Peter B. Reiner

β-Amyloid efflux mediated by p-glycoprotein

A large body of evidence suggests that an increase in the brain β‐amyloid (Aβ) burden contributes to the etiology of Alzheimers disease (AD). Much is now known about the intracellular processes regulating the production of Aβ, however, less is known regarding its secretion from cells. We now report that p‐glycoprotein (p‐gp), an ATP‐binding cassette (ABC) transporter, is an Aβ efflux pump. Pharmacological blockade of p‐gp rapidly decrease extracellular levels of Aβ secretion. In vitro binding studies showed that addition of synthetic human Aβ1–40 and Aβ1–42 peptides to hamster mdr1‐enriched vesicles labeled with the fluorophore MIANS resulted in saturable quenching, suggesting that both peptides interact directly with the transporter. Finally, we were able to directly measure transport of Aβ peptides across the plasma membranes of p‐gp enriched vesicles, and showed that this phenomenon was both ATP‐ and p‐gp‐dependent. Taken together, our study suggests a novel mechanism of Aβ detachment from cellular membranes, and represents an obvious route towards identification of such a mechanism in the brain.

Ca2+ channels: diversity of form and function

The past year has seen some significant advances in our understanding of the structural and functional properties of neuronal voltage-gated Ca2+ channels. Molecular cloning and protein purification studies have identified structural components, and expression studies are beginning to define the biophysical and pharmacological properties of the cloned channels. A number of studies of native Ca2+ channels show that the concept of channel modulation includes gating by both voltage and ligands.

Regulation of Amyloid Precursor Protein Cleavage

Abstract : Multiple lines of evidence suggest that increased production and/or deposition of the β‐amyloid peptide, derived from the amyloid precursor protein, contributes to Alzheimers disease. A growing list of neuro‐transmitters, growth factors, cytokines, and hormones have been shown to regulate amyloid precursor protein processing. Although traditionally thought to be mediated by activation of protein kinase C, recent data have implicated other signaling mechanisms in the regulation of this process. Moreover, novel mechanisms of regulation involving cholesterol‐, apolipoprotein E‐, and stress‐activated pathways have been identified. As the phenotypic changes associated with Alzheimers disease encompass many of these signaling systems, it is relevant to determine how altered cell signaling may be contributing to increasing brain amyloid burden. We review the myriad ways in which first messengers regulate amyloid precursor protein catabolism as well as the signal transduction cascades that give rise to these effects.

The immunohistochemical localization of choline acetyltransferase in the cat brain

The distribution of neurons displaying choline acetyltransferase (ChAT) immunoreactivity was examined in the feline brain using a monoclonal antibody. Groups of ChAT-immunoreactive neurons were detected that have not been identified previously in the cat or in any other species. These included small, weakly stained cells found in the lateral hypothalamus, distinct from the magnocellular rostral column cholinergic neurons. Other small, lightly stained cells were also detected in the parabrachial nuclei, distinct from the caudal cholinergic column. Many small ChAT-positive cells were also found in the superficial layers of the superior colliculus. Other ChAT-immunoreactive neurons previously detected in rodent and primate, but not in cat, were observed in the present study. These included a dense cluster of cells in the medial habenula, together with outlying cells in the lateral habenula. Essentially all of the cells in the parabigeminal nucleus were found to be ChAT-positive. Additional ChAT-positive neurons were detected in the periolivary portion of the superior olivary complex, and scattered in the medullary reticular formation. In addition to these new observations, many of the cholinergic cell groups that have been previously identified in the cat as well as in rodent and primate brain such as motoneurons, striatal interneurons, the magnocellular rostral cholinergic column in the basal forebrain and the caudal cholinergic column in the midbrain and pontine tegmentum were confirmed. Together, these observations suggest that the feline central cholinergic system may be much more extensive than previous studies have indicated.

SINGLE CHOLINERGIC MESOPONTINE TEGMENTAL NEURONS PROJECT TO BOTH THE PONTINE RETICULAR FORMATION AND THE THALAMUS IN THE RAT

Microinjections of the cholinergic agonist carbachol into a caudal part of the pontine reticular formation of the rat induce a rapid eye movement sleep-like state. This carbachol-sensitive region of the pontine reticular formation is innervated by cholinergic neurons in the pedunculopontine and laterodorsol tegmental nuclei. The same population of cholinergic neurons also project heavily to the thalamus, where there is good evidence that acetylcholine facilitates sensory transmission and blocks rhythmic thalamocortical activity. The present study was undertaken to examine the degree to which single cholinergic neurons in the mesopontine tegmentum project to both the carbachol-sensitive region of the pontine reticular formation and the thalamus, by combining double fluorescent retrograde tracing and immunofluorescence with a monoclonal antibody to choline acetyltransferase in the rat. The results indicated that a subpopulation (5-21% ipsilaterally) of cholinergic neurons in the mesopontine tegmentum projects to both the thalamus and the carbachol-sensitive site of the pontine reticular formation, and these neurons represented the majority (45-88%) of cholinergic neurons projecting to the pontine reticular formation site. The percentage of cholinergic neurons with dual projections was higher in the pedunculopontine tegmental nucleus (6-27%) than in the laterodorsal tegmental nucleus (4-11%). In addition, mixed with cholinergic neurons in the mesopontine tegmentum, there was a small population of dually projecting neurons that did not appear to be cholinergic. Mesopontine cholinergic neurons with dual projections may simultaneously modulate neuronal activity in the pontine reticular formation and the thalamus, and thereby have the potential of concurrently regulating different aspects of rapid eye movement sleep.

