Exploring the pathological mechanisms underlying Cohen syndrome.

Cohen Syndrome (CS) is a rare autosomal recessive disorder caused by biallelic mutations in the VPS13B gene. It is characterized by multiple clinical features, including acquired microcephaly, developmental delay, intellectual disability, neutropenia, and retinal degeneration. VPS13B is part of the bridge-like lipid transport (BLTP) protein family, which in mammals also includes VPS13A, -C, and -D. The proteins of this family are peripheral membrane proteins with different sub-cellular localization, but all share similar structural features and have been proposed to act as lipid transport proteins at organellar membrane contact sites. VPS13B is localized at the Golgi apparatus and is essential for the maintenance of organelle architecture. Here we present a review of the experimental data on the function of the protein at the cellular level, discussing the potential link with disease phenotype and review the studies on animal models recapitulating features of the human disease.

Drug-induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann-Pick type C cells and mice.

Most cells acquire cholesterol by endocytosis of circulating low-density lipoproteins (LDLs). After cholesteryl ester de-esterification in endosomes, free cholesterol is redistributed to intracellular membranes via unclear mechanisms. Our previous work suggested that the unconventional phospholipid lysobisphosphatidic acid (LBPA) may play a role in modulating the cholesterol flux through endosomes. In this study, we used the Prestwick library of FDA-approved compounds in a high-content, image-based screen of the endosomal lipids, lysobisphosphatidic acid and LDL-derived cholesterol. We report that thioperamide maleate, an inverse agonist of the histamine H3 receptor HRH3, increases highly selectively the levels of lysobisphosphatidic acid, without affecting any endosomal protein or function that we tested. Our data also show that thioperamide significantly reduces the endosome cholesterol overload in fibroblasts from patients with the cholesterol storage disorder Niemann-Pick type C (NPC), as well as in liver of Npc1-/- mice. We conclude that LBPA controls endosomal cholesterol mobilization and export to cellular destinations, perhaps by fluidifying or buffering cholesterol in endosomal membranes, and that thioperamide has repurposing potential for the treatment of NPC.

Cyclodextrin triggers MCOLN1-dependent endo-lysosome secretion in Niemann-Pick type C cells.

In specialized cell types, lysosome-related organelles support regulated secretory pathways, whereas in nonspecialized cells, lysosomes can undergo fusion with the plasma membrane in response to a transient rise in cytosolic calcium. Recent evidence also indicates that lysosome secretion can be controlled transcriptionally and promote clearance in lysosome storage diseases. In addition, evidence is also accumulating that low concentrations of cyclodextrins reduce the cholesterol-storage phenotype in cells and animals with the cholesterol storage disease Niemann-Pick type C, via an unknown mechanism. Here, we report that cyclodextrin triggers the secretion of the endo/lysosomal content in nonspecialized cells and that this mechanism is responsible for the decreased cholesterol overload in Niemann-Pick type C cells. We also find that the secretion of the endo/lysosome content occurs via a mechanism dependent on the endosomal calcium channel mucolipin-1, as well as FYCO1, the AP1 adaptor, and its partner Gadkin. We conclude that endo-lysosomes in nonspecialized cells can acquire secretory functions elicited by cyclodextrin and that this pathway is responsible for the decrease in cholesterol storage in Niemann-Pick C cells.

Wnt directs the endosomal flux of LDL-derived cholesterol and lipid droplet homeostasis.

The Wnt pathway, which controls crucial steps of the development and differentiation programs, has been proposed to influence lipid storage and homeostasis. In this paper, using an unbiased strategy based on high-content genome-wide RNAi screens that monitored lipid distribution and amounts, we find that Wnt3a regulates cellular cholesterol. We show that Wnt3a stimulates the production of lipid droplets and that this stimulation strictly depends on endocytosed, LDL-derived cholesterol and on functional early and late endosomes. We also show that Wnt signaling itself controls cholesterol endocytosis and flux along the endosomal pathway, which in turn modulates cellular lipid homeostasis. These results underscore the importance of endosome functions for LD formation and reveal a previously unknown regulatory mechanism of the cellular programs controlling lipid storage and endosome transport under the control of Wnt signaling.

Endosome maturation, transport and functions.

