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Tesi etd-03272018-130419

Thesis type
Tesi di dottorato di ricerca
Evaluation of nuclear factor erythroid 2-related factor 2 gene as a genic modulator of response to oxidative stress in neurodegenerative diseases.
Settore scientifico disciplinare
Corso di studi
tutor Prof. Siciliano, Gabriele
Parole chiave
  • neurodegenerative diseases
  • oxidative stress
  • ALS
  • PD
  • AD
Data inizio appello
Riassunto analitico
To maintain redox homeostasis is imperative for normal function of the brain. This process is<br>regulated by antioxidants, detoxifying proteins and other molecules. With age, genetic and<br>environmental risk factors, the oxidative-redox system becomes imbalanced and oxidative stress<br>(OS) ensues through increased levels of reactive oxygen species (ROS) and reactive nitrogen<br>species (RNS). The rate of ROS/RNS production eventually overwhelms our endogenous<br>antioxidant defenses leading to the accumulation of oxidative damage such as post-translational<br>modifications of lipids, proteins and DNA/RNA, a common feature of many neurodegenerative<br>diseases. The oxidative modifications affect the physiological functions of these cell components<br>and cause abnormal deposits in neurons and/or glia in diseases such as Alzheimer&#39;s disease<br>(AD), Parkinson&#39;s disease (PD) and amyotrophic lateral sclerosis (ALS). Although it remains<br>hard to understand whether or no OS is the cause or effect of the disease, also because of the<br>multifactorial nature of neuronal death and additive effects of pathogenic mechanisms on<br>neuronal vitality along disease course, the association between oxidative damage and the disease<br>makes therapeutic targeting of the antioxidant systems an attractive option. The nuclear factor<br>erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a primary<br>sensor and a master regulator of OS via its ability to modulate the expression of hundreds of<br>antioxidant and detoxifying genes. Activation of the Nrf2-ARE pathway has shown benefits in<br>animal models of many neurodegenerative disorders supporting the concept of developing<br>pharmaceuticals to activate the Nrf2-ARE pathway in the brain.<br>Nrf2 belongs to the Cap’n’collar (Cnc) transcription factor family and is considered the “master regulator” of the antioxidant response since it modulates the expression and the coordinated induction of an array of defensive genes encoding phase II detoxifying enzymes and antioxidant proteins, such as NAD(P)H: quinine oxidoreductases (NQOs), heme oxygenase- 1 (HO-1), the glutathione S-transferase (GST) family, multidrug resistance-associated proteins (Mrps).<br>TheNrf2 is a very unstable protein, typically present in association with its negative regulator<br>Kelch-like ECH-associated protein 1 (Keap1), which acts as a molecular sensor of cellular redox<br>homeostasis disturbance. Under basal condition, Keap1 retains Nrf2 in the cytoplasm, linking<br>this transcriptional factor to the actin cytoskeleton and driving its degradation. Specifically,<br>Keap1 acts as a linker protein between Nrf2 and the Cul3-based E3-ubiquitin ligase complex,<br>promoting Nrf2 ubiquitination and consequent degradation by the 26S proteasome.<br>This quenching interaction between the two proteins is a dynamic process controlled by specific<br>intracellular cascades that allow for a fine-tuned regulation of inducible expression of Nrf2 target<br>genes under OS or after exposure to toxic electrophiles. In fact, activation of Nrf2 requires its<br>cytosolic stabilization via oxidative modification of distinct Keap1 cysteine residues and/or<br>Keap1 ubiquitination and proteasomal degradation. It has been largely demonstrated that also<br>Nrf2 phosphorylation facilitates its dissociation from Keap1. Therefore, several signaling<br>pathways, such as the activation of mitogen-activated protein kinase (MAPK) cascade,<br>phosphatidylinositol 3-kinase (PI3K), and protein kinaseC (PKC), favour Nrf2 detachment from<br>its repressors and the consequent translocation to the nucleus. In the nuclear compartment Nrf2<br>forms a heterodimer with its partner small Maf and binds specific cis-acting antioxidant response<br>element (ARE) sequences, ultimately transactivating a battery of highly inducible cytoprotective<br>genes thus allowing cell to efficiently cope with endogenous stress and exogenous toxicants.<br>Nrf2 has also been shown to modulate the transcription of genes promoting mitochondrial<br>biogenesis, such as mitochondrial transcription factors (TFAM), and consequently to be directly<br>involved in mitochondrial maintenance.<br>Considering the pivotal defensive role exerted by the Nrf2/ARE pathway, it is evident that the<br>dysregulation of Nrf2-regulated genes offers a logical explanation for the direct and indirect<br>association between OS and several neurodegenerative conditions, such as PD, AD and ALS.