PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor.

Four mutant PC12 pheochromocytoma cell lines that are nerve growth factor (NGF)-nonresponsive (PC12nnr) have been selected from chemically mutagenized cultures by a double selection procedure: failure both to grow neurites in the presence of NGF and to survive in NGF-supplemented serum-free medium. The PC12nnr cells were deficient in all additional NGF responses surveyed: abatement of cell proliferation, changes in glycoprotein composition, induction of ornithine decarboxylase, rapid changes in protein phosphorylation, and cell surface ruffling. However, PC12nnr cells closely resembled non-NGF-treated PC12 cells in most properties tested: cell size and shape; division rate; protein, phosphoprotein, and glycoprotein composition; and cell surface morphology. All four PC12nnr lines differed from PC12 cells in three ways in addition to failure of NGF response: PC12nnr cells failed to internalize bound NGF by the normal, saturable, high-affinity mechanism present in PC12 cells. The PC12nnr cells bound NGF but entirely, or nearly entirely, at low-affinity sites only, whereas PC12 cells possess both high- and low-affinity NGF binding sites. The responses to dibutyryl cyclic AMP that were tested appeared to be enhanced or altered in the PC12nnr cells compared to PC12 cells. Internalization of, and responses to, epidermal growth factor were normal in the PC12nnr cells ruling out a generalized defect in hormonal binding, uptake, or response mechanisms. These findings are consistent with a causal association between the presence of high-affinity NGF receptors and of NGF responsiveness and internalization. A possible relationship is also suggested between regulation of cAMP responses and regulation of NGF responses or NGF receptor affinity.

Evidence for excitoprotective and intraneuronal calcium-regulating roles for secreted forms of the beta-amyloid precursor protein.

The beta-amyloid precursor protein (beta APP) is a membrane-spanning glycoprotein that is the source of the beta-amyloid peptide (beta AP) which accumulates as senile plaques in the brains of patients with Alzheimer's disease. beta APP is normally processed such that a cleavage occurs within the beta AP, liberating secreted forms of beta APP (APPss) from the cell. The neuronal functions of these forms are unknown. We now report that APPss have a potent neuroprotective action in cultured rat hippocampal and septal neurons and in human cortical neurons. APPs695 and APPs751 protected neurons against hypoglycemic damage, and the neuroprotection was abolished by antibodies to a specific region common to both APPs695 and APPs751. APPss caused a rapid and prolonged reduction in [Ca2+]i and prevented the rise in [Ca2+]i that normally mediated hypoglycemic damage. APPss also protected neurons against glutamate neurotoxicity, effectively raising the excitotoxic threshold. APPss may normally play excitoprotective and neuromodulatory roles. Alternative processing of APPss in Alzheimer's disease may contribute to neuronal degeneration by compromising the normal function of APPss and by promoting the deposition of beta AP.

beta-Amyloid precursor protein metabolites and loss of neuronal Ca2+ homeostasis in Alzheimer's disease.

Recent findings link altered processing of beta-amyloid precursor protein (beta APP) to disruption of neuronal Ca2+ homeostasis and an excitotoxic mechanism of cell death in Alzheimer's disease. A major pathway of beta APP metabolism results in the release of secreted forms of beta APP, APPss. These secreted forms are released in response to electrical activity and can modulate neuronal responses to glutamate, suggesting roles in developmental and synaptic plasticity. beta APP is upregulated in response to neural injury and APPss can protect neurons against excitotoxic or ischemic insults by stabilizing the intracellular Ca2+ concentration [Ca2+]i. An alternative beta APP processing pathway liberates intact beta-amyloid peptide, which can form aggregates that disrupt Ca2+ homeostasis and render neurons vulnerable to metabolic or excitotoxic insults. Genetic abnormalities (e.g. certain beta APP mutations or Down syndrome) and age-related changes in brain metabolism (e.g. reduced energy availability or increased oxidative stress) may favor accumulation of [Ca2+]i-destabilizing beta-amyloid peptide and diminish the release of [Ca2+]i-stabilizing, neuroprotective APPss.

Mechanism of interleukin-1- and tumor necrosis factor alpha-dependent regulation of the alpha 1-antichymotrypsin gene in human astrocytes.

