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Neuron-like staining by different GFAP antibodies. Neuron-like structures are stained by the GFAP +1 (A), GFAP C-terminal (B) and GFAP N-terminal (C) antibody (arrows). The C-terminal and N-terminal antibodies also stain astrocytes (B-C, arrowheads). Double-labeling of panGFAP (black) and neurofilament (brown) (D-E) shows that neuron-like structures stained by GFAP (E, arrowheads) are surrounded by neurofilament positive neurons (E, arrow), but do not co-localize. NBB 96-058 (A-C), NBB 88-073 (D-E). Scale bars represent 100 mm (A-C), 200 mm (D), 25 mm (E). doi:10.1371/journal.pone.0007663.g001
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The human GFAP splice variants GFAPDelta164 and GFAPDeltaexon6 both result in a GFAP protein isoform with a unique out-of-frame carboxy-terminus that can be detected by the GFAP+1 antibody. We previously reported that GFAP+1 was expressed in astrocytes and in degenerating neurons in Alzheimer's disease brains. In this study we aimed at further inve...
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... structures were not only stained by the GFAP +1 antibody (Fig. 1A, arrows), but also by an antibody against the C- terminus (Fig. 1B, arrows) and N-terminus (Fig. 1C, arrows) of GFAPa. In addition, as expected these last two antibodies also stained the GFAP-IF network of astrocytes in the same area (Fig. 1B-C, arrowheads). Since the C-terminal GFAP antibody should not stain the GFAP +1 protein, as the ...
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... structures were not only stained by the GFAP +1 antibody (Fig. 1A, arrows), but also by an antibody against the C- terminus (Fig. 1B, arrows) and N-terminus (Fig. 1C, arrows) of GFAPa. In addition, as expected these last two antibodies also stained the GFAP-IF network of astrocytes in the same area (Fig. 1B-C, arrowheads). Since the C-terminal GFAP antibody should not stain the GFAP +1 protein, as the C-terminus of the GFAPD164 and GFAPDexon6 isoforms differ completely from ...
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... structures were not only stained by the GFAP +1 antibody (Fig. 1A, arrows), but also by an antibody against the C- terminus (Fig. 1B, arrows) and N-terminus (Fig. 1C, arrows) of GFAPa. In addition, as expected these last two antibodies also stained the GFAP-IF network of astrocytes in the same area (Fig. 1B-C, arrowheads). Since the C-terminal GFAP antibody should not stain the GFAP +1 protein, as the C-terminus of the GFAPD164 and GFAPDexon6 isoforms differ completely from the C-terminus of GFAPa, we ...
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... structures were not only stained by the GFAP +1 antibody (Fig. 1A, arrows), but also by an antibody against the C- terminus (Fig. 1B, arrows) and N-terminus (Fig. 1C, arrows) of GFAPa. In addition, as expected these last two antibodies also stained the GFAP-IF network of astrocytes in the same area (Fig. 1B-C, arrowheads). Since the C-terminal GFAP antibody should not stain the GFAP +1 protein, as the C-terminus of the GFAPD164 and GFAPDexon6 isoforms differ completely from the C-terminus of GFAPa, we concluded that GFAPa is present in the neuronal structures. These data confirm our earlier results with the pan-GFAP antibodies from Dako, Sigma and Dahl ...
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... not stain the GFAP +1 protein, as the C-terminus of the GFAPD164 and GFAPDexon6 isoforms differ completely from the C-terminus of GFAPa, we concluded that GFAPa is present in the neuronal structures. These data confirm our earlier results with the pan-GFAP antibodies from Dako, Sigma and Dahl [5], which also stained these neuron-like structures (Fig. 1E, arrowheads). However, doublestaining with a pan-neuronal neurofilament antibody ( Fig. 1D-E) showed that these GFAP positive neuronal structures (grey) were different from the neurons stained by the neurofilament antibody (brown, Fig. 1E, arrow). Although the neuronal structures were present in the CA1 area of the hippocampus, no co-localization ...
