Rat Brain Lysates Research Articles

Rab3A interacts with spastin to regulate neurite outgrowth in hippocampal neurons

Investigating novel mechanisms of neurite outgrowth via cytoskeleton is critical for developing therapeutic strategies against neural disorders. Rab3A is a vesicle-related protein distributed throughout the nervous system, but the detailed mechanism related to cytoskeleton remains largely unknown. Our previous reports show that spastin serves microtubule to regulate neurite outgrowth. Here, we asked whether Rab3A could function via modulating spastin during neuronal development. The results revealed that Rab3A colocalized with spastin in cultured hippocampal neurons. Immunoprecipitation assays showed that Rab3A physically interacted with spastin in rat brain lysates. Rab3A overexpression significantly induced spastin degradation; this effect was reversed by leupeptin- or MG-132- administration, suggesting the lysosomal and ubiquitin-mediated degradation system. Immunofluorescence staining further confirmed that Rab3A and spastin immune-colocalized with the lysosome marker lysotracker. In COS7 cells, Rab3A overexpression significantly downregulated spastin expression and abolished the spastin-mediated microtubule severing. Furthermore, overexpression inhibited while genetic knockdown of Rab3A promoted neurite outgrowth. However, this inhibitory effect on neurite outgrowth in hippocampal neurons could be reversed via co-transfection of spastin, indicating that Rab3A functions via its interaction protein spastin. In general, our data identify an interaction between Rab3A and spastin, and this interaction affects the protein stability of spastin and eliminates its microtubule severing function, thereby modulating neurite outgrowth.

Identification of Glutamine Synthetase as a Contryphan-Bt Binding Protein by His-Tag Pull-Down.

Contryphan-Bt is a D-tryptophan-containing disulfide-constrained decapeptide recently isolated from the venom of Conus betulinus. The molecular targets of contryphans are controversial, and the identification of its interacting proteins may be of great importance. His-tag pull-down assays were performed to investigate intracellular binding proteins of contryphan-Bt from rat brain lysate. Bt-Acp-[His]6, a contryphan-Bt derivative containing hexahistidine tag, was synthesized and used as the bait. As a control, Acp-[His]6 was used to exclude nonspecific bindings. Glutamine synthetase was identified as a potential contryphan-Bt binding protein by pull-- down assays and subsequent LC-MS/MS. The binding of contryphan-Bt to glutamine synthetase was confirmed and determined using microscale thermophoresis, with a Kd of 74.02 ± 2.8 μM. The binding did not affect glutamine synthetase activity, suggesting that the interaction site was distinct from the catalytic center. Glutamine synthetase was identified as a novel contryphan-Bt binding protein. This is the first report in which the conopeptide binds to an intracellular protein.

In Situ Controllable Fabrication of Two-Dimensional Magnetic Fe3O4/TiO2@Ti3C2Tx Composites for Highly Efficient Phosphopeptides Enrichment.

Highly efficient enrichment of phosphopeptides is of great significance for phosphoproteomics-related biological and pathological processes research, but it remains challenging due to the lack of affinity materials which hold high enrichment efficiency and capacity. Ti3C2Tx MXene, a novel two-dimensional material with outstanding physicochemical properties, has attracted wide research interests for application in various fields. However, there are few reports on the use of MXene-derived materials for phosphopeptides separation in the biomedical field. In this work, we proposed a facile one-pot method that in situ oxidation and modification of Ti3C2Tx MXene, to prepare two-dimensional (2D) magnetic Fe3O4/TiO2@Ti3C2Tx composites for potential application in phosphopeptides enrichment. Benefiting from the outstanding magnetic responsiveness and multiaffinity sites (Ti-O, Fe-O, and NH2 groups), the Fe3O4/TiO2@Ti3C2Tx composites possessed excellent enrichment performance with high sensitivity (0.1 fmol μL-1), excellent selectivity (β-casein: bovine serum albumin = 1:5000, molar ratio), good repeatability (5 times), and high enrichment capacity (200 mg g-1). Moreover, the results of selective enrichment of phosphopeptides from nonfat milk, human saliva, human serum, and rat brain lysates indicated the great potential of Fe3O4/TiO2@Ti3C2Tx composites in low-abundance phosphopeptides enrichment from complex biological samples. This work has put forward a versatile method to prepare magnetic MXene composites and promoted the use of MXene composites in phosphoproteome in biomedicine.

