44 research outputs found
Blockade of the Interaction of Calcineurin with FOXO in Astrocytes Protects Against Amyloid-βInduced Neuronal Death
Astrocytes actively participate in neuro-inflammatory processes associated to Alzheimer’s disease (AD), and other brain pathologies. We recently showed that an astrocyte-specific intracellular signaling pathway involving an interaction of the phosphatase calcineurin with the transcription factor FOXO3 is a major driver in AD associated pathological inflammation, suggesting a potential new druggable target for this devastating disease. We have now developed decoy molecules to interfere with calcineurin/FOXO3 interactions, and tested them in astrocytes and neuronal co-cultures exposed to amyloid-β (Aβ) toxicity.
We observed that interference of calcineurin/FOXO3 interactions exerts a protective action against A-induced neuronal death and favors the production of a set of growth factors that we hypothesize form part of a cytoprotective pathway to resolve inflammation. Furthermore, interference of the A-induced interaction of calcineurin with FOXO3 by decoy compounds significantly decreased amyloid-β protein precursor (AβPP) synthesis, reduced the AβPP amyloidogenic pathway, resulting in lower Alevels, and blocked the expression of pro-inflammatory cytokines TNFα and IL-6 in astrocytes. Collectively, these data indicate that interrupting pro-inflammatory calcineurin/FOXO3 interactions in astrocytes triggered by Aβ accumulation in brain may constitute an effective new therapeutic approach in AD. Future studies with intranasal delivery, or brain barrier
permeable decoy compounds, are warranted
Amyloid-β reduces the expression of neuronal FAIM-L, thereby shifting the inflammatory response mediated by TNFα from neuronal protection to death
The brains of patients with Alzheimer's disease (AD) present elevated levels of tumor necrosis factor-α (TNFα), a cytokine that has a dual function in neuronal cells. On one hand, TNFα can activate neuronal apoptosis, and on the other hand, it can protect these cells against amyloid-β (Aβ) toxicity. Given the dual behavior of this molecule, there is some controversy regarding its contribution to the pathogenesis of AD. Here we examined the relevance of the long form of Fas apoptotic inhibitory molecule (FAIM) protein, FAIM-L, in regulating the dual function of TNFα. We detected that FAIM-L was reduced in the hippocampi of patients with AD. We also observed that the entorhinal and hippocampal cortex of a mouse model of AD (PS1M146LxAPP751sl) showed a reduction in this protein before the onset of neurodegeneration. Notably, cultured neurons treated with the cortical soluble fractions of these animals showed a decrease in endogenous FAIM-L, an effect that is mimicked by the treatment with Aβ-derived diffusible ligands (ADDLs). The reduction in the expression of FAIM-L is associated with the progression of the neurodegeneration by changing the inflammatory response mediated by TNFα in neurons. In this sense, we also demonstrate that the protection afforded by TNFα against Aβ toxicity ceases when endogenous FAIM-L is reduced by short hairpin RNA (shRNA) or by treatment with ADDLs. All together, these results support the notion that levels of FAIM-L contribute to determine the protective or deleterious effect of TNFα in neuronal cell
Molecular and pharmacological characterization of native cortical γ- aminobutyric acids receptors containing both α1 and α3 subunits
We have investigated the existence, molecular composition, and benzodiazepine binding properties of native cortical α1-α3 γ- aminobutyric acid(A) (GABA(A)) receptors using subunit-specific antibodies. The co-existence of α1 and α3 subunits in native GABA(A) receptors was demonstrated by immunoblot analysis of the anti-α1- or anti-α3- immunopurified receptors and by immunoprecipitation experiments of the [3H]zolpidem binding activity. Furthermore, immunodepletion experiments indicated that the α1-α3 GABA(A) receptors represented 54.7 ± 5.0 and 23.6 ± 3.3% of the α3 and α1 populations, respectively. Therefore, α1 and α3 subunits are associated in the same native GABA(A) receptor complex, but, on the other hand, these α1-α3 GABA(A) receptors from the cortex constitute a large proportion of the total α3 population and a relatively minor component of the α1 population. The pharmacological analysis of the α1- or α3-immunopurified receptors demonstrated the presence of two different benzodiazepine binding sites in each receptor population with high (type I binding sites) and low (type II binding sites) affinities for zolpidem and Cl 218,872. These results indicate the existence of native GABA(A) receptors possessing both α1 and α3 subunits, with α1 and α3 