Osmotin attenuates LPS-induced neuroinflammation and memory impairments via the TLR4/NFκB signaling pathway

Haroon Badshah, Tahir Ali, Myeong Ok Kim, Haroon Badshah, Tahir Ali, Myeong Ok Kim

Abstract

Toll-like receptor 4 (TLR4) signaling in the brain mediates autoimmune responses and induces neuroinflammation that results in neurodegenerative diseases, such as Alzheimer's disease (AD). The plant hormone osmotin inhibited lipopolysaccharide (LPS)-induced TLR4 downstream signaling, including activation of TLR4, CD14, IKKα/β, and NFκB, and the release of inflammatory mediators, such as COX-2, TNF-α, iNOS, and IL-1β. Immunoprecipitation demonstrated colocalization of TLR4 and AdipoR1 receptors in BV2 microglial cells, which suggests that osmotin binds to AdipoR1 and inhibits downstream TLR4 signaling. Furthermore, osmotin treatment reversed LPS-induced behavioral and memory disturbances and attenuated LPS-induced increases in the expression of AD markers, such as Aβ, APP, BACE-1, and p-Tau. Osmotin improved synaptic functionality via enhancing the activity of pre- and post-synaptic markers, like PSD-95, SNAP-25, and syntaxin-1. Osmotin also prevented LPS-induced apoptotic neurodegeneration via inhibition of PARP-1 and caspase-3. Overall, our studies demonstrated that osmotin prevented neuroinflammation-associated memory impairment and neurodegeneration and suggest AdipoR1 as a therapeutic target for the treatment of neuroinflammation and neurological disorders, such as AD.

