Biliverdin reductase A (BVRA), the terminal enzyme in heme catabolism, generates the neuroprotective and lipophilic antioxidant bilirubin. Here, we identify a nonenzymatic role for BVRA in redox regulation. Through phylogenetic, genetic, biochemical, and enzymatic assays, we found that BVRA exerts critical nonenzymatic antioxidant activity. Transcriptomic analyses further revealed that BVRA physically and genetically interacts with nuclear factor erythroid-derived factor-like 2 (NRF2), a major transcriptional regulator of cellular redox signaling. ChIP-seq and RNA-seq analyses reveal that BVRA and NRF2 coordinate the expression of antioxidant genes, many of which are typically dysregulated in neurodegenerative conditions such as Alzheimer's disease. Thus, this noncanonical BVRA-NRF2 axis controls an essential pathway of redox signaling in neuroprotection. Our findings position BVRA as a dual-function integrator of antioxidant defense across both lipophilic and hydrophilic compartments, bridging these two distinct modes of redox protection in the brain.

Cocaine exerts its stimulant effect by inhibiting dopamine (DA) reuptake, leading to increased dopamine signaling. This action is thought to reflect the binding of cocaine to the dopamine transporter (DAT) to inhibit its function. However, cocaine is a relatively weak inhibitor of DAT, and many DAT inhibitors do not share cocaine’s behavioral actions. Further, recent reports show more potent actions of the drug, implying the existence of a high-affinity receptor for cocaine. We now report high-affinity binding of cocaine associated with the brain acid soluble protein 1 (BASP1) with a dissociation constant (Kd) of 7 nM. Knocking down BASP1 in the striatum inhibits [3H]cocaine binding to striatal synaptosomes. Depleting BASP1 in the nucleus accumbens but not the dorsal striatum diminishes locomotor stimulation in mice. Our findings imply that BASP1 is a pharmacologically relevant receptor for cocaine.

Trigeminal neuralgia, historically dubbed the “suicide disease,” is an exceedingly painful neurologic condition characterized by sudden episodes of intense facial pain. Unfortunately, the only U.S. Food and Drug Administration (FDA)–approved medication for trigeminal neuralgia carries substantial side effects, with many patients requiring surgery. Here, we identify the NRF2 transcriptional network as a potential therapeutic target. We report that cerebrospinal fluid from patients with trigeminal neuralgia accumulates reactive oxygen species, several of which directly activate the pain-transducing channel TRPA1. Similar to our patient cohort, a mouse model of trigeminal neuropathic pain also exhibits notable oxidative stress. We discover that stimulating the NRF2 antioxidant transcriptional network is as analgesic as inhibiting TRPA1, in part by reversing the underlying oxidative stress. Using a transcriptome-guided drug discovery strategy, we identify two NRF2 network modulators as potential treatments. One of these candidates, exemestane, is already FDA-approved and may thus be a promising alternative treatment for trigeminal neuropathic pain.

Synapses connect discrete neurons into vast networks that send, receive, and encode diverse forms of information. Synaptic function and plasticity, the neuronal process of adapting to diverse and variable inputs, depend on the dynamic nature of synaptic molecular components, which is mediated in part by cell adhesion signaling pathways. Here, we found that the enzyme biliverdin reductase (BVR) physically links together key focal adhesion signaling molecules at the synapse. BVR -null ( BVR −/− ) mice exhibited substantial deficits in learning and memory on neurocognitive tests, and hippocampal slices in which BVR was postsynaptically depleted showed deficits in electrophysiological responses to stimuli. RNA sequencing, biochemistry, and pathway analyses suggested that these deficits were mediated through the loss of focal adhesion signaling at both the transcriptional and biochemical level in the hippocampus. Independently of its catalytic function, BVR acted as a bridge between the primary focal adhesion signaling kinases FAK and Pyk2 and the effector kinase Src. Without BVR, FAK and Pyk2 did not bind to and stimulate Src, which then did not phosphorylate the N -methyl- d -aspartate (NMDA) receptor, a critical posttranslational modification for synaptic plasticity. Src itself is a molecular hub on which many signaling pathways converge to stimulate NMDAR-mediated neurotransmission, thus positioning BVR at a prominent intersection of synaptic signaling.

