Dysregulation of S-adenosylmethionine Metabolism in Nonalcoholic Steatohepatitis Leads to Polyamine Flux and Oxidative Stress

Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease worldwide, with 25% of these patients developing nonalcoholic steatohepatitis (NASH). NASH significantly increases the risk of cirrhosis and decompensated liver failure. Past studies in rodent models have shown that glycine-N-methyltransferase (GNMT) knockout results in rapid steatosis, fibrosis, and hepatocellular carcinoma progression. However, the attenuation of GNMT in subjects with NASH and the molecular basis for its impact on the disease process is still unclear. To address this knowledge gap, we show the reduction of GNMT protein levels in the liver of NASH subjects compared to healthy controls. To gain insight into the impact of decreased GNMT in the disease process, we performed global label-free proteome studies on the livers from a murine modified amylin diet-based model of NASH. Histological and molecular characterization of the animal model demonstrate a high resemblance to human disease. We found that a reduction of GNMT leads to a significant increase in S-adenosylmethionine (AdoMet), an essential metabolite for transmethylation reactions and a substrate for polyamine synthesis. Further targeted proteomic and metabolomic studies demonstrated a decrease in GNMT transmethylation, increased flux through the polyamine pathway, and increased oxidative stress production contributing to NASH pathogenesis.     

Recurrent Herpes Simplex Virus Type 1 (HSV-1) Infection Modulates Neuronal Aging Marks in In Vitro and In Vivo Models

Herpes simplex virus 1 (HSV-1) is a widespread neurotropic virus establishing a life-long latent infection in neurons with periodic reactivations. Recent studies linked HSV-1 to neurodegenerative processes related to age-related disorders such as Alzheimer’s disease. Here, we explored whether recurrent HSV-1 infection might accelerate aging in neurons, focusing on peculiar marks of aged cells, such as the increase in histone H4 lysine (K) 16 acetylation (ac) (H4K16ac); the decrease of H3K56ac, and the modified expression of Sin3/HDAC1 and HIRA proteins. By exploiting both in vitro and in vivo models of recurrent HSV-1 infection, we found a significant increase in H4K16ac, Sin3, and HDAC1 levels, suggesting that the neuronal response to virus latency and reactivation includes the upregulation of these aging markers. On the contrary, we found a significant decrease in H3K56ac that was specifically linked to viral reactivation and apparently not related to aging-related markers. A complex modulation of HIRA expression and localization was found in the brain from HSV-1 infected mice suggesting a specific role of this protein in viral latency and reactivation. Overall, our results pointed out novel molecular mechanisms through which recurrent HSV-1 infection may affect neuronal aging, likely contributing to neurodegeneration.     

S-腺苷酰-L-甲硫氨酸对肝癌细胞HepG2增殖、迁移及癌基因C-myc表达的影响

目的探讨S-腺苷酰-L-甲硫氨酸(S-adenosyl-L-methionine,SAMe)对肝癌细胞HepG2增殖、迁移及癌基因C-myc表达的影响,为深入研究SAMe在肝癌治疗中的作用奠定基础。方法体外培养HepG2细胞,MTT法检测不同浓度SAMe作用不同时间对HepG2细胞增殖的影响;细胞划痕法检测最适浓度SAMe对HepG2细胞迁移能力的影响;RT-PCR、免疫细胞化学法及Westernblot法检测SAMe对HepG2细胞癌基因C-myc转录、蛋白定位及表达的影响。结果终浓度为15.0μmol/L的SAMe处理细胞5d,细胞抑制率达54.6%,且抑制作用呈时间与剂量依赖性;经15.0μmol/LSAMe处理的细胞迁移能力明显下降,愈合速率为对照组的76.78%;细胞中癌基因C-myc的转录和蛋白表达均明显下降,蛋白定位无明显变化。结论 SAMe可通过降低C-myc的表达,抑制HepG2细胞的增殖和迁移,为肝癌治疗性药物的研究提供了一个新的方向。     

Description of Osmolyte Pathways in Maturing Mdx Mice Reveals Altered Levels of Taurine and Sodium/Myo-Inositol Co-Transporters

