Дисертації з теми "Glial Fibrillary Acidic Protein (GFAP)"

Müller glial cells can regenerate the retina in zebrafish throughout life. However, in the damaged adult human retina, upregulation of inflammatory cytokines, including TNF-α, leads to Müller cell gliosis, a hallmark of which is an increase in intermediate filament glial fibrillary acid protein (GFAP) production. However, a subset of these human cells has stem cell characteristics in vitro. This study investigated the role that inflammatory cytokines may play in regulating Müller cell gliosis-associated proteins and the implications this could have on the neurogenic ability of Müller glia in vitro. As determined by gene and protein analysis, culture of the human Müller glial cell line MIO-M1 in the presence of TNF-α, causes downregulation of GFAP expression. Through upregulation of TNF-receptor2 and downstream activation of the NFκB signalling pathway, a cell survival signal is initiated. MIO-M1 cells co-cultured with TNF-α and factors known to induce rod photoreceptor precursor differentiation, showed increased expression of the photoreceptor marker NR2E3. These observations suggest that TNF-α may not inhibit the neurogenic ability of these cells. A retroviral transfection method was developed to overexpress GFAP in MIO-M1 cells using molecular cloning techniques. Overexpression of GFAP resulted in no phenotypic changes as Müller cells maintained their stem cell characteristics. Culturing these transfected cells with TNF-α revealed differential transcriptional regulation of endogenous and exogenous GFAP. This indicates the importance of the GFAP promoter and transcriptional response elements in responding to TNF-α during gliosis. In conclusion, the present study has identified the downregulation of GFAP expression by TNF-α in Müller glial cells as a target that could be further explored to control scarring of the human retina. These observations pave the way for further investigations to promote endogenous regeneration of the adult human retina by Müller glia.

Après une lesion médullaire, les astrocytes réactionnels surexpriment les proteines GFAP et Vimentine, formant ainsi une cicatrice gliale qui constitue un obstacle majeur à la régénération axonale. Les animaux doublement invalidés pour les gènes de la GFAP et de la vimentine présentent, après hémisection de la moelle épinière, une réduction de la cicatrice gliale, une repousse axonale significative ainsi qu’un recouvrement des fonctions locomotrices (Menet et al. 2003). Sur la base de ces résultats, nous avons développé une stratégie de transfert de gènes qui a pour objectif d’inhiber localement l’expression de GFAP et de vimentine par ARN interférence (ARNi) de manière à limiter la formation de cette cicatrice. Dans un premier temps nous avons développé des vecteurs lentiviraux capables de réprimer efficacement l’expression de GFAP et de Vimentine par ARNi in vitro et in vivo. Dans différents modèles cellulaires, nous avons mis en évidence la capacité de ces vecteurs à limiter la formation de cicatrice gliale d’une part, et d’autre part à favoriser la survie neuronale ainsi que la croissance axonale. Enfin, dans un modèle murin d’hémisection médullaire, nous avons montré que l’application de ces vecteurs dans le parenchyme médullaire permet d’améliorer la récupération fonctionnelle et favorise la repousse des fibres sérotoninergiques impliquées dans la locomotion.

La maladie d’Alexander est une maladie neurodégénérative due à des mutations hétérozygotes dans la GFAP, filament intermédiaire majoritaire de l’astrocyte mature, conduisant à la formation d’agrégats La conséquence des mutations de la GFAP sur la formation du cytosquelette astrocytaire, la façon dont les agrégats se forment et l’induction potentielle de processus de défense cellulaires restent à déterminer. Nous avons étudié différentes mutations de la GFAP dans des astrocytes murins sauvages ou dépourvus de GFAP et/ou de vimentine et dans des cellules SW13. Dans les astrocytes, la GFAP mutée produit un réseau ou des agrégats, tandis que la protéine sauvage produit un réseau. La dynamique de la GFAP mutée a été étudiée en vidéo-microscopie. Dans les astrocytes, les agrégats peuvent se déplacer de façon centrifuge et disparaître. Le plus souvent ils se rassemblent près du noyau, ce qui est fréquemment associé à la mort cellulaire et correspond à la formation d’aggresomes. Les petites protéines du stress et le système ubiquitine-protéasome pourraient être impliqués dans la désagrégation, tandis que l’autophagie ne semble pas sollicitée. Nous montrons par ailleurs que l’intégration de GFAP mutée dans un réseau de filament formé par la vimentine prévient la formation des agrégats d’une manière dépendante de la quantité de GFAP exprimée. En conclusion, l’agrégation de la GFAP mutée semble dépendre de la mise en place de mécanismes de défense cellulaire, dont les protéines chaperonnes et le système ubiquitine-protéasome, et de la quantité de GFAP et de vimentine produite par l’astrocyte.