Assembly of Taurine Transporter (Slc6a6) with Na+-H+ Exchanger Regulatory Factor 1 (Slc9a3r1) Improves GABA Transport Activity by Increasing the Maximum Transport Velocity.

Regulating γ-aminobutyric acid (GABA) uptake transport on the plasma membranes is required for its efficient clearance from the brain interstitial fluid. The purpose of this study was to clarify the assembly of taurine transporter (TauT/Slc6a6) and PSD-95/Disc-large/Zo-1 (PDZ) domain of Na+-H+ exchanger regulatory factor 1 (NHERF1) as a regulatory mechanism of TauT-mediated GABA transport activity. In vitro glutathione S-transferase (GST)-pull down assay and immunoblotting with anti-NHERF1 antibody revealed that NHERF1 protein was present in rat brain lysates as the binding protein of the GST-fusion TauT C-terminal protein with the PDZ-binding ETMM motif but not its corresponding deletion mutant lacking the motif. [3H]GABA uptake by TauT-NHERF1-coexpressing oocytes and TauT-singly expressing oocytes exhibited saturable kinetics with Michaelis-Menten constant values of 0.835±0.288 and 0.982±0.569 mM and a maximal transport velocity of 206±37 and 283±28 pmol/(h·oocyte), respectively. These results suggest that the assembly of TauT PDZ-binding motif and NHERF1 increases the maximal transport velocity of GABA rather than changes the affinity.

Graft modification of cotton with phosphate group and its application to the enrichment of phosphopeptides

Grafting copolymerization, especially “grafting from” approach, has attracted a great attention for the preparation of cellulose-based materials with various functionalities. In this study, a novel phosphate group-containing cotton fiber-based material, CF-NH2-AZO-p(VPA-x), was successfully synthesized by a “grafting from” radical polymerization approach using vinyl phosphonic acid (VPA) as monomer. The phosphate group content of CF-NH2-AZO-p(VPA-x) could be easily regulated by adjusting the concentration of VPA monomer. Subsequently, titanium ions immobilized CF-NH2-AZO-p(VPA-x) (CF-NH2-AZO-p(VPA-x)-Ti4+) was prepared and used as an immobilized metal affinity chromatography (IMAC) adsorbent for the selective enrichment of phosphopeptides from various biological samples. The relationship between enrichment efficiency and phosphate group content of CF-NH2-AZO-p(VPA-x)-Ti4+ was investigated. The optimized CF-NH2-AZO-p(VPA-2)-Ti4+ (x=2) exhibited high selectivity and extraction capacity in phosphopeptides enrichment from standard peptides mixture, non-fat milk digests and protein-rich human serum. In addition, CF-NH2-AZO-p(VPA-2)-Ti4+ was further applied for the specific capture of phosphopeptides from tryptic digests of rat brain lysate, and 3241 unique phosphopeptides were identified from 0.5mg of rat brain by combining CF-NH2-AZO-p(VPA-2)-Ti4+ pretreatment with liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. We expect the proposed method can promote the development of “grafting from” approach in the preparation of cellulose-based adsorbents and broaden the application of cellulose-based adsorbents for biological analysis.

Synthesis of Polyethylenimine Functionalized Mesoporous Silica for In-Pipet-Tip Phosphopeptide Enrichment.

Synthesis of functionalized mesoporous silica material with large particle size remains a chanllenge. In this work, polyethylenimine (PEI) functionalized mesoporous silica (PFMS) with particle size as large as 100 μm was successfully synthesized by a facile method. In the synthesis process, PEI served as four roles simultaneously, including functionalized reagent, alkaline catalyst, template for particle formation, and pore-structure-directing agent. The surface areas of the products were higher than 260 m2/g. Benefiting from the large particle size and high surface area, PFMS was packed in a pipet tip to fabricate a convenient and miniaturized solid phase extraction apparatus for sample preparation. Additionally, based on the extremely abundant basic sites in the organic units of PFMS, the in-pipet-tip system was used as an anion-exchanger for phosphopeptide enrichment. The specificity of the developed method was investigated by capture of phosphopeptides from tryptic digests of standard protein mixtures, tryptic digests of nonfat milk, and human serum. Furthermore, the method was utilized to analyze phosphopeptides in tryptic digests of rat brain lysate, and 2251 unique phosphopeptides were successfully detected.

