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The necroptotic effector molecule MLKL accumulates in neurons over the lifespan of mice, and its downregulation has the potential to improve cognition through neuroinflammation, and changes in the abundance of synaptic proteins and enzymes in the central nervous system. Notwithstanding, direct evidence of cell-autonomous effects of MLKL expression on neuronal physiology and metabolism are lacking. Here, we tested whether the overexpression of MLKL in the absence of cell death in the neuronal cell line Neuro-2a recapitulates some of the hallmarks of aging at the cellular level. Using genetically-encoded fluorescent biosensors, we monitored the cytosolic and mitochondrial Ca 2+ levels, along with the cytosolic concentrations of several metabolites involved in energy metabolism (lactate, glucose, ATP) and oxidative stress (oxidized/reduced glutathione). We found that MLKL overexpression marginally decreased cell viability, however, it led to reduced cytosolic and mitochondrial Ca 2+ elevations in response to Ca 2+ influx from the extracellular space. On the contrary, Ca 2+ signals were elevated after mobilizing Ca 2+ from the endoplasmic reticulum. Transient elevations in cytosolic Ca 2+ , mimicking neuronal stimulation, lead to higher lactate levels and lower glucose concentrations in Neuro-2a cells when overexpressing MLKL, which suggest enhanced neuronal glycolysis. Despite these alterations, energy levels and glutathione redox state in the cell bodies remained largely preserved after inducing MLKL overexpression for 24-48 h. Taken together, our proof-of-concept experiments are consistent with the hypothesis that MLKL overexpression in the absence of cell death contributes to both Ca 2+ and metabolic dyshomeostasis, which are cellular hallmarks of brain aging., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Aging has a significant impact on the immune system, leading to a gradual decline in immune function and changes in the body's ability to respond to bacterial infections. Non-tuberculous mycobacteria (NTM), also known as atypical mycobacteria or environmental mycobacteria, are commonly found in soil, water, and various environmental sources. While many NTM species are considered opportunistic pathogens, some can cause significant infections, particularly in individuals with compromised immune systems, such as older individuals. When mycobacteria enter the body, macrophages are among the first immune cells to encounter them and attempt to engulf mycobacteria through a process called phagocytosis. Some NTM species, including Mycobacterium avium (M. avium) can survive and replicate within macrophages. However, little is known about the interaction between NTM and macrophages in older individuals. In this study, we investigated the response of bone marrow-derived macrophage (BMMs) isolated from young (5 months) and old (25 months) mice to M. avium serotype 4, one of the main NTM species in patients with pulmonary NTM diseases. Our results demonstrated that BMMs from old mice have an increased level of intracellular iron and are more susceptible to M. avium serotype 4 infection compared to BMMs from young mice. The whole-cell proteomic analysis indicated a dysregulated expression of iron homeostasis-associated proteins in old BMMs regardless of mycobacterial infection. Deferoxamine, an iron chelator, significantly rescued mycobacterial killing and phagolysosome maturation in BMMs from old mice. Therefore, our data for the first time indicate that an intracellular iron accumulation improves NTM survival within macrophages from old mice and suggest a potential application of iron-chelating drugs as a host-directed therapy for pulmonary NTM infection in older individuals., (© 2023. The Author(s).)

Chronic inflammation is a key player in metabolic dysfunction-associated fatty liver disease (MAFLD) progression. Necroptosis, an inflammatory cell death pathway, is elevated in MAFLD patients and mouse models, yet its role is unclear due to the diverse mouse models and inhibition strategies. In our study, we inhibited necroptosis by targeting mixed lineage kinase domain-like pseudokinase (MLKL), the terminal effector of necroptosis, in a high-fat, high-fructose, high-cholesterol (HFHFrHC) mouse model of diet-induced MAFLD. Despite the HFHFrHC diet upregulating MLKL (2.5-fold), WT mice livers showed no increase in necroptosis markers or associated proinflammatory cytokines. Surprisingly, Mlkl -/- mice experienced exacerbated liver inflammation without protection from diet-induced liver damage, steatosis, or fibrosis. In contrast, Mlkl +/- mice showed a significant reduction in these parameters that was associated with elevated Pparα and Pparγ levels. Both Mlkl -/- and Mlkl +/- mice on the HFHFrHC diet resisted diet-induced obesity, attributed to the increased beiging, enhanced oxygen consumption, and energy expenditure due to adipose tissue, and exhibited improved insulin sensitivity. These findings highlight the tissue-specific effects of MLKL on the liver and adipose tissue, and they suggest a dose-dependent effect of MLKL on liver pathology.

To study the impact of necroptosis-induced chronic inflammation on age-related diseases and aging, two knockin mouse models (Ripk3-KI and Mlkl-KI) were generated that overexpress two genes involved in necroptosis (Ripk3 or Mlkl) when crossed to Cre transgenic mice. Crossing Ripk3-KI or Mlkl-KI mice to albumin-Cre transgenic mice produced hepatocyte specific hRipk3-KI or hMlkl-KI mice, which express the two transgenes only in the liver. Ripk3 and Mlkl proteins were overexpressed 10- and fourfold, respectively, in the livers of the hRipk3-KI or hMlkl-KI mice. Treating young (2-month) hRipk3-KI or hMlkl-KI mice with carbon tetrachloride (CCl 4 ), a chemical inducer of oxidative stress, resulted in increased necroptosis (Mlkl-oligomers) and inflammation in the liver compared to control mice receiving CCl 4 . Mlkl-oligomerization also was significantly increased in old (18-month) hRipk3-KI and hMlkl-KI mice compared to old control (Cre negative, Ripk3-KI and Mlkl-KI) mice. The increase in necroptosis was associated with an increase in inflammation, e.g., inflammatory cytokines (TNFα, IL-6) and macrophage markers (F4/80, CD68). Importantly, steatosis (triglycerides) and fibrosis (e.g., picrosirius red staining, hydroxyproline levels, and transcripts for TGFβ, Col1α1, and Col3α1) that increase with age were significantly higher in the livers of the old hRipk3-KI or hMlkl-KI mice compared to old control mice. In addition, markers of cellular senescence were significantly increased in the livers of the old hRipk3-KI and hMlkl-KI mice. Thus, the first mouse models have been developed that allow researchers to study the impact of inducing necroptosis in specific cells/tissues on chronic inflammation in aging and age-related diseases., (© 2023. The Author(s).)

Nonalcoholic fatty liver disease (NAFLD) is one of the etiologies that contribute to hepatocellular carcinoma (HCC), and chronic inflammation is one of the proposed mediators of HCC. Because necroptosis is a cell death pathway that induces inflammation, we tested whether necroptosis-induced inflammation contributes to the progression of NAFLD to HCC in a mouse model of diet-induced HCC. Male and female wild-type (WT) mice and mouse models where necroptosis is blocked (Ripk3-/- or Mlkl-/- mice) were fed either a control diet, choline-deficient low-fat diet or choline-deficient high-fat diet. Blocking necroptosis reduced markers of inflammation [proinflammatory cytokines (TNFα, IL6, and IL1β), F4/80+ve macrophages, CCR2+ve infiltrating monocytes], inflammation-associated oncogenic pathways (JNK, PD-L1/PD-1, β-catenin), and HCC in male mice. We demonstrate that hepatic necroptosis promotes recruitment and activation of liver macrophages leading to chronic inflammation, which in turn trigger oncogenic pathways leading to the progression of NAFLD to HCC in male mice. Whereas in female mice, blocking necroptosis reduced HCC independent of inflammation. Our data show a sex-specific difference in the development of inflammation, fibrosis, and HCC in WT mice. However, blocking necroptosis reduced HCC in both males and females without altering liver fibrosis. Thus, our study suggests that necroptosis is a valid therapeutic target for NAFLD-mediated HCC., Implications: Necroptosis is a major contributor to hepatic inflammation that drives the progression of NAFLD to HCC and therefore represents a valid target for NAFLD-mediated HCC., (©2023 The Authors; Published by the American Association for Cancer Research.)

