Dennis A. Aquino

Quantitative aspects of reactive gliosis a review

Recent studies of gliosis in a variety of animal models are reviewed. The models include brain injury, neurotoxic damage, genetic diseases and inflammatory demyelination. These studies show that reactive gliosis is not a stereotypic response, but varies widely in duration, degree of hyperplasia, and time course of expression of GFAP immunostaining, content and mRNA. We conclude that there are different biological mechanisms for induction and maintenance of reactive gliosis, which, depending on the kind of tissue damage, result in different expressions of the gliotic response.

Heat Shock Protein 70 Suppresses Astroglial-inducible Nitric-oxide Synthase Expression by Decreasing NFκB Activation

In brain glial cells, expression of calcium independent nitric-oxide synthase (NOS-2) is induced following stimulation with bacterial endotoxin (lipopolysaccharide (LPS)) and/or pro-inflammatory cytokines. We have investigated the effects of heat shock (HS), which can reduce inflammatory responses in several cell types, on the induction of glial NOS-2 expression. Preincubation of cells for 20-60 min at 43°C decreased subsequent levels of NOS-2 induction, with a maximal 80% reduction after 60 min of HS. Following HS, cells were refractory to NOS inducers for up to 4 h, after which time little or no suppression was observed. HS reduced cytosolic NOS-2 enzymatic activity (3-fold), steady state mRNA levels (2-3-fold), and gene promoter activity (by 50%). HS also reduced LPS-induced nuclear accumulation of transcription factor NFκB p65 subunit, suggesting perturbation of NFκB activation. A role for HS protein (HSP) 70 in NOS-2 suppression by HS is supported by the demonstration that 1) transfection with human HSP70 cDNA partially replicated HS effects; 2) antisense, but not sense, oligonucleotides directed against rat HSP70 partially blocked HS effects; and 3) rat fibroblasts stably expressing human HSP70 did not express NOS-2 in response to LPS plus cytokines. As with heat-shocked cells, HSP70-expressing cells also exhibited decreased NFκB p65 subunit nuclear accumulation. These results demonstrate that in glial cells, as well as other cell types, NOS-2 induction can be modulated by the HS response, mediated at least in part by HSP70 expression.

Multiple sclerosis: altered expression of 70- and 27-kDa heat shock proteins in lesions and myelin.

Recent studies have implicated heat shock proteins (HSP) in the pathogenesis of the multiple sclerosis (MS) lesion. Expression of the 73 kDa constitutive HSP (HSC70), the 72 kDa stress-inducible HSP (HSP70), and the 27 kDa small HSP (HSP27) was analyzed in white matter and myelin from central nervous system (CNS) tissue of MS and normal subjects using a combination of immunocytochemistry and quantitative immunoblotting. Plaques of all types were sharply defined by reduced immunostaining for HSC70, and shown by immunoblotting to contain 30 to 50% less HSC70 than surrounding white matter or normal tissue. In contrast, HSP27 was markedly enhanced 2.5- to 4-fold in plaque regions, especially in fibrous astrocytes and in hyperplastic interfascicular oligodendrocytes at the lesion edge. HSP70 was less abundant than HSC70, and no significant differences in HSP70 levels were noted between MS and normal white matter. Myelin isolated from active plaques contained 3- to 4-fold more HSC70 than normal myelin. Pronounced expression of HSP70 and HSP27 was also found in MS myelin, although neither protein was detected in normal myelin. Thus, white matter undergoing immune-mediated destruction in MS was associated with altered distribution and expression of HSC70 and HSP27. These changes may initially serve to protect myelin from further destruction and facilitate repair; however, enhanced expression of HSC70, HSP70, and HSP27 in myelin may subsequently present as additional immune targets involved in the progression of disease.

Glial fibrillary acidic protein increases in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis.

Abstract: Glial fibrillary acidic protein (GFAP) in the spinal cords of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) was quantitated by densi‐tometry of both stained gels and immunoblots of electro‐phoretically separated cytoskeletal proteins. The experimental period ranged from 7 to 65 days postinocula‐tion (dpi). Greater than 92% of the total spinal cord GFAP was recovered in the Triton‐insoluble cytoskeletal pellet; less than 2% was truly soluble. GFAP increased gradually and significantly with time, reaching a level one‐and‐a‐half to two times greater than that of controls by 35 dpi and remaining elevated at 65 dpi. In EAE animals, GFAP was 33% of the total Triton‐insoluble protein (excluding his‐tones and other small basic proteins) at 7 dpi, rising to 48% at 65 dpi. Increases in vimentin were also noted, following a time course similar to that of GFAP. An increase in immu‐nocytochemical staining of GFAP was noticeable at 10 dpi and became marked at 14 dpi, a time before GFAP levels had increased significantly. Thus, enhanced staining at the peak of the disease cannot be explained simply by an increase in antigen protein. Other possible explanations, such as an increase in soluble GFAP content, proteolytic degradation, or modifications in the immunochemical properties of GFAP in EAE animals, were ruled out. Both the biochemical and immunocytochemical increases in GFAP persisted through 65 dpi, even though the animals recovered from clinical signs at approximately 18 dpi.

