Janna L. Harris

Altered Neurochemical Profile after Traumatic Brain Injury: 1H-MRS Biomarkers of Pathological Mechanisms

Specific neurochemicals measured with proton magnetic resonance spectroscopy (1H-MRS) may serve as biomarkers of pathological mechanism in the brain. We used high field in vivo 1H-MRS to measure a detailed neurochemical profile after experimental traumatic brain injury (TBI) in rats. We characterized neurochemical changes in the contused cortex and the normal-appearing perilesional hippocampus over a time course from 1 hour to 2 weeks after injury. We found significant changes in 19 out of 20 neurochemicals in the cortex, and 9 out of 20 neurochemicals in the hippocampus. These changes provide evidence of altered cellular metabolic status after TBI, with specific compounds proposed to reflect edema, excitotoxicity, neuronal and glial integrity, mitochondrial status and bioenergetics, oxidative stress, inflammation, and cell membrane disruption. Our results support the utility of 1H-MRS for monitoring cellular mechanisms of TBI pathology in animal models, and the potential of this approach for preclinical evaluation of novel therapies.

Oxaloacetate Activates Brain Mitochondrial Biogenesis, Enhances the Insulin Pathway, Reduces Inflammation, and Stimulates Neurogenesis

Brain bioenergetic function declines in some neurodegenerative diseases, this may influence other pathologies and administering bioenergetic intermediates could have therapeutic value. To test how one intermediate, oxaloacetate (OAA) affects brain bioenergetics, insulin signaling, inflammation and neurogenesis, we administered intraperitoneal OAA, 1-2 g/kg once per day for 1-2 weeks, to C57Bl/6 mice. OAA altered levels, distributions or post-translational modifications of mRNA and proteins (proliferator-activated receptor-gamma coactivator 1α, PGC1 related co-activator, nuclear respiratory factor 1, transcription factor A of the mitochondria, cytochrome oxidase subunit 4 isoform 1, cAMP-response element binding, p38 MAPK and adenosine monophosphate-activated protein kinase) in ways that should promote mitochondrial biogenesis. OAA increased Akt, mammalian target of rapamycin and P70S6K phosphorylation. OAA lowered nuclear factor κB nucleus-to-cytoplasm ratios and CCL11 mRNA. Hippocampal vascular endothelial growth factor mRNA, doublecortin mRNA, doublecortin protein, doublecortin-positive neuron counts and neurite length increased in OAA-treated mice. (1)H-MRS showed OAA increased brain lactate, GABA and glutathione thereby demonstrating metabolic changes are detectable in vivo. In mice, OAA promotes brain mitochondrial biogenesis, activates the insulin signaling pathway, reduces neuroinflammation and activates hippocampal neurogenesis.

High-field proton magnetic resonance spectroscopy reveals metabolic effects of normal brain aging.

Altered brain metabolism is likely to be an important contributor to normal cognitive decline and brain pathology in elderly individuals. To characterize the metabolic changes associated with normal brain aging, we used high-field proton magnetic resonance spectroscopy in vivo to quantify 20 neurochemicals in the hippocampus and sensorimotor cortex of young adult and aged rats. We found significant differences in the neurochemical profile of the aged brain when compared with younger adults, including lower aspartate, ascorbate, glutamate, and macromolecules, and higher glucose, myo-inositol, N-acetylaspartylglutamate, total choline, and glutamine. These neurochemical biomarkers point to specific cellular mechanisms that are altered in brain aging, such as bioenergetics, oxidative stress, inflammation, cell membrane turnover, and endogenous neuroprotection. Proton magnetic resonance spectroscopy may be a valuable translational approach for studying mechanisms of brain aging and pathology, and for investigating treatments to preserve or enhance cognitive function in aging.

Probing astrocyte metabolism in vivo: proton magnetic resonance spectroscopy in the injured and aging brain

Following a brain injury, the mobilization of reactive astrocytes is part of a complex neuroinflammatory response that may have both harmful and beneficial effects. There is also evidence that astrocytes progressively accumulate in the normal aging brain, increasing in both number and size. These astrocyte changes in normal brain aging may, in the event of an injury, contribute to the exacerbated injury response and poorer outcomes observed in older traumatic brain injury (TBI) survivors. Here we present our view that proton magnetic resonance spectroscopy (1H-MRS), a neuroimaging approach that probes brain metabolism within a defined region of interest, is a promising technique that may provide insight into astrocyte metabolic changes in the injured and aging brain in vivo. Although 1H-MRS does not specifically differentiate between cell types, it quantifies certain metabolites that are highly enriched in astrocytes (e.g., Myo-inositol, mlns), or that are involved in metabolic shuttling between astrocytes and neurons (e.g., glutamate and glutamine). Here we focus on metabolites detectable by 1H-MRS that may serve as markers of astrocyte metabolic status. We review the physiological roles of these metabolites, discuss recent 1H-MRS findings in the injured and aging brain, and describe how an astrocyte metabolite profile approach might be useful in clinical medicine and clinical trials.

