BAD-LAMP defines a subset of early endocytic organelles in subpopulations of cortical projection neurons

Abstract

The brain-associated LAMP-like molecule (BAD-LAMP) is a new member of the family of lysosome associated membrane proteins (LAMPs). In contrast to other LAMPs, which show a widespread expression, BAD-LAMP expression in mice is confined to the postnatal brain and therein to neuronal subpopulations in layers II/III and V of the neocortex. Onset of expression strictly parallels cortical synaptogenesis. In cortical neurons, the protein is found in defined clustered vesicles, which accumulate along neurites where it localizes with phosphorylated epitopes of neurofilament H. In primary neurons, BAD-LAMP is endocytosed, but is not found in classical lysosomal/endosomal compartments. Modification of BAD-LAMP by addition of GFP revealed a cryptic lysosomal retention motif, suggesting that the cytoplasmic tail of BAD-LAMP is actively interacting with, or modified by, molecules that promote its sorting away from lysosomes. Analysis of BAD-LAMP endocytosis in transfected HeLa cells provided evidence that the protein recycles to the plasma membrane through a dynamin/AP2-dependent mechanism. Thus, BAD-LAMP is an unconventional LAMP-like molecule and defines a new endocytic compartment in specific subtypes of cortical projection neurons. The striking correlation between the appearance of BAD-LAMP and cortical synatogenesis points towards a physiological role of this vesicular determinant for neuronal function.

BAD-LAMP is a novel biomarker of nonactivated human plasmacytoid dendritic cells

“…On this basis, UNC-46/BAD-LAMP has been proposed to act as a sorting chaperone addressing the membrane-associated GABA transporter (UNC-47) to synaptic vesicles. 31 Although human BAD-LAMP, like its murine homolog, 28 is principally expressed in the brain, we show here that among blood cells, it is also specifically found in primary CD123 ϩ pDCs and BPDCNs. BAD-LAMP mRNA and protein levels are downregulated upon CpG DNA stimulation of freshly isolated primary BDCA-4 ϩ human blood pDCs.…”

“…BAD-LAMP mutants and tagged forms were as described previously. 28 The human brain, spleen, and skin total RNA extracts were obtained from Zyagen. The human UNC93B1-His cDNA construct was a kind gift from Dr J.-L. Casanova (Rockefeller University, New York, NY).…”

“…28 BAD-LAMP is a transmembrane glycosylated protein that shares sequence and structural homology with the canonical LAMP1 and LAMP2 molecules (CD107). 29,30 BAD-LAMP harbors an endosomal addressing signal within its cytoplasmic tail, and contains several conserved cysteine residues that allow for the formation of particular structural loops known as LAMP folds.…”

“…Mouse BAD-LAMP was shown to accumulate in a novel endocytic compartment in specific subtypes of cortical projection neurons. 28 At the functional level, mutations in UNC-46, the Caenorhabditis elegans ortholog of BAD-LAMP, cause defects in most neurotransmitter GABA-mediated behaviors in the nematode. On this basis, UNC-46/BAD-LAMP has been proposed to act as a sorting chaperone addressing the membrane-associated GABA transporter (UNC-47) to synaptic vesicles.…”

NCAM is ubiquitylated, endocytosed and recycled in neurons

“…NCAM-deficient mice have been described previously (Cremer et al, 1994). Primary cortical neurons from mice embryonic day 15.5 (E15.5) were prepared as described (David et al, 2007). Cells were then transiently transfected using the Mouse Neuron Nucleofector Kit (amaxa biosystems, Köln, Germany) slightly modified according to the manufacturer's instructions using program Q-05 and plated at a density of 300,000 cells per 24-well plate on pLL-coated coverslips.…”