Sandrine Arbogast is post-doctoral researcher in Ana Ferreiro research group (U787). In July 2009, she published an original article* in Annals of Neurology and a review** in Antioxidants and Redox Signaling, articles about selenoproteins and protection against oxidative stress, in particular the role of selenoprotein N in myopathy related to SEPN1 (SEPN1-related myopathy or selenoproteinopathy.
 
What was the aim of your study?
I wanted to clarify the unknown role of selenoprotein N (SelN) and the pathophysiological mechanisms of this myopathy to identify potential therapeutic targets. This muscle disease is caused by mutations in the SEPN1 gene, which induces a deficiency or loss of function of this selenoprotein. Affected children have muscle weakness, scoliosis and respiratory failure that can be fatal. I have demonstrated for the first time that oxidative stress plays a key role in selenoproteinopathy. I performed ex vivo studies of cultures of myoblasts and myotubes from patients deficient in SelN and found that these cells have an increased production of free radicals that induce an oxidation process (carbonylation) of specific proteins. Actin and myosin, two proteins particularly important in muscle, are oxidized, which could explain the muscle weakness observed in patients. The survival of these cells in stress conditions is very reduced (up to 50% less than control cells). These results have allowed to demonstrate that SelN plays a major role in protecting human cells against oxidative stress.
 
Which techniques allow these oxidative phenomena to be demonstrated?
I study the biology of free oxygen radicals in living and intact cells. For this, I use chemical probes (such as dichlorofluoroscein diacetate), which by reacting with free radicals present in cells (H2O2, NO and the superoxide ion O2-) become fluorescent and allow to measure the intracellular oxidative activity using a fluorescence microscope. This oxidative activity can be modulated by various antioxidants. This original but difficult technique is only practiced in a few laboratories and this is the first time that it has been used on human cells. It has allowed to reveal an increase in oxidative activity in cells lacking SelN. At the molecular level, we can highlight the effects of oxidants on proteins by measuring protein carbonylation by Western blot by using oxyblot.
 
You then measured the intracellular calcium, what is the link with SelN?
SelN is localized to the endoplasmic reticulum (ER), as well as the ryanodine receptor RyR1, which is the main calcium channel of the muscle cell. Moreover, RyR1 is a protein that is sensitive to free radicals, whose oxidation which causes a leak of Ca2+ from the ER towards the cytosol. We therefore analyzed the flow of calcium into muscle cells of patients in collaboration with B. Fraysse, and observed an increase in cytoplasmic Ca2+, after leaking from the ER. This suggests that, in the absence of SelN, RyR1 is too oxidized, and confirms that SelN has a key role at the crossroads of calcium homeostasis and oxidative stress. Consequently, calcium homeostasis is a second potential therapeutic target in SEPN1-RM.
 
Have you identified a therapeutic approach to treat this myopathy?
Knowing that a failure in the antioxidant defense is the primary pathogenic mechanism in this myopathy, the therapeutic approach should aim to limit or correct the oxidative stress in the affected muscles, thereby protecting them from muscle weakness. We studied the ex vivo effect of various antioxidants on cells of patients lacking SelN. Some naturally occurring antioxidants (carotenoids and flavonoids) provide partial improvement, but only treatment with N-acetyl cysteine (NAC) results in a spectacular total correction of the cellular phenotype. NAC is an antioxidant drug already used in human pathology, inexpensive and with few side effects. So, on the basis of our very promising ex vivo results, and after pre-clinical studies in progress on SEPN1 KO mice, we are going to set up an international clinical trial in SEPN1-RM patients in the near future. It will be the first pharmacological treatment aimed at correcting the primary defect in a hereditary myopathy.
 
October 2009
Interview by Anne Berthomier, translation by Racquel N. Cooper
 

* Arbogast S, Beuvin M, Fraysse B, Zhou H, Muntoni F, Ferreiro A. Oxidative stress in SEPN1-related myopathy: from pathophysiology to treatment. Ann Neurol. 2009 Jun;65(6):677-86.
** Arbogast S, Ferreiro A. Selenoproteins and protection against oxidative stress: selenoprotein N as a novel player at the crossroads of redox signalling and calcium homeostasis. Antioxid Redox Signal, 2009 Sep 21. [Epub ahead of print]