Photochemical activation of TRPA1 channels in neurons and animals

Optogenetics is a powerful research tool because it enables high-resolution optical control of neuronal activity. However, current optogenetic approaches are limited to transgenic systems expressing microbial opsins and other exogenous photoreceptors. Here, we identify optovin, a small molecule that enables repeated photoactivation of motor behaviors in wild type animals. Surprisingly, optovin's behavioral effects are not visually mediated. Rather, photodetection is performed by sensory neurons expressing the cation channel TRPA1. TRPA1 is both necessary and sufficient for the optovin response. Optovin activates human TRPA1 via structure-dependent photochemical reactions with redox-sensitive cysteine residues. In animals with severed spinal cords, optovin treatment enables control of motor activity in the paralyzed extremities by localized illumination. These studies identify a light-based strategy for controlling endogenous TRPA1 receptors in vivo, with potential clinical and research applications in non-transgenic animals, including humans

Metabolic State Determines Sensitivity to Cellular Stress in Huntington Disease: Normalization by Activation of PPARγ

Impairments in mitochondria and transcription are important factors in the pathogenesis of Huntington disease (HD), a neurodegenerative disease caused by a polyglutamine expansion in the huntingtin protein. This study investigated the effect of different metabolic states and peroxisome proliferator-activated receptor γ (PPARγ) activation on sensitivity to cellular stressors such as H2O2 or thapsigargin in HD. Striatal precursor cells expressing wild type (STHdhQ7) or mutant huntingtin (STHdhQ111) were prepared in different metabolic conditions (glucose vs. pyruvate). Due to the fact that STHdhQ111 cells exhibit mitochondrial deficits, we expected that in the pyruvate condition, where ATP is generated primarily by the mitochondria, there would be greater differences in cell death between the two cell types compared to the glucose condition. Intriguingly, it was the glucose condition that gave rise to greater differences in cell death. In the glucose condition, thapsigargin treatment resulted in a more rapid loss of mitochondrial membrane potential (ΔΨm), a greater activation of caspases (3, 8, and 9), and a significant increase in superoxide/reactive oxygen species (ROS) in STHdhQ111 compared to STHdhQ7, while both cell types showed similar kinetics of ΔΨm-loss and similar levels of superoxide/ROS in the pyruvate condition. This suggests that bioenergetic deficiencies are not the primary contributor to the enhanced sensitivity of STHdhQ111 cells to stressors compared to the STHdhQ7 cells. PPARγ activation significantly attenuated thapsigargin-induced cell death, concomitant with an inhibition of caspase activation, a delay in ΔΨm loss, and a reduction of superoxide/ROS generation in STHdhQ111 cells. Expression of mutant huntingtin in primary neurons induced superoxide/ROS, an effect that was significantly reduced by constitutively active PPARγ. These results provide significant insight into the bioenergetic disturbances in HD with PPARγ being a potential therapeutic target for HD

Primers used for real-time PCR.

<p>PGC-1α, peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α; SIRT1, sirtuin 1; CytC, cytochrome C; RXRα, retinoid X receptor α; UCP, uncoupling protein; SOD, superoxide dismutase; SERCA2, sarco(endo)plasmic reticulum Ca²⁺-ATPase 2; CypD, cyclophilin D; TBP, TATA binding protein.</p

Metabolic conditions differentiate ΔΨm-loss in response to TG.

<p><i>A</i>, In the glucose condition STHdh^Q111 cells undergo ΔΨm-loss in response to TG at a significantly faster rate than STHdh^Q7 cells. In the pyruvate condition, ΔΨm of both cell types shows similar kinetics in response to TG. Arrows indicate the point at which ΔΨm begins to drop below the baseline. <i>n</i> = 4. <i>B</i>, RSG slightly but significantly delays TG-induced ΔΨm-loss in glucose and pyruvate conditions. GW9662 abrogates the delayed ΔΨm-loss by RSG treatment. <i>n</i> = 5–9. Data shown are mean ± SE. * <i>P</i><0.05.</p

Mutant huntingtin expression sensitizes striatal cells to stressors in the glucose condition.

