THE ROLE OF PEROXISOME PROLIFERATOR ACTIVATED RECEPTORS IN AMYOTROPHIC LATERAL SCLEROSIS: POTENTIAL MECHANISMS FOR NEUROPROTECTION

The vast majority of neurodegenerative disorders are adult-onset, incurable diseases. Understanding the pathogenetic mechanisms underlying these disorders and finding molecules apt to correct such processes are, therefore, among the hottest topics of biomedical research. Amyotrophic Lateral Sclerosis is one of the most common adult-onset neurodegenerative diseases characterized by progressive degeneration of upper and lower motor neurons leading to paralysis and death due to respiratory failure within 3-5 years from the onset. Only one drug, riluzole, has proved effective in extending the lifespan of patients with ALS, but only by 3-6 months. For this reason the development of effective therapies for this pathology is highly invocated, but to date all attempts to develop novel treatments have failed. In this context, two recent reports on the neuroprotective activity of the PPAR\uf067 agonist Pioglitazione in ALS mice result of considerable interest: in these studies, two independent groups demonstrated that Pioglitazone, an agent which is currently used in therapy for the treatment of type II diabetes, is neuroprotective in a mouse model of Amyotrophic Lateral Sclerosis, the hSOD1-G93A transgenic mice. Pioglitazone treatment started before the appearance of the symptoms, improved the motor performance and reduced the weight loss, attenuated motor neuron death and increased the survival. In addition, Pioglitazone reduced microglial activation and gliosis in the spinal cord, decreasing the production of pro-inflammatory mediators, such as iNOS, NF-kB and COX2. On this ground, we decided to investigate the transcriptional activity of PPARs in the central nervous system of the hSOD1-G93A mouse line, a well-characterized animal model of Amyotrophic Lateral Sclerosis, with the aim of identifying the stage of the disease at which the activity of PPARs becomes relevant to the pathology. To this end, we took advantage of the transgenic mouse PPRE-Luc, available in the laboratory, in which the reporter gene luciferase is expressed under the control of a promoter responsive to PPARs. Thus, we crossed the PPRE-Luc mice with the hSOD1-G93A animals to obtain mice that are heterozygous for the PPRE-Luc transgene and heterozygous or null for the hSOD1-G93A transgene. The analysis of the enzymatic activity of luciferase in the spinal cord and the brain areas of PPRE-Luc;hSOD1-G93A mice shows an abrupt increase of PPAR activity at the end stage of the disease in the spinal cord, which is the organ principally involved in the pathology, and in all the brain areas analysed. We demonstrated that this phenomenon clearly depends on the pathology because it is not shared by the peripheral organs (e.g. kidney and liver). Furthermore, it is not dependent on the metabolic modifications induced from the starvation that the animals experience during the last days of their life when they are almost completely paralysed and, thus, unable to reach for food and water. We subsequently decided to further investigate this mechanism by identifying the isoform(s) responsible for the increase of PPARs activity at the last stage of the disease and the cell type(s) involved. We first analysed the nuclear translocation of PPAR\u3b1, PPAR\u3b2/\u3b4 and PPAR\u3b3 in the spinal cord of hSOD1-G93A mice with an ELISA-based Transcription Factor Assay. The results obtained from