Lipocalin-2: a therapeutic target to overcome neurodegenerative diseases by regulating reactive astrogliosis.

Glial cell activation precedes neuronal cell death during brain aging and the progression of neurodegenerative diseases. Under neuroinflammatory stress conditions, lipocalin-2 (LCN2), also known as neutrophil gelatinase-associated lipocalin or 24p3, is produced and secreted by activated microglia and reactive astrocytes. Lcn2 expression levels are known to be increased in various cells, including reactive astrocytes, through the activation of the NF-κB signaling pathway. In the central nervous system, as LCN2 exerts neurotoxicity when secreted from reactive astrocytes, many researchers have attempted to identify various strategies to inhibit LCN2 production, secretion, and function to minimize neuroinflammation and neuronal cell death. These strategies include regulation at the transcriptional, posttranscriptional, and posttranslational levels, as well as blocking its functions using neutralizing antibodies or antagonists of its receptor. The suppression of NF-κB signaling is a strategy to inhibit LCN2 production, but it may also affect other cellular activities, raising questions about its effectiveness and feasibility. Recently, LCN2 was found to be a target of the autophagy‒lysosome pathway. Therefore, autophagy activation may be a promising therapeutic strategy to reduce the levels of secreted LCN2 and overcome neurodegenerative diseases. In this review, we focused on research progress on astrocyte-derived LCN2 in the central nervous system.

Reduced secretion of LCN2 (lipocalin 2) from reactive astrocytes through autophagic and proteasomal regulation alleviates inflammatory stress and neuronal damage.

LCN2/neutrophil gelatinase-associated lipocalin/24p3 (lipocalin 2) is a secretory protein that acts as a mammalian bacteriostatic molecule. Under neuroinflammatory stress conditions, LCN2 is produced and secreted by activated microglia and reactive astrocytes, resulting in neuronal apoptosis. However, it remains largely unknown whether inflammatory stress and neuronal loss can be minimized by modulating LCN2 production and secretion. Here, we first demonstrated that LCN2 was secreted from reactive astrocytes, which were stimulated by treatment with lipopolysaccharide (LPS) as an inflammatory stressor. Notably, we found two effective conditions that led to the reduction of induced LCN2 levels in reactive astrocytes: proteasome inhibition and macroautophagic/autophagic flux activation. Mechanistically, proteasome inhibition suppresses NFKB/NF-κB activation through NFKBIA/IκBα stabilization in primary astrocytes, even under inflammatory stress conditions, resulting in the downregulation of Lcn2 expression. In contrast, autophagic flux activation via MTOR inhibition reduced the intracellular levels of LCN2 through its pre-secretory degradation. In addition, we demonstrated that the N-terminal signal peptide of LCN2 is critical for its secretion and degradation, suggesting that these two pathways may be mechanistically coupled. Finally, we observed that LPS-induced and secreted LCN2 levels were reduced in the astrocyte-cultured medium under the above-mentioned conditions, resulting in increased neuronal viability, even under inflammatory stress.Abbreviations: ACM, astrocyte-conditioned medium; ALP, autophagy-lysosome pathway; BAF, bafilomycin A1; BTZ, bortezomib; CHX, cycloheximide; CNS, central nervous system; ER, endoplasmic reticulum; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; JAK, Janus kinase; KD, knockdown; LCN2, lipocalin 2; LPS, lipopolysaccharide; MACS, magnetic-activated cell sorting; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MTOR, mechanistic target of rapamycin kinase; NFKB/NF-κB, nuclear factor of kappa light polypeptide gene enhancer in B cells 1, p105; NFKBIA/IκBα, nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha; OVEX, overexpression; SLC22A17, solute carrier family 22 member 17; SP, signal peptide; SQSTM1, sequestosome 1; STAT3, signal transducer and activator of transcription 3; TNF/TNF-α, tumor necrosis factor; TUBA, tubulin, alpha; TUBB3/ß3-TUB, tubulin, beta 3 class III; UB, ubiquitin; UPS, ubiquitin-proteasome system.