Tumor necrosis factor-induced proliferation of astrocytes from mature brain is associated with down-regulation of glial fibrillary acidic protein mRNA

Previous results from this laboratory have shown that tumor necrosis factor (TNF) is mitogenic for bovine astrocytes in chemically defined (CO) medium. The maximum mitogenic response was detected with 200 U/ml at 48 h. We have now extended these studies to assess the effect of TNF on message levels for the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The results have shown that, whereas TNF had only a slight effect on vimentin mRNA, TNF induced a marked decrease to 4.3 ± 2.0% of controls in GFAP mRNA which was both time and dose dependent. The lowest effective dose was 50 U/ml and the maximal effective dose was 200 U/ml. Kinetic analysis of this response demonstrated that a marked decrease in GFAP mRNA was present at 12 h and continued to decrease through 72 h. To determine the reversibility of the TNF effect, astrocyte cultures were exposed to 200 U/ml TNF for varying periods of tee and then cultured in fresh CD medium. A 1‐h pulse with TNF was sufficient to reduce GFAP mRNA levels when measured 24 h later. However, cultures incubated with 200 U/ml TNF for 48 h followed by incubation in CD medium without TNF for 7 days showed that GFAP mRNA levels had returned to 60% of the control values. Nuclear runoff assays showed that the effect of TNF on GFAP mRNA was at the posttranscriptional level. Polyacrylamide gel electrophoretic analysis of astrocyte cytoskeletal proteins demonstrated that GFAP levels were reduced after a 5‐day incubation with 200 U/ml TNF whereas protein levels of vimentin and actin were not significantly changed. Western blots confirmed that GFAP levels were reduced to 36% of the control values. Thus the effect of TNF on GFAP mRNA expression was not due to a generalized effect on intermediate filament metabolism.

GFAP mRNA levels following stab wounds in rat brain

We previously reported that glial fibrillary acidic protein (GFAP) levels increased significantly at 3 days after stab wounds, relative to sham-operated controls, reaching a maximum of 200% of control value at 5-7 days. They then fell to near-normal values by 21 days. To determine whether these protein changes correlated with changes in GFAP mRNA we performed Northern blot analyses. Total RNA, isolated from lesioned, sham-operated and intact rat forebrains, was hybridized with 32P-labeled mouse GFAP cDNA and quantified by densitometry. The maximum increase in total RNA content in lesioned animals was only 20% over controls at 12 h. GFAP mRNA levels increased to 2-fold control values at 6 h and reached 5-fold at 12 h. Thereafter they remained at 3.5- to 6-fold until 5 days and then declined to 1.5-fold by 21 days. The rapid increase of GFAP message at 12 h preceded a significant increase in GFAP by 2 days and the decrease of message after 5 days was more precipitate than the slow decrease in GFAP content. Sham-operated animals showed no significant changes in GFAP mRNA, compared to intact controls, during the period 3 h to 14 days postoperation. GFAP mRNA and GFAP in the stab-wound model reached levels similar to those found in the experimental autoimmune encephalomyelitis (EAE) model, but returned to normal much more rapidly.

Procedure for isolation of gangliosides in high yield and purity: simultaneous isolation of neutral glycosphingolipids

While several methods for ganglioside extraction and isolation have been described, relatively little attention has been given to the effectiveness of separation from peptides, phospholipids, and various low-molecular-weight contaminants. A procedure is described for isolation of gangliosides in high purity and good yield from 1- to 400-mg samples (wet wt). A key step was mild acidification following homogenization, designed to dissociate gangliosides from lipophilic peptides which coextracted into organic solvents. This has proved particularly helpful for myelin and myelin-containing tissues (e.g., white matter, nerve) whose proteins have presented special problems in ganglioside isolation. In this study isolation was effected by consecutive chromatographies on Sephadex LH-20, DEAE-Sephadex, and silica gel following the initial acidification. The method applied to bovine white matter gave tissue concentrations (calculated from yields and radiolabeled tracer recoveries) that were similar to those obtained with three previously described procedures; however, peptide contaminants were an order of magnitude lower. Removal of low-molecular-weight contaminants, including nucleotide sugars, was virtually complete. In addition to ganglioside isolation the method can be used to obtain neutral glycosphingolipids as well. It is believed to have broad applicability to a diversity of tissues.

