Paul E. Gottschall

A|[beta]| peptide vaccination prevents memory loss in an animal model of Alzheimer's disease

Vaccinations with amyloid-β peptide (AB) can dramatically reduce amyloid deposition in a transgenic mouse model of Alzheimers disease. To determine if the vaccinations had deleterious or beneficial functional consequences, we tested eight months of Aβ vaccination in a different transgenic model for Alzheimers disease in which mice develop learning deficits as amyloid accumulates . Here we show that vaccination with Aβ protects transgenic mice from the learning and age-related memory deficits that normally occur in this mouse model for Alzheimers disease. During testing for potential deleterious effects of the vaccine, all mice performed superbly on the radial-arm water-maze test of working memory. Later, at an age when untreated transgenic mice show memory deficits, the Aβ-vaccinated transgenic mice showed cognitive performance superior to that of the control transgenic mice and, ultimately, performed as well as nontransgenic mice. The Aβ-vaccinated mice also had a partial reduction in amyloid burden at the end of the study. This therapeutic approach may thus prevent and, possibly, treat Alzheimers dementia.

Cytokines regulate gelatinase A and B (matrix metalloproteinase 2 and 9) activity in cultured rat astrocytes.

Abstract: Under a tightly regulated expression mechanism, matrix metalloproteinases degrade extracellular matrix proteins and are though to play a role in injury repair and tumor metastasis in peripheral tissues. Little is known about the function of matrix metalloproteinases or agents that regulate their production in adult brain; however, it has been shown that the activity of a calcium‐dependent metalloproteinase is elevated in Alzheimers hippocampus. The goals of this study were to determine whether cultured rat astrocytes produce matrix metalloproteinases and to identify agents that regulate protease activity. Enriched astrocyte cultures were prepared from brains of 1‐day‐old rat pups, and experiments were performed 13 days later. Gelatinase activity in astrocyte conditioned medium was determined using zymography with gelatin copolymerized with acrylamide in the gel. Under basal conditions after a 24‐h incubation, rat astrocytes produce gelatinases of 58 and 66 kDa. On stimulation of astrocytes with lipopolysaccharide, interleukin‐1α or ‐β, or tumor necrosis factor‐α for 24 h, a dose‐dependent increase in the activity of the 58‐ and 66‐kDa gelatinases and the induction of a 94‐kDa gelatinase occurred. All three astrocyte‐derived proteases showed maximal activity in the presence of millimolar levels of Ca2+, their activity was inhibited in the presence of 1,10‐phenanthroline, and their proenzymes were cleaved and activated after incubation with p‐aminophenylmercuric acetate. Using immunoblotting, immunopositive bands at the respective molecular sizes indicated that the 58‐kDa gelatinase was gelatinase A (matrix metalloproteinase 2) and the 94‐kDa activity was gelatinase B (matrix metalloproteinase 9). Induction of the 94‐kDa gelatinase by lipopolysaccharide was not influenced when interleukin‐1 receptor antagonist was included during the 24‐h incubation period; however, the antagonist completely blocked interleukin‐1β‐induced 94‐kDa activity and diminished the activity of the 58‐ and 66‐kDa gelatinases. Dexamethasone inhibited both lipopolysaccharide and interleukin‐1β stimulation of the 94‐kDa gelatinase. These results indicate that cytokines regulate matrix metalloproteinase expression in cultured rat astrocytes. Because astrocytes become “activated” (are hypertrophic and express increased levels of glial fibrillary acidic protein) in the presence of several inflammatory cytokines, it is possible that these astrocyte‐derived enzymes contribute to the activation process and may participate in tissue remodeling after brain injury.

Activation of the proteolytic activity of ADAMTS4 (aggrecanase-1) by C-terminal truncation.

