Drp1/Fis1-Dependent Pathologic Fission and Associated Damaged Extracellular Mitochondria Contribute to Macrophage Dysfunction in Endotoxin Tolerance.

OBJECTIVES: Recent publications have shown that mitochondrial dynamics can govern the quality and quantity of extracellular mitochondria subsequently impacting immune phenotypes. This study aims to determine if pathologic mitochondrial fission mediated by Drp1/Fis1 interaction impacts extracellular mitochondrial content and macrophage function in sepsis-induced immunoparalysis. DESIGN: Laboratory investigation. SETTING: University laboratory. SUBJECTS: C57BL/6 and BALB/C mice. INTERVENTIONS: Using in vitro and murine models of endotoxin tolerance (ET), we evaluated changes in Drp1/Fis1-dependent pathologic fission and simultaneously measured the quantity and quality of extracellular mitochondria. Next, by priming mouse macrophages with isolated healthy mitochondria (MC) and damaged mitochondria, we determined if damaged extracellular mitochondria are capable of inducing tolerance to subsequent endotoxin challenge. Finally, we determined if inhibition of Drp1/Fis1-mediated pathologic fission abrogates release of damaged extracellular mitochondria and improves macrophage response to subsequent endotoxin challenge. MEASUREMENTS AND MAIN RESULTS: When compared with naïve macrophages (NMs), endotoxin-tolerant macrophages (ETM) demonstrated Drp1/Fis1-dependent mitochondrial dysfunction and higher levels of damaged extracellular mitochondria (Mitotracker-Green + events/50 µL: ETM = 2.42 × 106 ± 4,391 vs NM = 5.69 × 105 ± 2,478; p < 0.001). Exposure of NMs to damaged extracellular mitochondria (MH) induced cross-tolerance to subsequent endotoxin challenge, whereas MC had minimal effect (tumor necrosis factor [TNF]-α [pg/mL]: NM = 668 ± 3, NM + MH = 221 ± 15, and NM + Mc = 881 ± 15; p < 0.0001). Inhibiting Drp1/Fis1-dependent mitochondrial fission using heptapeptide (P110), a selective inhibitor of Drp1/Fis1 interaction, improved extracellular mitochondrial function (extracellular mitochondrial membrane potential, JC-1 [R/G] ETM = 7 ± 0.5 vs ETM + P110 = 19 ± 2.0; p < 0.001) and subsequently improved immune response in ETMs (TNF-α [pg/mL]; ETM = 149 ± 1 vs ETM + P110 = 1,150 ± 4; p < 0.0001). Similarly, P110-treated endotoxin tolerant mice had lower amounts of damaged extracellular mitochondria in plasma (represented by higher extracellular mitochondrial membrane potential, TMRM/MT-G: endotoxin tolerant [ET] = 0.04 ± 0.02 vs ET + P110 = 0.21 ± 0.02; p = 0.03) and improved immune response to subsequent endotoxin treatment as well as cecal ligation and puncture. CONCLUSIONS: Inhibition of Drp1/Fis1-dependent mitochondrial fragmentation improved macrophage function and immune response in both in vitro and in vivo models of ET. This benefit is mediated, at least in part, by decreasing the release of damaged extracellular mitochondria, which contributes to endotoxin cross-tolerance. Altogether, these data suggest that alterations in mitochondrial dynamics may play an important role in sepsis-induced immunoparalysis.

δPKC-Mediated DRP1 Phosphorylation Impacts Macrophage Mitochondrial Function and Inflammatory Response to Endotoxin.

