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Qian Sun, Ruifeng Mao, Dongling Wang, Changyan Hu, Yang Zheng, Dequn Sun Department of Pharmacy, Marine College, Shandong University, Weihai, People’s Republic of China Abstract: Praziquantel (PZQ) is prescribed as a racemic mixture (racemic-PZQ, rac-PZQ), which is composed of (R)-PZQ and (S)-PZQ. In this work, the cytotoxicity of rac-PZQ and its two enantiomers (R)-PZQ and (S)-PZQ on eight cell lines (L-02, HepG2, prf-plc-5, SH-SY5Y, HUVEC, A549, HCT-15, Raw264.7) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays. The morphology of apoptotic cells was studied by fluorescence microscope using Hoechst 33342 staining, and the cytotoxicity of the compounds was also tested by lactate dehydrogenase assay. Results revealed that (R)-PZQ had negligible cytotoxicity against L-02, SH-SY5Y, HUVEC, A549, HCT-15, and Raw264.7 cells but selectively inhibited tumor cell lines (prf-plc-5 and HepG2). However, in contrast to (R)-PZQ, the (S)-isomer showed higher cytotoxicity against L-02 cells and lower inhibition on prf-plc-5 and HepG2 cells. Besides, (R)-PZQ showed lower cytotoxicity on SH-SY5Y cells than (S)-PZQ. Meanwhile, (R)-PZQ at

Background: Ethanol exposure induces apoptotic neurodegeneration in the developing rodent brain during synaptogenesis. This process has been studied as a model for fetal alcohol syndrome. Previously, we have shown that gangliosides and LIGA20 (a semisynthetic derivative of GM1 ganglioside) attenuate ethanol-induced apoptosis in cultured neurons. In the present study, the effects of GM1 and LIGA20 on ethanol-induced apoptotic neurodegeneration were examined using an in vivo neonatal mouse model. Methods: Seven-day-old C57BL/6By (B6By) mice were pretreated twice with intraperitoneal administration of GM1 (30 mg/kg), LIGA20 (2.5 mg/kg), or saline, followed by subcutaneous injection of either saline or ethanol (2.5 g/kg) twice with a 2 hours interval. Then the brains were: (1) perfusion-fixed 24 hours after the first ethanol injection, and the extent of neurodegeneration was assessed by cupric silver staining of the brain sections, or (2) perfusion-fixed 8 hours after the first ethanol injection, and the sections were immunostained with anti-cleaved (activated) caspase-3 antibody to evaluate caspase-3 activation. Results: The comparison of cupric silver stained coronal sections indicates that ethanol-induced widespread neurodegeneration in the forebrains of B6By mice was reduced overall by GM1 and LIGA20 pretreatments. The extent of neurodegeneration detected by silver impregnation and activated caspase-3 immunostaining was quantified in the cingulate and retrosplenial cortices, which were the regions most severely affected by ethanol. The results indicate that GM1 and LIGA20 pretreatments induced statistically significant reductions-approximately 50% of the ethanol-treated samples-in silver impregnation and activated caspase-3 immunostaining. No significant differences were observed between saline controls and samples treated with GM1 or LIGA20 alone. Conclusions: These results indicate that GM1 and LIGA20, which have been shown to be neuroprotective against insults caused by various agents, partially attenuate ethanol-induced apoptotic neurodegeneration in the developing mouse brain. [ABSTRACT FROM AUTHOR]

In this editorial, we commented on two articles published online in August and September 2024 in the World Journal of Diabetes , which focused on modifying the gut microbiota (GM) to prevent or delay the progression of diabetes mellitus (DM) and DM-related complications. Numerous studies, many of which are animal studies, have indicated the potential role of GM in the pathogenesis of DM. However, the detailed causality and mechanisms between GM and DM have not been fully clarified. Although there have been some reports of a potential role of modifying the GM in treating DM, most lack long-term observations and are not mechanistic. Additionally, the GM and its role in DM might vary among individuals; therefore, GM-targeted interventions should be individualized to realize their therapeutic potential., Competing Interests: Conflict-of-interest statement: Authors declare no conflict of interests for this article., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