Neuronal oxidative stress has been implicated in aging and neurodegenerative disease. Here we investigated the impact of elevated oxidative stress induced in mouse spinal cord by deletion of Mn-Superoxide dismutase (MnSOD) using a neuron specific Cre recombinase in Sod2 floxed mice (i-mn-Sod2 KO). Sod2 deletion in spinal cord neurons was associated with mitochondrial alterations and peroxide generation. Phenotypically, i-mn-Sod2 KO mice experienced hindlimb paralysis and clasping behavior associated with extensive demyelination and reduced nerve conduction velocity, axonal degeneration, enhanced blood brain barrier permeability, elevated inflammatory cytokines, microglia activation, infiltration of neutrophils and necroptosis in spinal cord. In contrast, spinal cord motor neuron number, innervation of neuromuscular junctions, muscle mass, and contractile function were not altered. Overall, our findings show that loss of MnSOD in spinal cord promotes a phenotype of demyelination, inflammation and progressive paralysis that mimics phenotypes associated with progressive multiple sclerosis., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The goal of this study was to test the role cellular senescence plays in the increased inflammation, chronic liver disease, and hepatocellular carcinoma seen in mice null for Cu/Zn-Superoxide dismutase (Sod1KO). To inhibit senescence, wildtype (WT) and Sod1KO mice were given the senolytics, dasatinib, and quercetin (D + Q) at 6 months of age when the Sod1KO mice begin exhibiting signs of accelerated aging. Seven months of D + Q treatment reduced the expression of p16 in the livers of Sod1KO mice to WT levels and the expression of several senescence-associated secretory phenotype factors (IL-6, IL-1β, CXCL-1, and GDF-15). D + Q treatment also reduced markers of inflammation in livers of the Sod1KO mice, for example, cytokines, chemokines, macrophage levels, and Kupffer cell clusters. D + Q treatment had no effect on various markers of liver fibrosis in the Sod1KO mice but reduced the expression of genes involved in liver cancer and dramatically reduced the incidence of hepatocellular carcinoma. Surprisingly, D + Q also reduced markers of necroptosis (phosphorylated and oligomerized MLKL) in the Sod1KO mice to WT levels. We also found that inhibiting necroptosis in the Sod1KO mice with necrostatin-1s reduced the markers of cellular senescence (p16, p21, and p53). Our study suggests that an interaction occurs between cellular senescence and necroptosis in the liver of Sod1KO mice. We propose that these two cell fates interact through a positive feedback loop resulting in a cycle amplifying both cellular senescence and necroptosis leading to inflammaging and age-associated pathology in the Sod1KO mice., (© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

We previously reported the remarkable potency of uttroside B (Utt-B), saponin-isolated and characterized in our lab from Solanum nigrum Linn, against HCC. Recently, the U.S. FDA approved Utt-B as an 'orphan drug' against HCC. The current study validates the superior anti-HCC efficacy of Utt-B over sorafenib, the first-line treatment option against HCC. The therapeutic efficacies of Utt-B vs. sorafenib against HCC were compared in vitro, using various liver cancer cell lines and in vivo, utilizing NOD.CB17-Prkdcscid/J mice bearing human HCC xenografts. Our data indicate that Utt-B holds an augmented anti-HCC efficacy over sorafenib. Our previous report demonstrated the pharmacological safety of Utt-B in Chang Liver, the normal immortalized hepatocytes, and in the acute and chronic toxicity murine models even at elevated Utt-B concentrations. Here, we show that higher concentrations of sorafenib induce severe toxicity, in Chang Liver, as well as in acute and chronic in vivo models, indicating that, apart from the superior therapeutic benefit over sorafenib, Utt-B is a pharmacologically safer molecule, and the drug-induced undesirable effects can, thus, be substantially alleviated in the context of HCC chemotherapy. Clinical studies in HCC patients utilizing Utt-B, is a contiguous key step to promote this drug to the clinic.

Inflammaging, characterized by an increase in low-grade chronic inflammation with age, is a hallmark of aging and is strongly associated with various age-related diseases, including chronic liver disease (CLD) and hepatocellular carcinoma (HCC). Because necroptosis is a cell death pathway that induces inflammation through the release of DAMPs, we tested the hypothesis that age-associated increase in necroptosis contributes to chronic inflammation in aging liver. Phosphorylation of MLKL and MLKL oligomers, markers of necroptosis, as well as phosphorylation of RIPK3 and RIPK1 were significantly upregulated in the livers of old mice relative to young mice and this increase occurred in the later half of life (i.e., after 18 months of age). Markers of M1 macrophages, expression of pro-inflammatory cytokines (TNFα, IL6 and IL1β), and markers of fibrosis were all significantly upregulated in the liver with age and the change in necroptosis paralleled the changes in inflammation and fibrosis. Hepatocytes and liver macrophages isolated from old mice showed elevated levels of necroptosis markers as well as increased expression of pro-inflammatory cytokines relative to young mice. Short-term treatment with the necroptosis inhibitor, necrostatin-1s (Nec-1s), reduced necroptosis, markers of M1 macrophages, fibrosis, and cell senescence as well as reducing the expression of pro-inflammatory cytokines in the livers of old mice. Thus, our data show for the first time that liver aging is associated with increased necroptosis and necroptosis contributes to chronic inflammation in the liver, which in turn appears to contribute to liver fibrosis and possibly CLD., (© 2021 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Chronic inflammation of the central nervous system (CNS), termed neuroinflammation, is a hallmark of aging and a proposed mediator of cognitive decline associated with aging. Neuroinflammation is characterized by the persistent activation of microglia, the innate immune cells of the CNS, with damage-associated molecular patterns (DAMPs) being one of the well-known activators of microglia. Because necroptosis is a cell death pathway that induces inflammation through the release of DAMPs, we hypothesized that an age-associated increase in necroptosis contributes to increased neuroinflammation with age. The marker of necroptosis, phosphorylated form of MLKL (P-MLKL), and kinases in the necroptosis pathway (RIPK1, RIPK3, and MLKL) showed a region-specific increase in the brain with age, specifically in the cortex layer V and the CA3 region of the hippocampus of mice. Similarly, MLKL-oligomers, which cause membrane binding and permeabilization, were significantly increased in the cortex and hippocampus of old mice relative to young mice. Nearly 70 to 80% of P-MLKL immunoreactivity was localized to neurons and less than 10% was localized to microglia, whereas no P-MLKL was detected in astrocytes. P-MLKL expression in neurons was detected in the soma, not in the processes. Blocking necroptosis using Mlkl -/- mice reduced markers of neuroinflammation (Iba-1 and GFAP) in the brains of old mice, and short-term treatment with the necroptosis inhibitor, necrostatin-1s, reduced expression of proinflammatory cytokines, IL-6 and IL-1β, in the hippocampus of old mice. Thus, our data demonstrate for the first time that brain necroptosis increases with age and contributes to age-related neuroinflammation in mice., (© 2021. The Author(s).)

