Association of Circulating Caprylic Acid with Risk of Mild Cognitive Impairment and Alzheimer's Disease in the Alzheimer's Disease Neuroimaging Initiative (ADNI) Cohort.

OBJECTIVE: Medium-chain fatty acids (MCFAs) can rapidly cross the blood-brain barrier and provide an alternative energy source for the brain. This study aims to determine 1) whether plasma caprylic acid (C8:0) is associated with risk of incident mild cognitive impairment (MCI) among baseline cognitively normal (CN) participants, and incident Alzheimer's Disease (AD) among baseline MCI participants; and 2) whether these associations differ by sex, comorbidity of cardiometabolic diseases, apolipoprotein E (APOE) ε4 alleles, and ADAS-Cog 13. METHODS: Within the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort, plasma C8:0 was measured at baseline in 618 AD-free participants aged 55 to 91. Logistic regression models were used to estimate odds ratios (ORs) and 95% CIs with incident MCI and AD as dependent variables, separately. RESULTS: The inverse association between circulating C8:0 and risk of incident MCI was of borderline significance. The inverse association between circulating levels of C8:0 and risk of incident MCI was significant among CN participants with ≥1 cardiometabolic diseases [OR (95% CI): 0.75 (0.58-0.98) (P=0.03)], those with one copy of APOE ε4 alleles [OR (95% CI): 0.43 (0.21-0.89) (P=0.02)], female [OR (95% CI): 0.60 (0.38-0.94) (P=0.02)], and ADAS-Cog 13 above the median [OR (95%CI): 0.69 (0.50-0.97)(P=0.03)] after adjusting for all covariates. CONCLUSION: The inverse associations were present only among subgroups of CN participants, including female individuals, those with one or more cardiometabolic diseases, or one APOE ε4 allele, or higher ADAS-Cog 13 scores. If confirmed, this finding will facilitate precision prevention of MCI, in turn, AD among CN older adults.

BMCP1: a mitochondrial uncoupling protein in neurons which regulates mitochondrial function and oxidant production.

Outside the nervous system, members of the mitochondrial uncoupling protein (UCP) family have been proposed to contribute to control of body temperature and energy metabolism, and regulation of mitochondrial production of reactive oxygen species (ROS). However, the function of brain mitochondrial carrier protein 1 (BMCP1), which is highly expressed in brain, remains to be determined. To study BMCP1 expression and function in the nervous system, a high-affinity antibody to BMCP1 was generated and used to analyze tissue expression of BMCP1 protein in mouse. BMCP1 protein was highly expressed in heart and kidney, but not liver or lung. In the nervous system, BMCP1 was present in cortex, basal ganglia, substantia nigra, cerebellum, and spinal cord. Both BMCP1 mRNA and protein expression was almost exclusively neuronal. To study the effect of BMCP1 expression on mitochondrial function, neuronal (GT1-1) cell lines with stable overexpression of BMCP1 were generated. Transfected cells had higher State 4 respiration and lower mitochondrial membrane potential (psi(m)), consistent with greater mitochondrial uncoupling. BMCP1 expression also decreased mitochondrial production of ROS. These data suggest that BMCP1 can modify mitochondrial respiratory efficiency and mitochondrial oxidant production, and raise the possibility that BMCP1 might alter the vulnerability of brain to both acute injury and to neurodegenerative conditions.

The mitochondrial permeability transition pore and nitric oxide synthase mediate early mitochondrial depolarization in astrocytes during oxygen-glucose deprivation.

