Abstracts A1-A5

Category A.  Basic and Translational Neuroscience


A1.  Heat Shock Protein 70 is Necessary to Improve Mitochondrial Bioenergetics and Reverse Diabetic Sensory Neuropathy Following KU-32 Therapy.

Jiacheng Ma1, Kevin L. Farmer1, Pan Pan1, Michael J. Urban1, 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


Impaired neuronal mitochondrial bioenergetics contributes to the pathophysiologic progression of diabetic peripheral neuropathy (DPN) and may be a focal point for disease management. We have 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 Type 1 diabetes. The current study used mouse models of Type 1 and Type 2 diabetes to determine the relationship of changes in sensory neuron mitochondrial bioenergetics to the onset of and recovery from DPN. The onset of DPN showed a tight temporal correlation with a decrease in mitochondrial bioenergetics in a genetic model of Type 2 diabetes. In contrast, sensory hypoalgesia developed 10 weeks before the occurrence of significant declines in sensory neuron mitochondrial bioenergetics in the Type 1 model. KU-32 therapy improved mitochondrial bioenergetics in both the Type 1 and Type 2 models and this tightly correlated with a decrease in DPN. Mechanistically, improved mitochondrial function following KU-32 therapy required Hsp70 since the drug was ineffective in diabetic Hsp70 KO mice. Our data indicate that changes in mitochondrial bioenergetics may rapidly contribute to nerve dysfunction in Type 2 diabetes, but not Type 1 diabetes, and that modulating Hsp70 offers an effective approach toward correcting sensory neuron bioenergetic deficits and DPN in both Type 1 and Type 2 diabetes.



A2.  Identification of Human ABAD Inhibitors for Rescuing Abeta Mediated Mitochondrial Dysfunction

Koteswara Rao Valasani, Qinru Sun, Gang Hu, Jianping Li, Fang Du, Yaopeng Guo, Emily A Carlson, Xueqi Gan, and Shirley ShiDu Yan*
Department of Pharmacology & Toxicology and Higuchi Bioscience Center, School of Pharmacy, University of Kansas, Lawrence, KS 66047 (USA)


Amyloid beta (A) binding alcohol dehydrogenase (ABAD) is a cellular cofactor for promoting (A)-mediated mitochondrial and neuronal dysfunction, and cognitive decline in transgenic Alzheimer’s disease (AD) mouse models. Targeting mitochondrial ABAD may represent a novel therapeutic strategy against AD. Here, we report the biological activity of small molecule ABAD inhibitors. Using in vitro surface plasmon resonance (SPR) studies, we synthesized com- pounds with strong binding affinities for ABAD. Further, these ABAD inhibitors (ABAD-4a and 4b) reduced ABAD enzyme activity and administration of phosphonate derivatives of ABAD inhibitors antagonized calcium-mediated mitochondrial swelling. Importantly, these compounds also abolished A-induced mitochondrial dysfunction as shown by increased cytochrome c oxidase activity and adenosine-5'-triphosphate levels, suggesting protective mitochondrial function effects of these synthesized compounds. Thus, these compounds are potential candidates for further pharmacologic development to target ABAD to improve mitochondrial function.



A3.  Identification of human presequence protease (hPreP) agonists for the treatment of Alzheimer’s disease

Jhansi Rani Vangavaragu, Koteswara Rao Valasani, Xueqi Gan, Shirley ShiDu Yan*
Department of Pharmacology and Toxicology, and Higuchi Bioscience Center, School of Pharmacy, University of Kansas, Lawrence, KS 66047, USA


Amyloid-β (Aβ), a neurotoxic peptide, is linked to the onset of Alzheimer’s disease(AD). Increased Aβ content within neuronal cell mitochondria is a pathological feature in both human and mouse models with AD. This accumulation of Aβ within the mitochondrial landscape perpetuates increased free radical production and activation of the apoptotic pathway. Human Presequence Protease (hPreP) is responsible for the degradation of mitochondrial amyloid-β peptide in human neuronal cells, and is thus an attractive target to increase the proteolysis of Aβ. Therefore, it offers a potential target for Alzheimer’s drug design, by identifying potential activators of hPreP. We applied structure-based drug design, combined with experimental methodologies to investigate the ability of various compounds to enhance hPreP proteolytic activity. Compounds 3c & 4c enhanced hPreP-mediated proteolysis of Aβ (1-42), pF1β (2-54) and fluorogenic-substrate V. These results suggest that activation of hPreP by small benzimidazole derivatives provide a promising avenue for AD treatment.



