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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 1,243 records · Page 69
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Cephalosporin antibiotics are weak blockers of GABAa receptor-mediated synaptic transmission in rat brain slices

Highlights: • Millimolar concentrations of cephalosporins are required to block eIPSCs. • Cefepime and ceftriaxone have different mechanisms of GABAa receptor inhibition. • Cephalosporins alone fail to induce consistent epileptiform activity in brain slices. Cephalosporins are beta-lactam antibiotics that are extensively used in medical practice and are reported to cause epileptic seizures in some patients. The primary cause of cephalosporin-induced convulsions is believed to be their ability to block GABAa receptors. However, direct evidence for the involvement of this mechanism has not yet been provided. The present study aims to investigate the ability of two cephalosporins – cefepime and ceftriaxone – to block inhibitory synaptic transmission in entorhinal cortex slices of rats. Using the whole-cell patch-clamp method, we found that millimolar concentrations of cefepime (IC{sub 50} = 1.6 ± 0.1 mM) and ceftriaxone (2.0 ± 0.1 mM) were required to block the evoked inhibitory postsynaptic currents (IPSCs). These concentrations are almost two orders of magnitude higher than cerebrospinal fluid concentrations of antibiotics achieved during treatment. We also found that while ceftriaxone did not affect the IPSC decay kinetics, cefepime significantly slowed the decays of the evoked currents, which may be attributed to the diverse mechanisms of the GABAa receptor inhibition of cefepime and ceftriaxone. The experiments involving the fast application of GABA at various concentrations to isolated neurons suggests that cefepime blocks receptors competitively, while ceftriaxone does so noncompetitively. Cefepime, at a concentration of up to 4 mM, was unable to produce seizure-like events in brain slices. However, this antibiotic could induce epileptiform activity in combination with the altered ionic composition of the perfusing media, which may be the case for patients with renal insufficiency. Our results suggest that cefepime and ceftriaxone are weak GABAa receptor blockers and that it is unlikely that the inhibition of GABAa receptors by antibiotics is the primary cause of the seizures.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Particulate matter increases beta-amyloid and activated glial cells in hippocampal tissues of transgenic Alzheimer's mouse: Involvement of PARP-1

Highlights: • Particulate matter (PM) activates PARP-1 in neuronal cells. • PM increases beta-amyloid in ex vivo hippocampus of 3xTg-AD mouse. • PM activates glial cells in ex vivo hippocampus of 3xTg-AD mouse. • Pharmacological inhibition of PARP-1 reverses PM-induced AD pathologies. • PARP-1 inhibition would be a promising approach counteracting PM's neurotoxicity. Exposure to air pollutants, such as particulate matter (PM), has been implicated in neurodegenerative disorders including Alzheimer's disease (AD). However, direct effects of PM on production of β-amyloid (Aβ), a key pathogenic molecule in AD, and its underlying mechanism are still elusive. Given PM's potential to induce oxidative stress in other tissues, we hypothesized that poly(ADP-ribose) polymerase (PARP-1) might be involved in PM-induced neurotoxicity. To address this, we used an ex vivo model of AD, the organotypic hippocampal slice tissue culture from old (12-14 months-of-age) triple transgenic 3xTg-AD mice. First, we observed that fine PM (aerodynamic diameter < 4 μm) can dose-dependently activate PARP-1 and decrease NAD{sup +} levels in Neuro2A cells. PARP-1 activation did occur under concentrations of PM which did not affect cell viability. Next, we observed that direct treatment of PM increased Aβ levels and activated glial cells in the ex vivo hippocampal tissues of 3xTg-AD mice. PM-induced glial activation was most prominent in CA1 region of the hippocampal tissue. Notably, we found that pharmacological inhibition of PARP-1 reversed both PM-induced Aβ increase and glial activation, arguing the possible involvement of PARP-1 in PM-induced AD pathogenesis. Our findings suggest that PARP-1 might be a potential molecular target, responsible for mediating negative effects of PM on the brain. Modulating PARP-1 activity could be a promising approach to prevent or alleviate PM-related environmental neurotoxicity which could initiate AD pathogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Investigation of TRPV1 loss-of-function phenotypes in TRPV1 Leu206Stop mice generated by N-ethyl-N-nitrosourea mutagenesis

