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Tesi etd-03272018-130419


Thesis type
Tesi di dottorato di ricerca
Author
LO GERFO, ANNALISA
URN
etd-03272018-130419
Title
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
MED/26
Corso di studi
SCIENZE CLINICHE E TRASLAZIONALI
Supervisors
tutor Prof. Siciliano, Gabriele
Parole chiave
  • neurodegenerative diseases
  • oxidative stress
  • ALS
  • PD
  • AD
Data inizio appello
09/04/2018;
Consultabilità
Completa
Riassunto analitico
To maintain redox homeostasis is imperative for normal function of the brain. This process is
regulated by antioxidants, detoxifying proteins and other molecules. With age, genetic and
environmental risk factors, the oxidative-redox system becomes imbalanced and oxidative stress
(OS) ensues through increased levels of reactive oxygen species (ROS) and reactive nitrogen
species (RNS). The rate of ROS/RNS production eventually overwhelms our endogenous
antioxidant defenses leading to the accumulation of oxidative damage such as post-translational
modifications of lipids, proteins and DNA/RNA, a common feature of many neurodegenerative
diseases. The oxidative modifications affect the physiological functions of these cell components
and cause abnormal deposits in neurons and/or glia in diseases such as Alzheimer's disease
(AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Although it remains
hard to understand whether or no OS is the cause or effect of the disease, also because of the
multifactorial nature of neuronal death and additive effects of pathogenic mechanisms on
neuronal vitality along disease course, the association between oxidative damage and the disease
makes therapeutic targeting of the antioxidant systems an attractive option. The nuclear factor
erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a primary
sensor and a master regulator of OS via its ability to modulate the expression of hundreds of
antioxidant and detoxifying genes. Activation of the Nrf2-ARE pathway has shown benefits in
animal models of many neurodegenerative disorders supporting the concept of developing
pharmaceuticals to activate the Nrf2-ARE pathway in the brain.
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).
TheNrf2 is a very unstable protein, typically present in association with its negative regulator
Kelch-like ECH-associated protein 1 (Keap1), which acts as a molecular sensor of cellular redox
homeostasis disturbance. Under basal condition, Keap1 retains Nrf2 in the cytoplasm, linking
this transcriptional factor to the actin cytoskeleton and driving its degradation. Specifically,
Keap1 acts as a linker protein between Nrf2 and the Cul3-based E3-ubiquitin ligase complex,
promoting Nrf2 ubiquitination and consequent degradation by the 26S proteasome.
This quenching interaction between the two proteins is a dynamic process controlled by specific
intracellular cascades that allow for a fine-tuned regulation of inducible expression of Nrf2 target
genes under OS or after exposure to toxic electrophiles. In fact, activation of Nrf2 requires its
cytosolic stabilization via oxidative modification of distinct Keap1 cysteine residues and/or
Keap1 ubiquitination and proteasomal degradation. It has been largely demonstrated that also
Nrf2 phosphorylation facilitates its dissociation from Keap1. Therefore, several signaling
pathways, such as the activation of mitogen-activated protein kinase (MAPK) cascade,
phosphatidylinositol 3-kinase (PI3K), and protein kinaseC (PKC), favour Nrf2 detachment from
its repressors and the consequent translocation to the nucleus. In the nuclear compartment Nrf2
forms a heterodimer with its partner small Maf and binds specific cis-acting antioxidant response
element (ARE) sequences, ultimately transactivating a battery of highly inducible cytoprotective
genes thus allowing cell to efficiently cope with endogenous stress and exogenous toxicants.
Nrf2 has also been shown to modulate the transcription of genes promoting mitochondrial
biogenesis, such as mitochondrial transcription factors (TFAM), and consequently to be directly
involved in mitochondrial maintenance.
Considering the pivotal defensive role exerted by the Nrf2/ARE pathway, it is evident that the
dysregulation of Nrf2-regulated genes offers a logical explanation for the direct and indirect
association between OS and several neurodegenerative conditions, such as PD, AD and ALS.
