Abstracts A1 - A7

Category A.  Neuroscience and Neurodegenerative Diseases


A1.  CypD Deficiency Attenuates Mitochondrial and Synaptic Dysfunction and Cognitive Decline in STZ-induced Type 1 Diabetic Mouse Model

Long Wu, Shijun Yan, Fang Du, Shirley ShiDu Yan

Department of Pharmacology and Toxicology, and Higuchi Biosciences Center, School of Pharmacy, University of Kansas, Lawrence, Kansas, USA

Diabetes mellitus is a heterogeneous metabolic disorder associated with an increased risk of synaptic injury and cognitive dysfunction. Diabetic patients have higher incidence of dementia including the Alzheimer’s type. However, the underlying mechanisms of diabetes-associated cognitive deficits remain to be elucidated. Here we demonstrated that Cyclophilin D (CypD), the modulatory component of mitochondrial permeability transition pore (mPTP) is underlying the diabetic mitochondrial abnormalities and related synaptic and cognitive dysfunction using streptozotocin (STZ)-induced type 1 diabetic mouse model. The absence of CypD reversed synaptic dysfunction as shown by the attenuation in the reduction of long-term potentiation (LTP) in the hippocampus of STZ mice. Notably, compared with STZ treated nontransgenic mice, CypD-deficient STZ mice exhibited substantial improvement in learning and memory function in Morris Water Maze navigation task. The potential mechanisms of reversing cognitive deficits by blocking CypD might be reducing oxidative stress-induced damage on mitochondrial respiratory function. Our results provide new insights into the role of CypD-dependent mitochondrial mPTP in brain mitochondrial malfunction, synaptic perturbation and cognitive impairment. Therefore, targeting CypD could be a potential therapeutic strategy for diabetes-associated cognitive dysfunction and dementia including Alzheimer’s disease.



A2.  Development of functionally selective agonists at the kappa opioid receptor (KOR)

Lei Zhou1, Kimberly M. Lovell1, Kevin J. Frankowski2, Stephen R. Slauson2, Sarah M. Scarry2, Angela M. Phillips1, John M. Streicher1, Edward Stahl1, Cullen L. Schmid1, Peter Hodder3, Franck Madoux3, Michael D. Cameron1, Thomas E. Prisinzano2, Jeffrey Aubé2, Laura M. Bohn1

1Department of Molecular Therapeutics and Neuroscience, The Scripps Research Institute, Jupiter, FL, 33458
2Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS, 66045
3Lead Identification, The Scripps Research Institute, Jupiter, FL 33458

The kappa opioid receptor (KOR) is widely expressed in the CNS and can serve as means to modulate pain perception, stress responses and affective reward states.  Therefore, the KOR has become a prominent drug discovery target towards treating pain, depression and drug addiction.  Agonists at KOR can promote G protein coupling and ßbarrestin2 recruitment as well as multiple downstream signaling pathways, including ERK1/2 MAP kinase activation.  It has been suggested that the physiological effects of KOR activation result from different signaling cascades, with analgesia being G protein-mediated and dysphoria being mediated through ßbarrestin2 recruitment.  Dysphoria associated with KOR activation limits the therapeutic potential in the use of KOR agonists as analgesics; therefore, it may be beneficial to develop KOR agonists that are biased towards G protein coupling and away from ßbarrestin2 recruitment.  Here we describe two classes of biased KOR agonists that potently activate G protein coupling but weakly recruit ßbarrestin2.  These potent functionally selective compounds may prove to be useful tools for refining the therapeutic potential of KOR-directed signaling in vivo.



A3.  Modulating Molecular Chaperones with KU-596 Suppresses c-jun Expression and Improves Motor Function in MPZ-Raf Mice

Xinyue Zhang1, Chengyuan Li1, Brain S. Blagg2, and Rick T. Dobrowsky1

1Department of Pharmacology and Toxicology and  2Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045, USA

Demyelinating neuropathies can result from Schwann cell (SC) dedifferentiation upon loss of axonal contact or injury. Recent evidence suggests that c-jun is at the center of promoting Schwann cell dedifferentiation. Elevated c-jun levels have been detected in a variety of human neuropathies suggesting that it may be a potential target for preventing or slowing the demyelination process. We previously demonstrated that modulation of heat shock protein 90 (Hsp90) with a small molecule drug called KU-32 could decrease c-jun expression and demyelination in Schwann cell-neuron co-cultures in an Hsp70-dependent manner. In the current study, we utilized a transgenic mouse model (MPZ-Raf) in which injection of tamoxifen (TMX) leads to activation of the Raf kinase pathway in SCs, demyelination and subsequent motor dysfunction. With this model, we sought to determine whether modulating heat shock proteins is sufficient to improve the motor neuropathy that develops in these mice. For this work, we used a new generation of Hsp90 inhibitor called KU-596. Our data indicate that modulating heat shock proteins could be beneficial in c-jun-induced demyelinating neuropathies.



