Recent studies using knock-out mice for various secreted phospholipase A2 (sPLA2) isoforms have revealed their non-redundant roles in diverse biological events. In the skin, group IIF sPLA2 ...(sPLA2-IIF), an “epidermal sPLA2” expressed in the suprabasal keratinocytes, plays a fundamental role in epidermal-hyperplasic diseases such as psoriasis and skin cancer. In this study, we found that group IIE sPLA2 (sPLA2-IIE) was expressed abundantly in hair follicles and to a lesser extent in basal epidermal keratinocytes in mouse skin. Mice lacking sPLA2-IIE exhibited skin abnormalities distinct from those in mice lacking sPLA2-IIF, with perturbation of hair follicle ultrastructure, modest changes in the steady-state expression of a subset of skin genes, and no changes in the features of psoriasis or contact dermatitis. Lipidomics analysis revealed that sPLA2-IIE and -IIF were coupled with distinct lipid pathways in the skin. Overall, two skin sPLA2s, hair follicular sPLA2-IIE and epidermal sPLA2-IIF, play non-redundant roles in distinct compartments of mouse skin, underscoring the functional diversity of multiple sPLA2s in the coordinated regulation of skin homeostasis and diseases.
Choline supplies methyl groups for regeneration of methionine and the methyl donor S-adenosylmethionine in the liver. Here, we report that the catabolism of membrane phosphatidylcholine (PC) into ...water-soluble glycerophosphocholine (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several metabolites related to the methionine cycle, accompanied by various signs of methionine insufficiency, including growth retardation, hypoglycemia, hypolipidemia, increased energy consumption, reduced adiposity, increased fibroblast growth factor 21 (FGF21), and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a methionine/choline-deficient diet, both knockout strains display decreased hepatic triglyceride, likely via reductions of lipogenesis and GPC-derived glycerol flux. Collectively, our findings highlight the biological importance of phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and triglyceride synthesis in the liver.
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•PNPLA7 is a major lysophospholipase that produces GPC in the liver•Mice deficient in PNPLA7 or PNPLA8 show various signs of methionine insufficiency•Choline mobilized from hepatic PC by the PNPLA8-PNPLA7 axis supplies methyl groups•This PC-catabolic pathway is also linked to hepatic TG synthesis via glycerol flux
Hirabayashi et al. demonstrate that hepatic phosphatidylcholine catabolism driven by two particular members of the phospholipase A2 family, PNPLA7 and PNPLA8, mobilizes endogenous choline to replenish the methionine cycle with methyl groups. Genetic deletion of these enzymes leads to systemic abnormalities reminiscent of methyl group deficiency.
Arrest of replication fork progression is one of the most common causes for increasing the genomic instability. In bacteria, PriA, a conserved DEXH-type helicase, plays a major role in recognition of ...the stalled forks and restart of DNA replication. We took advantage of PriA's ability to specifically recognize stalled replication forks to determine the genomic loci where replication forks are prone to stall on the Escherichia coli genome. We found that PriA binds around oriC upon thymine starvation which reduces the nucleotide supply and causes replication fork stalling. PriA binding quickly disappeared upon readdition of thymine. Furthermore, BrdU was incorporated at around oriC upon release from thymine starvation. Our results indicate that reduced supply of DNA replication precursors causes replication fork stalling preferentially in the 600 kb segment centered at oriC. This suggests that replication of the vicinity of oriC requires higher level of nucleotide precursors. The results also point to a possibility of slow fork movement and/or the presence of multiple fork arrest signals within this segment. Indeed, we have identified rather strong fork stall/pausing signals symmetrically located at ∼50 kb away from oriC. We speculate that replication pausing and fork-slow-down shortly after initiation may represent a novel checkpoint that ensures the presence of sufficient nucleotide supply prior to commitment to duplication of the entire genome.
•Upon thymine starvation, PriA protein binds around oriC, suggesting replication forks stall preferentially near oriC shortly after initiation.•PriA dissociates from oriC upon readdition of thymine to thymine-starved cells.•BrdU incorporation assays show fork restart at around oriC after release, consistent with the fork stall near oriC.•Specific replication fork pause/arrest sites were detected at ∼50 kb away from oriC in unperturbed growth.