AD is probably the most common neurodegenerative disease, accounting for 60% to 70% of
cases of dementia with nearly 44 million affected people worldwide, and although its etiology is
still unclear, it is characterized by the presence of brain amyloid plaques and neurofibrillary
tangles whose accumulation ultimately leads to extensive neuronal loss and progressive decline
of cognitive function. They are deposits of proteins distributed throughout the brain of AD
patients, particularly in the entorhinal cortex, hippocampus, and temporal, frontal, and inferior
parietal lobes. Some of the major risk factors for AD are unhealthy aging in sporadic AD cases,
the presence of ApoE-4 alleles in both sporadic and familial AD and genetic factors, such as
mutation in amyloid precursor protein (APP) and presenilin-1 (PS1) in familial AD among
others. AD brain is characterized by mitochondrial dysfunction, reactive gliosis and oxidative
damage to lipids and proteins.
Growing evidence demonstrates that the AD brain is under tremendous OS. A significantly
increased HO-1 expression was reported in post-mortem AD temporal cortex and hippocampus
compared to aged-matched control. Additionally, an increased Nqo1 activity and expression was
found in astrocytes and neurons of AD brain and Nrf2 was predominantly localized in cytoplasm
in AD hippocampal neurons. Furthermore, there is increased protein oxidation and lipid
peroxidation in AD brain when compared to aged matched controls. Recent studies in aged
APP/PS1 AD mouse models showed reduced Nrf2, Nqo1, GCL catalytic subunit (GCLC) and
GCL modifier subunit (GCLM) mRNA and Nrf2 protein levels.
PD affects more than 1% of the population over 60 years of age and is the second most common
neurodegenerative disorder after AD. The majority of cases (90%) are sporadic, while about 10%
show monogenic inheritance.
PD is caused by the degeneration of dopaminergic neurons within the substantia nigra pars
compacta (SNc) and although there is still no clear explanation for the intrinsic vulnerability of
these neurons, it is known that they are more prone and susceptible to OS. Data indicate that OS
plays an important role in αSyn proteostasis. As the master regulator of the cellular antioxidant
defense system, the Nrf2-ARE pathway is a logical target to examine for neuroprotection against
misfolded proteins induced pathology.
Activation of theNrf2-ARE pathway has been shown to be protective against the toxic forms of
αSyn in several studies. In SK-N-SH neuroblastoma cells, ferrous iron promotes αSyn
aggregation through inhibiting Nrf2 pathway. αSyn aggregation exacerbates ferrous iron-induced
oxidative damage, mitochondrial impairment and apoptosis.
Recently studies have identified the importance of astrocytic Nrf2 regulating αSyn proteostasis.
Astrocytic over expression of Nrf2 (GFAP-Nrf2) can reduce αSyn aggregates in the central
nervous system of a PD mouse model with neuronal over expression of human αSyn mutant
A53T. These observations are not due to Nrf2-mediated down regulation of the hαSynA53T
transgene levels in the mice.
ALS is a rare adult-onset neurodegenerative disease characterized by the selective degeneration
of motor neurons in the motor cortex, brainstem, and spinal cord. Most of the cases (90%) are
sporadic (SALS), while the remainder presents a family history (FALS). Although the exact
cause of ALS is still unknown, a major step forward in the understanding of the pathogenetic
events involved in ALS was provided in 1993 by the observation that mutations in the gene
coding for the antioxidant enzyme Cu/Zn superoxide dismutase (SOD1) are carried by the 15–
20% of FALS patients.
ALS is a complex and multifactorial disease characterized by the involvement of several
interconnected pathogenic events, such as OS, mitochondrial dysfunction, inflammation,
glutamate excitotoxicity, protein misfolding and aggregation, aberrant RNA metabolism, and
altered gene expression. In particular, OS is one of the most detrimental contributors of disease
onset and progression. In fact, several distinctive oxidation markers have been observed in both
nervous and peripheral tissues in SALS and FALS patients. Elevated protein carbonyl and 3-
nitrotyrosine levels have been detected in spinal cord and motor cortex from SALS and FALS
patients, particularly in large ventral motor neurons. Lipid oxidation has also been identified in
motor neurons, astrocytes, and microglia of SALS patients compared to control individuals.
Elevated levels of HNE have been detected also in CSF and in sera from ALS patients.
Additionally, mitochondrial defects have been reported as a major hallmark in motor neuron
degeneration in ALS. These dysfunctions are tightly interrelated with OS cascades, activating
overlapping molecular pathways in a vicious cycle of harmful events. Notably, impairment in
defensive mechanisms has also been revealed in ALS, including downregulation of members of
glutathione S-transferase family, peroxiredoxins, and, in particular, the transcriptional factor
Nrf2.
