Genetic ablation of Saposin-D in Krabbe disease eliminates psychosine accumulation but does not significantly improve demyelination.

Krabbe disease is an inherited demyelinating disease caused by a genetic deficiency of the lysosomal enzyme galactosylceramide (GalCer) ß-galactosidase (GALC). The Twitcher (Twi) mouse is a naturally occurring, genetically and enzymatically authentic mouse model that mimics infantile-onset Krabbe disease. The major substrate for GALC is the myelin lipid GalCer. However, the pathogenesis of Krabbe disease has long been explained by the accumulation of psychosine, a lyso-derivative of GalCer. Two metabolic pathways have been proposed for the accumulation of psychosine: a synthetic pathway in which galactose is transferred to sphingosine and a degradation pathway in which GalCer is deacylated by acid ceramidase (ACDase). Saposin-D (Sap-D) is essential for the degradation of ceramide by ACDase in lysosome. In this study, we generated Twi mice with a Sap-D deficiency (Twi/Sap-D KO), which are genetically deficient in both GALC and Sap-D and found that very little psychosine accumulated in the CNS or PNS of the mouse. As expected, demyelination with the infiltration of multinucleated macrophages (globoid cells) characteristic of Krabbe disease was milder in Twi/Sap-D KO mice than in Twi mice both in the CNS and PNS during the early disease stage. However, at the later disease stage, qualitatively and quantitatively comparable demyelination occurred in Twi/Sap-D KO mice, particularly in the PNS, and the lifespans of Twi/Sap-D KO mice were even shorter than that of Twi mice. Bone marrow-derived macrophages from both Twi and Twi/Sap-D KO mice produced significant amounts of TNF-α upon exposure to GalCer and were transformed into globoid cells. These results indicate that psychosine in Krabbe disease is mainly produced via the deacylation of GalCer by ACDase. The demyelination observed in Twi/Sap-D KO mice may be mediated by a psychosine-independent, Sap-D-dependent mechanism. GalCer-induced activation of Sap-D-deficient macrophages/microglia may play an important role in the neuroinflammation and demyelination in Twi/Sap-D KO mice.

Detection of Arc/Arg3.1 oligomers in rat brain: constitutive and synaptic activity-evoked dimer expression in vivo.

The immediate early gene product activity-regulated cytoskeleton-associated protein (Arc or Arg3.1) is a major regulator of long-term synaptic plasticity with critical roles in postnatal cortical development and memory formation. However, the molecular basis of Arc function is undefined. Arc is a hub protein with interaction partners in the postsynaptic neuronal compartment and nucleus. Previous in vitro biochemical and biophysical analysis of purified recombinant Arc showed formation of low-order oligomers and larger particles including retrovirus-like capsids. Here, we provide evidence for naturally occurring Arc oligomers in the mammalian brain. Using in situ protein crosslinking to trap weak Arc-Arc interactions, we identified in various preparations a prominent Arc immunoreactive band on SDS-PAGE of molecular mass corresponding to a dimer. While putative trimers, tetramers and heavier Arc species were detected, they were of lower abundance. Stimulus-evoked induction of Arc expression and dimer formation was first demonstrated in SH-SY5Y neuroblastoma cells treated with the muscarinic cholinergic agonist, carbachol, and in primary cortical neuronal cultures treated with brain-derived neurotrophic factor (BDNF). In the dentate gyrus (DG) of adult anesthetized rats, induction of long-term potentiation (LTP) by high-frequency stimulation (HFS) of medial perforant synapses or by brief intrahippocampal infusion of BDNF led to a massive increase in Arc dimer expression. Arc immunoprecipitation of crosslinked DG tissue showed enhanced dimer expression during 4 h of LTP maintenance. Mass spectrometric proteomic analysis of immunoprecipitated, gel-excised bands corroborated detection of Arc dimer. Furthermore, Arc dimer was constitutively expressed in naïve cortical, hippocampal and DG tissue, with the lowest levels in the DG. Taken together the results implicate Arc dimer as the predominant low-oligomeric form in mammalian brain, exhibiting regional differences in its constitutive expression and enhanced synaptic activity-evoked expression in LTP.

The Urinary Bladder is Rich in Glycosphingolipids Composed of Phytoceramides.

