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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 promisin

[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.

[EN] The present invention relates to a neuroprotective peptide, which is characterized in that it comprises the 11 C-terminal amino acids of TrkB-T1 (residues 466-476) (SEQ ID No. 1) and is capable of impeding and/or inhibiting binding between TrkB-T1 and the molecules that interact therewith in a neurone and/or glial cell. The present invention also protects the use of said neuroprotective peptide for preventing and/or treating neuronal damage in mammals caused by a cerebrovascular disease or cerebral ischemia and/or by another pathological condition of the CNS associated with excitotoxicity, and/or traumatic cerebral and/or medullary damage. Also protected is a pharmaceutical composition or drug, which is characterized in that it comprises said neuroprotective peptide, and also the use thereof in the treatment of cerebrovascular diseases and other pathological conditions of the CNS. Lastly, the method for obtaining said neuroprotective peptide is protected. (FR): La présente invention concerne un peptide neuroprotecteur caractérisé en ce qu'il contient les 11 acides aminés C-terminaux de TrkB-T1 (résidus 466-476) (SEQ ID NO: 1), et en ce qu'il est capable d'interférer avec la liaison entre TrkB-T1 et ses molécules d'interaction dans un neurone et/ou une cellule gliale et/ou d'inhiber ladite liaison. La présente invention s'étend en outre à l'utilisation dudit peptide neuroprotecteur pour la prévention et/ou le traitement des lésions neuronales causées par une maladie cérébrovasculaire ou une ischémie cérébrale et/ou par une autre pathologie du SNC associée à une excitotoxicité et/ou par une lésion traumatique cérébrale et/ou médullaire, chez les mammifères. L'invention concerne également une composition pharmaceutique ou un médicament ayant pour caractéristique de contenir ledit peptide neuroprotecteur, ainsi que leur utilisation pour le traitement de maladies cérébrovasculaires et d'autres pathologies du SNC. Finalement, l'invention couvre le procédé d'obtention, [ES] La presente invención hace referencia a un péptido neuroprotector, caracterizado por comprender los 11 aminoácidos C-terminalesde TrkB-T1 (residuos 466-476) (SEQ ID NO: 1) y ser capaz de interferir y/o inhibir la unión entre TrkB-T1 y sus moléculas interaccionantes en una neurona y/o célula glial. Así mismo, en la presente invención se protege el uso de dicho péptido neuroprotector para prevenir y/o tratar el daño neuronal en mamíferos causado por una enfermedad cerebrovascular o isquemia cerebral y/o por otra patología del SNC asociada a excitotoxicidad, y/o daño traumático cerebral y/o medular. También se protege una composición farmacéutica o medicamento caracterizado por comprender dicho péptido neuroprotector, así como el uso de los mismos en el tratamiento de enfermedades cerebrovasculares y otras patologías del SNC. Finalmente, se protege el método de obtención de dicho péptido neuroprotector.

La presente invención hace referencia a un péptido neuroprotector, caracterizado por comprender los 11 aminoácidos C-terminales de TrkB-T1 (residuos 466- 476) (SEQ ID NO: 1) y ser capaz de interferir y/o inhibir la unión entre TrkB-T1 y sus moléculas interaccionantes en una neurona y/o célula glial. Así mismo, en la presente invención se protege el uso de dicho péptido neuroprotector para prevenir y/o tratar el daño neuronal en mamíferos causado por una enfermedad cerebrovascular o isquemia cerebral y/o por otra patología del SNC asociada a excitotoxicidad, y/o daño traumático cerebral y/o medular. También se protege una composición farmacéutica o medicamento caracterizado por comprender dicho péptido neuroprotector, así como el uso de los mismos en el tratamiento de enfermedades cerebrovasculares y otras patologías del SNC. Finalmente, se protege el método de obtención de dicho péptido neuroprotector

La presente invención hace referencia a un péptido neuroprotector, caracterizado por comprender los 11 aminoácidos C-terminales de TrkB-T1 (residuos 466- 476) (SEQ ID NO: 1) y ser capaz de interferir y/o inhibir la unión entre TrkB-T1 y sus moléculas interaccionantes en una neurona y/o célula glial. Así mismo, en la presente invención se protege el uso de dicho péptido neuroprotector para prevenir y/o tratar el daño neuronal en mamíferos causado por una enfermedad cerebrovascular o isquemia cerebral y/o por otra patología del SNC asociada a excitotoxicidad, y/o daño traumático cerebral y/o medular. También se protege una composición farmacéutica o medicamento caracterizado por comprender dicho péptido neuroprotector, así como el uso de los mismos en el tratamiento de enfermedades cerebrovasculares y otras patologías del SNC. Finalmente, se protege el método de obtención de dicho péptido neuroprotector

A better understanding of the mechanisms underlying neuronal death in cerebral ischemia is required for the development of stroke therapies. Here we analyze the contribution of the tropomyosin-related kinase B (TrkB) neurotrophin receptor to excitotoxicity, a primary pathological mechanism in ischemia, which is induced by overstimulation of glutamate receptors of the N-methyl-D-aspartate type. We demonstrate a significant modification of TrkB expression that is strongly associated with neurodegeneration in models of ischemia and in vitro excitotoxicity. Two mechanisms cooperate for TrkB dysregulation: (1) calpain-processing of full-length TrkB (TrkB-FL), high-affinity receptor for brain-derived neurotrophic factor, which produces a truncated protein lacking the tyrosine-kinase domain and strikingly similar to the inactive TrkB-T1 isoform and (2) reverse regulation of the mRNA of these isoforms. Collectively, excitotoxicity results in a decrease of TrkB-FL, the production of truncated TrkB-FL and the upregulation of TrkB-T1. A similar neuro-specific increase of the TrkB-T1 isoform is also observed in stroke patients. A lentivirus designed for both neuro-specific TrkB-T1 interference and increased TrkB-FL expression allows recovery of the TrkB-FL/TrkB-T1 balance and protects neurons from excitotoxic death. These data implicate a combination of TrkB-FL downregulation and TrkB-T1 upregulation as significant causes of neuronal death in excitotoxicity, and reveal novel targets for the design of stroke therapies.

Functional and protein interactions between the N-methyl-D-aspartate type of glutamate receptor (NMDAR) and neurotrophin or ephrin receptors play essential roles in neuronal survival and differentiation. A shared downstream effector for neurotrophin- and ephrin-receptor signaling is kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS). Because this molecule is obligatory for neurotrophin-induced differentiation, we investigated whether Kidins220/ARMS and NMDAR functions were related. Here, we identify an association between these proteins and discover that excitotoxicity, a specific form of neuronal death induced by NMDAR overstimulation, dramatically decreases Kidins220/ARMS levels in cortical neurons and in a model of cerebral ischemia. Kidins220/ARMS downregulation is triggered by overactivation of NMDARs containing NR2B subunits and subsequent Ca2+ influx, and involves a dual mechanism: rapid cleavage by the Ca2+-dependent protease calpain and calpain-independent silencing of Kidins220/Arms gene transcription. Additionally, Kidins220/ARMS knockdown decreases ERK activation and basal neuronal viability, and enhances neuronal death under excitotoxic conditions. Our results demonstrate Kidins220/ARMS participation in neuronal life and death pathways, and constitute the first report of its regulation under pathological conditions.