Browsing by Subject "mTOR signaling"

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Open Access
Dietary and pharmacological interventions that inhibit mammalian target of rapamycin activity alter the brain expression levels of neurogenic and glial markers in an age-and treatment-dependent manner
(Mary Ann Liebert, 2020) Çelebi-Birand, Dilan; Ardıç, Narin İlgim; Karoğlu-Eravşar, Elif Tuğçe; Şengül, Göksemin Fatma; Kafalıgönül, Hulusi; Adams, Michelle M.
Intermittent fasting (IF) and its mimetic, rapamycin extend lifespan and healthspan through mechanisms that are not fully understood. We investigated different short-term durations of IF and rapamycin on cellular and molecular changes in the brains of young (6–10 months) and old (26–31 months) zebrafish. Interestingly, our results showed that IF significantly lowered glucose levels while increasing DCAMKL1 in both young and old animals. This proliferative effect of IF was supported by the upregulation of foxm1 transcript in old animals. Rapamycin did not change glucose levels in young and old animals but had differential effects depending on age. In young zebrafish, proliferating cell nuclear antigen and the LC3-II/LC3-I ratio was decreased, whereas glial fibrillary acidic protein and gephyrin were decreased in old animals. The changes in proliferative markers and a marker of autophagic flux suggest an age-dependent interplay between autophagy and cell proliferation. Additionally, changes in glia and inhibitory tone suggest a suppressive effect on neuroinflammation but may push the brain toward a more excitable state. Mammalian target of rapamycin (mTOR) activity in the brain following the IF and rapamycin treatment was differentially regulated by age. Interestingly, rapamycin inhibited mTOR more potently in young animals than IF. Principal component analysis supported our conclusion that the regulatory effects of IF and rapamycin were age-specific, since we observed different patterns in the expression levels and clustering of young and old animals. Taken together, our results suggest that even a short-term duration of IF and rapamycin have significant effects in the brain at young and old ages, and that these are age and treatment dependent.
Open Access
Elucidating immunomodulatory effects of telomeric repeat mimicking synthetic A151 oligodeoxynucleotide on immune cell transcriptome
(2019-09) Yazar, Volkan
Recent evidence revealed that DNA is beyond just the blueprint of life; it is also involved in immunomodulation. Unmethylated Cytosine-phosphate-Guanine (CpG) motifs of prokaryotic DNA stimulate immune response by interacting with Toll-like receptor 9 (TLR9). This interaction is mimicked using synthetic oligodeoxynucleotides (ODN) bearing similar DNA motifs to boost vaccinedriven immune response in human. Conversely, mammalian telomeric ends expressing TTAGGG repeats suppress immune response and contribute to fine-tuning of delicate immune balance. In this respect, suppressive ODN A151 with such G-rich telomeric repeats has proven useful in downregulating immune response; an overly active immune response is just as harmful to the host, as in the case of autoimmune disorders. Both CpG ODN and A151 are currently under preclinical/clinical trials with the aim of averting or medically treating a wide range of conditions from cancer to infectious disease or from autoimmune to autoinflammatory conditions. Contrary to CpG ODN, A151 literature is very limited and its modus operandi at gene level remains more of a mystery. Additionally, the degree, duration and breath of A151-induced alterations in immune transcriptome appear partially understood. Given the medical potential A151 holds for immunosuppressive therapy in human as a “self-molecule”, understanding the underlying molecular mechanisms via which A151 operates is invaluable. Toward this end, we attempted to uncover the unidentified features lying behind A151 ODNs immunosuppressive effects on immune cell transcriptome using a combined analysis approach of microarray data in this thesis. We demonstrated for the first time that A151 ODN deprives the cells energy by ceasing cellular uptake of fundamental molecules into the immune cells after derailing the entire intracellular trafficking. Putting it another way, A151 does not directly act on immune system cells but actually suffocates the cells by messing with intracellular trafficking, thereby blocking cellular uptake of fundamental molecules like glucose and glutamine. As such, immune suppression is just an indirect consequence of this larger cellular chaos. Our results indicated that this phenomenon occurs independent of CpG ODN stimulation of the cells and in a timely manner. Most, if not all, regulators of intracellular trafficking, vesicle signaling, and membrane protein transportation were found downregulated after incubation of cells with A151 at a physiologically relevant concentration, as well, implying full-blown entry to this intracellular turmoil at cellular level. The A151 effect on immune transcriptome was not just restricted to setting off a chaos for intracellular dynamics; novel long non-coding RNAs (lncRNAs) with immunometabolic activities were identified within the scope of this study among elements potentially regulated by A151, such as Lncpint, Malat1 and H2-T10 just to name a few. The involvement of lncRNAs in immune regulation is a well-documented phenomenon. Finally, our data showed that as an epiphenomenon of the intracellular turmoil mentioned above A151 has a deep impact in immune cells on mTOR network, the cardinal network of cellular energetics, growth, proliferation, and survival. A major shift in expression profile of relevant genes, i.e. downregulation of many activators of mTOR signaling along with core mTOR components, was validated on the benchtop after different layers of experimental validation using a wide range of marker genes and functional assays, reflecting A151’s ability to vastly shape dynamics of metabolism in favor of a metabolically inert state in macrophages and in B-cells. This knowledge will expand the breadth of A151 therapy in the clinics.
