Inhibition of LTP-induced translation by IL-1Î² ... - ACS Publications

Subscriber access provided by Iowa State University | Library

Letter

Inhibition of LTP-induced translation by IL-1# reduces the level of newly synthesized proteins in hippocampal dendrites G. Aleph Prieto, Erica D. Smith, Liqi Tong, Michelle Nguyen, and Carl W Cotman ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00511 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

Inhibition of LTP-induced translation by IL-1 reduces the level of newly synthesized proteins in hippocampal dendrites

G. Aleph Prieto1*, Erica D. Smith1, Liqi Tong1, Michelle Nguyen1, Carl W. Cotman1,2

1Institute

for Memory Impairments and Neurological Disorders, University of California-Irvine, Irvine, CA 92697 USA 2Department

of Neurobiology and Behavior, University of California-Irvine, Irvine, CA 92697 USA

*Correspondence: G. Aleph Prieto, PhD; [email protected] University of California-Irvine, Irvine, California 92697, USA, TEL: (949) 824-6071, FAX: (949) 824-2071

Total words: 5,444 Figures: 4 Tables: 0 Pages: 24 Running title: IL-1 suppresses cLTP-driven translation

1 ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 26

Abstract In rodent hippocampus, the inflammatory cytokine interleukin-1 (IL-1) impairs memory and long-term potentiation (LTP), a major form of plasticity that depends on protein synthesis. A better understanding of the mechanisms by which IL-1 impairs LTP may help identify targets for preventing cognitive deterioration. We tested whether IL-1 inhibits protein synthesis in hippocampal neuron cultures following chemically-induced LTP (cLTP). Fluorescent-tagging using click-chemistry showed that IL-1 reduces the level of newly synthesized proteins in proximal dendrites of cLTP stimulated neurons.

Relative to controls, in cLTP stimulated

neurons IL-1 inhibited Akt/mTOR signaling, as well as the upregulation of GluA1, an AMPA receptor subunit, and LIMK1, a kinase that promotes actin polymerization. Notably, a novel TIR domain peptidomimetic (EM163) blocked both the activation of p38 and the suppression of cLTP-dependent protein synthesis by IL-1. Our data support a model where IL-1 suppresses LTP directly in neurons by inhibiting mTOR-dependent translation.

Keywords: cLTP, Akt, mTOR, GluA1, LIMK1, translation


Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A growing literature indicates that the inflammatory cytokine interleukin-1β (IL-1β) has a crucial role in the etiology and progression of Alzheimer’s disease (AD),1 the most prevalent form of dementia. The idea that IL-1β contributes to memory impairments in humans is consistent with the finding that transgenic overexpression of IL-1β in the hippocampus induces memory deficits in mice,2 and is further supported by the incidence of memory impairments in animals models of neuroinflammation induced by obesity,3 nerve injury,4 and aging.5 In looking for neuronal mechanisms affected by IL-1β, we have previously found that this cytokine suppresses long-term potentiation (LTP),6 the cellular correlate of learning and memory.7

In rodents, we have

demonstrated that IL-1 impairs theta burst-induced LTP and brain derived neurotrophic factor (BDNF)-dependent LTP in hippocampal slices,6 as well as GluA1 surface expression following chemically-induced LTP (cLTP) in hippocampal neuronal cultures8 and in synaptosomes isolated from the hippocampus of adult animals.9, 10 However, the identity of LTP-related biochemical pathways vulnerable to IL-1 has not been fully defined.

A better understanding of the

molecular mechanisms by which IL-1β impairs LTP may help in identifying potential therapeutic targets to alleviate cognitive decline in AD and age-related dementias. LTP is characterized by a rapid and remarkably persistent increase in synaptic transmission elicited by brief patterns of afferent activity.7 LTP induction by calcium influx through NMDA receptors (NMDAR) is coupled to the PI3K/Akt/mTOR cascade, which induces protein synthesis in dendrites and spines.11 Protein synthesis drives molecular and morphological adaptations in spines critical for the consolidation of LTP (late-phase) and memory.11-13 As IL-1β impairs the consolidation but not the induction of LTP in acute hippocampal slices,6 we hypothesized that IL-1 suppresses activity-dependent protein synthesis, thus affecting late-phase LTP.



