Theses supervised by Prof. Dr. Mıchelle Marıe Adams
12 theses · İhsan Doğramacı Bilkent University
Fare serebral korteksinde hiperlipidemi kaynaklı organel stresini ve nöroinflamasyonunun incelenmesi: Perk yolağına müdahaleye ilişkin anlayışlar
Deficits in the metabolism of lipids called hyperlipidemia have been linked to a higher risk of developing neurodegenerative diseases. Protein Kinase RNA-like Endoplasmic Reticulum Kinase (PERK) signaling is crucial in cellular homeostasis. Abnormalities in the PERK have been associated with neurodegeneration. Mitophagy and the PERK pathway emphasize how cellular stress responses are regulated to preserve cellular homeostasis and mitochondrial quality control. The activity of main mitophagy regulators, such as Parkin and PINK1 (PTEN-induced kinase 1), is regulated by the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) by PERK. If lipid metabolism is at a high level, abnormalities in the mitochondria and endoplasmic stress (ER) emerge. During the ER stress activation, the PERK pathway is induced, and mitophagy is blocked, causing an enhancement in the neuroinflammation. The underlying molecular mechanism by which hyperlipidemia impacts the PERK pathway and mitophagy in the cerebral cortex, as well as the relationship between mitophagy and neuroinflammation, is not fully understood. In this study, Apoe-/- and C57BL/6 mice were given a chow or western diet to stimulate hyperlipidemia. Moreover, western diet-fed Apoe-/- mice were injected with PERK inhibitors, GSK2606414 and Trans-ISRIB, intraperitoneally for six weeks to suppress the PERK pathway. This study explores the effects of hyperlipidemia on the PERK pathway, inflammatory and mitophagy markers in the cerebral cortex of chow and western diet-fed C57BL/6 and Apoe-/- mice and investigates whether the inhibition of the PERK pathway can change the levels of inflammatory and mitochondrial markers in the cerebral cortex of hyperlipidemic mice subjects. mRNA and protein expression levels of mitophagy and inflammatory markers were assessed using the RT-qPCR and western blot, respectively. PERK pathway activation under hyperlipidemia conditions was not determined. Nevertheless, significant alterations in mitophagy markers and inflammation were detected in Apoe-/- mice apart from the diet. Furthermore, significant alterations were not seen in the PERK pathway markers; however, mitophagy was stimulated, and some inflammation markers were significantly decreased mildly at the cortical tissue of WD-fed Apoe-/- mice administrated with PERK pathway inhibitors, GSK2606414 and Trans-ISRIB. Besides, no statistically significant changes were observed in the transcript levels of the inflammatory markers. Taken together, hyperlipidemia did not cause the PERK pathway to be activated in the cerebral cortex of mice; nevertheless, it mildly altered inflammation and caused mild effects of the dysregulation of the mitochondria by hyperlipidemia independent from the PERK pathway. Furthermore, although the PERK pathway was not inhibited by the administration of PERK pathway inhibitors, mitophagy was induced, and inflammation was decreased mildly. Targeting the PERK pathway with GSK2606414 and Trans-ISRIB inhibitors from the cerebral cortex would not be a therapeutic approach for neurodegenerative diseases.
Yüksek yağlı diyet ile genotipin farenin serebral korteksinde kan-beyin bariyeri ve sinaptik bütünlüğe etkileri: PERK yolağının araştırılması
High-fat diet intake can induce hyperlipidemia and result in cognitive decline by causing endoplasmic reticulum stress, decreased blood-brain barrier, and synaptic integrity. The protein kinase R-like endoplasmic reticulum kinase (PERK) pathway is one of the arms of the unfolded protein response, which is activated by endoplasmic reticulum stress. The PERK inhibits the global protein translation while allowing the translation of certain proteins that are involved in inflammation and apoptosis. Due to its apoptotic properties, it is thought that the PERK pathway causes neurodegeneration. To study the effects of hyperlipidemia, a high-fat diet-fed Apoe knock-out mice model (Apoe-/-) is appropriate. Knocking out the Apoe in mice makes the animal model more prone to high-fat diet-induced hyperlipidemia. In the cerebral cortex of these animals, endoplasmic reticulum stress, blood-brain barrier, and synaptic integrity markers are checked at protein and mRNA levels. No changes are observed in the PERK pathway markers besides phosphorylated eukaryotic Initiation Factor 2a. Additionally, there is a significant increase in blood-brain barrier marker Claudin-5 levels in Apoe-/- mice fed with a high-fat diet. There is also no significant change in synaptic integrity markers. In the second part, the effects of the PERK pathway inhibition are checked with integrated stress response inhibitor and GSK2606414 in the high-fat diet-fed Apoe-/- mice cerebral cortex. There are no significant alterations in BBB and synaptic integrity when the animals are injected with inhibitors. In conclusion, this study investigates the effects of high-fat diet induced hyperlipidemia in the cerebral cortex of Apoe-/- mice on ER stress, blood-brain barrier, and synaptic integrity. In the cerebral cortex region, the PERK pathway-related ER stress is not observed, and synaptic integrity remained unchanged while the blood-brain barrier is affected. Moreover, the effects of the PERK pathway inhibition are researched, and there is no inhibition effect observed in the cerebral cortex region.