Recent studies suggest that the degree of mitochondrial dysfunction in cerebral ischemia may be an important determinant of the final extent of tissue injury. Although loss of mitochondrial membrane potential (psi(m)), one index of mitochondrial dysfunction, has been documented in neurons exposed to ischemic conditions, it is not yet known whether astrocytes, which are relatively resistant to ischemic injury, experience changes in psi(m) under similar conditions. To address this, we exposed cortical astrocytes cultured alone or with neurons to oxygen-glucose deprivation (OGD) and monitored psi(m) using tetramethylrhodamine ethyl ester. Both neurons and astrocytes exhibited profound loss of psi(m) after 45-60 min of OGD. However, although this exposure is lethal to nearly all neurons, it is hours less than that needed to kill astrocytes. Astrocyte psi(m) was rescued during OGD by cyclosporin A, a permeability transition pore blocker, and (G)N-nitro-arginine, a nitric oxide synthase inhibitor. Loss of mitochondrial membrane potential in astrocytes was not accompanied by depolarization of the plasma membrane. Recovery of astrocyte psi(m) after reintroduction of O(2) and glucose occurred over a surprisingly long period (>1 hr), suggesting that OGD caused specific, reversible changes in astrocyte mitochondrial physiology beyond the simple lack of O(2) and glucose. Decreased psi(m) was associated with a cyclosporin A-sensitive loss of cytochrome c but not with activation of caspase-3 or caspase-9. Our data suggest that astrocyte mitochondrial depolarization could be a previously unrecognized event early in ischemia and that strategies that target the mitochondrial component of ischemic injury may benefit astrocytes as well as neurons.

Superoxide stress identifies neurons at risk in a model of ataxia-telangiectasia.

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder caused by mutations in the ATM gene. A-T children demonstrate sensitivity to ionizing radiation, predisposition to hematological malignancies, and telangiectasias. However, the hallmark of A-T is fulminant degeneration of cerebellar Purkinje cells accompanied by a progressive ataxia with features of both cerebellar and basal ganglia dysfunction. Although the ATM gene product (ATM) is known to be involved in DNA repair, the mechanisms that link loss of ATM with neurodegeneration remain unknown. Recently, it has been suggested that abnormalities in redox status contribute to the A-T phenotype. To address this question in the nervous system, we measured reactive oxygen species (ROS) in brain regions and specific neuronal populations in ATM-/- mice. We found increased ROS levels in cerebellum and striatum but not cortex of ATM-/- mice compared to ATM+/+ mice. Confocal microscopic examination revealed elevated superoxide levels in cerebellar Purkinje cells and nigral dopaminergic neurons but not cortical neurons, thus mapping increased superoxide levels onto the neuronal populations selectively affected in A-T. These data are the first demonstration of elevated levels of ROS in neurons at risk in any genetic neurodegenerative disorder and, furthermore, suggest that ATM acts as a pro-survival signal in post-mitotic Purkinje cells and dopaminergic neurons by modifying superoxide radical handling in these selectively vulnerable neurons.

Fullerene-based antioxidants and neurodegenerative disorders.

Water-soluble derivatives of buckminsterfullerene (C(60)) derivatives are a unique class of compounds with potent antioxidant properties. Studies on one class of these compounds, the malonic acid C(60) derivatives (carboxyfullerenes), indicated that they are capable of eliminating both superoxide anion and H(2)O(2), and were effective inhibitors of lipid peroxidation, as well. Carboxyfullerenes demonstrated robust neuroprotection against excitotoxic, apoptotic and metabolic insults in cortical cell cultures. They were also capable of rescuing mesencephalic dopaminergic neurons from both MPP(+) and 6-hydroxydopamine-induced degeneration. Although there is limited in vivo data on these compounds to date, we have previously reported that systemic administration of the C(3) carboxyfullerene isomer delayed motor deterioration and death in a mouse model of familial amyotrophic lateral sclerosis (FALS). Ongoing studies in other animal models of CNS disease states suggest that these novel antioxidants are potential neuroprotective agents for other neurodegenerative disorders, including Parkinson's disease.

Clusterin contributes to caspase-3-independent brain injury following neonatal hypoxia-ischemia.

Clusterin, also known as apolipoprotein J, is a ubiquitously expressed molecule thought to influence a variety of processes including cell death. In the brain, it accumulates in dying neurons following seizures and hypoxic-ischemic (H-I) injury. Despite this, in vivo evidence that clusterin directly influences cell death is lacking. Following neonatal H-I brain injury in mice (a model of cerebral palsy), there was evidence of apoptotic changes (neuronal caspase-3 activation), as well as accumulation of clusterin in dying neurons. Clusterin-deficient mice had 50% less brain injury following neonatal H-I. Surprisingly, the absence of clusterin had no effect on caspase-3 activation, and clusterin accumulation and caspase-3 activation did not colocalize to the same cells. Studies with cultured cortical neurons demonstrated that exogenous purified astrocyte-secreted clusterin exacerbated oxygen/glucose-deprivation-induced necrotic death. These results indicate that clusterin may be a new therapeutic target to modulate non-caspase-dependent neuronal death following acute brain injury.

