Catherine J. Baty

Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes

Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.

Mutation in Glycerol-3-Phosphate Dehydrogenase 1–Like Gene (GPD1-L) Decreases Cardiac Na+ Current and Causes Inherited Arrhythmias

Background— Brugada syndrome is a rare, autosomal-dominant, male-predominant form of idiopathic ventricular fibrillation characterized by a right bundle-branch block and ST elevation in the right precordial leads of the surface ECG. Mutations in the cardiac Na+ channel SCN5A on chromosome 3p21 cause ≈20% of the cases of Brugada syndrome; most mutations decrease inward Na+ current, some by preventing trafficking of the channels to the surface membrane. We previously used positional cloning to identify a new locus on chromosome 3p24 in a large family with Brugada syndrome and excluded SCN5A as a candidate gene. Methods and Results— We used direct sequencing to identify a mutation (A280V) in a conserved amino acid of the glycerol-3-phosphate dehydrogenase 1–like (GPD1-L) gene. The mutation was present in all affected individuals and absent in >500 control subjects. GPD1-L RNA and protein are abundant in the heart. Compared with wild-type GPD1-L, coexpression of A280V GPD1-L with SCN5A in HEK cells reduced inward Na+ currents by ≈50% (P<0.005). Wild-type GPD1-L localized near the cell surface to a greater extent than A280V GPD1-L. Coexpression of A280V GPD1-L with SCN5A reduced SCN5A cell surface expression by 31±5% (P=0.01). Conclusions— GPD1-L is a novel gene that may affect trafficking of the cardiac Na+ channel to the cell surface. A GPD1-L mutation decreases SCN5A surface membrane expression, reduces inward Na+ current, and causes Brugada syndrome.

Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells

Recognition of injured mitochondria for degradation by macroautophagy is essential for cellular health, but the mechanisms remain poorly understood. Cardiolipin is an inner mitochondrial membrane phospholipid. We found that rotenone, staurosporine, 6-hydroxydopamine and other pro-mitophagy stimuli caused externalization of cardiolipin to the mitochondrial surface in primary cortical neurons and SH-SY5Y cells. RNAi knockdown of cardiolipin synthase or of phospholipid scramblase-3, which transports cardiolipin to the outer mitochondrial membrane, decreased the delivery of mitochondria to autophagosomes. Furthermore, we found that the autophagy protein microtubule-associated-protein-1 light chain 3 (LC3), which mediates both autophagosome formation and cargo recognition, contains cardiolipin-binding sites important for the engulfment of mitochondria by the autophagic system. Mutation of LC3 residues predicted as cardiolipin-interaction sites by computational modelling inhibited its participation in mitophagy. These data indicate that redistribution of cardiolipin serves as an ‘eat-me’ signal for the elimination of damaged mitochondria from neuronal cells.

Noncytotoxic Lytic Granule–Mediated CD8+ T Cell Inhibition of HSV-1 Reactivation from Neuronal Latency

Reactivation of herpes simplex virus type 1 (HSV-1) from neuronal latency is a common and potentially devastating cause of disease worldwide. CD8+ T cells can completely inhibit HSV reactivation in mice, with interferon-γ affording a portion of this protection. We found that CD8+ T cell lytic granules are also required for the maintenance of neuronal latency both in vivo and in ex vivo ganglia cultures and that their directed release to the junction with neurons in latently infected ganglia did not induce neuronal apoptosis. Here, we describe a nonlethal mechanism of viral inactivation in which the lytic granule component, granzyme B, degrades the HSV-1 immediate early protein, ICP4, which is essential for further viral gene expression.

Lipotoxicity Causes Multisystem Organ Failure and Exacerbates Acute Pancreatitis in Obesity