Dss kaynakli er stresin ire1 ve jnk sinyal yolaklarinin fare serebral korteksinde incelenmesi̇
Endoplasmic reticulum (ER) stress plays a critical role in cellular homeostasis and is implicated in various neurodegenerative diseases. The inositol-requiring enzyme 1 (IRE1) is one of the arms of the unfolded protein response (UPR), which is activated in the presence of ER stress. IRE1 pathway activates c-Jun Nterminal kinase (JNK) downstream under prolonged stress. This thesis explores the activation of the IRE1 and JNK signaling pathways in the mouse cerebral cortex following DSS-induced colitis, focusing on their roles as markers of ER stress in the context of the gut-brain axis. While DSS-induced intestinal inflammation and ER stress are well-established, neurological effects remain less understood. Employing a murine model, the study explored ER stress markers in the cerebral cortex resulting from intestinal pathology. Despite evidence of DSS-triggered systemic inflammation and ER stress in intestinal tissues, our study revealed no significant differences in the expression levels of IRE1, p-IRE1, or the p-IRE1/IRE1, nor in JNK, p-JNK, or the p-JNK/JNK between the control and DSS-treated groups. Additionally, these results are supported with correlational and linear discriminant analyses (LDA). These findings suggest that acute DSS-induced colitis did not elicit a detectable ER stress response in the mouse cerebral cortex under the conditions used. Possible explanations include tissue-specific reactions to the ER stress, transient activation of the IRE1-JNK pathway that returned to baseline by the time of analysis, or potential survivor's bias. Despite its limitations, this thesis provides a novel investigation into the effects of DSS-induced colitis on ER stress in the cerebral cortex.
Sacsin'in glial ara filament organizasyonu ve inflamatuar sinyal yolağındaki rolünün erken dönem zebrafish gelişiminde araştırılması
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare neurodegenerative disorder characterized by early-onset ataxia, spasticity, and peripheral neuropathy. While ARSACS has been primarily studied in neurons, recent findings implicate glial cells in disease pathology. Sacsin, the protein encoded by the SACS gene and mutated in ARSACS, is shown to be expressed in both astrocytes and microglia while playing key roles in cytoskeletal integrity and cellular stress signaling. When adding the growing recognition of ARSACS as a disorder with neurodevelopmental components, investigating glial contributions to the disease pathogenesis during early development is becoming a requirement. This study aimed to investigate the impact of sacsin deletion on developmental and glial markers in zebrafish embryos and larvae, using a sacs-null mutant model of ARSACS. To this end, we quantified gene and protein expression profiles of sacsin and key glial markers across developmental time points (60–180 hours post-fertilization, hpf). RT-qPCR was used to assess the expression of sacs, s100b (astrocytic inflammatory marker), and aif1l (microglial inflammatory marker IBA-1), while Western blotting was used to examine protein levels of STAT3 (developmental marker), vimentin (early glial intermediate filament marker), and GFAP (astrocytic intermediate filament marker). Zebrafish sacs-null mutants showed a significant decrease in sacs mRNA expression only at 180 hpf, while the lack of earlier differences is possibly due to the stable mutant transcripts. Among glial inflammatory markers, s100b expression was significantly upregulated in sacs-null mutants; however, aif1l showed no genotype-dependent changes, reflecting a compensatory response of astrocytes that may even occur earlier than the microglial response. Additionally, no significant correlations were found between sacs and these glial inflammatory markers, indicating selective, possibly indirect, molecular interactions. Vimentin protein expression was significantly higher in sacs-null mutants, supporting the notion that sacsin loss disrupts intermediate filament dynamics, not just structurally, but in quantity. However, GFAP expression did not differ between genotypes, unlike the literature based on cellular imaging, suggesting the necessity of anatomical tissue-specific assays. Together, these results demonstrate that sacsin loss leads to selective and temporally defined alterations in glial development, particularly affecting cytoskeletal and inflammatory markers. The zebrafish model effectively captured these dynamics, validating its utility for studying glial contributions to early-onset neurodegenerative diseases. In conclusion, this study advances our understanding of glial dysregulation in ARSACS and underscores the importance of considering non-neuronal mechanisms in early disease progression. In future studies, tissue-specific analyses based on the imaging of glial cells in the central nervous system will be of critical importance for uncovering the molecular foundations of glial involvement in ARSACS and related disorders.
