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. 2011;6(6):e21108.
doi: 10.1371/journal.pone.0021108. Epub 2011 Jun 21.

Blockade of gap junction hemichannel suppresses disease progression in mouse models of amyotrophic lateral sclerosis and Alzheimer's disease

Hideyuki Takeuchi  1 , Hiroyuki Mizoguchi, Yukiko Doi, Shijie Jin, Mariko Noda, Jianfeng Liang, Hua Li, Yan Zhou, Rarami Mori, Satoko Yasuoka, Endong Li, Bijay Parajuli, Jun Kawanokuchi, Yoshifumi Sonobe, Jun Sato, Koji Yamanaka, Gen Sobue, Tetsuya Mizuno, Akio Suzumura
Affiliations

Blockade of gap junction hemichannel suppresses disease progression in mouse models of amyotrophic lateral sclerosis and Alzheimer's disease

Hideyuki Takeuchi et al. PLoS One. 2011.

Abstract

Background: Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases.

Methods and findings: In this study, we generated a novel blood-brain barrier permeable gap junction hemichannel blocker based on glycyrrhetinic acid. We found that pharmacologic blockade of gap junction hemichannel inhibited excessive glutamate release from activated microglia in vitro and in vivo without producing notable toxicity. Blocking gap junction hemichannel significantly suppressed neuronal loss of the spinal cord and extended survival in transgenic mice carrying human superoxide dismutase 1 with G93A or G37R mutation as an amyotrophic lateral sclerosis mouse model. Moreover, blockade of gap junction hemichannel also significantly improved memory impairments without altering amyloid β deposition in double transgenic mice expressing human amyloid precursor protein with K595N and M596L mutations and presenilin 1 with A264E mutation as an Alzheimer's disease mouse model.