Figures

Figure 1
Figure 1
Osmotin prevents LPS-induced neuroinflammatory processes via inhibition of the TLR4-NFκB pathway (A) Shown are representative Western blots probed with TLR4, CD14, p-IKKα/β, and p-NFκB antibodies from the hippocampus of adult mice. The density values are expressed in arbitrary units as the mean ± SEM of the indicated proteins (n = 5 animals per group). (B) Shown are representative photomicrographs of immunofluorescence analyses of p-NFκB-positive cells in the experimental groups. Images are representative stains obtained in sections prepared from at least 5 animals per group. All of the panels representing the CA1, CA3, and DG regions of the hippocampus show p-NFκB-stained brain tissue at a magnification of 10× objective field (scale bar = 100 μm) and magnification of 40× objective field (scale bar = 20 μm). Symbol representation for the treatment groups and level of significance are described in the data analysis section of the Materials and Methods.
Figure 2
Figure 2
Anti-inflammatory mechanism of osmotin against LPS-induced neuroinflammation in BV2 microglial cells (A) Shown are representative histograms for (a) Cell Viability, (b) Cytotoxicity, and (c) Caspase-3/7 assays performed under experimental conditions as mentioned in the Materials and Methods section. (B) Shown are representative Western blots probed with TLR4, p-NFκB, and TNF-α antibodies in microglial cells. (C) Shown are immunoprecipitation results followed by representative immunoblots as mentioned in the Materials and Methods section. AdipoR1 colocalizes with the TLR4/CD14 signaling complex in microglial cells. (D) Shown are representative Western blots probed with TLR4 and p-NFκB antibodies in microglial cells. AdipoR1 siRNA transfection significantly inhibited LPS-induced inflammation. The density values are expressed in arbitrary units as the mean ± SEM for the indicated proteins (n = 5 per group). (E) The immunofluorescence images indicate localization of TLR4 (red panels), AdipoR1 (green panels) and merged TLR4 and AdipoR1 (merge panels). The images are representative of staining obtained in sections prepared from at least 5 animals per group (low magnification = 10x, scale bar: low zoom = 50 μm and high zoom = 20 μm). Symbols for treatment groups and levels of significance are mentioned in the data analysis section of the Materials and Methods.
Figure 3
Figure 3
Osmotin inhibits LPS-induced COX-2, iNOS, TNF-α and IL-1β expression (A) Shown are representative western blots probed with antibodies of COX-2, iNOS, TNF-α and IL-1β in the hippocampus of adult mice. The protein bands were quantified using sigma gel software. The density values are expressed in arbitrary units as the mean ± SEM for the indicated proteins (n = 5 animals per group). (B) Showed are representative photomicrographs of immunofluorescence analysis of Tnfα positive cells in the experimental groups. Images are representative of staining obtained in sections prepared from at least 5 animals per group. Panels representing DG, CA1 and CA3 region of hippocampus showed TNF-α stained brain tissue at magnification 10× objective field, scale bar= 100 µm. Symbols for treatment groups and level of significance are mentioned in data analysis section of material methods.
Figure 4. Effect of osmotin on LPS-induced…
Figure 4. Effect of osmotin on LPS-induced activation of astrocytes and microglia in the hippocampus of adult mice.
Shown are representative photomicrographs of immunofluorescence analyses of (A) astrocytes (GFAP-positive cells) and (B) microglia (Iba-1-positive cells) in the experimental groups. Images are representative of staining obtained in sections prepared from at least 5 animals per group. Panels representing the hippocampal fissure and DG region of the hippocampus show GFAP and Iba-1 stained brain tissue, respectively, at a magnification of 10x objective field, scale bar = 100 μm. Symbols for the treatment groups and levels of significance are mentioned in the data analysis section of the Materials and Methods.
Figure 5. Osmotin inhibits LPS-induced protein expression…
Figure 5. Osmotin inhibits LPS-induced protein expression of Aβ, APP, BACE-1, and p-Tau.
(A) Shown are representative Western blots probed with Aβ, APP, BACE-1, and p-Tau antibodies in the hippocampus of adult mice. The density values are expressed in arbitrary units as the mean ± SEM for the indicated proteins (n = 5 animals per group). (B) Shown are representative photomicrographs of immunofluorescence analyses of Aβ-positive cells in the experimental groups. Images are representative of staining obtained in sections prepared from at least 5 animals per group. All of the panels representing the CA1, CA3, and DG regions of the hippocampus show Aβ-stained brain tissue at a magnification of 10x objective field, scale bar = 50 μm. Symbols for treatment groups and levels of significance are mentioned in the data analysis section of the Materials and Methods.
Figure 6. Effect of osmotin on LPS-induced…
Figure 6. Effect of osmotin on LPS-induced synaptic dysfunction and memory impairment.
(A) Shown are representative Western blots probed with PSD-95, SNAP-25, SYP, and syntaxin-1 antibodies in the hippocampus of adult mice. The density values are expressed in arbitrary units as the mean ± SEM for the indicated proteins (n = 5 animals per group). (B) Shown are representative photomicrographs of immunofluorescence analysis of PSD-95- and SNAP-25-positive cells in the experimental groups. Images are representative of staining obtained in sections prepared from at least 5 animals per group. All of the panels representing the hippocampus show PSD-95- and SNAP-25-stained brain tissue at a magnification of 10x objective field, scale bar = 50 μm. Symbols for treatment groups and level of significance are mentioned in the data analysis section of the Materials and Methods. (C) Behavioral studies show osmotin improves memory impairment in LPS-treated mice (n = 5 per group). (a) Average escape latency time for experimental mice to reach the hidden platform from day 3 to day 8. (b) The number of crossings at the hidden platform during the probe test of the Morris water maze experiment. (c) Time spent in the platform quadrant, where the hidden platform was placed during the trial session. Symbols for treatment groups and levels of significance are mentioned in the data analysis section of the Materials and Methods.
Figure 7
Figure 7
Osmotin attenuated LPS-induced apoptotic neurodegeneration in the hippocampus of adult mice (A) Shown are representative Western blots probed with caspase-3 and PARP-1 antibodies in the hippocampus of adult mice. The density values are expressed in arbitrary units as the mean ± SEM for the indicated proteins (n = 5 animals per group). Shown are representative photomicrographs of (B) FJB staining (magnification 40× objective field, scale bar = 100 μm) and (C) Nissl staining (magnification 20× objective field, scale bar = 200 μm) for dead and damaged neurons. Images are representative of staining obtained in sections prepared from at least 5 animals per group. Symbols for treatment groups and level of significance are mentioned in the data analysis section of the Materials and Methods.