Zebrabalığı (Danıo rerio) modelinde yetişkin nörogenezi ve astositlerin yaşlanmaya bağlı değişimleri
Brain aging is marked by a decline in cognitive abilities and associated with neurodegenerative disorders. In order to identify appropriate interventions to change the course of brain aging and age-related neurological disorders, we should first understand the normal age-related changes. Previous studies claimed that there was a correlation between cognitive capacities and number of neurons. However, recent studies have shown no statistically significant change in total neuron number during healthy aging. Therefore, further studies are required to understand the reasons behind these changes in the brain. One possibility could be the age-related alterations in neuronal lineage and glial markers. Thus, this study aims to show the protein levels, distributions, and localizations of key neuronal lineage and glial markers, which include neural progenitor, early neuronal, immature neuron, and mature neuron and glial markers during healthy aging of the zebrafish brain. For this aim, we measured NeuN (Fox-3, Rbfox3, or Hexaribonucleotide Binding Protein-3), MAP-2 (Microtubule-associated protein 2), HuC (ELAV like neuron-specific RNA binding protein 3), DCAMKL-1 (Doublecortin-like kinase 1), and GFAP (Glial fibrillary acidic protein) with immunohistochemistry and western blot techniques. First, the immunohistochemistry technique was applied on two specific proliferation areas, pallium and optic tectum, to detect the changes in the number of neuronal lineages and glial marker. The results indicated no statistically significant changes between young and old groups. Secondly, we performed whole-brain immunohistochemistry of all markers and quantified every image by manually counting the positive signal. We found that aging did not have an effect on the distribution and expression of the markers, even in the whole brain. Finally, Western-blot was performed in whole brain lysates to compare neuron number and protein level changes. Western-blot results indicated an age-related statistically significant decline in immature neuron marker for specifically males and glial marker for specifically females. The protein level of neural progenitor marker showed the significant decline in males during aging but no change between two age groups. Results of the mature neuron antibody revealed that the protein levels were consistent through aging and did not show variation. Our results overall support the finding that the number of neurons and glia do not change during aging since the numbers of markers were not show statistically significant changes during the aging process in the proliferation areas of the zebrafish brain. However, protein levels showed changes between age and gender groups. Thus, this study shows that understanding changes in the number of cells need to count; protein level is not representative, and zebrafish is an appropriate model for brain aging studies.
