A systematic structure-function characterization of a human mutation in Neurexin-3α reveals an extracellular modulatory sequence that stabilizes neuroligin-1 binding to enhance the postsynaptic properties of excitatory synapses.

α-neurexins are essential and highly expressed presynaptic cell-adhesion molecules that are frequently linked to neuropsychiatric and neurodevelopmental disorders. Despite their importance, how the elaborate extracellular sequences of α-neurexins contribute to synapse function is poorly understood. We recently characterized the presynaptic gain-of-function phenotype caused by a missense mutation in an evolutionarily conserved extracellular sequence of neurexin-3α (A687T) that we identified in a patient diagnosed with profound intellectual disability and epilepsy. The striking A687T gain-of-function mutation on neurexin-3α prompted us to systematically test using mutants whether the presynaptic gain-of-function phenotype is a consequence of the addition of side-chain bulk (i.e., A687V) or polar/hydrophilic properties (i.e., A687S). We used multidisciplinary approaches in mixed-sex primary hippocampal cultures to assess the impact of the neurexin-3αA687 residue on synapse morphology, function and ligand binding. Unexpectedly, neither A687V nor A687S recapitulated the neurexin-3α A687T phenotype. Instead, distinct from A687T, molecular replacement with A687S significantly enhanced postsynaptic properties exclusively at excitatory synapses and selectively increased binding to neuroligin-1 and neuroligin-3 without changing binding to neuroligin-2 or LRRTM2. Importantly, we provide the first experimental evidence supporting the notion that the position A687 of neurexin-3α and the N-terminal sequences of neuroligins may contribute to the stability of α-neurexin-neuroligin-1 trans-synaptic interactions and that these interactions may specifically regulate the postsynaptic strength of excitatory synapses.Significance Statement Although neurexins were discovered over 30 years ago, our understanding of how the complex extracellular sequences unique to α-neurexins participate in synapse function remains incomplete. We leveraged a previously studied human missense mutation, located in a conserved extracellular region of neurexin-3α  and linked to profound intellectual disability and epilepsy, to systematically assess the tolerance of neurexin-3α function to mutations within this region. Using molecular replacement, we assessed how single amino acid substitutions in this extracellular region alters synapse morphology, presynaptic calcium dynamics, and synaptic transmission. We reveal that multiple neurexin ligands unexpectedly use this region to modulate trans-synaptic binding and that different amino acid substitutions in place of the disease mutation result in dramatically different changes to synaptic transmission.

MDGAs perform activity-dependent synapse type-specific suppression via distinct extracellular mechanisms.

MDGA (MAM domain containing glycosylphosphatidylinositol anchor) family proteins were previously identified as synaptic suppressive factors. However, various genetic manipulations have yielded often irreconcilable results, precluding precise evaluation of MDGA functions. Here, we found that, in cultured hippocampal neurons, conditional deletion of MDGA1 and MDGA2 causes specific alterations in synapse numbers, basal synaptic transmission, and synaptic strength at GABAergic and glutamatergic synapses, respectively. Moreover, MDGA2 deletion enhanced both N-methyl-D-aspartate (NMDA) receptor- and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated postsynaptic responses. Strikingly, ablation of both MDGA1 and MDGA2 abolished the effect of deleting individual MDGAs that is abrogated by chronic blockade of synaptic activity. Molecular replacement experiments further showed that MDGA1 requires the meprin/A5 protein/PTPmu (MAM) domain, whereas MDGA2 acts via neuroligin-dependent and/or MAM domain-dependent pathways to regulate distinct postsynaptic properties. Together, our data demonstrate that MDGA paralogs act as unique negative regulators of activity-dependent postsynaptic organization at distinct synapse types, and cooperatively contribute to adjustment of excitation-inhibition balance.

LPS induces microglial activation and GABAergic synaptic deficits in the hippocampus accompanied by prolonged cognitive impairment.

Neuroinflammation impacts the brain and cognitive behavior through microglial activation. In this study, we determined the temporal sequence from microglial activation to synaptic dysfunction and cognitive behavior induced by neuroinflammation in mice. We found that LPS injection activated microglia within a short period, followed by impairments in GABAergic synapses, and that these events led to long-term cognitive impairment. We demonstrated that, 3 days after LPS injection, microglia in the hippocampus were significantly activated due to the LPS-induced inflammation in association with alterations in cellular morphology, microglial density, and expression of phagocytic markers. GABAergic synaptic impairments were detected at 4-6 days after LPS treatment, a time when microglia activity had returned to normal. Consequently, memory impairment persisted for 6 days after injection of LPS. Our results suggest that neuroinflammation induces microglia activation, GABAergic synaptic deficits and prolonged memory impairment over a defined temporal sequence. Our observations provide insight into the temporal sequence of neuroinflammation-associated brain pathologies. Moreover, the specific loss of inhibitory synapses accompanying the impaired inhibitory synaptic transmission provides mechanistic insight that may explain the prolonged cognitive deficit observed in patients with neuroinflammation. Thus, this study provides essential clues regarding early intervention strategies against brain pathologies accompanying neuroinflammation.

Novel insertion/deletion polymorphisms and genetic features of the shadow of prion protein gene (SPRN) in dogs, a prion-resistant animal.

Prion diseases are fatal infectious neurodegenerative disorders that are induced by misfolded prion protein (PrPSc). Previous studies have reported that the shadow of prion protein (Sho) encoded by the shadow of prion protein gene (SPRN) plays a critical role in stimulating the conversion process of normal PrP (PrPC) into PrPSc, and genetic polymorphisms of the SPRN gene are significantly related to susceptibility to prion diseases. Recent studies have reported that dogs show prion resistance, and there have been several attempts to identify resistance factors to prion diseases in dogs. However, there has been no study of the canine SPRN gene thus far. We investigated genetic polymorphisms of the canine SPRN gene in 201 dogs using amplicon sequencing and compared the number of SPRN polymorphisms among prion-related species. In addition, we performed multiple sequence alignments of the amino acid sequences of Sho among prion-related species by ClustalW and analyzed the 3D structure of Sho using AlphaFold. Furthermore, we assessed the protein-protein interaction of canine PrP with canine Sho carrying wild-type and mutant alleles using HawkDock. We found four novel insertion/deletion polymorphisms of the SPRN gene in 201 dogs and identified a significant difference in the number of SPRN polymorphisms between prion-susceptible and prion-resistant animals. In addition, Sho has two α-helixes linked with the coil. Furthermore, we found different binding complexes and binding free energies between canine Sho and PrP according to SPRN polymorphisms. To the best of our knowledge, this is the first report of canine SPRN polymorphisms.

The First Report of Polymorphisms and Genetic Characteristics of the Shadow of Prion Protein (SPRN) in Prion Disease-Resistant Animal, Chickens.

Prion diseases are irreversible neurodegenerative disorders caused by the aggregated form of prion protein (PrPSc) derived from the normal form of prion protein (PrPC). Previous studies have reported that shadow of prion protein (Sho) interacts with prion protein (PrP) and accelerates the conversion of PrPC to PrPSc. In addition, genetic polymorphisms of the shadow of the prion protein gene (SPRN) are related to the vulnerability of prion diseases in various hosts. However, to date, polymorphisms and genetic features of the SPRN gene have not been investigated in chickens, which are prion disease-resistant animals. We investigated genetic polymorphisms of the SPRN gene in 2 breeds of chickens, i.e., Dekalb White and Ross, using amplicon sequencing. We analyzed genotype, allele and haplotype frequencies and linkage disequilibrium (LD) among the genetic polymorphisms. In addition, we compared the amino acid sequences of Sho among several prion-related species to identify the unique genetic features of chicken Sho using ClustalW. Furthermore, we evaluated the N-terminal signal peptide and glycosylphosphatidylinositol (GPI)-anchor using SignalP and PredGPI, respectively. Finally, we compared the number of SPRN polymorphisms between prion disease-resistant and prion disease-susceptible animals. We identified 7 novel single nucleotide polymorphisms (SNPs), including 1 synonymous SNP in the open reading frame (ORF) of the chicken SPRN gene. We also found significantly different genotypes, allele frequencies and haplotypes between the 2 chicken breeds. In addition, we found that the interaction regions between Sho and PrP and the NXT glycosylation motif were conserved among all species. Notably, sequence similarity was extremely low in the N-terminal and C-terminal regions between mammals and chickens. Furthermore, we found that chicken Sho was the longest N-terminal signal peptide, and the amino acids of the cutting site of chicken are different from those of mammals. Last, unlike other species investigated, omega-site and signal sequences of the GPI-anchor were not found in chickens. To the best of our knowledge, this is the first report of genetic polymorphisms of the SPRN gene in chickens.

SLITRK2 variants associated with neurodevelopmental disorders impair excitatory synaptic function and cognition in mice.

SLITRK2 is a single-pass transmembrane protein expressed at postsynaptic neurons that regulates neurite outgrowth and excitatory synapse maintenance. In the present study, we report on rare variants (one nonsense and six missense variants) in SLITRK2 on the X chromosome identified by exome sequencing in individuals with neurodevelopmental disorders. Functional studies showed that some variants displayed impaired membrane transport and impaired excitatory synapse-promoting effects. Strikingly, these variations abolished the ability of SLITRK2 wild-type to reduce the levels of the receptor tyrosine kinase TrkB in neurons. Moreover, Slitrk2 conditional knockout mice exhibited impaired long-term memory and abnormal gait, recapitulating a subset of clinical features of patients with SLITRK2 variants. Furthermore, impaired excitatory synapse maintenance induced by hippocampal CA1-specific cKO of Slitrk2 caused abnormalities in spatial reference memory. Collectively, these data suggest that SLITRK2 is involved in X-linked neurodevelopmental disorders that are caused by perturbation of diverse facets of SLITRK2 function.

Novel Polymorphisms and Genetic Characteristics of the Shadow of Prion Protein Gene (SPRN) in Cats, Hosts of Feline Spongiform Encephalopathy.

Prion diseases are transmissible spongiform encephalopathies (TSEs) caused by pathogenic prion protein (PrPSc) originating from normal prion protein (PrPC) and have been reported in several types of livestock and pets. Recent studies have reported that the shadow of prion protein (Sho) encoded by the shadow of prion protein gene (SPRN) interacts with prion protein (PrP) and accelerates prion diseases. In addition, genetic polymorphisms in the SPRN gene are related to susceptibility to prion diseases. However, genetic polymorphisms in the feline SPRN gene and structural characteristics of the Sho have not been investigated in cats, a major host of feline spongiform encephalopathy (FSE). We performed amplicon sequencing to identify feline SPRN polymorphisms in the 623 bp encompassing the open reading frame (ORF) and a small part of the 3' untranslated region (UTR) of the SPRN gene. We analyzed the impact of feline SPRN polymorphisms on the secondary structure of SPRN mRNA using RNAsnp. In addition, to find feline-specific amino acids, we carried out multiple sequence alignments using ClustalW. Furthermore, we analyzed the N-terminal signal peptide and glycosylphosphatidylinositol (GPI)-anchor using SignalP and PredGPI, respectively. We identified three novel SNPs in the feline SPRN gene and did not find strong linkage disequilibrium (LD) among the three SNPs. We found four major haplotypes of the SPRN polymorphisms. Strong LD was not observed between PRNP and SPRN polymorphisms. In addition, we found alterations in the secondary structure and minimum free energy of the mRNA according to the haplotypes in the SPRN polymorphisms. Furthermore, we found four feline-specific amino acids in the feline Sho using multiple sequence alignments among several species. Lastly, the N-terminal signal sequence and cutting site of the Sho protein of cats showed similarity with those of other species. However, the feline Sho protein exhibited the shortest signal sequence and a unique amino acid in the omega-site of the GPI anchor. To the best of our knowledge, this is the first report on genetic polymorphisms of the feline SPRN gene.

IQSEC3 Deletion Impairs Fear Memory Through Upregulation of Ribosomal S6K1 Signaling in the Hippocampus.

BACKGROUND: IQSEC3, a gephyrin-binding GABAergic (gamma-aminobutyric acidergic) synapse-specific guanine nucleotide exchange factor, was recently reported to regulate activity-dependent GABAergic synapse maturation, but the underlying signaling mechanisms remain incompletely understood. METHODS: We generated mice with conditional knockout (cKO) of Iqsec3 to examine whether altered synaptic inhibition influences hippocampus-dependent fear memory formation. In addition, electrophysiological recordings, immunohistochemistry, and behavioral assays were used to address our question. RESULTS: We found that Iqsec3-cKO induces a specific reduction in GABAergic synapse density, GABAergic synaptic transmission, and maintenance of long-term potentiation in the hippocampal CA1 region. In addition, Iqsec3-cKO mice exhibited impaired fear memory formation. Strikingly, Iqsec3-cKO caused abnormally enhanced activation of ribosomal P70-S6K1-mediated signaling in the hippocampus but not in the cortex. Furthermore, inhibiting upregulated S6K1 signaling by expressing dominant-negative S6K1 in the hippocampal CA1 of Iqsec3-cKO mice completely rescued impaired fear learning and inhibitory synapse density but not deficits in long-term potentiation maintenance. Finally, upregulated S6K1 signaling was rescued by IQSEC3 wild-type, but not by an ARF-GEF (adenosine diphosphate ribosylation factor-guanine nucleotide exchange factor) inactive IQSEC3 mutant. CONCLUSIONS: Our results suggest that IQSEC3-mediated balanced synaptic inhibition in hippocampal CA1 is critical for the proper formation of hippocampus-dependent fear memory.