Glycosphingolipids (GSLs) are composed of a polar glycan chain and a hydrophobic tail known as ceramide. Together with variation in the glycan chain, ceramides exhibit tissue-specific structural variation in the long-chain base (LCB) and N-acyl chain moieties in terms of carbon chain length, degree of desaturation, and hydroxylation. Here, we report the structural variation in GSLs in the urinary bladders of mice and humans. Using TLC, we showed that the major GSLs are hexosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, Neu5Ac-Gal-Glc-Ceramide, and Neu5Ac-Neu5Ac-Gal-Glc-Ceramide. Our LC-MS analysis indicated that phytoceramide structures with a 20-carbon LCB (4-hydroxyeicosasphinganine) and 2-hydroxy fatty acids are abundant in hexosylceramide and Neu5Ac-Gal-Glc-Ceramide in mice and humans. In addition, quantitative PCR demonstrated that DES2 and FA2H, which are responsible for the generation of 4-hydroxysphinganine and 2-hydroxy fatty acid, respectively, and SPTLC3 and SPTSSB, which are responsible for the generation of 20-carbon LCBs, showed significant expressions in the epithelial layer than in the subepithelial layer. Immunohistochemically, dihydroceramide:sphinganine C4-hydroxylase (DES2) was expressed exclusively in urothelial cells of the urinary bladder. Our findings suggest that these ceramide structures have an impact on membrane properties of the stretching and shrinking in transitional urothelial cells.

Development and Validation of Arc Nanobodies: New Tools for Probing Arc Dynamics and Function.

Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-tagged ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N- and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and C-terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc, and interrogation of Arc capsid domain specific functions.

In vitro and in vivo functions of T cells produced in complemented thymi of chimeric mice generated by blastocyst complementation.

Blastocyst complementation is an intriguing way of generating humanized animals for organ preparation in regenerative medicine and establishing novel models for drug development. Confirming that complemented organs and cells work normally in chimeric animals is critical to demonstrating the feasibility of blastocyst complementation. Here, we generated thymus-complemented chimeric mice, assessed the efficacy of anti-PD-L1 antibody in tumor-bearing chimeric mice, and then investigated T-cell function. Thymus-complemented chimeric mice were generated by injecting C57BL/6 (B6) embryonic stem cells into Foxn1nu/nu morulae or blastocysts. Flow cytometry data showed that the chimeric mouse thymic epithelial cells (TECs) were derived from the B6 cells. T cells appeared outside the thymi. Single-cell RNA-sequencing analysis revealed that the TEC gene-expression profile was comparable to that in B6 mice. Splenic T cells of chimeric mice responded very well to anti-CD3 stimulation in vitro; CD4+ and CD8+ T cells proliferated and produced IFNγ, IL-2, and granzyme B, as in B6 mice. Anti-PD-L1 antibody treatment inhibited MC38 tumor growth in chimeric mice. Moreover, in the chimeras, anti-PD-L1 antibody restored T-cell activation by significantly decreasing PD-1 expression on T cells and increasing IFNγ-producing T cells in the draining lymph nodes and tumors. T cells produced by complemented thymi thus functioned normally in vitro and in vivo. To successfully generate humanized animals by blastocyst complementation, both verification of the function and gene expression profiling of complemented organs/cells in interspecific chimeras will be important in the near future.

Herpes Simplex Virus Type 1 Neuronal Infection Triggers the Disassembly of Key Structural Components of Dendritic Spines.

Herpes simplex virus type 1 (HSV-1) is a widespread neurotropic virus. Primary infection of HSV-1 in facial epithelium leads to retrograde axonal transport to the central nervous system (CNS) where it establishes latency. Under stressful conditions, the virus reactivates, and new progeny are transported anterogradely to the primary site of infection. During the late stages of neuronal infection, axonal damage can occur, however, the impact of HSV-1 infection on the morphology and functional integrity of neuronal dendrites during the early stages of infection is unknown. We previously demonstrated that acute HSV-1 infection in neuronal cell lines selectively enhances Arc protein expression - a major regulator of long-term synaptic plasticity and memory consolidation, known for being a protein-interaction hub in the postsynaptic dendritic compartment. Thus, HSV-1 induced Arc expression may alter the functionality of infected neurons and negatively impact dendritic spine dynamics. In this study we demonstrated that HSV-1 infection induces structural disassembly and functional deregulation in cultured cortical neurons, an altered glutamate response, Arc accumulation within the somata, and decreased expression of spine scaffolding-like proteins such as PSD-95, Drebrin and CaMKIIß. However, whether these alterations are specific to the HSV-1 infection mechanism or reflect a secondary neurodegenerative process remains to be determined.