The expression of alpha(1)-antichymotrypsin (ACT) is significantly enhanced in affected brain regions in Alzheimer's disease. This serine proteinase inhibitor specifically colocalizes with filamentous beta-amyloid deposits and recently has been shown to influence both formation and destabilization of beta-amyloid fibrils. In the brain, ACT is expressed in astrocytes, and interleukin-1 (IL-1), tumor necrosis factor alpha (TNF), oncostatin M (OSM), and IL-6/soluble IL-6 receptor complexes control synthesis of this inhibitor. Here, we characterize a molecular mechanism responsible for both IL-1 and TNF-induced expression of ACT gene in astrocytes. We identify the 5' distal IL-1/TNF-responsive enhancer of the ACT gene located 13 kb upstream of the transcription start site. This 413-bp-long enhancer contains three elements, two of which bind nuclear factor kB (NF-kB) and one that binds activating protein 1 (AP-1). All of these elements contribute to the full responsiveness of the ACT gene to both cytokines, as determined by deletion and mutational analysis. The 5' NF-kB high-affinity binding site and AP-1 element contribute most to the enhancement of gene transcription in response to TNF and IL-1. In addition, we demonstrate that the 5' untranslated region of the ACT mRNA does not contribute to cytokine-mediated activation. Finally, we find that overexpression of the NF-kB inhibitor (IkB) totally inhibits any activation mediated by the newly identified IL-1/TNF enhancer of the ACT gene.

The plasmin system is induced by and degrades amyloid-beta aggregates.

Amyloid-beta (Abeta) appears critical to Alzheimer's disease. To clarify possible mechanisms of Abeta action, we have quantified Abeta-induced gene expression in vitro by using Abeta-treated primary cortical neuronal cultures and in vivo by using mice transgenic for the Abeta precursor (AbetaP). Here, we report that aggregated, but not nonaggregated, Abeta increases the level of the mRNAs encoding tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Moreover, tPA and uPA were also upregulated in aged AbetaP overexpressing mice. Because others have reported that Abeta aggregates can substitute for fibrin aggregates in activating tPA post-translationally, the result of tPA induction by Abeta would be cleavage of plasminogen to the active protease plasmin. To gain insights into the possible actions of plasmin, we evaluated the hypotheses that tPA and plasmin may mediate Abeta in vitro toxicity or, alternatively, that plasmin activation may lead to Abeta degradation. In evaluating these conflicting hypotheses, we found that purified plasmin degrades Abeta with physiologically relevant efficiency, i.e., approximately 1/10th the rate of plasmin on fibrin. Mass spectral analyses show that plasmin cleaves Abeta at multiple sites. Electron microscopy confirms indirect assays suggesting that plasmin degrades Abeta fibrils. Moreover, exogenously added plasmin blocks Abeta neurotoxicity. In summation, we interpret these results as consistent with the possibility that the plasmin pathway is induced by aggregated Abeta, which can lead to Abeta degradation and inhibition of Abeta actions.

Inflammation and Alzheimer's disease.

Inflammation clearly occurs in pathologically vulnerable regions of the Alzheimer's disease (AD) brain, and it does so with the full complexity of local peripheral inflammatory responses. In the periphery, degenerating tissue and the deposition of highly insoluble abnormal materials are classical stimulants of inflammation. Likewise, in the AD brain damaged neurons and neurites and highly insoluble amyloid beta peptide deposits and neurofibrillary tangles provide obvious stimuli for inflammation. Because these stimuli are discrete, microlocalized, and present from early preclinical to terminal stages of AD, local upregulation of complement, cytokines, acute phase reactants, and other inflammatory mediators is also discrete, microlocalized, and chronic. Cumulated over many years, direct and bystander damage from AD inflammatory mechanisms is likely to significantly exacerbate the very pathogenic processes that gave rise to it. Thus, animal models and clinical studies, although still in their infancy, strongly suggest that AD inflammation significantly contributes to AD pathogenesis. By better understanding AD inflammatory and immunoregulatory processes, it should be possible to develop anti-inflammatory approaches that may not cure AD but will likely help slow the progression or delay the onset of this devastating disorder.

Altered calcium signaling and neuronal injury: stroke and Alzheimer's disease as examples.

