Noncanonical translation via deadenylated 3' UTRs maintains primordial germ cells.

Primordial germ cells (PGCs) form during early embryogenesis with a supply of maternal mRNAs that contain shorter poly(A) tails. How translation of maternal mRNAs is regulated during PGC development remains elusive. Here we describe a small-molecule screen with zebrafish embryos that identified primordazine, a compound that selectively ablates PGCs. Primordazine's effect on PGCs arises from translation repression through primordazine-response elements in the 3' UTRs. Systematic dissection of primordazine's mechanism of action revealed that translation of mRNAs during early embryogenesis occurs by two distinct pathways, depending on the length of their poly(A) tails. In addition to poly(A)-tail-dependent translation (PAT), early embryos perform poly(A)-tail-independent noncanonical translation (PAINT) via deadenylated 3' UTRs. Primordazine inhibits PAINT without inhibiting PAT, an effect that was also observed in quiescent, but not proliferating, mammalian cells. These studies reveal that PAINT is an alternative form of translation in the early embryo and is indispensable for PGC maintenance.

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 zebrafish and mice. To our surprise, 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 nontransgenic animals, including humans.

Glutathione metabolism-related gene signature predicts prognosis and treatment response in low-grade glioma.

Cancer cells can induce molecular changes that reshape cellular metabolism, creating specific vulnerabilities for targeted therapeutic interventions. Given the importance of reactive oxygen species (ROS) in tumor development and drug resistance, and the abundance of reduced glutathione (GSH) as the primary cellular antioxidant, we examined an integrated panel of 56 glutathione metabolism-related genes (GMRGs) across diverse cancer types. This analysis revealed a remarkable association between GMRGs and low-grade glioma (LGG) survival. Unsupervised clustering and a GMRGs-based risk score (GS) categorized LGG patients into two groups, linking elevated glutathione metabolism to poorer prognosis and treatment outcomes. Our GS model outperformed established clinical prognostic factors, acting as an independent prognostic factor. GS also exhibited correlations with pro-tumor M2 macrophage infiltration, upregulated immunosuppressive genes, and diminished responses to various cancer therapies. Experimental validation in glioma cell lines confirmed the critical role of glutathione metabolism in glioma cell proliferation and chemoresistance. Our findings highlight the presence of a unique metabolic susceptibility in LGG and introduce a novel GS system as a highly effective tool for predicting the prognosis of LGG.

Chemical Genetics: Manipulating the Germline with Small Molecules.

Primordial germ cells (PGCs) are the precursor cells that form during early embryogenesis and later differentiate into oocytes or spermatozoa. Abnormal development of PGCs is frequently a causative factor of infertility and germ cell tumors. However, our understanding of PGC development remains insufficient, and we have few pharmacological tools for manipulating PGC development for biological study or therapy. The zebrafish (Danio rerio) embryos provide an excellent in vivo animal model to study PGCs, because zebrafish embryos are transparent and develop outside the mother. Importantly, the model is also amenable to facile chemical manipulations, including scalable screening to discover novel compounds that alter PGC development. This chapter describes methodologies for manipulating the germline (i.e., PGCs) with small molecules and for monitoring PGC development. Utilizing the 3'UTR of PGC marker genes such as nanos3 and ddx4/vasa is a key component of these methodologies, which consist of expressing fluorescent or luminescent proteins in PGCs, treatment with small molecules, and quantitative observation of PGC development.

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 Ca(2+)-induced decreases in state 3 respiration and mitochondrial permeability transition pore (mPTP) opening concurrent with a reduction in mitochondrial Ca(2+) uptake capacity. Treatment of striatal cells expressing mHtt with thapsigargin results in a decrease in mitochondrial Ca(2+) uptake and membrane potential and an increase in reactive oxygen species (ROS) production. Transcriptional processes regulated by peroxisome proliferator-activated receptor gamma (PPAR gamma) coactivator-1 alpha (PGC-1 alpha), which are critical for mitochondrial biogenesis, have been shown to be impaired in HD. In addition, the PPAR gamma signaling pathway is impaired by mHtt and the activation of this pathway ameliorates many of the mitochondrial deficits, suggesting that PPAR gamma agonists may represent an important treatment strategy for HD.

PTPMT1 Inhibition Lowers Glucose through Succinate Dehydrogenase Phosphorylation.

Virtually all organisms seek to maximize fitness by matching fuel availability with energy expenditure. In vertebrates, glucose homeostasis is central to this process, with glucose levels finely tuned to match changing energy requirements. To discover new pathways regulating glucose levels in vivo, we performed a large-scale chemical screen in live zebrafish and identified the small molecule alexidine as a potent glucose-lowering agent. We found that alexidine inhibits the PTEN-like mitochondrial phosphatase PTPMT1 and that other pharmacological and genetic means of inactivating PTPMT1 also decrease glucose levels in zebrafish. Mutation of ptpmt1 eliminates the effect of alexidine, further confirming it as the glucose-lowering target of alexidine. We then identified succinate dehydrogenase (SDH) as a substrate of PTPMT1. Inactivation of PTPMT1 causes hyperphosphorylation and activation of SDH, providing a possible mechanism by which PTPMT1 coordinates glucose homeostasis. Therefore, PTPMT1 appears to be an important regulator of SDH phosphorylation status and glucose concentration.

