Utility of Liquid-Based Cytology and Cell Block Procedure Obtained by Vitrectomy to Diagnose Ocular Sarcoidosis-The Significance of Epithelioid Granuloma and Epithelioid Cells.

Purpose: The eyes are one of the most frequently involved organs in sarcoidosis in Asia, including Japan. Sarcoid uveitis is the major complaint of ocular sarcoidosis. The detection of epithelioid granuloma (EG) requires histological biopsy of the uvea for the precise diagnosis of sarcoid uveitis, because it is challenging to diagnose sarcoid uveitis without a history of systemic sarcoidosis. To diagnose sarcoid uveitis, we have established novel methods. Patients and Methods: In this study, we included 30 eyes of 21 patients with granulomatous uveitis diagnosed via slit-lamp examinations, gonioscopy, fundus photography, and fluorescein angiography. Vitrectomy was performed to remove the vitreous opacity with vision loss. To examine vitreous cell components, we used liquid-based cytology (LBC). To detect EG in an intraocular irrigating solution, we collected vitreous cell components, and then the cell pellets were embedded in the cell block procedure. Results: Here, we demonstrated the usefulness of the histological detection of EG and epithelioid cells (ECs) in LBC from vitreous body specimens and in the cell block procedure from vitreous cell components in an intraocular irrigating solution. Our results showed that the detection rates of EG were 6.3% (1/16) in LBC and 9.1% (1/11) in the cell block procedure in the sarcoid uveitis-suspected group and 7.7% (1/13) in LBC and 28.6% (2/7) in the cell block procedure in the sarcoidosis group. We would discuss the specificity of the EG/EC detection of ocular sarcoidosis. Conclusion: Our methods are helpful in the precise diagnosis of ocular sarcoidosis and the control of the development of systemic sarcoidosis.

Synaptic plasticity in hippocampal CA1 neurons of mice lacking inositol-1,4,5-trisphosphate receptor-binding protein released with IP3 (IRBIT).

In hippocampal CA1 neurons of wild-type mice, a short tetanus (15 or 20 pulses at 100 Hz) or a standard tetanus (100 pulses at 100 Hz) to a naive input pathway induces long-term potentiation (LTP) of the responses. Low-frequency stimulation (LFS; 1000 pulses at 1 Hz) 60 min after the standard tetanus reverses LTP (depotentiation [DP]), while LFS applied 60 min prior to the standard tetanus suppresses LTP induction (LTP suppression). We investigated LTP, DP, and LTP suppression of both field excitatory postsynaptic potentials and population spikes in CA1 neurons of mice lacking the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-binding protein released with IP3 (IRBIT). The mean magnitudes of LTP induced by short and standard tetanus were not different in mutant and wild-type mice. In contrast, DP and LTP suppression were attenuated in mutant mice, whereby the mean magnitude of responses after LFS or tetanus were significantly greater than in wild-type mice. These results suggest that, in hippocampal CA1 neurons, IRBIT is involved in DP and LTP suppression, but is not essential for LTP. The attenuation of DP and LTP suppression in mice lacking IRBIT indicates that this protein, released during or after priming stimulations, determines the direction of LTP expression after the delivery of subsequent stimulations.

A Farnesyltransferase Inhibitor Restores Cognitive Deficits in Tsc2+/- Mice through Inhibition of Rheb1.

