Higuchi Biosciences Center - University of Kansas

Fall 2008 Science Talks
December 5, 2008

A1. Cocaine-induced increases in 5-HT_2A receptor function in frontal cortex is associated with increased levels of Gβ subunits and decreased levels of GRK5 proteins
Gonzalo A. Carrasco, Ph.D.
¹Dept. of Pharmacol. & Toxicol., School of Pharmacy, University of Kansas, KS 66045

We previously demonstrated that early withdrawal from cocaine induces supersensitivity of serotonin 2A (5-HT_2A) receptor signaling. This supersensitivity is associated with region-specific increases in the levels of Gα_q and Gα₁₁ proteins in hypothalamic paraventricular nucleus, and basolateral and central amygdala; and with increased 5-HT_2A receptor-mediated phospholipase C Beta (PLCβ) activity in frontal cortex. The cocaine withdrawal-associated increase in 5-HT_2A-stimulated PLCβ activity in cortex occurs independent of changes in the protein levels of Gα_q, Gα11 or 5-HT_2A receptors. In this study, we examined the effects of cocaine withdrawal on several intracellular signaling proteins that associate with 5-HT_2A receptors and could mediate the increase in PLCβ activity in frontal cortex. Adult male Spague-Dawley rats were treated with either saline (1ml/kg, b.i.d) or cocaine (15 mg/kg, b.i.d) for 7 days. After 48 hours of withdrawal, rats were sacrificed and the brains were immediately removed and the frontal cortex was dissected and frozen in liquid nitrogen for subsequent determination of PLCβ activity and Western blot analysis.

Rats withdrawn from cocaine showed increased 5-HT_2A-stimulated PLCβ activity (↑80%) compared to saline-treated rats. This increased PLCβ activity was associated with 2-fold increases in levels of membrane-associated G-protein Beta (Gβ) subunits and reduced (↓30%) levels of membrane-associated G-protein receptor kinase 5 (GRK5) compared to saline controls. No changes were detected in the membrane-associated levels of G-protein receptor kinase 2 (GRK2), p90 ribosomal S6 kinase (RSK-2), caveolin-1, and Beta(β)-arrestin 2. Consistent with our previous studies, we did not detect changes in the levels of membrane-associated Gαq, Gα11 or 5-HT_2A receptors. Previous reports demonstrated that Gβ subunits can directly stimulate PLCβ activity and that GRK5-induced phosphorylation of 5-HT_2A receptors is associated with desensitization of 5-HT_2A receptor signaling. Therefore, cocaine withdrawal-induced increases in cortical 5-HT_2A–stimulated PLCβ activity could be mediated by the observed increases in Gβ subunits and decreases in GRK5.

In summary, our results reveal unique neuroadaptations of 5-HT_2A receptors in frontal cortex associated with cocaine withdrawal. As 5-HT_2A receptors regulate mood and impulse control, and 5-HT_2A receptor antagonists block cocaine relapse, we hypothesize that withdrawal-associated increases in 5-HT_2A receptor function in various limbic regions may be clinically relevant with respect to drug relapse.

This research was supported by the University of Kansas startup funds (GAC).

-top-

A2. Design and Synthesis of Ring-constrained Derivatives of Salvinorin A
Kimberly Lovell^†*, Denise S. Simpson^†, Christina M. Dersch^¦, Richard B. Rothman^¦, Thomas E. Prisinzano^†
^†Division of Medicinal Chemistry, The University of Kansas., Lawrence, Kansas; ^¦Clinical Psychopharmacology Section, IRP, NIDA, NIH, DHHS, Baltimore, MD

Substance abuse disorders result in significant personal and economic impact of many Americans. Currently, 9.3% of the US population suffers from a substance abuse disorder. Substance abuse contributes to the spread of HIV-1, Hepatitis B and C, as well as drug-resistant tuberculosis. A growing amount of evidence suggests that κ opioid (KOP) receptors are involved in the control of several abuse related effects of central nervous system (CNS) stimulants. KOP receptor agonists modulate the activity of dopamine neurons and decrease self-administration of cocaine in non-human primates. KOP receptor antagonists have the potential to be utilized as opioid abuse therapies and in the treatment of stress-induced reinstatement (a model of drug relapse). Salvinorin A (SVA) is the first non-nitrogenous scaffold having high affinity and efficacy at KOP receptors. In addition to a unique structure, SVA induces hallucinations through a mechanism different from classical hallucinogens. Past structure-activity relationship (SAR) studies have shown that modification of the C-2 acetate to a methoxymethyl (MOM) moiety leads to increased potency at KOP receptors. However, the reason for the increase in potency remains unclear. Further SAR studies were conducted to investigate these findings. Here, we report our efforts towards the synthesis and evaluation of ring-constrained analogues of SVA.