Efficient sorting of the material internalized by endocytosis is essential for key cellular functions and represents a, if not the, major trafficking pathway in mammalian cells. Incoming material - solutes, receptors and cargos, lipids and even pathogenic agents - are routed to various destinations within mammalian cells at two major sorting stations: the early and late endosome. The early endosome receives all manner of incoming material from the plasma membrane, as well as from the Golgi, and serves as an initial sorting nexus routing molecules back to the cell surface through recycling endosomes, to the trans-Golgi network by retrograde transport, or on to the late endosome/lysosome. The early endosome also regulates cell signaling, through the downregulation of internalized receptors, which are packaged into intralumenal vesicles that arise from inward invaginations of the limiting membrane. These multivesicular regions detach or mature from early endosomes and become free endocytic carrier vesicle/multivesicular body, which transports cargoes to late endosomes. The late endosome provides a central hub for incoming traffic from the endocytic, biosynthetic and autophagic pathways and outgoing traffic to the lysosomes, the Golgi complex or the plasma membrane. They also function as a key sensing/signaling platform that inform the cell about the nutrient situation. Herein we summarize the current understanding of the organization and functions of the endocytic pathway, differences across species, and the process of endosome maturation.

PNPLA3/adiponutrin functions in lipid droplet formation.

BACKGROUND INFORMATION: In animals, adipose tissue contains the main energy store as lipid droplets (LDs) composed of esterified cholesterol (CE) and triacylglycerol (TAG) enveloped in a mono-layer of phospholipid and decorated by a coat of proteins. Upon increased energy demand, dedicated lipases hydrolyse TAG stepwise into free fatty acids that are released in circulation and made available to peripheral tissue. In case of aberrant caloric load, TAGs are deposited into non-adipocyte tissues, primarily liver cells. For instance, non-alcoholic fatty liver disease (NAFLD) is a common chronic disorder characterised by an excess of TAG in the liver of patients regardless of their susceptibility to obesity, diabetes or exposure to alcohol. Several independent linkage studies have associated NAFLD with a non-synonymous variant of patatin-like phospholipase domain-containing 3 (PNPLA3/adiponutrin) encoding an isoleucine to methionine substitution at position 148 (I148M) (see Cohen et al., 2011 for review). However, the mechanism by which a variation in PNPLA3 gives susceptibility to NAFLD is not known, primarily because the physiological role of PNPLA3 still needs to be elucidated. RESULTS: We have identified PNPLA3 in a screen for genes upregulated by intracellular lipid accumulation. We investigated the sub-cellular distribution and potential function of PNPLA3 in fibroblast-like cells supplemented with lipids. We demonstrate that PNPLA3 is targetted to LDs in a process that requires an intact Brummer box domain, which is conserved in the patatin-like phospholipase family. We show that increased levels of the NAFLD-linked PNPLA3 isoform leads to larger LDs, whereas decreased levels of PNPLA3 had the opposite effect. Interestingly, however, PNPLA3 induced a reduction in LD size upon co-expression with ABDH5/CGI-58, an activator of the TAG lipase PNPLA2, which is the closest homolog of PNPLA3. By investigating LD populations according to their size and composition, we show that perturbing intracellular lipid trafficking drastically modifies LD nature. CONCLUSIONS: Taken together, our results suggest that PNPLA3 exhibits a dual function in LD metabolism, and that it participates in the restoration of lipid homeostasis upon aberrant intracellular lipid accumulation.

N-Glycans mutations rule oligomeric assembly and functional expression of P2X3 receptor for extracellular ATP.

N-Glycosylation affects the function of ion channels at the level of multisubunit assembly, protein trafficking, ligand binding and channel opening. Like the majority of membrane proteins, ionotropic P2X receptors for extracellular ATP are glycosylated in their extracellular moiety. Here, we used site-directed mutagenesis to the four predicted N-glycosylation sites of P2X(3) receptor (Asn(139), Asn(170), Asn(194) and Asn(290)) and performed comparative analysis of the role of N-glycans on protein stability, plasma membrane delivery, trimer formation and inward currents. We have found that in transiently transfected HEK293 cells, Asn(170) is apparently the most important site for receptor stability, since its mutation causes a primary loss in protein content and indirect failure in membrane expression, oligomeric association and inward current responses. Even stronger effects are obtained when mutating Thr(172) in the same glycosylation consensus. Asn(194) and Asn(290) are the most dispensable, since even their simultaneous mutation does not affect any tested receptor feature. All double mutants containing Asn(170) mutation or the Asn(139)/Asn(290) double mutant are instead almost unable to assemble into a functional trimeric structure. The main emerging finding is that the inability to assemble into trimers might account for the impaired function in P2X(3) mutants where residue Asn(170) is replaced. These results improve our knowledge about the role of N-glycosylation in proper folding and oligomeric association of P2X(3) receptor.

Rapid constitutive and ligand-activated endocytic trafficking of P2X receptor.

