Desarrollo de péptidos neuroprotectores frente a la isquemia cerebral basados en el receptor de glutamato de tipo NMDA y su proteína interaccionante PSD-95

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 27-04-2017Esta tesis tiene embargado el acceso al texto completo hasta el 27-10-201

Péptido derivado de TrkB-FL y su uso como neuroprotector

[EN] The invention relates to a neuroprotective peptide characterised in that it consists of 15 amino acids of the human TrkB-FL sequence corresponding to positions 458 to 472 of the canonical sequence, and can exert a neuroprotective effect when the pro-survival response time is increased. The invention also relates to the use of said peptide for preventing and/or treating neuronal damage in mammals caused by a pathology of the central nervous system associated with excitotoxicity. The invention further relates to a pharmaceutical composition characterised in that it comprises said neuroprotective peptide, and to the use of same in the treatment of cerebrovascular diseases and other pathologies of the CNS.[ES] La presente invención hace referencia a un péptido neuroprotector, caracterizado por que consiste en15 aminoácidos de la secuencia de TrkB-FL humana correspondiente a las posiciones 458 hasta 472 de la secuencia canónica y que es capaz de ejercer un efecto neuroprotector al aumentar el tiempode respuesta pro-supervivencia. Asímismo, en la presente invención, se protege el uso de dicho péptido para prevenir y/o tratar el daño neuronal en mamíferos causado por una patología del sistema nervioso central asociada a excitotoxicidad. También se protege una composición farmaceútica caracterizada por comprender dicho péptido neuroprotector, así como el uso de los mismos en el tratamiento de enfermedades cerebrovasculares y otras patologías del SNC.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad Autónoma de MadridA1 Solicitud de patente con informe sobre el estado de la técnic

Development of a neuroprotective peptide that preserves survival pathways by preventing Kidins220/ARMS calpain processing induced by excitotoxicity

Kinase D-interacting substrate of 220kDa (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.This work was supported by the Ministerio de Economía y Competitividad (SAF2011-26233 and SAF2014-52737- P to TI, BFU2010-18380/BFI and BFU2013-43808- R to MD-G.); Comunidad de Madrid (P2010/BMD-2331-Neurodegmodels to TI) and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, Instituto de Salud Carlos III, to TI). CL-M was a recipient of a contract from SAF2011-26233; AG-M has been funded by contracts from P2010/BMD-2331, SAF2011-26233 and CIBERNED; SAD is a recipient of a FPI pre-doctoral fellowship associated to BFU2010-18380/BFI and GST has been funded by contracts from Consejo Superior de Investigaciones Cientıficas associated to projects BFU2010-18380/BFI and BFU2013-43808- R.Peer Reviewe

A novel cell-penetrating peptide targeting calpain-cleavage of PSD-95 induced by excitotoxicity improves neurological outcome after stroke

© The author(s).Postsynaptic density protein-95 (PSD-95) is a multidomain protein critical to the assembly of signaling complexes at excitatory synapses, required for neuronal survival and function. However, calpain-processing challenges PSD-95 function after overactivation of excitatory glutamate receptors (excitotoxicity) in stroke, a leading cause of death, disability and dementia in need of efficient pharmacological treatments. A promising strategy is neuroprotection of the infarct penumbra, a potentially recoverable area, by promotion of survival signaling. Interference of PSD-95 processing induced by excitotoxicity might thus be a therapeutic target for stroke and other excitotoxicity-associated pathologies. [Methods]: The nature and stability of PSD-95 calpain-fragments was analyzed using in vitro assays or excitotoxic conditions induced in rat primary neuronal cultures or a mouse model of stroke. We then sequenced PSD-95 cleavage-sites and rationally designed three cell-penetrating peptides (CPPs) containing these sequences. The peptides effects on PSD-95 stability and neuronal viability were investigated in the cultured neurons, subjected to acute or chronic excitotoxicity. We also analyzed the effect of one of these peptides in the mouse model of stroke by measuring infarct size and evaluating motor coordination and balance. [Results]: Calpain cleaves three interdomain linker regions in PSD-95 and produces stable fragments corresponding to previously described PSD-95 supramodules (PDZ1-2 and P-S-G) as well as a truncated form SH3-GK. Peptide TP95414, containing the cleavage site in the PDZ3-SH3 linker, is able to interfere PSD-95 downregulation and reduces neuronal death by excitotoxicity. Additionally, TP95414 is delivered to mice cortex and, in a severe model of permanent ischemia, significantly improves the neurological outcome after brain damage. [Conclusions]: Interference of excitotoxicity-induced PSD-95-processing with specific CPPs constitutes a novel and promising therapeutic approach for stroke treatment.We acknowledge funding from Ministerio de Economía y Competitividad (BFU2013-43808-R and BFU2016-75973-R) and from Agencia Estatal de Investigación (PID2019-105784RB-100/AEI/10.13039/501100011033). The cost of publication has been paid in part by FEDER funds. Contracts were funded associated to projects BFU2013-43808-R (G.M. E-O and S.A-D) and BFU2016-75973-R (G.M.E-O). We are grateful to Dr. Sobrado (HU La Princesa) and Drs. Avendaño and Negredo (Departamento de Anatomía, Histología y Neurociencia, UAM, Spain) for technical advice with the ischemia model, and Dr. Scheiffele for sharing plasmid pNice-PSD-95-YF

Brain ischaemia induces shedding of a BDNF-scavenger ectodomain from TrkB receptors by excitotoxicity activation of metalloproteinases and γ-secretases

Stroke remains a leading cause of death and disability in the world with limited therapies available to restrict brain damage or improve functional recovery after cerebral ischaemia. A promising strategy currently under investigation is the promotion of brain‐derived neurotrophic factor (BDNF) signalling through tropomyosin‐related kinase B (TrkB) receptors, a pathway essential for neuronal survival and function. However, TrkB and BDNF‐signalling are impaired by excitotoxicity, a primary pathological process in stroke also associated with neurodegenerative diseases. Pathological imbalance of TrkB isoforms is critical in neurodegeneration and is caused by calpain processing of BDNF high affinity full‐length receptor (TrkB‐FL) and an inversion of the transcriptional pattern of the Ntrk2 gene, to favour expression of the truncated isoform TrkB‐T1 over TrkB‐FL. We report here that both TrkB‐FL and neuronal TrkB‐T1 also undergo ectodomain shedding by metalloproteinases activated after ischaemic injury or excitotoxic damage of cortical neurons. Subsequently, the remaining membrane‐bound C‐terminal fragments (CTFs) are cleaved by γ‐secretases within the transmembrane region, releasing their intracellular domains (ICDs) into the cytosol. Therefore, we identify TrkB‐FL and TrkB‐T1 as new substrates of regulated intramembrane proteolysis (RIP), a mechanism that highly contributes to TrkB‐T1 regulation in ischaemia but is minor for TrkB‐FL which is mainly processed by calpain. However, since the secreted TrkB ectodomain acts as a BDNF scavenger and significantly alters BDNF/TrkB signalling, the mechanism of RIP could contribute to neuronal death in excitotoxicity. These results are highly relevant since they reveal new targets for the rational design of therapies to treat stroke and other pathologies with an excitotoxic component