-top-

A3. Exploring mechanisms of selective neuronal vulnerability to oxidative stress by genomic and biochemical approaches
Xinkun Wang^1, 2, Asma Zaidi1, ²*, Ranu Pal1, ², Alexander S. Garrett³, Rogelio Braceras^2, 4, Xue-wen Chen³, Mary L. Michaelis^1, 2, Elias K. Michaelis^1, 2
¹ Higuchi Biosciences Center, 2099 Constant Avenue, University of Kansas, Lawrence, KS 66047, ² Department of Pharmacology and Toxicology, 1251 Wescoe Dr., University of Kansas, Lawrence, KS 66045, ³ Bioinformatics and Computational Life Sciences Laboratory, Department of Electrical Engineering and Computer Science, University of Kansas, Lawrence, KS 66045, ⁴ Novartis Pharmaceuticals Corporation, One Health Plaza, East Hanover, NJ 07936, * Current address: Department of Biochemistry, Kansas City University of Medicine and Biosciences,1750 Independence Avenue, Kansas City, MO 64106

Background. Oxidative stress (OS) is an important factor in brain aging and neurodegenerative diseases. Neurons in different brain regions exhibit differential vulnerability to OS. For example, the CA1 neurons of the hippocampus are much more vulnerable to OS than are the CA3 hippocampal cells. We have also found that cerebellar granule cells (CbG) are much more sensitive to oxidative stress than are neurons of the frontal cerebral cortex. Development of strategies to protect such OS-sensitive neurons requires new insights into the basis of selective vulnerability. However, currently little is known about the underlying mechanisms of selective neuronal vulnerability to OS.
Objective. The goal of our studies was to explore the mechanisms of differential neuronal vulnerability by integrating functional genomics and detailed biochemical analyses.
Methods. In this study, we first determined the vulnerability to paraquat-induced OS, both in vivo and ex vivo (organotypic slice culture), of CbG, cortical, and hippocampal CA1 and CA3 neurons. We subsequently performed comparative GeneChip analyses on individual neurons collected by laser capture microdissection (LCM) from the four regions of rat brain mentioned above. The cells were harvested under basal conditions, i.e., no OS induction.
Results. Whereas CbG and CA1 neurons were highly susceptible to paraquat-induced OS ex vivo, CA3 and cortical neurons were mostly resistant under the same conditions. In vivo studies confirmed the differential vulnerability of CA1 vs. CA3 neurons. GeneChip analyses identified 994 genes that were differentially expressed between the OS-vulnerable (CA1 and CbG) and resistant (CA3 and cortical) neurons. Gene Ontology, pathway and networking analyses revealed higher expression of stress and immune response, and lower expression of energy generation and signal transduction genes in vulnerable compared with resistant neurons. Targeted biochemical analyses confirmed the lower energy levels (in the form of ATP) in primary CbG neurons compared with cortical neurons.
Conclusion. Low energy reserves and high intrinsic stress levels are two underlying factors for neuronal selective vulnerability to OS. These mechanisms may be targeted in the future for the protection of vulnerable neurons. This work was supported by grants from NIA AG12993, NIA AG025350, NICHD HD02528, RR-P20 17708, the Kansas Technology Enterprise Corporation, and the Miller-Hadwiger Fund.