Mesmo a isquemia sendo a terceira causa de morte em países industrializados, os mecanismos relacionados a esta doença ainda continuam polêmicos e obscuros. Utilizou-se a técnica de privação de oxigênio e glicose (OGD) em fatias do hipocampo de rato para investigar parâmetros mitocondriais, neurais, astrogliais e metabólicos no período de isquemia e durante o período de reoxigenação. Os resultados mostraram uma diminuição na atividade mitocondrial durante o período isquêmico que foi mantido durante todo o período de reoxigenação. Analisando o sobrenadante destas fatias submetidas à OGD, foi observado que os níveis de LDH, NSE e GFAP se elevaram. Com relação aos níveis de lactato, verificou-se sua diminuição durante todos os períodos. Os níveis de S100B estavam elevados somente durante o período de reoxigenação. Este aumento pode ser tanto um mecanismo de neuroproteção desta proteína frente ao insulto ou ainda uma liberação por dano celular astrocitário. Além disso, foi observado um grande aumento nos níveis de glutamato durante a isquemia e este aumento retornou no período de reoxigenação. Por fim, houve uma diminuição na captação de glutamato somente no período de reoxigenação. Todos estes resultados podem ser conseqüência de uma hiper-estimulação dos receptores glutamatérgicos devido ao insulto isquêmico. Em resumo, nosso estudo mostrou alterações em diversos parâmetros neuroquímicos específicos tanto no período isquêmico quanto na reoxigenação, mostrando que cada tipo celular, reage diferentemente frente ao insulto isquêmico na técnica de OGD in vitro.
Stroke is the third cause of mortality in industrialized countries, and the mechanisms related to this disease are polemic and unclear. Oxygen and glucose deprivation (OGD) in acute rat hippocampal slices was performed to investigate mitochondrial, neural, astroglial and metabolic neurochemical parameters at different ischemic and reoxygenation periods. Results showed the mitochondrial activity decrease due energy failure during ischemic insult and reoxygenation time. In the supernatant medium, LDH, NSE and glutamate levels were increased and the lactate decrease by the lack of energy observed in the ischemic period. Parameters such as GFAP, S100B and glutamate uptake suffered alterations only at the reoxygenation period. These results have shown the vulnerability of neurons facing ischemic insult. Meanwhile, it was also observed a delayed injure of astrocytes only at reoxygenation time, which demonstrate the difference between cell types at OGD. In summary, our finding has shown altered at specific neurochemical parameters in OGD in vitro which features the ischemic episodes and reoxygenation periods.

This thesis assessed the suitability of unilateral, electrolytic lesions as a model of spinal cord damage and repair in the adult rat. This type of lesion resulted acutely in localized damage in the upper motor neuron at the L2-L3 level of the spinal cord. Minimal acute damage to ascending sensory pathways was indicated by preserved somatosensory evoked potentials elicited by stimulation of the tibial nerve. Immediately after lesion generation one of several substrates was injected into the lesion cavity. These substrates were saline buffer, liquid collagen solution, foetal spinal cord cells from 14 day old rat embryos, and a mixture of collagen and E 14 foetal spinal cord cells. The 4 groups were compared for functional recovery over 3 months using the inclined plane test and a Tarlov movement scale. After sacrifice, the tibialis anterior muscles were dissected and weighed to assess atrophy due to lower motor neuron injury. After removing and embedding the spinal cords in paraffin, transverse and longitudinal sections were taken for cytoarchitectural investigation. Cresyl violet was used to indicate Nissl substance, Luxol fast blue stained for myelin and anti - glial fibrillary acidic protein (GFAP) antibody revealed the expression of GFAP in the cord sections. Chronic electrolytic lesions were characterized by the highly variable degree of cavitation, demyelination and macrophage infiltration that was present. There was no significant performance deficit on the inclined plane test in any of the lesioned groups when compared to unoperated animals. The tibialis muscles from all groups were of normal weight, indicating that the lower motor neurons were not significantly damaged by the lesions used. There was, however, a marked decrease in the number of GFAP reactive astrocytes in the lesioned animals when compared to unlesioned controls (P < 0.01, Wilcoxon test). Moreover, this reduction of GFAP - like immunoreactivity was not prevented by implants of foetal neurons, collagen or foetal neurons suspended in collagen. Possible explanations for the reduced GFAP - like immunoreactivity seen in all electrolytically lesioned cords are discussed.
Medicine, Faculty of
Cellular and Physiological Sciences, Department of
Graduate

