Succinate supplementation improves metabolic performance of mixed glial cell cultures with mitochondrial dysfunction

Abstract

Mitochondrial dysfunction, the inability to efficiently utilise metabolic fuels and oxygen, contributes to pathological changes following traumatic spinal cord or traumatic brain injury (TBI). In the present study, we tested the hypothesis that succinate supplementation can improve cellular energy state under metabolically stressed conditions in a robust, reductionist in vitro model of mitochondrial dysfunction in which primary mixed glial cultures (astrocytes, microglia and oligodendrocytes) were exposed to the mitochondrial complex I inhibitor rotenone. Cellular response was determined by measuring intracellular ATP, extracellular metabolites (glucose, lactate, pyruvate), and oxygen consumption rate (OCR). Rotenone produced no significant changes in glial ATP levels. However, it induced metabolic deficits as evidenced by lactate/pyruvate ratio (LPR) elevation (a clinically-established biomarker for poor outcome in TBI) and decrease in OCR. Succinate addition partially ameliorated these metabolic deficits. We conclude that succinate can improve glial oxidative metabolism, consistent our previous findings in TBI patients’ brains. The mixed glial cellular model may be useful in developing therapeutic strategies for conditions involving mitochondrial dysfunction, such as TBI.

AKAP1 Protects from Cerebral Ischemic Stroke by Inhibiting Drp1-Dependent Mitochondrial Fission

“…For delayed calcium deregulation (DCD) and DHE experiments a baseline was established with a 2 min perfusion of HH buffer of the following composition (in mM): 140 NaCl, 5 KCl, 1.3 CaCl 2 , 0.5 MgCl 2 , 0.4 MgSO 4 , 0.4 KH 2 PO 4 , 0.6 Na 2 HPO 4 , 3 NaHCO 3 , 10 HEPES, 10 D-glucose, and at 310 mOsm, pH 7.4. The perfusion was then switched to a solution containing 100 M glutamate, 10 M glycine, and 200 nM tetrodotoxin in HH (Castilho et al, 1999).…”

“…A-kinase anchoring proteins (AKAPs) are a group of proteins defined by their ability to bind regulatory subunits of protein kinase A (PKA) enabling targeting of the PKA holoenzyme to various subcellular compartments (Herberg et al, 2000;Carlson et al, 2003). The first AKAP to be identified, AKAP1 has an N-terminal transmembrane domain and tethers PKA to the cytosolic surface of the outer mitochondrial membrane (OMM) to promote localized cAMP signaling (Affaitati et al, 2003;Ma and Taylor, 2008;Merrill and Strack, 2014;Jun et al, 2016). In addition to anchoring PKA, AKAP1 was shown to bind other signaling molecules, including Src kinase, protein phosphatase 1, calcineurin, and phosphodiesterase 4.…”

“…Precise regulation of mitochondrial fission/fusion is particularly important in neurons as mutations in these membrane-sculpting enzymes manifest with neurological symptoms in humans (Flippo and Strack, 2017a,b). Drp1 activity is regulated through a variety of posttranslational modifications including nitrosylation, SUMOylation, and phosphorylation (Chang and Blackstone, 2010). Perhaps best characterized, PKA-mediated phosphorylation of Drp1 at Serine 637 (S637 in human isoform 1 and S656 in rat isoform 1) inhibits its fission activity by as yet incompletely understood mechanisms (Chang and Blackstone, 2007;Cribbs and Strack, 2007), whereas dephosphorylation of the same residue by the Ca 2ϩ -dependent phosphatase calcineurin (PP2B) or PP2A activates the enzyme (Cribbs and Strack, 2007;Cereghetti et al, 2008;Slupe et al, 2013).…”

“…Neuroprotection by AKAP1 required PKA anchoring and Drp1 phosphorylation at Ser637 Dickey and Strack, 2011;Merrill et al, 2011;Pryde et al, 2016). During in vitro ischemia/reperfusion injury or oxygen glucose deprivation, AKAP1 is degraded through ubiquitination via the E3 ligase Siah2, enhancing cell death in several culture models (Carlucci et al, 2008;Kim et al, 2011). According to transcriptomics studies, AKAP1 mRNA levels in the rodent hippocampus and cortex are unaltered 24 h after mid-cerebral artery occlusion (MCAO;Hori et al, 2012;, indicating that injury regulates AKAP1 expression mainly through posttranscriptional mechanisms.…”

‘SNO’-Storms Compromise Protein Activity and Mitochondrial Metabolism in Neurodegenerative Disorders

“…Importantly, NADH from the TCA cycle interacts with complex I of the ETC to drive transfer of electrons from complex I to complex III and then IV. In addition, a component of complex II in the ETC, succinate dehydrogenase, converts succinate to fumarate in the TCA cycle and actuates the ETC to run from complex II, while bypassing NADH-activated complex I [33]. Thus, the enzyme succinate dehydrogenase represents a key component in both the ETC and TCA cycle and is critical in integrating their coordinated action.…”