A Neuroprotective Dose of Isatin Causes Multilevel Changes Involving the Brain Proteome: Prospects for Further Research

Abstract

Isatin (indole-2,3-dione) is an endogenous regulator, exhibiting a wide range of biological and pharmacological activities. At doses of 100 mg/kg and above, isatin is neuroprotective in different experimental models of neurodegeneration. Good evidence exists that its effects are realized via interaction with numerous isatin-binding proteins identified in the brain and peripheral tissues studied. In this study, we investigated the effect of a single dose administration of isatin to mice (100 mg/kg, 24 h) on differentially expressed proteins and a profile of the isatin-binding proteins in brain hemispheres. Isatin administration to mice caused downregulation of 31 proteins. However, these changes cannot be attributed to altered expression of corresponding genes. Although at this time point isatin influenced the expression of more than 850 genes in brain hemispheres (including 433 upregulated and 418 downregulated genes), none of them could account for the changes in the differentially expressed proteins. Comparative proteomic analysis of brain isatin-binding proteins of control and isatin-treated mice revealed representative groups of proteins sensitive to isatin administration. Control-specific proteins (n = 55) represent specific targets that interact directly with isatin. Appearance of brain isatin-binding proteins specific to isatin-treated mice (n = 94) may be attributed to the formation of new clusters of protein-protein interactions and/or novel binding sites induced by a high concentration of this regulator (ligand-induced binding sites). Thus, isatin administration produces multiple effects in the brain, which include changes in gene expression and also profiles of isatin-binding proteins and their interactomes. Further studies are needed for deeper insight into the mechanisms of the multilevel changes in the brain proteome induced by isatin. In the context of the neuroprotective action, these changes may be aimed at interruption of pathological links that begin to form after initiation of pathological processes.

Keywords: differentially expressed proteins; interactome; isatin-binding proteins; molecular targets; neuroprotector isatin; proteome analysis.

Figure 1

Functional annotation of transcriptome data in terms of Gene Ontology (GO) biological processes. Color intensity reflects the significance of enrichment: darker colors indicate higher statistical significance. The biological process in terms of GO supported the prevalence of downregulated transcripts related to the regulation and development of neurons including those guiding gliogenesis and myelination (A). The upregulated transcripts are related to processes involved in the response to DNA damage and consequent regulation of cell death as well as negative regulation of common cellular processes in the population of upregulated transcripts (B). Since each gene may be involved in several biological processes, the number of up- and downregulated transcripts does not match the sum of biological processes shown in the Figure. Other explanations are given in the text.

Figure 2

Cellular localization and functional links of brain isatin-binding proteins specific to isatin-treated mice. Details are given in the Materials and Methods Section.

Figure 3

Functional links between control-specific isatin-binding proteins of the mouse brain hemispheres. Line thickness and type (solid or dashed) reflect combined score and evidence suggesting a functional link. Bold lines indicate direct interactions between the identified proteins, annotated in String (v. 11.0 of January 19, 2019). Bold dashed lines show experimentally proven interactions between the identified proteins. Colors indicate different functional clusterization: red color shows lack of functional interactions, green color designates interaction between structural and regulatory proteins, blue color indicates interaction of proteins involved redox-dependent regulation.

Figure 4

Functional links between isatin-binding proteins specific to the brain hemispheres of isatin-treated mice. Line thickness and type (solid or dashed) reflect combined score and evidence suggesting a functional link. Bold lines indicate direct interactions between the identified proteins, annotated in String (v. 11.0 of January 19, 2019). Bold dashed lines show experimentally proven interactions between the identified proteins. Colors indicate different functional clusterization: red color shows intracellular molecular scaffolds with various intracellular proteins, aquamarine designates proteins involved in regulations of RNA splicing, green color shows enzymes, involved in metabolic processes, blue color shows proteins involved in regulation of neuron-specific proliferation.

Figure 5

The scheme illustrating the multilevel effects of the neuroprotective dose of isatin (100 mg/kg, 24 h). Isatin has a significant impact on the expression of responsive genes and thus influences the cell transcriptome. Acting on isatin-binding proteins, isatin changes protein–protein interactions (PPI), which are also influenced by the altered proteome, thus causing global changes in the PPI network.