P2X receptors mediate a variety of physiological actions, including smooth muscle contraction, neuro-endocrine secretion and synaptic transmission. Among P2X receptors, the P2X(3) subtype is expressed in sensory neurons of dorsal root- and trigeminal-ganglia, where it performs a well-recognized role in sensory and pain transmission. Recent evidence indicates that the strength of P2X(3)-mediated responses is modulated in vivo by altering the number of receptors at the plasma membrane. In the present study, we investigate the trafficking properties of P2X(3) receptor in transfected HEK293 cells and in primary cultures of dorsal root ganglion neurons, finding that P2X(3) receptor undergoes rapid constitutive and cholesterol-dependent endocytosis. We also show that endocytosis is accompanied by preferential targeting of the receptor to late endosomes/lysosomes, with subsequent degradation. Furthermore, we observe that at steady state the receptor localizes predominantly in lamp1-positive intracellular structures, with a minor fraction present at the plasma membrane. Finally, the level of functional receptor expressed on the cell surface is rapidly up-regulated in response to agonist stimulation, which also augments receptor endocytosis. The findings presented in this work underscore a very dynamic trafficking behavior of P2X(3) receptor and disclose a possible mechanism for the rapid modulation of ATP-mediated responses potentially relevant during physiological and pathological conditions.

P2Y1 receptor switches to neurons from glia in juvenile versus neonatal rat cerebellar cortex.

BACKGROUND: In the CNS, several P2 receptors for extracellular nucleotides are identified on neurons and glial cells to participate to neuron-neuron, glia-glia and glia-neuron communication. RESULTS: In this work, we describe the cellular and subcellular presence of metabotropic P2Y1 receptor in rat cerebellum at two distinct developmental ages, by means of immunofluorescence-confocal and electron microscopy as well as western blotting and direct membrane separation techniques. At postnatal day 21, we find that P2Y1 receptor in addition to Purkinje neurons, is abundant on neuronal specializations identified as noradrenergic by anatomical, morphological and biochemical features. P2Y1 receptor immunoreactivity colocalizes with dopamine beta-hydroxylase, tyrosine hydroxylase, neurofilament light chain, synaptophysin and flotillin, but not with glial fibrillary acidic protein for astrocytes. P2Y1 receptor is found enriched in membrane microdomains such as lipid rafts, in cerebellar synaptic vesicles, and is moreover visualized on synaptic varicosities by electron microscopy analysis. When examined at postnatal day 7, P2Y1 receptor immunoreactivity is instead predominantly expressed only on Bergmann and astroglial cells, as shown by colocalization with glial fibrillary acidic protein rather then neuronal markers. At this age, we moreover identify that P2Y1 receptor-positive Bergmann fibers wrap up doublecortin-positive granule cells stretching along them, while migrating through the cerebellar layers. CONCLUSION: Membrane components including purinergic receptors are already known to mediate cellular contact and aggregation in platelets. Our results suggesting a potential role for P2Y1 protein in cell junction/communication and development, are totally innovative for the CNS.

The P2Y4 receptor forms homo-oligomeric complexes in several CNS and PNS neuronal cells.

It is well established that several cell surface receptors interact with each other to form dimers and oligomers, which are essential for their activation. Since little is known about the quaternary structure of P2Y receptors, in the present work, we investigated the expression of the G-protein-coupled P2Y4 subunit as monomeric or higher-order complex protein. We examined both endogenously expressed P2Y4 subtype with the aid of specific anti-P2Y4 antiserum, and heterologously transfected P2Y4-tagged receptors with the use of antitag antibodies. In both cases, we found the P2Y4 receptor displaying molecular masses corresponding to monomeric, dimeric and oligomeric structures. Experiments performed in the absence of reducing agents demonstrated that there is a strict correlation among the multiple protein bands and that the multimeric forms are at least partially assembled by disulphide bonds. The direct demonstration of P2Y4 homodimerisation comes instead from co-transfection and differential co-immunoprecipitation experiments, with the use of differently tagged P2Y4 receptors and antitag antibodies. The structural propensity of the P2Y4 protein to form homo-oligomers may open the possibility of a novel regulatory mechanism of physiopathological functions for this and additional P2Y receptors.

Cellular localization of TRPC3 channel in rat brain: preferential distribution to oligodendrocytes.

In the present work we describe the cellular localization of TRPC3 in non-excitable cells as compared to the neurons in normal rat brain. We performed a double labeling study for TRPC3 and one of the following cell-specific markers: mouse anti-glial fibrillary acidic protein (GFAP) for astrocytes; mouse anti-RIP for oligodendrocytes, or mouse anti-OX42 for microglia, or mouse anti-NeUN for neuronal nuclei or mouse anti-tyrosine hydroxylase (TH) for detection of dopaminergic neurons of the substantia nigra. Our double label immunofluorescence study showed that that TRPC3 is mainly localized in oligodendrocytes. These result were confirmed by the electron microscopy study, which showed TRPC3 immunoreactivity in oligodendrocytes. Consistent with the evidence that calcium homeostasis is important to oligodendrocytes for development, myelination, and demyelination [Microsc. Res. Tech. 52 (2001) 672], we can speculate that the distribution of TRPC3 in oligodendrocytes plays a role in myelination and or demyelination processes.