Highlights: • N-ethyl-N-nitrosourea mutagenesis can generate mouse models for pain research. • TRPV1 Leu206Stop mice closely resemble the phenotype of TRPV1 knockout mice. • Mechanical hypersensitivity is not impaired in TRPV1 Leu206Stop mice. N-ethyl-N-nitrosourea (ENU) random mutagenesis was used to generate a mouse model for the analysis of the transient receptor potential vanilloid 1 (TRPV1) cation channel. A transversion from T→A in exon 4 led to a Leu206Stop mutation generating a loss-of-function mutant. The TRPV1 agonist capsaicin was used to analyze functional and nociceptive parameters in vitro and in vivo in TRPV1 Leu206Stop mice and congenic C3HeB/FeJ controls. Capsaicin-induced [Ca{sup 2+}]{sub i} changes in small diameter DRG neurons were significantly diminished in TRPV1 Leu206Stop mice and administration of capsaicin induced neither hypothermia nor nocifensive behaviour in vivo. TRPV1 Leu206Stop mice were tested in the spinal nerve ligation of mononeuropathic pain and developed mechanical hypersensitivity two weeks after nerve injury. In the open field test, a significant increase in spontaneous locomotion was detected in TRPV1 Leu206Stop mice as compared to wildtype controls. TRPV1 knockout mice have been reported to carry a similar phenotype regarding capsaicin-evoked responses in vitro and in vivo. However, in contrast to TRPV1 Leu206Stop mice, TRPV1 knockout mice did not differ in spontaneous locomotion as compared to congenic C57BL/6 mice, suggesting subtle ENU-dependent or independent strain differences between TRPV1 Leu206Stop mice and their wildtype controls. In summary, these data revealed a target-related (i.e. capsaicin-evoked) phenotype of TRPV1 Leu206Stop mice closely resembling that of published TRPV1 knockout mice. However, since ENU-mutant mice are congenic with the mouse strain initially used in random mutagenesis, direct phenotypic comparison with the respective wildtype controls is possible, and the time-consuming backcrossing in lines with targeted mutations is avoided.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Aβ1-40 mediated aggregation of proteins and metabolites unveils the relevance of amyloid cross-seeding in amyloidogenesis

The multicomponent nature of neuronal plaques in Alzheimer's disease signifies the possible recruitment of non-Aβ candidates during the amyloid growth of Aβ peptides. Here, we show that amyloid fibrils of Aβ{sub 1-40} peptide can effectively initiate amyloid formation in different globular proteins and metabolites, converting native structures into β-sheet rich assemblies. Structural and biophysical properties of the resultant protein fibrils display amyloid like characteristic features. Viable contacts between Aβ peptide's cross-β architecture and the native structure of proteins, mediated through H-bonds and hydrophobic interactions seem crucial for the onset of amyloid cross-seeding. Results reveal the inherent cross-seeding potential of Aβ amyloids to initiate amyloid formation process in proteins and metabolites and revelation of such a property may further our mechanistic understanding of amyloid pathologies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Circular RNA expression profiles in hippocampus from mice with perinatal glyphosate exposure