Page 4 of 26

Hippocampal LTP can be induced by a train of electrical stimulation bursts (e.g., theta bursts)7 or by chemical stimulation (chemical-LTP, cLTP).14 A well-established protocol to induce cLTP is based on a brief application of the NMDAR co-agonist glycine, which facilitates NMDAR activation.15 This cLTP protocol induces a potentiated state that parallels the essential features of electrically-induced LTP, including the insertion of AMPA receptors (AMPAR) into the postsynaptic surface, F-actin formation and protein synthesis induction, as well as growth and remodeling of spines.14, 15 Importantly, cLTP is occluded in hippocampal slice cultures by prior induction of theta-burst LTP, thus confirming that glycine-induced cLTP and theta-burst LTP approaches share underlying cellular processes16. To test whether IL-1 impairs activitydependent translation in hippocampal neurons we used a neuron culture model of glycineinduced cLTP. We tracked protein synthesis by click-chemistry, an approach where newly synthesized proteins incorporate the non-canonical amino acid azidohomoalanine (AHA), which is next selectively tagged with a fluorescent compound by means of click chemical reactions17 (Figure 1A). First, we examined the dynamic range of the click-chemistry assay in the 0-120 min interval, in non-stimulated hippocampal cultured neurons. Levels of fluorescence-tagged newly synthetized proteins were analyzed by confocal microscopy in the soma and in straightened images of dendrites, using the area defined by neuronal mark staining as a mask (see Figure S1). During the 0-120 min interval we found a linear increase in the AHA-labeling in proximal dendrites (0-60 µm), but not in distal dendrites (61-100 µm), which showed low levels of fluorescence (Figure S1). Consistent with these observations, direct visualization of nascent peptides on translating mRNA via SINAPS (single-molecule imaging of nascent peptides) has demonstrated that dendritic mRNAs are mainly translated in proximal dendrites within 30 m of 4 ACS Paragon Plus Environment

Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the soma, with fewer translating mRNA detected in distal dendrites.18 At the soma, we detected a strong AHA-labeling; however, fluorescence signal was found to be saturated at 60 min (Figure S1). As our study aims to dissect the effect of IL-1 on protein synthesis in late-phase cLTP (after 2 h of cLTP induction), click-chemistry analysis focused only on proximal dendrites, which are the only neuron compartment exhibiting a reliable 2h-AHA-labeling for quantifying protein synthesis in our experimental conditions (Figure S1). Notably, proximal dendrites are essential structures for active translation in hippocampal neurons.18 To induce cLTP, we treated neurons with glycine for 10 min in Mg2+-free external solution supplemented with antagonists for GABA (bicuculline) and glycine (strychnine) receptors, as previously described.19 After cLTP stimulation, neurons were maintained in standard external solution for 2 h (Figure 1B). Analysis of fluorescence intensity showed an increase in the level of fluorescence-tagged AHA in proximal segments of dendrites of cLTP stimulated neurons, as compared to control cultures treated with external solution (p < 0.0001, 0-20 m dendritic segment; Figure 1C and D). Consistent with our hypothesis, IL-1 pretreatment significantly reduced cLTP-induced increase in dendritic AHA fluorescence levels (p < 0.0001, 0-20 m dendritic segment; Figure 1C and D), thus suggesting that translation of synaptic markers of plasticity may be affected by IL-1. As learning and LTP are thought to be dependent on synthesis of synaptic receptors,20 we evaluated protein levels of the GluA1 AMPAR subunit, the BDNF receptor TrkB, and the NR2B subunit of NMDAR in cLTP stimulated neuronal cultures (Figure 2A). Western blot analysis on cell lysates showed that cLTP increases GluA1 and TrkB but not NR2B levels after 3h of stimulation (Figure 2A and B), a finding that closely matches the increased synthesis of GluA1



Page 6 of 26

and GluA2/3 but not NR2B receptor subunits after 3 h of electrical-induced LTP in hippocampal slices.21

Notably, IL-1 totally blocked the increase in GluA1 levels in cLTP stimulated

neurons, while partially reduced TrkB upregulation (Figure 2A and B). Previous evidence indicates that neuron activity increases GluA1 mRNAs targeting to dendrites where they are locally translated.22 Taken together with our click-chemistry data showing reduced levels of newly-synthesized proteins in proximal dendrites of cultures treated with IL-1 (Figure 1C and D), our data suggest that IL-1 locally impairs activity-dependent GluA1 translation in dendrites. Next, we evaluated the effect of IL-1β on cLTP signaling, in particular on the activation of Akt and its downstream effector mTOR (Figure 3A), a kinase that plays a critical role in plasticity by integrating converging signals from glutamate and BDNF receptors.11 Consistent with the major role of the PI3K/Akt pathway in synaptic plasticity,11 we found that cLTP in neuronal cultures increases phosphorylation levels of both Akt and mTOR (p < 0.05, Figure 3B and C) at sites associated with the activity of these kinases (p-Akt, Akt phosphorylated at Ser473; p-mTOR, mTOR phosphorylated at Ser-2448). Notably, cLTP stimulation was unable to increase p-Akt or p-mTOR levels in neurons pretreated with IL-1 for 2 h (Figure 3A-C), a finding consistent with the suppression of BDNF-induced mTOR phosphorylation in neurons pretreated with IL-1 for 2 h.23

Overall, these data indicate that IL-1β impairs activity-

dependent Akt/mTOR signaling. Activation of Akt and mTOR by cLTP was also blocked by two pharmacological inhibitors of electrical-induced LTP: the NMDA receptor antagonist AP5, and the tyrosine kinase inhibitor K252a, thus confirming that cLTP parallels the molecular events associated with electrical-induced LTP (Figure 3D).


Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Long persistence of LTP and memory depends on physical changes on synapses via both protein synthesis and rearrangement of cytoskeleton.13 For the stabilization of LTP the kinase LIMK1 promotes actin polymerization by inactivating cofilin, a protein that severs actin filaments. We tested LIMK1 levels after 3 h of cLTP stimulation, as it has been shown that 4 htreatment with the activity-dependent neurotrophin BDNF induces LIMK1 mRNA translation via mTOR in neuronal cultures.24 We found an increase in LIMK1 levels after cLTP in hippocampal neurons (p < 0.05, Figure 3E and F). However, LIMK1 levels did not change when cLTPstimulated neurons were pretreated either with IL-1 or with the mTOR inhibitor rapamycin (Figure 3E and F), a finding consistent with the idea that IL-1 inhibits mTOR-dependent translation in late-phase cLTP. These results further suggest that the modulation of LIMK1 levels by IL-1 may contribute to the IL-1-induced suppression of F-actin formation in latephase LTP.6, 8 A main goal of aging research is to identify therapeutic interventions that can counteract cognitive decline. For optimal penetration of brain, ideal chemical properties of pharmaceutics include small size and high lipophilicity. These chemical properties are characteristics of novel IL-1β inhibitors known as TIR peptidomimetics, which target TIR (Toll/interleukin-1 receptor) domains in IL-1 receptors, and block the interaction of TIR domains with MyD8825,

26,

an

adaptor protein that provides the initial platform for downstream molecules involved in IL-1 signaling such as the stress kinase p38.27 In cultured hippocampal neurons, we found that the TIR peptidomimetic EM16325 blocks IL-1-induced activation of p38 (phosphorylation at Thr180/Tyr182, p-p38) (p < 0.0006, Figure 4A and B), a downstream effector for the suppression of LTP consolidation by IL-1.6,

9

Click-chemistry analysis showed that cLTP-

induced translation in dendrites was not affected by IL-1 if neurons were pretreated with 7 ACS Paragon Plus Environment


Page 8 of 26

EM163 (p < 0.0001, Figure 4C and D). Further, we found an increase in GluA1 levels after cLTP in neurons pretreated with EM163 before IL-1 (p < 0.05, Figure 4E). As GluA1 upregulation by cLTP was found to be mTOR-dependent (inhibited by rapamycin, Figure 4E), overall these data support the idea that EM163 can block IL-1 signaling and rescue mTORdependent translation in late-phase cLTP in hippocampal neurons. LTP is characterized by three stages: induction, expression, and consolidation. LTP consolidation closely parallels memory in that an initially unstable event becomes increasingly resistant to disruption.7 We have previously shown that via p38 IL-1β impairs F-actin formation in spines, and the consolidation of LTP in hippocampal slices6. Recently, using cLTP in neuron hippocampal cultures, we confirmed that IL-1β impairs F-actin formation, thus suppressing GluA1 surface insertion and spine growth8. Our current study provides novel information by identifying that IL-1 suppresses activity-dependent protein synthesis in hippocampal neurons. Here, we show for the first time that IL-1 suppresses cLTP-induced protein synthesis in neurons. Further, we found that IL-1 impairs the upregulation of LIMK1 and GluA1, as well as the activation of the Akt/mTOR pathway in cLTP stimulated neurons. How can all these findings be integrated in a mechanistic model for the suppression of LTP by IL-1? We propose that, first, by inhibiting activity-dependent dendritic protein synthesis IL-1 contributes to reduce F-actin formation, as several lines of evidence support a link between protein synthesis and Factin formation during LTP.13 For instance, cLTP increases the availability and local translation of -actin mRNA in dendrites of cultured hippocampal neurons28, and translation of dendritically localized Arc mRNA is required for stabilization of nascent polymerized actin and consolidation of LTP.13

Moreover, recent evidence suggests that LTP is stabilized by coupling mTOR-

dependent de novo protein synthesis with F-actin formation, via the RhoA/ROCK/LIMK/cofilin 8 ACS Paragon Plus Environment

Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pathway.29 Second, by inhibiting translation and F-actin formation IL-1 may indirectly block GluA1-AMPA expression at the spine surface, as it is known that myosin Vb mobilizes recycling endosomes and AMPA receptors along actin cytoskeleton for postsynaptic plasticity.30 Third, based on the fundamental role of BDNF in synaptic plasticity,13 the strong suppression of BDNF signaling by IL-1 (via p38)6, 23 may significantly contribute to the impairments in translation, Factin formation, and GluA1-AMPA surface expression. Hence, together with previous work from our laboratory6, 8-10, 23 and others,5 we propose a working model in which increased brain IL-1 levels impair hippocampal-dependent memory by suppressing activity-dependent translation and actin polymerization, two mechanisms essential for synapse growth and LTP consolidation.

