Genetic Approach to Elucidate the Role of Cyclophilin D in Traumatic Brain Injury Pathology.

Cyclophilin D (CypD) has been shown to play a critical role in mitochondrial permeability transition pore (mPTP) opening and the subsequent cell death cascade. Studies consistently demonstrate that mitochondrial dysfunction, including mitochondrial calcium overload and mPTP opening, is essential to the pathobiology of cell death after a traumatic brain injury (TBI). CypD inhibitors, such as cyclosporin A (CsA) or NIM811, administered following TBI, are neuroprotective and quell neurological deficits. However, some pharmacological inhibitors of CypD have multiple biological targets and, as such, do not directly implicate a role for CypD in arbitrating cell death after TBI. Here, we reviewed the current understanding of the role CypD plays in TBI pathobiology. Further, we directly assessed the role of CypD in mediating cell death following TBI by utilizing mice lacking the CypD encoding gene Ppif. Following controlled cortical impact (CCI), the genetic knockout of CypD protected acute mitochondrial bioenergetics at 6 h post-injury and reduced subacute cortical tissue and hippocampal cell loss at 18 d post-injury. The administration of CsA following experimental TBI in Ppif-/- mice improved cortical tissue sparing, highlighting the multiple cellular targets of CsA in the mitigation of TBI pathology. The loss of CypD appeared to desensitize the mitochondrial response to calcium burden induced by TBI; this maintenance of mitochondrial function underlies the observed neuroprotective effect of the CypD knockout. These studies highlight the importance of maintaining mitochondrial homeostasis after injury and validate CypD as a therapeutic target for TBI. Further, these results solidify the beneficial effects of CsA treatment following TBI.

High glucose induces mitochondrial dysfunction independently of protein O-GlcNAcylation.

Diabetes is characterized by hyperglycaemia and perturbations in intermediary metabolism. In particular, diabetes can augment flux through accessory pathways of glucose metabolism, such as the hexosamine biosynthetic pathway (HBP), which produces the sugar donor for the ß-O-linked-N-acetylglucosamine (O-GlcNAc) post-translational modification of proteins. Diabetes also promotes mitochondrial dysfunction. Nevertheless, the relationships among diabetes, hyperglycaemia, mitochondrial dysfunction and O-GlcNAc modifications remain unclear. In the present study, we tested whether high-glucose-induced increases in O-GlcNAc modifications directly regulate mitochondrial function in isolated cardiomyocytes. Augmentation of O-GlcNAcylation with high glucose (33 mM) was associated with diminished basal and maximal cardiomyocyte respiration, a decreased mitochondrial reserve capacity and lower Complex II-dependent respiration (P<0.05); however, pharmacological or genetic modulation of O-GlcNAc modifications under normal or high glucose conditions showed few significant effects on mitochondrial respiration, suggesting that O-GlcNAc does not play a major role in regulating cardiomyocyte mitochondrial function. Furthermore, an osmotic control recapitulated high-glucose-induced changes to mitochondrial metabolism (P<0.05) without increasing O-GlcNAcylation. Thus, increased O-GlcNAcylation is neither sufficient nor necessary for high-glucose-induced suppression of mitochondrial metabolism in isolated cardiomyocytes.

Treatment with amnion-derived cellular cytokine solution (ACCS) induces persistent motor improvement and ameliorates neuroinflammation in a rat model of penetrating ballistic-like brain injury.

