Pioglitazone restores mitochondrial function but does not spare cortical tissue following mild brain contusion.

Pioglitazone interacts through the mitochondrial protein mitoNEET to improve brain bioenergetics following traumatic brain injury. To provide broader evidence regarding the therapeutic effects of pioglitazone after traumatic brain injury, the current study is focused on immediate and delayed therapy in a model of mild brain contusion. To assess pioglitazone therapy on mitochondrial bioenergetics in cortex and hippocampus, we use a technique to isolate subpopulations of total, glia-enriched and synaptic mitochondria. Pioglitazone treatment was initially administered at either 0.25, 3, 12 or 24 h following mild controlled cortical impact. At 48 h post-injury, ipsilateral cortex and hippocampus were dissected and mitochondrial fractions were isolated. Maximal mitochondrial respiration injury-induced deficits were observed in total and synaptic fractions, and 0.25 h pioglitazone treatment following mild controlled cortical impact was able to restore respiration to sham levels. While there are no injury-induced deficits in hippocampal fractions, we do find that 3 h pioglitazone treatment after mild controlled cortical impact can significantly increase maximal mitochondrial bioenergetics compared to vehicle-treated mild controlled cortical impact group. However, delayed pioglitazone treatment initiated at either 3 or 24 h after mild brain contusion does not improve spared cortical tissue. We demonstrate that synaptic mitochondrial deficits following mild focal brain contusion can be restored with early initiation of pioglitazone treatment. Further investigation is needed to determine functional improvements with pioglitazone beyond that of overt cortical tissue sparing following mild contusion traumatic brain injury.

Clinically relevant mitochondrial-targeted therapy improves chronic outcomes after traumatic brain injury.

Pioglitazone, an FDA-approved compound, has been shown to target the novel mitochondrial protein mitoNEET and produce short-term neuroprotection and functional benefits following traumatic brain injury. To expand on these findings, we now investigate the dose- and time-dependent effects of pioglitazone administration on mitochondrial function after experimental traumatic brain injury. We then hypothesize that optimal pioglitazone dosing will lead to ongoing neuroprotection and cognitive benefits that are dependent on pioglitazone-mitoNEET signalling pathways. We show that delayed intervention is significantly more effective than early intervention at improving acute mitochondrial bioenergetics in the brain after traumatic brain injury. In corroboration, we demonstrate that mitoNEET is more heavily expressed, especially near the cortical contusion, in the 18 h following traumatic brain injury. To explore whether these findings relate to ongoing pathological and behavioural outcomes, mice received controlled cortical impact followed by initiation of pioglitazone treatment at either 3 or 18 h post-injury. Mice with treatment initiation at 18 h post-injury exhibited significantly improved behaviour and tissue sparing compared to mice with pioglitazone initiated at 3 h post-injury. Further using mitoNEET knockout mice, we show that this therapeutic effect is dependent on mitoNEET. Finally, we demonstrate that delayed pioglitazone treatment improves serial motor and cognitive performance in conjunction with attenuated brain atrophy after traumatic brain injury. This study illustrates that mitoNEET is the critical target for delayed pioglitazone intervention after traumatic brain injury, mitochondrial-targeting is highly time-dependent after injury and there is an extended therapeutic window to effectively treat mitochondrial dysfunction after brain injury.