In particular, our current finding that IL-1 reduces the level of newly-

synthesized proteins locally in proximal dendrites, a major site for translation in hippocampal neurons,18 suggests that IL-1 may disrupt mTOR-dependent spatial controlled translation and the strengthening of circuitries relevant for memory consolidation. Quantification of protein synthesis by methods such as incorporation of radioactive

35S-

methionine coupled to Western blot analysis has provided relevant information on the role of protein synthesis in both LTP12 and cLTP.31 However, in studies based on these approaches it is uncertain which brain cell(s) incorporate

35S-methionine

(e.g., neurons, microglia and

astrocytes). The high cell heterogeneity in the brain also affects the study of cytokines’ effects. In particular, because most brain cells express IL-1 receptors and IL-1β increases the expression of multiple cytokines in their target cells, it has been challenging to elucidate neuron-specific effects of IL-1β in hippocampal slices or in in vivo experiments. In contrast, to selectively focus on MAP2 (or neuron marker)-identified neurons, we detected newly-synthesized proteins by a fluorescence-based method, using a cLTP protocol in cultures of primary hippocampal neurons, 9 ACS Paragon Plus Environment


Page 10 of 26

an experimental system enriched in neurons. Thus, our results likely reflect neuron-specific effects of IL-1β on activity-dependent translation. The idea that IL-1β suppresses plasticityrelated mechanisms directly in neurons is further supported by the activation of p389 and the inhibition of cLTP9,

10

in mouse hippocampal synaptosomes briefly treated with IL-1β.

Importantly, our neuron-specific analysis may uncover potential targets for intervention. In agreement with previous reports showing that TIR peptidomimetics block IL-1 signaling in rodent lymphocytes,26 here we provide experimental evidence that IL-1β signaling in rat hippocampal neurons can be blocked by EM163, a novel low-molecular weight TIR mimetic. In IL-1β treated neurons, we found that EM163 strongly blocks both the activation of p38 activation and the suppression of cLTP-induced translation. However, a limitation of our study is the absence of in vivo validation of these results. In vivo validation of our data is timely and relevant, as IL-1β accumulates in the brain and periphery with age and AD, and it has been suggested that blocking brain IL-1β signaling may improve cognition in AD.1 Thus, it remains to be tested whether EM163 crosses the blood-brain barrier (BBB) and inhibits hippocampal IL1β signaling, thus rescuing protein synthesis and memory; similar to AS1, a structurally related TIR mimetic that targets the brain following systemic delivery26 and improves hippocampaldependent memory in aged mice challenged by inflammation.9 In summary, our data indicate that IL-1 inhibits cLTP-dependent signaling, and reduces the level of newly-synthesized proteins in proximal dendrites of cLTP stimulated hippocampal neurons. This mechanism may contribute to impairing consolidation of hippocampal-dependent memory in animal models of IL-1-related neuroinflammation (e.g., obesity, aging and AD).


Page 11 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Methods All procedures used in the present study followed the Principles of Laboratory Animal Care from NIH and were approved by the University of California, Irvine, Institutional Animal Care and Use Committee. Cell Culture—Primary cultures of dissociated hippocampal neurons were prepared from E18 Sprague-Dawley rats as described previously.23 Cells were maintained in serum-free Neurobasal supplemented with B27, GlutaMAX, and penicillin/streptomycin (all culture reagents from Life Sciences). Unless otherwise specified, 3 nM (50 ng/ml) IL-1 (PeproTech) was used to be consistent with previous reports from our laboratory9, and rapamycin was 200 nM (Cell Signaling). cLTP. Extracellular solution contained (in mM): 120 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 15 glucose, and 15 Hepes, pH 7.4; whereas cLTP solution was Mg2+-free and contained (in mM): 150 NaCl, 2 CaCl2, 5 KCl, 10 Hepes, and 30 glucose, pH 7.4. cLTP solution was supplemented with 0.001 mM strychnine and 0.02 mM bicuculline methiodide. For cytokine treatment, 3 pM IL-1β was added 30 min before cLTP; same IL-1β concentration was maintained during and after cLTP. Click chemistry and Immunocytochemistry.

Click chemistry is a metabolic pulse labeling

method that measures protein synthesis with excellent linearity. In neuronal cultures (7-10 DIV), endogenous methionine was depleted by 60 min incubation in medium lacking methionine or cysteine. Metabolic labeling was performed by 200 µM L-azidohomoalanine17 during and after 2 h of cLTP in medium without methionine (i.e., external physiological solution or cLTP buffer with no Mg2+) but containing treatments as indicated in Figures 1 and 4. For cycloaddition reaction, cells previously fixed with paraformaldehyde (4%) for 20 min at room temperature 11 ACS Paragon Plus Environment