BACKGROUND: Recent studies have demonstrated that alterations in mitochondrial dynamics can impact innate immune function. However, the upstream mechanisms that link mitochondrial dynamics to innate immune phenotypes have not been completely elucidated. This study asks if Protein Kinase C, subunit delta (δPKC)-mediated phosphorylation of dynamin-related protein 1 (Drp1), a key driver of mitochondrial fission, impacts macrophage pro-inflammatory response following bacterial-derived lipopolysaccharide (LPS) stimulation. METHODS: Using RAW 264.7 cells, bone marrow-derived macrophages from C57BL/6J mice, as well as human monocyte-derived macrophages, we first characterized changes in δPKC-mediated phosphorylation of Drp1 following LPS stimulation. Next, using rationally designed peptides that inhibit δPKC activation (δV1-1) and δPKC-Drp1 interaction (ψDrp1), we determined whether δPKC-mediated phosphorylation of Drp1 impacts LPS-induced changes in mitochondrial morphology, mitochondrial function, and inflammatory response. RESULTS: Our results demonstrated that δPKC-dependent Drp1 activation is associated with increased mitochondrial fission, impaired cellular respiration, and increased mitochondrial reactive oxygen species in LPS-treated macrophages. This is reversed using a rationally designed peptide that selectively inhibits δPKC phosphorylation of Drp1 (ψDrp1). Interestingly, limiting excessive mitochondrial fission using ψDrp1 reduced LPS-triggered pro-inflammatory response, including a decrease in NF-κB nuclear localization, decreased iNOS induction, and a reduction in pro-inflammatory cytokines (IL-1ß, TNFα, IL-6). CONCLUSION: These data suggest that inhibiting Drp1 phosphorylation by δPKC abates the excessive mitochondrial fragmentation and mitochondrial dysfunction that is seen following LPS treatment. Furthermore, these data suggest that limiting δPKC-dependent Drp1 activation decreases the pro-inflammatory response following LPS treatment. Altogether, δPKC-dependent Drp1 phosphorylation might be an upstream mechanistic link between alterations in mitochondrial dynamics and innate immune phenotypes, and may have therapeutic potential.

Restoring metabolism of myeloid cells reverses cognitive decline in ageing.

Ageing is characterized by the development of persistent pro-inflammatory responses that contribute to atherosclerosis, metabolic syndrome, cancer and frailty1-3. The ageing brain is also vulnerable to inflammation, as demonstrated by the high prevalence of age-associated cognitive decline and Alzheimer's disease4-6. Systemically, circulating pro-inflammatory factors can promote cognitive decline7,8, and in the brain, microglia lose the ability to clear misfolded proteins that are associated with neurodegeneration9,10. However, the underlying mechanisms that initiate and sustain maladaptive inflammation with ageing are not well defined. Here we show that in ageing mice myeloid cell bioenergetics are suppressed in response to increased signalling by the lipid messenger prostaglandin E2 (PGE2), a major modulator of inflammation11. In ageing macrophages and microglia, PGE2 signalling through its EP2 receptor promotes the sequestration of glucose into glycogen, reducing glucose flux and mitochondrial respiration. This energy-deficient state, which drives maladaptive pro-inflammatory responses, is further augmented by a dependence of aged myeloid cells on glucose as a principal fuel source. In aged mice, inhibition of myeloid EP2 signalling rejuvenates cellular bioenergetics, systemic and brain inflammatory states, hippocampal synaptic plasticity and spatial memory. Moreover, blockade of peripheral myeloid EP2 signalling is sufficient to restore cognition in aged mice. Our study suggests that cognitive ageing is not a static or irrevocable condition but can be reversed by reprogramming myeloid glucose metabolism to restore youthful immune functions.

Novel and prevalent non-East Asian ALDH2 variants; Implications for global susceptibility to aldehydes' toxicity.

BACKGROUND: Aldehyde dehydrogenase 2 (ALDH2) catalyzes the detoxification of aliphatic aldehydes, including acetaldehyde. About 45% of Han Chinese (East Asians), accounting for 8% of humans, carry a single point mutation in ALDH2*2 (E504K) that leads to accumulation of toxic reactive aldehydes. METHODS: Sequencing of a small Mexican cohort and a search in the ExAC genomic database for additional ALDH2 variants common in various ethnic groups was set to identify missense variants. These were evaluated in vitro, and in cultured cells expressing these new and common variants. FINDINGS: In a cohort of Hispanic donors, we identified 2 novel mutations in ALDH2. Using the ExAC genomic database, we found these identified variants and at least three other ALDH2 variants with a single point mutation among Latino, African, South Asian, and Finnish ethnic groups, at a frequency of >5/1000. Although located in different parts of the ALDH2 molecule, these common ALDH2 mutants exhibited a significant reduction in activity compared with the wild type enzyme in vitro and in 3T3 cells overexpressing each of the variants, and a greater ethanol-induced toxicity. As Alda-1, previously identified activator, did not activate some of the new mutant ALDH2 enzymes, we continued the screen and identified Alda-64, which is effective in correcting the loss of activity in most of these new and common ALDH2 variants. INTERPRETATION: Since ~80% of the world population consumes ethanol and since acetaldehyde accumulation contributes to a variety of diseases, the identification of additional inactivating variants of ALDH2 in different ethnic groups may help develop new 'precision medicine' for carriers of these inactive ALDH2.