<br>AD is probably the most common neurodegenerative disease, accounting for 60% to 70% of<br>cases of dementia with nearly 44 million affected people worldwide, and although its etiology is<br>still unclear, it is characterized by the presence of brain amyloid plaques and neurofibrillary<br>tangles whose accumulation ultimately leads to extensive neuronal loss and progressive decline<br>of cognitive function. They are deposits of proteins distributed throughout the brain of AD<br>patients, particularly in the entorhinal cortex, hippocampus, and temporal, frontal, and inferior<br>parietal lobes. Some of the major risk factors for AD are unhealthy aging in sporadic AD cases,<br>the presence of ApoE-4 alleles in both sporadic and familial AD and genetic factors, such as<br>mutation in amyloid precursor protein (APP) and presenilin-1 (PS1) in familial AD among<br>others. AD brain is characterized by mitochondrial dysfunction, reactive gliosis and oxidative<br>damage to lipids and proteins.<br>Growing evidence demonstrates that the AD brain is under tremendous OS. A significantly<br>increased HO-1 expression was reported in post-mortem AD temporal cortex and hippocampus<br>compared to aged-matched control. Additionally, an increased Nqo1 activity and expression was<br>found in astrocytes and neurons of AD brain and Nrf2 was predominantly localized in cytoplasm<br>in AD hippocampal neurons. Furthermore, there is increased protein oxidation and lipid<br>peroxidation in AD brain when compared to aged matched controls. Recent studies in aged<br>APP/PS1 AD mouse models showed reduced Nrf2, Nqo1, GCL catalytic subunit (GCLC) and<br>GCL modifier subunit (GCLM) mRNA and Nrf2 protein levels.<br>PD affects more than 1% of the population over 60 years of age and is the second most common<br>neurodegenerative disorder after AD. The majority of cases (90%) are sporadic, while about 10%<br>show monogenic inheritance.<br>PD is caused by the degeneration of dopaminergic neurons within the substantia nigra pars<br>compacta (SNc) and although there is still no clear explanation for the intrinsic vulnerability of<br>these neurons, it is known that they are more prone and susceptible to OS. Data indicate that OS<br>plays an important role in αSyn proteostasis. As the master regulator of the cellular antioxidant<br>defense system, the Nrf2-ARE pathway is a logical target to examine for neuroprotection against<br>misfolded proteins induced pathology.<br>Activation of theNrf2-ARE pathway has been shown to be protective against the toxic forms of<br>αSyn in several studies. In SK-N-SH neuroblastoma cells, ferrous iron promotes αSyn<br>aggregation through inhibiting Nrf2 pathway. αSyn aggregation exacerbates ferrous iron-induced<br>oxidative damage, mitochondrial impairment and apoptosis.<br>Recently studies have identified the importance of astrocytic Nrf2 regulating αSyn proteostasis.<br>Astrocytic over expression of Nrf2 (GFAP-Nrf2) can reduce αSyn aggregates in the central<br>nervous system of a PD mouse model with neuronal over expression of human αSyn mutant<br>A53T. These observations are not due to Nrf2-mediated down regulation of the hαSynA53T<br>transgene levels in the mice.<br>ALS is a rare adult-onset neurodegenerative disease characterized by the selective degeneration<br>of motor neurons in the motor cortex, brainstem, and spinal cord. Most of the cases (90%) are<br>sporadic (SALS), while the remainder presents a family history (FALS). Although the exact<br>cause of ALS is still unknown, a major step forward in the understanding of the pathogenetic<br>events involved in ALS was provided in 1993 by the observation that mutations in the gene<br>coding for the antioxidant enzyme Cu/Zn superoxide dismutase (SOD1) are carried by the 15–<br>20% of FALS patients.<br>ALS is a complex and multifactorial disease characterized by the involvement of several<br>interconnected pathogenic events, such as OS, mitochondrial dysfunction, inflammation,<br>glutamate excitotoxicity, protein misfolding and aggregation, aberrant RNA metabolism, and<br>altered gene expression. In particular, OS is one of the most detrimental contributors of disease<br>onset and progression. In fact, several distinctive oxidation markers have been observed in both<br>nervous and peripheral tissues in SALS and FALS patients. Elevated protein carbonyl and 3-<br>nitrotyrosine levels have been detected in spinal cord and motor cortex from SALS and FALS<br>patients, particularly in large ventral motor neurons. Lipid oxidation has also been identified in<br>motor neurons, astrocytes, and microglia of SALS patients compared to control individuals.<br>Elevated levels of HNE have been detected also in CSF and in sera from ALS patients.<br>Additionally, mitochondrial defects have been reported as a major hallmark in motor neuron<br>degeneration in ALS. These dysfunctions are tightly interrelated with OS cascades, activating<br>overlapping molecular pathways in a vicious cycle of harmful events. Notably, impairment in<br>defensive mechanisms has also been revealed in ALS, including downregulation of members of<br>glutathione S-transferase family, peroxiredoxins, and, in particular, the transcriptional factor<br>Nrf2.