Although AP-1 has been ascribed many other roles, particularly in transport between the TGN and endosomes in undifferentiated cells and unicellular organisms ( Robinson, 2004), mounting evidence indicates that this protein complex functions as a regulator

of polarized sorting in differentiated cells and multicellular organisms. Consistent with the critical role of AP-1 in polarized sorting in many cell types, null mutations in AP-1 subunit genes cause embryonic lethality in multicellular organisms such as C. elegans ( Shim et al., 2000), zebrafish ( Zizioli et al., 2010), and mouse ( Zizioli et al., 1999; Meyer et al., AP24534 concentration 2000). This is in contrast to the viability of AP-1 null mutant yeast ( Phan et al., 1994), Dictyostelium ( Lefkir et al., 2003), and mouse embryonic fibroblasts ( Meyer et al., 2000) grown in single-cell cultures. Mutations in AP-1 subunit genes also cause two human developmental disorders, the MEDNIK syndrome and

a form of X-linked mental retardation (XLMR) that is also referred to as Fried syndrome. MEDNIK syndrome is a neurocutaneous disorder caused by mutation of the gene encoding σ1A ( Montpetit et al., 2008), one of three isoforms of the σ1 subunit of AP-1 (i.e., σ1A, PCI-32765 cost σ1B, and σ1C) ( Boehm and Bonifacino, 2001; Mattera et al., 2011). Fried syndrome is a neurodevelopmental disorder that results from mutations in σ1B ( Tarpey et al., 2006). Both disorders present with mental retardation and a range of other anatomical and functional abnormalities of the central nervous system. It is currently unclear how deficiency of a σ1 isoform could cause these diseases. One possibility is that σ1 isoforms are differentially expressed in different cell populations. Alternatively, σ1 isoforms could endow AP-1 with different cargo-recognition specificities, as recently shown for the binding of proteins with dileucine-based sorting signals ( Mattera et al., 2011). In either case, our findings suggest that these disorders may arise from failure to sort certain cargoes to the somatodendritic domain of specific neuronal populations.

Primary cultures of rat hippocampal neurons were prepared as previously described (Caceres et al., 1984). Briefly, hippocampi were dissected from Sprague-Dawley rats on embryonic day SPTLC1 18 and dissociated with trypsin. Cells were plated onto poly-L-lysine-treated plates and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% v/v horse serum for 2–3 hr. The culture medium was then substituted with Neurobasal medium supplemented with B-27 and Glutamax (Invitrogen). After 3–4 days in culture, neurons were transfected with different plasmid constructs (see Supplemental Experimental Procedures) using Lipofectamine 2000 (Invitrogen), except for biochemical studies in which nucleofection was performed in suspension using the Amaxa system (Lonza).