Open Access
Impact of the inflammatory process in the aging brain: evidence from in Vitro and Ex Vivo models
(2023-08) Aktürk, Serena Sevdiye
Aging is a complex and dynamic process that is characterized by a gradual decline over time in the physiological integrity of organisms. Several cellular mechanisms contribute to aging, including telomere shortening, damage accumulation in DNA, disabled macroautophagy, mitochondrial dysfunction, and cellular senescence. These processes, consecutively, lead to impaired cellular function, declined tissue repair, and stem cell exhaustion and are seen in the development of neurodegenerative disorders and healthy aging. One of the hallmarks of brain aging is the altered chronic inflammatory status of the brain. The over-activation and polarization of microglia, increased secretion of pro-inflammatory cytokines and reactive oxygen species, inflammasome activation, and the upregulation of the NF- κB signaling pathway are among the markers of neuroinflammation. This mechanism's anticipated effects include dysregulated nutrition sensing via the mTOR (mammalian target of rapamycin) pathway, decreased neurogenesis, and synaptic integrity over time. Another element that contributes to vulnerability to inflammation is genetic predisposition. Hence, additional research endeavors are required to investigate the influence of dietary interventions and therapeutic modalities targeting inflammation on genetic pathways. Thus, this study aimed to understand how inflammation can be triggered on different models, investigate potential inflammation-related biomarkers with meta-analysis and observe the effect of inflammation for both zebrafish primary brain cells and murine microglial cells. We conducted short-term copper sulfate treatments on both models for this objective. Moreover, to examine the effects of intermittent fasting, an mTOR downregulator, and high-fat diet, an inflammation inducer, on the brain of zebrafish at the molecular level by primary cell culture method. Finally, we applied rapamycin+DMSO treatment to primary cells to assess the possibility of reversing the progression of inflammation. The results showed that copper sulfate is an efficient oxidative stress-induced inflammatory reagent for zebrafish; however, it did not cause a direct inflammatory response in murine microglial cells. For zebrafish, in the copper sulfate+DMSO treated group, age affected Nrf2a mRNA, altering oxidative stress in old animals. Regardless of diet and treatment group, inflammation markers were higher in old animals, which underscores the association between aging and chronic inflammation. Elevated Lc3b levels in young and old animals captured that high copper concentrations can trigger autophagy. Results for neurogenesis markers revealed that overfeeding or acute inflammation could contribute to compromised neurogenesis in advanced stages of life. On the contrary, the enhanced neurogenesis potential of intermittent fasting in old animals was revealed. In conclusion, this study has demonstrated that the modulation of neuroinflammatory responses, as well as oxidative stress, neurogenesis, and autophagy, occurs in an age-related manner. Moreover, dietary or pharmaceutical interventions could yield comprehensive outcomes in perceiving the brain's neuroinflammatory profile during aging.
Open Access
Inositol‐requiring enzyme‐1 regulates phosphoinositide signaling lipids and macrophage growth
(Wiley-VCH Verlag, 2020-11) Hamid, S. M.; Çıtır, M.; Terzi, E. M.; Çimen, İ.; Yıldırım, Zehra; Doğan, Aslı Ekin; Kocatürk, B.; Onat, Umut Inci; Arditi, M.; Weber, C.; Traynor‐Kaplan, A.; Schultz, C.; Erbay, E.
