Intraperitoneal Administration of Etizolam Improves Locomotor Function in Mice After Spinal Cord Injury.

Neuroinflammation occurs in the acute phase of spinal cord injury (SCI) and inhibits neural regeneration. In mouse models, etizolam (ETZ) is a strong anxiolytic with unclear effects on SCI. This study investigated the effects of short-term administration of ETZ on neuroinflammation and behavior in mice after SCI. We administrated an ETZ (0.5 mg/kg) daily intraperitoneal injection from the day after SCI for 7 days. Mice were randomly divided into three groups (sham group: only laminectomy, saline group, and ETZ group). Inflammatory cytokine concentrations in the injured spinal cord epicenter were measured using an enzyme-linked immunosorbent assay on day 7 after SCI to evaluate spinal cord inflammation in the acute phase. Behavior analysis was performed the day before surgery and on days 7, 14, 28, and 42 after surgery. The behavioral analysis included anxiety-like behavior using the open field test, locomotor function using the Basso Mouse Scale, and sensory function using the mechanical and heat test. Inflammatory cytokine concentrations were significantly lower in the ETZ group than in the saline group in the acute phase after spinal surgery. After SCI, anxiety-like behaviors and sensory functions were comparable between the ETZ and saline groups. ETZ administration reduced neuroinflammation in the spinal cord and improved locomotor function. Gamma-amino butyric acid type A receptor stimulants may be effective therapeutic agents for patients with SCI.

Pathological substrate of memory impairment in multiple system atrophy.

AIMS: Synaptic dysfunction in Parkinson's disease is caused by propagation of pathogenic α-synuclein between neurons. Previously, in multiple system atrophy (MSA), pathologically characterised by ectopic deposition of abnormal α-synuclein predominantly in oligodendrocytes, we demonstrated that the occurrence of memory impairment was associated with the number of α-synuclein-positive neuronal cytoplasmic inclusions (NCIs) in the hippocampus. In the present study, we aimed to investigate how abnormal α-synuclein in the hippocampus can lead to memory impairment. METHODS: We performed pathological and biochemical analyses using a mouse model of adult-onset MSA and human cases (MSA, N = 25; Parkinson's disease, N = 3; Alzheimer's disease, N = 2; normal controls, N = 11). In addition, the MSA model mice were examined behaviourally and physiologically. RESULTS: In the MSA model, inducible human α-synuclein was first expressed in oligodendrocytes and subsequently accumulated in the cytoplasm of excitatory hippocampal neurons (NCI-like structures) and their presynaptic nerve terminals with the development of memory impairment. α-Synuclein oligomers increased simultaneously in the hippocampus of the MSA model. Hippocampal dendritic spines also decreased in number, followed by suppression of long-term potentiation. Consistent with these findings obtained in the MSA model, post-mortem analysis of human MSA brain tissues showed that cases of MSA with memory impairment developed more NCIs in excitatory hippocampal neurons along with α-synuclein oligomers than those without. CONCLUSIONS: Our results provide new insights into the role of α-synuclein oligomers as a possible pathological cause of memory impairment in MSA.

The role of neutrophil gelatinase-associated lipocalin and iron homeostasis in object recognition impairment in aged sepsis-survivor rats.

Older adult patients with sepsis frequently experience cognitive impairment. The roles of brain neutrophil gelatinase-associated lipocalin (NGAL) and iron in older sepsis patients remain unknown. We investigated the effects of lipopolysaccharide-induced sepsis on novel object recognition test, NGAL levels, an inflammatory mediator tumor necrosis factor-α (TNFα) levels, and iron ion levels in the hippocampus and cortex of young and aged rats. The effect of an iron chelator deferoxamine pretreatment on aged sepsis rats was also examined. Young sepsis-survivor rats did not show impaired novel object recognition, TNFα responses, or a Fe2+/Fe3+ imbalance. They showed hippocampal and cortical NGAL level elevations. Aged sepsis-survivor rats displayed a decreased object discrimination index, elevation of NGAL levels and Fe2+/Fe3+ ratio, and no TNFα responses. Pretreatment with deferoxamine prevented the reduction in the object recognition of aged sepsis-survivor rats. The elevation in hippocampal and cortical NGAL levels caused by lipopolysaccharide was not influenced by deferoxamine pretreatment. The lipopolysaccharide-induced Fe2+/Fe3+ ratio elevation was blocked by deferoxamine pretreatment. In conclusion, our findings suggest that iron homeostasis in the cortex and hippocampus contributes to the maintenance of object recognition ability in older sepsis survivors.

