Q134R: Small chemical compound with NFAT inhibitory properties improves behavioral performance and synapse function in mouse models of amyloid pathology

Inhibition of the protein phosphatase calcineurin (CN) ameliorates pathophysiologic and cognitive changes in aging rodents and mice with aging‐related Alzheimer''s disease (AD)‐like pathology. However, concerns over adverse effects have slowed the transition of common CN‐inhibiting drugs to the clinic for the treatment of AD and AD‐related disorders. Targeting substrates of CN, like the nuclear factor of activated T cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. However, small chemical inhibitors of NFATs have only rarely been described. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that suppresses NFAT signaling, without inhibiting CN activity. Q134R partially inhibited NFAT activity in primary rat astrocytes, but did not prevent CN‐mediated dephosphorylation of a non‐NFAT target, either in vivo, or in vitro. Acute (≤1 week) oral delivery of Q134R to APP/PS1 (12 months old) or wild‐type mice (3–4 months old) infused with oligomeric Aβ peptides led to improved Y maze performance. Chronic (≥3 months) oral delivery of Q134R appeared to be safe, and, in fact, promoted survival in wild‐type (WT) mice when given for many months beyond middle age. Finally, chronic delivery of Q134R to APP/PS1 mice during the early stages of amyloid pathology (i.e., between 6 and 9 months) tended to reduce signs of glial reactivity, prevented the upregulation of astrocytic NFAT4, and ameliorated deficits in synaptic strength and plasticity, without noticeably altering parenchymal Aβ plaque pathology. The results suggest that Q134R is a promising drug for treating AD and aging‐related disorders.     

Glutamate Postsynaptic Receptors under the Cap: Strictly Limited Access to Receptors in the Postsynaptic Density by Non-Synaptically Released Glutamate

Astroglia is capable of releasing glutamate (Glu) in concentrations sufficient to activate ionotropic Glu receptors. Provided the released Glu reaches the receptors in the postsynaptic density, it can desensitize them. We have tested this possibility in the hippocampal CA1 synapses of rats either by applying exogenous Glu to the CA1 neurons or activating Glu release by astroglia. We found that Glu does not reach the synapses due to the existence of a protective uptake cap, which is sensitive to dihydrokainate, an inhibitor of GLT-type Glu transporter(s). Our results suggest that extrasynaptic and postsynaptic densities of the membranes of CA1 neurons form separate compartments differing from each other in the mechanisms and efficiency of processing external Glu. This provides additional diversity to specialized regulation of synaptic transmission and electrical excitation of pyramidal neurons.     

Protein processing and releases of neuregulin-1 are regulated in an activity-dependent manner

Identification of the key molecules that bridge presynaptic neuronal events and long-term modification of the postsynaptic process is an important challenge which will have to be met in order to further our understanding of the mechanisms for learning and memory. This study is focused on neuregulin-1 (NRG-1), a neurotrophic factor, that is known to regulate the development of various tissues and/or the life/death of cells through activation of the ErbB family receptor tyrosine kinases. It was discovered that the soluble form of NRG-1 (sNRG-1) is produced from the transmembrane form of NRG through proteolytic cleavage during electrical stimulation of either cultured cerebellar granule cells (GCs) or pontine nucleus neurons (PNs) that are presynaptic to GCs. sNRG-1 was assayed by measuring the phosphorylation of both the ErbB receptors and cyclic AMP-responsive element-binding protein (CREB), and by means of antibodies to sNRG-1. The cleavage and release of NRG-1 depended on the frequency of electrical stimulation; the peak effect was at 50 Hz in both GCs and PNs. Activation of protein kinase C (PKC) mimicked this effect. The culture apparatus provided along with the mass-electrical stimulation that was employed proved to be a powerful tool for combining neuronal electrical events and chemical events. We conclude from the results that, in mossy fibre (PN axon)-GC synapses, electrical activity controls the proteolytic processing of NRG-1 in a frequency-dependent fashion and involves PKC. Furthermore, cleaved sNRG-1 plays an important functional role in regulating transmission across these synapses.     