Temporal changes in inflammatory mitochondria-enriched microRNAs following traumatic brain injury and effects of miR-146a nanoparticle delivery.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate post-transcriptional gene expression and contribute to all aspects of cellular function. We previously reported that the activities of several mitochondria-enriched miRNAs regulating inflammation (i.e., miR-142-3p, miR-142-5p, and miR-146a) are altered in the hippocampus at 3-12 hours following a severe traumatic brain injury. In the present study, we investigated the temporal expression profile of these inflammatory miRNAs in mitochondria and cytosol fractions at more chronic post-injury times following severe controlled cortical impact injury in rats. In addition, several inflammatory genes were analyzed in the cytosol fractions. The analysis showed that while elevated levels were observed in cytoplasm, the mitochondria-enriched miRNAs, miR-142-3p and miR-142-5p continued to be significantly reduced in mitochondria from injured hippocampi for at least 3 days and returned to near normal levels at 7 days post-injury. Although not statistically significant, miR-146a also remained at reduced levels for up to 3 days following controlled cortical impact injury, and recovered by 7 days. In contrast, miRNAs that are not enriched in mitochondria, including miR-124a, miR-150, miR-19b, miR-155, and miR-223 were either increased or demonstrated no change in their levels in mitochondrial fractions for 7 days. The one exception was that miR-223 levels were reduced in mitochondria at 1 day following injury. No major alterations were observed in sham operated animals. This temporal pattern was unique to mitochondria-enriched miRNAs and correlated with injury-induced changes in mitochondrial bioenergetics as well as expression levels of several inflammatory markers. These observations suggested a potential compartmental re-distribution of the mitochondria-enriched inflammatory miRNAs and may reflect an intracellular mechanism by which specific miRNAs regulate injury-induced inflammatory signaling. To test this, we utilized a novel peptide-based nanoparticle strategy for in vitro and in vivo delivery of a miR-146a mimic as a potential therapeutic strategy for targeting nuclear factor-kappaB inflammatory modulators in the injured brain. Nanoparticle delivery of miR-146a to BV-2 or SH-SY5Y cells significantly reduced expression of TNF receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1), two important modulators of the nuclear factor-kappaB (NF-κB) pro-inflammatory pathway. Moreover, injections of miR-146a containing nanoparticles into the brain immediately following controlled cortical impact injury significantly reduced hippocampal TNF receptor-associated factor 6 and interleukin-1 receptor-associated kinase 1 levels. Taken together, our studies demonstrate the subcellular alteration of inflammatory miRNAs after traumatic brain injury and establish proof of principle that nanoparticle delivery of miR-146a has therapeutic potential for modulating pro-inflammatory effectors in the injured brain. All of the studies performed were approved by the University of Kentucky Institutional Animal Care and Usage Committee (IACUC protocol # 2014-1300) on August 17, 2017.

Formoterol, a ß2-adrenoreceptor agonist, induces mitochondrial biogenesis and promotes cognitive recovery after traumatic brain injury.

Traumatic brain injury (TBI) leads to acute necrosis at the site of injury followed by a sequence of secondary events lasting from hours to weeks and often years. Targeting mitochondrial impairment following TBI has shown improvements in brain mitochondrial bioenergetics and neuronal function. Recently formoterol, a highly selective ß2-adrenoreceptor agonist, was found to induce mitochondrial biogenesis (MB) via Gßγ-Akt-eNOS-sGC pathway. Activation of MB is a novel approach that has been shown to restore mitochondrial function in several disease and injury models. We hypothesized that activation of MB as a target of formoterol after TBI would mitigate mitochondrial dysfunction, enhance neuronal function and improve behavioral outcomes. TBI-injured C57BL/6 male mice were injected (i.p.) with vehicle (normal saline) or formoterol (0.3 mg/kg) at 15 min, 8 h, 16 h, 24 h and then daily after controlled cortical impact (CCI) until euthanasia. After CCI, mitochondrial copy number and bioenergetic function were decreased in the ipsilateral cortex of the CCI-vehicle group. Compared to CCI-vehicle, cortical and hippocampal mitochondrial respiration rates as well as cortical mitochondrial DNA copy number were increased in the CCI-formoterol group. Mitochondrial Ca2+ buffering capacity in the hippocampus was higher in the CCI-formoterol group compared to CCI-vehicle group. Both assessments of cognitive performance, novel object recognition (NOR) and Morris water maze (MWM), decreased following CCI and were restored in the CCI-formoterol group. Although no changes were seen in the amount of cortical tissue spared between CCI-formoterol and CCI-vehicle groups, elevated levels of hippocampal neurons and improved white matter sparing in the corpus callosum were observed in CCI-formoterol group. Collectively, these results indicate that formoterol-mediated MB activation may be a potential therapeutic target to restore mitochondrial bioenergetics and promote functional recovery after TBI.

Acute Mitochondrial Impairment Underlies Prolonged Cellular Dysfunction after Repeated Mild Traumatic Brain Injuries.

