Saravanan S. Karuppagounder

Dietary supplementation with resveratrol reduces plaque pathology in a transgenic model of Alzheimer's disease.

Resveratrol, a polyphenol found in red wine, peanuts, soy beans, and pomegranates, possesses a wide range of biological effects. Since resveratrols properties seem ideal for treating neurodegenerative diseases, its ability to diminish amyloid plaques was tested. Mice were fed clinically feasible dosages of resveratrol for forty-five days. Neither resveratrol nor its conjugated metabolites were detectable in brain. Nevertheless, resveratrol diminished plaque formation in a region specific manner. The largest reductions in the percent area occupied by plaques were observed in medial cortex (-48%), striatum (-89%) and hypothalamus (-90%). The changes occurred without detectable activation of SIRT-1 or alterations in APP processing. However, brain glutathione declined 21% and brain cysteine increased 54%. The increased cysteine and decreased glutathione may be linked to the diminished plaque formation. This study supports the concept that onset of neurodegenerative disease may be delayed or mitigated with use of dietary chemo-preventive agents that protect against beta-amyloid plaque formation and oxidative stress.

Thiamine deficiency induces oxidative stress and exacerbates the plaque pathology in Alzheimer’s mouse model

Mitochondrial dysfunction, oxidative stress and reductions in thiamine-dependent enzymes have been implicated in multiple neurological disorders including Alzheimers disease (AD). Experimental thiamine deficiency (TD) is an established model for reducing the activities of thiamine-dependent enzymes in brain. TD diminishes thiamine-dependent enzymes throughout the brain, but produces a time-dependent selective neuronal loss, glial activation, inflammation, abnormalities in oxidative metabolism and clusters of degenerating neurites in only specific thalamic regions. The present studies tested how TD alters brain pathology in Tg19959 transgenic mice over expressing a double mutant form of the amyloid precursor protein (APP). TD exacerbated amyloid plaque pathology in transgenic mice and enlarged the area occupied by plaques in cortex, hippocampus and thalamus by 50%, 200% and 200%, respectively. TD increased Abeta(1-42) levels by about three fold, beta-CTF (C99) levels by 33% and beta-secretase (BACE1) protein levels by 43%. TD-induced inflammation in areas of plaque formation. Thus, the induction of mild impairment of oxidative metabolism, oxidative stress and inflammation induced by TD alters metabolism of APP and/or Abeta and promotes accumulation of plaques independent of neuron loss or neuritic clusters.

Hypoxia-inducible factor prolyl hydroxylase inhibition: robust new target or another big bust for stroke therapeutics?

A major challenge in developing stroke therapeutics that augment adaptive pathways to stress has been to identify targets that can activate compensatory programs without inducing or adding to the stress of injury. In this regard, hypoxia-inducible factor prolyl hydroxylases (HIF PHDs) are central gatekeepers of posttranscriptional and transcriptional adaptation to hypoxia, oxidative stress, and excitotoxicity. Indeed, some of the known salutary effects of putative ‘antioxidant’ iron chelators in ischemic and hemorrhagic stroke may derive from their abilities to inhibit this family of iron, 2-oxoglutarate, and oxygen-dependent enzymes. Evidence from a number of laboratories supports the notion that HIF PHD inhibition can improve histological and functional outcomes in ischemic and hemorrhagic stroke models. In this review, we discuss this evidence and highlight important gaps in our understanding that render HIF PHD inhibition a promising but not yet preclinically validated target for protection and repair after stroke.

Exercise promotes the expression of brain derived neurotrophic factor (BDNF) through the action of the ketone body β-hydroxybutyrate

Exercise induces beneficial responses in the brain, which is accompanied by an increase in BDNF, a trophic factor associated with cognitive improvement and the alleviation of depression and anxiety. However, the exact mechanisms whereby physical exercise produces an induction in brain Bdnf gene expression are not well understood. While pharmacological doses of HDAC inhibitors exert positive effects on Bdnf gene transcription, the inhibitors represent small molecules that do not occur in vivo. Here, we report that an endogenous molecule released after exercise is capable of inducing key promoters of the Mus musculus Bdnf gene. The metabolite β-hydroxybutyrate, which increases after prolonged exercise, induces the activities of Bdnf promoters, particularly promoter I, which is activity-dependent. We have discovered that the action of β-hydroxybutyrate is specifically upon HDAC2 and HDAC3, which act upon selective Bdnf promoters. Moreover, the effects upon hippocampal Bdnf expression were observed after direct ventricular application of β-hydroxybutyrate. Electrophysiological measurements indicate that β-hydroxybutyrate causes an increase in neurotransmitter release, which is dependent upon the TrkB receptor. These results reveal an endogenous mechanism to explain how physical exercise leads to the induction of BDNF. DOI: http://dx.doi.org/10.7554/eLife.15092.001

