Abstracts A1-A4

Category A.  Neuroscience and Neurodegenerative Diseases

 

A1.  Mutant Huntingtin-Calmodulin Interaction: Potential Therapeutic Target for Huntington’s Disease

Khushboo Kapadia1, Anuradha Roy2, Peter McDonald2, Jeff Aube3, Nancy Muma1
 

1Department of Pharmacology and Toxicology, The University of Kansas, Lawrence, KS, USA.
2 High Throughput Screening Laboratory, The University of Kansas, Lawrence, KS, USA.
3Department of Chemical Biology and Medicinal Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Huntington’s disease (HD) is a neurodegenerative disorder caused by an autosomal dominant mutation in the huntingtin (htt) gene. Previous studies in our lab demonstrated that disrupting the binding of mutant huntingtin (mhtt) to calmodulin (CaM) had beneficial effects in cell culture and the R6/2 transgenic animal model. The goal of the current study is to identify and develop small chemical compounds that are non-toxic and can selectively disrupt the binding of mhtt to CaM. To this end, we screened ~ 199,270 compounds from various chemical libraries using a high throughput AlphaScreen assay. The primary AlphaScreen assay along with counter-screening assays have identified 481 hit compounds that disrupt the interaction between (His)htt-CaM(GST). The structures of the hits obtained were analyzed and 8 structurally diverse representative compounds were chosen. These compounds were re-screened in the primary AlphaScreen assay and also counter-screened in the alpha screen His-GST assay to eliminate compounds that disrupt the His-GST interaction without the presence of protein. Three out of the eight compounds have shown preferential activity in disrupting the (His)htt-CaM(GST) interaction. Currently, secondary assays are being employed to determine if the compounds identified in the primary screen can selectively disrupt the mhtt-CaM interaction without affecting other functions of CaM. The compound selectivity is being determined using two CaM dependent enzymes which are abundantly expressed in neuronal cells and play an important role in neuronal function; Ca+2/CaM dependent protein kinase kinase alpha and Ca+2/CaM dependent protein kinase 2 gamma (CAMK2ϒ). To date, kinase inhibition assay results indicate that one compound has ~ 75 fold selectivity and another compound has a 8 fold selectivity for inhibition of the CAMK2ϒ enzyme when compared to the IC50 value obtained in the primary screen. The selective compounds once identified will further be tested for cytotoxicity and neuroprotective effects in cells expressing mhtt. These studies will aid in identifying compounds that will serve as novel and promising biological probes for drug development in HD.

-top-

 

A2.  Neuronal overexpression of Cyclophilin D promotes tau pathology, mitochondrial dysfunction, and anxiety-like behavior in AD model

Erika Northcutt, Qing Yu, Fang Du, Firoz Akhter, ShiDu Yan

Department of Pharmacology and Toxicology, The University of Kansas, Lawrence, KS, USA.

Alzheimer’s disease (AD) affects 5 million Americans and costs the country $236 billion. Over 200 drugs addressing other aspects of the disease have failed to show safe, therapeutic effect in clinical trials. Cyclophilin D (CypD) is a key modulator of mitochondrial homeostasis and is a novel therapeutic target for neurodegeneration. Overexpression of CypD increases accumulation of hyperphosphorylated tau, increases synaptic and neuronal loss, reduces mitochondrial function, and induces an anxiety phenotype in an animal model of mutant tau-induced Alzheimer’s disease. This research provides a basis for future pharmacological interventions into this system.

-top-

 

A3.  KU596 Improves Mitochondrial Bioenergetics and Decreases Oxidative Stress in Diabetic Sensory Neurons via Hsp70

Zhenyuan You1, Brian Blagg2, Rick T. Dobrowsky1

1Department of Pharmacology & Toxicology, University of Kansas, Lawrence, KS, USA;
2Department of Medicinal Chemistry, University of Kansas, Lawrence, KS, USA

Neuronal mitochondrial dysfunction is a key pathophysiologic mechanism of diabetic peripheral neuropathy (DPN). In vivo treatment with the heat shock protein (Hsp) 90 modulator KU596 can reverse symptoms of DPN and this correlates with improving mitochondrial bioenergetics (mtBE) in diabetic sensory neurons in an Hsp70 dependent manner. However, we have not determined the mechanism by which KU596 improves mtBE. The goal of the current work is to determine if KU596 improves mtBE by decreasing glucose-induced oxidative stress. In WT sensory neurons, KU596 significantly improved mtBE in non-diabetic and diabetic neurons under both normal and hyperglycemic conditions. Surprisingly, although KU596 increased mtBE in Hsp70 KO neurons isolated from diabetic mice and maintained under normal glucose conditions, the drug was not able to improve mtBE when these neurons where placed in high glucose medium. These results suggest that KU596 does not require Hsp70 to improve mtBE in cells incubated in low glucose but that induction of hyperglycemia requires Hsp70 for the drug to improve mtBE. We next determined if this effect was related to an inability of KU596 to decrease oxidative stress under hyperglycemic conditions by measuring mitochondrial superoxide with fluorescent microscopy. In WT and Hsp70 KO neurons, superoxide levels were significantly increased in cell incubated with high glucose. Although KU596 decreased the ability of hyperglycemia to increase superoxide levels in WT neurons, this effect was lost in the hyperglycemically stressed Hsp70 KO neurons. These data means that the ability of KU596 to increase mtBE in diabetically stressed neurons is linked to an Hsp70-dependent inhibition of glucose-induced superoxide production. However, the cause and effect relationship between improved respiration and decreased oxidative stress has not been determined. Since MnSOD is the main mechanism to detoxify mitochondrial superoxide radicals, we knocked it down by using MnSOD shRNA adenovirus to increase mitochondrial oxidative stress in sensory neurons. Comparing with scrambled shRNA, MnSOD downregulation blocked KU596 effect on enhancing mtBE in diabetic sensory neurons. This means the ability of KU596 to improve mitochondrial bioenergetics is through reducing mitochondrial oxidative stress.

-top-

 

A4.  Modulating Molecular Chaperones with KU-596 Suppresses c-jun Expression and Improves Motor Function in Demyelinating Mouse Models

Xinyue Zhang1, Brian Blagg2, Rick T. Dobrowsky1

1Department of Pharmacology & Toxicology, University of Kansas, Lawrence, KS, USA;
2Department of Medicinal Chemistry, University of Kansas, Lawrence, KS, 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 the small molecule Hsp90 modulator KU-32 could decrease c-jun expression and demyelination in SC-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, elevated c-jun expression, demyelination and subsequent motor dysfunction. With this model, we sought to determine whether modulating heat shock proteins with KU596, a second generation Hsp90 modulator was sufficient to improve the motor neuropathy that develops in these mice. Treating MPZ-Raf mice with KU-596 decreased the induction of c-jun, improved motor performance on the rotorod, decreased the onset of rear-limb paralysis and improved the extent of peripheral nerve demyelination. In addition, KU596 treatment showed similar effects in connexin 32 deficient (Cx32def) mice, a model for X-linked Charcot-Marie-Tooth disease (CMT1X). One-month treatment with KU596 decreased the c-jun expression and improved nerve function in male Cx32def mice. Collectively, our data indicate that modulating heat shock proteins could be beneficial in attenuating the severity of demyelinating neuropathies.

-top-


One of 34 U.S. public institutions in the prestigious Association of American Universities
Nearly $290 million in financial aid annually
44 nationally ranked graduate programs.
—U.S. News & World Report
Top 50 nationwide for size of library collection.
—ALA
23rd nationwide for service to veterans —"Best for Vets," Military Times
KU Today