Category A. Basic and Translational Neuroscience
A1. Small Hsp90 Inhibitors Improves Symptoms of Diabetic Peripheral Neuropathy through Restoring Mitochondrial Bioenergetics in an Hsp70-dependent Manner
Jiacheng Ma, Rick T. Dobrowsky*
Department of Pharmacology and Toxicology, The University of Kansas, Lawrence, KS
Diabetic peripheral neuropathy (DPN) is one of the most prevalent diabetic complications that affects about 60-70% of all diabetic patients. Due to the high complexity of the disease etiology, therapies that target at pathogenic pathways have gain little success so far. Thus our innovative strategy explores the therapeutic potential of reinforcing innate cellular protective mechanisms-molecular chaperpones. Molecular chaperones, such as heat shock protein 90 and 70 (Hsp90, Hsp70) are essential, widely distributed proteins that are involved in cellular stress responses. The chaperone machinery is designed to prevent and reverse protein aggregation, as well as target misfolded/aggregated proteins for proteolytic degradation. Our previous studies using KU32, a small molecule inhibitor of Hsp90, have shown that pharmacological inhibition of Hsp90 reverses multiple clinical indices of DPN in wild type (WT) but not Hsp70 Knockout (Hsp70 KO) mice. However, the mechanisms underlying the protective effects of KU32 are yet to be determined.
Mitochondrial dysfunction has been established as one fundamental factor that contributes to the pathogenesis of DPN. HSPs, especially Hsp70, are important for the maintenance of mitochondrial function and cell viability. Thus, we evaluated the possibility that KU32 improves clinical indices of DPN through restoring mitochondrial function in sensory neurons. Using a streptozotocin (STZ)-induced type I diabetic mouse model (Swiss Webster mice), we observed that KU32 improved mitochondrial bioenergetics, reversed sensory hypoalgesia and nerve conduction deficits in a time-dependent manner. The restoration in mitochondrial bioenergetics correlated with improved sensory nerve performance in KU32-treated diabetic mice, suggesting that KU32 might reverse sensory deficits of DPN through improving mitochondrial function. Further, age-matched WT C57Bl/6 and Hsp70 KO mice were rendered diabetic with STZ injection. We observed that KU32 reversed diabetes-induced decrease in mitochondrial spare respiratory capacity (SRC) in WT but not Hsp70 KO mice. Taken together, these data suggest that KU32 might reverse sensory hypoalgesia and nerve conduction deficits at chronic stages of DPN by improving mitochondrial function, and the effect of KU32 on mitochondrial function is Hsp70-dependent.
A2. Effects of glutamate dehydrogenase overexpression and glutamate binding protein knockout on locomotor behavior of mice
Ranu Pal, Dongwei Hui, Alexandra Akhunova, Xinkun Wang and Elias K. Michaelis
Higuchi Biosciences Center, University of Kansas, Lawrence, KS 66047
Glutamate (Glu), a major excitatory neurotransmitter and a signaling molecule in the brain, has long lasting effects on neuronal structure, function and synaptic plasticity. Acute, excess release of Glu in the environment of neurons can lead to neuronal toxicity and death. To assess the effects of moderate, chronic excess release of Glu we created a mouse model where glutamate dehydrogenase 1 (GLUD1), a mitochondrial metabolic enzyme is over-expressed under the control of the neuron-specific enolase promoter. In the transgenic (Tg) mice, moderate neuronal loss occurs in certain regions of the brain such as the somatosensory cortex, CA1 region of the hippocampus, and the striatum (Bao et al, 2009). We previously showed that down-regulation of the Glu Binding Protein (GBP) provides protection against Glu-induced neurotoxicity. Therefore, we generated both hetero- and homozygous null mutants of the GBP and have cross bred them to Balb/c mice as well as to homozygous Glud1 Tg mice. In terms of the behavior of the Glud1 Tg mice, their overall locomotor activity was significantly higher than that of the wt mice. The locomotor activity of each mouse was sub-classified into fine or ambulatory movements, and as occurring in the center or the periphery of the open field. The Glud1 Tg mice exhibited significantly higher levels of central ambulatory and of peripheral fine and ambulatory movements as compared with the wt mice. Following the measurement of baseline locomotor activities, both Tg and wt mice were injected with MK801 (an NMDA receptor antagonist, 0.15mg/kg). After an initial post-injection suppression of activity, the wt mice steadily recovered their peripheral ambulatory activity, whereas the Glud1 Tg mice did not. The Glud1 Tg mice were cross-bred with GBP homo- and heterozygous null mutants in order to determine whether decreased presence of GBP would protect the Glud1 mice from neuronal loss. Here we report on the locomotor activity of the GBP null mutant and the Glud1/GBP hybrid mice. The baseline locomotor activity of the GBP null mutant mice was lower than that of wt C57Bl/6 or of the Glud1 Tg mice on a C57Bl/6 background. The hybrid mice with Glud1 Tg and either GBP homo- or hetero-zygous null mutations gene exhibited significantly higher locomotor activity than the null mutants without the Glud1 transgene. We now plan to perform careful microanatomical characterization of the GBP null mutant mice with or without the Glud1 transgene to determine whether suppression of GBP expression has protected neurons from excess Glu release.
