Epithelial-to-mesenchymal transition involves triacylglycerol accumulation in DU145 prostate cancer cells

Epithelial to mesenchymal transition (EMT) is a biological process that plays a crucial role in cancer metastasis. Although studies regarding the EMT mechanisms are usual in terms of gene expression and protein functions, little is known about the involvement of lipids in EMT. In this work, an untargeted lipidomic analysis was performed to reveal which lipids are involved in the EMT process. DU145 prostate cancer cells were treated with TNFα, a well-known EMT inducer. After 6 hours of treatment, a decrease of cell membrane E-cadherin as well as a reduction in its gene expression were observed. Also, the mesenchymal markers Vimentin and Snail were up-regulated, suggesting that EMT started below 6 hours of treatment. Lipid extracts of untreated and TNFα-treated cells at short times were analyzed using ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-MS). Multivariate data analysis methods were applied to decipher which lipids presented significant changes after EMT induction. Among the results obtained, a significant increase of twelve unsaturated triacylglycerides (TAGs) was observed. This increase of TAGs was also observed for cells treated with TGFβ (another EMT inducer), suggesting that this feature is a common mechanism in the EMT process. In conclusion, this work reported for the first time a TAG accumulation through EMT induction. These TAG lipids could play a key role in providing cells with the energy, cell membrane components and signaling lipids necessary to guarantee the enhanced cell migration and proliferation of metastatic cells.This work was supported by the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement 320737. J.J acknowledges a CSIC JAE-Doc contract cofounded by FSE.Peer reviewe

GM2-GM3 gangliosides ratio is dependent on GRP94 through down-regulation of GM2-AP cofactor in brain metastasis cells

GRP94 is an ATP-dependent chaperone able to regulate pro-oncogenic signaling pathways. Previous studies have shown a critical role of GRP94 in brain metastasis (BrM) pathogenesis and progression. In this work, an untargeted lipidomic analysis revealed that some lipid species were altered in GRP94-deficient cells, specially GM2 and GM3 gangliosides. The catalytic pathway of GM2 is affected by the low enzymatic activity of beta-Hexosaminidase (HexA), responsible for the hydrolysis of GM2 to GM3. Moreover, a deficiency of the GM2-activator protein (GM2-AP), the cofactor of HexA, is observed without alteration of gene expression, indicating a post-transcriptional alteration of GM2-AP in the GRP94-ablated cells. One plausible explanation of these observations is that GM2-AP is a client of GRP94, resulting in defective GM2 catabolic processing and lysosomal accumulation of GM2 in GRP94-ablated cells. Overall, given the role of gangliosides in cell surface dynamics and signaling, their imbalance might be linked to modifications of cell behaviour acquired in BrM progression. This work indicates that GM2-AP could be an important factor in ganglioside balance maintenance. These findings highlight the relevance of GM3 and GM2 gangliosides in BrM and reveal GM2-AP as a promising diagnosis and therapeutic target in BrM research

3-Ketosphinganine provokes the accumulation of dihydroshingolipids and induces autophagy in cancer cells.

Although several reports describe the metabolic fate of sphingoid bases and their analogs, as well as their action and that of their phosphates as regulators of sphingolipid metabolizing-enzymes, similar studies for 3-ketosphinganine (KSa), the product of the first committed step in de novo sphingolipid biosynthesis, have not been reported. In this article we show that 3-ketosphinganine (KSa) and its dideuterated analog at C4 (d2KSa) are metabolized to produce high levels of dihydrosphingolipids in HGC27, T98G and U87MG cancer cells. In contrast, either direct C1 O-phosphorylation or N-acylation of d2KSa to produce dideuterated ketodihydrosphingolipids does not occur. We also show that cells respond to d2KSa treatment with induction of autophagy. Time-course experiments agree with sphinganine, sphinganine 1-phosphate and dihydroceramides being the mediators of autophagy stimulated by d2KSa. Enzyme inhibition studies support that inhibition of Des1 by 3-ketobases is caused by their dihydroceramide metabolites. However, this effect contributes to increasing dihydrosphingolipid levels only at short incubation times, since cells respond to long time exposure to 3-ketobases with Des1 overexpression. The translation of these overall effects into cell fate is discussed.Partial financial support from the ‘‘Ministerio de Ciencia e Innovación’’, Spain (Grants SAF2011-22444), ‘‘Ministerio de Economía y Competitividad’’ (CTQ2014-54743-R), CSIC (Grant PIE 2008801034) and Fundacio´ La Marato´ TV3 (Grant 112130 and 112132) is acknowledged. A PhD fellowship from SENESCYTEcuador to Y. F. O. is also acknowledged. We thank Pedro Rayo for his excellent technical assistance.Peer reviewe