Tuberous sclerosis complex (TSC) is caused by mutations in Tsc1 or Tsc2, whose gene products inhibit the small G-protein Rheb1. Rheb1 activates mTORC1, which may cause refractory epilepsy, intellectual disability, and autism. The mTORC1 inhibitors have been used for TSC patients with intractable epilepsy. However, its effectiveness for cognitive symptoms remains unclear. We found a new signaling pathway for synapse formation through Rheb1 activation, but not mTORC1. Here, we show that treatment with the farnesyltransferase inhibitor lonafarnib increased unfarnesylated (inactive) Rheb1 levels and restored synaptic abnormalities in cultured Tsc2+/- neurons, whereas rapamycin did not enhance spine synapse formation. Lonafarnib treatment also restored the plasticity-related Arc (activity-regulated cytoskeleton-associated protein) expression in cultured Tsc2+/- neurons. Lonafarnib action was partly dependent on the Rheb1 reduction with syntenin. Oral administration of lonafarnib increased unfarnesylated protein levels without affecting mTORC1 and MAP (mitogen-activated protein (MAP)) kinase signaling, and restored dendritic spine morphology in the hippocampi of male Tsc2+/- mice. In addition, lonafarnib treatment ameliorated contextual memory impairments and restored memory-related Arc expression in male Tsc2+/- mice in vivo Heterozygous Rheb1 knockout in male Tsc2+/- mice reproduced the results observed with pharmacological treatment. These results suggest that the Rheb1 activation may be responsible for synaptic abnormalities and memory impairments in Tsc2+/- mice, and its inhibition by lonafarnib could provide insight into potential treatment options for TSC-associated neuropsychiatric disorders.SIGNIFICANCE STATEMENT Tuberous sclerosis complex (TSC) is an autosomal-dominant disease that causes neuropsychiatric symptoms, including intractable epilepsy, intellectual disability (ID) and autism. No pharmacological treatment for ID has been reported so far. To develop a pharmacological treatment for ID, we investigated the mechanism of TSC and found that Rheb1 activation is responsible for synaptic abnormalities in TSC neurons. To inhibit Rheb1 function, we used the farnesyltransferase inhibitor lonafarnib, because farnesylation of Rheb1 is required for its activation. Lonafarnib treatment increased inactive Rheb1 and recovered proper synapse formation and plasticity-related Arc (activity-regulated cytoskeleton-associated protein) expression in TSC neurons. Furthermore, in vivo lonafarnib treatment restored contextual memory and Arc induction in TSC mice. Together, Rheb1 inhibition by lonafarnib could provide insight into potential treatments for TSC-associated ID.

Depotentiation depends on IP3 receptor activation sustained by synaptic inputs after LTP induction.

In CA1 neurons of guinea pig hippocampal slices, long-term potentiation (LTP) was induced in field excitatory postsynaptic potentials (EPSPs) or population spikes (PSs) by the delivery of high-frequency stimulation (HFS, 100 pulses at 100 Hz) to CA1 synapses, and was reversed by the delivery of a train of low-frequency stimulation (LFS, 1000 pulses at 2 Hz) at 30 min after HFS (depotentiation), and this effect was inhibited when test synaptic stimulation was halted for a 19-min period after HFS or for a 20-min period after LFS or applied over the same time period in the presence of an antagonist of N-methyl-D-aspartate receptors (NMDARs), group I metabotropic glutamate receptors (mGluRs), or inositol 1, 4, 5-trisphosphate receptors (IP3Rs). Depotentiation was also blocked by the application of a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor or a calcineurin inhibitor applied in the presence of test synaptic input for a 10-min period after HFS or for a 20-min period after LFS. These results suggest that, in postsynaptic neurons, the coactivation of NMDARs and group I mGluRs due to sustained synaptic activity following LTP induction results in the activation of IP3Rs and CaMKII, which leads to the activation of calcineurin after LFS and depotentiation of CA1 synaptic responses.

Scalaradial Is a Potent Inhibitor of Transient Receptor Potential Melastatin 2 (TRPM2) Ion Channels.