A3. Imbalanced neuregulin-1 isoforms and down-regulated erbin expression are associated with enhanced erb b2 receptor activation in diabetic peripheral neuropathy
P. Pan and R. T. Dobrowsky
Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS
Growth factors mediate numerous interactions between neurons and glia. Impaired trophic support and aberrant neuron/glia interaction can contribute to various neurodegenerative diseases, including diabetic peripheral neuropathy (DPN). In peripheral nerves, neuregulins (NRGs) regulate Schwann Cell (SC) growth, migration and differentiation through interacting with Erb B receptors, which are members of epidermal growth factor receptor (EGFR) family. NRG-1 family comprises three major isoforms (I, II and III) with distinct functions: NRG-1 type I and NRG-1 type II can induce SC dedifferentiation, whereas NRG-1 type III promotes myelination and regulates myelin thickness. As an adapter protein downstream of Erb B2 receptor, Erbin has been reported to bind and stabilize inactive Erb B2 receptor and is critical to normal myelination of peripheral nerves. The demyelination-related mitogen-activated protein kinase (MAPK) pathway can be inhibited by Erbin. Abnormal activation of Erb B2 receptor can contribute to the development of DPN since administration of the Erb B2 receptor inhibitor, erlotinib, partially reversed several pathophysiologic aspects of DPN, including the sensory hypoalgesia, nerve conduction velocity deficits, and the diabetes-induced reduction in intra-epidermal nerve innervation at the distal ends of axons. However, whether enhanced Erb B2 activation was associated with changes in the expression of NRG-1 isoforms, Erbin, or MAPK activation remained unclear. Using a streptozotocin (STZ)-induced type I diabetic mouse model, we observed a 2.6-fold decrease in NRG-1 type III but a 2-fold increase in NRG-1 type I level in sural nerve of diabetic mice. We also detected a 2-fold lower expression of Erbin in sciatic nerve of diabetic mice. In myelinated SC/DRG neuron co-cultures, hyperglycemia sensitized NRG-induced demyelination and this correlated with a decrease in Erbin expression and an elevated MAPK activation compared to normoglycemic control cultures. These results support that hyperglycemia may impair NRG/Erb B2 signaling by affecting the expression of NRG-1 isoforms in peripheral nerve and decreasing the expression of Erbin, a negative regulator of Erb B2. Together, imbalanced NRG-1 isoforms and down-regulated Erbin may contribute to the altered neuregulinism in DPN.
Support: NIH Grant
Juvenile Diabetes Research Foundation
A4. Analytical Methodology for the Investigation of the Dopamine Metabolic Pathway using Microchip Electrophoresis with Electrochemical Detection
Rachel A. Saylor, Erin A. Reid, and Susan M. Lunte
Department of Chemistry and Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, KS, United States of America
The dopamine metabolic pathway involves many biogenic amines, including L-DOPA and dopamine, that have been implicated in numerous neurological processes and disease states, including Parkinson’s disease. In order to study the transport of L-DOPA across the blood-brain barrier, its conversion into dopamine, and the subsequent degradation processes in vivo, an analytical method must be developed that has fast analysis times and the ability to analyze the small sample volumes (100 nL – 1 µL) characteristic of brain microdialysate samples. Towards this end, a method for the separation and detection of L-Tyr, L-DOPA, dopamine, DOPAC, and HVA by microchip electrophoresis and electrochemical detection was developed. Separation conditions were optimized with a run buffer consisting of 15 mM phosphate, 15 mM SDS, and 2.5 mM boric acid providing the best separation. Both PDMS/PDMS and hybrid PDMS/glass microchips with in-channel detection at carbon fiber and pyrolyzed carbon electrodes, respectively, were evaluated. While hybrid microchips generated a much more reproducible separation, the efficiency was much lower with 43,100 (±1,100) plates/meter, compared to 228,000 (±13,000) plates/meter for PDMS/PDMS chips. The ultimate goal is to integrate this system with a miniaturized analysis system and telemetry for on-animal sensing in freely roaming animals.