Magnetic graphitic carbon nitride anion exchanger for specific enrichment of phosphopeptides

Anion-exchange chromatography (AEX) is one of the chromatography-based methods effectively being used for phosphopeptide enrichment. However, the development of AEX materials with high specificity toward phosphopeptides is still less explored as compared to immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC). In this work, magnetic graphitic carbon nitride (MCN) was successfully prepared and introduced as a promising AEX candidate for phosphopeptide enrichment. Due to the extremely abundant content of nitrogen with basic functionality on the surface, this material kept excellent retention for phosphopeptides at pH as low as 1.8. Benefiting from the large binding capacity at such low pH, MCN showed remarkable specificity to capture phosphopeptides from tryptic digests of standard protein mixtures as well as nonfat milk and human serum. In addition, MCN was also applied to selective enrichment of phosphopeptides from the tryptic digests of rat brain lysate and 2576 unique phosphopeptides were successfully identified.

Protein purification and cloning of diacylglycerol lipase from rat brain.

Diacylglycerol (DG) lipase, which hydrolyses 1-stearoyl-2-arachidonyl-sn-glycerol to produce an endocannabinoid, 2-arachidonoylglycerol, was purified from the soluble fraction of rat brain lysates. DG lipase was purified about 1,200-fold by a sequential column chromatographic procedure. Among proteins identified by mass spectrometry analysis in the partially purified DG lipase sample, only DDHD domain containing two (DDHD2), which was formerly regarded as a phospholipase A1, exhibited significant DG lipase activity. Rat DDHD2 expressed in Chinese hamster ovary cells showed similar enzymatic properties to partially purified DG lipase from rat brain. The source of DG lipase activity in rat brain was immunoprecipitated using anti-DDHD2 antibody. Thus, we concluded that the DG lipase activity in the soluble fraction of rat brain is derived from DDHD2. DDHD2 is distributed widely in the rat brain. Immunohistochemical analysis revealed that DDHD2 is expressed in hippocampal neurons, but not in glia.

Abstract B171: Anti-oncogenic effects of RP10107, a novel and potent glutaminase inhibitor, in solid cancer cell lines

Abstract Background: Cancer cells preferentially channel glucose towards lactate production even when oxygen is plentiful, a process termed “aerobic glycolysis” or the Warburg effect. The entire metabolic machinery is reprogrammed in cancer cells wherein glutamine utilization is increased via elevation of glutaminase activity thereby generating the necessary substrates required for eventual ATP synthesis and energy production. Targeting glutaminase therefore represents a potential therapeutic strategy to curb malignant transformation of cells and combat cancer progression. Herein, we describe the biological and pharmacokinetic properties of RP10107, a novel small molecule glutaminase inhibitor, with scope to be further developed as a clinical candidate for solid tumors. Methods: Inhibition of glutaminase (GLS1) activity was determined in rodent brain lysates and recombinant human enzyme preparations using a colorimetric or fluorometric procedure. Selectivity over liver glutaminase (GLS2) was confirmed using mouse liver mitochondrial lysates. Viability was assessed using the colorimetric MTT reagent after incubation of RP10107 with cell lines representative of breast, lung, and colorectal cancer. Glutamate concentrations in cell lines following treatment with RP10107 was estimated by LC-MS/MS. Expression of GLS-1, cMyc, pS6, and p-mTOR were estimated by Western Blotting and bands were quantified using ImageJ after normalization with Actin. Pharmacokinetic profile of RP10107 in plasma after single dose oral administration (10 mg/kg) or IV injection (1 mg/kg) was determined in mice. Results: RP10107 inhibited glutaminase (GLS1) activity in mouse brain lysate, rat brain lysate, and recombinant human enzyme with IC50 of 21.2, 18.2, and 26.4 nM, respectively. In contrast, activity in liver mitochondrial preparations (GLS2) was minimal (<10% inhibition) even at 10 μM concentration. RP10107 caused a dose-dependent inhibition in proliferation of breast, lung, and colorectal cell lines. Response was the highest in MDA-MB-231 (GI50 = 27 nM) with half-maximal growth inhibition for ZR.75.1, MCF-7, A549, H441, HCT-8, and HCT-116 observed at <2 μM RP10107. Reduction in cell viability was accompanied by a dose-dependent increase in the ratio of glutamine to glutamate with >50% response noticed at 100 nM in MDA-MB-231 cells. While p-mTOR expression levels were not affected, addition of RP10107 caused a reduction in cMyc and pS6 (>50% at 100-1000 nM) in lung and breast cancer cell lines. Pharmacokinetic studies of RP10107 in mice indicated high oral bioavailability (62%) with maximum plasma concentrations (Cmax) exceeding 10 μM. Conclusions: RP10107 is a potent and selective GLS1 inhibitor that inhibited growth of solid tumor cell lines with a concomitant increase in glutamine to glutamate ratio. Reduction in glutaminase activity resulted in downregulation of cMyc and pS6 in breast and lung cancer cells. Based on the in vitro efficacy and excellent pharmacokinetic profile, RP10107 is current being tested in various mouse xenograft models. Citation Format: Srikant Viswanadha, Prashant Bhavar, Gayatriswaroop Merikapudi, Babu Govindrajulu, Satyanarayana Eleswarapu, Sridhar Veeraraghavan, Swaroop Vakkalanka. Anti-oncogenic effects of RP10107, a novel and potent glutaminase inhibitor, in solid cancer cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B171.