Mitochondrial dysfunction mediated through dynamin-related protein 1 (Drp1) propagates impairment in blood brain barrier in septic encephalopathy.

BACKGROUND: Out of the myriad of complications associated with septic shock, septic-associated encephalopathy (SAE) carries a significant risk of morbidity and mortality. Blood-brain-barrier (BBB) impairment, which subsequently leads to increased vascular permeability, has been associated with neuronal injury in sepsis. Thus, preventing BBB damage is an attractive therapeutic target. Mitochondrial dysfunction is an important contributor of sepsis-induced multi-organ system failure. More recently, mitochondrial dysfunction in endothelial cells has been implicated in mediating BBB failure in stroke, multiple sclerosis and in other neuroinflammatory disorders. Here, we focused on Drp1-mediated mitochondrial dysfunction in endothelial cells as a potential target to prevent BBB failure in sepsis. METHODS: We used lipopolysaccharide (LPS) to induce inflammation and BBB disruption in a cell culture as well as in murine model of sepsis. BBB disruption was assessed by measuring levels of key tight-junction proteins. Brain cytokines levels, oxidative stress markers, and activity of mitochondrial complexes were measured using biochemical assays. Astrocyte and microglial activation were measured using immunoblotting and qPCR. Transwell cultures of brain microvascular endothelial cells co-cultured with astrocytes were used to assess the effect of LPS on expression of tight-junction proteins, mitochondrial function, and permeability to fluorescein isothiocyanate (FITC) dextran. Finally, primary neuronal cultures exposed to LPS were assessed for mitochondrial dysfunction. RESULTS: LPS induced a strong brain inflammatory response and oxidative stress in mice which was associated with increased Drp1 activation and mitochondrial localization. Particularly, Drp1-(Fission 1) Fis1-mediated oxidative stress also led to an increase in expression of vascular permeability regulators in the septic mice. Similarly, mitochondrial defects mediated via Drp1-Fis1 interaction in primary microvascular endothelial cells were associated with increased BBB permeability and loss of tight-junctions after acute LPS injury. P110, an inhibitor of Drp1-Fis1 interaction, abrogated these defects, thus indicating a critical role for this interaction in mediating sepsis-induced brain dysfunction. Finally, LPS mediated a direct toxic effect on primary cortical neurons, which was abolished by P110 treatment. CONCLUSIONS: LPS-induced impairment of BBB appears to be dependent on Drp1-Fis1-mediated mitochondrial dysfunction. Inhibition of mitochondrial dysfunction with P110 may have potential therapeutic significance in septic encephalopathy.

Aldehyde dehydrogenase 2 activity and aldehydic load contribute to neuroinflammation and Alzheimer's disease related pathology.

