Kohlschutter-Tonz syndrome (KTS) is a rare genetic disorder with neurological dysfunctions including seizure and intellectual impairment. Mutations at the Rogdi locus have been linked to development of KTS, yet the underlying mechanisms remain elusive. This study demonstrates that a Drosophila homolog of Rogdi acts as a novel sleep-promoting factor by supporting a specific subset of gamma-aminobutyric acid (GABA) transmission. Rogdi mutant flies displayed insomnia-like behaviors accompanied by sleep fragmentation and delay in sleep initiation. The sleep suppression phenotypes were rescued by sustaining GABAergic transmission primarily via metabotropic GABA receptors or by blocking wake-promoting dopaminergic pathways. Transgenic rescue further mapped GABAergic neurons as a cell-autonomous locus important for Rogdi-dependent sleep, implying metabotropic GABA transmission upstream of the dopaminergic inhibition of sleep. Consistently, an agonist specific to metabotropic but not ionotropic GABA receptors titrated the wake-promoting effects of dopaminergic neuron excitation. Taken together, these data provide the first genetic evidence that implicates Rogdi in sleep regulation via GABAergic control of dopaminergic signaling. Given the strong relevance of GABA to epilepsy, it is proposed that similar mechanisms might underlie the neural pathogenesis of Rogdi-associated KTS (Kim, 2017).

Neurological disorders caused by single-gene mutations are important genetic models to understand how individual genes execute their roles to support the development and function of the brain, as in the case of Kohlschutter-Tönz syndrome (KTS). KTS patients display developmental delays and psychomotor regression. The most prominent symptoms include amelogenesis imperfecta, early-onset seizures, and intellectual disabilities. Linkage analyses followed by genomic sequencing have revealed that most, if not all, KTS patients have homozygous nonsense, frameshift deletion, or splicing site mutations at the Rogdi locus. ROGDI protein expression is not detectable in affected individuals, indicating that the loss of Rogdi function is responsible for the pathogenesis of KTS. In wild-type human tissues, Rogdi transcripts are ubiquitously expressed while the highest enrichment is evident in adult brain and spinal cord. This observation is consistent with the neurological phenotypes observed in KTS patients (Kim, 2017).

Given that Rogdi homologs are relatively well conserved in higher eukaryotes, animal models may facilitate understanding of Rogdi-dependent neural processes. In fact, Rogdi was initially identified in a Drosophila genetic screen as a memory-relevant gene and was thus named after one of Pavlov’s dogs. Sequence analyses of Rogdi homologs revealed a putative leucine zipper (ZIP) motif, which could mediate the dimerization of DNA-binding basic ZIP (bZIP) transcription factors. Interestingly, ROGDI proteins localize to the nuclear envelope in cultured human cells, although they lack basic amino acid residues that are typically located at the N-terminus of the ZIP domain and are required for the DNA-binding and nuclear localization of the bZIP transcription factors. Nonetheless, few or no studies have demonstrated the biological activity of Rogdi and genetic models for Rogdi homologs have not been reported yet. Therefore, how Rogdi exerts its physiological roles particularly in the central nervous system and how its mutation leads to the development of KTS are largely unknown (Kim, 2017).

In the course of genetic studies to elucidate genes and regulatory pathways involved in sleep behaviors, novel sleep mutant alleles were identified in the Drosophila Rogdi gene. This study employed the sleep-promoting effects of Rogdi as a readout of its neural function and demonstrated that Rogdi acts cell-autonomously in GABAergic neurons to enhance metabotropic GABA transmission and thereby sustain sleep. In addition, dopaminergic rescue of Rogdi mutant sleep revealed a novel sleep-regulatory mechanism that functionally links a specific subset of sleep-promoting GABAergic neurons to a wake-promoting dopaminergic pathway. Since epilepsy, a well penetrated phenotype in KTS patients, implicates GABAergic transmission, the current findings provide an important genetic clue to understanding the molecular and neural pathogenesis of KTS (Kim, 2017).