Hydrophilic Carboxyl Cotton Chelator for Titanium(IV) Immobilization and Its Application as Novel Fibrous Sorbent for Rapid Enrichment of Phosphopeptides.

Sample preparation methods with high selectivity, efficiency, and matrix resistance are essential for phosphoproteomic analysis. In this study, carboxyl cotton chelator-titanium(IV) (CCC-Ti4+) fibers, a novel CCC-based fibrous sorbent with excellent biocompatibility, were successfully synthesized on the basis of the coordination effect between double carboxyl groups on CCC and Ti4+. The synthesis of CCC-Ti4+ fibers was easy, and the incorporated titanium content was high. On the basis of immobilized metal ion affinity chromatography (IMAC), CCC-Ti4+ fibers were used for specific capture of phosphopeptides using a lab-in-syringe solid-phase extraction (SPE) from multiple biological samples, including standard protein digests, nonfat milk digests, human serum, and animal tissue. The proposed sorbent exhibited high selectivity (β-casein/BSA=1:1000) and good sensitivity (10 fmol) in phosphopeptides analysis. Meanwhile, the lab-in-syringe SPE greatly simplified the entire process of enrichment. Thanks to the good biocompatibility of CCC-based material, CCC-Ti4+ fibers showed excellent performance in phosphopeptide enrichment from protein-rich human serum. Finally, CCC-Ti4+ fibers were applied for selective capture of phosphopeptides from tryptic digests of rat brain lysate followed by LC-MS/MS analysis. Using the proposed method, we identified 3950 unique phosphosites from 1 mg of rat brain in a single experiment, which is much better than previously reported IMAC-based strategies. Taken together, this efficient method will find broad application in large-scale phosphoproteomics analysis because of the rapid (3 min) convenient procedure and excellent performance.

Involvement of fatty acid amide hydrolase and fatty acid binding protein 5 in the uptake of anandamide by cell lines with different levels of fatty acid amide hydrolase expression: a pharmacological study.

BackgroundThe endocannabinoid ligand anandamide (AEA) is removed from the extracellular space by a process of cellular uptake followed by metabolism. In many cells, such as the RBL-2H3 cell line, inhibition of FAAH activity reduces the observed uptake, indicating that the enzyme regulates uptake by controlling the intra- : extracellular AEA concentration gradient. However, in other FAAH-expressing cells, no such effect is seen. It is not clear, however, whether these differences are methodological in nature or due to properties of the cells themselves. In consequence, we have reinvestigated the role of FAAH in gating the uptake of AEA.Methodology/Principal FindingsThe effects of FAAH inhibition upon AEA uptake were investigated in four cell lines: AT1 rat prostate cancer, RBL-2H3 rat basophilic leukaemia, rat C6 glioma and mouse P19 embryonic carcinoma cells. Semi-quantitative PCR for the cells and for a rat brain lysate confirmed the expression of FAAH. No obvious expression of a transcript with the expected molecular weight of FLAT was seen. FAAH expression differed between cells, but all four could accumulate AEA in a manner inhibitable by the selective FAAH inhibitor URB597. However, there was a difference in the sensitivities seen in the reduction of uptake for a given degree of FAAH inhibition produced by a reversible FAAH inhibitor, with C6 cells being more sensitive than RBL-2H3 cells, despite rather similar expression levels and activities of FAAH. The four cell lines all expressed FABP5, and AEA uptake was reduced in the presence of the FABP5 inhibitor SB-FI-26, suggesting that the different sensitivities to FAAH inhibition for C6 and RBL2H3 cells is not due to differences at the level of FABP-5.Conclusions/SignificanceWhen assayed using the same methodology, different FAAH-expressing cells display different sensitivities of uptake to FAAH inhibition.

