ROGDI defines a GABAergic input to a dopaminergic neural circuit to promote sleep in Drosophila

Kohlschutter-Tönz syndrome (KTS) is a rare genetic disorder characterized by severe developmental delay with neurological dysfunction such as epilepsy, psychomotor regression and intellectual disability. While genetic mutations in a human homolog of Rogdi gene have been linked to the development of KTS, its neural basis remains elusive. Here we establish a Drosophila model of KTS to demonstrate a novel role of Rogdi in GABAergic transmission. Our genetic screen initially identified a hypomorphic mutation in Drosophila Rogdi that supressed daily baseline sleep. The short sleep phenotypes were rescued by transgenic Rogdi expression in GABAergic neurons or by the oral administration of a GABA transaminase inhibitor that enhances GABAergic signaling. An enhancer trap line originated from Rogdi locus displayed its expression in sleep-regulatory neurons of adult fly brains, including GABA-positive neurons. Furthermore, RNA interference-mediated depletion of vesicular GABA transporter in Rogdi-expressing neurons decreased sleep, largely phenocoping Rogdi mutants. Notably, Rogdi effects on sleep were masked by genetic or pharmacological inhibition of dopaminergic transmission, newly defining a dopaminergic circuit as a downstream target of Rogdi-dependent sleep. Taken together, these data suggest that ROGDI sustains GABAergic transmission to promote sleep. Given the strong relevance of KTS phenotypes to GABA, our findings provide the first neural clues important for understanding KTS pathogenesis

Role of cyclooxygenase-2 in tetrahydrobiopterin-induced dopamine oxidation

Dopamine is considered one of the main contributing factors in the induction of oxidative stress and selective dopaminergic neurodegeneration in Parkinson's disease. We have previously reported that tetrahydrobiopterin (BH4) leads to dopamine oxidation and renders dopamine-producing cells vulnerable. In the present study, we found that BH4 selectively upregulates cyclooxygenase-2 (COX-2) expression in dopaminergic cells. BH4 caused an induction of COX-2 mRNA, and a critical regulatory motif for BH4-induced transcriptional activation of COX-2 is CRE/AP-1. COX-2 can oxidize dopamine and cause oxidative stress, which is evidenced by the findings that significant increase in dopamine-chrome formation and protein carbonyl contents by BH4-induced COX-2 up-regulation, and the increases are abolished by COX-2 selective inhibitor meloxicam. Increased COX-2 promotes dopaminergic neurodegeneration in both SH-SY5Y cells and rat mesencephalic neurons. These data suggest that BH4-induced COX-2 expression is responsible for dopamine oxidation, leading to the preferential vulnerability of dopaminergic cells in Parkinson's disease

Amelioration of Behavioral Abnormalities in BH₄-deficient Mice by Dietary Supplementation of Tyrosine

<div><p>This study reports an amelioration of abnormal motor behaviors in tetrahydrobiopterin (BH₄)-deficient <i>Spr</i>^−/− mice by the dietary supplementation of tyrosine. Since BH₄ is an essential cofactor for the conversion of phenylalanine into tyrosine as well as the synthesis of dopamine neurotransmitter within the central nervous system, the levels of tyrosine and dopamine were severely reduced in brains of BH₄-deficient <i>Spr</i>^−/− mice. We found that <i>Spr</i>^−/− mice display variable ‘open-field’ behaviors, impaired motor functions on the ‘rotating rod’, and dystonic ‘hind-limb clasping’. In this study, we report that these aberrant motor deficits displayed by <i>Spr</i>^−/− mice were ameliorated by the therapeutic tyrosine diet for 10 days. This study also suggests that dopamine deficiency in brains of <i>Spr</i>^−/− mice may not be the biological feature of aberrant motor behaviors associated with BH₄ deficiency. Brain levels of dopamine (DA) and its metabolites in <i>Spr</i>^−/− mice were not substantially increased by the dietary tyrosine therapy. However, we found that mTORC1 activity severely suppressed in brains of <i>Spr</i>^−/− mice fed a normal diet was restored 10 days after feeding the mice the tyrosine diet. The present study proposes that brain mTORC1 signaling pathway is one of the potential targets in understanding abnormal motor behaviors associated with BH₄-deficiency.</p> </div

Hind-limb clasping test.

<p>Each experimental group of mice was suspended by the tail for 25 sec, and their abilities of hind-limb clasping were monitored by video recording. (A) Representative hind-limb clasping observed in <i>Spr</i>^−/− mice fed a normal diet (ND) (b) but not in <i>Spr</i>^+/+ mice fed a normal diet (ND) (a) and <i>Spr</i>^−/− mice fed the tyrosine diet (+Tyr) (c) is shown. (B) Total duration of hind-limb clasping during a 25 sec in <i>Spr</i>^+/+ mice fed a normal diet, <i>Spr</i>^+/+ mice fed the tyrosine diet and <i>Spr</i>^−/− mice fed either a normal or the tyrosine diet (n  = 5 each) is shown. Values represent average time of duration ± SD, *** P<0.001.</p

Levels of brain DA and its metabolites are irrelevant to the dietary tyrosine supplementation in <i>Spr</i>^−/− mice.

<p>(A), (B), and (C) <i>Spr</i>^+/+ or <i>Spr</i>^−/− mice were fed a normal (ND) or the tyrosine diet (+Tyr). Mice were analyzed for brain levels of DA (A), DOPAC (B), and HVA (C) in their caudate putamen (n ≥3). Values are means ± SD, ** P<0.01, *** P<0.001, compared with respective values in <i>Spr</i>^+/+ mice. (D) The brain levels of TH proteins in experimental mice were monitored by Western blot analysis. <i>Spr</i>^+/+ or <i>Spr</i>^−/− mice were fed a normal diet or treated with the dietary tyrosine, L-DOPA, or leucine therapy for 10 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060803#s2" target="_blank">Materials and Methods</a>.</p