Adaptor protein (AP)-1/σ1B(-/-) mice have reduced synaptic-vesicle (SV) recycling and increased endosomes. Mutant mice have impaired spatial memory, and σ1B-deficient humans have a severe mental retardation. In order to define these σ1B(-/-) 'bulk' endosomes and to determine their functions in SV recycling, we developed a protocol to separate them from the majority of the neuronal endosomes. The σ1B(-/-) 'bulk' endosomes proved to be classic early endosomes with an increase in the phospholipid phosphatidylinositol 3-phosphate (PI-3-P), which recruits proteins mediating protein sorting out of early endosomes into different routes. σ1B deficiency induced alterations in the endosomal proteome reveals two major functions: SV protein storage and sorting into endolysosomes. Alternative endosomal recycling pathways are not up-regulated, but certain SV proteins are misrouted. Tetraspanins are enriched in σ1B(-/-) synaptosomes, but not in their endosomes or in their clathrin-coated-vesicles (CCVs), indicating AP-1/σ1B-dependent sorting. Synapses contain also more AP-2 CCV, although it is expected that they contain less due to reduced SV recycling. Coat composition of these AP-2 CCVs is altered, and thus, they represent a subpopulation of AP-2 CCVs. Association of calmodulin-dependent protein kinase (CaMK)-IIα, -δ and casein kinase (CK)-IIα with the endosome/SV pool is altered, as well as 14-3-3η, indicating changes in specific signalling pathways regulating synaptic plasticity. The accumulation of early endosomes and endocytotic AP-2 CCV indicates the regulation of SV recycling via early endosomes by the interdependent regulation of AP-2-mediated endocytosis and AP-1/σ1B-mediated SV reformation.

AP-1/σ1B-deficiency causes X-linked intellectual disability. AP-1/σ1B −/− mice have impaired synaptic vesicle recycling, fewer synaptic vesicles and enhanced endosome maturation mediated by AP-1/σ1A. Despite defects in synaptic vesicle recycling synapses contain two times more endocytic AP-2 clathrin-coated vesicles. We demonstrate increased formation of two classes of AP-2/clathrin coated vesicles. One which uncoats readily and a second with a stabilised clathrin coat. Coat stabilisation is mediated by three molecular mechanisms: reduced recruitment of Hsc70 and synaptojanin1 and enhanced μ2/AP-2 phosphorylation and activation. Stabilised AP-2 vesicles are enriched in the structural active zone proteins Git1 and stonin2 and synapses contain more Git1. Endocytosis of the synaptic vesicle exocytosis regulating Munc13 isoforms are differentially effected. Regulation of synaptic protein endocytosis by the differential stability of AP-2/clathrin coats is a novel molecular mechanism of synaptic plasticity.

Cancers of unknown primary (CUPs), featuring metastatic dissemination in the absence of a primary tumor, are a biological enigma and a fatal disease. We propose that CUPs are a distinct, yet unrecognized, pathological entity originating from stem-like cells endowed with peculiar and shared properties. These cells can be isolated in vitro (agnospheres) and propagated in vivo by serial transplantation, displaying high tumorigenicity. After subcutaneous engraftment, agnospheres recapitulate the CUP phenotype, by spontaneously and quickly disseminating, and forming widespread established metastases. Regardless of different genetic backgrounds, agnospheres invariably display cell-autonomous proliferation and self-renewal, mostly relying on unrestrained activation of the MAP kinase/MYC axis, which confers sensitivity to MEK inhibitors in vitro and in vivo. Such sensitivity is associated with a transcriptomic signature predicting that more than 70% of CUP patients could be eligible to MEK inhibition. These data shed light on CUP biology and unveil an opportunity for therapeutic intervention.

The σ1 subunit of the AP-1 clathrin-coated-vesicle adaptor-protein complex is expressed as three isoforms. Tissues express σ1A and one of the σ1B and σ1C isoforms. Brain is the tissue with the highest σ1A and σ1B expression. σ1B-deficiency leads to severe mental retardation, accumulation of early endosomes in synapses and fewer synaptic vesicles, whose recycling is slowed down. AP-1/σ1A and AP-1/σ1B regulate maturation of these early endosomes into multivesicular body late endosomes, thereby controlling synaptic vesicle protein transport into a degradative pathway. σ1A binds ArfGAP1, and with higher affinity brain-specific ArfGAP1, which bind Rabex-5. AP-1/σ1A-ArfGAP1-Rabex-5 complex formation leads to more endosomal Rabex-5 and enhanced, Rab5GTP-stimulated Vps34 PI3-kinase activity, which is essential for multivesicular body endosome formation. Formation of AP-1/σ1A-ArfGAP1-Rabex-5 complexes is prevented by σ1B binding of Rabex-5 and the amount of endosomal Rabex-5 is reduced. AP-1 complexes differentially regulate endosome maturation and coordinate protein recycling and degradation, revealing a novel molecular mechanism by which they regulate protein transport besides their established function in clathrin-coated-vesicle formation.

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