Background Multiple sclerosis (MS) is an autoimmune disease characterised by inflammatory damage to the central nervous system (CNS). Accumulated CNS injury can be quantified as brain atrophy, definable as a reduction in brain parenchymal fraction (BPF). BPF correlate with disability in MS and is used routinely as an endpoint in clinical trials. In 2009/2010, a new MS clinical care program, that includes follow-up of BPF, was introduced at Umeå University Hospital (NUS). Levels of neurofilament light polypetide (NFL) and glial fibrillary acidic protein (GFAP) in cerebrospinal fluid (CSF) are markers of axonal and astrocytic injury, respectively, and also potential surrogate biomarkers for BPF decline. The goals of this thesis were to establish age-adjusted values of BPF in healthy individuals and to relate these to the BPF values from individuals with MS as well as to the levels of NFL and GFAP in CSF. Another goal was to investigate if expanded disability status scale (EDSS)-worsening could be predicted in a clinical MS cohort and if BPF measurements could contribute to such predictions. Methods A group of 111 healthy individuals volunteered to participate in the studies. A total of 106 of these underwent MRI with BPF measurements, 53 underwent lumbar puncture (LP) with measurement of NFL and GFAP and 48 underwent both MRI and LP. Three different automatic and one manual method were utilised to determine BPF. A literature search on BPF in healthy individuals was performed for the purpose of a systematic review. For studying disability progression in MS, all individuals with MS followed at NUS and included in the Swedish MS registry were included if they had matched data on BPF, EDSS and lesion load as part of clinical follow-up (n=278). Results BPF as well as NFL and GFAP levels in CSF were all associated with age. NFL was associated with BPF and GFAP, but only the association with GFAP was retained when adjusting for age. Significant differences were found between different methods for BPF determination. In the MS population, BPF was associated with EDSS. Only progressive disease course could predict EDSS worsening. Conclusion The data on BPF and levels of NFL and GFAP in CSF of healthy individuals can aid in the interpretation of these variables in the setting of MS. Knowledge on differences in BPF data from different methods for BPF determination can be useful in comparing data across studies, but also highlights the need for a commonly accepted gold standard. The correlation between GFAP and NFL levels in CSF may indicate an association between glial and axonal turnover that is independent of the aging effect on the brain. However, the low number of volunteers for LP precluded clear conclusions. An association between BPF and EDSS was seen in the MS group. The ability to predict EDSS worsening in the clinical MS cohort was limited.

Thesis (Ph.D.)--University of Florida, 2004.
Typescript. Title from title page of source document. Document formatted into pages; contains 87 pages. Includes Vita. Includes bibliographical references.