Oxidant‐induced Changes in Mitochondria and Calcium Dynamics in the Pathophysiology of Alzheimer's Disease

Considerable data support the hypothesis that mitochondrial abnormalities link gene defects and/or environmental insults to the neurodegenerative process. The interaction of oxidants with calcium and the mitochondrial enzymes of the tricarboxylic acid cycle are central to that relationship. Abnormalities that were discovered in brains or fibroblasts from patients with Alzheimers disease (AD) have been modeled in vitro and in vivo to assess their pathophysiological importance and to determine how they might be reversed. The conclusions are consistent with the hypothesis that the AD‐related abnormalities result from oxidative stress. The selection of compounds for reversal is complex because the actions of the relevant compounds vary under different conditions, such as cell redox states and acute versus chronic changes. However, the models that have been developed are useful for testing the effectiveness of the potential medications. The results suggest that the reversal of mitochondrial deficits and a reduction in oxidative stress will reduce clinical and pathological changes and benefit patients.

Responses of the mitochondrial alpha-ketoglutarate dehydrogenase complex to thiamine deficiency may contribute to regional selective vulnerability

Thiamine-dependent enzymes are diminished in multiple neurodegenerative diseases. Thiamine deficiency (TD) reduces the activity of thiamine dependent-enzymes [e.g., the alpha-ketoglutarate dehydrogenase complex (KGDHC)], induces regional selective neurodegeneration and serves as a model of a mild impairment of oxidative metabolism. The current experiments tested whether changes in KGDHC protein subunits (E1k, E2k and E3) or activity or message levels underlie the selective loss of neurons in particular brain regions. Thus, TD-induced changes in these variables in the brain region most vulnerable to TD [the sub-medial thalamic nucleus (SmTN)] were compared to those in a region that is relatively resistant to TD (cortex) at stages of TD when the neuron loss in SmTN is not present, minimal or severe. Impaired motor performance on rotarod was apparent by 8 days of TD (-32%) and was severe by 10 days of TD (-97%). At TD10, the overall KGDHC activity measured by an in situ histochemical staining method declined 52% in SmTN but only 20% in cortex. Reductions in the E2k and E3 mRNA in SmTN occurred as early as TD6 (-28 and -18%, respectively) and were more severe by TD10 (-61 and -66%, respectively). On the other hand, the level of E1k mRNA did not decline in SmTN until TD10 (-48%). In contrast, TD did not alter mRNA levels of the subunits in cortex at late stages. Western blots and immunocytochemistry revealed different aspects of the changes in protein levels. In SmTN, the immunoreactivity of E1k and E3 by Western blotting increased 34 and 40%, respectively, only at TD8. In cortex, the immunoreactivity of the three subunits was not altered. Immunocytochemical staining of brain sections from TD10 mice indicated a reduction in the immunoreactivity of all subunits in SmTN, but not in cortex. These findings demonstrate that the response of the KGDHC activity, mRNA and immunoreactivity of E1k, E2k and E3 to TD is region and time dependent. Loss of KGDHC activity in cortex is likely related to post-translational modification rather than a loss of protein, whereas in SmTN transcriptional and post-translational modifications may account for diminished KGDHC activity. Moreover, the earlier detection in TD induced-changes of the transcripts of KGDHC indicates that transcriptional modification of the two subunits (E2k and E3) of KGDHC may be one of the early events in the cascade leading to selective neuronal death.

Hypoxia-inducible factor prolyl hydroxylases as targets for neuroprotection by "antioxidant" metal chelators: From ferroptosis to stroke.

Neurologic conditions including stroke, Alzheimer disease, Parkinson disease, and Huntington disease are leading causes of death and long-term disability in the United States, and efforts to develop novel therapeutics for these conditions have historically had poor success in translating from bench to bedside. Hypoxia-inducible factor (HIF)-1α mediates a broad, evolutionarily conserved, endogenous adaptive program to hypoxia, and manipulation of components of the HIF pathway is neuroprotective in a number of human neurological diseases and experimental models. In this review, we discuss molecular components of one aspect of hypoxic adaptation in detail and provide perspective on which targets within this pathway seem to be ripest for preventing and repairing neurodegeneration. Further, we highlight the role of HIF prolyl hydroxylases as emerging targets for the salutary effects of metal chelators on ferroptosis in vitro as well in animal models of neurological diseases.