Several cellular signaling systems have been implicated in the neuronal death that occurs both in development ("natural" cell death) or in pathological conditions such as stroke and Alzheimer's disease (AD). Here we consider the possibility that neuronal degeneration in an array of disorders including stroke and AD arises from one or more alterations in calcium-regulating systems that result in a loss of cellular calcium homeostasis. A long-standing hypothesis of neuronal injury, the excitatory amino acid (EAA) hypothesis, is revisited in light of new supportive data concerning the roles of EAAs in stroke and the neurofibrillary degeneration in AD. Two quite new concepts concerning mechanisms of neuronal injury and death are presented, namely: 1) growth factors normally "stabilize" intracellular free calcium levels ([Ca2+]i) and protect neurons against ischemic/excitotoxic injury, and 2) aberrant processing of beta-amyloid precursor protein (APP) can cause neurodegeneration by impairing a neuroprotective function of secreted forms of APP (APPs) which normally regulate [Ca2+]i. Altered APP processing also results in the accumulation of beta-amyloid peptide which contributes to neuronal damage by destabilizing calcium homeostasis; in AD beta-amyloid peptide may render neurons vulnerable to excitotoxic conditions that accrue with increasing age (e.g., altered glucose metabolism, ischemia). Growth factors may normally protect neurons against the potentially damaging effects of calcium influx resulting from energy deprivation and overexcitation. For example, bFGF, NGF and IGFs can protect neurons from several brain regions against excitotoxic/ischemic insults. Growth factors apparently stabilize [Ca2+]i by several means including: a reduction in calcium influx; enhanced calcium extrusion or buffering; and maintenance or improvement of mitochondrial function. For example, bFGF can suppress the expression of a N-methyl-D-aspartate (NMDA) receptor protein that mediates excitotoxic damage in hippocampal neurons. Growth factors may also prevent the loss of neuronal calcium homeostasis and the increased vulnerability to neuronal injury caused by beta-amyloid peptide. Since elevated [Ca2+]i can elicit cytoskeletal alterations similar to those seen in AD neurofibrillary tangles, we propose that neuronal damage in AD results from a loss of calcium homeostasis. The data indicate that a variety of alterations in [Ca2+]i regulation may contribute to the neuronal damage in stroke and AD, and suggest possible means of preventing neuronal damage in these disorders.

beta-Amyloid precursor protein mismetabolism and loss of calcium homeostasis in Alzheimer's disease.

The suspected involvement of the beta-amyloid precursor protein (beta APP) in the etiology of Alzheimer's disease (AD) has been strengthened by recent genetic evidence, but pursuit of the mechanisms involved will initially require basic cell biology approaches. Several studies have concentrated on toxic activities of beta-amyloid peptide (beta AP) itself, illuminating its contributions to excitotoxicity and calcium-mediated degeneration in general. We now know that generation of beta AP from beta APP also compromises the production of an important set of trophic factors: the secreted forms of beta APP (APPS), which may act--ironically--by conferring protection from calcium-mediated insults. Therefore, conditions which contribute to the formation of beta AP (possibly including ischemia) not only produce an agent which exacerbates calcium-mediated cell death, but also reduce the levels of one of the few factors able to rescue calcium homeostasis. The implications of these postulates and their relationship to the process of aging are discussed.

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

The mechanisms that contribute to neuronal degeneration in Alzheimer's 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.

Species and structural specificity of the lipopigment accumulation and neuronal destruction induced by N-(2-guanidinoethyl)-4-methyl-1,2,5,6-tetrahydropyridine (guanacline).

Guanacline, a guanidinium adrenergic neuron blocking agent similar to guanethidine, was studied clinically and experimentally during the late 1960s. Like guanethidine, it has been reported to produce sympathetic neuronal destruction in rats. Unlike guanethidine, it has been reported to produce irreversible sympathetic deficits in man and to produce fluorescent lipopigment in rat sympathetic neurons. Guanacline and its derivative in which the double bond of the tetrahydropyridine ring is reduced (saturated analog of guanacline, SAG) were prepared. Several species were treated chronically with varying doses of guanethidine, guanacline or SAG; the superior cervical ganglia were examined light microscopically for neuronal destruction and for osmiophilic fluorescent lipopigment accumulation. All 3 drugs produced rapid neuronal destruction in rats accompanied by massive small-cell infiltration. In striking contrast, treatment for many weeks with doses up to 100 mg/kg/day produced no small-cell infiltration or apparent neuronal destruction in mice or guinea pigs. The neuronal destruction produced by guanacline and SAG in the rat, like that caused by guanethidine, was prevented by immunosuppression or gamma-irradiation, indicating that all 3 agents produce neuronal destruction in rats by an immune-mediated mechanism. Thus, the ability of the drug to produce sympathectomy is species specific but not drug specific. The opposite was found with respect to fluorescent lipopigment accumulation. Guanacline, but not guanethidine or SAG, produced fluorescent lipopigment in all species examined. Therefore, the double bond of the tetrahydropyridine ring plays a critical role in the production of the fluorescent lipopigment.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

cAMP analogs promote survival and neurite outgrowth in cultures of rat sympathetic and sensory neurons independently of nerve growth factor.

