Hippocampal expression of myelin-associated inhibitors is induced with age-related cognitive decline and correlates with deficits of spatial learning and memory

Impairment of cognitive functions including hippocampus-dependent spatial learning and memory affects nearly half of the aged population. Age-related cognitive decline is associated with synaptic dysfunction that occurs in the absence of neuronal cell loss, suggesting that impaired neuronal signaling and plasticity may underlie age-related deficits of cognitive function. Expression of myelin-associated inhibitors (MAIs) of synaptic plasticity, including the ligands myelin-associated glycoprotein, neurite outgrowth inhibitor A, and oligodendrocyte myelin glycoprotein, and their common receptor, Nogo-66 receptor, was examined in hippocampal synaptosomes and Cornu ammonis area (CA)1, CA3 and dentate gyrus subregions derived from adult (12-13 months) and aged (26-28 months) Fischer 344 × Brown Norway rats. Rats were behaviorally phenotyped by Morris water maze testing and classified as aged cognitively intact (n = 7-8) or aged cognitively impaired (n = 7-10) relative to adults (n = 5-7). MAI protein expression was induced in cognitively impaired, but not cognitively intact, aged rats and correlated with cognitive performance in individual rats. Immunohistochemical experiments demonstrated that up-regulation of MAIs occurs, in part, in hippocampal neuronal axons and somata. While a number of pathways and processes are altered with brain aging, we report a coordinated induction of myelin-associated inhibitors of functional and structural plasticity only in cognitively impaired aged rats. Induction of MAIs may decrease stimulus-induced synaptic strengthening and structural remodeling, ultimately impairing synaptic mechanisms of spatial learning and memory and resulting in cognitive decline.     

Identification of HSP90 as a new GABARAPL1 (GEC1)-interacting protein

GABARAPL1 belongs to the small family of GABARAP proteins (including GABARAP, GABARAPL1 and GABARAPL2/GATE-16), one of the two subfamilies of the yeast Atg8 orthologue. GABARAPL1 is involved in the intracellular transport of receptors, via an interaction with tubulin and GABA(A) or kappa opioid receptors, and also participates in autophagy and cell proliferation. In the present study, we identify the HSP90 protein as a novel interaction partner for GABARAPL1 using GST pull-down, mass spectrometry and coimmunoprecipitation experiments. GABARAPL1 and HSP90 partially colocalize in MCF-7 breast cancer cells overexpressed Dsred-GABARAPL1 and in rat brain. Moreover, treatment of MCF-7 cells overexpressed FLAG-GABARAPL1-6HIS with the HSP90 inhibitor 17-AAG promotes the GABARAPL1 degradation, a process that is blocked by proteasome inhibitors such as MG132, bortezomib and lactacystin. Accordingly, we demonstrate that HSP90 interacts and protects GABARAPL1 from its degradation by the proteasome.     

Biophysical characterization of glycosaminoglycan-IL-7 interactions using SPR

Glycosaminoglycans (GAGs) interact with a number of cytokines and growth factors thereby playing an essential role in the regulation of many physiological processes. These interactions are important for both normal signal transduction and the regulation of the tissue distribution of cytokines/growth factors. In the present study, we employed surface plasmon resonance (SPR) spectroscopy to dissect the binding interactions between GAGs and murine and human forms of interleukin-7 (IL-7). SPR results revealed that heparin binds with higher affinity to human IL-7 than murine IL-7 through a different kinetic mechanism. The optimal oligosaccharide length of heparin for the interactions to human and murine IL-7 involves a sequence larger than a tetrasaccharide. These results further demonstrate that while IL-7 is principally a heparin/heparan sulfate binding protein, it also interacts with dermatan sulfate, chondroitin sulfates C, D, and E, indicating that this cytokine preferentially interacts with GAGs having a higher degree of sulfation.     

Extracellular HspBP1 inhibits formation of a cytotoxic Tag7-Hsp70 complex in vitro and in human serum

Tag7 (PGRP-S) was described as an innate immunity protein. Earlier we have shown that Tag7 forms with Hsp70 a stable complex with cytotoxic and antitumor activity. The same complex is formed in and secreted by cytotoxic T-lymphocytes. We have also found that Hsp-binding protein HspBP1 incapacitates the Tag7-Hsp70 complex. Here we have studied the interaction of extracellular Tag7 and HspBP1. We have shown that HspBP1 binds Tag7 in the conditioned medium of tumor CSML0 cells, thereby preventing formation of the cytotoxic Tag7-Hsp70 complex. We have also found that Tag7, if present in serum (in every third donor on average), is always in complex with HspBP1. This may be a protective measure against indiscriminate attack of the cytotoxic complex on normal cells.     

