Although a neurotoxic role has been postulated for the beta-amyloid protein (beta AP), which accumulates in brain tissues in Alzheimer's disease, a precise mechanism underlying this toxicity has not been identified. The peptide fragment consisting of amino acid residues 25 through 35 (beta AP25-35), in particular, has been reported to be toxic in cultured neurons. We report that beta AP25-35, applied to rat hippocampal neurons in culture, caused reversible and repeatable increases in the intracellular Ca2+ concentration ([Ca2+]i), as measured by fura 2 fluorimetry. Furthermore, beta AP25-35 induced bursts of excitatory potentials and action potential firing in individual neurons studied with whole cell current clamp recordings. The beta AP25-35-induced [Ca2+]i elevations and electrical activity were enhanced by removal of extracellular Mg2+, and they could be blocked by tetrodotoxin, by non-N-methyl-D-aspartate (NMDA) and NMDA glutamate receptor antagonists, and by the L-type Ca2+ channel antagonist nimodipine. Similar responses of bursts of action potentials and [Ca2+]i increases were evoked by beta AP1-40. Responses to beta AP25-35 were not prevented by pretreatment with pertussis toxin. Excitatory responses and [Ca2+]i elevations were not observed in cerebellar neuron cultures in which inhibitory synapses predominate. Although the effects of beta AP25-35 depended on the activation of glutamatergic synapses, there was no enhancement of kainate- or NMDA-induced currents by beta AP25-35 in voltage-clamp studies. We conclude that beta AP25-35 enhances excitatory activity in glutamatergic synaptic networks, causing excitatory potentials and Ca2+ influx. This property may explain the toxicity of beta AP25-35.
Parathyroid hormone-related protein (PTHrP) is synthesized in the brain, and a single type of cloned receptor for the N-terminal portion ofPTHrP and PTH is present in the central nervous system. Nothing is known about the physiological actions or signaling pathways used by PTHrP in the brain. Using cultured rat hippocampal neurons, we demonstrate that N-terminal PTHrP The gene encoding the human parathyroid hormone-related protein (hPTHrP), a polypeptide of 141 amino acid residues, was first identified and cloned from malignant tumor cells and tissues from patients with the syndrome of humoral hypercalcemia of malignancy (1-4). Subsequently, it was shown that the PTHrP gene is expressed at the protein and/or mRNA level in several normal tissues, including bone, kidney, brain, placenta, skin, and certain fetal organs (5-9). N-terminal PTHrP and PTH , which have a high degree of sequence identity in their most N-terminal portions, act via a single species of cloned PTH/PTHrP receptor (PTH/ PTHrPR) (10-15), although certain PTHrP-specific actions have been described (16-21). The PTH/PTHrPR is also widely distributed (22, 23), implying that PTHrP acts physiologically in normal tissues (24). It has recently been shown that the PTH/PTHrP receptor and/or its mRNA are expressed in the rat brain (25,26). Targeted disruption of the PTHrP gene is lethal (27).Biological responses to PTHrP can be elicited by the Nterminal 1-34 or 1-36 fragments, although multiple forms including mid-portion and C-terminal fragments have been identified in the circulation (7, 9). These PTHrP fragments are derived from the cleavage of the intact 1-141 molecule by specific enzymes (7, 9). It is not known whether the fragments have physiological actions, although some unique effects of the C-terminal 107-139 peptide, which does not bind to the cloned PTH/PTHrPR (10, 15), have been described (28,29). No mechanistic studies of PTHrP signaling in the central nervous system have been reported. In this paper, we demonstrate that both N-and C-terminal PTHrP peptides act directly on hippocampal neurons to cause cAMP production and/or Ca2+ influx. Unlike classical PTH/PTHrP target tissues, there was no acute homologous desensitization of neuronal Ca2+ responses to these peptides; in fact, preincubation with PTHrP dramatically recruited additional responsive neurons.MATERIALS AND METHODS Primary Cell Culture. Neurons, dissociated from newborn rat brains (<48 h old) were maintained in primary cell culture as described (30, 31) with minor modifications. Briefly, pups were decapitated and their brains were placed in oxygenated HBSS buffer (118 mM NaCl/4.6 mM KC1/0.4 mM CaCl2/10 mM D-glucose/20 mM Hepes, pH adjusted to 7.2). The hippocampal formation (including the dentate gyrus) was dissected, stripped of meninges and blood vessels, and incubated for 30 min (37°C, 5% C02) with gentle shaking in HBSS buffer containing 1.5 mM CaCl2, 0.5 mM EDTA, 0.2 mg of L-cysteine per ml, and 7.5 units of papain per ml. The tissue was then rinsed and gently tritu...
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