Discovery and biological characterization of a novel scaffold for potent inhibitors of peripheral serotonin synthesis.

Aim: Tryptophan hydroxylase 1 (TPH1) catalyzes serotonin synthesis in peripheral tissues. Selective TPH1 inhibitors may be useful for treating disorders related to serotonin dysregulation. Results & methodology: Screening using a thermal shift assay for TPH1 binders yielded Compound 1 (2-(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one), which showed high potency (50% inhibition at 98 ± 30 nM) and selectivity for inhibiting TPH over related aromatic amino acid hydroxylases in enzyme activity assays. Structure-activity relationships studies revealed several analogs of 1 showing comparable potency. Kinetic studies suggested a noncompetitive mode of action of 1, with regards to tryptophan and tetrahydrobiopterin. Computational docking studies and live cell assays were also performed. Conclusion: This TPH1 inhibitor scaffold may be useful for developing new therapeutics for treating elevated peripheral serotonin.

A simple DMSO-based method for cryopreservation of primary hippocampal and cortical neurons.

BACKGROUND: Primary neuronal cultures are widely used to elucidate fundamental aspects of neuronal anatomy, physiology, cell biology, and neuronal dysfunction in animal models of disease. However, preparation of primary neuronal cultures from rodent embryos is labor-intensive, and it is often difficult to produce high-quality cultures consistently in a single laboratory, and to compare results between laboratories. To overcome these issues, cryopreservation can be used to obtain more standardized, high-quality banks of neuronal cultures. NEW METHOD: In this study, we present a simplified cryopreservation method for rodent primary hippocampal and cortical neurons from embryonic day 18.5 fetuses, using DMSO-containing traditional cell freezing medium. RESULTS: Cryopreserved neurons stored for more than 1 year in liquid nitrogen were assessed by cell imaging, as well as biochemical signaling transduction and gene expression in response to pharmacological treatments. Cryopreserved neuronal cultures were comparable to freshly prepared cultures in terms of: (1) neuronal viability, (2) neuronal morphology and maturation, (3) functional synapse formation, (4) stimulus responsiveness. These results indicate that DMSO-cryopreserved neurons are equivalent to freshly prepared neurons both developmentally and functionally. COMPARISON WITH EXISTING METHODS: Our method is simple and does not require special reagents or equipment. CONCLUSIONS: Introduction of the cryopreserved neurons as a standard laboratory practice has the potential to increase the robustness and reproducibility of findings between laboratories and reduce the number of animals used in research.

Drebrin in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder accompanied by severe progressive memory and cognitive impairment. The brain of AD patients has an abundance of two abnormal structures, amyloid plaques (senile plaques) and neurofibrillary tangles. In addition, drebrin loss is another hallmark of AD brains, which is a common feature in the brain of both AD patients and AD mouse models. Strong evidence from human genetics and transgenic mouse models has indicated that amyloid ß (Aß) is part of the etiology and pathogenesis of AD. Recently, it has become clear that synaptic dysfunction, including reduced synaptic transmission and loss of dendritic spines, occurs prior to the formation of amyloid plaques and neuronal cell loss. Furthermore, immunohistochemistry using postmortem human brains and AD mouse models has shown that drebrin loss in postsynaptic sites occurs earlier than the presynaptic change in AD brains. In addition, dysregulation of glutamate receptor trafficking and the p21-activated kinase/LIM kinase pathway has been observed in AD brains. It is now believed that soluble Aß oligomers, namely, Aß-derived diffusible ligands (ADDLs), but not insoluble Aß aggregation mediates Aß toxicity. ADDLs bind to the postsynaptic site and induce the aberrant morphology and density of dendritic spines. Consistent with the AD mouse models, the surface expression of glutamate receptors decreases after ADDL exposure. Importantly, the ADDL-induced drebrin loss in dendritic spines occurs prior to aberrations in dendritic spine morphology and density. These observations indicate that drebrin loss in dendritic spines occurs at the prodromal stage of AD, before the density and morphology of dendritic spines change. Quantitation of drebrin may be a possible tool for diagnosing the prodromal stage of AD, before dementia development in AD.

The role of drebrin in neurons.