Mice deficient in the antioxidant enzyme Cu/Zn-superoxide dismutase (Sod1 -/- or Sod1KO mice) have increased oxidative stress, show accelerated aging and develop spontaneous hepatocellular carcinoma (HCC) with age. Similar to humans, HCC development in Sod1KO mice progresses from non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH) with fibrosis, which eventually progresses to HCC. Oxidative stress plays a role in NAFLD to NASH progression, and liver inflammation is the main mechanism that drives the disease progression from NASH to fibrosis. Because necroptosis is a major source of inflammation, we tested the hypothesis that increased necroptosis in the liver plays a role in increased inflammation and fibrosis in Sod1KO mice. Phosphorylation of MLKL (P-MLKL), a well-accepted marker of necroptosis, and expression of MLKL protein were significantly increased in the livers of Sod1KO mice compared to wild type (WT) mice indicating increased necroptosis. Similarly, phosphorylation of RIPK3 and RIPK3 protein levels were also significantly increased. Markers of pro-inflammatory M1 macrophages, NLRP3 inflammasome, and transcript levels of pro-inflammatory cytokines and chemokines, e.g., TNFα, IL-6, IL-1β, and Ccl2 that are associated with human NASH, were significantly increased. Expression of antioxidant enzymes and heat shock proteins, and markers of fibrosis and oncogenic transcription factor STAT3 were also upregulated and autophagy was downregulated in the livers of Sod1KO mice. Short term treatment of Sod1KO mice with necrostatin-1s (Nec-1s), a necroptosis inhibitor, reversed these conditions. Our data show for the first time that necroptosis-mediated inflammation contributes to fibrosis in a mouse model of increased oxidative stress and accelerated aging, that also exhibits progressive HCC development., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

An age-associated increase in chronic, low-grade sterile inflammation termed "inflammaging" is a characteristic feature of mammalian aging that shows a strong association with occurrence of various age-associated diseases. However, the mechanism(s) responsible for inflammaging and its causal role in aging and age-related diseases are not well understood. Age-associated accumulation of damage-associated molecular patterns (DAMPs) is an important trigger in inflammation and has been proposed as a potential driver of inflammaging. DAMPs can initiate an inflammatory response by binding to the cell surface receptors on innate immune cells. Programmed necrosis, termed necroptosis, is one of the pathways that can release DAMPs, and cell death due to necroptosis is known to induce inflammation. Necroptosis-mediated inflammation plays an important role in a variety of age-related diseases such as Alzheimer's disease, Parkinson's disease, and atherosclerosis. Recently, it was reported that markers of necroptosis increase with age in mice and that dietary restriction, which retards aging and increases lifespan, reduces necroptosis and inflammation. Genetic manipulations that increase lifespan (Ames Dwarf mice) and reduce lifespan (Sod1 -/- mice) are associated with reduced and increased necroptosis and inflammation, respectively. While necroptosis evolved to protect cells/tissues from invading pathogens, e.g., viruses, we propose that the age-related increase in oxidative stress, mTOR signaling, and cell senescence results in cells/tissues in old animals being more prone to undergo necroptosis thereby releasing DAMPs, which contribute to the chronic inflammation observed with age. Approach to decrease DAMPs release by reducing/blocking necroptosis is a potentially new approach to reduce inflammaging, retard aging, and improve healthspan.

Mice deficient in the antioxidant enzyme Cu/Zn-superoxide dismutase (Sod1KO mice) have a significant reduction in lifespan, exhibit many phenotypes of accelerated aging, and have high levels of oxidative stress in various tissues. Age-associated cognitive decline is a hallmark of aging and the increase in oxidative stress/damage with age is one of the mechanisms proposed for cognitive decline with age. Therefore, the goal of this study was to determine if Sod1KO mice exhibit an accelerated loss in cognitive function similar to that observed in aged animals. Cognition was assessed in Sod1KO and wild type (WT) mice using an automated home-cage testing apparatus (Noldus PhenoTyper) that included an initial discrimination and reversal task. Comparison of the total distance moved by the mice during light and dark phases of the study demonstrated that the Sod1KO mice do not show a deficit in movement. Assessment of cognitive function showed no significant difference between Sod1KO and WT mice during the initial discrimination phase of learning. However, during the reversal task, Sod1KO mice showed a significantly greater number of incorrect entries compared to WT mice indicating a decline in cognition similar to that observed in aged animals. Markers of oxidative stress (4-Hydroxynonenal, 4-HNE) and neuroinflammation [proinflammatory cytokines (IL6 and IL-1β) and neuroinflammatory markers (CD68, TLR4, and MCP1)] were significantly elevated in the hippocampus of male and female Sod1KO compared to WT mice. This study provides important evidence that increases in oxidative stress alone are sufficient to induce neuroinflammation and cognitive dysfunction that parallels the memory deficits seen in advanced aging and neurodegenerative diseases.

Mice lacking Cu/Zn-superoxide dismutase (Sod1 -/- or Sod1KO mice) show high levels of oxidative stress/damage and a 30% decrease in lifespan. The Sod1KO mice also show many phenotypes of accelerated aging with the loss of muscle mass and function being one of the most prominent aging phenotypes. Using various genetic models targeting the expression of Cu/Zn-superoxide dismutase to specific tissues, we evaluated the role of motor neurons and skeletal muscle in the accelerated loss of muscle mass and function in Sod1KO mice. Our data are consistent with the sarcopenia in Sod1KO mice arising through a two-hit mechanism involving both motor neurons and skeletal muscle. Sarcopenia is initiated in motor neurons leading to a disruption of neuromuscular junctions that results in mitochondrial dysfunction and increased generation of reactive oxygen species (ROS) in skeletal muscle. The mitochondrial ROS generated in muscle feedback on the neuromuscular junctions propagating more disruption of neuromuscular junctions and more ROS production by muscle resulting in a vicious cycle that eventually leads to disaggregation of neuromuscular junctions, denervation, and loss of muscle fibers., (Published by Elsevier Inc.)

Necroptosis is a newly identified programmed cell death pathway that is highly proinflammatory due to the release of cellular components that promote inflammation. To determine whether necroptosis might play a role in inflammaging, we studied the effect of age and dietary restriction (DR) on necroptosis in the epididymal white adipose tissue (eWAT), a major source of proinflammatory cytokines. Phosphorylated MLKL and RIPK3, markers of necroptosis, were increased 2.7- and 1.9-fold, respectively, in eWAT of old mice compared to adult mice, and DR reduced P-MLKL and P-RIPK3 to levels similar to adult mice. An increase in the expression of RIPK1 (1.6-fold) and MLKL (2.7-fold), not RIPK3, was also observed in eWAT of old mice, which was reduced by DR in old mice. The increase in necroptosis was paralleled by an increase in 14 inflammatory cytokines, including the pro-inflammatory cytokines IL-6 (3.9-fold), TNF-α (4.7-fold), and IL-1β (5.1-fold)], and 11 chemokines in old mice. DR attenuated the expression of IL-6, TNF-α, and IL-1β as well as 85% of the other cytokines/chemokines induced with age. In contrast, inguinal WAT (iWAT), which is less inflammatory, did not show any significant increase with age in the levels of P-MLKL and MLKL or inflammatory cytokines/chemokines. Because the changes in biomarkers of necroptosis in eWAT with age and DR paralleled the changes in the expression of pro-inflammatory cytokines, our data support the possibility that necroptosis might play a role in increased chronic inflammation observed with age., (© 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Loss of SURF1, a Complex IV assembly protein, was reported to increase lifespan in mice despite dramatically lower cytochrome oxidase (COX) activity. Consistent with this, our previous studies found advantageous changes in metabolism (reduced adiposity, increased insulin sensitivity, and mitochondrial biogenesis) in Surf1 -/- mice. The lack of deleterious phenotypes in Surf1 -/- mice is contrary to the hypothesis that mitochondrial dysfunction contributes to aging. We found only a modest (nonsignificant) extension of lifespan (7% median, 16% maximum) and no change in healthspan indices in Surf1 -/- vs. Surf1 +/+ mice despite substantial decreases in COX activity (22%-87% across tissues). Dietary restriction (DR) increased median lifespan in both Surf1 +/+ and Surf1 -/- mice (36% and 19%, respectively). We measured gene expression, metabolites, and targeted expression of key metabolic proteins in adipose tissue, liver, and brain in Surf1 +/+ and Surf1 -/- mice. Gene expression was differentially regulated in a tissue-specific manner. Many proteins and metabolites are downregulated in Surf1 -/- adipose tissue and reversed by DR, while in brain, most metabolites that changed were elevated in Surf1 -/- mice. Finally, mitochondrial unfolded protein response (UPR mt )-associated proteins were not uniformly altered by age or genotype, suggesting the UPR mt is not a key player in aging or in response to reduced COX activity. While the changes in gene expression and metabolism may represent compensatory responses to mitochondrial stress, the important outcome of this study is that lifespan and healthspan are not compromised in Surf1 -/- mice, suggesting that not all mitochondrial deficiencies are a critical determinant of lifespan., (© 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