Proteolysis of the hyalectans (aggrecan, versican, brevican) in vivo appears to result from the activity of ADAMTS4 (aggrecanase-1, herein referred to as an hyalectanase). To examine the mode of activation of ADAMTS4, a human chondrosarcoma cell line, JJ012, has been stably transfected with the full-length c-DNA for human ADAMTS4. The cells synthesized a high molecular weight form of the enzyme (p100), which in serum-free culture was processed to three truncated forms, p75, p60, and p50. Treatment of the p100 form with recombinant furin indicated that the p75 form is generated by the removal of the prodomain by a furin-like activity. Analysis with domain-specific antisera showed that the p60 and p50 forms are generated by C-terminal truncation of the p75 form. The appearance of the p60 and p50 forms in culture medium was prevented by inclusion of a furin inhibitor, inhibitors of glycosylphosphatidylinositol synthesis, glucosamine, a hydroxamate-based matrix metalloproteinase (MMP) inhibitor, and TIMP-1, but not by AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride) or E64. Only medium samples containing the p60/p50 forms exhibited aggrecanase activity, and isolation of the p75, p60, and p50 forms by preparative SDS-PAGE showed that only p60 and p50 were active in aggrecanase and versicanase assays. Pig synovium and human cartilages also contained ADAMTS4 in the p75, p60, and p50 forms. We suggest thatin vivo production of proteolytically active ADAMTS4 requires not only removal of the prodomain by a furin-like activity but also MMP-mediated removal of a portion of the C-terminal spacer domain.

Deglycosylated anti-amyloid-beta antibodies eliminate cognitive deficits and reduce parenchymal amyloid with minimal vascular consequences in aged amyloid precursor protein transgenic mice.

Systemic administration of anti-amyloid-β (Aβ) antibodies results in reduced parenchymal amyloid but increased vascular amyloid and microhemorrhage in amyloid precursor protein (APP) transgenic mice. Here, we evaluate the effects of reducing effector interactions of the antibody via deglycosylation. Mice aged 20 months were treated weekly for 4 months and tested behaviorally before they were killed. APP transgenic mice receiving either anti-Aβ (2H6) or deglycosylated anti-Aβ (de-2H6) showed significant improvement in radial arm water maze performance compared with mice receiving a control antibody. Both groups receiving anti-Aβ antibodies showed significant reductions in total Aβ immunochemistry and Congo red. Significantly fewer vascular amyloid deposits and microhemorrhages were observed in mice administered the de-2H6 antibody compared with those receiving unmodified 2H6 antibody. Deglycosylated anti-Aβ antibodies may be preferable to unmodified IgG because they retain the cognition-enhancing and amyloid-reducing properties of anti-Aβ immunotherapy, while greatly attenuating the increased vascular amyloid deposition and microhemorrhage observed with unmodified IgG.

Identification of a high-affinity receptor for interleukin-1 beta in rat brain

A single type of high-affinity binding sites for IL-1 beta was identified in the rat hypothalamus (Kd = 1.0 +/- 0.2 nM) and cerebral cortex (Kd = 1.3 +/- 0.2 nM), but not in the pituitary. The maximum binding capacity (Bmax) in the hypothalamus (Bmax = 75.4 +/- 10.8 fmol/mg protein) was 4 times greater than in the cerebral cortex (Bmax = 17.2 +/- 1.5 fmol/mg protein). Neither various neuropeptides nor IL-2 appeared to influence the binding of [125I]IL-1 beta to the hypothalamic membrane preparations. The potency of unlabeled IL-1 alpha to replace the binding of [125I]IL-1 beta to the hypothalamic membrane preparations was considerably less than that of unlabeled IL-1 beta. These findings indicate that IL-1 beta receptors are heterogeneously distributed in the central nervous system and that IL-1 alpha does not bind with IL-1 beta receptors in the brain.

Increased production of matrix metalloproteinases in enriched astrocyte and mixed hippocampal cultures treated with β-amyloid peptides

Abstract: Growing evidence supports the notion of a functional relationship between the presence of the β‐amyloid (Aβ) peptide and the production of inflammatory mediators in and around neuritic plaques of Alzheimers disease. Tissue remodeling enzymes that are critical in peripheral inflammatory responses are the matrix metalloproteinases (MMPs), enzymes produced by neurons and glia. Thus, it was of interest to determine whether Aβ may alter the expression of MMPs in glial and neuronal cultures. It was demonstrated that Aβ (1–40) is a potent stimulator of MMP‐9 and MMP‐2 activity in addition to inducing the expression of a lower molecular weight, unidentified gelatinase activity in mixed hippocampal and astrocyte cultures. Shorter fragments of Aβ were less effective in stimulating the production of these enzymes. The lower molecular weight activity was observed only in response to Aβ, and not after treatment with various cytokines. In addition, both cultures express MMP‐3 (stromelysin‐1) in response to Aβ peptides. These results suggest that MMPs may play a role in the development or progression of neuritic plaques, i.e., abnormal neurite outgrowth.

Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes

The high and low affinity binding sites for PACAP were identified in rat astrocytes using [125I]PACAP27 as the labeled ligand. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites, with the dissociation constant (Kd) = 1.22 +/- 0.4 nM, the binding maximal capacity (Bmax) = 821 +/- 218 fmols/mg protein for the high affinity binding site, and Kd = 0.59 +/- 0.06 microM, Bmax = 563 +/- 12 pmols/mg protein for the low affinity binding site, respectively. The specificity of [125I]PACAP27 binding was tested using PACAP38 and peptides structurally related to PACAP, such as VIP, GHRF, PHI, secretin and glucagon. PACAP38 completely displaced the binding of [125I]PACAP27 and Scatchard analysis also indicated the presence of two classes of binding sites with similar Kd and Bmax to those for PACAP27. VIP and GHRF competed with [125I]PACAP27, but to a much lesser extent than unlabeled PACAP27 in binding. Other peptides tested did not displace the binding of [125I]PACAP27 at 10(-6) M.

Regulation of interleukin-6 (IL-6) secretion in primary cultured rat astrocytes: synergism of interleukin-1 (IL-1) and pituitary adenylate cyclase activating polypeptide (PACAP)

Interleukin-6 (IL-6) is a pleiotropic cytokine that is produced by astrocytes and microglia and may act as a trophic factor in the nervous system. These experiments were intended to identify neuroactive agents that regulate IL-6 production in primary cultured rat astrocytes. Addition of either lipopolysaccharide (LPS) or human recombinant interleukin-1 beta (IL-1 beta) to rat astrocytes in culture stimulated IL-6 secretion. However, LPS was significantly more efficacious in eliciting IL-6 production compared to IL-1 beta. Co-addition of the specific IL-1 receptor antagonist (IL-1ra) completely inhibited IL-1 beta-induced IL-6 secretion but did not affect LPS-stimulated IL-6 production during a 6 h incubation period. Two neuroactive peptides, pituitary adenylate cyclase activating polypeptide (PACAP38) and vasoactive intestinal peptide (VIP), stimulated IL-6 production either alone or in combination with IL-1 beta. PACAP38 was significantly more potent in stimulating IL-6 compared to VIP. Results from these experiments indicate that LPS is an effective inducer of IL-6 production in rat astrocytes. This effect of LPS is independent of astrocyte IL-1 production since the IL-1ra was unable to inhibit LPS-stimulated IL-6 secretion. Also, the neuropeptides PACAP38 and VIP are potential secretagogues for IL-6 secretion, and both peptides synergize with IL-1 to stimulate IL-6 secretion in rat astrocytes.

Zymographic measurement of gelatinase activity in brain tissue after detergent extraction and affinity-support purification

Several methods have been developed for the measurement of gelatinase activity from various tissues using detergent extraction. Gelatin-affinity chromatography has been employed for the large-scale purification of gelatinases from conditioned medium obtained from cultured cells. The objective of this paper was to develop a rapid method whereby gelatinase activity could be extracted from regional brain tissues without tedious, intervening purification steps. After Triton X-100 extraction and gelatin-Sepharose 4B purification of rat brain tissue extracts, two major activities were observed on gelatin zymograms. These were identified as gelatinase A and B using co-migration with astrocyte-derived enzymes and inhibition of activity by tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). The non-ionic detergents, Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) were equally effective in extracting activities from brain tissue. Little difference in recovery was observed among 0.1, 1 and 10% concentrations of Triton X-100. The method developed here was capable of recovering gelatinase activities from rat brain tissue over a 4-10-fold range using gelatin zymography for the measurement of activity. It is possible that this method may be modified for the measurement of gelatinases in tissues such as biopsy samples of gliomas or astrocytomas or other cancers where gelatinases are thought to play a role in tumor invasion and/or metastasis.

Stimulation of ACTH release by human interleukin-1β, but not by interleukin-1α, in conscious, freely-moving rats

Abstract Ever since two distinct molecules of human interleukin-1 (termed interleukin-1α and interleukin-1β) were cloned, sequencd and expressed, it has been a matter of investigation whether these two forms of interleukin-1 possess an identical spectrum of biological activities. Our current studies of interleukin-1 and its involvement in the hypothalamic-pituitary-adrenal axis have indicated that there is a clear-cut differential response to interleukin-1α and interleukin-1β. The intravenous injection of human recombinant interleukin-1β significantly increased the plasma levels of adrenocorticotropic hormone in a dose-related manner, whereas interleukin-1α did not. This observation suggests for the first time that the two members of the interleukin-1 family may have a different spectrum of biological actions.