Excitotoxicity induced by kainic acid provokes glycogen synthase kinase-3 truncation in the hippocampus

© 2015 Elsevier B.V. In neuronal cultures, glycogen synthase kinase 3(GSK3) is truncated at the N-terminal end by calpain downstream of activated glutamate receptors. However, the in vivo biological significance of that truncation has not been explored. In an attempt to elucidate if GSK3 truncation has a pathophysiological relevance, we have used intraperitoneal injections of kainic acid (KA) in rats and intra-amygdala KA microinjections in mice as in vivo models of excitotoxicity. Spectrin cleavage analyzed by immunohistochemistry was observed in the CA1 hippocampal field in KA-intraperitoneal treated rats while the CA3 region was the hippocampal area affected after intra-amygdala KA microinjections. GSK3β immunofluorescence did not colocalize with truncated spectrin in both treatments using an antibody that recognize the N-terminal end of GSK3β. Thus, those neurons which are spectrin-positive do not show GSK3β immunolabelling. To study GSK3β truncation in vitro, we exposed organotypic hippocampal slices and cultured cortical neurons to KA leading to the truncation of GSK3 and we found that truncation was blocked by the calpain inhibitor calpeptin. These data suggest a relationship between N-terminal GSK3β truncation and excitotoxicity. Overall, our data reinforces the important relationship between glutamate receptors and GSK3 and their role in neurodegenerative processes in which excitotoxicity is involved.Ministerio de Educa ción y Ciencia (SAF2010-15525 and BUF2013-40664-P) and Comunidad de Madrid (S2010/BMD-2331). We also acknowl- edge institutional support from Fundación Ramón ArecesPeer Reviewe

Uso de compuestos que se unen al dominio de unión a microtúbulos de tau en la elaboración de composiciones farmacéuticas, dichas composiciones farmacéuticas y su aplicación en el tratamiento de tauopatías

Uso de compuestos que se unen al dominio de unión a microtúbulos de tau en la elaboración de composiciones farmacéuticas, dichas composiciones farmacéuticas y su aplicación en el tratamiento de tauopatías. La presente invención se basa en el hecho que la heparina inhibe la toxicidad neuronal de la proteína tau. Así, la invención describe el uso de un compuesto que se une al dominio de unión a microtúbulos de la proteína tau, preferentemente heparina, en la elaboración de medicamentos; y una composición farmacéutica para la profilaxis y/o el tratamiento de tauopatías, preferentemente para el tratamiento de la enfermedad de Alzheimer en humanos.Consejo Superior de Investigaciones Científicas (España), Universidad Autónoma de MadridB1 Patente con informe sobre el estado de la ténic

CHOP regulates the p53-MDM2 axis and is required for neuronal survival after seizures

Hippocampal sclerosis is a frequent pathological finding in patients with temporal lobe epilepsy and can be caused by prolonged single or repeated brief seizures. Both DNA damage and endoplasmic reticulum stress have been implicated as underlying molecular mechanisms in seizure-induced brain injury. The CCAAT/enhancer-binding protein homologous protein (CHOP) is a transcriptional regulator induced downstream of DNA damage and endoplasmic reticulum stress, which can promote or inhibit apoptosis according to context. Recent work has proposed inhibition of CHOP as a suitable neuroprotective strategy. Here, we show that transcript and protein levels of CHOP increase in surviving subfields of the hippocampus after prolonged seizures (status epilepticus) in mouse models. CHOP was also elevated in the hippocampus from epileptic mice and patients with pharmacoresistant epilepsy. The hippocampus of CHOP-deficient mice was much more vulnerable to damage in mouse models of status epilepticus. Moreover, compared with wild-type animals, CHOP-deficient mice subject to status epilepticus developed more spontaneous seizures, displayed protracted hippocampal neurodegeneration and a deficit in a hippocampus-dependent object–place recognition task. The absence of CHOP was associated with a supra-maximal induction of p53 after status epilepticus, and inhibition of p53 abolished the cell death-promoting consequences of CHOP deficiency. The protective effect of CHOP could be partly explained by activating transcription of murine double minute 2 that targets p53 for degradation. These data demonstrate that CHOP is required for neuronal survival after seizures and caution against inhibition of CHOP as a neuroprotective strategy where excitotoxicity is an underlying pathomechanism.Health Research Board (PD/2009/31, HRA_POR/2011/41); Science Foundation Ireland (08/IN.1./B1875); Spanish Ministry of Science MEC/MICINN/MINECO; CiberNed; Fundación Ramón ArecesPeer Reviewe

Full reversal of Alzheimer's disease-like phenotype in a mouse model with conditional overexpression of glycogen synthase kinase-3

Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine kinase that is particularly abundant in the CNS. Dysregulation of GSK-3 activity is believed to play a key role in the pathogenesis of CNS chronic disorders such as Alzheimer's disease (AD), bipolar disorder, and Huntington's disease, and of metabolic disorders such as type II diabetes. Accordingly, GSK-3 inhibitors have been postulated as therapeutic tools for these diseases. Interestingly, pathophysiological and pharmacological regulation of GSK-3 is affected by an amplification mechanism that applies both to inhibition and activation. The possibility therefore exists that sustained inhibition or activation might persist after cessation of the initial trigger. Regarding AD, GSK-3 has been shown to accumulate in pretangle neurons. Furthermore, GSK-3 phosphorylates tau in most serine and threonine residues hyperphosphorylated in PHF (paired helical filament)-tau and GSK-3 activity contributes both to β-amyloid production and to β-amyloid-mediated neuronal death. In good agreement, mice with conditional overexpression of GSK-3 in forebrain neurons (Tet/GSK-3β mice) recapitulate aspects of AD neuropathology such as tau hyperphosphorylation, apoptotic neuronal death, and reactive astrocytosis as well as spatial learning deficit. Here, we exploit the conditional system used to generate Tet/GSK-3β mice to explore whether the biochemical, histopathological, and behavioral consequences of increased GSK-3 activity are susceptible to revert after restoration of normal GSK-3 levels. Here, we show that transgene shutdown in symptomatic mice leads to normal GSK-3 activity, normal phospho-tau levels, diminished neuronal death, and suppression of the cognitive deficit, thus further supporting the potential of GSK-3 inhibitors for AD therapeutics.This work was supported by grants from Comunidad de Madrid, Fundación “La Caixa,” and the Spanish Ministry of Science and by institutional grants from Fundación Ramón Areces and from Fondo de Investigaciones Sanitarias.Peer reviewe

Characterization of Alzheimer paired helical filaments by electron microscopy

We show how electron microscopy can be used to answer several critical issues in neurodegenerative disorders that course with the formation of aberrant filamentous structures. Thus, electron microscopy is a useful technique to study in vitro assembly of pathogenic proteins, to map the regions involved in filament formation, as well as to detect by immunoelectron microscopy which proteins bind to the filaments. Furthermore, electron microscopy is the main technique used to discover if an animal model develops fibrillar pathology and if those filaments are similar to those found in human patients. This review focuses on Alzheimer's disease and related tauopathies, although similar studies have been done with other neurodegenerative disorders as, for example, Huntington's disease.Contract grant sponsors: La Caixa, Neuropharma, Comunidad de Madrid, Spanish CICYT, Fundacion Ramon Areces.Peer reviewe

Chronic lithium administration to FTDP-17 tau and GSK-3β overexpressing mice prevents tau hyperphosphorylation and neurofibrillary tangle formation, but pre-formed neurofibrillary tangles do not revert

Glycogen synthase kinase-3 (GSK-3) has been proposed as the main kinase able to aberrantly phosphorylate tau in Alzheimer's disease (AD) and related tauopathies, raising the possibility of designing novel therapeutic interventions for AD based on GSK-3 inhibition. Lithium, a widely used drug for affective disorders, inhibits GSK-3 at therapeutically relevant concentrations. Therefore, it was of great interest to test the possible protective effects of lithium in an AD animal model based on GSK-3 overexpression. We had previously generated a double transgenic model, overexpressing GSK-3β in a conditional manner, using the Tet-off system and tau protein carrying a triple FTDP-17 (frontotemporal dementia and parkinsonism linked to chromosome 17) mutation. This transgenic line shows tau hyperphosphorylation in hippocampal neurones accompanied by neurofibrillary tangles (NFTs). We used this transgenic model to address two issues: first, whether chronic lithium treatment is able to prevent the formation of aberrant tau aggregates that result from the overexpression of FTDP-17 tau and GSK-3β; second, whether lithium is able to change back already formed NFTs in aged animals. Our data suggest that progression of the tauopathy can be prevented by administration of lithium when the first signs of neuropathology appear. Furthermore, it is still possible to partially reverse tau pathology in advanced stages of the disease, although NFT-like structures cannot be changed. The same results were obtained after shut-down of GSK-3β overexpression, supporting the possibility that GSK-3 inhibition is not sufficient to reverse NFT-like aggregates.This work was supported by grants from Fundación La Caixa, Comunidad de Madrid, Neuropharma, Spanish CICYT, and by institutional grants from Fundación Ramón Areces and from Fondo de Investigaciones Sanitarias.Peer reviewe

