Cross-talk between inflammation and mitochondria in x-linked adrenoleukodystrophy (x-ald)an integrative approach towards different therapies

Resumen

Project Title “Cross-talk between inflammation and mitochondria in X-linked adrenoleukodystrophy (X-ALD): an integrative approach towards different therapies¨ Background: X-adrenoleukodystrophy (X-ALD) is a metabolic, peroxisomal disease affecting the nervous system, adrenal cortex and testis resulting from inactivating mutations in ABCD1 gene, whose defect is associated with impaired peroxisomal β-oxidation and accumulation of saturated very long-chain fatty acids (VLCFA) in tissues and body fluids. The main accumulated VLCFA is the hexacosanoic acid, or C26:0, which serves as a pathognomonic biomarker for the biochemical diagnosis of the disease and we have formerly described that an excess of the VLCFA C26:0 induces oxidative damage, which underlies the axonal degeneration (Fourcade et al., 2008; López-Erauskin et al., 2011; López-Erauskin et al., 2013. Despite being a single-gene disease, X-ALD is a complex inherited syndrome in which the same mutation in the ABCD1 gene can lead to clinically very distinct phenotypes from adrenal insufficiency to fatal cerebral demyelination. Abcd1 null mice (Abcd1-) develop a late onset axonal degeneration in the spinal cord and locomotor disability without active signs of inflammatory demyelination in brain resembling the most common phenotype in humans, adrenomyeloneuropathy (AMN). The animals show early oxidative damage, mitochondrial dysfunction and impaired bioenergetic and redox homeostasis. As most the findings from our laboratory were performed in human fibroblasts or in total spinal cord samples derived from the Abcd1 null mice, in the first part of the present study, we will take the advantage of primary glial cultures to identify the molecular mechanism involved in different cell types in brain (mainly astrocytes and microglia) where their dysfunction could lead to neurodegeneration. Also we had shown that there is mitochondrial dysfunction in fibroblasts of X-ALD patients and in X-ALD mouse model associated with impaired oxidative phosphorylation,(Lopez erauskin., et al 2013, Morato et al., 2013) therefore we are interested to find the new therapeutic targets by using different drugs and finally to investigate the working mechanism underlying them. Aims and Objectives: 1. The main aim of my project is to decipher the cell type responsible of the disease onset. We have formerly identified axonal degeneration prior to myelin degeneration in spinal cords and also have seen oxidative damage in motor neurons. We will test the hypothesis of glial cells (microglia and astrocytes) as target of C26:0 driven toxicity and identify the effectors cell type for the cytotoxic action. Further we explore the novel therapeutic targets with the existing and new studies derived from X-ALD mouse model. To address these questions, we propose the following approaches: - Using primary cultures derived from brain of Abcd1 knockout mouse and wild-type littermates we characterized the phenotype of microglia cells after exposition to C26:0. Microglia can be identified as a cytotoxic (M1) or neuroprotective (M2) using specific markers, thus we will determine its phenotype by qRT PCR. At the end, we will corroborate these results with in vivo studies. - We checked the noxious effects of C26:0 on different parameters like ROS, source of ROS and inflammation profile in both WT and Abcd1- mice microglia and astrocytes after C26:0 insult. 2. We proposed to test the efficacy of FDA-approved, blood brain barrier permeable drugs which protect mitochondria, using both in vitro and in vivo models of X-ALD, for testing their ability to halt axonal degeneration and associated disability. Our research will facilitate to gaining insight into the mechanisms of action of the chosen drugs. This will widen the impact of our results to major neurodegenerative diseases in which mitochondria dysfunction plays a pivotal role in the pathogenic equation. For this aim, we have selected the following compounds: - CB2 agonist (JWH133). CB2 receptors are also Gi/o protein-coupled receptors that share 44% protein identity with CB1 receptors and display a distinct pharmacological profile and expression pattern. Many laboratories reported that CB2 receptors are not expressed in healthy brain but can be induced in microglia and infiltrating monocytes upon inflammation or activation. Microglial cells in primary cultures are intrinsically activated or “primed” because of the procedure involved in transferring these cells into culture (Becher and Antel, 1996). Many laboratories have shown that “primed” microglia prepared from human, rat or mouse tissue express CB2 receptors (Carlisle et al., 2002; Facchinetti et al., 2003; Klegeris et al., 2003;Ramirez et al., 2005; Rock et al., 2007; Walter et al., 2003). The expression level of these receptors can be further induced or inhibited by certain pathogens and cytokines. Researchers have shown that CB2 selective agonist to be beneficial in several animal models of neurodegenerative diseases, as well as neuropathic and inflammatory pain by inhibiting microglia activation. Thus our aim is to check whether Cb2 agonist modulates the progression of disease in our X-ALD mouse model. - Methylene blue. Due to its unique redox properties, MB directly accepts electrons from NADH, NADPH, and FADH2 and mediates the flow of electrons between the mitochondrial respiratory complexes and regulates mitochondrial respiration. Importantly, it has been demonstrated that the MB-induces the increase of mitochondrial complex I and I-III activities and is insensitive to complex I and III inhibition. Furthermore, reduced form MB (MBH2) found to be able to deliver the electrons to cytochrome c in the presence of oxygen in mitochondria in an alternate route despite the inhibition of complex I and III (Atamna