Kidins220 correlates with tau in Alzheimer's disease brain and cerebrospinal fluid

Identification of neurodegeneration-monitoring biomarkers would be of great clinical value for Alzheimer's disease (AD) diagnosis. Using N- or C-terminal antibodies, we studied the pro-survival synaptic effector, Kidins220, in the brain and cerebrospinal fluid (CSF) of controls and AD patients. Only the N-terminal antibody showed a positive correlation between Kidins220 and phosphorylated tau in AD brains. Using this antibody, Kidins220 was detected in CSF from AD patients where it positively correlated with CSF phosphorylated tau and tau. This study highlights the potential of Kidins220 as a CSF biomarker in AD

Neuroprotective role of PKD1 against ischemic and kainic acid-induced brain injury

Trabajo presentado en el Second Spanish Molecular Imaging Network (SMIN) Meeting, celebrado en Madrid (España) el 26 de febrero de 2018

Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1

Excitotoxicity, a critical process in neurodegeneration, induces oxidative stress and neuronal death through mechanisms largely unknown. Since oxidative stress activates protein kinase D1 (PKD1) in tumor cells, we investigated the effect of excitotoxicity on neuronal PKD1 activity. Unexpectedly, we find that excitotoxicity provokes an early inactivation of PKD1 through a dephosphorylation-dependent mechanism mediated by protein phosphatase-1 (PP1) and dual specificity phosphatase-1 (DUSP1). This step turns off the IKK/NF-kappa B/SOD2 antioxidant pathway. Neuronal PKD1 inactivation by pharmacological inhibition or lentiviral silencing in vitro, or by genetic inactivation in neurons in vivo, strongly enhances excitotoxic neuronal death. In contrast, expression of an active dephosphorylation-resistant PKD1 mutant potentiates the IKK/NF-kappa B/SOD2 oxidative stress detoxification pathway and confers neuroprotection from in vitro and in vivo excitotoxicity. Our results indicate that PKD1 inactivation underlies excitotoxicity-induced neuronal death and suggest that PKD1 inactivation may be critical for the accumulation of oxidation-induced neuronal damage during aging and in neurodegenerative disorders

Effects of thioflavin T and GSK-3 inhibition on lifespan and motility in a Caenorhabditis elegans model of tauopathy

The nematode Caenorhabditis elegans (C. elegans) is a powerful model organism to study lifespan and aging, protein aggregation, and neurodegeneration, as well as to carry out drug screenings. The C. elegans strain aex-3/T337 expresses human pathogenic V337M mutant tau under a pan-neuronal promoter and presents uncoordinated locomotion, accumulation of phosphorylated insoluble tau, and shortened lifespan. Herein we have used this strain to assay two compounds that could affect tau aggregation and/or phosphorylation, and looked for phenotypic changes in their lifespan and motility. The first compound is Thioflavin T (ThT), a member of the tetracycline family with protein antiaggregant properties, yet to be tested in a tauopathy model. The second is a novel small molecule, NP103, a highly selective inhibitor of glycogen synthase kinase-3 (GSK-3), the main kinase contributing to pathogenic tau hyperphosphorylation. Importantly, we find that ThT extends lifespan of aex-3/T337 worms as it does with control N2 animals, showing both strains similar locomotion features under this treatment. By contrast, NP103 improves the paralysis phenotype of aex-3/T337 mutants but not their lifespan. Our results show that both treatments present beneficial effects for this model of tauopathy and encourage pursuing further investigations on their therapeutic potential for AD and other tauopathies.This work was supported by grants SAF2014-52737-P and SAF2017-88885-R to T.I., SAF2016-78603-R to M.M. from Ministerio de Ciencia, Innovación y Universidades (Spain). It was also funded by B2017/BMD-3700 (NEUROMETAB-CM) from Comunidad de Madrid to T.I., and Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED, Instituto de Salud Carlos III, Spain) to T.I. and M.M. A.G.M was funded by a contract from CIBERNED. The cost of this publication has been paid in part by FEDER (European Funds for Regional Development) funds.Peer reviewe

Analysis of Kidins220 levels in human AD samples and its correlation with biomarkers

Resumen del póster presentado al 3rd Symposium on Biomedical Research: "Advances and Perspectives in Neuroscience", celebrado en la Universidad Autónoma de Madrid el 22 de abril de 2016.Kidins220 (Kinase D interacting substrate) also known as ARMS (Ankyrin rich-repeat membrane spanning), is an integral membrane protein with unique features which interacts with Eph, neurotrophin and N-methyl-D-aspartate (NMDA) receptors. We previously demonstrated that downregulation of Kidins220 by excitotoxicity and cerebral ischemia contributed to neuronal death. As excitotoxicity has been proposed to be associated to Alzheimer’s disease (AD) and other neurodegenerative disorders, we expected to find a downregulation of Kidins220 in this disease. Contrary to our hypothesis, we observed an increase in Kidins220 levels in necropsies from AD patients where it accumulates with hyperphosphorylated tau. This increase correlated with higher Kidins220 resistance to calpain processing. We also observed that GSK3-β phosphorylation decreases Kidins220 susceptibility to calpain proteolysis, while protein phosphatase 1 (PP1) action has the opposite effect. In this study we have used a larger number of human brain necropsies from control individuals and AD patients in order to corroborate the increase of Kidins220 levels in this disease with two different antibodies recognizing this protein. Here we demonstrate that the analysis of this type of samples renders more accurate results when using the antibody against Kidins220 N-terminal region, as the C-terminal antibody gives higher variability in the results and lower significance. With this N-terminal antibody we have found a positive correlation between p-tau and Kidins220 levels in human brain necropsies. As many biochemical changes in AD occur in cerebrospinal fluid (CSF), we also analyzed Kidins220 by immunoblot in CSF samples from control individuals and AD patients. Some of these AD samples were positive for Kidins220 signal, further determining a correlation with parameters defined as AD biomarkers. Finally, we highlight the importance of the use of Kidins220 N-terminal antibody when analyzing this type of human samples, and the need to generate a monoclonal antibody recognizing the N-terminal region of this protein in order to develop an ELISA that could be used for AD diagnosis and prognosis studies.Peer reviewe

