Compartmentalized role of xCT in supporting pancreatic tumor growth, inflammation and mood disturbance in mice.

xCT (Slc7a11), the specific subunit of the cystine/glutamate antiporter system xc-, is present in the brain and on immune cells, where it is known to modulate behavior and inflammatory responses. In a variety of cancers -including pancreatic ductal adenocarcinoma (PDAC)-, xCT is upregulated by tumor cells to support their growth and spread. Therefore, we studied the impact of xCT deletion in pancreatic tumor cells (Panc02) and/or the host (xCT-/- mice) on tumor burden, inflammation, cachexia and mood disturbances. Deletion of xCT in the tumor strongly reduced tumor growth. Targeting xCT in the host and not the tumor resulted only in a partial reduction of tumor burden, while it did attenuate tumor-related systemic inflammation and prevented an increase in immunosuppressive regulatory T cells. The latter effect could be replicated by specific xCT deletion in immune cells. xCT deletion in the host or the tumor differentially modulated neuroinflammation. When mice were grafted with xCT-deleted tumor cells, hypothalamic inflammation was reduced and, accordingly, food intake improved. Tumor bearing xCT-/- mice showed a trend of reduced hippocampal neuroinflammation with less anxiety- and depressive-like behavior. Taken together, targeting xCT may have beneficial effects on pancreatic cancer-related comorbidities, beyond reducing tumor burden. The search for novel and specific xCT inhibitors is warranted as they may represent a holistic therapy in pancreatic cancer.

Metformin confers sensitisation to syrosingopine in multiple myeloma cells by metabolic blockage and inhibition of protein synthesis.

Multiple myeloma (MM) remains an incurable haematological malignancy despite substantial advances in therapy. Hypoxic bone marrow induces metabolic rewiring in MM cells contributing to survival and drug resistance. Therefore, targeting metabolic pathways may offer an alternative treatment option. In this study, we repurpose two FDA-approved drugs, syrosingopine and metformin. Syrosingopine was used as a dual inhibitor of monocarboxylate transporter 1 and 4 (MCT1/4) and metformin as an inhibitor for oxidative phosphorylation (OXPHOS). Anti-tumour effects were evaluated for single agents and in combination therapy. Survival and expression data for MCT1/MCT4 were obtained from the Total Therapy 2, Mulligan, and Multiple Myeloma Research Foundation cohorts. Cell death, viability, and proliferation were measured using Annexin V/7-AAD, CellTiterGlo, and BrdU, respectively. Metabolic effects were assessed using Seahorse Glycolytic Rate assays and LactateGlo assays. Differential protein expression was determined using western blotting, and the SUnSET method was implemented to quantify protein synthesis. Finally, the syngeneic 5T33MMvv model was used for in vivo analysis. High-level expression of MCT1 and MCT4 both correlated with a significantly lower overall survival of patients. Lactate production as well as MCT1/MCT4 expression were significantly upregulated in hypoxia, confirming the Warburg effect in MM. Dual inhibition of MCT1/4 with syrosingopine resulted in intracellular lactate accumulation and reduced cell viability and proliferation. However, only at higher doses (>10 µm) was syrosingopine able to induce cell death. By contrast, combination treatment of syrosingopine with metformin was highly cytotoxic for MM cell lines and primary patient samples and resulted in a suppression of both glycolysis and OXPHOS. Moreover, pathway analysis revealed an upregulation of the energy sensor p-AMPKα and more downstream a reduction in protein synthesis. Finally, the combination treatment resulted in a significant reduction in tumour burden in vivo. This study proposes an alternative combination treatment for MM and provides insight into intracellular effects. © 2023 The Pathological Society of Great Britain and Ireland.

Targeting the ß2 -adrenergic receptor increases chemosensitivity in multiple myeloma by induction of apoptosis and modulating cancer cell metabolism.

