Generation And Phenotypic Analyses Of Dopaminergic Neuron-specific Cul4b Knockout Mice

Psychiatric disorders and drug addiction has become a public health and social problem in many countries. Although the clinical manifestations of these diseases are significantly different, all these diseases are related to the abnormalities of neurotransmitter.In1958, Swedish scientist Arvid Carlsson firstly identified dopamine as an important neurotransmitter in the central nervous system, which is widely involved in regulation of learning and memory, motor control, emotion, motivation and neuroendocrine. During recent thirty years, dopaminergic system has become one of the hotspots in neuroscience field. Many neuropsychiatric disorders are associated with dysfunction of the dopaminergic neurotransmission, including Parkinson’s disease, Schizophrenia, Tourette syndrome, Huntington’s disease, attention deficit hyperactivity disorder and drug addiction. The dopaminergic signaling depends on the concentration of dopamine in the synaptic cleft. The dopamine transporter (DAT) on presynaptic neurons is the key player in regulation of dopamine concentration in the synaptic cleft. The dopamine transporter is a member of the Na+, Cl-dependent substrate-specific neuronal membrane transporter and its major function is the reuptake of extracellular dopamine thereby regulating the concentration of dopamine in the synaptic cleft and maintaining effective dopaminergic signaling.Recent studies have shown that the function of DAT can be regulated through direct protein-protein interactions by several proteins and receptors. D2receptors recruit DAT to plasma membrane and enhance DAT-mediated dopamine uptake through the D2-DAT direct protein-protein interaction. Alpha-synuclein directly couples to the carboxyl tail of the dopamine transporter and the alpha-synuclein/DAT protein complex accelerates DAT-mediated cellular dopamine uptake and dopamine-induced cellular apoptosis. Parkin impairs the alpha-synuclein/DAT coupling by interacting with the carboxyl-terminus of the DAT thereby protecting against DA-induced cell toxicity. Carboxypeptidase E (CPE) interacts with the DAT carboxyl terminus and enhances DAT-mediated dopamine uptake activity.Mutations of CUL4B in human are one of the major causes of X-linked mental retardation (XLMR) syndrome. Those patients manifested significant mental retardation, seizure, tremor, ataxia, impaired speech, short stature, central obesity and other abnormalities. We found that those patients’ features are consistent with abnormal dopaminergic neurotransmission.CUL4B is a member of CULLIN gene family. Serving as the scaffold protein, cullins are crucial components of the CULLIN-RING ubiquitin ligase (E3) complexes (CRLs). CRLs constitute a major subclass of ubiquitin ligases that regulate diverse cellular processes, including cell cycle regulation, signal transduction, transcription, development and DNA repair.To investigate the roles of CUL4B in dopaminergic neurotransmission, we generated dopaminergic neuron specific Cul4b knockout mice model and analysed the phenotypes of the conditional knockout mice.PART ONEGeneration of dopaminergic neuron-specific Cul4b knockout miceThe exploiting of the Cre/LoxP strategy marks the knockout technology into a new era. Using this technology, we can knock out a gene in particular tissue, cell and developmental stages. We have successfully generated Cul4b gene targeted mice, Cul4b floxed mice, using Cre/LoxP strategy. Mating this Cul4b floxed mice with different types of Cre transgenic mice could generate Cul4b conditional knockout mice in specific tissues or specific stages. Previously, we genereated constitutive Cul4b knockout mice by mating Cul4b floxed mice with EIIα-Cre transgenic mice, and found that the Cul4b null mice were embryonic lethal. This study indicates that CUL4B plays an important role during mouse development. We also generted nervous system-specific Cul4b knockout mice by mating Cul4b floxed mice with Nestin-Cre transgenic mice. To understand the roles of CUL4B in dopaminergic neurotransmission, we generated dopaminergic neuron-specific Cul4b knockout mice.1. Two-step strategy was used to generate dopaminergic neuron-specific Cul4b knockout mice. Firstly, Cul4b floxed mice (Cul4bflox/flox) were crossed with Slc6a3-Cre transgenic mice, in which Cre recombinase was under the control of the Slc6a3promoter and enhancer, to generate dopaminergic neuron-specific Cul4b knockout male mice (Slc6a3-cre+/-; Cul4bflox/Y). Then these males were crossed to Cul4bflox/flox female. Fifty percent of their offsprings were knockout. Their genotypes were confirmed by PCR assay.2. To determine the efficiency and specificity of Cul4b deletion in dopamine neurons, we examined the expression of Cul4b in substantia nigra, which was indicated by TH-positive cells, by immunofluorescence assay. We found no Cul4b expressing in TH positive cells in dopaminergic neuron-specific Cul4b knockout mice. The result revealed that this mice model was successfully generated.PART TWOPhenotypic analyses of dopaminergic neuron-specific Cul4b knockout miceWe successfully generated dopaminergic neuron-specific Cul4b knockout mice (Cul4bSlc6a3-Cre mice). Next, we investigated the phenotypes of the conditional knockout mice.1. Histological analyses of the midbrain and striatum by H&E staining did not detect any significant different between Cul4bSlc6a3-Cre and control mice. 2. Anti-TH antibody was used to label dopamine neurons in substantia nigra. No difference in the morphology and distribution of TH positive neurons was detected in Cul4bSlc6a3-Cre mice compared with the littermate controls.3. TUNEL assay was used to evaluate the apoptotic cells in the midbrain of Cul4bSlc6a3-Cre mice. Few apoptotic cells were detected in the midbrain of Cul4bSlc6a3-Cre mice, which was consistent with the littermate controls.4. We performed immunohistochemistry to examine the expression levels of DAT in both substantia nigra and striatum. We did not find any significant changes in the levels of DAT in Cul4bSlc6a-Cre mice. Western blotting analysis was also performed to detect the expression levels of TH and DAT in substantia nigra and striatum. We found no significant difference in the levels of these proteins between Cul4bSlc6a3-Cre mice and the littermate controls.5. Cul4bSlc6a3-Cre mice did not show any apparent developmental abnormalities till1.5years old. Morris water maze was used to evaluate the spatial learning and memory abilities of mice. Open field test was used to evaluate the spontaneous locomotor activity and the anxiety levels. Rotarod test was used to measue the sport coordinate ability and resistance to fatigue. All the three tests revealed no behavioral changes in Cul4bSlc6a3-Cre mice comparing to the littermate controls.Taken together, we successfully generated a dopaminergic neuron-specific Cul4b knockout mice model by using Cre/LoxP system. Analyses of their phenotypes revealed no significant differences between Cul4bSlc6a3-Cre mice and the littermate controls. These results implicated that knockout of Cul4b gene in dopaminergic neuron may not influence the dopaminergic neurotransmission.