Aldehyde dehydrogenase 2 deficiency (ALDH2*2) causes facial flushing in response to alcohol consumption in approximately 560 million East Asians. Recent meta-analysis demonstrated the potential link between ALDH2*2 mutation and Alzheimer's Disease (AD). Other studies have linked chronic alcohol consumption as a risk factor for AD. In the present study, we show that fibroblasts of an AD patient that also has an ALDH2*2 mutation or overexpression of ALDH2*2 in fibroblasts derived from AD patients harboring ApoE ε4 allele exhibited increased aldehydic load, oxidative stress, and increased mitochondrial dysfunction relative to healthy subjects and exposure to ethanol exacerbated these dysfunctions. In an in vivo model, daily exposure of WT mice to ethanol for 11 weeks resulted in mitochondrial dysfunction, oxidative stress and increased aldehyde levels in their brains and these pathologies were greater in ALDH2*2/*2 (homozygous) mice. Following chronic ethanol exposure, the levels of the AD-associated protein, amyloid-ß, and neuroinflammation were higher in the brains of the ALDH2*2/*2 mice relative to WT. Cultured primary cortical neurons of ALDH2*2/*2 mice showed increased sensitivity to ethanol and there was a greater activation of their primary astrocytes relative to the responses of neurons or astrocytes from the WT mice. Importantly, an activator of ALDH2 and ALDH2*2, Alda-1, blunted the ethanol-induced increases in Aß, and the neuroinflammation in vitro and in vivo. These data indicate that impairment in the metabolism of aldehydes, and specifically ethanol-derived acetaldehyde, is a contributor to AD associated pathology and highlights the likely risk of alcohol consumption in the general population and especially in East Asians that carry ALDH2*2 mutation.

Fragmented mitochondria released from microglia trigger A1 astrocytic response and propagate inflammatory neurodegeneration.

In neurodegenerative diseases, debris of dead neurons are thought to trigger glia-mediated neuroinflammation, thus increasing neuronal death. Here we show that the expression of neurotoxic proteins associated with these diseases in microglia alone is sufficient to directly trigger death of naive neurons and to propagate neuronal death through activation of naive astrocytes to the A1 state. Injury propagation is mediated, in great part, by the release of fragmented and dysfunctional microglial mitochondria into the neuronal milieu. The amount of damaged mitochondria released from microglia relative to functional mitochondria and the consequent neuronal injury are determined by Fis1-mediated mitochondrial fragmentation within the glial cells. The propagation of the inflammatory response and neuronal cell death by extracellular dysfunctional mitochondria suggests a potential new intervention for neurodegeneration-one that inhibits mitochondrial fragmentation in microglia, thus inhibiting the release of dysfunctional mitochondria into the extracellular milieu of the brain, without affecting the release of healthy neuroprotective mitochondria.

Proteasome-Dependent Regulation of Distinct Metabolic States During Long-Term Culture of Human iPSC-Derived Cardiomyocytes.

RATIONALE: The immature presentation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is currently a challenge for their application in disease modeling, drug screening, and regenerative medicine. Long-term culture is known to achieve partial maturation of iPSC-CMs. However, little is known about the molecular signaling circuitries that govern functional changes, metabolic output, and cellular homeostasis during long-term culture of iPSC-CMs. OBJECTIVE: We aimed to identify and characterize critical signaling events that control functional and metabolic transitions of cardiac cells during developmental progression, as recapitulated by long-term culture of iPSC-CMs. METHODS AND RESULTS: We combined transcriptomic sequencing with pathway network mapping in iPSC-CMs that were cultured until a late time point, day 200, in comparison to a medium time point, day 90, and an early time point, day 30. Transcriptomic landscapes of long-term cultured iPSC-CMs allowed mapping of distinct metabolic stages during development of maturing iPSC-CMs. Temporally divergent control of mitochondrial metabolism was found to be regulated by cAMP/PKA (protein kinase A)- and proteasome-dependent signaling events. The PKA/proteasome-dependent signaling cascade was mediated downstream by Hsp90 (heat shock protein 90), which in turn modulated mitochondrial respiratory chain proteins and their metabolic output. During long-term culture, this circuitry was found to initiate upregulation of iPSC-CM metabolism, resulting in increased cell contractility that reached a maximum at the day 200 time point. CONCLUSIONS: Our results reveal a PKA/proteasome- and Hsp90-dependent signaling pathway that regulates mitochondrial respiratory chain proteins and determines cardiomyocyte energy production and functional output. These findings provide deeper insight into signaling circuitries governing metabolic homeostasis in iPSC-CMs during developmental progression.

Drp1/Fis1 interaction mediates mitochondrial dysfunction in septic cardiomyopathy.

