Hepatic extracellular ATP/adenosine dynamics in zebrafish models of alcoholic and metabolic steatotic liver disease.

Steatotic liver disease (SLD) is a burgeoning health problem predominantly associated with excessive alcohol consumption, which causes alcohol-related liver disease (ALD), and high caloric intake, which results in metabolic dysfunction-associated SLD (MASLD). The pathogenesis of ALD and MASLD, which can progress from steatohepatitis to more severe conditions such as liver fibrosis, cirrhosis, and hepatocellular carcinoma, is complicated by several factors. Recently, extracellular ATP and adenosine (Ado), as damage-associated molecular patterns, were reported to promote inflammation and liver fibrosis, contributing to SLD pathogenesis. Here, we explored the in vivo dynamics of hepatic extracellular ATP and Ado during the progression of steatohepatitis using a genetically encoded GPCR-activation-based sensor (GRAB) in zebrafish models. We established hepatocyte-specific GRABATP and GRABAdo in zebrafish and investigated the changes in in vivo hepatic extracellular ATP and Ado levels under ALD or MASLD conditions. Disease-specific changes in hepatocyte extracellular ATP and Ado levels were observed, clearly indicating a correlation between hepatocyte extracellular ATP/Ado dynamics and disease progression. Furthermore, clodronate, a vesicular nucleotide transporter inhibitor, alleviated the MASLD phenotype by reducing the hepatic extracellular ATP and Ado content. These findings provide deep insights into extracellular ATP/Ado dynamics in disease progression, suggesting therapeutic potential for ALD and MASLD.

DHCR7 links cholesterol synthesis with neuronal development and axonal integrity.

The DHCR7 enzyme converts 7-DHC into cholesterol. Mutations in DHCR7 can block cholesterol production, leading to abnormal accumulation of 7-DHC and causing Smith-Lemli-Opitz syndrome (SLOS). SLOS is an autosomal recessive disorder characterized by multiple malformations, including microcephaly, intellectual disability, behavior reminiscent of autism, sleep disturbances, and attention-deficit/hyperactivity disorder (ADHD)-like hyperactivity. Although 7-DHC affects neuronal differentiation in ex vivo experiments, the precise mechanism of SLOS remains unclear. We generated Dhcr7 deficient (dhcr7-/-) zebrafish that exhibited key features of SLOS, including microcephaly, decreased neural stem cell pools, and behavioral phenotypes similar to those of ADHD-like hyperactivity. These zebrafish demonstrated compromised myelination, synaptic anomalies, and neurotransmitter imbalances. The axons of the dhcr7-/- zebrafish showed increased lysosomes and attenuated autophagy, suggesting that autophagy-related neuronal homeostasis is disrupted.

Behavioral and neurotransmitter changes on antiepileptic drugs treatment in the zebrafish pentylenetetrazol-induced seizure model.

Epilepsy, a recurrent neurological disorder involving abnormal neurotransmitter kinetics in the brain, has emerged as a global health concern. The mechanism of epileptic seizures is thought to involve a relative imbalance between excitatory and inhibitory neurotransmitters. Despite the recent advances in clinical and basic research on the pathogenesis of epilepsy, the complex relationship between the neurotransmitter changes and behavior with and without antiepileptic drugs (AEDs) during seizures remains unclear. To investigate the effects of AEDs such as levetiracetam (LEV), carbamazepine (CBZ), and fenfluramine (FFR) on key neurotransmitters in the pentylenetetrazol (PTZ)-induced seizures in adult zebrafish, we examined the changes in glutamic acid, gamma-aminobutyric acid (GABA), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), choline, acetylcholine, norepinephrine, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and adenosine. In this study, we observed that 5-HT and DA levels in the brain increased immediately after PTZ-induced seizures. Behavioral tests clearly showed that all of these AEDs suppressed the PTZ-induced seizures. Upon treatment of PTZ-induced seizures with these AEDs, CBZ decreased the glutamic acid and FFR increased the GABA levels; however, no neurotransmitter changes were observed in the brain after LEV administration. Thus, we demonstrated a series of neurotransmitter changes linked to behavioral changes during PTZ-induced epileptic seizures when LEV, CBZ, or FFR were administered. These findings will lead to a more detailed understanding of the pathogenesis of epilepsy associated with behavioral and neurotransmitter changes under AED treatment.