As a common hyperglycemic disease, type 1 diabetes mellitus (T1DM) is a complicated disorder that requires a lifelong insulin supply due to the immune-mediated destruction of pancreatic β cells. Although it is an organ-specific autoimmune disorder, T1DM is often associated with multiple other autoimmune disorders. The most prevalent concomitant autoimmune disorder occurring in T1DM is autoimmune thyroid disease (AITD), which mainly exhibits two extremes of phenotypes: hyperthyroidism [Graves' disease (GD)] and hypo-thyroidism [Hashimoto's thyroiditis, (HT)]. However, the presence of comorbid AITD may negatively affect metabolic management in T1DM patients and thereby may increase the risk for potential diabetes-related complications. Thus, routine screening of thyroid function has been recommended when T1DM is diagnosed. Here, first, we summarize current knowledge regarding the etiology and pathogenesis mechanisms of both diseases. Subsequently, an updated review of the association between T1DM and AITD is offered. Finally, we provide a relatively detailed review focusing on the application of thyroid ultrasonography in diagnosing and managing HT and GD, suggesting its critical role in the timely and accurate diagnosis of AITD in T1DM., Competing Interests: Conflict-of-interest statement: Authors declare no conflict of interests for this article., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

A Gram-negative, facultative anaerobic bacterial strain, designated YIM B02556 T , was isolated from the root of Paris polyphylla Smith var. yunnanensis collected from Yunnan Province, southwest China. By using a polyphasic approach, its taxonomic position was investigated. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YIM B02556 T belonged to the genus Azospirillum and the 16S rRNA gene sequence similarity values of strain YIM B02556 T to the type strains of members of this genus ranged from 94.9 to 98.3%. Overall genome relatedness index (OGRI) analysis estimated based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between YIM B02556 T and other Azospirillum species type strains were <90.8% and <37.8%, lower than the limit of species circumscription. Cells of the strain were characterized as oxidase- and catalase-positive, with motility provided by flagella. The growth conditions of the strain were found to occur at 20-40 °C (optimum, 35 °C), and pH 6.0-9.5 (optimum, pH 7.5). Strain YIM B02556 T can tolerate 2% NaCl concentration. Strain YIM B02556 T contained Q-10 as the major ubiquinone. The major fatty acids were C 18:1 ω7c and summed feature three (C 16:1 ω7c and/or C 16:1 ω6c). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. Based on polyphasic analysis, strain YIM B02556 T could be differentiated genotypically and phenotypically from recognized species of the genus Azospirillum. Therefore, the isolate represents a novel species, for which the name Azospirillum endophyticum is proposed. The type strain is YIM B02556 T (=JCM 34631 T =CGMCC 1.18654 T )., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Clonorchiasis, an infectious disease caused by the liver fluke Clonorchis sinensis, may lead to the development of liver and gallbladder diseases, and even cholangiocarcinoma (CCA). However, the pathogenesis, host-pathogen interaction, and diagnostic markers for clonorchiasis remain unclear., Methods: Eighteen rabbits were randomly divided into control group (n = 9) and C. sinensis-infected group (n = 9), and their plasma samples were collected at 7, 14, 28, and 63 days post-infection (dpi). Biochemical indices and metabolites in different infection periods were detected. A non-targeted ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach was employed to investigate the metabolic profiles of plasma in rabbits, and related metabolic pathways of differential metabolites and correlation between candidate biochemical indices and differential metabolites were analyzed. Finally, the candidate biomarkers were verified with human samples using a targeted metabolomics method., Results: The result of biochemical indices indicated C. sinensis infection would affect the liver function biochemical indices, especially alanine aminotransferase, aspartate transaminase (AST), glutamyl transpeptidase (GGT), total bile acid, high-density lipoprotein, and cholinesterase. The metabonomic results showed that 58, 212, 23, and 21 differential metabolites were identified in different phases of the infection. Multivariate statistical analysis of differential metabolites revealed distinct metabolic signatures during different phases of infection, with most of these signatures being observed at 14 dpi, which mainly influences the amino acid metabolisms. For metabolites and biochemical indices, AST, GGT, hypoxanthine, L-pipecolic acid, and D-glucuronate represented potential noninvasive biomarkers for the diagnosis of C. sinensis (P < 0.05 and AUC > 0.8). Furthermore, GGT and D-glucuronate levels were positively correlated with the infection (r(28) = 0.98, P < 0.0001) and showed excellent diagnostic performance (AUC = 0.972; 95% confidence interval, 0.921 to 1.000)., Conclusions: The present results provide new insights into plasma metabolic changes in rabbits during C. sinensis infection, and the potential biomarker may be used for developing an effective method to diagnose clonorchiasis in the future., (© 2022. The Author(s).)