Glyphosate is the active ingredient in numerous herbicide formulations. The roles of glyphosate in embryo-toxicity and neurotoxicity have been reported in human and animal models. Recently, several studies have reported evidence linking neurodevelopmental disorders (NDDs) with gestational glyphosate exposure. However, the role of glyphosate in neuronal development is still not fully understood. Our previous study found that perinatal glyphosate exposure resulted in differential microRNA expression in the prefrontal cortex of mouse offspring. However, the mechanism of glyphosate-induced neurotoxicity in the developing brain is still not fully understood. Considering the pivotal role of Circular RNAs (circRNAs) in the regulation of gene expression, a circRNA microarray method was used in this study to investigate circRNA expression changes in the hippocampus of mice with perinatal glyphosate exposure. The circRNA microarrays revealed that 663 circRNAs were significantly altered in the perinatal glyphosate exposure group compared with the control group. Among them, 330 were significantly upregulated, and the other 333 were downregulated. Furthermore, the relative expression levels of mmu-circRNA-014015, mmu-circRNA-28128 and mmu-circRNA-29837 were verified using quantitative real-time polymerase chain reaction (qRT-PCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses demonstrated that stress-associated steroid metabolism pathways, such as aldosterone synthesis and secretion pathways, may be involved in the neurotoxicity of glyphosate. These results showed that circRNAs are aberrantly expressed in the hippocampus of mice with perinatal glyphosate exposure and play potential roles in glyphosate-induced neurotoxicity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Modulation of Angiopoietin 2 release from endothelial cells and angiogenesis by the synaptic protein Neuroligin 2

The synaptic protein Neuroligin 2, similarly to its isoform Neuroligin 1, is produced by endothelial cells, but its activity in the vascular context remains unknown. This study aimed at verifying the hypothesis that Neuroligin 2, in parallel with its extraneuronal involvement in pancreatic beta cells exocytosis, modulated cytokine release from endothelial cells and consequently angiogenesis. }We used in vitro approaches to modulate Neuroligin 2 expression and Neuroligin 2 null mice to test our hypotheses. In vitro, upon VEGF stimulation, Neuroligin 2 silencing strongly reduces Angiopoietin 2 release in the medium and increases the endothelial cell retention of Weibel Palade Bodies, the specialized organelles that store Angiopoietin 2 and various other cytokines. On the contrary, Neuroligin 2 overexpression almost depletes cells of Weibel Palade Bodies, independent of VEGF. In vivo, both the retina and tumor xenografts grown in NLGN2- null mice display an immature vasculature, with lower pericyte coverage and lower Tie2 phosphorylation. At the molecular level NLGN2 colocalizes with its neuronal partner collibystin, a CDC42 guanine nucleotide exchange factor, which is also expressed by endothelial cells and in turn modulates Angiopoietin 2 release. }Neuroligin 2, an inhibitory synaptic protein, modulates a peculiar aspect of vascular function and could represent a novel target of therapy in various fields, from tumor angiogenesis to vascular diseases.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Small GTPase R-Ras participates in neural tube formation in zebrafish embryonic spinal cord

Ras related (R-Ras), a small GTPase, is involved in the maintenance of apico-basal polarity in neuroepithelial cells of the zebrafish hindbrain, axonal collapse in cultured murine hippocampal neurons, and maturation of blood vessels in adult mice. However, the role of R-Ras in neural tube formation remains unknown. Using antisense morpholino oligonucleotides (AMOs), we found that in the spinal cord of zebrafish embryos, the lumen was formed bilaterally in rras morphants, whereas it was formed at the midline in control embryos. As AMO can cause off-target effects, we generated rras mutant zebrafish lines using CRISPR/Cas9 technology. Although these rras mutant embryos did not have a bilateral lumen in the spinal cord, the following findings suggest that the phenotype is unlikely due to an off-target effect of rras AMO: 1) The rras morphant phenotype was rescued by an injection of AMO-resistant rras mRNA, and 2) a bilaterally segregated spinal cord was not observed in rras mutant embryos injected with rras AMO. The results suggest that the function of other ras family genes may be redundant in rras mutants. Previous research reported a bilaterally formed lumen in the spinal cord of zebrafish embryos with a mutation in a planar cell polarity (PCP) gene, van gogh-like 2 (vangl2). In the present study, in cultured cells, R-Ras was co-immunoprecipitated with Vangl2 but not with another PCP regulator, Pricke1. Interestingly, the interaction between R-Ras and Vangl2 was stronger in guanine-nucleotide free point mutants of R-Ras than in wild-type or constitutively active (GTP-bound) forms of R-Ras. R-Ras may regulate neural tube formation in cooperation with Vangl2 in the developing zebrafish spinal cord.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

GAPDH-targeted therapy – A new approach for secondary damage after traumatic brain injury on rats