Anti-hypersensitive effect of angiotensin (1-7) on streptozotocin-induced diabetic neuropathic pain in mice.

BACKGROUND: We have recently reported that the spinal angiotensin (Ang) converting enzyme (ACE)/Ang II/AT1 receptor axis and downstream p38 MAPK phosphorylation are activated in streptozotocin (STZ)-induced diabetic mice and lead to tactile hypersensitivity. Moreover, our previous results suggested that the intrathecal (i.t.) administration of Ang (1-7), an N-terminal fragment of Ang II, may attenuate the Ang II-induced nociceptive behaviour through the inhibition of p38 MAPK phosphorylation via Mas receptors. Here, we investigated whether the i.t. administration of Ang (1-7) can attenuate STZ-induced diabetic neuropathic pain. METHODS: Tactile and thermal hypersensitivities were determined using the von Frey filament and Hargreaves tests, respectively. The protein expression of ACE2, Mas receptors and phospho-p38 MAPK was measured by western blotting. Spinal ACE2 activity was determined using ACE2 activity assay kit. RESULTS: The i.t. administration of Ang (1-7) significantly reduced the tactile and thermal hypersensitivities on day 14 after STZ injection, and these effects were significantly prevented by the Mas receptor antagonist A779. The expression of ACE2 and Mas receptors in the plasma membrane fraction of the lumbar dorsal spinal cord was both significantly decreased in STZ mice. Spinal ACE2 activity was also decreased while p38 MAPK phosphorylation was increased in the lumbar dorsal region of these mice. This phosphorylation was attenuated by the injection of Ang (1-7), whose effect was reversed by A779. CONCLUSIONS: Our data demonstrate that Ang (1-7) attenuates STZ-induced diabetic neuropathic pain and that this occurs through a mechanism involving spinal Mas receptors and he inhibition of p38 MAPK phosphorylation. SIGNIFICANCE: The ACE2/Ang (1-7)/Mas receptor axis was down-regulated in the spinal cord of STZ mice and the i.t. administration of Ang (1-7) attenuated the STZ-induced diabetic neuropathic pain via Mas receptors. Therefore, the activation of this axis could be an effective therapeutic target to alleviate the neuropathic pain in diabetic patients.

HSP90 Regulation of P2X7 Receptor Function Requires an Intact Cytoplasmic C-Terminus.

P2X7 receptors (P2X7Rs) are ATP-gated ion channels that display the unusual property of current facilitation during long applications of agonists. Here we show that facilitation disappears in chimeric P2X7Rs containing the C-terminus of the P2X2 receptor (P2X2R), and in a truncated P2X7R missing the cysteine-rich domain of the C-terminus. The chimeric and truncated receptors also show an apparent decreased permeability to N-methyl-d-glucamine(+) (NMDG(+)). The effects of genetic modification of the C-terminus on NMDG(+) permeability were mimicked by preapplication of the HSP90 antagonist geldanamycin to the wild-type receptor. Further, the geldanamycin decreased the shift in the reversal potential of the ATP-gated current measured under bi-ionic NMDG(+)/Na(+) condition without affecting the ability of the long application of agonist to facilitate current amplitude. Taken together, the results suggest that HSP90 may be essential for stabilization and function of P2X7Rs through an action on the cysteine-rich domain of the cytoplasmic the C-terminus.

The mycotoxin patulin decreases expression of density-enhanced phosphatase-1 by down-regulating PPARγ in human colon cancer cells.

