EFA6A, an Exchange Factor for Arf6, Regulates NGF-Dependent TrkA Recycling From Early Endosomes and Neurite Outgrowth in PC12 Cells.

Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling.

Methiothepin downregulates SNAP-23 and inhibits degranulation of rat basophilic leukemia cells and mouse bone marrow-derived mast cells.

In the present study, we found that methiothepin (a nonselective 5-hydroxytryptamine [5-HT] receptor antagonist) inhibited antigen-induced degranulation in rat basophilic leukemia cells and mouse bone marrow-derived mast cells. Although antigen stimulation induces release of histamine and serotonin (5-HT) by exocytosis and mast cells express several types of 5-HT receptor, the detailed role of these receptors remains unclear. Here, pretreatment of cells with methiothepin attenuated increased intracellular Ca2+ concentration, phosphorylated critical upstream signaling components (Src family tyrosine kinases, Syk, and PLCγ1), and suppressed TNF-α secretion via inhibition of Akt (a Ser/Thr kinase activated by PI3K)and ERK phosphorylation. Furthermore, it inhibited PMA/ionomycin-induced degranulation; this finding suggested that methiothepin affected downstream signaling. IκB kinase ß phosphorylates synaptosomal associated protein 23, which regulates the fusion events of the secretory granule/plasma membrane after mast cell activation, resulting in degranulation. We showed that methiothepin blocked PMA/ionomycin-induced phosphorylation of synaptosomal associated protein 23 by inhibiting its interaction with IκB kinase ß. Together with the results of selective 5-HT antagonists, it is suggested that methiothepin inhibits mast cell degranulation by downregulating upstream signaling pathways and exocytotic fusion machinery through mainly 5-HT1A receptor. Our findings provide that 5-HT antagonists may be used to relieve allergic reactions.

Medermycin Inhibits TNFα-Promoted Inflammatory Reaction in Human Synovial Fibroblasts.

Synovial inflammation plays a crucial role in the destruction of joints and the experience of pain in osteoarthritis (OA). Emerging evidence suggests that certain antibiotic agents and their derivatives possess anti-inflammatory properties. Medermycin (MED) has been identified as a potent antibiotic, specifically active against Gram-positive bacteria. In this study, we aimed to investigate the impact of MED on TNFα-induced inflammatory reactions in a synovial cell line, SW-982, as well as primary human synovial fibroblasts (HSF) using RNA sequencing, rtRT-PCR, ELISA, and western blotting. Through the analysis of differentially expressed genes (DEGs), we identified a total of 1478 significantly upregulated genes in SW-982 cells stimulated with TNFα compared to the vehicle control. Among these upregulated genes, MED treatment led to a reduction in 1167 genes, including those encoding proinflammatory cytokines such as IL1B, IL6, and IL8. Pathway analysis revealed the enrichment of DEGs in the TNF and NFκB signaling pathway, further supporting the involvement of MED in modulating inflammatory responses. Subsequent experiments demonstrated that MED inhibited the expression of IL6 and IL8 at both the mRNA and protein levels in both SW982 cells and HSF. Additionally, MED treatment resulted in a reduction in p65 phosphorylation in both cell types, indicating its inhibitory effect on NFκB activation. Interestingly, MED also inhibited Akt phosphorylation in SW982 cells, but not in HSF. Overall, our findings suggest that MED suppresses TNFα-mediated inflammatory cytokine production and p65 phosphorylation. These results highlight the potential therapeutic value of MED in managing inflammatory conditions in OA. Further investigations utilizing articular chondrocytes and animal models of OA may provide valuable insights into the therapeutic potential of MED for this disease.

TNF-α Suppresses Apelin Receptor Expression in Mouse Quadriceps Femoris-Derived Cells.

Expression of the apelin receptor, APJ, in skeletal muscle (SM) is known to decrease with age, but the underlying mechanism remains unclear. Increased tumor necrosis factor (TNF)-α levels are observed in SM with age and are associated with muscle atrophy. To investigate the possible interconnection between TNF-α elevation and APJ reduction with aging, we investigated the effect of TNF-α on APJ expression in cells derived from the quadriceps femoris of C57BL/6J mice. Expression of Tnfa and Apj in the quadriceps femoris was compared between 4- (young) and 24-month-old (old) C57BL/6J mice (n = 10 each) using qPCR. Additionally, APJ-positive cells and TNF-α protein were analyzed by flow cytometry and Western blotting, respectively. Further, quadricep-derived cells were exposed to 0 (control) or 25 ng/mL TNF-α, and the effect on Apj expression was examined by qRT-PCR. Apj expression and the ratio of APJ-positive cells among quadricep cells were significantly lower in old compared to young mice. In contrast, levels of Tnfa mRNA and TNF-α protein were significantly elevated in old compared to young mice. Exposing young and old derived quadricep cells to TNF-α for 8 and 24 h caused Apj levels to significantly decrease. TNF-α suppresses APJ expression in muscle cells in vitro. The increase in TNF-α observed in SM with age may induce a decrease in APJ expression.