Highlights: • Exogenous GAPDH interact with SH-SY5Y cells and penetrate them. • Exogenous GAPDH induce death of SH-SY5Y cells. • Hydrocortysone-21-hemisuccinate reduced cytotoxic effect of exogenous GAPDH. • Hydrocortisone derivative rescue motor function of rats after traumatic brain injure. Massive neuronal death caused by a neurodegenerative pathology or damage due to ischaemia or traumatic brain injury leads to the appearance of cytosolic proteins in the extracellular space. We found that one of the most abundant cellular polypeptides, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), appearing in the medium of dying cells or body fluids is able to form aggregates that are cytotoxic to adjacent cells. Since we previously showed that the hydrocortisone derivative RX624 can inhibit the ability of GAPDH to transport the enzyme complex with polyglutamine and reduce the cytotoxicity of the complex, we explored the effects of GAPDH on SH-SY5Y neuroblastoma cells. We found that the latter treated with particular forms of GAPDH molecules die with a high efficiency, suggesting that the exogenous enzyme does kill adjacent cells. RX624 prevented the interaction of exogenous GAPDH with the cell membrane and reduced the level of death by more than 10%. We also demonstrated the efficiency of RX624 treatment in a rat model of traumatic brain injury. The chemical blocked the formation of GAPDH aggregates in the brain, inhibited the cytotoxic effects of cerebrospinal fluid and rescued the motor function of injured rats. Importantly, RX624 treatment of rats had a similar effect as the intracranial injection of anti-GAPDH antibodies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Expression of ZO1, vimentin, pan-cadherin and AGTR1 in tanycyte-like cells of the sulcus medianus organum

Highlights: • Sulcus Medianus Organum contains ependyma with distinct tanycyte-like population. • These cells express ZO1, vimentin, pan-cadherin and angiotensin II type 1 receptors. • Long basal processes bridge these tanycyte-like cells to deeper blood vessels. Tanycytes are a specialized ependymal lining of brain ventricles with exceptional features of having long basal processes and junctional complexes between cell bodies. These tanycytes are present at the regions of circumventricular organs (CVOs) which possess common morphological and functional features enabling them to be described as the brain windows where the barrier systems have special properties. Previous studies detailed seven of these CVOs but little information is available regarding another putative site at the rostral part of the median sulcus of the 4th ventricle, or the sulcus medianus organum (SMO). Here we performed a pilot immunohistochemical study to support earlier observations suggesting the SMO as a novel CVO. We labeled rat brain with ZO1, vimentin, pan-cadherin and angiotensin II type 1 receptors markers which showed a morphologically distinct population of cells at the region of the SMO similar to tanycytes present in the median eminence, a known CVO. These cells had basal processes reaching the deeply seated blood vessels while the caudal part of the median sulcus did not show similar long cellular extensions. We concluded that tanycyte-like cells are present in the SMO in a pattern resembling that of other CVOs where the strategic location of the SMO is probably for signal integration between brainstem nuclei and the rostrally located neuronal centers.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Activation of β- and α2-adrenergic receptors stimulate tubulin polymerization and promote the association of Gβγ with microtubules in cultured NIH3T3 cells

