Youngnam N. Jin

Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52.

Nuclear factor E2-related factor 2 (Nrf2) is a pivotal transcription factor in the defense against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its over-expression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2 knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

Rosiglitazone Treatment Prevents Mitochondrial Dysfunction in Mutant Huntingtin-expressing Cells: POSSIBLE ROLE OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-γ (PPARγ) IN THE PATHOGENESIS OF HUNTINGTON DISEASE*

Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the PPAR family of transcription factors. Synthetic PPARγ agonists are used as oral anti-hyperglycemic drugs for the treatment of non-insulin-dependent diabetes. However, emerging evidence indicates that PPARγ activators can also prevent or attenuate neurodegeneration. Given these previous findings, the focus of this report is on the potential neuroprotective role of PPARγ activation in preventing the loss of mitochondrial function in Huntington disease (HD). For these studies we used striatal cells that express wild-type (STHdhQ7/Q7) or mutant (STHdhQ111/Q111) huntingtin protein at physiological levels. Treatment of mutant cells with thapsigargin resulted in a significant decrease in mitochondrial calcium uptake, an increase in reactive oxygen species production, and a significant decrease in mitochondrial membrane potential. PPARγ activation by rosiglitazone prevented the mitochondrial dysfunction and oxidative stress that occurred when mutant striatal cells were challenged with pathological increases in calcium. The beneficial effects of rosiglitazone were likely mediated by activation of PPARγ, as all protective effects were prevented by the PPARγ antagonist GW9662. Additionally, the PPARγ signaling pathway was significantly impaired in the mutant striatal cells with decreases in PPARγ expression and reduced PPARγ transcriptional activity. Treatment with rosiglitazone increased mitochondrial mass levels, suggesting a role for the PPARγ pathway in mitochondrial function in striatal cells. Altogether, this evidence indicates that PPARγ activation by rosiglitazone attenuates mitochondrial dysfunction in mutant huntingtin-expressing striatal cells, and this could be an important therapeutic avenue to ameliorate the mitochondrial dysfunction that occurs in HD.

Truncated tau and Aβ cooperatively impair mitochondria in primary neurons

Mitochondrial dysfunction is likely a significant contributing factor to Alzheimer disease pathogenesis, and both amyloid peptide (Aβ) and pathological forms of tau may contribute to this impairment. Cleavage of tau at Asp421 occurs early in Alzheimer disease, and Asp421-cleaved tau likely negatively impacts neuronal function. Previously we showed that expression of Asp421-cleaved tau in a neuronal cell model resulted in mitochondrial impairment. To extend these findings we expressed either full length tau or Asp421-cleaved tau (truncated tau) in primary cortical neurons and measured different aspects of mitochondrial function with or without the addition of sublethal concentrations of Aβ. The expression of truncated tau alone induced significant mitochondrial fragmentation in neurons. When truncated tau expression was combined with Aβ at sublethal concentrations, increases in the stationary mitochondrial population and the levels of oxidative stress in cortical neurons were observed. Truncated tau expression also enhanced Aβ-induced mitochondrial potential loss in primary neurons. These new findings show that Asp421-cleaved tau and Aβ cooperate to impair mitochondria, which likely contributes to the neuronal dysfunction in Alzheimer disease.

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

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. STHdhQ111/Q111 cells, striatal cells expressing mHtt, display more fragmented mitochondria compared to STHdhQ7/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 STHdhQ111/Q111 cells. Oxidative stress leads to dramatic increases in the number of STHdhQ111/Q111 cells containing swollen mitochondria, while STHdhQ7/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 STHdhQ111/Q111 cells. Nrf2 expression does not differ between the two cell types, but STHdhQ111/Q111 cells show reduced expression of Keap1 and p62, key modulators of Nrf2 signaling. In addition, STHdhQ111/Q111 cells exhibit increases in autophagy, whereas the basal level of autophagy activation is low in STHdhQ7/Q7 cells. These results suggest that mHtt disrupts Nrf2 signaling which contributes to impaired mitochondrial dynamics and may enhance susceptibility to oxidative stress in STHdhQ111/Q111 cells.

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, optovins 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.

Mitochondrial permeability transition pore induces mitochondria injury in Huntington disease

