Abstract
The primary pathological hallmark of Parkinson disease (PD) is the profound loss of dopaminergic neurons in the substantia nigra pars compacta. To facilitate the understanding of the underling mechanism of PD, several zebrafish PD models have been generated to recapitulate the characteristics of dopaminergic (DA) neuron loss. In zebrafish studies, tyrosine hydroxylase 1 (th1) has been frequently used as a molecular marker of DA neurons. However, th1 also labels norepinephrine and epinephrine neurons. Recently, a homologue of th1, named tyrosine hydroxylase 2 (th2), was identified based on the sequence homology and subsequently used as a novel marker of DA neurons. In this study, we present evidence that th2 co-localizes with serotonin in the ventral diencephalon and caudal hypothalamus in zebrafish embryos. In addition, knockdown of th2 reduces the level of serotonin in the corresponding th2-positive neurons. This phenotype can be rescued by both zebrafish th2 and mouse tryptophan hydroxylase 1 (Tph1) mRNA as well as by 5-hydroxytryptophan, the product of tryptophan hydroxylase. Moreover, the purified Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro. Based on these in vivo and in vitro results, we conclude that th2 is a gene encoding for tryptophan hydroxylase and should be used as a marker gene of serotonergic neurons.
Highlights
Zebrafish tyrosine hydroxylase 2 has been considered as a marker of dopaminergic (DA) neurons in the investigation of DA neuron development and the pathological mechanism of Parkinson disease
5-HT and th2 Are Co-localized in the Ventral Diencephalon and Caudal Hypothalamus—As reported, 5-HT distributes in almost all brain regions, whereas in embryos, there are three major clusters of 5-HT-positive neurons in the diencephalon, which are the ventral part of the posterior tuberculum, the intermediate hypothalamus, and the caudal most aspect of the hypothalamus [30]. th2 is expressed in the preoptic region (3b), the anterior part of the paraventricular organ (8b), the intermediate part of the paraventricular organ (9b), and the posterior part of the paraventricular organ (10b) during embryogenesis [17, 26]
We found that purified Th2 had a tryptophan hydroxylase activity of 14.4 Ϯ 1.14 nM 5-HTP/min/mg, which was comparable with that of mouse TPH1 (17.6 Ϯ 1.44 nM 5-HTP/min/mg, p ϭ 0.554) (Fig. 6B)
Summary
Zebrafish tyrosine hydroxylase 2 (th2) has been considered as a marker of dopaminergic (DA) neurons in the investigation of DA neuron development and the pathological mechanism of Parkinson disease. In the adult zebrafish, other DA neuron markers (L-3,4-dihydroxyphenylalanine (L-DOPA) decarboxylase (ddc; Gene ID 406651), vesicle monoamine transporter 2 (vmat; Gene ID 553304), dopamine transporter (dat; Gene ID 80787), and dopamine) can only be detected in some but not all th2-positive neurons [28, 29] Based on these results, we reasoned that th should not be considered as a tyrosine hydroxylase gene merely based on sequence homology. The purified zebrafish Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro Based on these findings, we suggest that th should be considered as a gene encoding tryptophan hydroxylase and used as a marker for serotonergic neurons
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