While multi-drug combinations and continuous treatment have become standard for multiple myeloma, the disease remains incurable. Repurposing drugs that are currently used for other indications could provide a novel approach to improve the therapeutic efficacy of standard multiple myeloma treatments. Here, we assessed the anti-tumor effects of cardiac drugs called ß-blockers as a single agent and in combination with commonly used anti-myeloma therapies. Expression of the ß2 -adrenergic receptor correlated with poor survival outcomes in patients with multiple myeloma. Targeting the ß2 -adrenergic receptor (ß2 AR) using either selective or non-selective ß-blockers reduced multiple myeloma cell viability, and induced apoptosis and autophagy. Blockade of the ß2 AR modulated cancer cell metabolism by reducing the mitochondrial respiration as well as the glycolytic activity. These effects were not observed by blockade of ß1 -adrenergic receptors. Combining ß2 AR blockade with the chemotherapy drug melphalan or the proteasome inhibitor bortezomib significantly increased apoptosis in multiple myeloma cells. These data identify the therapeutic potential of ß2 AR-blockers as a complementary or additive approach in multiple myeloma treatment and support the future clinical evaluation of non-selective ß-blockers in a randomized controlled trial. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Effects of lactate, super-GDF9, and low oxygen tension during bi-phasic in vitro maturation on the bioenergetic profiles of mouse cumulus-oocyte complex†.

In vitro maturation (IVM) is an alternative assisted reproductive technology with reduced hormone-related side effects and treatment burden compared to conventional IVF. Capacitation (CAPA)-IVM is a bi-phasic IVM system with improved clinical outcomes compared to standard monophasic IVM. Yet, CAPA-IVM efficiency compared to conventional IVF is still suboptimal in terms of producing utilizable blastocysts. Previously, we have shown that CAPA-IVM leads to a precocious increase in cumulus cell (CC) glycolytic activity during cytoplasmic maturation. In the current study, considering the fundamental importance of CCs for oocyte maturation and cumulus-oocyte complex (COC) microenvironment, we further analyzed the bioenergetic profiles of maturing CAPA-IVM COCs. Through a multi-step approach, we (i) explored mitochondrial function of the in vivo and CAPA-IVM matured COCs through real-time metabolic analysis with Seahorse analyzer, and to improve COC metabolism (ii) supplemented the culture media with lactate and/or super-GDF9 (an engineered form of growth differentiation factor 9) and (iii) reduced culture oxygen tension. Our results indicated that the pre-IVM step is delicate and prone to culture-related disruptions. Lactate and/or super-GDF9 supplementations failed to eliminate pre-IVM-induced stress on COC glucose metabolism and mitochondrial respiration. However, when performing pre-IVM culture under 5% oxygen tension, CAPA-IVM COCs showed similar bioenergetic profiles compared to in vivo matured counterparts. This is the first study providing real-time metabolic analysis of the COCs from a bi-phasic IVM system. The currently used analytical approach provides the quantitative measures and the rational basis to further improve IVM culture requirements.

Lifespan extension with preservation of hippocampal function in aged system xc--deficient male mice.

The cystine/glutamate antiporter system xc- has been identified as the major source of extracellular glutamate in several brain regions as well as a modulator of neuroinflammation, and genetic deletion of its specific subunit xCT (xCT-/-) is protective in mouse models for age-related neurological disorders. However, the previously observed oxidative shift in the plasma cystine/cysteine ratio of adult xCT-/- mice led to the hypothesis that system xc- deletion would negatively affect life- and healthspan. Still, till now the role of system xc- in physiological aging remains unexplored. We therefore studied the effect of xCT deletion on the aging process of mice, with a particular focus on the immune system, hippocampal function, and cognitive aging. We observed that male xCT-/- mice have an extended lifespan, despite an even more increased plasma cystine/cysteine ratio in aged compared to adult mice. This oxidative shift does not negatively impact the general health status of the mice. On the contrary, the age-related priming of the innate immune system, that manifested as increased LPS-induced cytokine levels and hypothermia in xCT+/+ mice, was attenuated in xCT-/- mice. While this was associated with only a very moderate shift towards a more anti-inflammatory state of the aged hippocampus, we observed changes in the hippocampal metabolome that were associated with a preserved hippocampal function and the retention of hippocampus-dependent memory in male aged xCT-/- mice. Targeting system xc- is thus not only a promising strategy to prevent cognitive decline, but also to promote healthy aging.

Genetic deletion of xCT attenuates peripheral and central inflammation and mitigates LPS-induced sickness and depressive-like behavior in mice.

