Aging and amyloid β oligomers enhance TLR4 expression, LPS-induced Ca2+ responses, and neuron cell death in cultured rat hippocampal neurons
© The Author(s). 2017
Received: 6 August 2016
Accepted: 23 January 2017
Published: 31 January 2017
Toll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system recognizing diverse pathogen-derived and tissue damage-related ligands. It has been suggested that TLR signaling contributes to the pathogenesis of age-related, neurodegenerative diseases, including Alzheimer’s disease (AD). AD is associated to oligomers of the amyloid β peptide (Aβo) that cause intracellular Ca2+ dishomeostasis and neuron cell death in rat hippocampal neurons. Here we assessed the interplay between inflammation and Aβo in long-term cultures of rat hippocampal neurons, an in vitro model of neuron aging and/or senescence.
Ca2+ imaging and immunofluorescence against annexin V and TLR4 were applied in short- and long-term cultures of rat hippocampal neurons to test the effects of TLR4-agonist LPS and Aβo on cytosolic [Ca2+] and on apoptosis as well as on expression of TLR4.
LPS increases cytosolic [Ca2+] and promotes apoptosis in rat hippocampal neurons in long-term culture considered aged and/or senescent neurons, but not in short-term cultured neurons considered young neurons. TLR4 antagonist CAY10614 prevents both effects. TLR4 expression in rat hippocampal neurons is significantly larger in aged hippocampal cultures. Treatment of aged hippocampal cultures with Aβo increases TLR4 expression and enhances LPS-induced Ca2+ responses and neuron cell death.
Aging and amyloid β oligomers, the neurotoxin involved in Alzheimer’s disease, enhance TLR4 expression as well as LPS-induced Ca2+ responses and neuron cell death in rat hippocampal neurons aged in vitro.
KeywordsAging TLR4 Alzheimer’s disease Hippocampal neurons Calcium Amyloid β oligomers
Toll-like receptors (TLRs) are a family of innate immune system receptors involved in sensing and response to pathogen-associated molecular patterns (PAMPs) and endogenous ligands known as damage-associated molecular patterns (DAMPs) that are released upon cell damage and necrosis . Toll proteins were discovered in Drosophila melanogaster . Later, a mammalian homologue for Toll was found, and TLR4, the receptor for lipopolysaccharide (LPS) present in Gram-negative bacteria, was identified [3, 4]. TLRs are widely expressed in a diversity of mammalian immune and non-immune cells, and they are present in the brain, where their expression is not restricted to microglia  but expands to astrocytes , oligodendrocytes , and neurons . The functional implication of TLR expression in neurons is not well understood yet. It has been proposed that TLR4 may contribute to neural plasticity and development in neurons . In addition, recent studies indicate that TLR4 expression is upregulated with normal aging , suggesting an altered regulation of the innate immune response in aging that may be relevant in different neurodegenerative disorders such as Alzheimer’s disease (AD). AD, the most common form of dementia, is strongly associated to aging and is characterized by the gradual loss of memory and cognitive function. The causes for AD are still not well understood. However, it is well known that AD is associated to formation of amyloid plaques made up of amyloid β peptide (Aβ), mainly Aβ1–42, derived from the altered metabolism of the amyloid precursor protein after being processed by β- and γ-secretases. AD is also linked to intracellular neurofibrillary tangles, composed of abnormally hyper phosphorylated tau protein .
