Caspase-Cleaved Glial Fibrillary Acidic Protein Within Cerebellar White Matter of the Alzheimer\u27s Disease Brain

Although the cerebellum is generally thought of as an area spared of Alzheimer\u27s disease (AD) pathology, recent evidence suggests that balance and mobility dysfunction may be magnified in affected individuals. In the present study, we sought to determine the degree of pathological changes within the cerebellum utilizing an antibody that specifically detects caspase-cleaved GFAP within degenerating astrocytes. Compared to control subjects, application of this antibody, termed the GFAP caspase-cleavage product (GFAPccp) antibody, revealed widespread labeling in cerebellar white matter with little staining observed in grey matter. Staining was observed within damaged astrocytes, was often localized near blood vessels and co-localized with other markers of apoptosis including TUNEL and caspase-cleaved tau. Of interest was the association of beta-amyloid deposition in white matter together with GFAPccp in cerebellar AD sections. In contrast, utilizing the tangle marker, PHF-1, neuritic pathology was completely absent in AD cerebellar sections. It is suggested that the observed pathological changes found in the white matter of the cerebellum may contribute to the declined motor performance in AD

Identification of an Amino-Terminal Fragment of Apolipoprotein E4 that Localizes to Neurofibrillary Tangles of the Alzheimer’s Disease Brain

Although the risk factor for harboring the apolipoprotein E4 (apoE4) allele in late-onset Alzheimer’s disease (AD) is well known, the mechanism by which apoE4 contributes to AD pathogenesis has yet to be clarified. Preferential cleavage of the ApoE4 isoform relative to other polymorphic forms appears to be significant, as the resulting fragments are associated with hallmarks of AD. To examine the possible role of apoE4 proteolysis in AD, we designed a site-directed antibody directed at position D172, which would yield a predicted amino-terminal fragment previously identified in AD brain extracts. Western blot analysis utilizing this novel antibody, termed the amino-terminal apoE4 cleavage fragment (nApoE4CF) Ab consistently identified the predicted amino-terminal fragment (~18 kDa) in several commercially available forms of human recombinant apoE4 purified from E. coli. Mass spectrometry confirmed the identity of this 18 kDa fragment as being an amino-terminal fragment of apoE4. Immunohistochemical experiments indicated the nApoE4CF Ab specifically labeled neurofibrillary tangles (NFTs) in AD frontal cortex sections that colocalized with the mature tangle marker PHF-1. Taken together, these results suggest a novel cleavage event of apoE4, generating an amino-terminal fragment that localizes within NFTs of the AD brain

Immunolocalization of an Amino-Terminal Fragment of Apolipoprotein E in the Pick\u27s Disease Brain

Although the risk factor for apolipoprotein E (apoE) polymorphism in Alzheimer\u27s disease (AD) has been well described, the role that apoE plays in other neurodegenerative diseases, including Pick\u27s disease, is not well established. To examine a possible role of apoE in Pick\u27s disease, an immunohistochemical analysis was performed utilizing a novel site-directed antibody that is specific for an amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE cleavage fragment (nApoECF) antibody, consistently labeled Pick bodies within area CA1 of the hippocampus in 4 of the 5 cases examined. Co-localization of the nApoECF antibody with PHF-1, a general marker for Pick bodies, as well as with an antibody to caspase-cleaved tau (TauC3) was evident within the hippocampus. While staining of the nApoECF antibody was robust in area CA1, little co-localization with PHF-1 in Pick bodies within the dentate gyrus was observed. A quantitative analysis indicated that approximately 86% of the Pick bodies identified in area CA1 labeled with the nApoECF antibody. The presence of truncated apoE within Pick bodies suggests a broader role of apoE beyond AD and raises the question as to whether this protein contributes to pathogenesis associated with Pick\u27s disease

Immunolocalization of Influenza A Virus and Markers of Inflammation in the Human Parkinson's Disease Brain

Although much is known regarding the molecular mechanisms leading to neuronal cell loss in Parkinson's disease (PD), the initiating event has not been identified. Prevailing theories including a chemical insult or infectious agent have been postulated as possible triggers, leading to neuroinflammation. We present immunohistochemical data indicating the presence of influenza A virus within the substantia nigra pars compacta (SNpc) from postmortem PD brain sections. Influenza A virus labeling was identified within neuromelanin granules as well as on tissue macrophages in the SNpc. Further supporting a role for neuroinflammation in PD was the identification of T-lymphocytes that colocalized with an antibody to caspase-cleaved Beclin-1 within the SNpc. The presence of influenza A virus together with macrophages and T-lymphocytes may contribute to the neuroinflammation associated with this disease

Caspase-cleaved glial fibrillary acidic protein within cerebellar white matter of the Alzheimer's disease brain