The Geropathology Grading Platform (GGP) that is being developed by the Geropathology Research Network provides a grading system that allows investigators to assess biological aging in mice by measuring the pathological status of a wide range of tissues in a standardized scoring system. The GGP is a grading system that generates a numerical score for the total lesions in each tissue, which when averaged over the mice in the cohort provides a composite lesion score (CLS) for each tissue and mouse. In this study, we tested ability of the GGP to predict accelerated aging in mice null for Cu/Zn-superoxide dismutase (Sod1KO mice), which have been shown to have reduced lifespan and healthspan. Using the GGP, we evaluated the pathological status of 11 tissues from male and female wild-type (WT) and Sod1KO mice at 9 to 10 months of age. The whole animal CLS was 2- to 3.5-fold higher for both male and female Sod1KO mice compared to WT mice. The tissues most affected in the Sod1KO mice were the liver, lung, and kidney. These data demonstrate that the GGP is able to predict the accelerated aging phenotype observed in the Sod1KO mice and correlates with the changes in healthspan that have been reported for Sod1KO mice. Thus, the GGP is a new paradigm for evaluating the effect of an intervention on the pathological status of an animal as well as the healthspan of the mice.

Over the past 50 years, dietary restriction (DR) has been shown to extend the life span of a wide variety of organisms. A hallmark feature of DR is improved glucose homeostasis resulting in increased glucose tolerance and insulin sensitivity of animals ranging from rodents to humans. In this study, we demonstrate the early effects of varying levels of DR on glucose tolerance. Within 10 days of 40% DR, glucose tolerance was significantly improved and by 120 days; 10 and 20% DR also showed enhanced glucose tolerance. All three levels of DR showed reduced adiposity, increased expression of genes involved in fat turnover, and a reduction in the expression for markers of inflammation. Studies have shown that mice fed a DR diet retained metabolic memory in terms of improved glucose tolerance even after DR is discontinued. We show that 40% DR not only has an early effect on glucose tolerance but also maintained it after DR was discontinued for 2 months. Therefore, improvement in glucose tolerance is brought about by all three levels of DR but the metabolic memory is not dose responsive.

Caseinolytic peptidase P (ClpP) is a mammalian quality control protease that is proposed to play an important role in the initiation of the mitochondrial unfolded protein response (UPR mt ), a retrograde signaling response that helps to maintain mitochondrial protein homeostasis. Mitochondrial dysfunction is associated with the development of metabolic disorders, and to understand the effect of a defective UPR mt on metabolism, ClpP knockout ( ClpP -/- ) mice were analyzed. ClpP -/- mice fed ad libitum have reduced adiposity and paradoxically improved insulin sensitivity. Absence of ClpP increased whole-body energy expenditure and markers of mitochondrial biogenesis are selectively up-regulated in the white adipose tissue (WAT) of ClpP -/- mice. When challenged with a metabolic stress such as high-fat diet, despite similar caloric intake, ClpP -/- mice are protected from diet-induced obesity, glucose intolerance, insulin resistance, and hepatic steatosis. Our results show that absence of ClpP triggers compensatory responses in mice and suggest that ClpP might be dispensable for mammalian UPR mt initiation. Thus, we made an unexpected finding that deficiency of ClpP in mice is metabolically beneficial., (© 2018 The Authors.)

Cytochrome c oxidase (COX) is an essential transmembrane protein complex (Complex IV) in the mitochondrial respiratory electron chain. Mutations in genes responsible for the assembly of COX are associated with Leigh syndrome, cardiomyopathy, spinal muscular atrophy and other fatal metabolic disorders in humans. Previous studies have shown that mice lacking the COX assembly protein Surf1 (Surf1 -/- mice) paradoxically show a number of beneficial metabolic phenotypes including increased insulin sensitivity, upregulation of mitochondrial biogenesis, induction of stress response pathways and increased lifespan. To determine whether these effects are specific to the Surf1 mutation or a more general effect of reduced COX activity, we asked whether a different mutation causing reduced COX activity would have similar molecular and physiologic changes. Sco2 knock-in/knock-out (KI/KO) mice in which one allele of the Sco2 gene that encodes a copper chaperone required for COX activity is deleted and the second allele is mutated, have previously been shown to be viable despite a 30-60% reduction in COX activity. In contrast to the Surf1 -/- mice, we show that Sco2 KI/KO mice have increased fat mass, associated with reduced β-oxidation and increased adipogenesis markers, reduced insulin receptor beta (IR-β levels in adipose tissue, reduced muscle glucose transporter 4 (Glut4) levels and a impaired response to the insulin tolerance test consistent with insulin resistance. COX activity and protein are reduced approximately 50% in adipose tissue from the Sco2 KI/KO mice. Consistent with the increase in adipose tissue mass, the Sco2 KI/KO mice also show increased hepatosteatosis, elevated serum and liver triglyceride and increased serum cholesterol levels compared to wild-type controls. In contrast to the Surf1 -/- mice, which show increased mitochondrial number, upregulation of the mitochondrial unfolded protein response (UPR MT ) pathway and no significant change in mitochondrial respiration in several tissues, Sco2 KI/KO mice do not upregulate the UPR MT , and tissue oxygen consumption and levels of several proteins involved in mitochondrial function are reduced in adipose tissue compared to wild type mice. Thus, the metabolic effects of the Sco2 and Surf1 -/- mutations are opposite, despite comparable changes in COX activity, illuminating the complex impact of mitochondrial dysfunction on physiology and pointing to an important role for complex IV in regulating metabolism., (Published by Elsevier B.V.)

White adipose tissue (WAT) mitochondrial dysfunction is linked to the pathogenesis of obesity driven insulin resistance. Dietary conditions that alter fat mass are known to affect white adipocyte mitochondrial function, however, the impact of high calorie diets on white adipocyte mitochondria is not fully understood. The aim of this study is to assess the effect of a diet rich in saturated or polyunsaturated fat on mitochondrial unfolded protein response (UPR mt ), a retrograde signaling response that maintains mitochondrial homeostasis, in epididymal WAT (eWAT). Mice were fed a low fat diet (LFD), saturated fat diet (SFD) or fish oil (unsaturated fat diet, UFD) and assessed changes in eWAT mitochondria. Compared to mice fed a LFD, SFD-fed mice have reduced mitochondrial biogenesis markers, mitochondrial fatty acid oxidation enzymes and TCA cycle enzymes, suggesting an impaired mitochondrial function that could contribute to increased fat mass. In contrast, isocaloric UFD-fed mice have increased expression of mitochondrial uncoupling protein 1 (UCP1) and peroxisomal fatty acid oxidation enzymes suggesting that elevated mitochondrial uncoupling and peroxisomal fatty acid oxidation could contribute to the reduction in fat mass. Interestingly, expression of UPR mt -associated proteins caseinolytic peptidase (ClpP) and heat shock protein 60 (Hsp60) are induced by UFD, whereas SFD reduced the expression of ClpP. Based on our data, we propose that induction of UPR mt helps to preserve a functional mitochondria and efficient utilization of fat by UFD whereas a dampened UPR mt response might impair mitochondrial function and promote fat accumulation by SFD. Thus, our findings suggest a potential role of UPR mt in mediating the beneficial effects of fish oil., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In contrast to other mouse models that are deficient in antioxidant enzymes, mice null for Cu/Zn-superoxide dismutase (Sod1 -/ - mice) show a major decrease in lifespan and several accelerated aging phenotypes. The goal of this study was to determine if cell senescence might be a contributing factor in the accelerated aging phenotype observed in the Sod1 -/ - mice. We focused on kidney because it is a tissue that has been shown to a significant increase in senescent cells with age. The Sod1 -/ - mice are characterized by high levels of DNA oxidation in the kidney, which is attenuated by DR. The kidney of the Sod1 -/ - mice also have higher levels of double strand DNA breaks than wild type (WT) mice. Expression (mRNA and protein) of p16 and p21, two of the markers of cellular senescence, which increased with age, are increased significantly in the kidney of Sod1 -/ - mice as is β-gal staining cells. In addition, the senescence associated secretory phenotype was also increased significantly in the kidney of Sod1 -/ - mice compared to WT mice as measured by the expression of transcripts for IL-6 and IL-1β. Dietary restriction of the Sod1 -/ - mice attenuated the increase in DNA damage, cellular senescence, and expression of IL-6 and IL-1β. Interestingly, the Sod1 -/ - mice showed higher levels of circulating cytokines than WT mice, suggesting that the accelerated aging phenotype shown by the Sod1 -/ - mice could result from increased inflammation arising from an accelerated accumulation of senescent cells. Based on our data with Sod1 -/ - mice, we propose that various bouts of increased oxidative stress over the lifespan of an animal leads to the accumulation of senescent cells. The accumulation of senescent cells in turn leads to increased inflammation, which plays a major role in the loss of function and increased pathology that are hallmark features of aging., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Frailty is a geriatric syndrome that is an important public health problem for the older adults living in the USA. Although several methods have been developed to measure frailty in humans, we have very little understanding of its etiology. Because the molecular basis of frailty is poorly understood, mouse models would be of great value in determining which pathways contribute to the development of frailty. More importantly, mouse models would be critical in testing potential therapies to treat and possibly prevent frailty. In this article, we present data showing that Sod1KO mice, which lack the antioxidant enzyme, Cu/Zn superoxide dismutase, are an excellent model of frailty, and we compare the Sod1KO mice to the only other mouse model of frailty, mice with the deletion of the IL-10 gene. Sod1KO mice exhibit four characteristics that have been used to define human frailty: weight loss, weakness, low physical activity, and exhaustion. In addition, Sod1KO mice show increased inflammation and sarcopenia, which are strongly associated with human frailty. The Sod1KO mice also show alterations in pathways that have been proposed to play a role in the etiology of frailty: oxidative stress, mitochondrial dysfunction, and cell senescence. Using Sod1KO mice, we show that dietary restriction can delay/prevent characteristics of frailty in mice.

