Methods to Study Inflammasome Activation in the Central Nervous System: Immunoblotting and Immunohistochemistry

Cyr, Brianna; Vaccari, Juan Pablo de Rivero

doi:10.1007/978-1-0716-3350-2_15

Cited by 3 publications

(2 citation statements)

References 28 publications

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“…Brain protein lysates were obtained, as described in [62,63], and 25 µg of protein lysates was then resolved via immunoblotting for the expression of inflammasome signaling proteins, as described in [64]. Primary antibodies used in this study were against the following proteins: NLRP1 (Novus Biologicals, Centennial, CO, USA), NLRP3 (Novus Biologicals Centennial, CO, USA), AIM2 (eBioscience, San Diego, CA, USA), NLRC4 (Novus Biologicals, Centennial, CO, USA), pyrin (Santa Cruz Biotechnology, Inc. Dallas, TX, USA), caspase-1 (Novus Biologicals, Centennial, CO, USA), ASC (Santa Cruz), IL-1β (Cell Signaling, Technology, Inc. Danvers, MA, USA), caspase-8 (Novus Biologicals, Centennial, CO, USA), caspase-11 (Novus Biologicals, Centennial, CO, USA), CD63 (Novus Biologicals), β-actin (Sigma-Aldrich, St. Louis, MO, USA) and GAPDH (Sigma Aldrich, St. Louis, MO, USA).…”

Section: Immunoblottingmentioning

confidence: 99%

Neural–Cardiac Inflammasome Axis after Traumatic Brain Injury

Keane,

Hadad,

Scott

et al. 2023

Pharmaceuticals

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Traumatic brain injury (TBI) affects not only the brain but also peripheral organs like the heart and the lungs, which influences long-term outcomes. A heightened systemic inflammatory response is often induced after TBI, but the underlying pathomechanisms that contribute to co-morbidities remain poorly understood. Here, we investigated whether extracellular vehicles (EVs) containing inflammasome proteins are released after severe controlled cortical impact (CCI) in C57BL/6 mice and cause activation of inflammasomes in the heart that result in tissue damage. The atrium of injured mice at 3 days after TBI showed a significant increase in the levels of the inflammasome proteins AIM2, ASC, caspases-1, -8 and -11, whereas IL-1β was increased in the ventricles. Additionally, the injured cortex showed a significant increase in IL-1β, ASC, caspases-1, -8 and -11 and pyrin at 3 days after injury when compared to the sham. Serum-derived extracellular vesicles (EVs) from injured patients were characterized with nanoparticle tracking analysis and Ella Simple Plex and showed elevated levels of the inflammasome proteins caspase-1, ASC and IL-18. Mass spectrometry of serum-derived EVs from mice after TBI revealed a variety of complement- and cardiovascular-related signaling proteins. Moreover, adoptive transfer of serum-derived EVs from TBI patients resulted in inflammasome activation in cardiac cells in culture. Thus, TBI elicits inflammasome activation, primarily in the atrium, that is mediated, in part, by EVs that contain inflammasome- and complement-related signaling proteins that are released into serum and contribute to peripheral organ systemic inflammation, which increases inflammasome activation in the heart.

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Section: Immunoblottingmentioning

confidence: 99%

Neural–Cardiac Inflammasome Axis after Traumatic Brain Injury

Keane,

Hadad,

Scott

et al. 2023

Pharmaceuticals

Self Cite

View full text Add to dashboard Cite

show abstract

“…Brain and heart protein lysates were obtained as described in ( Mejias et al, 2018 ), and lysates were then resolved by immunoblotting for the expression of inflammasome signaling proteins as in ( Cyr and de Rivero Vaccari, 2023a ). Briefly, lysates were resolved in 4–20% Criterion TGX Stain-Free precast gels (Bio-Rad), using antibodies at a dilution of 1: 1,000.…”

Section: Methodsmentioning

confidence: 99%

Extracellular vesicles mediate inflammasome signaling in the brain and heart of Alzheimer’s disease mice

Cyr,

Cabrera Ranaldi,

Hadad

et al. 2024

Front. Mol. Neurosci.

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IntroductionAlzheimer’s disease (AD) is an inflammatory neurodegenerative disease characterized by memory loss and cognitive impairment that worsens over time. AD is associated with many comorbidities, including cardiovascular disease that are associated with poorer outcomes. Comorbidities, especially heart disease and stroke, play a significant role in the demise of AD patients. Thus, it is important to understand how comorbidities are linked to AD. We have previously shown that extracellular vesicle (EV)-mediated inflammasome signaling plays an important role in the pathogenesis of brain injury and acute lung injury after traumatic brain injury.MethodsWe analyzed the cortical, hippocampal, ventricular, and atrial protein lysates from APP/PS1 mice and their respective controls for inflammasome signaling activation. Additionally, we analyzed serum-derived EV for size, concentration, and content of inflammasome proteins as well as the EV marker CD63. Finally, we performed conditioned media experiments of EV from AD patients and healthy age-matched controls delivered to cardiovascular cells in culture to assess EV-induced inflammation.ResultsWe show a significant increase in Pyrin, NLRP1, caspase-1, and ASC in the brain cortex whereas caspase-8, ASC, and IL-1β were significantly elevated in the heart ventricles of AD mice when compared to controls. We did not find significant differences in the size or concentration of EV between groups, but there was a significant increase of caspase-1 and IL-1β in EV from AD mice compared to controls. In addition, conditioned media experiments of serum-derived EV from AD patients and age-matched controls delivered to cardiovascular cells in culture resulted in inflammasome activation, and significant increases in TNF-α and IL-2.ConclusionThese results indicate that EV-mediated inflammasome signaling in the heart may play a role in the development of cardiovascular diseases in AD patients.

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Catecholamine-Induced Inflammasome Activation in the Heart Following Photothrombotic Stroke

Scott,

Kerr,

Sanchez-Molano

et al. 2024

Transl. Stroke Res.

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Cerebrovascular stroke patients exhibit an increased incidence of cardiac arrhythmias. The pathomechanisms underlying post-traumatic cardiac dysfunction include a surge of catecholamines and an increased systemic inflammatory response, but whether inflammasome activation contributes to cardiac dysfunction remains unexplored. Here, we used a mouse model of photothrombotic stroke (PTS) to investigate the role of inflammasome activation in post-stroke cardiac dysfunction by catecholamines and to evaluate the effectiveness of the inflammasome inhibitor IC100 on inflammasome activation. To evaluate functional electrophysiological changes in the heart by catecholamine treatment, we recorded action potential duration in excised zebrafish hearts with and without IC100 treatment. We show that PTS induced AIM2 inflammasome activation in atria and ventricles that was significantly reduced by administration of IC100. Injection of epinephrine into naïve mice induced a significant increase in AIM2, IL-1b and caspase-8 in atria. Treatment of excised zebrafish hearts with epinephrine shortened the action potential duration and this shortening that was reduced by IC100. These findings indicate that stroke initiates a catecholamine surge that induces inflammasome activation and pyroptosis in the heart that is blocked by IC100, thus providing a framework for the development of therapeutics for stroke-related cardiovascular injury.

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Methods to Study Inflammasome Activation in the Central Nervous System: Immunoblotting and Immunohistochemistry

Cited by 3 publications

References 28 publications

Neural–Cardiac Inflammasome Axis after Traumatic Brain Injury

Neural–Cardiac Inflammasome Axis after Traumatic Brain Injury

Extracellular vesicles mediate inflammasome signaling in the brain and heart of Alzheimer’s disease mice

Catecholamine-Induced Inflammasome Activation in the Heart Following Photothrombotic Stroke

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