Expression and potential role of apolipoprotein D on the death–survival balance of human colorectal cancer cells under oxidative stress conditions

Producción CientíficaPurpose Inverse correlations of apolipoprotein D (ApoD) expression with tumor growth have been shown, therefore proposing ApoD as a good prognostic marker for diverse cancer types, including colorectal cancer (CRC). Besides, ApoD expression is boosted upon oxidative stress (OS) in many pathological situations. This study aims at understanding the role of ApoD in the progression of human CRC. Methods Samples of CRC and distant normal tissue (n051) were assayed for levels of lipid peroxidation, expression profile of OS-dependent genes, and protein expression. Three single-nucleotide polymorphisms in the ApoD gene were analyzed (n0139), with no significant associations found. Finally, we assayed the effect of ApoD in proliferation and apoptosis in the CRC HT-29 cell line

Cannabinoid-induced motor dysfunction via autophagy inhibition

The recreational and medical use of cannabis is largely increasing worldwide. Cannabis use, however, can cause adverse side effects, so conducting innovative studies aimed to understand and potentially reduce cannabis-evoked harms is important. Previous research conducted on cultured neural cells had supported that CNR1/CB1R (cannabinoid receptor 1), the main molecular target of cannabis, affects macroautophagy/autophagy. However, it was not known whether CNR1 controls autophagy in the brain in vivo, and, eventually, what the functional consequences of a potential CNR1-autophagy connection could be. We have now found that Δ9-tetrahydrocannabinol (THC), the major intoxicating constituent of cannabis, impairs autophagy in the mouse striatum. Administration of autophagy activators (specifically, the rapalog temsirolimus and the disaccharide trehalose) rescues THC-induced autophagy inhibition and motor dyscoordination. The combination of various genetic strategies in vivo supports the idea that CNR1 molecules located on neurons belonging to the direct (striatonigral) pathway are required for the autophagy- and motor-impairing activity of THC. By identifying autophagy as a mechanistic link between THC and motor performance, our findings may open a new conceptual view on how cannabis acts in the brain

Estudio de los mecanismos moleculares y celulares de la función protectora de la Apolipoproteína D