Drebrin is an actin-binding protein that changes the helical pitch of actin filaments (F-actin), and drebrin-decorated F-actin shows slow treadmilling and decreased rate of depolymerization. Moreover, the characteristic morphology of drebrin-decorated F-actin enables it to respond differently to the same signals from other actin cytoskeletons. Drebrin consists of two major isoforms, drebrin E and drebrin A. In the developing brain, drebrin E appears in migrating neurons and accumulates in the growth cones of axons and dendrites. Drebrin E-decorated F-actin links lamellipodium F-actin to microtubules in the growth cones. Then drebrin A appears at nascent synapses and drebrin A-decorated F-actin facilitates postsynaptic molecular assembly. In the adult brain, drebrin A-decorated F-actin is concentrated in the central region of dendritic spines. During long-term potentiation initiation, NMDA receptor-mediated Ca2+ influx induces the transient exodus of drebrin A-decorated F-actin via myosin II ATPase activation. Because of the unique physical characteristics of drebrin A-decorated F-actin, this exodus likely contributes to the facilitation of F-actin polymerization and spine enlargement. Additionally, drebrin reaccumulation in dendritic spines is observed after the exodus. In our drebrin exodus model of structure-based synaptic plasticity, reestablishment of drebrin A-decorated F-actin is necessary to keep the enlarged spine size during long-term potentiation maintenance. In this review, we introduce the genetic and biochemical properties of drebrin and the roles of drebrin in early stage of brain development, synaptic formation and synaptic plasticity. Further, we discuss the pathological relevance of drebrin loss in Alzheimer's disease. This article is part of the mini review series "60th Anniversary of the Japanese Society for Neurochemistry".

X Irradiation Induces Acute Cognitive Decline via Transient Synaptic Dysfunction.

Cranial X irradiation can severely impair higher brain function, resulting in neurocognitive deficits. Radiation-induced brain injury is characterized by acute, early and late delayed changes, and morbidity is evident more than 6 months after irradiation. While the acute effects of radiation exposure on the brain are known, the underlying mechanisms remain unclear. In this study, we examined the acute effect of X radiation on synaptic function using behavioral analysis and immunohistochemistry. We found that 10 Gy whole-brain irradiation immediately after conditioning (within 30 min) impaired the formation of fear memory, whereas irradiation 24 h prior to conditioning did not. To investigate the mechanisms underlying these behavioral changes, we irradiated one hemisphere of the brain and analyzed synaptic function and adult neurogenesis immunohistochemically. We focused on drebrin, whose loss from dendritic spines is a surrogate marker of synaptopathy. The intensity of drebrin immunoreactivity started to decrease in the irradiated hemisphere 2 h after exposure. The immunostaining intensity recovered to preirradiation levels by 24 h, indicating that X radiation induced transient synaptic dysfunction. Interestingly, the number of newly generated neurons was not changed at 2 h postirradiation, whereas it was significantly decreased at 8 and 24 h postirradiation. Because irradiation 24 h prior to conditioning had no effect on fear memory, our findings suggest that radiation-induced death of newly-generated neurons does not substantially impact fear memory formation. The radiation-induced synaptic dysfunction likely caused a transient memory deficit during the critical period for fear memory formation (approximately 1-3 h after conditioning), which coincides with a change in drebrin immunostaining in the hippocampus, a structure critical for fear memory formation.

Nuclear Translocation of Calcium/Calmodulin-dependent Protein Kinase IIδ3 Promoted by Protein Phosphatase-1 Enhances Brain-derived Neurotrophic Factor Expression in Dopaminergic Neurons.

We have reported previously that dopamine D2 receptor stimulation activates calcium/calmodulin-dependent protein kinase II (CaMKII) δ3, a CaMKII nuclear isoform, increasing BDNF gene expression. However, the mechanisms underlying that activity remained unclear. Here we report that CaMKIIδ3 is dephosphorylated at Ser(332) by protein phosphatase 1 (PP1), promoting CaMKIIδ3 nuclear translocation. Neuro-2a cells transfected with CaMKIIδ3 showed cytoplasmic and nuclear staining, but the staining was predominantly nuclear when CaMKIIδ3 was coexpressed with PP1. Indeed, PP1 and CaMKIIδ3 coexpression significantly increased nuclear CaMKII activity and enhanced BDNF expression. In support of this idea, chronic administration of the dopamine D2 receptor partial agonist aripiprazole increased PP1 activity and promoted nuclear CaMKIIδ3 translocation and BDNF expression in the rat brain substantia nigra. Moreover, aripiprazole treatment enhanced neurite extension and inhibited cell death in cultured dopaminergic neurons, effects blocked by PP1γ knockdown. Taken together, nuclear translocation of CaMKIIδ3 following dephosphorylation at Ser(332) by PP1 likely accounts for BDNF expression and subsequent neurite extension and survival of dopaminergic neurons.

