Katsuhiko Tabuchi

Nicastrin Functions as a γ-Secretase-Substrate Receptor

Summary γ-secretase catalyzes the intramembrane cleavage of amyloid precursor protein (APP) and Notch after their extracellular domains are shed by site-specific proteolysis. Nicastrin is an essential glycoprotein component of the γ-secretase complex but has no known function. We now show that the ectodomain of nicastrin binds the new amino terminus that is generated upon proteolysis of the extracellular APP and Notch domains, thereby recruiting the APP and Notch substrates into the γ-secretase complex. Chemical- or antibody-mediated blocking of the free amino terminus, addition of purified nicastrin ectodomain, or mutations in the ectodomain markedly reduce the binding and cleavage of substrate by γ-secretase. These results indicate that nicastrin is a receptor for the amino-terminal stubs that are generated by ectodomain shedding of type I transmembrane proteins. Our data are consistent with a model where nicastrin presents these substrates to γ-secretase and thereby facilitates their cleavage via intramembrane proteolysis.

Neuroligin-1 Deletion Results in Impaired Spatial Memory and Increased Repetitive Behavior

Neuroligins (NLs) are a family of neural cell-adhesion molecules that are involved in excitatory/inhibitory synapse specification. Multiple members of the NL family (including NL1) and their binding partners have been linked to cases of human autism and mental retardation. We have now characterized NL1-deficient mice in autism- and mental retardation-relevant behavioral tasks. NL1 knock-out (KO) mice display deficits in spatial learning and memory that correlate with impaired hippocampal long-term potentiation. In addition, NL1 KO mice exhibit a dramatic increase in repetitive, stereotyped grooming behavior, a potential autism-relevant abnormality. This repetitive grooming abnormality in NL1 KO mice is associated with a reduced NMDA/AMPA ratio at corticostriatal synapses. Interestingly, we further demonstrate that the increased repetitive grooming phenotype can be rescued in adult mice by administration of the NMDA receptor partial coagonist d-cycloserine. Broadly, these data are consistent with a role of synaptic cell-adhesion molecules in general, and NL1 in particular, in autism and implicate reduced excitatory synaptic transmission as a potential mechanism and treatment target for repetitive behavioral abnormalities.

Disruption of LGI1–linked synaptic complex causes abnormal synaptic transmission and epilepsy

Epilepsy is a devastating and poorly understood disease. Mutations in a secreted neuronal protein, leucine-rich glioma inactivated 1 (LGI1), were reported in patients with an inherited form of human epilepsy, autosomal dominant partial epilepsy with auditory features (ADPEAF). Here, we report an essential role of LGI1 as an antiepileptogenic ligand. We find that loss of LGI1 in mice (LGI1−/−) causes lethal epilepsy, which is specifically rescued by the neuronal expression of LGI1 transgene, but not LGI3. Moreover, heterozygous mice for the LGI1 mutation (LGI1+/−) show lowered seizure thresholds. Extracellularly secreted LGI1 links two epilepsy-related receptors, ADAM22 and ADAM23, in the brain and organizes a transsynaptic protein complex that includes presynaptic potassium channels and postsynaptic AMPA receptor scaffolds. A lack of LGI1 disrupts this synaptic protein connection and selectively reduces AMPA receptor–mediated synaptic transmission in the hippocampus. Thus, LGI1 may serve as a major determinant of brain excitation, and the LGI1 gene-targeted mouse provides a good model for human epilepsy.

Autism-linked neuroligin-3 R451C mutation differentially alters hippocampal and cortical synaptic function

