Binding and Activation of LRP1-Dependent Cell Signaling in Schwann Cells Using a Peptide Derived from the Hemopexin Domain of MMP-9.

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following a peripheral nervous system injury. The low-density lipoprotein receptor-related protein-1 (LRP1) is significantly upregulated in SCs in response to acute injury, activating cJun and promoting SC survival. Matrix-metalloproteinase-9 (MMP-9) is an LRP1 ligand that binds LRP1 through its hemopexin domain (PEX) and activates SC survival signaling and migration. To identify novel peptide mimetics within the hemopexin domain of MMP-9, we examined the crystal structure of PEX, synthesized four peptides, and examined their potential to bind and activate LRP1. We demonstrate that a 22 amino acid peptide, peptide 2, was the only peptide that activated Akt and ERK1/2 signaling in SCs, similar to a glutathione s-transferase (GST)-fused holoprotein, GST-PEX. Intraneural injection of peptide 2, but not vehicle, into crush-injured sciatic nerves activated cJun greater than 2.5-fold in wild-type mice, supporting that peptide 2 can activate the SC repair signaling in vivo. Peptide 2 also bound to Fc-fusion proteins containing the ligand-binding motifs of LRP1, clusters of complement-like repeats (CCRII and CCRIV). Pulldown and computational studies of alanine mutants of peptide 2 showed that positively charged lysine and arginine amino acids within the peptide are critical for stability and binding to CCRII. Collectively, these studies demonstrate that a novel peptide derived from PEX can serve as an LRP1 agonist and possesses qualities previously associated with LRP1 binding and SC signaling in vitro and in vivo.

Impact of implementing structured note templates on data capture for hernia surgery.

BACKGROUND: Electronic medical record notes have been determined to be lacking in quality, accessibility and content. Structured note templates could provide a way to improve these aspects, particularly with regard to data availability for research and quality improvement. OBJECTIVE: To determine whether the implementation of a standardised template for hernia documentation can improve data completeness and timeliness. METHOD: Retrospective review of clinic notes of 30 patients, 15 prior to implementation of a standardised note template and 15 after implementation of the template. The number of the 21 Americas Hernia Society Quality Collaborative (AHSQC) variables which were present in the notes was recorded, as was the time that the consultation ended and the time that the note was submitted. RESULTS: Mean number of variables collected prior to implementation of the template was 5.9 ± 1.6 vs. 20 ± 0.4 after implementation (p < 0.001). In the pre-implementation group, 20% of the notes were completed after the day of the visit, while all of the notes in the post-implementation group were completed on the same day as the visit (p = 0.367). CONCLUSION: Implementation of a structured note template resulted in significantly improved capture of specific database variables within clinical notes. Structured note templates are an effective tool to improve data capture from the clinical setting for research and quality improvement.

SMG-6 mRNA cleavage stalls ribosomes near premature stop codons in vivo.

Nonsense-mediated mRNA decay (NMD) protects cells from the toxic and potentially dominant effects of truncated proteins. Targeting of mRNAs with early stop codons is mediated by the ribosome and spatiotemporally aligned with translation termination. Previously we identified a novel NMD intermediate: ribosomes stalled on cleaved stop codons, raising the possibility that NMD begins even prior to ribosome removal from the stop codon. Here we show that this intermediate is the result of mRNA cleavage by the endonuclease SMG-6. Our work supports a model in which ribosomes stall secondary to SMG-6 mRNA cleavage in Caenorhabditis elegans and humans, i.e. that the novel NMD intermediate occurs after a prior ribosome elicits NMD. Our genetic analysis of C. elegans' SMG-6 supports a central role for SMG-6 in metazoan NMD, and provides a context for evaluating its function in other metazoans.

A model for DHX15 mediated disassembly of A-complex spliceosomes.

