Image2_NQO1 protects against clioquinol toxicity.TIF

Clioquinol (CQ) was widely used as oral antibiotic before being taken off the market in many countries in 1970, after it was linked to subacute myelo-optic neuropathy (SMON) in Japan, leading to vision loss with many patients left wheelchair-bound. The common pathology of CQ-associated SMON was reproduced in animals but none of the proposed modes of toxicity explained the restriction of CQ-induced SMON to Japan. Given a re-emergence of CQ and related analogues as neuroprotectants, it is crucial to understand the underlying mechanism of CQ-induced toxicity to prevent any potential CQ-associated risks to future patients. A small molecule screen to find drugs that induce mitochondrial dysfunction in vitro identified CQ and the structurally related 8-hydroxyquinoline (8-OHQ). Their mitochondrial liability, pro-oxidative and cytotoxic activity was subsequently confirmed in some cell lines but surprisingly not in others. Subsequent studies in isogenic cell lines demonstrated that the antioxidant protein NQO1 is differentially expressed in the cell lines tested and potently protects against CQ toxicity. CQ-induced reduction of cellular ATP levels, increased lipid peroxidation and elevated cell death was also attenuated by antioxidants, implicating oxidative stress as the core mechanism of CQ-induced toxicity. These in-vitro findings were replicated in zebrafish. Visual acuity in zebrafish larvae that do not express NQO1, was reduced by CQ in a dose-dependent manner, while CQ did not affect visual function in the adult zebrafish that express NQO1. Similarly, pharmacological inhibition of NQO1 activity resulted in CQ-induced oxidative stress in the retina and severe acute systemic toxicity in the adult fish. Given the much higher prevalence of the inactivating C609T NQO1 polymorphism in the Japanese population compared to the European population, the results of this study could for the first time indicate how the geographic restriction of SMON cases to Japan could be explained. Importantly, if CQ or its derivatives are to be used safely for the treatment of neurodegenerative diseases, it seems imperative that NQO1 levels and activity of prospective patients should be ascertained.</p

Fucoidan Extracts Ameliorate Acute Colitis

<div><p>Inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn’s disease, are an important cause of morbidity and impact significantly on quality of life. Overall, current treatments do not sustain a long-term clinical remission and are associated with adverse effects, which highlight the need for new treatment options. Fucoidans are complex sulphated, fucose-rich polysaccharides, found in edible brown algae and are described as having multiple bioactivities including potent anti-inflammatory effects. Therefore, the therapeutic potential of two different fucoidan preparations, fucoidan-polyphenol complex (Maritech <i>Synergy</i>) and depyrogenated fucoidan (DPF) was evaluated in the dextran sulphate sodium (DSS) mouse model of acute colitis. Mice were treated once daily over 7 days with fucoidans via oral (<i>Synergy</i> or DPF) or intraperitoneal administration (DPF). Signs and severity of colitis were monitored daily before colons and spleens were collected for macroscopic evaluation, cytokine measurements and histology. Orally administered <i>Synergy</i> and DPF, but not intraperitoneal DPF treatment, significantly ameliorated symptoms of colitis based on retention of body weight, as well as reduced diarrhoea and faecal blood loss, compared to the untreated colitis group. Colon and spleen weight in mice treated with oral fucoidan was also significantly lower, indicating reduced inflammation and oedema. Histological examination of untreated colitis mice confirmed a massive loss of crypt architecture and goblet cells, infiltration of immune cells and oedema, while all aspects of this pathology were alleviated by oral fucoidan. Importantly, in this model, the macroscopic changes induced by oral fucoidan correlated significantly with substantially decreased production of at least 15 pro-inflammatory cytokines by the colon tissue. Overall, oral fucoidan preparations significantly reduce the inflammatory pathology associated with DSS-induced colitis and could therefore represent a novel nutraceutical option for the management of IBD.</p></div

Dose-effect of idebenone treatment on multiple endpoints in an <i>in vivo</i> mouse model for LHON.

<p>Dose-effect of idebenone treatment on multiple endpoints in an <i>in vivo</i> mouse model for LHON.</p

Effect of fucoidan extracts during acute colitis.

<p>(A) Daily changes of body weight during colitis induction in C57BL/6 mice with and without fucoidan extracts treatment versus healthy control. Body weight changes, expressed as percentage, were calculated by dividing body weight on each day with the initial body weight before the start of DSS treatment. Stool samples were scored for (B) consistency and (C) occult blood on a daily basis during experiment. Data represent percentage or mean ± SD of n = 6–10 animals. Significance is indicated by *<i>p</i> < 0.05 and **<i>p</i> < 0.01 using two-way ANOVA followed by Tukey’s post-test. Healthy control (HC); untreated colitis (DSS); intraperitoneal injection of depyrogenated fucoidan (IPDPF); oral treatment of depyrogenated fucoidan (ODPF); oral treatment of Maritech <i>Synergy</i> (OS).</p

Idebenone Protects against Retinal Damage and Loss of Vision in a Mouse Model of Leber’s Hereditary Optic Neuropathy