Multiple independent mutations in neuroligin genes were identified in patients with familial autism, including the R451C substitution in neuroligin-3 (NL3). Previous studies showed that NL3R451C knock-in mice exhibited modestly impaired social behaviors, enhanced water maze learning abilities, and increased synaptic inhibition in the somatosensory cortex, and they suggested that the behavioral changes in these mice may be caused by a general shift of synaptic transmission to inhibition. Here, we confirm that NL3R451C mutant mice behaviorally exhibit social interaction deficits and electrophysiologically display increased synaptic inhibition in the somatosensory cortex. Unexpectedly, however, we find that the NL3R451C mutation produced a strikingly different phenotype in the hippocampus. Specifically, in the hippocampal CA1 region, the NL3R451C mutation caused an ∼1.5-fold increase in AMPA receptor-mediated excitatory synaptic transmission, dramatically altered the kinetics of NMDA receptor-mediated synaptic responses, induced an approximately twofold up-regulation of NMDA receptors containing NR2B subunits, and enhanced long-term potentiation almost twofold. NL3 KO mice did not exhibit any of these changes. Quantitative light microscopy and EM revealed that the NL3R451C mutation increased dendritic branching and altered the structure of synapses in the stratum radiatum of the hippocampus. Thus, in NL3R451C mutant mice, a single point mutation in a synaptic cell adhesion molecule causes context-dependent changes in synaptic transmission; these changes are consistent with the broad impact of this mutation on murine and human behaviors, suggesting that NL3 controls excitatory and inhibitory synapse properties in a region- and circuit-specific manner.

A Genetic Approach to Visualization of Multisynaptic Neural Pathways Using Plant Lectin Transgene

The wiring patterns among various types of neurons via specific synaptic connections are the basis of functional logic employed by the brain for information processing. This study introduces a powerful method of analyzing the neuronal connectivity patterns by delivering a tracer selectively to specific types of neurons while simultaneously transsynaptically labeling their target neurons. We developed a novel genetic approach introducing cDNA for a plant lectin, wheat germ agglutinin (WGA), as a transgene under the control of specific promoter elements. Using this method, we demonstrate three examples of visualization of specific transsynaptic neural pathways: the mouse cerebellar efferent pathways, the mouse olfactory pathways, and the Drosophila visual pathways. This strategy should greatly facilitate studies on the anatomical and functional organization of the developing and mature nervous system.

Increased Anxiety-like Behavior in Mice Lacking the Inhibitory Synapse Cell Adhesion Molecule Neuroligin 2

Neuroligins (NL) are postsynaptic cell adhesion molecules that are thought to specify synapse properties. Previous studies showed that mutant mice carrying an autism‐associated point mutation in NL3 exhibit social interaction deficits, enhanced inhibitory synaptic function and increased staining of inhibitory synaptic puncta without changes in overall inhibitory synapse numbers. In contrast, mutant mice lacking NL2 displayed decreased inhibitory synaptic function. These studies raised two relevant questions. First, does NL2 deletion impair inhibitory synaptic function by altering the number of inhibitory synapses, or by changing their efficacy? Second, does this effect of NL2 deletion on inhibition produce behavioral changes? We now show that although NL2‐deficient mice exhibit an apparent decrease in number of inhibitory synaptic puncta, the number of symmetric synapses as determined by electron microscopy is unaltered, suggesting that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers. This decrease in inhibitory synaptic function in NL2‐deficient mice correlates with a discrete behavioral phenotype that includes a marked increase in anxiety‐like behavior, a decrease in pain sensitivity and a slight decrease in motor co‐ordination. This work confirms that NL2 modulates inhibitory synaptic function and is the first demonstration that global deletion of NL2 can lead to a selective behavioral phenotype.

Presynaptic Neurexin-3 Alternative Splicing trans-Synaptically Controls Postsynaptic AMPA Receptor Trafficking

Neurexins are essential presynaptic cell adhesion molecules that are linked to schizophrenia and autism and are subject to extensive alternative splicing. Here, we used a genetic approach to test the physiological significance of neurexin alternative splicing. We generated knockin mice in which alternatively spliced sequence #4 (SS4) of neuexin-3 is constitutively included but can be selectively excised by cre-recombination. SS4 of neurexin-3 was chosen because it is highly regulated and controls neurexin binding to neuroligins, LRRTMs, and other ligands. Unexpectedly, constitutive inclusion of SS4 in presynaptic neurexin-3 decreased postsynaptic AMPA, but not NMDA receptor levels, and enhanced postsynaptic AMPA receptor endocytosis. Moreover, constitutive inclusion of SS4 in presynaptic neurexin-3 abrogated postsynaptic AMPA receptor recruitment during NMDA receptor-dependent LTP. These phenotypes were fully rescued by constitutive excision of SS4 in neurexin-3. Thus, alternative splicing of presynaptic neurexin-3 controls postsynaptic AMPA receptor trafficking, revealing an unanticipated alternative splicing mechanism for trans-synaptic regulation of synaptic strength and long-term plasticity.

