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Tyrosine hydroxylase (TH) is a rate-limiting enzyme in the biosynthesis of catecholamines. In zebrafish, two genes encoding TH have been identified. We cloned them and studied their expression in zebrafish. In adult tissues, th1 mRNA was more abundant than th2 mRNA in the brain and eyes, whereas th2 mRNA was more abundant in the liver, kidney, heart and gills. In developing brain, th1 mRNA was readily detected at 1 day post-fertilization using qPCR and in situ hybridization, whereas th2 mRNA appeared later. th1 was found in 17 catecholaminergic groups in larval brain, whereas th2 was found in four additional groups. A monoclonal antibody commonly used against TH detected preferentially TH1 protein. The two th genes, probably originated as a result of genome duplication, thus show complementary expression, although th1 is predominant in the brain and th2 in the periphery. th2 may be a novel essential factor in regulation of catecholamine synthesis in zebrafish.

Mitochondrial dysfunction plausibly underlies the aging-associated brain degeneration. Mitochondria play a pivotal role in cellular bioenergetics and cell-survival. Oxidative stress consequent to chronic hypoperfusion induces mitochondrial damage, which is implicated as the primary cause of cerebrovascular accidents (CVA) mediated Alzheimer's disease (AD). The mitochondrial function deteriorates with aging, and the mitochondrial damage correlates with increased intracellular production of oxidants and pro-oxidants. The prolonged oxidative stress and the resultant hypoperfusion in the brain tissues stimulate the expression of nitric oxide synthase (NOS) enzymes, which further drives the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The ROS and RNS collectively contributes to the dysfunction of the blood-brain barrier (BBB) and damage to the brain parenchymal cells. Delineating the molecular mechanisms of these processes may provide clues for the novel therapeutic targets for CVA and AD patients.

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Mesencephalic astrocyte derived neurotrophic factor (MANF) is recognized as a dopaminergic neurotrophic factor, which can protect dopaminergic neurons from neurotoxic damage. However, little is known about the function of MANF during the vertebrate development. Here, we report that MANF expression is widespread during embryonic development and in adult organs analyzed by qPCR and in situ hybridization in zebrafish. Knockdown of MANF expression with antisense splice-blocking morpholino oligonucleotides resulted in no apparent abnormal phenotype. Nevertheless, the dopamine level of MANF morphants was lower than that of the wild type larvae, the expression levels of the two tyrosine hydroxylase gene transcripts were decreased and a decrease in neuron number in certain groups of th1 and th2 cells in the diencephalon region in MANF morphants was observed. These defects were rescued by injection of exogenous manf mRNA. Strikingly, manf mRNA could partly restore the decrease of th1 positive cells in Nr4a2-deficient larvae. These results suggest that MANF is involved in the regulation of the development of dopaminergic system in zebrafish.

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Oxidative stress-mediated neuronal dysfunction is characteristic of several neurodegenerative disorders, including Parkinson’s disease (PD). The enzyme tyrosine hydroxylase (TH) catalyzes the formation of L-DOPA, the rate-limiting step in the biosynthesis of dopamine. A lack of dopamine in the striatum is the most characteristic feature of PD, and the cause of the most dominant symptoms. Loss of function mutations in the PTEN-induced putative kinase (PINK1) gene cause autosomal recessive PD. This study explored the basic mechanisms underlying the involvement of pink1 in oxidative stress-mediated PD pathology using zebrafish as a tool. We generated a transgenic line, Tg(pink1:EGFP), and used it to study the effect of oxidative stress (exposure to H2O2) on pink1 expression. GFP expression was enhanced throughout the brain of zebrafish larvae subjected to oxidative stress. In addition to a widespread increase in pink1 mRNA expression, mild oxidative stress induced a clear decline in tyrosine hydroxylase 2 (th2), but not tyrosine hydroxylase 1 (th1) expression, in the brain of wild-type larvae. The drug L-Glutathione Reduced (LGR) has been associated with anti-oxidative and possible neuroprotective properties. Administration of LGR normalized the increased fluorescence intensity indicating pink1 transgene expression and endogenous pink1 mRNA expression in larvae subjected to oxidative stress by H2O2. In the pink1 morpholino oliogonucleotide-injected larvae, the reduction in the expression of th1 and th2 was partially rescued by LGR. The pink1 gene is a sensitive marker of oxidative stress in zebrafish, and LGR effectively normalizes the consequences of mild oxidative stress, suggesting that the neuroprotective effects of pink1 and LGR may be significant and useful in drug development.

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