Summary The DENN domain is an evolutionarily ancient protein module. Mutations in the DENN domain cause developmental defects in plants and human diseases, yet the function of this common module is unknown. We now demonstrate that the connecdenn DENN domain functions as a guanine nucleotide exchange factor for Rab35 to regulate endosomal trafficking. Loss of Rab35 activity causes an enlargement of early endosomes, inhibits MHCI recycling, and prevents the early endosomal recruitment of EHD1, a common component recycling tubules on endosomes. Our data are the first to reveal an enzymatic activity for a DENN domain and demonstrate that distinct Rab GTPases can recruit a common protein machinery to various sites within the endosomal network to establish cargo-selective recycling pathways.

Macroautophagy is an essential cellular pathway mediating the lysosomal degradation of defective organelles, long-lived proteins and a variety of protein aggregates. Similar to other intracellular trafficking pathways, macroautophagy involves a complex sequence of membrane remodeling and trafficking events. These include the biogenesis of autophagosomes (APs), which engulf portions of cytoplasm at specific subcellular locations, and their subsequent maturation into autophagolysosomes through fusion with the endo-lysosomal compartment. Although the formation and maturation of APs are controlled by molecular reactions occurring at the membrane-cytosol interface, little is known about the role of lipids and their metabolizing enzymes in this process. Historically dominated by studies on class III phosphatidylinositol 3-kinase (PI3K) (also known as Vps34), its product PI3P, as well as on the lipidation of Atg8/LC3-like proteins, this area of research has recently expanded, implicating a variety of other lipids, such as phosphatidic acid and diacylglycerol, and their metabolizing enzymes in macroautophagy. This review summarizes this progress and highlights the role of specific lipids in the various steps of macroautophagy, including the signaling processes underlying macroautophagy initiation, AP biogenesis and maturation.

Defects in endosomal sorting have been implicated in Alzheimer’s disease (AD). Endosomal traffic is largely controlled by phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide synthesized primarily by lipid kinase Vps34. Here we show that PI3P is selectively deficient in brain tissue from humans with AD and AD mouse models. Silencing Vps34 causes an enlargement of neuronal endosomes, enhances the amyloidogenic processing of amyloid precursor protein (APP) in these organelles and reduces APP sorting to intraluminal vesicles. This trafficking phenotype is recapitulated by silencing components of the ESCRT pathway, including the PI3P effector Hrs and Tsg101. APP is ubiquitinated, and interfering with this process by targeted mutagenesis alters sorting of APP to the intraluminal vesicles of endosomes and enhances amyloid-beta peptide generation. In addition to establishing PI3P deficiency as a contributing factor in AD, these results clarify the mechanisms of APP trafficking through the endosomal system in normal and pathological states.

Synaptic dysfunction caused by oligomeric assemblies of amyloid-β peptide (Aβ) has been linked to cognitive deficits in Alzheimer's disease. Here we found that incubation of primary cortical neurons with oligomeric Aβ decreases the level of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ), a phospholipid that regulates key aspects of neuronal function. The destabilizing effect of Aβ on PtdIns (4,5)P 2 metabolism was Ca 2+ -dependent and was not observed in neurons that were derived from mice that are haploinsufficient for Synj1. This gene encodes synaptojanin 1, the main PtdIns(4,5) P 2 phosphatase in the brain and at the synapses. We also found that the inhibitory effect of Aβ on hippocampal long-term potentiation was strongly suppressed in slices from Synj1 +/− mice, suggesting that Aβ-induced synaptic dysfunction can be ameliorated by treatments that maintain the normal PtdIns(4,5)P 2 balance in the brain.

Cytosolic proteins can be selectively delivered to lysosomes for degradation through a type of autophagy known as chaperonemediated autophagy (CMA). CMA contributes to intracellular quality control and to the cellular response to stress. Compromised CMA has been described in aging and in different age-related disorders. CMA substrates cross the lysosomal membrane through a translocation complex; consequently, changes in the properties of the lysosomal membrane should have a marked impact on CMA activity. In this work, we have analyzed the impact that dietary intake of lipids has on CMA activity. We have found that chronic exposure to a high-fat diet or acute exposure to a cholesterolenriched diet both have an inhibitory effect on CMA. Lysosomes from livers of lipid-challenged mice had a marked decrease in the levels of the CMA receptor, the lysosome-associated membrane protein type 2A, because of loss of its stability at the lysosomal membrane. This accelerated degradation of lysosome-associated membrane protein type 2A, also described as the mechanism that determines the decline in CMA activity with age, results from its increased mobilization to specific lipid regions at the lysosomal membrane. Comparative lipidomic analyses revealed qualitative and quantitative changes in the lipid composition of the lysosomal membrane of the lipid-challenged animals that resemble those observed with age. Our findings identify a previously unknown negative impact of high dietary lipid intake on CMA and underscore the importance of diet composition on CMA malfunction in aging.cathepsins | lipid load | lyso-bis phosphatidic acid | membrane microdomains | membrane proteins