A4.  Small Molecular HSP90 Inhibitor Improves Mitochondrial Bioenergetics and Reverses Inflammatory Responses in Sensory Neurons of Diabetic Mice

Jiacheng Ma1, Huiping Zhao2, Brain S.J. Blagg2, and Rick T. Dobrowsky1

1Department of Pharmacology and Toxicology and  2Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045, USA

Diabetic peripheral neuropathy (DPN) is one of the most prevalent diabetic complications that at affects about 60-70% of diabetic patients. We have previously demonstrated that modulating heat shock protein 90 (Hsp90) and Hsp70 with the small molecule drug, KU-32, ameliorates psychosensory, electrophysiologic, morphologic and bioenergetic deficits of DPN in animal models of both Type 1 and Type 2 diabetes. In the current study, we sought to determine whether KU-596, an analogue of KU-32 with higher synthetic efficiency, has similar effects in ameliorating symptoms of DPN. Our results indicate that similar to KU-32, KU-596 could improve psychosensory and bioenergetics deficits of DPN in a dose-dependent manner. In addition, a subsequent RNA sequencing analysis using RNA isolated from sensory neurons suggests that KU-596 might also ameliorate symptoms of DPN through mediating the inflammatory response pathways: multiple genes/gene networks involved in inflammatory responses that are upregulated/downregulated in diabetic mice have been shown to be regulated to an opposite direction by KU-596 treatment.



A5.  Synergistic exacerbation of mitochondrial and synaptic dysfunction and resultant learning and memory deficit in a mouse model of diabetic Alzheimer’s disease

Yongfu Wang, Long Wu, Jianping Li, Shirley ShiDu Yan*

Department of Pharmacology and Toxicology, and Higuchi Biosciences Center, School of Pharmacy, University of Kansas, Lawrence, Kansas, 66047

Diabetes is considered to be a risk factor in Alzheimer's disease (AD) pathogenesis. Although recent evidence indicates that diabetes exaggerates pathologic features of AD, the underlying mechanisms are not well understood. To determine whether mitochondrial perturbation is associated with the contribution of diabetes to AD progression, we characterized mouse models of streptozotocin (STZ)-induced type 1 diabetes and transgenic AD mouse models with diabetes. Brains from mice with STZ-induced diabetes revealed a significant increase of cyclophilin D (CypD) expression, reduced respiratory function, and decreased hippocampal long-term potentiation (LTP); these animals had impaired spatial learning and memory. Hyperglycemia exacerbated the upregulation of CypD, mitochondrial defects, synaptic injury, and cognitive dysfunction in the brains of transgenic AD mice overexpressing amyloid-β as shown by decreased mitochondrial respiratory complex I and IV enzyme activity and greatly decreased mitochondrial respiratory rate. Concomitantly, hippocampal LTP reduction and spatial learning and memory decline, two early pathologic indicators of AD, were enhanced in the brains of diabetic AD mice. Our results suggest that the synergistic interaction between effects of diabetes and AD on mitochondria may be responsible for brain dysfunction that is in common in both diabetes and AD.

Key words: Alzheimer’s disease, diabetes, mitochondria, LTP, synaptic injury, cognitive impairment



A6.  Polydiallyldimethylammonium chloride (PDADMAC) coated capillaries for the separation of opioid peptides by capillary electrophoresis

Abdullah M. Al-Hossaini1,3, Leena Suntornsuk3,4 and Susan M. Lunte1,2,3

1Department of Pharmaceutical Chemistry, 2Department of Chemistry, 3Adams Institute for Bioanalytical Chemistry. The University   of Kansas, Lawrence, KS
4Department of Pharmaceutical Chemistry, Mahidol University, Thailand