References

    1. Badshah H. et al. Protective effect of lupeol against lipopolysaccharide-induced neuroinflammation via the p38/c-Jun N-terminal kinase pathway in the adult mouse brain J. Neuroimmune Pharmacol. 11, 48–60 (2016).
    1. Parajuli B. et al. GM-CSF increase LPS-induced production of proinflammatory mediators via upregulation of TLR4 and CD14 in murine microglia. J. Neuroinflammation 9, 268 (2012).
    1. Font-Nieves M. et al. Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes. J. Biol. Chem. 287, 6564–68 (2012).
    1. Rosi S. et al. Memantine protects against LPS-induced neuroinflammation, restores behaviourally-induced gene expression and spatial learning in the rat. Neuroscience 142, 1303–15 (2006).
    1. Okun E., Griffioen K. J. & Mattson M. P. Toll-like receptor signaling in neural plasticity and disease. Trends Neurosci. 34, 269–81 (2011).
    1. Garate I. et al. Toll-like receptor inhibitor TAK242 decreases neuroinflammation in rat brain frontal cortex after stress. J. Neuroinflammation 11, 8 (2014).
    1. Sambamurti K. et al. Gene structure and organization of the human beta-secretase (BACE) promoter. FASEB J. 18, 1034–6 (2004).
    1. Qin L. et al. Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration. Glia 55, 453–62 (2007).
    1. Sastre M. et al. Nonsteroidal anti-inflammatory drugs and peroxisome proliferatoractivated receptor-gamma agonists modulate immunostimulated processing of amyloid precursor protein through regulation of beta-secretase. J. Neurosci. 23, 9796–804 (2003).
    1. McGeer E. G. & McGeer P. L. Inflammatory processes in Alzheimer’s disease. Prog. neuropsychopharmacol. Biol. Psychiatry 27, 741–9 (2003).
    1. Pratico D. & Trojanowski J. Q. Inflammatory hypotheses: novel mechanisms of Alzheimer’s neurodegeneration and new therapeutic targets? Neurobiol. Aging 21, 441–3 (2000).
    1. Akiyama H. et al. Inflammation and Alzheimer’s disease. Neurobiol. Aging 21, 383–421 (2000).
    1. Griffin W. S. et al. Glial-neuronal interactions in Alzheimer’s disease: the potential role of a ‘cytokine cycle’ in disease progression. Brain Pathol. 8, 65–72 (1998).
    1. Sambamurti K. et al. Gene structure and organization of the human beta-secretase (BACE) promoter. FASEB J. 18, 1034–6 (2004).
    1. Deng X. et al. Lipopolysaccharide-induced neuroinflammation is associated with Alzheimer-like amyloidogenic axonal pathology and dendritic degeneration in rats. Adv. Alzheimer Dis. 3, 78–93 (2014).
    1. Lee Y. J. et al. Epigallocatechin-3-gallate prevents systemic inflammation-induced memory deficiency and amyloidogenesis via its anti-neuroinflammatory properties. J. Nutr. Biochem. 24, 296–310 (2013).
    1. Teeling J. L. et al. The effect of non-steroidal anti-inflammatory agents on behavioral changes and cytokine production following systemic inflammation: Implications for a role of COX-1. Brain Behav. Immun. 24, 409–19 (2010).
    1. Jenke A. et al. Adiponectin protects against Toll-like receptor 4-mediated cardiac neuroinflammation and injury. Cardiovas. Res. 99, 422–31 (2013).
    1. Narasimhan M. L. et al. Osmotin is a homolog of mammalian adiponectin and controls apoptosis in yeast through a homolog of a mammalian adiponectin receptor. Mol. Cell 17, 171–80 (2005).
    1. Miele M., Costantini S. & Colonna G. Structural and functional similarities between osmotin from Nicotiana tabacum seeds and human adiponectin. PLos One 6, e16690 (2011).
    1. Naseer M. I. et al. Neuroprotective effect of osmotin against ethanol-induced apoptotic neurodegeneration in the developing brain. Cell Death Dis. 5, e1150 (2014).
    1. Ali T. et al. Osmotin attenuates amyloid beta-induced memory impairment, tau phosphorylation and neurodegeneration in the mouse hippocampus. Sci. Rep. 5, 11708 (2015).
    1. Park B. S. & Lee J. O. Recognition of lipopolysaccharide pattern by TLR4 complexes. Exp. Mol. Med. 45, e66 (2013).
    1. Zanoni I. et al. CD14 controls the LPS-induced endocytosis of Toll-like receptor 4. Cell 147, 868–80 (2011).
    1. Gorina R. et al. Astrocyte TLR4 activation induces a proinflammatory environment through the interplay between My88-dependent NFκB signaling, MAPK, and Jak1/Stat1 pathways. Glia 59, 242–55 (2011).
