Browsing by Subject "Zebrafish"

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Open Access
Age and gender alter synaptic proteins in zebrafish (Danio Rerio) models of normal and delayed aging
(2017-07) Karoğlu, Elif Tuğçe
Cognitive decline occurs during normal aging in some specific domains of cognitive abilities including but not limited to episodic memory, divided attention and executive functions, however, it is not a unitary decline since some cognitive domains, including vocabulary and implicit memory tend to be preserved and even improved at older ages. Normal aging is not associated with global and significant neuronal and synapse loss, yet subtle molecular alterations occurring in gene expression patterns, protein homeostasis, mitochondrial dynamics and hypofunction in the cholinergic system may account for the age related decline in some cognitive abilities. Additionally, males and females showed differential vulnerabilities against age-related alterations in the cognitive abilities, physiological integrity and subtle molecular dynamics. More direct relationships can be established between the age-related cognitive decline and subtle molecular changes by analyzing the elements of synaptic integrity, which could alter synaptic plasticity and result in the changes in learning and memory abilities. Post-synaptic 95 (PSD-95), gephyrin (GEP) and synaptophysin (SYP) are integral synaptic proteins and they could be attributed as indicators of excitatory post-synaptic, inhibitory post-synaptic and pre-synaptic integrities, respectively. The first aim of this study was to show effects of age and gender on the expression levels of PSD-95, GEP and SYP in young, middle-aged and old, female and male zebrafish cohorts. Significant age by gender interactions were revealed in the levels of PSD-95 and SYP. It was shown that PSD-95 and SYP levels tend to be preserved and increased in the female groups throughout the aging process, whereas, in male groups, expression levels of these proteins tend to be reduced at older ages. The second aim was to investigate whether ameliorating the cholinergic hypofunction might have beneficial effects on the aging-related protein expression alterations and check for sexually dimorphic patterns. For this aim old male and female zebrafish from a mutant line (ache), which has decreased levels of acetylcholinesterase and increased levels of acetylcholine, were compared with old male and female wildtype animals. In the ache old groups, significant increases in the expression levels of SYP and GEP were revealed compared to the wildtype, and also in the old ache females SYP expression was higher than the other groups. These studies emphasized the importance of gender and sexually dimorphic patterns in the context of aging andcholinergic manipulations could be a promising target of intervention to attenuate the effects of age-related synaptic alterations, which could have possible contributions to age-related cognitive decline. .
Open Access
Age related alterations of adult neurogenesis and astrocytes in Zebrafish (Danio Rerio)
(2019-09) Ardıç, Narin Ilgım
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.
Open Access
Age-dependent effects of short-term intermittent fasting and rapamycin treatment in Zebrafish (Danio Rerio) brain
(2020-05) Birand, Ergül Dilan Çelebi
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.
Open Access
Aging alters the molecular dynamics of synapses in a sexually dimorphic pattern in zebrafish (Danio rerio)
(Elsevier, 2017-06) Karoglu, Elif Tugce; Halim, Dilara Ozge; Erkaya, Bahriye; Altaytas, Ferda; Arslan-Ergul, Ayca; Konu, Ozlen; Adams, Michelle M.
The zebrafish has become a popular model for studying normal brain aging due to its large fecundity, conserved genome, and available genetic tools; but little data exists about neurobiological age-related alterations. The current study tested the hypothesis of an association between brain aging and synaptic protein loss across males and females. Western blot analysis of synaptophysin (SYP), a presynaptic vesicle protein, and postsynaptic density-95 (PSD-95) and gephyrin (GEP), excitatory and inhibitory postsynaptic receptor-clustering proteins, respectively, was performed in young, middle-aged, and old male and female zebrafish (Danio rerio) brains. Univariate and multivariate analyses demonstrated that PSD-95 significantly increased in aged females and SYP significantly decreased in males, but GEP was stable. Thus, these key synaptic proteins vary across age in a sexually dimorphic manner, which has been observed in other species, and these consequences may represent selective vulnerabilities for aged males and females. These data expand our knowledge of normal aging in zebrafish, as well as further establish this model as an appropriate one for examining human brain aging.
Open Access
Aging, neurogenesis, and caloric restriction in different model organisms
(International Society on Aging and Disease, 2013) Ergul, A. A.; Ozdemir A.T.; Adams, M. M.
