| Parkinson's disease (PD) is one of the most common progressive neurodegenerative disorders in the world. It affects about 1% of the population above the age of 60. Clinically, PD is characterized by slowness of movement, rest tremor, rigidity, and disturbances in balance. Pathologically, PD is characterized by the presence of Lewy bodies and selective loss of dopaminergic neuron in the substantia nigra pars compacta (SNc). The dopaminergic neuronal cell death leads to the loss of dopamine (DA) in the striatum and in the SNc. Recent research has enhanced our understanding in PD and this will certainly improve the approaches that can be used in the treatment of PD. Advances in human genetics have enabled us to identify genes that are linked to rare forms of familial PD. DJ-1 is the third gene discovered that to be linked to familial Parkinson's disease by linkage analysis in 2003. These important findings serve as the foundation for discovering new pathways that may lead to the development of new therapies for PD.DJ-1 belongs to the ThiJ/PfpI superfamily and is well conserved among species. Human DJ-1 coding region consists of 567 nucleotides and encodes a 20kd protein. Mouse DJ-1 shares 90% amino acid homology with human DJ-1. DJ-1 deficient mice are fertile and viable, They display mild movement impairment, dopaminergic dysfunction without dopamine neuron degeneration. The molecular mechanism for DJ-1 mutation related familial Parkinson's disease is still unknown. Several studies suggested DJ-1 may be involved in mitochondria, oxidative sensoring, ubiquitin proteasome system and post-transcriptional regulation control. Moreover, DJ-1, initially discovered as an oncogene and oxidative stress sensor, is increased in serum and cerebrospinal fluid (CSF) of several types of cancers and neurological disorders and might be a novel biomarker for these disease conditions. Northern blot showed DJ-1 mRNA present in all tissues examined, suggesting ubiquitous function of DJ-1. Several studies have examined the expression pattern of DJ-1 in brains of both human and rodents. Surprisingly, these studies generated considerably conflicting results. The major controversy is whether DJ-1 is expressed in neurons or astrocytes and whether DJ-1 is expressed in dopamine neuron. In human brain, it is rather controversial whether DJ-1 is expressed in astrocytes or in neurons. In rodent brains, it is not clear whether DJ-1 is expressed in dopamine neuron in substantia nigra.Using DJ-1 deficient mouse as negative control, we examined DJ-1 mRNA expression in mouse brains. By sequential double labeling on the same sections, in situ hybridization of DJ-1 mRNA was followed by immunofluorescence detection for TH, NeuN and GFAP. We found DJ-1 mRNA was expressed by greater than 96% neurons but undetectable in astrocytes in most brain regions. All dopamine neurons in ventral midbrain expressed DJ-1 mRNA. Interestingly, choroid plexus and ependymal cells lining ventricles strongly expressed DJ-1 mRNA. The ubiquitous neuronal expression of DJ-1 suggests its fundamental roles underlying neuronal functions. By immunohistochemistry, we found DJ-1 protein is not co-localized with astrocytes. To our surprise, DJ-1 protein is not colocalized with neurons, and the DJ-1 staining is diffused in mouse brain, we then proposed that DJ-1 is axonal transported and secreted by neurons. By colchicine treatment, CSF, and primary neuron culture experiments, we confirmed our hypothesis.Molecular and genetic studies have made significant progress in revealing protective or risk factors for dopamine neuron survival and dysfunction in Parkinson's disease. However, there is no good genetic approach for high level gene expression in dopamine neuron to test their biological significance in living animals. To overexpress transgene in dopamine neurons, we propose to gene target (knockin) "positive-feedback cassette" into endogenous locus of dopamine transporter (DAT). Positive-feedback cassette will serve as an amplifier to boost transgene expression using bi-directional tetO promoter and DAT locus will favor transgene expression in dopamine neuron. We expect this unique "in situ amplifier" design will achieve doxycycline regulated transgene overexpression specifically in dopamine neurons. The proposed project aims to lay the groundwork for making regulatable tissue specific transgenic overexpression models for future projects in our lab as well as for the entire scientific community.We constructed ES cell targeting vector, successfully performed ES cell targeting and identified positive targeted ES clones with high targeting efficiency. The identified positive clones were re-amplified and confirmed by southern blot with both 5'and 3'probes. Microinjection is done after we prepared the positive ES cell clones. Chimera mice was got, and we then crossed with C57BL/6 mice, the heterozygous mice with agouti fur will be used to characterize tTA expression pattern and characterize the time course of transgene expression induction by dietary doxycycline in the future.In summary, we found DJ-1mRNA was expressed by greater than 96% neurons but undetectable in astrocytes in most brain regions. All dopamine neurons in ventral midbrain expressed DJ-1 mRNA. DJ-1 is axonal transported and secreted by neurons.And we generated a knockin mice, the expression pattern of tTA and ChR2-mCherry selectively in dopamine neurons via regulated expression will be done in the future.
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