Polyubiquitin gene Ubb is required for upregulation of Piwi protein level during mouse testis development.

Testis development, including early embryonic gonad formation and late postnatal spermatogenesis, is essential for the reproduction of higher metazoans to generate fertile gametes, called sperm. We have previously reported that the polyubiquitin gene Ubb is required for fertility in both male and female mice. In particular, the Ubb-null male mice showed an azoospermia phenotype due to arrest of spermatogenesis at the pachytene stage. Here, we analyzed the whole testis proteome at postnatal day 20 to define the molecular mediators of the male-infertility phenotype caused by Ubb knockout. From the identified proteome, 564 proteins were significantly and differentially expressed in Ubb-knockout testes and, among these, 36 downregulated proteins were involved at different stages of spermatogenesis. We also found that levels of piRNA metabolic process-related proteins, including Piwil2 and Tdrd1, were downregulated in Ubb-null testes through functional gene ontology analysis. Further, protein-protein interaction mapping revealed that 24 testis development-related proteins, including Hsp90aa1, Eef1a1, and Pabpc1, were directly influenced by the depletion of ubiquitin. In addition, the reduced mRNA levels of these proteins were observed in Ubb-knockout testes, which closely resembled the global downregulation of piRNA-metabolic gene expression at the transcriptional and post-transcriptional levels. Together with proteomic and transcriptional analyses, our data suggest that Ubb expression is essential for the maintenance of testicular RNA-binding regulators and piRNA-metabolic proteins to complete spermatogenesis in mice.

Regulation of polyubiquitin genes to meet cellular ubiquitin requirement.

Ubiquitin (Ub) is one of the proteins that are highly conserved from yeast to humans. It is an essential core unit of the welldefined post-translational modification, called ubiquitination, which is involved in a variety of biological processes. In metazoans, Ub is encoded by two monoubiquitin genes and two polyubiquitin genes, in which a single Ub is fused to a ribosomal protein or Ub coding units are arranged in tandem repeats. In mice, polyubiquitin genes (Ubb and Ubc) play a pivotal role to meet the requirement of cellular Ub pools during embryonic development. In addition, expression levels of polyubiquitin genes are increased to adapt to environmental stimuli such as oxidative, heat-shock, and proteotoxic stress. Several researchers have reported about the perturbation of Ub pools through genetic alteration or exogenous Ub delivery using diverse model systems. To study Ub pool changes in a physiologically relevant manner, changing Ub pools via the regulation of endogenous polyubiquitin gene expression has recently been introduced. Furthermore, to understand the regulation of polyubiquitin gene expression more precisely, cis-acting elements and trans-acting factors, which are regulatory components of polyubiquitin genes, have been analyzed. In this review, we discuss how the role of polyubiquitin genes has been studied during the past decade, especially focusing on their regulation. [BMB Reports 2021; 54(4): 189-195].

Quorum Sensing System Affects the Plant Growth Promotion Traits of Serratia fonticola GS2.

Quorum sensing (QS) enables bacteria to organize gene expression programs, thereby coordinating collective behaviors. It involves the production, release, and population-wide detection of extracellular signaling molecules. The cellular processes regulated by QS in bacteria are diverse and may be used in mutualistic coordination or in response to changing environmental conditions. Here, we focused on the influence of the QS-dependent genes of our model bacterial strain Serratia fonticola GS2 on potential plant growth promoting (PGP) activities including indole-3-acetic acid (IAA) production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, and biofilm formation. Based on genomic and phenotypic experimental data we identified and investigated the function of QS genes in the genome of the model strain. Our gene deletion study confirmed the biological functionality of the QS auto-inducer (gloI) and receptor (gloR) on potential PGP activities of GS2. A transcriptomic approach was also undertaken to understand the role of QS genes in regulation of genes primarily involved in PGP activities (IAA, ACC deaminase activity, and biofilm formation). Both transcriptomic and phenotypic data revealed that the QS-deletion mutants had considerably less PGP activities, as compared to the wild type. In addition, in vivo plant experiments showed that plants treated with GS2 had significantly higher growth rates than plants treated with the QS-deletion mutants. Overall, our results showed how QS-dependent genes regulate the potential PGP activities of GS2. This information may be helpful in understanding the relationship between QS-dependent genes and the PGP activity of bacteria, which aid in the production of practical bio-fertilizers for plant growth promotion.