Interestingly, McGeer and McGeer proposed in the late 1980s that innate immunity had an important role in neurodegenerative diseases (revised in [11, 12]). Although this theory was not well accepted initially, consensus is growing about the involvement of an inflammatory component in AD. Microglia is responsible for immunity in the brain and becomes activated by signals released by surrounding cells. In the AD brain, the sites of neuroinflammation are surrounding senile plaques, which show increased levels of pro-inflammatory factors, such as pro-inflammatory cytokines, complement components, and proteases [12, 13]. Recently, the tlr4 gene has emerged as a candidate susceptibility gene for AD. For example, a genetic study proposed that a polymorphism in TLR4 (Asp299Gly) may decrease the risk of AD independently of a polymorphism in apolipoprotein E, suggesting the involvement of the innate immunity in neurodegeneration in general, and of TLR4 in AD, in particular [14, 15]. Consistently, AD brains show increased expression of TLR4 . Furthermore, this receptor plays an important role in microglial neurotoxicity, since LPS binding induces its activation, thus releasing toxic substances to neurons . Consequently, instead of counteracting the damage caused by pathogens, TLR4 activation may lead to increased damage due to the release of toxic factors such as nitric oxide and oxygen free radicals . In this manner, it appears that Aβ may sensitize microglia to stimulation by some TLR ligands like LPS  since co-administration of Aβ and LPS increases activation of TLR4, leading to increased release of nitric oxide and tumor necrosis factor α . However, the possible interactions of LPS and Aβ on hippocampal neurons have not been assessed yet. In this work, we aimed at investigating the interplay between neuroinflammation and AD in the context of aging.
To accomplish the above goal, we have employed here aged cultures of rat hippocampal neurons that are considered a model of aging and/or senescence, since some of the changes occurring in the elderly in vivo are mimicked in neurons aged in vitro [20, 21]. Our results show that rat hippocampal neurons express TLR4 and expression increases with time in culture consistently with in vivo aging. We also found that LPS increases cytosolic [Ca2+] and promotes neuron cell death only in aged cultures. Treatment with AD-related oligomers of the amyloid β peptide (Aβo) further enhanced TLR4 expression, Ca2+ responses induced by LPS and neuron cell death, suggesting the interplay between TLR4 and Aβo in neuron cell death associated to aging and AD.
Animals and reagents
Wistar rat pups were obtained from the Valladolid University animal facility. Fura2/AM was from Invitrogen (Carlsbad, CA). Fetal bovine serum was from Lonza (Basel, Switzerland). Neurobasal medium, Hank’s balanced salt solution, minimal essential medium, B27, l-glutamine, and gentamicin were from Gibco (Carlsbad, CA). Papain solution was from Worthington (Lakewood, NJ). Aβ1–42 peptides were purchased from Bachem AG (Bubendorf, Switzerland). TLR4 (H80) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against β III tubulin is from Covance (Princeton, NJ). Antibodies against NR1 (AB9864), NR2A (AB1555P), and NR2B (AB1557P) are from Chemicon International (Millerica, MA). CAY10614 was from Cayman Chem. (Ann Arbor, MI). MK801 is from Sigma (St. Louis, MO). Poly-d-lysine and Annexin V were from BD (Franklin Lakes, NJ). DNase I and LPS from Escherichia coli O111B4 were from Sigma (St. Louis, MO). LPS, solved in ultrapure water, was boiled for 1 h to avoid protein contaminants, as reported previously . Other reagents and chemicals were obtained either from Sigma or Merck.
Primary hippocampal neuron culture
Hippocampal neurons were prepared from P0 Wistar rat pups under sterile conditions as reported by Brewer et al.  with further modifications . Briefly, rat pups were sacrificed and, after brain removal, meninges were discarded and hippocampi were separated from the cortex. Hippocampal tissue was cut in small pieces, transferred to papain solution (20 u/ml), and incubated at 37 °C for 30 min with occasional gentle shaking. After 15 min, DNase I (50 μg/ml final concentration) was added. Tissue pieces were washed with Neurobasal medium and cell suspension was obtained using a fire-polished pipette in Neurobasal supplemented with 10% fetal bovine serum. Cells were centrifuged at 160 g for 5 min and pellet was suspended in Neurobasal medium. Hippocampal cells were plated onto poly-d-lysine-coated, 12-mm diameter glass coverslips at 30 × 103 cells/dish, cultured in the same medium supplemented with 2 mM l-glutamine, 1 μg/ml gentamicin, 2% B27, and 10% FBS, and maintained in a humidified 37 °C incubator with 5% CO2. Cells were either cultured for 4–8 days in vitro (DIV) for short-term cultures (young neurons), 9–10 DIV for mature neurons, or >18 DIV for long-term cultures (aged and/or senescent neurons) as in previous studies [25, 26].