Page 12 of 26

(RT), permeabilized in 0.25% Triton x-100 in PBS for 15 min, and washed in 2.5%BSA in PBS (pH = 7.1-7.4). Next, cells were incubated in the Click-iT reaction buffer mix with 2µM 488alkyne (reagents from Life Sciences) for 1 h at RT, in a foil covered humid box under gentle agitation. Cells were washed (x3, 2.5% BSA in PBS), permeabilized/blocked for 30 min in 0.1% Triton-X, 10% BSA in PBS. Cells were incubated with primary antibodies: chicken anti-MAP2 (Abcam, 1:200), and mouse pan neuronal marker (Millipore, 1:200) in blocking buffer overnight at 4°C. After washes (×3, blocking buffer), secondary antibodies conjugated to Alexa Fluor 488, Alexa 555, and Alexa 647 (Life Sciences) were incubated 2 h at RT, and washed (×3, PBS). For p-p38 detection, after treatment neurons were washed in cold PBS, fixed with paraformaldehyde (4%) for 20 min at room temperature (RT), permeabilized/blocked for 60 min in 5% goat serum and 0.3% Triton-X in PBS at RT, washed twice with PBS, and incubated for 30 min at RT in blocking buffer (5% goat serum and 0.1% Triton X-100 in PBS) with primary antibodies: rabbit anti-p-p38 (Cell signaling, 1:100), chicken anti-MAP2 (Abcam, 1:200) in blocking buffer overnight. After washes (×3, blocking buffer), secondary antibodies conjugated to Alexa Fluor 488 and Alexa 647 were incubated 2 h at RT, and washed (×3, PBS). Slides were coverslipped with Gold Prolonged Antifade reagent (Life Sciences) and imaged on an IX70 Olympus confocal microscope with a x20/0.70 objective. ImageJ software was used for analysis. Signal from green, red and blue channels was quantified in neurons from five to six fields per condition, acquired from a consistent starting point within each well. Western blot. After treatment, cells were washed in ice-cold PBS, and lysed in RIPA/Nonidet P40 buffer containing protease and phosphatase inhibitor mixtures (Thermo), and immediately frozen. Neurons were harvested, and protein concentration was determined by BCA assay (Pierce; 23227). Lysates were supplemented with Laemmli buffer, boiled (7 min), run on


Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Criterion XT gels, transferred to PVDF membranes (Bio-Rad), which were blocked in 5% BSA for 1 h and probed with primary antibody (4 °C, overnight). The membranes were washed (4 × 10 min in TBS with 0.1% Tween-20, vol/vol), probed with HRP-conjugated secondary antibody for 1 h, and developed by using Pierce Chemiluminescent Substrate (Pierce; 32106). Multiple film exposures were used to verify linearity. Blots were washed, stripped, and reprobed with antibodies to GAPDH, β-actin, or total levels of specific proteins. Membranes were incubated in stripping buffer (Pierce, 46430; Bioland Scientific, SB01-01; or Millipore, WB59) according to the manufacturer’s instructions. Statistical analysis. Sample sizes were chosen on the basis of previous experience with neuronal cultures6, 8-10, 23. ANOVA was used where assumptions of normality (Kolmogorov–Smirnov) and equal variance (Bartlett’s test) were met, and was replaced by Kruskal–Wallis and Mann– Whitney test for nonparametric datasets. For mean comparisons of three or more groups, oneway ANOVA was followed by post hoc Tukey’s test; whereas Kruskal–Wallis was followed by Dunn’s post hoc test. Two-way ANOVAs were followed by Tukey’s test. Statistical tests were performed using GraphPad Prism 5.0. Data are presented as mean ± s.e.m. A value of p < 0.05 was considered significant.



Page 14 of 26

Figure 1

Figure 1. IL-1 suppresses activity-dependent protein synthesis in hippocampal neurons. For labeling of newly synthesized proteins, AHA (1 hr incubation) was fluorescence tagged via click-chemistry; as staining and experimental controls, parallel neuron cultures were not supplemented with AHA (no AHA), or they were treated with 40 µM anisomycin (AHA + aniso). Signals of AHA and pan-neuronal marker are displayed with the ImageJ heatmap LUTs fire and yellow, respectively. Color lookup table indicates AHA fluorescence intensity (pixel intensities 0–255); scale bar, 20 μm. Mean AHA intensities ± s.e.m. of the soma are shown at the left (A). Outline of the experiment (B). For testing IL-1β effects, neurons (7-10 DIV) were pretreated with 3 nM IL-1β for 30 min or vehicle before cLTP stimulation. After cLTP (10 min) (controls were treated with physiological solution), neurons were maintained in physiological solution with 2 µM AHA for 2 h. AHA was quantified in neuron dendrites identified by MAP2 (C). For analysis, primary dendrites of randomly selected neurons were straightened and fluorescent intensities of binned 20 μm segments were measured using ImageJ. Left, proximal; 14 ACS Paragon Plus Environment

Page 15 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


right, distal. Scale bar, 20 μm (C). Quantification of fluorescence intensities (mean ± s.e.m.) of 17–23 proximal dendrites per group, in 20-μm bins; n = 4 independent experiments (neurons from different embryo litters) (D). (#, p < 0.0001 cLTP vs. control, and vs. cLTP+IL-1β; Tukey posthoc test following two-way ANOVA, effect of treatment, p < 0.0001, F2,138 = 14.1).