Zinc-induced neuronal death in cortical neurons.

Although Zn2+ is normally stored and released in the brain, excessive exposure to extracellular Zn2+ can be neurotoxic. The purpose of the present study was to determine the type of neuronal cell death, necrosis versus apoptosis, induced by Zn2+ exposure. Addition of 10-50 microM ZnCl2 to the bathing medium of murine neuronal and glial cell cultures induced, over the next 24 hrs., Zn2+-concentration-dependent neuronal death; some glial death also occurred with Zn2+ concentrations above 30 microM. The neuronal death induced by 20 microM Zn2+ was characterized by coarse chromatin condensation, the formation of apoptotic bodies, and internucleosomal DNA fragmentation. It was attenuated in cortical cell cultures prepared from mice null for the bax gene, and by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-CH2F (ZVAD, 100 microM), but not by the NMDA receptor antagonist, D-2-amino-5-phosphonovalerate (D-APV, 200 microM ). In contrast, the neuronal death induced by 50 microM Zn2+ was characterized by plasma membrane disruption and random DNA fragmentation; this death was attenuated by D-APV, but exhibited little sensitivity to ZVAD or deletion of bax. These results suggest that Zn2+ can induce cell death with characteristics of either apoptosis or necrosis, depending on the intensity of the Zn2+ exposure.

Rapid microplate assay for superoxide scavenging efficiency.

Here we report a method to determine superoxide scavenging efficiency, using kinetic analysis of cytochrome c reduction and an automated UV/vis microtiter plate reader. Superoxide (O(2)(-&z. rad;)) was generated by xanthine oxidase metabolism of hypoxanthine, and quantified by following reduction of cytochrome c by O(2)(-&z. rad;) as increasing absorbance at 550 nm. Reaction conditions were established that provided a linear increase in O(2)(-&z.rad;) generation for more than 20 min, and good reproducibility over time. The majority of cytochrome c reduction was blocked by superoxide dismutase, indicating cytochrome c reduction derived predominantly from O(2)(-&z.rad;). Although EDTA is commonly included in this assay to eliminate undesirable Fenton side-reactions with H(2)O(2) (a co-product of reactions that use xanthine oxidase to produce O(2)(-&z.rad;)), we found that catalase, but not EDTA, blocked suicide elimination of cytochrome c from the reaction. Finally, we demonstrate the feasibility of evaluating superoxide scavenging abilities on small samples extracted from two types of neuronal cultures, a hypothalamic neuronal cell line (GT1 trk cells) and primary mouse cortical cell cultures. This assay allows rapid, high throughput assessments of superoxide scavenging efficacy for small molecules of interest, as well as for cell or tissue extracts.

Elevation of reactive oxygen species following ischemia-reperfusion in mouse cochlea observed in vivo.

An in vivo method for assessment of changes in hydroxyl radical levels in cochlear perilymphatic spaces is described and applied to cochlear ischemia-reperfusion in the mouse. Cochlear blood flow was reversibly reduced by compression of the anterior inferior cerebellar arterial network. Changes in the production of hydroxyl radicals, used as an index of tissue production of reactive oxygen species (ROS), were determined by measuring the conversion of salicylate to 2,3-dihydroxybenzoic acid. Low levels of salicylate (0.1 mM) in artificial perilymph were applied by perfusion of the cochlea using a round window entry and apical exit. Perfusate was collected and analyzed by high-performance liquid chromatography. Forty minutes of partial ischemia led to a > 10-fold average increase over baseline in the concentration of hydroxyl radical, which increase persisted for at least 40-80 min following reperfusion. Our observations support previous results obtained using less direct methods, indicating that cochlear ischemia-reperfusion and related damage is associated with elevated ROS levels. Development of an in vivo method for assessing changes in cochlear ROS in mice will facilitate the study of the relation between deafness genes, vulnerability to insults and dynamics of cellular processes that produce and regulate ROS.