Modeling of neurological diseases and disease-relevant genes has greatly advanced understanding of the fundamental principles that underlie disease pathogenesis as well as brain function. This study has established the first genetic model of the KTS-associated disease gene Rogdi to demonstrate that Rogdi functions as a novel sleep-promoting factor in GABAergic neurons by promoting GABA transmission. While GABA-dependent sleep regulation via ionotropic GABA receptors have been well documented in Drosophila, the data suggest that GABAergic transmission via metabotropic GABA receptors might be primarily compromised by Rogdi mutation. Furthermore, the wake-promoting DA pathway was identified as a neural locus downstream of Rogdi-dependent GABA signaling given that Rogdi mutant sleep could be rescued by pharmacological or genetic manipulation of dopaminergic transmission. This sleep-regulatory pathway was further supported by the observation that wake-promoting effects of TH-expressing dopaminergic neurons could be selectively titrated by an agonist of metabotropic GABA receptors. Rogdi thus defines a novel pathway coupling these two neurotransmitters to promote baseline sleep in Drosophila as exemplified in other behavioral paradigms across species. On the other hand, Rogdi-dependent GABA transmission might have inhibitory effects on a sleep-promoting neural pathway for sleep homeostasis to suppress recovery sleep after sleep loss (Kim, 2017).

What is the molecular basis by which Rogdi supports GABAergic transmission and promotes sleep? A possible role of ROGDI as a transcription factor has been suggested by the nuclear localization of human ROGDI protein, particularly in the nuclear envelope of blood mononuclear cells and dermal fibroblasts, and by the conservation of a putatively dimerizing leucine zipper (ZIP) motif among ROGDI homologs. Several lines of evidence, however, argue against this possibility. bZIP transcription factors possess basic residues followed by their ZIP domains whereas ROGDI protein lacks the canonical motif (i.e., basic residues) for DNA-binding activity and nuclear localization. Drosophila ROGDI actually displays its subcellular distribution in both nucleus and cytoplasm of cultured cells or adult fly neurons although the exclusive nuclear localization might not be a prerequisite for transcriptional activities. The crystal structure of human ROGDI protein (Lee, 2017) showed that, unlike other bZIP transcription factors, human ROGDI protein exists as a monomer containing two structurally distinguishable domains (designated as α and β domains, respectively). The α domain exhibits an α-helical bundle that consists of H1, H2, H3, and H6 helices. In fact, the ZIP-like motif in the α domain appears to mediate their intramolecular interactions, contributing to the overall structure and stability of a monomeric ROGDI protein (Lee, 2017). Based on sequence homology between Drosophila and human ROGDI proteins, it is predicted that Rogdi[del] allele removes the majority of the first helix including the repeated leucine residues in the α domain and the first three strands in the β domain, explaining the instability of ROGDI^del proteins. A smaller but comparable deletion of the ZIP-like motif has been reported in a KTS patient with a splicing mutation in human Rogdi gene. In addition, a functional study in cervical cancer cell lines demonstrated Rogdi effects on cell cycle progression and radio-sensitivity. However, further investigations will be required to understand how these cellular phenotypes could be linked to the molecular and neural function of ROGDI protein (Kim, 2017).

What will be the relevance of these findings to KTS pathogenesis? Genetic heterogeneity has been reported among KTS patients, indicating that Rogdi-independent genetic mutations could contribute to KTS pathogenesis. A recent study indeed showed that familial mutations in a sodium-citrate transporter gene SLC13A5 are the second genetic cause of KTS. The pathogenic phenotypes commonly found in Rogdi- and SLC13A5-associated KTS gives rise to the intriguing possibility that these two genes might work together to control the intracellular levels of citrate. This idea is further supported by the relevance of citrate metabolism to neurological phenotypes in KTS patients. Neurons are energetically dependent on astrocytes because neurons lack pyruvate carboxylase, an enzyme that converts pyruvate to oxaloacetate in the citric acid cycle. SLC13A5 plays an important role in the transport of glial citrate into neurons to supplement the neuronal citric acid cycle and thereby supply cellular energy. Furthermore, citrate, an intermediate in the citric acid cycle, acts as a precursor of α-ketoglutarate, which can be metabolized to glutamate and GABA, implicating SLC13A5 in the biogenesis of GABA. Consistently, anti-epileptic drugs that elevate GABAergic transmission rescued the seizure phenotypes in SLC13A-associated KTS patients. In addition, this study showed that the pharmacological enhancement of GABAergic transmission by oral administration of GABA-T or GAT inhibitors was sufficient to rescue the short sleep phenotypes in Rogdi mutant flies (Kim, 2017).