Figure 2

Figure 2. IL-1 suppresses cLTP-induced upregulation of GluA1 levels in hippocampal neurons. GluA1, NR2B and TrkB total protein levels were assessed by Western blot in lysates of cultured rat hippocampal neurons (7-10 DIV) after 10 min or 3h of cLTP induction. Neurons were pretreated for 30 min with 3 nM IL-1β or vehicle, as control, as well as during and after cLTP stimulation (A). Quantitative analysis of eight independent experiments (neurons from different embryo litters) showing GluA1 (n = 8), TrkB (n = 6), and NR2B (n = 3) levels normalized to GAPDH (loading control), and relative to vehicle-treated neurons (B). (*p < 0.05 by post hoc Dunns’s test following Kruskal-Wallis test).



Page 16 of 26

Figure 3

Figure 3. IL-1 suppresses cLTP-induced increase in p-Akt, p-mTOR and LIMK1 levels in hippocampal neurons. After 10 min or 3h of cLTP stimulation, phosphorylated and total levels of Akt and mTOR were evaluated by Western blot in rat hippocampal neurons (7-10 DIV). Neurons were pretreated with 3 nM IL-1β or vehicle for 2 h, as well as during and after cLTP stimulation (A). Analysis of nine independent experiments, normalized to vehicle-treated neurons (control): p-Akt/Akt (10 min, n = 5; 3 h, n = 8, B), p-mTOR/mTOR (10 min, n = 3; 3h, n = 9, C). (*p < 0.05 by post hoc Dunns’s test following Kruskal-Wallis test). Similar analysis was done after 10 min (for Akt) or 3h (for mTOR) of cLTP in neurons pretreated with 3 nM IL-1β for 2 h, or with K252a (20 and 50 nM) or AP5 (100 µM) for 3 min, or with vehicle (2 h). Treatments were maintained during and after cLTP. Representative results are of two independent experiments (D). Total LIMK1 levels in neurons after 3 h of cLTP stimulation. Neurons were pretreated with 3 nM IL-1β for 30 min or 200 nM rapamaycin for 15 min, as well as during and after cLTP stimulation (E). Densitometry values of LIMK1/GAPDH were 16 ACS Paragon Plus Environment

Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


normalized to unstimulated neurons (control) (n = 9, F). (*p < 0.05 vs control by post hoc Dunns’s test following Kruskal-Wallis test, F).

Figure 4

Figure 4. TIR mimetic EM163 blocks IL-1 signaling. Neurons (7-10 DIV) were treated with 3 nM IL-1 for 20 min; EM163 (20 µM) was added 15 min before IL-1. Insets indicate quantification area using the square macro of Image J software. Red, MAP2; green, p-p38, scale bar, 20 μm (A). Levels of p-p38/MAP2 were normalized to vehicle-treated neurons (B); n = 4 independent experiments. #p < 0.0006 by post hoc Dunns’ test following Kruskal-Wallis test. 17 ACS Paragon Plus Environment


Page 18 of 26

Vehicle (n = 165 neurons), IL-1 (n = 236), EM163 (n = 181), EM163 + IL-1 (n = 191). In clickchemistry experiments (C and D), 7-10 DIV neurons were pretreated with vehicle or with 20 M EM163 for 15 min before adding 3 nM IL-1β for 30 min, and cLTP stimulated (controls treated with physiological solution), and maintained in physiological solution containing 200 M AHA for 2h. After click-chemistry reaction, analysis of randomly selected neurons focused on primary dendrites (identified by MAP staining), which were straightened. Quantification of fluorescent intensities of binned 20 μm segments was carried out using ImageJ. Left, proximal; right, distal. Scale bar, 20 μm (C). Graph shows the quantification of AHA signal in proximal dendritic segments (mean ± s.e.m.) of 18-25 primary dendrites per group; two independent experiments. For the 0-20 m segment: #, p < 0.0001, control vs cLTP; p < 0.0001, control vs cLTP+IL1+EM163; Tukey posthoc test following two-way ANOVA, effect of treatment, p < 0.0001, F4,298 = 9.23) (D). Western blot analysis of GluA1 total levels in hippocampal neurons (7-10 DIV) following 3h of cLTP stimulation (E). Neurons were pretreated with IL-1β (3 nM) for 30 min before cLTP stimulation. EM163 (20 µM) was added 15 min before IL-1β. Rapamycin (200 nM) was added 30 min before cLTP induction; n = 6 of four independent experiments.