Neurotoxic potential of polystyrene nanoplastics in primary cells originating from mouse brain.

Polystyrene (PS) and chemically modified compounds in the PS family have long been used in commercial and industrial fields. However, it is poorly understood whether nanoscale-PS microplastic or PS nanoplastic exposure leads to perturbations in fundamental cellular functions, such as proliferation, differentiation, and apoptosis. Herein, we cultured three types of primary cells, including mouse embryonic fibroblasts (MEFs), mixed neuronal cells isolated from embryonic cortex, and cortical astrocytes, and investigated the effects of their exposure to PS nanoplastics with a 100 nm diameter. Although PS nanoplastic exposure did not affect the viability of MEFs or astrocytes, it significantly reduced the viability of mixed neuronal cells. Consistent with the observed effect on cellular viability, levels of the apoptosis marker, cleaved caspase-3, were elevated exclusively in mixed neuronal cells. To investigate whether cells uptake PS nanoplastics into the cytoplasm, we exposed MEFs and neurons to fluorescent PS latex beads and monitored fluorescence over time. We found that PS nanoplastics were deposited and accumulated in the cytoplasm in a concentration-dependent manner. Although astrocytes were not apoptotic upon exposure to PS nanoplastics, they underwent reactive astrocytosis, with increased levels of lipocalin-2 and proinflammatory cytokines. Therefore, our findings suggested that the vulnerability of cells to the deposition and accumulation of PS nanoplastics in the cytoplasm was dependent on cell type. Furthermore, based on our data from primary cells originating from mouse brains, we suggest that reactive astrocytosis may contribute to the neuronal apoptosis seen in defective neurons with PS nanoplastics accumulated in the cell body.

Reduced free ubiquitin levels and proteasome activity in cultured neurons and brain tissues treated with amyloid beta aggregates.

Neurodegenerative diseases are characterized by progressive cognitive decline and the loss of neurons in the central nervous system; many are also characterized by abnormal aggregation of misfolded proteins. Ubiquitin (Ub) is a eukaryotic protein that plays pivotal roles in protein degradation and cellular signaling. Ubiquitinated aggregates are observed in neurodegenerative diseases; this ultimately results in reduced levels of available or free Ub. However, it remains unclear whether neurotoxicity arises from the aggregates or a deficiency of free Ub. To investigate this, we treated primary neurons of mouse embryonic brains with amyloid beta (Aß) 42 and found that free Ub levels were decreased and cell viability was reduced in Aß42-treated neurons. As reduced levels of free Ub are closely related to impaired function of the proteasome, we evaluated proteasome activity and found that proteasome activity was reduced upon treatment of primary neurons and mouse brain slices with Aß42. Therefore, we conclude that proteotoxic stress from Aß42 treatment reduced the levels of available Ub and decreased proteasome activity, resulting in inflammatory stress and apoptosis of neurons.

Characterization of Clostridium novyi isolated from a sow in a sudden death case in Korea.

BACKGROUND: Multifocal spherical nonstaining cavities and gram-positive, rod-shaped, and endospore-forming bacteria were found in the liver of a sow that died suddenly. Clostridium novyi type B was identified and isolated from the sudden death case, and the isolate was characterized by molecular analyses and bioassays in the current study. RESULTS: C. novyi was isolated from the liver of a sow that died suddenly and was confirmed as C. novyi type B by differential PCR. The C. novyi isolate fermented glucose and maltose and demonstrated lecithinase activity, and the cell-free culture supernatant of the C. novyi isolate exhibited cytotoxicity toward Vero cells, demonstrating that the isolate produces toxins. In addition, whole-genome sequencing of the C. novyi isolate was performed, and the complete sequences of the chromosome (2.29 Mbp) and two plasmids (134 and 68 kbp) were identified for the first time. Based on genome annotation, 7 genes were identified as glycosyltransferases, which are known as alpha toxins; 23 genes were found to be related to sporulation; 12 genes were found to be related to germination; and 20 genes were found to be related to chemotaxis. CONCLUSION: C. novyi type B was isolated from a sow in a sudden death case and confirmed by biochemical and molecular characterization. Various virulence-associated genes were identified for the first time based on whole-genome sequencing.