A novel endophytic strain, designated YIM B02564 T , was isolated from the root of Paris polyphylla Smith var. yunnanensis obtained from Yunnan Province, southwest China. By using a polyphasic approach, cells of the strain were characterized as facultative anaerobic, Gram-positive and rod-shaped. The growth conditions of the strain were found to occur at 20-55 °C (optimum, 30 °C), pH 6.0-9.0 (optimum, pH 7.0). Strain YIM B02564 T can tolerate 2% NaCl concentration. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YIM B02564 T belonged to the genus Neobacillus and the 16S rRNA gene sequence similarity values of strain YIM B02564 T to the type strains of members of this genus ranged from 95.6 to 97.8%. The DNA G+C content of strain YIM B02564 T calculated from the whole genome sequence was 41.6 mol%. Values of the ANI and the dDDH between strain YIM B02564 T and its closely related Neobacillus species were below 77.9% and 21.5%. Strain YIM B02564 T contained MK-7 as the major menaquinone, iso-C 15:0 and anteiso-C 15:0 as the major fatty acids. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminophospholipid and four unidentified lipids. It contained meso-diaminopimelic acid in the cell-wall peptidoglycan. On the basis of polyphasic analysis, strain YIM B02564 T could be differentiated genotypically and phenotypically from recognized species of the genus Neobacillus. The isolate therefore represents a novel species, for which the name Neobacillus paridis is proposed. The type strain is YIM B02564 T (= JCM 34668 T  = CGMCC 1.18655 T )., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A novel endophytic actinobacterium, designated as strain YIM B02568 T , was isolated from the root of Paris polyphylla Smith var. Yunnanensis obtained from Yunnan Province, southwest China. Strain YIM B02568 T was characterized using a polyphasic approach. Phylogenetic analysis indicated that this isolate belonged to the genus Janibacter. The 16S rRNA gene sequence similarity values of strain YIM B02568 T to the type strains of members of this genus ranged from 95.8 to 98.6%. However, overall genome relatedness indices were significantly lower than the widely accepted species-defined threshold. The cell wall of strain YIM B02568 T contained meso-diaminopimelic acid. The major menaquinone was MK-8(H 4 ). The main polar lipids were phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylinositol. The major cellular fatty acids were comprised of iso-C 16:0 and C 18:1 ω9c. The DNA G + C content was 71.6 mol%. Based on the data from the polyphasic studies, we propose that strain YIM B02568 T represents a novel species within the genus Janibacter, Janibacter endophyticus sp. nov. The type strain is YIM B02568 T (= JCM 34639 T  = CGMCC 1.18658 T )., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Tetrameres grusi is a significant parasitic nematode of cranes that is classified into suborder Spirurina. However, for more than a century, this classification has been controversial. Mitochondrial genomes are valuable resources for parasite taxonomy, population genetics and systematics studies. Here, the mitochondrial genome of T. grusi was determined and subsequently compared with those from Spirurina species using concatenated datasets of amino acid sequences predicted from mitochondrial protein-coding genes. The complete mitochondrial genome of T. grusi is circular with 13,709 bp, and it contains 12 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one non-coding region. All of the protein-coding genes are transcribed in the same direction. There were 18 intergenic spacers of 1-44 bp, and six locations with gene overlaps, ranging from 1 bp to 28 bp, in the mitochondrial genome of T. grusi . The AT content of this mitochondrial genome was 71.56%. This was similar to mitochondrial genomes of other Spirurina species, which also exhibited strong AT content bias, not only in the nucleotide composition but also in codon usage. The sequenced mitogenomes of the 25 Spirurina nematodes showed three classes of gene arrangements based on the 12 protein-coding genes, and the gene arrangement of the T. grusi mitochondrial genome belonged to the Class I. Phylogenetic analyses using mitochondrial genomes of 25 Spirurina nematodes revealed that T. grusi (Habronematoidea) was closer to Gongylonema pulchrum (Spiruroidea) than Spirocerca lupi (Thelazioidea). The availability of the complete mitochondrial genome sequence of T. grusi provides new and useful genetic markers for further studies on Spirurina nematodes., Competing Interests: The authors report no conflicts of interests., (© 2021 The Authors.)