Acknowledgements We thank Brandon Nguyen for helping on ImageJ-assisted analysis of fluorescence. EM163 was kindly provided by Drs. Dariush Ajami and Julius Rebek Jr from The Scripps Research Institute (La Jolla, CA). Work in the authors’ lab is supported by National Institutes of Health Grants R21-AG048506, P01-AG000538 and RO1-AG34667 (to C.W.C.), as well as by UC MEXUSCONACYT Grant CN-16-170 (to G.A.P. and C.W.C.). 18 ACS Paragon Plus Environment

Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Author Contributions G.A.P. designed the study, and performed and analyzed all experiments with assistance from M.N. E.D.S. set experimental conditions of click-chemistry assay. L.T. provided cultured cells. M.N. performed the ImageJ-assisted analysis of fluorescence. C.W.C. supervised all the work. G.A.P. and C.W.C. prepared the manuscript. All authors read and approved the final version.

Supporting Information: Quantification of newly-synthesized proteins by click-chemistry (20, 60 and 120 min).

References [1] Heneka, M. T., Kummer, M. P., Stutz, A., Delekate, A., Schwartz, S., Vieira-Saecker, A., Griep, A., Axt, D., Remus, A., Tzeng, T. C., Gelpi, E., Halle, A., Korte, M., Latz, E., and Golenbock, D. T. (2013) NLRP3 is activated in Alzheimer's disease and contributes to pathology in APP/PS1 mice, Nature 493, 674-678. [2] Hein, A. M., Stasko, M. R., Matousek, S. B., Scott-McKean, J. J., Maier, S. F., Olschowka, J. A., Costa, A. C., and O'Banion, M. K. (2010) Sustained hippocampal IL-1beta overexpression impairs contextual and spatial memory in transgenic mice, Brain Behav Immun 24, 243-253. [3] Erion, J. R., Wosiski-Kuhn, M., Dey, A., Hao, S., Davis, C. L., Pollock, N. K., and Stranahan, A. M. (2014) Obesity elicits interleukin 1-mediated deficits in hippocampal synaptic plasticity, J Neurosci 34, 2618-2631. [4] Gui, W. S., Wei, X., Mai, C. L., Murugan, M., Wu, L. J., Xin, W. J., Zhou, L. J., and Liu, X. G. (2016) Interleukin-1beta overproduction is a common cause for neuropathic pain, memory deficit, and depression following peripheral nerve injury in rodents, Mol Pain 12. [5] Murray, C. A., and Lynch, M. A. (1998) Evidence that increased hippocampal expression of the cytokine interleukin-1 beta is a common trigger for age- and stress-induced impairments in long-term potentiation, J Neurosci 18, 2974-2981. [6] Tong, L., Prieto, G. A., Kramar, E. A., Smith, E. D., Cribbs, D. H., Lynch, G., and Cotman, C. W. (2012) Brain-derived neurotrophic factor-dependent synaptic plasticity is suppressed by interleukin-1beta via p38 mitogen-activated protein kinase, J Neurosci 32, 17714-17724. [7] Lynch, G., Rex, C. S., and Gall, C. M. (2007) LTP consolidation: substrates, explanatory power, and functional significance, Neuropharmacology 52, 12-23. 19 ACS Paragon Plus Environment