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

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence 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 overexpression 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.

Mitochondrial permeability transition pore induces mitochondria injury in Huntington disease.

BACKGROUND: Mitochondrial 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. RESULTS: Depolarization 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. CONCLUSIONS: Expression of mutant huntingtin alters mitochondrial and cell viability through mPTP opening in striatal cells and cortical neurons.

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. 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.

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 H(2)O(2) or thapsigargin in HD. Striatal precursor cells expressing wild type (STHdh(Q7)) or mutant huntingtin (STHdh(Q111)) were prepared in different metabolic conditions (glucose vs. pyruvate). Due to the fact that STHdh(Q111) 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 STHdh(Q111) compared to STHdh(Q7), 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 STHdh(Q111) cells to stressors compared to the STHdh(Q7) 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 STHdh(Q111) 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.

Truncated tau and Aß cooperatively impair mitochondria in primary neurons.

Mitochondrial dysfunction is likely a significant contributing factor to Alzheimer disease pathogenesis, and both amyloid peptide (Aß) 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 Aß. The expression of truncated tau alone induced significant mitochondrial fragmentation in neurons. When truncated tau expression was combined with Aß 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 Aß-induced mitochondrial potential loss in primary neurons. These new findings show that Asp421-cleaved tau and Aß cooperate to impair mitochondria, which likely contributes to the neuronal dysfunction in Alzheimer disease.

Usp14 deficiency increases tau phosphorylation without altering tau degradation or causing tau-dependent deficits.

Regulated protein degradation by the proteasome plays an essential role in the enhancement and suppression of signaling pathways in the nervous system. Proteasome-associated factors are pivotal in ensuring appropriate protein degradation, and we have previously demonstrated that alterations in one of these factors, the proteasomal deubiquitinating enzyme ubiquitin-specific protease 14 (Usp14), can lead to proteasome dysfunction and neurological disease. Recent studies in cell culture have shown that Usp14 can also stabilize the expression of over-expressed, disease-associated proteins such as tau and ataxin-3. Using Usp14-deficient ax(J) mice, we investigated if loss of Usp14 results in decreased levels of endogenous tau and ataxin-3 in the nervous system of mice. Although loss of Usp14 did not alter the overall neuronal levels of tau and ataxin-3, we found increased levels of phosphorylated tau that correlated with the onset of axonal varicosities in the Usp14-deficient mice. These changes in tau phosphorylation were accompanied by increased levels of activated phospho-Akt, phosphorylated MAPKs, and inactivated phospho-GSK3ß. However, genetic ablation of tau did not alter any of the neurological deficits in the Usp14-deficient mice, demonstrating that increased levels of phosphorylated tau do not necessarily lead to neurological disease. Due to the widespread activation of intracellular signaling pathways induced by the loss of Usp14, a better understanding of the cellular pathways regulated by the proteasome is required before effective proteasomal-based therapies can be used to treat chronic neurological diseases.

Decreases in valosin-containing protein result in increased levels of tau phosphorylated at Ser262/356.

VCP/p97 is a multifunctional AAA+-ATPase involved in vesicle fusion, proteasomal degradation, and autophagy. Reported dysfunctions of these processes in Alzheimer disease (AD), along with the linkage of VCP/p97 to inclusion body myopathy with Paget's disease and frontotemporal dementia (IBMPFD) led us to examine the possible linkage of VCP to the AD-relevant protein, tau. VCP levels were reduced in AD brains, but not in the cerebral cortex of an AD mouse model, suggesting that VCP reduction occurs upstream of tau pathology. Genetic reduction of VCP in a primary neuronal model led to increases in the levels of tau phosphorylated at Ser(262/356), indicating that VCP may be involved in regulating post-translational processing of tau in AD, demonstrating a possible functional linkage between tau and VCP.

Rosiglitazone treatment prevents mitochondrial dysfunction in mutant huntingtin-expressing cells: possible role of peroxisome proliferator-activated receptor-gamma (PPARgamma) in the pathogenesis of Huntington disease.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the PPAR family of transcription factors. Synthetic PPARgamma agonists are used as oral anti-hyperglycemic drugs for the treatment of non-insulin-dependent diabetes. However, emerging evidence indicates that PPARgamma activators can also prevent or attenuate neurodegeneration. Given these previous findings, the focus of this report is on the potential neuroprotective role of PPARgamma activation in preventing the loss of mitochondrial function in Huntington disease (HD). For these studies we used striatal cells that express wild-type (STHdh(Q7/Q7)) or mutant (STHdh(Q111/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. PPARgamma 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 PPARgamma, as all protective effects were prevented by the PPARgamma antagonist GW9662. Additionally, the PPARgamma signaling pathway was significantly impaired in the mutant striatal cells with decreases in PPARgamma expression and reduced PPARgamma transcriptional activity. Treatment with rosiglitazone increased mitochondrial mass levels, suggesting a role for the PPARgamma pathway in mitochondrial function in striatal cells. Altogether, this evidence indicates that PPARgamma 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.