Jeffrey R. Liddell

Endogenous TDP-43 localized to stress granules can subsequently form protein aggregates.

TDP-43 proteinopathies are characterized by loss of nuclear TDP-43 and accumulation of the protein in the cytosol as ubiquitinated protein aggregates. These protein aggregates may have an important role in subsequent neuronal degeneration in motor neuron disease, frontotemporal dementia and potentially other neurodegenerative diseases. Although the cellular mechanisms driving the abnormal accumulation of TDP-43 are not understood, recent studies have shown that an early change to TDP-43 metabolism in disease may be accumulation in cytosolic RNA stress granules (SGs). However, it is unclear whether the TDP-43 in these SGs progresses to become irreversible protein aggregates as observed in patients. We have shown recently that paraquat-treated cells are a useful model for examining TDP-43 SG localization. In this study, we used the paraquat model to examine if endogenous TDP-43 in SGs can progress to more stable protein aggregates. We found that after treatment of HeLa cells overnight with paraquat, TDP-43 co-localized to SGs together with the ubiquitous SG marker, human antigen R (HuR). However, after a further incubation in paraquat-free, conditioned medium for 6h, HuR-positive SGs were rarely detected yet TDP-43 positive aggregates remained present. The majority of these TDP-43 aggregates were positive for ubiquitin. Further evidence for persistence of TDP-43 aggregates was obtained by treating cultures with cycloheximide after paraquat treatment. Cycloheximide abolished nearly all cytosolic HuR aggregation (SGs) but large TDP-43-positive aggregates remained. Finally, we showed that addition of ERK and JNK inhibitors together with paraquat blocked TDP-43-positive SG formation, while treatment with inhibitors after 24h paraquat exposure failed to reverse the TDP-43 accumulation. This failure was most likely due to the addition of inhibitors after maximal activation of the kinases at 4h post-paraquat treatment. These findings provide strong evidence that once endogenous TDP-43 accumulates in SGs, it has the potential to progress to stable protein aggregates as observed in neurons in TDP-43 proteinopathies. This may provide a therapeutic opportunity to inhibit the transition of TDP-43 from SG protein to aggregate.

C-Jun N-terminal kinase controls TDP-43 accumulation in stress granules induced by oxidative stress

BackgroundTDP-43 proteinopathies are characterized by loss of nuclear TDP-43 expression and formation of C-terminal TDP-43 fragmentation and accumulation in the cytoplasm. Recent studies have shown that TDP-43 can accumulate in RNA stress granules (SGs) in response to cell stresses and this could be associated with subsequent formation of TDP-43 ubiquinated protein aggregates. However, the initial mechanisms controlling endogenous TDP-43 accumulation in SGs during chronic disease are not understood. In this study we investigated the mechanism of TDP-43 processing and accumulation in SGs in SH-SY5Y neuronal-like cells exposed to chronic oxidative stress. Cell cultures were treated overnight with the mitochondrial inhibitor paraquat and examined for TDP-43 and SG processing.ResultsWe found that mild stress induced by paraquat led to formation of TDP-43 and HuR-positive SGs, a proportion of which were ubiquitinated. The co-localization of TDP-43 with SGs could be fully prevented by inhibition of c-Jun N-terminal kinase (JNK). JNK inhibition did not prevent formation of HuR-positive SGs and did not prevent diffuse TDP-43 accumulation in the cytosol. In contrast, ERK or p38 inhibition prevented formation of both TDP-43 and HuR-positive SGs. JNK inhibition also inhibited TDP-43 SG localization in cells acutely treated with sodium arsenite and reduced the number of aggregates per cell in cultures transfected with C-terminal TDP-43 162-414 and 219-414 constructs.ConclusionsOur studies are the first to demonstrate a critical role for kinase control of TDP-43 accumulation in SGs and may have important implications for development of treatments for FTD and ALS, targeting cell signal pathway control of TDP-43 aggregation.

