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.
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•Immunocal® 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.
Mitochondrial oxidative stress and dysfunction are strongly implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Glutathione (GSH) is an endogenous antioxidant that exists as ...distinct cytosolic and mitochondrial pools. The status of the mitochondrial GSH pool is reliant on transport from the cytosol through the 2-oxoglutarate carrier (OGC), an inner membrane anion carrier. We have previously reported that the outer mitochondrial membrane protein, Bcl-2, directly binds GSH and is a key regulator of OGC-dependent mitochondrial GSH transport. Here, we show that G93A mutant SOD1 (Cu, Zn-superoxide dismutase) reduces the binding of GSH to Bcl-2 and disrupts mitochondrial GSH uptake in vitro. In the G93A mutant hSOD1 mouse model of ALS, mitochondrial GSH is significantly depleted in spinal cord of end-stage mice. Finally, we show that OGC is heavily S-nitrosylated in the spinal cord of end-stage mice and consequently, the GSH uptake capacity of spinal cord mitochondria isolated from these mutant mice is significantly diminished. Collectively, these findings suggest that spinal cord GSH depletion, particularly at the level of the mitochondria, plays a significant role in ALS pathogenesis induced by mutant SOD1. Furthermore, the depletion of mitochondrial GSH in the G93A mutant hSOD1 mouse model may be caused by the S-nitrosylation of OGC and the capacity of mutant SOD1 to disrupt the Bcl-2/GSH interaction, resulting in a disruption of mitochondrial GSH transport.
•Patients with a history of mTBI have significant and prolonged antioxidant biomarker depletion.•Sex-specific differences exist in antioxidant biomarker depletion due to history of mTBI.•The data ...support the use of antioxidant supplementation (e.g., PUFAs) following mTBI.
Individuals with a history of mild traumatic brain injury (mTBI) are at an increased risk for neurodegenerative disease, suggesting that intrinsic neuroprotective mechanisms, such as the endogenous antioxidant reservoir, may be depleted long-term after mTBI. Here, we retrospectively analyzed symptoms and blood antioxidants in patients with a history of mTBI who presented to Resilience Code, a sports medicine clinic in Colorado. Significant decreases in alpha-tocopherol, selenium, linoleic acid, taurine, docosahexaenoic acid, and total omega-3 were measured in the total mTBI population versus controls. Male mTBI patients showed depletion of a larger array of antioxidants than females. Patients with a history of mTBI also reported significantly worsened emotional, energy, head, and cognitive symptoms, with males displaying more extensive symptomology. Multiple or chronic mTBI patients had worsened symptoms than single or acute/subchronic mTBI patients, respectively. Finally, male mTBI patients with the largest reductions in polyunsaturated fatty acids (PUFAs) displayed worse symptomology than male mTBI patients with less depletion of this antioxidant reservoir. These results demonstrate that antioxidant depletion persists in patients with a history of mTBI and these deficits are sex-specific and associated with worsened symptomology. Furthermore, supplementation with specific antioxidants, like PUFAs, may diminish symptom severity in patients suffering from chronic effects of mTBI.
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Concussion or mild traumatic brain injury (mTBI) in athletes can cause persistent symptoms, known as post-concussion syndrome (PCS), and repeated injuries may increase the long-term risk for an ...athlete to develop neurodegenerative diseases such as chronic traumatic encephalopathy (CTE), and Alzheimer's disease (AD). The Center for Disease Control estimates that up to 3.8 million sport-related mTBI are reported each year in the United States. Despite the magnitude of the phenomenon, there is a current lack of comprehensive prognostic indicators and research has shown that available monitoring tools are moderately sensitive to short-term concussion effects but less sensitive to long-term consequences. The overall aim of this review is to discuss novel, quantitative, and objective measurements that can predict long-term outcomes following repeated sports-related mTBIs. The specific objectives were (1) to provide an overview of the current clinical and biomechanical tools available to health practitioners to ensure recovery after mTBIs, (2) to synthesize potential biological mechanisms in animal models underlying the long-term adverse consequences of mTBIs, (3) to discuss the possible link between repeated mTBI and neurodegenerative diseases, and (4) to discuss the current knowledge about fluid biomarkers for mTBIs with a focus on novel exosomal biomarkers. The conclusions from this review are that current post-concussion clinical tests are not sufficiently sensitive to injury and do not accurately quantify post-concussion alterations associated with repeated mTBIs. In the current review, it is proposed that current practices should be amended to include a repeated symptom inventory, a cognitive assessment of executive function and impulse control, an instrumented assessment of balance, vestibulo-ocular assessments, and an improved panel of blood or exosome biomarkers.
Amyloid-β (Aβ) is a principal cleavage product of amyloid-β protein precursor (AβPP) and is widely recognized as a key pathogenic player in Alzheimer's disease (AD). Yet, there is increasing evidence ...of a neurotoxic role for the AβPP intracellular domain (AICD) which has been proposed to occur through its nuclear function. Intriguingly, there is a γ-secretase resident at the mitochondria which could produce AICD locally.