A critical step of pre-mRNA splicing is the recruitment of U2 snRNP to the branch point sequence of an intron. U2 snRNP conformation changes extensively during branch helix formation, and several RNA-dependent ATPases are implicated in the process. However, the molecular mechanisms involved remain to be fully dissected. We took advantage of the differential nucleotide triphosphates requirements for DExD/H-box enzymes to probe their contributions to in vitro spliceosome assembly. Both ATP and GTP hydrolysis support the formation of A-complex, indicating the activity of a DEAH-enzyme because DEAD-enzymes are selective for ATP. We immunodepleted DHX15 to assess its involvement, and although splicing efficiency decreases with reduced DHX15, A-complex accumulation incongruently increases. DHX15 depletion also results in the persistence of the atypical ATP-independent interaction between U2 snRNP and a minimal substrate that is otherwise destabilized in the presence of either ATP or GTP. These results lead us to hypothesize that DHX15 plays a quality control function in U2 snRNP's engagement with an intron. In efforts to identify the RNA target of DHX15, we determined that an extended polypyrimidine tract is not necessary for disruption of the atypical interaction between U2 snRNP and the minimal substrate. We also examined U2 snRNA by RNase H digestion and identified nucleotides in the branch binding region that become accessible with both ATP and GTP hydrolysis, again implicating a DEAH-enzyme. Together, our results demonstrate that multiple ATP-dependent rearrangements are likely involved in U2 snRNP addition to the spliceosome and that DHX15 may have an expanded role in maintaining splicing fidelity.

ARCAM-1 Facilitates Fluorescence Detection of Amyloid-Containing Deposits in the Retina.

Purpose: To investigate the use of an amyloid-targeting fluorescent probe, ARCAM-1, to identify amyloid-containing deposits in the retina of a transgenic mouse model of Alzheimer's disease (AD) and in human postmortem AD patients. Methods: Aged APP/PS1 transgenic AD and wild-type (WT) mice were given an intraperitoneal (IP) injection of ARCAM-1 and their retinas imaged in vivo using a fluorescence ophthalmoscope. Eyes were enucleated and dissected for ex vivo inspection of retinal amyloid deposits. Additionally, formalin-fixed eyes from human AD and control patients were dissected, and the retinas were stained using ARCAM-1 or with an anti-amyloid-ß antibody. Confocal microscopy was used to image amyloid-containing deposits stained with ARCAM-1 or with immunostaining. Results: Four out of eight APP/PS1 mice showed the presence of amyloid aggregates in the retina during antemortem imaging. Retinas from three human AD patients stained with ARCAM-1 showed an apparent increased density of fluorescently labeled amyloid-containing deposits compared to the retinas from two healthy, cognitively normal (CN) patients. Immunolabeling confirmed the presence of amyloid deposits in both the retinal neuronal layers and in retinal vasculature. Conclusions: ARCAM-1 facilitates antemortem detection of amyloid aggregates in the retina of a mouse model for AD, and postmortem detection of amyloid-containing deposits in human retinal tissues from AD patients. These results support the hypothesis of AD pathology manifesting in the eye and highlight a novel area for fluorophore development for the optical detection of retinal amyloid in AD patients. Translational Relevance: This paper represents an initial examination for potential translation of an amyloid-targeting fluorescent probe to a retinal imaging agent for aiding in the diagnosis of Alzheimer's disease.

Agricultural acceleration of soil carbonate weathering.

Soil carbonates (i.e., soil inorganic carbon or SIC) represent more than a quarter of the terrestrial carbon pool and are often considered to be relatively stable, with fluxes significant only on geologic timescales. However, given the importance of climatic water balance on SIC accumulation, we tested the hypothesis that increased soil water storage and transport resulting from cultivation may enhance dissolution of SIC, altering their local stock at decadal timescales. We compared SIC storage to 7.3 m depth in eight sites, each having paired plots of native vegetation and rain-fed croplands, and half the sites having additional irrigated cropland plots. Rain-fed and irrigated croplands had 328 and 730 Mg C/ha less SIC storage, respectively, compared to their native vegetation (grassland or woodland) pairs, and irrigated croplands had 402 Mg C/ha less than their rain-fed pairs (p < .0001). SIC contents were negatively correlated with estimated groundwater recharge, suggesting that dissolution and leaching may be responsible for SIC losses observed. Under croplands, the remaining SIC had more modern radiocarbon and a δ13 C composition that was closer to crop inputs than under native vegetation, suggesting that cultivation has led to faster turnover and incorporation of recent crop carbon into the SIC pool (p < .0001). The losses occurred just 30-100 years after land-use changes, indicating SIC stocks that were stable for millennia can rapidly adjust to increased soil water flows. Large SIC losses (194-242 Mg C/ha) also occurred below 4.9 m deep under irrigated croplands, with SIC losses lagging behind the downward-advancing wetting front by ~30 years, suggesting that even deep SIC were affected. These observations suggest that the vertical distribution of SIC in dry ecosystems is dynamic on decadal timescales, highlighting its potential role as a carbon sink or source to be examined in the context of land use and climate change.