The increased glucose flux into the polyol pathway via aldose reductase (AR) is recognized as a major contributing factor for the pathogenesis of diabetic neuropathy, whereas little is known about ...the functional significance of AR in the peripheral nervous system. Spontaneously immortalized Schwann cell lines established from long‐term cultures of AR‐deficient and normal C57BL/6 mouse dorsal root ganglia and peripheral nerves can be useful tools for studying the physiological and pathological roles of AR. These cell lines, designated as immortalized knockout AR Schwann cells 1 (IKARS1) and 1970C3, respectively, demonstrated distinctive Schwann cell phenotypes, such as spindle‐shaped morphology and immunoreactivity to S100, p75 neurotrophin receptor, and vimentin, and extracellular release of neurotrophic factors. Conditioned media obtained from these cells promoted neuronal survival and neurite outgrowth of cultured adult mouse dorsal root ganglia neurons. Microarray and real‐time RT‐PCR analyses revealed significantly down‐regulated mRNA expression of polyol pathway‐related enzymes, sorbitol dehydrogenase and ketohexokinase, in IKARS1 cells compared with those in 1970C3 cells. In contrast, significantly up‐regulated mRNA expression of aldo‐keto reductases (AKR1B7 and AKR1B8) and aldehyde dehydrogenases (ALDH1L2, ALDH5A1, and ALDH7A1) was detected in IKARS1 cells compared with 1970C3 cells. Exposure to reactive aldehydes (3‐deoxyglucosone, methylglyoxal, and 4‐hydroxynonenal) significantly up‐regulated the mRNA expression of AKR1B7 and AKR1B8 in IKARS1 cells, but not in 1970C3 cells. Because no significant differences in viability between these two cell lines after exposure to these aldehydes were observed, it can be assumed that the aldehyde detoxification is taken over by AKR1B7 and AKR1B8 in the absence of AR.
Aldose reductase (AR) is involved in the pathogenesis of diabetic neuropathy via activating polyol pathway, whereas it plays a role in aldehyde detoxification. We observed down‐regulation of polyol pathway‐related enzyme mRNA expression, and up‐regulation of aldo‐keto reductase and aldehyde dehydrogenase mRNA expression in AR‐deficient Schwann cells IKARS1 as compared with those in wild‐type Schwann cells 1970C3. The up‐regulated enzymes might take over AR detoxifying function.
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that is associated with neurological symptoms, including autism spectrum disorder. Tuberous sclerosis complex is caused by ...pathogenic germline mutations of either the TSC1 or TSC2 gene, but somatic mutations were identified in both genes, and the combined effects of TSC1 and TSC2 mutations have been unknown.
The present study investigated social behaviors by the social interaction test and three-chambered sociability tests, effects of rapamycin treatment, and gene expression profiles with a gene expression microarray in Tsc1 and Tsc2 double heterozygous mutant (TscD
) mice.
TscD
mice exhibited impairments in social behaviors, and the severity of impairments was similar to Tsc2
mice rather than Tsc1
mice. Impairments in social behaviors were rescued by rapamycin treatment in all mutant mice. Gene expression profiles in the brain were greatly altered in TscD
mice more than in Tsc1
and Tsc2
mice. The gene expression changes compared with wild type (WT) mice were similar between TscD
and Tsc2
mice, and the overlapping genes whose expression was altered in mutant mice compared with WT mice were enriched in the neoplasm- and inflammation-related canonical pathways. The "signal transducer and activator of transcription 3, interferon regulatory factor 1, interferon regulatory factor 4, interleukin-2R α chain, and interferon-γ" signaling pathway, which is initiated from signal transducer and activator of transcription 4 and PDZ and LIM domain protein 2, was associated with impairments in social behaviors in all mutant mice.
It is unclear whether the signaling pathway also plays a critical role in autism spectrum disorders not caused by Tsc1 and Tsc2 mutations.
These findings suggest that TSC1 and TSC2 double mutations cause autistic behaviors similarly to TSC2 mutations, although significant changes in gene expression were attributable to the double mutations. These findings contribute to the knowledge of genotype-phenotype correlations in TSC and suggest that mutations in both the TSC1 and TSC2 genes act in concert to cause neurological symptoms, including autism spectrum disorder.
Hypoxic-ischemic brain injury induces metabolic dysfunction that ultimately leads to neuronal cell death. Astrocytes, a type of glial cell, play a key role in brain metabolism; however, their ...response to hypoxic-ischemic brain injury is not fully understood. Microglia were removed from murine primary mixed glial cultures to enrich astrocytes. Next, we explored genes whose expression is altered following oxygen-glucose deprivation using a microarray. Microarray analysis revealed that the expression of
and
is markedly increased in astrocyte-enriched cultures after 15 h of oxygen-glucose deprivation. The expression of both
and
was regulated by HIF-1α. At the protein level, NR4A1 was translocated from the nucleus to the cytoplasm following oxygen-glucose deprivation and co-localized with mitochondria in apoptotic cells; however, its localization was restored to the nucleus after reoxygenation. Oxygen-glucose deprivation causes an increase in NR4A1 mRNA in astrocytes as well as its nuclear to cytoplasmic transfer. Furthermore, reoxygenation enhances NR4A1 transcription and promotes its nuclear translocation.
