Endothelial cell junctions.

In the course of a freeze-cleave study on intercellular junctions in the regenerating rat liver, we observed an unusual array of intramembranous particles located in regions of contact between endothelial cells lining the hepatic sinusoids. These arrays were characterized by an accumulation of particles which resembled a zonula occludens in their linear deployment but differed in that the contact regions were composed of individual particles which remained separated from each other by regular particle-free intervals.

Loss and reappearance of gap junctions in regenerating liver.

Changes in intercellular junctional morphology associated with rat liver regeneration were examined in a freeze-fracture study. After a two-thirds partial hepatectomy, both gap junctions and zonulae occludentes were drastically altered. Between 0 and 20 h after partial hepatectomy, the junctions appeared virtually unchanged. 28 h after partial hepatectomy, however, the large gap junctions usually located close to the bile canaliculi and the small gap junctions enmeshed within the strands of the zonulae occudentes completely disappeared. Although the zonulae occludentes bordering the bile canaliculi apparently remained intact, numerous strands could now be found oriented perpendicular to the canaliculi. In some instances, the membrane outside the canaliculi was extensively filled with isolated junctional strands, often forming very complex configurations. About 40 h after partial hepatectomy, very many small gap junctions reappeared in close association with the zonulae occludentes. Subsequently, gap junctions increased in size and decreased in number until about 48 h after partial hepatectomy when gap junctions were indistinguishable in size and number from those of control animals. The zonulae occludentes were again predominantly located around the canalicular margins. These studies provide further evidence for the growth of gap junctions by the accretion of particles and of small gap junctions to form large maculae.

ARIA is concentrated in the synaptic basal lamina of the developing chick neuromuscular junction.

ARIA is a member of a family of polypeptide growth and differentiation factors that also includes glial growth factor (GGF), neu differentiation factor, and heregulin. ARIA mRNA is expressed in all cholinergic neurons of the central nervous systems of rats and chicks, including spinal cord motor neurons. In vitro, ARIA elevates the rate of acetylcholine receptor incorporation into the plasma membrane of primary cultures of chick myotubes. To study whether ARIA may regulate the synthesis of junctional synaptic acetylcholine receptors in chick embryos, we have developed riboprobes and polyclonal antibody reagents that recognize isoforms of ARIA that include an amino-terminal immunoglobulin C2 domain and examined the expression and distribution of ARIA in motor neurons and at the neuromuscular junction. We detected significant ARIA mRNA expression in motor neurons as early as embryonic day 5, around the time that motor axons are making initial synaptic contacts with their target muscle cells. In older embryos and postnatal animals, we found ARIA protein concentrated in the synaptic cleft at neuromuscular junctions, consistent with transport down motor axons and release at nerve terminals. At high resolution using immunoelectron microscopy, we detected ARIA immunoreactivity exclusively in the synaptic basal lamina in a pattern consistent with binding to synapse specific components on the presynaptic side of the basal lamina. These results support a role for ARIA as a trophic factor released by motor neuron terminals that may regulate the formation of mature neuromuscular synapses.

The synaptic vesicle proteins SV2, synaptotagmin and synaptophysin are sorted to separate cellular compartments in CHO fibroblasts.

We expressed the synaptic vesicle proteins SV2, synaptotagmin, and synaptophysin in CHO fibroblasts to investigate the targeting information contained by each protein. All three proteins entered different cellular compartments. Synaptotagmin was found on the plasma membrane. Both SV2 and synaptophysin were sorted to small intracellular vesicles, but synaptophysin colocalized with early endosomal markers, while SV2 did not. SV2-containing vesicles did not have the same sedimentation characteristics as authentic synaptic vesicles, even though transfected SV2 was sorted from endosomal markers. We also created cell lines expressing both SV2 and synaptotagmin, both synaptotagmin and synaptophysin, and lines expressing all three synaptic vesicle proteins. In all cases, the proteins maintained their distinct compartmentalizations, were not found in the same organelle, and did not created synaptic vesicle-like structures. These results have important implications for models of synaptic vesicle biogenesis.

Unconventional myosins in inner-ear sensory epithelia.