Expression of Glial Fibrillary Acidic Protein and Neurofilament mRNA in Gliosis Induced by Experimental Autoimmune Encephalomyelitis

We have previously shown that the content of glial fibrillary acidic protein (GFAP) gradually increases in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE), reaching a level 1.5–2 times greater than that in controls by 35 days postimmunization (dpi). We report here that the increase in GFAP mRNA level followed a completely different time course and reached higher levels relative to controls than did that of the protein. Total RNA was isolated using a modified version of current methods using phenol/chloroform extractions to ensure optimal recovery from spinal cord. Control animals yielded 323 ± 35 μg (mean ± SD; n = 34) of total RNA/spinal cord throughout the experimental period. EAE animals contained up to three times as much total RNA during 11–14 dpi, a finding largely reflecting the infiltration of inflammatory cells. By 65 dpi, total RNA levels closely approached control values. As early as 10 dpi, increased amounts of GFAP mRNA were detected in EAE animals relative to controls. During 11–14 dpi, GFAP mRNA levels reached six‐ to eightfold greater than values in controls and then slowly declined throughout the remainder of the time course, with a fourfold increase still detected at 65 dpi. However, coinciding with the height of inflammation and clinical signs at 12 dpi, the GFAP mRNA content dropped to ∼50% of the level at 11 dpi but rose again at 13 dpi. This dip was mirrored by a similar decrease in neurofilament mRNA content, but otherwise the level of this message remained relatively constant and equal to that in controls. The changes in GFAP message content contrasted sharply with those in protein content, which increases slowly and does not reach maximal levels until after 4 weeks. Elevated levels of GFAP mRNA, protein, and immunostaining persisted through 65 dpi, >40 days after clinical signs had resolved.

The constitutive heat shock protein-70 is required for optimal expression of myelin basic protein during differentiation of oligodendrocytes

To further elucidate the role of the constitutive heat shock protein-70 (HSC70) as a chaperone for the synthesis of myelin basic protein (MBP), HSC70 content was decreased in oligodendrocyte precursor cells prior to MBP expression either by transfection with an antisense oligonucleotide specific for HSC70, or by exposure to low levels of quercetin, a bioflavonoid known to decrease synthesis of HSC70. As these cells underwent differentiation in vitro, antisense treatment decreased HSC70 levels to 66% of controls. At the same time, a sharp induction resulted in the stress-inducible heat shock protein-70 (HSP70). Levels of two other stress proteins increased as well, namely, the 25-kDa heat shock protein (HSP25) and the 78-kDa glucose regulated protein (GRP78). MBP synthesis proceeded over a normal time course, but at only 50% of control values. As HSC70 content returned to normal, MBP synthesis was also restored to normal levels. Quercetin reduced the expression of HSC70 to an even greater extent than transfection, and prevented the induction of HSP70. In contrast to antisense-treated cells, MBP synthesis was essentially blocked in quercetin-treated cells even though levels of HSP25 and GRP78 increased. Taken together, these observations (a) indicate that HSP70 partially compensates for decreased chaperoning of nascent MBP by HSC70 (HSC70 and HSP70 are closely related and perform similar functions); (b) preclude the involvement of HSP25 and GRP78 in MBP synthesis; and (c) emphasize the requirement of HSC70 for optimal synthesis of MBP.

The 70‐kDa Heat Shock Cognate Protein (HSC70) Is a Major Constituent of the Central Nervous System and Is Up‐Regulated Only at the mRNA Level in Acute Experimental Autoimmune Encephalomyelitis

Abstract: The expression of the 70‐kDa heat shock cognate (HSC70) and stress‐inducible (HSP70) proteins, and their mRNAs, was examined in experimental autoimmune encephalomyelitis, a model of inflammatory demyelination in the CNS. This study was undertaken as an extension of previous work demonstrating an abrupt decline in mRNA levels of both glial fibrillary acidic protein and the low‐molecular‐weight neurofilament subunit in experimental autoimmune encephalomyelitis spinal cord at 12 days after inoculation, the height of inflammation and clinical signs. Using the same total RNA preparations as our previous study, we report here that mRNA levels for HSC70 increased approximately sixfold over control values at the same time that glial fibrillary acidic protein and low‐molecular‐weight neurofilament subunit messages decreased and were similar to controls by 21 days after inoculation. In situ hybridization experiments showed that HSC70 mRNA was predominantly expressed in neurons and that the influx of inflammatory cells into the CNS was not responsible for the large increase in HSC70 message. Despite this elevation in mRNA, only small (if any) increases in protein levels for HSC70 were detected by both western blotting and in vitro cell‐free translation systems. However, by quantitative immunoblotting, we determined that constitutive levels of HSC70 comprised a substantial portion of CNS proteins, representing 2–3% of the total protein content of spinal cord. Immunohistochemical staining illustrated that the distribution of HSC70 was consistent with that of its message. In contrast, no HSP70 mRNA or protein was detected in either control or experimental animals.