Pharmacological Induction of Mitochondrial Biogenesis for the Treatment of Spinal Cord Injury

Spinal cord injury (SCI) is devastating disorder defined by direct trauma to the spinal cord, which disrupts the vasculature, leading to decreased oxygen delivery within the area and reducing the ability of mitochondria to maintain cellular energetics. Neuronal loss of mitochondrial function leads to excitotoxicity and oxidative stress, emphasizing the critical nature of restoration of mitochondrial homeostasis following SCI. Studies investigating mitochondria as a therapeutic target for SCI have primarily addressed individual aspects of mitochondrial function and have proven largely inefficacious. Therapeutics pursuing reestablishment of mitochondrial homeostasis through increased MB, however, could alleviate multiple facets of injury progression. Our previous studies found that the FDA-approved beta2-adrenergic receptor (ADRB2) agonist, formoterol, is not only a potent inducer of MB in multiple organ systems in mice, but is also capable of restoring mitochondrial and organ function following ischemic kidney injury. Our preliminary data indicate that formoterol induces MB in the spinal cord of naïve mice and following SCI. Additionally, mice treated daily with formoterol following SCI exhibited improved mitochondrial homeostasis and decreased lesion volume by 3 d after injury, as well as increased vascular and locomotor recovery by 7 days post-SCI. Previous studies showed that treatment with a 20- fold higher dose of the less selective ADRB2 agonist, clenbuterol, improved locomotor recovery in vivo beginning three weeks post-SCI, yet clenbuterol has not been clinically successful. Unlike formoterol, clenbuterol does not induce MB. We have recently attributed this difference to unique structural features of the two agonists resulting in divergent signaling pathways; formoterol activates the G??-Akt-eNOS-sGC pathway and induces MB, while clenbuterol does not. Therefore, formoterol has greater therapeutic potential for the treatment of SCI. We hypothesize that treatment with formoterol following SCI will increase MB, resulting in decreased spinal cord neuron death/dysfunction, increased vascular repair and locomotor recovery post-SCI in mice. We propose the following specific aims: Specific Aim 1: Determine MB, mitochondrial homeostasis (e.g. fission/fusion) and mitochondrial function in response to formoterol post-SCI in male and female mice; Specific Aim 2: Determine the role of vascular recovery and endothelial cells in response to formoterol post-SCI in male and female mice. We will use a force-controlled impactor-induced contusion mouse model of SCI, with sham mice undergoing laminectomy only. Because delayed treatment initiation is important clinically, formoterol treatment will begin at least 8 h post?injury. Several markers of MB will be investigated in the injury site, including mitochondrial proteins, mtDNA and mitochondrial function using a Seahorse Bioscience XF96 instrument. Lesion volume and inflammation will also be assessed, as well as functional recovery. To thoroughly investigate the effect of formoterol and ADRB2 on vascular recovery, we will assess BSCB integrity and functional vasculature around the injury site. Additionally, we are currently breeding conditional knockout mice lacking ADRB2 in endothelial cells. These mice will be subjected to SCI to discern the role of endothelial ADRB2 in SCI recovery, with and without formoterol. Despite the knowledge that mitochondrial dysfunction is a known mediator of SCI, no studies have investigated the therapeutic efficacy of pharmacological activation of MB on SCI pathology. Importantly, formoterol is an FDA-approved drug, increasing the possibility of repurposing it for SCI. Furthermore, we will assess treatment initiation up to 24 h after injury, increasing clinical applicability and attending to the "Preserving and protecting tissue early after injury" and "Rehabilitation and regeneration" FY18 SCIRP IIRA Focus Areas. The VA is the largest healthcare network for SCI victims, providing care for 25% of total sufferers in the United States, and the incidence of SCI among military is increasing. Furthermore, the cost for SCI treatment is 6.5 times more than the average veteran healthcare, with more than 42,000 American veterans receiving over $30,000 a year each in disability compensation, which still equates to less than the estimated $3 million lifetime cost of care post-SCI. Therefore, successful completion of these studies could substantially advance therapeutic treatment of SCI, which would not only benefit patients and their families, but also the military health care system.