Brain aging is a multifactorial process that is occurring across multiple cognitive domains. A significant complaint that occurs in the elderly is a decrement in learning and memory ability. Both rodents and zebrafish exhibit a similar problem with memory during aging. The neurobiological changes that underlie this cognitive decline are complex and undoubtedly influenced by many factors. Alterations in the birth of new neurons and neuron turnover may contribute to age-related cognitive problems. Caloric restriction is the only non-genetic intervention that reliably increases life span and healthspan across multiple organisms although the molecular mechanisms are not well-understood. Recently the zebrafish has become a popular model organism for understanding the neurobiological consequences but to date very little work has been performed. Similarly, few studies have examined the effects of dietary restriction in zebrafish. Here we review the literature related to memory decline, neurogenesis, and caloric restriction across model organisms and suggest that zebrafish has the potential to be an important animal model for understanding the complex interactions between age, neurobiological changes in the brain, and dietary regimens or their mimetics as interventions.
Open Access
Alterations in the molecular properties of neural stem cells from aged brains and brain tumors
(2017-06) Burhan, Özge Pelin
It is known that new neuron formation in the brain continues throughout the life of an organism. In the adult human brain, it was proven that neurogenesis in the hippocampus is higher than expected, almost 700 new neurons are formed in a day. The formation of new neurons is supported by the stem cell subpopulation in the brain. With learning and the formation of new memories, the neuron production increases. However, changes in the cognitive abilities with advancing age are thought to be caused by the functional and molecular alterations in the stem cell populations. Molecular changes in neural stem cells throughout aging were found to be deterrents of the increased risk of cancer with age, such as tumor suppressor mechanisms. However, the activation and overlap of tumor suppressing mechanisms result in senescence in stem cells that have accumulated oncogenic mutations, which causes the stem cell pool exhaustion. It is thought that cancer cells acquire stem cell-like properties in order to have the unlimited proliferation and self-renewal properties, which are characteristics of both healthy and cancer stem cells. Neural cancer stem cells have the ability to produce glial and neural cells, like normal stem cells. The cancer stem cell subpopulations are implicated in the growth of tumor tissues. Hence, it is important to identify and characterize cancer stem cells and make a distinction between cancer and non-cancer stem cells. In this project, this issue was addressed by studying the marker expressions of brain tumor tissues obtained from humans, which confirmed that the cancer cells do express stem cell and progenitor cell markers, such as Sox2 and Vimentin. The presence of mature neurons was also established by the mature neuronal marker NeuN. In order to determine whether these stem cells may be different in young and old subjects, a study was also carried out in young and old zebrafish neural stem cells in order to identify the expression differences between the groups. The presence of proliferating stem cells and differentiated cells were identified in cell culture. This analysis of neural stem cells in old and young zebrafish revealed 18 differentially-expressed genes. The results indicated a higher differentiation rate in old zebrafish stem cells, which may be due to the increased loss of neural cells in the old zebrafish brain. The development of markers that could be widely used for the diagnosis of cancer and the identification of cell types is important. For reliable diagnosis and identification of cancer cells, multiple cellular markers are used. Hence the distinction of cell types based on light scattering differences would speed up the process of diagnosis, and the elimination of marker used for the distinction of cell types would be beneficial. The final project mentioned in this thesis involves the analysis of C6 (rat glioma) cell line for scattering properties and cell cycle arrest. A general method for definition of a scatter data interval for C6 cells in different stages was developed and can be applied to other cell types and diseases. These studies show that the proliferation and stem cell markers’ expressions differ between cancer and healthy stem cells, and the expression of neuroprotective genes is differentially upregulated in old zebrafish neural stem cells compared to the young. This data could contribute to the knowledge on normal and cancer stem cell expression differences, as well as how age affects the expression, and supply information required for the development of a cancer stem cell identification and targeting methods.
Open Access
Caloric restriction reinforces the stem cell pool in the aged brain without affecting overall proliferation status
(Elsevier BV, 2022-11-01) Erbaba, Begün; Macaroğlu, Duygu; Avcı, N. İlgim Ardıç; Ergül , Ayça Arslan; Adams, Michelle M.