The aim of this research project has been to evaluate a possible association between -653 A> G, -
651 G> A and -617 C> functional polymorphisms in the NRF2 promoter gene, and the
respective risk of ALS, PD and AD disease and their possible implication in molecular
mechanisms of cellular response to oxidative damage. In particular, the following evaluations
were assessed:
• the distribution of allelic and genotypic frequencies of the three functional single nucleotide
polymorphisms (SNPs) -653 A> G, -651 G> A and -617 C> A in 154 ALS patients, 172 PD
patients, 240 AD patients and 186 healthy controls; genotyping was carried out by DNA direct
sequencing.
• the plasma levels of some oxidative stress biomarkers in 73 ALS patients, 47 PD patients,
139 AD patients and 68 healthy controls ; in particular, as oxidative damage markers, we
evaluated the protein oxidation products (AOPP) and, as non-enzymatic antioxidant markers we
evaluated the Antioxidant Iron Reduction Capacity (FRAP) and total plasma thiol groups. The
biomarkers levels were carried out by spectrophotometric methods
• the mRNA expression level by Real Time PCR in 13 ALS patients, 15 PD patients and 14
AD patients
• the possible association of the functional polymorphisms in the NRF2 gene promoter with
the clinical features of the patients.
• the possible association of the functional polymorphisms in the NRF2 gene promoter with
the NRF2 transcript levels and oxidative stress biomarkers
The analysis of AD population shows that the allelic variant -653G is associated with increased risk
of disease (OR 1.27 IC95% 1.01-1.59); relative to the polymorphisms -651 G> A and -617 C> A,
any significant differences in the genotypic distribution and allelic frequencies of patients with AD
compared to the controls has been found. The evaluation of peripheral oxidative stress biomarkers
shows a significant decrease in FRAP (p<0.01) and thiol groups levels (p<0.001). We not found any
imbalance in the AOPP level of AD patients compared to controls. mRNA expression in individuals
carrying one (AG) or two (GG) mutated alleles of the -653 A>G SNP promoter was significantly
decreased (p<0.01) compared to wild-type (AA) carriers at this position, this both for AG and GG
carriers.
Analysis of PD population data shows that the allelic variant -653G is associated with increased risk
of disease (OR 1.34 IC95% 1.08-1.67); with respect to the polymorphisms -651 G>A and -617 C>
A, any significant differences has been found in the genotypic distribution and in allelic frequencies
of PD patients compared to the controls. The evaluation of peripheral oxidative stress biomarkers
shows a significant decrease in FRAP (p< 0.0001) and thiol groups levels (p<0.001). Any
imbalance in the AOPP level of PD patients compared to controls has been found. mRNA
expression in individuals carrying one (AG) or two (GG) mutated alleles of the -653 A>G promoter
SNP was significantly decreased (p<0.01) compared to wild-type (AA) carriers at this position.
Also in this case the difference was significant difference or both AG and GG carriers. Finally, also
in PD patients, a correlation between -653G variant, mRNA expression level and oxidative stress
biomarkers has been found.
Analysis of ALS population data shows that the allelic -653G variant is associated with increased
risk of disease (OR 1.71 IC95% 1.18-2.48); in relation to the polymorphisms -651 G> A and -617
C> A, no significant differences has been found in the genotypic distribution and in the allelic
frequencies of patients with ALS compared to the controls. The evaluation of peripheral oxidative
stress biomarkers shows a significant increase in AOPP levels (p< 0.001) and a significant decrease
in thiol groups levels (p<0.01) in ALS patients; we did not found any imbalance in the FRAP level
of ALS patients compared to controls. mRNA expression in individuals carrying one (AG) or two
(GG) mutated alleles of the -653 A>G SNP promoter was significantly decreased (p>0.05)
compared to wild-type (AA) carriers at this position; this observed between the G non carriers and
G carriers, although at of genotypic levels, AG and GG. Finally, the data obtained showed a
correlation between -653G variant, mRNA expression level and oxidative stress biomarkers.
The data obteined suggest that the -653G variant in NRF2 promoter gene is a common risk factor
for AD or PD and ALS. This variant is always associated to decreased level of NRF2 mRNA as
evaluated in peripheral lymphocytes.
All together these underlines that Nrf2-ARE pathway can be one of the molecular mechanisms
commonly involved in neurodegeneration, although the different profile of alteration of redox
balance indicates that the effects are different in each one of the neurodegenerative disorders.
In any case, conclusive remarks can be assumed in terms of relevance of oxidative stress events as
integral part of the pathogenic complex of these diseases.
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