Photoactivatable glycopolymers for the proteome-wide identification of fucose-α(1-2)-galactose binding proteins.

Although fucose-α(1-2)-galactose (Fucα(1-2)Gal)-containing glycans have been implicated in cognitive processes such as learning and memory, their precise molecular mechanisms are poorly understood. Here we employed the use of multivalent glycopolymers to enable the first proteome-wide identification of weak affinity, low abundance Fucα(1-2)Gal glycan-binding proteins (GBPs). Biotin-terminated glycopolymers containing photoactivatable cross-linking groups were designed to capture and enrich GBPs from rat brain lysates. Candidate proteins were tested for their ability to bind Fucα(1-2)Gal, and the functional significance of the interaction was investigated for the synaptic vesicle protein SV2a using a knockout mouse system. The results suggest a role for SV2a-Fucα(1-2)Gal interactions in SV2a trafficking and synaptic vesicle recycling. More broadly, our studies outline a general chemical approach for the systems-level discovery of novel GBPs.

Stable over‐expression of the 2‐oxoglutarate carrier enhances neuronal cell resistance to oxidative stress via Bcl‐2‐dependent mitochondrial GSH transport

Mitochondrial glutathione (GSH) is a key endogenous antioxidant and its maintenance is critical for cell survival. Here, we generated stable NSC34 motor neuron-like cell lines over-expressing the mitochondrial GSH transporter, the 2-oxoglutarate carrier (OGC), to further elucidate the importance of mitochondrial GSH transport in determining neuronal resistance to oxidative stress. Two stable OGC cell lines displayed specific increases in mitochondrial GSH content and resistance to oxidative and nitrosative stressors, but not staurosporine. Inhibition of transport through OGC reduced levels of mitochondrial GSH and resensitized the stable cell lines to oxidative stress. The stable OGC cell lines displayed significant up-regulation of the anti-apoptotic protein, B cell lymphoma 2 (Bcl-2). This result was reproduced in parental NSC34 cells by chronic treatment with GSH monoethylester, which specifically increased mitochondrial GSH levels. Knockdown of Bcl-2 expression decreased mitochondrial GSH and resensitized the stable OGC cells to oxidative stress. Finally, endogenous OGC was co-immunoprecipitated with Bcl-2 from rat brain lysates in a GSH-dependent manner. These data are the first to show that increased mitochondrial GSH transport is sufficient to enhance neuronal resistance to oxidative stress. Moreover, sustained and specific enhancement of mitochondrial GSH leads to increased Bcl-2 expression, a required mechanism for the maintenance of increased mitochondrial GSH levels. Stable over-expression of the 2-oxoglutarate carrier (OGC) in a motor neuronal cell line induced a specific increase in mitochondrial GSH and markedly enhanced resistance to oxidative stress. Over-expression of OGC also induced Bcl-2 expression which was owing to the specific increase in mitochondrial GSH. Intriguingly, enhanced expression of Bcl-2 was required to sustain OGC-dependent GSH transport into the mitochondria. Thus, OGC and Bcl-2 work in a concerted manner to maintain the mitochondrial GSH pool which is crucial for neuronal survival.

