Nuclear factor erythroid 2-related factor 2 come modulatore genico di risposta allo stress ossidativo in pazienti affetti da Sclerosi Laterale Amiotrofica. A. Lo Gerfo; L. Chico; L. Petrozzi; E. Caldarazzo-Ienco; C. Simoncini; V. Montano; C. Carlesi; E. Schirinzi; A. Galgani; F. Baldacci; F. Coppedè; A. Stoccoro; S. Daniele; G. Ricci; M. Mancuso e G. Siciliano

ALS is a NDD characterized by progressive muscular paralysis, caused by the degeneration of motor neurons in the motor cortex primary, in the corticospinal tract and in the spinal cord. Most ALS is sporadic; however, a small fraction of ALS is familial in origin. To date, around 20 genes are associated with ALS, with the most common causes of typical ALS associated with mutations in: SOD1 TARDBP FUS C9orf72

Carbonylated proteins Nitrotyrosine 8-OHdG 8-NitrodG Carbonylated proteins Nitrotyrosine Advanced protein oxidation products (AOPP) Proteins Lipids Nucleic acids ROS Sod Iron-reducing capacity of plasma (FRAP) Catalase Bilirubin GPx GSH/GSSG Plasma thiols (-SH) ANTIOXIDANTS

To evaluate a possible association between -653 A>G, - 651 G>A and -617 C>A functional polymorphisms in the NRF2 promoter gene, and the risk of ALS and their possible implication in molecular mechanisms that underlie the oxidative stress response.

QIAamp DNA blood MINI kit STUDY DESIGN 2. the plasma levels of some oxidative stress biomarkers in 73 ALS patients, 47 PD patients, 139 AD patients and 68 healthy controls ; in particular, we evaluated AOPP, FRAP and total plasma thiol groups. The biomarker levels were carried out by spectrophotometric methods 10’ 1000 x g 4°C - 80° C Samples collection AOPP -SH FRAP Evaluation of: 1. 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 DNA extraction Polymerase Chain Reaction (PCR) DNA sequencing QIAamp DNA blood MINI kit (Qiagen) 4. the possible association of the polymorphisms in the NRF2 gene promoter with the NRF2 transcript levels and oxidative stress biomarkers -like 3. the mRNA expression level by Real Time PCR in 13 ALS patients, 15 PD patients and 14 AD patients Sample amplification Cycles

Results

CONCLUSION Strong involvement of oxidative stress and a deficit of the Nrf2-ARE system in the pathogenesis of ALS; It is possible to hypothesize that the -653G variant is able to attenuate the defence system directly controlled by Nrf2, making the G carriers more susceptible to oxidative insult; -653G variant in NRF2 promoter gene is a common risk factor for ALS. -653G 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. In agreement with other studies present in the literature, the results of this work indicate the Nrf2 pathway / ARE as an important mediator of neuroprotection and suggest this way as a possible therapeutic target for neurodegenerative diseases.