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration. Osmotic stress participates to DMD pathology and altered levels of osmolyte pathway members have been reported. The goal of this study was to gain insight in osmoregulatory changes in the mdx mouse model by examining the expression of osmolyte pathway members, including taurine transporter (TauT), sodium myo-inositol co-transporter (SMIT), betaine GABA transporter (BGT), and aldose reductase (AR) in the skeletal muscles and diaphragm of mdx mice aged 4, 8, 12, and 26 weeks. Necrosis was most prominent in 12 week-old mdx mice, whereas the amount of regenerated fibers increased until week 26 in the tibialis anterior. TauT protein levels were downregulated in the tibialis anterior and gastrocnemius of 4 to 12 week-old mdx mice, but not in 26 week-old mice, whereas TauT levels in the diaphragm remained significantly lower in 26 week-old mdx mice. In contrast, SMIT protein levels were significantly higher in the muscles of mdx mice when compared to controls. Our study revealed differential regulation of osmolyte pathway members in mdx muscle, which points to their complex involvement in DMD pathogenesis going beyond general osmotic stress responses. These results highlight the potential of osmolyte pathway members as a research interest and future therapeutic target in dystrophinopathy.     

Identification of 1,2,3,4,6-Penta-O-galloyl-β-d-glucopyranoside as a Glycine N-Methyltransferase Enhancer by High-Throughput Screening of Natural Products Inhibits Hepatocellular Carcinoma

Glycine N-methyltransferase (GNMT) expression is vastly downregulated in hepatocellular carcinomas (HCC). High rates of GNMT knockout mice developed HCC, while overexpression of GNMT prevented aflatoxin-induced carcinogenicity and inhibited liver cancer cell proliferation. Therefore, in this study, we aimed for the identification of a GNMT inducer for HCC therapy. We established a GNMT promoter-driven luciferase reporter assay as a drug screening platform. Screening of 324 pure compounds and 480 crude extracts from Chinese medicinal herbs resulted in the identification of Paeonia lactiflora Pall (PL) extract and the active component 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranoside (PGG) as a GNMT inducer. Purified PL extract and PGG induced GNMT mRNA and protein expression in Huh7 human hepatoma cells and in xenograft tumors. PGG and PL extract had potent anti-HCC effects both in vitro and in vivo. Furthermore, PGG treatment induced apoptosis in Huh7 cells. Moreover, PGG treatment sensitized Huh7 cells to sorafenib treatment. Therefore, these results indicated that identifying a GNMT enhancer using the GNMT promoter-based assay might be a useful approach to find drugs for HCC. These data also suggested that PGG has therapeutic potential for the treatment of HCC.     

Brain Neurons during Physiological Aging: Morphological Features,Autophagic and Mitochondrial Contribution

Accumulating data suggest that the brain undergoes various changes during aging. Among them are loss of both white and gray matter, neurons and synapses degeneration, as well as oxidative, inflammatory, and biochemical changes. The above-mentioned age-related features are closely related to autophagy and mitochondria. Therefore, we aimed to reveal the most peculiar morphological features of brain nervous tissue and to characterize the expression of autophagy and mitochondrial immunohistochemical biomarkers in neurons of different human brain zones during aging. Counting the number of neurons as well as Microtubule-associated proteins 1A/1B light chain 3B (LC3B), Heat shock protein 70 (HSP70), Lysosome-associated membrane protein type 2A (LAMP2A), Alpha subunit of ATP synthase (ATP5A), and Parkinson disease protein 7 (DJ1) immunohistochemical staining were performed on FFPE samples of human prefrontal cortex, corpus striatum, and hippocampus obtained from autopsy. Statistical analysis revealed a loss of neurons in the studied elderly group in comparison to the young group. When the expression of macroautophagy (LC3B), chaperon-mediated autophagy (HSP70, LAMP2A), and mitochondrial respiratory chain complex V (ATP5A) markers for the young and elderly groups were compared, the latter was found to have a significantly higher rate of optical density, whilst there was no significance in DJ1 expression. These findings, while preliminary, suggest that both autophagy and mitochondria are involved in neuronal maintenance during aging and could indicate their potential role in adaptive mechanisms that occur in aging.     

Physiological Ovarian Aging Is Associated with Altered Expression of Post-Translational Modifications in Mice

