KATP Channel Opener Diazoxide Prevents Neurodegeneration: A New Mechanism of Action via Antioxidative Pathway Activation

Pharmacological modulation of ATP-sensitive potassium channels has become a promising new therapeutic approach for the treatment of neurodegenerative diseases due to their role in mitochondrial and cellular protection. For instance, diazoxide, a well-known ATP-sensitive potassium channel activator with high affinity for mitochondrial component of the channel has been proved to be effective in animal models for different diseases such as Alzheimer's disease, stroke or multiple sclerosis. Here, we analyzed the ability of diazoxide for protecting neurons front different neurotoxic insults in vitro and ex vivo. Results showed that diazoxide effectively protects NSC-34 motoneurons from glutamatergic, oxidative and inflammatory damage. Moreover, diazoxide decreased neuronal death in organotypic hippocampal slice cultures after exicitotoxicity and preserved myelin sheath in organotypic cerebellar cultures exposed to pro-inflammatory demyelinating damage. In addition, we demonstrated that one of the mechanisms of actions implied in the neuroprotective role of diazoxide is mediated by the activation of Nrf2 expression and nuclear translocation. Nrf2 expression was increased in NSC-34 neurons in vitro as well as in the spinal cord of experimental autoimmune encephalomyelitis animals orally administered with diazoxide. Thus, diazoxide is a neuroprotective agent against oxidative stress-induced damage and cellular dysfunction that can be beneficial for diseases such as multiple sclerosis

Correction: KATP Channel Opener Diazoxide Prevents Neurodegeneration: A New Mechanism of Action via Antioxidative Pathway Activation.

[This corrects the article DOI: 10.1371/journal.pone.0075189.]

K(ATP) channel opener diazoxide prevents neurodegeneration: a new mechanism of action via antioxidative pathway activation.

Pharmacological modulation of ATP-sensitive potassium channels has become a promising new therapeutic approach for the treatment of neurodegenerative diseases due to their role in mitochondrial and cellular protection. For instance, diazoxide, a well-known ATP-sensitive potassium channel activator with high affinity for mitochondrial component of the channel has been proved to be effective in animal models for different diseases such as Alzheimer's disease, stroke or multiple sclerosis. Here, we analyzed the ability of diazoxide for protecting neurons front different neurotoxic insults in vitro and ex vivo. Results showed that diazoxide effectively protects NSC-34 motoneurons from glutamatergic, oxidative and inflammatory damage. Moreover, diazoxide decreased neuronal death in organotypic hippocampal slice cultures after exicitotoxicity and preserved myelin sheath in organotypic cerebellar cultures exposed to pro-inflammatory demyelinating damage. In addition, we demonstrated that one of the mechanisms of actions implied in the neuroprotective role of diazoxide is mediated by the activation of Nrf2 expression and nuclear translocation. Nrf2 expression was increased in NSC-34 neurons in vitro as well as in the spinal cord of experimental autoimmune encephalomyelitis animals orally administered with diazoxide. Thus, diazoxide is a neuroprotective agent against oxidative stress-induced damage and cellular dysfunction that can be beneficial for diseases such as multiple sclerosis

A Gain-of-Function Suppressor Screen for Genes Involved in Dorsal–Ventral Boundary Formation in the Drosophila Wing

The Drosophila wing primordium is subdivided into a dorsal (D) and a ventral (V) compartment by the activity of the LIM-homeodomain protein Apterous in D cells. Cell interactions between D and V cells induce the activation of Notch at the DV boundary. Notch is required for the maintenance of the compartment boundary and the growth of the wing primordium. Beadex, a gain-of-function allele of dLMO, results in increased levels of dLMO protein, which interferes with the activity of Apterous and results in defects in DV axis formation. We performed a gain-of-function enhancer-promoter (EP) screen to search for suppressors of Beadex when overexpressed in D cells. We identified 53 lines corresponding to 35 genes. Loci encoding for micro-RNAs and proteins involved in chromatin organization, transcriptional control, and vesicle trafficking were characterized in the context of dLMO activity and DV boundary formation. Our results indicate that a gain-of-function genetic screen in a sensitized background, as opposed to classical loss-of-function-based screenings, is a very efficient way to identify redundant genes involved in a developmental process

Diazoxide effects in NMDA-induced neurodegeneration in hippocampal slice culture.

<p>At 7 DIV, treatment with 10 µM NMDA for 4 h induced a region-specific increase neuronal cell death of the CA1 layer compared to undamaged slices, as determined as PI uptake (PI, red) in colocalization with the neuronal nuclear marker NeuN (green) (A, B). Treatment 30 min before NMDA lesion with diazoxide prevented cell death, significantly at 100 µM but also at lowest dose 1 µM (C, D). Upon quantification, results showed a significant decrease at 100 µM (65±5% in PI uptake compared to control) and 1 µM (67±7% in PI uptake compared to control (E). When microglia was removed from the slices, cell protection remained at the lowest dose 1 µM (62±10% in PI uptake compared to control) (F-J). Data are a summary of four to seven individual experiments with 6 slices per conditions analyzed in each experiment. Results are shown as mean ± SEM. * p<0.05, ** p<0.01. Scale bar 300 µm</p

Diazoxide prevents neuronal damage after different neurotoxic insults.

<p>Representative images of differentiated NSC-34 motoneuron cells line after glutamate damage and diazoxide treatment (A). Diazoxide inhibited glutamate mediated cell death by 24 h in differentiated NSC-34 motoneuron cell line (B). Diazoxide inhibited H₂O₂ mediated cell death by 24 h (C) and also when used as pretreatment during 2 h (survival analysis performed 24 h after toxic insult) (D). Diazoxide inhibited inflammatory BV2 microglial mediated cell death by 24 h in NSC-34 motoneuron. Conditioned Medium (CM): Neurons damaged with activated BV2 medium. CM (Dxz to NSC-34): Neurons treated with diazoxide and damaged with activated BV2 medium afterwards. CM (Dzx to BV2): Neurons damaged with diazoxide treated BV2 activated medium (E). Diazoxide treatment 100 µM. Results expressed as mean ± SEM. n≥4 experiments. *: p < 0.05. Scale bar =  30 µm</p

Diazoxide increases Nrf2 nuclear translocation in NSC-34 motoneurons and prevents endogenous oxidative damage.

<p>Western blot showed an increase of Nrf2 signaling in the nuclear extracts from NSC-34 neurons treated with different doses of diazoxide for 24 h. The higher increase of Nrf2 was found at lower doses (10 and 1 µM) (A). Cell viability of NSC-34 cells was measured after 24 h AAPH oxidative stress activation and results demonstrated that diazoxide treatment effectively ameliorates cell viability at low doses (B). Results expressed as mean ± SEM. n≥4 experiments. *: p < 0.05, ** p<0.01. Scale bar  =  30 µm</p