Peroxiredoxin I expression in oral cancer: a potential new tumor marker

This study investigates the applicability of the novel antioxidant protein, peroxiredoxin (Prx) I as a marker for tumor status in oral squamous cell carcinoma (SCC). Samples from 53 patients with SCC in the oral cavity were examined by immunohistochemistry. Correlations between the expression level of Prx I and proliferating cell nuclear antigen (PCNA), the clinical features of tumors, and their histopathological classifications were statistically analyzed. Cases exhibiting low Prx I expression level included significantly more with larger tumor mass cases (T-category, P=0.004), positive lymph node metastasis (N-category, P=0.015), advanced stage (P=0.002), and poorly differentiated cells (P=0.020). There was no significant difference between Prx I expression and the other indices.

Localization of 5-HT2A Receptor in rat cerebral cortex and olfactory system revealed by immunohistochemistry using two antibodies raised in rabbit and chicken

Serotonin 2A receptor (5-HT2A receptor) is widely distributed in the central nervous system, and has been suggested to be involved in a variety of behavioral conditions and neuropsychiatric disorders. Two polyclonal antibodies were raised against the N-terminus peptide of rat 5-HT2A receptor in chickens (5-HT2A-N) and a glutathione S-transferase fusion protein that contained the C-terminus of the mouse 5-HT2A receptor in rabbits (5-HT2A-C). Affinity-purified 5-HT2A-N and -C antibodies reacted strongly with a single band of 77-78 kDa in postsynaptic density proteins prepared from the rat cortex. The distribution pattern of immunoreactive structures in the rat brain was virtually the same for the two antibodies. The highest levels of immunoreactivity were observed in the olfactory bulb, neocortex, claustrum, piriform cortex, mamillary bodies, pontine nuclei, red nucleus and cranial motor nuclei. In the olfactory bulb, mitral cells were intensely labeled. In the neocortex, many immunoreactive neurons were found in layers II-VI. In layer IV of the neocortex, strong neuropil labeling was observed. In a double-labeling study using chicken 5-HT2A-N and rabbit anti-glial fibrillary acidic protein (GFAP) antibody, a considerable number of GFAP positive cells also showed 5-HT2A immunoreactivity. By using an immunoelectron microscopic technique, 5-HT2A receptor immunoreaction was shown to be localized just beneath the postsynaptic membrane thickening of asymmetric synapses.

An autism-associated point mutation in the neuroligin cytoplasmic tail selectively impairs AMPA receptor-mediated synaptic transmission in hippocampus

Neuroligins are evolutionarily conserved postsynaptic cell‐adhesion molecules that function, at least in part, by forming trans‐synaptic complexes with presynaptic neurexins. Different neuroligin isoforms perform diverse functions and exhibit distinct intracellular localizations, but contain similar cytoplasmic sequences whose role remains largely unknown. Here, we analysed the effect of a single amino‐acid substitution (R704C) that targets a conserved arginine residue in the cytoplasmic sequence of all neuroligins, and that was associated with autism in neuroligin‐4. We introduced the R704C mutation into mouse neuroligin‐3 by homologous recombination, and examined its effect on synapses in vitro and in vivo. Electrophysiological and morphological studies revealed that the neuroligin‐3 R704C mutation did not significantly alter synapse formation, but dramatically impaired synapse function. Specifically, the R704C mutation caused a major and selective decrease in AMPA receptor‐mediated synaptic transmission in pyramidal neurons of the hippocampus, without similarly changing NMDA or GABA receptor‐mediated synaptic transmission, and without detectably altering presynaptic neurotransmitter release. Our results suggest that the cytoplasmic tail of neuroligin‐3 has a central role in synaptic transmission by modulating the recruitment of AMPA receptors to postsynaptic sites at excitatory synapses.