Nerve growth factor (NGF) is a neurotrophic agent for sympathetic and embryonic sensory neurons both in vivo and in vitro. We report here that the membrane-permeant cAMP analogs, 8-(4-chlorophenylthio)-cAMP and 8-bromo-cAMP, can replace NGF in promoting long-term survival and neurite outgrowth in cultures of rat neonatal sympathetic and embryonic sensory neurons. N6-substituted analogs, including the more commonly used N6,O2'-dibutyryl-cAMP, are less efficacious. Additivity and switching experiments indicate that the cAMP analogs affect the same neuronal population as that maintained by NGF. However, unlike NGF, the cAMP analogs do not evoke somatic hypertrophy. Moreover, studies with sympathetic neurons reveal that the neurotrophic actions of the cAMP analogs, but not of NGF, are blocked by the axial diastereoisomer of adenosine 3',5'-phosphorothioate, a competitive cAMP antagonist. Thus, the mechanism by which cAMP analogs promote neuronal survival and differentiation appears to involve activation of cAMP-dependent protein kinases, whereas, in contrast, the same effects of NGF neither require nor are mediated by such a pathway. Furthermore, the different efficacies observed with N6- and C8-substituted cAMP analogs suggest that this neurotrophic pathway may involve differential activation of the regulatory subunits of cAMP-dependent protein kinases. The presence of this parallel, cAMP-responsive, neurotrophic pathway in at least two types of NGF-responsive neurons may be developmentally important and has the potential to be exploited for the treatment of injuries or diseases affecting these and possibly other nerve cells.

Acidic and basic fibroblast growth factors promote stable neurite outgrowth and neuronal differentiation in cultures of PC12 cells.

Acidic (aFGF) and basic (bFGF) fibroblast growth factors are well-characterized peptide hormones that have potent angiogenic activity and that are mitogenic for a variety of cell types. The present findings demonstrate that FGFs can reproduce the entire spectrum of rat pheochromocytoma PC12 cell responses previously shown to be elicited by NGF. These include responses that are rapid (cell flattening, enhanced phosphorylation of tyrosine hydroxylase) or delayed (neurite outgrowth, induction of phosphorylated MAP 1.2, regulation of NILE and Thy-1 glycoproteins, cessation of mitosis, elevation of AChE activity), as well as responses that have been shown to be either transcription-independent (neurite regeneration, promotion of survival) or transcription-dependent (priming, regulation of NILE and Thy-1 glycoproteins, elevation of AChE activity). The only responses for which the FGFs and NGF consistently showed quantitative differences were in the rates for neurite initiation and elongation in serum-containing medium. Thus, while all 3 factors promoted the formation of stable neurites, the network of outgrowth elicited by NGF at any given time of treatment was always of greater density. Togari et al. (1985) have previously reported that bFGF can initiate transient neurite formation in PC12 cell cultures. The present observations describe a variety of additional actions of bFGF on a neuronal cell line, and demonstrate that aFGF is capable of mimicking many, if not all, of these actions. These observations thus extend the range of actions that aFGF and bFGF may potentially exert on nerve cells, either during their development, repair, or maintenance. In addition, this work suggests that the PC12 cell line may serve as a useful model system with which to study the mechanism of action of FGFs on neurons. Since all 3 factors appear capable of eliciting the same wide spectrum of responses, molecular events specifically associated with FGFs and NGF in PC12 cells may prove illuminating of the causal steps involved in neuronal differentiation.

Multiple agents rescue PC12 cells from serum-free cell death by translation- and transcription-independent mechanisms.

Past studies revealed that NGF and fibroblast growth factor (FGF) prevent the death of PC 12 pheochromocytoma cells that otherwise occurs in serum-free medium. Additional agents were tested here for their abilities to promote long-term survival of naive and NGF-pretreated (primed) PC 12 cells in serum-free conditions. Forskolin and permeant cAMP analogs effectively prevented serum-free cell death, as did micromolar levels of insulin and 10-100-nM levels of insulin-like growth factors I and II. In contrast to NGF and FGF, none of these agents caused neuronal differentiation of naive cells or neurite regeneration by primed cells. Each of the agents also prevented rapid cell death in a balanced salt solution, thus apparently ruling out a mechanism dependent on regulation of nutrient uptake. Epidermal growth factor and elevated K+ appeared to slow the rate of cell death, but did not promote long-term survival; phorbol ester, dexamethasone, or vanadate did not prevent cell death. Each of the survival-promoting agents was effective even when macromolecular synthesis was blocked. Because the synthesis inhibitors themselves did not significantly prevent cell death, such findings indicate that survival was promoted by mechanisms that do not require synthesis of RNA or protein. In addition, various lines of experimental evidence (using the kinase inhibitor K-252a or PC 12 cell variants deficient either in protein kinase A activity or in responsiveness to NGF) further suggested that the effective agents maintain survival by independent initial pathways. Regulation of protein kinase activity appears to be a common feature of each pathway and may therefore play a key convergent role in mediating prevention of cell death.

beta-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity.