Hippocampal neurons at different DIV were treated with or without 1 μg/ml LPS, 0.25 μM CAY10614, or 1 μM Aβo, and incubated for 48 h at 37 °C/5% CO2. Then, cells were washed with phosphate buffered saline once and apoptosis was evaluated using Annexin V (1:20, 10 min) in annexing binding buffer (140 mM NaCl; 2.5 mM CaCl2; 10 mM Hepes pH 7.4) and assessed by fluorescence microscopy using a Nikon Eclipse TS100 microscope (objective ×40).
Hippocampal cells at different days in vitro (DIV) (4–8, 9–10, and >18 DIV) were fixed with 4% p-formaldehyde and incubated with antibodies against TLR4 (1:200) or antibodies against NR1, NR2A, and NR2B (1:100). Immunopositive cells were revealed using an Alexa Fluor 488-tagged antibody (1:300). Optical density was measured in individual neurons using the ImageJ software (National Institute of Mental Health, Bethesda, MA, USA) as reported previously . In some experiments, cells were co-stained with anti-β-tubulin III (1:400) for detection of TLR4 in identified neurons and incubated with 1:100 labeled anti-IgG antibodies as reported previously .
Preparation of Aβ1–42 oligomers (Aβo)
Aβo were prepared as reported previously . Briefly, Aβ1–42 was initially solved to 1 mM in cold hexafluoroisopropanol and separated into aliquots in sterile microcentrifuge tubes. Hexafluoroisopropanol was removed under vacuum in a Speed Vac concentrator, and the peptide film was stored desiccated at −20 °C. For aggregation, the peptide was first suspended in dry dimethyl sulfoxide to a concentration of 5 mM. For complete resuspension of the peptide, it was subjected to a 10 min of ultrasound, aliquoted in propylene non-siliconized vials, and stored at −20 °C. Minimal essential medium supplemented with 0.5 mg/ml Fe2+, 0.5 mg/ml Cu2+, and 0.5 mg/ml Zn2+ was added to bring the peptide to a concentration of 80 μM, and it was incubated at 37 °C for 24 h. For experiments, Aβo were solved in MEM at a final concentration of 1 μM. Oligomer formation was tested using western blotting as previously reported .
Fluorescence imaging of cytosolic [Ca2+]
Cells were incubated in standard external medium (SEM) containing 145 mM NaCl, KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM Hepes 10, pH 7.42), and later loaded with the fura2/AM dye (4 μM) for 60 min at room temperature. Then, coverslips were placed on the perfusion chamber of a Zeiss Axiovert 100 TV, perfused continuously with the same standard medium pre-warmed at 37 °C and epi-illuminated alternately at 340 and 380 nm. Light emitted at 520 nm was recorded every 5 s with a Hamamatsu ER camera (Hamamatsu Photonics France). Perfusion was stopped before adding vehicle or LPS during times indicated in the figures. Pixel by pixel ratios of consecutive frames were captured, and [Ca2+]cyt of regions of interest corresponding to individual neurons selected by morphometric characteristics were expressed as the ratio of emission following excitation at 340 and 380 nm (Ratio F340/F380), as reported in detail previously . Analysis of responses in non-neuronal cells was also carried out by selecting cells not showing the morphology of neurons. In addition, the glial phenotype was confirmed by the lack of Ca2+ responses to N-methyl d-aspartate (NMDA).
Changes in fluorescence ratio are expressed as Δ[Ca2+]cyt (Ratio F340/F380). Calculation of Δ[Ca2+]cyt was performed using Origin Lab 7.0. Data are presented as mean ± SEM. When only two means were compared, Student’s t test was used. For comparing more than two groups, statistical significance of the data was assessed by one-way ANOVA and compared using Bonferroni’s multiple comparison tests. Differences were considered significant at p < 0.05.
LPS induces apoptosis only in aged cultures of rat hippocampal neurons
Rat hippocampal neurons express TLR4 and its level of expression increases significantly with in vitro aging and/or senescence
LPS increases cytosolic [Ca2+] in aged cultured hippocampal neurons but not in young cultured neurons
LPS may contribute to cell death in neurons by modulating expression of NMDA receptor subunits in neurons, particularly aged neurons. We have addressed this possibility by testing the effects of LPS treatment (1 μg/m, 24 h) on expression of NMDA receptor subunits in aged neurons using immunofluorescence followed by optical density analysis. Our results indicate that LPS has minor or no effects on expression of NMDA receptor subunits NR1, NR2A, and NR2B in aged neurons (Additional file 1: Figure S1).