TRPM2 is a Ca2+-permeable, nonselective cation channel that plays a role in oxidant-induced cell death, insulin secretion, and cytokine release. Few TRPM2 inhibitors have been reported, which hampers the validation of TRPM2 as a drug target. While screening our in-house marine-derived chemical library, we identified scalaradial and 12-deacetylscalaradial as the active components within an extract of an undescribed species of Cacospongia (class Demospongiae, family Thorectidae) that strongly inhibited TRPM2-mediated Ca2+ influx in TRPM2-overexpressing HEK293 cells. In whole-cell patch-clamp experiments, scalaradial (and similarly 12-deacetylscalaradial) inhibited TRPM2-mediated currents in a concentration- and time-dependent manner (∼20 min to full onset; IC50 210 nM). Scalaradial inhibited TRPM7 with less potency (IC50 760 nM) but failed to inhibit CRAC, TRPM4, and TRPV1 currents in whole-cell patch clamp experiments. Scalaradial's effect on TRPM2 channels was shown to be independent of its well-known ability to inhibit secreted phospholipase A2 (sPLA2) and its reported effects on extracellular signal-regulated kinases (ERK) and Akt pathways. In addition, scalaradial was shown to inhibit endogenous TRPM2 currents in a rat insulinoma cell line (IC50 330 nM). Based on its potency and emerging specificity profile, scalaradial is an important addition to the small number of known TRPM2 inhibitors.

Prior activation of inositol 1,4,5-trisphosphate receptors suppresses the subsequent induction of long-term potentiation in hippocampal CA1 neurons.

We investigated the role of inositol 1,4,5-trisphosphate receptors (IP3Rs) activated by preconditioning low-frequency afferent stimulation (LFS) in the subsequent induction of long-term potentiation (LTP) in CA1 neurons in hippocampal slices from mature guinea pigs. Induction of LTP in the field excitatory postsynaptic potential or the population spike by the delivery of high-frequency stimulation (HFS, a tetanus of 100 pulses at 100 Hz) to the Schaffer collateral-commissural pathway to CA1 neuron synapses was suppressed when group I metabotropic glutamate receptors (mGluRs) were activated prior to the delivery of HFS. LTP induction was also suppressed when CA1 synapses were preconditioned 60 min before HFS by LFS of 1000 pulses at 1 Hz and this effect was inhibited when the test stimulation delivered at 0.05 Hz was either halted or applied in the presence of an antagonist ofN-methyl-d-aspartate receptors, group I mGluRs, or IP3Rs during a 20-min period from 20 to 40 min after the end of LFS. Furthermore, blockade of group I mGluRs or IP3Rs immediately before the delivery of HFS overcame the effects of the preconditioning LFS on LTP induction. These results suggest that, in CA1 neurons, after a preconditioning LFS, activation of group I mGluRs caused by the test stimulation results in IP3Rs activation that leads to a failure of LTP induction.

Role of postsynaptic inositol 1, 4, 5-trisphosphate receptors in depotentiation in guinea pig hippocampal CA1 neurons.

The long-term potentiation (LTP) in the field excitatory postsynaptic potential (EPSP) induced at hippocampal CA1 pyramidal neuron synapses by delivery of high frequency stimulation (HFS), a tetanus of 100 pulses at 100Hz, is decreased (depotentiation) by a train of low frequency stimulation (LFS) of 1000 pulses at 2Hz applied 30min later. Inositol 1, 4, 5-trisphosphate receptors (IP3Rs) activated both during the HFS and after the LFS are involved in this depotentiation, the former triggering, and the latter modifying, LTP induction (decreasing the amplitude of the LTP established by the priming HFS). Furthermore, the decrease in the LTP at CA1 synapses requires activation of IP3Rs during LFS and activation of calcineurin after LFS. These results suggest that, at hippocampal CA1 neuron synapses, HFS-induced IP3R activation, which is modulated by the subsequent LFS, results in postsynaptic protein dephosphorylation after the LFS, leading to a decrease in the field EPSP and in the HFS-induced LTP.

Potent functional uncoupling between STIM1 and Orai1 by dimeric 2-aminodiphenyl borinate analogs.