Tau phosphorylation in hippocampus results in toxic gain-of-function

The MAP (microtubule-associated protein) tau binds to tubulin, the main component of MTs (microtubules), which results in the stabilization of MT polymers. Tau binds to the C-terminal of tubulin, like other MAPs (including motor proteins such as kinesin) and it therefore may compete with these proteins for the same binding site in the tubulin molecule. In pathological conditions, tau is the main component of aberrant protein aggregates found in neurodegenerative disorders known as tauopathies where tau is present in its hyperphosphorylated form. GSK3 (glycogen synthase kinase 3, also known as tau kinase I) has been described as one of the main kinases involved in tau modifications. We have analysed the role of phospho-tau as a neurotoxic agent. We have analysed a transgenic mouse model which overexpresses GSK3beta. In this transgenic mouse, a clear degeneration of the dentate gyrus, which increases with age, was found. In a double transgenic mouse, which overexpresses GSK3 and tau at the same time, dentate gyrus degeneration was dramatically increased. This result may suggest that phospho-tau may be toxic inside neurons of the dentate gyrus. Once neuronal degeneration takes place, intracellular tau is secreted to the extracellular space. The present review discusses the toxicity of this extracellular tau for surrounding neurons.Peer reviewe

Haploinsufficient TNAP Mice Display Decreased Extracellular ATP Levels and Expression of Pannexin-1 Channels

Hypophosphatasia (HPP) is a rare heritable metabolic bone disease caused by hypomorphic mutations in the ALPL (in human) or Akp2 (in mouse) gene, encoding the tissue-nonspecific alkaline phosphatase (TNAP) enzyme. In addition to skeletal and dental malformations, severe forms of HPP are also characterized by the presence of spontaneous seizures. Initially, these seizures were attributed to an impairment of GABAergic neurotransmission caused by altered vitamin B6 metabolism. However, recent work by our group using knockout mice null for TNAP (TNAP-/-), a well-described model of infantile HPP, has revealed a deregulation of purinergic signaling contributing to the seizure phenotype. In the present study, we report that adult heterozygous (TNAP+/-) transgenic mice with decreased TNAP activity in the brain are more susceptible to adenosine 5′-triphosphate (ATP)-induced seizures. Interestingly, when we analyzed the extracellular levels of ATP in the cerebrospinal fluid, we found that TNAP+/- mice present lower levels than control mice. To elucidate the underlying mechanism, we evaluated the expression levels of other ectonucleotidases, as well as different proteins involved in ATP release, such as pannexin, connexins, and vesicular nucleotide transporter. Among these, Pannexin-1 (Panx1) was the only one showing diminished levels in the brains of TNAP+/- mice. Altogether, these findings suggest that a physiological regulation of extracellular ATP levels and Panx1 changes may compensate for the reduced TNAP activity in this model of HPP

Profiling of Argonaute-2-loaded microRNAs in a mouse model of frontotemporal dementia with parkinsonism-17

Tauopathies are a group of neurodegenerative diseases characterized by the pathological aggregation of the microtubule-associated protein tau. These include more than 20 diseases, with Alzheimer's disease being the most frequent. While pathological and neurotoxic effects of tau are well documented, the mechanisms by which tau can promote neurodegeneration are less clear. Increasing evidence suggests a functional role for microRNAs in the pathogenesis of tauopathies, with altered expression and function of microRNAs in experimental models and patient brain. To determine whether a pathological expression of tau leads to altered microRNA expression, we investigated a mouse model (VLW), which overexpresses tau carrying three mutations identified in patients suffering from frontotemporal dementia with parkinsonism-17. Argonaute-2-bound microRNAs were co-immunoprecipitated using hippocampal tissue to identify active microRNAs within the model and quantified using a genome-wide high-throughput qPCR-based microRNA platform. While similar numbers of microRNAs are present between wild-type and VLW mice, a prominent increase in Argonaute-2-bound levels of microRNAs could be observed in VLW mice. This included microRNA-134, microRNA-99a and microRNA-101. Subsequent experiments revealed this increase in Argonaute-2 loading of microRNAs to correlate with increased microRNA expression. Our in vivo study suggests that a pathological tau overexpression may lead to an increase in active microRNAs, possibly contributing to dysregulation of gene expression and tau-induced pathology.Science Foundation Ireland (12/COEN/18 to JHMP., DCH and TE, 13/IA/1891 to DCH; 13/SIRG/2114 to EMJM, 13/SIRG/2098 and 17/CDA/4708 to T.E and co-funded under the European Regional Development Fund and by FutureNeuro industry partners 16/RC/3948 to DCH) and the European Union’s ‘Seventh Framework’ Programme (FP7