Aβ oligomers enhance LPS-induced Ca2+ responses and neuron cell death in aged cultured neurons
To appreciate best the effect of each treatment on apoptosis in the context of aging and/or senescence, we calculated the rise in apoptotic cells (fold increase) induced by each stimuli relative to the percent of apoptotic cells in young cultures treated with vehicle (Fig. 7c). The results show that in young neurons, LPS and Aβo showed a negligible effect on apoptosis. Only when LPS and Aβo were added together, they were able to promote a small, but statistically significant rise in apoptosis, which was similar to the apoptosis observed in untreated aged cultures of neurons (Aging) (Fig. 7c). Remarkably, any of the above treatments added in aged cultures of neurons induced a much larger fold increase in apoptosis, being this effect even significantly larger when both stimuli are present simultaneously in aged neurons. Thus, aging negatively influences hippocampal neuron survival and seems to be a critical factor required for exacerbating LPS- and Aβo-induced neuron death. However, the coincidence of both LPS and Aβo may promote apoptosis also in young neurons, yet to a limited extent.
Aβo enhance expression of TLR4 in aged cultured neurons
The present study reveals aging and/or senescence as a critical factor required for a marked LPS- and Aβ oligomer-induced neuron cell death as well as for TLR4 expression increase in rat hippocampal neurons. In addition, it discloses an exacerbated interplay between TLR4 and Aβ in the context of aging and/or senescence with consequences in neuronal damage. Our results show that the endotoxin associated to Gram-negative bacteria LPS, by acting on TLR4, increases [Ca2+]cyt and promotes cell death in aged cultures of rat hippocampal neurons, but not in young cultures of hippocampal neurons. Several evidences support this conclusion. First, CAY16014, an specific inhibitor of TLR4 activation, inhibits LPS-induced [Ca2+]cyt increase and LPS-induced neuron cell death. Second, LPS promotes neuron cell death only in aged cultures of hippocampal neurons that show Ca2+ responses to LPS and increased expression of TLR4. In fact, in young cultured neurons expressing low levels of TLR4, LPS did not increase [Ca2+] and neither promoted neuronal apoptosis. Thus, aging and/or senescence correlates with enhanced TLR4 expression underlying LPS-mediated, hippocampal neuron death.
We must stress that our results have been obtained in an in vitro model of aging and/or senescence that may not reflect entirely in vivo aging. Evidently, long-term cultured neurons do not undergo the complex processes and interactions involved in in vivo aging. For example, actual young and aged neurons are very different from the metabolic point of view and only limited evidence suggests that these differences remain between short- and long-term cultured hippocampal neurons. However, long-term cultured neurons are amenable for single-cell studies such as the ones performed in this study. In addition, aged cultures of rat hippocampal neurons display many of the hallmarks of aging in vivo, including accumulation of reactive oxygen species, increased oxidative damage of cell proteins, protein carbonyl formation lipofuscin granules, heterochromatic foci, activation of the Jun N-terminal protein kinase and p53/p21 pathways, gradual loss of cholesterol, and changes in Ca2+ channel density and NMDA receptor subunits similar to those found in in vivo aging [20, 21]. In addition, Letiembre et al.  reported that several TLR mRNAs are upregulated in the mouse-aged brain including TLR4 and its co-receptor CD14, as well as TLR1/2/5/7, while in contrast, transcripts of TLR3/6/8 did not change or even decreased as in the case of tlr9. Therefore, long-term cultures of rat hippocampal neurons may provide a reasonable as well as amenable model to investigating aging and/or senescence-related changes in Ca2+ signaling in individual neurons. In addition, our results are consistent with increased expression of TLR4 in aged brain reported in vivo .