Comparative study with two different enrichments in the culture media used in the disinfectant efficacy assay

Recent changes in Brazilian legislation for commercial disinfectants have been published due to the recent epidemic of nosocomial infections caused by rapidly growing mycobacteria (RGM) in many states of Brazil over the last 8 years. One of these documents requires that all the manufacturers provide evidence of efficacy of sterilizing and disinfectant products, used for semi critical medical devices, against the Mycobacterium bovis BCG Moreau and Mycobacterium abscessus subsp. bolletii INCQS 00594 strains by using the Confirmative in vitro Test for Determining Tuberculocidal Activity of Disinfectants recommended by the Association of Official Analytical Chemists. These changes have caused additional costs and increased problems for importation of enrichment products at national laboratories where disinfectant efficacy assay service is performed. Middlebrook ADC Enrichment (ADC) is provided by a unique manufacturer and used in the official protocol. The aim of the present study was to evaluate an alternative in house low-cost enrichment composed of fetal bovine serum and glucose (FBSG) with ADC for performance of disinfectant efficacy assay against mycobacteria. After obtaining the growth curves for M. abscessus ATCC 19977, M. abscessus subsp. bolletii INCQS 00594, Mycobacterium chelonae ATCC 35752, and Mycobacterium fortuitum ATCC 6841 by using ADC enrichment and FBSG in Kirchners and 7H9 culture media. Through statistical analysis via the Kruskal-Wallis test on the evaluation of microorganism growth rate, it was observed that there was no inhibition of RGM growth by any of the enrichments used. These results suggest that low-cost enrichment FBSG may be used as a potential substitute of ADC for composition of media for mycobacterial growth, including in disinfectant tests.     

GABA transporters mediate glycine release from cerebellum nerve endings: roles of Ca(2+)channels, mitochondrial Na(+)/Ca(2+) exchangers, vesicular GABA/glycine transporters and anion channels

GABA transporters accumulate GABA to inactivate or reutilize it. Transporter-mediated GABA release can also occur. Recent findings indicate that GABA transporters can perform additional functions. We investigated how activation of GABA transporters can mediate release of glycine. Nerve endings purified from mouse cerebellum were prelabeled with [(3)H]glycine in presence of the glycine GlyT1 transporter inhibitor NFPS to label selectively GlyT2-bearing terminals. GABA was added under superfusion conditions and the mechanisms of the GABA-evoked [(3)H]glycine release were characterized. GABA stimulated [(3)H]glycine release in a concentration-dependent manner (EC(50) = 8.26 μM). The GABA-evoked release was insensitive to GABA(A) and GABA(B) receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca(2+) entering the nerve terminals through VSCCs sensitive to ω-conotoxins. The external Ca(2+)-independent release involved mitochondrial Ca(2+), as it was prevented by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca(2+) did not trigger exocytotic release because the [(3)H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [(3)H]glycine so that less [(3)H]glycine can become cytosolic when GABA taken up exchanges with [(3)H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [(3)H]glycine efflux could be prevented by niflumic acid or NPPB indicating that the evoked release occurred essentially by permeation through anion channels. In conclusion, GABA uptake into GlyT2-bearing cerebellar nerve endings triggered glycine release which occurred essentially by permeation through Ca(2+)-dependent anion channels. Glial GABA release mediated by anion channels was proposed to underlie tonic inhibition in the cerebellum; the present results suggest that glycine release by neuronal anion channels also might contribute to tonic inhibition.     

