Cross-talk between sarmoptosis and ferroptosis in hemorrhagic stroke - Project Summary/Abstract Brain bleeding in the parenchyma, also known as intracerebral hemorrhage (ICH), represents 10 to 30 percent of strokes in distinct countries around the world. Despite its lower incidence than ischemic stroke, ICH carries higher rates of mortality and morbidity. Worse outcomes in humans with ICH have been correlated with damage to axons in areas of the brain like the thalamus or the striatum where bleeding from small, penetrating arteries (often affected by longstanding hypertension) tends to occur. Red blood cells accumulate in ICH and lyse and release toxins. These include hemoglobin (Hgb) and its breakdown product hemin . Additional understanding of how Hgb/hemin leads axons to degenerate in ICH is a strategy to fully assess and overcome disability from this condition. Excitingly, a molecular path for programmed axonal destruction has been defined involving the protein, sterile alpha and Toll/Interleukin receptor-1 (TIR) motif containing 1 (SARM1). SARM1's degenerative activities in the axon emerge from its NADase (NAD+ degrading) activity in the TIR domain. This domain's importance is highlighted by its conservation from plants to humans. In preliminary studies, we found that germline deletion of full length SARM1 prevents axonal damage in the striatum following ICH. Since previous studies from our lab have shown that hemin induces ferroptosis, an iron-dependent, caspase-independent form of programmed necrosis, following experimental ICH, we are seeking to understand the cross talk between ferroptosis and SARM1-mediated axonal destruction (sarmoptosis). Of note, we found that cortical neurons cultured from the SARM1 knockout mice are partially resistant to ferroptotic stimuli (hemin and erastin). Moreover, agents such as P7C3 that augment the cell's ability to make NAD+ protect against ferroptosis; conversely, FK866, which impedes the same NAD+ synthesizing pathway makes ferroptosis worse. From these preliminary studies we propose three aims designed to understand the interplay between ferroptosis and sarmoptosis in ICH. In the first aim, we will leverage mice that possess a germline, homozygous, single amino acid mutation (E642A) in the NADase domain of SARM1. Like the SARM1-/- mice, the neurons of these mice are resistant to axotomy in vitro and in vivo. They will be used to probe whether the NADase activity is critical for ferroptosis in vitro and ICH in vivo. In the second aim, we will address the hypothesis that hemin-induces axonal degeneration via local ferroptotic pathways that trigger SARM1 activation. We will use sensory neurons cultured in Campenot Chambers to isolate cell bodies from axons and query their possibly distinct responses to lethal concentrations of hemin. We will also probe the role of ferroptosis and sarmoptosis in the destruction of corticospinal tract axons in vivo. In the last aim, we will seek to understand whether interventions that target ferroptosis by acting in the nucleus (cell body) can be combined with SARM1 deletion (that acts primarily in axons) in mice to reduce damage and improve behavioral recovery over each agent tested alone. At the culmination of these studies, new mechanistic information will emerge regarding axonal and cell body loss following ICH with clear therapeutic implications.