Our results pose the question on whether age-related changes in TLR4 gene expression contribute to susceptibility to neurodegeneration in the elderly. Consistent with this possibility, it has been reported that activation of the central innate immune system may lead to exacerbated neuroinflammation and prolonged sickness behavior in response to LPS in aged mice compared to adult mice . In addition, we have recently reported that increased susceptibility to excitotoxicity and brain damage in aging are also reflected in in vitro aged cultures of rat hippocampal neurons that are much more sensitive to glutamate-induced neuron cell death than young neurons . This effect is induced by dramatic increases in the rise in [Ca2+]cyt induced by the glutamate receptor-agonist N-methyl d-aspartate associated to changes in receptor subunits  similar to those reported in in vivo aging . Therefore, the aged culture of rat hippocampal neurons may be a good model to assess aging and/or senescence-related changes in the susceptibility to brain damage induced by excitotoxicity and neuroinflammation induced by Ca2+ signals that may contribute largely to age-related neurodegeneration. In this regard, it is relevant the mechanism by which LPS increases [Ca2+]cyt in hippocampal neurons. In macrophages, it is well established that intracellular Ca2+ participates as a second messenger in TLR4-dependent signaling that increases [Ca2+]cyt by activating a vanilloid member of the transient receptor potential superfamily of cation channels (TRP channels). In fact, TRPV2 is involved in the LPS-induced Ca2+ mobilization from intracellular Ca2+ store and extracellular Ca2+ . In sensory neurons, however, it has been recently reported that LPS exerts fast, membrane delimited, excitatory actions via TRPA1 , another TRP cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Surprisingly, the effects of LPS on nociceptors were independent of TLR4 activation . In hippocampal neurons, both LPS-induced [Ca2+]cyt rises and neuron cell death were prevented by TLR4 antagonist and the effects correlated with changes in expression of TLR4 suggesting that LPS-induced effects in aged hippocampal neurons depend on TLR4.
We show here that glial cells also show Ca2+ responses induced by LPS, sometimes in the form of [Ca2+]cyt oscillations preceding Ca2+ responses in neurons. These results invite speculation on whether neuronal Ca2+ responses to LPS may be secondary to activated glia. For example, TLR4 activation in astrocytes might promote release of glutamate or other molecules that indirectly affect Ca2+ currents in neurons. Further research is required to fully exploring this and other possibilities. In any case, direct responses to LPS are definitely involved as most often neuronal Ca2+ responses are observed in the absence of glial responses. In addition, TLR4 expression is observed in morphologically and immunologically identified neurons, LPS-induced Ca2+ responses in neurons correlated with increased TLR4 expression in aged neurons and are prevented by inhibitors of TLR4 activation.
It could be claimed that LPS effects in aging cultures may be mediated by excitotoxicity events related to changes in NMDA receptor expression. However, several evidences are against this possibility. First, LPS has no or minor effects on expression of NMDA receptor subunits. Second, Ca2+ responses to NMDA and LPS are dissociated. Thus, Ca2+ responses to LPS are missing in 9–10 DIV neurons that display large Ca2+ responses to NMDA. Finally, MK801, a NMDA receptor antagonist, did not inhibit Ca2+ responses to LPS at concentrations that inhibit largely Ca2+ responses to NMDA.