Rats deficient in the GAD65 isoform exhibit epilepsy and premature lethality.

GABA is synthesized by glutamate decarboxylase (GAD), which has two isoforms, namely, GAD65 and GAD67, encoded by the Gad2 and Gad1 genes, respectively. GAD65-deficient (Gad2-/- ) mice exhibit a reduction in brain GABA content after 1 month of age and show spontaneous seizures in adulthood. Approximately 25% of Gad2-/- mice died by 6 months of age. Our Western blot analysis demonstrated that the protein expression ratio of GAD65 to GAD67 in the brain was greater in rats than in mice during postnatal development, suggesting that the contribution of each GAD isoform to GABA functions differs between these two species. To evaluate whether GAD65 deficiency causes different phenotypes between rats and mice, we generated Gad2-/- rats using TALEN genome editing technology. Western blot and immunohistochemical analyses with new antibodies demonstrated that the GAD65 protein was undetectable in the Gad2-/- rat brain. Gad2-/- pups exhibited spontaneous seizures and paroxysmal discharge in EEG at postnatal weeks 3-4. More than 80% of the Gad2-/- rats died at postnatal days (PNDs) 17-23. GABA content in Gad2-/- brains was significantly lower than those in Gad2+/- and Gad2+/+ brains at PND17-19. These results suggest that the low levels of brain GABA content in Gad2-/- rats may lead to epilepsy followed by premature death, and that Gad2-/- rats are more severely affected than Gad2-/- mice. Considering that the GAD65/GAD67 ratio in human brains is more similar to that in rat brains than in mouse brains, Gad2-/- rats would be useful for further investigating the roles of GAD65 in vivo.

BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3.

Brefeldin A-resistant ArfGEF 2 (BRAG2) [or Iqsec1 (IQ motif and Sec7 domain-containing protein 1)] is a guanine nucleotide exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes. BRAG2 regulates Arf6-dependent endocytosis of AMPA receptors (AMPARs) through the direct interaction during the hippocampal long-term depression. However, the molecular mechanism by which the BRAG2-Arf6 pathway links AMPARs to the endocytic machinery remains elusive. Herein, using mouse brains of both sexes, we demonstrated that BRAG2a, an alternative isoform with a long C-terminal insert containing a proline-rich domain and type I PDZ-binding motif, was selectively localized to the excitatory postsynaptic density (PSD). Using yeast two-hybrid screening, we identified PSD-95 and endophilin 1/3 as BRAG2a-binding partners in the brain. The interaction with PSD-95 was required for synaptic targeting of BRAG2a. In cultured hippocampal neurons, stimulation of group I metabotropic glutamate receptors (mGluRs) increased the interaction of BRAG2a with endophilin 3 and concomitant Arf6 activation in a time-dependent manner. Knockdown of BRAG2 in cultured hippocampal neurons blocked the mGluR-dependent decrease in surface AMPAR levels, which was rescued by introducing wild-type BRAG2a, but not wild-type BRAG2b or BRAG2a mutants lacking the ability to activate Arf6 or to interact with endophilin 3 or PSD-95. Further postembedding immunoelectron microscopic analysis revealed the preorganized lateral distribution of BRAG2a, Arf6, and endophilin 3 for efficient endocytosis at the postsynaptic membrane. Together, the present findings unveiled a novel molecular mechanism by which BRAG2a links AMPARs to the clathrin-dependent endocytic pathway through its interaction with PSD-95 and endophilin 3.SIGNIFICANCE STATEMENT BRAG2/Iqsec1 is a GDP/GTP exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes, and regulates Arf6-dependent endocytosis of AMPARs through direct interaction during hippocampal long-term depression, one of the mechanisms of synaptic plasticity related to learning and memory. However, the molecular mechanism by which the BRAG2-Arf6 pathway links AMPARs to the endocytic machinery remains elusive. Here, we identified isoform-specific mechanisms of BRAG2-mediated AMPAR internalization. We demonstrated that the interaction of BRAG2a isoform with PSD-95 and endophilin 3 was required for the mGluR-dependent decrease in surface AMPARs in hippocampal neurons. These results unveiled a novel molecular mechanism by which BRAG2 links AMPARs to the clathrin-mediated endocytic machinery at postsynaptic sites.