To understand how cells differentially use the dozens of myosin isozymes present in each genome, we examined the distribution of four unconventional myosin isozymes in the inner ear, a tissue that is particularly reliant on actin-rich structures and unconventional myosin isozymes. Of the four isozymes, each from a different class, three are expressed in the hair cells of amphibia and mammals. In stereocilia, constructed of cross-linked F-actin filaments, myosin-Ibeta is found mostly near stereociliary tips, myosin-VI is largely absent, and myosin-VIIa colocalizes with crosslinks that connect adjacent stereocilia. In the cuticular plate, a meshwork of actin filaments, myosin-Ibeta is excluded, myosin-VI is concentrated, and modest amounts of myosin-VIIa are present. These three myosin isozymes are excluded from other actin-rich domains, including the circumferential actin belt and the cortical actin network. A member of a fourth class, myosin-V, is not expressed in hair cells but is present at high levels in afferent nerve cells that innervate hair cells. Substantial amounts of myosins-Ibeta, -VI, and -VIIa are located in a pericuticular necklace that is largely free of F-actin, squeezed between (but not associated with) actin of the cuticular plate and the circumferential belt. Our localization results suggest specific functions for three hair-cell myosin isozymes. As suggested previously, myosin-Ibeta probably plays a role in adaptation; concentration of myosin-VI in cuticular plates and association with stereociliary rootlets suggest that this isozyme participates in rigidly anchoring stereocilia; and finally, colocalization with cross-links between adjacent stereocilia indicates that myosin-VIIa is required for the structural integrity of hair bundles.

Ultrastructure of electrophysiologically-characterized synapses formed by serotonergic raphe neurons in culture.

Recent electrophysiological investigations in this laboratory have shown that cultured mesopontine serotonergic neurons from neonatal rats evoke serotonergic and/or glutamatergic responses in themselves and in non-serotonergic neurons. Serotonergic nerve terminals in vivo are heterogeneous with respect to vesicle type, synaptic structure, and the frequency with which they form conventional synaptic contacts, but the functional correlates of this heterogeneity are unclear. We have therefore examined the ultrastructure of electrophysiologically-characterized synapses formed by cultured serotonergic neurons, and have compared the findings with the ultrastructural characteristics of serotonergic synapses reported in vivo. Dissociated rat serotonergic neurons in microcultures were identified by serotonin immunocytochemistry or by uptake of the autofluorescent serotonin analogue 5,7-dihydroxytryptamine, and were subsequently processed for electron microscopy. Unlabeled axon terminals formed numerous synapses on serotonin-immunoreactive somata and dendrites. Serotonin-immunoreactive axon terminals formed synapses on the somata, dendrites and somatodendritic spine-like appendages of serotonergic and non-serotonergic neurons. In microcultures containing a solitary serotonergic neuron that evoked glutamatergic or serotonergic/glutamatergic autaptic responses, both symmetric and asymmetric synapses were present. In addition to large dense core vesicles, individual neurons contained either microcanaliculi and microvesicles, clear round vesicles, or clear pleiomorphic vesicles. For a given cell, however, the subtypes of vesicles present in each axon terminal were similar. Thus, dissociated serotonergic and non-serotonergic raphe neurons formed functional, morphological synapses in culture. A direct examination of both the synaptic physiology and ultrastructure of single cultured serotonergic neurons indicated that these cells released serotonin and glutamate at synapses that were morphologically similar to synapses formed by serotonergic neurons in vivo. The findings also suggested that individual serotonergic neurons differ with respect to synaptic vesicle morphology, and are capable of simultaneously forming symmetric and asymmetric synapses with target cells.

Clusters of intramembranous particles on cultured myotubes at sites that are highly sensitive to acetylcholine.

Electrophysiological and autoradiographic studies have shown that the distribution of acetylcholine (AcCho) receptors on uninnervated cultured chicken muscle cells is not uniform. Regions of high receptor density (hot spots 10--40 times more sensitive than surrounding areas are localized as discrete patches or clusters about 10 micrometer in diameter o myotube muscle membranes. Hot spots were also found on fusion-arrested mononucleated myoblasts. We have developed a method for freeze-fracturing monolayer cultures that allows the unambiguous reidentification of membrane patches previously assayed for ACCho sensitivity. The freeze-fractured membranes at physiologically defined hot spots contain aggregates of many (10--20) small clusters of large (10--19 nm in diameter) intramembranous particles. Clusters are found on both fracture faces, but the particle density is much greater on the protoplasmic (P) face than on the extracellular (E) face (about 2000/micrometer2 vs. 700/micrometer2). Some of the particles appear to be composed of five or six "subunits" arranged cylindrically around a central dark dot. Because the aggregates are present at sites of high AcCho sensitivity, it is likely that the intramembranous particles are in some way related to the AcCho receptor molecule.

Gap junctions between electrotonically coupled cells in tissue culture and in brown fat.