Overfeeding (OF) and obesity increase the risk for brain aging and neurodegenerative diseases due to increased oxidative stress and neuroinflammation, which likely contribute to cellular dysfunction. In contrast, caloric restriction (CR) is an intervention known for its effects on extending both life- and health-span. In the current study, the effects on the aging brain of two short-term feeding regimens, OF and CR, were investigated. We applied these diets for 12 weeks to both young and aged zebrafish. We performed protein and mRNA level analysis to examine diet-mediated effects on any potential age-related alterations in the brain. Markers implicated in the regulation of brain aging, cell cycle, proliferation, inflammation, and cytoskeleton were analyzed. The most prominent result observed was a downregulation in the expression levels of the stem cell marker, Sox2, in CR-fed animals as compared to OF-fed fish. Furthermore, our data highlighted significant age-related downregulations in Tp53, Myca, and L-plastin levels. The multivariate analyses of all datasets suggested that as opposed to OF, the adaptive mechanisms increasing lifespan via CR are likely exerting their effects by reinforcing the stem cell pool and downregulating inflammation. The data reveal important therapeutic targets with respect to the state of nutrient uptake for the slowing down of the detrimental effects of aging, resulting in a healthy and extended lifespan, as well as lowering the risk for neurodegenerative disease.
Open Access
The characterization and potential functional role of wdr81, a novel zebrafish gene, associated with cerebellar ataxia, mental retardation and dysequilibrium syndrome (camrq) in humans
(2016-04) Doldur Ballı, Füsun
Cerebellar ataxia, mental retardation and dysequilibrium syndrome (CAMRQ) is a neurodevelopmental disorder. The gene encoding WD repeat containing protein 81 (WDR81) was reported to be associated with CAMRQ2 [MIM 610185]. Human and mouse studies indicated the potential importance of WDR81 in neurodevelopment. The first aim in this study was to characterize the transcript and to reveal the expression profile of wdr81 in zebrafish. The second aim was to perform the initial characterization of wdr81 morphants. In silico analysis indicated that the conserved domains are shared in human, mouse and zebrafish orthologous proteins, implying a conserved function of WDR81 in three species. The characterization of the transcript revealed that wdr81 possessed one ORF and one 5’UTR structure. The predicted sequence for 3’UTR was confirmed along with detection of some variants and an insertion site in samples from ten developmental timepoints and in several adult tissues. This region was not detected in kidney, intestine and gills, which might be pointing out an alternative polyadenylation event. wdr81 appeared to be maternally supplied. 5 hpf and 18 hpf were detected as crucial timepoints regarding wdr81 expression. Expression of wdr81 was found to be increased in the eye and brain regions at 18 hpf and 48 hpf. wdr81 was found to be ubiquitously expressed in the adult zebrafish. The expression of wdr81 in the adult brain and eye was detected in several regions including retinal layers, presumptive Purkinje cells and some proliferative zones. The splice blocking morpholino which targets the exon 2-intron 2 junction of wdr81 worked at 3 tested doses; 2 ng, 4 ng and 8 ng. The effect of the wdr81 morpholino was detected to add the intron, which is downstream of the target exon, to the transcript and introduce a stop codon. Preliminary results indicated a significant reduction in the head sizes at a ratio of 3.88% (p:0.027) in the wdr81 morphant group compared to uninjected group and gbx2 expression was observed to be higher in wdr81 morphants compared to the control groups. In short, findings of this study emphasize the significance of wdr81 in neurodevelopment and suggest a potential role in neuronal proliferation. This study also serves as a basis for future functional studies.
Open Access
Characterization of a novel zebrafish (Danio rerio) gene, wdr81, associated with cerebellar ataxia, mental retardation and dysequilibrium syndrome (CAMRQ)
(BioMed Central Ltd., 2015) Doldur-Balli, F.; Ozel, M. N.; Gulsuner, S.; Tekinay, A. B.; Ozcelik, T.; Konu, O.; Adams, M. M.