Conclusions: Our results suggest that gap junction hemichannel blockers may represent a new therapeutic strategy to target neurotoxic microglia specifically and prevent microglia-mediated neuronal death in various neurodegenerative diseases.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. A novel gap junction hemichannel blocker INI-0602 effectively inhibits neurotoxic microglial glutamate release.
(A) Structure of carbenoxolone (CBX) and INI-0602. (B) Assessment of microglial hemichannel inhibition by EtBr dye uptake. Scale bar, 100 µm. Data represent the means ± SD (n = 6 per group). *, P<0.05 vs. PBS. (C–D) TNF-α production (C) and NO production (D) by microglia treated with 1 µg/ml LPS for 24 h. Data represent the means ± SD (n = 3 per group). *, P<0.05 vs. PBS. (E) Fluorescent microscopic images of live/dead staining of mouse primary cortical neuron cultures in microglia-conditioned media. Neurons in microglia-conditioned media containing 1 µg/ml LPS (LPS) underwent cell death with neuritic beading, whereas neurons in microglia-conditioned media containing PBS (PBS), 1 µg/ml LPS plus 100 µM CBX (100 µM CBX), or 1 µg/ml LPS plus 100 µM INI-0602 (100 µM INI-0602) appeared healthy. Green, neuron (MAP2); red, dead cell (PI); blue, nucleus (Hoechst). Scale bar, 20 µm. (F) Glutamate concentrations in media. (G) The percentages of dead neurons. Data represent the means ± SD (n = 6 per group). *, P<0.05 vs. PBS; **, P<0.05 vs. LPS; †, P<0.05 vs. 1 µM; §, P<0.05 vs. 10 µM.
Figure 2
Figure 2. INI-0602 effectively inhibits microglial glutamate release in vivo.
(A) Glutamate concentrations in microglia conditioned media. Cx32 mimetic peptide reduced microglial glutamate release, which revealed that activated microglia release glutamate from Cx32 hemichannels. Data represent the means ± SD (n = 6 per group). *, P<0.05 vs. PBS. (B) Glutamate concentrations in media from primary cell cultures of mouse brain. Only the microglia released a significant amount of glutamate after stimulation with 1 µg/ml LPS for 24 h. Neu, neuron; Ast, astrocyte; Mi, microglia. Data represent the means ± SD (n = 6 per group). *, P<0.05 vs. fresh medium. (C) Real-time glutamate concentration monitoring in the hippocampi of mice. All mice were injected LPS at time zero except PBS-treated mice. Blue, PBS-treated mice; black, LPS-treated mice; orange, LPS plus 20 mg/kg CBX-treated mice; red, LPS plus 20 mg/kg INI-0602-treated mice. Data represent the means ± SE (n = 8 per group). *, P<0.05 vs. LPS.
Figure 3
Figure 3. INI-0602 ameliorates disease symptoms in both SOD1 G93A Tg mice and SOD1 G37R Tg mice.
(A) A representative photograph of 20-week-old SOD1 G93A Tg mice treated with PBS (left) or 10 mg/kg INI-0602 (right). (B) Survival rates of SOD1 G93A Tg mice (n = 24 per group). Black, PBS; light blue, 5 mg/kg (P<0.05); red, 10 mg/kg (P<0.00001); green, 20 mg/kg (P<0.00001); purple, 40 mg/kg INI-0602 (P<0.05). (C–G) Microscopic images of L5 roots from SOD1 G93A Tg mice treated with PBS (C), 5 mg/kg (D), 10 mg/kg (E), 20 mg/kg (F), or 40 mg/kg INI-0602 (G). (H) Body weights of SOD1 G93A Tg mice (n = 24 per group). Black, PBS; light blue, 5 mg/kg (P<0.05); red, 10 mg/kg (P<0.05); green, 20 mg/kg (P<0.05); purple, 40 mg/kg INI-0602 (P<0.05). (I) Survival rates and (J) body weights of SOD1 G37R Tg mice (n = 8 per group). Black, PBS; light blue, 5 mg/kg; red, 10 mg/kg (P<0.05); green, 20 mg/kg INI-0602 (P<0.05).
Figure 4
Figure 4. INI-0602 prevents neuronal damage and gliosis in lumber spinal cords of SOD1 G93A Tg mice.
(A–B) Fluorescent microscopic images of the lumber spinal cords of 20-week-old SOD1 G93A Tg mice treated with PBS (A) or 10 mg/kg INI-0602 (B). Green, neurons (MAP2); red, microglia (CD11b); blue, astrocytes (GFAP). Scale bar, 200 µm. (C–J) Higher magnification images of the boxed areas in a (C, E, G, and I) and b (D, F, H, and J). Scale bar, 30 µm. Each graph shows the percentage of area occupied by neurons, microglia, and astrocytes, respectively. Data represent the means ± SD (n = 6 per group). *, P<0.05 vs. PBS. (K, L) Fluorescent microscopic images of MAP2 staining in the anterior horns of lumber spinal cords from 14-week-old SOD1 G93A Tg mice treated with PBS (K) or 10 mg/kg INI-0602 (L). Arrows indicate neuritic bead formation, an early marker of neuronal dysfunction. Scale bar, 10 µm.
Figure 5
Figure 5. INI-0602 improves memory deficits in APP/PS1 Tg mice.
(A) Object recognition memory assessment by a novel-object recognition test using 9-month-old mice. Data represent the means ± SD (WT, wild-type mice, n = 22; Tg-PBS, PBS-treated Tg mice, n = 22; Tg-10 mg/kg, 10 mg/kg INI-0602-treated Tg mice, n = 16; Tg-20 mg/kg, 20 mg/kg INI-0602-treated Tg mice, n = 15). *, P<0.05 vs. WT; †, P<0.05 vs. Tg-PBS. (B) Associative learning assessment by cued and contextual fear conditioning tests using 10-month-old mice. We assessed the same cohort of mice used in A. Data represent the means ± SD (WT, n = 13; Tg-PBS, n = 13; Tg-10 mg/kg, n = 16; Tg-20 mg/kg, n = 15). *, P<0.05 vs. WT; †, P<0.05 vs. Tg-PBS. (C–D) Reference memory assessment using the distance moved (C) and the percentage of time spent searching (D) during a 60-sec session in a Morris water maze test using 11-month-old mice. We assessed the same cohort of mice used in A. Data represent the means ± SD (WT, n = 22; Tg-PBS, n = 22; Tg-10 mg/kg, n = 16; Tg-20 mg/kg, n = 15). *, P<0.05 vs. WT; †, P<0.05 vs. Tg-PBS.
Figure 6
Figure 6. INI-0602 does not affect Aβ deposition or glial activation.
(A–T) Fluorescent microscopic images of hippocampi from 11-month-old APP/PS1 Tg mice. Scale bar, 200 µm. (U–W) Percentage of area occupied by Aß (U), microglia (V), and astrocytes (W). Data represent the means ± SD (n = 6 per group). (X) Human Aß1–40- and Aß1–42-specific ELISAs using homogenized brains from 11-month-old APP/PS1 Tg mice. Data represent the means ± SD (n = 6 per group). (Y) Western blot analysis of oligomeric Aβ extracted from 11-month-old APP/PS1 Tg mice. *, oligomeric Aβ. Data represent the means ± SD (n = 3 per group). WT, wild-type mice; Tg-PBS, PBS-treated Tg mice; Tg-10 mg/kg, 10 mg/kg INI-0602-treated Tg mice; Tg-20 mg/kg, 20 mg/kg INI-0602-treated Tg mice.
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