Kısa süreli aralı oruç ve rapamisin uygulamalarının zebra balığı (danio rerio) beynindeki yaşa bağlı etkileri
World populations are rapidly aging, and there is an urgent need to develop interventions that prevent or reverse age-related deterioration of health. To date, several approaches have been developed to extend health span. Among these, non-genetic interventions have a higher potential to be utilized in translational studies. Caloric restriction (CR) and its pharmacological mimetic rapamycin, are two applications that have been shown to reliably extend life and health span across species. Despite a growing body of knowledge on how CR and rapamycin show their beneficial effects, their molecular mechanisms in the brain are not completely understood. Furthermore, most studies applied life-long CR, which is not suitable for translational research. To fill this gap, we investigated whether short-term durations of a CR approach intermittent fasting (IF) or rapamycin altered cellular and molecular markers of critical processes in the brain as well as metabolic parameters in the body. To assess how the age of the subjects affect the outcome of the treatments, we included young (6-10 months old) and old (26-31 months) zebrafish, which has recently emerged as a suitable model for gerontological research. Our results demonstrated that IF decreased whole-body glucose and cortisol levels, and increased neural progenitor marker DCAMKL1 in young and old animals. While this proliferation-promoting effect was preceded by suppression of mTOR activity in young, the upregulation of foxm1 and reduced autophagic flux as measured by LC3-II/LC3-I ratio were observed in old animals. Rapamycin, on the other hand, did not alter the metabolic parameters and induced entirely different molecular profiles at young and old ages. The most notable changes in young animals were reduced mTOR activity, LC3-II/LC3-I ratio and expression levels of a global proliferation marker PCNA. In old animals, the marker of activated astrocytes (i.e. GFAP) was decreased, indicating lower neuroinflammation, whereas excitatory-inhibitory balance as measured by PSD-95/Gephyrin ratio was shifted towards a more excitatory state. These results suggested that IF and rapamycin induced distinct metabolic profiles in young and old animals. Furthermore, there was an age-dependent reciprocal relationship between proliferation and autophagy, which might be partly due to differential regulation of mTOR activity. Interestingly, rapamycin treatment was more effective in suppressing mTOR activity in young animals, and compared to IF. Nevertheless, these results suggested that rapamycin crosses the blood-brain barrier in zebrafish, and that short-term durations of IF or rapamycin were sufficient to alter the expression levels of key proteins involved in critical mechanisms in the brain.
Zebrabalığı (danio rerio) beyninde yaşlanma, diyet ve olası genetik müdahalelerin SMURF2 ve etkileşim ortaklarının ifadesine etkileri
Aging is a natural process that is ultimate combination of numerous intrinsic and extrinsic changes in an organism. Contrary the common belief, brain aging is not a loss of neurons while it has been shown that subtle cellular and synaptic alterations have contribution to brain aging. Therefore, the molecular and cellular alterations may give more insight into the brain aging process. There are some hallmarks of aging that are common features in different organisms including genomic instability, telomere attrition, cellular senescence. There are some common factors with the ability to regulate more than one of the hallmarks of aging such as Smurf2. HECT-domain E3 ubiquitin ligase Smurf2 has several roles in the cellular processes for example, telomere attrition and cellular senescence. Moreover, its gene expression is higher in the aged brain. Although there are several publications about Smurf2, most of them focused on its role in cancer. We believed that Smurf2 levels should be examined in terms of brain aging. The first aim of the study was to examine the levels of Smurf2 and its interacting partners across lifespan. Although the Smurf2 protein level was not increased significantly in the whole zebrafish brain, its protein level was upregulated significantly in telencephalon and cerebellum. Also, subcellular protein fractionation demonstrated an enriched Smurf2 level in the cytosolic part. In the case of gene expression levels, smurf2 level was significantly higher in aged whole brain although its expression was downregulated during aging in telencephalon and cerebellum. In addition, the levels of mdm2, ep300a and sirt1 were lower in the aged telencephalon. According to multivariate analysis there is a potential balance between Smurf2-mediated ubiquitination, ep300a-mediated acetylation and Sirt1-mediated deacetylation but with advancing age, this balance may disrupt and other regulatory genes should also take a role to sustain cellular stability. The second aim was to investigate the roles of Smurf2 on brain aging with the help of genetic interventions including inducible knockin, stable knockout or transient knockdown. Since stable knockin and knockout models should be genotyped before further investigations, the genotyping and phenotyping methods were employed to find an efficient and reliable way. Also, transient knockdown via Vivo-morpholino was applied to adult brain and efficient post injection times of two different morpholinos were identified in order to examine the effects of Smurf2 knockdown in both young and old zebrafish. Lastly, it was aimed to examine the effects of non-genetic interventions including dietary regimens and pharmacological compounds on the gene expression of smurf2 and its interacting partners and the levels of the neuronal proteins and proliferation/senescence proteins. The opposing short-term dietary regimens, overfeeding and caloric restriction, were altered the levels of neuronal proteins, HuC and DCAMKL1, and their relation with proliferation and senescence proteins during aging. Also, the gene expression levels of smurf2 and interacting partners except tp53 was not influenced by dietary regimens and aging in terms of whole brain. Also, multivariate analysis indicated that the correlations among smurf2, mdm2, ep300a and sirt1 were conserved in both young and old ages independent to dietary regimen which may imply that the balance between ubiquitination, acetylation and deacetylation is maintained in order to provide cellular stability during aging. Heclin, an inhibitor of HECT E3 ligases, were employed to inhibit Smurf2 activity. Before using in adult zebrafish, heclin was applied to embryos to see its effects. The higher dose of heclin decreased the survival ratio and altered the gene expression levels of downstream gene drastically. So, moderate dose of heclin should be applied to the adult brain and neuronal markers should be examined to observe target effects rather than off-target, unspecific impacts. Taken together, Smurf2 has potential roles during aging and it could be a promising target to delay the brain aging process and probably the onset of age-related cognitive decline.