Unsaturated fatty acids cause lipotoxicity and mediate acute adverse outcomes in obese individuals with pancreatitis. The Burden of Adiposity As if diabetes and heart disease were not burden enough, obese people who suffer trauma, burns, or other critical conditions have an increased likelihood of death. During these exacerbated illnesses, multiple organs can fail, a situation that is particularly hard to reverse. How the presence of excess adipose tissue contributes to the severity of these diseases is not clear, but understanding the mechanisms could provide clues for possible treatments. Pancreatitis is a relatively well-defined disease that tends to be worse in the obese and, in its most severe form, is accompanied by multi-organ failure. By using a combination of patient investigation, in vitro cell studies, and an animal model, Navina et al. have assembled evidence that pinpoints the culprits in the obesity-related complications of this disease: unsaturated fatty acids liberated by lipolysis from adipose tissue. The authors carefully examine the pancreases of 24 patients who had died of pancreatitis. The staining patterns indicated that nonesterified fatty acids, derived by lipolysis of excess intrapancreatic fat, contributed to the pancreatic necrosis in these patients. To test this idea, the authors used a cell culture system and showed that it is unsaturated fatty acids that do the damage, impairing acinar cell activities, inhibiting mitochondrial function, releasing calcium, and causing cell death. But what about the failure of other organs? To answer this question, the authors used obese mice with pancreatitis and, by inhibiting lipolysis with the drug orlistat, were able to prevent the pancreatic-associated rise in serum unsaturated fatty acids and, of most importance, to reduce damage to the lung and kidney, as well as mortality. It is not yet clear which lipase is the critical one for multiorgan failure or where it is located. But once revealed, this potential therapeutic target may specify a treatment that enhances the survival of critically ill obese patients. Obesity increases the risk of adverse outcomes during acute critical illnesses such as burns, severe trauma, and acute pancreatitis. Although individuals with more body fat and higher serum cytokines and lipase are more likely to experience problems, the roles that these characteristics play are not clear. We used severe acute pancreatitis as a representative disease to investigate the effects of obesity on local organ function and systemic processes. In obese humans, we found that an increase in the volume of intrapancreatic adipocytes was associated with more extensive pancreatic necrosis during acute pancreatitis and that acute pancreatitis was associated with multisystem organ failure in obese individuals. In vitro studies of pancreatic acinar cells showed that unsaturated fatty acids were proinflammatory, releasing intracellular calcium, inhibiting mitochondrial complexes I and V, and causing necrosis. Saturated fatty acids had no such effects. Inhibition of lipolysis in obese (ob/ob) mice with induced pancreatitis prevented a rise in serum unsaturated fatty acids and prevented renal injury, lung injury, systemic inflammation, hypocalcemia, reduced pancreatic necrosis, and mortality. Thus, therapeutic approaches that target unsaturated fatty acid–mediated lipotoxicity may reduce adverse outcomes in obese patients with critical illnesses such as severe acute pancreatitis.

GJC2 Missense Mutations Cause Human Lymphedema

Lymphedema is the clinical manifestation of defects in lymphatic structure or function. Mutations identified in genes regulating lymphatic development result in inherited lymphedema. No mutations have yet been identified in genes mediating lymphatic function that result in inherited lymphedema. Survey microarray studies comparing lymphatic and blood endothelial cells identified expression of several connexins in lymphatic endothelial cells. Additionally, gap junctions are implicated in maintaining lymphatic flow. By sequencing GJA1, GJA4, and GJC2 in a group of families with dominantly inherited lymphedema, we identified six probands with unique missense mutations in GJC2 (encoding connexin [Cx] 47). Two larger families cosegregate lymphedema and GJC2 mutation (LOD score = 6.5). We hypothesize that missense mutations in GJC2 alter gap junction function and disrupt lymphatic flow. Until now, GJC2 mutations were only thought to cause dysmyelination, with primary expression of Cx47 limited to the central nervous system. The identification of GJC2 mutations as a cause of primary lymphedema raises the possibility of novel gap-junction-modifying agents as potential therapy for some forms of lymphedema.

Spatial localization of m-calpain to the plasma membrane by phosphoinositide biphosphate binding during epidermal growth factor receptor-mediated activation.

ABSTRACT Calpain activity is required for de-adhesion of the cell body and rear to enable productive locomotion of adherent cells during wound repair and tumor invasion. Growth factors activate m-calpain (calpain 2, CAPN2) via ERK/mitogen-activated protein kinases, but only when these kinases are localized to the plasma membrane. We thus hypothesized that m-calpain is activated by epidermal growth factor (EGF) only when it is juxtaposed to the plasma membrane secondary to specific docking. Osmotic disruption of NR6 fibroblasts expressing the EGF receptor demonstrated m-calpain being complexed with the substratum-adherent membrane with this increasing in an EGF-dependent manner. m-Calpain colocalized with phosphoinositide biphosphate (PIP2) with exogenous phospholipase C removal of phosphoinositides, specifically, PI(4,5)P2 but not PI(4)P1 or PIP3, releasing the bound m-calpain. Downregulation of phosphoinositide production by 1-butanol resulted in diminished PIP2 in the plasma membrane and eliminated EGF-induced calpain activation. This PIP2-binding capacity resided in domain III of calpain, which presents a putative C2-like domain. This active conformation of this domain appears to be partially masked in the holoenzyme as both activation of m-calpain by phosphorylation at serine 50 and expression of constitutively active phosphorylation mimic glutamic acid-increased m-calpain binding to the membrane, consistent with blockade of this cascade diminishing membrane association. Importantly, we found that m-calpain was enriched toward the rear of locomoting cells, which was more pronounced in the plasma membrane footprints; EGF further enhanced this enrichment, in line with earlier reports of loss of PIP2 in lamellipodia of motile cells. These data support a model of m-calpain binding to PIP2 concurrent with and likely to enable ERK activation and provides a mechanism by which cell de-adhesion is directed to the cell body and tail as phospholipase C-γ hydrolyzes PIP2 in the protruding lamellipodia.