Background: WDR81 (WD repeat-containing protein 81) is associated with cerebellar ataxia, mental retardation and disequilibrium syndrome (CAMRQ2, [MIM 610185]). Human and mouse studies suggest that it might be a gene of importance during neurodevelopment. This study aimed at fully characterizing the structure of the wdr81 transcript, detecting the possible transcript variants and revealing its expression profile in zebrafish, a powerful model organism for studying development and disease. Results: As expected in human and mouse orthologous proteins, zebrafish wdr81 is predicted to possess a BEACH (Beige and Chediak-Higashi) domain, a major facilitator superfamily domain and WD40-repeats, which indicates a conserved function in these species. We observed that zebrafish wdr81 encodes one open reading frame while the transcript has one 5' untranslated region (UTR) and the prediction of the 3' UTR was mainly confirmed along with a detected insertion site in the embryo and adult brain. This insertion site was also found in testis, heart, liver, eye, tail and muscle, however, there was no amplicon in kidney, intestine and gills, which might be the result of possible alternative polyadenylation processes among tissues. The 5 and 18 hpf were critical timepoints of development regarding wdr81 expression. Furthermore, the signal of the RNA probe was stronger in the eye and brain at 18 and 48 hpf, then decreased at 72 hpf. Finally, expression of wdr81 was detected in the adult brain and eye tissues, including but not restricted to photoreceptors of the retina, presumptive Purkinje cells and some neurogenic brains regions. Conclusions: Taken together these data emphasize the importance of this gene during neurodevelopment and a possible role for neuronal proliferation. Our data provide a basis for further studies to fully understand the function of wdr81.
Embargo
Development of a new Parkinson’s disease model using zebrafish to study alphasynuclein aggregation
(2024-08) Akış, Elif
Parkinson's Disease is a neurodegenerative disease mainly caused by the dopaminergic neuron loss in the CNS, specifically substantia nigra. It progresses through three stages: preclinical, prodromal, and clinical. Patients with Parkinson’s disease experience motor symptoms like tremors, postural problems and rigidity as well as non-motor symptoms such as autonomic dysfunction, sleep disorders, and depression. Various genetic and environmental factors influence the disease's progression, making classifying patients based on disease pathology challenging. The SNCA gene encodes the alpha-synuclein protein and is a primary risk factor for Parkinson’s disease. Mutant forms of alphasynuclein can form insoluble fibrils and Lewy bodies, affecting the transmission of healthy proteins between cells. Recent hypotheses suggest that the location of disease emergence in the body (brain-first or body-first) influences disease progression and resulting pathology. Existing zebrafish models have the potential to provide insights into neurodegenerative diseases due to their ease of handling, large population size, and genetic manipulability. This study aims to investigate the relationship between the location of alpha-synuclein emergence and the progression of Parkinson’s disease. In this study, a new zebrafish model expressing the human alpha-synuclein coding sequence was developed using the Tol2 transposase-based recombination system. The initial expression vector was created using multisite gateway cloning methodology, and the constructs were validated at each step using PCR and sequencing. The final construct was co-injected with transposase mRNA into one-cell stage zebrafish embryos to facilitate the formation of a stable line. In addition, human alpha-synuclein fibril injections were performed on young and old zebrafish, either brain or gut. This way, we ensured the transmission of α-syn between the central nervous system and peripheral organs.
Open Access
Development of a specialized zebrafish xenotransplantation database and establishment of ALU-based tumor DNA quantification method in zebrafish: focus on models of overexpression and microenvironment
(2020-09) Targen, Seniye
Successful xenotransplantation of human cancer cells into zebrafish host marked a new era in cancer research enabling high throughput in vivo screens. Zebrafish xenotransplantation literature continues to rapidly accumulate, and this necessitates the development of an interactive database for accommodating the collective data for fined-tune search, visualization and statistical representation purposes. Herein, I have introduced an interactive database, ZenoFishDb v1.1 (https://konulab.shinyapps.io/zenofishdb), housing manually curated details on molecularly-modified cell transplantations, PDXs, stem cell and cancer stem cell transplantation studies as well as transplantation studies bearing modified host details. The database projects collected data in a table format via various attributes and provides graphical representation of the curated details as well as statistical analyses yielding information