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Mutations in the amyloid precursor protein (APP) and presenilin-1 and -2 genes (PS-1, -2) cause Alzheimer's disease (AD). Mice carrying both mutant genes (PS/APP) develop AD-like deposits composed of beta-amyloid (Abeta) at an early age. In this study, we have examined how Abeta deposition is associated with immune responses. Both fibrillar and nonfibrillar Abeta (diffuse) deposits were visible in the frontal cortex by 3 months, and the amyloid load increased dramatically with age. The number of fibrillar Abeta deposits increased up to the oldest age studied (2.5 years old), whereas there were less marked changes in the number of diffuse deposits in mice over 1 year old. Activated microglia and astrocytes increased synchronously with amyloid burden and were, in general, closely associated with deposits. Cyclooxygenase-2, an inflammatory response molecule involved in the prostaglandin pathway, was up-regulated in astrocytes associated with some fibrillar deposits. Complement component 1q, an immune response component, strongly colocalized with fibrillar Abeta, but was also up-regulated in some plaque-associated microglia. These results show: i) an increasing proportion of amyloid is composed of fibrillar Abeta in the aging PS/APP mouse brain; ii) microglia and astrocytes are activated by both fibrillar and diffuse Abeta; and iii) cyclooxygenase-2 and complement component 1q levels increase in response to the formation of fibrillar Abeta in PS/APP mice.

Neuropathy target esterase (NTE) is a neuronal membrane protein originally identified for its property to be modified by organophosphates (OPs), which in humans cause neuropathy characterized by axonal degeneration. Drosophila mutants for the homolog gene of NTE, swisscheese (sws), indicated a possible involvement of sws in the regulation of axon-glial cell interaction during glial wrapping. However, the role of NTE͞sws in mammalian brain pathophysiology remains unknown. To investigate NTE function in vivo, we used the cre͞loxP site-specific recombination strategy to generate mice with a specific deletion of NTE in neuronal tissues. Here we show that loss of NTE leads to prominent neuronal pathology in the hippocampus and thalamus and also defects in the cerebellum. Absence of NTE resulted in disruption of the endoplasmic reticulum, vacuolation of nerve cell bodies, and abnormal reticular aggregates. Thus, these results identify a physiological role for NTE in the nervous system and indicate that a loss-of-function mechanism may contribute to neurodegenerative diseases characterized by vacuolation and neuronal loss.cre͞lox P ͉ vacuolation ͉ thalamus ͉ hippocampus ͉ Purkinje cells N europathy target esterase (NTE) is a 150-kDa transmembrane protein that is highly conserved among species including insects, nematodes, yeast, and bacteria (1). Mutations in the Drosophila homolog, swisscheese (sws), produced glial-axonal hyperwrapping, degeneration, and a shortened life span (2). Both neurons and glia underwent cell death, accompanied by extensive vacuolation in the nervous system of Drosophila sws mutants. The NTE protein represents the mammalian homolog of the Drosophila swisscheese gene (3). The swisscheese and NTE proteins share a number of motifs including several hydrophobic transmembrane sequences, a serine esterase domain, and a sequence similar to the regulatory subunit of protein kinase A. A variety of pharmacological experiments have established the involvement of NTE in organophosphate (OP)-induced delayed neuropathy in humans and in animal models (3, 4), presumably through inactivation of the serine esterase activity. The protein NTE was initially identified as a target of OPs, found in pesticides and chemical warfare agents that cause peripheral neuropathy in humans (5, 6). OP-induced neuropathy has been implicated in Gulf War disease and other toxic neuropathies. However, the physiological functions of NTE, as well as the downstream events of NTE-induced neuropathy after OP exposure, remain unknown.We therefore sought to examine the physiological role of NTE in the central nervous system. Knockout of the NTE gene is embryonic lethal in mice at an early embryonic age (4, 19), thereby precluding the study of the role of NTE in the adult brain. We therefore constructed a conditional mutant NTE strain and deleted NTE specifically in neuronal tissues. We show here that neuron-specific deletion of NTE causes vacuolation of neuronal bodies and dendrites. The NTE protein was found to associate with the endopla...

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The Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs, also known as Epac1 and Epac2) mediate stimulatory actions of the second messenger cAMP on insulin secretion from pancreatic β cells. Because Epac2 is reported to interact in vitro with the isolated nucleotide-binding fold-1 (NBF-1) of the β-cell sulphonylurea receptor-1 (SUR1), we hypothesized that cAMP might act via Epac1 and/or Epac2 to inhibit β-cell ATP-sensitive K + channels (K ATP channels; a hetero-octomer of SUR1 and Kir6.2). If so, Epac-mediated inhibition of K ATP channels might explain prior reports that cAMP-elevating agents promote β-cell depolarization, Ca

The regulation of ATP-sensitive potassium (K ATP ) channel activity is complex and a multitude of factors determine their open probability. Physiologically and pathophysiologically, the most important of these are intracellular nucleotides, with a long-recognized role for glycolytically derived ATP in regulating channel activity. To identify novel regulatory subunits of the K ATP channel complex, we performed a two-hybrid protein-protein interaction screen, using as bait the mouse Kir6.2 C terminus. Screening a rat heart cDNA library, we identified two potential interacting proteins to be the glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triose-phosphate isomerase. The veracity of interaction was verified by co-immunoprecipitation techniques in transfected mammalian cells. We additionally demonstrated that pyruvate kinase also interacts with Kir6.2 subunits. The physiological relevance of these interactions is illustrated by the demonstration that native Kir6.2 protein similarly interact with GAPDH and pyruvate kinase in rat heart membrane fractions and that Kir6.2 protein co-localize with these glycolytic enzymes in rat ventricular myocytes. The functional relevance of our findings is demonstrated by the ability of GAPDH or pyruvate kinase substrates to directly block the K ATP channel under patch clamp recording conditions. Taken together, our data provide direct evidence for the concept that key enzymes involved in glycolytic ATP production are part of a multisubunit K ATP channel protein complex. Our data are consistent with the concept that the activity of these enzymes (possibly by ATP formation in the immediate intracellular microenvironment of this macromolecular K ATP channel complex) causes channel closure.K ATP channels act as metabolic sensors of a large diversity of cell types by directly coupling their energy metabolism to cellular excitability. This function serves as a crucial regulatory mechanism in the responses of various cell types to their metabolic demand. For example, K ATP channels mediate insulin release from pancreatic ␤ cells, control the firing rate of glucose-responsive neurons in the ventromedial hypothalamus, and protect neurons during hypoxia. K ATP channels also have unique roles in the cardiovascular system. In the coronary vasculature, they participate in the maintenance of the coronary vascular tone, whereas in cardiac myocytes, K ATP channel modulation causes alterations in action potential duration and induction of arrhythmias during cardiac ischemia (1).The minimum requirement for the formation of a heterologously expressed K ATP channel appears to be the presence of two types of subunits, namely a pore-forming subunit (Kir6.x) belonging to the family of inward rectifying K ϩ channel subunits and a sulfonylurea receptor regulatory subunit, which is a member of the family of ABC-cassette proteins (2). However, ion channels are increasingly realized to be multisubunit macromolecular complexes (3-5). Recent evidence suggest that the K ATP channel pro...