ABSTRACT
Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), has a central role in the coordination of receptor crosstalk and the integration of signaling pathways essential for neuronal differentiation, survival and function. This protein is a shared downstream effector for neurotrophin- and ephrin-receptors signaling that also interacts with the N-methyl-d-aspartate type of glutamate receptors (NMDARs). Failures in neurotrophic support and glutamate signaling are involved in pathologies related to excitotoxicity and/or neurodegeneration, where different components of these dynamic protein complexes result altered by a combination of mechanisms. In the case of Kidins220/ARMS, overactivation of NMDARs in excitotoxicity and cerebral ischemia triggers its downregulation, which contributes to neuronal death. This key role in neuronal life/death decisions encouraged us to investigate Kidins220/ARMS as a novel therapeutic target for neuroprotection. As the main mechanism of Kidins220/ARMS downregulation in excitotoxicity is proteolysis by calpain, we decided to develop cell-penetrating peptides (CPPs) that could result in neuroprotection by interference of this processing. To this aim, we first analyzed in detail Kidins220/ARMS cleavage produced in vitro and in vivo, identifying a major calpain processing site in its C-terminal region (between amino acids 1669 and 1670) within a sequence motif highly conserved in vertebrates. Then, we designed a 25-amino acids CPP (Tat-K) containing a short Kidins220/ARMS sequence enclosing the identified calpain site (amino acids 1668-1681) fused to the HIV-1 Tat protein basic domain, able to confer membrane permeability to attached cargoes. Transduction of cortical neurons with Tat-K reduced Kidins220/ARMS calpain processing in a dose- and time-dependent manner upon excitotoxic damage and allowed preservation of the activity of pERK1/2 and pCREB, signaling molecules central to neuronal survival and functioning. Importantly, these effects were associated to a significant increase in neuronal viability. This Kidins220/ARMS-derived peptide merits further research to develop novel neuroprotective therapies for excitotoxicity-associated pathologies.
Subject(s)
Calpain/metabolism , Cell Death , Cell-Penetrating Peptides/chemistry , Membrane Proteins/chemistry , Nerve Tissue Proteins/chemistry , Neurons/cytology , Neuroprotective Agents/chemistry , Animals , Binding Sites , Cell-Penetrating Peptides/pharmacology , Cells, Cultured , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP Response Element-Binding Protein/physiology , Down-Regulation , HEK293 Cells , Humans , MAP Kinase Signaling System , Male , Mice, Inbred BALB C , Neuroprotective Agents/pharmacology , Protein Engineering , Protein Structure, Tertiary , Receptors, N-Methyl-D-Aspartate/metabolism , Signal Transduction , Transduction, Genetic , tat Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
The Ts65Dn (TS) mouse is the most widely used model of Down syndrome (DS). This mouse shares many phenotypic characteristics with the human condition including cognitive and neuromorphological alterations. In this study the effects of physical exercise on hippocampal neurogenesis and behavior in TS mice were assessed. 10-12 month-old male TS and control (CO) mice were submitted to voluntary physical exercise for 7 weeks and the effects of this protocol on hippocampal morphology, neurogenesis and apoptosis were evaluated. Physical exercise improved performance in the acquisition sessions of the Morris water maze in TS but not in CO mice. Conversely, it did not have any effect on anxiety or depressive behavior in TS mice but it did reduce the cognitive components of anxiety in CO mice. TS mice presented a reduced dentate gyrus (DG) volume, subgranular zone area and number of granule neurons. Hippocampal neurogenesis was reduced in TS mice as shown by the reduced number of 5-bromo-2-deoxyuridine (BrdU) positive cells. Voluntary physical exercise did not rescue these alterations in TS mice but it did increase the number of doublecortin (DCX)-and phospho histone 3 (PH3)-positive neurons in CO mice. It is concluded that physical exercise produced a modest anxiolytic effect in CO mice and that this was accompanied by an increased number of immature cells in the hippocampal DG. On the other hand, voluntary physical exercise exerted a positive effect on TS mice learning of the platform position in the Morris water maze that seems to be mediated by a neurogenesis-independent mechanism.