Early elevation of cochlear reactive oxygen species following noise exposure.

Reactive oxygen species (ROS) have been implicated in a growing number of neurological disease states, from acute traumatic injury to neurodegenerative conditions such as Alzheimer's disease. Considerable evidence suggests that ROS also mediate ototoxicant- and noise-induced cochlear injury, although most of this evidence is indirect. To obtain real-time assessment of noise-induced cochlear ROS production in vivo, we adapted a technique which uses the oxidation of salicylate to 2,3-dihydroxybenzoic acid as a probe for the generation of hydroxyl radical. In a companion paper we described the development and characterization of this method in cochlear ischemia-reperfusion. In the present paper we use this method to demonstrate early elevations in ROS production following acute noise exposure. C57BL/6J mice were exposed for 1 h to intense broad-band noise sufficient to cause permanent threshold shift (PTS), as verified by auditory brainstem responses. Comparison of noise-exposed animals with unexposed controls indicated that ROS levels increase nearly 4-fold in the period 1-2 h following exposure and do not decline over that time. Our ROS measures extend previous results indicating that noise-induced PTS is associated with elevated cochlear ROS production and ROS-mediated injury. Persistent cochlear ROS elevation following noise exposure suggests a sustained process of oxidative stress which might be amenable to intervention with chronic antioxidant therapy.

Differential effects of cAMP in neurons and astrocytes. Role of B-raf.

Mitogen-activated protein kinase (MAPK) activation provides cell type-specific signals important for cellular differentiation, proliferation, and survival. Cyclic AMP (cAMP) has divergent effects on MAPK activity depending on whether signaling is through Ras/Raf-1 or Rap1/B-raf. We found that central nervous system-derived neurons, but not astrocytes, express B-raf. In neurons, cAMP activated MAPK in a Rap1/B-raf-dependent manner, while in astrocytes, cAMP decreased MAPK activity. Inhibition of MAPK in neurons decreased neuronal growth factor-mediated survival, and activation of MAPK by cAMP analogues rescued neurons from death. Furthermore, constitutive expression of B-raf in astrocytoma cells increased MAPK activation, as seen in neurons, and enhanced proliferation. These data provide the first experimental evidence that B-raf is the molecular switch which dominantly permits differential cAMP-dependent regulation of MAPK in neurons versus astrocytes, with important implications for both survival and proliferation.

Neurotrophin-mediated potentiation of neuronal injury.

The neurotrophins are a diverse family of peptides which activate specific tyrosine kinase-linked receptors. Over the past five decades, since the pioneering work of Levi-Montalcini and colleagues, the critical role that neurotrophins play in shaping the developing nervous system has become increasingly established. These molecules, which include the nerve growth factor (NGF)-related peptides, NGF, brain-derived neurotrophic factor (BDNF), NT-4/5 and NT-3, promote differentiation and survival in the developing nervous system, and to a lesser extent in the adult nervous system. As survival-promoting molecules, neurotrophins have been studied as potential neuroprotective agents, and have shown beneficial effects in many model systems. However, a surprising "dark side" to neurotrophin behavior has emerged from some of these studies implying that, under certain pathological conditions, neurotrophins may exacerbate, rather than alleviate, injury. How neurotrophins cause these deleterious consequences is a question which is only beginning to be answered, but initial work supports altered free radical handling or modification of glutamate receptor expression as possible mechanisms underlying these effects. This review will focus on evidence suggesting that neurotrophins may enhance injury under certain circumstances and on the mechanisms behind these injury-promoting aspects.

Distinct mechanisms underlie neurotoxin-mediated cell death in cultured dopaminergic neurons.

