Tadr is an axonal histidine transporter required for visual neurotransmission in Drosophila

Neurotransmitters are generated by de novo synthesis and are essential for sustained, high-frequency synaptic transmission. Histamine, a monoamine neurotransmitter, is synthesized through decarboxylation of histidine by histidine decarboxylase (Hdc). However, little is known about how histidine is presented to Hdc as a precursor. Here, we identified a specific histidine transporter, TADR (torn and diminished rhabdomeres), which is required for visual transmission in Drosophila. Both TADR and Hdc localized to neuronal terminals, and mutations in tadr reduced levels of histamine, thus disrupting visual synaptic transmission and phototaxis behavior. These results demonstrate that a specific amino acid transporter provides precursors for monoamine neurotransmitters, providing the first genetic evidence that a histidine amino acid transporter plays a critical role in synaptic transmission. These results suggest that TADR-dependent local de novo synthesis of histamine is required for synaptic transmission.

44 Lebrand et al., 1996). However, to date, amino acid transporters specific for the synthesis of

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Histamine was first identified as a neurotransmitter that localized to the tuberomamillary 51 nucleus where it was synthesized from the amino acid histidine through a reaction catalyzed 52 by the enzyme histidine decarboxylase (HDC), which removes a carboxyl group from histidine 53 Watanabe et al., 1984). As a neurotransmitter, histamine plays important 54 roles in regulating multiple physiological processes, including cognition, sleep, synaptic 55 plasticity, and feeding behaviors (Bekkers, 1993

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Histidine decarboxylase (Hdc) localizes to neuronal terminals 94 Histamine acts as major neurotransmitter at photoreceptor synaptic terminals, transmitting 95 visual information to interneurons (Hardie, 1989). Further, histamine de novo synthesis in 96 photoreceptor cells is essential for maintaining visual transmission (Burg et al., 1993).

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Interestingly, we have identified a vesicle transporter specific for histamine, LOVIT, which is 98 concentrated exclusively in photoreceptor terminals and helps to maintain levels of histamine 99 at synapses (Xu and Wang, 2019). Together with the fact that visual neurotransmission 100 requires rapid and high-frequency firing, we hypothesize that the fast neurotransmitter

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1D). The finding that Hdc protein was enriched in photoreceptor terminals is consistent with 118 the assumption that the neurotransmitter histamine is synthesized directly in axon terminals.

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TADR is required for visual synaptic transmission 120 Given that the enzyme responsible for catalyzing the biosynthesis of histamine localized to 121 pre-synaptic regions, we next sought to determine how histidine, an Hdc substrate, is 122 transported to neuronal terminals. We hypothesized that an amino acid transporter resided on 123 the plasma membrane of photoreceptor synaptic terminals, and that this transporter would be 124 responsible for histidine uptake and required for rapid histamine synthesis and visual 125 transmission. Among ~600 putative transmembrane transporters encoded by the Drosophila 126 genome (Ren et al., 2007), we identified 42 genes that could potentially encode an amino acid 127 transporter. These we tested as candidate histamine transporters (Table S1).

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To examine whether these putative transporters were involved in visual neurotransmission, 129 each candidate gene was knocked down individually via the eye-specific expression of RNAi

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To further confirm that tadr was the causal gene, we generated a null mutation in the tadr gene 146 by deleting ~700-bp genomic fragment using the CRISPR-associated single-guide RNA  TADR, suggesting that TADR is a bona fide plasma membrane histidine transporter (Bröer, 176 2014) ( Figure 3B).

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Considering that transporters related to histamine recycling are necessary for synaptic 178 transmission, we next sought to determine whether TADR specifically transports histidine in 179 Drosophila. Because a histamine transporter has not yet been identified, we first asked 180 whether TADR can transport histamine. Histamine uptake assays revealed that TADR does 181 not exhibit histamine uptake activity. As a control, the human Organic Cation Transporter 182 (OCT2), which is known to take up histamine, exhibited high levels of histamine transport 183 when expressed in S2 cells (Busch et al., 1998) ( Figure 3C). Next, we found that TADR did not

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The full raw unedited gels for PCR products obtained from tadr 2 mutants.