Neurotransmitters and neurotrophins regulate nervous system development, and preservation and remodeling of adult neural circuits. Prominent roles are played by glutamate, major excitatory neurotransmitter, and brain-derived neurotrophic factor (BDNF). By activating their receptors and signaling pathways, these ligands regulate multiple neuronal processes, including survival. Physiological stimulation of N-methyl-d-aspartate type of glutamate receptors (NMDARs) induces neuronal survival through extracellular signal-regulated kinases (ERKs) activation, antioxidant defenses induction, and cAMP response element-binding protein (CREB) phosphorylation. In turn, CREB increases expression of BDNF and its receptor tropomyosin-related kinase B (TrkB). Neurotrophin receptors enhance neuronal survival via signaling cascades involving PI3K-Akt, ERK, CREB and nuclear factor kappa-B (NF-κB). Signaling requires large complexes formed at postsynaptic membranes by receptors and effectors. For instance, NMDAR association with ephrin receptor (Eph)B is critical for synaptic function, while EphB activation by ephrin-B modulates NMDAR-dependent calcium influx and receptor expression. Likewise, TrkB interacts with ephrin-A7 and EphA. In addition, Fyn tyrosine-kinase associates to TrkB and NMDAR-GluN2B, while neural Shc (N-Shc), a neurotrophin signaling adaptor, also regulates NMDAR function. Coordination of these receptors crosstalk requires their shared effector Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS). Kidins220 bears twelve ankyrin repeats, four trans-membrane (TM) segments forming a KAP-NTPase domain, a proline-rich region, a sterile alpha domain (SAM) and a PDZ ligand (PDZ-L) at the C-terminus. It interacts with Trk receptors and is obligatory for neurotrophin-sustained ERK activation and neuronal differentiation. In addition, Kidins220 associates with NMDARs and modulates their ERK signaling and neuronal survival.
Excitotoxicity is a specific neuronal-death process due to NMDAR overstimulation that contributes to neurodegeneration in acute disorders (ischemia, trauma and epilepsy) or chronic neurodegenerative diseases (Alzheimer’s, Parkinson’s or Huntington’s). Defective neurotrophic support and signaling are also involved in neurodegeneration. Altogether, these conditions cause high mortality and/or neurological impairment representing a social and health challenge. As there are no satisfactory treatments, it is capital to characterize excitotoxic mechanisms and how they affect proteins fundamental to survival/death choices in order to develop novel therapies. We discovered decreases in NMDAR and TrkB signaling during excitotoxicity due to activation of the Ca 2+-dependent protease calpain and transcriptional inhibition. Similar mechanisms control Kidins220 downregulation that contributes to neuronal death after NMDAR overactivation. Indeed, Kidins220 is crucial for neuronal survival as its knockdown decreases ERK activation and neuronal viability and enhances excitotoxic death. This key role in neuronal life/death decisions points to Kidins220 as a novel therapeutic target for neuroprotection. As calpain processing is the major mechanism of Kidins220 downregulation in excitotoxicity, we approach here the design of a neuroprotective peptide able to prevent Kidins220 calpain-dependent loss. First, we analyzed Kidins220 processing and identified a major calpain cleavage site within its C-terminus. We then designed a cell-penetrating peptide (CPP) containing the identified calpain site fused to a HIV-1 Tat protein basic domain, which confers membrane permeability and capability of crossing the blood–brain barrier (BBB). Transduction of this peptide reduced Kidins220 calpain processing upon excitotoxicity and significantly increased neuronal viability.
To develop a neuroprotective strategy based on prevention of Kidins220 calpain processing induced by excitotoxicity, we first characterized cleavage topology and kinetics. We treated mature cortical neurons of 14 days in vitro (DIVs) with high concentrations of NMDAR co-agonists, NMDA and glycine, and analyzed Kidins220 by immunoblot using antibodies recognizing a C-terminal peptide (Kid-Ct) or the N-terminal region (Kid-Nt).
