Maya Ota

Selective control of inhibitory synapse development by Slitrk3-PTPδ trans-synaptic interaction

Balanced development of excitatory and inhibitory synapses is required for normal brain function, and an imbalance in this development may underlie the pathogenesis of many neuropsychiatric disorders. Compared with the many identified trans-synaptic adhesion complexes that organize excitatory synapses, little is known about the organizers that are specific for inhibitory synapses. We found that Slit and NTRK-like family member 3 (Slitrk3) actS as a postsynaptic adhesion molecule that selectively regulates inhibitory synapse development via trans-interaction with axonal tyrosine phosphatase receptor PTPδ. When expressed in fibroblasts, Slitrk3 triggered only inhibitory presynaptic differentiation in contacting axons of co-cultured rat hippocampal neurons. Recombinant Slitrk3 preferentially localized to inhibitory postsynaptic sites. Slitrk3-deficient mice exhibited decreases in inhibitory, but not excitatory, synapse number and function in hippocampal CA1 neurons and exhibited increased seizure susceptibility and spontaneous epileptiform activity. Slitrk3 required trans-interaction with axonal PTPδ to induce inhibitory presynaptic differentiation. These results identify Slitrk3-PTPδ as an inhibitory-specific trans-synaptic organizing complex that is required for normal functional GABAergic synapse development.

Slitrk1-deficient mice display elevated anxiety-like behavior and noradrenergic abnormalities

Mutations in SLITRK1 are found in patients with Tourettes syndrome and trichotillomania. SLITRK1 encodes a transmembrane protein containing leucine-rich repeats that is produced predominantly in the nervous system. However, the role of this protein is largely unknown, except that it can modulate neurite outgrowth in vitro. To clarify the role of Slitrk1 in vivo, we developed Slitrk1-knockout mice and analyzed their behavioral and neurochemical phenotypes. Slitrk1-deficient mice exhibited elevated anxiety-like behavior in the elevated plus-maze test as well as increased immobility time in forced swimming and tail suspension tests. Neurochemical analysis revealed that Slitrk1-knockout mice had increased levels of norepinephrine and its metabolite 3-methoxy-4-hydroxyphenylglycol. Administration of clonidine, an α2-adrenergic agonist that is frequently used to treat patients with Tourettes syndrome, attenuated the anxiety-like behavior of Slitrk1-deficient mice in the elevated plus-maze test. These results lead us to conclude that noradrenergic mechanisms are involved in the behavioral abnormalities of Slitrk1-deficient mice. Elevated anxiety due to Slitrk1 dysfunction may contribute to the pathogenesis of neuropsychiatric diseases such as Tourettes syndrome and trichotillomania.

Zic1 and Zic3 Regulate Medial Forebrain Development through Expansion of Neuronal Progenitors

The medial telencephalon is a source of neurons that follow distinct tangential trajectories of migration to various structures such as the cerebral cortex, striatum, and olfactory bulb. In the present study, we characterized the forebrain anomalies in Zic1/Zic3 compound mutant mice. Zic1 and Zic3 were strongly expressed in the medial structures, including the septum, medial cerebral cortex, and choroid plexus. Mice homozygous for the Zic1 mutant allele together with the null Zic3 allele showed medial forebrain defects, which were not obvious in either Zic1 or Zic3 single mutants. Absence of both Zic1 and Zic3 caused hypoplasia of the hippocampus, septum, and olfactory bulb. Analysis of the cell cycle revealed that the cell cycle exit rate was increased in the septa of double mutants. Misexpression of Zic3 in the ventricular layer of the cerebral cortex inhibited neuronal differentiation. These results indicated that both Zic1 and Zic3 function in maintaining neural precursor cells in an undifferentiated state. The functions of these genes may be essential to increasing neural cell numbers regionally in the medial telencephalon and to proper mediolateral patterning of the telencephalon.

Disorganized Innervation and Neuronal Loss in the Inner Ear of Slitrk6-Deficient Mice

Slitrks are type I transmembrane proteins that share conserved leucine-rich repeat domains similar to those in the secreted axonal guidance molecule Slit. They also show similarities to Ntrk neurotrophin receptors in their carboxy-termini, sharing a conserved tyrosine residue. Among 6 Slitrk family genes in mammals, Slitrk6 has a unique expression pattern, with strong expression in the sensory epithelia of the inner ear. We generated Slitrk6-knockout mice and investigated the development of their auditory and vestibular sensory organs. Slitrk6-deficient mice showed pronounced reduction in the cochlear innervation. In the vestibule, the innervation to the posterior crista was often lost, reduced, or sometimes misguided. These defects were accompanied by the loss of neurons in the spiral and vestibular ganglia. Cochlear sensory epithelia from Slitrk6-knockout mice have reduced ability in promoting neurite outgrowth of spiral ganglion neurons. Indeed the Slitrk6-deficient inner ear showed a mild but significant decrease in the expression of Bdnf and Ntf3, both of which are essential for the innervation and survival of sensory neurons. In addition, the expression of Ntrk receptors, including their phosphorylated forms was decreased in Slitrk6-knockout cochlea. These results suggest that Slitrk6 promotes innervation and survival of inner ear sensory neurons by regulating the expression of trophic and/or tropic factors including neurotrophins from sensory epithelia.