these experiments showed that the overall nuclear presence of the different isoforms of PPARs does not change during the course of the disease. In order to obtain a cell specific information about the distribution of PPARs in the spinal cord, we next analysed the localization of PPAR\u3b1, PPAR\u3b2/\u3b4 and PPAR\u3b3 by immunohistochemistry on sections from the lumbar spinal cord of hSOD1-G93A at the different stages of the pathology using primary antibodies for the specific isoforms of PPARs and cell specific markers. Our stainings revealed that all the three isoforms of PPARs are expressed in spinal cord motor neurons; PPAR\u3b1 and PPAR\u3b2/\u3b4 are localized prevalently into the nucleus but show also a cytoplasmic staining, while PPAR\u3b3 is exclusively nuclear. All the three isoforms are present also in astrocytes where they are exclusively nuclear while only PPAR\u3b3 was detectable in microglia, and was localized into the nucleus. Immunohistochemical analysis confirms that the increase in PPAR activity at the end stage of the disease is not dependent on the increase in the nuclear presence of the receptors in the different cell types of the spinal cord, suggesting that it possibly derives from ligand-dependent effects and/or the differential recruitment of co-regulators. To identify the specific isoform whose activity is important during the pathology we analysed the expression of isoform-specific target genes, i.e. MCAD for PPAR\u3b1, Acsl6 for PPAR\u3b2/\u3b4 and LPL for PPAR\u3b3. Only the expression of LPL abruptly increases at the end stage of the disease strongly suggesting that the increase in luciferase activity detected at the later stage of ALS is due to the activation of PPAR\u3b3. To confirm this result we analysed other PPAR\u3b3 target genes, i.e. Catalase, Glutathione S-tranferase alpha-2 and Peroxisome Proliferator Activated Receptor gamma coactivator 1-alpha. The RT-PCR analysis of the expression of Cat, Gsta2 and PGC1\u3b1 showed that Cat and PGC1\u3b1 show a similar trend of reduction till the onset of the disease, 100 days, then the levels of PGC1\u3b1 slightly increase while the Cat expression increases in a significant manner. Gsta2 expression remains fairly constant till the end stage when it increases significantly. On these bases we decided to further investigate the mechanisms of PPAR\u3b3 activation at the end stage of the disease by identifying the cell type involved. The analysis of the fluorescence intensity into the cellular nuclei of lumbar spinal cord sections stained for PPAR\u3b3 demonstrated that the intensity of the receptor signal is greater in motor neurons than in non-neuronal cells. This data led us to hypothesize that motor neurons could be the most likely cell type involved in the activation of PPAR\u3b3 at the end stage of the disease in vivo. Thus, we decided to analyse the expression of the PPAR\u3b3 target genes previously analysed in the spinal cords of hSOD1-G93A mice in an immortalized motor neuronal cell line, the NSC-34 cells. The expression of LPL, Cat and PGC1\u3b1 in NSC-34 cells transiently transfected with the hSOD1-G93A- encoding expression vector is significantly increased as compared to the NSC-34 cells transfected with the empty vector. These data clearly confirm the involvement of motor neurons in PPAR\u3b3 activation at the last stage of the disease; on these bases future studies will be aimed to further elucidate the mechanisms of PPAR\u3b3 protective activity on motor neurons in ALS