The caseinolytic peptidase P (ClpP) is the endopeptidase component of the mitochondrial matrix ATP-dependent ClpXP protease. ClpP degrades unfolded proteins to maintain mitochondrial protein homeostasis and is involved in the initiation of the mitochondrial unfolded protein response (UPR(mt)). Outside of an integral role in the UPR(mt), the cellular function of ClpP is not well characterized in mammalian cells. To investigate the role of ClpP in mitochondrial function, we generated C2C12 muscle cells that are deficient in ClpP using siRNA or stable knockdown using lentiviral transduction. Reduction of ClpP levels by ~70% in C2C12 muscle cells resulted in a number of mitochondrial alterations including reduced mitochondrial respiration and reduced oxygen consumption rate in response to electron transport chain (ETC) complex I and II substrates. The reduction in ClpP altered mitochondrial morphology, changed the expression level of mitochondrial fission protein Drp1 and blunted UPR(mt) induction. In addition, ClpP deficient cells showed increased generation of reactive oxygen species (ROS) and decreased membrane potential. At the cellular level, reduction of ClpP impaired myoblast differentiation, cell proliferation and elevated phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) suggesting an inhibition of translation. Our study is the first to define the effects of ClpP deficiency on mitochondrial function in muscle cells in vitro. In addition, we have uncovered novel effects of ClpP on mitochondrial morphology, cell proliferation and protein translation pathways in muscle cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mutations in SURF1 (surfeit locus protein 1) COX (cytochrome c oxidase) assembly protein are associated with Leigh's syndrome, a human mitochondrial disorder that manifests as severe mitochondrial phenotypes and early lethality. In contrast, mice lacking the SURF1 protein (Surf1-/-) are viable and were previously shown to have enhanced longevity and a greater than 50% reduction in COX activity. We measured mitochondrial function in heart and skeletal muscle, and despite the significant reduction in COX activity, we found little or no difference in ROS (reactive oxygen species) generation, membrane potential, ATP production or respiration in isolated mitochondria from Surf1-/- mice compared with wild-type. However, blood lactate levels were elevated and Surf1-/- mice had reduced running endurance, suggesting compromised mitochondrial energy metabolism in vivo. Decreased COX activity in Surf1-/- mice is associated with increased markers of mitochondrial biogenesis [PGC-1α (peroxisome-proliferator-activated receptor γ co-activator 1α) and VDAC (voltage-dependent anion channel)] in both heart and skeletal muscle. Although mitochondrial biogenesis is a common response in the two tissues, skeletal muscle has an up-regulation of the UPRMT (mitochondrial unfolded protein response) and heart exhibits induction of the Nrf2 (nuclear factor-erythroid 2-related factor 2) antioxidant response pathway. These data are the first to show induction of the UPRMT in a mammalian model of decreased COX activity. In addition, the results of the present study suggest that impaired mitochondrial function can lead to induction of mitochondrial stress pathways to confer protective effects on cellular homoeostasis.

Recent studies have challenged the prevailing view that reduced mitochondrial function and increased oxidative stress are correlated with reduced longevity. Mice carrying a homozygous knockout (KO) of the Surf1 gene showed a significant decrease in mitochondrial electron transport chain Complex IV activity, yet displayed increased lifespan and reduced brain damage after excitotoxic insults. In the present study, we examined brain metabolism, brain hemodynamics, and memory of Surf1 KO mice using in vitro measures of mitochondrial function, in vivo neuroimaging, and behavioral testing. We show that decreased respiration and increased generation of hydrogen peroxide in isolated Surf1 KO brain mitochondria are associated with increased brain glucose metabolism, cerebral blood flow, and lactate levels, and with enhanced memory in Surf1 KO mice. These metabolic and functional changes in Surf1 KO brains were accompanied by higher levels of hypoxia-inducible factor 1 alpha, and by increases in the activated form of cyclic AMP response element-binding factor, which is integral to memory formation. These findings suggest that Surf1 deficiency-induced metabolic alterations may have positive effects on brain function. Exploring the relationship between mitochondrial activity, oxidative stress, and brain function will enhance our understanding of cognitive aging and of age-related neurologic disorders.

Excess nutrient uptake leads to obesity, insulin resistance, and type 2 diabetes. Mammalian target of the rapamycin (mTOR), a major component of the nutrient-sensing pathway also regulates mitochondrial oxidative function. Rapamycin, a pharmacological inhibitor of mTOR, causes glucose intolerance and inhibits mitochondrial oxidative function. While a number of studies have focused on the effect of rapamycin on control wild-type mice, ours is the first to study the effect of rapamycin on mitochondrial gene expression and insulin sensitivity in the db/db mouse, a model of diabetic dyslipidemia. Female db/+ and db/db mice were fed ad libitum a rapamycin-containing diet or a control diet for 6 months, starting at two months of age. Body weight, fat mass, lean mass and food intake were measured monthly. Effect of rapamycin or control diet on markers of adipogenesis, fatty acid oxidation and mitochondrial biogenesis in the gonadal white adipose tissue (WAT) as well as different serum parameters were assessed. Whole body insulin sensitivity was measured by insulin tolerance test. Rapamycin feeding to db/db mice decreased body weight (58%) and fat mass (33%), elevated markers of fatty acid oxidation and mitochondrial biogenesis in WAT, reduced circulating non-esterified free fatty acids (NEFA), elevated circulating adiponectin and improved insulin sensitivity, compared to control diet fed db/db mice. These data demonstrate that rapamycin exhibits an anti-obesity effect and improves whole body insulin sensitivity in db/db mice and suggest an unexpected effect of simultaneous inhibition mTOR and leptin signaling in mice.