Neuroinflammation is a prominent feature of most neurodegenerative and affective disorders and has been increasingly implicated as a contributing factor in the disease development. The inflammatory process is closely linked with multiple neurodegenerative pathways and represents an important therapeutic target in halting or reversing disease progression. This study explored the cognitive and behavioural effects of chronic neuroinflammation in the glial fibrillary acidic protein promoter-interleukin 6 (GFAP-IL6) transgenic mouse in which the pro-inflammatory cytokine interleukin 6 (IL-6) is overexpressed causing low level, chronic inflammation localised to the brain. Additionally the anti-inflammatory, anti-oxidant and neuroprotective potential of the dietary bioflavanoid apigenin was investigated to determine whether any deficits in behaviour and cognition could be rescued. Male and female heterozygous GFAP-IL6 mice (n=32) and their non-transgenic littermates (C57/BL6J) (n=36) were introduced to either an apigenin enriched pellet diet (40mg/kg daily dose) or control pellet diet at the age of 3 months. After 3 months of feeding (6 months of age) the mice were subjected to a behavioural test battery including the elevated plus maze (EPM), open field test (OF), Barnes maze (BM) and functional observational tests. The results showed that at 6 months of age, GFAP-IL6 mice exhibited alterations in anxiety-related behaviour in the EPM and OF, presenting a tendency toward an anxiolytic-like phenotype and demonstrated impairments in memory and spatial learning in the BM. GFAP-IL6 mice also displayed an ataxic phenotype and exhibited reduced locomotor activity compared to wild-type (WT) controls. The effect of apigenin on anxiety-related behaviours were mixed, being sedative-like in reducing locomotion and explorative behaviour in the EPM and OF, and anxiolytic-like in reducing risk assessment behaviour. Cognition improving properties are indicated for apigenin in the BM probe trial, enhancing recall of the target hole location, particularly among GFAP-IL6 mice. Interestingly, apigenin was additionally associated with increased food consumption and weight gain, suggesting potential as an appetite stimulant.

碩士
國立臺灣大學
解剖學研究所
93
Some intermediate filament (IF) proteins expressed in the development of glia include nestin, vimentin, and glial fibrillary acidic protein (GFAP). Yet GFAP is the major intermediate filament protein of mature astrocytes. To determine the organization of GFAP in glial cells, the rat GFAP cDNA tagged with enhanced green fluorescent protein (EGFP) was transfected into rat C6 glioma cell line. After selection of neomycin analogue G418, two stable C6-EGFP-GFAP cell lines were established under. Stable C6-EGFP-GFAP cell lines with or without heat shock treatment were analyzed by immunocytochemistry, electron microscopy, and Western blot analysis. In transient transfection study, EGFP-GFAP transiently expressed in C6 cells formed punctate aggregations in the cytoplasm right after transfection, but gradually the filamentous structure of EGFP-GFAP was observed. Comparing the C6-EGFP-GFAP stable clone with pEGFP-C1 transfected C6 stable clones and non-transfected C6 cells, the protein level of nestin in C6-EGFP-GFAP was similar to others; where as the level of vimentin was reduced in Western blotting. Interestingly, the expression level of small heat shock protein αB-crystallin in C6-EGFP-GFAP cells was also enhanced after transfection. Immunostaining patterns of C6-EGFP-GFAP cells showed that the distribution of GFAP was dispersed, as a pattern of little fine filamentous structure. However, after heat shock treatment, GFAP formed IF bundles in C6-EGFP-GFAP cells significantly. Meantime, αB-crystallin also colocalized with IF bundles of GFAP in C6-EGFP-GFAP cells. From our observations in this study, it could be suggested that the organization of GFAP in glial cells was dynamic and regulated by several different mechanisms. The heat-induced GFAP reorganization we found suggested that small heat shock protein aB-crystallin may play a functional role to regulate the cytoarchitecture of GFAP.

碩士
國立成功大學
生物學系碩博士班
92
Astrocytes, the most abundant cells in the central nervous system (CNS), form the blood-brain barrier (BBB) with brain capillaries. BBB protects the neurons from microorganisms and toxicants. Astrocytes also function in supplying the energy to neurons and modulating ion levels to make the neuronal homeostasis. However, in the injured CNS they become reactive and highly express glial fibrillary acidic protein (GFAP) and vimentin that are involved in the formation of glial scar, a physical barrier for CNS repair. The up-regulated of astrocytic GFAP and vimentin in the injured CNS is thought to be induced by pro-inflammatory mediators. Microglia, CNS resident macrophages, produce pro-inflammatory mediators during CNS infection or injury including pro-inflammatory cytokines, reactive oxygen species, and nitric oxide. Among pro-inflammatory cytokine, interleukin-18 (IL-18) is known as microglial interferon-�� inducing factor. The action of IL-18 on neurons and astrocytes are not yet known. In this study, we found that IL-18 induced an increase astrocytic GFAP and vimentin in rat primary astrocytes. Interestingly, treatment of rat astrocytes with the culture supernatant of IL-18 stimulated microglial cell line BV2 also caused an increase in GFAP and vimentin. In consistence with in vitro study, the in vivo study indicated that the injection of IL-18 into the adult rat cortical areas above corpus callosum resulted in the up-regulation of GFAP and vimentin in astrocytes at 3 day post injection. Together, our results indicated that IL-18 action may act as modulatory factor for glial scar formation through increasing astrocytic GFAP and vimentin.