Key words: selective neuronal vulnerability, oxidative stress, energy generation, stress response, functional genomics

-top-

A4. In vitro and in vivo effects of a microtubule-stabilizing small molecule in P301L mutant Tau mice
M. L. Michaelis¹, S. Ansar¹, G. Georg²
¹Dept Pharmacol, Univ. Kansas, Lawrence, KS; ²Univ. of Minnesota, Minneapolis, MN

Abstract:

Most drug discovery efforts for Alzheimer disease (AD) are focused on preventing or clearing oligomers and aggregates of the amyloid peptides, with much less emphasis on targeting neurofibrillary Tau pathology, the second brain lesion. We have previously reported that microtubule (MT)-stabilizing drugs such as Taxol very effectively prevent Aß-induced cell death and abnormal hyperphosphorylation of Tau in primary neurons. The purpose of the present project was to identify neuroprotective MT-stabilizing agents that cross the blood brain barrier (BBB) to enable in vivo proof-of-concept studies in an animal model of AD pathology. Methods: Several structurally diverse MT-interacting agents were synthesized and evaluated for their neuroprotective properties in primary neuronal cultures. The pharmacokinetic (PK) and BBB permeability properties of the most promising candidates were characterized, and the optimal agent, TH-237A, selected for in vivo testing in the P301L Tau mutant mouse. The drug was administered at 10 mg/Kg daily for a period of 12 weeks. The mice were euthanized; brains and spinal cords recovered, and the tissues fractionated into soluble and insoluble Tau components. The relative levels of insoluble, hyperphosphorylated Tau were determined using quantitative densitometric analyses of the immunoreactivity with a panel of 3 antibodies selective for the abnormally phosphorylated Tau protein. Results: The TH-237A was found to be a very potent (low nM EC50) neuroprotective agent in primary neurons exposed to amyloid peptides as well as to other toxic stimuli such as oxidative stress. The PK profile, the BBB permeability, and the t½ for the compound in brain indicated that the TH-237A concentration in brain remained well above the EC50 for in vitro efficacy for up to 5 h. Comparison of the levels of insoluble phospho-Tau in the brains and spinal cords of the TH-237A-treated vs vehicle-treated mice showed a significantly lower amount of the abnormal Tau in both brains and spinal cords of treated mice with all 3 phospho-Tau antibodies (p<0.05 to 0.01). Conclusions: The demonstration that TH-237A permeated the BBB and reduced the formation of Tau aggregates in the mutant mice supports the concept that agents that help to preserve the integrity of the cytoskeletal network may slow progression of neuritic dystrophy and degeneration in AD brain. If amyloid peptides initiate the disruption of the cytoskeletal network, drugs that preserve that network may prove to be valuable therapeutic agents in combination with drugs directly targeting amyloid pathology.

-top-

A5. Modification to the Furan Ring of Salvinorin A in the Effort to Identify Novel Biological Probes
Anthony Lozama¹, Denise S. Simpson¹, Kimberly M. Lovell1, Richard B. Rothman² and Thomas E. Prisinzano¹
¹The Department of Medicinal Chemistry, School of Pharmacy, The University of Kansas, Lawrence, Kansas 66045: ²Clinical Psychopharmacology Section, IRP, NIDA, NIH, DHHS, Baltimore, MD

The treatment of pain is a major focus in medicine. While there has been a litany of analgesics that have come and gone, the “gold standard” for treatment of non-inflammatory pain remains opioid ligands such as morphine. Despite its therapeutic utility, the use of morphine is hindered by detrimental side-effects such as respiratory depression, constipation and addiction. Therefore, there remains a vested interest in the development of analgesics with morphine-like benefits without these drawbacks. Salvinorin A, the active component of the plant Salvia divinorum, is an intriguing lead compound, as it produces opioid-mediated analgesia with no structural resemblance to morphine. Salvinorin A does not cause diuresis and mediates cocaine seeking behavior in laboratory animals, all without bearing any structural resemblance to other opioids that display similar characteristics. These unique and exciting characteristics have led to exploration by our research group, as well as others, to elucidate the pharmacophore of salvinorin A through structure activity relationship studies. Recent studies have shown the furan ring of salvinorin A may play a role in binding to the kappa opioid receptor. However, many compounds containg furan rings have shown hepatotoxicity. In order to explore the role of the furan ring in binding, and to reduce potential hepatotoxicity, chemical modifications were made to salvinorin A to replace the furan ring. It is our belief that a better understanding of the interactions with salvinorin A and its derivatives at opioid receptors will yield valuable information about the opioid system. This information has the potential to be used in the development of novel analgesics that do not posses the negative consequences of morphine. Synthesis and biological results to date will be presented.