The coupling of ER Ca(2+)-sensing STIM proteins and PM Orai Ca(2+) entry channels generates "store-operated" Ca(2+) signals crucial in controlling responses in many cell types. The dimeric derivative of 2-aminoethoxydiphenyl borinate (2-APB), DPB162-AE, blocks functional coupling between STIM1 and Orai1 with an IC50 (200 nM) 100-fold lower than 2-APB. Unlike 2-APB, DPB162-AE does not affect L-type or TRPC channels or Ca(2+) pumps at maximal STIM1-Orai1 blocking levels. DPB162-AE blocks STIM1-induced Orai1 or Orai2, but does not block Orai3 or STIM2-mediated effects. We narrowed the DPB162-AE site of action to the STIM-Orai activating region (SOAR) of STIM1. DPB162-AE does not prevent the SOAR-Orai1 interaction but potently blocks SOAR-mediated Orai1 channel activation, yet its action is not as an Orai1 channel pore blocker. Using the SOAR-F394H mutant which prevents both physical and functional coupling to Orai1, we reveal DPB162-AE rapidly restores SOAR-Orai binding but only slowly restores Orai1 channel-mediated Ca(2+) entry. With the same SOAR mutant, 2-APB induces rapid physical and functional coupling to Orai1, but channel activation is transient. We infer that the actions of both 2-APB and DPB162-AE are directed toward the STIM1-Orai1 coupling interface. Compared to 2-APB, DPB162-AE is a much more potent and specific STIM1/Orai1 functional uncoupler. DPB162-AE provides an important pharmacological tool and a useful mechanistic probe for the function and coupling between STIM1 and Orai1 channels.

Impairment of hippocampal long-term potentiation and failure of learning in mice treated with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.

Gangliosides (sialic acid-containing glycosphingolipids) play important roles in many physiological functions, including synaptic plasticity in the hippocampus, which has been suggested as the basal cellular process of learning and memory in the brain. In the present study, long-term potentiation (LTP) and long-term depression (LTD) in CA1 hippocampal neurons and learning behavior were examined in mice treated with (D)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol ((D)-PDMP), an inhibitor of ganglioside biosynthesis. Mice treated with (D)-PDMP, but not those treated with (L)-PDMP, showed impairment of LTP induction in hippocampal CA1 neurons without any significant change in LTD formation and also showed a failure of learning in the 4-pellet taking test. These results indicate that de novo synthesis of gangliosides in the brain is involved in synaptic plasticity of LTP in mouse hippocampal CA1 neurons and plays important roles in learning and memory.

Suppressive effect of preconditioning low-frequency stimulation on subsequent induction of long-term potentiation by high frequency stimulation in hippocampal CA3 neurons.

We investigated the role of inositol 1, 4, 5-trisphosphate receptors (IP3Rs), activated during preconditioning low-frequency afferent stimulation (LFS), in the subsequent induction of long-term potentiation (LTP) in CA3 neurons in hippocampal slices from mature guinea pigs. Induction of LTP in the field excitatory postsynaptic potential (EPSP) by the delivery of high-frequency stimulation (HFS, a tetanus of two trains of 100 pulses at 100Hz with a 10s interval) to mossy fiber-CA3 neuron synapses was suppressed when CA3 synapses were preconditioned by the LFS of 1000 pulses at 2Hz and this effect was inhibited when the LFS preconditioning was performed in the presence of an IP3R antagonist or a protein phosphatase inhibitor. Furthermore, activation of group 1 metabotropic glutamate receptors (mGluRs) during HFS canceled the effects of an IP3R antagonist given during preconditioning LFS on the subsequent LTP induction at mossy fiber-CA3 synapses. These results suggest that, in hippocampal mossy fiber-CA3 neuron synapses, activation of IP3Rs during a preconditioning LFS results in dephosphorylation events that lead to failure of the HFS to induce subsequent LTP.

Mechanistic basis of bell-shaped dependence of inositol 1,4,5-trisphosphate receptor gating on cytosolic calcium.

