for 1 h prior to virus addition at an MOI of 50. Virus was removed after 16?0 h and cells were analyzed 48 h post infection. For transfection, cells were transfected using lipofectamine 2000 (Thermo Fisher).Antibodies for western blotting (WB) and immunocytochemistry (ICC)protein/sample was loaded into precast 4?2 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17139194 bis-tris gels and run with MES buffer (Invitrogen). Gels were transferred onto 0.2 m nitrocellulose (BioRad) and incubated with antigen-specific primary antibodies at 4 overnight. For LiCor detection, membranes were incubated with species-specific IR Dye antibodies (LiCor) and.