Mild traumatic brain injuries (mTBI), accounting for more than 80% of TBIs, can cause cognitive and behavioral impairments, the severity and duration of which increase after additional mTBIs. While mTBI does not cause widespread neuronal death, the mechanisms underlying increased cellular susceptibility to subsequent head impacts remain unknown. To investigate the hypothesis that altered mitochondrial bioenergetics underlie cellular vulnerability to repeated insults, we employed a mouse model of mild closed head injury (CHI) to examine mitochondrial function and oxidative stress, because these mechanisms are often intertwined. Mitochondrial respiration was assayed (Seahorse XFe24 Flux Analyzer) from cortex and hippocampus collected at 6 h, 24 h, 48 h, and 96 h post-injury. State III (adenosine diphosphate [ADP]-mediated) respiration was significantly decreased in the hippocampal mitochondria of the CHI group compared with sham at 48 h post-injury. Further, cortex-derived mitochondria exhibited a decrease in State III respiration at 24 h and 48 h post-injury. No significant differences were observed at 6 h or 96 h post-injury in either region of interest. A second CHI repeated either 48 h or 96 h after the first did not worsen State III respiration at 48 h after the final injury compared with a single CHI, but CHI repeated at a 48 h interval prolonged cortical mitochondrial dysfunction to 96 h after the final injury. Markers of oxidative stress were significantly elevated after two CHIs delivered 48 h apart, but not after single CHI or two CHI delivered 96 h apart. This study establishes that mTBI results in early mitochondrial dysfunction, which may be a determinant for cellular vulnerability to repeated head impacts. Thus, therapies targeting mitochondrial impairment could improve outcomes after repeated mTBI.

Effects of a docosahexaenoic acid-rich microalgae nutritional product on insulin sensitivity after prolonged dexamethasone treatment in healthy mature horses.

OBJECTIVE: To determine effects of a microalgae nutritional product on insulin sensitivity in horses. ANIMALS: 8 healthy mature horses. PROCEDURES :Horses (n = 4/group) received a basal diet without (control diet) or with docosahexaenoic acid-rich microalgae meal (150 g/d) for 49 days (day 0 = first day of diet). On day 28, an isoglycemic hyperinsulinemic clamp procedure was performed. Horses then received dexamethasone (0.04 mg/kg/d) for 21 days. On day 49, the clamp procedure was repeated. After a 60-day washout, horses received the alternate diet, and procedures were repeated. Plasma fatty acid, glucose, and insulin concentrations and glucose and insulin dynamics during the clamp procedure were measured on days 28 and 49. Two estimates of insulin sensitivity (reciprocal of the square root of the insulin concentration and the modified insulin-to-glucose ratio for ponies) were calculated. RESULTS: Baseline glucose and insulin concentrations or measures of insulin sensitivity on day 28 did not differ between horses when fed the control diet or the basal diet plus microalgae meal. On day 49 (ie, after dexamethasone administration), the microalgae meal was associated with lower baseline insulin and glucose concentrations and an improved modified insulin-to-glucose ratio for ponies, compared with results for the control diet. CONCLUSIONS AND CLINICAL RELEVANCE: Although the microalgae meal had no effect on clamp variables following dexamethasone treatment, it was associated with improved plasma glucose and insulin concentrations and insulin sensitivity estimates. A role for microalgae in the nutritional management of insulin-resistant horses warrants investigation.

Stem cell factor rescues tyrosinase expression and pigmentation in discreet anatomic locations in albino mice.

The K14-SCF transgenic murine model of variant pigmentation is based on epidermal expression of stem cell factor (SCF) on the C57BL/6J background. In this system, constitutive expression of SCF by epidermal keratinocytes results in retention of melanocytes in the interfollicular basal layer and pigmentation of the epidermis itself. Here, we extend this animal model by developing a compound mutant transgenic amelanotic animal defective at both the melanocortin 1 receptor (Mc1r) and tyrosinase (Tyr) loci. In the presence of K14-Scf, tyrosinase-mutant animals (previously thought incapable of synthesizing melanin) exhibited progressive robust epidermal pigmentation with age in the ears and tails. Furthermore, K14-SCF Tyr(c2j/c2j) animals demonstrated tyrosinase expression and enzymatic activity, suggesting that the c2j Tyr defect can be rescued in part by SCF in the ears and tail. Lastly, UV sensitivity of K14-Scf congenic animals depended mainly on the amount of eumelanin present in the skin. These findings suggest that c-kit signaling can overcome the c2j Tyr mutation in the ears and tails of aging animals and that UV resistance depends on accumulation of epidermal eumelanin.

In vitro skin diffusion study of pure forskolin versus a forskolin-containing Plectranthus barbatus root extract.

An in vitro skin diffusion study of pure forskolin (1) versus a 1-containing Plectranthus barbatus root extract (P. barbatus extract) in hairless guinea pig skin and human skin in a flow-through diffusion cell system was conducted and is being reported for the first time. Both topical agents were formulated in a solution of 70% ethanol and 30% propylene glycol (v/v). The results showed that forskolin can be delivered through the stratum corneum and that the flux of this compound was enhanced when 1 was delivered as a constituent of the P. barbatus extract as compared to an equivalent amount in pure form. These results suggest that the P. barbatus extract used contains permeation enhancement activity from other compound(s) contained in the crude root extract. It is possible that P. barbatus root extract may be used as an economical source of 1 to perform topical chemical manipulation of pigmentation in high-risk populations.