Mice lacking Surf1, a complex IV assembly protein, have ∼50-70% reduction in cytochrome c oxidase activity in all tissues yet a paradoxical increase in lifespan. Here we report that Surf1(-/-) mice have lower body (15%) and fat (20%) mass, in association with reduced lipid storage, smaller adipocytes, and elevated indicators of fatty acid oxidation in white adipose tissue (WAT) compared with control mice. The respiratory quotient in the Surf1(-/-) mice was significantly lower than in the control animals (0.83-0.93 vs. 0.90-0.98), consistent with enhanced fat utilization in Surf1(-/-) mice. Elevated fat utilization was associated with increased insulin sensitivity measured as insulin-stimulated glucose uptake, as well as an increase in insulin receptor levels (∼2-fold) and glucose transporter type 4 (GLUT4; ∼1.3-fold) levels in WAT in the Surf1(-/-) mice. The expression of peroxisome proliferator-activated receptor γ-coactivator 1-α (PGC-1α) mRNA and protein was up-regulated by 2.5- and 1.9-fold, respectively, in WAT from Surf1(-/-) mice, and the expression of PGC-1α target genes and markers of mitochondrial biogenesis was elevated. Together, these findings point to a novel and unexpected link between reduced mitochondrial complex IV activity, enhanced insulin sensitivity, and increased mitochondrial biogenesis that may contribute to the increased longevity in the Surf1(-/-) mice.

Mutation in the BRAF gene (BRAFV600E) exists in nearly 70% of human melanomas. Targeted therapy against BRAFV600E kinase using a recently identified RAF-selective inhibitor, PLX4032, has been successful in early clinical trials. However, in patients with the normal BRAF allele (wild-type), PLX4032 is protumorigenic. This conundrum identifies the unmet need for novel therapeutic agents to target BRAFV600E kinase that are not counterproductive. We have identified gossypin, a pentahydroxy flavone, as a potent antimelanoma agent. Gossypin inhibited human melanoma cell proliferation, in vitro, in melanoma cell lines that harbor both BRAFV600E kinase and cyclin-dependent kinase 4 (CDK4) as well as in cells with BRAF wild-type allele. Gossypin inhibited kinase activities of BRAFV600E and CDK4, in vitro, possibly through direct binding of gossypin with these kinases, as confirmed by molecular docking studies. For cells harboring the BRAFV600E, gossypin inhibited cell proliferation through abrogation of the MEK-ERK-cyclin D1 pathway and in cells with BRAF wild-type allele, through attenuation of the retinoblastoma-cyclin D1 pathway. Furthermore, gossypin significantly inhibited melanoma growth in an organotypic three-dimensional skin culture mimicking human skin. Gossypin (10 and 100 mg/kg) treatment for 10 days in human melanoma (A375) cell xenograft tumors harboring BRAFV600E significantly reduced tumor volume through induction of apoptosis and increased survival rate in mice, and the effect was significantly superior to that of PLX4032 (10 mg/kg) or roscovitine 10 mg/kg. In summary, this study identified gossypin as a novel agent with dual inhibitory effects for BRAFV600E kinase and CDK4 for treatment of melanoma.

Chondroitin sulfate (CS) chains regulate the development of the central nervous system in vertebrates and are linear polysaccharides consisting of variously sulfated repeating disaccharides, [-4GlcUAβ1-3GalNAcβ1-](n), where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. CS chains containing D-disaccharide units [GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)] are involved in the development of cerebellar Purkinje cells and neurite outgrowth-promoting activity through interaction with a neurotrophic factor, pleiotrophin, resulting in the regulation of signaling. In this study, to obtain further structural information on the CS chains containing d-disaccharide units involved in brain development, oligosaccharides containing D-units were isolated from a shark fin cartilage. Seven novel hexasaccharide sequences, ΔO-D-D, ΔA-D-D, ΔC-D-D, ΔE-A-D, ΔD-D-C, ΔE-D-D and ΔA-B-D, in addition to three previously reported sequences, ΔC-A-D, ΔC-D-C and ΔA-D-A, were isolated from a CS preparation of shark fin cartilage after exhaustive digestion with chondroitinase AC-I, which cannot act on the galactosaminidic linkages bound to D-units. The symbol Δ stands for a 4,5-unsaturated bond of uronic acids, whereas A, B, C, D, E and O represent [GlcUA-GalNAc(4-O-sulfate)], [GlcUA(2-O-sulfate)-GalNAc(4-O-sulfate)], [GlcUA-GalNAc(6-O-sulfate)], [GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)], [GlcUA-GalNAc(4-O-, 6-O-sulfate)] and [GlcUA-GalNAc], respectively. In binding studies using an anti-CS monoclonal antibody, MO-225, the epitopes of which are involved in cerebellar development in mammals, novel epitope structures, ΔA-D-A, ΔA-D-D and ΔA-B-D, were revealed. Hexasaccharides containing two consecutive D-units or a B-unit will be useful for the structural and functional analyses of CS chains particularly in the neuroglycobiological fields.

We recently found that the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif (APPL)1 is essential for mediating adiponectin signal to induce liver kinase B (LKB)1 cytosloic translocation, an essential step for activation of AMP-activated protein kinase (AMPK) in cells. However, the underlying molecular mechanisms remain unknown. Here, we demonstrate that treating C2C12 myotubes with adiponectin promoted APPL1 interaction with protein phosphatase 2A (PP2A) and protein kinase Cζ (PKCζ), leading to the activation of PP2A and subsequent dephosphorylation and inactivation of PKCζ. The adiponectin-induced inactivation of PKCζ results in dephosphorylation of LKB1 at Ser(307) and its subsequent translocation to the cytosol, where it stimulates AMPK activity. Interestingly, we found that metformin also induces LKB1 cytosolic translocation, but the stimulation is independent of APPL1 and the PP2A-PKCζ pathway. Together, our study uncovers a new mechanism underlying adiponectin-stimulated AMPK activation in muscle cells and shed light on potential targets for prevention and treatment of insulin resistance and its associated diseases.

Chondroitin sulphate proteoglycans in the extracellular matrix restrict plasticity in the adult central nervous system and their digestion with chondroitinase reactivates plasticity. However the structures in the extracellular matrix that restrict plasticity are unknown. There are many changes in the extracellular matrix as critical periods for plasticity close, including changes in chondroitin sulphate proteoglycan core protein levels, changes in glycosaminoglycan sulphation and the appearance of dense chondroitin sulphate proteoglycan-containing perineuronal nets around many neurons. We show that formation of perineuronal nets is triggered by neuronal production of cartilage link protein Crtl1 (Hapln1), which is up-regulated in the visual cortex as perineuronal nets form during development and after dark rearing. Mice lacking Crtl1 have attenuated perineuronal nets, but the overall levels of chondroitin sulphate proteoglycans and their pattern of glycan sulphation are unchanged. Crtl1 knockout animals retain juvenile levels of ocular dominance plasticity and their visual acuity remains sensitive to visual deprivation. In the sensory pathway, axons in knockout animals but not controls sprout into the party denervated cuneate nucleus. The organization of chondroitin sulphate proteoglycan into perineuronal nets is therefore the key event in the control of central nervous system plasticity by the extracellular matrix.

The binding of the adaptor protein APPL1 to adiponectin receptors is necessary for adiponectin-induced AMP-activated protein kinase (AMPK) activation in muscle, yet the underlying molecular mechanism remains unknown. Here we show that in muscle cells adiponectin and metformin induce AMPK activation by promoting APPL1-dependent LKB1 cytosolic translocation. APPL1 mediates adiponectin signaling by directly interacting with adiponectin receptors and enhances LKB1 cytosolic localization by anchoring this kinase in the cytosol. Adiponectin also activates another AMPK upstream kinase Ca2+/calmodulin-dependent protein kinase kinase by activating phospholipase C and subsequently inducing Ca2+ release from the endoplasmic reticulum, which plays a minor role in AMPK activation. Our results show that in muscle cells adiponectin is able to activate AMPK via two distinct mechanisms as follows: a major pathway (the APPL1/LKB1-dependent pathway) that promotes the cytosolic localization of LKB1 and a minor pathway (the phospholipase C/Ca2+/Ca2+/calmodulin-dependent protein kinase kinase-dependent pathway) that stimulates Ca2+ release from intracellular stores.