Metorchis orientalis is a neglected zoonotic parasite of the gallbladder and bile duct of poultry, mammals, and humans. It has been widely reported in Asian, including China, Japanese, and Korea, where it is a potential threat to public health. Despite its significance as an animal and human pathogen, there are few published transcriptomic and proteomics data available. Transcriptome Illumina RNA sequencing and label-free protein quantification were performed to compare the gene and protein expression of adult and metacercariae-stage M. orientalis , resulting in 100,234 unigenes and 3,530 proteins. Of these, 13,823 differentially expressed genes and 1,445 differentially expressed proteins were identified in adult versus metacercariae. In total, 570 genes were differentially expressed consistent with the mRNA and protein level in the adult versus metacercariae stage. Differential gene transcription analyses revealed 34,228 genes to be expressed in both stages, whereas 66,006 genes showed stage-specific expression. Compared with adults, the metacercariae stage was highly transcriptional. GO and KEGG analyses based on transcriptome and proteome revealed numerous up-regulated genes in adult M. orientalis related to microtubule-based processes, microtubule motor activity, and nucleocytoplasmic transport. The up-regulated genes in metacercariae M. orientalis were mainly related to transmembrane receptor protein serine/threonine kinase activity, transmembrane receptor protein serine/threonine kinase signaling pathway. Transcriptome and proteome comparative analyses showed numerous up-regulated genes in adult stage were mainly enriched in actin filament capping, spectrin, and glucose metabolic process, while up-regulated genes in metacercariae stage were mainly related to cilium assembly, cilium movement, and motile cilium. These results highlight changes in protein and gene functions during the development of metacercariae into adults, and provided evidence for the mechanisms involved in morphological and metabolic changes at both the protein and gene levels. Interestingly, many genes had been proved associated with liver fibrosis and carcinogenic factors were identified highly expressed in adult M. orientalis , which suggests that M. orientalis is a neglected trematode with potential carcinogenic implications. These data provide attractive targets for the development of therapeutic or diagnostic interventions for controlling M. orientalis ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gao, Lv, Mao, Sun, Chen, Qiu, Chang and Wang.)

As a T cell-mediated autoimmune disease, type 1 diabetes mellitus (T1DM) is marked by insulin defect resulting from the destruction of pancreatic β-cells. The understanding of various aspects of T1DM, such as its epidemiology, pathobiology, pathogenesis, clinical manifestations, and complications, has been greatly promoted by valuable research performed during the past decades. However, these findings have not been translated into an effective treatment. The ideal treatment should safely repair the destroyed immune balance in a long-lasting manner, preventing or stopping the destruction of β-cells. As a type of immune hypo-responsiveness to the orally administrated antigen, oral tolerance may be induced by enhancement of regulatory T cells (Tregs) or by anergy/deletion of T cells, depending on the dosage of orally administrated antigen. Acting as an antigen-specific immunotherapy, oral tolerance therapy for T1DM has been mainly performed using animal models and some clinical trials have been completed or are still ongoing. Based on the review of the proposed mechanism of the development of T1DM and oral tolerance, we give a current overview of oral tolerance therapy for T1DM conducted in both animal models and clinical trials., Competing Interests: Conflict-of-interest statement: The authors declare no conflicts of interest., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Microplastic pollution is an increasingly important environment problem. Many studies show the occurrence of microplastics in our environmental system. However, the freshwater system is less understood, especially in northwest China. We investigated the occurrence and characteristics of microplastics in the Wei River Basin, which is located in northwestern China. The Wei River is the largest tributary of the Yellow River and runs through three major provinces. In the Wei River, the concentration of microplastics in the surface waters varied from 3.67 to 10.7 items /L and in the sediments, the abundance of microplastics varied from 360 to 1320 items/kg. Fiber (50.1%)was the dominant types in water samples and sediments. The small size (<0.5 mm)(68.1%)were the main size of microplastics in Wei River. The types of microplastics were polyethylene, Polyvinyl chloride and polystyrene, as identified using a Fourier transform infrared spectrometer. This study could be a valuable reference for better understanding the microplastics pollution in inland northwestern China., (Copyright © 2019 Elsevier B.V. All rights reserved.)

