Howard Gu, Ph.D.
Department of Pharmacology and Psychiatry
OSU Program in Pharmacogenomics
5184B Graves Hall
333 W Tenth Avenue
Columbus, OH 43210
Neurotransmitters are chemical messengers released by presynaptic neurons to communicate with other neurons and cells. Neurotransmitter transporters (NTTs) terminate neurotransmission by the re-uptaking and recycling the released neurotransmitters from the synaptic cleft and surrounding areas. Dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) are high affinity targets for the psycho-stimulants cocaine and amphetamines. The transporters are also molecular targets for therapeutic drugs, such as Ritalin (methylphenidate), Prozac (fluoxetine), Zoloft (Sertraline), Paxil (paroxetine),Wellbutrin (bupropion), etc. These drugs are used to treat several neurological and mental disorders, including attention-deficit/hyperactivity disorder, depression, anxiety, Autism, obsessive-compulsive disorder, and minimal brain dysfunction. My laboratory focuses on these important transporter proteins and studies their structures and functions, their roles in drug addiction and mental disorders.
Current Research Projects
Knock-in mice with a cocaine resistant dopamine transporter
Cocaine is an addictive psychostimulant. The abuse of cocaine is a world wide problem. Cocaine blocks DAT, SERT, and NET with similar potencies, elevating the extracellular concentrations of all three neurotransmitters in the brain and producing complex neurochemical and behavioral effects. Based on abundant indirect evidence, it was hypothesized and generally accepted that the addictive property of cocaine is mediated predominantly through its inhibition of DAT. Contrary to predictions, knockout mice with DAT deleted still display the reward effect of cocaine, suggesting that the cocaine target DAT is not necessary for cocaine reward at least in DAT knockout mice. This led to the re-evaluation of the dopamine hypothesis and the proposal of redundant pathways for cocaine reward. However, the adaptive changes in the knockout mice that compensate for the lack of the deleted DAT might have altered the reward pathways. Therefore, we employ a more controlled and specific approach – to engineer a mouse model that carries functional but cocaine-resistant DAT. Using species-scanning mutagenesis and random mutagenesis, we have made a DAT mutant that is 70-fold less sensitive to cocaine while retaining DA uptake function. We have generated a knock-in mouse line with the endogenous DAT replaced by this DAT mutant. We have found that in our knock-in mice cocaine does not elevate the DA level in nucleus accumbens, does not stimulate locomotion, and does not produce reward as measured by conditioned place preference test. Our results demonstrate that the inhibition of DAT is necessary for cocaine reward in mice with a functional DAT and cocaine reward seen in DAT knockout mice is due to the adaptive changes in the knockout mice to compensate for the absence of DAT. Currently, we are using this mouse line as a unique tool to dissect out the contributions of DAT to the short and long term effects of cocaine on neurochemistry, gene expression, and animal behavior.
Knock-in mice with cocaine insensitive NET or SERT
In addition to DAT, NET and SERT are the other two high affinity targets of cocaine. The serotonin (5HT) and norepinephrine (NE) systems play important roles in mood, sleep, arousal, attention, vigilance, learning and memory, etc. Therefore, it is likely that the blockade of SERT and NET by cocaine and the subsequent modulation of 5HT and NE pathways also play crucial roles in mediating cocaine effects. Using a similar approach, we have generated mutants of NET and SERT that are functional and cocaine-insensitive. We plan to generate one knock-in mouse model carrying a cocaine-insensitive NET and another with a cocaine-insensitive SERT. We will then breed among these mouse lines and produce mice with two or all three cocaine targets that are resistant to cocaine. These mouse lines will allow us to dissect out the roles of each transporter or neurotransmitter system in the complex effects of cocaine.