Behavioral and neurological effects of Vrk1 deficiency in zebrafish.

Vaccinia-related kinase 1 (VRK1) is a serine/threonine kinase, for which mutations have been reported cause to neurodegenerative diseases, including spinal muscular atrophy, characterized by microcephaly, motor dysfunction, and impaired cognitive function, in humans. Partial Vrk1 knockdown in mice has been associated with microcephaly and impaired motor function. However, the pathophysiological relationship between VRK1 and neurodegenerative disorders and the precise mechanism of VRK1-related microcephaly and motor function deficits have not been fully investigated. To address this, in this study, we established vrk1-deficient (vrk1-/-) zebrafish and found that they show mild microcephaly and impaired motor function with a low brain dopamine content. Furthermore, vrk1-/- zebrafish exhibited decreased cell proliferation, defects in nuclear envelope formation, and heterochromatin formation in the brain. To our knowledge, this is the first report demonstrating the important role of VRK1 in microcephaly and motor dysfunction in vivo using vrk1-/- zebrafish. These findings contribute to elucidating the pathophysiological mechanisms underlying VRK1-mediated neurodegenerative diseases associated with microcephaly.

Ankle2 deficiency-associated microcephaly and spermatogenesis defects in zebrafish are alleviated by heterozygous deletion of vrk1.

Autosomal recessive primary microcephaly (MCPH) is a rare congenital disorder characterized by a below average brain volume at birth and is associated with neurodevelopmental disorders such as growth retardation and intellectual disability. Mutations in ANKLE2 have been identified as one of the causes of MCPH (MCPH16). ANKLE2 is a target molecule of the Zika virus NS4a protein that interferes with ANKLE2 function, resulting in severe microcephaly. ANKLE2 is essential for organizing the nuclear envelope and chromatin structures during the mitotic-end process via barrier to autointegration factor (BAF) dephosphorylation. However, the precise mechanism by which the loss of ANKLE2 function causes the pathogenesis of microcephaly remains unclear. In this study, we generated Ankle2-deficient zebrafish (ankle2-/-) with a significant reduction in brain size compared with that of their control siblings. The ankle2-/- brain showed a significant decrease in the number of radial glial progenitor cells, suggesting that Ankle2 deficiency in zebrafish causes neurogenesis defects. Furthermore, ankle2-/- male zebrafish showed infertility owing to defects in spermatogenesis. Notably, microcephaly was overcome by vrk1 morpholino knockdown or vrk1 heterozygous deletion. In addition, spermatogenesis in ankle2-/- zebrafish males was partially restored by the vrk1 heterozygous deletion, although infertility was not resolved. These results indicate that ANKLE2 and VRK1 coordinate with each other for BAF phosphorylation to maintain normal mitosis during neurogenesis and spermatogenesis.

Protective effect of UV-irradiated red perilla (Perilla frutescens (L.) Britton) on carbon tetrachloride-induced liver injury in mice.

UV-irradiated red perilla demonstrated promising protective effects against carbon tetrachloride-induced liver injury in mice. UV exposure significantly enhanced the accumulation of rosmarinic acid, malonylshisonin, and shisonin in red perilla, and increased 1,1-diphenyl-2-picrylhydrazyl radical scavenging capacity. The hepatoprotective effect of UV-irradiated red perilla may be attributed to the high level of its polyphenolic compounds, which exhibit antioxidant activity.

Vrk2 deficiency elicits aggressive behavior in female zebrafish.

Vaccinia-related kinase 2 (VRK2) is a serine/threonine kinase initially identified in highly proliferative cells such as thymocytes and fetal liver cells, and it is involved in cell proliferation and survival. VRK2 is also expressed in the brain; however, its molecular function in the central nervous system is mostly unknown. Many genome-wide association studies (GWASs) have reported that VRK2 is a potential candidate molecule for neuropsychiatric diseases such as schizophrenia in humans. However, the pathophysiological relationship between VRK2 and neuropsychiatric disorders has not been fully investigated. In this study, we evaluated vrk2-deficient (vrk2-/- ) zebrafish and found that vrk2-/- female zebrafish showed aggressive behavior and different social preference compared with control (vrk2+/+ ) zebrafish, with low gamma-aminobutyric acid (GABA) content in the brain and high density of neuronal dendrites when compared to vrk2+/+ zebrafish. These findings suggest that female vrk2-/- zebrafish were indeed a model of malbehavior characterized by aggression and social interaction, which can be attributed to the low levels of GABA content in their brain.

