Oligodendroglial exosomes in glia to neuron signaling

In the CNS, myelinating oligodendrocytes and axons form a functional unit based on intimate cell-cell interactions. In addition to axonal insulation serving to increase the conduction velocity of electrical impulses, oligodendrocytes provide trophic support to neurons essential for the long-term functional integrity of axons. The glial signals maintaining axonal functions are just at the beginning to become uncovered. Yet, their determination is highly relevant for all types of demyelinating diseases, where lack of glial support significantly contributes to pathology. rnThe present PhD thesis uncovers exosomes as a novel signaling entity in the CNS by which cargo can be transferred from oligodendrocytes to neurons. Exosomes are small membranous vesicles of endocytic origin, which are released by almost every cell type and have been implicated in intercellular communication. Oligodendrocytes secrete exosomes containing a distinct set of proteins as well as mRNA and microRNA. Intriguingly, oligodendroglial exosome release is stimulated by the neurotransmitter glutamate indicating that neuronal electrical activity controls glial exosome release. In this study, the role of exosomes in neuron-glia communication and their implications on glial support was examined. Cortical neurons internalized and accumulated oligodendroglial exosomes in the neuronal cell soma in a time-dependent manner. Moreover, uptake occurred likewise at the somatodendritic and axonal compartment of the neurons via dynamin and clathrin dependent endocytosis. Intriguingly, neuronal internalization of exosomes resulted in functional retrieval of exosomal cargo in vitro and in vivo upon stereotactic injection of Cre recombinase bearing exosomes. Functional recovery of Cre recombinase from transferred exosomes was indicated by acquired reporter recombination in the target cell. Electrophysiological analysis showed an increased firing rate in neurons exposed to oligodendroglial exosomes. Moreover, microarray analysis revealed differentially expressed genes after exosome treatment, indicating functional implications on neuronal gene expression and activity. rnTaken together, the results of this PhD thesis represent a proof of principle for exosome transmission from oligodendrocytes to neurons suggesting a new route of horizontal transfer in the CNS.rn

Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset neurological disease resulting from mutations in the SACS gene encoding sacsin, a 4,579-aa protein of unknown function. Originally identified as a founder disease in Québec, ARSACS is now recognized worldwide. Prominent features include pyramidal spasticity and cerebellar ataxia, but the underlying pathology and pathophysiological mechanisms are unknown. We have generated an animal model for ARSACS, sacsin knockout mice, that display age-dependent neurodegeneration of cerebellar Purkinje cells. To explore the pathophysiological basis for this observation, we examined the cell biological properties of sacsin. We show that sacsin localizes to mitochondria in non-neuronal cells and primary neurons and that it interacts with dynamin-related protein 1, which participates in mitochondrial fission. Fibroblasts from ARSACS patients show a hyperfused mitochondrial network, consistent with defects in mitochondrial fission. Sacsin knockdown leads to an overly interconnected and functionally impaired mitochondrial network, and mitochondria accumulate in the soma and proximal dendrites of sacsin knockdown neurons. Disruption of mitochondrial transport into dendrites has been shown to lead to abnormal dendritic morphology, and we observe striking alterations in the organization of dendritic fields in the cerebellum of knockout mice that precedes Purkinje cell death. Our data identifies mitochondrial dysfunction/mislocalization as the likely cellular basis for ARSACS and indicates a role for sacsin in regulation of mitochondrial dynamics.

THE ROLE OF RECEPTOR TYROSINE KINASE AXL IN PANCREATIC DUCTAL ADENOCARCINOMA AND ITS REGULATION BY HEMATOPOIETIC PROGENITOR KINASE 1

Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive malignancies with less than 5% of five year survival rate. New molecular markers and new therapeutic targets are urgently needed for patients with PDA. Oncogenic receptor tyrosine kinase Axl has been reported to be overexpressed in many types of human malignancies, including diffuse glioma, melanoma, osteosarcoma, and carcinomas of lung, colon, prostate, breast, ovary, esophagus, stomach, and kidney. However, the expression and functions of Axl in PDA are unclear. We hypothesized that Axl contributes to the development and progression of PDA. We examined Axl expression in 54 human PDA samples and their paired benign pancreatic tissue by immunohistochemistry, we found that Axl was overexpressed in 70% of stage II PDAs, but only 22% of benign ducts (P=0.0001). Axl overexpression was associated with higher frequencies of distant metastasis and was an independent prognostic factor for both poor overall and recurrence-free survivals in patients with stage II PDA (p = 0.03 and 0.04). Axl silencing by shRNA in pancreatic cancer cell lines, panc-28 and Panc-1, decreased tumor cell migration and invasion and sensitized PDA cells to apoptosis stimuli such as γ-irradiation and serum starvation. In addition, we found that Axl-mediated Akt and NF-κB activation and up regulation of MMP2 were involved in the invasion, migration and survival of PDA cells. Thus, we demonstrate that Axl plays an important role in the development and progression of PDA. Targeting Axl signaling pathway may represent a new approach for the treatment of PDA. To understand the molecular mechanisms of Axl overexpression in PDA, we found that Axl expression was down-regulated by hematopoietic progenitor kinase 1 (HPK1), a newly identified tumor suppressor in PDA. HPK1 is lost in over 95% of PDAs. Restoration of HPK1 in PDA cells down-regulated Axl expression. HPK1-mediated Axl degradation was inhibited by leupeptin, baflomycin A1, and monensin, suggesting that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway. HPK1 interacted with and phosphorylated dynamin, a critical component of endocytosis pathway. Overexpression of dominant negative form of dynamin blocked the HPK1-mediated Axl degradation. Therefore we concluded that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway and loss of HPK1 expression may contribute to Axl overexpression in PDAs.

Ligand-stimulated internalization of a Dictyostelium discoideum G protein-coupled cAMP receptor

The social amoeba, Dictyostelium discoideum, undergoes a remarkable starvation-induced program of development that transforms a population of unicellular amoebae into a fruiting body composed of resistant spores suspended on a stalk. During this development, secreted cAMP drives chemotaxis of the amoebae, leading to their aggregation, and subsequent differentiation and morphogenesis. Four sequentially expressed G protein-coupled receptors (GPCRs) for cAMP play critical roles in this process. The first of these, cAR1, is essential for aggregation as it mediates chemotaxis as well as the propagation of secreted cAMP waves throughout aggregating populations. Ligand-induced internalization has been shown to regulate a variety of GPCRs. However, little was known at the outset of this study about the role of internalization in the regulation of cAR1 function or, for that matter, in developmental systems in general. For this study, cAMP-induced cAR1 internalization was assessed by measuring (1) the reduction of cell surface binding sites for [ 3H]cAMP and (2) the redistribution of YFP-tagged receptors to the cell's interior, cAMP was found to induce little or no loss of ligand binding (LLB) in vegetative cells. However, the ability to induce LLB increased progressively over the initial 6 hrs of development, reaching ∼70% in cells undergoing aggregation. Despite these reductions in surface binding, detectable cAR1-YFP redistribution could be induced by cAMP only after the cells reached the mound stage (10 hrs) and was found to occur naturally by the ensuing slug stage (18 hrs). Site-directed substitution of a cluster of 5 serines in the receptor's cytoplasmic tail that was previously shown to be the principal site of cAMP-induced cAR1 phosphorylation impaired both LLB and receptor redistribution and furthermore resulted in mound-stage developmental arrest, suggesting that phosphorylation of cAR1 is a prerequisite for its internalization and that cAR1 internalization is required for post-aggregative development. To assess the involvement of clathrin mediated endocytosis, Dictyostelium cells lacking the clathrin light chain gene (clc-) or either of two dynamin genes were examined and found to be defective in LLB and, in the case of clc- cells, also cAR1 redistribution and turnover. Furthermore, cAR1 overexpression in clc- cells (like the serine mutant in wild-type cells) promoted developmental arrest in mounds. The mound-arrest phenotype was also recapitulated in a wild-type background by the specific expression of cAR1 in prestalk cells (but not prespore cells), suggesting that development depends critically on internalization and clearance of cAR1 from these cells. Persistent cAR1 expression following aggregation was found to be associated with aberrant expression of prestalk and prespore genes, which may adversely affect development in the prestalk cell lineage. The PI3 kinase-TORC2 signal transduction pathway, known to be important for Dictyostelium chemotaxis and internalization of yeast pheromone receptors, was examined using chemical inhibitors and null cells and found to be necessary for cAR1 internalization. In conclusion, cAR1 was shown to be similar to other GPCRs in that its internalization depends on phosphorylation of cytoplasmic domain serines, utilizes clathrin and dynamin, and involves the TORC2 complex. In addition, the findings presented here that cAR1 internalization is both developmentally regulated and required for normal development represent a novel regulatory paradigm that might pertain to other GPCRs known to play important roles in the development of humans and other metazoans. ^

