PLoS One. 2009;4(5):e5518. Epub 2009 May 13.

IL-6 mediated degeneration of forebrain GABAergic interneurons and cognitive impairment in aged mice through activation of neuronal NADPH oxidase.

Dugan LL, Ali SS, Shekhtman G, Roberts AJ, Lucero J, Quick KL, Behrens MM.

Source

Division of Geriatric Medicine, Department of Medicine, University of California San Diego, La Jolla, California, USA. ladugan@ucsd.edu

Abstract

BACKGROUND:

Multiple studies have shown that plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) are elevated in patients with important and prevalent adverse health conditions, including atherosclerosis, diabetes, obesity, obstructive sleep apnea, hypertension, and frailty. Higher plasma levels of IL-6, in turn, increase the risk of many conditions associated with aging including age-related cognitive decline. However, the mechanisms underlying this association between IL-6 and cognitive vulnerability remain unclear.

METHODS AND FINDINGS:

We investigated the role of IL-6 in brain aging in young (4 mo) and aged (24 mo) wild-type C57BL6 and genetically-matched IL-6(-/-) mice, and determined that IL-6 was necessary and sufficient for increased neuronal expression of the superoxide-producing immune enzyme, NADPH-oxidase, and this was mediated by non-canonical NFkappaB signaling. Furthermore, superoxide production by NADPH-oxidase was directly responsible for age-related loss of parvalbumin (PV)-expressing GABAergic interneurons, neurons essential for normal information processing, encoding, and retrieval in hippocampus and cortex. Targeted deletion of IL-6 or elimination of superoxide by chronic treatment with a superoxide-dismutase mimetic prevented age-related loss of PV-interneurons and reversed age-related cognitive deficits on three standard tests of spatial learning and recall.

CONCLUSIONS:

Present results indicate that IL-6 mediates age-related loss of critical PV-expressing GABAergic interneurons through increased neuronal NADPH-oxidase-derived superoxide production, and that rescue of these interneurons preserves cognitive performance in aging mice, suggesting that elevated peripheral IL-6 levels may be directly and mechanistically linked to long-lasting cognitive deficits in even normal older individuals. Further, because PV-interneurons are also selectively affected by commonly used anesthetic agents and drugs, our findings imply that IL-6 levels may predict adverse CNS effects in older patients exposed to these compounds through specific derangements in inhibitory interneurons, and that therapies directed at lowering IL-6 may have cognitive benefits clinically.

PMID:: 19436757; [PubMed - indexed for MEDLINE]
PMCID:: PMC2678193

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Figure 2

Imaging of in vivo superoxide production in brain of young and old animals: role of Nox2 and IL-6.

(a) Confocal imaging of in vivo DHE oxidation reveals that superoxide-mediated DHE oxidation is increased in neurons in old mice, and this increase is blocked by the NADPH oxidase inhibitor, apocynin, or by the SOD mimetic, C₃. Young (4 mo) and old (24 mo) mice were treated orally for 7 days with the Nox inhibitor, apocynin, or for 21 days with the SOD mimetic, C₃. (b) Quantitative analyses of fluorescence in hippocampal CA1 and cortex were performed as described in detail in the methods section. Images were obtained using a 40× WI objective. Values are mean±SEM, n = 3. Significance was determined by ANOVA: for CA1 (*old versus young, P = 0.01;**old versus old+apocynin (P = 0.02) or old+C₃ (P = 0.01) and cortex (Ctx) (*old versus young, P = 0.01;**old versus old+apocynin (P = 0.01) or old+C₃ (P = 0.006) by Tukey's. (c) Live animal imaging of DHE oxidation using a GE eXplore Optix™-MX2 whole animal scanner also demonstrates increased DHE oxidation in brain of old (24 mo) relative to young (4 mo) mice. Fluorescence images of brain through intact skull and skin are shown on the left , and fluorescence intensity from ox-DHE is mapped onto a linear pseudocolor scale. Images to the right are ex vivo scans of the same brains showing greater anatomical detail, including fluorescence from the cortical hemispheres and cerebellum. (d) Repeated injections of IL-6 on two consecutive days (5 µg/kg) in old mice induced Nox2 expression (top images, green), and produced a small increase in DHE oxidation (red). However, repeated injections of IL-6 every 12 h (5 µg/kg, times three) to produce sustained systemic levels resulted in a dramatic increased in ox-DHE fluorescence compared to saline-injected old controls (bottom images). Note that the laser excitation power was decreased from 260 mW in (c) to 100 mW (d) to avoid saturating the camera in the scanner.

IL-6 Mediated Degeneration of Forebrain GABAergic Interneurons and Cognitive Impairment in Aged Mice through Activation of Neuronal NADPH Oxidase

PLoS ONE. PLoS ONE;4(5):e5518.

Figure 3

Increased synaptosomal Nox-2 activation and superoxide production in old wild-type mice is absent in old IL-6-/- mice; determination by electrochemical oxygen consumption analysis and EPR spin-trapping spectroscopy.

(a) NADPH-induced oxygen consumption is significantly decreased by the Nox inhibitor apocynin (300 µM, black bars). Oxygen consumption by synaptosomal Nox from the cortex of young (3–4 M, n = 9) and old (23–24 M, n = 9) animals at 37°C was induced by the addition of 5 mM NADPH to samples containing 2–5 mg synaptosomal protein. The observed Nox specific activity was significantly higher in IL-6 injected young animals (white bars, p = 0.04, n = 5) and in old brains (light gray bar, p = 0.01, n = 9) and was significantly lower in IL-6-/- old mice (dark gray bar, p = 0.04, n = 4). IL-6 treatment: Two IL-6 i.p. injections (5 mg/kg), separated by 12 hrs were administered to young and old animals before the groups were sacrificed 12 hrs later for synaptosomal isolation. (b) Representative EPR spectra recorded 3 minutes after mixing 5 mM NADPH with 0.2–0.5 mg synaptosomal protein isolated from brain of old mice at 37°C with 70 mM DEPMPO in the absence or in the presence of 200 µM AEBSF or 300 µM apocynin. In each incubation, 10 mM DETC was included to inhibit the SOD enzyme, resulting in enhanced and quantifiable EPR signals. Using computer simulations we concluded that the observed signals are attributable to a mixture of adducts of DEPMPO with hydroxyl radical and lipid derived carbon centered radical and both radicals are derived from superoxide since the signals were completely abolished in the presence of SOD; not shown. (c) Quantification of the observed EPR signals accumulated over the first 3 minutes from NADPH addition, n = 4–9 per group. Superoxide yield in synaptosomes isolated from different groups follows essentially the same activity pattern seen by oxygraphy and shown in (a). The asterisks (*) designate statistical significance by ANOVA followed by Tukey test with respect to young control while (#) indicate statistical significance with respect to old control. EPR settings are described fully in methods. Data are mean ± SEM. (d) Nox-generated superoxide burst in synaptosomes isolated from IL-6-/- KO mice is significantly lower than that in their old control brains.

IL-6 Mediated Degeneration of Forebrain GABAergic Interneurons and Cognitive Impairment in Aged Mice through Activation of Neuronal NADPH Oxidase

PLoS ONE. PLoS ONE;4(5):e5518.

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IL-6 mediated degeneration of forebrain GABAergic interneurons and cognitive impairment in aged mice through activation of neuronal NADPH oxidase.

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