John J. Nunnari

Nuclear Envelope Budding Enables Large Ribonucleoprotein Particle Export during Synaptic Wnt Signaling

Localized protein synthesis requires assembly and transport of translationally silenced ribonucleoprotein particles (RNPs), some of which are exceptionally large. Where in the cell such large RNP granules first assemble was heretofore unknown. We previously reported that during synapse development, a fragment of the Wnt-1 receptor, DFrizzled2, enters postsynaptic nuclei where it forms prominent foci. Here we show that these foci constitute large RNP granules harboring synaptic protein transcripts. These granules exit the nucleus by budding through the inner and the outer nuclear membranes in a nuclear egress mechanism akin to that of herpes viruses. This budding involves phosphorylation of A-type lamin, a protein linked to muscular dystrophies. Thus nuclear envelope budding is an endogenous nuclear export pathway for large RNP granules.

Mechanism of Evenness Interrupted (Evi)-Exosome Release at Synaptic Boutons

Background: Release of microvesicles, including exosomes, is a novel mechanism of intercellular communication. At Drosophila synapses, the transmembrane Wnt-binding protein Evi/Wls is released in vesicles. Results: Evi-exosome release requires Rab11, Syntaxin 1A, and Myosin5. Conclusion: We established an in vivo system to elucidate the mechanisms of exosomal release. Significance: This is the first in vivo characterization of exosomal communication in the nervous system. Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released and travel to target cells are unresolved. During synaptic development, the secretion of Drosophila Wnt1, Wingless, requires the function of Evenness Interrupted (Evi)/Wls, a Wingless-binding protein that is secreted along with Wingless at the neuromuscular junction. Given that Evi is a transmembrane protein, these studies suggested the presence of a novel vesicular mechanism of trans-synaptic communication, potentially in the form of exosomes. To establish the mechanisms for the release of Evi vesicles, we used a dsRNA assay in cultured cells to screen for genes that when down-regulated prevent the release of Evi vesicles. We identified two proteins, Rab11 and Syntaxin 1A (Syx1A), that were required for Evi vesicle release. To determine whether the same mechanisms were used in vivo at the neuromuscular junction, we altered the activity of Rab11 and Syx1A in motoneurons and determined the impact on Evi release. We found that Syx1A, Rab11, and its effector Myosin5 were required for proper Evi vesicle release. Furthermore, ultrastructural analysis of synaptic boutons demonstrated the presence of multivesicular bodies, organelles involved in the production and release of exosomes, and these multivesicular bodies contained Evi. We also used mass spectrometry, electron microscopy, and biochemical techniques to characterize the exosome fraction from cultured cells. Our studies revealed that secreted Evi vesicles show remarkable conservation with exosomes in other systems. In summary, our observations unravel some of the in vivo mechanisms required for Evi vesicle release.

Quantitation of oil red O staining of the aorta in hypercholesterolemic rats

A technique is described which provides morphologic and quantitative data on the amount of oil red O (ORO) staining in thoracic aortas of rats fed a high cholesterol diet. Samples are stained with ORO, the dye is extracted, and the concentration of ORO in the extract is measured colorimetrically. Wistar rats fed ad libitum either standard chow (control group: n = 15) or chow supplemented with 4% cholesterol, 1% cholic acid, and 0.5% thiouracil (CCT group: n = 23) were maintained on these diets for 1, 3, 6, 9, or 12 months. Plasma cholesterol levels averaged overall 87 and 737 mg/dl for the control and CCT groups, respectively. Animals were killed under anesthesia by perfusion fixation with formalin or glutaraldehyde, and samples of thoracic aorta were stained with ORO. After microscopic study en face and measurement of surface area, the ORO was extracted in chloroform-methanol (2:1). Concentrations of ORO (microM) were determined from a standard curve and expressed as microM/mm2 of aorta. Aortas of CCT animals showed progressive diet- and time-dependent increases in the amount of ORO staining compared to controls. We conclude that this method yields reliable quantitative data applicable to studying atherosclerosis in small animals.

Endothelium and “silver lines”

The significance of endothelial “silver lines” was studied by TEM in rat aortas after perfusion with glutaraldehyde followed by silver nitrate. Standard TEM technique proved unsatisfactory (coarse silver granules, imprecise localization, artefacts). Exposure of the silver-treated aortas to photographic fixer markedly improved the image of the deposits leaving fine, stable, uniform “residual granules” about 100 Å in diameter. Most of these granules were localized along the intercellular junctions; they also tended to pool in the basement membrane beneath each junction. This image suggests that the Ag+ ions pass through the junction, and react with its contents as well as with the basement membrane beyond it. A scheme is proposed to explain the reaction of Ag+ ions with anions and negatively charged radicals within the junction. It is concluded that the “silver lines” represent not only a histochemical effect, but also the visualization of a transendothelial electrolyte pathway.