on incorporated numbers and frequencies of selected attributes; hence can be used for reviews and designing new experiments. Zebrafish PDX studies are separately conceptualized and displayed through ZenoFishDb v1.1. Development of the ZenoFishDb v1.1 leads to a better understanding of tumor analysis methods such as assessment of proliferation and/or tumor growth in xenotransplantation studies and further marks the need for development of novel methods for precise quantification of tumor size. In the light of these findings, I have helped establish a novel qRT-PCRbased proliferation assessment method for xenografts in zebrafish, adapted from previous mouse xenotransplantation studies. Herein, the use and precision of ALU repeat-based quantification of transplanted liver cancer cells in genotyped zebrafish ache mutants and wildtype siblings was shown exemplifying microenvironment as an important factor for tumor growth. I further demonstrated the power of ALU repeatbased quantification in Mineralocorticoid Receptor (MR) overexpressing breast cancer cells (MCF7) injected to the transparent casper zebrafish as a case study. First, I demonstrated that MR expression and signaling was important in breast cancer biology and prognosis based on in silico TCGA and custom RNA sequencing as well as other in vitro and ex vivo assays. I further showed that results obtained from ALU repeatbased quantification of tumor growth in MR-overexpressing MCF7 cells paralleled fluorescent image-based intensity measurements while the former being relatively less time-consuming and more high-throughput. In this study, accurate quantification of MR overexpression in xenografts was also successfully performed by a cDNA-specific primer pair; and the rate of tumor growth based on image analysis, did not correlate with the amount of MR DNA in casper fish xenografts. However, MCF7 cells overexpressing MR exhibited lower cell viability in vitro although some of these effects were due to empty vector (EV) integration. Accordingly, tumor size in xenografts of naïve, EV- and MR-transfected MCF7 cells injected into pigmented AB larvae were quantified for ALU-repeats yet no significant difference was observed due to high within-group variability in vivo. Future studies are needed to assess the role of varying the volume and placement of injected cells along with the amount of MR gene transfected on tumor growth in vivo.
Open Access
Diet-induced changes in mouse cells in vitro and in vivo zebrafish models of angiogenesis
(2024-01) Yıldız, Selvin
Cardiovascular disorders rank as the primary cause of global mortality. Being overweight or obese impacts the pathogenesis of cardiovascular disease, resulting in an imbalance in endothelial function, cell growth, and inflammatory activation. Disruption of these factors resulting from endothelial cell dysfunction serves as both an outcome and a catalyst for vascular disease processes. Endothelial cells (ECs) are a natural barrier between circulating blood and vessel components. They also play critical roles in multiple physiological and pathophysiological processes, such as angiogenesis, vascular permeability, and inflammation. Amelioration of endothelial dysfunction may be attained by weight loss; however, complementary in vitro and in vivo studies are needed to establish the effects of weight loss on endothelial function and angiogenesis. This study developed an in vitro model to understand better the diet-induced changes in angiogenesis for mouse endothelial cells. In addition, a novel in vivo model of diet-induced vascular changes and its potential reversal with a return to regular diet in a zebrafish model was also studied. In vitro studies showed that a serum from mice fed a high-fat diet (HFD) might lead to proliferation of endothelial cells, yet weight loss did not compensate for prior stress induced by HFD. In vivo, studies in adult zebrafish showed that egg yolk-based high-fat diet might affect cytological architecture in the adult fish liver. Switching to a normal diet could effectively reverse these changes. Moreover, a caudal fin inter-ray vascularization assay was developed and used to test whether vessel sprouting was affected by different diets. Overfeeding resulted in a higher number of vessels, yet future studies with higher sample sizes are needed. Similarly, the expressions of several angiogenesis-related genes, which were quantified using cDNAs from the whole larvae and adult caudal fin treated with different diets, showed significant changes in vcam in larvae and cdh5 in adult fin by diet. However, further experiments are needed due to high individual variability and low sample size. The findings herein show that in vitro mouse endothelial cells and zebrafish larvae and adults could be used as valuable models for studies involving reversal/weight loss of high fat or overfeeding dietary regimes. Furthermore, the caudal fin vascularization assay in Tg(fli1:eGFP) Casper fish could be a promising preclinical model for testing the effects of different diets on angiogenesis and endothelial dysfunction.