Highly accurate and precise quantification strategy using stable isotope dimethyl labeling coupled with GeLC-MS/MS.

Shotgun proteomics is a powerful method for comprehensively identifying and quantifying tryptic peptides, but it is difficult to analyze proteolytic events. One-dimensional gel and liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) enables the separation of proteolytic fragments using SDS-PAGE followed by identification using LC-MS/MS. GeLC-MS/MS is thus an excellent method for identifying fragmentation. However, the lower reproducibility of gel extraction and nano flow LC-MS/MS can produce inaccurate results in comparative analyses of protein quantification among samples. In this study, a novel GeLC-MS/MS method coupled with stable isotope dimethyl labeling was developed. In the method, a mixture of light- and heavy-labeled samples is loaded onto an SDS-PAGE gel, and proteins with different isotopes in one extracted band are quantitatively analyzed by one-shot injection. This procedure enables accurate determination of the abundance ratio of peptides between two samples, even in cases of low peptide abundance, and it is not affected by the reproducibility of the gel extraction or LC-MS procedures. Therefore, our new GeLC-MS/MS method coupled with stable isotope dimethyl labeling provides high accuracy and comprehensive peptide comparisons, enabling the detection of proteolysis events caused by disease or physiological processes.

A highly efficient method for extracting peptides from a single mouse hypothalamus.

Living organisms contain a variety of endogenous peptides that function as significant regulators of many biological processes. Endogenous peptides are typically analyzed using liquid chromatography-mass spectrometry (LC-MS). However, due to the low efficiency of peptide extraction and low abundance of peptides in a single animal, LC-MS-based peptidomics studies have not facilitated an understanding of the individual differences and tissue specificity of peptide abundance. In this study, we developed a peptide extraction method followed by nano-flow LC-MS/MS analysis. This method enabled highly efficient and reproducible peptide extraction from sub-milligram quantities of hypothalamus dissected from a single animal. Diverse bioactive and authentic peptides were detected from a sample volume equivalent to 135 µg of hypothalamus. This method may be useful for elucidating individual differences and tissue specificity, as well as for facilitating the discovery of novel bioactive peptides and biomarkers and developing peptide therapeutics.

TGF-ß regulates nerve growth factor expression in a mouse intervertebral disc injury model.

BACKGROUND: Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). Following disc injury, nerve growth factor (NGF) concentrations rise in IVDs, and anti-NGF therapy has been shown to attenuate LBP in humans. Increased levels of tumor necrosis factor-α (TNF-α) and transforming growth factor-ß (TGF-ß) in degenerative IVDs and in in vitro studies suggest that these factors promote NGF production. However, whether these factors regulate NGF in vivo remains unclear. Thus, we studied NGF regulation in a mouse model of IVD injury. METHODS: After inducing IVD injury, we examined mRNA levels of Tnfa, Tgfb, and Ngf in IVDs from control and IVD-injured mice across 7 days. To do this, we used magnetic cell separation to isolate CD11b ( +) (macrophage-rich) and CD11b (-) (IVD cell-rich) cell fractions from injured IVDs. To study the effect of TNF-α on Ngf expression, we examined Ngf expression in injured IVDs from C57BL/6 J and Tnfa-knockout (KO) mice (C57BL/6 J background). To study the effect of TGF-ß on Ngf expression, C57/BL6J mice were given an intraperitoneal injection of either the TGF-ß inhibitor SB431542 or DMSO solution (vehicle) one and two days before harvesting IVDs. RESULTS: mRNA expression of Tnfa, Tgfb, and Ngf was significantly increased in injured IVDs. Tnfa was predominantly expressed in the CD11b ( +) fraction, and Tgfb in the CD11b (-) fraction. Ngf expression was comparable between CD11b ( +) and CD11b (-) fractions, and between wild-type and Tnfa-KO mice at post-injury day (PID) 1, 3, and 7. SB431542 suppressed TGF-ß-mediated Ngf expression and NGF production in vitro. Further, administration of SB431542 significantly reduced Ngf expression in IVDs such that levels were below those observed in vehicle-treated animals at PID3 and PID7. CONCLUSION: A TGF-ß inhibitor reduced Ngf expression in a mouse model of IVD injury, suggesting that TGF-ß may regulate NGF expression in vivo.