Tesis Doctoral presentada por Raquel Bajo Grañeras para optar al grado de doctora por la Universidad de Valladolid, Facultad de Medicina (Departamento de Bioquímica y Biología y Fisiología).-- Sujeta a Licencia Creative Commons.[EN]: ApoD is a secreted Lipocalin that has been functionally associated with aging, degeneration and nervous system damage, and with many cancer types as well. Recent work in model organisms like plants, flies and mice1-5 has shown that ApoD participates as a survival mechanism, conserved across species, against diverse oxidative stress situations. In this thesis we propose four objectives aiming to understand the ApoD protective role in various physiological and pathological situations. To carry out these objectives we have used biological models with high sensitivity to and/or high levels of oxidative stress. The experimental approach includes analyses at the following levels: 1) gene expression assays (microarrays or qPCR arrays), 2) biochemical assays (enzyme activity, lipid peroxidation, or dopamine levels), 3) cell cultures (cell lines or primary cultures), 4) tissue analyses (mouse nervous system tissue or human colorectal adenocarcinoma), and 5) locomotor behavior analyses in the whole organism (in the mouse experimental model). We further ask whether ApoD function changes in different tissues or whether it works through a general mechanism of action everywhere it is expressed. The first three objectives of this work have been performed in the nervous system, an essentially post-mitotic tissue highly sensitive to stress. We have focused on glial cells (astrocytes), because this cell type predominantly responds against pro-oxidative situations; and on dopaminergic neurons because they are particularly sensitive to this type of stress. The last thesis objective aims at studying ApoD function in a proliferating tissue that is able to support high levels and tolerance to the oxidative stress caused by its high rate of ROS production (human colorectal cancer). Thus, we have been able to contrast the similarities and differences between both physiological situations to contextualize the relevance and impact of ApoD.In summary, the presence of ApoD in the neuronal environment is necessary for an adequate protection against oxidative damage in the nervous system since it affects the transcriptional profile of the early response to this kind of stress. We have shown that ApoD preferentially alters the neuronal and oligodendroglial transcriptome with changes in expression of genes involved in neuronal excitability, synaptic transmission, management of myelin and the response to oxidative stress (Objective 1). After demonstrating the influence of ApoD in a proper glial response that could cushion the neurodegeneration associated with oxidative stress, we directed our study to the role of ApoD in the important glia-glia and glia-neuron interactions within the nervous system. We show that ApoD is secreted by astrocytes in response to the ROS-generator paraquat, and that it has a beneficial effect on the functionality of the locomotor system in the mouse, particularly on the dopaminergic system. Our data demonstrate that ApoD expression is induced by the activation of the JNK signaling pathway and that it functions as an autocrine mechanism to protect astrocytes against oxidative stress. In addition, ApoD modulates astroglial reactivity and alters the astrocytes transcriptional response upon oxidative stress. The addition of human ApoD to mouse astrocytes promotes their survival, further indicating the existence of mechanisms conserved across species. ApoD contributes to the endurance of astrocytes and reduces their reactivity both in vitro and in vivo. These two effects are sufficient to improve the functionality of the nigrostriatal dopaminergic system (Objective 2). The observed decrease in the impact of damage in neurons of the substantia nigra could be due to a combination of the benefits of a healthy surrounding glia and the direct effects of ApoD on neurons. Among other glial factors released to extracellular medium, ApoD could perform direct effects on the viability of neurons. We tested this hypothesis and found that ApoD is effective even in PINK1 deficient dopaminergic neurons (a Parkinson's disease model) and that these beneficial effects are mediated by ERK signaling pathway activation which promotes cell survival (Objective 3).After observing what happens in the nervous system, where ApoD plays a protective role both for glia and for damaged neurons, we wanted to study a very different model to confirm if ApoD protective effects are applicable. For this purpose we have used human colorectal cancer tissues and a cell line of colon cancer. Both strategies have allowed us to observe a negative correlation between the ApoD expression and cancer progression. This represents a paradox because oxidative stress increases along cancer progression. Our study shows that cancer cells are able to respond to prooxidant stimuli. Even though ApoD expression is low in the stromal cells, it increases in the dysplastic epithelium. Finally, ApoD modifies neither the proliferation rate nor apoptosis levels in control conditions, but it promotes apoptosis under oxidative stress conditions. Therefore ApoD might become a therapeutic resource to promote cancer cell death when they are under stress (Objective 4). The general conclusion extracted from these results is that ApoD is a protein able to perform protective effects in different systems upon oxidative stress, promoting cell survival in glial and neurons (essentially post-mitotic cells), but promoting cell death in neoplastic cells under oxidative stress. Our work also uncovers some of the mechanisms by which this apparently pleiotropic protein is able to control the survival/death balance in both physiological and pathological conditions of diverse etiology.Peer reviewe

Apolipoprotein D mediates autocrine protection of astrocytes and controls their reactivity level, contributing to the functional maintenance of paraquat-challenged dopaminergic systems

16 páginas, 9 figuras, 1 tabla.The study of glial derived factors induced by injury and degeneration is important to understand the nervous system response to deteriorating conditions. We focus on Apolipoprotein D (ApoD), a Lipocalin expressed by glia and strongly induced upon aging, injury or neurodegeneration. Here we study ApoD function in the brain of wild type and ApoD-KO mice by combining in vivo experiments with astrocyte cultures. Locomotor performance, dopamine concentration, and gene expression levels in the substantia nigra were assayed in mice treated with paraquat (PQ). The regulation of ApoD transcription, a molecular screening of oxidative stress (OS)-related genes, cell viability and oxidation status, and the effects of adding human ApoD were tested in astrocyte cultures. We demonstrate that (1) ApoD is required for an adequate locomotor performance, modifies the gene expression profile of PQ-challenged nigrostriatal system, and contributes to its functional maintenance; (2) ApoD expression in astrocytes is controlled by the OS-responsive JNK pathway; (3) ApoD contributes to an autocrine protecting mechanism in astrocytes, avoiding peroxidated lipids accumulation and altering the PQ transcriptional response of genes involved in ROS managing and the inflammatory response to OS; (4) Addition of human ApoD to ApoD-KO astrocytes promotes survival through a mechanism accompanied by protein internalization and modulation of astroglial reactivity. Our data support that ApoD contributes to the endurance of astrocytes and decreases their reactivity level in vitro and in vivo. ApoD function as a maintenance factor for astrocytes would suffice to explain the observed protection by ApoD of OS-vulnerable dopaminergic circuits in vivo.Grant sponsor: MEC; Grant number: BFU2005-00522; Grant sponsor: JCyL; Grant number: VA049A05; Grant sponsor: MICINN; Grant number: BFU2008-01170; Grant sponsor: JCyL; Grant number: GRS/278/A/08.Peer reviewe