Structure and Function of the dopamine transporter
Despite intense studies, our knowledge about the structures of neurotransmitter transporters (NTT) is still limited. Recently, a crystal structure of leucine transporter from bacterium Aquifex aeolicus (LeuTAa) has been solved. This transporter is a remote homolog of the mammalian NTT with only 20-25% homology in primary sequences. It is believed that the structure of LeuTAa provide a guideline for the structures of DAT and other NTTs. However, the LeuTAa structure does not provide structural information about the molecular mechanisms of NTTs in transport function or drug binding because of the differences in primary sequences (75-80%), substrate structures, ion requirements, electrogenicity, etc. Further structure-function studies are needed. One of the approaches we will use is employing cysteine specific reagents which can be used to cross-link two cysteine residues placed in different parts of a protein to determine whether the two parts are close to each other in the 3-D structure. However, NTTs all have many cysteines. Removing all cysteines usually results in nonfunctional transporters. By using species scanning mutagenesis and random mutagenesis, we have successfully engineered a functional DAT with all cysteine residues removed except two cysteines in an extracellular loop. We have shown that the two cysteines are required for cell surface expression and they form a disulfide bond. The disulfide bond prevents the two cysteines from reacting to cysteine-specific reagents. Therefore, we have successfully engineered a functional DAT with all reactive cysteines removed. In addition, we have inserted a Factor Xa (a protease) site in different locations of the Cys-less DAT. The Factor-Xa site allows us to cut DAT into two fragments and examine whether the fragments are linked by a disulfide bond between two cysteines. We are now inserting cysteines back into the C-less DAT construct at desired positions. We are ready to probe the 3-dimentional structure of DAT. In addition, we are also working on identifying amino residues that are involved in cocaine binding. The knowledge of cocaine binding site provides critical information for the rational design of drugs treating cocaine addiction.
High throughput screening of cocaine antagonists and the development of drugs treating cocaine addiction
Currently, there are no effective pharmacological treatments for cocaine addiction. We have shown that cocaine-inhibition of DAT is necessary for the rewarding effect of cocaine. Therefore, compounds that reduce cocaine inhibition of DAT have great potential as effective drugs to treat cocaine addiction. We have made a stable cell line expressing human DAT at very high level. We will screen large compound libraries for cocaine antagonists in collaboration with an NIH sponsored screening center. We will measure DA uptake into these cells in the presence of cocaine plus a test compound and compare it with DA uptake in the presence of cocaine alone. Any compounds that increase DA uptake in the presence of cocaine will be a hit compound. In follow up studies, we will characterize the hit compounds and improve their ability to counter cocaine actions in cultured cells and in animals. If successful, this project may lead to the development of the first specific pharmacological treatment ..for cocaine addiction.
Projects for graduate students:
Highly motivated and enthusiastic graduate students are welcomed and encouraged to rotate through the laboratory. They will learn the basic techniques in molecular biology and work on one or more aspects of the above projects, or initiate a new project. I hope you would choose to join our research team and enjoy science and medical discovery...
Selected Recent Publications:
Naughton BJ, Thirtamara-Rajamani K, Wang C, During MJ, Gu HH. (2012) Specific knockdown of the D2 long dopamine receptor variant. Neuroreport 23:1-5.
Castelli M, Federici M, Rossi S, De Chiara V, Napolitano F, Studer V, Motta C, Sacchetti L, Romano R, Musella A, Bernardi G, Siracusano A, Gu HH, Mercuri NB, Usiello A, Centorza D. (2011) Loss of striatal cannabinoid CB1 receptor function in attention-deficit/hyperactivity disorder mice with point-mutation of the dopamine transporter. Eur J Neurosci 34:1369-1377.
Naughton BJ, Han DD, Gu HH. (2011) Fluorescence-based evaluation of shRNA efficacy. Anal Biochem 417:162-164.
Pinsonneault JK, Han DD, Burdick KE, Kataki M, Bertolino A, Malhotra AK, Gu HH, Sadee W. (2011) Dopamine transporter gene variant affecting expression in human brain is associated with bipolar disorder. Neuropsychopharmacology 36:1644:1655.