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
Drug repurposing and investigation of novel combinations for glioblastoma therapeutics using in vitro and zebrafish in vivo models
(2024-01) Tok, Güneş
Glioblastoma is the most common and aggressive brain cancer type with the survival rate less than 2 years after diagnosis. Yet, potent drug treatments used in patients are limited and the field is in need of development of new potential drugs. In this study, repurposing of approved drugs alone or in combination and novel drugs are investigated in terms of inhibition of cell viability, glial fluorescent signals and their effects on behavior in zebrafish larval models. The main aim of this study was to test whether phenothiazines, trifluoperazine and a novel molecule 10, could be repurposed for glioblastoma treatment with lower dosages and more potency when combined with Sorafenib, an approved drug, in glioblastoma cell lines and zebrafish larvae. Those drug combinations were not found as toxic in the dosages studied while acted on glia cells in zebrafish transgenic larval models. Last but not least, behavior and stress response of the wild type and heterozygous mutant ache larvae in comparison with homozygous siblings were tested upon drug administration to assess genotype by drug interactions. Combination treatments exhibited higher efficacies suggesting phenothiazines with sorafenib could have potential in glioblastoma treatment. Genotype specific effects of individual and combination treatments on larval light-dark behavior, stress response and recovery exhibited potential for passage of blood brain barrier by the tested drugs. The established protocols for genotype and drug interactions could be applied to other kinases in combination with phenothiazines.
Open Access
Effects of aging and short-term dietary restriction on neurogenesis and cellular senescence in the zebrafish (Danio Reio) brain
(2016-09) Erbaba, Begün
Currently we know from rodent and fish studies that adult neuron generation is reduced but still continues in old animals with a dynamic change throughout aging. This process occurs mainly in hippocampal region, which is thought to be analogous to a region in telencephalon of the zebrafish brain. Changes in this neuron turnover are thought to be one contributing factor to cognitive change occuring with advanced age. Since we know that external factors can affect the process of neurogenesis, and as previous studies showed, dietary restriction (DR) extends life span; here, we hypothesized that DR should also alleviate several age associated alterations. In order to test this, we applied a 10-week feeding regimen to young (8-9 months) and old (26-32.5 months) male and female fish. We had two dietary regimen groups, one fed Ad libitum and one fed with a DR that was a pattern of every-other-day feeding, which is a widely accepted method of DR. A total of 124 animals were used in this study. As a result, a significant loss of body weight in both young and old DR groups was observed without an effect on body lengths. To be able to label actively dividing cells we used Bromodeoxyuridine (BrdU), which is a thymidine analog. It is injected into the fish intraperitoneally prior to euthanasia. Four hours later the brains were dissected and fixed for sectioning. We obtained cross-sectional slices of 50 m thickness with a vibratome, performed immunostaining with antibodies against BrdU, NeuN (neuronal marker), HuC (neuronal marker); and visualized the brain sections with confocal microscopy forming 3D reconstructed pictures. We counted the BrdU positive cells in all brain slices, forming a regional map of the telencephalic region of zebrafish brain, in which we documented the specific regions where the adult neurogenesis dominates the most and least. Our results confirmed that there are more BrdU positive cells in young animals than olds, and that age is correlated with an increased senescence associated fi-galactosidase (SA-fi-gal) activity, along with shortened telomere lengths. The 10-week diet was not found to be creating a significant change in cell proliferation rates, cellular senescence, or the differentiation pattern of glial cells. However, it was demonstrated to have a shortening effect on telomere lengths. Our data suggest that the potential effects of DR could be related to telomere regulation. Therefore, in order to detect differentially expressed genes that could be related to this mechanism between the groups, we performed microarray analysis with differing DR regimens. Initial data indicated no significant effects of a 4-week diet on gene expression differences among aged fish. Further analysis of the different periods of DR will be performed. Taken together, the effects of age are more robust than a short-term DR.
Open Access
Effects of aging, diet and potential genetic interventions on the levels of Smurf2 and its interacting partners in Zebrafish (Danio Rerio) brain
(2020-09) Şaşik, Melek Umay Tüz
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. HECTdomain 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.