<p><i>A</i>, H₂O₂ treatment in the glucose condition results in significantly greater cell death in STHdh^Q111 than STHdh^Q7 cells, while both cell types show similar cell death responses to H₂O₂ in pyruvate condition. <i>n</i> = 4. <i>B</i>, RSG treatment does not protect striatal cells from H₂O₂ toxicity. <i>n</i> = 3–4. <i>C</i>, TG treatment in the glucose condition results in much greater cell death in STHdh^Q111 than STHdh^Q7 cells, while both cell types show similar cell death responses to TG in the pyruvate condition. <i>n</i> = 4–6. <i>D</i>, RSG significantly attenuates TG-induced cell death in the glucose and pyruvate conditions. <i>n</i> = 3–4. RP, rolipram. Data shown are mean ± SE. * <i>P</i><0.05, ** <i>P</i><0.01.</p

Mutant huntingtin results in repressed transcriptional activities, and reduced PPARγ activity is independent of the protein level.

<p>B3 and E4 STHdh^Q7 cells and 1A and 6L STHdh^Q111 cells were transiently transfected with PPRE, CRE, or PGC-1α promoter luciferase reporter plasmids. The basal activities of PPRE (<i>A</i>), CRE (<i>B</i>), and PGC-1α promoter (<i>C</i>) reporters were dramatically reduced in both 1A and 6L STHdh^Q111 cells. <i>n</i> = 3–4 Data shown are mean ± SE. <i>D</i>, PPARγ protein levels were variable in B3 and E4 STHdh^Q7 cells and 1A and 6L STHdh^Q111 cells, while the original STHdh^Q111 cells exhibited lower levels of PPARγ than the original STHdh^Q7 cells. Sixty micrograms of protein was run in each lane.</p

Metabolic conditions differentially affect superoxide/ROS generation.

<p><i>A</i>, In the glucose condition TG-induced superoxide/ROS generation in STHdh^Q111 cells was greater compared to STHdh^Q7 cells, while in the pyruvate condition TG induced superoxide/ROS generation to a similar extent in both cell types. <i>B</i>, The percentage of DHE positive cells was calculated. Quantitative data shows the differential effects of metabolic conditions on TG-induced superoxide/ROS generation as shown in <i>A</i>. <i>n</i> = 6. STHdh^Q111 cells display significantly higher basal levels of superoxide/ROS compared to STHdh^Q7 cells. Data shown are mean ± SE. ** <i>P</i><0.01, *** <i>P</i><0.001.</p

The protective effect of RSG is due to the specific activation of PPARγ.

<p><i>A</i>, GW9662 (GW), a PPARγ antagonist, abolishes the protective effect of RSG on TG-induced cell death in the glucose and pyruvate conditions. <i>n</i> = 4–6. 40 µM GW9662 was added in the presence or absence of 20 µM RSG for 24 h prior to TG treatment. <i>B</i>, RSG increases the expression of genes involved in mitochondrial function (CytC, UCP2, UCP4, UCP5), calcium regulation (SERCA2), and ROS response (SOD1, SOD2), but does not change the expression of PPARγ and SIRT1. <i>n</i> = 4–6. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 vs. control of STHdh^Q7; ^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001 vs. control of each cell type. Statistical significance was determined by one-way ANOVA followed by Student-Newman-Keuls multiple comparisons test. <i>C</i>, PGC-1α promoter is slightly but significantly activated by RSG in STHdh^Q111 cells. The mutation at PPRE but not CRE sites in PGC-1α promoter completely abolishes PGC-1α promoter activation induced by RSG treatment. <i>n</i> = 4–6. ** <i>P</i><0.01, *** <i>P</i><0.001 vs. control; ^#<i>P</i><0.05 vs. RSG. <i>D</i>, RSG increases the protein level of CytC in STHdh^Q111 but not STHdh^Q7 cells. Data shown are mean ± SE.</p

RSG reduces TG-induced caspase activation.