Post-translational modifications (PTMs) have been confirmed to be involved in multiple female reproductive events, but their role in physiological ovarian aging is far from elucidated. In this study, mice aged 3, 12 or 17 months (3M, 12M, 17M) were selected as physiological ovarian aging models. The expression of female reproductive function-related genes, the global profiles of PTMs, and the level of histone modifications and related regulatory enzymes were examined during physiological ovarian aging in the mice by quantitative real-time PCR and western blot, respectively. The results showed that the global protein expression of Kbhb (lysineβ-hydroxybutyryllysine), Khib (lysine 2-hydroxyisobutyryllysine), Kglu (lysineglutaryllysine), Kmal (lysinemalonyllysine), Ksucc (lysinesuccinyllysine), Kcr (lysinecrotonyllysine), Kbu (lysinebutyryllysine), Kpr (lysinepropionyllysine), SUMO1 (SUMO1 modification), ub (ubiquitination), P-Typ (phosphorylation), and 3-nitro-Tyr (nitro-tyrosine) increased significantly as mice aged. Moreover, the modification level of Kme2 (lysinedi-methyllysine) and Kac (lysineacetyllysine) was the highest in the 3M mice and the lowest in 12M mice. In addition, only trimethylation of histone lysine was up-regulated progressively and significantly with increasing age (p < 0.001), H4 ubiquitination was obviously higher in the 12M and 17M mice than 3M (p < 0.001), whereas the modification of Kpr (lysinepropionylation) and O-GlcNA in 17M was significantly decreased compared with the level in 3M mice (p < 0.05, p < 0.01). Furthermore, the expression levels of the TIP60, P300, PRDM9, KMT5B, and KMT5C genes encoding PTM regulators were up-regulated in 17M compared to 3M female mice (p < 0.05). These findings indicate that altered related regulatory enzymes and PTMs are associated with physiological ovarian aging in mice, which is expected to provide useful insights for the delay of ovarian aging and the diagnosis and treatment of female infertility.     

Effects of Tenascin C on the Integrity of Extracellular Matrix and Skin Aging

Tenascin C (TNC) is an element of the extracellular matrix (ECM) of various tissues, including the skin, and is involved in modulating ECM integrity and cell physiology. Although skin aging is apparently associated with changes in the ECM, little is known about the role of TNC in skin aging. In this study, we found that the Tnc mRNA level was significantly reduced in the skin tissues of aged mice compared with young mice, consistent with reduced TNC protein expression in aged human skin. TNC-large (TNC-L; 330-kDa) and -small (TNC-S; 240-kDa) polypeptides were observed in conditional media from primary dermal fibroblasts. Both recombinant TNC polypeptides, corresponding to TNC-L and TNC-S, increased the expression of type I collagen and reduced the expression of matrix metalloproteinase-1 in fibroblasts. Treatment of fibroblasts with a recombinant TNC polypeptide, corresponding to TNC-L, induced phosphorylation of SMAD2 and SMAD3. TNC increased the level of transforming growth factor-β1 (TGF-β1) mRNA and upregulated the expression of type I collagen by activating the TGF-β signaling pathway. In addition, TNC also promoted the expression of type I collagen in fibroblasts embedded in a three-dimensional collagen matrix. Our findings suggest that TNC contributes to the integrity of ECM in young skin and to prevention of skin aging.     

Proteomic-Based Approach Reveals the Involvement of Apolipoprotein A-I in Related Phenotypes of Autism Spectrum Disorder in the BTBR Mouse Model

Autism spectrum disorder (ASD) is a neurodevelopmental disorder. Abnormal lipid metabolism has been suggested to contribute to its pathogenesis. Further exploration of its underlying biochemical mechanisms is needed. In a search for reliable biomarkers for the pathophysiology of ASD, hippocampal tissues from the ASD model BTBR T+ Itpr3tf/J (BTBR) mice and C57BL/6J mice were analyzed, using four-dimensional (4D) label-free proteomic analysis and bioinformatics analysis. Differentially expressed proteins were significantly enriched in lipid metabolic pathways. Among them, apolipoprotein A-I (ApoA-I) is a hub protein and its expression was significantly higher in the BTBR mice. The investigation of protein levels (using Western blotting) also confirmed this observation. Furthermore, expressions of SphK2 and S1P in the ApoA-I pathway both increased. Using the SphK inhibitor (SKI-II), ASD core phenotype and phenotype-related protein levels of P-CREB, P-CaMKII, and GAD1 were improved, as shown via behavioral and molecular biology experiments. Moreover, by using SKI-II, we found proteins related to the development and function of neuron synapses, including ERK, caspase-3, Bax, Bcl-2, CDK5 and KCNQ2 in BTBR mice, whose levels were restored to protein levels comparable to those in the controls. Elucidating the possible mechanism of ApoA-I in ASD-associated phenotypes will provide new ideas for studies on the etiology of ASD.     