Opioid peptides are endogenous molecules that are found in blood and CNS tissue samples at very low concentrations (nanomolar - picomolar) and are known to play a key role in a number of physiological functions including the physiological response to pain and stress. They are also believed to be involved in drug abuse. The opioid peptides dynorphin A, substance P and their metabolites are linked to a number of normal physiological and disease states as elevated levels of these peptides are linked with neurological disease such as Alzheimer’s and Parkinson’s disease. These peptides have an isoelectric points above 8.5 and are highly positively charged at physiological pH. Microdialysis is an in vivo sampling technique that allows for continuous monitoring of neurotransmitters and peptides in the brain and other tissues. Analyte recovery in microdialysis is dependent on flow rate. Therefore methods that require very small sample volumes make it possible to use submicroliter flow rates in microdialysis to enhance recovery and still maintain reasonable temporal resolution. Other more commonly used methods such as HPLC require large sample volumes. Capillary electrophoresis (CE) is a powerful separation technique that can achieve high efficiency separations of charged analytes. However, one limitation of CE is the adsorption of cationic proteins and peptides onto the inner surface of the capillary wall, due to the negatively charged silanol groups present on the bare fused silica capillaries. This can lead to poor separation efficiencies, band broadening and low analyte recovery. Highly charged polymers have been used as static coatings to improve CE separation and efficiencies of analytes of the same charge. A positively charged polydiallyldimethylammonium chloride (PDADMAC) polymer (MW 200-350 KDa) is being evaluated as a static coating for CE. This is a polymer coats the capillary walls to produce a layer of positive charge that repels cationic peptides (fragments of the peptides dynorphin A & substance P) at low pH BGE, reducing peptides adsorption onto the capillary wall. This polymer coating lead to an improvement in separation efficiency and increased analytes recovery. Electroosmotic flow, apparent electrophoretic mobilities, resolution and plate numbers of the analytes of interest was compared to those obtained by conventional capillary zone electrophoresis on bare silica.



A7.  Use of Griess assay and ME-LIF for detection and quantification of nitrite and nitric oxide produced by RAW 264.7 macrophages

Claudia G. Fresta1,2, Giuseppe Caruso2,4, Richard P. S. de Campos1,2,3, Joseph M. Siegel1,2, and Susan M. Lunte1,2,5

1Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, Kansas
2Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas
3Department of Chemistry, State University of Campinas, Campinas, Brazil
4Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas
5Department of Chemistry, University of Kansas, Lawrence, Kansas

Since the discovery that nitric oxide (NO) is the endothelium-derived relaxing factor in 1986, this molecule has been shown to exhibit numerous other physiological functions. NO is produced from arginine by a group of enzymes known as nitric oxide synthases. Mammalian cells are endowed with three genes encoding distinct isoforms of NOS - nNOS (neuronal), eNOS (endothelial), and iNOS (inducible). The production of NO is substantially increased in immune and other cell types through the upregulation of iNOS caused by exposure to stimulating agents such as lipopolysaccharide (LPS). The reaction of NO with O2 or O2- can result in the formation of reactive nitrogen species (RNS), such as dinitrogen trioxide (N2O3) and peroxynitrite (ONOO-).

In order to prove the connection between NO production and iNOS activation in macrophages, two different iNOS inhibitors were investigated. In addition, the effect of ascorbic acid and L-carnosine on RNS production was investigated. Carnosine is a hydroxyl scavenger of and superoxide radicals as well as a strong quencher of singlet oxygen. Ascorbic acid has been shown to be important for immune response. Macrophages under stressed condition (eg. LPS stimulation) increase their uptake of ascorbic acid 100-fold compared to untreated ones.

The goals of this research were to (1) determine the intracellular and extracellular concentrations of the primary degradation product of NO, nitrite (NO2-), in macrophages cells under different stress conditions and (2) investigate the effect of antioxidants on extracellular NO2- production under stressful conditions. Our results indicate that macrophages stimulated by LPS and IFN-γ produce a higher amount of intracellular and extracellular nitrite along and exhibit a pronounced cell differentiation. To verify that iNOS was the source of nitrite production, it was found that the specific inhibitors of iNOS, L-NAME and L-NMMA, drastically reduced the amount nitrite produced inside the cell. The two antioxidants exhibited converse behaviour with regards to extracellular nitrite production. Ascorbic acid was found to decrease extracellular nitrite, while carnosine increased extracellular nitrite production in a concentration-dependent manner. Lastly, analysis by microchip electrophoresis using DAF-FM DA and LIF detection confirmed the production of a higher amount of NO by macrophages under simulated inflammation conditions.


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