Cysteine analogs with a free thiol group promote fertilization by reducing disulfide bonds in the zona pellucida of mice.

Archives of cryopreserved sperm harvested from genetically engineered mice, in mouse resource centers, are a readily accessible genetic resource for the scientific community. We previously reported that exposure of oocytes to reduced glutathione (GSH) greatly improves the fertilization rate of frozen-thawed mouse sperm. Application of GSH to in vitro fertilization techniques is widely accepted as a standard protocol to produce sufficient numbers of mice from cryopreserved sperm. However, the detailed mechanism of the enhancement of fertilization mediated by GSH in vitro is not fully understood. Here we focused on the chemical by determining the effects of its amino acid constituents and cysteine analogs on the fertilization of oocytes by frozen-thawed sperm. Furthermore, we determined the stability of these compounds in aqueous solution. We show here that l-cysteine (l-Cys), d-cysteine (d-Cys), or N-acetyl-l-cysteine (NAC) increased the rate of fertilization when added to the medium but did not adversely affect embryo development in vitro or in vivo. The levels of thiol groups of proteins in the zona pellucida (ZP) and the expansion of the ZP were increased by l-Cys, d-Cys, and NAC. These effects were abrogated by the methylation of the thiol group of l-Cys. NAC was the most stable of these compounds in the fertilization medium at 4°C. These results suggest that the thiol groups of cysteine analogs markedly enhance the fertilization rate of mouse oocytes.

Prolonged exposure to hyaluronidase decreases the fertilization and development rates of fresh and cryopreserved mouse oocytes.

Hyaluronidase is generally used to remove cumulus cells from mouse oocytes before oocyte cryopreservation, intracytoplasmic sperm injection or DNA injection. In general, use of cumulus-free mouse oocytes decreases in vitro fertilizing ability compared with cumulus-surrounded oocytes. The effect of hyaluronidase exposure on the quality of mouse oocytes is not fully understood. Here, we investigated the effect of hyaluronidase exposure time on the fertilization rate of fresh and vitrified mouse oocytes and their subsequent developmental ability in vitro. We found that the fertilization rate decreased with hyaluronidase treatments. This reduction in the fertilization rate following treatment with hyaluronidase was fully reversed by removal of the zona pellucida. In addition, oocytes treated with hyaluronidase for 5 min or longer had a reduced capacity to develop to the morula and blastocyst stage. The survival, fertilization, and developmental rates of vitrified-warmed oocytes were also reduced by longer exposure to hyaluronidase. In conclusion, these results suggest that prolonged exposure to hyaluronidase decreases the quality of mouse oocytes and shorter hyaluronidase treatment times may help achieve a stable and high fertilization rate in fresh and cryopreserved oocytes.

Histone deacetylase mediates the decrease in drebrin cluster density induced by amyloid beta oligomers.

Dendritic spine defects are found in a number of cognitive disorders, including Alzheimer's disease (AD). Amyloid beta (Aß) toxicity is mediated not only by the fibrillar form of the protein, but also by the soluble oligomers (Aß-derived diffusible ligands, ADDLs). Drebrin is an actin-binding protein that is located at mature dendritic spines. Because drebrin expression is decreased in AD brains and in cultured neurons exposed to Aß, it is thought that drebrin is closely associated with cognitive functions. Recent studies show that histone deacetylase (HDAC) activity is elevated in the AD mouse model, and that memory impairments in these animals can be ameliorated by HDAC inhibitors. In addition, spine loss and memory impairment in HDAC2 over-expressing mice are ameliorated by chronic HDAC inhibitor treatment. Therefore, we hypothesized that the regulation of histone acetylation/deacetylation is critical to synaptic functioning. In this study, we examined the relationship between HDAC activity and synaptic defects induced by ADDLs using an HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA). We show that ADDLs reduce the cluster density of drebrin along dendrites without reducing drebrin expression. SAHA markedly increased the acetylation of histone proteins, and it simultaneously attenuated the ADDL-induced decrease in drebrin cluster density. In comparison, SAHA treatment did not affect the density of drebrin clusters or dendritic protrusions in control neurons. Therefore, SAHA likely inhibits ADDL-induced drebrin loss from dendritic spines by stabilizing drebrin in these structures, rather than by increasing drebrin clusters or dendritic protrusions. Taken together, our findings suggest that HDAC is involved in ADDL-induced synaptic defects, and that the regulation of histone acetylation plays an important role in modulating actin cytoskeletal dynamics in dendritic spines under cellular stress conditions, such as ADDL exposure.