The mammalian target of rapamycin (mTOR) signaling pathway plays a crucial role in cell metabolism, growth, and proliferation. The overactivation of mTOR has been implicated in the pathogenesis of ...syndromic autism spectrum disorder (ASD), such as tuberous sclerosis complex (TSC). Treatment with the mTOR inhibitor rapamycin improved social interaction deficits in mouse models of TSC. Prenatal exposure to valproic acid (VPA) increases the incidence of ASD. Rodent pups that are exposed to VPA in utero have been used as an animal model of ASD. Activation of the mTOR signaling pathway was recently observed in rodents that were exposed to VPA in utero, and rapamycin ameliorated social interaction deficits. The present study investigated the effect of rapamycin on social interaction deficits in both adolescence and adulthood, and gene expressions in mice that were exposed to VPA in utero. We subcutaneously injected 600 mg/kg VPA in pregnant mice on gestational day 12.5 and used the pups as a model of ASD. The pups were intraperitoneally injected with rapamycin or an equal volume of vehicle once daily for 2 consecutive days. The social interaction test was conducted in the offspring after the last rapamycin administration at 5-6 weeks of ages (adolescence) or 10-11 weeks of age (adulthood). Whole brains were collected after the social interaction test in the adulthood, and microarray and Western blot analyses were performed. Mice that were exposed to VPA and treated with vehicle exhibited a decrease in social interaction compared with control mice that were treated with vehicle. Rapamycin treatment in VPA-exposed mice improved social deficits. Mice that were exposed to VPA and treated with vehicle exhibited the aberrant expression of genes in the mTOR signaling pathway, and rapamycin treatment recovered changes in the expression of some genes, including Fyb and A330094K24Rik. Rapamycin treatment suppressed S6 phosphorylation in VPA-exposed mice. Aberrant gene expression was associated with social interaction deficits in VPA-exposed mice. Rapamycin may be an effective treatment for non-syndromic ASD in adolescent and adult patients who present impairments in the mTOR signaling pathway.
The epidermis is the outermost layer of the skin and the body's primary barrier to external pathogens; however, the early epidermal immune response remains to be mechanistically understood. We show ...that the chemokine CXCL14, produced by epidermal keratinocytes, exhibits robust circadian fluctuations and initiates innate immunity. Clearance of the skin pathogen
in nocturnal mice was associated with CXCL14 expression, which was high during subjective daytime and low at night. In contrast, in marmosets, a diurnal primate, circadian CXCL14 expression was reversed. Rhythmically expressed CXCL14 binds to
DNA and induces inflammatory cytokine production by activating Toll-like receptor (TLR)9-dependent innate pathways in dendritic cells and macrophages underneath the epidermis. CXCL14 also promoted phagocytosis by macrophages in a TLR9-independent manner. These data indicate that circadian production of the epidermal chemokine CXCL14 rhythmically suppresses skin bacterial proliferation in mammals by activating the innate immune system.
Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs) relative to insect nAChRs. However, because of importance of nAChRs in ...mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children's health. Here we examined the effects of longterm (14 days) and low dose (1 μM) exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold) between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain.
Hyperserotonemia in the brain is suspected to be an endophenotype of autism spectrum disorder (ASD). Reducing serotonin levels in the brain through modulation of serotonin transporter function may ...improve ASD symptoms.
We analyzed behavior and gene expression to unveil the causal mechanism of ASD-relevant social deficits using serotonin transporter (
) knockout mice.
Social deficits were observed in both heterozygous knockout mice (HZ) and homozygous knockout mice (KO), but increases in general anxiety were only observed in KO mice. Two weeks of dietary restriction of the serotonin precursor tryptophan ameliorated both brain hyperserotonemia and ASD-relevant social deficits in
HZ and KO mice. The expression of rather distinct sets of genes was altered in
HZ and KO mice, and a substantial portion of these genes was also affected by tryptophan depletion. Tryptophan depletion in
HZ and KO mice was associated with alterations in the expression of genes involved in signal transduction pathways initiated by changes in extracellular serotonin or melatonin, a derivative of serotonin. Only expression of the
gene was altered in both
HZ and KO mice.
expression and ASD-relevant social deficits normalized after dietary tryptophan restriction.
These findings reveal a
gene dose-dependent effect on brain hyperserotonemia and related behavioral sequelae in ASD and a possible therapeutic target to normalize brain hyperserotonemia and ASD-relevant social deficits.
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