Mutations in the postsynaptic density signaling hub TNIK disrupt PSD signaling in human models of neurodevelopmental disorders.

A large number of synaptic proteins have been recurrently associated with complex brain disorders. One of these proteins, the Traf and Nck interacting kinase (TNIK), is a postsynaptic density (PSD) signaling hub, with many variants reported in neurodevelopmental disorder (NDD) and psychiatric disease. While rodent models of TNIK dysfunction have abnormal spontaneous synaptic activity and cognitive impairment, the role of mutations found in patients with TNIK protein deficiency and TNIK protein kinase activity during early stages of neuronal and synapse development has not been characterized. Here, using hiPSC-derived excitatory neurons, we show that TNIK mutations dysregulate neuronal activity in human immature synapses. Moreover, the lack of TNIK protein kinase activity impairs MAPK signaling and protein phosphorylation in structural components of the PSD. We show that the TNIK interactome is enriched in NDD risk factors and TNIK lack of function disrupts signaling networks and protein interactors associated with NDD that only partially overlap to mature mouse synapses, suggesting a differential role of TNIK in immature synapsis in NDD.

Spatiotemporal profile of postsynaptic interactomes integrates components of complex brain disorders.

The postsynaptic density (PSD) contains a collection of scaffold proteins used for assembling synaptic signaling complexes. However, it is not known how the core-scaffold machinery associates in protein-interaction networks or how proteins encoded by genes involved in complex brain disorders are distributed through spatiotemporal protein complexes. Here using immunopurification, proteomics and bioinformatics, we isolated 2,876 proteins across 41 in vivo interactomes and determined their protein domain composition, correlation to gene expression levels and developmental integration to the PSD. We defined clusters for enrichment of schizophrenia, autism spectrum disorders, developmental delay and intellectual disability risk factors at embryonic day 14 and adult PSD in mice. Mutations in highly connected nodes alter protein-protein interactions modulating macromolecular complexes enriched in disease risk candidates. These results were integrated into a software platform, Synaptic Protein/Pathways Resource (SyPPRes), enabling the prioritization of disease risk factors and their placement within synaptic protein interaction networks.

Long-term potentiation modulates synaptic phosphorylation networks and reshapes the structure of the postsynaptic interactome.

The postsynaptic site of neurons is composed of more than 1500 proteins arranged in protein-protein interaction complexes, the composition of which is modulated by protein phosphorylation through the actions of complex signaling networks. Components of these networks function as key regulators of synaptic plasticity, in particular hippocampal long-term potentiation (LTP). The postsynaptic density (PSD) is a complex multicomponent structure that includes receptors, enzymes, scaffold proteins, and structural proteins. We triggered LTP in the mouse hippocampus CA1 region and then performed large-scale analyses to identify phosphorylation-mediated events in the PSD and changes in the protein-protein interactome of the PSD that were associated with LTP induction. Our data indicated LTP-induced reorganization of the PSD. The dynamic reorganization of the PSD links glutamate receptor signaling to kinases (writers) and phosphatases (erasers), as well as the target proteins that are modulated by protein phosphorylation and the proteins that recognize the phosphorylation status of their binding partners (readers). Protein phosphorylation and protein interaction networks converged at highly connected nodes within the PSD network. Furthermore, the LTP-regulated phosphoproteins, which included the scaffold proteins Shank3, Syngap1, Dlgap1, and Dlg4, represented the "PSD risk" for schizophrenia and autism spectrum disorder, such that without these proteins in the analysis, the association with the PSD and these two psychiatric diseases was not present. These data are a rich resource for future studies of LTP and suggest that the PSD holds the keys to understanding the molecular events that contribute to complex neurological disorders that affect synaptic plasticity.

Engineering a therapeutic IgG molecule to address cysteinylation, aggregation and enhance thermal stability and expression.

Antibodies can undergo a variety of covalent and non-covalent degradation reactions that have adverse effects on efficacy, safety, manufacture and storage. We had identified an antibody to Angiopoietin 2 (Ang2 mAb) that neutralizes Ang2 binding to its receptor in vitro and inhibits tumor growth in vivo. Despite favorable pharmacological activity, the Ang2 mAb preparations were heterogeneous, aggregated rapidly and were poorly expressed. Here, we report the engineering of the antibody variable and constant domains to generate an antibody with reduced propensity to aggregate, enhanced homogeneity, 11°C elevated T(m), 26-fold improved level of expression and retained activity. The engineered molecule, MEDI-3617, is now compatible with the large scale material supply required for clinical trials and is currently being evaluated in Phase 1 in cancer patients. This is the first report to describe the stability engineering of a therapeutic antibody addressing non canonical cysteine residues and the design strategy reported here is generally applicable to other therapeutic antibodies and proteins.

Application of phage display to high throughput antibody generation and characterization.

We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation.

Retortamonas intestinalis: un parásito poco conocido / Retortamonas intestinalis: a little-known parasite

Se realizaron sesenta y cuatro estudios enteroparasitológicos. Las personas examinadas residían en el conurbano bonaerense, y contaban con condiciones sanitarias satisfactorias. Los síntomas que las había llevado a la consulta médica eran compatibles con parasitosis intestinales. En dos de ellas se hallaron Retortamonas intestinalis. Estos pacientes padecían alteraciones gastrointestinales leves que remitieron con el tratamiento. En los dos casos, R. intestinalis, fue el único parásito hallado. No se realizaron estudios complementarios para detectar bacterias enteropatógenas ni Rotavirus. Los trofozoitos y quistes, que se observaron en las preparaciones microscópicas, sólo fueron hallados por el método de Sedimentación-Centrifugación. Las mediciones micrométricas del parásito revelaron una discordancia importante con los valores hasta aquí informados. Se obtuvo una media, para el largo del trofozoito, de 10,6 µ (DE = 2,12; CV = 20 por ciento), y para el quiste, de 10,2 µ (DE = 1,66; CV = 16,2 por ciento). De las observaciones microscópicas realizadas se hicieron tomas fotográficas de los detalles morfológicos característicos del parásito; y también, dibujos de las imágenes proyectadas por cámara clara, con el propósito de recalcar las anomalías más frecuentes. En el plano teórico se discute acerca de las causas del porcentaje hallado, relacionándolo con el de otras encuestas; se plantea la relación de éste flagelo con la patología humana; y por último, se pone a consideración la posibilidad de confusión diagnóstica de R. intestinalis con otros flagelados