Adiponectin, an adipokine secreted by the white adipose tissue, plays an important role in regulating glucose and lipid metabolism and controlling energy homeostasis in insulin-sensitive tissues. A decrease in the circulating level of adiponectin has been linked to insulin resistance, type 2 diabetes, atherosclerosis, and metabolic syndrome. Adiponectin exerts its effects through two membrane receptors, AdipoR1 and AdipoR2. APPL1 is the first identified protein that interacts directly with adiponectin receptors. APPL1 is an adaptor protein with multiple functional domains, the Bin1/amphiphysin/rvs167, pleckstrin homology, and phosphotyrosine binding domains. The PTB domain of APPL1 interacts directly with the intracellular region of adiponectin receptors. Through this interaction, APPL1 mediates adiponectin signaling and its effects on metabolism. APPL1 also functions in insulin-signaling pathway and is an important mediator of adiponectin-dependent insulin sensitization in skeletal muscle. Adiponectin signaling through APPL1 is necessary to exert its anti-inflammatory and cytoprotective effects on endothelial cells. APPL1 also acts as a mediator of other signaling pathways by interacting directly with membrane receptors or signaling proteins, thereby playing critical roles in cell proliferation, apoptosis, cell survival, endosomal trafficking, and chromatin remodeling. This review focuses mainly on our current understanding of adiponectin signaling in various tissues, the role of APPL1 in mediating adiponectin signaling, and also its role in the cross-talk between adiponectin/insulin-signaling pathways.

Chondroitin sulfate (CS) proteoglycans are major components of cartilage and other connective tissues. The monoclonal antibody WF6, developed against embryonic shark cartilage CS, recognizes an epitope in CS chains, which is expressed in ovarian cancer and variably in joint diseases. To elucidate the structure of the epitope, we isolated oligosaccharide fractions from a partial chondroitinase ABC digest of shark cartilage CS-C and established their chain length, disaccharide composition, sulfate content, and sulfation pattern. These structurally defined oligosaccharide fractions were characterized for binding to WF6 by enzyme-linked immunosorbent assay using an oligosaccharide microarray prepared with CS oligosaccharides derivatized with a fluorescent aminolipid. The lowest molecular weight fraction recognized by WF6 contained octasaccharides, which were split into five subfractions. The most reactive subfraction contained several distinct octasaccharide sequences. Two octasaccharides, DeltaD-C-C-C and DeltaC-C-A-D (where A represents GlcUAbeta1-3GalNAc(4-O-sulfate), C is GlcUAbeta1-3Gal-NAc(6-O-sulfate), D is GlcUA(2-O-sulfate)beta1-3GalNAc(6-O-sulfate), DeltaCis Delta(4,5)HexUAalpha1-3GalNAc(6-O-sulfate), and DeltaDis Delta(4,5)HexUA(2-O-sulfate)alpha1-3GalNAc(6-O-sulfate)), were recognized by WF6, but other related octasaccharides, DeltaC-A-D-C and DeltaC-C-C-C, were not. The structure and sequences of both the binding and nonbinding octasaccharides were compared by computer modeling, which revealed a remarkable similarity between the shape and distribution of the electrostatic potential in the two different octasaccharide sequences that bound to WF6 and that differed from the nonbinding octasaccharides. The strong similarity in structure predicted for the two binding CS octasaccharides (DeltaD-C-C-C and DeltaC-C-A-D) provided a possible explanation for their similar affinity for WF6, although they differed in sequence and thus form two specific mimetopes for the antibody.

Twelve octasaccharide fractions were obtained from chondroitin sulfate C derived from shark cartilage after hyaluronidase digestion. Their sugar and sulfate composition was assigned by matrix-assisted laser desorption ionization time of flight mass spectrometry. The sequences were determined at low picomole amounts by a combination of enzymatic digestions with high-performance liquid chromatography, and were composed of disaccharide building units including O [GlcUAbeta1-3GalNAc], C [GlcUAbeta1-3GalNAc(6S)], A [GlcUAbeta1-3GalNAc(4S)], and/or D [GlcUA(2S)beta1-3GalNAc(6S)], where 2S, 4S, and 6S represent 2-O-, 4-O-, and 6-O-sulfate, respectively. As many as 24 different sequences including minor ones were revealed, exhibiting a high degree of structural diversity reflecting the enormous heterogeneity of the parent polysaccharides. Nineteen of them were novel, with the other four reported previously as unsaturated counterparts obtained after digestion with chondroitinase. Microarrays of these structurally defined octasaccharide fractions were prepared using low picomole amounts of their lipid-derivatives to investigate the binding specificity of four commercial anti-chondroitin sulfate antibodies CS-56, MO-225, 2H6, and LY111. The results revealed that multiple unique sequences were recognized by each antibody, which implies that the common conformation shared by the multiple primary sequences in the intact chondroitin sulfate chains is important as an epitope for each monoclonal antibody. Comparison of the specificity of the tested antibodies indicates that CS-56 and MO-225 specifically recognize octasaccharides containing an A-D tetrasaccharide sequence, whereas 2H6 and LY111 require a hexasaccharide as a minimum size for their binding, and prefer sequences with A- and C-units such as C-C-A-C (2H6) or C-C-A-O, C-C-A-A, and C-C-A-C (LY111) for strong binding but require no D-unit.

A mixture of octa- and decasaccharides obtained by the digestion with the hyaluronidase of chondroitin sulfate E derived from squid cartilage was subfractionated into 20 and 23 different components, respectively, by anion-exchange HPLC. MALDI-TOF/MS was used to assign the sugar and sulfate composition of the putative octa- and decasaccharides, and a disaccharide composition analysis revealed the building blocks to be A- [GlcUAbeta1-3GalNAc(4S)], C- [GlcUAbeta1-3GalNAc(6S)], and E- [GlcUAbeta1-3GalNAc(4S,6S)] units, where 4S and 6S represent 4-O- and 6-O-sulfate, respectively. The sequences of these octa- and decasaccharides were determined at low picomole amounts by a combination of enzymatic digestions with chondroitinases in conjunction with anion-exchange HPLC. Sequencing revealed that each fraction is a mixture of a major component together with one to three minor components, reflecting the heterogeneity of the parent polysaccharide. Among the 11 different octasaccharide sequences reported here, 8 are novel, while all of the 6 decasaccharide sequences are novel, and this is the first report of the sequencing of CS oligosaccharides longer than octasaccharides. The reactivity of the monoclonal antibody MO-225 with octa- and decasaccharides tested with an oligosaccharide microarray revealed that a CS-E decasaccharide is the minimal requirement for antibody recognition. Among the 6 decasaccharides, only E-E-E-E-C was recognized by MO-225, suggesting the requirement of a C-unit at the reducing end and also the importance of chain length, which in turn may indicate the importance of the conformation acquired by this specific sequence for antibody recognition.

We developed a method to extract differentially chondroitin sulfate proteoglycans (CSPGs) that are diffusely present in the central nervous system (CNS) matrix and CSPGs that are present in the condensed matrix of perineuronal nets (PNNs). Adult rat brain was sequentially extracted with Tris-buffered saline (TBS), TBS-containing detergent, 1 m NaCl, and 6 m urea. Extracting tissue sections with these buffers showed that the diffuse and membrane-bound CSPGs were extracted in the first three buffers, but PNN-associated CSPGs remained and were only removed by 6 m urea. Most of the CSPGs were extracted to some degree with all the buffers, with neurocan, brevican, aggrecan, and versican particularly associated with the stable urea-extractable PNNs. The CSPGs in stable complexes only extractable in urea buffer are found from postnatal day 7-14 coinciding with PNN formation. Disaccharide composition analysis indicated a different glycosaminoglycan (GAG) composition for PGs strongly associated with extracellular matrix (ECM). For CS/dermatan sulfate (DS)-GAG the content of nonsulfated, 6-O-sulfated, 2,6-O-disulfated, and 4,6-O-disulfated disaccharides were higher and for heparan sulfate (HS)-GAG, the content of 6-O-sulfated, 2-N-, 6-O-disulfated, 2-O-, 2-N-disulfated, and 2-O-, 2-N-, 6-O-trisulfated disaccharides were higher in urea extract compared with other buffer extracts. Digestions with chondroitinase ABC and hyaluronidase indicated that aggrecan, versican, neurocan, brevican, and phosphacan are retained in PNNs through binding to hyaluronan (HA). A comparison of the brain and spinal cord ECM with respect to CSPGs indicated that the PNNs in both parts of the CNS have the same composition.