Glutathione peroxidase 1 and glutathione are required to protect mouse astrocytes from iron-mediated hydrogen peroxide toxicity

The enzyme glutathione peroxidase 1 (GPx1) is involved in the cellular detoxification of peroxides. To test for the consequences of GPx deficiency in astrocytes, astrocyte‐rich primary cultures from wild‐type and GPx1‐deficient [GPx1(–/–)] mice were exposed to H2O2. In GPx1(–/–) astrocytes, the clearance rate of H2O2 was slower than in wild‐type cells. In contrast to GPx1‐deficient astrocytes, wild‐type cells exhibited, within 2 min of H2O2 application, a rapid and transient accumulation of cellular glutathione disulfide that amounted to 60% of total glutathione. The peroxide treatment did not affect the viability of wild‐type astrocytes, whereas 45% of the GPx1(–/–) cells died within 8 hr. However, the viability of both types of astrocytes was strongly compromised by lowering cellular glutathione content before peroxide application. In contrast, inactivation of catalase caused substantial cell death only in GPx1(–/–) cells but not in wild‐type astrocytes. The cell death observed was prevented by the iron chelators deferoxamine, 1,10‐phenathroline, or 2,2′‐dipyridyl, whereas preincubation with ferric ammonium citrate increased the toxicity of peroxide treatments. These results demonstrate that GPx1 contributes to the rapid clearance of H2O2 by mouse astrocytes and that both GPx1 and a high concentration of glutathione are required to protect these cells from iron‐dependent peroxide damage.

An impaired mitochondrial electron transport chain increases retention of the hypoxia imaging agent diacetylbis(4-methylthiosemicarbazonato)copperII

Radiolabeled diacetylbis(4-methylthiosemicarbazonato)copperII [CuII(atsm)] is an effective positron-emission tomography imaging agent for myocardial ischemia, hypoxic tumors, and brain disorders with regionalized oxidative stress, such as mitochondrial myopathy, encephalopathy, and lactic acidosis with stroke-like episodes (MELAS) and Parkinson’s disease. An excessively elevated reductive state is common to these conditions and has been proposed as an important mechanism affecting cellular retention of Cu from CuII(atsm). However, data from whole-cell models to demonstrate this mechanism have not yet been provided. The present study used a unique cell culture model, mitochondrial xenocybrids, to provide whole-cell mechanistic data on cellular retention of Cu from CuII(atsm). Genetic incompatibility between nuclear and mitochondrial encoded subunits of the mitochondrial electron transport chain (ETC) in xenocybrid cells compromises normal function of the ETC. As a consequence of this impairment to the ETC we show xenocybrid cells upregulate glycolytic ATP production and accumulate NADH. Compared to control cells the xenocybrid cells retained more Cu after being treated with CuII(atsm). By transfecting the cells with a metal-responsive element reporter construct the increase in Cu retention was shown to involve a CuII(atsm)-induced increase in intracellular bioavailable Cu specifically within the xenocybrid cells. Parallel experiments using cells grown under hypoxic conditions confirmed that a compromised ETC and elevated NADH levels contribute to increased cellular retention of Cu from CuII(atsm). Using these cell culture models our data demonstrate that compromised ETC function, due to the absence of O2 as the terminal electron acceptor or dysfunction of individual components of the ETC, is an important determinant in driving the intracellular dissociation of CuII(atsm) that increases cellular retention of the Cu.

Glutathione peroxidase 1 and a high cellular glutathione concentration are essential for effective organic hydroperoxide detoxification in astrocytes.

Organic hydroperoxides are produced in the eicosanoid metabolism and by lipid peroxidation. To examine the contribution of glutathione peroxidase‐1 (GPx1) and glutathione (GSH) in the disposal of organic hydroperoxides in brain astrocytes, primary astrocyte cultures from wild type or GPx1‐deficient (GPx1(−/−)) mice were exposed to cumene hydroperoxide (CHP). After application of 100 μM CHP, the peroxide disappeared quickly from the incubation medium of wild type cells with a half‐life of 9 min, whereas CHP clearance was strongly retarded in GPx1(−/−) astrocytes. Depletion of GSH by pre‐incubation with buthionine sulfoximine (BSO) significantly slowed CHP clearance by wild type astrocytes, while almost completely preventing peroxide disposal by GPx1(−/−) cells. In contrast, the catalase inhibitor 3‐aminotriazole (3AT) had no effect on CHP clearance. Application of CHP to wild type astrocytes was followed by a rapid and transient accumulation of GSSG, whereas in GPx1(−/−) cells no increase in the GSSG content was detected. Astrocytes from both mouse lines remained viable for up to 24 h following CHP exposure, however depletion of cellular GSH by pre‐treatment with BSO compromised the viability of astrocytes, an effect that was stronger in GPx1(−/−) than in wild type cells. This cell death was almost completely prevented by iron chelators, whereas pre‐incubation with iron increased CHP toxicity. These novel data demonstrate that the toxicity of organic hydroperoxides in astrocytes is iron‐mediated, and that an intact GSH system is required for the effective removal of organic hydroperoxides and for protection from these peroxides.