Highlights: • GPCR activation stimulates microtubule assembly in NIH3T3 cells. • GPCR activation increases Gβγ association with microtubules. • β- and α2-adrenergic receptors have similar effects on microtubule assembly. • Signaling via GPCR is important for MT assembly and regulation. Microtubules (MTs) constitute a crucial part of the cytoskeleton and are essential for cell division and differentiation, cell motility, intracellular transport, and cell morphology. Precise regulation of MT assembly and dynamics is essential for the performance of these functions. Although much progress has been made in identifying and characterizing the cellular factors that regulate MT assembly and dynamics, signaling events in this process is not well understood. Gβγ, an important component of the G protein-coupled receptor (GPCR) signaling pathway, has been shown to promote MT assembly in vitro and in cultured NIH3T3 and PC12 cells. Using the MT depolymerizing agent nocodazole, it has been demonstrated that the association of Gβγ with polymerized tubulin is critical for MT assembly. More recently, Gβγ has been shown to play a key role in NGF-induced neuronal differentiation of PC12 cells through its interaction with tubulin/MTs and modulation of MT assembly. Although NGF is known to exert its effect through tyrosine kinase receptor TrkA, the result suggests a possible involvement of GPCRs in this process. The present study was undertaken to determine whether agonist activation of GPCR utilizes Gβγ to promote MT assembly. We used isoproterenol and UK 14,304, agonists for two different GPCRs (β- and α2-adrenergic receptors, respectively) known to activate Gs and Gi respectively, with an opposing effect on production of cAMP. The results demonstrate that the agonist activation of β- and α2-adrenergic receptors promotes the association of Gβγ with MTs and stimulates MT assembly in NIH3T3 cells. Interestingly, the effects of these two agonists were more prominent when the cellular level of MT assembly was low (30% or less). In contrast to MT assembly, actin polymerization was not affected by isoproterenol or UK 14, 304 indicating that the effects of these agonists are limited to MTs. Thus, it appears that, upon cellular demand, GPCRs may utilize Gβγ to promote MT assembly. Stimulation of MT assembly appears to be a novel function of G protein-mediated signaling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

UCH-L1 inhibition aggravates mossy fiber sprouting in the pentylenetetrazole kindling model

Highlights: • UCH-L1 mainly locates in cytomembrane and cytoplasm of neuron, while rarely located in gliocyte. • UCH-L1 is increased in PTZ-induced epilepsy rats and co-relates with tau protein. • Inhibition of UCH-L1 aggravates the severity of seizures, the accumulation of hyperphosphorylated tau and MSF. Mossy fiber sprouting (MFS) is a pathological phenomenon that is commonly observed in epilepsy, and plentiful data reveal that abnormal phosphorylated modification of tau protein plays a critical role in MSF by the regulation of microtubule dynamics and axonal transport. Ubiquitin C-terminal hydrolase L1 (UCH-L1), a proteasomal deubiquitinating enzyme, has been proved to be associated with tau aggregation through mediating degradation of ubiquitinated and hyperphosphorylated tau. Thus, this study aimed to determine the expression of UCH-L1 in the rat hippocampus during the pentylenetetrazole (PTZ)-induced process and to demonstrate the possible correlation with MFS in epileptogenesis. Seizures were established by intraperitoneal injection of PTZ and LDN-57444 was used to inhibit the hydrolase activity of UCH-L1. We used western blot, immunofluorescence, immunoprecipitation, and timm staining to detect phosphorylated modification of tau and MSF. The results presented that LDN-57444 induced the deteriorated severity of seizures, increased phosphorylation of tau and increased distribution of Timm granules in both the supragranular region of the dentate gyrus (DG) and the stratum pyramidale of CA3 subfield. Our results suggest that UCH-L1 may be associated with hippocampal MSF followed the epileptogenesis through mediating phosphorylation of tau. UCH-L1 may be a potential and novel therapeutic target to limit epileptogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Natronomonas pharaonis halorhodopsin Ser81 plays a role in maintaining chloride ions near the Schiff base