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... completely from the C-terminus of GFAPa, we concluded that GFAPa is present in the neuronal structures. These data confirm our earlier results with the pan-GFAP antibodies from Dako, Sigma and Dahl [5], which also stained these neuron-like structures (Fig. 1E, arrowheads). However, doublestaining with a pan-neuronal neurofilament antibody ( Fig. 1D-E) showed that these GFAP positive neuronal structures (grey) were different from the neurons stained by the neurofilament antibody (brown, Fig. 1E, arrow). Although the neuronal structures were present in the CA1 area of the hippocampus, no co-localization was seen between pan-GFAP and neurofilament. This made us question the specificity ...
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... with the pan-GFAP antibodies from Dako, Sigma and Dahl [5], which also stained these neuron-like structures (Fig. 1E, arrowheads). However, doublestaining with a pan-neuronal neurofilament antibody ( Fig. 1D-E) showed that these GFAP positive neuronal structures (grey) were different from the neurons stained by the neurofilament antibody (brown, Fig. 1E, arrow). Although the neuronal structures were present in the CA1 area of the hippocampus, no co-localization was seen between pan-GFAP and neurofilament. This made us question the specificity of the neuronal GFAP +1 staining, which appeared to be different from the pan-GFAP neuron-like ...
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... western blot on human spinal cord filaments stained for GFAP +1 resulted in two bands; one band migrating at approxi- mately 70 kDa and one band at approximately 50 kDa ( Fig. 2A, unpur). After affinity purification of the antibody only the 50 kDa band remained visible (Fig. 1A, pur), which is the expected height of GFAP +1 protein as we confirmed by recombinant protein (not shown). Two bands at the same heights were also visible on the Coomassie protein gel (not shown) and these bands were isolated and analyzed by Maldi analysis. Peptide Mass Fingerprinting analysis of the band migrating at approximately 50 kDa ...
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Citations
... References: (1) (Lewis et al., 1984), (2) (Brenner et al., 1990), (3) , (4) (Feinstein et al., 1992), (5) (Galea et al., 1995), (6) (Condorelli et al., 1999b), (7) , (8) (Roelofs et al., 2005), (9) , (10) , (11) (Middeldorp et al., 2009), (12) (Stassen et al., 2017), (13) (Blechingberg et al., 2007a), (14) (Zelenika et al., 1995), (15) , (16) (Hol et al., 2003), (17) transcript of GFAPδ has an alternative 3 prime untranslated region (3 ′ UTR) and polyadenylation site. A study by Blechingberg and colleagues showed that the activity of the polyadenylation signal rather than the 3 ′ -splice site usage was the primary determent for processing into the GFAPα or GFAPδ/κ mRNA (Blechingberg et al., 2007b). ...
Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is characteristic for astrocytes and neural stem cells, and their malignant analogues in glioma. Since the discovery of the protein 50 years ago, multiple alternative splice variants of the GFAP gene have been discovered, leading to different GFAP isoforms. In this review, we will describe GFAP isoform expression from gene to protein to network, taking the canonical isoforms GFAPα and the main alternative variant GFAPδ as the starting point. We will discuss the relevance of studying GFAP and its isoforms in disease, with a specific focus on diffuse gliomas.
... However, it remains unclear to date whether the neuronal positivity for GFAP is an exclusive observation for the hippocampus or also affects other brain regions. In light of previous observations of cross-reactions between GFAP antibodies and epitopes of neurofilament-L in Alzheimer's disease (AD) patients [24], this given study will particularly focus on the possibility that neuronal GFAP is a processing artefact, rather than a reactive process following brain injuries. ...
... CB cerebellum, CLC contralateral cortex, FC frontal cortex, HC hippocampus, PCZ pericontusional zone AD patients [28], which further substantiates that neuronal GFAP might be related to neuron degeneration or damage, thereby corroborating the findings in TBI cases [22]. However, the immunopositivity for GFAP in AD patients was shown to be related to a cross-reactivity of the GFAP antibody with neurofilament-L [24]. This was attributed to a homology between amino acids of the GFAP +1 peptide and the tail domain of neurofilament-L [24]. ...