Tissue-type plasminogen activator-primed human iPSC-derived neural progenitor cells promote motor recovery after severe spinal cord injury.

The goal of stem cell therapy for spinal cord injury (SCI) is to restore motor function without exacerbating pain. Induced pluripotent stem cells (iPSC) may be administered by autologous transplantation, avoiding immunologic challenges. Identifying strategies to optimize iPSC-derived neural progenitor cells (hiNPC) for cell transplantation is an important objective. Herein, we report a method that takes advantage of the growth factor-like and anti-inflammatory activities of the fibrinolysis protease, tissue plasminogen activator tPA, without effects on hemostasis. We demonstrate that conditioning hiNPC with enzymatically-inactive tissue-type plasminogen activator (EI-tPA), prior to grafting into a T3 lesion site in a clinically relevant severe SCI model, significantly improves motor outcomes. EI-tPA-primed hiNPC grafted into lesion sites survived, differentiated, acquired markers of motor neuron maturation, and extended ßIII-tubulin-positive axons several spinal segments below the lesion. Importantly, only SCI rats that received EI-tPA primed hiNPC demonstrated significantly improved motor function, without exacerbating pain. When hiNPC were treated with EI-tPA in culture, NMDA-R-dependent cell signaling was initiated, expression of genes associated with stemness (Nestin, Sox2) was regulated, and thrombin-induced cell death was prevented. EI-tPA emerges as a novel agent capable of improving the efficacy of stem cell therapy in SCI.

Mutant UBQLN2 promotes toxicity by modulating intrinsic self-assembly.

UBQLN2 is one of a family of proteins implicated in ubiquitin-dependent protein quality control and integrally tied to human neurodegenerative disease. Whereas wild-type UBQLN2 accumulates in intraneuronal deposits in several common age-related neurodegenerative diseases, mutations in the gene encoding this protein result in X-linked amyotrophic lateral sclerosis/frontotemporal dementia associated with TDP43 accumulation. Using in vitro protein analysis, longitudinal fluorescence imaging and cellular, neuronal, and transgenic mouse models, we establish that UBQLN2 is intrinsically prone to self-assemble into higher-order complexes, including liquid-like droplets and amyloid aggregates. UBQLN2 self-assembly and solubility are reciprocally modulated by the protein's ubiquitin-like and ubiquitin-associated domains. Moreover, a pathogenic UBQLN2 missense mutation impairs droplet dynamics and favors amyloid-like aggregation associated with neurotoxicity. These data emphasize the critical link between UBQLN2's role in ubiquitin-dependent pathways and its propensity to self-assemble and aggregate in neurodegenerative diseases.

Ionotropic glutamate receptors activate cell signaling in response to glutamate in Schwann cells.

In the peripheral nervous system, Schwann cells (SCs) demonstrate surveillance activity, detecting injury and undergoing trans-differentiation to support repair. SC receptors that detect peripheral nervous system injury remain incompletely understood. We used RT-PCR to profile ionotropic glutamate receptor expression in cultured SCs. We identified subunits required for assembly of N-methyl-d-aspartic acid (NMDA) receptors (NMDA-Rs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and kainate receptors. Treatment of SCs with 40-100 µM glutamate or with 0.5-1.0 µM NMDA robustly activated Akt and ERK1/2. The response was transient and bimodal; glutamate concentrations that exceeded 250 µM failed to activate cell signaling. Phosphoprotein profiling identified diverse phosphorylated proteins in glutamate-treated SCs in addition to ERK1/2 and Akt, including p70 S6-kinase, glycogen synthase kinase-3, ribosomal S6 kinase, c-Jun, and cAMP response element binding protein. Activation of SC signaling by glutamate was blocked by EGTA and dizocilpine and by silencing expression of the NMDA-R NR1 subunit. Phosphoinositide 3-kinase/PI3K functioned as an essential upstream activator of Akt and ERK1/2 in glutamate-treated SCs. When glutamate or NMDA was injected directly into crush-injured rat sciatic nerves, ERK1/2 phosphorylation was observed in myelinated and nonmyelinating SCs. Glutamate promoted SC migration by a pathway that required PI3K and ERK1/2. These results identified ionotropic glutamate receptors and NMDA-Rs, specifically, as potentially important cell signaling receptors in SCs.-Campana, W. M., Mantuano, E., Azmoon, P., Henry, K., Banki, M. A., Kim, J. H., Pizzo, D. P., Gonias, S. L. Ionotropic glutamate receptors activate cell signaling in response to glutamate in Schwann cells.