Zebra balığı (Danio rerio) model organizması kullanılarak yaşlanmanın beyindeki inflamatuar, hücre iskeleti, ve mikroglia belirteçlerindeki gen ifade düzeyleri üzerine etkileri
Age-related cognitive decline burdens the elderly population, limiting their ability to socialize and be independent. To be able to develop proper treatments, healthy aging should be examined. Previous studies focusing on healthy brain aging revealed that abnormal microglial activation was observed. Aging microglia exhibits 0partial loss of motility due to cytoskeletal changes, leading to decreases in their ability to respond to environmental cues. Thus, a more inflammatory phenotype was observed in microglia. These disruptions of the previously established homeostasis in the brain could be the underlying reason for cognitive decline experienced during aging. To understand these changes during aging in the brain, cytoskeletal, microglial, and inflammation-related markers were investigated by using both in silico and in vivo approaches. In silico analyses were performed on mice hippocampus and the whole brain revealed that the genes involved in the actin cytoskeleton reorganization (Arpc1b), neurogenesis (Erbb4), and proinflammatory related pathways (Il1b, P2x7r, Elf2b) showed differential gene expression levels among different age groups, genders, and tissue of origin. On the other hand, no differential expression was observed in microglial (Coro1a and Aif1) and anti-inflammatory markers (Tgfb1 and Il10). To further validate these results in vivo, quantitative polymerase chain reaction (qPCR) was performed on young and old zebrafish brains. According to the results, only two genes showed marginally significant differences among young and old brains: arpc1b and p2x7r. These results collectively could mean 1) the overall microglia population does not change during aging, 2) the brain does not exhibit imbalances in terms of pro- and anti-inflammatory cytokines, and 3) neurogenesis. Furthermore, the significant changes observed in arpc1b and p2x7r indicated the iii iv importance of the cytoskeleton and inflammation-related pathways in the correct functioning of the cells. Therefore, this study showed that in silico analysis are the reliable indicators of in vivo experiments, zebrafish can be used as a gerontological model, and the importance of cytoskeleton in motile cells. However, to understand these described relations, further investigation on the protein level of these genes should be done.
Diyet ve diyet-mimetiği manipülasyonların gen anlatımı açısından yaşlanan beyne etkileri
Aside from many genetic and environmental influences on the brain, aging itself is a significant risk factor for accelerated cognitive decline, making aging research crucial due to the increasing population age in our era. We aimed to discover gene expression differences in the aging zebrafish brain using three age groups in the first aim. We identified gjc2 (CX47) and alcamb (ALCAM) cell adhesion genes showing consistent downregulation with age across all experiments. ALCAM is also known to be associated with neuroinflammation, which has been implicated to be lowered using anti-aging, non-genetic nutrient interventions. In the second aim, we applied 12 weeks of two opposing nutrient interventions, caloric restriction (CR) and overfeeding (OF) in aging zebrafish, in order to be able to propose a reliable therapeutic approach for reversing age-related neurobiological changes. We measured protein and expression level differences of selected genes related to proliferation to inflammation with these diets. The results showed that sox2 gene expression was significantly upregulated following OF treatment than CR diet, and myca and tp53 mRNA levels were significantly downregulated with advanced age. Alcamb and tfdp1 expression levels were also marginally significantly lowered with CR compared to other groups. Meanwhile, we also conducted another transcriptomic approach using microarray to assess gene expression differences with CR compared to Ad-libitum (AL) feeding. Thus, lastly, in the third part, we found that CR causes changes in cell cycle regulation among several other functional regulatory pathways in zebrafish brains. We identified the tfdp1 gene, which showed downregulation with CR, as a possible CR regulator. Then, to create a CR mimic, we performed morpholino oligo (MO) injections to zebrafish embryos and adult brains to knock down tfdp1 gene expression levels. The injections were not successful in altering Tfdp1 protein levels in neither embryos and adults. However, 8ng tfdp1-MO injections in embryos significantly increased myca and tp53 expression levels, which are among the downstream targets of tfdp1. Our examinations shed light on healthy brain aging and possibly propose new drug targets.