Leucyl-tRNA synthetase deficiency systemically induces excessive autophagy in zebrafish.

Leucyl-tRNA synthetase (LARS) is an enzyme that catalyses the ligation of leucine with leucine tRNA. LARS is also essential to sensitize the intracellular leucine concentration to the mammalian target of rapamycin complex 1 (mTORC1) activation. Biallelic mutation in the LARS gene causes infantile liver failure syndrome type 1 (ILFS1), which is characterized by acute liver failure, anaemia, and neurological disorders, including microcephaly and seizures. However, the molecular mechanism underlying ILFS1 under LARS deficiency has been elusive. Here, we generated Lars deficient (larsb-/-) zebrafish that showed progressive liver failure and anaemia, resulting in early lethality within 12 days post fertilization. The atg5-morpholino knockdown and bafilomycin treatment partially improved the size of the liver and survival rate in larsb-/- zebrafish. These findings indicate the involvement of autophagy in the pathogenesis of larsb-/- zebrafish. Indeed, excessive autophagy activation was observed in larsb-/- zebrafish. Therefore, our data clarify a mechanistic link between LARS and autophagy in vivo. Furthermore, autophagy regulation by LARS could lead to development of new therapeutics for IFLS1.

Exosc2 deficiency leads to developmental disorders by causing a nucleotide pool imbalance in zebrafish.

Exosc2 is one of the components of the exosome complex involved in RNA 3' end processing and degradation of various RNAs. Recently, EXOSC2 mutation has been reported in German families presenting short stature, hearing loss, retinitis pigmentosa, and premature aging. However, the in vivo function of EXOSC2 has been elusive. Herein, we generated Exosc2 knockout (exosc2-/-) zebrafish that showed larval lethality 13 days post fertilization, with microcephaly, loss of spinal motor neurons, myelin deficiency, and retinitis pigmentosa. Mechanistically, Exosc2 deficiency caused impaired mRNA turnover, resulting in a nucleotide pool imbalance. Rapamycin, which modulated mRNA turnover by inhibiting the mTOR pathway, improved nucleotide pool imbalance in exosc2-/- zebrafish, resulting in prolonged survival and partial rescue of neuronal defects. Taken together, our findings offer new insights into the disease pathogenesis caused by Exosc2 deficiency, and might help explain fundamental molecular mechanisms in neuronal diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, and spinal muscular atrophy.

Community composition and methane oxidation activity of methanotrophs associated with duckweeds in a fresh water lake.

Methanotrophs are the only biological sink of the greenhouse gas methane. To understand the ecological features of methanotrophs in association with plants in the methane emitting environments, we investigated the community composition and methane oxidation of methanotrophs associated with duckweeds in a fresh water lake. Duckweeds collected from Lake Biwa, Japan over three summers showed methane consumption activity between 0.0067 and 0.89 µmol h-1 g-1 (wet weight), with the highest values occurring from the end of July to August. The methanotrophic community on duckweeds consisted primarily of γ-proteobacterial groups including the genera Methylomonas and Methylocaldum. Further analysis of co-cultures of a methanotroph isolate with sterilized duckweed revealed that the duckweed plant as well as the duckweed spent culture supernatant exerted an enhancing effect on methane oxidation. These results indicate that duckweeds not only provide a habitat for methanotrophs but also stimulate methanotrophic growth.

Direct free radical scavenging effects of water-soluble HMG-CoA reductase inhibitors.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, statins, are widely used for preventing cardiovascular and cerebrovascular diseases by controlling blood cholesterol level. Additionally, previous studies revealed the scavenging effects of statins on free radicals. We assessed direct scavenging activities of two water-soluble statins, fluvastatin and pravastatin, on multiple free radicals using electron spin resonance spectrometry with spin trapping method. We estimated reaction rate constants (k fv for fluvastatin, and k pv for pravastatin). Superoxide anion was scavenged by fluvastatin and pravastatin with k fv and k pv of 4.82 M-1s-1 and 49.0 M-1s-1, respectively. Scavenging effects of fluvastatin and pravastatin on hydroxyl radical were comparable; both k fv and k pv were >109 M-1s-1. Fluvastatin also eliminated tert-butyl peroxyl radical with relative k fv of 2.63 to that of CYPMPO, whereas pravastatin did not affect tert-butyl peroxyl radical. Nitric oxide was scavenged by fluvastatin and pravastatin with k fv and k pv of 68.6 M-1s-1 and 701 M-1s-1, respectively. Both fluvastatin and pravastatin had scavenging effects on superoxide anion, hydroxyl radical and nitric oxide radical. On the other hand, tert-butyl peroxyl radical was scavenged only by fluvastatin, suggesting that fluvastatin might have more potential effect than pravastatin to prevent atherosclerosis and ischemia/reperfusion injury via inhibiting oxidation of lipids.

