Lea Oron

The effects of apolipoprotein e deficiency on braincholinergic neurons

Previous studies utilizing apolipoprotein E (apoE)‐deficient mice revealed distinctdecreases in the levels of cholinergic synaptic markers of projecting basal forebrain cholinergicneurons and no such alterations in other brain cholinergic systems. In order to investigate themechanisms underlying these neuron‐specific cholinergic effects, primary neuronal cultures fromapoE‐deficient and control mice were prepared and characterized. These include basal forebraincultures, which are enriched in projecting cholinergic neurons, and cortical cultures, which containcholinergic interneurons. The levels of cholinergic nerve terminals in these cultures were assessedby ligand binding measurements of the levels of the vesicular acetylcholine transporter (VAChT).This revealed that basal forebrain cultures of apoE‐deficient mice contain markedly lower VAChTlevels (∼50%) than do control cultures, but that VAChT levels of the corresponding corticalcultures of the apoE‐deficient and control mice were the same. Time course studies revealed thatVAChT levels of the basal forebrain cultures increased with culture age, but that the relativereduction in VAChT levels of the apoE‐deficient cholinergic neurons was unaltered and was thesame for freshly prepared and for 96 h old cultures. These in vitro observations are inaccordance with the in vivo findings and suggest that projecting basal forebraincholinergic neurons, but not cholinergic interneurons, are markedly dependent on apoE and thatsimilar mechanisms mediate the in vivo and in vitro effects of apoE deficiencyon cholinergic function.

Effect of opioid peptides on electrically evoked acetylcholine release from Torpedo electromotor neurons

The opioid peptide dynorphin A(1-8) (1 micron) increased acetylcholine release from the Torpedo electric organ by approximately twofold. This effect was reversed by the opiate antagonist naloxone. The effect of Dyn A(1-8) on acetylcholine release was found to vary in magnitude with the seasons of the year, with maximal enhancement being observed in the summer and none in winter. Dynorphin B, methionine-enkephalin and leucine-enkephalin also increased acetylcholine release and showed similar seasonal variations. These findings suggest that acetylcholine release from Torpedo electromotor neurons is regulated by opiate receptors. The physiological significance of these observations is discussed in view of the previous findings that the Torpedo neurons contain an endogenous enkephalin-like peptide.

Animal model and in vitro studies of anti neurofilament antibodies mediated neurodegeneration in Alzheimer’s disease

Alzheimers disease (AD) is associated with serum antibodies directed specifically against phosphorylated epitopes highly enriched in the heavy neurofilament protein NF-H of cholinergic neurons. Prolonged immunization of rats with these molecules but not with other NF-H isoforms results in cognitive impairments. This animal model, termed experimental autoimmune dementia (EAD), supports a role for such antibodies in neurodegeneration in AD. In the present study we investigated the cellular and immunological mechanisms underlying the cognitive defects in EAD. Immunohistochemical studies revealed that IgG accumulate in the septum, hippocampus and in the entorhinal cortex of the EAD rats. This is accompanied by a marked reduction in the density of septal cholinergic neurons. An inverse correlation was observed between the level of IgG in the septum of individual EAD rats and the density of their septal cholinergic neurons. Time course studies revealed that the decrease in the density of cholinergic neurons in the septum of EAD rats and the accumulation of IgG in this brain area have the same time course and are both significant by three to four months following the initiation of immunization with cholinergic NF-H. The cognitive deficits of the EAD rats evolve more slowly and are pronounced only after six months following the initation of immunization. In vitro studies revealed that anti NF-H IgG bind to the outer surface of neurons in tissue cultures of rat forebrain and can affect neuronal viability. These AD and in vitro findings provide model systems for studying the mechanisms underlying the neuropathological effects of specific anti NF-H antibodies.

Secretion of the amyloid precursor protein is elevated isoform specifically by apolipoprotein E4

Genetic studies suggest that the neuropathology and etiology of Alzheimers disease (AD) are associated with several genotypes including mutations in the amyloid precursor protein (APP) gene and the allele E4 of apolipoprotein E (apoE). The present study investigated the possibility that cross talk interactions exist between APP and apoE and the extent to which they are affected by the apoE genotype. This was pursued by cell culture and immunoblot experiments utilizing neuroblastoma N2a cells in which the effects of distinct apoE isoforms on the levels of intracellular APP and of secreted APPs were determined. This revealed that treatment of the cells with apoE4, the AD risk factor, resulted in a marked increase in the levels of secreted APPs. This effect was dose dependent (ED50 approximately/= 2.5 microg/ml) and isoform specific in that apoE3 had virtually no effect on the secretion of APPs.