The communication between the immune and central nervous system (CNS) is affected in many neurological disorders. Peripheral injections of the endotoxin lipopolysaccharide (LPS) are widely used to study this communication: an LPS challenge leads to a biphasic syndrome that starts with acute sickness and is followed by persistent brain inflammation and chronic behavioral alterations such as depressive-like symptoms. In vitro, the response to LPS treatment has been shown to involve enhanced expression of system x c - . This cystine-glutamate antiporter, with xCT as specific subunit, represents the main glial provider of extracellular glutamate in mouse hippocampus. Here we injected male xCT knockout and wildtype mice with a single intraperitoneal dose of 5 mg/kg LPS. LPS-injection increased hippocampal xCT expression but did not alter the mainly astroglial localization of the xCT protein. Peripheral and central inflammation (as defined by cytokine levels and morphological activation of microglia) as well as LPS-induced sickness and depressive-like behavior were significantly attenuated in xCT-deficient mice compared with wildtype mice. Our study is the first to demonstrate the involvement of system x c - in peripheral and central inflammation in vivo and the potential therapeutic relevance of its inhibition in brain disorders characterized by peripheral and central inflammation, such as depression.

Functional cardiotoxicity assessment of cosmetic compounds using human-induced pluripotent stem cell-derived cardiomyocytes.

There is a large demand of a human relevant in vitro test system suitable for assessing the cardiotoxic potential of cosmetic ingredients and other chemicals. Using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we have already established an in vitro cardiotoxicity assay and identified genomic biomarkers of anthracycline-induced cardiotoxicity in our previous work. Here, five cosmetic ingredients were studied by the new hiPSC-CMs test; kojic acid (KJA), triclosan (TS), triclocarban (TCC), 2,7-naphthalenediol (NPT), and basic red 51 (BR51) based on cytotoxicity as well as ATP assays, beating rate, and genomic biomarkers to determine the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC). The LOEC for beating rate were 400, 10, 3, >400, and 3 µM for KJA, TS, TCC, NPT, and BR51, respectively. The corresponding concentrations for cytotoxicity or ATP depletion were similar, with the exception of TS and TCC, where the cardiomyocyte-beating assay showed positive results at non-cytotoxic concentrations. Functional analysis also showed that the individual compounds caused different effects on hiPSC-CMs. While exposure to KJA, TS, TCC, and BR51 induced significant arrhythmic beating, NPT slightly decreased cell viability, but did not influence beating. Gene expression studies showed that TS and NPT caused down-regulation of cytoskeletal and cardiac ion homeostasis genes. Moreover, TS and NPT deregulated genomic biomarkers known to be affected also by anthracyclines. The present study demonstrates that hiPSC-CMs can be used to determine LOECs and NOECs in vitro, which can be compared to human blood concentrations to determine margins of exposure. Our in vitro assay, which so far has been tested with several anthracyclines and cosmetics, still requires validation by larger numbers of positive and negative controls, before it can be recommended for routine analysis.

Comparison of base-line and chemical-induced transcriptomic responses in HepaRG and RPTEC/TERT1 cells using TempO-Seq.

The utilisation of genome-wide transcriptomics has played a pivotal role in advancing the field of toxicology, allowing the mapping of transcriptional signatures to chemical exposures. These activities have uncovered several transcriptionally regulated pathways that can be utilised for assessing the perturbation impact of a chemical and also the identification of toxic mode of action. However, current transcriptomic platforms are not very amenable to high-throughput workflows due to, high cost, complexities in sample preparation and relatively complex bioinformatic analysis. Thus, transcriptomic investigations are usually limited in dose and time dimensions and are, therefore, not optimal for implementation in risk assessment workflows. In this study, we investigated a new cost-effective, transcriptomic assay, TempO-Seq, which alleviates the aforementioned limitations. This technique was evaluated in a 6-compound screen, utilising differentiated kidney (RPTEC/TERT1) and liver (HepaRG) cells and compared to non-transcriptomic label-free sensitive endpoints of chemical-induced disturbances, namely phase contrast morphology, xCELLigence and glycolysis. Non-proliferating cell monolayers were exposed to six sub-lethal concentrations of each compound for 24 h. The results show that utilising a 2839 gene panel, it is possible to discriminate basal tissue-specific signatures, generate dose-response relationships and to discriminate compound-specific and cell type-specific responses. This study also reiterates previous findings that chemical-induced transcriptomic alterations occur prior to cytotoxicity and that transcriptomics provides in depth mechanistic information of the effects of chemicals on cellular transcriptional responses. TempO-Seq is a robust transcriptomic platform that is well suited for in vitro toxicity experiments.