We examined the potential of AICD to induce neuronal apoptosis when targeted specifically to the mitochondria and compared its mechanism of neurotoxicity to that of Aβ.
We utilized transient transfection of HT22 neuronal cells with bicistronic plasmids coding for DsRed and either empty vector (Ires), Aβ, AICD59, or mitochondrial-targeted AICD (mitoAICD) in combination with various inhibitors of pathways involved in apoptosis.
AICD induced significant neuronal apoptosis only when targeted to the mitochondria. Apoptosis required functional mitochondria as neither Aβ nor mitoAICD induced significant toxicity in cells devoid of mitochondrial DNA. Both glutathione and a Bax inhibitor protected HT22 cells from either peptide. However, inhibition of the mitochondrial permeability transition pore only protected from Aβ, while pan-caspase inhibitors uniquely rescued cells from mitoAICD.
Our results show that AICD displays a novel neurotoxic function when targeted to mitochondria. Moreover, mitoAICD induces apoptosis via a mechanism that is distinct from that of Aβ. These findings suggest that AICD produced locally at mitochondria via organelle-specific γ-secretase could act in a synergistic manner with Aβ to cause mitochondrial dysfunction and neuronal death in AD.
Mitochondrial oxidative stress significantly contributes to the underlying pathology of several devastating neurodegenerative disorders. Mitochondria are highly sensitive to the damaging effects of ...reactive oxygen and nitrogen species; therefore, these organelles are equipped with a number of free radical scavenging systems. In particular, the mitochondrial glutathione (GSH) pool is a critical antioxidant reserve that is derived entirely from the larger cytosolic pool via facilitated transport. The mechanism of mitochondrial GSH transport has not been extensively studied in the brain. However, the dicarboxylate (DIC) and 2-oxoglutarate (OGC) carriers localized to the inner mitochondrial membrane have been established as GSH transporters in liver and kidney. Here, we investigated the role of these carriers in protecting neurons from oxidative and nitrosative stress. Immunoblot analysis of DIC and OGC in primary cultures of rat cerebellar granule neurons (CGNs) and cerebellar astrocytes showed differential expression of these carriers, with CGNs expressing only DIC and astrocytes expressing both DIC and OGC. Consistent with these findings, butylmalonate specifically reduced mitochondrial GSH in CGNs, whereas both butylmalonate and phenylsuccinate diminished mitochondrial GSH in astrocytes. Moreover, preincubation with butylmalonate but not phenylsuccinate significantly enhanced susceptibility of CGNs to oxidative and nitrosative stressors. This increased vulnerability was largely prevented by incubation with cell-permeable GSH monoethylester but not malate. Finally, knockdown of DIC with adenoviral siRNA also rendered CGNs more susceptible to oxidative stress. These findings demonstrate that maintenance of the mitochondrial GSH pool via sustained mitochondrial GSH transport is essential to protect neurons from oxidative and nitrosative stress.
Background: Mitochondrial glutathione transport has not been extensively studied within the CNS.
Results: Cerebellar neurons and astrocytes use distinct mechanisms of mitochondrial glutathione transport.
Conclusion: Inhibition of a single mitochondrial glutathione transporter renders neurons more susceptible to oxidative and nitrosative stress.
Significance: Mitochondrial glutathione transport is essential to protect neurons from oxidative and nitrosative stress conditions common to neurodegenerative diseases.
Neuroinflammation occurs in Alzheimer's disease (AD). While AD genetic studies implicate inflammation-relevant genes and fibrillar amyloid-β protein promotes inflammation, our understanding of AD ...neuroinflammation nevertheless remains incomplete. In this study we hypothesized damage-associated molecular pattern (DAMP) molecules arising from mitochondria, intracellular organelles that resemble bacteria, could contribute to AD neuroinflammation. To preliminarily test this possibility, we exposed neuronal and microglial cell lines to enriched mitochondrial lysates. BV2 microglial cells treated with mitochondrial lysates showed decreased TREM2 mRNA, increased TNFα mRNA, increased MMP-8 mRNA, increased IL-8 mRNA, redistribution of NFκB to the nucleus, and increased p38 MAPK phosphorylation. SH-SY5Y neuronal cells treated with mitochondrial lysates showed increased TNFα mRNA, increased NFκB protein, decreased IκBα protein, increased AβPP mRNA, and increased AβPP protein. Enriched mitochondrial lysates from SH-SY5Y cells lacking detectable mitochondrial DNA (ρ0 cells) failed to induce any of these changes, while mtDNA obtained directly from mitochondria (but not PCR-amplified mtDNA) increased BV2 cell TNFα mRNA. These results indicate at least one mitochondrial-derived DAMP molecule, mtDNA, can induce inflammatory changes in microglial and neuronal cell lines. Our data are consistent with the hypothesis that a mitochondrial-derived DAMP molecule or molecules could contribute to AD neuroinflammation.