Oxidative-Stress-Associated Proteostasis Disturbances and Increased DNA Damage in the Hippocampal Granule Cells of the Ts65Dn Model of Down Syndrome

Oxidative stress (OS) is one of the neuropathological mechanisms responsible for the deficits in cognition and neuronal function in Down syndrome (DS). The Ts65Dn (TS) mouse replicates multiple DS phenotypes including hippocampal-dependent learning and memory deficits and similar brain oxidative status. To better understand the hippocampal oxidative profile in the adult TS mouse, we analyzed cellular OS-associated alterations in hippocampal granule cells (GCs), a neuronal population that plays an important role in memory formation and that is particularly affected in DS. For this purpose, we used biochemical, molecular, immunohistochemical, and electron microscopy techniques. Our results indicate that TS GCs show important OS-associated alterations in the systems essential for neuronal homeostasis: DNA damage response and proteostasis, particu larly of the proteasome and lysosomal system. Specifically, TS GCs showed: (i) increased DNA damage, (ii) reorganization of nuclear proteolytic factories accompanied by a decline in proteasome activity and cytoplasmic aggregation of ubiquitinated proteins, (iii) formation of lysosomal-related structures containing lipid droplets of cytotoxic peroxidation products, and (iv) mitochondrial ultrastructural defects. These alterations could be implicated in enhanced cellular senescence, accelerated aging and neurodegeneration, and the early development of Alzheimer?s disease neuropathology present in TS mice and the DS population.Funding: This work was supported by the following grants: “Instituto de Investigación Valdecilla” (IDIVAL; NVAL 19/23), Santander, Spain; “Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas” (CIBERNED; CB06/05/0037) Spain; and “Agencia Estatal de Investicación, MICIN” (grant number: PID2020-117601RB-I00). Acknowledgments: The authors would like to thank Raquel García-Ceballos and Eva García Iglesias for their technical assistance

Nuclear Reorganization in Hippocampal Granule Cell Neurons from a Mouse Model of Down Syndrome: Changes in Chromatin Configuration, Nucleoli and Cajal Bodies

Down syndrome (DS) or trisomy of chromosome 21 (Hsa21) is characterized by impaired hippocampal-dependent learning and memory. These alterations are due to defective neurogenesis and to neuromorphological and functional anomalies of numerous neuronal populations, including hippocampal granular cells (GCs). It has been proposed that the additional gene dose in trisomic cells induces modifications in nuclear compartments and on the chromatin landscape, which could contribute to some DS phenotypes. The Ts65Dn (TS) mouse model of DS carries a triplication of 92 genes orthologous to those found in Hsa21, and shares many phenotypes with DS individuals, including cognitive and neuromorphological alterations. Considering its essential role in hippocampal memory formation, we investigated whether the triplication of this set of Hsa21 orthologous genes in TS mice modifies the nuclear architecture of their GCs. Our results show that the TS mouse presents alterations in the nuclear architecture of its GCs, affecting nuclear compartments involved in transcription and pre-rRNA and pre-mRNA processing. In particular, the GCs of the TS mouse show alterations in the nucleolar fusion pattern and the molecular assembly of Cajal bodies (CBs). Furthermore, hippocampal GCs of TS mice present an epigenetic dysregulation of chromatin that results in an increased heterochromatinization and reduced global transcriptional activity. These nuclear alterations could play an important role in the neuromorphological and/or functional alterations of the hippocampal GCs implicated in the cognitive dysfunction characteristic of TS mice

Bexarotene Impairs Cognition and Produces Hypothyroidism in a Mouse Model of Down Syndrome and Alzheimer’s Disease

