Aimee N. Winter

Neuroprotective effects of anthocyanins on apoptosis induced by mitochondrial oxidative stress

Abstract Objectives Mitochondrial oxidative stress (MOS) is a major factor in the underlying pathology of many neurodegenerative diseases. Here, we investigated the neuroprotective effects of a unique class of nutraceutical antioxidants, anthocyanins, against MOS-induced death of cultured cerebellar granule neurons (CGNs). Callistephin and kuromanin are anthocyanins derived from strawberries and black rice, respectively, whose neuroprotective properties have yet to be examined in detail. Methods Glutathione (GSH)-sensitive MOS and intrinsic apoptosis were induced in CGNs by incubation with the Bcl-2 inhibitor, HA14-1. The effects of anthocyanin co-incubation on CGN survival were assessed. Results The anthocyanins demonstrated significant protection from MOS-induced apoptosis which was equivalent to that provided by the green tea polyphenol, epigallocatechin 3-gallate; however, neither anthocyanin was as effective as GSH at rescuing CGNs. Inhibition of Bcl-2 caused a significant reduction of mitochondrial GSH which was prevented by the anthocyanins. Furthermore, the anthocyanins inhibited iron-induced lipid peroxidation in rat brain homogenates and prevented cardiolipin oxidation induced by MOS in CGNs. MOS-induced mitochondrial fragmentation and proteolytic cleavage of the optic atrophy 1 (OPA1) fusion GTPase were also attenuated by the anthocyanins. Finally, the anthocyanins significantly enhanced GSH peroxidase activity in a cell-free assay. Discussion These data show that anthocyanins suppress MOS-induced apoptosis by preserving mitochondrial GSH and inhibiting cardiolipin oxidation and mitochondrial fragmentation. These nutraceutical antioxidants warrant further study as potential therapeutic agents for neurodegenerative diseases caused by MOS.

C-terminal binding proteins: central players in development and disease.

Abstract C-terminal binding proteins (CtBPs) were initially identified as binding partners for the E1A-transforming proteins. Although the invertebrate genome encodes one CtBP protein, two CtBPs (CtBP1 and CtBP2) are encoded by the vertebrate genome and perform both unique and duplicative functions. CtBP1 and CtBP2 are closely related and act as transcriptional corepressors when activated by nicotinamide adenine dinucleotide binding to their dehydrogenase domains. CtBPs exert transcriptional repression primarily via recruitment of a corepressor complex to DNA that consists of histone deacetylases (HDACs) and histone methyltransferases, although CtBPs can also repress transcription through HDAC-independent mechanisms. More recent studies have demonstrated a critical function for CtBPs in the transcriptional repression of pro-apoptotic genes such as Bax, Puma, Bik, and Noxa. Nonetheless, although recent efforts have characterized the essential involvement of CtBPs in promoting cellular survival, the dysregulation of CtBPs in both neurodegenerative disease and cancers remains to be fully elucidated.

Neuroprotection comparison of chlorogenic acid and its metabolites against mechanistically distinct cell death-inducing agents in cultured cerebellar granule neurons

While the number of patients diagnosed with neurodegenerative disorders like Alzheimers disease, amyotrophic lateral sclerosis, and Parkinsons disease is increasing, there are currently no effective treatments that significantly limit the neuronal cell death underlying these diseases. Chlorogenic acid (CGA), a polyphenolic compound found in high concentration in coffee, is known to possess antioxidant and free radical scavenging activity. In this study, we investigated the neuroprotective effects of CGA and its major metabolites in primary cultures of rat cerebellar granule neurons. We show that CGA and caffeic acid displayed a dramatic protective effect against the nitric oxide donor, sodium nitroprusside. In marked contrast, ferulic acid and quinic acid had no protective effect against this nitrosative stress. While CGA and quinic acid had no protective effect against glutamate-induced cell death, caffeic acid and ferulic acid significantly protected neurons from excitotoxicity. Finally, caffeic acid was the only compound to display significant protective activity against hydrogen peroxide, proteasome inhibition, caspase-dependent intrinsic apoptosis, and endoplasmic reticulum stress. These results indicate that caffeic acid displays a much broader profile of neuroprotection against a diverse range of stressors than its parent polyphenol, CGA, or the other major metabolites, ferulic acid and quinic acid. We conclude that caffeic acid is a promising candidate for testing in pre-clinical models of neurodegeneration.