A novel genotoxin-specific qPCR array based on the metabolically competent human HepaRG™ cell line as a rapid and reliable tool for improved in vitro hazard assessment.

Although the value of the regulatory accepted batteries for in vitro genotoxicity testing is recognized, they result in a high number of false positives. This has a major impact on society and industries developing novel compounds for pharmaceutical, chemical, and consumer products, as afflicted compounds have to be (prematurely) abandoned or further tested on animals. Using the metabolically competent human HepaRG™ cell line and toxicogenomics approaches, we have developed an upgraded, innovative, and proprietary gene classifier. This gene classifier is based on transcriptomic changes induced by 12 genotoxic and 12 non-genotoxic reference compounds tested at sub-cytotoxic concentrations, i.e., IC10 concentrations as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The resulting gene classifier was translated into an easy-to-handle qPCR array that, as shown by pathway analysis, covers several different cellular processes related to genotoxicity. To further assess the predictivity of the tool, a set of 5 known positive and 5 known negative test compounds for genotoxicity was evaluated. In addition, 2 compounds with debatable genotoxicity data were tested to explore how the qPCR array would classify these. With an accuracy of 100%, when equivocal results were considered positive, the results showed that combining HepaRG™ cells with a genotoxin-specific qPCR array can improve (geno)toxicological hazard assessment. In addition, the developed qPCR array was able to provide additional information on compounds for which so far debatable genotoxicity data are available. The results indicate that the new in vitro tool can improve human safety assessment of chemicals in general by basing predictions on mechanistic toxicogenomics information.

Indocyanine green-mediated photobiomodulation on human osteoblast cells.

Photobiomodulation (PBM) and photodynamic therapy (PDT) share similar mechanisms but have opposite aims. Increased levels of reactive oxygen species (ROS) in the target tissue in response to light combined photosensitizer (PS) application may lead to cell proliferation or oxidative damage depending on the ROS amount. The purpose of the present study is to investigate the effect of indocyanine green (ICG)-mediated PBM on osteoblast cells by measuring cell viability, proliferation, alkaline phosphatase (ALP) activity, mineralization, and gene expressions of three phenotypic osteoblast markers. A diode laser irradiating at 809 nm (10 W output power, 50 mW/cm2 power density) was used at 0.5, 1, and 2 J/cm2 energy densities (10, 20, and 40 s respectively) was applied following ICG incubation. No inhibitory effect was observed in cell viability and proliferation according to the (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Alamar Blue assays. ICG-mediated PBM did not alter cell viability but increased ALP activity and enhanced mineralization of existing osteoblasts. These results were also confirmed by real-time polymerase chain reaction (RT-PCR) analysis of osteoblastic markers. PS can be combined to PBM not only to damage the malignant cells as aimed in PDT studies, but also to promote cellular activity. The findings of this in vitro study may contribute to in vivo studies and ICG-mediated PBM can have promising outcomes in bone healing and regeneration therapies in future.

Methylene blue mediated photobiomodulation on human osteoblast cells.