... However, the immunopositivity for GFAP in AD patients was shown to be related to a cross-reactivity of the GFAP antibody with neurofilament-L [24]. This was attributed to a homology between amino acids of the GFAP +1 peptide and the tail domain of neurofilament-L [24]. Therefore, the current study not only performed a standard negative control, but also confirmed the findings applying another antibody batch and even an additional immunofluorescence stain. ...
Glial fibrillary acidic protein (GFAP) is a well-established astrocytic biomarker for the diagnosis, monitoring and outcome prediction of traumatic brain injury (TBI). Few studies stated an accumulation of neuronal GFAP that was observed in various brain pathologies, including traumatic brain injuries. As the neuronal immunopositivity for GFAP in Alzheimer patients was shown to cross-react with non-GFAP epitopes, the neuronal immunopositivity for GFAP in TBI patients should be challenged. In this study, cerebral and cerebellar tissues of 52 TBI fatalities and 17 controls were screened for immunopositivity for GFAP in neurons by means of immunohistochemistry and immunofluorescence. The results revealed that neuronal immunopositivity for GFAP is most likely a staining artefact as negative controls also revealed neuronal GFAP staining. However, the phenomenon was twice as frequent for TBI fatalities compared to non-TBI control cases (12 vs. 6%). Neuronal GFAP staining was observed in the pericontusional zone and the ipsilateral hippocampus, but was absent in the contralateral cortex of TBI cases. Immunopositivity for GFAP was significantly correlated with the survival time (r = 0.306, P = 0.015), but no correlations were found with age at death, sex nor the post-mortem interval in TBI fatalities. This study provides evidence that the TBI-associated neuronal immunopositivity for GFAP is indeed a staining artefact. However, an absence post-traumatic neuronal GFAP cannot readily be assumed. Regardless of the particular mechanism, this study revealed that the artefact/potential neuronal immunopositivity for GFAP is a global, rather than a regional brain phenomenon and might be useful for minimum TBI survival time determinations, if certain exclusion criteria are strictly respected.
... GFAP and neurofilament primary antibodies were selected as the immunohistochemical markers to establish the formation degree of axonal regrowth. When other studies related to neuronal GFAP and neurofilament staining are investigated, 43,44 it has been seen that any labeling associated with newly-formed axons were not achieved ( Figure 6). ...
In this study, we aimed at generating 3‐dimensional (3D) decellularized bovine spinal cord extracellular matrix‐based scaffolds (3D‐dCBS) for neural tissue engineering applications. Within this scope, bovine spinal cord tissue pieces were homogenized in 0.1 M NaOH and this viscous mixture was molded to attain 3D bioscaffolds. After resultant bioscaffolds were chemically crosslinked, the decellularization process was conducted with detergent, buffer, and enzyme solutions. Nuclear remnants in the native tissue and 3D‐dCBS were determined with DNA content analysis and agarose gel electrophoresis. Afterward, 3D‐dCBS were biochemically characterized in depth via glycosaminoglycan (GAG) content, hydroxyproline (HYP) assay, and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Cellular survival of human adipose‐derived mesenchymal stem cells (hAMSCs) on the 3D‐dCBS for 3rd, 7th, and 10th days was assessed via MTT assay. Scaffold and cell/scaffold constructs were also evaluated with scanning electron microscopy and histochemical studies. DNA contents for native and 3D‐dCBS were respectively found to be 520.76 ± 18.11 and 28.80 ± 0.20 ng/mg dry weight (n = 3), indicating a successful decellularization process. GAG content, HYP assay, and SDS‐PAGE results proved that the extracellular matrix was substantially preserved during the decellularization process. In conclusion, it is believed that the novel decellularization method may allow fabricating 3D bioscaffolds with desired geometry from soft nervous system tissues.
... Several studies indicate that astrocytes consist of functionally and morphologically heterogeneous populations of cells, that develop at different times and different locations in the central nervous system (CNS) under the influence of different intrinsic and environmental factors (Bayraktar et al., 2014;Molofsky and Deneen, 2015). Neurological disorders show defects in specific astrocyte subtypes (Martinian et al., 2009;Middeldorp et al., 2009;Diaz-Amarilla et al., 2011). This is especially clear in Vanishing White Matter (VWM), one of the more prevalent leukodystrophies, which is caused by mutations in the EIF2B1-5 genes and for which no treatment is available (Van der Knaap, 2016). ...