LRRK2 phosphorylates Snapin and inhibits interaction of Snapin with SNAP-25

Leucine-rich repeat kinase 2 (LRRK2) is a gene that, upon mutation, causes autosomal-dominant familial Parkinson's disease (PD). Yeast two-hybrid screening revealed that Snapin, a SNAP-25 (synaptosomal-associated protein-25) interacting protein, interacts with LRRK2. An in vitro kinase assay exhibited that Snapin is phosphorylated by LRRK2. A glutathione-S-transferase (GST) pull-down assay showed that LRRK2 may interact with Snapin via its Ras-of-complex (ROC) and N-terminal domains, with no significant difference on interaction of Snapin with LRRK2 wild type (WT) or its pathogenic mutants. Further analysis by mutation study revealed that Threonine 117 of Snapin is one of the sites phosphorylated by LRRK2. Furthermore, a Snapin T117D phosphomimetic mutant decreased its interaction with SNAP-25 in the GST pull-down assay. SNAP-25 is a component of the SNARE (Soluble NSF Attachment protein REceptor) complex and is critical for the exocytosis of synaptic vesicles. Incubation of rat brain lysate with recombinant Snapin T117D, but not WT, protein caused decreased interaction of synaptotagmin with the SNARE complex based on a co-immunoprecipitation assay. We further found that LRRK2-dependent phosphorylation of Snapin in the hippocampal neurons resulted in a decrease in the number of readily releasable vesicles and the extent of exocytotic release. Combined, these data suggest that LRRK2 may regulate neurotransmitter release via control of Snapin function by inhibitory phosphorylation.

PSD‐95 interacts with NBCn1 and enhances channel‐like activity without affecting cotransport function

NBCn1 plays an essential role in intracellular pH regulation and transepithelial bicarbonate movement in the body. NBCn1 also has sodium channel‐like activity uncoupled to Na/HCO3 cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD‐95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD‐95 interaction. In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C‐terminal 131 amino acids of NBCn1 pulled down PSD‐95 from rat brain lysates, whereas GST/NBCn1‐ΔETSL (deletion of the last four amino acids) and GST/NBCn2 lacking the same ETSL did not. NBCn1 and PSD‐95 were coimmunoprecipitated in HEK 293 cells. PSD‐95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD‐95 increased an ionic conductance produced by NBCn1 channel‐like activity. This increase was abolished by NBCn1‐ΔETSL or by the peptide containing the last 15 amino acids of NBCn1. Our data suggest that PSD‐95 interacts with NBCn1 and increases its channel‐like activity while negligibly affecting Na/HCO3 cotransport. We propose that the channel‐like activity may occur via an intermolecular cavity of multimeric NBCn1 proteins.

Putative Agmatinase Inhibitor for Hypoxic-Ischemic New Born Brain Damage

Agmatine is an endogenous brain metabolite, decarboxylated arginine, which has neuroprotective properties when injected intraperitoneally (i.p.) into rat pups following hypoxic-ischemia. A previous screen for compounds based on rat brain lysates containing agmatinase with assistance from computational chemistry, led to piperazine-1-carboxamidine as a putative agmatinase inhibitor. Herein, the neuroprotective properties of piperazine-1-carboxamidine are described both in vitro and in vivo. Organotypic entorhinal-hippocampal slices were firstly prepared from 7-day-old rat pups and exposed in vitro to atmospheric oxygen depletion for 3h. Upon reoxygenation, the slices were treated with piperazine-1-carboxamidine or agmatine (50μg/ml agents), or saline, and 15h later propidium iodine was used to stain. Piperazine-1-carboxamidine or agmatine produced substantial in vitro protection compared to post-reoxygenated saline-treated controls. An in vivo model involved surgical right carotid ligation followed by exposure to hypoxic-ischemia (8% oxygen) for 2.5h. Piperazine-1-carboxamidine at 50mg/kg i.p. was given 15min post-reoxygenation and continued twice daily for 3days. Cortical agmatine levels were elevated (+28.5%) following piperazine-1-carboxamidine treatment with no change in arginine or its other major metabolites. Histologic staining with anti-Neun monoclonal antibody also revealed neuroprotection of CA1-3 layers of the hippocampus. Until endpoint at 22days of age, no adverse events were observed in treated pups' body weights, rectal temperatures, or prompted ambulation. Piperazine-1-carboxamidine therefore appears to be a neuroprotective agent of a new category, agmatinase inhibitor.

LIM Kinase 1 (LIMK1) Interacts with Tropomyosin-related Kinase B (TrkB) and Mediates Brain-derived Neurotrophic Factor (BDNF)-induced Axonal Elongation

BDNF/TrkB signaling plays critical roles in axonal outgrowth of neurons, the process of which requires the remodeling of the cytoskeleton structure, including microtubules and filamentous actin. However, the mechanism by which BDNF/TrkB signaling regulates cytoskeleton reorganization is still unclear. Here, we identified a novel interaction between LIMK1 and TrkB, which is required for the BDNF-induced axonal elongation. We demonstrated that BDNF-induced TrkB dimerization led to LIMK1 dimerization and transphosphorylation independent of TrkB kinase activity, which could further enhance the activation and stabilization of LIMK1. Moreover, activated LIMK1 translocated to the membrane fraction and phosphorylated its substrate cofilin, thus promoting actin polymerization and axonal elongation. Our findings provided evidence of a novel mechanism for the BDNF-mediated signal transduction leading to axonal elongation.