A high fat diet alters metabolic and bioenergetic function in the brain: A magnetic resonance spectroscopy study.

Diet-induced obesity and associated metabolic effects can lead to neurological dysfunction and increase the risk of developing Alzheimers disease (AD) and Parkinsons disease (PD). Despite these risks, the effects of a high-fat diet on the central nervous system are not well understood. To better understand the mechanisms underlying the effects of high fat consumption on brain regions affected by AD and PD, we used proton magnetic resonance spectroscopy ((1)H-MRS) to measure neurochemicals in the hippocampus and striatum of rats fed a high fat diet vs. normal low fat chow. We detected lower concentrations of total creatine (tCr) and a lower glutamate-to-glutamine ratio in the hippocampus of high fat rats. Additional effects observed in the hippocampus of high fat rats included higher N-acetylaspartylglutamic acid (NAAG), and lower myo-inositol (mIns) and serine (Ser) concentrations. Post-mortem tissue analyses revealed lower phosphorylated AMP-activated protein kinase (pAMPK) in the striatum but not in the hippocampus of high fat rats. Hippocampal pAMPK levels correlated significantly with tCr, aspartate (Asp), phosphoethanolamine (PE), and taurine (Tau), indicating beneficial effects of AMPK activation on brain metabolic and energetic function, membrane turnover, and edema. A negative correlation between pAMPK and glucose (Glc) indicates a detrimental effect of brain Glc on cellular energy response. Overall, these changes indicate alterations in neurotransmission and in metabolic and bioenergetic function in the hippocampus and in the striatum of rats fed a high fat diet.

Ubiquinol treatment for TBI in male rats: Effects on mitochondrial integrity, injury severity, and neurometabolism

Following traumatic brain injury (TBI), there is significant secondary damage to cerebral tissue from increased free radicals and impaired mitochondrial function. This imbalance between reactive oxygen species (ROS) production and the effectiveness of cellular antioxidant defenses is termed oxidative stress. Often there are insufficient antioxidants to scavenge ROS, leading to alterations in cerebral structure and function. Attenuating oxidative stress following a TBI by administering an antioxidant may decrease secondary brain injury, and currently many drugs and supplements are being investigated. We explored an over‐the‐counter supplement called ubiquinol (reduced form of coenzyme Q10), a potent antioxidant naturally produced in brain mitochondria. We administered intra‐arterial ubiquinol to rats to determine if it would reduce mitochondrial damage, apoptosis, and severity of a contusive TBI. Adult male F344 rats were randomly assigned to one of three groups: (1) Saline‐TBI, (2) ubiquinol 30 minutes before TBI (UB‐PreTBI), or (3) ubiquinol 30 minutes after TBI (UB‐PostTBI). We found when ubiquinol was administered before or after TBI, rats had an acute reduction in brain mitochondrial damage, apoptosis, and two serum biomarkers of TBI severity, glial fibrillary acidic protein (GFAP) and ubiquitin C‐terminal hydrolase‐L1 (UCH‐L1). However, in vivo neurometabolic assessment with proton magnetic resonance spectroscopy did not show attenuated injury‐induced changes. These findings are the first to show that ubiquinol preserves mitochondria and reduces cellular injury severity after TBI, and support further study of ubiquinol as a promising adjunct therapy for TBI.

IS NON-INVASIVE PROTON MAGNETIC RESONANCE SPECTROSCOPY SENSITIVE TO ALTERED BRAIN BIOENERGETICS?

P4-201 IS NON-INVASIVE PROTON MAGNETIC RESONANCE SPECTROSCOPY SENSITIVE TO ALTERED BRAIN BIOENERGETICS? WilliamMiles Brooks, Janna Harris, Hung-Wen Yeh, JeffreyM. Burns, Russell H. Swerdlow, University of Kansas Alzheimer’s Disease Center, Kansas City, Kansas, United States; University of Kansas, Kansas City, Kansas, United States; University of Kansas, Kansas City, Kansas, United States; University of Kansas School of Medicine, Fairway, Kansas, United States. Contact e-mail: wbrooks@kumc.edu