A novel neuroprotection target with distinct regulation in stroke and Alzheimer’s disease

Stroke and Alzheimer’s disease (AD) share a common mechanism of neuronal death known as excitotoxicity. Therapeutic approaches blocking excitotoxic responses, with agents, such as the NMDA receptor antagonist memantine, inhibitors of the protease calpain or GSK3 have been tested in poststroke and AD treatments. Novel therapies for neuroprotection are based on the use of cell-penetrating peptides (CPPs) targeting molecules and signaling pathways involved in neurodegeneration. The study of the molecular mechanisms that regulate the levels of the prosurvival protein Kidins220/ARMS in acute versus chronic AD-related neurodegeneration has established a clear parallelism with those controlling tau proteostasis that involve changes in GSK3 phosphorylation and calpain processing. The development of a neuroprotective CPP derived from this molecule, effective against acute excitotoxicity provide new evidence to support the use of GSK3 inhibitors in combination with specific CPPs as a novel strategy to reduce neuronal damage in AD.Peer reviewe

Kidins220 correlates with tau in Alzheimer's disease brain and cerebrospinal fluid

Identification of neurodegeneration-monitoring biomarkers would be of great clinical value for Alzheimer's disease (AD) diagnosis. Using N- or C-terminal antibodies, we studied the pro-survival synaptic effector, Kidins220, in the brain and cerebrospinal fluid (CSF) of controls and AD patients. Only the N-terminal antibody showed a positive correlation between Kidins220 and phosphorylated tau in AD brains. Using this antibody, Kidins220 was detected in CSF from AD patients where it positively correlated with CSF phosphorylated tau and tau. This study highlights the potential of Kidins220 as a CSF biomarker in AD.T.I. is funded by SAF2014-52737-P (Ministerio de Economía y Competitividad, Spain), P2010/BMD-2331-Neurodegmodels (Comunidad de Madrid, Madrid, Spain); A.L. is funded by PI11/3035 and PI14/1561 provided by FEDER (European Funds for Regional Development) and Instituto de Salud Carlos III (Ministerio de Economía y Competitividad, Spain). T.I. and A.L. are also funded by Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) and CIBERNED cooperarive project 2013/07 (Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain). A.G.M is funded by a contract from CIBERNED-2013/07; O.B. is funded by the Miguel Servet Associate Investigator Project Grant (CP13/0091) and FIS (PI15/00058) provided by FEDER and Instituto de Salud Carlos III (Ministerio de Economía y Competitividad, Spain). The cost of this publication has been paid in part by FEDER funds.Peer Reviewe

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

PKD1 as a possible therapeutic target in neurodegenerative diseases

Resumen del póster presentado al 3rd Symposium on Biomedical Research: "Advances and Perspectives in Neuroscience", celebrado en la Universidad Autónoma de Madrid el 22 de abril de 2016.-- et al.Recent studies implicate excitotoxicity in a variety of neuropathological conditions, suggesting that neurodegenerative diseases with distinct genetic etiologies may share excitotoxicity as a common pathogenic pathway. Excessive activation of glutamate receptors, both NMDA and AMPA/KA subtypes leads to a number of deleterious consequences, including impairment of calcium buffering, generation of free radicals, activation of the mitochondrial permeability transition and resulting in neuronal apoptosis and necrosis. PKD1 is a serine-threonine protein kinase that is involved in multiple biological processes. Because PKD1 has been implicated in ROS-detoxification and survival in cancer cells we hypothesized that this isoform could confer neuroprotection under excitotoxic conditions. We first investigated the possible regulation of PKD in response to overstimulation of NMDA and AMPA/KA receptors. We found a fast PKC- and Srcdependent activation of the kinase followed by a PP1-dependent inactivation during excitotoxicity. We also found that pharmacological inhibition or silencing of PKD1 decreased neuronal survival, supporting the fact that PKD inactivation is linked to neuronal death. To analyse the possible neuroprotective effects of PKD1 we generated a constitutively active mutant and cloned it in a lentiviral vector under the control of the synapsin promoter for its neurospecific expression. Transduction with this virus strongly protected neurons from excitotoxic insults by molecular mechanisms that involve potentiation of IKK/NFkB-pathway that in turn increased MnSOD levels and decreased ROS production. Together, our results demonstrate that PKD1 signaling plays a survival and detoxifications role in cortical neurons and therefore, selective and neurospecific PKD1 activators could constitute potent tools to induce neuroprotection in neurodegenerative diseases.Peer Reviewe

Kidins220 correlates with tau in Alzheimer's disease brain and cerebrospinal fluid

Identification of neurodegeneration-monitoring biomarkers would be of great clinical value for Alzheimer's disease (AD) diagnosis. Using N- or C-terminal antibodies, we studied the pro-survival synaptic effector, Kidins220, in the brain and cerebrospinal fluid (CSF) of controls and AD patients. Only the N-terminal antibody showed a positive correlation between Kidins220 and phosphorylated tau in AD brains. Using this antibody, Kidins220 was detected in CSF from AD patients where it positively correlated with CSF phosphorylated tau and tau. This study highlights the potential of Kidins220 as a CSF biomarker in AD