Highlights: • S81A mutant of NpHR lose light-induced photocurrent. • The direction of the Thr126 side chain was fixed by a hydroxyl group of Ser81. • S81A mutation decrease the interaction between Thr126 and a chloride ion. • Ser81 plays a key role in chloride migration. Optogenetic technologies have often been used as tools for neuronal activation or silencing by light. Natronomonas pharaonis halorhodopsin (NpHR) is a light-driven chloride ion pump. Upon light absorption, a chloride ion passes through the cell membrane, which is accompanied by the temporary binding of a chloride ion with Thr126 at binding site-1 (BS1) near the protonated Schiff base in NpHR. However, the mechanism of stabilization of the binding state between a chloride ion and BS1 has not been investigated. Therefore, to identify a key component of the chloride ion transport pathway as well as to acquire dynamic information about the chloride ion-BS1 binding state, we performed a rough analysis of the chloride ion pathway shape followed by molecular dynamics (MD) simulations for both wild-type and mutant NpHR structures. The MD simulations showed that the hydrogen bond between Thr126 and the chloride ion was retained in the wild-type protein, while the chloride ion could not be retained at and tended to leave BS1 in the S81A mutant. We found that the direction of the Thr126 side chain was fixed by a hydroxyl group of Ser81 through a hydrogen bond and that Thr126 bound to a chloride ion in the wild-type protein, while this interaction was lost in the S81A mutant, resulting in rotation of the Thr126 side chain and reduction in the interaction between Thr126 and a chloride ion. To confirm the role of S81, patch clamp recordings were performed using cells expressing NpHR S81A mutant protein. Considered together with the results that the NpHR S81A-expressing cells did not undergo hyperpolarization under light stimulation, our results indicate that Ser81 plays a key role in chloride migration. Our findings might be relevant to ongoing clinical trials using optogenetic gene therapy in blind patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Palmitate induces lipoapoptosis in Schwann cells through ROS generation-mediated STAMP2 downregulation

Free fatty acids (FFAs) are considered the principal inducers of lipotoxicity, leading to cell dysfunction and/or cell death. Lipotoxicity in Schwann cells (SCs) damages neurons, which may be associated with peripheral neuropathies and axon degeneration. However, the molecular mechanism by which FFAs exert lipotoxicity in SCs remains to be established. In the present study, we demonstrate that palmitate exerts lipotoxicity in SCs through apoptosis and that palmitate-induced lipotoxicity in SCs is mediated through reactive oxygen species (ROS) generation. We observed that the six-transmembrane protein of prostate 2 (STAMP2), which plays a pivotal role in lipid homeostasis, is expressed in SCs. We further demonstrate that palmitate induces lipoapoptosis in SCs through ROS generation-mediated STAMP2 downregulation and that STAMP2 depletion accelerates the palmitate-exerted lipoapoptosis in SCs, indicating that STAMP2 confers on SCs the ability to resist palmitate-induced lipotoxicity. In conclusion, palmitate induces lipoapoptosis in SCs through ROS generation-mediated STAMP2 downregulation. Our findings indicate that ROS and STAMP2 may represent suitable targets for pharmacological interventions targeting lipotoxicity-associated peripheral neuropathies and axon degeneration.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Microglia support neural stem cell maintenance and growth

Increasing evidence suggests that disease-associated microglia play a protective role in neurodegenerative diseases. Microglia are known to polarize into two reciprocate forms in response to external cues - inflammatory M1 state and anti-inflammatory M2 state. These cells perform key functions in the development of the brain, such as circuit refinement, neurogenesis, and neuronal growth. In this study, we analyzed the secretion effect of microglia on neural stem/progenitor cell (NSPC) proliferation and differentiation. We cultured adult mouse-derived NSPCs in a conditioned medium from BV2 immortalized microglia without growth factors and evaluated their differentiation. When cultivated with BV2-derived soluble factors in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), NSPCs were able to maintain Nestin expression and showed increased proliferation compared with those cells cultivated with bFGF and EGF only. Moreover, conditioned media from M2-polarized primary microglia, stimulated by IL-10/IL-13, showed supportive effect on NSPC proliferation. These data suggest that microglia support neural stem cell proliferation through secreting neuro-nutritious soluble factors.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

CTCF is required for maintenance of auditory hair cells and hearing function in the mouse cochlea