Although the cerebellum is generally thought of as an area spared of Alzheimer’s disease (AD) pathology, recent evidence suggests that balance and mobility dysfunction may be magnified in affected individuals. In the present study, we sought to determine the degree of pathological changes within the cerebellum utilizing an antibody that specifically detects caspase-cleaved GFAP within degenerating astrocytes. Compared to control subjects, application of this antibody, termed the GFAP caspase-cleavage product (GFAPccp) antibody, revealed widespread labeling in cerebellar white matter with little staining observed in grey matter. Staining was observed within damaged astrocytes, was often localized near blood vessels and co-localized with other markers of apoptosis including TUNEL and caspase-cleaved tau. Of interest was the association of beta-amyloid deposition in white matter together with GFAPccp in cerebellar AD sections. In contrast, utilizing the tangle marker, PHF-1, neuritic pathology was completely absent in AD cerebellar sections. It is suggested that the observed pathological changes found in the white matter of the cerebellum may contribute to the declined motor performance in AD

Optimal 3D Culture of Primary Articular Chondrocytes for Use in the Rotating Wall Vessel Bioreactor

Introduction: Reliable culturing methods for primary articular chondrocytes are essential to study the effects of loading and unloading on joint tissue at the cellular level. Due to the limited proliferation capacity of primary chondrocytes and their tendency to dedifferentiate in conventional culture conditions, long-term culturing conditions of primary chondrocytes can be challenging. The goal of this study was to develop a suspension culturing technique that not only would retain the cellular morphology, but also maintain the gene expression characteristics of primary articular chondrocytes. Methods: Three-dimensional culturing methods were compared and optimized for primary articular chondrocytes in the rotating wall vessel bioreactor, which changes the mechanical culture conditions to provide a form of suspension culture optimized for low shear and turbulence. We performed gene expression analysis and morphological characterization of cells cultured in alginate beads, Cytopore-2 microcarriers, primary monolayer culture, and passaged monolayer cultures using reverse transcription-PCR and laser scanning confocal microscopy. Results: Primary chondrocytes grown on Cytopore-2 microcarriers maintained the phenotypical morphology and gene expression pattern observed in primary bovine articular chondrocytes, and retained these characteristics for up to 9 d. Discussion: Our results provide a novel and alternative culturing technique for primary chondrocytes suitable for studies that require suspension such as those using the rotating wall vessel bioreactor. In addition, we provide an alternative culturing technique for primary chondrocytes that can impact future mechanistic studies of osteoarthritis progression, treatments for cartilage damage and repair, and cartilage tissue engineering

Immunolocalization of an Amino-Terminal Fragment of Apolipoprotein E in the Pick's Disease Brain

Although the risk factor for apolipoprotein E (apoE) polymorphism in Alzheimer's disease (AD) has been well described, the role that apoE plays in other neurodegenerative diseases, including Pick's disease, is not well established. To examine a possible role of apoE in Pick's disease, an immunohistochemical analysis was performed utilizing a novel site-directed antibody that is specific for an amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE cleavage fragment (nApoECF) antibody, consistently labeled Pick bodies within area CA1 of the hippocampus in 4 of the 5 cases examined. Co-localization of the nApoECF antibody with PHF-1, a general marker for Pick bodies, as well as with an antibody to caspase-cleaved tau (TauC3) was evident within the hippocampus. While staining of the nApoECF antibody was robust in area CA1, little co-localization with PHF-1 in Pick bodies within the dentate gyrus was observed. A quantitative analysis indicated that approximately 86% of the Pick bodies identified in area CA1 labeled with the nApoECF antibody. The presence of truncated apoE within Pick bodies suggests a broader role of apoE beyond AD and raises the question as to whether this protein contributes to pathogenesis associated with Pick's disease

Co-localization of the nApoECF antibody with caspase-cleaved tau.

<p>(<b>A–C</b>): Representative immunofluorescence double-labeling in Case #4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080180#pone-0080180-t001" target="_blank">Table 1</a>) within the CA1 region of the hippocampus in Pick's disease utilizing the nApoECF antibody (green, Panel A), caspase-cleaved tau (red, Panel B) and with the overlap image shown in Panel C. All scale bars represent 10 µm. (<b>D</b>): Quantification of Pick bodies double-labeled by TauC3 (caspase-cleaved tau) and nApoECF in area CA1 of the hippocampus. Data show the number of Pick bodies labeled with nApoECF alone (blue bar), Tauc3 alone (green bar) or those Pick bodies that were labeled with both antibodies (red bar). Pick bodies were identified in a 20× field within area CA1 by immunofluorescence overlap microscopy (n = 3 fields for 4 different Pick cases) ±S.D., *p<0.05. Data indicated that roughly 75% of all labeled Pick bodies within area CA1 were co-localized with both antibodies. (<b>E–G</b>): Double-labeled confocal immunofluorescence images with the nApoECF antibody (green, E), PHF-1 (red, F), and the two images overlapped (G). (<b>H</b>): High magnification confocal overlapped image representing nApoECF (green channel, left), TauC3 (red channel, middle), and overlapped image (yellow/orange, right). Scale bars in Panel H represent 5 µm.</p

Case Demographics.

<p>PMI, postmortem interval in hours; NPD, neuropathological diagnosis.</p