Differences in kainate receptor involvement in hippocampal mossy fibre long-term potentiation depending on slice orientation

Long-term potentiation (LTP) is a well-established experimental model used to investigate the synaptic basis of learning and memory. LTP at mossy fibre - CA3 synapses in the hippocampus is unusual because it is normally N-methyl-d-aspartate (NMDA) receptor-independent. Instead it seems that the trigger for mossy fibre LTP involves kainate receptors (KARs). Although it is generally accepted that pre-synaptic KARs play an essential role in frequency facilitation and LTP, their subunit composition remains a matter of significant controversy. We have reported previously that both frequency facilitation and LTP can be blocked by selective antagonism of GluK1 (formerly GluR5/Glu(K5))-containing KARs, but other groups have failed to reproduce this effect. Moreover, data from receptor knockout and mRNA expression studies argue against a major role of GluK1, supporting a more central role for GluK2 (formerly GluR6/Glu(K6)). A potential reason underlying the controversy in the pharmacological experiments may reside in differences in the preparations used. Here we show differences in pharmacological sensitivity of synaptic plasticity at mossy fibre - CA3 synapses depend critically on slice orientation. In transverse slices, LTP of fEPSPs was invariably resistant to GluK1-selective antagonists whereas in parasagittal slices LTP was consistently blocked by GluK1-selective antagonists. In addition, there were pronounced differences in the magnitude of frequency facilitation and the sensitivity to the mGlu2/3 receptor agonist DCG-IV. Using anterograde labelling of granule cells we show that slices of both orientations possess intact mossy fibres and both large and small presynaptic boutons. Transverse slices have denser fibre tracts but a smaller proportion of giant mossy fibre boutons. These results further demonstrate a considerable heterogeneity in the functional properties of the mossy fibre projection.     

Coumarin-3-carboxylic acid derivatives as potentiators and inhibitors of recombinant and native N-methyl-d-aspartate receptors

N-Methyl-d-aspartate receptors (NMDARs) are known to be involved in a range of neurological and neurodegenerative disorders and consequently the development of compounds that modulate the function of these receptors has been the subject of intense interest. We have recently reported that 6-bromocoumarin-3-carboxylic acid (UBP608) is a negative allosteric modulator with weak selectivity for GluN2A-containing NMDARs. In the present study, a series of commercially available and newly synthesized coumarin derivatives have been evaluated in a structure-activity relationship (SAR) study as modulators of recombinant NMDAR activity. The main conclusions from this SAR study were that substituents as large as iodo were accommodated at the 6-position and that 6,8-dibromo or 6,8-diiodo substitution of the coumarin ring enhanced the inhibitory activity at NMDARs. These coumarin derivatives are therefore excellent starting points for the development of more potent and GluN2 subunit selective inhibitors, which may have application in the treatment of a range of neurological disorders such as neuropathic pain, epilepsy and depression. Surprisingly, 4-methyl substitution of UBP608 to give UBP714, led to conversion of the inhibitory activity of UBP608 into potentiating activity at recombinant GluN1/GluN2 receptors. UBP714 also enhanced NMDAR mediated field EPSPs in the CA1 region of the hippocampus. UBP714 is therefore a novel template for the development of potent and subunit selective NMDAR potentiators that may have therapeutic applicability in the treatment of patients with cognitive deficits or schizophrenia.     

鸡β-防御素7在大肠杆菌中的表达及活性分析

【目的】根据鸡β-防御素7(Gal-7)的成熟肽基因序列合成基因,构建表达Gal-7的大肠杆菌工程菌,研究重组鸡防御素Gal-7成熟肽的体外生物活性。【方法】将合成的gal-7基因克隆到大肠杆菌表达载体pGEX-6p-1中,得到重组质粒pGEX-6p-gal7,转化大肠杆菌BL21(DE3),经IPTG诱导表达得到含GST标签的融合蛋白GST-Gal7;之后用Prescission蛋白酶将GST标签切除,并对成熟肽进行质谱分析;再利用琼脂打孔扩散法检测Gal-7成熟肽的体外抑菌活性,用2倍稀释法测定对指示菌的最低抑菌浓度。【结果】成功构建Gal-7大肠杆菌异源表达工程菌,表达纯化的重组Gal-7成熟肽质谱鉴定分子量为5 516 Da,其对黄色微球菌(NCIB 8166)、金黄色葡萄球菌(ATCC 25923)、粪肠球菌(ATCC 29212)、大肠杆菌(CMCC 44102)均有抑菌活性,最低抑菌浓度分别为16.875、67.5、67.5、135 mg/L。【结论】获得表达鸡Gal-7成熟肽的大肠杆菌工程菌,并且切除GST标签的Gal-7成熟肽具有生物活性。