Neuroinflammation has been associated to neurodegeneration and AD pathology, particularly in the elderly. For example, healthy aged individuals are more likely to suffer profound memory impairments following a challenging life event such as a severe bacterial infection than younger counterparts . Since TLR4 can sense not only molecular patterns derived from bacteria but also from necrotic damage, these data support the hypothesis of a decisive role of aging in exacerbating TLR4-mediated effects. Our data discloses an exacerbated interplay between Aβo and TLR4 in context of aging with consequences in neuronal damage. Specifically, we show here that treatment of rat hippocampal neurons with Aβo increased expression of TLR4, enhanced [Ca2+] responses to LPS and increased LPS-induced neuron cell death. Most importantly, all these effects were observed only in aged cultures of rat hippocampal neurons but not in young cultures, indicating that aging and/or senescence is the critical factor required for Aβo-mediated enhancement of LPS-induced damage. It may be argued that coincidence of the neuron damaging effect by LPS and Aβo is very unlikely. However, a non-infectious tissue injury can release TLR4 endogenous ligands called DAMPs that trigger sterile inflammation in the nervous system. Therefore, in the brain, the simultaneous accumulation of Aβ and DAMP-induced TLR4 activation is plausible upon stress and/or brain damage, particularly in the context of aging. In fact, human aging is associated to increased serum levels of pro-inflammatory cytokines, a chronic subclinical condition named as “inflammaging”, as well as increased NF-кB signaling, a transcription factor that is a master regulator of TLR-mediated inflammation. Recent evidences in rat models show that elevation in the levels of inflammatory cytokines induced by TLR4 activation may promote the accumulation of Aβ, which may in turn increase TLR4 levels, thus creating a positive feedback loop that may contribute largely to the progression of AD . This feedback loop may be also amplified further by the age-associated increased expression of NMDA receptors that could be targeted simultaneously by LPS (or DAMPs) and Aβo. Our data is consistent with the report and further demonstrates an exacerbated crosstalk between Aβo and TLR4 on [Ca2+] responses and cell death in aged hippocampal neurons, which might be relevant to the pathogenesis of age-related neurodegenerative diseases. Based on this, it is tempting to speculate that aging is the critical contributing factor that enables the vicious cycle between TLR4 and Aβo to promote AD.
LPS increases cytosolic [Ca2+] and promotes apoptosis in rat hippocampal neurons aged in vitro. This effect depends on the age-dependent increase in TLR4 expression that is further enhanced by oligomers of the amyloid β peptide. Thus, TLR4 and amyloid β oligomers cross talk in neuron cell death associated to aging, which might be relevant to AD.
- [Ca2+]cyt :
Cytosolic-free calcium concentration
Oligomers of the amyloid β peptide
Days in vitro
Standard external medium
Toll-like receptor 4
Transient receptor potential
We thank Mr. David del Bosque for technical support.
This work was supported by grants (VA145U13, BIO/VA33/13, BIO103/VA45/11, BIO/VA36/15) from Regional Government of Castilla y León, Spain and (BFU2012-37146, BFU2015-70131R and SAF2013-44521-R) from Ministry of Economy and Competitivity of Spain, cofunded by the European Social Fund. MCR was supported by a pre-doctoral fellowship from Regional Government of Castilla y León and the European Social Fund. None of these public agencies has any role in the design of the study and collection, analysis, and interpretation or writing of data.
Availability of data and materials
All data are available upon request.
MCR, CDF, and MGD carried out all the experiments and data analysis. MCR, CGR, LN, and CV designed experiments, analyzed and discussed results, and wrote the paper. All authors read and approved the final manuscript.
Present address for María Calvo-Rodríguez: Alzheimer Research Unit, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, 114, 16th St. Charlestown, MA 02129, USA. firstname.lastname@example.org
Present address for Carmen de la Fuente. Centre for Translational Omics Genetics and Genomic Medicine. Institute of Child Health. London, UK. email@example.com
The authors declare that they have no competing interests.
Consent for publication
The experiments carried out in this work have been approved by the ethics committees of Valladolid University School of Medicine and Spanish National Research Council (CSIC).