Lactobacillus brevis KB290 With Vitamin A Ameliorates Murine Intestinal Inflammation Associated With the Increase of CD11c+ Macrophage/CD103- Dendritic Cell Ratio.

Background: The ratio of colonic anti-inflammatory CD11c+ macrophages (MPs) to inflammatory CD103- dendritic cells (DCs) plays pivotal roles in intestinal inflammation. Little is known about how the ratio is regulated by lactic acid bacteria (LAB) and bifidobacteria (Bif). We investigated the contribution of LAB/Bif to this ratio. Methods: We established an in vitro experimental system using human myeloblastic KG-1 cells, which differentiate into CD11c+ MP-like (CD11c+ MPL) and CD103- DC-like (CD103- DCL) cells, and explored effective LAB/Bif strains. The selected strain's effect on the colonic CD11c+ MP/CD103- DC ratio and intestinal inflammation was examined in mice, and the strain's underlying mechanisms were investigated in vitro. Results: We screened 19 strains of LAB/Bif, and found that Lactobacillus brevis KB290 (KB290) increased the CD11c+ MPL/CD103- DCL cell ratio only in the presence of a vitamin A (VA) metabolite, retinoic acid (RA). Supplementation of KB290 with VA increased the CD11c+ MP/CD103- DC ratio in healthy mouse and prevented the disruption of the ratio during colitis. Supplementation of KB290 with pro-VA (ß-carotene) also increased the ratio in healthy mouse and ameliorated the development of colitis. The ratio was increased by reduction of CD103- DCs (or CD103- DCL cells). Our in vitro data suggested that KB290 induced cell death in CD103- DCL cells in the presence of RA signaling. Conclusions: Supplementation of KB290 with VA increases the colonic CD11c+ MP/CD103- DC ratio associated with the amelioration of murine colitis, suggesting a possible way to control intestinal inflammation by LAB.

Glucoraphanin Ameliorates Obesity and Insulin Resistance Through Adipose Tissue Browning and Reduction of Metabolic Endotoxemia in Mice.

Low-grade sustained inflammation links obesity to insulin resistance and nonalcoholic fatty liver disease (NAFLD). However, therapeutic approaches to improve systemic energy balance and chronic inflammation in obesity are limited. Pharmacological activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) alleviates obesity and insulin resistance in mice; however, Nrf2 inducers are not clinically available owing to safety concerns. Thus, we examined whether dietary glucoraphanin, a stable precursor of the Nrf2 inducer sulforaphane, ameliorates systemic energy balance, chronic inflammation, insulin resistance, and NAFLD in high-fat diet (HFD)-fed mice. Glucoraphanin supplementation attenuated weight gain, decreased hepatic steatosis, and improved glucose tolerance and insulin sensitivity in HFD-fed wild-type mice but not in HFD-fed Nrf2 knockout mice. Compared with vehicle-treated controls, glucoraphanin-treated HFD-fed mice had lower plasma lipopolysaccharide levels and decreased relative abundance of the gram-negative bacteria family Desulfovibrionaceae in their gut microbiomes. In HFD-fed mice, glucoraphanin increased energy expenditure and the protein expression of uncoupling protein 1 (Ucp1) in inguinal and epididymal adipose depots. Additionally, in this group, glucoraphanin attenuated hepatic lipogenic gene expression, lipid peroxidation, classically activated M1-like macrophage accumulation, and inflammatory signaling pathways. By promoting fat browning, limiting metabolic endotoxemia-related chronic inflammation, and modulating redox stress, glucoraphanin may mitigate obesity, insulin resistance, and NAFLD.