Astrocytes gained attention as important players in neurological disease, including a number of leukodystrophies. Several studies explored the generation of induced pluripotent stem cell-derived astrocytes for drug screening and regenerative studies. Developing robust models of patient induced pluripotent stem cells is challenged by high variability due to diverse genetic backgrounds and long-term culture procedures. While human models are of special interest, mouse-based models have the advantage that for them these issues are less pronounced. Here we present astrocyte differentiation protocols for both human and mouse induced pluripotent stem cells to specifically induce grey and white matter astrocytes. Both subtypes expressed astrocyte-associated markers, had typical astrocyte morphologies, and gave a reactive response to stress. Importantly, the grey and white matter-like astrocytes differed in size, complexity of processes, and expression profile, conform primary grey and white matter astrocytes. The newly presented mouse and human stem cell-based models for the leukodystrophy Vanishing White Matter replicated earlier findings, such as increased proliferation, decreased OPC maturation and modulation by hyaluronidase. We studied intrinsic astrocyte subtype vulnerability in Vanishing White Matter in both human and mouse cells. Oligodendrocyte maturation was specifically inhibited in cultures with Vanishing White Matter white matter-like astrocytes. By performing RNA sequencing, we found more differentially regulated genes in the white than in the grey matter-like astrocytes. Human and mouse astrocytes showed the same affected pathways, although human white matter-like astrocytes presented human-specific disease mechanisms involved in Vanishing White Matter. Using both human and mouse induced pluripotent stem cells, our study presents protocols to generate white and grey matter-like astrocytes, and shows astrocyte subtype-specific defects in Vanishing White Matter. While mouse induced pluripotent stem cell-based cultures may be less suitable to mimic human astrocyte subtype- or patient-specific changes, they might more robustly represent disease mutation-related cellular phenotypes as the cells are derived from inbred mice and the protocols are faster. The presented models give new tools to generate astrocyte subtypes for in vitro disease modeling and in vivo regenerative applications.
... Although it was originally considered an astrocyte-specific marker (169), GFAP has subsequently been demonstrated in glial and non-glial cells of the periphery (170)(171)(172)(173). GFAP has been observed in virtually all areas of the brain but is mainly expressed in hippocampal regions (174)(175)(176) as well as the subventricular zone and olfactory system of both non-demented elders and patients with dementia (174)(175)(176)(177). Multiple splice variants exist and in human hippocampal AD tissue many of these isoforms show differential transcript levels (176). ...
... Although it was originally considered an astrocyte-specific marker (169), GFAP has subsequently been demonstrated in glial and non-glial cells of the periphery (170)(171)(172)(173). GFAP has been observed in virtually all areas of the brain but is mainly expressed in hippocampal regions (174)(175)(176) as well as the subventricular zone and olfactory system of both non-demented elders and patients with dementia (174)(175)(176)(177). Multiple splice variants exist and in human hippocampal AD tissue many of these isoforms show differential transcript levels (176). ...
The diagnosis of dementia is challenging and early stages are rarely detected limiting the possibilities for early intervention. Another challenge is the overlap in the clinical features across the different dementia types leading to difficulties in the differential diagnosis. Identifying biomarkers that can detect the pre-dementia stage and allow differential diagnosis could provide an opportunity for timely and optimal intervention strategies. Also, such biomarkers could help in selection and inclusion of the right patients in clinical trials of both Alzheimer’s disease and other dementia treatment candidates. The cerebrospinal fluid (CSF) has been the most investigated source of biomarkers and several candidate proteins have been identified. However, looking solely at protein levels is too simplistic to provide enough detailed information to differentiate between dementias, as there is a significant crossover between the proteins involved in the different types of dementia. Additionally, CSF sampling makes these biomarkers challenging for presymptomatic identification. We need to focus on disease-specific protein fragmentation to find a fragment pattern unique for each separate dementia type – a form of protein fragmentology. Targeting protein fragments generated by disease-specific combinations of proteins and proteases opposed to detecting the intact protein could reduce the overlap between diagnostic groups as the extent of processing as well as which proteins and proteases constitute the major hallmark of each dementia type differ. In addition, the fragments could be detectable in blood as they may be able to cross the blood–brain barrier due to their smaller size. In this review, the potential of the fragment-based biomarker discovery for dementia diagnosis and prognosis is discussed, especially highlighting how the knowledge from CSF protein biomarkers can be used to guide blood-based biomarker development.
