Russell E. Rydel

Calcium-destabilizing and neurodegenerative effects of aggregated β-amyloid peptide are attenuated by basic FGF

The mechanisms that contribute to neuronal degeneration in Alzheimers disease (AD) are not understood. Abnormal accumulations of beta-amyloid peptide (beta AP) are thought to be involved in the neurodegenerative process, and recent studies have demonstrated neurotoxic actions of beta APs. We now report that the mechanism of beta AP-mediated neurotoxicity in hippocampal cell culture involves a destabilization of neuronal calcium homeostasis resulting in elevations in intracellular calcium levels ([Ca2+]i) that occur during exposure periods of 6 hr to several days. Both the elevations of [Ca2+]i and neurotoxicity were directly correlated with aggregation of the peptide as assessed by beta AP immunoreactivity and confocal laser scanning microscopy. Exposure of neurons to beta AP resulted in increased sensitivity to the [Ca2+]i-elevating and neurodegenerative effects of excitatory amino acids. Moreover, [Ca2+]i responses to membrane depolarization and calcium ionophore were greatly enhanced in beta AP-treated neurons. Neurons in low cell density cultures were more vulnerable to beta AP toxicity than were neurons in high cell density cultures. Basic fibroblast growth factor (bFGF), but not nerve growth factor (NGF), significantly reduced both the loss of calcium homeostasis and the neuronal damage otherwise caused by beta AP. In AD, beta AP may endanger neurons by destabilizing calcium homeostasis and bFGF may protect neurons by stabilizing intracellular calcium levels. Aggregation of beta AP seems to be a major determinant of its [Ca2+]i-destabilizing and neurotoxic potency.

Secreted Forms of β-Amyloid Precursor Protein Protect Against Ischemic Brain Injury

Abstract: The β‐amyloid precursor protein (βAPP) is the source of the amyloid β‐peptide that accumulates in the brain in Alzheimers disease. A major processing pathway for βAPP involves an enzymatic cleavage within the amyloid β‐peptide sequence that liberates secreted forms of βAPP (APPSs) into the extracellular milieu. We now report that postischemic administration of these APPSs intracerebroventricularly protects neurons in the CA1 region of rat hippocampus against ischemic injury. Treatment with APPS695 or APPS751 resulted in increased neuronal survival, and the surviving cells were functional as demonstrated by their ability to synthesize protein. These data provide direct evidence for a neuroprotective action of APPSs in vivo.

Tissue Plasminogen Activator Requires Plasminogen to Modulate Amyloid-β Neurotoxicity and Deposition

Abstract: Tissue plasminogen (plgn) activator (tPA) modulates neuronal death in models of stroke, excitotoxicity, and oxidative stress. Amyloid‐β (Aβ) appears central to Alzheimers disease and is neurotoxic to neurons in vitro. Here, we evaluate tPA effects on Aβ toxicity. We report that tPA alone had no effect on Aβ toxicity. However, in combination with plgn, tPA reduced Aβ toxicity in a robust fashion. Moreover, the combined tPA and plgn treatment markedly inhibited Aβ accumulation. The addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to a sample of tPA, plgn, and Aβ resulted in a marked reduction of Aβ degradation. We interpret the actions of tPA and plgn within the context of the ability of plasmin to degrade Aβ.

α2β1 and αVβ1 integrin signaling pathways mediate amyloid-β-induced neurotoxicity

Pathological hallmarks of Alzheimers disease are the presence of extracellular amyloid plaques, intracellular neurofibrillary tangles, and neurodegeneration. The principal component of amyloid plaques is the amyloid-β peptide (Aβ). Accumulating evidence indicates that Aβ may play a causal role in Alzheimers disease. In this report, we demonstrate that Aβ deposition and neurotoxicity in human cortical primary neurons are mediated through α2β1 and αVβ1 integrins using specific integrin-blocking antibodies. An aberrant integrin signaling pathway causing the neurotoxicity is mediated through Pyk2. The role of α2β1 and αVβ1 integrins can be extended to another amyloidosis using an amylin in vitro neurotoxicity model. These results indicate that the α2β1 and αVβ1 integrin signaling pathway may be critical components of neurodegeneration in Alzheimers disease and that integrins may recognize and be activated by a shared structural motif of polymerizing amyloidogenic proteins.