Immunological evidence for phenobarbital-stimulated synthesis of cytochrome P-450 in primary cultures of chick embryo hepatocytes

The induction of cytochrome P-450 by phenobarbital was studied in primary cultures of chick embryo hepatocytes. The rate of the de novo synthesis of the induced form of cytochrome P-450 was measured directly and specifically, using form-specific anti-cytochrome antibodies that quantitatively immunoprecipitated this form from the radiolabeled hepatocytes. Additionally, the steady-state levels of the cytochrome were estimated spectrophotometrically and electrophoretically. In the presence of phenobarbital the synthesis of cytochrome P-450PB by cultured hepatocytes was markedly accelerated. Furthermore, the same cytochrome P-450PB form was induced by phenobarbital in vivo in chicken liver and in the cultured chick embryo hepatocytes. Their identity was judged from immunological and electrophoretic properties of these induced cytochromes. Immunological cross-reactivity was also detected between the cytochrome P-450PB forms from chick embryo hepatocytes and from adult rat liver. The immunological cross-reactivity observed between the phenobarbital-induced cytochrome P-450 forms from different species was not observed between the different cytochrome forms with the same liver (Thomas, P.E., Reik, L.M., Ryan, D.E. and Levin, W. (1981) J. Biol. Chem. 256, 1044-1052). Implications as to the evolutionary origin of the different cytochrome forms are discussed.

Evidence that 2-allyl-2-isopropylacetamide and phenobarbital induce the same cytochrome P-450 in cultured chick embryo hepatocytes

The induction of cytochrome P-450 in cultured chick embryo hepatocytes was studied using two structurally unrelated compounds, 2-allyl-2-isopropylacetamide and phenobarbital. Pulse-labeling of these cells showed enhanced de novo synthesis of cytochrome P-450. The cytochrome induced by 2-allyl-2-isopropylacetamide, as well as the one induced by phenobarbital, reacted immunologically with antibodies raised against the major hepatic phenobarbital-induced isozyme. Additional form of cytochrome P-450 is induced exclusively by phenobarbital. These results clearly demonstrate that these two drugs induce at least one form of cytochrome P-450 in common.

Nonopioid Effect of Morphine on Electrically Evoked Acetylcholine Release from Torpedo Electromotor Neurons

Abstract: The release of acetylcholine from Torpedo electric organ slices following their electrical stimulation was modulated by morphine, by the muscarinic antagonist atropine, and by the nicotinic antagonist tubocurarine. Addition of either atropine or tubocurarine in the presence of the acetylcholinesterase inhibitor phospholine iodide enhanced acetylcholine release. The effects of the two antagonists were additive, a result suggesting that the secreted acetylcholine regulates its own release by activating both muscarinic and nicotinic cholinergic receptors and that these receptors inhibit acetylcholine release by different mechanisms. The effects of opiates on acetylcholine release were examined under conditions in which the cholinergic modulation of release is blocked, i.e., in the presence of atropine and tubocurarine. These experiments revealed that electrically evoked release of acetylcholine is blocked by the opiate agonists morphine and levorphanol. However, the inhibitory effect of morphine on acetylcholine release was not reversed by the opioid antagonist naloxone. Furthermore, dextrorphan, the nonopioid stereoisomer of levorphanol, had the same inhibitory effect as its opioid counterpart. These findings suggest that the effects of opiates on electrically evoked release of acetylcholine are not mediated by opioid receptors. The possible mechanisms underlying these nonopioid effects of morphine and levorphanol are discussed.

Model Studies of the Role of Anti-Neurofilament Antibodies in Neurodegeneration in Alzheimer’s Disease

Recent studies suggest that the heavy neurofilament protein (NF-H) of cholinergic neurons differs biochemically and antigenically from that of chemically heterogeneous NF-H (Faigon et al., 1991; Soussan et al., 1994) and that degeneration of brain cholinergic neurons in Alzheimer’s disease (AD) and in Down Syndrome is associated with serum IgG directed specifically against Torpedo and mammalian cholinergic NF-H (Chapman et al., 1989; Hassin-Baer et al., 1992; Tchernakov et al., 1992). In order to examine whether the anticholinergic NF-H antibodies (Abs) associated with AD and Down Syndrome could play a role in neuronal degeneration in these diseases, we developed a rat model in which the cellular and cognitive effects of such Abs are being examined. This model is termed Experimental Autoimmune Dementia (EAD). In the following we review the evidence that the immune response of EAD rats to prolonged immunization with cholinergic NF-H induces memory and other cognitive deficits. In addition we present new EAD and in vitro data which suggest that these behavioral changes are due to IgG mediated damage to brain cholinergic neurons.