Photobiomodulation (PBM) and photodynamic therapy (PDT) are two major methods, which use light in medicine and dentistry. PBM uses low-level laser light to induce cell proliferation and activity. In contrast, PDT use laser light combined with a photosensitizer (PS) to cause cell death. Due to similar, not fully understood mechanisms and biphasic response of light, unexpected and complex outcomes may be observed. In the present study, the effect of 635 nm laser light, with power density 50 mW/cm2, at three different energy densities (0.5, 1, and 2 J/cm2 which last 10, 20, and 40 s, respectively) mediated by methylene blue (MB) on the human osteoblast cell line (ATCC-CRL-11372, Rockville, MD, USA) was investigated. Cell viability (MTT assay and acridine orange/propidium iodide staining) and proliferation (Alamar Blue assay) were assessed at 24, 48, and 72 h post irradiation. Alkaline phosphatase (ALP) activity, mineralization (Alizarin Red staining) and gene expressions (RT-PCR analysis) were analyzed at 7th and 14th days after treatment. Five groups were formed as the control group (no MB, no irradiation), MB (only 0.05 µM MB), MB + 0.5 J/cm2, MB + 1 J/cm2, and MB + 2 J/cm2. Cell viability was decreased at 72 h (ANOVA; p < 0.05) for MB + 0.5 J/cm2, MB + 1 J/cm2, and MB + 2 J/cm2 groups. Although proliferation does not seem to be effected by MB-mediated laser application, osteo-anabolic activity is altered. ALP activity was significantly increased at day 7 (ANOVA; p < 0.05) for MB-combined laser groups; on the other hand, mineralization was significantly decreased (ANOVA; p < 0.05) in all treatment groups. Alkaline phosphatase and collagen-I expressions were upregulated in MB + 2 J/cm2 group at 7th and 14th days, respectively. These results may contribute to the low-dose PDT researches and understanding PBM effects on osteoblast behavior but further studies are needed since inappropriate conditions may lead to undesirable results for both therapies.

Investigation of photobiomodulation potentiality by 635 and 809 nm lasers on human osteoblasts.

Photobiomodulation (PBM) describes light-induced photochemical reactions achieved by the application of red or near infrared lasers/LED light with low energy densities. This noninvasive and painless method has been used in some clinical areas but controversial outcomes demand a skeptical look for its promising and potential effects. In this detailed in vitro study, the osteoblast cells were irradiated with 635 and 809 nm diode lasers at energy densities of 0.5, 1, and 2 J/cm2. Cell viability, proliferation, bone formation, and osteoblast differentiation were evaluated by methylthiazole tetrazolium (MTT) assay, Alamar Blue assay, acridine orange/propidium iodide staining, alkaline phosphatase (ALP) activity, Alizarin red staining, and reverse-transcription polymerase chain reaction (RT-PCR) to test the expression of collagen type I, ALPL, and osteocalcin. The results indicate that studied energy doses have a transient effect (48 h after laser irradiation) on the osteoblast viability and proliferation. Similarly, laser irradiation did not appear to have any effect on ALP activity. These results were confirmed by RT-PCR analysis of osteoblast markers. This study suggests that several irradiation parameters and variations in the methods should be clearly established in the laboratory before laser treatment becomes a postulated application for bone tissue regeneration in clinical level.

In silico tools and transcriptomics analyses in the mutagenicity assessment of cosmetic ingredients: a proof-of-principle on how to add weight to the evidence.

Prior to the downstream development of chemical substances, including pharmaceuticals and cosmetics, their influence on the genetic apparatus has to be tested. Several in vitro and in vivo assays have been developed to test for genotoxicity. In a first tier, a battery of two to three in vitro tests is recommended to cover mutagenicity, clastogenicity and aneugenicity as main endpoints. This regulatory in vitro test battery is known to have a high sensitivity, which is at the expense of the specificity. The high number of false positive in vitro results leads to excessive in vivo follow-up studies. In the case of cosmetics it may even induce the ban of the particular compound since in Europe the use of experimental animals is no longer allowed for cosmetics. In this article, an alternative approach to derisk a misleading positive Ames test is explored. Hereto we first tested the performance of five existing computational tools to predict the potential mutagenicity of a data set of 132 cosmetic compounds with a known genotoxicity profile. Furthermore, we present, as a proof-of-principle, a strategy in which a combination of computational tools and mechanistic information derived from in vitro transcriptomics analyses is used to derisk a misleading positive Ames test result. Our data shows that this strategy may represent a valuable tool in a weight-of-evidence approach to further evaluate a positive outcome in an Ames test.

Linking existing in vitro dermal absorption data to physicochemical properties: Contribution to the design of a weight-of-evidence approach for the safety evaluation of cosmetic ingredients with low dermal bioavailability.