PURPOSE: The present work compared the behavioral outcomes of ACCS therapy delivered either intravenously (i.v.) or intracerebroventricularly (i.c.v.) after penetrating ballistic-like brain injury (PBBI). Histological markers for neuroinflammation and neurodegeneration were employed to investigate the potential therapeutic mechanism of ACCS. METHODS: Experiment-1, ACCS was administered either i.v. or i.c.v. for 1 week post-PBBI. Outcome metrics included behavioral (rotarod and Morris water maze) and gross morphological assessments. Experiment-2, rats received ACCS i.c.v for either 1 or 2 weeks post-PBBI. The inflammatory response was determined by immunohistochemistry for neutrophils and microglia reactivity. Neurodegeneration was visualized using silver staining. RESULTS: Both i.v. and i.c.v. delivery of ACCS improved motor outcome but failed to improve cognitive outcome or tissue sparing. Importantly, only i.c.v. ACCS treatment produced persistent motor improvements at a later endpoint. The i.c.v. ACCS treatment significantly reduced PBBI-induced increase in myeloperoxidase (MPO) and ionized calcium binding adaptor molecule 1 (Iba1) expression. Concomitant reduction of both Iba1 and silver staining were detected in corpus callosum with i.c.v. ACCS treatment. CONCLUSIONS: ACCS, as a treatment for TBI, showed promise with regard to functional (motor) recovery and demonstrated strong capability to modulate neuroinflammatory responses that may underline functional recovery. However, the majority of beneficial effects appear restricted to the i.c.v. route of ACCS delivery, which warrants future studies examining delivery routes (e.g. intranasal delivery) which are more clinically viable for the treatment of TBI.

N-acetylcysteine amide confers neuroprotection, improves bioenergetics and behavioral outcome following TBI.

Traumatic brain injury (TBI) has become a growing epidemic but no approved pharmacological treatment has been identified. Our previous work indicates that mitochondrial oxidative stress/damage and loss of bioenergetics play a pivotal role in neuronal cell death and behavioral outcome following experimental TBI. One tactic that has had some experimental success is to target glutathione using its precursor N-acetylcysteine (NAC). However, this approach has been hindered by the low CNS bioavailability of NAC. The current study evaluated a novel, cell permeant amide form of N-acetylcysteine (NACA), which has high permeability through cellular and mitochondrial membranes resulting in increased CNS bioavailability. Cortical tissue sparing, cognitive function and oxidative stress markers were assessed in rats treated with NACA, NAC, or vehicle following a TBI. At 15days post-injury, animals treated with NACA demonstrated significant improvements in cognitive function and cortical tissue sparing compared to NAC or vehicle treated animals. NACA treatment also was shown to reduce oxidative damage (HNE levels) at 7days post-injury. Mechanistically, post-injury NACA administration was demonstrated to maintain levels of mitochondrial glutathione and mitochondrial bioenergetics comparable to sham animals. Collectively these data provide a basic platform to consider NACA as a novel therapeutic agent for treatment of TBI.

Cardiomyocyte Ogt is essential for postnatal viability.

The singly coded gene O-linked-ß-N-acetylglucosamine (O-GlcNAc) transferase (Ogt) resides on the X chromosome and is necessary for embryonic stem cell viability during embryogenesis. In mature cells, this enzyme catalyzes the posttranslational modification known as O-GlcNAc to various cellular proteins. Several groups, including our own, have shown that acute increases in protein O-GlcNAcylation are cardioprotective both in vitro and in vivo. Yet, little is known about how OGT affects cardiac function because total body knockout (KO) animals are not viable. Presently, we sought to establish the potential involvement of cardiomyocyte Ogt in cardiac maturation. Initially, we characterized a constitutive cardiomyocyte-specific (cm)OGT KO (c-cmOGT KO) mouse and found that only 12% of the c-cmOGT KO mice survived to weaning age (4 wk old); the surviving animals were smaller than their wild-type littermates, had dilated hearts, and showed overt signs of heart failure. Dysfunctional c-cmOGT KO hearts were more fibrotic, apoptotic, and hypertrophic. Several glycolytic genes were also upregulated; however, there were no gross changes in mitochondrial O2 consumption. Histopathology of the KO hearts indicated the potential involvement of endoplasmic reticulum stress, directing us to evaluate expression of 78-kDa glucose-regulated protein and protein disulfide isomerase, which were elevated. Additional groups of mice were subjected to inducible deletion of cmOGT, which did not produce overt dysfunction within the first couple of weeks of deletion. Yet, long-term loss (via inducible deletion) of cmOGT produced gradual and progressive cardiomyopathy. Thus, cardiomyocyte Ogt is necessary for maturation of the mammalian heart, and inducible deletion of cmOGT in the adult mouse produces progressive ventricular dysfunction.

High fat feeding in mice is insufficient to induce cardiac dysfunction and does not exacerbate heart failure.