BackgroundMitochondrial impairment has been implicated in the pathogenesis of Huntington’s disease (HD). However, how mutant huntingtin impairs mitochondrial function and thus contributes to HD has not been fully elucidated. In this study, we used striatal cells expressing wild type (STHdhQ7/Q7) or mutant (STHdhQ111/Q111) huntingtin protein, and cortical neurons expressing the exon 1 of the huntingtin protein with physiological or pathological polyglutamine domains, to examine the interrelationship among specific mitochondrial functions.ResultsDepolarization induced by KCl resulted in similar changes in calcium levels without compromising mitochondrial function, both in wild type and mutant cells. However, treatment of mutant cells with thapsigargin (a SERCA antagonist that raises cytosolic calcium levels), resulted in a pronounced decrease in mitochondrial calcium uptake, increased production of reactive oxygen species (ROS), mitochondrial depolarization and fragmentation, and cell viability loss. The mitochondrial dysfunction in mutant cells was also observed in cortical neurons expressing exon 1 of the huntingtin protein with 104 Gln residues (Q104-GFP) when they were exposed to calcium stress. In addition, calcium overload induced opening of the mitochondrial permeability transition pore (mPTP) in mutant striatal cells. The mitochondrial impairment observed in mutant cells and cortical neurons expressing Q104-GFP was prevented by pre-treatment with cyclosporine A (CsA) but not by FK506 (an inhibitor of calcineurin), indicating a potential role for mPTP opening in the mitochondrial dysfunction induced by calcium stress in mutant huntingtin cells.ConclusionsExpression of mutant huntingtin alters mitochondrial and cell viability through mPTP opening in striatal cells and cortical neurons.

The interrelationship between mitochondrial dysfunction and transcriptional dysregulation in Huntington disease

Huntington disease (HD) is an inherited neurodegenerative disease caused by an abnormal expansion of the CAG repeat region in the huntingtin (Htt) gene. Although the pathogenic mechanisms by which mutant Htt (mHtt) causes HD have not been fully elucidated, it is becoming increasingly apparent that mHtt can impair mitochondrial function directly, as well as indirectly by dysregulation of transcriptional processes. mHtt causes increased sensitivity to Ca2+-induced decreases in state 3 respiration and mitochondrial permeability transition pore (mPTP) opening concurrent with a reduction in mitochondrial Ca2+ uptake capacity. Treatment of striatal cells expressing mHtt with thapsigargin results in a decrease in mitochondrial Ca2+ uptake and membrane potential and an increase in reactive oxygen species (ROS) production. Transcriptional processes regulated by peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α), which are critical for mitochondrial biogenesis, have been shown to be impaired in HD. In addition, the PPARγ signaling pathway is impaired by mHtt and the activation of this pathway ameliorates many of the mitochondrial deficits, suggesting that PPARγ agonists may represent an important treatment strategy for HD.

Pharmacological HIF2α inhibition improves VHL disease-associated phenotypes in zebrafish model.

Patients with a germline mutation in von Hippel-Lindau (VHL) develop renal cell cancers and hypervascular tumors of the brain, adrenal glands, and pancreas as well as erythrocytosis. These phenotypes are driven by aberrant expression of HIF2α, which induces expression of genes involved in cell proliferation, angiogenesis, and red blood cell production. Currently, there are no effective treatments available for VHL disease. Here, using an animal model of VHL, we report a marked improvement of VHL-associated phenotypes following treatment with HIF2α inhibitors. Inactivation of vhl in zebrafish led to constitutive activation of HIF2α orthologs and modeled several aspects of the human disease, including erythrocytosis, pathologic angiogenesis in the brain and retina, and aberrant kidney and liver proliferation. Treatment of vhl(-/-) mutant embryos with HIF2α-specific inhibitors downregulated Hif target gene expression in a dose-dependent manner, improved abnormal hematopoiesis, and substantially suppressed erythrocytosis and angiogenic sprouting. Moreover, pharmacologic inhibition of HIF2α reversed the compromised cardiac contractility of vhl(-/-) embryos and partially rescued early lethality. This study demonstrates that small-molecule targeting of HIF2α improves VHL-related phenotypes in a vertebrate animal model and supports further exploration of this strategy for treating VHL disease.

An In Vivo Zebrafish Screen Identifies Organophosphate Antidotes with Diverse Mechanisms of Action

Organophosphates are a class of highly toxic chemicals that includes many pesticides and chemical weapons. Exposure to organophosphates, either through accidents or acts of terrorism, poses a significant risk to human health and safety. Existing antidotes, in use for over 50 years, have modest efficacy and undesirable toxicities. Therefore, discovering new organophosphate antidotes is a high priority. Early life stage zebrafish exposed to organophosphates exhibit several phenotypes that parallel the human response to organophosphates, including behavioral deficits, paralysis, and eventual death. Here, we have developed a high-throughput zebrafish screen in a 96-well plate format to find new antidotes that counteract organophosphate-induced lethality. In a pilot screen of 1200 known drugs, we identified 16 compounds that suppress organophosphate toxicity in zebrafish. Several in vitro assays coupled with liquid chromatography/tandem mass spectrometry–based metabolite profiling enabled determination of mechanisms of action for several of the antidotes, including reversible acetylcholinesterase inhibition, cholinergic receptor antagonism, and inhibition of bioactivation. Therefore, the in vivo screen is capable of discovering organophosphate antidotes that intervene in distinct pathways. These findings suggest that zebrafish screens might be a broadly applicable approach for discovering compounds that counteract the toxic effects of accidental or malicious poisonous exposures.

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.