Alzheimer's disease (AD), the most common cause of dementia and neurodegeneration in the elderly, is characterized by deterioration of memory and executive and motor functions. Neuropathologic hallmarks of AD include neurofibrillary tangles (NFTs), paired helical filaments, and amyloid plaques. Mutations in the microtubule-associated protein Tau, a major component of the NFTs, cause its hyperphosphorylation in AD. We have shown that signaling by the gaseous molecule hydrogen sulfide (H2S) is dysregulated during aging. H2S signals via a posttranslational modification termed sulfhydration/persulfidation, which participates in diverse cellular processes. Here we show that cystathionine γ-lyase (CSE), the biosynthetic enzyme for H2S, binds wild type Tau, which enhances its catalytic activity. By contrast, CSE fails to bind Tau P301L, a mutant that is present in the 3xTg-AD mouse model of AD. We further show that CSE is depleted in 3xTg-AD mice as well as in human AD brains, and that H2S prevents hyperphosphorylation of Tau by sulfhydrating its kinase, glycogen synthase kinase 3β (GSK3β). Finally, we demonstrate that sulfhydration is diminished in AD, while administering the H2S donor sodium GYY4137 (NaGYY) to 3xTg-AD mice ameliorates motor and cognitive deficits in AD.

Alzheimer's disease (AD), the most common cause of dementia and neurodegeneration in the elderly, is characterized by deterioration of memory and executive and motor functions. Neuropathologic hallmarks of AD include neurofibrillary tangles (NFT5), paired helical filaments, and amyloid plaques. Mutations in the microtubule-associated protein Tau, a major component of the NFT5, cause its hyperphosphorylation in AD. We have shown that signaling by the gaseous molecule hydrogen sulfide (H2S) is dysregulated during aging. H2S signals via a posttranslational modification termed sulfhydration/persulfidation, which participates in diverse cellular processes. Here we show that cystathionine gamma-lyase (CSE), the biosynthetic enzyme for H2S, binds wild type Tau, which enhances its catalytic activity. By contrast, CSE fails to bind Tau P301L, a mutant that is present in the 3xTg-AD mouse model of AD. We further show that CSE is depleted in 3xTg-AD mice as well as in human AD brains, and that H2S prevents hyperphosphorylation of Tau by sulfhydrating its kinase, glycogen synthase kinase 3 beta (GSK3 beta). Finally, we demonstrate that sulfhydration is diminished in AD, while administering the H2S donor sodium GYY4137 (NaGYY) to 3xTg-AD mice ameliorates motor and cognitive deficits in AD.

Bilirubin is amongst the most frequently measured peripheral metabolites, yet its roles in vivo are not totally understood. Believed to be a marker for liver function, it is now increasingly evident that bilirubin plays vital roles in the brain. Bilirubin is synthesized by Biliverdin reductase (BVR) from biliverdin, which in turn is derived from heme. BVR exists as two isoforms, BVR‐A and BVR‐B, of which BVR‐A is enriched in the brain. In addition to its role in bilirubin generation, BVR‐A is a kinase and transcription factor, and participates in several signaling pathways. Besides, these activities, the BVR pathway plays significant roles in maintenance of redox homeostasis in cells. Cell culture studies have shown that depletion of BlvrA, the gene encoding BVRA causes elevated oxidative stress and associated cellular damage. Using BlvrA−/− mice, we show that BVRA is the major enzyme responsible for bilirubin production. The gall bladders of these mice are green due to accumulation of biliverdin and inability to produce bilirubin. We analyzed the effects of bilirubin depletion in the brain using these mice. Paucity of bilirubin leads to elevated superoxide production in the brain and altered cytoprotective response to stress stimuli. These mice are defective in several antioxidant defense pathways. BlvrA mice also display a variety of behavioral abnormalities including cognitive deficits. We further show that bilirubin is a potent and direct superoxide scavenger in vivo, which accounts for its role in maintenance of redox balance. Treatment of mice with N‐methyl D‐aspartate (NMDA), an excitotoxin, results in increased neuronal death and larger lesions in BlvrA−/− mice. NMDA receptor activation is known to elevate superoxide production via the NADPH oxidases, enzymes which are known to be inhibited by the BVR‐bilirubin pathway. Taken together, our findings uncover important roles for BVR in neuroprotective processes. Modulating the BVR/bilirubin pathway may offer therapeutic benefits in neurodegenerative diseases involving redox imbalance.Support or Funding InformationNIH grants MH18501(to S.H.S.)