Rumpshaker Behaves like Juvenile-Lethal Plp Mutations When Combined with Shiverer in Double Mutant Mice

The phenotypes of double mutant mice whose genomes are homozygous for an Mbp (myelin basic protein) mutation and hemizygous for a juvenile-lethal Plp (proteolipid protein) mutation were compared in earlier studies. The results suggested that the shiverer Mpb mutation might have some unexplained ability to partially rescue oligodendrocytes (OLs) from the ‘death sentence’ that is imposed by the Plp mutations. Conversely, they also indicated that the juvenile-lethal Plp mutations may normalize shiverer OL morphology by reducing the numbers of microprocesses. The Plp mutation rumpshaker produces a mild hypomyelination without reduction in OL numbers and a normal lifespan. This report describes double mutant mice combining two Mbp mutations with rumpshaker, utilizing a common B6C3F1 hybrid-based genetic background. Initial studies on B6C3F1 rumpshaker optic nerve and spinal cord white matter showed unanticipated signs of OL death, with morphologic criteria suggestive of an apoptotic mechanism. In shiverer*rumpshaker double mutant mice, this small class of dying cells could not be identified. White matter morphology was similar to that of mice expressing only the shiverer mutation, except that OL microprocesses were far less abundant. This evidence suggests that, despite their distinctive phenotypic differences, rumpshaker may share more characteristics with the juvenile-lethal Plp mutations than has previously been recognized.

Carvedilol Inhibits Aortic Lipid Deposition in the Hypercholesterolemic Rat

The effects of carvedilol, a vasodilating beta-blocker with antioxidant activity, and nifedipine, a calcium channel blocker, were investigated on aortic lipid deposition and the accumulation of monocytes and foam cells at the sites of atherosclerotic lesions in rats subjected to a hypercholesterolemic diet. Fifty rats were randomly assigned to the following experimental groups: (1) regular rat chow (n = 5); (2) regular rat chow supplemented with a high-cholesterol diet (1% cholesterol and 1% cholic acid; n = 15); (3) a high-cholesterol diet plus nifedipine (n = 15), and (4) a high-cholesterol diet plus carvedilol (n = 15). Animals were maintained on these diets for 12 weeks. None of the treatment groups had blood pressures that were outside the normotensive range, and no significant differences in plasma lipid levels were observed among the high-cholesterol diet and drug-treated groups. There was a significantly lower lipid content (p < 0.001) in the thoracic aortas of the nifedipine-treated (211 +/- 23 nmol/mm2) and carvedilol-treated (182 +/- 23 nmol/mm2) groups compared to cholesterol-fed controls (242 +/- 27 nmol/mm2). Furthermore, carvedilol-treated animals showed significantly less (p < 0.001) lipid accumulation than did the nifedipine-treated animals. The number of monocytes and foam cells were decreased in both drug-treated groups compared to animals receiving high-cholesterol diets without drug treatment. The results demonstrate that treatment with carvedilol or nifedipine can significantly inhibit lipid deposition in the aorta and reduce monocyte and foam cell accumulation, and that carvedilol is significantly more effective than nifedipine in inhibiting lipid deposition.

Intimal Responses to Shear Stress, Hypercholesterolemia, and Hypertension

From the perspective of pathology, the three most common challenges to the intima of larger arteries are local disturbances of flow, hypercholesterolemia, and hypertension. We have studied all three in one experimental system, the aorta of the rat. This chapter is a critical summary of these studies. As an introduction, we will examine some relevant features of our experimental model.

Inhibition of lipid deposition in the hypercholesterolemic rat by clentiazem, a calcium channel blocker

We studied the effects of clentiazem, a calcium channel blocker (1) on the accumulation of lipid in the aorta, (2) on the level of plasma lipids, and (3) on the number of adherent intimal monocytes and foam cells. Seventy Wistar rats were assigned to one of the following groups: (1) regular diet, (2) an atherogenic diet consisting of regular chow with 2% cholesterol, 1% cholic acid, and 0.5% thiouracil (CCT), (3) CCT supplemented with 5 mg/kg/day clentiazem, and (4) CCT with 25 mg/kg/day clentiazem. Animals were sacrificed after 6 or 12 weeks of diet. Aortas were studied by light microscopy after staining with oil red O (ORO) and/or hematoxylin. ORO staining was quantified in both abdominal and thoracic regions of the aorta. The aortas of the clentiazem groups demonstrated significantly less ORO staining than CCT diet controls in thoracic aorta after 6 weeks and abdominal aorta after 12 weeks. There was no significant difference in the plasma lipid concentrations. The clentiazem-treated groups had fewer numbers of adherent monocytes and foam cells. We conclude that clentiazem inhibits lipid deposition in cholesterol-fed rats without lowering plasma lipid concentrations and that the number of intimal monocytes and foam cells is decreased in the presence of this calcium antagonist.