Rita Azzam

Organization of the core structure of the postsynaptic density

Much is known about the composition and function of the postsynaptic density (PSD), but less is known about its molecular organization. We use EM tomography to delineate the organization of PSDs at glutamatergic synapses in rat hippocampal cultures. The core of the PSD is dominated by vertically oriented filaments, and ImmunoGold labeling shows that PSD-95 is a component of these filaments. Vertical filaments contact two types of transmembrane structures whose sizes and positions match those of glutamate receptors and intermesh with two types of horizontally oriented filaments lying 10–20 nm from the postsynaptic membrane. The longer horizontal filaments link adjacent NMDAR-type structures, whereas the smaller filaments link both NMDA- and AMPAR-type structures. The orthogonal, interlinked scaffold of filaments at the core of the PSD provides a structural basis for understanding dynamic aspects of postsynaptic function.

PSD-95 Is Required to Sustain the Molecular Organization of the Postsynaptic Density

PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength. Here we show that PSD-95 is in an extended configuration and positioned into regular arrays of vertical filaments that contact both glutamate receptors and orthogonal horizontal elements layered deep inside the PSD in rat hippocampal spine synapses. RNA interference knockdown of PSD-95 leads to loss of entire patches of PSD material, and electron microscopy tomography shows that the patchy loss correlates with loss of PSD-95-containing vertical filaments, horizontal elements associated with the vertical filaments, and putative AMPA receptor-type, but not NMDA receptor-type, structures. These observations show that the orthogonal molecular scaffold constructed from PSD-95-containing vertical filaments and their associated horizontal elements is essential for sustaining the three-dimensional molecular organization of the PSD. Our findings provide a structural basis for understanding the functional role of PSD-95 at the PSD.

Novel Cell Types, Neurosecretory Cells, and Body Plan of the Early-Diverging Metazoan Trichoplax adhaerens

BACKGROUND Trichoplax adhaerens is the best-known member of the phylum Placozoa, one of the earliest-diverging metazoan phyla. It is a small disk-shaped animal that glides on surfaces in warm oceans to feed on algae. Prior anatomical studies of Trichoplax revealed that it has a simple three-layered organization with four somatic cell types. RESULTS We reinvestigate the cellular organization of Trichoplax using advanced freezing and microscopy techniques to identify localize and count cells. Six somatic cell types are deployed in stereotyped positions. A thick ventral plate, comprising the majority of the cells, includes ciliated epithelial cells, newly identified lipophil cells packed with large lipid granules, and gland cells. Lipophils project deep into the interior, where they alternate with regularly spaced fiber cells whose branches contact all other cell types, including cells of the dorsal and ventral epithelium. Crystal cells, each containing a birefringent crystal, are arrayed around the rim. Gland cells express several proteins typical of neurosecretory cells, and a subset of them, around the rim, also expresses an FMRFamide-like neuropeptide. CONCLUSIONS Structural analysis of Trichoplax with significantly improved techniques provides an advance in understanding its cell types and their distributions. We find two previously undetected cell types, lipohil and crystal cells, and an organized body plan in which different cell types are arranged in distinct patterns. The composition of gland cells suggests that they are neurosecretory cells and could control locomotor and feeding behavior.

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

Significance The postsynaptic density (PSD) at the glutamatergic excitatory synapse is a macromolecular machine that underlies synaptic transmission and information storage. Membrane-associated guanylate kinases (MAGUKs), the major scaffolding proteins at the PSD, are positively correlated with synaptic maturation and strength, but how MAGUKs sustain the strength of synaptic transmission remains unclear. Here, we remove three MAGUK proteins from neurons and find significant reductions in synaptic transmission by AMPARs and NMDARs with a concomitant reduction in PSD sizes and core scaffold and transmembrane structures. Our results show how MAGUKs anchor and organize both types of glutamate receptors, thereby regulating the strength of excitatory synapses. The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95–like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.