CHI3L1 (YKL40) is a secreted glycoprotein and elevated serum CHI3L1 level has been proved to be associated with poor prognosis in many human cancers. However, the mechanism of how CHI3L1 causes poor prognosis in cancers is still unknown. Here, considering that CHI3L1 is a liver specific/enriched protein, we use hepatocellular carcinoma as a model to study the function of CHI3L1. We showed that, both in vivo and in vitro, overexpression of CHI3L1 could promote liver cancer cells growth, migration and invasion. We then used RNA-seq to analyze the expression profiles of CHI3L1 overexpressed in two HCC cell lines and found that CHI3L1 overexpression affected genes that were involved in cell-cell adhesion, extracellular exosome and adherens junction. Western blot analysis further revealed that CHI3L1 could activate TGF-β signal pathways. Our data added new understanding of the mechanism of CHI3L1's action. 1) CHI3L1 promoted cancer cell proliferation by regulating cell cycles; 2) CHI3L1 promoted cancer cell invasion and metastasis; 3) CHI3L1 regulate liver cancer potentially by regulating the TGF-β signaling pathways; 4) CHI3L1 has direct kinase activities or activate kinase to phosphorylate SMAD2, SMAD3.

The heat-shock response is an evolutionarily conserved cellular defense mechanism against environmental stresses, characterized by the rapid synthesis of heat-shock proteins (HSPs). HSP70, a highly inducible molecular chaperone, assists in refolding or clearance of damaged proteins, thereby having a central role in maintaining intracellular homeostasis and thermotolerance. To date, induction of HSP70 expression has been described extensively at the transcriptional level. However, post-translational regulation of HSP70, such as protein stability, is only partially understood. In this study, we investigated the role of OLA1 (Obg-like ATPase 1), a previously uncharacterized cytosolic ATPase, in regulating the turnover of HSP70. Downregulation of OLA1 in mammalian cells by either RNAi or targeted gene disruption results in reduced steady-state levels of HSP70, impaired HSP70 induction by heat, and functionally, increased cellular sensitivity to heat shock. Conversely, overexpression of OLA1 correlates with elevated HSP70 protein levels and improved thermal resistance. Protein-protein interaction assays demonstrated that binding of OLA1 to the HSP70 carboxyl terminus variable domain hinders the recruitment of CHIP (C-terminus of Hsp70-binding protein), an E3 ubiquitin ligase for HSP70, and thus prevents HSP70 from the CHIP-mediated ubiquitination. These findings suggest a novel molecular mechanism by which OLA1 stabilizes HSP70, leading to upregulation of HSP70 as well as increased survival during heat shock.

Our previous studies have indicated that de novo ceramide synthesis plays a critical role in ethanol-induced apoptotic neurodegeneration in the 7-day-old mouse brain. In this study, we examined whether the formation of sphingosine 1-phosphate (S1P), a ceramide metabolite, is associated with this apoptotic pathway. Analyses of basal levels of S1P-related compounds indicated that S1P, sphingosine, sphingosine kinase 2, and S1P receptor 1 increased significantly during postnatal brain development. In the 7-day-old mouse brain, sphingosine kinase 2 was localized mainly in neurons. Subcellular fractionation studies of the brain homogenates showed that sphingosine kinase 2 was enriched in the plasma membrane and the synaptic membrane/synaptic vesicle fractions, but not in the nuclear and mitochondrial/lysosomal fractions. Ethanol exposure in 7-day-old mice induced sphingosine kinase 2 activation and increased the brain level of S1P transiently 2-4 h after exposure, followed by caspase 3 activation that peaked around 8 h after exposure. Treatment with dimethylsphingosine, an inhibitor of sphingosine kinases, attenuated the ethanol-induced caspase 3 activation and the subsequent neurodegeneration. These results indicate that ethanol activates sphingosine kinase 2, leading to a transient increase in S1P, which may be involved in neuroapoptotic action of ethanol in the developing brain., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