SLITRK6 mutations cause myopia and deafness in humans and mice

Myopia is by far the most common human eye disorder that is known to have a clear, albeit poorly defined, heritable component. In this study, we describe an autosomal-recessive syndrome characterized by high myopia and sensorineural deafness. Our molecular investigation in 3 families led to the identification of 3 homozygous nonsense mutations (p.R181X, p.S297X, and p.Q414X) in SLIT and NTRK-like family, member 6 (SLITRK6), a leucine-rich repeat domain transmembrane protein. All 3 mutant SLITRK6 proteins displayed defective cell surface localization. High-resolution MRI of WT and Slitrk6-deficient mouse eyes revealed axial length increase in the mutant (the endophenotype of myopia). Additionally, mutant mice exhibited auditory function deficits that mirrored the human phenotype. Histological investigation of WT and Slitrk6-deficient mouse retinas in postnatal development indicated a delay in synaptogenesis in Slitrk6-deficient animals. Taken together, our results showed that SLITRK6 plays a crucial role in the development of normal hearing as well as vision in humans and in mice and that its disruption leads to a syndrome characterized by severe myopia and deafness.

Rines E3 ubiquitin ligase regulates MAO-A levels and emotional responses.

Monoamine oxidase A (MAO-A), the catabolic enzyme of norepinephrine and serotonin, plays a critical role in emotional and social behavior. However, the control and impact of endogenous MAO-A levels in the brain remains unknown. Here we show that the RING finger-type E3 ubiquitin ligase Rines/RNF180 regulates brain MAO-A subset, monoamine levels, and emotional behavior. Rines interacted with MAO-A and promoted its ubiquitination and degradation. Rines knock-out mice displayed impaired stress responses, enhanced anxiety, and affiliative behavior. Norepinephrine and serotonin levels were altered in the locus ceruleus, prefrontal cortex, and amygdala in either stressed or resting conditions, and MAO-A enzymatic activity was enhanced in the locus ceruleus in Rines knock-out mice. Treatment of Rines knock-out mice with MAO inhibitors showed genotype-specific effects on some of the abnormal affective behaviors. These results indicated that the control of emotional behavior by Rines is partly due to the regulation of MAO-A levels. These findings verify that Rines is a critical regulator of the monoaminergic system and emotional behavior and identify a promising candidate drug target for treating diseases associated with emotion.

Lrfn2/SALM1, a synaptic leucine-rich repeat transmembrane molecule is required for proper synapse maturation and brain function

O2-D1-2 GABAergic inhibition onto Purkinje cell soma is crucial for climbing fiber synapse elimination in developing cerebellum Hisako Nakayama1, Taisuke Miyazaki2, Kouichi Hashimoto1,3, Yuchio Yanagawa4, Kunihiko Obata5, Masahiko Watanabe2, Masanobu Kano1 1 Dept. Neurophysiol., Univ. of Tokyo, Tokyo, Japan; 2 Dept. Anat., Hokkaido Univ., Sapporo, Japan; 3 CREST, Tokyo, Japan; 4 Dept. Genetic and Behavioral Neurosci., Gunma Univ., Maebashi, Japan; 5 Obata. Res. Unit, RIKEN BSI, Wako, Japan

Lrfn2/SALM1, a synaptic leucine-rich repeat-containing transmembrane protein regulates synapse formation and function

We have been investigating the role of a neuronal LRR transmembrane family, Lrfn1-5, in vertebrate CNS. Here we focused on the synaptic function of Lrfn2/SALM1. Immunofluorescence staining and subcellular fractionation studies indicated that Lrfn2 is an excitatory postsynaptic protein. Lrfn2 co-precipitated PSD-95 and NR1 subunit of NMDA receptor in an immunoprecipitation assay using mouse brain lysates. Coexpression of Lrfn2 and PSD-95 in cultured hippocampal neurons resulted in the activation of the synaptic activity, while an Lrfn2 mutant lacking PSD-95-binding ability did not show such activation in the same assay. To explore the role of Lrfn2 in synaptic function in vivo, we generated Lrfn2 knockout mice. Hippocampal neurons from Lrfn2 KO mice showed a reduced clustering of both PSD-95 and NR1, suggesting that Lrfn2 is required for the proper formation of the excitatory synapses.