Prolonged treatment of fair-skinned mice with topical forskolin causes persistent tanning and UV protection.

We previously reported that topical application of forskolin to the skin of fair-skinned MC1R-defective mice with epidermal melanocytes resulted in accumulation of eumelanin in the epidermis and was highly protective against UV-mediated cutaneous injury. In this report, we describe the long-term effects of chronic topical forskolin treatment in this animal model. Forskolin-induced eumelanin production persisted through 3 months of daily applications, and forskolin-induced eumelanin remained protective against UV damage as assessed by minimal erythematous dose (MED). No obvious toxic changes were noted in the skin or overall health of animals exposed to prolonged forskolin therapy. Body weights were maintained throughout the course of topical forskolin application. Topical application of forskolin was associated with an increase in the number of melanocytes in the epidermis and thickening of the epidermis due, at least in part, to an accumulation of nucleated keratinocytes. Together, these data suggest in this animal model, short-term topical regular application of forskolin promotes eumelanin induction and that over time, topical forskolin treatment is associated with persistent melanization, epidermal cell accumulation, and skin thickening.

DNA repair pathways and hereditary cancer susceptibility syndromes.

Every living organism is exposed to numerous genomic insults on a daily basis as a consequence of cellular metabolism and exposure to environmental agents capable of interacting with the genome (e.g. chemicals, toxins, pollutants, UV and ionizing radiation) (1). Maintenance of the integrity of the genome is paramount to the survival and propagation of a species and involves the continuous activity of a variety of DNA repair pathways. Inherited mutations in genes involved in DNA damage recognition and repair lead to disease by destabilization of the genome and increased mutagenesis. In fact, it is common for cancer cells to exhibit loss of genomic stability presumably as a result of clonally acquired mutations in DNA repair genes (2). Currently, roughly 150 DNA repair genes have been identified in humans (3) and a variety of familial cancer predisposition and/or premature aging syndromes are now linked to various loss-of-function mutations in these genes (4). Genetic interaction between DNA repair pathways and global cell differentiation pathways is supported by phenotypic similarities between inactivating mutations in a DNA repair, cell cycle arrest and apoptosis proteins. Though there is clearly some degree of functional redundancy between DNA repair pathways for correction of specific DNA lesions, the particular clinical characteristics of a repair defect can be predicted by the specific repair pathway affected (5). Patients with cancer predisposition syndromes often have multiple family members affected by cancer, develop cancer at an early age, and are at risk for developing multiple primary tumors over time (6, 7). Though patients with identifiable cancer predisposition syndromes are rare, defining their molecular defects has led to widespread applicability by uncovering relevant molecular pathways that are perturbed via somatic (non-inherited) mutations in the majority of sporadic cancers. In this review, we describe general molecular mechanisms of major forms of DNA repair and illustrate clinical consequences of deficiencies in these pathways. For more in depth detail, the reader is referred to several recent reviews and texts (2, 8-13).

Topical drug rescue strategy and skin protection based on the role of Mc1r in UV-induced tanning.

Ultraviolet-light (UV)-induced tanning is defective in numerous 'fair-skinned' individuals, many of whom contain functional disruption of the melanocortin 1 receptor (MC1R). Although this suggested a critical role for the MC1R ligand melanocyte stimulating hormone (MSH) in this response, a genetically controlled system has been lacking in which to determine the precise role of MSH-MC1R. Here we show that ultraviolet light potently induces expression of MSH in keratinocytes, but fails to stimulate pigmentation in the absence of functional MC1R in red/blonde-haired Mc1r(e/e) mice. However, pigmentation could be rescued by topical application of the cyclic AMP agonist forskolin, without the need for ultraviolet light, demonstrating that the pigmentation machinery is available despite the absence of functional MC1R. This chemically induced pigmentation was protective against ultraviolet-light-induced cutaneous DNA damage and tumorigenesis when tested in the cancer-prone, xeroderma-pigmentosum-complementation-group-C-deficient genetic background. These data emphasize the essential role of intercellular MSH signalling in the tanning response, and suggest a clinical strategy for topical small-molecule manipulation of pigmentation.