Trafficking of AMPA Receptors at Plasma Membranes of Hippocampal Neurons

The number of AMPA receptors at synapses depends on receptor cycling. Because receptors diffuse rapidly in plasma membranes, their exocytosis and endocytosis need not occur near synapses. Here, pre-embedding immunogold electron microscopy is applied to dissociated rat hippocampal cultures to provide sensitive, high-resolution snapshots of the distribution of surface AMPA receptors in spines, dendrites, and cell bodies that will be informative about trafficking of AMPA receptors. The density of the label for GluR2 varies, but is consistent throughout cell body and dendrites in each individual neuron, except at postsynaptic densities (PSDs), where it is typically higher. Glutamate receptor 2 (GluR2) labels at PSDs significantly increase after synaptic activation by glycine treatment and increase further upon depolarization by high K+. Islands of densely packed labels have consistent size and density but vary in frequency under different experimental conditions. These patches of label, which occur on plasma membranes of cell bodies and dendrites but not near PSDs, are taken to be the aftermath of exocytosis of AMPA receptors. A subpopulation of clathrin-coated pits in cell bodies and dendrites label for GluR2, and the number and amount of label in individual pits increase after NMDA treatment. Coated pits near synapses typically lack GluR2 label under basal conditions, but ∼40% of peri-PSD pits label for GluR2 after NMDA treatment. Thus, exocytosis and endocytosis of AMPA receptors occur mainly at extrasynaptic locations on cell bodies and dendrites. Receptors are not preferentially exocytosed near PSDs, but may be removed via endocytosis at peri-PSD locations after activation of NMDA receptors.

Regenerating Axons Are Not Required to Induce the Formation of a Schwann Cell Cable in a Silicone Chamber

After suture of proximal and distal nerve stumps into the ends of a silicone chamber, a tissue cable forms inside the chamber through which axons regenerate. Schwann cells are a critical cellular component of the cable because in their absence axons fail to regenerate into the cable. In this study, we sought to determine whether axons were needed to induce the formation of a Schwann cell-containing cable. Transected stumps of sciatic nerves of adult rats were sutured into the ends of silicone chambers prefilled with phosphate-buffered saline or dialyzed plasma, leaving a 10-mm interstump gap. In order to eliminate any axonal influence in the chamber, the proximal sciatic nerve was further transected, ligated, and reflected, leaving a 4-mm piece of denervated nerve in the proximal chamber. A tissue cable formed at 4 weeks only in those chambers prefilled with dialyzed plasma. Light and electron microscopy revealed a central core of Schwann cells and fibroblasts within the cable that were collectively surrounded by a circumferential layer of fibroblasts and collagen. Blood vessels were randomly located throughout the cable. The Schwann cells extended numerous processes that were confined within a basal lamina-like membrane. Many of these processes contained microtubules and resembled unmyelinated axons. The ultrastructure of the processes, however, differed from that of axons in that some of the processes were in direct contact with the basal lamina of the Schwann cells and not surrounded by any other cell extensions. However, since these processes neither stained with silver nor disappeared after transection of the nerves entering or leaving the chamber, we conclude that they are not axons but in fact Schwann cell processes. In other animals bearing 4-week cables, the reflected nerve stump was reattached to the nerve piece in the proximal end of the chamber. Four weeks later, all the cables and varying lengths of the distal nerve trunks were filled with numerous myelinated and unmyelinated axons. The Schwann cell cable that forms within a dialyzed plasma prefilled chamber presents a useful system for basic research concerning the molecular mechanisms of Schwann cell or Schwann cell-axonal interactions and for applied research involving the clinical repair of human peripheral nerve injuries. Since a cable formed by our surgical method supports axonal regeneration, it has the potential to eliminate the need for a nerve graft to repair a gap in a nerve that requires delayed surgical intervention.

RAPID TURNOVER OF SPINULES AT SYNAPTIC TERMINALS

Spinules found in brain consist of small invaginations of plasma membranes which enclose membrane evaginations from adjacent cells. Here, we focus on the dynamic properties of the most common type, synaptic spinules, which reside in synaptic terminals. In order to test whether depolarization triggers synaptic spinule formation, hippocampal slice cultures (7-day-old rats, 10-14 days in culture) were exposed to high K+ for 0.5-5 min, and examined by electron microscopy. Virtually no synaptic spinules were found in control slices representing a basal state, but numerous spinules appeared at both excitatory and inhibitory synapses after treatment with high K+. Spinule formation peaked with approximately 1 min treatment at 37 degrees C, decreased with prolonged treatment, and disappeared after 1-2 min of washout in normal medium. The rate of disappearance of spinules was substantially slower at 4 degrees C. N-methyl-D-aspartic acid (NMDA) treatment also induced synaptic spinule formation, but to a lesser extent than high K+ depolarization. In acute brain slices prepared from adult mice, synaptic spinules were abundant immediately after dissection at 4 degrees C, extremely rare in slices allowed to recover at 28 degrees C, but frequent after high K(+) depolarization. High pressure freezing of acute brain slices followed by freeze-substitution demonstrated that synaptic spinules are not induced by chemical fixation. These results indicate that spinules are absent in synapses at low levels of activity, but form and disappear quickly during sustained synaptic activity. The rapid turnover of synaptic spinules may represent an aspect of membrane retrieval during synaptic activity.