... GFAP is an intermediate filament protein which has been described as a marker of reactive astrocytes as well as adult neural stem cells (NSCs) in vivo (Doetsch, Hermann, 2009). The GFAP antiserum (sc-6171, Santa Cruz) stained a major band around 50 kDa in western blot (manufacturer's technical information), and showed a pattern of staining in the rat brain that is similar to that described in a previous report using the same antiserum (Middeldorp et al., 2009). ...
Urotensin II (UII) and Urotensin II-related peptide (URP) are structurally related paralogue peptides which exert peripheral and central effects. UII binding sites have been partly described in brain, and those of URP have never been reported. We exhaustively compared [(125) I]-UII and -URP binding site distributions in the adult rat brain, and found that they fully overlapped at the regional level. We observed UII/URP binding sites in structures lining ventricles, comprising the sphenoid nucleus and cell rafts scattered on a line joining the fourth ventricle and its lateral recess. After injection of UII and URP in the lateral ventricle, we observed c-Fos-positive cell nuclei in areas close to the fourth ventricle, indicating that these receptors are functional. Different c-Fos-containing cell populations were activated. They were all positive for vimentin and GFAP, excluding the possibility of their ependymal nature. In conclusion, this study demonstrated that UII and URP binding sites are totally overlapping and unraveled that these sites were functional in a number of regions bordering the third and fourth ventricles. These data support a role for UII/URP at the interface between brain parenchyma and cerebrospinal fluid. J. Comp. Neurol., 2014. © 2014 Wiley Periodicals, Inc.
... GFAPg lacks exon 1 and includes the last 126 bp of intron 1-2 (Zelenika et al., 1995). Four splice variants, GFAPDEx6, GFAPD164, GFAPD135, and GFAPDEx7, skip sequences in exon 6/7, and were identified in AD tissue, focal lesions assocated with chronic epilepsy, and a specific human astrocyte subtype (Boer et al., 2010; Hol et al., 2003; Kamphuis et al., 2012; Middeldorp et al., 2009b). These splice variants encode for a frameshifted GFAP protein, termed GFAP þ1 , with a frameshifted carboxyterminus against which we have raised a specific antibody named GFAP þ1 (Hol et al., 2003; Middeldorp et al., 2009b). ...
... Four splice variants, GFAPDEx6, GFAPD164, GFAPD135, and GFAPDEx7, skip sequences in exon 6/7, and were identified in AD tissue, focal lesions assocated with chronic epilepsy, and a specific human astrocyte subtype (Boer et al., 2010; Hol et al., 2003; Kamphuis et al., 2012; Middeldorp et al., 2009b). These splice variants encode for a frameshifted GFAP protein, termed GFAP þ1 , with a frameshifted carboxyterminus against which we have raised a specific antibody named GFAP þ1 (Hol et al., 2003; Middeldorp et al., 2009b). In an article by Zelenika et al., a transcript including the last 284 bp of intron 8e9 was reported, which we found to be highly expressed in the mouse brain and which we termed GFAP-zeta (GFAPz) (Kamphuis et al., 2012; Zelenika et al., 1995). ...
... In previous articles, we reported observations on the differential expression of GFAPd (Roelofs et al., 2005; van den Berge et al., 2010) and GFAP þ1 (Boer et al., 2010; Hol et al., 2003; Middeldorp et al., 2009b) in distinct types of astrocytes in the human brain; however, a systematic description of all human GFAP isoforms in relation to AD pathology has not yet been published. To this end, we investigated the changes in GFAP isoform transcript levels and protein distribution in AD tissue and at different stages of the disease. ...