Although circumstantial evidence has suggested that gap junctions mediate intercellular electrotonic coupling, it has not been possible in most tissues to exclude the involvement of other, coexisting cell junctions. We have made an electron microscopic study of replicas of frozen-fractured BHK21 cells (from tissue culture) and of brown fat cells of newborn mice. Both of these cell types are known to exhibit intercellular electrical coupling. In each case, the only junctions found between the cells are small macular gap junctions (less than 1 mum in diameter) characterized by clusters of 6-nm (60 A) particles or depressions on membrane cleavage faces. Several replicas confirm the association of these particles and depressions with regions of narrowing of the intercellular space, i.e., with the sites of cell junctions. We have also determined the frequency of occurrence of gap junctions on the membrane cleavage faces of both cell types. Gap junctions occupy about 1-2% of the surface area of brown fat cells, but only 0.05% of the surface area of BHK21 cells. These observations indicate that gap junctions, when they are the only intercellular junctions present, are sufficient to account for electronic coupling between cells.

Localization of myosin-Ibeta near both ends of tip links in frog saccular hair cells.

Current evidence suggests that the adaptation motor of mechanoelectrical transduction in vertebrate hair cells is myosin-Ibeta. Previously, confocal and electron microscopy of bullfrog saccular hair cells using an anti-myosin-Ibeta antibody labeled the tips of stereocilia. We have now done quantitative immunoelectron microscopy to test whether myosin-Ibeta is enriched at or near the side plaques of tip links, the proposed sites of adaptation, using hair bundles that were serially sectioned parallel to the macular surface. The highest particle density occurred at stereocilia bases, close to the cuticular plate. Also, stereocilia of differing lengths had approximately the same number of total particles, suggesting equal targeting of myosin-Ibeta to all stereocilia. Finally, particles tended to clump in clusters of two to five particles in the distal two-thirds of stereocilia, suggesting a tendency for self-assembly of myosin-Ibeta. As expected from fluorescence microscopy, particle density was high in the distal 1 micrometer of stereocilia. If myosin-Ibeta is the adaptation motor, a difference should exist in particle density between regions containing the side plaque and those excluding it. Averaging of particle distributions revealed two regions with approximately twice the average density: at the upper ends of tip links in a 700-nm-long region centered approximately 100 nm above the side plaque, and at the lower ends of tip links within the tip plaques. Controls demonstrated no such increase. The shortest stereocilia, which lack side plaques, showed no concentration rise on their sides. Thus, the specific localization of myosin-Ibeta at both ends of tip links supports its role as the adaptation motor.

Inhibition of the p44/42 MAP kinase pathway protects hippocampal neurons in a cell-culture model of seizure activity.

Excessive release of glutamate and the subsequent influx of calcium are associated with a number of neurological insults that result in neuronal death. The calcium-activated intracellular signaling pathways responsible for this excitotoxic injury are largely unknown. Here, we report that PD098059, a selective inhibitor of the calcium-activated p44/42 mitogen-activated protein kinase (MAP kinase) pathway, reduces neuronal death in a cell-culture model of seizure activity. Dissociated hippocampal neurons grown chronically in the presence of kynurenate, a broad spectrum glutamate-receptor antagonist, and elevated amounts of magnesium exhibit intense seizure-like activity after the removal of these blockers of excitatory synaptic transmission. A 30-min removal of the blockers produced extensive neuronal death within 24 h as assayed by the uptake of trypan blue and the release of lactate dehydrogenase. Phospho-p44/42 MAP kinase immunoreactivity after 30 min of seizure-like activity was present in many neuronal somata and dendrites as well as some synaptic terminals, consistent with both the presynaptic and postsynaptic effects of this pathway. The addition of PD098059 (40 microM; EC50 = 10 microM) during a 30-min washout of synaptic blockers inhibited the phosphorylation of p44/42 MAP kinase and reduced both the trypan-blue staining (n = 13) and the release of lactate dehydrogenase (n = 16) by 73% +/- 18% and 75% +/- 19% (mean +/- SD), respectively. The observed neuroprotection could be caused by an effect of PD098059 on seizure-like events or on downstream signaling pathways activated by the seizure-like events. Either possibility suggests a heretofore unknown function for the p44/42 MAP kinase pathway in neurons.

Cytoskeletal changes in the brains of mice lacking calcineurin A alpha.