Neuronal Death After Hemorrhagic Stroke In Vitro and In Vivo Shares Features of Ferroptosis and Necroptosis

Background and Purpose— Intracerebral hemorrhage leads to disability or death with few established treatments. Adverse outcomes after intracerebral hemorrhage result from irreversible damage to neurons resulting from primary and secondary injury. Secondary injury has been attributed to hemoglobin and its oxidized product hemin from lysed red blood cells. The aim of this study was to identify the underlying cell death mechanisms attributable to secondary injury by hemoglobin and hemin to broaden treatment options. Methods— We investigated cell death mechanisms in cultured neurons exposed to hemoglobin or hemin. Chemical inhibitors implicated in all known cell death pathways were used. Identified cell death mechanisms were confirmed using molecular markers and electron microscopy. Results— Chemical inhibitors of ferroptosis and necroptosis protected against hemoglobin- and hemin-induced toxicity. By contrast, inhibitors of caspase-dependent apoptosis, protein or mRNA synthesis, autophagy, mitophagy, or parthanatos had no effect. Accordingly, molecular markers of ferroptosis and necroptosis were increased after intracerebral hemorrhage in vitro and in vivo. Electron microscopy showed that hemin induced a necrotic phenotype. Necroptosis and ferroptosis inhibitors each abrogated death by >80% and had similar therapeutic windows in vitro. Conclusions— Experimental intracerebral hemorrhage shares features of ferroptotic and necroptotic cell death, but not caspase-dependent apoptosis or autophagy. We propose that ferroptosis or necroptotic signaling induced by lysed blood is sufficient to reach a threshold of death that leads to neuronal necrosis and that inhibition of either of these pathways can bring cells below that threshold to survival.

Sodium salicylate protects against rotenone-induced Parkinsonism in rats

Complex I deficiency culminating in oxidative stress is proposed as one of the upstream mechanisms of nigral neuronal death in Parkinsons disease. We investigated whether sodium salicylate, an active metabolite of aspirin, could afford protection against rotenone‐induced oxidative stress, neuronal degeneration, and behavioral dysfunction in rats, because it has the potential to accept a molecule each of hydroxyl radical (•OH) at the third or fifth position of its benzyl ring. Rotenone caused dose‐dependent increase in •OH in isolated mitochondria from the cerebral cortex and time‐ (24–48h) and dose‐dependent (0.1–100 µM) increase in the substantia nigra and the striatum, ipsilateral to the side of rotenone infusion. Administration of sodium salicylate at 12‐h intervals for 4 days showed dose‐dependent (50–100 mg/kg, i.p) reductions in the levels of •OH in the nigra on the fifth day. These animals showed significant attenuation in rotenone‐induced loss in striatal dopamine levels, number of nigral dopaminergic neurons, reduced and oxidized glutathione levels, and complex I activity loss, but superoxide dismutase activity was increased further. Amphetamine‐ or apomorphine‐induced ipsilateral rotations in rotenone‐treated rats were significantly reduced in rats treated with sodium salicylate. Our results indicate a direct role of •OH in mediating nigral neuronal death by rotenone and confirm the neuroprotective potential of salicylate in a rodent model of parkinsonism. Synapse 67:502–514, 2013.

Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models.

Blocking oxygen-sensing prolyl hydroxylases in the rodent CNS enhances functional recovery after brain hemorrhage. Beating back damage from brain bleeding Brain bleeding is associated with stroke, anticoagulant use, amyloid angiopathy, and brain trauma. Blood in the brain leads to the deposition of toxic iron, and as expected, chelators of iron can enhance functional recovery after stroke. Here, Karuppagounder et al. show that iron chelators protect from a bleeding stroke not by binding all iron but rather by targeting a small family of iron-containing enzymes, the hypoxia-inducible factor prolyl hydroxylases. The target enzymes are oxygen sensors that, when inhibited, engage a broad homeostatic response to low oxygen and oxidative stress. The authors characterize and validate a selective, brain-penetrant inhibitor of brain oxygen sensors, which they call adaptaquin, as a new candidate treatment for brain bleeding in several rodent models. Protective doses of adaptaquin were used in combination with unbiased RNA profiling to identify an unexpected hypoxia-inducible factor–independent pathway mediated by the prodeath transcription factor ATF4. Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron, and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. We show that the hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) family of iron-dependent, oxygen-sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in the mouse striatum improved functional recovery after ICH. A low-molecular-weight hydroxyquinoline inhibitor of the HIF-PHD enzymes, adaptaquin, reduced neuronal death and behavioral deficits after ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of activity of the prodeath factor ATF4 rather than activation of an HIF-dependent prosurvival pathway. Together, these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier–permeable inhibitor adaptaquin can improve functional outcomes after ICH in several rodent models.