In Alzheimer's disease (AD), amyloid plaques are surrounded by reactive astrocytes with an increased expression of intermediate filaments including glial fibrillary acidic protein (GFAP). Different GFAP isoforms have been identified that are differentially expressed by specific subpopulations of astrocytes and that impose different properties to the intermediate filament network. We studied transcript levels and protein expression patterns of all known GFAP isoforms in human hippocampal AD tissue at different stages of the disease. Ten different transcripts for GFAP isoforms were detected at different abundancies. Transcript levels of most isoforms increased with AD progression. GFAPδ-immunopositive astrocytes were observed in subgranular zone, hilus, and stratum-lacunosum-moleculare. GFAPδ-positive cells also stained for GFAPα. In AD donors, astrocytes near plaques displayed increased staining of both GFAPα and GFAPδ. The reading-frame-shifted isoform, GFAP(+1), staining was confined to a subset of astrocytes with long processes, and their number increased in the course of AD. In conclusion, the various GFAP isoforms show differential transcript levels and are upregulated in a concerted manner in AD. The GFAP(+1) isoform defines a unique subset of astrocytes, with numbers increasing with AD progression. These data indicate the need for future exploration of underlying mechanisms concerning the functions of GFAPδ and GFAP(+1) isoforms in astrocytes and their possible role in AD pathology.
... The rabbit anti-cow GFAP antibody reacted with the homogenates to reveal several bands in each lane (rGFAP, Figure 4.4). Two bands were detected in the rat lane, the major band being ≈52 kDa, the other close to 70 kDa, possibly corresponding with neurofilament L which polyclonal GFAP antibodies are known to cross-react with [399]. A 50 kDa band is detected in the zebrafish lane with additional larger bands. ...
... However, the polyclonal anti-GFAP does cross-react with proteins of approximately 61, 69 and 114 kDa as predicted from [398,401]. Middledorp et al. [399] observed GFAP expression in degenerating neurons in culture using the Z0334 polyclonal GFAP antibody. This antibody was reacting against neurofilament L, a ≈68 kDa protein in humans. ...
... Antibodies specific to each band are essential to allow for cytological studies, not only to observe where the various isoforms localize to but how they interact with the cytoskeleton. More interesting, is determining whether there are radial glia subtypes [26,399] in Nothobranchius which may play different physiological roles. In light of the glial simplicity and accessibility of this model organism it could be exceedingly useful in learning the roles of the various glial subtypes (should they exist in the fish as in mammals). ...
This thesis details a study into aging-related neurodegeneration of Nothobranchius guentheri and the affect of resveratrol-treatment on this neurodegeneration. The goal of the study was to identify cytological probes by whichto study neurodegeneration and use these to deliver novel findings pertaining to Nothobranchius aging and resveratrol induced neuroprotection. The SMI31 (against phosphorylated neurofilamant protein), GA-5 anti-
GFAP (against glial fibrillary acid protein in radial glia), DAKO Z0334 anti-GFAP, anti-TNR antibodies (against Tenascin-R on oligodendrocytes) and E587 antiserum (against goldfish L1 which is implicated in synaptic plasticity) were shown to react against Nothobranchius tissue with high fidelity. By western blot and immunofluorescent microscopy evidence is presented that these antibodies are identifying the corresponding epiptopes in Nothobranchius as other species. BS-I Isolectin was shown to bind to microglia in the optic nerve and retina but not the brain (where no microglia have thus far been detected). Using the SMI31 antibody we demonstrate that neuronal density declines with age in the stratum griseum superficale of the optic tectum. Resveratrol-treatment did not decrease this neuron loss even though it extended lifespan of N. guentheri. Using the E587 antiserum it was shown that resveratrol-treated fish had more E587 immunoreactive neurons than age-matched controls. The E587 antiserum was used in conjunction with the GA-5 antibody to label and
count radial glia in the stratum griseum superficiale. It was found that the density of radial glia declined with age but was rescued by resveratrol-treatment. Western Blots indicated that the ratios of the GFAP isoforms change with age and resveratrol-treatment maintains youthful GFAP isoform proportions. The density of perineuronal nets, using the anti-TNR antibody, was also observed to decline with age. Resveratrol-treatment protected against this decline. Five antibodies and a lectin were tested against Nothobranchius guentheri tissue and found to be useful. Using these antibodies novel data was gathered suggesting that resveratrol lifespan extension is not strictly associated with the preservation of neurons into old age but is associated with the maintenance of synaptic plasticity.