Preclinical studies of animals with risk factors, and how those risk factors contribute to the development of cardiovascular disease and cardiac dysfunction, are clearly needed. One such approach is to feed mice a diet rich in fat (i.e. 60%). Here, we determined whether a high fat diet was sufficient to induce cardiac dysfunction in mice. We subjected mice to two different high fat diets (lard or milk as fat source) and followed them for over six months and found no significant decrement in cardiac function (via echocardiography), despite robust adiposity and impaired glucose disposal. We next determined whether antecedent and concomitant exposure to high fat diet (lard) altered the murine heart's response to infarct-induced heart failure; high fat feeding during, or before and during, heart failure did not significantly exacerbate cardiac dysfunction. Given the lack of a robust effect on cardiac dysfunction with high fat feeding, we then examined a commonly used mouse model of overt diabetes, hyperglycemia, and obesity (db/db mice). db/db mice (or STZ treated wild-type mice) subjected to pressure overload exhibited no significant exacerbation of cardiac dysfunction; however, ischemia-reperfusion injury significantly depressed cardiac function in db/db mice compared to their non-diabetic littermates. Thus, we were able to document a negative influence of a risk factor in a relevant cardiovascular disease model; however, this did not involve exposure to a high fat diet. High fat diet, obesity, or hyperglycemia does not necessarily induce cardiac dysfunction in mice. Although many investigators use such diabetes/obesity models to understand cardiac defects related to risk factors, this study, along with those from several other groups, serves as a cautionary note regarding the use of murine models of diabetes and obesity in the context of heart failure.

Standardized bioenergetic profiling of adult mouse cardiomyocytes.

Mitochondria are at the crux of life and death and as such have become ideal targets of intervention in cardiovascular disease. Generally, current methods to measure mitochondrial dysfunction rely on working with the isolated organelle and fail to incorporate mitochondrial function in a cellular context. Extracellular flux methodology has been particularly advantageous in this respect; however, certain primary cell types, such as adult cardiac myocytes, have been difficult to standardize with this technology. Here, we describe methods for using extracellular flux (XF) analysis to measure mitochondrial bioenergetics in isolated, intact, adult mouse cardiomyocytes (ACMs). Following isolation, ACMs were seeded overnight onto laminin-coated (20 µg/ml) microplates, which resulted in high attachment efficiency. After establishing seeding density, we found that a commonly used assay medium (containing a supraphysiological concentration of pyruvate at 1 mmol/l) produced a maximal bioenergetic response. After performing a pyruvate dose-response, we determined that pyruvate titrated to 0.1 mmol/l was optimal for examining alternative substrate oxidation. Methods for measuring fatty acid oxidation were established. These methods lay the framework using XF analysis to profile metabolism of ACMs and will likely augment our ability to understand mitochondrial dysfunction in heart failure and acute myocardial ischemia. This platform could easily be extended to models of diabetes or other metabolic defects.

Post-injury administration of the mitochondrial permeability transition pore inhibitor, NIM811, is neuroprotective and improves cognition after traumatic brain injury in rats.

Mitochondrial dysfunction is known to play a pivotal role in cell death mechanisms following traumatic brain injury (TBI). N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclosporin A (CsA) analog, inhibits the mitochondrial permeability transition pore (mPTP) and has been shown to be neuroprotective following TBI in mice. However, the translation of the neuroprotective effects of mPTP inhibitors, including CsA and NIM811, into improved cognitive end points has yet to be fully investigated. Therefore, to build upon these results, a severe unilateral controlled cortical impact model of TBI was used in the present study to establish a dose-response curve for NIM811 in rats. The findings demonstrate that the neuroprotection afforded by NIM811 is dose dependent, with the 10 mg/kg dose being the most effective dose. Once the dose response was established, we evaluated the effect of the optimal dose of NIM811 on behavior, mitochondrial bioenergetics, and mitochondrial oxidative damage following TBI. For behavioral studies, rats were administered NIM811 at 15 min and 24 h post-injury, with cognitive testing beginning 10 days post-injury. Mitochondrial studies involved a single injection of NIM811 at 15 min post-injury followed by mitochondrial isolation at 6 h post-injury. The results revealed that the optimal dose of NIM811 improves cognition, improves mitochondrial functioning, and reduces oxidative damage following TBI.