Immunocal® and preservation of glutathione as a novel neuroprotective strategy for degenerative disorders of the nervous system.

Oxidative stress and glutathione (GSH) depletion are both recognized as significant contributors to the pathogenesis of many devastating neurodegenerative diseases. In particular, mitochondrial dysfunction leads to the aberrant production and accumulation of reactive oxygen species (ROS), which are capable of oxidizing key cellular proteins, lipids, and DNA, ultimately triggering cell death. In addition to other roles that it plays in the cell, GSH functions as a critical scavenger of these ROS. Therefore, GSH depletion exacerbates cell damage due to free radical generation. Strategies that increase or preserve the levels of intracellular GSH have been shown to act in a neuroprotective manner, suggesting that augmentation of the available GSH pool may be a promising therapeutic target for neurodegeneration. This review discusses the capacity of a cystine-rich, whey protein supplement (Immunocal®) to enhance the de novo synthesis of GSH in neurons, and highlights its potential as a novel therapeutic approach to mitigate the oxidative damage that underlies the pathogenesis of various neurodegenerative diseases. Additionally, this review discusses various patents from 1993 to 2012 both with Immunocal® and other methods that modulate GSH in neurodegeneration.

A Cystine-Rich Whey Supplement (Immunocal ® ) Delays Disease Onset and Prevents Spinal Cord Glutathione Depletion in the hSOD1 G93A Mouse Model of Amyotrophic Lateral Sclerosis

Depletion of the endogenous antioxidant, glutathione (GSH), underlies progression of the devastating neurodegenerative disease, amyotrophic lateral sclerosis (ALS). Thus, strategies aimed at elevating GSH may yield new therapeutics for ALS. Here, we investigated the effects of a unique non-denatured whey protein supplement, Immunocal®, in the transgenic Gly position 93 to Ala (G93A) mutant hSOD1 (hSOD1G93A) mouse model of ALS. Immunocal® is rich in the GSH precursor, cystine, and is therefore capable of bolstering GSH content. Transgenic hSOD1G93A mice receiving Immunocal® displayed a significant delay in disease onset compared to untreated hSOD1G93A controls. Additionally, Immunocal® treatment significantly decreased the rate of decline in grip strength and prevented disease-associated reductions in whole blood and spinal cord tissue GSH levels in end-stage hSOD1G93A mice. However, Immunocal® did not extend survival, likely due to its inability to preserve the mitochondrial GSH pool in spinal cord. Combination treatment with Immunocal® and the anti-glutamatergic compound, riluzole, delayed disease onset and extended survival in hSOD1G93A mice. These findings demonstrate that sustaining tissue GSH with Immunocal® only modestly delays disease onset and slows the loss of skeletal muscle strength in hSOD1G93A mice. Moreover, the inability of Immunocal® to rescue mitochondrial GSH in spinal cord provides a possible mechanism for its lack of effect on survival and is a limiting factor in the potential utility of this supplement as a therapeutic for ALS.

Chemical basis for the disparate neuroprotective effects of the anthocyanins, callistephin and kuromanin, against nitrosative stress