Amyloid β-Mediated Oxidative and Metabolic Stress in Rat Cortical Neurons: No Direct Evidence for a Role for H2O2 Generation

Abstract: H2O2 and free radical‐mediated oxidative stresses have been implicated in mediating amyloid β(1–40) [Aβ(1–40)] neurotoxicity to cultured neurons. In this study, we confirm that addition of the H2O2‐scavenging enzyme catalase protects neurons in culture against Aβ‐mediated toxicity; however, it does so by a mechanism that does not involve its ability to scavenge H2O2. Aβ‐mediated elevation in intracellular H2O2 production is suppressed by addition of a potent H2O2 scavenger without any significant neuroprotection. Three intracellular biochemical markers of H2O2‐mediated oxidative stress were unchanged by Aβ treatment: (a) glyceraldehyde‐3‐phosphate dehydrogenase activity, (b) hexose monophosphate shunt activity, and (c) glucose oxidation via the tricarboxylic acid cycle. Ionspray mass spectra of Aβ in the incubation medium indicated that Aβ itself is an unlikely source of reactive oxygen species. In this study we demonstrate that intracellular ATP concentration is compromised during the first 24‐h exposure of neurons to Aβ. Our results challenge a pivotal role for H2O2 generation in mediating Aβ toxicity, and we suggest that impairment of energy homeostasis may be a more significant early factor in the neurodegenerative process.

Characterization of the effects of autoimmune nerve growth factor deprivation in the developing guinea-pig

Abstract We have previously reported that female guinea-pigs immunized with mouse nerve growth factor (NGF) make antibodies which cross-react with guinea-pig NGF. Offspring born to the immunized female guinea-pig were born with marked decreases in numbers of sympathetic and dorsal root ganglion sensory neurons. In this paper, we describe the degree and permanence of the destruction of these components in the nervous system of the offspring. Considerable variability is seen in the immunological response to NGF. Only those animals that generate a high titer antibody against mouse NGF which cross-reacts well with guinea-pig NGF produce offspring with deleterious effects on the developing nervous system. Female guinea-pigs with titers of approximately 4000 against guinea-pig NGF produce ‘severely’ affected offspring with gross sensory deficits which were easily observed as a complete lack of response to several painful stimuli. These ‘severely’ affected animals showed a virtual absence of unmyelinated axons in the sciatic nerve. These animals did not survive beyond a few days of age. Animals born to females with titers of 1000–2000 were ‘moderately’ affected; responses to painful stimuli were blunted but not absent. ‘Moderately’ affected animals survived and grew normally; maternal antibody in their circulation disappeared by six weeks of age. None of the moderately affected animals exhibited obvious deficits in proprioception and general coordination. Analysis of catecholamine levels showed that in severely and moderately affected animals norepinephrine levels were permanently reduced to almost non-detectable levels in heart and spleen. Less severe permanent effects were observed in small intestine and transient effects were seen in the sympathetic innervation of the male urogenital system. No adverse effects were seen upon light-microscopic examination of the adrenal medulla, nor was there any decrease in tyrosine hydroxylase activity in this tissue; thus, we found no evidence for an adverse effect of anti-NGF on the developing adrenal medulla. We conclude that varying degrees of destruction of the sympathetic and sensory nervous systems are produced by in utero exposure to maternal anti-NGF in the guinea-pig. The sources of the variability include differences in immunological responses among animals and varying degrees of susceptibility to NGF deprivation among neuronal types.

Effects of autoimmune NGF deprivation in the adult rabbit and offspring

An experimental autoimmune approach to the production of nerve growth factor deprivation, which we have previously described in the rat and guinea pig, has been applied to the rabbit. This species was chosen for study because of several potential advantages. The rabbit produces large litters and has a relatively short gestation period. More importantly, rabbits generate high titers of antibody against mouse NGF and large amounts of maternal antibody are passively transferred to the developing rabbit fetus compared to most other species, particularly the rat. The sympathetic nervous system of adult rabbit immunized against mouse NGF underwent degeneration with up to an 85% decrease in neuronal numbers in the superior cervical ganglion after 10 months of immunization, thus providing further evidence that NGF is required for the survival of mature sympathetic neurons. Despite the fact that newborn rabbits born to anti-NGF producing mothers had much higher titers of anti-NGF than did rats, the effects on the developing sympathetic and sensory nervous systems were not found to be any greater than in rats. Reductions in norepinephrine levels in the heart and spleen of adult rabbits born to anti-NGF producing mothers were greater than in small intestine. Prenatal exposure to maternal anti-NGF caused reductions (up to 70%) in the number of neurons in the dorsal root ganglia. Substance-P immunoreactivity was reduced in the substantia gelatinosa of the spinal cord of rabbit exposed to maternal anti-NGF. These changes, however, were not greater than seen in the rat. We conclude that although the rabbits offers some advantage in the study of the effects of NGF deprivation in the adult animal, it appears less well suited than the rat or guinea pig to the study of the effects of NGF deprivation on development.