Trade-offs in water and carbon ecosystem services with land-use changes in grasslands.

Increasing pressures for food, fiber, and fuel continue to drive global land-use changes. Efforts to optimize ecosystem services under alternative land uses are often hampered by the complex interactions and trade-offs among them. We examined the effects of land-use changes on ecosystem carbon storage and groundwater recharge in grasslands of Argentina and the United States to (1) understand the relationships between both services, (2) predict their responses to vegetation shifts across environmental gradients, and (3) explore how market or policy incentives for ecosystem services could affect land-use changes. A trade-off of ecosystem services was evident in most cases, with woody encroachment increasing carbon storage (+29 Mg C/ha) but decreasing groundwater recharge (-7.3 mm/yr) and conversions to rain-fed cultivation driving opposite changes (-32 Mg C/ha vs. +13 mm/yr). In contrast, crops irrigated with ground water tended to reduce both services compared to the natural grasslands they replaced. Combining economic values of the agricultural products together with the services, we highlight potentials for relatively modest financial incentives for ecosystem services to abate land-use changes and for incentives for carbon to drive land-use decisions over those of water. Our findings also identify key opportunities and caveats for some win-win and lose-lose land-use changes for more integrative and sustainable strategies for land management.

The effect of hydraulic lift on organic matter decomposition, soil nitrogen cycling, and nitrogen acquisition by a grass species.

Hydraulic lift (HL) is the passive movement of water through plant roots, driven by gradients in water potential. The greater soil-water availability resulting from HL may in principle lead to higher plant nutrient uptake, but the evidence for this hypothesis is not universally supported by current experiments. We grew a grass species common in North America in two-layer pots with three treatments: (1) the lower layer watered, the upper one unwatered (HL), (2) both layers watered (W), and (3) the lower layer watered, the upper one unwatered, but with continuous light 24 h a day to limit HL (no-HL). We inserted ingrowth cores filled with enriched-nitrogen organic matter ((15)N-OM) in the upper layer and tested whether decomposition, mineralization and uptake of (15)N were higher in plants performing HL than in plants without HL. Soils in the upper layer were significantly wetter in the HL treatment than in the no-HL treatment. Decomposition rates were similar in the W and HL treatments and lower in no-HL. On average, the concentration of NH(4)(+)-N in ingrowth cores was highest in the W treatment, and NO(3)(-)-N concentrations were highest in the no-HL treatment, with HL having intermediate values for both, suggesting differential mineralization of organic N among treatments. Aboveground biomass, leaf (15)N contents and the (15)N uptake in aboveground tissues were higher in W and HL than in no-HL, indicating higher nutrient uptake and improved N status of plants performing HL. However, there were no differences in total root nitrogen content or (15)N uptake by roots, indicating that HL affected plant allocation of acquired N to photosynthetic tissues. Our evidence for the role of HL in organic matter decomposition and nutrient cycling suggests that HL could have positive effects on plant nutrient dynamics and nutrient turnover.

Changes in hydrology and salinity accompanying a century of agricultural conversion in Argentina.