Transforming growth factor-ß stimulates nerve growth factor production in osteoarthritic synovium.

BACKGROUND: Nerve growth factor (NGF) contributes to pain in knee osteoarthritis (KOA) patients. Transforming growth factor-beta (TGF-ß) stimulates NGF expression in chondrocytes from KOA patients. However, the correlation between synovial TGF-ß and NGF levels has not been sufficiently studied in human KOA patients. Further, the mechanism governing NGF regulation by TGF-ß in synovial cells is unclear. METHODS: During total knee arthroplasty, we extracted the synovial tissue (SYT) of 107 subjects with unilateral Kellgren/Lawrence grade 3-4 KOA confirmed by radiography. We examined the distribution of TGF-ß and NGF using immunohistochemistry, and analyzed the relationship between NGF and TGFB mRNA levels. Cultured synovial cells extracted from SYT were exposed to culture medium (control), human recombinant TGF-ß (rhTGF-ß), rhTGF-ß + ALK5 inhibitor SB505124, rhTGF-ß + transforming growth factor activating kinase 1 (TAK1) inhibitor (5Z)-7-oxozeaenol, or rhTGF-ß + p38 inhibitor SB203580 for 30 min, 6 h and 24 h. NGF mRNA expressed by the cultured cells and NGF protein levels in the cell supernatant were detected by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Phosphorylation of p38 was evaluated by western blotting. RESULTS: NGF mRNA levels were positively correlated with those of TGFB. Cells expressing TGF-ß and NGF protein were observed in the lining layer of SYT. TGF-ß stimulated increased NGF mRNA expression and NGF protein production. The ALK5 inhibitor completely suppressed the TGF-ß-mediated increase in NGF expression and NGF production in synovial cells. ALK5, TAK1 and p38 inhibitors inhibited the TGF-ß-induced phosphorylation of p38, and TAK1 and p38 inhibitors partially inhibited the TGF-ß-mediated increase in NGF expression and NGF production in synovial cells. CONCLUSION: TGF-ß regulates NGF production via the TGF-ß/ALK5 signaling pathway in osteoarthritic synovium. This effect may partially occur through inhibition of the TAK1/p38 pathway in the SYT of KOA patients.

Transforming growth factor activating kinase 1 regulates extracellular matrix degrading enzymes and pain-related molecule expression following tumor necrosis factor-α stimulation of synovial cells: an in vitro study.

BACKGROUND: Recent studies have suggested that the tumor necrosis factor-α (TNF-α) pathway is a potential target for the management of osteoarthritis (OA). Transforming growth factor (TGF)-ß-activated kinase 1 (TAK1) is essential in several cytokine-mediated cascades, including the TNF-α, interleukin-1 (IL-1), and TGF-ß pathways. The role of TAK1 in synovial tissue in OA is not fully understood. Using synovial cells harvested from OA patients during surgery, we investigated whether TAK1 inhibition suppresses production of TNF-α-induced extracellular matrix degrading enzymes and expression of pain-related molecules. METHODS: Synovial tissues were harvested from ten subjects with radiographic evidence of osteoarthritis (OA) during total knee arthroplasty. Synovial cells were cultured and stimulated with control (culture media), 10 ng/mL human recombinant TNF-α, or 10 ng/mL TNF-α and 10 µM of the TAK1 inhibitor (5Z)-7-oxozeaenol for 24 h. Real-time polymerase chain reaction (PCR) analysis was used to monitor expression of mRNA of the extracellular matrix degrading enzymes matrix metalloproteinase-3 (MMP-3) and a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 4 (ADAMTS-4); and of the pain-related molecules cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), and nerve growth factor (NGF). MMP-3 and NGF protein concentrations in cell supernatant were measured by enzyme-linked immunosorbent assay (ELISA). COX-2, mPGES-1 and ADAMTS-4 protein expression was also evaluated by western blotting. RESULTS: TNF-α stimulated increases in ADAMTS-4 and MMP3 mRNA (2.0-fold and 1.6-fold, respectively, p < 0.05) and protein expression (21.5-fold and 2.0-fold, respectively). Treatment with the TAK1 inihibitor (5Z)-7-oxozeaenol reduced ADAMTS-4 and MMP3 mRNA (0.5-fold and 0.6-fold, respectively) and protein expression (1.4-fold and 0.5-fold, respectively) in OA synovial cells. COX-2, mPGES-1 and NGF mRNA (11.2-fold, 3.1-fold and 2.7-fold, respectively) and protein expression (3.0-fold, 2.7-fold and 2.2-fold, respectively) were increased by TNF-α. (5Z)-7-oxozeaenol treatment reduced mPGES1 and NGF mRNA (1.5-fold and 0.8-fold, respectively) and protein (1.5-fold and 0.5-fold, respectively). CONCLUSION: TAK1 plays an important role in the regulation of TNF-α induced extracellular matrix degrading enzymes and pain-related molecule expression. TAK1 may be a potential target for therapeutic strategies aimed at preventing osteoarthritis progression and pain.