Bcl-2 protects cells against mitochondrial oxidative stress and subsequent apoptosis. However, the mechanism underlying the antioxidant function of Bcl-2 is currently unknown. Recently, Bax and ...several Bcl-2 homology-3 domain (BH3)-only proteins (Bid, Puma, and Noxa) have been shown to induce a pro-oxidant state at mitochondria (1-4). Given the opposing effects of Bcl-2 and Bax/BH3-only proteins on the redox state of mitochondria, we hypothesized that the antioxidant function of Bcl-2 is antagonized by its interaction with the BH3 domains of pro-apoptotic family members. Here, we show that BH3 mimetics that bind to a hydrophobic surface (the BH3 groove) of Bcl-2 induce GSH-sensitive mitochondrial dysfunction and apoptosis in cerebellar granule neurons. BH3 mimetics displace a discrete mitochondrial GSH pool in neurons and suppress GSH transport into isolated rat brain mitochondria. Moreover, BH3 mimetics and the BH3-only protein, Bim, inhibit a novel interaction between Bcl-2 and GSH in vitro. These results suggest that Bcl-2 regulates an essential pool of mitochondrial GSH and that this regulation may depend upon Bcl-2 directly interacting with GSH via the BH3 groove. We conclude that this novel GSH binding property of Bcl-2 likely plays a central role in its antioxidant function at mitochondria.
Amyotrophic lateral sclerosis (ALS) is a devastating disorder characterized by motor neuron apoptosis and subsequent skeletal muscle atrophy caused by oxidative and nitrosative stress, mitochondrial ...dysfunction, and neuroinflammation. Anthocyanins are polyphenolic compounds found in berries that possess neuroprotective and anti-inflammatory properties. Protocatechuic acid (PCA) is a phenolic acid metabolite of the parent anthocyanin, kuromanin, found in blackberries and bilberries. We explored the therapeutic effects of PCA in a transgenic mouse model of ALS that expresses mutant human Cu, Zn-superoxide dismutase 1 with a glycine to alanine substitution at position 93. These mice display skeletal muscle atrophy, hindlimb weakness, and weight loss. Disease onset occurs at approximately 90 days old and end stage is reached at approximately 120 days old. Daily treatment with PCA (100 mg/kg) by oral gavage beginning at disease onset significantly extended survival (121 days old in untreated vs. 133 days old in PCA-treated) and preserved skeletal muscle strength and endurance as assessed by grip strength testing and rotarod performance. Furthermore, PCA reduced astrogliosis and microgliosis in spinal cord, protected spinal motor neurons from apoptosis, and maintained neuromuscular junction integrity in transgenic mice. PCA lengthens survival, lessens the severity of pathological symptoms, and slows disease progression in this mouse model of ALS. Given its significant preclinical therapeutic effects, PCA should be further investigated as a treatment option for patients with ALS.
Mitochondrial glutathione (GSH) is a key endogenous antioxidant and its maintenance is critical for cell survival. Here, we generated stable NSC34 motor neuron‐like cell lines over‐expressing the ...mitochondrial GSH transporter, the 2‐oxoglutarate carrier (OGC), to further elucidate the importance of mitochondrial GSH transport in determining neuronal resistance to oxidative stress. Two stable OGC cell lines displayed specific increases in mitochondrial GSH content and resistance to oxidative and nitrosative stressors, but not staurosporine. Inhibition of transport through OGC reduced levels of mitochondrial GSH and resensitized the stable cell lines to oxidative stress. The stable OGC cell lines displayed significant up‐regulation of the anti‐apoptotic protein, B cell lymphoma 2 (Bcl‐2). This result was reproduced in parental NSC34 cells by chronic treatment with GSH monoethylester, which specifically increased mitochondrial GSH levels. Knockdown of Bcl‐2 expression decreased mitochondrial GSH and resensitized the stable OGC cells to oxidative stress. Finally, endogenous OGC was co‐immunoprecipitated with Bcl‐2 from rat brain lysates in a GSH‐dependent manner. These data are the first to show that increased mitochondrial GSH transport is sufficient to enhance neuronal resistance to oxidative stress. Moreover, sustained and specific enhancement of mitochondrial GSH leads to increased Bcl‐2 expression, a required mechanism for the maintenance of increased mitochondrial GSH levels.
Stable over‐expression of the 2‐oxoglutarate carrier (OGC) in a motor neuronal cell line induced a specific increase in mitochondrial GSH and markedly enhanced resistance to oxidative stress. Over‐expression of OGC also induced Bcl‐2 expression which was owing to the specific increase in mitochondrial GSH. Intriguingly, enhanced expression of Bcl‐2 was required to sustain OGC‐dependent GSH transport into the mitochondria. Thus, OGC and Bcl‐2 work in a concerted manner to maintain the mitochondrial GSH pool which is crucial for neuronal survival.
Stable over‐expression of the 2‐oxoglutarate carrier (OGC) in a motor neuronal cell line induced a specific increase in mitochondrial GSH and markedly enhanced resistance to oxidative stress. Over‐expression of OGC also induced Bcl‐2 expression which was owing to the specific increase in mitochondrial GSH. Intriguingly, enhanced expression of Bcl‐2 was required to sustain OGC‐dependent GSH transport into the mitochondria. Thus, OGC and Bcl‐2 work in a concerted manner to maintain the mitochondrial GSH pool which is crucial for neuronal survival.
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