Brian Malester

Hypercholesterolemia accelerates the Alzheimer's amyloid pathology in a transgenic mouse model.

Recent data suggest that cholesterol metabolism is linked to susceptibility to Alzheimers disease (AD). However, no direct evidence has been reported linking cholesterol metabolism and the pathogenesis of AD. To test the hypothesis that amyloid β-peptide (Aβ) deposition can be modulated by diet-induced hypercholesterolemia, we used a transgenic-mouse model for AD amyloidosis and examined the effects of a high-fat/high-cholesterol diet on central nervous system (CNS) Aβ accumulation. Our data showed that diet-induced hypercholesterolemia resulted in significantly increased levels of formic acid-extractable Aβ peptides in the CNS. Furthermore, the levels of total Aβ were strongly correlated with the levels of both plasma and CNS total cholesterol. Biochemical analysis revealed that, compared with control, the hypercholesterolemic mice had significantly decreased levels of sAPPα and increased levels of C-terminal fragments (β-CTFs), suggesting alterations in amyloid precursor protein processing in response to hypercholesterolemia. Neuropathological analysis indicated that the hypercholesterolemic diet significantly increased β-amyloid load by increasing both deposit number and size. These data demonstrate that high dietary cholesterol increases Aβ accumulation and accelerates the AD-related pathology observed in this animal model. Thus, we propose that diet can be used to modulate the risk of developing AD.

Inflammatory Responses to Amyloidosis in a Transgenic Mouse Model of Alzheimer’s Disease

Mutations in the amyloid precursor protein (APP) and presenilin-1 and -2 genes (PS-1, -2) cause Alzheimers 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.

cAMP sensor Epac as a determinant of ATP-sensitive potassium channel activity in human pancreatic β cells and rat INS-1 cells

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 (KATP channels; a hetero‐octomer of SUR1 and Kir6.2). If so, Epac‐mediated inhibition of KATP channels might explain prior reports that cAMP‐elevating agents promote β‐cell depolarization, Ca2+ influx and insulin secretion. Here we report that Epac‐selective cAMP analogues (2′‐O‐Me‐cAMP; 8‐pCPT‐2′‐O‐Me‐cAMP; 8‐pMeOPT‐2′‐O‐Me‐cAMP), but not a cGMP analogue (2′‐O‐Me‐cGMP), inhibit the function of KATP channels in human β cells and rat INS‐1 insulin‐secreting cells. Inhibition of KATP channels is also observed when cAMP, itself, is administered intracellularly, whereas no such effect is observed upon administration N6‐Bnz‐cAMP, a cAMP analogue that activates protein kinase A (PKA) but not Epac. The inhibitory actions of Epac‐selective cAMP analogues at KATP channels are mimicked by a cAMP agonist (8‐Bromoadenosine‐3′, 5′‐cyclic monophosphorothioate, Sp‐isomer, Sp‐8‐Br‐cAMPS), but not a cAMP antagonist (8‐Bromoadenosine‐3′, 5′‐cyclic monophosphorothioate, Rp‐isomer, Rp‐8‐Br‐cAMPS), and are abrogated following transfection of INS‐1 cells with a dominant‐negative Epac1 that fails to bind cAMP. Because both Epac1 and Epac2 coimmunoprecipitate with full‐length SUR1 in HEK cell lysates, such findings delineate a novel mechanism of second messenger signal transduction in which cAMP acts via Epac to modulate ion channel function, an effect measurable as the inhibition of KATP channel activity in pancreatic β cells.

The Glycolytic Enzymes, Glyceraldehyde-3-phosphate Dehydrogenase, Triose-phosphate Isomerase, and Pyruvate Kinase Are Components of the KATP Channel Macromolecular Complex and Regulate Its Function

The regulation of ATP-sensitive potassium (KATP) 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 KATP 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 KATP 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 KATP 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 KATP channel complex) causes channel closure.