The ER‐bound kinase/endoribonuclease (RNase), inositol‐requiring enzyme‐1 (IRE1), regulates the phylogenetically most conserved arm of the unfolded protein response (UPR). However, the complex biology and pathology regulated by mammalian IRE1 cannot be fully explained by IRE1’s one known, specific RNA target, X box‐binding protein‐1 (XBP1) or the RNA substrates of IRE1‐dependent RNA degradation (RIDD) activity. Investigating other specific substrates of IRE1 kinase and RNase activities may illuminate how it performs these diverse functions in mammalian cells. We report that macrophage IRE1 plays an unprecedented role in regulating phosphatidylinositide‐derived signaling lipid metabolites and has profound impact on the downstream signaling mediated by the mammalian target of rapamycin (mTOR). This cross‐talk between UPR and mTOR pathways occurs through the unconventional maturation of microRNA (miR) 2137 by IRE1’s RNase activity. Furthermore, phosphatidylinositol (3,4,5) phosphate (PI(3,4,5)P3) 5‐phosphatase‐2 (INPPL1) is a direct target of miR‐2137, which controls PI(3,4,5)P3 levels in macrophages. The modulation of cellular PI(3,4,5)P3/PIP2 ratio and anabolic mTOR signaling by the IRE1‐induced miR‐2137 demonstrates how the ER can provide a critical input into cell growth decisions.
Open Access
Modulation of the neuroinflammatory response following genetic and environmental manipulations in the zebrafish (Danio Rerio)
(2022-05) Özen, Beyza
Aging is an inevitable process through which organisms experience functional and physical decline. Cellular changes such as mitochondrial dysfunction, telomere attrition, and loss of proteostasis constitute the main components of this process. One of the hallmarks of brain aging is the increased inflammatory status of the brain. This process is named neuroinflammation and is seen both in the development of neurodegenerative disorders and in healthy aging. The over-activation of microglia and astrocytes, increased secretion of pro-inflammatory cytokines and reactive oxygen species, NLRP3/NALP3 inflammasome activation, and the upregulation of NF-κB signaling pathway are among the markers of neuroinflammation. Deregulated nutrient sensing through mammalian target of rapamycin pathway(mTOR), impaired neurogenesis, and synaptic integrity over time are among the common outcomes of this process. Genetic susceptibility is also another factor to contribute the vulnerability to inflammation. Therefore, further studies are necessary to investigate the effects of the inflammation-stimulating agents and the genetic susceptibility. Thus, this study aimed to develop copper sulfate as an inflammatory stimulating agent for both zebrafish embryos and adults. For this objective, we conducted long-term and short-term copper sulfate embryo treatment studies. The gene expression results showed that the inflammatory response was quite predictable in embryos by increasing pro-inflammatory markers early on and later increasing anti-inflammatory cytokines. Secondly, we conducted copper sulfate and rapamycin treatment in very old (38 months) zebrafish animals to investigate the impact of the inflammation on mTOR signaling and synaptic integrity. The protein expression results indicated that rapamycin was an effective for mTOR suppression in very old animals but copper sulfate was not able to stimulate a robust inflammatory response. Also, synaptic integrity markers were mostly stable among treatment groups in very old animals. Finally, we used tsc2+/- adult animals and applied rapamycin treatment to very old (33 months) tsc2+/- adults to assess the effect of overactive mTOR signaling and the possibility of reversing this in the progression of inflammation. Comparatively, we wanted to understand the effect of copper sulfate exposure in very old (43 months) ztor+/- animals that have a downregulated mTOR pathway. The results showed that the rapamycin effect was not significant between wild-type and tsc2+/- animals in terms of pro-inflammatory cytokines and autophagy markers. Similarly, the copper sulfate effect was not different between wild type and ztor+/- animals for pro-inflammatory markers. However, autophagy markers decreased significantly in mutants. In conclusion, this study showed that aging affects the regulation of the inflammatory response within the brain. Also, genetic manipulations on the mTOR pathway would provide crucial insights to investigate the neuroinflammatory profile of the brain in the course of aging.