Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism

Small GTPases play a critical role in membrane traffic. Among them, Arf6 mediates transport to and from the plasma membrane, as well as phosphoinositide signalling and cholesterol homeostasis. Here we delineate the molecular basis for the link between Arf6 and cholesterol homeostasis using an inducible knockout (KO) model of mouse embryonic fibroblasts (MEFs). We find that accumulation of free cholesterol in the late endosomes/lysosomes of Arf6 KO MEFs results from mistrafficking of Niemann–Pick type C protein NPC2, a cargo of the cation-independent mannose-6-phosphate receptor (CI-M6PR). This is caused by a selective increase in an endosomal pool of phosphatidylinositol-4-phosphate (PI4P) and a perturbation of retromer, which controls the retrograde transport of CI-M6PR via sorting nexins, including the PI4P effector SNX6. Finally, reducing PI4P levels in KO MEFs through independent mechanisms rescues aberrant retromer tubulation and cholesterol mistrafficking. Our study highlights a phosphoinositide-based mechanism for control of cholesterol distribution via retromer

Different interactions between MT7 toxin and the human muscarinic M1 receptor in its free and N-methylscopolamine-occupied

ABSTRACT Muscarinic MT7 toxin is a highly selective and potent antagonist of the M 1 subtype of muscarinic receptor and acts by binding to an allosteric site. To identify the molecular determinants by which MT7 toxin interacts with this receptor in its free and NMS-occupied states, the effect on toxin potency of alanine substitution was evaluated in equilibrium and kinetic binding experiments as well as in functional assays. The determination of the crystallographic structure of an MT7-derivative (MT7-diiodoTyr51) allowed the selection of candidate residues that are accessible and present on both faces of the three toxin loops. The equilibrium binding data are consistent with negative cooperativity between N-methylscopolamine (NMS) and wild-type or modified MT7 and highlight the critical role of the tip of the central loop of the toxin (Arg34, Met35 Tyr36) in its interaction with the unoccupied receptor. Examination of the potency of wild-type and modified toxins to allosterically decrease the dissociation rate of [ 3 H]NMS allowed the identification of the MT7 residues involved in its interaction with the NMSoccupied receptor. In contrast to the results with the unoccupied receptor, the most important residue for this interaction was Tyr36 in loop II, assisted by Trp10 in loop I and Arg52 in loop III. The critical role of the tips of the MT7 loops was also confirmed in functional experiments. The high specificity of the MT7-M 1 receptor interaction exploits several MT7-specific residues and reveals a different mode of interaction of the toxin with the free and NMS-occupied states of the receptor. Muscarinic neurotoxins, small peptides of 64 to 66 residues derived from the venom of African mambas (Dendroaspis angusticeps and Dendroaspis polylepis), are well known for their ability to interact with different muscarinic receptor subtypes. NMR and X-ray studies of the MT2 toxin have shown that muscarinic toxins have the three-finger fold structure, characteristic of the large superfamily of toxins that act at cholinergic synapses There is a limited understanding of the specificity, selectivity, and mechanism of action of the muscarinic toxins at Article, publication date, and citation information can be found a

Tectonic interleaving along the Main Central Thrust, Sikkim Himalaya

Geochemical and geochronological analyses provide quantitative evidence about the origin, development and motion along ductile faults, where kinematic structures have been overprinted. The Main Central Thrust is a key structure in the Himalaya that accommodated substantial amounts of the India–Asia convergence. This structure juxtaposes two isotopically distinct rock packages across a zone of ductile deformation. Structural analysis, whole-rock Nd isotopes, and U–Pb zircon geochronology reveal that the hanging wall is characterized by detrital zircon peaks at c. 800–1000 Ma, 1500–1700 Ma and 2300–2500 Ma and an εNd(0) signature of –18.3 to –12.1, and is intruded by c. 800 Ma and c. 500–600 Ma granites. In contrast, the footwall has a prominent detrital zircon peak at c. 1800–1900 Ma, with older populations spanning 1900–3600 Ma, and an εNd(0) signature of –27.7 to –23.4, intruded by c. 1830 Ma granites. The data reveal a c. 5 km thick zone of tectonic imbrication, where isotopically out-of-sequence packages are interleaved. The rocks became imbricated as the once proximal and distal rocks of the Indian margin were juxtaposed by Cenozoic movement along the Main Central Thrust. Geochronological and isotopic characterization allows for correlation along the Himalayan orogen and could be applied to other cryptic ductile shear zones

Excess Synaptojanin 1 Contributes to Place Cell Dysfunction and Memory Deficits in the Aging Hippocampus in Three Types of Alzheimer’s Disease

The phosphoinositide phosphatase synaptojanin 1 (SYNJ1) is a key regulator of synaptic function. We first tested whether SYNJ1 contributes to phenotypic variations in familial Alzheimer’s disease (FAD) and show that SYNJ1 polymorphisms are associated with age of onset in both early- and late-onset human FAD cohorts. We then interrogated whether SYNJ1 levels could directly affect memory. We show that increased SYNJ1 levels in autopsy brains from adults with Down syndrome (DS/AD) are inversely correlated with synaptophysin levels, a direct readout of synaptic integrity. We further report age-dependent cognitive decline in a mouse model overexpressing murine Synj1 to the levels observed in human sporadic AD, triggered through hippocampal hyperexcitability and defects in the spatial reproducibility of place fields. Taken together, our findings suggest that SYNJ1 contributes to memory deficits in the aging hippocampus in all forms of AD

Cholesterol and ApoE in Alzheimer’s disease

Genetic, neuropathological and biochemical studies suggest strong links between cholesterol, the apolipoprotein E (APOE) and Alzheimer’s disease (AD), both in humans and in animal models of the disease. From the literature and our work, we can predict that transient increase of the levels of cholesterol at the membrane of neurons would profoundly affect the processing of the transmembrane Amyloid Precursor Protein (APP) by triggering its clathrin dependent endocytosis and the resulting production of amyloid-β (Aβ) peptides. Here, we will review these data together with structural and molecular dynamic studies that characterized the role of cholesterol on APP conformation and positioning at the membrane. Specifically decreasing brain cholesterol or replacing it with plant sterols crossing the blood brain barrier appear like promising strategies to either delay or counteract the development of sporadic AD

Cholesterol and ApoE in Alzheimer’s disease

Genetic, neuropathological and biochemical studies suggest strong links between cholesterol, the apolipoprotein E (APOE) and Alzheimer’s disease (AD), both in humans and in animal models of the disease. From the literature and our work, we can predict that transient increase of the levels of cholesterol at the membrane of neurons would profoundly affect the processing of the transmembrane Amyloid Precursor Protein (APP) by triggering its clathrin dependent endocytosis and the resulting production of amyloid-β (Aβ) peptides. Here, we will review these data together with structural and molecular dynamic studies that characterized the role of cholesterol on APP conformation and positioning at the membrane. Specifically decreasing brain cholesterol or replacing it with plant sterols crossing the blood brain barrier appear like promising strategies to either delay or counteract the development of sporadic AD

Identification of Various Allosteric Interaction Sites on M 1 Muscarinic Receptor Using 125 I-Met35-Oxidized Muscarinic Toxin 7

International audienc

Different Interactions between MT7 Toxin and the Human Muscarinic M 1 Receptor in Its Free and N -Methylscopolamine-Occupied States

International audienc