Pharmacological properties of homomeric and heteromeric pannexin hemichannels expressed in Xenopus oocytes

Several new findings have emphasized the role of neuron-specific gap junction proteins (connexins) and electrical synapses in processing sensory information and in synchronizing the activity of neuronal networks. We have recently shown that pannexins constitute an additional family of proteins that can form gap junction channels in a heterologous expression system and are also widely expressed in distinct neuronal populations in the brain, where they may represent a novel class of electrical synapses. In this study, we have exploited the hemichannel-forming properties of pannexins to investigate their sensitivity to well-known connexin blockers. By combining biochemical and electrophysiological approaches, we report here further evidence for the interaction of pannexin1 (Px1) with Px2 and demonstrate that the pharmacological sensitivity of heteromeric Px1/Px2 is similar to that of homomeric Px1 channels. In contrast to most connexins, both Px1 and Px1/Px2 hemichannels were not gated by external Ca2+. In addition, they exhibited a remarkable sensitivity to blockade by carbenoxolone (with an IC50 of approximately 5 microm), whereas flufenamic acid exerted only a modest inhibitory effect. The opposite was true in the case of connexin46 (Cx46), thus indicating that gap junction blockers are able to selectively modulate pannexin and connexin channels.     

Conical tomography II: A method for the study of cellular organelles in thin sections

We have used conical electron tomography in order to reconstruct neuronal organelles in thin sections of plastic embedded rat somato-sensory cortical tissue. The conical tilt series were collected at a 55 degrees tilt and at 5 degrees rotations, aligned using gold particles as fiduciary markers, and reconstructed using the weighted back projection algorithm. After a refinement process based on projection matching, the 3D maps showed the "unit membrane pattern" along the entire reconstructed volume. This pattern is indicative of the bilayer arrangement of phospholipids in biological membranes. Based on Fourier correlation methods as well as the visualization of the "unit membrane" pattern, we estimated resolutions of approximately 4 nm. To illustrate the prospective advantages of conical tomography, we segmented "coated" vesicles in the reconstructed volumes. These vesicles were comprised of a central core enclosing a small lumen, and a protein "coating" extending into the cytoplasm. The "coated" vesicle was attached to the plasma membrane through a complex structure shaped as an arch where the ends are attached to the membrane and the crook is connected to the vesicle. We concluded that conical electron tomography of thin-sectioned specimens provides a powerful experimental approach for studying thin-sectioned neuronal organelles at resolution levels of approximately 4 nm.     

Spontaneous Vesicle Fusion Is Differentially Regulated at Cholinergic and GABAergic Synapses

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The oculomotor system of Daphnia magna

Summary The highly mobile cyclopic compound eye of Daphnia magna is rotated by six muscles arranged as three bilateral pairs. The three muscles on each side of the head share a common origin on the carapace and insert dorsally, laterally and ventrally on the eye. The dorsal and ventral muscles are each composed of two muscle fibers and the lateral muscle is composed of from two to five fibers, with three the most common number. Individual muscle fibers are spindle-shaped mononucleated cells with organized bundles of myofilaments. Lateral eye-muscle fibers are thinner than those of the other muscles but are otherwise similar in ultrastructure. Two motor neurons innervate each dorsal and each ventral muscle and one motor neuron innervates each lateral muscle. The cell bodies of the motor neurons are situated dorsally in the supraesophageal ganglion (SEG) and are ipsilateral to the muscles they innervate. The dendritic fields of the dorsal-muscle motor neurons are ipsilateral to their cell bodies; those of the ventral-muscle motor neurons are bilateral though predominantly contralateral. The central projections of the lateral-muscle motor neurons are unknown. In the dorsal and ventral muscles one motor axon synapses principally with one muscle fiber; in each lateral muscle the single motor axon branches to, and forms synapses with, all the fibers. The neuromuscular junctions, characterized by pre- and postsynaptic densities and clear vesicles, are similar in all the eye muscles.     

Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae

The innervation of ventral longitudinal abdominal muscles (muscles 6, 7, 12, and 13) of third-instar Drosophila larvae was investigated with Nomarski, confocal, and electron microscopy to define the ultrastructural features of synapse-bearing terminals. As shown by previous workers, muscles 6 and 7 receive in most abdominal segments “Type I” endings, which are restricted in distribution and possess relatively prominent periodic terminal enlargements (“boutons”); whereas muscles 12 and 13 have in addition “Type II” terminals, which are more widely distributed and have smaller “boutons.” Serial sectioning of the Type I innervation of muscles 6 and 7 showed that two axons with distinctive endings contribute to it. One axon (termed Axon 1) has somewhat larger boutons, containing numerous synapses and presynaptic dense bodies (putative active zones for transmitter release). This axon also has more numerous intraterminal mitochondria, and a profuse subsynaptic reticulum around or under the synaptic boutons. The second axon (Axon 2) provides somewhat smaller boutons, with fewer synapses and dense bodies per bouton, fewer intraterminal mitochondria, and less-developed subsynaptic reticulum. Both axons contain clear synaptic vesicles, with occasional large dense vesicles. Approximately 800 synapses are provided by Axon 1 to muscles 6 and 7, and approximately 250 synapses are provided by Axon 2. In muscles 12 and 13, endings with predominantly clear synaptic vesicles, generally similar to the Type I endings of muscles 6 and 7, were found, along with another type of ending containing predominantly dense-cored vesicles, with small clusters of clear synaptic vesicles. This second type of ending was found most frequently in muscle 12, and probably corresponds to a subset of the “Type II” endings seen in the light microscope. Type I endings are thought to generate the ?fast’? and ?slow’? junctional potentials seen in electrophysiological recordings, whereas the physiological actions of Type II endings are presently not known. © 1993 John Wiley & Sons, Inc.     

Ion channel involvement in the temperature-sensitive response of the rabbit corneal endothelial cell resting membrane potential

Previous studies have shown that the resting potential (E _m) of the corneal endothelium hyperpolarizes following an increase in temperature above 24°C. Whole-cell studies using the perforated-patch technique were used to compare currents and E _mvalues from isolated corneal endothelial cells at 24 and 32°C. These studies revealed a small, outwardly rectifying, slowly activating, weakly voltage-dependent current with a reversal potential showing K⁺ selectivity (E _rev = –80 mV). This current had features similar to the whole-cell current seen following addition of HCO₃ ^– to these cells. E _mmeasurements found an average 24 mV hyperpolarization following temperature elevation in NaCl Ringer. Single channel studies found the only change in channel activity following an elevation in temperature to be an increase in the open probability (P _o) of a K⁺ channel previously reported in this cell type to be activated by external anions. P _o(–30 mV) at 24 and 32°C equaled 0.003 and 0.06, respectively. Increases in P _owere found at all voltages examined. This increased P _ocan account for the magnitude of the hyperpolarization seen in these cells following temperature elevation. Addition of HCO₃ ^– along with elevated temperature produced a synergistic effect on the increase in P _oalong with an increased hyperpolarization of the cell, pointing to separate mechanisms of activation from these two stimuli.The authors would like to thank Ms. Helen Hendrickson for her technical support and Drs. Gianrico Farrugia and Adam Rich for their helpful comments. This work was supported by NIH grants EY09673, EY03282, EY06005, and an unrestricted award from Research to Prevent Blindness.     

Inactivation of voltage-dependent calcium current in an insulinoma cell line

We have studied the mechanism of Ca current inactivation in the -cell line HIT-T15 by conventional and perforated patch recording techniques, using two pulse voltage protocols and a combination of current and tail current measurements. In 5 mM Ca, from a holding potential of - 80 mV, the maximum current showed a complex time course of inactivation: a relatively fast, double exponential inactivation (_h1 12 ms and _h2 60 ms) and a very slowly inactivating component ( > 1 s). The faster component (_h1) was due to the voltage-dependent inactivation of a low-threshold-activated (LVA), T-type current, which deactivates more slowly ( 3–5 ms) than the other components ( 0.2–0.3 ms). The intermediate component (_h2) was due to the Ca-dependent inactivation of a portion of the high-threshold-activated (HVA) current. A saturating dose of the dihydropyridine (DHP) nifedipine (10 M) did not affect the LVA current, but inhibited by 68 ± 5% the transient, Ca-sensitive portion of the HVA current and by 33 ± 12% the long lasting component. We suggest that three components of the calcium current can be resolved in HIT cells and the main target of DHPs is a HVA current, which inactivates faster than the DHP-resistant HVA component and does so primarily through calcium influx. Correspondence to: C. Marchetti