Disruption of the polyubiquitin gene Ubb reduces the self-renewal capacity of neural stem cells.

Ubiquitin (Ub) is a highly conserved eukaryotic protein that plays pivotal roles in cellular signal transduction, differentiation, and proteolysis. Although we have previously reported that disruption of the polyubiquitin gene Ubb is associated with the dysregulated differentiation of neural stem cells (NSCs) into neurons, it is unclear how gene expression patterns are altered in Ubb knockout (KO) NSCs, and whether this altered gene expression contributes to Ubb KO neural phenotypes. To answer these questions, we used RNA-Seq to compare the transcriptomes of Ubb KO NSCs and Ubb heterozygous (HT) controls. We found that the expression levels of most proliferation markers were decreased in Ubb KO NSCs. To determine whether the reduced levels of proliferation markers were due to reduced self-renewal of NSCs, such as radial glia, we measured the levels of the radial glia marker, Pax6, in mouse embryonic brains at 14.5 dpc. We found that Pax6 levels were decreased and the ventricular zone was thinner in the embryonic brains of Ubb KO mice compared to those of wild-type (WT) control mice. To determine whether the decreased self-renewal of Ubb KO NSCs was caused by cell-autonomous defects and not due to their microenvironment, we transplanted NSCs into WT mouse brains using a cannula system. In mouse brain sections, immunoreactivity of the NSC marker, nestin, was much lower in Ubb KO NSCs than in Ubb HT controls. Therefore, our data suggest that cell-autonomous defects, due to the disruption of Ubb, lead to a decrease in the self-renewal capacity of NSCs and may contribute to their dysregulated differentiation into neurons.

Reversible Regulation of Polyubiquitin Gene UBC via Modified Inducible CRISPR/Cas9 System.

Ubiquitin is required under both normal and stress conditions. Under stress conditions, upregulation of the polyubiquitin gene UBC is essential to meet the requirement of increased ubiquitin levels to confer stress resistance. However, UBC upregulation is usually observed only under stress conditions and not under normal conditions. Therefore, it has not been possible to upregulate UBC under normal conditions to study the effect of excess ubiquitin on cellular machinery. Recently, the CRISPR/Cas9 system has been widely used in biological research as a useful tool to study gene disruption effects. In this study, using an inducible CRISPR/Cas9 variant, a dCas9-VP64 fusion protein, combined with a single guide RNA (sgRNA) containing MS2 aptamer loops and MS2-p65-HSF1, we developed a system to increase the ubiquitin pool via upregulation of UBC. Although it is challenging to upregulate the expression of a gene that is already expressed at high levels, the significance of our system is that UBC upregulation can be induced in an efficient, reversible manner that is compatible with cellular processes, even under normal conditions. This system can be used to study ubiquitin pool dynamics and it will be a useful tool in identifying the role of ubiquitin under normal and stress conditions.

Expression and Characterization of Calcium- and Zinc-Tolerant Xylose Isomerase from Anoxybacillus kamchatkensis G10.