Open Access
Expression levels of SMAD specific E3 ubiquitin protein ligase 2 (Smurf2) and its interacting partners show region-specific alterations during brain aging
(Elsevier, 2020) Tüz-Şaşik, Melek Umay; Karoğlu-Eravşar, Elif Tuğce; Kınalı, M.; Arslan-Ergül, A.; Adams, Michelle Marie
Aging occurs due to a combination of several factors, such as telomere attrition, cellular senescence, and stem cell exhaustion. The telomere attrition-dependent cellular senescence is regulated by increased levels of SMAD specific E3 ubiquitin protein ligase 2 (smurf2). With age smurf2 expression increases and Smurf2 protein interacts with several regulatory proteins including, Smad7, Ep300, Yy1, Sirt1, Mdm2, and Tp53, likely affecting its function related to cellular aging. The current study aimed at analyzing smurf2 expression in the aged brain because of its potential regulatory roles in the cellular aging process. Zebrafish were used because like humans they age gradually and their genome has 70% similarity. In the current study, we demonstrated that smurf2 gene and protein expression levels altered in a region-specific manner during the aging process. Also, in both young and old brains, Smurf2 protein was enriched in the cytosol. These results imply that during aging Smurf2 is regulated by several mechanisms including post-translational modifications (PTMs) and complex formation. Also, the expression levels of its interacting partners defined by the STRING database, tp53, mdm2, ep300a, yy1a, smad7, and sirt1, were analyzed. Multivariate analysis indicated that smurf2, ep300a, and sirt1, whose proteins regulate ubiquitination, acetylation, and deacetylation of target proteins including Smad7 and Tp53, showed age- and brain region-dependent patterns. Our data suggest a likely balance between Smurf2- and Mdm2-mediated ubiquitination, and Ep300a-mediated acetylation/Sirt1-mediated deacetylation, which most possibly affects the functionality of other interacting partners in regulating cellular and synaptic aging and ultimately cognitive dysfunction.
Open Access
Expression of key synaptic proteins in Zebrafish (Danio Rerio) brain following caloric restriction and its mimetic and their relationship with gender
(2017-01) Dede, Ayşegül
Aging is a progressive decline of physiological functioning and metabolic processes. Among all the organs, the brain seems to be the most vulnerable part of the body to the age-related changes because of the relatively high consumption of oxygen and glucose as compared to other organs. Both structural and cognitive changes occur during the aging process. A great effort has been spent to ameliorate the outcomes occurring within the brain as a result of aging. Caloric restriction (CR) is considered to be the only non-genetic intervention which decreases age-related cognitive decline. Rapamycin (RAP) has become a candidate drug which was shown to mimic the effects of CR by blocking the nutrient-sensing pathway, the mammalian target of Rapamycin, (mTOR) pathway. The first aim of this study was to investigate the expressions of key synaptic proteins; gephyrin, PSD-95 and synaptophysin, which are involved in the synaptic plasticity, after short-term (4 weeks) CR and RAP interventions in young and old, male and female zebrafish. The second aim was to investigate whether the expression of glutamate receptor subunits, NR2B and GluR2/3, display a sexually dimorphic pattern in middle age zebrafish. It was found that there was no significant difference in the expression of key synaptic proteins between the CR and RAP animal groups as compared to the ad libitium (AL) fed group and also no significance was found in the expression of NR2B and GluR2/3 in middle-aged male and female zebrafish. Highlighted studies in this thesis demonstrate that short-term (4 weeks) of CR and RAP treatments were too short to observe an effect in the expression level of gephyrin, synaptophysin, and PSD-95, and in the middle age, expression of NR2B and GluR2/3 did not display sexually dimorphic pattern. Our initial results of key synaptic protein levels indicate that they are stable throughout aging with respect to gender and CR interventions.
Open Access
Functionally conserved effects of rapamycin exposure on zebrafish
(Spandidos Publications, 2016-03) Sucularli, C.; Shehwana, H.; Kuscu, C.; Dungul, D. C.; Ozdag, H.; Konu, O.
Mechanistic target of rapamycin (mTOR) is a conserved serine/threonine kinase important in cell proliferation, growth and protein translation. Rapamycin, a well-known anti-cancer agent and immunosuppressant drug, inhibits mTOR activity in different taxa including zebrafish. In the present study, the effect of rapamycin exposure on the transcriptome of a zebrafish fibroblast cell line, ZF4, was investigated. Microarray analysis demonstrated that rapamycin treatment modulated a large set of genes with varying functions including protein synthesis, assembly of mitochondrial and proteasomal machinery, cell cycle, metabolism and oxidative phosphorylation in ZF4 cells. A mild however, coordinated reduction in the expression of proteasomal and mitochondrial ribosomal subunits was detected, while the expression of numerous ribosomal subunits increased. Meta-analysis of heterogeneous mouse rapamycin microarray datasets enabled the comparison of zebrafish and mouse pathways modulated by rapamycin, using Kyoto Encyclopedia of Genes and Genomes and Gene Ontology pathway analysis. The analyses demonstrated a high degree of functional conservation between zebrafish and mice in response to rapamycin. In addition, rapamycin treatment resulted in a marked dose-dependent reduction in body size and pigmentation in zebrafish embryos. The present study is the first, to the best of our knowledge, to evaluate the conservation of rapamycin-modulated functional pathways between zebrafish and mice, in addition to the dose-dependent growth curves of zebrafish embryos upon rapamycin exposure.