Mitochondrial dysfunction is a key contributor to septic cardiomyopathy. Although recent literature implicates dynamin related protein 1 (Drp1) and its mitochondrial adaptor fission 1 (Fis1) in the development of pathologic fission and mitochondrial failure in neurodegenerative disease, little is known about the role of Drp1/Fis1 interaction in the context of sepsis-induced cardiomyopathy. Our study tests the hypothesis that Drp1/Fis1 interaction is a major driver of sepsis-mediated pathologic fission, leading to mitochondrial dysfunction in the heart. METHODS: H9C2 cardiomyocytes were treated with lipopolysaccharide (LPS) to evaluate changes in mitochondrial membrane potential, oxidative stress, cellular respiration, and mitochondrial morphology. Balb/c mice were treated with LPS, cardiac function was measured by echocardiogaphy, and mitochondrial morphology determined by electron microscopy (EM). Drp1/Fis1 interaction was inhibited by P110 to determine whether limiting mitochondrial fission can reduce LPS-induced oxidative stress and cardiac dysfunction. RESULTS: LPS-treated H9C2 cardiomyocytes demonstrated a decrease in mitochondrial respiration followed by an increase in mitochondrial oxidative stress and a reduction in membrane potential. Inhibition of Drp1/Fis1 interaction with P110 attenuated LPS-mediated cellular oxidative stress and preserved membrane potential. In vivo, cardiac dysfunction in LPS-treated mice was associated with increased mitochondrial fragmentation. Treatment with P110 reduced cardiac mitochondrial fragmentation, prevented decline in cardiac function, and reduced mortality. CONCLUSIONS: Sepsis decreases cardiac mitochondrial respiration and membrane potential while increasing oxidative stress and inducing pathologic fission. Treatment with P110 was protective in both in vitro and in vivo models of septic cardiomyopathy, suggesting a key role of Drp1/Fis1 interaction, and a potential target to reduce its morbidity and mortality.

Macrophage de novo NAD+ synthesis specifies immune function in aging and inflammation.

Recent advances highlight a pivotal role for cellular metabolism in programming immune responses. Here, we demonstrate that cell-autonomous generation of nicotinamide adenine dinucleotide (NAD+) via the kynurenine pathway (KP) regulates macrophage immune function in aging and inflammation. Isotope tracer studies revealed that macrophage NAD+ derives substantially from KP metabolism of tryptophan. Genetic or pharmacological blockade of de novo NAD+ synthesis depleted NAD+, suppressed mitochondrial NAD+-dependent signaling and respiration, and impaired phagocytosis and resolution of inflammation. Innate immune challenge triggered upstream KP activation but paradoxically suppressed cell-autonomous NAD+ synthesis by limiting the conversion of downstream quinolinate to NAD+, a profile recapitulated in aging macrophages. Increasing de novo NAD+ generation in immune-challenged or aged macrophages restored oxidative phosphorylation and homeostatic immune responses. Thus, KP-derived NAD+ operates as a metabolic switch to specify macrophage effector responses. Breakdown of de novo NAD+ synthesis may underlie declining NAD+ levels and rising innate immune dysfunction in aging and age-associated diseases.

Mortal engines: Mitochondrial bioenergetics and dysfunction in neurodegenerative diseases.

Mitochondria are best known for their role in ATP generation. However, studies over the past two decades have shown that mitochondria do much more than that. Mitochondria regulate both necrotic and apoptotic cell death pathways, they store and therefore coordinate cellular Ca2+ signaling, they generate and metabolize important building blocks, by-products and signaling molecules, and they also generate and are targets of free radical species that modulate many aspects of cell physiology and pathology. Most estimates suggest that although the brain makes up only 2 percent of body weight, utilizes about 20 percent of the body's total ATP. Thus, mitochondrial dysfunction greatly impacts brain functions and is indeed associated with numerous neurodegenerative diseases. Furthermore, a number of abnormal disease-associated proteins have been shown to interact directly with mitochondria, leading to mitochondrial dysfunction and subsequent neuronal cell death. Here, we discuss the role of mitochondrial dynamics impairment in the pathological processes associated with neurodegeneration and suggest that a therapy targeting mitochondrialdysfunction holds a great promise.

Drp1/Fis1 interaction mediates mitochondrial dysfunction, bioenergetic failure and cognitive decline in Alzheimer's disease.