The regulation of ion channels and transporters by glycolytically derived ATP

Abstract.Glycolysis is an evolutionary conserved metabolic pathway that provides small amounts of energy in the form of ATP when compared to other pathways such as oxidative phosphorylation or fatty acid oxidation. The ATP levels inside metabolically active cells are not constant and the local ATP level will depend on the site of production as well as the respective rates of ATP production, diffusion and consumption. Membrane ion transporters (pumps, exchangers and channels) are located at sites distal to the major sources of ATP formation (the mitochondria). We review evidence that the glycolytic complex is associated with membranes; both at the plasmalemma and with membranes of the endo/sarcoplasmic reticular network. We examine the evidence for the concept that many of the ion transporters are regulated preferentially by the glycolytic process. These include the Na+/K+-ATPase, the H+-ATPase, various types of Ca2+-ATPases, the Na+/H+ exchanger, the ATP-sensitive K+ channel, cation channels, Na+ channels, Ca2+ channels and other channels involved in intracellular Ca2+ homeostasis. Regulation of these pumps, exchangers and ion channels by the glycolytic process has important consequences in a variety of physiological and pathophysiological processes, and a better understanding of this mode of regulation may have important consequences for developing future strategies in combating disease and developing novel therapeutic approaches.

Transgenic expression of a dominant negative KATP channel subunit in the mouse endothelium: effects on coronary flow and endothelin-1 secretion

KATP channels are involved in regulating coronary function, but the contribution of endothelial KATP channels remains largely uncharacterized. We generated a transgenic mouse model to specifically target endothelial KATP channels by expressing a dominant negative Kir6.1 subunit only in the endothelium. These animals had no obvious overt phenotype and no early mortality. Histologically, the coronary endothe‐lium in these animals was preserved. There was no evidence of increased susceptibility to ergonovine‐in‐duced coronary vasospasm. However, isolated hearts from these animals had a substantially elevated basal coronary perfusion pressure. The KATP channel openers, adenosine and levcromakalim, decreased the perfusion pressure whereas the KATP channel blocker glibenclamide failed to produce a vasoconstrictive response. The inducible endothelial nitric oxide pathway was intact, as evidenced by vasodilation caused by bradykinin. In contrast, basal endothelin‐1 release was significantly elevated in the coronary effluent from these hearts. Treatment of mice with bosentan (endothelin‐1 receptor antagonist) normalized the coronary perfusion pressure, demonstrating that the elevated endothelin‐1 release was sufficient to account for the increased coronary perfusion pressure. Pharmacological blockade of KATP channels led to elevated endothe‐lin‐1 levels in the coronary effluent of isolated mouse and rat hearts as well as enhanced endothelin‐1 secretion from isolated human coronary endothelial cells. These data are consistent with a role for endothelial KATP channels to control the coronary blood flow by modulating the release of the vasoconstrictor, endothe‐lin‐1.–Malester, B., Tong, X.Y., Ghiu, I., Kontogeorgis, A., Gutstein, D. E., Xu, J., Hendricks‐Munoz, K. D., Coetzee, W. A. Transgenic expression of a dominant negative KATP channel subunit in the mouse endothelium: effects on coronary flow and endothelin‐1 secretion. FASEB J. 21, 2162–2172 (2007)

Cardiac ATP-sensitive K+ channel associates with the glycolytic enzyme complex

Being gated by high‐energy nucleotides, cardiac ATP‐sensitive potassium (KATP) channels are exquisitely sensitive to changes in cellular energy metabolism. An emerging view is that proteins associated with the KATP channel provide an additional layer of regulation. Using putative sulfonylurea receptor (SUR) coiled‐coil domains as baits in a 2‐hybrid screen against a rat cardiac cDNA library, we identified glycolytic enzymes (GAPDH and aldolase A) as putative interacting proteins. Interaction between aldolase and SUR was confirmed using GST pulldown assays and coimmunoprecipitation assays. Mass spectrometry of proteins from KATP channel immunoprecipitates of rat cardiac membranes identified glycolysis as the most enriched biological process. Coimmunoprecipitation assays confirmed interaction for several glycolytic enzymes throughout the glycolytic pathway. Immunocytochemistry colocalized many of these enzymes with KATP channel subunits in rat cardiac myocytes. The catalytic activities of aldolase and pyruvate kinase functionally modulate KATP channels in patch‐clamp experiments, whereas D‐glucose was without effect. Overall, our data demonstrate close physical association and functional interaction of the glycolytic process (particularly the distal ATP‐generating steps) with cardiac KATP channels.—Hong, M., Kefaloyianni, E., Bao, L., Malester, B., Delaroche, D., Neubert, T. A., Coetzee, W. A. Cardiac ATP‐sensitive K+ channel associates with the glycolytic enzyme complex. FASEB J. 25, 2456–2467 (2011). www.fasebj.org