Open Access
Genetic and environmental interventions altering the course of brain aging: evidence from the zebrafish (Danio Rerio) model
(2021-04) Eravşar, Elif Tuğce Karoğlu
Age-related cognitive decline occurs during normal aging, although there is no prominent neural loss in the brain. Subtle molecular alterations in synaptic and cellular dynamics are likely underlying these cognitive alterations. One challenge is the widely heterogeneous profile regarding age-related behavioral changes and neurobiological underpinnings. Therefore, it is crucial to characterize how individual factors can contribute to successful or unsuccessful aging and whether these factors can induce shared patterns of alterations in the cellular and synaptic dynamics. Three different intervention approaches were utilized in the current study. The first intervention was a genetic manipulation in the cholinergic system component acetylcholinesterase (AChE), which results in reduced levels of this enzyme in the achesb55/+ mutants. Previous studies have characterized this model as a delayed aging model because of its preserved cognitive abilities at an older age. The current study was the first study analyzing the neurobiological changes in this mutant model within the context of aging. It was shown that reduced brain AChE activity levels persist in different age groups, including old age in the mutant animals. This reduction was accompanied by subtle decreases in the other elements of the cholinergic system, including acetylcholine and nicotinic acetylcholine receptor subunit alpha-7. Genotype significantly altered key glutamatergic receptor subunits such as N-methyl D-aspartate-type receptor subunit 2B (NR2B) and glutamate receptor subunits 2 and 3 (GluR2/3), with these markers significantly reduced in the achesb55/+ mutants and likely maintaining homeostatic synaptic scaling. At old age, a significant age-related elevation was observed in the synaptophysin levels (SYP) of the old achesb55/+ mutants, and this mutation prevented an age-related decline in the gephyrin (GEP) levels which was evident in the wild-type controls. This mutation also altered the cellular dynamics; an immature neuronal marker, embryonic lethal abnormal-vision (ELAV Drosophila) like-3 (HuC) was significantly upregulated in the achesb55/+ mutants at all ages. In contrast, the levels of inflammation-related markers, glial fibrillary acidic protein (GFAP) and reactive oxygen species (ROS), were downregulated subtly in the mutants. It can be concluded that reduced levels of brain AChE can be associated with altered excitatory homeostasis and preserved levels of GEP and SYP through aging. At the same time, the neuronal marker was upregulated, and inflammation-related markers were downregulated. The second intervention was applying short-term environmental enrichment using the sensory cues to young and old zebrafish to induce successful aging. It was shown that environmental enrichment increases the brain weight in old zebrafish, prevents age-related decrements in the levels of synaptic proteins, including SYP and NR2B, and doublecortin-like kinase (DCAMKL1). Additionally, environmentally enriched old zebrafish had elevated levels of GEP while applying this environmental intervention did not modulate the age-related increases in oxidative stress indicators. The third intervention was also a non-genetic approach. Two short-term opposing dietary treatments, such as caloric restriction (CR) and over-feeding (OF), were applied to young and old zebrafish and an ad-libitum diet. It was demonstrated that a short-term CR regimen upregulated the glutamatergic components of neurotransmission such as GluR2/3 and post-synaptic density 95 (PSD95). Significant age-related decline in GEP levels was observed in old zebrafish in the OF dietary condition. Expression levels of synaptic and regulatory genes were relatively stable, while inflammation-related gene tnfa was altered in an age-dependent manner. Additionally, in the young zebrafish, a significant elevation of trunk cortisol was demonstrated in the OF group compared to CR-fed young zebrafish. Taken together, evaluating different components such as the cholinergic system, diet, and environment can provide us insights into the neurobiological underpinning of successful aging and possible determinants of unhealthy aging.