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular Ca(2+) release channel, and its opening is controlled by IP(3) and Ca(2+). A single IP(3) binding site and multiple Ca(2+) binding sites exist on single subunits, but the precise nature of the interplay between these two ligands in regulating biphasic dependence of channel activity on cytosolic Ca(2+) is unknown. In this study, we visualized conformational changes in IP(3)R evoked by various concentrations of ligands by using the FRET between two fluorescent proteins fused to the N terminus of individual subunits. IP(3) and Ca(2+) have opposite effects on the FRET signal change, but the combined effect of these ligands is not a simple summative response. The bell-shaped Ca(2+) dependence of FRET efficiency was observed after the subtraction of the component corresponding to the FRET change evoked by Ca(2+) alone from the FRET changes evoked by both ligands together. A mutant IP(3)R containing a single amino acid substitution at K508, which is critical for IP(3) binding, did not exhibit this bell-shaped Ca(2+) dependence of the subtracted FRET efficiency. Mutation at E2100, which is known as a Ca(2+) sensor, resulted in ∼10-fold reduction in the Ca(2+) dependence of the subtracted signal. These results suggest that the subtracted FRET signal reflects IP(3)R activity. We propose a five-state model, which implements a dual-ligand competition response without complex allosteric regulation of Ca(2+) binding affinity, as the mechanism underlying the IP(3)-dependent regulation of the bell-shaped relationship between the IP(3)R activity and cytosolic Ca(2+).

Impaired hippocampal long-term potentiation and failure of learning in ß1,4-N-acetylgalactosaminyltransferase gene transgenic mice.

Gangliosides (sialic acid-containing glycosphingolipids) play important roles in many physiological functions, including synaptic plasticity in the hippocampus, which is considered as a cellular mechanism of learning and memory. In the present study, three types of synaptic plasticity, long-term potentiation (LTP), long-term depression (LTD) and reversal of LTP (depotentiation, DP), in the field excitatory post-synaptic potential in CA1 hippocampal neurons and learning behavior were examined in ß1,4-N-acetylgalactosaminyltransferase (ß1,4 GalNAc-T; GM2/GD2 synthase) gene transgenic (TG) mice, which showed a marked decrease in b-pathway gangliosides (GQ1b, GT1b and GD1b) in the brain and isolated hippocampus compared with wild-type (WT) mice. The magnitude of the LTP induced by tetanus (100 pulses at 100 Hz) in TG mice was significantly smaller than that in control WT mice, whereas there was no difference in the magnitude of the LTD induced by three short trains of low-frequency stimulation (LFS) (200 pulses at 1 Hz) at 20 min intervals between the two groups of mice. The reduction in the LTP produced by delivering three trains of LFS (200 pulses at 1 Hz, 20 min intervals) was significantly greater in the TG mice than in the WT mice. Learning was impaired in the four-pellet taking test (4PTT) in TG mice, with no significant difference in daily activity or activity during the 4PTT between TG and WT mice. These results suggest that the overexpression of ß1,4 GalNAc-T resulted in altered synaptic plasticity of LTP and DP in hippocampal CA1 neurons and learning in the 4PTT, and this is attributable to the shift from b-pathway gangliosides to a-pathway gangliosides.

Inositol 1,4,5-trisphosphate receptor-mediated calcium release in Purkinje cells: from molecular mechanism to behavior.

The inositol 1,4,5-trisphosphate (IP(3)) receptor is highly expressed in cerebellar Purkinje cells and mediates conspicuous calcium release from intracellular calcium stores. Receptor stimulation, such as through mGluR1, activates the G(q)-PLC pathway, which leads to IP(3)-induced calcium release and subsequent cellular responses, including cerebellar long-term depression in Purkinje cells. Recent studies have demonstrated the regulatory mechanisms of IP(3) receptor, revealing activation via IP(3) and Ca(2+), inactivation via high concentrations of Ca(2+), and modulation by various proteins that bind to the IP(3) receptor. Novel calcium imaging techniques and caged compounds provide analysis of calcium signals at the single spine level in relation to the induction of long-term depression. Genetically encoded indicators for calcium or IP(3) could provide alternate Ca(2+) or IP(3) imaging, in particular, for in vivo observations. IP(3)-induced calcium release participates in early development of dendritic branch formation, and loss-of-function mutations or hyper-activation could result various diseases. The IP(3) receptor plays a central role in calcium signaling in Purkinje cells, affecting a wide variety of cellular functions, including development, plasticity, maintenance of synaptic functions, and cerebellar motor control.