In Alzheimer's disease (AD), abnormal accumulations of beta-amyloid are present in the brain and degenerating neurons exhibit cytoskeletal aberrations (neurofibrillary tangles). Roles for beta-amyloid in the neuronal degeneration of AD have been suggested based on recent data obtained in rodent studies demonstrating neurotoxic actions of beta-amyloid. However, the cellular mechanism of action of beta-amyloid is unknown, and there is no direct information concerning the biological activity of beta-amyloid in human neurons. We now report on experiments in human cerebral cortical cell cultures that tested the hypothesis that beta-amyloid can destabilize neuronal calcium regulation and render neurons more vulnerable to environmental stimuli that elevate intracellular calcium levels. Synthetic beta-amyloid peptides (beta APs) corresponding to amino acids 1-38 or 25-35 of the beta-amyloid protein enhanced glutamate neurotoxicity in cortical cultures, while a peptide with a scrambled sequence was without effect. beta APs alone had no effect on neuronal survival during a 4 d exposure period. beta APs enhanced both kainate and NMDA neurotoxicity, indicating that the effect was not specific for a particular subtype of glutamate receptor. The effects of beta APs on excitatory amino acid (EAA)-induced neuronal degeneration were concentration dependent and required prolonged (days) exposures. The beta APs also rendered neurons more vulnerable to calcium ionophore neurotoxicity, indicating that beta APs compromised the ability of the neurons to reduce intracellular calcium levels to normal limits. Direct measurements of intracellular calcium levels demonstrated that beta APs elevated rest levels of calcium and enhanced calcium responses to EAAs and calcium ionophore. The neurotoxicity caused by EAAs and potentiated by beta APs was dependent upon calcium influx since it did not occur in calcium-deficient culture medium. Finally, the beta APs made neurons more vulnerable to neurofibrillary tangle-like antigenic changes induced by EAAs or calcium ionophore (i.e., increased staining with tau and ubiquitin antibodies). Taken together, these data suggest that beta-amyloid destabilizes neuronal calcium homeostasis and thereby renders neurons more vulnerable to environmental insults.

Tissue plasminogen activator requires plasminogen to modulate amyloid-beta neurotoxicity and deposition.

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

Amyloid beta peptide potentiates cytokine secretion by interleukin-1 beta-activated human astrocytoma cells.

Neurodegenerative processes in Alzheimer disease (AD) are thought to be driven in part by the deposition of amyloid beta (A beta), a 39- to 43-amino acid peptide product resulting from an alternative cleavage of amyloid precursor protein. Recent descriptions of in vitro neurotoxic effects of A beta support this hypothesis and suggest toxicity might be mediated by A beta-induced neuronal calcium disregulation. In addition, it has been reported that "aging" A beta results in increased toxic potency due to peptide aggregation and formation of a beta-sheet secondary structure. In addition, A beta might also promote neuropathology indirectly by activating immune/inflammatory pathways in affected areas of the brain (e.g., cortex and hippocampus). Here we report that A beta can modulate cytokine secretion [interleukins 6 and 8 (IL-6 and IL-8)] from human astrocytoma cells (U-373 MG). Freshly prepared and aged A beta modestly stimulated IL-6 and IL-8 secretion from U-373 MG cells. However, in the presence of interleukin-1 beta (IL-1 beta), aged, but not fresh, A beta markedly potentiated (3- to 8-fold) cytokine release. In contrast, aged A beta did not potentiate substance P (NK-1)- or histamine (H1)-stimulated cytokine production. Further studies showed that IL-1 beta-induced cytokine release was potentiated by A beta-(25-35), while A beta-(1-16) was inactive. Calcium disregulation may be responsible for the effects of A beta on cytokine production, since the calcium ionophore A23187 similarly potentiated IL-1 beta-induced cytokine secretion and EGTA treatment blocked either A beta or A23187 activity. Thus, chronic neurodegeneration in AD-affected brain regions may be mediated in part by the ability of A beta to exacerbate inflammatory pathways in a conformation-dependent manner.