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- Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 2010;11(5):373–84.View ArticlePubMedGoogle Scholar
- Hashimoto C, Hudson KL, Anderson KV. The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell. 1988;52(2):269–79.View ArticlePubMedGoogle Scholar
- Medzhitov R, Preston-Hurlburt P, Janeway Jr CA. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 1997;388(6640):394–7.View ArticlePubMedGoogle Scholar
- Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science. 1998;282(5396):2085–8.8.View ArticlePubMedGoogle Scholar
- Olson JK, Miller SD. Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs. J Immunol. 2004;173(6):3916–24.View ArticlePubMedGoogle Scholar
- Bowman CC, Rasley A, Tranguch SL, Marriott I. Cultured astrocytes express toll-like receptors for bacterial products. Glia. 2003;43(3):281–91.View ArticlePubMedGoogle Scholar
- Aravalli RN, Peterson PK, Lokensgard JR. Toll-like receptors in defense and damage of the central nervous system. J Neuroimmune Pharmacol. 2007;4:297–312.View ArticleGoogle Scholar
- Tang SC, Arumugam TV, Xu X, Cheng A, Mughal MR, Jo DG, et al. Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits. Proc Natl Acad Sci U S A. 2007;104(34):13798–803.View ArticlePubMedPubMed CentralGoogle Scholar
- Okun E, Griffioen KJ, Mattson MP. Toll-like receptor signaling in neural plasticity and disease. Trends Neurosci. 2011;34(5):269–81.View ArticlePubMedPubMed CentralGoogle Scholar
- Letiembre M, Hao W, Liu Y, Walter S, Mihaljevic I, Rivest S, et al. Innate immune receptor expression in normal brain aging. Neuroscience. 2007;146(1):248–54.View ArticlePubMedGoogle Scholar
- McGeer PL, McGeer EG. The amyloid cascade-inflammatory hypothesis of Alzheimer disease: implications for therapy. Acta Neuropathol. 2013;126(4):479–97.View ArticlePubMedGoogle Scholar
- McGeer PL, McGeer EG. History of innate immunity in neurodegenerative disorders. Front Pharmacol. 2011;2:77.View ArticlePubMedPubMed CentralGoogle Scholar
- Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, et al. Inflammation and Alzheimer’s disease. Neurobiol Aging. 2000;21(3):383–421.View ArticlePubMedPubMed CentralGoogle Scholar
- McGeer PL, Rogers J, McGeer EG. Inflammation, anti-inflammatory agents and Alzheimer disease: the last 12 years. J Alzheimers Dis. 2006;9(3 Suppl):271–6.PubMedGoogle Scholar
- Minoretti P, Gazzaruso C, Vito CD, Emanuele E, Bianchi M, Coen E, et al. Effect of the functional toll-like receptor 4 Asp299Gly polymorphism on susceptibility to late-onset Alzheimer's disease. Neurosci Lett. 2006;391(3):147–9.View ArticlePubMedGoogle Scholar
- Walter S, Letiembre M, Liu Y, Heine H, Penke B, Hao W, et al. Role of the toll-like receptor 4 in neuroinflammation in Alzheimer’s disease. Cell Physiol Biochem. 2007;20(6):947–56.View ArticlePubMedGoogle Scholar
- Lehnardt S, Massillon L, Follett P, Jensen FE, Ratan R, Rosenberg PA, et al. Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway. Proc Natl Acad Sci U S A. 2003;100(14):8514–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Michelucci A, Heurtaux T, Grandbarbe L, Morga E, Heuschling P. Characterization of the microglial phenotype under specific pro-inflammatory and anti-inflammatory conditions: effects of oligomeric and fibrillar amyloid-beta. J Neuroimmunol. 2009;210(1–2):3–12.View ArticlePubMedGoogle Scholar
- Gasic-Milenkovic J, Dukic-Stefanovic S, Deuther-Conrad W, Gärtner U, Münch G. Beta-amyloid peptide potentiates inflammatory responses induced by lipopolysaccharide, interferon -gamma and ‘advanced glycation endproducts’ in a murine microglia cell line. Eur J Neurosci. 2003;17(4):813–21.View ArticlePubMedGoogle Scholar
- Brewer LD, Thibault O, Staton J, Thibault V, Rogers JT, García-Ramos G, et al. Increased vulnerability of hippocampal neurons with age in culture: temporal association with increases in NMDA receptor current, NR2A subunit expression and recruitment of L-type calcium channels. Brain Res. 2007;1151:20–31.View ArticlePubMedGoogle Scholar