Hyperphosphorylated tau, the major component of the paired helical filaments of Alzheimer's disease, was found to accumulate in the brains of mice in which the calcineurin A alpha gene was disrupted [calcineurin A alpha knockout (CNA alpha -/-)]. The hyperphosphorylation involved several sites on tau, especially the Ser396 and/or Ser404 recognized by the PHF-1 monoclonal antibody. The increase in phosphorylated tau content occurred primarily in the mossy fibers of the CNA alpha -/- hippocampus, which contained the highest level of calcineurin in brains of wild-type mice. The CNA alpha -/- mossy fibers also contained less neurofilament protein than normal, although the overall level of neurofilament phosphorylation was unchanged. In the electron microscope, the mossy fibers of CNA alpha -/- mice exhibited abnormalities in their cytoskeleton and a lower neurofilament/microtubule ratio than those of wild-type animals. These findings indicate that hyperphosphorylated tau can accumulate in vivo as a result of reduced calcineurin activity and is accompanied by cytoskeletal changes that are likely to have functional consequences on the affected neurons. The CNA alpha -/- mice were found in a separate study to have deficits in learning and memory that may result in part from the cytoskeletal changes in the hippocampus.

Laminin inhibition of beta-amyloid protein (Abeta) fibrillogenesis and identification of an Abeta binding site localized to the globular domain repeats on the laminin a chain.

beta-Amyloid protein (Abeta) is a major component of neuritic plaques and cerebrovascular amyloid deposits in the brains of patients with Alzheimer's disease (AD). Inhibitors of Abeta fibrillogenesis are currently sought as potential future therapeutics for AD and related disorders. In the present study, the basement membrane protein laminin was found to bind Abeta 1-40 with a single dissociation constant, K(d) = 2.7 x 10(-9) M, and serve as a potent inhibitor of Abeta fibril formation. 25 microM of Abeta 1-40 was incubated at 37 degrees C for 1 week in the presence of 100 nM of laminin or other basement membrane components, including perlecan, type IV collagen, and fibronectin to determine their effects on Abeta fibril formation as evaluated by thioflavin T fluorometry. Of all the basement membrane components tested, laminin demonstrated the greatest inhibitory effect on Abeta-amyloid fibril formation, causing a ninefold inhibition at 1 and 3 days and a 21-fold inhibition at 1 week. The inhibitory effects of laminin on Abeta fibrillogenesis occurred in a dose-dependent manner and were still effective at lower concentrations. The inhibitory effects of laminin on Abeta 1-40 fibril formation was confirmed by negative stain electron microscopy, whereby laminin caused an almost complete inhibition of Abeta fibril formation and assembly by 3 days, resulting in the appearance of primarily amorphous nonfibrillar material. Laminin also caused partial disassembly of preformed Abeta-amyloid fibrils following 4 days of coincubation. Laminin was not effective as an inhibitor of islet amyloid polypeptide fibril formation, suggesting that laminin's amyloid inhibitory effects were Abeta-specific. To identify a potential Abeta-binding site(s) on laminin, laminin was first digested with V8, trypsin, or elastase. An Abeta-binding elastase digestion product of approximately 120-130 kDa was found. In addition, a approximately 55 kDa fragment derived from V8 and elastase-digested laminin interacted with biotinylated Abeta 1-40. Amino acid sequencing of the approximately 55 kDa fragment identified a conformationally dependent Abeta-binding site within laminin localized to the globular repeats on the laminin A chain. These studies demonstrate that laminin not only binds Abeta with relatively high affinity but is a potent inhibitor of Abeta-amyloid fibril formation. In addition, further identification of an Abeta-binding domain within the globular repeats on the laminin A chain may lead to the design of new therapeutics for the inhibition of Abeta fibrillogenesis.

Expression patterns of transmembrane and released forms of neuregulin during spinal cord and neuromuscular synapse development.

We mapped the distribution of neuregulin and its transmembrane precursor in developing, embryonic chick and mouse spinal cord. Neuregulin mRNA and protein were expressed in motor and sensory neurons shortly after their birth and levels steadily increased during development. Expression of the neuregulin precursor was highest in motor and sensory neuron cell bodies and axons, while soluble, released neuregulin accumulated along early motor and sensory axons, radial glia, spinal axonal tracts and neuroepithelial cells through associations with heparan sulfate proteoglycans. Neuregulin accumulation in the synaptic basal lamina of neuromuscular junctions occurred significantly later, coincident with a reorganization of muscle extracellular matrix resulting in a relative concentration of heparan sulfate proteoglycans at endplates. These results demonstrate an early axonal presence of neuregulin and its transmembrane precursor at developing synapses and a role for heparan sulfate proteoglycans in regulating the temporal and spatial sites of soluble neuregulin accumulation during development.