Nitric Oxide–Dependent Bone Marrow Progenitor Mobilization by Carbon Monoxide Enhances Endothelial Repair After Vascular Injury

Background— Carbon monoxide (CO) has emerged as a vascular homeostatic molecule that prevents balloon angioplasty–induced stenosis via antiproliferative effects on vascular smooth muscle cells. The effects of CO on reendothelialization have not been evaluated. Methods and Results— Exposure to CO has diametrically opposite effects on endothelial cell (EC) and vascular smooth muscle cell proliferation in rodent models of carotid injury. In contrast to its effect of blocking vascular smooth muscle cell growth, CO administered as a gas or as a CO-releasing molecule enhances proliferation and motility of ECs in vitro by >50% versus air controls, and in vivo, it accelerates reendothelialization of the denuded artery by day 4 after injury versus day 6 in air-treated animals. CO enhanced EC proliferation via rapid activation of RhoA (Ras homolog gene family, member A), followed by downstream phosphorylation of Akt, endothelial nitric oxide (NO) synthase phosphorylation, and a 60% increase in NO generation by ECs. CO drives cell cycle progression through phosphorylation of retinoblastoma, which is dependent in part on endothelial NO synthase–generated NO. Similarly, endothelial repair in vivo requires NO-dependent mobilization of bone marrow–derived EC progenitors, and CO yielded a 4-fold increase in the number of mobilized green fluorescent protein–Tie2–positive endothelial progenitor cells versus controls, with a corresponding accelerated deposition of differentiated green fluorescent protein–Tie2–positive ECs at the site of injury. CO was ineffective in augmenting EC repair and the ensuing development of intimal hyperplasia in eNOS−/− mice. Conclusions— Collectively, the present data demonstrate that CO accelerates EC proliferation and vessel repair in a manner dependent on NO generation and enhanced recruitment of bone marrow–derived endothelial progenitor cells.

AMPK agonists ameliorate sodium and fluid transport and inflammation in cystic fibrosis airway epithelial cells.

The metabolic sensor AMP-activated kinase (AMPK) inhibits both the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl(-) channel and epithelial Na(+) channel (ENaC), and may inhibit secretion of proinflammatory cytokines in epithelia. Here we have tested in primary polarized CF and non-CF human bronchial epithelial (HBE) cells the effects of AMPK activators, metformin and 5-aminoimidazole-4-carboxamide-1-beta-D-riboside (AICAR), on various parameters that contribute to CF lung disease: ENaC-dependent short-circuit currents (I(sc)), airway surface liquid (ASL) height, and proinflammatory cytokine secretion. AMPK activation after overnight treatment with either metformin (2-5 mM) or AICAR (1 mM) substantially inhibited ENaC-dependent I(sc) in both CF and non-CF airway cultures. Live-cell confocal images acquired 60 minutes after apical addition of Texas Red-dextran-containing fluid revealed significantly greater ASL heights after AICAR and metformin treatment relative to controls, suggesting that AMPK-dependent ENaC inhibition slows apical fluid reabsorption. Both metformin and AICAR decreased secretion of various proinflammatory cytokines, both with and without prior LPS stimulation. Finally, prolonged exposure to more physiologically relevant concentrations of metformin (0.03-1 mM) inhibited ENaC currents and decreased proinflammatory cytokine levels in CF HBE cells in a dose-dependent manner. These findings suggest that novel therapies to activate AMPK in the CF airway may be beneficial by blunting excessive sodium and ASL absorption and by reducing excessive airway inflammation, which are major contributors to CF lung disease.

PKA regulates vacuolar H+-ATPase localization and activity via direct phosphorylation of the a subunit in kidney cells.

The vacuolar H+-ATPase (V-ATPase) is a major contributor to luminal acidification in epithelia of Wolffian duct origin. In both kidney-intercalated cells and epididymal clear cells, cAMP induces V-ATPase apical membrane accumulation, which is linked to proton secretion. We have shown previously that the A subunit in the cytoplasmic V1 sector of the V-ATPase is phosphorylated by protein kinase A (PKA). Here we have identified by mass spectrometry and mutagenesis that Ser-175 is the major PKA phosphorylation site in the A subunit. Overexpression in HEK-293T cells of either a wild-type (WT) or phosphomimic Ser-175 to Asp (S175D) A subunit mutant caused increased acidification of HCO3−-containing culture medium compared with cells expressing vector alone or a PKA phosphorylation-deficient Ser-175 to Ala (S175A) mutant. Moreover, localization of the S175A A subunit mutant expressed in HEK-293T cells was more diffusely cytosolic than that of WT or S175D A subunit. Acute V-ATPase-mediated, bafilomycin-sensitive H+ secretion was up-regulated by a specific PKA activator in HEK-293T cells expressing WT A subunit in HCO3−-free buffer. In cells expressing the S175D mutant, V-ATPase activity at the membrane was constitutively up-regulated and unresponsive to PKA activators, whereas cells expressing the S175A mutant had decreased V-ATPase activity that was unresponsive to PKA activation. Finally, Ser-175 was necessary for PKA-stimulated apical accumulation of the V-ATPase in a polarized rabbit cell line of collecting duct A-type intercalated cell characteristics (Clone C). In summary, these results indicate a novel mechanism for the regulation of V-ATPase localization and activity in kidney cells via direct PKA-dependent phosphorylation of the A subunit at Ser-175.