Cleavage of the NR2B Subunit Amino Terminus of N-Methyl-d-aspartate (NMDA) Receptor by Tissue Plasminogen Activator: IDENTIFICATION OF THE CLEAVAGE SITE AND CHARACTERIZATION OF IFENPRODIL AND GLYCINE AFFINITIES ON TRUNCATED NMDA RECEPTOR

Thrombolysis using tissue plasminogen activator (tPA) has been the key treatment for patients with acute ischemic stroke for the past decade. Recent studies, however, suggest that this clot-busting protease also plays various roles in brain physiological and pathophysiological glutamatergic-dependent processes, such as synaptic plasticity and neurodegeneration. In addition, increasing evidence implicates tPA as an important neuromodulator of the N-methyl-d-aspartate (NMDA) receptors. Here, we demonstrate that recombinant human tPA cleaves the NR2B subunit of NMDA receptor. Analysis of NR2B in rat brain lysates and cortical neurons treated with tPA revealed concentration- and time-dependent degradation of NR2B proteins. Peptide sequencing studies performed on the cleaved-off products obtained from the tPA treatment on a recombinant fusion protein of the amino-terminal domain of NR2B revealed that tPA-mediated cleavage occurred at arginine 67 (Arg(67)). This cleavage is tPA-specific, plasmin-independent, and removes a predicted ~4-kDa fragment (Arg(27)-Arg(67)) from the amino-terminal domain of the NR2B protein. Site-directed mutagenesis of putative cleavage site Arg(67) to Ala(67) impeded tPA-mediated degradation of recombinant protein. This analysis revealed that NR2B is a novel substrate of tPA and suggested that an Arg(27)-Arg(67)-truncated NR2B-containing NMDA receptor could be formed. Heterologous expression of NR2B with Gln(29)-Arg(67) deleted is functional but exhibits reduced ifenprodil inhibition and increased glycine EC(50) with no change in glutamate EC(50). Our results confirmed NR2B as a novel proteolytic substrate of tPA, where tPA may directly interact with NR2B subunits leading to a change in pharmacological properties of NR2B-containing NMDA receptors.

Short peptide tools for monitoring caspase and proteasome activities in embryonal and adult rat brain lysates: an approach for the differential identification of proteases

The numerous caspase-like activities present in nervous tissue can be investigated with labelled peptides. However, the cross-reactivities of peptides with both proteasomes and caspases complicate the analysis of protease activity. The pharmacological features of substrates and inhibitors specific for either caspases or proteasome caspase-like proteases in rat brain lysates were similar or identical to the profiles of commercially purified proteasome preparations. Caspase inhibitors bind directly to active proteasome centres, thus competing with selective antagonists of proteasomes. Separation of lysates by molecular weight does not separate active caspases from proteasomes because these enzymes co-localize under native electrophoresis. The addition of ATP or its analogues is associated with the differential modulation of proteasomal activity, which also leads to ambiguity in the data. However, induced caspase activity could be successfully differentiated from proteasome activity in embryonal brain lysates with the non-selective caspase inhibitors Z-VAD-FMK and Q-VD-OPh and the proteasome inhibitor AdaAhx(3)L(3)VS that are not cross-reactive. This strategy is proposed for the simultaneous examination of caspases and proteasomes using proteolysis experiments. The present study reveals that all of the caspase-like activities in the tissue lysates of non-injured adult rat brains were related to proteasomal caspase-like activities.

Synthesis and protein binding studies of a peptide fragment of clathrin assembly protein AP180 bearing an O-linked β-N-acetylglucosaminyl-6-phosphate modification

A novel post-translational modification of threonine, β-N-acetylglucosaminyl-phosphate, was recently discovered on assembly protein AP180, a protein which plays a crucial role in clathrin coated vesicle formation in synaptic vesicle endocytosis (SVE). Herein, we report studies aimed at probing the effect of this modification on binding to proteins in rat brain lysate using pull down experiments with peptide fragments of AP180.