Role for G protein-coupled receptor kinase in agonist-specific regulation of μ-opioid receptor responsiveness

The G protein-coupled μ-opioid receptor (μOR) mediates the physiological effects of endogenous opioid peptides as well as the structurally distinct opioid alkaloids morphine and etorphine. An intriguing feature of μOR signaling is the differential receptor trafficking and desensitization properties following activation by distinct agonists, which have been proposed as possible mechanisms related to opioid tolerance. Here we report that the ability of distinct opioid agonists to differentially regulate μOR internalization and desensitization is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the μOR. Although both etorphine and morphine effectively activate the μOR, only etorphine elicits robust μOR phosphorylation followed by plasma membrane translocation of β-arrestin and dynamin-dependent receptor internalization. In contrast, corresponding to its inability to cause μOR internalization, morphine is unable to either elicit μOR phosphorylation or stimulate β-arrestin translocation. However, upon the overexpression of GRK2, morphine gains the capacity to induce μOR phosphorylation, accompanied by the rescue of β-arrestin translocation and receptor sequestration. Moreover, overexpression of GRK2 also leads to an attenuation of morphine-mediated inhibition of adenylyl cyclase. These findings point to the existence of marked differences in the ability of different opioid agonists to promote μOR phosphorylation by GRK. These differences may provide the molecular basis underlying the different analgesic properties of opioid agonists and contribute to the distinct ability of various opioids to induce drug tolerance.

Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.

In Vitro Reconstitution of Microtubule Plus End-directed, GTPγS-sensitive Motility of Golgi MembranesV⃞

Purified Golgi membranes were mixed with cytosol and microtubules (MTs) and observed by video enhanced light microscopy. Initially, the membranes appeared as vesicles that moved along MTs. As time progressed, vesicles formed aggregates from which membrane tubules emerged, traveled along MTs, and eventually generated extensive reticular networks. Membrane motility required ATP, occurred mainly toward MT plus ends, and was inhibited almost completely by the H1 monoclonal antibody to kinesin heavy chain, 5′-adenylylimidodiphosphate, and 100 μM but not 20 μM vanadate. Motility was also blocked by GTPγS or AlF4− but was insensitive to AlCl3, NaF, staurosporin, or okadaic acid. The targets for GTPγS and AlF4− were evidently of cytosolic origin, did not include kinesin or MTs, and were insensitive to several probes for trimeric G proteins. Transport of Golgi membranes along MTs mediated by a kinesin has thus been reconstituted in vitro. The motility is regulated by one or more cytosolic GTPases but not by protein kinases or phosphatases that are inhibited by staurosporin or okadaic acid, respectively. The pertinent GTPases are likely to be small G proteins or possibly dynamin. The in vitro motility may correspond to Golgi-to-ER or Golgi-to-cell surface transport in vivo.

Amphiphysin Heterodimers: Potential Role in Clathrin-mediated Endocytosis

Amphiphysin (Amph) is a src homology 3 domain-containing protein that has been implicated in synaptic vesicle endocytosis as a result of its interaction with dynamin. In a screen for novel members of the amphiphysin family, we identified Amph2, an isoform 49% identical to the previously characterized Amph1 protein. The subcellular distribution of this isoform parallels Amph1, both being enriched in nerve terminals. Like Amph1, a role in endocytosis at the nerve terminal is supported by the rapid dephosphorylation of Amph2 on depolarization. Importantly, the two isoforms can be coimmunoprecipitated from the brain as an equimolar complex, suggesting that the two isoforms act in concert. As determined by cross-linking of brain extracts, the Amph1–Amph2 complex is a 220- to 250-kDa heterodimer. COS cells transfected with either Amph1 or Amph2 show greatly reduced transferrin uptake, but coexpression of the two proteins rescues this defect, supporting a role for the heterodimer in clathrin-mediated endocytosis. Although the src homology 3 domains of both isoforms interact with dynamin, the heterodimer can associate with multiple dynamin molecules in vitro and activates dynamin’s GTPase activity. We propose that it is an amphiphysin heterodimer that drives the recruitment of dynamin to clathrin-coated pits in endocytosing nerve terminals.

Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors

Acting through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the α2A adrenergic receptor (α2A AR), with ERK activation mediated by the β2 adrenergic receptor (β2 AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the α2A AR, like that mediated by both the β2 AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant β-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both α2A and β2 AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the β2 AR, the α2A AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although β2 AR stimulation leads to redistribution of both the β2 AR and EGFR, activation of the α2A AR leads to redistribution of the EGFR but the α2A AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.

Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids

Interferon-induced human MxA protein belongs to the dynamin superfamily of large GTPases. It exhibits antiviral activity against a variety of RNA viruses, including Thogoto virus, an influenza virus-like orthomyxovirus transmitted by ticks. Here, we report that MxA blocks the transport of Thogoto virus nucleocapsids into the nucleus, thereby preventing transcription of the viral genome. This interaction can be abolished by a mAb that neutralizes the antiviral activity of MxA. Our results reveal an antiviral mechanism whereby an interferon-induced protein traps the incoming virus and interferes with proper transport of the viral genome to its ultimate target compartment within the infected cell.

Division of Mitochondria Requires a Novel DNM1-interacting Protein, Net2p

Mitochondria are dynamic organelles that undergo frequent division and fusion, but the molecular mechanisms of these two events are not well understood. Dnm1p, a mitochondria-associated, dynamin-related GTPase was previously shown to mediate mitochondrial fission. Recently, a genome-wide yeast two-hybrid screen identified an uncharacterized protein that interacts with Dnm1p. Cells disrupted in this new gene, which we call NET2, contain a single mitochondrion that consists of a network formed by interconnected tubules, similar to the phenotype of dnm1Δ cells. NET2 encodes a mitochondria-associated protein with a predicted coiled-coil region and six WD-40 repeats. Immunofluorescence microscopy indicates that Net2p is located in distinct, dot-like structures along the mitochondrial surface, many of which colocalize with the Dnm1 protein. Fluorescence and immunoelectron microscopy shows that Dnm1p and Net2p preferentially colocalize at constriction sites along mitochondrial tubules. Our results suggest that Net2p is a new component of the mitochondrial division machinery.

Profilin I is essential for cell survival and cell division in early mouse development

Profilins are thought to play a central role in the regulation of de novo actin assembly by preventing spontaneous actin polymerization through the binding of actin monomers, and the adding of monomeric actin to the barbed actin-filament ends. Other cellular functions of profilin in membrane trafficking and lipid based signaling are also likely. Binding of profilins to signaling molecules such as Arp2/3 complex, Mena, VASP, N-WASP, dynamin I, and others, further implicates profilin and actin as regulators of diverse motile activities. In mouse, two profilins are expressed from two distinct genes. Profilin I is expressed at high levels in all tissues and throughout development, whereas profilin II is expressed in neuronal cells. To examine the function of profilin I in vivo, we generated a null profilin I (pfn1ko) allele in mice. Homozygous pfn1ko/ko mice are not viable. Pfn1ko/ko embryos died as early as the two-cell stage, and no pfn1ko/ko blastocysts were detectable. Adult pfn1ko/wt mice show a 50% reduction in profilin I expression with no apparent impairment of cell function. However, pfn1ko/wt embryos have reduced survival during embryogenesis compared with wild type. Although weakly expressed in early embryos, profilin II cannot compensate for lack of profilin I. Our results indicate that mouse profilin I is an essential protein that has dosage-dependent effects on cell division and survival during embryogenesis.