<p><i>A</i>, TG induces the cleaved forms of caspase 3 and PARP in both cell types, responses that were reduced by RSG. <i>B</i>, TG increases caspase 3 activity of STHdh^Q111 cells to a significantly greater extent compared to STHdh^Q7 cells, a response that was significantly attenuated by RSG. Basal activity of caspase 3 in STHdh^Q111 cells was significantly higher compared to STHdh^Q7 cells. <i>n</i> = 5. <i>C</i>, TG increases caspase 8 activity in STHdh^Q111 but not STHdh^Q7 cells, a response that was significantly attenuated by RSG. Basal activity of caspase 8 of STHdh^Q111 cells is significantly higher compared to STHdh^Q7 cells. <i>n</i> = 5. <i>D</i>, TG increases caspase 9 activity in STHdh^Q111 but not STHdh^Q7 cells, a response that was significantly attenuated by RSG. Basal activity (<i>D</i>) and basal level (<i>E</i>) of caspase 9 in STHdh^Q111 cells are significantly higher compared to STHdh^Q7 cells. <i>n</i> = 4–5. Data shown are mean ± SE. ** <i>P</i><0.01, *** <i>P</i><0.001 vs. control of each cell type; ^##<i>P</i><0.01, ^###<i>P</i><0.001 vs. TG of each cell type; ⁺<i>P</i><0.05, ⁺⁺<i>P</i><0.01 vs. control of STHdh^Q7. Statistical significance was determined by one-way ANOVA followed by Student-Newman-Keuls multiple comparisons test (<i>B</i>, <i>C</i>, and <i>D</i>) or by Student's <i>t</i> test (<i>E</i>).</p

Impaired mitochondrial dynamics and Nrf2 signaling contribute to compromised responses to oxidative stress in striatal cells expressing full-length mutant huntingtin.

<div><p>Huntington disease (HD) is an inherited neurodegenerative disease resulting from an abnormal expansion of polyglutamine in huntingtin (Htt). Compromised oxidative stress defense systems have emerged as a contributing factor to the pathogenesis of HD. Indeed activation of the Nrf2 pathway, which plays a prominent role in mediating antioxidant responses, has been considered as a therapeutic strategy for the treatment of HD. Given the fact that there is an interrelationship between impairments in mitochondrial dynamics and increased oxidative stress, in this present study we examined the effect of mutant Htt (mHtt) on these two parameters. STHdh^Q111/Q111 cells, striatal cells expressing mHtt, display more fragmented mitochondria compared to STHdh^Q7/Q7 cells, striatal cells expressing wild type Htt, concurrent with alterations in the expression levels of Drp1 and Opa1, key regulators of mitochondrial fission and fusion, respectively. Studies of mitochondrial dynamics using cell fusion and mitochondrial targeted photo-switchable Dendra revealed that mitochondrial fusion is significantly decreased in STHdh^Q111/Q111 cells. Oxidative stress leads to dramatic increases in the number of STHdh^Q111/Q111 cells containing swollen mitochondria, while STHdh^Q7/Q7 cells just show increases in the number of fragmented mitochondria. mHtt expression results in reduced activity of Nrf2, and activation of the Nrf2 pathway by the oxidant tBHQ is significantly impaired in STHdh^Q111/Q111 cells. Nrf2 expression does not differ between the two cell types, but STHdh^Q111/Q111 cells show reduced expression of Keap1 and p62, key modulators of Nrf2 signaling. In addition, STHdh^Q111/Q111 cells exhibit increases in autophagy, whereas the basal level of autophagy activation is low in STHdh^Q7/Q7 cells. These results suggest that mHtt disrupts Nrf2 signaling which contributes to impaired mitochondrial dynamics and may enhance susceptibility to oxidative stress in STHdh^Q111/Q111 cells.</p> </div