Patulin is a mycotoxin that is found mainly in apple products and causes symptoms such as bleeding from the digestive tract and diarrhea. Efforts to elucidate the mechanism of its toxicity have focused on protein tyrosine phosphatases (PTPs), which regulate the function of tight junctions (TJs) in colon epithelial cells. Patulin reacts with the conserved cysteine residues in the catalytic domains of PTP isoforms. Treatment of Caco-2 human colon cancer cells, used as a colon epithelial model, with 50 µM patulin decreased the level of density-enhanced phosphatase-1 (DEP-1) protein to 30% of the control level after 6 h. The level of DEP-1 mRNA was also decreased during 24 h after treatment with patulin. Moreover, knockdown of DEP-1 increased the level of phosphorylated claudin-4. Destruction of TJs by patulin treatment was observed by immunostaining with an antibody against zonula occludens (ZO)-1. To better understand the mechanistic basis of the decrease in DEP-1 mRNA levels, we searched for a cis-element upstream of the DEP-1 gene and found an element responsive to the peroxisome proliferator-activated receptor gamma (PPARγ) protein. Using a PPARγ-specific antibody, we showed a decrease in PPARγ abundance to 42% of the control level within 6 h after treatment with patulin. PPARγ has four cysteine residues that are involved in zinc finger formation. Our data suggest that DEP-1 affects TJ function and that PPARγ might control DEP-1 expression. Therefore, the toxicity of patulin to cellular functions might be attributable to its ability to down-regulate the expression of DEP-1 and PPARγ.

Dephosphorylation of Sp1 at Ser-59 by protein phosphatase 2A (PP2A) is required for induction of CYP1A1 transcription after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin or omeprazole.

The aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or omeprazole (OP). Activated AhR can induce CYP1A1 transcription by binding to the xenobiotic responsive element (XRE). However, the mechanism of activation of the CYP1A1 promoter region is poorly understood. Previous reports showed that Sp1 could bind to a GC-rich region near the CYP1A1 promoter. This study sought to clarify the function of Sp1 in CYP1A1 transcription. Phosphorylation of Sp1 at Ser-59 (pSer-59) was previously reported to be closely related to transcriptional regulation. We used a site-specific phospho-antibody to show that treatment with TCDD or OP drastically reduced the level of pSer-59 in Sp1 from HepG2 cells. This reduction was too much, we hypothesized that the reduced phosphorylation level resulted from activation of phosphatase activity. Given that pSer-59 is dephosphorylated by PP2A, we examined the effect of a PP2A inhibitor, okadaic acid (OA), on pSer-59 and transcription of CYP1A1. The results showed that OA blocked dephosphorylation of Ser-59 and drastically inhibited transcription of CYP1A1. Similar results were obtained after knockdown of PP2A. Treatment with OA had no effect on the expression of AhR, its nuclear translocation, or its ability to bind to the XRE. Furthermore, dephosphorylation of Sp1 at Ser-59 was not affected by knockdown of AhR. These results indicate that the signals from TCDD or OP caused PP2A-mediated dephosphorylation of Sp1 at Ser-59 and induced CYP1A1 transcription. This signaling pathway was independent of the AhR-mediated pathway.

Involvement of CREM in CYP1A1 induction through ligand-independent activation pathway of aryl hydrocarbon receptor in HepG2 cells.

Using a radiation-hybrid cell, E11, produced by the fusion of Hepa-1c1c7 and X-ray irradiated HepG2 cells, we located the omeprazole (OP) responsive region on chromosome 10p. The cDNA of 12 genes expressed in E11 cells were cloned from HepG2 mRNA by RT-PCR. The cDNA was transfected into Hepa-1c1c7 cells to check the increased expression of Cyp1a1 by reporter gene (luciferase) assay. Finally, one gene (tauCREM: cAMP responsive element modulator, tau-isoform) was identified as a candidate gene for the gene responsive to OP. The regulatory sequence of Cyp1a1 in response to tauCREM transfection was identified in one region (-60 to -52bp relative to the transcription start site), which was the basic transcription element (BTE). Electromobility shift assay with the BTE sequence showed an increase in the band intensity when the cells were treated with OP. Decreased level of endogenous CREM by siRNA transfection inhibited the induction of CYP1A1-mRNA in HepG2 cells by OP.