A comparative analysis was carried out of heparan sulfate (HS) and chondroitin sulfate (CS) chains of the ectodomains of hybrid type transmembrane proteoglycans, syndecan-1 and -4, synthesized simultaneously by normal murine mammary gland epithelial cells. Although the HS chains were structurally indistinguishable, intriguingly the CS chains were structurally and functionally distinct, probably reflecting the differential regulation of sulfotransferases involved in the synthesis of HS and CS. The CS chains of the two syndecans comprised nonsulfated, 4-O-, 6-O-, and 4,6-O-disulfated N-acetylgalactosamine-containing disaccharide units and were significantly different, with a higher degree of sulfation for syndecan-4. Functional analysis using a BIAcore system showed that basic fibroblast growth factor (bFGF) specifically bound only to the HS chains of both syndecans, whereas midkine (MK) and pleiotrophin (PTN) bound not only to the HS but also to the CS chains. Stronger binding of MK and PTN to the CS chains of syndecan-4 than those of syndecan-1 was revealed, supporting the structural and functional differences. Intriguingly, removal of the CS chains decreased the association and dissociation rate constants of MK, PTN, and bFGF for both syndecans, suggesting the simultaneous binding of these growth factors to both types of chains, producing a ternary complex that transfers the growth factors to the corresponding cell surface receptors more efficiently compared with the HS chains alone. The involvement of the core protein was also shown in the binding of MK and PTN to syndecan-1, suggesting the possibility of cooperation with the HS and/or CS chains in the binding of these growth factors and their delivery to the cell surface receptors.

We previously observed that the cortical neuronal cell adhesion mediated by midkine (MK), a heparin (Hep)-binding growth factor, is specifically inhibited by oversulfated chondroitin sulfate-E (CS-E) (Ueoka, C., Kaneda, N., Okazaki, I., Nadanaka, S., Muramatsu, T., and Sugahara, K. (2000) J. Biol. Chem. 275, 37407-37413) and that CS-E exhibits neurite outgrowth promoting activities toward embryonic rat hippocampal neurons. We have also shown oversulfated CS chains in embryonic chick and rat brains and demonstrated that the CS disaccharide composition changes during brain development. In view of these findings, here we tested the possibility of CS-E interacting with Hep-binding growth factors during development, using squid cartilage CS-E. The binding ability of Hep-binding growth factors (MK, pleiotrophin (PTN), fibroblast growth factor-1 (FGF-1), FGF-2, Hep-binding epidermal growth factor-like growth factor (HB-EGF), FGF-10, FGF-16, and FGF-18) toward [(3)H]CS-E was first tested by a filter binding assay, which demonstrated direct binding of all growth factors, except FGF-1, to CS-E. The bindings were characterized further in an Interaction Analysis system, where all of the growth factors, except FGF-1, gave concentration-dependent and specific bindings. The kinetic constants k(a), k(d), and K(d) suggested that MK, PTN, FGF-16, FGF-18, and HB-EGF bound strongly to CS-E, in comparable degrees to the binding to Hep, whereas the intensity of binding of FGF-2 and FGF-10 toward CS-E was lower than that for Hep. These findings suggest the possibility of CS-E being a binding partner, a coreceptor, or a genuine receptor for various Hep-binding growth factors in the brain and possibly also in other tissues.

Soluble peroxidase (POD) from oil palm leaf was purified by (NH(4))(2)SO(4) precipitation, anion exchange chromatography and molecular exclusion chromatography. The purification grade obtained was 429 yielding 54% of the enzyme activity. Electrophoresis of purified enzyme under denatured conditions revealed M(r) of 48+/-2 kDa. It has an optimum pH of 5 and it exhibited very high pH and thermal stabilities. K(m) for guaiacol, ABTS and pyrogallol were 3.96, 1 and 0.84 mM, respectively. Immunocytochemical localization studies showed that soluble POD was mainly located in the vascular bundles and epidermis of leaf.

Caenorhabditis elegans and Drosophila melanogaster are relevant models for studying the roles of glycosaminoglycans (GAG) during the development of multicellular organisms. The genome projects of these organisms have revealed the existence of multiple genes related to GAG-synthesizing enzymes. Although the putative genes encoding the enzymes that synthesize the GAG-protein linkage region have also been identified, there is no direct evidence that the GAG chains bind covalently to core proteins. This study aimed to clarify whether GAG chains in these organisms are linked to core proteins through the conventional linkage region tetrasaccharide sequence found in vertebrates and whether modifications by phosphorylation and sulfation reported for vertebrates are present also in invertebrates. The linkage region oligosaccharides were isolated from C. elegans chondroitin in addition to D. melanogaster heparan and chondroitin sulfate after digestion with the respective bacterial eliminases and were then derivatized with a fluorophore 2-aminobenzamide. Their structures were characterized by gel filtration and anion-exchange high performance liquid chromatography in conjunction with enzymatic digestion and matrix-assisted laser desorption ionization time-of-flight spectrometry, which demonstrated a uniform linkage tetrasaccharide structure of -GlcUA-Gal-Gal-Xyl- or -GlcUA-Gal-Gal-Xyl(2-O-phosphate)- for C. elegans chondroitin and D. melanogaster CS, respectively. In contrast, the unmodified and phosphorylated counterparts were demonstrated in heparan sulfate of adult flies at a molar ratio of 73:27, and in that of the immortalized D. melanogaster S2 cell line at a molar ratio of 7:93, which suggests that the linkage region in the fruit fly first becomes phosphorylated uniformly on the Xyl residue and then dephosphorylated. It has been established here that GAG chains in both C. elegans and D. melanogaster are synthesized on the core protein through the ubiquitous linkage region tetrasaccharide sequence, suggesting that indispensable functions of the linkage region in the GAG synthesis have been well conserved during evolution.

Red palm oil (5 ml and 10 ml), ground nut oil fortified with 400 and 800 retinol equivalent retinol palmitate, and ground nut oil (5 and 10 ml), were administered to six groups of preschool children (four experimental and two control groups) in randomly assigned balwadis of Ramanathapuram District of Tamil Nadu for a period of 7 months, to monitor the difference in the efficacy of the mode of supplementation and the optimum dose for improving vitamin A status. Results show that red palm oil groups recorded more gain in retinol and beta-carotene levels compared to other dosage groups, and that administration of 10 ml did not offer any substantial improvement over the 5-ml daily dose.

A 10-month long feeding trial was conducted to assess the impact of beta-carotene supplementation through red palm oil (RPO) with the focus on vitamin A status, morbidity status and acceptability of an RPO-incorporated noon-meal as a dietary supplement among two cohorts of 409 (experimental) and 346 (control) preschool children in two southern districts of rural Tamil Nadu, selected by stratified random sampling. Information was gathered on ocular symptoms of vitamin A deficiency and anthropometry, and blood samples were drawn at baseline and final rounds for estimation of serum beta-carotene, retinol and tocopherol. Data about Socioeconomic Status (SES) were collected once during the study period, while information on attendance, consumption and morbidity was recorded by preschool teachers. The results showed the following. 1. Significant improvement in the vitamin A status of children in terms of disappearance of Bitot's spots (50.0 per cent) in the experimental group vs. 28.0 per cent in the control group. 2. After feeding of RPO, incidence rate of new Bitot's spots cases was low at 2.13 in the experimental children vs. 4.78 in control children. 3. Marked improvement in the serum beta-carotene levels after 10 months of feeding. 4. RPO is acceptable to children as an edible grade oil as there is no perceptible difference in the consumption pattern between experimental and control children.