Cocaine is a behavioral stimulant with substantial abuse potential related to its positively rewarding actions. Definitive molecular mechanisms of action have not been established, though cocaine is known to inhibit the reuptake inactivation of monoamine neurotransmission at high nanomolar to low micromolar concentrations. There is evidence for significantly more potent neurochemical and behavioral actions of cocaine that could not be attributed to the inhibition of monoamine transporters. Thus, evidence for a specific, high affinity cocaine receptor that mediates its behavioral actions has been lacking. In the present study we report high affinity binding of cocaine to the membrane‐associated protein BASP1, which appears to be a pharmacologically relevant cocaine receptor. Utilizing affinity pull‐down, mass spectrometry and ligand binding assays, we establish that BASP1 is a high‐affinity cocaine binding protein (Kd 7 nM). Our data show that knocking down BASP1 in the nucleus accumbens or striatum (pre‐ and post‐synaptic) inhibits behavioral actions of cocaine and reduces [3H]cocaine binding to striatal synaptosomes respectively. Taken together, our data suggest that BASP1 is a high‐affinity receptor for cocaine.Support or Funding Information Fundingthis work was supported by U.S. Public Health Service Grants DA00266 and DA044123 to SHS and a NARSAD Young Investigator Grant (grant # 25360) from the Brain & Behavior Foundation to MMH.

Bilirubin is one of the most frequently measured metabolites in medicine, yet its physiologic roles remain unclear. Bilirubin can act as an antioxidant in vitro, but whether its redox activity is physiologically relevant is unclear because many other antioxidants are far more abundant in vivo. Here, we report that depleting endogenous bilirubin renders mice hypersensitive to oxidative stress. We find that mice lacking bilirubin are particularly vulnerable to superoxide (O-2(center dot-)) over other tested reactive oxidants and electrophiles. Whereas major antioxidants such as glutathione and cysteine exhibit little to no reactivity toward O-2(center dot-), bilirubin readily scavenges O-2(center dot-). We find that bilirubin's redox activity is particularly important in the brain, where it prevents excitotoxicity and neuronal death by scavenging O-2(center dot-) during NMDA neurotransmission. Bilirubin's unique redox activity toward O-2(center dot-) may underlie a prominent physiologic role despite being significantly less abundant than other endogenous and exogenous antioxidants.

Glutamate is the most abundant excitatory neurotransmitter, present at the bulk of cortical synapses, and participating in many physiologic and pathologic processes ranging from learning and memory to stroke. The tripeptide, glutathione, is one-third glutamate and present at up to low millimolar intracellular concentrations in brain, mediating antioxidant defenses and drug detoxification. Because of the substantial amounts of brain glutathione and its rapid turnover under homeostatic control, we hypothesized that glutathione is a relevant reservoir of glutamate and could influence synaptic excitability. We find that drugs that inhibit generation of glutamate by the glutathione cycle elicit decreases in cytosolic glutamate and decreased miniature excitatory postsynaptic potential (mEPSC) frequency. In contrast, pharmacologically decreasing the biosynthesis of glutathione leads to increases in cytosolic glutamate and enhanced mEPSC frequency. The glutathione cycle can compensate for decreased excitatory neurotransmission when the glutamate-glutamine shuttle is inhibited. Glutathione may be a physiologic reservoir of glutamate neurotransmitter.

Rationale: Inositol polyphosphate multikinase (IPMK) and its major product inositol pentakisphosphate (IP5) regulate a variety of cellular functions, but their role in vascular biology remains unexplored. Objective: We have investigated the role of IPMK in regulating angiogenesis. Methods and Results: Deletion of IPMK in fibroblasts induces angiogenesis in both in vitro and in vivo models. IPMK deletion elicits a substantial increase of VEGF (vascular endothelial growth factor), which mediates the regulation of angiogenesis by IPMK. The regulation of VEGF by IPMK requires its catalytic activity. IPMK is predominantly nuclear and regulates gene transcription. However, IPMK does not apparently serve as a transcription factor for VEGF. HIF (hypoxia-inducible factor)-1 is a major determinant of angiogenesis and induces VEGF transcription. IPMK deletion elicits a major enrichment of HIF-1 protein and thus VEGF. HIF-1 alpha is constitutively ubiquitinated by pVHL (von Hippel-Lindau protein) followed by proteasomal degradation under normal conditions. However, HIF-1 alpha is not recognized and ubiquitinated by pVHL in IPMK KO (knockout) cells. IP5 reinstates the interaction of HIF-1 and pVHL. HIF-1 alpha prolyl hydroxylation, which is prerequisite for pVHL recognition, is interrupted in IPMK-deleted cells. IP5 promotes HIF-1 alpha prolyl hydroxylation and thus pVHL-dependent degradation of HIF-1 alpha Deletion of IPMK in mouse brain increases HIF-1/VEGF levels and vascularization. The increased VEGF in IPMK KO disrupts blood-brain barrier and enhances brain blood vessel permeability. Conclusions: IPMK, via its product IP5, negatively regulates angiogenesis by inhibiting VEGF expression. IP5 acts by enhancing HIF-1 alpha hydroxylation and thus pVHL-dependent degradation of HIF-1 alpha.