Acceleration of Acid-Catalyzed Hydrolysis in a Biphasic System by Sodium Tetracyanocyclopentadienides.

The hydrolysis of tert-butyldimethylsilyl L-menthyl ether (3) in a CH2Cl2-1 M HCl biphasic solvent system was accelerated by the addition of sodium tetracyanocyclopentadienides 1. Particularly, the reaction rate was enhanced using sodium salt 1a-c with a lipophilic substituent on the cyclopentadienide ring. From the results obtained by a triphasic experiment, hydrolysis proceeds via the formation of hydronium ion 2 in the aqueous phase by ion exchange, followed by the transfer of 2 to the CH2Cl2 phase.

Accumulation of α-synuclein triggered by presynaptic dysfunction.

Pathological examination of dementia with Lewy bodies patients identified the presence of abnormal α-synuclein (αSyn) aggregates in the presynaptic terminals. αSyn is involved in the regulation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Importantly, αSyn-transgenic mouse and postmortem examination of patients with Parkinson's disease have demonstrated the abnormal distribution of SNARE protein in presynaptic terminals. In this study, we investigated the effects of SNARE dysfunction on endogenous αSyn using Snap25(S187A/S187A) mutant mice. These mice have homozygous knock-in gene encoding unphosphorylatable S187A-substituted synaptosomal-associated protein of 25 kDa (SNAP-25). The mice displayed a significant age-dependent change in the distribution of αSyn and its Ser(129)-phosphorylated form in abnormally hypertrophied glutamatergic nerve terminals in the striatum. Electron-microscopic analysis revealed the abnormally condensed synaptic vesicles with concomitant mislocalization of αSyn protein to the periactive zone in the glutamatergic nerve terminals. However, the Snap25(S187A/S187A) mutant mouse harbored no abnormalities in the nigrostriatal dopaminergic neurons. Our present results suggest that SNARE dysfunction is the initial trigger of mislocalization and accumulation of αSyn, and probably is an important pathomechanism of α-synucleinopathies.

Hippocampal CA3 and CA2 have distinct bilateral innervation patterns to CA1 in rodents.

Ipsilateral and contralateral hippocampal CA3-CA1 and CA2-CA1 projections were investigated in adult male Long-Evans rats by retrograde tracing. Injection of the retrograde tracer cholera toxin subunit B in the strata oriens and radiatum of dorsal CA1 resulted in labeling of predominantly pyramidal cells in ipsilateral and contralateral CA3 and CA2. The contralateral and ipsilateral anterior-posterior extents of CA3 innervation to CA1 were similar. Fifteen to twenty per cent of the hippocampus proper cells that give rise to CA1 stratum oriens innervation were CA2 pyramidal cells, whereas CA2 cells were a mere 3% for CA1 stratum radiatum innervation. The preferred projection of CA2 pyramidal cells to the CA1 stratum oriens was also manifested in transgenic mice that express GFP under the control of the CACNG5 promoter, in which CA2 cells express high amounts of GFP. The ratios of ipsilateral to contralateral projections were compared. For the CA3-CA1 connection, we found that dorsal CA1 stratum radiatum received more ipsilateral projections whereas CA1 stratum oriens received more contralateral innervation. Interestingly, ipsilateral connections dominated for both CA2-CA1 stratum oriens and CA2-CA1 stratum radiatum. These results demonstrate that the primary intrahippocampal target of CA2 pyramidal cells is the ipsilateral CA1 stratum oriens, in contrast to CA3 cells which project more diversely to bilateral CA1 regions. Such innervation patterns may suggest differential dendritic information processing in apical and basal dendrites of CA1 pyramidal cells.