et al., 2012, Wen et al., 2011). These properties together with the ability to cross BBB made MB as a good candidate for targeting mitochondria dysfunction and oxidative stress in many neurological disoders. With the existing knowledge of MB, here we evaluate the efficacy of MB in physiopathogenesis of X-ALD. Results: 1.1 Microglia from in Abcd1- mice co-expresses potentially neuroprotective and toxic factors: In the first study, with the application of both in-vivo and in-vitro approaches, we identified a general inflammatory imbalance in X-ALD mice. We analyzed the M1 and M2 inflammatory profile in our mouse model with background knowledge of M1 and M2 induction in PBMC and plasma from AMN patients. We identified a mixed inflammatory profile in Abcd1- mice spinal cord associated with upregulation of most of the pro and anti-inflammatory cytokines and chemokines. Further, we speculate whether these inflammatory responses come from microglia, as they are key players of many neurodegenerative disorders that contribute the major role in neurodegeneration. We found a similar inflammatory pattern like spinal cord as these cultures display an inflammatory imbalance at baseline and show more severe inflammatory profile with exogenous C26:0. These inflammatory responses are characterized by up-regulation of some cytokines and chemokines in Abcd1- mice microglia like Tnf-α, Il1β and Ccl5 at baseline and further elevation of cytotoxic factors like Cd86, iNOS and Cox2 upon C26:0. However, no baseline differences were seen in case of astrocytes on inflammation indicating microglia could play an important role in progression of the disease. 1.2 Mitochondria, as the major source of ROS production in Abcd1- mice microglia after C26:0 insult: By using the probe carboxy-H2DCFDA (dichlorofluorescence), which detects reactive oxygen species (ROS) of any intracellular origin, we formerly reported that an excess of C26:0 produced oxidative lesions in proteins and generated ROS in human fibroblasts (Lopez erauskin., et al 2013). Here we checked the ROS levels in both WT and Abcd1- mice microglia and astrocytes after C26:0 treatment. We found that there is slight increase in total ROS levels in Abcd1- microglia and further increase after C26:0 in both genotypes. Similar toxic effects of C26:0 was found, when treated on astrocytes cultures. However, no baseline differences were seen in case of astrocytes in total ROS levels. Next we sought to investigate whether mitochondria could be the major source of ROS production like seen in X-ALD human fibroblasts. We thus used the two fluorescent dyes DHE (dihydroethidium) and MitoSOX (DHE covalently bonded to hexyl triphenylphosphonium cation to measure the superoxide levels (total Vs mitochondria). The levels of ROS induced by C26:0, detected using either DHE or MitoSOX probes, were similar, which indicates that mitochondria are the major sources of ROS in X-ALD mice microglia. 2 CB2 agonist, JWH133 halts axonal degeneration and inflammation in X-ALD mice: We found an altered ECS with upregulated CB2 levels in X-ALD spinal cord and in primary microglia. With these data, we hypothesized that modulation of inflammatory responses and regulating microglia activation via CB2 would be a beneficial therapeutic approach for treating X-ALD. We found that CB2 agonist blocks microgliosis and prevents axonal degeneration and stops the progression of locomotor deficits in Abcd1-/Abcd2-/- mice model. Also it prevents inflammation by normalizing the levels of pro inflammatory cytokines in Abcd1- mice spinal cord. It also normalizes ATP and mtDNA levels in Abcd1- mice spinal cord. This induction of mtDNA is correlated with an increase of mitochondrial biogenesis characterized by an up-regulation of Pgc-1α and Tfam in Abcd1- mice. Moreover, we found that CB2 activation also inhibits C26:0 driven inflammation and ROS production in primary Abcd1- mice microglia. 3 Methylene blue halts axonal degeneration and induces mitochondrial biogenesis in X-ALD mice. Earlier we have reported that X-linked adrenoleukodystrophy mice exhibit mitochondrial depletion at 12 months of age, concomitant with a downregulation (PGC-1_) pathway leading to a mitochondrial biogenesis defect, metabolic failure and oxidative stress which further leads to axonal degeneration and locomotor disability. On treatment with Methylene blue, reversed the axonal degeneration and associated locomotors disability in Abcd1-/Abcd2-/- mouse model. Also we found that MB increases anti inflammatory response and inhibits the alternative NFkB pathway in Abcd1- mice thus reducing the inflammation. Moreover, MB prevents mitochondria depletion and induces the biogenesis and thus prevents the metabolic failure. Further MB treatment reduces the activated DRP1 levels in Abcd1- mice spinal cord. To corroborate these results with in-vitro studies, we checked the effect of MB on activity of DRP1 on mitochondria network. We demonstrated that MB treatment inhibits C26:0 induced ROS production and prevents the translocation of DRP1 to mitochondria, thus maintaining the integrity of mitochondria network. Collectively, the findings of this doctoral thesis suggest that the above two drugs targeting inflammation and mitochondria may be potential therapies for the treatment of X-ALD and also for the other neurodegenerative disorders that share the common pathogenic features. References: • Fourcade, S. et al. (2008). Hum Mol Genet, 17, 1762-73. • Galino, J. et al. (2011). Antioxid Redox Signal, 15, 2095-107. • Lopez-Erauskin, J. et al. (2011). Ann Neurol, 70, 84-92. • Launay, N. et al. (2013). Brain, 136, 891-904. • Lopez-Erauskin, J. et al. (2012). Brain, 135, 3584-98. • Lopez-Erauskin, J. et al. (2013). Hum Mol Genet, 22, 3296-305. • Molina-Holgado E, et al., (2002) Journal of Neurosci • Pujol, A. et al. (2004). Hum.Mol.Genet., 13, 2997-3006. • Saura et al., Glia. 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