The enzyme xylose isomerase (E.C. 5.3.1.5, XI) is responsible for the conversion of an aldose to ketose, especially xylose to xylulose. Owing to the ability of XI to isomerize glucose to fructose, this enzyme is used in the food industry to prepare high-fructose corn syrup. Therefore, we studied the characteristics of XI from Anoxybacillus kamchatkensis G10, a thermophilic bacterium. First, the gene coding for XI (xylA) was inserted into the pET-21a(+) expression vector and the construct was transformed into the Escherichia coli competent cell BL21 (DE3). The expression of recombinant XI was induced in the absence of isopropyl-thio-ß-galactopyranoside and purified using Ni-NTA affinity chromatography. The optimum temperature of recombinant XI was 80°C and measurement of the heat stability indicated that 55% of residual activity was maintained after 2 h incubation at 60°C. The optimum pH was found to be 7.5 in sodium phosphate buffer. Magnesium, manganese, and cobalt ions were found to increase the enzyme activity; manganese was the most effective. Additionally, recombinant XI was resistant to the presence of Ca²âº and Zn²âº ions. The kinetic properties, Km and Vmax, were calculated as 81.44 mM and 2.237 µmol/min/mg, respectively. Through redundancy analysis, XI of A. kamchatkensis G10 was classified into a family containing type II XIs produced by the genera Geobacillus, Bacillus, and Thermotoga. These results suggested that the thermostable nature of XI of A. kamchatkensis G10 may be advantageous in industrial applications and food processing.

Temporal downregulation of the polyubiquitin gene Ubb affects neuronal differentiation, but not maturation, in cells cultured in vitro.

Reduced levels of cellular ubiquitin (Ub) pools due to disruption of the polyubiquitin gene Ubb lead to dysregulation of neural stem cell (NSC) differentiation and impaired neuronal maturation in cells isolated from Ubb -/- mouse embryonic brains. However, it is currently unknown whether Ub is required for the specific stage of neuronal development or whether it plays a pleiotropic role throughout the process. To answer this question, we aimed to downregulate Ubb expression temporally during neuronal development, which could not be achieved in Ubb -/- cells. Therefore, we exploited lentivirus-mediated knockdown (KD) of Ubb at different stages of neuronal development, and investigated their phenotypes. Here, we report the outcome of Ubb KD on two independent culture days in vitro (DIV): DIV1 and DIV7. We observed that NSCs did not differentiate properly via Ubb KD on DIV1, but the maturation of already differentiated neurons was intact via Ubb KD on DIV7. Intriguingly, Ubb KD activated Notch signaling when it had been suppressed, but exerted no effect when it had already been activated. Therefore, our study suggests that Ub plays a pivotal role in NSC differentiation to suppress Notch signaling, but not in the subsequent maturation stages of neurons that had already been differentiated.

Genome sequencing to develop Paenibacillus donghaensis strain JH8T (KCTC 13049T=LMG 23780T) as a microbial fertilizer and correlation to its plant growth-promoting phenotype.

Paenibacillus donghaensis JH8T (KCTC 13049T=LMG 23780T) is a Gram-positive, mesophilic, endospore-forming bacterium isolated from East Sea sediment at depth of 500m in Korea. The strain exhibited plant cell wall hydrolytic and plant growth promoting abilities. The complete genome of P. donghaensis strain JH8T contains 7602 protein-coding sequences and an average GC content of 49.7% in its chromosome (8.54Mbp). Genes encoding proteins related to the degradation of plant cell wall, nitrogen-fixation, phosphate solubilization, and synthesis of siderophore were existed in the P. donghaensis strain JH8T genome, indicating that this strain can be used as an eco-friendly microbial agent for increasing agricultural productivity.

Whole genome sequence of lactic acid bacterium Pediococcus acidilactici strain S1

Abstract Pediococcus acidilactici strain S1, a lactic acid-fermenting bacterium, was isolated from makgeolli-a Korean traditional fermented alcoholic beverage. Here we report the 1,980,172 bp (G + C content, 42%) genome sequence of Pediococcus acidilactici strain S1 with 1,525 protein-coding sequences (CDS), of which 47% could be assigned to recognized functional genes. The genome sequence of the strain S1 might provide insights into the genetic basis of the lactic acid bacterium with alcohol-tolerant.

Draft genome sequence of a caprolactam degrader bacterium: Pseudomonas taiwanensis strain SJ9

Abstract Pseudomonas taiwanensis strain SJ9 is a caprolactam degrader, isolated from industrial wastewater in South Korea and considered to have the potential for caprolactam bioremediation. The genome of this strain is approximately 6.2 Mb (G + C content, 61.75%) with 6,010 protein-coding sequences (CDS), of which 46% are assigned to recognized functional genes. This draft genome of strain SJ9 will provide insights into the genetic basis of its caprolactam-degradation ability.