GM2 ganglioside in the brain increased during ethanol-induced acute apoptotic neurodegeneration in 7-day-old mice. A small but a significant increase observed 2 h after ethanol exposure was followed by a marked increase around 24 h. Subcellular fractionation of the brain 24 h after ethanol treatment indicated that GM2 increased in synaptic and non-synaptic mitochondrial fractions as well as in a lysosome-enriched fraction characteristic to the ethanol-exposed brain. Immunohistochemical staining of GM2 in the ethanol-treated brain showed strong punctate staining mainly in activated microglia, in which it partially overlapped with staining for LAMP1, a late endosomal/lysosomal marker. Also, there was weaker neuronal staining, which partially co-localized with complex IV, a mitochondrial marker, and was augmented in cleaved caspase 3-positive neurons. In contrast, the control brain showed only faint and diffuse GM2 staining in neurons. Incubation of isolated brain mitochondria with GM2 in vitro induced cytochrome c release in a manner similar to that of GD3 ganglioside. Because ethanol is known to trigger mitochondria-mediated apoptosis with cytochrome c release and caspase 3 activation in the 7-day-old mouse brain, the GM2 elevation in mitochondria may be relevant to neuroapoptosis. Subsequently, activated microglia accumulated GM2, indicating a close relationship between GM2 and ethanol-induced neurodegeneration., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Lys63-linked polyubiquitination of transforming growth factor-β-activated kinase 1 (TAK1) has an important role in tumor necrosis factor-α (TNFα)-induced NF-κB activation. Using a functional genomic approach, we have identified ubiquitin-specific peptidase 4 (USP4) as a deubiquitinase for TAK1. USP4 deubiquitinates TAK1 in vitro and in vivo. TNFα induces association of USP4 with TAK1 to deubiquitinate TAK1 and downregulate TAK1-mediated NF-κB activation. Overexpression of USP4 wild type, but not deuibiquitinase-deficient C311A mutant, inhibits both TNFα- and TAK1/TAB1 co-overexpression-induced TAK1 polyubiquitination and NF-κB activation. Notably, knockdown of USP4 in HeLa cells enhances TNFα-induced TAK1 polyubiquitination, IκB kinase phosphorylation, IκBα phosphorylation and ubiquitination, as well as NF-κB-dependent gene expression. Moreover, USP4 negatively regulates IL-1β-, LPS- and TGFβ-induced NF-κB activation. Together, our results demonstrate that USP4 serves as a critical control to downregulate TNFα-induced NF-κB activation through deubiquitinating TAK1.

Previous studies indicated that ethanol-induced neurodegeneration in postnatal day 7 (P7) mice, widely used as a model for the fetal alcohol spectrum disorders, was accompanied by glycogen synthase kinase-3beta (GSK-3beta) and caspase-3 activation. Presently, we examined whether tau, a microtubule associated protein, is modified by GSK-3beta and caspase-3 in ethanol-treated P7 mouse forebrains. We found that ethanol increased phosphorylated tau recognized by the paired helical filament (PHF)-1 antibody and by the antibody against tau phosphorylated at Ser199. Ethanol also generated tau fragments recognized by an antibody against caspase-cleaved tau (C-tau). C-tau was localized in neurons bearing activated caspase-3 and fragmented nuclei. Over time, cell debris and degenerated projections containing C-tau appeared to be engulfed by activated microglia. A caspase-3 inhibitor partially blocked C-tau formation. Lithium, a GSK-3beta inhibitor, blocked ethanol-induced caspase-3 activation, phosphorylated tau elevation, C-tau formation, and microglial activation. These results indicate that tau is phosphorylated by GSK-3beta and cleaved by caspase-3 during ethanol-induced neurodegeneration in the developing brain.

