Acute cerebrovascular disease still represents a very frequent condition. Stroke, a leading cause of death and disability, has a multifactorial etiopathogenesis. Understanding the molecular mechanisms underlying cerebrovascular injury represents a major target of research to develop novel preventive and therapeutic strategies able to reduce stroke occurrence and its fearful consequences. The rat model of stroke-prone spontaneously hypertensive rat (SHRSP), which shares several similarities with the human disease, shows higher predisposition to stroke, particularly when fed with a high-salt/low potassium (Japanese style) diet. The Ndufc2 gene encodes a subunit of the mitochondrial OXPHOS complex I. Downregulation of Ndufc2 gene and protein expression, responsible of significantly impaired complex I assembly and activity with consequent reduction of ATP synthesis, was found to exert a major role on stroke occurrence in high-salt fed SHRSP. This project will characterize the impact of mitochondrial dysfunction, due to Ndufc2 gene downregulation and consequent OXPHOS complex I deficiency, on mitophagy as a potential contributory molecular mechanism to stroke. In our model we will define the mechanisms underlying mitophagy and the precise step of mitophagy inhibition dependent on Ndufc2 gene downregulation. In Ndufc2 gene silenced cells and in a rat model of Ndufc2 gene knock-out we will explore the modulation of a downstream signaling pathway (SIRT1/mTOR) potentially dependent on Ndufc2 deficiency and on the lack of NAD+ (the main product of OXPHOS complex I activity). We will finally test the impact of NAD+ restoration on cell damage and on stroke occurrence in the presence of Ndufc2 gene inhibition. Our study will reveal if the modulation of mitophagy may represent a therapeutic strategy for the treatment of stroke. The suitability of SHRSP as a model for human stroke provides a promising background for the subsequent translation of our findings to humans.
We expect to identify that decreased mitophagy and increased mitochondrial DNA damage occur only in HS fed SHRSP, and that the inhibition occurs at a precise step of the mitophagy machinery.
Since Ndufc2 is a component of complex I, it is likely that the depletion of NAD+ (the main product of complex I activity) may contribute to mitophagy inhibition via SIRT1/mTOR axis in HS fed SHRSP, as well as in both silenced cells and in the heterozygous knock-out animal model. We expect that the restoration of NAD+ increases mitophagy and prevents stroke occurrence in our rat models. Activation of autophagy through trehalose is also expected to produce a beneficial effect towards stroke occurrence in our models.
The achievements of these studies will improve the knowledge on the molecular mechanisms that contribute to stroke predisposition. The experimental tools used in this project (the HS fed SHRSP rat model of human hypertensive stroke, the heterozygous rat model of Ndufc2 gene deletion, the Ndufc2 gene silenced vascular cell) represent robust experimental tools to test the hypothesis that a defective defense cellular mechanism is involved in stroke predisposition in the presence of Ndufc2 gene downregulation, causing complex I deficiency. We will assess the potential protective role of therapies able to restore NAD+ levels and to induce mitophagy towards cellular damage and stroke occurrence. In summary, our data will reveal how mitochondrial dynamics are responsible for stroke and if the restoration of NAD+ levels may be considered as a new strategy to reduce neuronal damage and to treat stroke.
The largely confirmed suitability of SHRSP as a model for the human disease represents a promising background for the successful translation of our findings to human stroke.