碩士
國立清華大學
分子醫學研究所
103
Alexander disease (AxD) is a primary genetic disorder of astrocytes caused by heterozygous mutations in GFAP, which encodes the major astrocyte intermediate filament protein, glial fibrillary acidic protein (GFAP). The mechanism of GFAP mutation causing the AxD remains unclear. The aim of this study is to investigate the effect of the novel AxD-causing mutation on GFAP filament formation and stability by using filament assembly in vitro and transient transfection in cultured cells. The results showed that all the GFAP mutations perturbed the filament assembly in vitro and in transiently transfected cells. The E312X GFAP caused the most dramatic effects on filament assembly both in vitro and in transiently transfected cells. This truncated mutant caused extensive filament aggregation coinciding with the activation of caspases, cleavage of GFAP, and a significant decrease in cell viability. These data provide a direct link of GFAP mutation on filament aggregation and loss of cell viability through the activation of caspases and cleavage of GFAP, suggesting that these could be contributing factors in the development of Alexander disease.

碩士
國防醫學院
生物及解剖學研究所
89
In the past, many studies were attempted to set up an animal model to explore the possible events of the spinal cord injury. Many models were therefore established to deal with the cord injury of different conditions. The authors also tried to create a reasonable and well-matched model for the physical changes of the observed factors. The more compatible normal physical phenomenon showed, the better animal model was. In the present animal model, a steel ball fixed on Nylon suture with Durelon, then implanted epidurally on the dorsolateral side of T10 spinal cord caused the constant compression injury. After the injury, the combined behavioral score (CBS) and foot print were carried out to evaluate the recovery of affected cords at day 1, 7 and 30 post injury. Then we applied Fast blue to follow the influenced spinal tracts to examine their integrity. Finally, the astroglial reactions in the compressed site (T10), the areas rostral to the compressed site (C2) and caudal to the compressed site (L2) would be examined with immunohistochemistries of glial fibrillary acidic protein (GFAP) for resting and activated astrocytes, and of calbindin for neuronal elements and activated astrocytes. The GFAP labeling showed that the astroglial activation was not quite related to the expected areas undergoing retrograde degeneration but widespread in the spinal cord. Concomitantly, calbindin immunoreactivity was increased in several regions merely related to the expected areas undergoing degeneration, but decreased in the spinal gray matter. These results suggested that cord astrocytes are highly sensitive to the changes of their ambient microenvironment. The significance of calbindin in the spinal cord is more complex than expected. It may be involved in the processes of neuronal degeneration and/or regeneration.