- Sodero AO, Weissmann C, Ledesma MD, Dotti CG. Cellular stress from excitatory neurotransmission contributes to cholesterol loss in hippocampal neurons aging in vitro. Neurobiol Aging. 2011;32:1043–53.View ArticlePubMedGoogle Scholar
- Dueñas A, Aceves M, Orduña A, Diaz R, Sánchez Crespo M, García-Rodríguez C. Francisella tularensis LPS induces the production of cytokines in human monocytes and signals via Toll-like receptor 4 with much lower potency than E. coli LPS. Int Immunol. 2006;18:785–95.View ArticlePubMedGoogle Scholar
- Brewer GJ, Torricelli JR, Evege EK, Price PJ. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res. 1993;35(5):567–76.View ArticlePubMedGoogle Scholar
- Pérez-Otaño I, Luján R, Tavalin SJ, Plomann M, Modregger J, Liu XB, Jones EG, Heinemann SF, Lo DC, Ehlers MD. Endocytosis and synaptic removal of NR3A-containing NMDA receptors by PACSIN1/syndapin1. Nat Neurosci. 2006;9(5):611–21.View ArticlePubMedPubMed CentralGoogle Scholar
- Calvo M, Sanz-Blasco S, Caballero E, Villalobos C, Núñez L. Susceptibility to excitotoxicity in aged hippocampal cultures and neuroprotection by non-steroidal anti-inflammatory drugs: role of mitochondrial calcium. J Neurochem. 2015;132(4):403–17.View ArticlePubMedGoogle Scholar
- Calvo-Rodríguez M, Villalobos C, Nuñez L. Fluorescence and bioluminescence imaging of subcellular Ca2+ in aged hippocampal neurons. J Vis Exp. 2015;106:e53330.Google Scholar
- Caballero E, Calvo-Rodriguez M, Gonzalo-Ruiz A, Villalobos C, Nuñez L. A new procedure for amyloid β oligomers preparation enables the unambiguous testing of their effects on cytosolic and mitochondrial Ca2+ entry and cell death in primary neurons. Neurosci Lett. 2016;612:66–73.View ArticlePubMedGoogle Scholar
- Núñez L, Valero RA, Senovilla L, Sanz-Blasco S, García-Sancho J, Villalobos C. Cell proliferation depends on mitochondrial Ca2+ uptake: inhibition by salicylate. J Physiol (Lond). 2006;571:57–73.View ArticleGoogle Scholar
- Calvo-Rodríguez M, García-Durillo M, Villalobos C, Nuñez L. In vitro aging promotes endoplasmic reticulum (ER)-mitochondria Ca2+ cross talk and loss of store-operated Ca2+ entry (SOCE) in rat hippocampal neurons. Biochim Biophys Acta Mol Cell Res. 2016;1863(11):2637–49.
- Sanz-Blasco S, Valero RA, Rodríguez-Crespo I, Villalobos C, Núñez L. Mitochondrial Ca2+ overload underlies Aβ oligomers neurotoxicity providing an unexpected mechanism of neuroprotection by NSAIDs. PLoS One. 2008;3(7):e2718.View ArticlePubMedPubMed CentralGoogle Scholar
- Calvo-Rodríguez M, Núñez L, Villalobos C. Non-steroidal anti-inflammatory drugs (NSAIDs) and neuroprotection in the elderly: a view from the mitochondria. Neural Regen Res. 2015;10(9):1371–2.View ArticlePubMedPubMed CentralGoogle Scholar
- Huang Y, Henry CJ, Dantzer R, Johnson RW, Godbout JP. Exaggerated sickness behavior and brain proinflammatory cytokine expression in aged mice in response to intracerebroventricular lipopolysaccharide. Neurobiol Aging. 2008;11:1744–53.View ArticleGoogle Scholar
- Yamashiro K, Sasano T, Tojo K, Namekata I, Kurokawa J, Sawada N, et al. Role of transient receptor potential vanilloid 2 in LPS-induced cytokine production in macrophages. Biochem Biophys Res Commun. 2010;398(2):284–9.View ArticlePubMedGoogle Scholar
- Meseguer V, Alpizar YA, Luis E, Tajada S, Denlinger B, Fajardo O, et al. TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins. Nat Commun. 2014;5:3125.View ArticlePubMedPubMed CentralGoogle Scholar
- Barrientos RM, Frank MG, Watkins LR, Maier SF. Aging-related changes in neuroimmune-endocrine function: implications for hippocampal-dependent cognition. Horm Behav. 2012;62(3):219–27.View ArticlePubMedPubMed CentralGoogle Scholar
- Wu D, Zhang X, Zhao M, Zhou AL. The role of the TLR4/NF-κB signaling pathway in Aβ accumulation in primary hippocampal neurons. Sheng Li Xue Bao. 2015;67(3):319–28.PubMedGoogle Scholar