To characterize the risk of cosmetic ingredients when threshold toxicity is assumed, often the "margin of safety" (MoS) is calculated. This uncertainty factor is based on the systemic no observable (adverse) effect level (NO(A)EL) which can be derived from in vivo repeated dose toxicity studies. As in vivo studies for the purpose of the cosmetic legislation are no longer allowed in Europe and a validated in vitro alternative is not yet available, it is no longer possible to derive a NO(A)EL value for a new cosmetic ingredient. Alternatively, cosmetic ingredients with a low dermal bioavailability might not need repeated dose data, as internal exposure will be minimal and systemic toxicity might not be an issue. This study shows the possibility of identifying compounds suspected to have a low dermal bioavailability based on their physicochemical properties (molecular weight, melting point, topological polar surface area and log P) and their in vitro dermal absorption data. Although performed on a limited number of compounds, the study suggests a strategic opportunity to support the safety assessor's reasoning to omit a MoS calculation and to focus more on local toxicity and mutagenicity/genotoxicity for ingredients for which limited systemic exposure is to be expected.

In vitro Dermal Absorption of Hydroquinone: Protocol Validation and Applicability on Illegal Skin-Whitening Cosmetics.

In Europe, hydroquinone is a forbidden cosmetic ingredient. It is, however, still abundantly used because of its effective skin-whitening properties. The question arises as to whether the quantities of hydroquinone used become systemically available and may cause damage to human health. Dermal absorption studies can provide this information. In the EU, dermal absorption has to be assessed in vitro since the Cosmetic Regulation 1223/2009/EC forbids the use of animals. To obtain human-relevant data, a Franz diffusion cell protocol was validated using human skin. The results obtained were comparable to those from a multicentre validation study. The protocol was applied to hydroquinone and the dermal absorption ranged between 31 and 44%, which is within the range of published in vivo human values. This shows that a well-validated in vitro dermal absorption study using human skin provides relevant human data. The validated protocol was used to determine the dermal absorption of illegal skin-whitening cosmetics containing hydroquinone. All samples gave high dermal absorption values, rendering them all unsafe for human health. These results add to our knowledge of illegal cosmetics on the EU market, namely that they exhibit a negative toxicological profile and are likely to induce health problems.

Retrospective analysis of the mutagenicity/genotoxicity data of the cosmetic ingredients present on the Annexes of the Cosmetic EU legislation (2000-12).

To evaluate the mutagenicity/genotoxicity of cosmetic ingredients at the regulatory level, usually a battery of three in vitro tests is applied. This battery, designed to be very sensitive, produces a high number of positive results, imposing the need for in vivo follow-up testing to clear the substance under study. In Europe, the use of experimental animals has become impossible for cosmetic ingredients due to the implementation of animal testing and marketing bans. Consequently, the possibility to 'de-risk' substances with positive in vitro results disappear and potentially safe cosmetic substances will be lost for the EU market unless currently used in vitro assays can be adapted or new non-animal mutagenicity/genotoxicity studies become available. Described strategies to improve the specificity of existing in vitro assays include optimisation of the used cell type and cytotoxicity assay and lowering of the applied top concentration. A reduction of the number of tests in the battery from three to two also has been suggested. In this study, the performance of the 'standard' in vitro mutagenicity/genotoxicity testing battery is analysed for a number of cosmetic ingredients. We composed a database with toxicological information on 249 cosmetic ingredients, mainly present on the Annexes of the European cosmetic legislation. Results revealed that the in vitro mutagenicity/genotoxicity tests showed a low specificity for the cosmetic ingredients concerned, comparable to the specificity published for chemicals. Non-confirmed or 'misleading' positive results amounted up to 93% for the in vitro test batteries. The cell type and top concentrations did not have a major impact on the specificity. With respect to cytotoxicity determinations, different end points were used, potentially leading to different testing concentrations, suggesting the need for a consensus in this matter. Overall, the results of this retrospective analysis point to an urgent need of better regulatory strategies to assess the potential mutagenicity/genotoxicity of cosmetic ingredients.

CMS121 Partially Attenuates Disease Progression in Mouse Models of Huntington's Disease.