Oxidative stress is thought to contribute to dopaminergic cell death in Parkinson's disease (PD). The neurotoxin 6-hydroxydopamine (6-OHDA), which is easily oxidized to reactive oxygen species (ROS), appears to induce neuronal death by a free radical-mediated mechanism, whereas the involvement of free radicals in N-methyl-4-phenylpyridinium (MPP+) toxicity is less clear. Using free radical-sensitive fluorophores and vital dyes with post hoc identification of tyrosine hydroxylase-positive neurons, we monitored markers of apoptosis and the production of ROS in dopaminergic neurons treated with either 6-OHDA or MPP+. Annexin-V staining suggested that 6-OHDA but not MPP+-mediated cell death was apoptotic. In accordance with this assignment, the general caspase inhibitor Boc-(Asp)-fluoromethylketone only blocked 6-OHDA neurotoxicity. Both toxins exhibited an early, sustained rise in ROS, although only 6-OHDA induced a collapse in mitochondrial membrane potential temporally related to the increase in ROS. Recently, derivatives of buckminsterfullerene (C60) molecules have been shown to act as potent antioxidants in several models of oxidative stress (Dugan et al., 1997). Significant, dose-dependent levels of protection were also seen in these in vitro models of PD using the C3 carboxyfullerene derivative. Specifically, C3 was fully protective in the 6-OHDA paradigm, whereas it only partially rescued dopaminergic neurons from MPP+-induced cell death. In either model, it was more effective than glial-derived neurotrophic factor. These data suggest that cell death in response to 6-OHDA and MPP+ may progress through different mechanisms, which can be partially or entirely saved by carboxyfullerenes.

Xanthine oxidase-derived superoxide causes reoxygenation injury of ischemic cerebral endothelial cells.

Oxygen free radicals, generated by cerebral ischemia, have been widely implicated in the damage of vascular endothelium. Endothelial cells have been proposed as a significant source of oxygen free radicals. In the present study, we developed an anoxia-reoxygenation (AX/RO) model using pure cultures of cerebral endothelial cells (CECs) isolated from piglet cortex to measure CEC oxygen free radical production and determine its role in AX/RO-induced CEC injury. CEC injury, as measured by lactate dehydrogenase efflux into the culture medium, increased progressively with the duration of anoxic exposure, becoming significant after 10 h. Reoxygenation significantly increased CEC anoxic injury in a time-dependent manner. A 55% increase in oxygen free radical production, determined by fluorescence detection of dihydroethidium oxidation, was measured at the end of 4-h reoxygenation in CECs subjected to AX/RO conditions that killed 40% of the cells. Blockade of oxygen free radical production with superoxide dismutase (SOD; 250 and 1000 U/ml) or oxypurinol (50 and 200 microM), a potent xanthine oxidase inhibitor, reduced this injury by 32-36% and 30-39%, respectively. Results from our in vitro model indicate that CECs produce significant amounts of oxygen free radicals following ischemia, primarily from the xanthine oxidase pathway. These radicals ultimately have a cytotoxic effect on the very cells that produced them. Thus, reductions in oxygen free radical-mediated vascular injury may contribute to improvements in neurophysiologic outcome following treatment with oxygen free radical inhibitors and scavengers.

Mediation of neuronal apoptosis by enhancement of outward potassium current.

Apoptosis of mouse neocortical neurons induced by serum deprivation or by staurosporine was associated with an early enhancement of delayed rectifier (IK) current and loss of total intracellular K+. This IK augmentation was not seen in neurons undergoing excitotoxic necrosis or in older neurons resistant to staurosporine-induced apoptosis. Attenuating outward K+ current with tetraethylammonium or elevated extracellular K+, but not blockers of Ca2+, Cl-, or other K+ channels, reduced apoptosis, even if associated increases in intracellular Ca2+ concentration were prevented. Furthermore, exposure to the K+ ionophore valinomycin or the K+-channel opener cromakalim induced apoptosis. Enhanced K+ efflux may mediate certain forms of neuronal apoptosis.

Carboxyfullerenes as neuroprotective agents.

Two regioisomers with C3 or D3 symmetry of water-soluble carboxylic acid C60 derivatives, containing three malonic acid groups per molecule, were synthesized and found to be equipotent free radical scavengers in solution as assessed by EPR analysis. Both compounds also inhibited the excitotoxic death of cultured cortical neurons induced by exposure to N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or oxygen-glucose deprivation, but the C3 regioisomer was more effective than the D3 regioisomer, possibly reflecting its polar nature and attendant greater ability to enter lipid membranes. At 100 microM, the C3 derivative fully blocked even rapidly triggered, NMDA receptor-mediated toxicity, a form of toxicity with limited sensitivity to all other classes of free radical scavengers we have tested. The C3 derivative also reduced apoptotic neuronal death induced by either serum deprivation or exposure to Abeta1-42 protein. Furthermore, continuous infusion of the C3 derivative in a transgenic mouse carrying the human mutant (G93A) superoxide dismutase gene responsible for a form of familial amyotrophic lateral sclerosis, delayed both death and functional deterioration. These data suggest that polar carboxylic acid C60 derivatives may have attractive therapeutic properties in several acute or chronic neurodegenerative diseases.