-top-

A6. Partitioning of the plasma membrane Ca²⁺-ATPase into lipid rafts in primary neurons: Effects of ganglioside depletion
Jiang L¹, Zaidi A², Bean JL¹, and Michaelis ML¹
¹ Department of Pharmacology/Toxicology, University of Kansas, Lawrence, KS, USA; ² Department of Biochemistry, Kansas City University of Medicine and Biosciences, Kansas City, MO, USA

Disruption in Ca²⁺ homeostasis has been implicated in the decline in neuronal function with brain aging and vulnerability to neurodegenerative disorders, though mechanisms are not yet fully delineated. The plasma membrane Ca²⁺-ATPase (PMCA), a calmodulin (CaM)-regulated, high affinity Ca²⁺ transporter crucial in the maintenance of low intracellular calcium [Ca²⁺]_i, is essential for signaling. We previously reported a decrease in both protein levels and the activity of PMCA in synaptic plasma membranes in aged animals (Neurobiol. Aging, 19, 487), a reduction that may contribute to the age-related [Ca²⁺]_i elevation observed in many laboratories. To investigate mechanisms underlying the PMCA decrease, we have examined the association of PMCA with different SPM microdomains and found both PMCA and CaM are present in specialized membrane microdomains or ‘lipid rafts’ highly enriched in cholesterol and sphingolipids. In aged rat brain, the greatest loss of PMCA was in the raft population, suggesting the membrane environment is critical for optimal PMCA function. In primary neuronal cultures, we confirmed the PMCA distribution in rafts and found that cholesterol depletion with Lovastatin (LS), significantly decreased the activity but not levels of the raft-associated PMCA (J Neurochem, 102, 378). In the present study, we probed the effect of depleting the major brain sphingolipid, ganglioside GM1, by treating neurons with the synthesis inhibitor D-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) in the presence or absence of LS. Exposure to 10 µM PDMP for 72 h led to an ~25% decrease in GM1 levels with no change in cholesterol. PDMP treatment led to a modest decrease (~10%) in PMCA protein level but a marked decrease in PMCA activity level (~40%). The PMCA activity is known to be regulated by specific phospholipids present in the membrane, particularly phosphatidylserine. Our findings that relatively modest alterations in the membrane cholesterol and GM1 have significant effects on the levels and activity of this critical Ca2+ transporter suggest that the age-dependent loss of PMCA from SPMs may be due to localized membrane lipid alterations. The association of PMCA with cholesterol and sphingolipid-enriched rafts may represent a novel mechanism for its regulation and, consequently, for Ca²⁺ signaling in the central nervous system. (This work was supported by NIH Grant AG12993).

-top-

A7. The methionine sulfoxide reductase A (MsrA) knockout mouse exhibits abnormal behavior and brain dopamine levels
Oien DB, Osterhaus GL, Latif SA, Pinkston JW, Fulks J, Johnson M, Fowler SC, Moskovitz J.
Department of Pharmacology and Toxicology, School of Pharmacy, University of Kansas, Lawrence, KS 66045, USA

Oxidative stress can cause methionine oxidation that has been implicated in various proteins malfunctions, if not adequately reduced by the methionine sulfoxide reductase system. Recent evidence has found oxidized methionine residues in neurodegenerative conditions. Previously, we have described elevated levels of brain pathologies and an abnormal walking pattern in the methionine sulfoxide reductase A knockout (MsrA(-/-)) mouse. Here we show that MsrA(-/-) mice have compromised complex task learning capabilities relative to wild-type mice. Likewise, MsrA(-/-) mice exhibit lower locomotor activity and altered gait that exacerbated with age. Furthermore, MsrA(-/-) mice were less responsive to amphetamine treatment. Consequently, brain dopamine levels were determined. Surprisingly, relative to wild-type mice, MsrA(-/-) brains contained significantly higher levels of dopamine up to 12 months of age, while lower levels of dopamine were observed at 16 months of age. Moreover, striatal regions of MsrA(-/-) mice showed an increase of dopamine release parallel to observed dopamine levels. Similarly, the expression pattern of tyrosine hydroxylase activating protein correlated with the age-dependent dopamine levels. Thus, it is suggested that dopamine regulation and signaling pathways are impaired in MsrA(-/-) mice, which may contribute to their abnormal behavior. These observations may be relevant to age-related neurological diseases associated with oxidative stress.

-top-

Higuchi Biosciences Center
University of Kansas
2099 Constant Avenue
Lawrence, KS 66047-2535
785-864-5183
hbc@ku.edu