RAC1 Regulates Adherens Junctions through Endocytosis of E-Cadherin

The establishment of cadherin-dependent cell–cell contacts in human epidermal keratinocytes are known to be regulated by the Rac1 small GTP-binding protein, although the mechanisms by which Rac1 participates in the assembly or disruption of cell–cell adhesion are not well understood. In this study we utilized green fluorescent protein (GFP)-tagged Rac1 expression vectors to examine the subcellular distribution of Rac1 and its effects on E-cadherin–mediated cell–cell adhesion. Microinjection of keratinocytes with constitutively active Rac1 resulted in cell spreading and disruption of cell–cell contacts. The ability of Rac1 to disrupt cell–cell adhesion was dependent on colony size, with large established colonies being resistant to the effects of active Rac1. Disruption of cell–cell contacts in small preconfluent colonies was achieved through the selective recruitment of E-cadherin–catenin complexes to the perimeter of multiple large intracellular vesicles, which were bounded by GFP-tagged L61Rac1. Similar vesicles were observed in noninjected keratinocytes when cell–cell adhesion was disrupted by removal of extracellular calcium or with the use of an E-cadherin blocking antibody. Moreover, formation of these structures in noninjected keratinocytes was dependent on endogenous Rac1 activity. Expression of GFP-tagged effector mutants of Rac1 in keratinocytes demonstrated that reorganization of the actin cytoskeleton was important for vesicle formation. Characterization of these Rac1-induced vesicles revealed that they were endosomal in nature and tightly colocalized with the transferrin receptor, a marker for recycling endosomes. Expression of GFP-L61Rac1 inhibited uptake of transferrin-biotin, suggesting that the endocytosis of E-cadherin was a clathrin-independent mechanism. This was supported by the observation that caveolin, but not clathrin, localized around these structures. Furthermore, an inhibitory form of dynamin, known to inhibit internalization of caveolae, inhibited formation of cadherin vesicles. Our data suggest that Rac1 regulates adherens junctions via clathrin independent endocytosis of E-cadherin.

Endothelial Transcytotic Machinery Involves Supramolecular Protein–Lipid Complexes

We have demonstrated that the plasmalemmal vesicles (caveolae) of the continuous microvascular endothelium function as transcytotic vesicular carriers for protein molecules >20 Å and that transcytosis is an N-ethylmaleimide–sensitive factor (NSF)-dependent, N-ethylmaleimide-sensitive process. We have further investigated NSF interactions with endothelial proteins to find out 1) whether a complete set of fusion and targeting proteins is present in the endothelium; 2) whether they are organized in multimolecular complexes as in neurons; and 3) whether the endothelial multimolecular complexes differ from their neuronal counterparts, because of their specialized role in transcytosis. To generate the complexes, we have used myc-NSF, cultured pulmonary endothelial cells, and rat lung cytosol and membrane preparations; to detect them we have applied coimmunoprecipitation with myc antibodies; and to characterize them we have used velocity sedimentation and cross-linking procedures. We have found that both cytosolic and membrane fractions contain complexes that comprise beside soluble NSF attachment proteins and SNAREs (soluble NSF attachment protein receptor), rab 5, dynamin, caveolin, and lipids. By immunogold labeling and negative staining we have detected in these complexes, myc-NSF, syntaxin, dynamin, caveolin, and endogenous NSF. Similar complexes are formed by endogenous NSF. The results indicate that complexes with a distinct protein–lipid composition exist and suggest that they participate in targeting, fusion, and fission of caveolae with the endothelial plasmalemma.

The dynamins: Redundant or distinct functions for an expanding family of related GTPases?

In the 7 years since dynamin was first isolated from bovine brain in search of novel microtubule-based motors, our understanding of this enzyme has expanded significantly. We now know that brain dynamin belongs to a family of large GTPases, which mediate vesicle trafficking. Furthermore, this enzymatic activity is markedly increased through association with microtubules, acidic phospholipids, and certain regulatory proteins that contain Src homology 3 (SH3) domains. From functional, genetic, and cellular manipulations, it is now generally accepted that dynamin participates in the endocytic uptake of receptors, associated ligands, and plasma membrane following an exocytic event. These observations have confirmed at least one function of dynamin that was predicted from seminal studies on a pleiotropic mutant, shibirets (shits) in Drosophila melanogaster. Of equal interest is the finding that there are multiple dynamin gene products, including two that are expressed in a tissue-specific manner, and they share marked homology with a larger family of distinct but related proteins. Therefore, it is attractive to speculate that the different dynamins may participate in related cellular functions, such as distinct endocytic processes and even secretion. In turn, dynamin could play an important role in cell growth, cell spreading, and neurite outgrowth. The purpose of this review is to enumerate on the expansive dynamin literature and to discuss the nomenclature, expression, and putative functions of this growing and interesting family of proteins.