Mitochondrial dynamics, involving a balance between fusion and fission, regulates mitochondrial quality and number. Increasing evidence suggests that dysfunctional mitochondria play a role in Alzheimer's disease (AD). We observed that Drp1 interaction with one of the adaptors, Fis1, is significantly increased in Aß-treated neurons and AD patient-derived fibroblasts. P110, a seven-amino acid peptide, which specifically inhibits Drp1/Fis1 interaction without affecting the interaction of Drp1 with its other adaptors, attenuated Aß42-induced mitochondrial recruitment of Drp1 and prevented mitochondrial structural and functional dysfunction in cultured neurons, in cells expressing mutant amyloid precursor protein (KM670/671NL), and in five different AD patient-derived fibroblasts. Importantly, sustained P110 treatment significantly improved behavioral deficits, and reduced Aß accumulation, energetic failure and oxidative stress in the brain of the AD mouse model, 5XFAD. This suggests that Drp1/Fis1 interaction and excessive mitochondrial fission greatly contribute to Aß-mediated and AD-related neuropathology and cognitive decline. Therefore, inhibiting excessive Drp1/Fis1-mediated mitochondrial fission may benefit AD patients.

Inhibition of Drp1/Fis1 interaction slows progression of amyotrophic lateral sclerosis.

Bioenergetic failure and oxidative stress are common pathological hallmarks of amyotrophic lateral sclerosis (ALS), but whether these could be targeted effectively for novel therapeutic intervention needs to be determined. One of the reported contributors to ALS pathology is mitochondrial dysfunction associated with excessive mitochondrial fission and fragmentation, which is predominantly mediated by Drp1 hyperactivation. Here, we determined whether inhibition of excessive fission by inhibiting Drp1/Fis1 interaction affects disease progression. We observed mitochondrial excessive fragmentation and dysfunction in several familial forms of ALS patient-derived fibroblasts as well as in cultured motor neurons expressing SOD1 mutant. In both cell models, inhibition of Drp1/Fis1 interaction by a selective peptide inhibitor, P110, led to a significant reduction in reactive oxygen species levels, and to improvement in mitochondrial structure and functions. Sustained treatment of mice expressing G93A SOD1 mutation with P110, beginning at the onset of disease symptoms at day 90, produced an improvement in motor performance and survival, suggesting that Drp1 hyperactivation may be an attractive target in the treatment of ALS patients.

Potential biomarkers to follow the progression and treatment response of Huntington's disease.

Huntington's disease (HD) is a rare genetic disease caused by expanded polyglutamine repeats in the huntingtin protein resulting in selective neuronal loss. Although genetic testing readily identifies those who will be affected, current pharmacological treatments do not prevent or slow down disease progression. A major challenge is the slow clinical progression and the inability to biopsy the affected tissue, the brain, making it difficult to design short and effective proof of concept clinical trials to assess treatment benefit. In this study, we focus on identifying peripheral biomarkers that correlate with the progression of the disease and treatment benefit. We recently developed an inhibitor of pathological mitochondrial fragmentation, P110, to inhibit neurotoxicity in HD. Changes in levels of mitochondrial DNA (mtDNA) and inflammation markers in plasma, a product of DNA oxidation in urine, mutant huntingtin aggregates, and 4-hydroxynonenal adducts in muscle and skin tissues were all noted in HD R6/2 mice relative to wild-type mice. Importantly, P110 treatment effectively reduced the levels of these biomarkers. Finally, abnormal levels of mtDNA were also found in plasma of HD patients relative to control subjects. Therefore, we identified several potential peripheral biomarkers as candidates to assess HD progression and the benefit of intervention for future clinical trials.

The entangled ER-mitochondrial axis as a potential therapeutic strategy in neurodegeneration: A tangled duo unchained.