Therapeutic effects of CuII(atsm) in the SOD1-G37R mouse model of amyotrophic lateral sclerosis

Abstract Our objective was to assess the copperII complex of diacetylbis(4-methylthiosemicarbazone) [CuII(atsm)] for its preclinical potential as a novel therapeutic for ALS. Experimental paradigms used were designed to assess CuII(atsm) efficacy relative to treatment with riluzole, as a function of dose administered, and when administered post symptom onset. Mice expressing human Cu/Zn superoxide dismutase harbouring the disease-causing G37R mutation (SOD1-G37R) were used and effects of CuII(atsm) determined by assessing mouse survival and locomotor function (rotarod assay). CuII(atsm) improved SOD1-G37R mouse survival and locomotor function in a dose-dependent manner. The highest dose tested improved survival by 26%. Riluzole had a modest effect on mouse survival (3.3%) but it did not improve locomotor function. Cotreatment with CuII(atsm) did not alter the protective activity of CuII(atsm) administered on its own. Commencing treatment with CuII(atsm) after the onset of symptoms was less effective than treatments that commenced before symptom onset but still significantly improved locomotor function and survival. Improved locomotor function and survival of SOD1-G37R mice supports the potential for CuII(atsm) as a novel treatment option for ALS.

The challenges of using a copper fluorescent sensor (CS1) to track intracellular distributions of copper in neuronal and glial cells

Copper is an essential biometal involved in critical cell functions including respiration. However, the mechanisms controlling its sub-cellular localization during health and disease remain poorly understood. This is partially due to the difficulty of detecting a metal ion that is bound tightly to metallo-chaperone and detoxification molecules in the cell. A BODIPY-based Cu fluorescent probe CS1 (Cu sensor 1) has been applied in innovative attempts to visualize monovalent Cu pools within cells (Zeng et al., J. Am. Chem. Soc., 2006, 128, 10–11). Inspired by this work, we sought to use CS1 to identify sub-cellular localization of Cu delivered to M17 neuronal or U87MG glial cells by a cell-permeable bis(thiosemicarbazonato)Cu(II) complex, CuII(gtsm). This complex increases cellular Cu concentrations by factors of 10–100 when compared to treatment with equivalent concentrations of CuCl2 (Donnelly et al., J. Biol. Chem., 2008, 283, 4568–4577). However, we were unable to identify any specific increase in CS1 fluorescence in neurons or glia treated with CuCl2 or with CuII(gtsm), despite controls revealing a large increase in total cellular Cu with the latter treatment. Further in vitro characterization of CS1 suggests that, consistent with its relatively weak affinity for CuI (KD ≈ 10−11 M), it is unlikely to compete with endogenous proteins with sub-picomolar affinities, nor with glutathione, the endogenous redox buffer essential for functional maintenance of many proteins, including those that bind CuI. Moreover, we show that CS1 is localized predominantly to lysosomes and that the observed background fluorescence may be attributed to increased concentrations of apo-CS1 in this organelle or to the probe gaining access to CuI made available via recycling of nutrient Cu in the acidic lysosome. It was possible to observe a consistent increase in CS1 fluorescence in neuronal cells exposed to stress. For example, treatment with buthionine sulfoximine decreased cellular glutathione levels and led to enhanced CS1 fluorescence, but the total cellular Cu levels did not correlate with the increased fluorescence. In addition, cells treated with reagents that are known to alter cellular pH homeostasis provided an enhanced fluorescence. Our findings demonstrate that the source of enhanced CS1 fluorescence in Cu-supplemented cells must be interpreted with caution. It may be a consequence of altered cell pH, compromised vesicle maturation, increased CS1 uptake and/or trapping of CS1 in the lysosomal compartment.