All individuals with Down syndrome (DS) eventually develop Alzheimer's disease (AD) neuropathology, including neurodegeneration, increases in ?-amyloid (A?) expression, and aggregation and neurofibrillary tangles, between the third and fourth decade of their lives. There is currently no effective treatment to prevent AD neuropathology and the associated cognitive degeneration in DS patients. Due to evidence that the accumulation of A? aggregates in the brain produces the neurodegenerative cascade characteristic of AD, many strategies which promote the clearance of A? peptides have been assessed as potential therapeutics for this disease. Bexarotene, a member of a subclass of retinoids that selectively activates retinoid receptors, modulates several pathways essential for cognitive performance and A? clearance. Consequently, bexarotene might be a good candidate to treat AD-associated neuropathology. However, the effects of bexarotene treatment in AD remain controversial. In the present study, we aimed to elucidate whether chronic bexarotene treatment administered to the most commonly used murine model of DS, the Ts65Dn (TS) mouse could reduce A? expression in their brains and improve their cognitive abilities. Chronic administration of bexarotene to aged TS mice and their CO littermates for 9 weeks diminished the reference, working, and spatial learning and memory of TS mice, and the spatial memory of CO mice in the Morris water maze. This treatment also produced marked hypoactivity in the plus maze, open field, and hole board tests in TS mice, and in the open field and hole board tests in CO mice. Administration of bexarotene reduced the expression of A?1-40, but not of A?1-42, in the hippocampi of TS mice. Finally, bexarotene increased Thyroid-stimulating hormone levels in TS mice and reduced Thyroid-stimulating hormone levels in CO mice, while animals of both karyotypes displayed reduced thyroxine levels after bexarotene administration. The bexarotene-induced hypothyroidism could be responsible for the hypoactivity of TS and CO mice and their diminished performance in the Morris water maze. Together, these results do not provide support for the use of bexarotene as a potential treatment of AD neuropathology in the DS population.FUNDING: This study was supported by the Institute of Research Valdecilla (IDIVAL) (NVAL 16/21 and NVAL 19/23) and the Consejería de Universidades, Igualdad, Cultura y Deporte del Gobierno de Cantabria (16. VP39.64662)

Prenatal, but not Postnatal, Curcumin Administration Rescues Neuromorphological and Cognitive Alterations in Ts65Dn Down Syndrome Mice

Background: The cognitive dysfunction in Down syndrome (DS) is partially caused by deficient neurogenesis during fetal stages. Curcumin enhances neurogenesis and learning and memory. Objectives: We aimed to test the ability of curcumin to rescue the neuromorphological and cognitive alterations of the Ts65Dn (TS) mouse model of DS when administered prenatally or during early postnatal stages, and to evaluate whether these effects were maintained several weeks after the treatment. Methods: To evaluate the effects of prenatal curcumin administration, 65 pregnant TS females were subcutaneously treated with curcumin (300 mg/kg) or vehicle from ED (Embryonic Day) 10 to PD (Postnatal Day) 2. All the analyses were performed on their TS and Control (CO) male and female progeny. At PD2, the changes in neurogenesis, cellularity, and brain weight were analyzed in 30 TS and CO pups. The long-term effects of prenatal curcumin were evaluated in another cohort of 44 TS and CO mice between PD30 and PD45. The neuromorphological effects of the early postnatal administration of curcumin were assessed on PD15 in 30 male and female TS and CO pups treated with curcumin (300 mg/kg) or vehicle from PD2 to PD15. The long-term neuromorphological and cognitive effects were assessed from PD60 to PD90 in 45 mice. Data was compared by ANOVAs. Results: Prenatal administration of curcumin increased the brain weight (+45%, P < 0.001), the density of BrdU (bromodeoxyuridine)- (+150%, P < 0.001) and DAPI (4',6-diamidino-2-phenylindole)- (+38%, P = 0.005) positive cells, and produced a long-term improvement of cognition in TS (+35%, P = 0.007) mice with respect to vehicle-treated mice. Postnatal administration of curcumin did not rescue any of the short- or long-term altered phenotypes of TS mice. Conclusion: The beneficial effects of prenatal curcumin administration to TS mice suggest that it could be a therapeutic strategy to treat DS cognitive disabilities.This study was supported by the “Fondazione Generali e Assicurazioni Generali”, Italy; Fundación Tatiana Pérez de Guzmán el Bueno, IDIVAL (NVAL 19/23), and the Spanish Ministry of Economy and Competitiveness (PSI-2016-76194-R, AEI/FEDER, EU)

Prenatal Administration of Oleic Acid or Linolenic Acid Reduces Neuromorphological and Cognitive Alterations in Ts65dn Down Syndrome Mice

Background: The cognitive impairments that characterize Down syndrome (DS) have been attributed to brain hypocellularity due to neurogenesis impairment during fetal stages. Thus, enhancing prenatal neurogenesis in DS could prevent or reduce some of the neuromorphological and cognitive defects found in postnatal stages. Objectives: As fatty acids play a fundamental role in morphogenesis and brain development during fetal stages, in this study, we aimed to enhance neurogenesis and the cognitive abilities of the Ts65Dn (TS) mouse model of DS by administering oleic or linolenic acid. Methods: In total, 85 pregnant TS females were subcutaneously treated from Embryonic Day (ED) 10 until Postnatal Day (PD) 2 with oleic acid (400 mg/kg), linolenic acid (500 mg/kg), or vehicle. All analyses were performed on their TS and Control (CO) male and female progeny. At PD2, we evaluated the short-term effects of the treatments on neurogenesis, cellularity, and brain weight, in 40 TS and CO pups. A total of 69 TS and CO mice were used to test the long-term effects of the prenatal treatments on cognition from PD30 to PD45, and on neurogenesis, cellularity, and synaptic markers, at PD45. Data were compared by ANOVAs. Results: Prenatal administration of oleic or linolenic acid increased the brain weight (+36.7% and +45%, P < 0.01), the density of BrdU (bromodeoxyuridine)- (+80% and +115%; P < 0.01), and DAPI (4',6-diamidino-2-phenylindole)-positive cells (+64% and +22%, P < 0.05) of PD2 TS mice with respect to the vehicle-treated TS mice. Between PD30 and PD45, TS mice prenatally treated with oleic or linolenic acid showed better cognitive abilities (+28% and +25%, P < 0.01) and a higher density of the postsynaptic marker PSD95 (postsynaptic density protein 95) (+65% and +44%, P < 0.05) than the vehicle-treated TS animals. Conclusion: The beneficial cognitive and neuromorphological effects induced by oleic or linolenic acid in TS mice suggest that they could be promising pharmacotherapies for DS-associated cognitive deficits.This study was supported by “Fondazione Generali e Assicurazioni Generali”, Italy; Fundación Tatiana Pérez de Guzmán el Bueno, IDIVAL (NVAL 19/23), and the Spanish Ministry of Economy and Competitiveness (PSI-2016- 76194-R, AEI/FEDER, EU)

Aprendizaje activo y autoevaluación a través de la implementación de la plataforma Kahoot! para la mejora e innovación docente en psicología y educación basado en la aplicación Q12 Trivia

Memoria ID-186. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2018-2019

Preclinical studies with glioblastoma brain organoid co-cultures show efficient 5-ALA photodynamic therapy

Abstract: Background: The high recurrence of glioblastoma (GB) that occurs adjacent to the resection cavity within two years of diagnosis urges an improvement of therapies oriented to GB local control. Photodynamic therapy (PDT) has been proposed to cleanse infiltrating tumor cells from parenchyma to ameliorate short long-term progression-free survival. We examined 5-aminolevulinic acid (5-ALA)- mediated PDT effects as therapeutical treatment and determined optimal conditions for PDT efficacy without causing phototoxic injury to the normal brain tissue. Methods: We used a platform of Glioma Initiation Cells (GICs) infiltrating cerebral organoids with two different glioblastoma cells, GIC7 and PG88. We measured GICs-5-ALA uptake and PDT/5-ALA activity in dose-response curves and the efficacy of the treatment by measuring proliferative activity and apoptosis. Results: 5-ALA (50 and 100  g/mL) was applied, and the release of protoporphyrin IX (PpIX) fluorescence measures demonstrated that the emission of PpIX increases progressively until its stabilization at 24 h. Moreover, decreased proliferation and increased apoptosis corroborated the effect of 5-ALA/PDT on cancer cells without altering normal cells. Conclusions: We provide evidence about the effectiveness of PDT to treat high proliferative GB cells in a complex in vitro system, which combines normal and cancer cells and is a useful tool to standardize new strategic therapies