Circuit-specific signaling in astrocyte-neuron networks in basal ganglia pathways

© 2015, American Association for the Advancement of Science. All rights reserved. Astrocytes are important regulatory elements in brain function. They respond to neurotransmitters and release gliotransmitters that modulate synaptic transmission. However, the cell- and synapse-specificity of the functional relationship between astrocytes and neurons in certain brain circuits remains unknown. In the dorsal striatum, which mainly comprises two intermingled subtypes (striatonigral and striatopallidal) of medium spiny neurons (MSNs) and synapses belonging to two neural circuits (the direct and indirect pathways of the basal ganglia), subpopulations of astrocytes selectively responded to specific MSN subtype activity. These subpopulations of astrocytes released glutamate that selectively activated N-methyl-D-aspartate receptors in homotypic, but not heterotypic, MSNs. Likewise, astrocyte subpopulations selectively regulated homotypic synapses through metabotropic glutamate receptor activation. Therefore, bidirectional astrocyte-neuron signaling selectively occurs between specific subpopulations of astrocytes, neurons, and synapses.Peer Reviewe

Apolipoprotein D alters the early transcriptional response to oxidative stress in the adult cerebellum

12 páginas, 5 figuras.-- et al.The lipocalin Apolipoprotein D (ApoD), known to protect the nervous system against oxidative stress (OS) in model organisms, is up-regulated early in the mouse brain in response to the ROS generator paraquat. However, the processes triggered by this up-regulation have not been explored. We present here a study of the effect of ApoD on the early transcriptional changes upon OS in the mouse cerebellum using microarray profiling. ApoD-KO and transgenic mice over-expressing ApoD in neurons are compared to wild-type controls. In control conditions, ApoD affects the transcriptional profile of neuron and oligodendrocyte-specific genes involved in neuronal excitability, synaptic function, and myelin homeostasis. When challenged with paraquat, the absence of ApoD modifies the response of genes mainly related to OS management and myelination. Interestingly, the over-expression of ApoD in neurons almost completely abolishes the early transcriptional response to OS. We independently evaluate the expression of protein kinase Cδ, a gene up-regulated by OS only in the ApoD-KO cerebellum, and find it over-expressed in cultured ApoD-KO primary astrocytes, which points to a role for ApoD in astrocyte-microglia signaling. Our results support the hypothesis that ApoD is necessary for a proper response of the nervous system against physiological and pathological OS.This work was supported by grant CIHR MOP 15677 to E.R.; FRSQ and CRSNG studentships to S.D.C.; grants BFU2007-61848 (DGICYT) and CIBER CB06/06/0050 (FISSICiii) to C.G.; and grants MEC BFU2005-00522, JCyL VA049A05, and MICINN BFU2008-01170 to M.D.G. and D.S.Peer reviewe

Aging without apolipoprotein D: Molecular and cellular modifications in the hippocampus and cortex

A detailed knowledge of the mechanisms underlying brain aging is fundamental to understand its functional decline and the baseline upon which brain pathologies superimpose. Endogenous protective mechanisms must contribute to the adaptability and plasticity still present in the healthy aged brain. Apolipoprotein D (ApoD) is one of the few genes with a consistent and evolutionarily conserved up-regulation in the aged brain. ApoD protecting roles upon stress or injury are well known, but a study of the effects of ApoD expression in the normal aging process is still missing.Using an ApoD-knockout mouse we analyze the effects of ApoD on factors contributing to the functional maintenance of the aged brain. We focused our cellular and molecular analyses in the cortex and hippocampus at an age representing the onset of senescence where mortality risks are below 25%, avoiding bias towards long-lived animals. Lack of ApoD causes a prematurely aged brain without altering lifespan. Age-dependent hyperkinesia and memory deficits are accompanied by differential molecular effects in the cortex and hippocampus. Transcriptome analyses reveal distinct effects of ApoD loss on the molecular age-dependent patterns of the cortex and hippocampus, with different cell-type contributions to age-regulated gene expression. Markers of glial reactivity, proteostasis, and oxidative and inflammatory damage reveal early signs of aging and enhanced brain deterioration in the ApoD-knockout brain. The lack of ApoD results in an age-enhanced significant reduction in neuronal calcium-dependent functionality markers and signs of early reduction of neuronal numbers in the cortex, thus impinging upon parameters clearly differentiating neurodegenerative conditions from healthy brain aging. Our data support the hypothesis that the physiological increased brain expression of ApoD represents a homeostatic anti-aging mechanism.This work was supported by grants: Junta de Castilla y León (JCyL) grant VA180A11-2, and Ministerio de Ciencia e Innovación (MICINN) grants BFU2008-01170 and BFU2011-23978 to M.D.G. and D.S.Peer Reviewe