<div><p>Leber’s hereditary optic neuropathy (LHON) is an inherited disease caused by mutations in complex I of the mitochondrial respiratory chain. The disease is characterized by loss of central vision due to retinal ganglion cell (RGC) dysfunction and optic nerve atrophy. Despite progress towards a better understanding of the disease, no therapeutic treatment is currently approved for this devastating disease. Idebenone, a short-chain benzoquinone, has shown promising evidence of efficacy in protecting vision loss and in accelerating recovery of visual acuity in patients with LHON. It was therefore of interest to study suitable LHON models <i>in vitro</i> and <i>in vivo</i> to identify anatomical correlates for this protective activity. At nanomolar concentrations, idebenone protected the rodent RGC cell line RGC-5 against complex I dysfunction <i>in vitro.</i> Consistent with the reported dosing and observed effects in LHON patients, we describe that in mice, idebenone penetrated into the eye at concentrations equivalent to those which protected RGC-5 cells from complex I dysfunction <i>in vitro</i>. Consequently, we next investigated the protective effect of idebenone in a mouse model of LHON, whereby mitochondrial complex I dysfunction was caused by exposure to rotenone. In this model, idebenone protected against the loss of retinal ganglion cells, reduction in retinal thickness and gliosis. Furthermore, consistent with this protection of retinal integrity, idebenone restored the functional loss of vision in this disease model. These results support the pharmacological activity of idebenone and indicate that idebenone holds potential as an effective treatment for vision loss in LHON patients.</p></div

Pharmacokinetic analysis of idebenone in eye fluids.

<p>Concentrations of idebenone in aqueous humor (open circle) and vitreous humor (filled triangle) following once daily administration of idebenone in the diet for 21 days (A) and following single oral administration of idebenone at 60 mg/kg (B) were determined. Concentrations of idebenone in the eye fluids are expressed as ng/ml (left y axis) and nM (right y axis). For (A), sampling time was more than 8h after last dose of idebenone, the values therefore represent trough levels.</p

Effect of fucoidan extracts on colon histology.

<p>Representative hematoxylin and eosin stained colon sections of healthy controls, untreated mice with colitis and colitic mice that received fucoidan extracts. Scale bars = 100 μm for 400× and 400 μm for 100× magnification. Healthy control (HC); untreated colitis (DSS); intraperitoneal injection of depyrogenated fucoidan (IPDPF); oral treatment of depyrogenated fucoidan (ODPF); oral treatment of Maritech <i>Synergy</i> (OS).</p

Effect of fucoidan extracts on colon-derived cytokine levels.

<p>Distal colon tissue samples were cultured for 24 hours. The supernatants were assessed for cytokine levels using a Bio-Plex assay kit. Cytokine levels in the supernatant were normalized to tissue weight to obtain pg / ml of cytokines/ 10 mg of tissue. Data represent minimum, 25th percentile, median, mean, 75 percentile and maximum of cytokine levels of n = 5 animals. Significance is indicated by *<i>p</i> < 0.05 and **<i>p</i> < 0.01 using one-way ANOVA followed by Dunnett’s post-test. Interleukin (IL); tumor necrosis factor-α (TNF-α); granulocyte colony-stimulating factor (G-CSF); granulocyte-macrophage colony-stimulating factor (GM-CSF); macrophage inflammatory protein (MIP); regulated and normal T cells expressed and secreted (RANTES); interferon-γ (IFN-γ); healthy control (open circle); untreated colitis (closed circle); intraperitoneal injection of depyrogenated fucoidan (closed square); oral treatment of depyrogenated fucoidan (closed diamond); oral treatment of Maritech <i>Synergy</i> (closed triangle).</p

Idebenone prevents rotenone-induced RGC death. Analysis of RGCs in the mouse retina 7 days after intravitreal injection of rotenone or DMSO (Sham).

<p>Mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-Brn3a primary antibody to visualize RGCs. Scale bar = 25 µm. (B) Quantification of RGCs (RGC number/mm) following idebenone treatment and rotenone injection (n = 6 to 10 per group). Data expressed as mean ± SEM.</p

Idebenone time-dependently restores rotenone-induced loss of vision.

<p>Visual acuity was evaluated by counting the number of head movements at a velocity of 3 rpm 7 days prior injection (day -7) and 1 to 70 days after injection (day 1, 7, 21, 35, 70) of rotenone (5 mM). Mice were treated with dietary idebenone 2000 mg/kg or vehicle for the time of the experiment. (A) Schematic representation of the experimental setup. After vehicle (DMSO) injection, head movements in both directions of drum rotation (clockwise: CW; counterclockwise CCW) remain intact. Injection of rotenone into the left eye however, affects only the CW responses (dashed arrow) whereas CCW responses remain unaffected. (B) Quantification of clockwise (CW) head movement (number of head movements/2 min) following vehicle treatment and rotenone injection (vehicle + rotenone, n = 10 animals), and idebenone 2000 mg/kg treatment and rotenone injection (IDE 2000+ rotenone, n = 11 animals). Data are expressed as mean ± SEM. Statistical significance relative to vehicle + rotenone group: p≤0.05 (*); (C) Percentage of mice showing clockwise (CW) head movements for each treatment group 70 days after injection. Responder: group of mice showing head movements; Non-responder: group of mice without head movements.</p