Estrogen anti-inflammatory activity in brain: a therapeutic opportunity for menopause and neurodegenerative diseases

Recent studies highlight the prominent role played by estrogens in protecting the central nervous system (CNS) against the noxious consequences of a chronic inflammatory reaction. The neurodegenerative process of several CNS diseases, including Multiple Sclerosis, Alzheimer's and Parkinson's Diseases, is associated with the activation of microglia cells, which drive the resident inflammatory response. Chronically stimulated during neurodegeneration, microglia cells are thought to provide detrimental effects on surrounding neurons. The inhibitory activity of estrogens on neuroinflammation and specifically on microglia might thus be considered as a beneficial therapeutic opportunity for delaying the onset or progression of neurodegenerative diseases; in addition, understanding the peculiar activity of this female hormone on inflammatory signalling pathways will possibly lead to the development of selected anti-inflammatory molecules. This review summarises the evidence for the involvement of microglia in neuroinflammation and the anti-inflammatory activity played by estrogens specifically in microglia

HelixComplex snail mucus as a potential technology against O3 induced skin damage

Mucus form H. aspersa muller has been reported to have several therapeutic proprieties, such as antimicrobial activity, skin protection and wound repair. In this study, we have analyzed H. aspersa mucus (Helixcomplex) bio-adhesive efficacy and its defensive properties against the ozone (O3) (0.5 ppm for 2 hours) exposure in human keratinocytes and reconstructed human epidermis models. Cytotoxicity, tissue morphology and cytokine levels were determined. We confirmed HelixComplex regenerative and bio-adhesive properties, the latter possibly via the characteristic mucopolysaccharide composition. In addition, HelixComplex was able to protect from O3 exposure by preventing oxidative damage and the consequent pro-inflammatory response in both 2D and 3D models. Based on this study, it is possible to suggest HelixComplex as a potentially new protective technology against pollution induced skin damage

Estrogen action in neuroprotection and brain inflammation

The fertile period of women's life compared to menopause is associated with a lower incidence of degenerative inflammatory diseases. In brain, estrogens ameliorate brain performance and have positive effects on selected neural pathologies characterized by a strong inflammatory component. We thus hypothesized that the inflammatory response is a target of estrogen action; several studies including ours provided strong evidence to support this prediction. Microglia, the brain's inflammatory cells, and circulating monocytes express the estrogen receptors ER-alpha and ER-beta and their responsiveness in vivo and in vitro to pro-inflammatory agents, such as lipopolysaccharide (LPS), is controlled by 17beta-estradiol (E(2)). Susceptibility of central nervous system (CNS) macrophage cells to E(2) is also preserved in animal models of neuroinflammatory diseases, in which ER-alpha seems to be specifically involved. At the molecular level, induction of inflammatory gene expression is blocked by E(2). We recently observed that, differently from conventional anti-inflammatory drugs, E(2) stimulates a nongenomic event that interferes with the LPS signal transduction from the plasma membrane to cytoskeleton and intracellular effectors, which results in the inhibition of the nuclear translocation of NF-kappaB, a transcription factor of inflammatory genes. Interference with NF-kappaB intracellular trafficking is selectively mediated by ER-alpha. In summary, evidence from basic research strongly indicates that the use of estrogenic drugs that can mimic the anti-inflammatory activity of E(2) might trigger beneficial effects against neurodegeneration in addition to carrying out their specific therapeutic functio

PLCB2 (phospholipase C, beta 2).

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The peroxisome proliferator-activated receptor γ (PPARγ) controls natural protective mechanisms against lipid peroxidation in amyotrophic lateral sclerosis

Recent evidence highlights the peroxisome proliferator-activated receptors (PPARs) as critical neuroprotective factors in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). To gain new mechanistic insights into the role of these receptors in the context of ALS, here we investigated how PPAR transcriptional activity varies in hSOD1(G93A) ALS transgenic mice. We demonstrate that PPAR\u3b3-driven transcription selectively increases in the spinal cord of symptomatic hSOD1(G93A) mice. This phenomenon correlates with the up-regulation of target genes, such as lipoprotein lipase and glutathione S-transferase \u3b1-2, which are implicated in scavenging lipid peroxidation by-products. Such events are associated with enhanced PPAR\u3b3 immunoreactivity within motor neuronal nuclei. This observation, and the fact that PPAR\u3b3 displays increased responsiveness in cultured hSOD1(G93A) motor neurons, points to a role for this receptor in neutralizing deleterious lipoperoxidation derivatives within the motor cells. Consistently, in both motor neuron-like cultures and animal models, we report that PPAR\u3b3 is activated by lipid peroxidation end products, such as 4-hydroxynonenal, whose levels are elevated in the cerebrospinal fluid and spinal cord from ALS patients. We propose that the accumulation of critical concentrations of lipid peroxidation adducts during ALS progression leads to the activation of PPAR\u3b3 in motor neurons. This in turn triggers self-protective mechanisms that involve the up-regulation of lipid detoxification enzymes, such as lipoprotein lipase and glutathione S-transferase \u3b1-2. Our findings indicate that anticipating natural protective reactions by pharmacologically modulating PPAR\u3b3 transcriptional activity may attenuate neurodegeneration by limiting the damage induced by lipid peroxidation derivatives