Myosin II ATPase activity mediates the long-term potentiation-induced exodus of stable F-actin bound by drebrin A from dendritic spines.

The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin) from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1) chemical long-term potentiation (LTP) stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2) Ca(2+) influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3) the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK) inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement.

AMP-activated protein kinase counteracts brain-derived neurotrophic factor-induced mammalian target of rapamycin complex 1 signaling in neurons.

Growth factors and nutrients, such as amino acids and glucose, regulate mammalian target of rapamycin complex 1 (mTORC1) signaling and subsequent translational control in a coordinated manner. Brain-derived neurotrophic factor (BDNF), the most prominent neurotrophic factor in the brain, activates mTORC1 and induces phosphorylation of its target, p70S6 kinase (p70S6K), at Thr389 in neurons. BDNF also increases mammalian target of rapamycin-dependent novel protein synthesis in neurons. Here, we report that BDNF-induced p70S6K activation is dependent on glucose, but not amino acids, sufficiency in cultured cortical neurons. AMP-activated protein kinase (AMPK) is the molecular background to this specific nutrient dependency. Activation of AMPK, which is induced by glucose deprivation, treatment with pharmacological agents such as 2-deoxy-D-glucose, metformin, and 5-aminoimidazole-4-carboxamide ribonucleoside or forced expression of a constitutively active AMPKα subunit, counteracts BDNF-induced phosphorylation of p70S6K and enhanced protein synthesis in cortical neurons. These results indicate that AMPK inhibits the effects of BDNF on mTORC1-mediated translation in neurons.

Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization.

Since the first successful reports into oocyte freezing, many papers concerning the cryopreservation of mouse oocytes have been published. However, a simple and practical cryopreservation method for unfertilized C57BL/6 mouse oocytes, and an IVF system using these cryopreserved oocytes have yet to be established, in spite of the fact that C57BL/6 is the prevalent inbred strain and is used for large-scale knockout programs. In this study, unfertilized C57BL/6 mouse oocytes were cryopreserved via a simple vitrification method. After warming, IVF was performed using cryopreserved unfertilized oocytes and fresh sperm, cryopreserved unfertilized oocytes and cold-stored sperm, cryopreserved unfertilized oocytes and frozen sperm (C57BL/6 strain sperm), and cryopreserved unfertilized oocytes and frozen sperm derived from GEM strains (C57BL/6 background GEM strains). Nearly all of the cryopreserved oocytes were recovered, of which over 90% were morphologically normal. Those oocytes were then used for in vitro fertilization, resulting in 72-97% of oocytes developing into 2-cell embryos. A portion of the 2-cell embryos were transferred to recipients, resulting in live young being produced from 32-49% of the embryos. In summary, we established the simple and practical method of mouse oocyte vitrification with high survivability and developmental ability and the IVF using the vitrified-warmed oocytes with fresh, cold-stored or cryopreserved sperm with high fertility.

Salicylic acid is involved in the regulation of starvation stress-induced flowering in Lemna paucicostata.

The short-day plant, Lemna paucicostata (synonym Lemna aequinoctialis), was induced to flower when cultured in tap water without any additional nutrition under non-inductive long-day conditions. Flowering occurred in all three of the tested strains, and strain 6746 was the most sensitive to the starvation stress conditions. For each strain, the stress-induced flowering response was weaker than that induced by short-day treatment, and the stress-induced flowering of strain 6746 was completely inhibited by aminooxyacetic acid and l-2-aminooxy-3-phenylpropionic acid, which are inhibitors of phenylalanine ammonia-lyase. Significantly higher amounts of endogenous salicylic acid (SA) were detected in the fronds that flowered under the poor-nutrition conditions than in the vegetative fronds cultured under nutrition conditions, and exogenously applied SA promoted the flowering response. The results indicate that endogenous SA plays a role in the regulation of stress-induced flowering.