Role of inositol 1, 4, 5-trisphosphate receptors in the postsynaptic expression of guinea pig hippocampal mossy fiber depotentiation.

Long-term potentiation (LTP) at hippocampal mossy fiber-CA3 pyramidal neuron synapses was induced in the field excitatory postsynaptic potential (EPSP) by the delivery of HFS (a tetanus of two trains of 100 pulses at 100 Hz with a 10s interval) and was reversed (depotentiated) by a train of LFS of 1000 pulses at 2 Hz applied 60 min later. This depotentiation was triggered by activation of inositol 1, 4, 5-trisphosphate receptors (IP3Rs) during HFS, which may increase the postsynaptic intracellular Ca(2+) concentration, leading to a cellular process responsible for modification of LTP expression at mossy fiber-CA3 synapses. Furthermore, we found that activation of IP3Rs or protein phosphatase during LFS was required for the reversal of LTP expressed at mossy fiber-CA3 synapses. These results suggest that, in hippocampal mossy fiber-CA3 neuron synapses, activation of IP3Rs by a preconditioning HFS results in modulation of IP3R activation and/or postsynaptic protein phosphorylation during a subsequent LFS, leading to a decrease in the field EPSP and the erasure of LTP.

Synthesis of bisboron compounds and their strong inhibitory activity on store-operated calcium entry.

Store-operated calcium entry (SOCE) is an important mechanism for replenishing intracellular calcium stores and for sustaining calcium signaling. We developed a method for synthesis of bisboron compounds that have two borinic acids or their esters in one molecule. These compounds are analogues of 2-APB, which is widely used as a membrane-permeable SOCE inhibitor. Further, we examined the effect of the newly synthesized bisboron compounds on SOCE in Jurkat T cells. All the bisboron compounds showed strong inhibitory activity on SOCE, with IC50 values of less than 1 microM, which were 20-45 times lower than observed with 2-APB.

Potent transglutaminase inhibitors, aryl beta-aminoethyl ketones.

Aryl beta-aminoethyl ketones were discovered as potent inhibitors of tissue transglutaminase. Heteroaryl-like thiophene groups and N-benzyl N-t-butyl aminoethyl group are critical to the strong inhibitory activity of aryl beta-aminoethyl ketones.

Two novel 2-aminoethyl diphenylborinate (2-APB) analogues differentially activate and inhibit store-operated Ca(2+) entry via STIM proteins.

Store-operated calcium entry (SOCE) or calcium release-activated calcium current (I(CRAC)) is a critical pathway to replenish intracellular calcium stores, and plays indispensable roles in cellular functions such as antigen-induced T lymphocyte activation. Despite the importance of I(CRAC) in cellular functions, lack of potent and specific inhibitor has limited the approaches to the function of I(CRAC) in native cells. 2-Aminoethyl diphenylborinate (2-APB) is a widely used SOCE/I(CRAC) inhibitor, while its effect is rather unspecific. In the attempt to develop more potent and selective compounds here we identified two structurally isomeric 2-APB analogues that are 100-fold more potent than 2-APB itself. One of the 2-APB analogues activates and inhibits endogenous SOCE depending on the concentration while the other only inhibits it. The 2-APB analogue inhibits store depletion-mediated STIM1 clustering as well as heterologously expressed CRAC current. Together with the observation that, unlike 2-APB, the analogue compounds failed to activate CRACM3/Orai3 current in the absence of STIM, our results suggest that inhibition and activation of SOCE/I(CRAC) by the 2-APB analogues is mediated by STIM.

Short-term potentiation at the parallel fiber-Purkinje cell synapse.

Changes in synaptic efficacy at the parallel fiber (PF)-Purkinje cell (PC) synapse are postulated to be a cellular basis for motor learning. Although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (STP) lasting for tens of minutes, have not been discussed to date. Here we report that this synapse shows an apparent STP reliably by a periodic burst pattern of homosynaptic stimulation. This STP is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic Ca(2+) reduction by BAPTA injection or in P/Q-type Ca channel knockout cerebella. This novel type of synaptic plasticity at the PF-PC synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse.