ABSTRACT
Neuronal synapses undergo structural and functional changes throughout life, which are essential for nervous system physiology. However, these changes may also perturb the excitatory-inhibitory neurotransmission balance and trigger neuropsychiatric and neurological disorders. Molecular tools to restore this balance are highly desirable. Here, we designed and characterized CPTX, a synthetic synaptic organizer combining structural elements from cerebellin-1 and neuronal pentraxin-1. CPTX can interact with presynaptic neurexins and postsynaptic AMPA-type ionotropic glutamate receptors and induced the formation of excitatory synapses both in vitro and in vivo. CPTX restored synaptic functions, motor coordination, spatial and contextual memories, and locomotion in mouse models for cerebellar ataxia, Alzheimer's disease, and spinal cord injury, respectively. Thus, CPTX represents a prototype for structure-guided biologics that can efficiently repair or remodel neuronal circuits.
Subject(s)
C-Reactive Protein/pharmacology , Nerve Tissue Proteins/pharmacology , Neural Pathways/drug effects , Protein Precursors/pharmacology , Receptors, AMPA/metabolism , Recombinant Proteins/pharmacology , Synapses/drug effects , Alzheimer Disease/therapy , Animals , C-Reactive Protein/chemistry , C-Reactive Protein/therapeutic use , Cerebellar Ataxia/therapy , Disease Models, Animal , HEK293 Cells , Hippocampus , Humans , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/therapeutic use , Protein Domains , Protein Precursors/chemistry , Protein Precursors/therapeutic use , Receptors, Glutamate/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/therapeutic use , Spine/drug effects , Spine/physiologyABSTRACT
The discovery of genetic variants influencing sleep patterns can shed light on the physiological processes underlying sleep. As part of a large clinical sequencing project, WGS500, we sequenced a family in which the two male children had severe developmental delay and a dramatically disturbed sleep-wake cycle, with very long wake and sleep durations, reaching up to 106-h awake and 48-h asleep. The most likely causal variant identified was a novel missense variant in the X-linked GRIA3 gene, which has been implicated in intellectual disability. GRIA3 encodes GluA3, a subunit of AMPA-type ionotropic glutamate receptors (AMPARs). The mutation (A653T) falls within the highly conserved transmembrane domain of the ion channel gate, immediately adjacent to the analogous residue in the Grid2 (glutamate receptor) gene, which is mutated in the mouse neurobehavioral mutant, Lurcher. In vitro, the GRIA3(A653T) mutation stabilizes the channel in a closed conformation, in contrast to Lurcher. We introduced the orthologous mutation into a mouse strain by CRISPR-Cas9 mutagenesis and found that hemizygous mutants displayed significant differences in the structure of their activity and sleep compared to wild-type littermates. Typically, mice are polyphasic, exhibiting multiple sleep bouts of sleep several minutes long within a 24-h period. The Gria3A653T mouse showed significantly fewer brief bouts of activity and sleep than the wild-types. Furthermore, Gria3A653T mice showed enhanced period lengthening under constant light compared to wild-type mice, suggesting an increased sensitivity to light. Our results suggest a role for GluA3 channel activity in the regulation of sleep behavior in both mice and humans.
Subject(s)
Intellectual Disability/genetics , Point Mutation , Receptors, AMPA/genetics , Receptors, AMPA/metabolism , Sleep Wake Disorders/genetics , Adult , Amino Acid Sequence , Animals , Base Sequence , Disease Models, Animal , Humans , Male , Mice , Mice, Inbred C57BLABSTRACT
Neuroligin-neurexin (NL-NRX) complexes are fundamental synaptic organizers in the central nervous system. An accurate spatial and temporal control of NL-NRX signaling is crucial to balance excitatory and inhibitory neurotransmission, and perturbations are linked with neurodevelopmental and psychiatric disorders. MDGA proteins bind NLs and control their function and interaction with NRXs via unknown mechanisms. Here, we report crystal structures of MDGA1, the NL1-MDGA1 complex, and a spliced NL1 isoform. Two large, multi-domain MDGA molecules fold into rigid triangular structures, cradling a dimeric NL to prevent NRX binding. Structural analyses guided the discovery of a broad, splicing-modulated interaction network between MDGA and NL family members and helped rationalize the impact of autism-linked mutations. We demonstrate that expression levels largely determine whether MDGAs act selectively or suppress the synapse organizing function of multiple NLs. These results illustrate a potentially brain-wide regulatory mechanism for NL-NRX signaling modulation.
Subject(s)
Dansyl Compounds/metabolism , Galactosamine/analogs & derivatives , Neurturin/metabolism , Signal Transduction/physiology , Synapses/physiology , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , COS Cells , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Adhesion Molecules, Neuronal/genetics , Cell Adhesion Molecules, Neuronal/metabolism , Chickens , Coculture Techniques , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Galactosamine/genetics , Galactosamine/metabolism , HEK293 Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Models, Molecular , Mutagenesis, Site-Directed , Mutation/genetics , Nerve Tissue Proteins/metabolism , Neurturin/genetics , Protein Interaction Maps , Receptors, N-Methyl-D-Aspartate/metabolism , Sequence AlignmentABSTRACT
Secretory and membrane-bound proteins are generally produced in lower amounts in insect cells compared with cytoplasmic and nuclear proteins. There may be many reasons for this, including degradation of recombinant proteins by proteases, competition for cellular resources between native and recombinant proteins, and physical blockage of the secretory pathways. In the present study, we describe the construction of a baculovirus in which chiA (chitinase) and cath (cathepsin) genes have been deleted and show improved recombinant protein expression using this vector. We confirmed the complete removal of both genes by PCR, restriction enzyme analysis and enzyme assays, and the modified virus DNA was shown to be stable in bacterial cells over multiple passages. A selection of recombinant genes were inserted into the double-deletion virus and their expression levels compared with recombinant viruses that had single or no gene deletions. In all instances, the double-deletion viruses showed greatly enhanced levels of protein production for both secreted and nuclear/cytoplasmic proteins. In summary, we have conclusively demonstrated the importance of this deletion vector for the high-level production of recombinant proteins.