Hill ER, Huang X, Zhan CG, Ivy Carroll F, Gu HH. (2011) Interaction of tyrosine 151 in norepinephrine transporter with the 2ß group of cocaine analog RTI-113. Neuropharmacology 61:112-120.
Martin BJ, Naughton BJ, Thirtamara-Rajamani K, Yoon DJ, Han DD, Devries AC, Gu HH. (2011) Dopamine transporter inhibition is necessary for cocaine-induced increases in dendritic spine density in the nucleus accumbens. Synapse 65:490-496.
Napolitano F, Bonito-Oliva A, Federici M, Carta M, Errico F, Magara S, Martella G, Nistico R, Centonze D, Pisani A, Gu HH, Meurcuri NB, Usiello A. (2010) Role of aberrant striatal dopmaine D1 receptor/cAMP/protein kinase A/DARPP32 signaling in the paradoxical calming effect of amphetamine.. J. Neurosci. 30:11043-1156.
Huang X, Gu HH, Zhang CG. (2009) Mechanism for cocaine blocking the transport of dopamine: Insights from molecular modeling and dynamics simulations. J Phys Chem B 45:15057-15066.
Hill ER, Tian J, Tilley MR, Zhu MX, Gu HH. (2009) Potencies of cocaine methiodide on major cocaine targets in mice. PLoS One 4:e7578.
Roe BE, Tilley MR, Gu HH, Beversdorf DQ, Sadee W, Haab TC, Papp AC. (2009) Financial and psychological risk attitudes associated with two single nucleotide polymorphisms in the nicotine receptor (CHRNA4) gene. PLoS One 4:E6704.
Thomsen M, Han DD, Gu HH, Caine SB. (2009) Lack of cocaine self-administration in mice expressing a cocaine-insensitive dopamine transporter. J Pharmacol Exp Ther 331:204-211.
Tilley MR, O'Neill B, Han DD, Gu HH. (2009) Cocaine does not produce reward in absence of dopamine transporter inhibition. Neuroreport 20:9-12.
Wei H, Hill ER, Gu HH. (2009) Functional mutations in mouse norepinephrine transporter reduce sensitivity to cocaine inhibition. Neuropharmacology 56:399-404.
Tilley MR, Gu H. (2008) The effects of methylphenidate on knock-in mice with a methylphenidate resistant dopamine transporter. J Pharmacol Exp Ther 327:554-560.
Tilley MR, Gu HH. (2008) Dopamine transporter inhibition is required for cocaine-induced stereotypy. Neuroreport 19:1137-1140.
Tilley MR, Cagniard B, Zhuang X, Han DD, Tiao N, Gu HH. (2007) Cocaine reward and locomotion stimulation in mice with reduced dopamine transporter expression. BMC Neurosci 28:42.
Chen R, Wei H, Hill ER, Chen L, Jiang L, Han DD, Gu HH. (2007) Direct evidence that two cysteines in the dopamine transporter form a disulfide bond. Mol Cell Biochem 298:41-48.
Chen R, Tilley MR, Wei H, Zhou F, Zhou FM, Ching S, Quan N, Stephens RL, Hill ER, Nottoli T, Han DD, Gu HH. (2007) Abolished cocaine reward in mice with a cocaine-insensitive dopamine transporter. Proc Natl Acad Sci USA 103:9333-9338.
Han DD, Chen R, Hill ER, Tilley MR Gu HH. (2006) Cause and solutions to the polymerase chain reaction smear problem in genotyping. Anal Biochem 353:296-298.
Gu HH, Wu X, Hann DD. (2006) Conserved serine residues in serotonin transporter contributes to high-affinity cocaine binding. Biochem Biophys Res Commun 343:1179-1185.
Han DD, Gu HH. (2006) Comparison of the monoamine transporters from human and mouse in their sensitivities to psychostimulant drugs. BMC Pharmacol 6:6.