    1. Delhase M., Hayakawa M., Chen Y. & Karin M. Positive and negative regulation of IkappaB kinase activity through IKKbeta subunit phosphorylation. Science 284, 309–13 (1999).
    1. Lawrence T. The nuclear factor NF-kappa B pathway in inflammation. Cold Spring Harb. Perspect. Biol. 1, a001651 (2009).
    1. Block M. L., Zecca L. & Hong J. S. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat. Rev. Neurosci. 8, 57–69 (2007).
    1. Brown G. C. & Neher J. J. Inflammatory neurodegeneration and mechanisms of microglial killing of neurons. Mol. Neurobiol. 41, 242–7 (2010).
    1. Lehnardt S. et al. Activation of innate imunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway. Proc. Natl. Acad. USA 100, 8514–9 (2003).
    1. Marcello E., Epis R., Saraceno C. & Di Luca M. Synaptic dysfunction in Alzheimer’s disease. Adv. Exp. Med. Biol. 970, 573–601 (2012).
    1. Xaus J. et al. LPS induces apoptosis in macrophages mostly through the autocrine production of TNF-alpha. Blood 95, 3823–31 (2000).
    1. Rupinder S. K., Gurpreet A. K. & Manjeet S. Cell suicide and caspases. Vascul. Pharmacol. 46, 383–39 (2007).
    1. Koh D. W., Dawson T. M. & Dawson V. L. Poly(ADP-ribosyl)ation regulation of life and death in the nervous system. Cell. Mol. Life Sci. 62, 760–768 (2005).
    1. Garden G. A. & Moller T. Microglia biology in health and disease. J.Neuroimmune Pharmacol. 1, 127–137 (2006).
    1. Kawai T. & Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11, 373–384 (2010).
    1. Lakhani S. A. & Boque C. W. Toll-like receptor signaling in sepsis. Curr. Opin. Pediatr. 15, 278–82 (2003).
    1. Chow J. C. et al. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J. Biol. Chem. 274, 10689–92 (1999).
    1. Akira S. & Takeda K. Toll-like receptor signalling. Nat. Rev. Immunol. 4, 499–511 (2004).
    1. Tsao T. S., Lodish H. F. & Fruebis J. ACRP30, a new hormone controlling fat and glucose metabolism. Eur. J. Pharmacol. 440, 213–21 (2002).
    1. Kadowaki T. & Yamauchi T. Adiponectin and adiponectin receptors. Endocr. Rev. 26, 439–51 (2005).
    1. Hwang D. Y. et al. Alterations in behavior, amyloid beta-42, caspase-3, and Cox-2 in mutant PS2 transgenic mouse model of Alzheimer’s disease. FASEB J. 16, 805–13 (2002).
    1. Nguyen M. D., Julien J. P. & Rivest S. Innate immunity: The missing link in neuroprotection and neurodegeneration? Nat. Rev. Neurosci. 3, 216–227 (2002).
    1. Tahara K. et al. Role of toll-like receptor signalling in Abeta uptake and clearance. Brain 129, 3006–19 (2006).
    1. Reed-Geaghan E. G., Savage J. C., Hise A. G. & Landreth G. E. CD14 and toll-like receptors 2 and 4 are required for fibrillar A{beta}-stimulated microglial activation. J. Neurosci. 29, 11982–92 (2009).
    1. Chan K. H. et al. Adiponectin is protective against oxidative stress induced cytotoxicity in amyloid-beta neurotoxicity. PLos One 7, e52354 (2012).
    1. Oh D. K., Ciaraldi T. & Henry R. R. Adiponectin in health and disease. Diabetes Obes. Metab. 9, 282–289 (2007).
    1. Hattori Y. et al. Insights into sepsis therapeutic design based on the apoptotic death pathway. J. Pharmacol. Sci. 114, 354–65 (2010).
    1. Cederbaum A. I., Yang L., Wang X. & Wu D. CYP2E1 sensitizes the liver to LPS- and Tnf α-induced toxicity via elevated oxidative and nitrosative stress and activation of ASK-1 and JNK mitogen-activated kinases. Int. J. Hepatol. 582790 (2012).
    1. Munshi N. et al. Lipopolysaccharide-induced apoptosis of endothelial cells and its inhibition by vascular endothelial growth factor. J. Immunol. 168, 5860–6 (2002).
    1. Dong M. et al. Chronic Akt activation attenuated lipopolysaccharide-induced cardiac dysfunction via Akt/GSK3β-dependent inhibition of apoptosis and ER stress. Biochem. Biophys. Acta 1832, 848–63 (2013).
    1. Putcha G. V. et al. JNK-mediated BIM phosphorylation potentiates BAX-dependent apoptosis. Neuron 19, 899–914 (2003).
    1. Tournier C. et al. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science 288, 870–874 (2000).

Source: PubMed

Patrocinadores y Colaboradores

Condiciones médicas

Intervenciones de drogas

3
Suscribir