Endoplasmic reticulum (ER) and mitochondrial function have both been shown to be critical events in neurodegenerative diseases. The ER mediates protein folding, maturation, sorting as well acts as calcium storage. The unfolded protein response (UPR) is a stress response of the ER that is activated by the accumulation of misfolded proteins within the ER lumen. Although the molecular mechanisms underlying ER stress-induced apoptosis are not completely understood, increasing evidence suggests that ER and mitochondria cooperate to signal cell death. Similarly, calcium-mediated mitochondrial function and dynamics not only contribute to ATP generation and calcium buffering but are also a linchpin in mediating cell fate. Mitochondria and ER form structural and functional networks (mitochondria-associated ER membranes [MAMs]) essential to maintaining cellular homeostasis and determining cell fate under various pathophysiological conditions. Regulated Ca(2+) transfer from the ER to the mitochondria is important in maintaining control of pro-survival/pro-death pathways. In this review, we summarize the latest therapeutic strategies that target these essential organelles in the context of neurodegenerative diseases.

Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Protein-Protein Interaction Inhibitor Reveals a Non-catalytic Role for GAPDH Oligomerization in Cell Death.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important glycolytic enzyme, has a non-catalytic (thus a non-canonical) role in inducing mitochondrial elimination under oxidative stress. We recently demonstrated that phosphorylation of GAPDH by δ protein kinase C (δPKC) inhibits this GAPDH-dependent mitochondrial elimination. δPKC phosphorylation of GAPDH correlates with increased cell injury following oxidative stress, suggesting that inhibiting GAPDH phosphorylation should decrease cell injury. Using rational design, we identified pseudo-GAPDH (ψGAPDH) peptide, an inhibitor of δPKC-mediated GAPDH phosphorylation that does not inhibit the phosphorylation of other δPKC substrates. Unexpectedly, ψGAPDH decreased mitochondrial elimination and increased cardiac damage in an animal model of heart attack. Either treatment with ψGAPDH or direct phosphorylation of GAPDH by δPKC decreased GAPDH tetramerization, which corresponded to reduced GAPDH glycolytic activity in vitro and ex vivo Taken together, our study identified the potential mechanism by which oxidative stress inhibits the protective GAPDH-mediated elimination of damaged mitochondria. Our study also identified a pharmacological tool, ψGAPDH peptide, with interesting properties. ψGAPDH peptide is an inhibitor of the interaction between δPKC and GAPDH and of the resulting phosphorylation of GAPDH by δPKC. ψGAPDH peptide is also an inhibitor of GAPDH oligomerization and thus an inhibitor of GAPDH glycolytic activity. Finally, we found that ψGAPDH peptide is an inhibitor of the elimination of damaged mitochondria. We discuss how this unique property of increasing cell damage following oxidative stress suggests a potential use for ψGAPDH peptide-based therapy.

The Role of Mitochondrial Aldehyde Dehydrogenase 2 (ALDH2) in Neuropathology and Neurodegeneration.

Aldehydes-induced toxicity has been implicated in many neurodegenerative diseases. Exposure to reactive aldehydes from (1) alcohol and food metabolism; (2) environmental pollutants, including car, factory exhausts, smog, pesticides, herbicides; (3) metabolism of neurotransmitters, amino acids and (4) lipid peroxidation of biological membrane from excessive ROS, all contribute to 'aldehydic load' that has been linked to the pathology of neurodegenerative diseases. In particular, the α, ß-unsaturated aldehydes derived from lipid peroxidation, 4-hydroxynonenal (4-HNE), DOPAL (MAO product of dopamine), malondialdehyde, acrolein and acetaldehyde, all readily form chemical adductions with proteins, DNA and lipids, thus causing neurotoxicity. Mitochondrial aldehyde dehydrogenase 2 (ALDH 2) is a major aldehyde metabolizing enzyme that protects against deleterious aldehyde buildup in brain, a tissue that has a particularly high mitochondrial content. In this review, we highlight the deleterious effects of increased aldehydic load in the neuropathology of ischemic stroke, Alzheimer's disease and Parkinson's disease. We also discuss evidence for the association between ALDH2 deficiency, a common East Asianspecific mutation, and these neuropathologies. A novel class of small molecule aldehyde dehydrogenase activators (Aldas), represented by Alda-1, reduces neuronal cell death in models of ischemic stroke, Alzheimer's disease and Parkinson's disease. Together, these data suggest that reducing aldeydic load by enhancing the activity of aldehyde dehydrogenases, such as ALDH2, represents as a therapeutic strategy for neurodegenerative diseases.