ATM drives DNA repair by phosphorylating the histone variant H2AX. While ATM mutations elicit prominent neurobehavioral phenotypes, neural roles for H2AX have been elusive. We report impaired motor learning and balance in H2AX-deficient mice. Mitigation of reactive oxygen species (ROS) with N-acetylcysteine (NAC) reverses the behavioral deficits. Mouse embryonic fibroblasts deficient for H2AX exhibit increased ROS production and failure to activate the antioxidant response pathway controlled by the transcription factor NRF2. The NRF2 targets GCLC and NQO1 are depleted in the striatum of H2AX knockouts, one of the regions most vulnerable to ROS-mediated damage. These findings establish a role for ROS in the behavioral deficits of H2AX knockout mice and reveal a physiologic function of H2AX in mediating influences of oxidative stress on NRF2-transcriptional targets and behavior.

Significance Inositol hexakisphosphate kinase 1 (IP6K1) regulates numerous physiological functions. We describe a role of IP6K1 in neural development, because its deletion results in defects in neuronal migration and brain malformation. IP6K1 physiologically interacts with α-actinin and localizes to focal adhesions. IP6K1 regulates phosphorylation of both focal adhesion kinase and α-actinin through its product 5-diphosphoinositol pentakisphosphate (5-IP7).

The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K3's role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells. SIGNIFICANCE STATEMENT We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function.

Cystathionine γ-lyase, which is responsible for the production of cysteine, is decreased in the striatum and cortex of mouse models of Huntington’s disease and in patients with Huntington’s disease, and cysteine supplementation in diet and drinking water partly rescues the phenotype and the diminished longevity of the mouse model. Huntington's disease is associated with polyglutamine expansion in the gene encoding huntingtin. Mutant huntingtin is expressed throughout the brain and rest of the body, but the striatum is the most affected brain region. Here it is shown that the enzyme cystathionine γ-lyase (CSE), responsible for cysteine biosynthesis, is decreased in the striatum and cortex of both mouse models and Huntington's disease patients. Mutant huntingtin inhibits the transcriptional activator Sp1, resulting in decreased CSE transcription. Cysteine supplementation in diet and drinking water partially rescues the phenotype and the diminished longevity in the mouse model, suggesting that cysteine supplementation might be beneficial for Huntington's disease patients. Huntington’s disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes1. Huntington’s disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington’s disease may reflect the striatally selective small G protein Rhes binding to mHtt and enhancing its neurotoxicity2. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington’s disease tissues, which may mediate Huntington’s disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington’s disease tissues and in intact mouse models of Huntington’s disease, suggesting therapeutic potential.

The tumor suppressor protein p53 is a critical stress response transcription factor that induces the expression of genes leading to cell cycle arrest, apoptosis, and tumor suppression. We found that mammalian inositol polyphosphate multikinase (IPMK) stimulated p53-mediated transcription by binding to p53 and enhancing its acetylation by the acetyltransferase p300 independently of its inositol phosphate and lipid kinase activities. Genetic or RNA interference (RNAi)-mediated knockdown of IPMK resulted in decreased activation of p53, decreased recruitment of p53 and p300 to target gene promoters, abrogated transcription of p53 target genes, and enhanced cell viability. Additionally, blocking the IPMK-p53 interaction decreased the extent of p53-mediated transcription. These results suggest that IPMK acts as a transcriptional coactivator for p53 and that it is an integral part of the p53 transcriptional complex facilitating cell death.

The tumor suppressor protein p53 is a critical stress response transcription factor that induces the expression of genes leading to cell cycle arrest, apoptosis, and tumor suppression. We found that mammalian inositol polyphosphate multikinase (IPMK) stimulated p53-mediated transcription by binding to p53 and enhancing its acetylation by the acetyltransferase p300 independently of its inositol phosphate and lipid kinase activities. Genetic or RNA interference (RNAi)-mediated knockdown of IPMK resulted in decreased activation of p53, decreased recruitment of p53 and p300 to target gene promoters, abrogated transcription of p53 target genes, and enhanced cell viability. Additionally, blocking the IPMK-p53 interaction decreased the extent of p53-mediated transcription. These results suggest that IPMK acts as a transcriptional coactivator for p53 and that it is an integral part of the p53 transcriptional complex facilitating cell death.