Astrocytes retain their antioxidant capacity into advanced old age

Oxidative stress has been implicated in the progression of ageing and in many age‐related neurodegenerative conditions. Astrocytes play a major role in the antioxidant protection of the brain, yet little is known about how the antioxidant defenses of astrocytes change across the lifespan. This study assessed the antioxidant capacity and glutathione metabolism of astrocytes cultured from the brains of neonatal (<24 h old), mature (12‐month‐old), old (25‐month‐old), and senescent (31‐month‐old) C57BL/6J mice. When exposed to 100 μM hydrogen peroxide, mature, old, and senescent astrocytes cleared the peroxide ∼30% more slowly than neonatal astrocytes. This difference persisted when catalase was inhibited by 3‐aminotriazole, but was abolished when glutathione was depleted by application of buthionine sulfoximine, suggesting a deficit in the glutathione system. Correspondingly, the specific glutathione reductase activity of mature, old, and senescent astrocytes was ∼30% lower than that of neonatal cultures, whereas no age‐related change was observed in the specific activities of glutathione peroxidase, catalase, or in total antioxidant capacity. In addition, the specific rate of glutathione export was almost identical in mature, old, and senescent astrocytes, but was more than double that of neonatal astrocytes. These results indicate that the antioxidant capacity and glutathione metabolism of astrocytes are preserved from mature adulthood into senescence. It is concluded that the oxidative stress seen in ageing brains is likely due to factors extrinsic to astrocytes, rather than to an impairment of the antioxidative functions of astrocytes.

Sustained hydrogen peroxide stress decreases lactate production by cultured astrocytes

Oxidative stress and disrupted energy metabolism are common to many pathological conditions of the brain. Because astrocytes play an important role in the glucose metabolism of the brain, we have investigated whether sustained oxidative stress affects astroglial glucose metabolism with cultured primary rat astrocytes as a model system. Cultured astrocytes were exposed to a sustained concentration of approximately 50 μM H2O2 in the presence of [U‐13C]glucose, and cellular and extracellular contents of lactate and glucose were analysed by enzymatic assays and NMR spectroscopy. Exposure of the cells to sustained H2O2 stress for up to 120 min significantly lowered the rate of lactate accumulation in the media to 61% ± 14% of that in cultures incubated without peroxide. In addition, the ratio of lactate release to glucose consumption was lowered in peroxide‐treated astrocytes to 77% ± 13% of that in control cells, and the specific activity of glyceraldehyde‐3‐phosphate dehydrogenase had declined to about 10% of control cells within 90 min. In addition, the 13C enrichment of intracellular and extracellular [13C]lactate was about 30% and 95%, respectively, and was not affected by the presence of peroxide, demonstrating that two metabolic pools of lactate are present in cultured astrocytes. The decreased rate of lactate production by astrocytes that have been exposed to peroxide stress is a new example of an alteration by oxidative stress of an important metabolic pathway in astrocytes. Such alterations could contribute to the pathological conditions that have been connected with oxidative stress and disrupted energy metabolism in the brain.

Endogenous glutathione and catalase protect cultured rat astrocytes from the iron-mediated toxicity of hydrogen peroxide

Primary astrocyte cultures from rat brain were exposed to hydrogen peroxide (H2O2) to investigate peroxide toxicity and clearance by astrocytes. After bolus application of H2O2 (100 microM), the peroxide was eliminated from the incubation medium following first-order kinetics with a half-time of approximately 4 min. The rate of peroxide detoxification was significantly slowed by pre-incubating the cells with the glutathione synthesis inhibitor buthionine sulfoximine (BSO), or the catalase inhibitor 3-amino-1,2,4-triazole (3AT), and was retarded further when both treatments were combined. H2O2 application killed a small proportion of cells, as indicated by the levels of the cytosolic enzyme lactate dehydrogenase in the media 1 and 24h later. In contrast, cell viability was strongly compromised when the cells were pre-incubated with 3AT and/or BSO before peroxide application. The iron chelator deferoxamine completely prevented this cell loss. These results demonstrate that chelatable iron is involved in the toxicity of H2O2 and that both the glutathione system and catalase protect astrocytes from this toxicity.