ABSTRACT Oxidative and nitrosative stress are major factors in neuronal cell death underlying neurodegenerative disease. Thus, supplementation of antioxidant defenses may be an effective therapeutic strategy for diseases such as amyotrophic lateral sclerosis, Parkinsons disease, and Alzheimers disease. In this regard, treatment with nutraceutical antioxidants has garnered increasing attention; however, the differential neuroprotective effects of structurally similar nutraceuticals, which may affect their suitability as therapeutic agents, has not been directly examined. In this study we compare the ability of two anthocyanins, callistephin (pelargonidin‐3‐O‐glucoside) and kuromanin (cyanidin‐3‐O‐glucoside) to protect cerebellar granule neurons from damage induced by either oxidative or nitrosative stress. These anthocyanins differ by the presence of a single hydroxyl group on the B‐ring of kuromanin, forming a catechol moiety. While both compounds protected neurons from oxidative stress induced by glutamate excitotoxicity, a stark contrast was observed under conditions of nitrosative stress. Only kuromanin displayed the capacity to defend neurons from nitric oxide (NO)‐induced apoptosis. This protective effect was blocked by addition of Cu, Zn‐superoxide dismutase, indicating that the neuroprotective mechanism is superoxide dependent. Based on these observations, we suggest a unique mechanism by which slight structural variances, specifically the absence or presence of a catechol moiety, lend kuromanin the unique ability to generate superoxide, which acts as a scavenger of NO. These findings indicate that kuromanin and compounds that share similar chemical characteristics may be more effective therapeutic agents for treating neurodegenerative diseases than callistephin and related (non‐catechol) compounds. HighlightsAnthocyanins are polyphenolic compounds with neuroprotective properties.Distinct anthocyanins display opposing neuroprotective effects against nitric oxide.Protection from nitric oxide relies on catechol‐dependent superoxide generation.Catecholicanthocyanins may be effective therapeutic agents for neurodegeneration.

Comparison of the Neuroprotective and Anti-Inflammatory Effects of the Anthocyanin Metabolites, Protocatechuic Acid and 4-Hydroxybenzoic Acid

Anthocyanins are being increasingly investigated for their neuroprotective and antineuroinflammatory effects; however, the overall bioavailability of many anthocyanins is relatively low. In contrast, phenolic acids, metabolites of many polyphenols, including anthocyanins, have been shown to accumulate in tissue at higher concentrations than those of parent compounds, suggesting that these metabolites may be the bioactive components of anthocyanin-rich diets. We examined the neuroprotective capacity of two common phenolic acids, 4-hydroxybenzoic acid (HBA) and protocatechuic acid (PCA), in primary cultures of cerebellar granule neurons. Both HBA and PCA are capable of mitigating oxidative stress induced by hydrogen peroxide, which is thought to contribute to neuronal cell death in neurodegeneration. Under conditions of nitrosative stress, which occur during inflammation in the central nervous system, only PCA was neuroprotective, despite similar structural characteristics between HBA and PCA. Intriguingly, this trend was reversed under conditions of excitotoxicity, in which only HBA was neuroprotective. Lastly, we explored the anti-inflammatory activity of these compounds in microglial cells stimulated with lipopolysaccharide. PCA was an effective anti-inflammatory agent, reducing nitric oxide production, while HBA had no effect. These data indicate that phenolic acids possess distinct neuroprotective and anti-inflammatory characteristics that could make them suitable for the treatment of neurodegeneration.

An anthocyanin-enriched extract from strawberries delays disease onset and extends survival in the hSOD1G93A mouse model of amyotrophic lateral sclerosis

Objective: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting from the death of motor neurons in the brain, brain stem, and spinal cord. Several processes such as oxidative stress, neuroinflammation, and neuronal apoptosis, contribute to disease progression. Anthocyanins are flavonoid compounds derived from fruits and vegetables that possess antioxidant, anti-inflammatory, and anti-apoptotic abilities. Thus, these unique compounds may provide therapeutic benefit for the treatment of ALS. Methods: We used the G93A mutant human SOD1 (hSOD1G93A) mouse model of ALS to assess the effects of an anthocyanin-enriched extract from strawberries (SAE) on disease onset and progression. Mice were administered SAE orally beginning at 60 days of age until end-stage such that mice received 2 mg/kg/day of the extracts primary anthocyanin constituent. Clinical indices of disease were assessed until mice were sacrificed at end-stage. Histopathological indices of disease progression were also evaluated at 105 days of age. Results: hSOD1G93A mice supplemented with SAE experienced a marked (∼17 day) delay in disease onset and a statistically significant (∼11 day) extension in survival in comparison to their untreated mutant counterparts. Additionally, SAE-treated hSOD1G93A mice displayed significantly preserved grip strength throughout disease progression. Histopathological analysis demonstrated that SAE supplementation significantly reduced astrogliosis in spinal cord, and preserved neuromuscular junctions (NMJs) in gastrocnemius muscle. Discussion: These data are the first to demonstrate that anthocyanins have significant potential as therapeutic agents in a preclinical model of ALS due to their ability to reduce astrogliosis in spinal cord and preserve NMJ integrity and muscle function. Therefore, further study of these compounds is warranted in additional preclinical models of ALS and other neurodegenerative diseases.