Conversions of natural woodlands to agriculture can alter the hydrologic balance, aquifer recharge, and salinity of soils and groundwater in ways that influence productivity and sustainable land use. Using a land-use change chronosequence in semiarid woodlands of Argentina's Espinal province, we examined the distribution of moisture and solutes and estimated recharge rates on adjacent plots of native woodlands and rain-fed agriculture converted 6-90 years previously. Soil coring and geoelectrical profiling confirmed the presence of spatially extensive salt accumulations in dry woodlands and pervasive salt losses in areas converted to agriculture. A 1.1-km-long electrical resistivity transect traversing woodland, 70-year-old agriculture, and woodland, for instance, revealed a low-resistivity (high-salinity) horizon between approximately 3 m and 13 m depth in the woodlands that was virtually absent in the agricultural site because of leaching. Nine-meter-deep soil profiles indicated a 53% increase in soil water storage after 30 or more years of cultivation. Conservative groundwater-recharge estimates based on chloride tracer methods in agricultural plots ranged from approximately 12 to 45 mm/yr, a substantial increase from the <1 mm/yr recharge in dry woodlands. The onset of deep soil moisture drainage and increased recharge led to >95% loss of sulfate and chloride ions from the shallow vadose zone in most agriculture plots. These losses correspond to over 100 Mg of sulfate and chloride salts potentially released to the region's groundwater aquifers through time with each hectare of deforestation, including a capacity to increase groundwater salinity to >4000 mg/L from these ions alone. Similarities between our findings and those of the dryland salinity problems of deforested woodlands in Australia suggest an important warning about the potential ecohydrological risks brought by the current wave of deforestation in the Espinal and other regions of South America and the world.

The sedative but not the memory-blocking properties of ethanol are modulated by α5-subunit-containing γ-aminobutyric acid type A receptors.

The precise mechanisms underlying the memory-blocking properties of ethanol are unknown, in part because ethanol targets a wide array of neurotransmitter receptors and transporters. The aim of this study was to determine whether the memory loss caused by ethanol is mediated, in part, by α5 subunit-containing γ-aminobutyric acid subtype A receptors. These receptors have been implicated in learning and memory processes and are targets for a variety of neurodepressive drugs. Also, since these receptors generate a tonic inhibitory current in hippocampal pyramidal neurons, we examined whether concentrations of ethanol that block memory in vivo increased the tonic current using whole-cell patch-clamp recordings in hippocampal neurons. Null mutant mice lacking the α5 subunit (Gabra5-/-) and wild-type mice were equally impaired in contextual fear conditioning by moderate (1mg/kg) and high (1.5mg/kg) doses of ethanol. The higher dose of ethanol also reduced auditory delay fear conditioning to the same extent in the two genotypes. Interestingly, wild-type mice were more sensitive than Gabra5-/- mice to the sedative effects of low (0.5mg/kg) and moderate (1mg/kg) doses of ethanol in the open-field task. Concentrations of ethanol that impaired memory performance in vivo did not increase the amplitude of the tonic current. Together, the results suggest that the α5-subunit containing γ-aminobutyric acid subtype A receptors are not direct targets for positive modulation by ethanol nor do they contribute to ethanol-induced memory loss. In contrast, these receptors may contribute to the sedative properties of ethanol.

Acquired infection with Toxoplasma gondii in adult mice results in sensorimotor deficits but normal cognitive behavior despite widespread brain pathology.

Toxoplasma gondii is a ubiquitous intracellular parasite which chronically infects 30-50% of the human population. While acquired infection is primarily asymptomatic several studies have suggested that such infections may contribute to neurological and psychiatric symptoms. Previous studies in rodents have demonstrated that T. gondii infection does not just kill its host, but alters the behavioral repertoire of an infected animal, making it more likely that predation with occur completing the parasite life cycle. The aim of the present study was to evaluate the behavioral changes in C57BL/6 mice chronically infected with the avirulent T. gondii (ME49, a Type II strain), in a comprehensive test battery. Infected mice demonstrated profound and widespread brain pathology, motor coordination and sensory deficits. In contrast, cognitive function, anxiety levels, social behavior and the motivation to explore novel objects were normal. The observed changes in behavior did not represent "gross" brain damage or dysfunction and were not due to targeted destruction of specific areas of the brain. Such changes point out the subtle interaction of this parasite with its intermediate hosts and are consistent with ideas about increased predation being an outcome of infection.

Brainstem concentrations of cholesterol are not influenced by genetic ablation of the low-density lipoprotein receptor.