A5. Intervention with the Heat Shock Protein 90 Inhibitor KU-32 Improves Chronic Experimental Diabetic Neuropathy
Michael J. Urban1, Pan Pan2, Kevin L. Farmer2, Brian S. J. Blagg3, and Rick T. Dobrowsky*1,2 1Neuroscience Graduate Program, The University of Kansas, Lawrence, KS 66045
2Department of Pharmacology and Toxicology, The University of Kansas, Lawrence, KS 66045
3Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045
Inducing the heat shock response (HSR) through Hsp90 inhibition augments heat shock protein support and may improve several aspects of neurodegenerative phenotypes. Heat shock proteins are molecular chaperones that assist in the folding of nascent polypeptides (client proteins) into their mature conformations. They also serve as intracellular triage units that refold damaged proteins, stabilize protein complexes, solubilize protein aggregates, and clear irreparable proteins. However, a confounding issue surrounding Hsp90 inhibitors is their inability to generate therapeutic windows that dissociate cytotoxic client protein degradation from HSR induction. We have developed a novel C-terminal Hsp90 inhibitor, KU-32, that induces the HSR while divesting client protein degradation, expanding the dose range for neuroprotection.
After 16 weeks of diabetes induced by streptozotocin in Swiss-Webster mice, the effects of weekly doses of KU-32 (20 mg/kg, intraperitoneal) on several standard indices of diabetic neuropathy were measured. Mice rendered diabetic after 16 weeks displayed clear deficits in thermal and mechanical sensitivity as well as motor and sensory nerve conduction velocities (MNCV and SNCV). Diabetic animals receiving weekly KU-32 treatments over 10 weeks exhibited a steady recovery to control levels in thermal and mechanical sensitivity, MNCV, and SNCV. Intra-epidermal nerve fiber (iENF) density analysis within the plantar surface of the hind paw revealed a ~31% reduction in cutaneous innervation in diabetic mice, a symptom commonly observed in diabetic patients. KU-32 treatment partially, but significantly, improved diabetic iENF density to within ~11% of untreated controls. Dorsal root ganglia (DRG) (3 mice per test group) were also collected and cultured to assess mitochondrial respiration (via XF96 Analyzer). After 26 weeks, oligomycin-induced reductions in oxygen consumption rates (OCRs) increased significantly in untreated diabetic DRG. This suggests that diabetic DRG devote most of their basal O2 consumption toward ATP synthesis, which may result from impaired electron transport capacities. KU-32 treatment of diabetic mice partially, but significantly, restored oligomycin-sensitive OCR, suggesting KU-32 improves ATP synthesis efficiency in diabetic DRG. Though both diabetic groups failed to generate spare respiratory capacities, KU-32-treated diabetic DRG exhibited an increased ability to rebound to near basal OCR values following FCCP treatment. Hence, KU-32 may improve resiliency to damage over extended periods of mitochondrial distress. These results suggest that KU-32 may reverse sensory hypoalgesia and nerve conduction deficits at more chronic stages of DPN by improving mitochondrial function and promoting nerve fiber survival/innervation. In vivo pharmacokinetic analyses have also established KU-32 bioavailability within the DRG, foot pads, and sciatic, tibial, and sural nerves, implicating several potential sites for drug efficacy.
A6. RNA-Seq analysis of two brain regions vulnerable to Alzheimer’s disease
1Genomics Facility, 2Higuchi Biosciences Center, 3Department of Pharmacology and Toxicology
The University of Kansas, Lawrence, KS 66047, USA
Alzheimer’s disease (AD), the most devastating neurodegenerative disease, does not affect different brain regions equally. Temporal and frontal lobes are among the brain regions most affected in AD. As the transcriptomic profile of neurons in a certain brain region largely affects their response to pathological conditions like AD, comparative transcriptomic analysis of these susceptible regions can be used to understand why they are particularly vulnerable to the disease. With the rapid advances in next-generation sequencing technologies, RNA-Seq, or whole transcriptome shotgun sequencing, has begun to become a mainstream approach to study brain regions that are affected by neurodegenerative diseases including AD.
In this study, RNA-Seq data that have recently become available from both normally aged and AD brain temporal and frontal lobes (SRA [http://www.ncbi.nlm.nih.gov/sra] Accession #: SRX035166, SRX035171, SRX035167, and SRX034874) were analyzed, in order to provide molecular insights into their common vulnerability while accounting for their regional specificities.
In this presentation, transcriptomic similarities and differences between temporal and frontal lobes as detected by RNA-Seq will be presented. Interpretation of their similarities helps understand their shared vulnerability to AD. Detection of their differences in both normal aging and AD helps elucidate the progression of this disease in the two different regions.
The study of AD from the perspective of selective regional vulnerability is the first step toward minimizing its devastating effects to patients through protecting vulnerable brain neurons.
The author is supported by NIH grants including NIGMS 1P20GM103638-01, NIA P30 AG035982, NIA P01 AG12993, and NICHD P30 HD02528, and the Miller-Hedwig and Wilbur Fund.