subunits expressing their characteristic benzodiazepine pharmacology. The molecular characterization of the anti-α1-anti-α3 double-Immunopurified receptors demonstrated the presence of stoichiometric amounts of α1 and α3 subunits, associated with α(2/3), and γ2 subunits. The pharmacological analysis of α1-α3 GABA(A) receptors demonstrated that, despite the fact that each α subunit retained its benzodiazepine binding properties, the relative proportion between type I and II binding sites or between 51- and 59-61-kDa [3H]Ro15-4513-photolabeled peptides was 70:30. Therefore, the α1 subunit is pharmacologically predominant over the α3 subunit. These results indicate the existence of active and nonactive α subunits in the native α1-α3 GABA(A) receptors from rat corte
GABAa and a-Amino-3-hydroxy-5-methylsoxazole-4-propionate receptors are differentially affected by aging in the rat hippocampus
We have investigated the age-dependent modifications in the expression of eight different subunits of the γ-aminobutyric acid, type A (GABAA) receptor (α1, α2, α3, α5, β2, β3, γ2S, and γ2L) and all four subunits of the α-amino-3-hydroxy-5-methylsoxazole-4-propionate (AMPA) receptor (GluR1–4) in the hippocampus of 24-month-old rats. All aged hippocampi displayed a remarkable increase (aged/adult ratio, 3.53 ± 0.54) in the mRNA levels of the short version of the γ2 subunit in parallel with a similar increase in the γ2 subunit protein (aged/adult ratio, 2.90 ± 0.62). However, this increase was not observed in the mature receptor. On the other hand, the expression of the different α subunit mRNAs increased moderately with aging, displaying a heterogeneous pattern. The most frequent modification consisted in an increase in the expression of the α1 subunit mRNA (aged/adult ratio, 1.26 ± 0.18), in parallel with a similar increase on the α1 protein (aged/adult ratio, 1.27 ± 0.12) and in the α1 incorporated to the assembled GABAA receptor (tested by immunoprecipitation; aged/adult ratio, = 1.20 ± 0.10). However, in the same hippocampal samples, no major modifications were observed on the expression of the AMPA receptor subunits. As a whole, these results indicated the existence of an increased expression of the GABAA receptor subunits and a preservation of the AMPA receptor at the hippocampal formation. These modifications could reflect the existence of specific deficiencies (neuronal loss and/or deafferentiation) on the GABAergic system in the aged rat
Glia and neurons from human iPSCs to address the pathology of Alzheimer´s disease
Alzheimer's disease (AD) is characterized by presenting a complex pathology, not fully resolved yet. This
fact, together with the lack of reliable models, has impeded the development of effective therapies.
Recently, several studies have shown that functional glial cell defects have a key role in the pathology of
AD. However, this glial dysfunction, currently, cannot be correctly modeled using the available animal
models, so we hypothesized that cells derived from Alzheimer's patients can serve as a better platform for
studying the disease. In this sense, human pluripotent stem cells (hPSC) allow the generation of different
types of neural cells, which can be used for disease modeling, identification of new targets and drugs
development.
Methods:
We have a collection of hiPSCs derived from patients with sporadic forms of AD stratified based on APOE
genotype. We have differentiated these cells towards neural cells and mature them to neurons or
astrocytes using a serum-free approach, to assess intrinsic differences between those derived from AD
patients or healthy controls.
Results:
We have implemented a serum-free approach and generated neural precursors and astrocytes from all the
lines tested. We observe differences at the phenotypic level and a reduced capacity to differentiate
towards neural lineage in those lines derived from APOE4 carriers.
Conclusions:
Our preliminary data suggest intrinsic differences in the neural differentiation capacity between cell lines
derived from APOE4 or APOE3 carrier subjects. Further experiments would contribute to elucidate novel
pathogenic pathways associated with neurodegeneration and susceptible of therapeutic modulation, likely
contributing to the development of new effective drugs against AD.This study was supported by Instituto de Salud Carlos III (ISCiii) of Spain, co-financed by FEDER funds from
European Union, through grants PI21/00915 (to AG) and PI21/00914 (to JV); by Junta de Andalucia through
Consejería de Economía y Conocimiento grants UMA20-FEDERJA-048 (to JAGL), PY18-RT-2233 (to AG) and
US-1262734 (to JV) co-financed by Programa Operativo FEDER 2014-2020, and Programa Operativo de
Empleo Juvenil SNGJ4-11 to LCP.
Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech
Neurons and astrocytes derived from human iPSCs to model Alzheimer´s disease
Alzheimer's disease (AD) is characterized by presenting a complex pathology, not fully resolved yet. This fact, together with the lack of reliable models, has impeded the development of effective therapies. Recently, several studies have shown that functional glial cell defects have a key role in the pathology of AD. However, this glial dysfunction, currently, cannot be correctly modeled using the available animal models, so we hypothesized that cells derived from Alzheimer's patients can serve as a better platform for studying the disease. In this sense, human pluripotent stem cells (hPSC) allow the generation of different types of neural cells, which can be used for disease modeling, identification of new targets and drugs development.
Methods: We have a collection of hiPSCs derived from patients with sporadic forms of AD stratified based on APOE genotype. We have differentiated these cells towards neural cells and mature them to neurons or astrocytes using a serum-free approach, to assess intrinsic differences between those derived from AD patients or healthy controls.
Results: We have implemented a serum-free approach and generated neural precursors and astrocytes from all the lines tested. We observe differences at the phenotypic level and a reduced capacity to differentiate towards neural lineage in those lines derived from APOE4 carriers.
Conclusions: Our preliminary data suggest intrinsic differences in the neural differentiation capacity between cell lines derived from APOE4 or APOE3 carrier subjects. Further experiments would contribute to elucidate novel pathogenic pathways associated with neurodegeneration and susceptible of therapeutic modulation, likely contributing to the development of new effective drugs against AD.This study was supported by Instituto de Salud Carlos III (ISCiii) of Spain, co-financed by FEDER funds from European Union, through grants PI21/00915 (to AG) and PI21/00914 (to JV); by Junta de Andalucia through Consejería de Economía y Conocimiento grants PY18-RT-2233 (to AG) and US-1262734 (to JV) co-financed by Programa Operativo FEDER 2014-2020, Consejeria de Salud grant PI-0276-2018 (to JAGL) and Programa Operativo de Empleo Juvenil SNGJ4-11 to LCP.
Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech
Co-cultures between neurons and astrocytes to address Alzheimer´s disease pathology.
Background: Alzheimer's disease (AD) is characterized by presenting a complex pathology, not fully resolved yet.
This fact, together with the lack of reliable models, has impeded the development of effective therapies. Recently,
several studies have shown that functional glial cell defects have a key role in the pathology of AD. However, this
glial dysfunction, currently, cannot be correctly modeled using the available animal models, so we hypothesized
that cells derived from Alzheimer's patients can serve as a better platform for studying the disease. In this sense,
human pluripotent stem cells (hPSC) allow the generation of different types of neural cells, which can be used for
disease modeling, identification of new targets and drugs development. Methods: We have a collection of hiPSCs
derived from patients with sporadic forms of AD stratified based on APOE genotype. We have differentiated these
cells towards neural cells and mature them to neurons or astrocytes using a serum-free approach, to assess
intrinsic differences between those derived from AD patients or healthy controls. Results: We have implemented a
serum-free approach and generated neural precursors and astrocytes from all the lines tested. We observe
differences at the phenotypic level and a reduced capacity to differentiate towards neural lineage in those lines
derived from APOE4 carriers. Conclusions: Our preliminary data suggest intrinsic differences in the neural
differentiation capacity between cell lines derived from APOE4 or APOE3 carrier subjects. Further experiments
would contribute to elucidate novel pathogenic pathways associated with neurodegeneration and susceptible of
therapeutic modulation, likely contributing to the development of new effective drugs against AD.This study was
supported by ISCiii (Spain), co-financed by FEDER funds, through grants PI21/00915 (AG) and PI21/00914 (JV);
by Junta de Andalucia through Consejería de Economía and Conocimiento grants UMA20-FEDERJA-048 (JAGL),
PY18-RT-2233 (to AG) and US-1262734 (JV), co-financed by Programa Operativo FEDER 2014-2020, and
SNGJ4-11 (LCP).
Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech
Disruption of Amyloid Plaques Integrity Affects the Soluble Oligomers Content from Alzheimer Disease Brains
The implication of soluble Abeta in the Alzheimer's disease (AD) pathology is currently accepted. In fact, the content of soluble extracellular Abeta species, such as monomeric and/or oligomeric Abeta, seems to correlate with the clinicopathological dysfunction observed in AD patients. However, the nature (monomeric, dimeric or other oligomers), the relative abundance, and the origin (extra-/intraneuronal or plaque-associated), of these soluble species are actually under debate. In this work we have characterized the soluble (defined as soluble in Trisbuffered saline after ultracentrifugation) Abeta, obtained from hippocampal samples of Braak II, Braak III-IV and Braak V-VI patients. Although the content of both Abeta40 and Abeta42 peptides displayed significant increase with pathology progression, our results demonstrated the presence of low, pg/mg protein, amount of both peptides. This low content could explain the absence (or below detection limits) of soluble Abeta peptides detected by western blots or by immunoprecipitation-western blot analysis. These data were in clear contrast to those published recently by different groups. Aiming to explain the reasons that determine these substantial differences, we also investigated whether the initial homogenization could mobilize Abeta from plaques, using 12-month-old PS1xAPP cortical samples. Our data demonstrated that manual homogenization (using Dounce) preserved the integrity of Abeta plaques whereas strong homogenization procedures (such as sonication) produced a vast redistribution of the Abeta species in all soluble and insoluble fractions. This artifact could explain the dissimilar and somehow controversial data between different groups analyzing human AD sample
Distinct Microglial Responses in Two Transgenic Murine Models of TAU Pathology
Microglial cells are crucial players in the pathological process of neurodegenerative
diseases, such as Alzheimer’s disease (AD). Microglial response in AD has been
principally studied in relation to amyloid-beta pathology but, comparatively, little is known
about inflammatory processes associated to tau pathology. In the hippocampus of
AD patients, where tau pathology is more prominent than amyloid-beta pathology,
a microglial degenerative process has been reported. In this work, we have directly
compared the microglial response in two different transgenic tau mouse models:
ThyTau22 and P301S. Surprisingly, these two models showed important differences
in the microglial profile and tau pathology. Where ThyTau22 hippocampus manifested
mild microglial activation, P301S mice exhibited a strong microglial response in parallel
with high phospho-tau accumulation. This differential phospho-tau expression could
account for the different microglial response in these two tau strains. However, soluble
(S1) fractions from ThyTau22 hippocampus presented relatively high content of soluble
phospho-tau (AT8-positive) and were highly toxic for microglial cells in vitro, whereas
the correspondent S1 fractions from P301S mice displayed low soluble phosphotau
levels and were not toxic for microglial cells. Therefore, not only the expression
levels but the aggregation of phospho-tau should differ between both models. In fact,
most of tau forms in the P301S mice were aggregated and, in consequence, forming
insoluble tau species.We conclude that different factors as tau mutations, accumulation,
phosphorylation, and/or aggregation could account for the distinct microglial responses
observed in these two tau models. For this reason, deciphering the molecular nature of
toxic tau species for microglial cells might be a promising therapeutic approach in order
to restore the deficient immunological protection observed in AD hippocampus.CIBERNEDJunta de Andalucía. Consejería de Economía, Innovación, Ciencia y Empleo CTS-2035Fundación Tatiana Pérez de Guzmán el BuenoMinisterio de Ciencia, Innovación y UniversidadesInstituto de Salud Carlos III. Fondo de Investigación Sanitaria. PI15/00957 PI15/00796Fondo Europeo de Desarrollo Regional PI15/00957 PI15/0079
Dysfunction of the unfolded protein response increases neurodegeneration in aged rat hippocampus following proteasome inhibition
Dysfunctions of the ubiquitin proteasome system (UPS) have been proposed to be involved in the aetiology and/or progression of several age-related neurodegenerative disorders. However, the mechanisms linking proteasome dysfunction to cell degeneration are poorly understood. We examined in young and aged rat hippocampus the activation of the unfolded protein response (UPR) under cellular stress induced by proteasome inhibition. Lactacystin injection blocked proteasome activity in young and aged animals in a similar extent and increased the amount of ubiquitinated proteins. Young animals activated the three UPR arms, IRE1α, ATF6α and PERK, whereas aged rats failed to induce the IRE1α and ATF6α pathways. In consequence, aged animals did not induce the expression of pro-survival factors (chaperones, Bcl-XL and Bcl-2), displayed a more sustained expression of proapoptotic markers (CHOP, Bax, Bak and JKN), an increased caspase-3 processing. At the cellular level, proteasome inhibition induced neuronal damage in young and aged animals as assayed using Fluorojade-B staining. However, degenerating neurons were evident as soon as 24 h postinjection in aged rats, but it was delayed up to 3 days in young animals. Our findings show evidence supporting age-related dysfunctions in the UPR activation as a potential mechanism linking protein accumulation to cell degeneration. An imbalance between pro-survival and pro-apoptotic proteins, because of noncanonical activation of the UPR in aged rats, would increase the susceptibility to cell degeneration. These findings add a new molecular vision that might be relevant in the aetiology of several age-related neurodegenerative disorders