Expression and potential role of apolipoprotein D on the death-survival balance of human colorectal cancer cells under oxidative stress conditions

et al.[Purpose]: Inverse correlations of apolipoprotein D (ApoD) expression with tumor growth have been shown, therefore proposing ApoD as a good prognostic marker for diverse cancer types, including colorectal cancer (CRC). Besides, ApoD expression is boosted upon oxidative stress (OS) in many pathological situations. This study aims at understanding the role of ApoD in the progression of human CRC. [Methods]: Samples of CRC and distant normal tissue (n = 51) were assayed for levels of lipid peroxidation, expression profile of OS-dependent genes, and protein expression. Three single-nucleotide polymorphisms in the ApoD gene were analyzed (n = 139), with no significant associations found. Finally, we assayed the effect of ApoD in proliferation and apoptosis in the CRC HT-29 cell line. [Results]: In CRC, lipid peroxides increase while ApoD messenger RNA and protein decrease through tumor progression, with a prominent decrease in stage I. In normal mucosa, ApoD protein is present in lamina propia and enteroendocrine cells. In CRC, ApoD expression is heterogeneous, with low expression in stromal cells commonly associated with high expression in the dysplastic epithelium. ApoD promoter is basally methylated in HT-29 cells but retains the ability to respond to OS. Exogenous addition of ApoD to HT-29 cells does not modify proliferation or apoptosis levels in control conditions, but it promotes apoptosis upon paraquat-induced OS. [Conclusion]: Our results show ApoD as a gene responding to OS in the tumor microenvironment. Besides using ApoD as marker of initial stages of tumor progression, it can become a therapeutic tool promoting death of proliferating tumor cells suffering OS. © 2013 Springer-Verlag Berlin Heidelberg.This work has been supported by grants to M.D.G. and D.S. (Ministerio de Educación y Ciencia (MEC), Spain, grant BFU2005-00522; Junta de Castilla y León (JCyL), grant VA049A05; and Ministerio de Ciencia e Innovación (MICINN), grant BFU2008-01170); and to R.B. (JCyL, GRS/278/A/08).Peer Reviewe

Early detection of high oxidative activity in patients with adenomatous intestinal polyps and colorectal adenocarcinoma: Myeloperoxidase and oxidized low-density lipoprotein in serum as new markers of oxidative stress in colorectal cancer

et al.[Objectives]: To detect whether signs of oxidative stress appear at early stages of colorectal adenocarcinoma (CRC), particularly in the polyp stage. We also aimed to evaluate the specific entities myeloperoxidase (MPO) and oxidized low-density lipoprotein (oxLDL) as novel markers of oxidation in the plasma of patients with CRC and to study the relationship between oxidative status in plasma and patient survival. [Methods]: We assayed serum or plasma specimens from healthy control subjects (n = 14), from patients with intestinal polyps (n = 39), and from patients with CRC (n = 128) to calculate the modified oxidative balance score (MOBS) using several serum markers (β-carotene, lycopene, vitamin A, vitamin E, MPO, and oxLDL). We also assayed the levels of C-reactive protein (CRP) and obtained lipid profiles. Finally, we studied the survival of patients in relationship to oxidative status (antioxidants and pro-oxidants) and inflammation markers, and added theses data to the lipid profile for each patient. [Results]: Oxidative stress levels increased as disease stage advanced. This increase was detected early in the polyp stage, before polyps progressed to cancer, and could be measured by the increase of such new markers as MPO and oxLDL, the decrease in antioxidants, and the MOBS value. Higher levels of oxidation correlated with lower survival. [Conclusion]: The oxidation process, which can cause mutations leading to CRC, begins development in the polyp stage. This process may be detected early by monitoring serum markers such as MPO and oxLDL.This work has been supported by grants to M.R.B. (JCyL GRS/278/A/08).Peer Reviewe