Whole genome sequence of lactic acid bacterium Pediococcus acidilactici strain S1.

Pediococcus acidilactici strain S1, a lactic acid-fermenting bacterium, was isolated from makgeolli-a Korean traditional fermented alcoholic beverage. Here we report the 1,980,172bp (G+C content, 42%) genome sequence of Pediococcus acidilactici strain S1 with 1,525 protein-coding sequences (CDS), of which 47% could be assigned to recognized functional genes. The genome sequence of the strain S1 might provide insights into the genetic basis of the lactic acid bacterium with alcohol-tolerant.

Draft genome sequence of sulfur-reducing archaeon Thermococcus thioreducens DSM 14981T

Abstract Thermococcus thioreducens DSM 14981T, a sulfur-reducing archaeon, was isolated from the rainbow hydrothermal vent site on the Mid-Atlantic Ridge. Herein, we report the draft genome sequence of T. thioreducens DSM 14981T; we obtained 41 contigs with a genome size of 2,052,483 bp and G + C content of 53.5%. This genome sequence will not only help understand how the archaeon adapts to the deep-sea hydrothermal environment but also aid the development of enzymes that are highly stable under extreme conditions for industrial applications.

Complete genome analysis of Serratia marcescens RSC-14: A plant growth-promoting bacterium that alleviates cadmium stress in host plants.

Serratia marcescens RSC-14 is a Gram-negative bacterium that was previously isolated from the surface-sterilized roots of the Cd-hyperaccumulator Solanum nigrum. The strain stimulates plant growth and alleviates Cd stress in host plants. To investigate the genetic basis for these traits, the complete genome of RSC-14 was obtained by single-molecule real-time sequencing. The genome of S. marcescens RSC-14 comprised a 5.12-Mbp-long circular chromosome containing 4,593 predicted protein-coding genes, 22 rRNA genes, 88 tRNA genes, and 41 pseudogenes. It contained genes with potential functions in plant growth promotion, including genes involved in indole-3-acetic acid (IAA) biosynthesis, acetoin synthesis, and phosphate solubilization. Moreover, annotation using NCBI and Rapid Annotation using Subsystem Technology identified several genes that encode antioxidant enzymes as well as genes involved in antioxidant production, supporting the observed resistance towards heavy metals, such as Cd. The presence of IAA pathway-related genes and oxidative stress-responsive enzyme genes may explain the plant growth-promoting potential and Cd tolerance, respectively. This is the first report of a complete genome sequence of Cd-tolerant S. marcescens and its plant growth promotion pathway. The whole-genome analysis of this strain clarified the genetic basis underlying its phenotypic and biochemical characteristics, underpinning the beneficial interactions between RSC-14 and plants.

Draft genome sequence of a caprolactam degrader bacterium: Pseudomonas taiwanensis strain SJ9.

Pseudomonas taiwanensis strain SJ9 is a caprolactam degrader, isolated from industrial wastewater in South Korea and considered to have the potential for caprolactam bioremediation. The genome of this strain is approximately 6.2 Mb (G+C content, 61.75%) with 6,010 protein-coding sequences (CDS), of which 46% are assigned to recognized functional genes. This draft genome of strain SJ9 will provide insights into the genetic basis of its caprolactam-degradation ability.

Draft genome sequence of sulfur-reducing archaeon Thermococcus thioreducens DSM 14981T.

Thermococcus thioreducens DSM 14981T, a sulfur-reducing archaeon, was isolated from the rainbow hydrothermal vent site on the Mid-Atlantic Ridge. Herein, we report the draft genome sequence of T. thioreducens DSM 14981T; we obtained 41 contigs with a genome size of 2,052,483bp and G+C content of 53.5%. This genome sequence will not only help understand how the archaeon adapts to the deep-sea hydrothermal environment but also aid the development of enzymes that are highly stable under extreme conditions for industrial applications.