There are currently no drugs that meaningfully slow down the progression of Huntington's disease (HD). Moreover, drug candidates against a single molecular target have not had significant success. Therefore, a different approach to HD drug discovery is needed. Previously we showed that the flavonol fisetin is efficacious in mouse and fly models of HD (Hum. Mol. Gen. 20:261, 2011). It is also effective in animal models of Alzheimer's disease (AD), ischemic stroke, and the CNS complications of diabetes, all of which share some pathological features with HD. Potent derivatives of fisetin with improved pharmacology were made that maintain its multiple biological activities (J. Med. Chem. 55:378, 2012). From 160 synthetic fisetin derivatives, one, CMS121, was selected for further study in the context of HD based on pharmacological parameters and its efficacy in animal models of AD. Both R6/2 and YAC128 mouse models of HD were used in these studies. We examined motor function using multiple assays as well as survival. In the R6/2 mice, we also looked at the effects of CMS121 on striatal gene expression. In both models, we found a slowing of motor dysfunction and an increase in median life span. Interestingly, in the YAC128 mice, the effects on the slowing in motor function loss became increasingly more pronounced as the mice aged. CMS121 also reduced HD-driven changes in the expression of genes associated with the proteasome and oxidative phosphorylation. Overall, these results suggest that CMS121 could provide some benefits for HD patients, particularly with regard to increasing health span.

Neutral Red Uptake Assay to Assess Cytotoxicity In Vitro.

The neutral red uptake (NRU) assay is a cell viability assay that can be used for the assessment of compound-induced cytotoxicity. It is based on the ability of living cells to incorporate neutral red, a weak cationic dye, in lysosomes. The quantification of xenobiotic-induced cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red when compared to cells exposed to corresponding vehicle controls. The NRU assay is mainly used for hazard assessment in in vitro toxicology applications. Hence, this method has been incorporated in regulatory recommendations such as the OECD test guideline TG 432, in which an in vitro 3T3-NRU-phototoxicityassay is described to assess the cytotoxicity of compounds in the presence or absence of UV light.This book chapter describes a detailed protocol to carry out the NRU assay using the human hepatoma cell line HepG2, which is frequently employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetylsalicylic acid is assessed.

AMPKα1 Deficiency in Astrocytes from a Rat Model of ALS Is Associated with an Altered Metabolic Resilience.

Alterations in the activity of the regulator of cell metabolism AMP-activated protein kinase (AMPK) have been reported in motor neurons from patients and animal models of amyotrophic lateral sclerosis (ALS). Considering the key role played by astrocytes in modulating energy metabolism in the nervous system and their compromised support towards neurons in ALS, we examined whether a putative alteration in AMPK expression/activity impacted astrocytic functions such as their metabolic plasticity and glutamate handling capacity. We found a reduced expression of AMPK mRNA in primary cultures of astrocytes derived from transgenic rats carrying an ALS-associated mutated superoxide dismutase (hSOD1G93A). The activation of AMPK after glucose deprivation was reduced in hSOD1G93A astrocytes compared to non-transgenic. This was accompanied by a lower increase in ATP levels and increased vulnerability to this insult, although the ATP production rate did not differ between the two cell types. Furthermore, soliciting the activity of glutamate transporters was found to induce similar AMPK activity in these cells. However, manipulation of AMPK activity did not influence glutamate transport. Together, these results suggest that the altered AMPK responsiveness in ALS might be context dependent and may compromise the metabolic adaptation of astrocytes in response to specific cellular stress.

Pancreatic acinar cell fate relies on system xC- to prevent ferroptosis during stress.

Acinar cell dedifferentiation is one of the most notable features of acute and chronic pancreatitis. It can also be the initial step that facilitates pancreatic cancer development. In the present study, we further decipher the precise mechanisms and regulation using primary human cells and murine experimental models. Our RNAseq analysis indicates that, in both species, early acinar cell dedifferentiation is accompanied by multiple pathways related to cell survival that are highly enriched, and where SLC7A11 (xCT) is transiently upregulated. xCT is the specific subunit of the cystine/glutamate antiporter system xC-. To decipher its role, gene silencing, pharmacological inhibition and a knock-out mouse model were used. Acinar cells with depleted or reduced xCT function show an increase in ferroptosis relating to lipid peroxidation. Lower glutathione levels and more lipid ROS accumulation could be rescued by the antioxidant N-acetylcysteine or the ferroptosis inhibitor ferrostatin-1. In caerulein-induced acute pancreatitis in mice, xCT also prevents lipid peroxidation in acinar cells. In conclusion, during stress, acinar cell fate seems to be poised for avoiding several forms of cell death. xCT specifically prevents acinar cell ferroptosis by fueling the glutathione pool and maintaining ROS balance. The data suggest that xCT offers a druggable tipping point to steer the acinar cell fate in stress conditions.