D1 Receptor Mediated Dopaminergic Neurotransmission Facilitates Remote Memory of Contextual Fear Conditioning.

Dopaminergic neurotransmission via dopamine D1 receptors (D1Rs) is considered to play an important role not only in reward-based learning but also in aversive learning. The contextual and auditory cued fear conditioning tests involve the processing of classical fear conditioning and evaluates aversive learning memory. It is possible to evaluate aversive learning memory in two different types of neural transmission circuits. In addition, when evaluating the role of dopaminergic neurotransmission via D1R, to avoid the effects in D1R-mediated neural circuitry alterations during development, it is important to examine using mice who D1R expression in the mature stage is suppressed. Herein, we investigated the role of dopaminergic neurotransmission via D1Rs in aversive memory formation in contextual and auditory cued fear conditioning tests using D1R knockdown (KD) mice, in which the expression of D1Rs could be conditionally and reversibly controlled with doxycycline (Dox) treatment. For aversive memory, we examined memory formation using recent memory 1 day after conditioning, and remote memory 4 weeks after conditioning. Furthermore, immunostaining of the brain tissues of D1RKD mice was performed after aversive footshock stimulation to investigate the distribution of activated c-Fos, an immediate-early gene, in the hippocampus (CA1, CA3, dentate gyrus), striatum, amygdala, and prefrontal cortex during aversive memory formation. After aversive footshock stimulation, immunoblotting was performed using hippocampal, striatal, and amygdalar samples from D1RKD mice to investigate the increase in the amount of c-Fos and phosphorylated SNAP-25 at Ser187 residue. When D1R expression was suppressed using Dox, behavioral experiments revealed impaired contextual fear learning in remote aversion memory following footshock stimulation. Furthermore, expression analysis showed a slight increase in the post-stimulation amount of c-Fos in the hippocampus and striatum, and a significant increase in the amount of phosphorylated SNAP-25 in the hippocampus, striatum, and prefrontal cortex before and after stimulation. These findings indicate that deficiency in D1R-mediated dopaminergic neurotransmission is an important factor in impairing contextual fear memory formation for remote memory.

ANGT_HUMAN[448-462], an Anorexigenic Peptide Identified Using Plasma Peptidomics.

The discovery of bioactive peptides is an important research target that enables the elucidation of the pathophysiology of human diseases and provides seeds for drug discovery. Using a large number of native peptides previously identified using plasma peptidomics technology, we sequentially synthesized selected sequences and subjected them to functional screening using human cultured cells. A 15-amino-acid residue proangiotensinogen-derived peptide, designated ANGT_HUMAN[448-462], elicited cellular responses and bound to cultured human cells. Synthetic fluorescent-labeled and biotinylated ANGT_HUMAN[448-462] peptides were rendered to bind to cell- and tissue-derived proteins and peptide-cell protein complexes were retrieved and analyzed using liquid chromatography-tandem mass spectrometry, revealing the ß-subunit of ATP synthase as its cell-surface binding protein. Because ATP synthase mediates the effects of anorexigenic peptides, the ability of ANGT_HUMAN[448-462] to modulate eating behavior in mice was investigated. Both intraperitoneal and intracerebroventricular injections of low doses of ANGT_HUMAN[448-462] suppressed spontaneous food and water intake throughout the dark phase of the diurnal cycle without affecting locomotor activity. Immunoreactive ANGT_HUMAN[448-462], distributed throughout human tissues and in human-derived cells, is mostly co-localized with angiotensin II and is occasionally present separately from angiotensin II. In this study, an anorexigenic peptide, ANGT_HUMAN[448-462], was identified by exploring cell surface target proteins of the human native peptides identified using plasma peptidomics.

Origin of M2 Mϕ and its macrophage polarization by TGF-ß in a mice intervertebral injury model.