碩士
國立臺灣大學
分子與細胞生物學研究所
99
In recent years, mitochondrial DNA and microsatellite sequences are often used as the molecular markers for studying species evolution and population structure. However, to date, appropriate molecular markers for distinguishing closely related species have not been well-established. Our previous study demonstrated that the gene sequences of intermediate filaments (IFs) proteins, one of the major types of cytoskeleton, could separate vertebrates and invertebrates into two different groups in phylogenetic reconstruction. In addition, each of the vertebrate IF genes was separated into different subgroups corresponding to their IF types. Furthermore, within each subgroup, the evolutionary relationship of different species is parallel to that of species. These results suggested that the evolution of IF genes is closely related to the evolution of vertebrate species. In this study, the gene of type III intermediate filament protein GFAP (glial fibrillary acidic protein) was selected as the molecular marker. Due to its specific expression in the central nervous system, it is postulated that its selective pressure from the external environment is minimum, thus it is more likely to maintain neutral. To prove that the GFAP gene is an appropriate molecular marker, we collected the GFAP sequences of each species from the Ensembl database. Subsequently, we generated the phylogenetic tree of the GFAP gene sequences of 19 animal species by Bayesian inference (BI). The results was in agree with the currently accepted classification. Therefore, the validity of GFAP gene as a molecular marker is confirmed. Recently, it has been shown that introns have relatively large nucleotide variability, and can be easily amplified with primers placed in the adjacent exons. Thus, there are increasing number of studies using intronic sequence to investigate the phylogenetic relationship of species. In 2010, Igea et al., by applying several reasonable filters, selected 224 intronic sequences that belongs to several mammalian species, including human, chimpanzee, macaque, dog and cattle, and successfully distinguish the phylogenetic relationship of these species. Therefore, in this study, it is anticipated to characterize the appropriate introns of GFAP gene for distinguishing the closely related species. By aligning the 8 intronic sequence of GFAP gene of five primate species, including human, chimpanzee, gorilla, gibbon and macaque, it was revealed that the length of the intron 2 fragment are the same among five primate species. In addition, the phylogenetic tree of these five primate species reconstructed with GFAP intron 2 by UPGMA method was also in consensus with currently established phylogenetic relationship. Thus, the intron 2 sequence was selected as the molecular marker for the current study. The traditional classification for mammals is based on fossil evidence. On the other hand, classification of fishes is mainly based on morphology of the extant species. Thus, the aim of this study is to examine the phylogenetic relationship of species under a unified standard by examining GFAP intron 2 sequences of primates and fishes. In our study, fishes of several different families and genera in the suborder Scombroidei were selected, and their GFAP genes sequences were amplified by PCR. After sequencing, their lengths and variation were compared. The results revealed the length of GFAP intron 2 is different in each of the five fish families. Surprisingly, although from the same family of Scombridae, the length of GFAP intron 2 of the yellowfin tuna, bigeye tuna, albacore tuna,, Pacific bluefin tuna (Thunnus) and the seer fish (Scomberomorus) is distinct from that of spotted mackerel (Scomber) and frigate mackerel (Auxis). Then, we reconstructed the phylogenetic tree by intron 2 sequence from each of the fishes, the results revealed one single group among the four fishes of Scombridae and fishes of Trichiuridae. Moreover, among the Scombridae, spotted mackerel and frigate mackerel are closer to each other, but are distinct from the Thunns and seer fish. Taken together, the results suggest that the evolutionary relationship of Scombridae and Trichiuridae are closer than the currently accepted phylogenetic classification. Furthermore, it is also proposed that spotted mackerel, frigate mackerel should be separated from Thunns, seer fish, and regrouped into a new family. Besides, the protein coding region of GFAP exon 1 was also included in our analysis. The results showed that the evolutionary rate of exon 1 is higher than intron 2, thus it can be used to clarify the phylogenetic relationship of closely related fish species. In summary, the current study confirmed the validity of GFAP intron 2 and exon 1 as the molecular marker for closely related species. We have also re-examined the phylogenetic relationship of mammals and fishes, and the results shows that GFAP may have the potential to serve as one of the species barcode genes.

Mild traumatic brain injury (mTBI), often referred to as concussion, has become increasingly recognized as a serious health issue in the general population. The prevalence of mTBI in athletes, particularly repeated injuries in young athletes, is of great concern as injuries to the developing brain can have long-term detrimental effects. In this study we used a novel awake closed-head injury (ACHI) model in rodents to examine repeated mTBI (rmTBI), to determine if repeated injuries produced the neurological and molecular changes evident with human concussion. Animals were administered 4, 8, and 16 rmTBIs and acute neurological assessments were performed after the injuries. Changes in glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1) levels were assessed using Western blot analysis at one day following rmTBI in the ipsilateral dentate gyrus (DG) and the cornu ammonis (CA) regions of the hippocampus and the cortex (CX) indicative of astrocyte and microglial cell reactivity. Results indicated that the ACHI model produces neurological deficits immediately after the injuries, with the most deficits arising in the rmTBI16 group. Despite deficits in all injury groups, histological staining with cresyl violet revealed no significant morphological tissue damage to the brain. Western blot analysis, however, showed a significant increase in DG and CX GFAP expression in the rmTBI16 group with no changes in Iba-1 levels. This suggests an acute activation of astrocytes in response to injury, with a delay or absence of microglial activation. Our findings show that with repetitive concussions, we are able to detect acute neurological and molecular changes in the juvenile female brain. However, further investigation is necessary to determine if these are transient changes.
Graduate