It has been shown that lipogenic enzymes, such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), are highly expressed in the rodent brain during the early neonatal period and decline thereafter. However, cellular localization of these enzymes is unknown. Presently, we examined developmental changes in the levels and cellular localization of FAS and ACC, and their putative regulators, sterol-regulatory element-binding protein (SREBP)-1 and AMP-activated protein kinase (AMPK) in the mouse brain. Levels of these proteins including phosphorylated forms of ACC and AMPK decreased between postnatal day 4 (P4) and P19. Immunohistochemical studies indicated that FAS, ACC, AMPK, and SREBP-1 were expressed in neurons at P7, while FAS was found mostly in cells of oligodendrocyte lineage at P19. These studies suggest that neurons in the early neonatal brain are involved in do novo fatty acid synthesis.

Lithium has been shown to be neuroprotective against various insults including ethanol exposure. We previously reported that ethanol-induced apoptotic neurodegeneration in the postnatal day 7 (P7) mice is associated with decreases in phosphorylation levels of Akt, glycogen synthase kinase-3beta (GSK-3beta), and AMP-activated protein kinase (AMPK), and alteration in lipid profiles in the brain. Here, P7 mice were injected with ethanol and lithium, and the effects of lithium on ethanol-induced alterations in phosphorylation levels of protein kinases and lipid profiles in the brain were examined. Immunoblot and immunohistochemical analyses showed that lithium significantly blocked ethanol-induced caspase-3 activation and reduction in phosphorylation levels of Akt, GSK-3beta, and AMPK. Further, lithium inhibited accumulation of cholesterol ester (ChE) and N-acylphosphatidylethanolamine (NAPE) triggered by ethanol in the brain. These results suggest that Akt, GSK-3beta, and AMPK are involved in ethanol-induced neurodegeneration and the neuroprotective effects of lithium by modulating both apoptotic and survival pathways.

Previously, we have shown that ethanol-induced apoptosis in cultured neurons is accompanied by changes in cellular lipid profiles. In the present study, the effects of ethanol on brain lipid metabolism were studied using 7-day-old C57BL/6ByJ mice, which display apoptotic neurodegeneration upon exposure to ethanol. The brain lipids were extracted 4-24 h after the ethanol or saline treatment, and analyzed by TLC. We found that the levels of triglyceride, cholesterol ester, ceramide, and N-acylphosphatidylethanolamine increased significantly in the brains of ethanol-treated mice compared to those of saline-treated mice. Concomitantly, ethanol reduced Thr172 phosphorylation of AMP-activated protein kinase (AMPK) alpha subunits. Ethanol also reduced phosphorylation of acetyl-CoA carboxylase, a substrate of AMPK and a lipogenic enzyme known to be activated by dephosphorylation. In contrast, lipid profiles of 19-day-old mouse brains, which scarcely manifested neurodegeneration upon ethanol exposure, were not significantly affected by ethanol. Also, the basal levels of Thr172-phosphorylated AMPK alpha were lower in these brains than in 7-day-old mouse brains, and no detectable changes in the phosphorylation status were observed by ethanol treatment. Our findings indicate that the ethanol-induced apoptotic neurodegeneration observed in mice during restricted developmental periods is accompanied by alterations in both the lipid content and the activity of AMPK in the brain.

Alcohol consumption by C57BL/6By background and BALB/cJ donor strains, and by two recently developed quasi-congenic QTL-introgression strains, which share about 96% of their genes with the background strain, was studied in a limited access paradigm. Alcohol and water were offered for 60 min per day using modified pipettes on a drinking cage. Increasing concentration of alcohol solutions, 3, 6, and 12%, were given for days 1-7, 8-14, and 15-22, respectively. Consumption of the 12% alcohol solution was highest in C57BL/6By (0.72 g/kg/h), lowest in BALB/cJ (0.14 g/kg/h). The B6.Cb4i5 beta 13 quasi-congenic strain, in spite of its genetic similarity to the C57BL/6By background strain, consumed significantly less alcohol (0.41 g/kg/h) than the background strain. The results suggest that polygenes that reduce alcohol consumption were introgressed from the BALB/cJ donor strain into the C57BL/6By background strain, and that the b4i5 series of the B6.C quasi-congenic QTL-introgression strains may be useful in mapping genes that influence alcohol-related behaviors. Locations of the introgressed candidate polygenes were tentatively identified by analyzing microsatellite maps of two of the quasi-congenic strains.