Auditory hair cells play an essential role in hearing. These cells convert sound waves, mechanical stimuli, into electrical signals that are conveyed to the brain via spiral ganglion neurons. The hair cells are located in the organ of Corti within the cochlea. They assemble in a special arrangement with three rows of outer hair cells and one row of inner hair cells. The proper differentiation and preservation of auditory hair cells are essential for acquiring and maintaining hearing function, respectively. Many genetic regulatory mechanisms underlying hair-cell differentiation and maintenance have been elucidated to date. However, the role of epigenetic regulation in hair-cell differentiation and maintenance has not been definitively demonstrated. CTCF is an essential epigenetic component that plays a primary role in the organization of global chromatin architecture. To determine the role of CTCF in mammalian hair cells, we specifically deleted Ctcf in developing hair cells by crossing Ctcf{sup fl/fl} mice with Gfi1{sup Cre/+} mice. Gfi1{sup Cre}; Ctcf{sup fl/fl} mice did not exhibit obvious developmental defects in hair cells until postnatal day 8. However, at 3 weeks, the Ctcf deficiency caused intermittent degeneration of the stereociliary bundles of outer hair cells, resulting in profound hearing impairment. At 5 weeks, most hair cells were degenerated in Gfi1{sup Cre}; Ctcf{sup fl/fl} mice, and defects in other structures of the organ of Corti, such as the tunnel of Corti and Nuel's space, became apparent. These results suggest that CTCF plays an essential role in maintaining hair cells and hearing function in mammalian cochlea.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

A revisit of the conformational dynamics of SNARE protein rYkt6

N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in the fusion of vesicles with their target membranes. R-SNARE protein Ykt6 is one of the most conserved SNARE in eukaryotes. The conformational state of Ykt6 is regulated by the lipidations at its C-terminal motif. Previous studies show that the binding of dodecylphosphocholine (DPC) can stabilize a closed conformation of rat Ykt6 (rYkt6) and mimic the farnesylated rYkt6. Despite this model, the detailed conformational dynamics of Ykt6 is still unclear. Here, we combined smFRET and MD simulation to demonstrate that the un-lipidated rYkt6 adopts five major conformational states. DPC binding shifts the conformational distribution toward the more closed states. At the same time, there remain considerable fractions of open and semi-open conformations in the presence of DPC. These newly revealed dynamic features of rYkt6 are consistent with its unique functional diversity in neuronal cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Protective effect of the imidazoline I2 receptor agonist 2-BFI on oxidative cytotoxicity in astrocytes

Highlights: • 2-BFI in H{sub 2}O{sub 2}-exposed astrocytes protects cell death. • 2-BFI in H{sub 2}O{sub 2}-exposed astrocytes increased lysosomal membrane stability. • 2-BFI in H{sub 2}O{sub 2}-exposed astrocytes activates autophagy and Cat D maturation. • The cytoprotective effects of 2-BFI may occurs through lysosomal stability. Astrocytes perform a variety of functions that are important for normal neuronal activity and recovery after brain injury. Because astrocytes are very vulnerable to H{sub 2}O{sub 2}, protection of astrocytes from oxidative damage in various neurological diseases is important in maintaining brain function and preventing brain damage. In this study, we investigated the characteristics and mechanisms of a specific imidazoline I2 receptor agonist 2-BFI-mediated cytoprotection using a rat astrocyte cultures of H{sub 2}O{sub 2}-exposed oxidative stress. Here we show that 2-BFI in H{sub 2}O{sub 2}-exposed astrocytes protects cell death through increased lysosomal membrane stability, LC3-II conversion, and subsequently suppresses accumulation of p62. These effects of 2-BFI were significantly reversed after treatment with the lysozyme activity inhibitor Bafilomycin A1. These results suggest that the cytoprotective effects of 2-BFI, which increases lysosomal stability in oxidative stress, may involve regulation of lysosomal-associated membrane protein-dependent autophagy and autolysosome degradation in astrocytes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Crystal structure of GSK3β in complex with the flavonoid, morin

GSK3β is a key kinase that plays a role in cellular signaling pathways. In Alzheimer's disease (AD), GSK3β has been implicated in hyperphosphorylation of tau proteins in the neuron, which is a hallmark of AD. Morin, a flavonoid that is abundant in nature, was found as an inhibitor of GSK3β that can reduce tau pathology in vivo and in vitro. In this study, we determined the crystal structure of GSK3β in complex with morin. The structure revealed that morin inhibits GSK3β by binding to the ATP binding pocket. Our findings augment the potential of morin as a functional food to help prevent AD, as well as to provide structural information to develop new therapeutics based on the morin skeleton.

60 APPLIED LIFE SCIENCES↗