Page 20 of 26

[8] Tong, L., Prieto, G. A., and Cotman, C. W. (2018) IL-1beta suppresses cLTP-induced surface expression of GluA1 and actin polymerization via ceramide-mediated Src activation, J Neuroinflammation 15, 127. [9] Prieto, G. A., Snighda, S., Baglietto-Vargas, D., Smith, E. D., Berchtold, N., Tong, L., Ajami, D., LaFerla, F. M., Rebek, J., and Cotman, C. W. (2015) Synapse-specific IL-1 receptor subunit reconfiguration augments vulnerability to IL-1 in the aged hippocampus, Proc Natl Acad Sci U S A 112, E5078-5087. [10] Prieto, G. A., Tong, L., Smith, E. D., and Cotman, C. W. (2018) TNFalpha and IL-1beta but not IL-18 Suppresses Hippocampal Long-Term Potentiation Directly at the Synapse, Neurochem Res. [11] Buffington, S. A., Huang, W., and Costa-Mattioli, M. (2014) Translational control in synaptic plasticity and cognitive dysfunction, Annu Rev Neurosci 37, 17-38. [12] Mullany, P., and Lynch, M. A. (1997) Changes in protein synthesis and synthesis of the synaptic vesicle protein, synaptophysin, in entorhinal cortex following induction of longterm potentiation in dentate gyrus: an age-related study in the rat, Neuropharmacology 36, 973-980. [13] Bramham, C. R. (2008) Local protein synthesis, actin dynamics, and LTP consolidation, Curr Opin Neurobiol 18, 524-531. [14] Molnar, E. (2011) Long-term potentiation in cultured hippocampal neurons, Seminars in cell & developmental biology 22, 506-513. [15] Lu, W., Man, H., Ju, W., Trimble, W. S., MacDonald, J. F., and Wang, Y. T. (2001) Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons, Neuron 29, 243-254. [16] Musleh, W., Bi, X., Tocco, G., Yaghoubi, S., and Baudry, M. (1997) Glycine-induced longterm potentiation is associated with structural and functional modifications of alphaamino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid receptors, Proc Natl Acad Sci U S A 94, 9451-9456. [17] Dieterich, D. C., Hodas, J. J., Gouzer, G., Shadrin, I. Y., Ngo, J. T., Triller, A., Tirrell, D. A., and Schuman, E. M. (2010) In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons, Nat Neurosci 13, 897-905. [18] Wu, B., Eliscovich, C., Yoon, Y. J., and Singer, R. H. (2016) Translation dynamics of single mRNAs in live cells and neurons, Science 352, 1430-1435. [19] Park, M., Salgado, J. M., Ostroff, L., Helton, T. D., Robinson, C. G., Harris, K. M., and Ehlers, M. D. (2006) Plasticity-induced growth of dendritic spines by exocytic trafficking from recycling endosomes, Neuron 52, 817-830. [20] Whitlock, J. R., Heynen, A. J., Shuler, M. G., and Bear, M. F. (2006) Learning induces long-term potentiation in the hippocampus, Science 313, 1093-1097. [21] Nayak, A., Zastrow, D. J., Lickteig, R., Zahniser, N. R., and Browning, M. D. (1998) Maintenance of late-phase LTP is accompanied by PKA-dependent increase in AMPA receptor synthesis, Nature 394, 680-683. [22] Ju, W., Morishita, W., Tsui, J., Gaietta, G., Deerinck, T. J., Adams, S. R., Garner, C. C., Tsien, R. Y., Ellisman, M. H., and Malenka, R. C. (2004) Activity-dependent regulation of dendritic synthesis and trafficking of AMPA receptors, Nat Neurosci 7, 244-253. [23] Smith, E. D., Prieto, G. A., Tong, L., Sears-Kraxberger, I., Rice, J. D., Steward, O., and Cotman, C. W. (2014) Rapamycin and Interleukin-1beta Impair Brain-derived


Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Neurotrophic Factor-dependent Neuron Survival by Modulating Autophagy, J Biol Chem 289, 20615-20629. [24] Schratt, G. M., Tuebing, F., Nigh, E. A., Kane, C. G., Sabatini, M. E., Kiebler, M., and Greenberg, M. E. (2006) A brain-specific microRNA regulates dendritic spine development, Nature 439, 283-289. [25] Kissner, T. L., Ruthel, G., Alam, S., Mann, E., Ajami, D., Rebek, M., Larkin, E., Fernandez, S., Ulrich, R. G., Ping, S., Waugh, D. S., Rebek, J., Jr., and Saikh, K. U. (2012) Therapeutic inhibition of pro-inflammatory signaling and toxicity to staphylococcal enterotoxin B by a synthetic dimeric BB-loop mimetic of MyD88, PLoS One 7, e40773. [26] Bartfai, T., Behrens, M. M., Gaidarova, S., Pemberton, J., Shivanyuk, A., and Rebek, J., Jr. (2003) A low molecular weight mimic of the Toll/IL-1 receptor/resistance domain inhibits IL-1 receptor-mediated responses, Proc Natl Acad Sci U S A 100, 7971-7976. [27] Weber, A., Wasiliew, P., and Kracht, M. (2010) Interleukin-1 (IL-1) pathway, Sci Signal 3, cm1. [28] Buxbaum, A. R., Wu, B., and Singer, R. H. (2014) Single beta-actin mRNA detection in neurons reveals a mechanism for regulating its translatability, Science 343, 419-422. [29] Briz, V., Zhu, G., Wang, Y., Liu, Y., Avetisyan, M., Bi, X., and Baudry, M. (2015) Activity-dependent rapid local RhoA synthesis is required for hippocampal synaptic plasticity, J Neurosci 35, 2269-2282. [30] Wang, Z., Edwards, J. G., Riley, N., Provance, D. W., Jr., Karcher, R., Li, X. D., Davison, I. G., Ikebe, M., Mercer, J. A., Kauer, J. A., and Ehlers, M. D. (2008) Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity, Cell 135, 535-548. [31] Haynes, K. A., Smith, T. K., Preston, C. J., and Hegde, A. N. (2015) Proteasome inhibition augments new protein accumulation early in long-term synaptic plasticity and rescues adverse Abeta effects on protein synthesis, ACS Chem Neurosci 6, 695-700.

For Table of Contents Only



Figure 1 134x86mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 22 of 26

Page 23 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2 168x36mm (300 x 300 DPI)



Figure 3 166x78mm (300 x 300 DPI)


Page 24 of 26

Page 25 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4 162x112mm (300 x 300 DPI)



Abstract Graphic 81x41mm (300 x 300 DPI)


Page 26 of 26

Inhibition of LTP-induced translation by IL-1Î² ... - ACS Publications

Recommend Documents