The Loss of Regenerated Host Axons in Nerve Allografts after Stopping Immunosuppression with Cyclosporin A Is Related to Immune Effects on Allogeneic Schwann Cells

After immunosuppressive therapy with Cyclosporin A (Cy-A) is stopped, nerve allograft rejection occurs. In addition to the loss of allogeneic perineurial, vascular, and Schwann cells, host axons that regenerate into the allograft disappear despite the fact that the axons are not foreign tissue. The present experiment was performed to correlate immune events and allogeneic cell and host axonal loss in nerve allografts after terminating Cy-A treatment. Nerve grafts (4 cm long) were taken from American Cancer Institute (ACI) rats and joined to the peroneal nerves of Fischer (FR) or ACI rats that received a daily dose of Cy-A (10 mg/kg, intraperitoneally). After one week, Cy-A therapy was stopped and the grafts were examined 2-6 weeks postoperatively by light and electron microscopy. No immune reaction nor destruction of perineural, vascular, or Schwann cells was found in 2- or 3-week-old allografts (i.e., ACI to FR grafts). These grafts underwent Wallerian degeneration and were invaded proximally by regenerating host axons, some of which were thinly myelinated. At 4 weeks, the perineurium of each allograft became infiltrated by mononuclear cells and was destroyed. Many of the endoneurial blood vessels of these grafts were occluded and their endothelial cells were degenerating or missing. Despite the immune reaction, allogeneic Schwann cells remained and continued to myelinate or ensheath host axons that had now grown up to 3 cm into the grafts.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of Tyrosine Phosphorylation of a Brain Protein by Hibernation

Mammalian hibernation is a state of natural tolerance to severely decreased brain blood flow. As protein tyrosine phosphorylation is believed to be involved in the development of resistance to potentially cell-damaging insults, we used immunoblotting for the phosphotyrosine moiety to analyze extracts from various tissues of hibernating and nonhibernating ground squirrels. A single, hibernation-specific phosphoprotein was detected in the brain, but not in any other tissue tested. This protein, designated pp98 to reflect its apparent molecular weight, is distributed throughout the brain, and is associated with the cellular membrane fraction. The presence of the protein is tightly linked to the hibernation state; it is not present in contemporaneously assayed animals that are exposed to the same cold temperature as the hibernators, is present for the duration of a hibernation bout (tested from 1 to 14 days), and disappears within 1 hour of arousal from hibernation. The close association of pp98 with the hibernation state, its presence in cellular membranes, and the known properties of membrane phosphotyrosine proteins suggest that it may transduce a signal for adaptation to the limited availability of oxygen and glucose and low cellular temperature that characterizes hibernation in the ground squirrel.

Depolarization of hippocampal neurons induces formation of nonsynaptic NMDA receptor islands resembling nascent postsynaptic densities

Abstract Depolarization of neurons in 3-week-old rat hippocampal cultures promotes a rapid increase in the density of surface NMDA receptors (NRs), accompanied by transient formation of nonsynaptic NMDA receptor clusters or NR islands. Islands exhibit cytoplasmic dense material resembling that at postsynaptic densities (PSDs), and contain typical PSD components, including MAGUKS (membrane-associated guanylate kinases), GKAP, Shank, Homer, and CaMKII detected by pre-embedding immunogold electron microscopy. In contrast to mature PSDs, islands contain more NMDA than AMPA receptors, and more SAP102 than PSD-95, features that are shared with nascent PSDs in developing synapses. Islands do not appear to be exocytosed or endocytosed directly as preformed packages because neurons lacked intracellular vacuoles containing island-like structures. Islands form and disassemble upon depolarization of neurons on a time scale of 2-3 min, perhaps representing an initial stage in synaptogenesis.