These genetic studies strongly implicate Rogdi function in GABAergic transmission, providing the first clue to understanding the neurological phenotypes observed in KTS patients. Molecular and neural deficits selectively caused by Rogdi mutation might explain why seizures in Rogdi-associated KTS are often resistant to anti-epileptic drugs. Future studies should thus address if Rogdi mutant flies display seizure-like behaviors similarly as in KTS patients and if Rogdi-dependent neural relay of GABAergic transmission controls seizure susceptibility in parallel with baseline sleep. In addition, it will be important to determine whether sleep deficiencies are also observed in KTS patients and whether reduced GABAergic transmission in Rogdi- and, possibly, SLC13A5-associated KTS patients is responsible for their neural dysfunctions, including early-onset seizures. Taken together, this genetic model would constitute an important platform for elucidating the molecular and neural pathogenesis underlying KTS and hint towards a precise development of a therapeutic strategy for KTS in the future (Kim, 2017).

Kohlschutter-Tonz syndrome (KTS) is a rare autosomal-recessive disorder of childhood onset characterized by global developmental delay, spasticity, epilepsy, and amelogenesis imperfecta. Rogdi, an essential protein, is highly conserved across metazoans, and mutations in Rogdi are linked to KTS. However, how certain mutations in Rogdi abolish its physiological functions and cause KTS is not known. This study determined the crystal structure of human Rogdi protein at atomic resolution. Rogdi forms a novel elongated curved structure comprising the alpha domain, a leucine-zipper-like four-helix bundle, and a characteristic beta-sheet domain. Within the alpha domain, the N-terminal H1 helix (residues 19-45) pairs with the C-terminal H6 helix (residues 252-287) in an antiparallel manner, indicating that the integrity of the four-helix bundle requires both N- and C-terminal residues. The crystal structure, in conjunction with biochemical data, indicates that the alpha domain might undergo a conformational change and provide a structural platform for protein-protein interactions. Disruption of the four-helix bundle by mutation results in significant destabilization of the structure. This study provides structural insights into how certain mutations in Rogdi affect its structure and cause KTS, which has important implications for the development of pharmaceutical agents against this debilitating neurological disease (Lee, 2017).

Mutations in the human homolog of the Drosophila gene Rogdi cause Kohlschutter-Tonz syndrome. This disorder is characterised by amelogenesis imperfecta, as well as severe neurological symptoms including epilepsy and psychomotor delay. However, little is known about the protein encoded by Rogdi, and hence the pathogenic mechanisms underlying Kohlschutter-Tonz syndrome have remained elusive. Using immunofluorescence of rat cultured hippocampal neurons and brain sections this study found that Rogdi is enriched at synaptic sites. In addition, recombinant GFP-Rogdi expressed in cultured neurons was efficiently targeted to presynaptic sites, where it colocalised with the presynaptic scaffolding protein Bassoon and the synaptic vesicle markers Synaptophysin, Synapsin-1, VAMP2/Synaptobrevin and Mover. These data indicate that GFP-Rogdi harbours efficient signals for presynaptic targeting, and that Rogdi is a presynaptic protein. Thus, the neurological symptoms associated with Kohlschutter-Tonz syndrome may arise from presynaptic dysfunction (Riemann, 2017).