P2X3 receptor localizes into lipid rafts in neuronal cells.

P2X receptors are a family of seven (P2X(1-7)) cation channels gated by extracellular ATP, widely expressed in neurons and nonneuronal cells. Lipid rafts are cholesterol/sphingolipid-rich membrane domains, involved in many cellular processes, including transmembrane receptor signaling, vesicle traffic, and protein sorting. We provide direct biochemical evidence that P2X3 receptor localizes into lipid rafts, in primary cultures of cerebellar granule neurons as well as in brain and dorsal root ganglia extracts. We show that P2X3 exhibits all the characteristics distinctive of a protein associated with lipid rafts. These characteristics include resistance to detergent extraction at 4 degrees C, solubility after extraction of cholesterol from membranes with either saponin or methyl-beta-cyclodextrin, and partitioning to low buoyant density fractions after sucrose gradient centrifugation in both detergent-containing and detergent-free conditions. Furthermore, P2X3 localizes in raft-containing fractions in transiently transfected SH-SY5Y neuroblastoma cells. The present finding contributes to the characterization of the functional localization of P2X3 in neurons and provides a novel potential mechanism for correct targeting and dynamic activation of this receptor.

Pathways of survival induced by NGF and extracellular ATP after growth factor deprivation.

In a previous work we demonstrated that extracellular adenosine-5'-triphosphate (ATP), acting on P2 receptors, exerts neuritogenic and trophic effects on the phaeochromocytoma PC12 cell line. These actions are comparable to those sustained by nerve growth factor (NGF) and involve several overlapping pathways. In this work, we describe some of the mechanisms recruited by ATP and NGF in maintaining PC12 cell survival after serum deprivation. We show that both ATP and NGF upregulate the expression of the stress-induced heat shock protein HSP70 and HSP90, whilst glucose-response protein GRP75 and GRP78 are not affected. In parallel with NGF, ATP prevents the cleavage and activation of caspase-2 and inhibits the release of cytochrome c from mitochondria into the cytoplasm. Finally, neither NGF, nor ATP directly modulate the expression of P2 receptors in the induction of cell survival. Our data contribute to dissect the biological mechanisms activated by extracellular purines exerting trophic actions and to establish that survival and neurite outgrowth lie on different mechanistic pathways.

Extracellular ATP and neurodegeneration.

ATP is a potent signaling molecule abundantly present in the CNS. It elicits a wide array of physiological effects and is regarded as the phylogenetically most ancient epigenetic factor playing crucial biological roles in several different tissues. These can range from neurotransmission, smooth muscle contraction, chemosensory signaling, secretion and vasodilatation, to more complex phenomena such as immune responses, pain, male reproduction, fertilization and embryonic development. ATP is released into the extracellular space either exocytotically or from damaged and dying cells. It is often co-released with other neurotransmitters and it can interact with growth factors at both receptor- and/or signal transduction-level. Once in the extracellular environment, ATP binds to specific receptors termed P2. Based on pharmacological profiles, on selectivity of coupling to second-messenger pathways and on molecular cloning, two main subclasses with multiple subtypes have been distinguished. They are P2X, i.e. fast cation-selective receptor channels (Na+, K+, Ca2+), possessing low affinity for ATP and responsible for fast excitatory neurotransmission, and P2Y, i.e. slow G protein-coupled metabotropic receptors, possessing higher affinity for the ligand. In the nervous system, they are broadly expressed in both neurons and glial cells and can mediate dual effects: short-term such as neurotransmission, and long-term such as trophic actions. Since massive extracellular release of ATP often occurs after metabolic stress, brain ischemia and trauma, purinergic mechanisms are also correlated to and involved in the etiopathology of many neurodegenerative conditions. Furthermore, extracellular ATP per se is toxic for primary neuronal dissociated and organotypic CNS cultures from cortex, striatum and cerebellum and P2 receptors can mediate and aggravate hypoxic signaling in many CNS neurons. Conversely, several P2 receptor antagonists abolish the cell death fate of primary neuronal cultures exposed to excessive glutamate, serum/potassium deprivation, hypoglycemia and chemical hypoxia. In parallel with these detrimental effects, also trophic functions have been extensively described for extracellular purines (both for neuronal and non-neuronal cells), but these might either aggravate or ameliorate the normal cellular conditions. In summary, extracellular ATP plays a very complex role not only in the repair, remodeling and survival occurring in the nervous system, but even in cell death and this can occur either after normal developmental conditions, after injury, or acute and chronic diseases.