The Lack of Amyloidogenic Activity Is Persistent in Old WT and APPswe/PS1ΔE9 Mouse Retinae

We have previously reported that vision decline was not associated with amyloidogenesis processing in aging C57BL/6J wild-type (WT) mice and in a mouse model of Alzheimer’s disease, the APP_swe/PS1ΔE9 transgenic mouse model (APP/PS1). This conclusion was drawn using middle-aged (10–13 months old) mice. Here, we hypothesized that compared with hippocampal and cortical neurons, the weak amyloidogenic activity of retinal neurons may result in a detectable release of amyloid β (Aβ) only in aged mice, i.e., between 14 and 24 months of age. The aim of the present study was thus to follow potential activity changes in the amyloidogenic and nonamyloidogenic pathways of young (4 months) and old (20–24 months) WT and APP/PS1 mice. Our results showed that in spite of retinal activity loss reported by electroretinogram (ERG) recordings, the level of amyloid beta precursor protein (APP) and its derivatives did not significantly vary in the eyes of old vs. young mice. Strikingly, the ectopic expression of human APP_swe in APP/PS1 mice did not allow us to detect Aβ monomers at 23 months. In contrast, Aβ was observed in hippocampal and cortical tissues at this age but not at 4 months of life. In contrast, optic nerve transection-induced retinal ganglion cell injury significantly affected the level of retinal APP and the secretion of soluble APP alpha in the vitreous. Collectively, these results suggest that the amyloidogenic and nonamyloidogenic pathways are not involved in visual function decline in aging mice. In WT and APP/PS1 mice, it is proposed that retinal neurons do not have the capacity to secrete Aβ in contrast with other cortical and hippocampal neurons.     

Age-Dependent Contributions of NMDA Receptors and L-Type Calcium Channels to Long-Term Depression in the Piriform Cortex

In the hippocampus, the contributions of N-methyl-D-aspartate receptors (NMDARs) and L-type calcium channels (LTCCs) to neuronal transmission and synaptic plasticity change with aging, underlying calcium dysregulation and cognitive dysfunction. However, the relative contributions of NMDARs and LTCCs in other learning encoding structures during aging are not known. The piriform cortex (PC) plays a significant role in odor associative memories, and like the hippocampus, exhibits forms of long-term synaptic plasticity. Here, we investigated the expression and contribution of NMDARs and LTCCs in long-term depression (LTD) of the PC associational fiber pathway in three cohorts of Sprague Dawley rats: neonatal (1–2 weeks), young adult (2–3 months) and aged (20–25 months). Using a combination of slice electrophysiology, Western blotting, fluorescent immunohistochemistry and confocal imaging, we observed a shift from an NMDAR to LTCC mediation of LTD in aged rats, despite no difference in the amount of LTD expression. These changes in plasticity are related to age-dependent differential receptor expression in the PC. LTCC Cav1.2 expression relative to postsynaptic density protein 95 is increased in the associational pathway of the aged PC layer Ib. Enhanced LTCC contribution in synaptic depression in the PC may contribute to altered olfactory function and learning with aging.     

Metabolic and Proteomic Profiles Associated with Immune Responses Induced by Different Inactivated SARS-CoV-2 Vaccine Candidates

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019, the virus has been mutating continuously, resulting in the continuous emergence of variants and creating challenges for epidemic prevention and control. Here, we immunized mice with different vaccine candidates, revealing the immune, protein, and metabolomic changes that take place in vaccines composed of different variants. We found that the prototype strain and Delta- and Omicron-variant inactivated vaccine candidates could all induce a high level of neutralizing antibodies and cellular immunity responses in mice. Next, we found that the metabolic and protein profiles were changed, showing a positive association with immune responses, and the level of the change was distinct in different inactivated vaccines, indicating that amino acid variations could affect metabolomics and proteomics. Our findings reveal differences between vaccines at the metabolomic and proteomic levels. These insights provide a novel direction for the immune evaluation of vaccines and could be used to guide novel strategies for vaccine design.     

Chemogenetic Depletion of Hypophysiotropic GnRH Neurons Does Not Affect Fertility in Mature Female Zebrafish

The hypophysiotropic gonadotropin-releasing hormone (GnRH) and its neurons are crucial for vertebrate reproduction, primarily in regulating luteinizing hormone (LH) secretion and ovulation. However, in zebrafish, which lack GnRH1, and instead possess GnRH3 as the hypophysiotropic form, GnRH3 gene knockout did not affect reproduction. However, early-stage ablation of all GnRH3 neurons causes infertility in females, implicating GnRH3 neurons, rather than GnRH3 peptides in female reproduction. To determine the role of GnRH3 neurons in the reproduction of adult females, a Tg(gnrh3:Gal4ff; UAS:nfsb-mCherry) line was generated to facilitate a chemogenetic conditional ablation of GnRH3 neurons. Following ablation, there was a reduction of preoptic area GnRH3 neurons by an average of 85.3%, which was associated with reduced pituitary projections and gnrh3 mRNA levels. However, plasma LH levels were unaffected, and the ablated females displayed normal reproductive capacity. There was no correlation between the number of remaining GnRH3 neurons and reproductive performance. Though it is possible that the few remaining GnRH3 neurons can still induce an LH surge, our findings are consistent with the idea that GnRH and its neurons are likely dispensable for LH surge in zebrafish. Altogether, our results resurrected questions regarding the functional homology of the hypophysiotropic GnRH1 and GnRH3 in controlling ovulation.     

Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1

The nontoxic heat-labile toxin (LT) B subunit (LTB) was used as mucosal adjuvant experimentally. However, the mechanism of LTB adjuvant was still unclear. The LTB and enterovirus 71 (EV71) VP1 subunit (EVP1) were constructed in pET32 and expressed in E. coli BL21, respectively. The immunogenicity of purified EVP1 and the adjuvanticity of LTB were evaluated via intranasal immunization EVP1 plus LTB in Balb/c mice. In order to elucidate the proteome change triggered by the adjuvant of LTB, the proteomic profiles of LTB, EVP1, and LTB plus EVP1 were quantitatively analyzed by iTRAQ-LC-MS/MS (isobaric tags for relative and absolute quantitation; liquid chromatography-tandem mass spectrometry) in murine macrophage RAW264.7. The proteomic data were analyzed by bioinformatics and validated by western blot analysis. The predicted protein interactions were confirmed using LTB pull-down and the LTB processing pathway was validated by confocal microscopy. The results showed that LTB significantly boosted EVP1 specific systematic and mucosal antibodies. A total of 3666 differential proteins were identified in the three groups. Pathway enrichment of proteomic data predicted that LTB upregulated the specific and dominant MAPK (mitogen-activated protein kinase) signaling pathway and the protein processing in endoplasmic reticulum (PPER) pathway, whereas LTB or EVP1 did not significantly upregulate these two signaling pathways. Confocal microscopy and LTB pull-down assays confirmed that the LTB adjuvant was endocytosed and processed through endocytosis (ENS)-lysosomal-endoplasmic reticulum (ER) system.     

Polyphenol Supplementation Reverses Age-Related Changes in Microglial Signaling Cascades

Microglial activity in the aging neuroimmune system is a central player in aging-related dysfunction. Aging alters microglial function via shifts in protein signaling cascades. These shifts can propagate neurodegenerative pathology. Therapeutics require a multifaceted approach to understand and address the stochastic nature of this process. Polyphenols offer one such means of rectifying age-related decline. Our group used mass spectrometry (MS) analysis to explicate the complex nature of these aging microglial pathways. In our first experiment, we compared primary microglia isolated from young and aged rats and identified 197 significantly differentially expressed proteins between these groups. Then, we performed bioinformatic analysis to explore differences in canonical signaling cascades related to microglial homeostasis and function with age. In a second experiment, we investigated changes to these pathways in aged animals after 30-day dietary supplementation with NT-020, which is a blend of polyphenols. We identified 144 differentially expressed proteins between the NT-020 group and the control diet group via MS analysis. Bioinformatic analysis predicted an NT-020 driven reversal in the upregulation of age-related canonical pathways that control inflammation, cellular metabolism, and proteostasis. Our results highlight salient aspects of microglial aging at the level of protein interactions and demonstrate a potential role of polyphenols as therapeutics for age-associated dysfunction.     

Characterization of the Skeletal Muscle Proteome in Undernourished Old Rats

Aging is associated with a progressive loss of skeletal muscle mass and function termed sarcopenia. Various metabolic alterations that occur with aging also increase the risk of undernutrition, which can worsen age-related sarcopenia. However, the impact of undernutrition on aged skeletal muscle remains largely under-researched. To build a deeper understanding of the cellular and molecular mechanisms underlying age-related sarcopenia, we characterized the undernutrition-induced changes in the skeletal muscle proteome in old rats. For this study, 20-month-old male rats were fed 50% or 100% of their spontaneous intake for 12 weeks, and proteomic analysis was performed on both slow- and fast-twitch muscles. Proteomic profiling of undernourished aged skeletal muscle revealed that undernutrition has profound effects on muscle proteome independently of its effect on muscle mass. Undernutrition-induced changes in muscle proteome appear to be muscle-type-specific: slow-twitch muscle showed a broad pattern of differential expression in proteins important for energy metabolism, whereas fast-twitch muscle mainly showed changes in protein turnover between undernourished and control rats. This first proteomic analysis of undernourished aged skeletal muscle provides new molecular-level insight to explain phenotypic changes in undernourished aged muscle. We anticipate this work as a starting point to define new biomarkers associated with undernutrition-induced muscle loss in the elderly.