MAGNETIC RESONANCE SPECTROSCOPY INVESTIGATIONS OF EFFECTS OF GLUCOSE IN NORMAL AGING BRAIN

Background:As the human brain ages, cellular mechanisms such as bioenergetics, inflammation, oxidative stress, and membrane turnover are altered, possibly contributing to the cognitive decline seen in otherwise healthy elderly. Accordingly, there is a need for non-invasive methods to provide quantitative assessment of the state of each of these mechanisms. Localized, non-invasive magnetic resonance spectroscopy (MRS) of the brain can quantify a neurochemical profile of at least 20 neurochemicals that have been associated with these cellular mechanisms. Methods: We used noninvasive high-field 9.4 Tesla short-echo STEAM MRS to quantify neurochemicals in the hippocampus and sensorimotor cortex of young adult (23mo, n1⁄430) and aged (20-22mo, n1⁄420) male Fischer 344 rats. Animals were housed in pairs on a 12 hour light-dark cycle with free access to standard rat chow and water. Animals were anesthetized with isoflurane for scanning in accordance with institutional guidelines. We used LCModel to calculate absolute concentrations of 20 neurochemicals using a weighted averages method to account for variable signal quality. We applied a mixedeffects ANOVA model (age, location, age*location) and implemented the Holm’s sequential Bonferroni procedure to control the family-wise type I error rate at the 0.05 level. Results: We found significant differences in the neurochemical profile of the aged brain when compared with younger adults, including higher glucose, myoinositol, and glutamine, and lower aspartate, ascorbate, and glutamate (all p<0.05). Across all spectra, we found that glucose was negatively correlated with lactate (r 2 1⁄40.32), possibly reflecting individual glycolytic state. We also found that in each brain location glucose was correlated with myo-inositol (r 2 1⁄40.37 in hippocampus and r 21⁄40.38 in cortex). Since myo-inositol is a marker of astrocytes, we speculate that this may reflect the pro-inflammatory effects of higher brain glucose. Conclusions: Although the specific mechanisms underlying altered spectroscopic biomarkers remain to be verified, MRSmight be a useful non-invasive approach to studying inflammation in normal aging and in disease. Although the current study was completed on a high-field pre-clinical scanner, novel techniques to quantify these biomarkers in humans are under development. Accordingly, MRS is a promising translational approach for assessing the state of the aging brain and for studying novel treatments.

IMPACT OF OXALOACETATE ON BRAIN BIOENERGETIC INFRASTRUCTURES, NEUROGENESIS, AND INFLAMMATION

Background: Reductions in sex steroids and neurosteroid production have been reported in AD brain. Hormonal supplementation has shown benefits in offering neuroprotection and reducing cerebral amyloid load in animal studies. Although requiring further investigation in clinical trials, concerns have been raised with long-term therapeutic application due to potential side-effects. Ligands of the translocator protein (TSPO) can stimulate synthesis of these protective hormones directly in the brain, therefore are promising candidates for the development of a targeted alternative to conventional hormone therapy for use in AD. Classic TSPO ligands have been previously shown to increase neurosteroid levels and reduce cerebralAb i n amousemodel ofAD.However, these ligands have limited clinical usefulness due to low specificity and poor blood-brain barrier penetration. We screened 14 novel TSPO ligands in vitro for safety and efficacy as potential therapeutic agents for AD.Methods: Fourteen novel TSPO ligands based on substituted 2’-phenyl imidazopyridine structure were prepared for evaluation. Structural variations on substituents on the 2’-phenyl ring and the acetamide side chain were made yielding compounds of varying affinity selectivity and biological function. These ligands were screened for toxicity (10 nM-100mM) by MTS and LDH release assay in the M17 human neuroblastoma cell line. Their neurosteroidogenic potency were assessed by measuring pregnenolone levels (the precursor to all other neurosteroids) by ELISA in M17, N2a (human and mouse neuroblastoma, respectively) and C6 (mouse glioma) cell lines. MA-10 Leydig cells were used t o identify ligands specific for inducing steroidogenesis in the brain and not the testes. Results: No toxicity was observed at the steroidogenic dose range (nm) for any of the ligands, although toxicity was observed in 6 out of the 14 ligands at high doses (100 uM). Three novel ligands were selected based on favourable toxicity and neurosteroidogenic potency profiles for assessment of anti-amyloidogenic efficacy. TSPO expression was confirmed by western blot. And finally, Ab and neurosteroid levels were measured by LC-MS/MS.Conclusions:New generation TSPO l igands are promising candidates for the development of a targeted alternative to conventional hormone therapy in AD. Our results represent an innovative model to identify novel TSPO ligands that specifically target neurosteroid production.