A4.  Increases in Axoplasmic Transport in Neurons of the Glutamate Dehydrogenase 1 (Glud1) Transgenic Mouse and the Effects of Ethanol on Transport.

Ranu Pal, Dongwei Hui, Xinkun Wang, and Elias K. Michaelis
Higuchi Biosciences Center, University of Kansas, Lawrence, KS 66047

Background: Glutamate (Glu) is the most widespread excitatory neurotransmitter in the vertebrate central nervous system (CNS) and plays an important role in synapse formation. But, hyperactivity at Glu synapses may also lead to neuronal damage. We recently developed transgenic (Tg) mice in which the gene for glutamate dehydrogenase 1 (Glud1) is over-expressed only in CNS neurons. The Glud1 mice exhibit moderate increases in synaptic release of Glu and have decreased numbers of synapses when compared with wild-type (wt) littermates. Analysis of whole genome transcription patterns in the hippocampus of Tg and wt mice revealed several molecular and cellular functional categories that differed significantly between Tg and wt mice, including those of microtubule-binding and molecular motors involved in organelle transport and neurite formation and elongation. Based on the observed changes in gene expression we surmised that increases in Glu transmitter activity, such as those that occur in the Glud1 Tg mice, might lead to altered intracellular trafficking of proteins, organelles, and membrane vesicles. To our knowledge, modifications in axoplasmic transport as a result of chronic, excess Glu release from neurons have not been reported previously.

Results: In the present study, we have measured axoplasmic transport in ex vivo preparations of hippocampus. The major findings of the present study were that a hyperglutamatergic state in CNS neurons of Glud1 mice led to increased rates of anterograde axoplasmic transport that was inhibited by a kinesin inhibitor and was insensitive to a dynein inhibitor. Exposure of hippocampus slices to low concentrations of ethanol inhibited transport in both wt and Tg mice, but more so in the Tg.

Conclusions: The up-regulation of genes related to intracellular transport of vesicles and other organelles, as well as the increases in the rate of axoplasmic transport that we observed, may represent a coordinated compensatory response of neurons in the Glud1 Tg mice to the damage that occurs in the brains of these animals at the terminal arbors of dendrites and axons. Furthermore, our studies appear to resolve a disagreement in the literature with regard to the effects of ethanol on neuronal tissues, i.e., whether ethanol inhibits anterograde transport or enhances retrograde transport.



A5.  Overexpression of Amyloid-β-binding Alcohol Dehydrogenase increases Breast Cancer cell growth

Emily A. Carlson1, Rebecca T. Marquez2, Liang Xu2, and Shirley ShiDu Yan1
1Department of Pharmacology & Toxicology and 2Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA


Amyloid- β-binding alcohol dehydrogenase (ABAD) has been shown to play a protective role in cells undergoing stress. Upregulation of ABAD under nutrient-limiting conditions leads to recovery of a homeostatic state. Across disease states, increased ABAD levels can have a profound and varied impact, such as being beneficial in Parkinson’s disease and harmful in Alzheimer’s disease. Recently, ABAD overexpression has been observed in some prostate and bone cancers. Additionally, our preliminary data revealed that ABAD is greatly increased in tumor tissue of breast cancer patients. As ABAD can catalyze the oxidation of a wide variety of fatty acids, alcohols, and steroids, we propose that ABAD overexpression promotes breast cancer cell growth and development. The main objectives of our study were to determine the effect of increased ABAD expression on breast cancer cellular growth and function, and to examine the consequence of ABAD inhibition on cell growth. We show that T47D breast cancer cells overexpressing ABAD grow faster in cell culture and an in vivo tumor mouse model. Mitochondrial complex IV enzyme activity and ATP levels were significantly increased in T47D cells overexpressing ABAD. We further demonstrate that the increased growth rate observed in T47D cells overexpressing ABAD is reduced by inhibition of ABAD with small molecule inhibitors generated in our laboratory. Overall, our results indicate that ABAD upregulation promotes breast cancer cell growth, and ABAD inhibition can reverse that growth. These results suggest that blockade of ABAD may have potential for limiting tumor growth, which could have a significant impact on breast cancer treatment.


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