INTRODUCTION: Studies have identified the presence of M1 and M2 macrophages (Mϕ) in injured intervertebral discs (IVDs). However, the origin and polarization-regulatory factor of M2 Mϕ are not fully understood. TGF-ß is a regulatory factor for M2 polarization in several tissues. Here, we investigated the source of M2 Mϕ and the role of TGF-ß on M2 polarization using a mice disc-puncture injury model. METHODS: To investigate the origin of M2 macrophages, 30 GFP chimeric mice were created by bone marrow transplantation. IVDs were obtained from both groups on pre-puncture (control) and post-puncture days 1, 3, 7, and 14 and CD86 (M1 marker)- and CD206 (M2 marker)-positive cells evaluated by flow cytometry (n = 5 at each time point). To investigate the role of TGF-ß on M2 polarization, TGF-ß inhibitor (SB431542) was also injected on post-puncture days (PPD) 5 and 6 and CD206 expression was evaluated on day 7 by flow cytometry (n = 5) and real time PCR (n = 10). RESULTS: The proportion of CD86+ Mϕ within the GFP+ population was significantly increased at PPD 1, 3, 7, and 14 compared to control. CD206-positive cells in GFP-populations were significantly increased on PPD 7 and 14. In addition, the percentage of CD206-positive cells was significantly higher in GFP-populations than in GFP+ populations. TGF-ß inhibitor reduced CD206-positive cells and Cd206 expression at 7 days after puncture. CONCLUSION: Our findings suggest that M2 Mϕ following IVD injury may originate from resident Mϕ. TGF-ß is a key factor for M2 polarization of macrophages following IVD injury.

Importance of the Q/N-rich segment for protein stability of endogenous mouse TDP-43.

TAR DNA-binding protein 43 kDa (TDP-43), a nuclear protein, plays an important role in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS). The long-disordered C-terminal region (CTR) of TDP-43 is known to be aggregation-prone and a hotspot for ALS mutations, so elucidation of the physiological function of CTR will provide insights into the pathogenesis of ALS. The CTR has two Gly, aromatic, and Ser-rich (GaroS) segments and an amyloidogenic core divided into a hydrophobic patch (HP) and a Gln/Asn (Q/N)-rich segment. Although TDP-43 lacking the CTR is known to be unstable, as observed in knock-in mice, it is unclear which of these segments contributes to the stability of TDP-43. Here, we generated 12 mouse lines lacking the various sub-regions of CTR by genome editing and compared the embryonic lethality of homozygotes, and protein and mRNA expression levels of TDP-43. We demonstrated the functional diversity of the four segments of CTR, finding that the presence of the Q/N-rich segment greatly restored the protein stability of TDP-43. In addition, we found that the second GaroS deletion did not affect protein stability and mouse development.

Jietacin Derivative Inhibits TNF-α-Mediated Inflammatory Cytokines Production via Suppression of the NF-κB Pathway in Synovial Cells.

Synovial inflammation plays a central role in joint destruction and pain in osteoarthritis (OA). The NF-κB pathway plays an important role in the inflammatory process and is activated in OA. A previous study reported that a jietacin derivative (JD), (Z)-2-(8-oxodec-9-yn-1-yl)-1-vinyldiazene 1-oxide, suppressed the nuclear translocation of NF-κB in a range of cancer cell lines. However, the effect of JD in synovial cells and the exact mechanism of JD as an NF-κB inhibitor remain to be determined. We investigated the effect of JD on TNF-α-induced inflammatory reaction in a synovial cell line, SW982 and human primary synovial fibroblasts (hPSFs). Additionally, we examined phosphorylated levels of p65 and p38 and expression of importin α3 and ß1 using Western blotting. RNA-Seq analysis revealed that JD suppressed TNF-α-induced differential expression: among 204 genes significantly differentially expressed between vehicle and TNF-α-stimulated SW982 (183 upregulated and 21 downregulated) (FC ≥ 2, Q < 0.05), expression of 130 upregulated genes, including inflammatory cytokines (IL1A, IL1B, IL6, IL8) and chemokines (CCL2, CCL3, CCL5, CCL20, CXCL9, 10, 11), was decreased by JD treatment and that of 14 downregulated genes was increased. KEGG pathway analysis showed that DEGs were increased in the cytokine−cytokine receptor interaction, TNF signaling pathway, NF-κB signaling pathway, and rheumatoid arthritis. JD inhibited IL1B, IL6 and IL8 mRNA expression and IL-6 and IL-8 protein production in both SW982 and hPSFs. JD also suppressed p65 phosphorylation in both SW982 and hPSFs. In contrast, JD did not alter p38 phosphorylation. JD may inhibit TNF-α-mediated inflammatory cytokine production via suppression of p65 phosphorylation in both SW982 and hPSFs. Our results suggest that JD may have therapeutic potential for OA due to its anti-inflammatory action through selective suppression of the NF-κB pathway on synovial cells.