<br>The aim of this research project has been to evaluate a possible association between -653 A&gt; G, -<br>651 G&gt; A and -617 C&gt; functional polymorphisms in the NRF2 promoter gene, and the<br>respective risk of ALS, PD and AD disease and their possible implication in molecular<br>mechanisms of cellular response to oxidative damage. In particular, the following evaluations<br>were assessed:<br>• the distribution of allelic and genotypic frequencies of the three functional single nucleotide<br>polymorphisms (SNPs) -653 A&gt; G, -651 G&gt; A and -617 C&gt; A in 154 ALS patients, 172 PD<br>patients, 240 AD patients and 186 healthy controls; genotyping was carried out by DNA direct<br>sequencing.<br>• the plasma levels of some oxidative stress biomarkers in 73 ALS patients, 47 PD patients,<br>139 AD patients and 68 healthy controls ; in particular, as oxidative damage markers, we<br>evaluated the protein oxidation products (AOPP) and, as non-enzymatic antioxidant markers we<br>evaluated the Antioxidant Iron Reduction Capacity (FRAP) and total plasma thiol groups. The<br>biomarkers levels were carried out by spectrophotometric methods<br>• the mRNA expression level by Real Time PCR in 13 ALS patients, 15 PD patients and 14<br>AD patients<br>• the possible association of the functional polymorphisms in the NRF2 gene promoter with<br>the clinical features of the patients.<br>• the possible association of the functional polymorphisms in the NRF2 gene promoter with<br>the NRF2 transcript levels and oxidative stress biomarkers<br>The analysis of AD population shows that the allelic variant -653G is associated with increased risk<br>of disease (OR 1.27 IC95% 1.01-1.59); relative to the polymorphisms -651 G&gt; A and -617 C&gt; A,<br>any significant differences in the genotypic distribution and allelic frequencies of patients with AD<br>compared to the controls has been found. The evaluation of peripheral oxidative stress biomarkers<br>shows a significant decrease in FRAP (p&lt;0.01) and thiol groups levels (p&lt;0.001). We not found any<br>imbalance in the AOPP level of AD patients compared to controls. mRNA expression in individuals<br>carrying one (AG) or two (GG) mutated alleles of the -653 A&gt;G SNP promoter was significantly<br>decreased (p&lt;0.01) compared to wild-type (AA) carriers at this position, this both for AG and GG<br>carriers.<br>Analysis of PD population data shows that the allelic variant -653G is associated with increased risk<br>of disease (OR 1.34 IC95% 1.08-1.67); with respect to the polymorphisms -651 G&gt;A and -617 C&gt;<br>A, any significant differences has been found in the genotypic distribution and in allelic frequencies<br>of PD patients compared to the controls. The evaluation of peripheral oxidative stress biomarkers<br>shows a significant decrease in FRAP (p&lt; 0.0001) and thiol groups levels (p&lt;0.001). Any<br>imbalance in the AOPP level of PD patients compared to controls has been found. mRNA<br>expression in individuals carrying one (AG) or two (GG) mutated alleles of the -653 A&gt;G promoter<br>SNP was significantly decreased (p&lt;0.01) compared to wild-type (AA) carriers at this position.<br>Also in this case the difference was significant difference or both AG and GG carriers. Finally, also<br>in PD patients, a correlation between -653G variant, mRNA expression level and oxidative stress<br>biomarkers has been found.<br>Analysis of ALS population data shows that the allelic -653G variant is associated with increased<br>risk of disease (OR 1.71 IC95% 1.18-2.48); in relation to the polymorphisms -651 G&gt; A and -617<br>C&gt; A, no significant differences has been found in the genotypic distribution and in the allelic<br>frequencies of patients with ALS compared to the controls. The evaluation of peripheral oxidative<br>stress biomarkers shows a significant increase in AOPP levels (p&lt; 0.001) and a significant decrease<br>in thiol groups levels (p&lt;0.01) in ALS patients; we did not found any imbalance in the FRAP level<br>of ALS patients compared to controls. mRNA expression in individuals carrying one (AG) or two<br>(GG) mutated alleles of the -653 A&gt;G SNP promoter was significantly decreased (p&gt;0.05)<br>compared to wild-type (AA) carriers at this position; this observed between the G non carriers and<br>G carriers, although at of genotypic levels, AG and GG. Finally, the data obtained showed a<br>correlation between -653G variant, mRNA expression level and oxidative stress biomarkers.<br>The data obteined suggest that the -653G variant in NRF2 promoter gene is a common risk factor<br>for AD or PD and ALS. This variant is always associated to decreased level of NRF2 mRNA as<br>evaluated in peripheral lymphocytes.<br>All together these underlines that Nrf2-ARE pathway can be one of the molecular mechanisms<br>commonly involved in neurodegeneration, although the different profile of alteration of redox<br>balance indicates that the effects are different in each one of the neurodegenerative disorders.<br>In any case, conclusive remarks can be assumed in terms of relevance of oxidative stress events as<br>integral part of the pathogenic complex of these diseases.<br>