Procyanidin B2 Protects Neurons from Oxidative, Nitrosative, and Excitotoxic Stress

The aberrant generation of oxygen and nitrogen free radicals can cause severe damage to key cellular components, resulting in cell apoptosis. Similarly, excitotoxicity leads to protease activation and mitochondrial dysfunction, which subsequently causes cell death. Each of these factors play critical roles in the neuronal cell death underlying various neurodegenerative diseases. Procyanidin B2 (PB2) is a naturally occurring polyphenolic compound found in high concentrations in cocoa, apples, and grapes. Here, we examine the neuroprotective effects of PB2 in primary cultures of rat cerebellar granule neurons (CGNs) exposed to various stressors. CGNs were pre-incubated with PB2 and then neuronal stress was induced as described below. Mitochondrial oxidative stress was triggered with HA14-1, an inhibitor of the pro-survival Bcl-2 protein which induces glutathione-sensitive apoptosis. Glutamate and glycine were used to induce excitotoxicity. Sodium nitroprusside, a nitric oxide generating compound, was used to induce nitrosative stress. We observed significant dose-dependent protection of CGNs with PB2 for all of the above insults, with the greatest neuroprotective effect being observed under conditions of nitrosative stress. Intriguingly, the neuroprotective effect of PB2 against nitric oxide was superoxide-dependent, as we have recently shown for other catechol antioxidants. Finally, we induced neuronal stress through the removal of depolarizing extracellular potassium and serum (5K conditions), which is a classical model of intrinsic apoptosis in CGNs. PB2 did not display any significant protection against 5K-induced apoptosis at any concentration tested. We conclude that PB2 offers neuronal protection principally as an antioxidant by scavenging reactive oxygen and nitrogen species instead of through modulation of pro-survival cell signaling pathways. These findings suggest that PB2 may be an effective neuroprotective agent for the treatment of neurodegenerative disorders.

The cysteine-rich whey protein supplement, Immunocal®, preserves brain glutathione and improves cognitive, motor, and histopathological indices of traumatic brain injury in a mouse model of controlled cortical impact

Abstract Traumatic brain injury (TBI) is a major public health problem estimated to affect nearly 1.7 million people in the United States annually. Due to the often debilitating effects of TBI, novel preventative agents are highly desirable for at risk populations. Here, we tested a whey protein supplement, Immunocal®, for its potential to enhance resilience to TBI. Immunocal® is a non‐denatured whey protein preparation which has been shown to act as a cysteine delivery system to increase levels of the essential antioxidant glutathione (GSH). Twice daily oral supplementation of CD1 mice with Immunocal® for 28 days prior to receiving a moderate TBI prevented an ˜ 25% reduction in brain GSH/GSSG observed in untreated TBI mice. Immunocal® had no significant effect on the primary mechanical injury induced by TBI, as assessed by MRI, changes in Tau phosphorylation, and righting reflex time or apnea. However, pre‐injury supplementation with Immunocal® resulted in statistically significant improvements in motor function (beam walk and rotarod) and cognitive function (Barnes maze). We also observed a significant preservation of corpus callosum width (axonal myelination), a significant decrease in degenerating neurons, a reduction in Iba1 (microglial marker), decreased lipid peroxidation, and preservation of brain‐derived neurotrophic factor (BDNF) in the brains of Immunocal®‐pretreated mice compared to untreated TBI mice. Taken together, these data indicate that pre‐injury supplementation with Immunocal® significantly enhances the resilience to TBI induced by a moderate closed head injury in mice. We conclude that Immunocal® may hold significant promise as a preventative agent for TBI, particularly in certain high risk populations such as athletes and military personnel. Graphical abstract Figure. No Caption available. HighlightsImmunocal® significantly enhanced resilience to TBI induced by closed head injury in mice.Immunocal® preserved brain GSH/GSSG and attenuated demyelination and neuronal degeneration.Most significantly, Immunocal® improved motor and cognitive deficits induced by TBI.Immunocal® holds significant promise as a preventative agent for TBI.