Zebra balığında (Danio rerio) genetik ve çevresel manipülasyonların ardından nöroinflamatuar yanıtın modülasyonu
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 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.
Yaşlanan beyinde inflamasyon sürecinin etkisi: In vıtro ve ex vıvo modellerinden elde edilen kanıtlar
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.
Beslenme şekillerinin zebra balığı beyin yaşlanmasında sox2 protein seviyesi ve yetişkin nörogenez üzerine etkisi
Aging in humans is described as the gradual decline in physiological functions due to alterations in complex biological processes. As the lifespan increases and the population shifts towards older age, health problems associated with age become a worldwide problem. Thus, understanding the molecular and cellular changes that contribute to the aging process is essential to ensure healthy aging. Nine hallmarks that describe these changes were identified, which are also observed in the aging brain, leading to structural and cognitive deficiencies. Stem cell exhaustion is one of these hallmarks that explains a loss of stem cell function due to brain aging. A transcription factor, Sox2 is one of the main responsible proteins ensuring the stem cell maintenance through regulation of self renewal and differentiation. Age-related changes in neural stem cells (NSCs) mediated by Sox2 can lead to a decline in new neuron formation. Neurogenesis is proven to continue in the defined neurogenic niches in the mammalian brain throughout adulthood and is considered to have a crucial role in the healthy functioning of the brain during aging. The regulation of the NSCs in neurogenic niches is achieved through diverse intrinsic and extrinsic factors that are affected by age-related changes. Dietary interventions are thought to modulate these factors and improve the age-related decline. Previous studies show that dietary restriction through lowering the calorie intake improves age-related impairment on neurogenesis, while high-calorie intake has a negative effect. Zebrafish, a small teleost fish, is a highly suitable model organism for investigations on neurogenesis with respect to aging due to exhibiting gradual decline with age, similar to humans and having a high capacity of neurogenesis in a widespread area. Hence, in the first part of the study, the influence of diet on age-associated changes in the brain was observed by following two short-term opposing dietary interventions. Using zebrafish as a model organism, dietary restriction (DR), over-feeding (OF), and ad libitum (AL) diets were included. These interventions demonstrated that short-term DR, compared to the AL, downregulated Sox2, as evidenced by western blot analysis. This might indicate a decreased self-renewal properties and activation of differentiation of NSCs. Short-term OF, on the other hand, did not change its expression levels. Moreover, aging did not alter the Sox2 expression levels. Further correlational and multivariate analyses were performed combining Sox2 with a proliferation marker proliferating cell nuclear antigen (PCNA) and neuronal lineage markers doublecortin-like kinase 1a (DCAMKL1) and ELAV-like neuron-specific RNA binding protein 3 (HuC). The analysis demonstrated a positive relationship between Sox2 and PCNA indicating their similar pattern in the aging process. Also, DCAMKL1 was shown to have a positive relationship with PCNA and a negative relationship with HuC. The multivariate analyses of all datasets exhibited age-specific effects of OF and DR by decreasing the survival of new neurons in old animals. Also, OF reinforced the commitment to neuronal fate with old age. The second part demonstrated a recombinant protein purification approach using immobilized-metal affinity chromatography to purify the His-tagged Sox2 protein. The recombinant Sox2 protein was successfully purified, and concentration values were measured. Taken together, these findings show that although older age led to changing dynamics in the neuronal lineage in the zebrafish brain, Sox2 managed to show a persistent expression. Furthermore, the short-term DR was shown to change the Sox2 expression levels, indicating the importance of calorie intake in alterations in neural stem cell properties. In conclusion, this study makes contributions to understanding cellular and biochemical changes occurring in the neurogenic niches of the aging brain and the altered neurogenic capacity due to dietary interventions.