Noradrenaline hyperpolarizes identified rat mesopontine cholinergic neurons in vitro

Inhibition of brainstem cholinergic neurons by noradrenergic neurons of the locus ceruleus has long been suggested as a key mechanism of behavioral state control. In particular, the commonly held view is that noradrenaline (NA) plays a permissive role in rapid eye movement (REM) sleep generation by disinhibiting brainstem cholinergic neurons. While this notion has been supported by numerous investigations, the inhibition of cholinergic neurons by NA has never been directly demonstrated. The purpose of this study was to investigate the effects of NA upon identified cholinergic neurons in the rat mesopontine tegmentum. Using whole-cell patch-clamp recordings in slices, 175 cells were studied during bath application of 50 microM NA. Cholinergic neurons were positively identified by intracellular labeling with biocytin and subsequent staining with NADPH-diaphorase, a reliable marker for brainstem cholinergic neurons (Vincent et al., 1983). Successful intracellular labeling was obtained in 96 cells. Ninety-two percent (36 of 39) of cholinergic neurons hyperpolarized in response to NA, while noncholinergic cells (n = 57) exhibited mixed responses. Application of NA in a low-Ca2+, high-Mg2+ solution elicited the same hyperpolarizing effect as in normal solution, which indicated that the effect of NA on cholinergic neurons was direct. The noradrenergic hyperpolarization was mimicked by the alpha 2-adrenoceptor agonist UK- 14,304, and was blocked by the alpha 2-adrenoceptor antagonist idazoxan, which suggested an alpha 2-mediated response. Finally, voltage-clamp experiments revealed that NA activates the inwardly rectifying potassium current, IKG.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Amyloid Precursor Protein Catabolism Involves the Mitogen-Activated Protein Kinase Signal Transduction Pathway

Catabolic processing of the amyloid precursor protein (APP) is subject to regulatory control by protein kinases. We hypothesized that this regulation involves sequential activation of the enzymes mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated protein kinase (ERK). In the present investigation, we provide evidence that MEK is critically involved in regulating APP processing by both nerve growth factor and phorbol esters. Western blot analysis of the soluble N-terminal APP derivative APPsdemonstrated that the synthetic MEK inhibitor PD 98059 antagonized nerve growth factor stimulation of both APPs production and ERK activation in PC12 cells. Moreover, PD 98059 inhibited phorbol ester stimulation of APPs production and activation of ERK in both human embryonic kidney cells and cortical neurons. Furthermore, overexpression of a kinase-inactive MEK mutant inhibited phorbol ester stimulation of APP secretion and activation of ERK in human embryonic kidney cell lines. Most important, PD 98059 antagonized phorbol ester-mediated inhibition of Aβ secretion from cells overexpressing human APP695 carrying the “Swedish mutation.” Taken together, these data indicate that MEK and ERK may be critically involved in protein kinase C and nerve growth factor regulation of APP processing. The mitogen-activated protein kinase cascade may provide a novel target for altering catabolic processing of APP.

Correlational analysis of central noradrenergic neuronal activity and sympathetic tone in behaving cats

The activity of the noradrenergic (NA) neurons of the feline locus coeruleus complex (LCx) were correlated with changes in peripheral sympathetic tone in behaving cats. LCx NA neurons exhibit stereotypical changes in discharge rate across behavioral states, falling virtually silent during paradoxical sleep (PS). Simultaneous recordings from LCx NA neurons and the cervical sympathetic trunk demonstrated a tonic reduction in nerve activity during PS as well. LCx NA neurons were also found to fall silent during induction of the scruff immobility reflex and significant pupillary miosis was seen during this behavior as well. These data support the hypothesis that NA neurons in the brain are a central analogue of the peripheral sympathetic system and demonstrate that the two systems operate in an integrated fashion in the behaving cat.

Electrophysiological properties of cortically projecting histamine neurons of the rat hypothalamus

The tuberomammillary (TM) nuclei of the hypothalamus appear to be the sole histaminergic cell group in the brain. The extracellular electrophysiological properties of cortically projecting TM neurons were studied in the urethane-anesthetized rat. TM neurons, antidromically activated from either ipsi- or contralateral cerebral cortex, displayed relatively slow conduction velocities, consistent with reports suggesting that TM neurons possess unmyelinated axons. Spontaneous activity was slow and regular, with action potentials of long duration. There was often a noticeable delay between initial segment and somatodendritic portions of spontaneous action potentials, and complete loss of the somatodendritic portion of the second antidromic action potential was commonly seen when double pulse paradigms were employed. These data demonstrate that in addition to anatomical and biochemical similarities, TM neurons share a constellation of physiological properties with other central aminergic neurons.

A pharmacological model of ischemia in the hippocampal slice.

The effects of various metabolic inhibitors on the time course of changes in membrane potential was studied using intracellular recordings from CA1 hippocampal neurons in vitro. Concurrent application of cyanide and iodoacetic acid, agents which block oxidative phosphorylation and glycolysis respectively, result in more rapid loss of membrane function than blockade of either pathway alone. This pharmacological regimen mimics the anoxia and the hypoglycemia encountered during ischemia in vivo, both in terms of the metabolic derangement as well as the time course of changes in membrane function. Thus, this treatment appears to represent a well-controlled pharmacological model of ischemia in vitro.