Mechanism of plasminogen activator inhibitor-1 regulation by oncostatin M and interleukin-1 in human astrocytes

Glial cells that produce and respond to various cytokines mediate inflammatory processes in the brain. Here, we show that oncostatin M (OSM) and interleukin‐1 (IL‐1) regulate the expression of plasminogen activator inhibitor‐1 (PAI‐1) and urokinase‐type plasminogen activator (uPA) in human astrocytes. Using the PAI‐1 reporter constructs we show that the −58 to −51 proximal element mediates activation by both cytokines. This element is already bound by c‐fos/c‐jun heterodimers in unstimulated astrocytes, and treatment with cytokine strongly stimulates both expression of c‐fos and binding of c‐fos/c‐jun heterodimers. In addition, IL‐1 activates an inhibitory mechanism that down‐regulates PAI‐1 expression after longer exposure to this cytokine. Overexpression of dominant‐negative signal transducer and activator of transcription‐1 (STAT1), STAT3, STAT5 and inhibitor of nuclear factor‐κB (IκB) suppressed OSM/IL‐1‐induced expression of the PAI‐1 reporter construct. We conclude that OSM and IL‐1 regulate the PAI‐1 gene expression via up‐regulating c‐fos levels and subsequent binding of c‐fos/c‐jun heterodimers to the proximal element of the PAI‐1 gene.

c‐Jun Contributes to Amyloid β‐Induced Neuronal Apoptosis but Is Not Necessary for Amyloid β‐Induced c‐jun Induction

Abstract : The role of gene expression in neuronal apoptosis may be cell‐ and apoptotic stimulus‐specific. Previously, we and others showed that amyloid β (Aβ)‐induced neuronal apoptosis is accompanied by c‐jun induction. Moreover, c‐Jun contributes to neuronal death in several apoptosis paradigms involving survival factor withdrawal. To evaluate the role of c‐Jun in Aβ toxicity, we compared Aβ‐induced apoptosis in neurons from murine fetal littermates that were deficient or wild‐type with respect to c‐Jun. We report that neurons deficient for c‐jun are relatively resistant to Aβ toxicity, suggesting that c‐Jun contributes to apoptosis in this model. When changes in gene expression were quantified in neurons treated in parallel, we found that Aβ treatment surprisingly led to an apparent activation of the c‐jun promoter in both c‐jun‐deficient and wild‐type neurons, suggesting that c‐Jun is not necessary for activation of the c‐jun promoter. Indeed, several genes induced by Aβ in wild‐type neurons were also induced in c‐jun‐deficient neurons, including c‐fos, fosB, ngfi‐B, and iκB. In summary, these results indicate that c‐Jun contributes to Aβ‐induced neuronal death but that c‐Jun is not necessary for c‐jun induction.

Astrocyte- and hepatocyte-specific expression of genes from the distal serpin subcluster at 14q32.1 associates with tissue-specific chromatin structures

The distal serpin subcluster contains genes encoding α1‐antichymotrypsin (ACT), protein C inhibitor (PCI), kallistatin (KAL) and the KAL‐like protein, which are expressed in hepatocytes, but only the act gene is expressed in astrocytes. We show here that the tissue‐specific expression of these genes associates with astrocyte‐ and hepatocyte‐specific chromatin structures. In hepatocytes, we identified 12 Dnase I‐hypersensitive sites (DHSs) that were distributed throughout the entire subcluster, with the promoters of expressed genes accessible to restriction enzyme digestion. In astrocytes, only six DHSs were located exclusively in the 5′ flanking region of the act gene, with its promoter also accessible to restriction enzyme digestion. The acetylation of histone H3 and H4 was found throughout the subcluster in both cell types but this acetylation did not correlate with the expression pattern of these serpin genes. Analysis of histone modifications at the promoters of the act and pci genes revealed that methylation of histone H3 on lysine 4 correlated with their expression pattern in both cell types. In addition, inhibition of methyltransferase activity resulted in suppression of ACT and PCI mRNA expression. We propose that lysine 4 methylation of histone H3 correlates with the tissue‐specific expression pattern of these serpin genes.