PURPOSE: The low-density lipoprotein receptor (LDLr) mediates the uptake of LDL particles enriched with cholesterol, into several tissues. In contrast to other tissues, the brain is thought to obtain cholesterol solely by de novo synthesis, yet certain brain regions such as the brainstem are highly enriched with the LDLr. The goal of the present study was to assess the role of the LDLr in maintaining cholesterol concentrations in the brainstem of wildtype and LDLr knockout (LDLr-/-) mice. Cholesterol concentrations were also measured in the cortex, which served as a reference point, due to the lower expression of the LDLr, as compared to the brainstem. METHODS: LDLr-/- and wildtype mice consumed an AIN-93G diet ad libitum until 7 weeks of age. After microwaving, the cortex and anterior brain stem were isolated for cholesterol analysis. Cholesterol was extracted into chloroform/methanol, derivatized in trimethylsilyl chloride and measured by gas chromatography/mass spectrometry. RESULTS: Concentrations of cholesterol in the brainstem did not differ statistically between LDLr-/- (18.8 +/- 1.6 mg/g wet weight brain) and wildtype (19.1 +/- 2.0). Cortical cholesterol concentrations also did not differ statistically between LDLr-/- (11.0 +/- 0.4 mg/g wet weight brain) and wildtype (11.1 +/- 0.2) mice. CONCLUSION: The LDLr is not necessary for maintaining cholesterol concentrations in the cortex or brainstem, suggesting that other mechanisms are sufficient to maintain brain cholesterol concentrations.

The low density lipoprotein receptor is not necessary for maintaining mouse brain polyunsaturated fatty acid concentrations.

The brain cannot synthesize n-6 or n-3 PUFAs de novo and requires their transport from the blood. Two models of brain fatty acid uptake have been proposed. One requires the passive diffusion of unesterified fatty acids through endothelial cells of the blood-brain barrier, and the other requires the uptake of lipoproteins via a lipoprotein receptor on the luminal membrane of endothelial cells. This study tested whether the low density lipoprotein receptor (LDLr) is necessary for maintaining brain PUFA concentrations. Because the cortex has a low basal expression of LDLr and the anterior brain stem has a relatively high expression, we analyzed these regions separately. LDLr knockout (LDLr(-/-)) and wild-type mice consumed an AIN-93G diet ad libitum until 7 weeks of age. After microwaving, the cortex and anterior brain stem (pons and medulla) were isolated for phospholipid fatty acid analyses. There were no differences in phosphatidylserine, phosphatidylinositol, ethanolamine, or choline glycerophospholipid esterified PUFA or saturated or monounsaturated fatty acid concentrations in the cortex or brain stem between LDLr(-/-) and wild-type mice. These findings demonstrate that the LDLr is not necessary for maintaining brain PUFA concentrations and suggest that other mechanisms to transport PUFAs into the brain must exist.

Alpha5GABAA receptors mediate the amnestic but not sedative-hypnotic effects of the general anesthetic etomidate.

A fundamental objective of anesthesia research is to identify the receptors and brain regions that mediate the various behavioral components of the anesthetic state, including amnesia, immobility, and unconsciousness. Using complementary in vivo and in vitro approaches, we found that GABAA receptors that contain the alpha5 subunit (alpha5GABAARs) play a critical role in amnesia caused by the prototypic intravenous anesthetic etomidate. Whole-cell recordings from hippocampal pyramidal neurons showed that etomidate markedly increased a tonic inhibitory conductance generated by alpha5GABAARs, whereas synaptic transmission was only slightly enhanced. Long-term potentiation (LTP) of field EPSPs recorded in CA1 stratum radiatum was reduced by etomidate in wild-type (WT) but not alpha5 null mutant (alpha5-/-) mice. In addition, etomidate impaired memory performance of WT but not alpha5-/- mice for spatial and nonspatial hippocampal-dependent learning tasks. The brain concentration of etomidate associated with memory impairment in vivo was comparable with that which increased the tonic inhibitory conductance and blocked LTP in vitro. The alpha5-/- mice did not exhibit a generalized resistance to etomidate, in that the sedative-hypnotic effects measured with the rotarod, loss of righting reflex, and spontaneous motor activity were similar in WT and alpha5-/- mice. Deletion of the alpha5 subunit of the GABAARs reduced the amnestic but not the sedative-hypnotic properties of etomidate. Thus, the amnestic and sedative-hypnotic properties of etomidate can be dissociated on the basis of GABAAR subtype pharmacology.