Mitochondria, Amyloid ß, and Alzheimer's Disease.

Hypometabolism is a hallmark of Alzheimer's disease (AD) and implicates a mitochondrial role in the neuropathology associated with AD. Mitochondrial amyloid-beta (Aß) accumulation precedes extracellular Aß deposition. In addition to increasing oxidative stress, Aß has been shown to directly inhibit mitochondrial enzymes. Inhibition of mitochondrial enzymes as a result of oxidative damage or Aß interaction perpetuates oxidative stress and leads to a hypometabolic state. Additionally, Aß has also been shown to interact with cyclophilin D, a component of the mitochondrial permeability transition pore, which may promote cell death. Therefore, ample evidence exists indicating that the mitochondrion plays a vital role in the pathophysiology observed in AD.

Increase in blood-brain barrier permeability, oxidative stress, and activated microglia in a rat model of blast-induced traumatic brain injury.

Traumatic brain injury (TBI) as a consequence of exposure to blast is increasingly prevalent in military populations, with the underlying pathophysiological mechanisms mostly unknown. In the present study, we utilized an air-driven shock tube to investigate the effects of blast exposure (120 kPa) on rat brains. Immediately following exposure to blast, neurological function was reduced. BBB permeability was measured using IgG antibody and evaluating its immunoreactivity in the brain. At 3 and 24 hr postexposure, there was a transient significant increase in IgG staining in the cortex. At 3 days postexposure, IgG immunoreactivity returned to control levels. Quantitative immunostaining was employed to determine the temporal course of brain oxidative stress following exposure to blast. Levels of 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT) were significantly increased at 3 hr postexposure and returned to control levels at 24 hr postexposure. The response of microglia to blast exposure was determined by autoradiographic localization of (3) H-PK11195 binding. At 5 days postexposure, increased binding was observed in the contralateral and ipsilateral dentate gyrus. These regions also displayed increased binding at 10 days postexposure; in addition to these regions there was increased binding in the contralateral ventral hippocampus and substantia nigra at this time point. By using antibodies against CD11b/c, microglia morphology characteristic of activated microglia was observed in the hippocampus and substantia nigra of animals exposed to blast. These results indicate that BBB breakdown, oxidative stress, and microglia activation likely play a role in the neuropathology associated with TBI as a result of blast exposure.

Fasting is neuroprotective following traumatic brain injury.

To determine the neuroprotective effect of fasting after traumatic brain injury (TBI) and to elucidate the potential underlying mechanisms, we used a controlled cortical impact (CCI) injury model to induce either a moderate or a severe injury to adult male Sprague Dawley rats. Tissue-sparing assessments were used to determine the level of neuroprotection of fasting, hypoglycemia (insulin 10 U), or ketone (1.66 mmoles/kg/day or 0.83 mmoles/kg/day; D-beta-hydroxtbutyrate) administration. Mitochondrial isolation and respiratory studies were utilized to determine the functionality of mitochondria at the site of injury. We also investigated biomarkers of oxidative stress, such as lipid/protein oxidation, reactive oxygen species (ROS) production, and intramitochondrial calcium load, as a secondary measure of mitochondrial homeostasis. We found that fasting animals for 24 hr, but not 48 hr, after a moderate (1.5 mm), but not severe (2.0 mm), CCI resulted in a significant increase in tissue sparing. This 24-hr fast also decreased biomarkers of oxidative stress and calcium loading and increased mitochondrial oxidative phosphorylation in mitochondria isolated from the site of injury. Insulin administration, designed to mimic the hypoglycemic effect seen during fasting did not result in significant tissue sparing after moderate CCI injury and in fact induced increased mortality at some injection time points. However, the administration of ketones resulted in increased tissue sparing after moderate injury. Fasting for 24 hr confers neuroprotection, maintains cognitive function, and improves mitochondrial function after moderate (1.5 mm) TBI. The underlying mechanism appears to involve ketosis rather than hypoglycemia.