The Alzheimer susceptibility gene BIN1 induces isoform-dependent neurotoxicity through early endosome defects

The Bridging Integrator 1 (BIN1) gene is a major susceptibility gene for Alzheimer’s disease (AD). Deciphering its pathophysiological role is challenging due to its numerous isoforms. Here we observed in Drosophila that human BIN1 isoform1 (BIN1iso1) overexpression, contrary to human BIN1 isoform8 (BIN1iso8) and human BIN1 isoform9 (BIN1iso9), induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level is responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs. Altogether, our data demonstrate that the AD susceptibility gene BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation, which is an early pathophysiological hallmark of AD pathology.

Plant trans-golgi network/early endosome pH regulation requires cation chloride cotransporter (CCC1)

Plant cells maintain a low luminal pH in the Trans-Golgi-Network/Early Endosome (TGN/EE), the organelle in which the secretory and endocytic pathways intersect. Impaired TGN/EE pH regulation translates into severe plant growth defects. The identity of the proton pump and proton/ion antiporters that regulate TGN/EE pH have been determined, but an essential component required to complete the TGN/EE membrane transport circuit remains unidentified - a pathway for cation and anion efflux. Here, we have used complementation, genetically encoded fluorescent sensors, and pharmacological treatments to demonstrate that Arabidopsis Cation Chloride Cotransporter (CCC1) is this missing component necessary for regulating TGN/EE pH and function. Loss of CCC1 function leads to alterations in TGN/EE-mediated processes including endocytic trafficking, exocytosis and response to abiotic stress, consistent with the multitude of phenotypic defects observed in ccc1 knockout plants. This discovery places CCC1 as a central component of plant cellular function.

The role of clathrin in exocytosis and the mutual regulation of endo- and exocytosis in plant cells

Within the plant endomembrane system, the vesicle coat protein clathrin localizes to the plasma membrane (PM) and the trans-Golgi Network/Early Endosome (TGN/EE). While the role of clathrin as a major component of endocytosis at the PM is well established, its function at TGN/EE, possibly in exocytosis or the vacuolar pathway, is a matter of debate. This shared function of clathrin also opens a question whether plant cells possess a homeostatic mechanisms that balance rates of opposite trafficking routes, such as endo- and exocytosis. Here we address these questions using lines inducibly silencing CLATHRIN HEAVY CHAIN (CHC). We find a relocation of exocytic soluble and integral membrane protein cargoes to the vacuole, supporting a function of clathrin in exocytosis. A comparison with lines overexpressing AUXILIN-LIKE1, where inhibition of CME precedes rerouting of secretory cargoes, does not support a homeostatic regulatory mechanism adjusting exocytosis to the rates of endocytosis. Complementary experiments reveal only minor and variably detectable reductions in the rates of CME in secretory mutants, also not indicative of a converse homeostatic mechanism adjusting rates of endocytosis to the rates of secretion.

The APPL1-Rab5 axis restricts NLRP3 inflammasome activation through early endosomal-dependent mitophagy in macrophages

Although mitophagy is known to restrict NLRP3 inflammasome activation, the underlying regulatory mechanism remains poorly characterized. Here we describe a type of early endosome-dependent mitophagy that limits NLRP3 inflammasome activation. Deletion of the endosomal adaptor protein APPL1 impairs mitophagy, leading to accumulation of damaged mitochondria producing reactive oxygen species (ROS) and oxidized cytosolic mitochondrial DNA, which in turn trigger NLRP3 inflammasome overactivation in macrophages. NLRP3 agonist causes APPL1 to translocate from early endosomes to mitochondria, where it interacts with Rab5 to facilitate endosomal-mediated mitophagy. Mice deficient for APPL1 specifically in hematopoietic cell are more sensitive to endotoxin-induced sepsis, obesity-induced inflammation and glucose dysregulation. These are associated with increased expression of systemic interleukin-1β, a major product of NLRP3 inflammasome activation. Our findings indicate that the early endosomal machinery is essential to repress NLRP3 inflammasome hyperactivation by promoting mitophagy in macrophages.

Immunofluorescence of RAB5 and FLAG-EEA1 puncta after Dynamin-1 and -2 inhibition with Dyngo4a v1

Selective purification of early endosomes can be achieved through affinity capture of the early endosome-associated protein EEA1 (termed Endo-IP) (Park et al. in submission). These purified endosomes can be used for proteomic and lipidomic studies to obtain snapshots of early endosomes. Here we present an immunofluorescence protocol to assess the extent of colocalization between FLAG-EEA1 and RAB5 with and without the Dynamin-1 and -2 (DNM1/2) inhibitor Dyngo4a.

ClCd and ClCf act redundantly at the TGN/EE and prevent acidification of the Golgi stack

The trans-Golgi network/early endosome (TGN/EE) serves as the central hub in which exo- and endocytic trafficking pathways converge and specificity of cargo routing needs to be achieved. Acidification is a hallmark of the TGN/EE and is maintained by the vacuolar H+-ATPase (V-ATPase) with support of proton-coupled antiporters. We show here that ClCd and ClCf, two distantly related members of the Arabidopsis chloride channel (ClC)-family co-localize in the TGN/EE, act redundantly, and are essential for male gametophyte development. Combining an inducible knock-down approach and in vivo pH-measurements, we show here that reduced ClC-activity does not affect pH in the TGN/EE but causes hyperacidification of trans-Golgi cisternae. Taken together, our results show that ClC-mediated anion transport into the TGN/EE is essential and affects spatio-temporal aspects of TGN/EE-maturation as well as its functional separation from the Golgi stack.

Revising Endosomal Trafficking under Insulin Receptor Activation

The endocytosis of ligand-bound receptors and their eventual recycling to the plasma membrane (PM) are processes that have an influence on signalling activity and therefore on many cell functions, including migration and proliferation. Like other tyrosine kinase receptors (TKR), the insulin receptor (INSR) has been shown to be endocytosed by clathrin-dependent and -independent mechanisms. Once at the early endosome (EE), the sorting of the receptor, either to the late endosome (LE) for degradation or back to the PM through slow or fast recycling pathways, will determine the intensity and duration of insulin effects. Both the endocytic and the endosomic pathways are regulated by many proteins, the Arf and Rab families of small GTPases being some of the most relevant. Here, we argue for a specific role for the slow recycling route, whilst we review the main molecular mechanisms involved in INSR endocytosis, sorting and recycling, as well as their possible role in cell functions.

Structure of the human FERRY Rab5 effector complex

Long-range mRNA transport is crucial for the spatio-temporal regulation of gene expression, and its malfunction is linked to neurological disorders. The pentameric FERRY Rab5 effector complex is the molecular link between mRNA and the early endosome in mRNA intracellular distribution. Here, we determine the cryo-EM structure of the human FERRY complex, composed of Fy-1 to Fy-5. The structure reveals a clamp-like architecture, in which two arm-like appendages, each consisting of Fy-2 and a Fy-5 dimer, protrude from the central Fy-4 dimer. We demonstrate that the coiled-coil domains of Fy-2 are flexible and project into opposite directions from the FERRY complex core. While the C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils together with Fy-5 bind mRNA. Thus, Fy-2 serves as binding hub that connects not only all five complex subunits, but also mediates the binding to mRNA and to the early endosome via Rab5. The FERRY structure provides novel mechanistic insight into long-distance mRNA transport.