Rapid suppression of free radical formation by nerve growth factor involves the mitogen-activated protein kinase pathway.

Neurotrophins such as nerve growth factor (NGF) regulate neuronal survival during development and are neuroprotective in certain models of injury to both the peripheral and the central nervous system. Although many effects of neurotrophins involve long-term changes in gene expression, several recent reports have focused on rapid effects of neurotrophins that do not involve synthesis of new gene products. Because enhanced formation of reactive oxygen species (ROS) represents one consequence of many insults that produce neuronal death, we hypothesized that neurotrophins might influence neuronal function and survival through acute alterations in the production of ROS. Using an oxidation-sensitive compound, dihydrorhodamine, we measured ROS formation in a central nervous system-derived neuronal cell line (GT1-1 trk) and in superior cervical ganglion neurons, both of which express the transmembrane NGF receptor tyrosine kinase, trkA. There was enhanced production of ROS in both cell types in the absence of NGF that was rapidly inhibited by application of NGF; complete inhibition of ROS generation in GT1-1 trk cells occurred within 10 min. NGF suppression of ROS formation was prevented by PD 098059, a specific inhibitor of MEK (mitogen/extracellular receptor kinase, which phosphorylates mitogen-activated protein kinase). The observation that NGF acutely blocks ROS formation in neurons through activation of the mitogen-activated protein kinase pathway suggests a novel mechanism for rapid neurotrophin signaling, and has implications for understanding neuroprotective and other effects of neurotrophins.

High density lipoprotein decreases beta-amyloid toxicity in cortical cell culture.

A hallmark of Alzheimer's disease (AD) is the extracellular deposition and accumulation of a 39-43 amino peptide, known as the amyloid beta (A beta) protein, within the brain. It has been postulated that A beta may in some way contribute directly to AD pathogenesis. The epsilon 4 allele of apolipoprotein E (apoE) is a major AD risk factor. Since both apoE and A beta are components of lipoproteins in plasma and cerebrospinal fluid, we asked whether lipoproteins and apoE isoforms would modify the toxicity of A beta (1-42) in cortical cell cultures. We show that high density lipoprotein with or without apoE reduces A beta toxicity and that apoE in the absence of lipoproteins does not affect A beta toxicity. These results suggest that interactions between A beta and lipoproteins in the brain could influence AD pathogenesis.

Vulnerability to glucose deprivation injury correlates with glutathione levels in astrocytes.

Astrocyte death from glucose deprivation appears to be mediated by free radicals. Reduced glutathione (GSH) was used as a measure of antioxidant defenses in primary cultures of cortical astrocytes. Glucose deprivation caused progressive, near complete loss of reduced glutathione (GSH). Astrocytes were protected by increasing endogenous GSH levels. Depletion of GSH to 21.4 +/- 3.3% of controls by the glutathione synthetase inhibitor buthionine sulfoximine resulted in more rapid injury by glucose deprivation, yet depletion of glutathione alone did not kill astrocytes. Both enhanced lipid peroxidation and membrane rigidification were caused by glucose deprivation, both indicators of oxidative damage. Membrane peroxidation was detected as a 24 +/- 2% decrease in cis-parinaric acid fluorescence, membrane rgidification as a 6.3 +/- 0.8% increase in fluorescence anisotropy using diphenylhexatriene. Glucose deprivation under normoxic conditions may occur clinically in patients such as diabetics. In addition, oxidative damage in the setting of energy depletion occurs with other insults, including ischemic brain injury. Glucose deprivation may thus be a clinically relevant model of hypoglycemic astrocyte injury, and may be useful to investigate the effects of glutathione and redox modulation on second messenger systems and gene regulation.