https://open.uct.ac.za/handle/11427/4401
... However, weak expression has been reported by others (Pirker et al., 2000). Cross-reactivity in the immunohistochemical techniques or the dissecting procedure could lead to false-positive detection (Dolman et al., 2004;Holmseth et al., 2005;Pláteník et al., 2005;Middeldorp et al., 2009). ...
The nucleus accumbens (NAc) has emerged as an important part of the neural circuitry regulating depressive-like behaviors. Given that the NAc GABAergic medium spiny neurons project to the ventral pallidum (VP), it is reasonable to suggest that the VP may also be involved in these behaviors. Consequently, we explored the role of the VP GABAergic terminals during depressive-like behaviors in rats using the forced swim test (FST) and the sucrose preference test (SPT). Microdialysis coupled with micellar electrokinetic chromatography was used to monitor in vivo changes of GABA in the VP during the FST. GABA levels significantly increased during day-1 and day-2 during swimming, returning to the pre-swimming levels after the test. Basal concentrations of GABA on day-2 of the FST significantly increased with respect to day-1. In another set of experiments, intra-VP injections of vigabatrin (a GABA transaminase inhibitor) increased extracellular GABA and immobility behaviors in the FST while the direct GABAA receptor antagonist bicuculline reduced immobility behaviors. In the SPT, intra-VP vigabatrin injection significantly reduced preference for sucrose while bicuculline did not produce any change. At the postsynaptic side, we used semiquantitative RT-PCR to measure mRNA expression of 17 GABAA receptor subunits (α1-α6, β1-β3, γ2, δ, ε, θ, π, and ρ1-ρ3) in rats subjected to the FST. We found a significant reduction of α3 and γ2 subunit expression and an increase of δ subunit expression after day-2 in rats subject to the FST which might enhance tonic inhibition of the VP. Furthermore, immunoblot experiments revealed that protein expression of γ2 and δ subunits changed 6 days after FST in a way similar to mRNA expression. These results suggest that the enhanced VP-GABAergic tone might trigger a low motivational state, anhedonia and a possible memory mechanism for unpleasant experiences.
... GFAP, the glial fibrillary acidic protein, encodes one of the major intermediate filament proteins of mature astrocytes. Besides its use as a marker to distinguish astrocytes from other glial cells during development [47,48] , GFAP is expressed in the neurons of hippocampus of AD patients495051. CTSB, the lysosomal cysteine proteinase, is known as an amyloid precursor protein (APP) secretase and it is involved in the proteolytic processing of APP. Indeed, the incomplete proteolytic processing of APP is the most known and important causative factor in AD [52]. ...
A molecular characterization of Alzheimer's Disease (AD) is the key to the identification of altered gene sets that lead to AD progression. We rely on the assumption that candidate marker genes for a given disease belong to specific pathogenic pathways, and we aim at unveiling those pathways stable across tissues, treatments and measurement systems. In this context, we analyzed three heterogeneous datasets, two microarray gene expression sets and one protein abundance set, applying a recently proposed feature selection method based on regularization.
For each dataset we identified a signature that was successively evaluated both from the computational and functional characterization viewpoints, estimating the classification error and retrieving the most relevant biological knowledge from different repositories. Each signature includes genes already known to be related to AD and genes that are likely to be involved in the pathogenesis or in the disease progression. The integrated analysis revealed a meaningful overlap at the functional level.
The identification of three gene signatures showing a relevant overlap of pathways and ontologies, increases the likelihood of finding potential marker genes for AD.
