Primary open-angle glaucoma (POAG) is one of the three principal subtypes of glaucoma and among the leading cause of blindness worldwide. POAG is defined by cell death of the retinal ganglion cells (RGCs) and surrounding neuronal cells at higher or normal intraocular pressure (IOP). Coded by one of the three genes responsible for POAG, WD repeat-containing protein 36 (WDR36) has two domains with a similar folding. To address whether WDR36 is functionally important in the retina, we developed four transgenic mice strains overexpressing a wild-type (Wt) and three mutant variants of D606G, deletion of amino acids at positions 605–607 (Del605–607) and at 601–640 (Del601–640) equivalent to the location of the D658G mutation observed in POAG patients. A triple amino acid deletion of mouse Wdr36 at positions 605–607 corresponding to the deletion at positions 657–659 in humans developed progressive retinal degeneration at the peripheral retina with normal IOP. RGCs and connecting amacrine cell synapses were affected at the peripheral retina. Axon outgrowth rate of cultured RGC directly isolated from transgenic animal was significantly reduced by the Wdr36 mutation compared with Wt. Molecular modeling of wild and mutant mouse Wdr36 revealed that deletion at positions 605–607 removed three residues and a hydrogen bond, required to stabilize anti-parallel β-sheet of the 6th β-propeller in the second domain. We concluded that WDR36 plays an important functional role in the retina homeostasis and mutation to this gene can cause devastating retinal damage. These data will improve understanding of the functional property of WDR36 in the retina and provide a new animal model for glaucoma therapeutics.

We demonstrated synchronous oscillation of intracellular Ca2+ in cultured-mouse mid-brain neurons. This synchronous oscillation was thought to result from spontaneous and synchronous neural bursts in a synaptic neural network. We also examined the role of endogenous dopamine in neural networks showing synchronous oscillation. Immunocytochemical study revealed a few tyrosine hydroxylase (TH)-positive dopaminergic neurons, and that cultured neurons expressed synaptophysin and synapsin I. Western blot analyses comfirmed synaptophysin, TH, and 2 types of dopamine receptor (DR), D1R and D2R expression. The synchronous oscillation in midbrain neurons was abolished by the application of R(-)-2-amino-5-phosphonopentanoic acid (AP-5) as an N-methyl-D-aspartate receptor (NMDAR) antagonist. This result suggests that the synchronous oscillation in midbrain neurons requires glutamatergic transmissions, as was the case in previously reported cortical neurons. SCH-12679, a D1R antagonist, inhibited synchronous oscillation in midbrain neurons, while raclopride, a D2R antagonist, induced a transient increase of intracellular Ca2+ and inhibited synchronous oscillation. We consider that endogenous dopamine maintains synchronous oscillation of intracellular Ca2+ through D1R and D2R, and that these DRs regulate intracellular Ca2+in distinctly different ways. Synchronous oscillation of midbrain neurons would be a useful tool for in vitro researches into various neural disorders directly or indirectly caused by dopaminergic neurons.

1. Synchronous oscillation of intracellular Ca2+ in the central nervous system is essential for neural development. We previously reported that endogenous dopamine was involved with synchronous Ca2+ oscillation of primary cultured midbrain neurons, and that regulation of dopamine in synchronous oscillation was distinctly different through dopamine receptor 1 (D1R) and 2 (D2R): the action of dopamine through D1R or D2R was facilitative or suppressive, respectively, to the Ca2+ influx of synchronous oscillation. 2. In the present study, we confirmed that the suppressive effects of D2R were mediated by the regulation of the L-type voltage-gated Ca2+ channel, not by the regulation of NMDA receptor on the Ca2+ influx in the midbrain neural network showing synchronous oscillation. 3. This evidence promotes better understanding of the regulation of neural activity by endogenous dopamine in networked neurons.