Maged M. Harraz

MicroRNA-223 is neuroprotective by targeting glutamate receptors

Stroke is a major cause of mortality and morbidity worldwide. Extracellular glutamate accumulation leading to overstimulation of the ionotropic glutamate receptors mediates neuronal injury in stroke and in neurodegenerative disorders. Here we show that miR-223 controls the response to neuronal injury by regulating the functional expression of the glutamate receptor subunits GluR2 and NR2B in brain. Overexpression of miR-223 lowers the levels of GluR2 and NR2B by targeting 3′-UTR target sites (TSs) in GluR2 and NR2B, inhibits NMDA-induced calcium influx in hippocampal neurons, and protects the brain from neuronal cell death following transient global ischemia and excitotoxic injury. MiR-223 deficiency results in higher levels of NR2B and GluR2, enhanced NMDA-induced calcium influx, and increased miniature excitatory postsynaptic currents in hippocampal neurons. In addition, the absence of MiR-223 leads to contextual, but not cued memory deficits and increased neuronal cell death following transient global ischemia and excitotoxicity. These data identify miR-223 as a major regulator of the expression of GluR2 and NR2B, and suggest a therapeutic role for miR-223 in stroke and other excitotoxic neuronal disorders.

MicroRNAs in Parkinson's disease.

MicroRNAs are small non-protein coding RNAs that regulate gene expression through post-transcriptional repression. Recent studies demonstrated the importance of microRNAs in the nervous system development, function and disease. Parkinsons disease is the second most prevalent neurodegenerative disease with only symptomatic treatment available. Recent success in using small RNAs as therapeutic targets hold a substantial promise for the Parkinsons disease field. Here we review recent work linking the microRNA pathway to Parkinsons disease.

A nuclease that mediates cell death induced by DNA damage and poly(ADP-ribose) polymerase-1

DNA damage-activated nuclease identified Cells that experience stresses and accumulate excessive damage to DNA undergo cell death mediated by a nuclear enzyme known as PARP-1. During this process, apoptosis-inducing factor (AIF) translocates to the nucleus and activates one or more nucleases to cleave DNA. Wang et al. found that macrophage migration inhibitory factor (MIF) is an AIF-associated endonuclease that contributes to PARP-1-induced DNA fragmentation (see the Perspective by Jonas). In mouse neurons in culture, loss of MIF protected neurons from cell death caused by excessive stimulation. Targeting MIF could thus provide a therapeutic strategy against diseases in which PARP-1 activation is excessive. Science, this issue p. 82; see also p. 36 An endonuclease that functions in a disease-associated form of cell death is identified. [Also see Perspective by Jonas] INTRODUCTION Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is a nuclear enzyme responding to oxidative stress and DNA damage. Excessive activation of PARP-1 causes an intrinsic caspase-independent cell death program designated parthanatos, which occurs in many organ systems because of toxic or stressful insults, including ischemia-reperfusion injury after stroke and myocardial infarction, inflammatory injury, reactive oxygen species–induced injury, glutamate excitotoxicity, and neurodegenerative diseases. Inhibition or genetic deletion of PARP-1 is profoundly protective against such cellular injury in models of human disease. RATIONALE The molecular mechanisms underlying parthanatos involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis into 20- to 50-kb large DNA fragments—a commitment point for parthanatos. Because AIF itself has no obvious nuclease activity, we propose that AIF recruits a nuclease or a nuclease complex to the nucleus to trigger DNA cleavage and parthanatos. Although the endonuclease G (EndoG) homolog may promote DNA degradation in Caenorhabditis elegans through cooperating with the AIF homolog, our group and others showed that EndoG does not have an essential role in PARP-dependent chromatinolysis and cell death in mammals. Thus, the identity of the nuclease responsible for large DNA fragmentation following AIF entry to the nucleus during parthanatos has been a long-standing mystery. RESULTS Using two sequential unbiased screens, including a human protein array and a small interfering RNA screen, we discovered that macrophage migration inhibitory factor (MIF) binds AIF and is required for parthanatos. Three-dimensional modeling of MIF revealed that the MIF trimer has the same core topology structure as PD-D/E(X)K superfamily nucleases. In the presence of Mg2+ or Ca2+, MIF has both 3′ exonuclease and endonuclease activity. It binds to 5′ unpaired bases of single-stranded DNA with stem loop structure and cleaves its 3′ unpaired bases. These nuclease activities allow MIF to cleave genomic DNA into large fragments. Depletion of MIF markedly reduced chromatinolysis and cell death induced by N-methyl-d-aspartate (NMDA) receptor–activated glutamate excitotoxicity in primary neuronal cultures, DNA damage caused by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or focal stroke in mice. Mutating key amino acid residues in the PD-D/E(X)K nuclease domain of MIF eliminated its nuclease activity and prevented parthanatos. Disrupting the AIF and MIF interaction prevented the translocation of MIF from the cytosol to the nucleus and protected against parthanatos. Moreover, depletion of MIF, disruption of AIF and MIF interaction, and eliminating MIF’s nuclease activity has long-lasting histological and behavioral rescue in the focal ischemia model of stroke. CONCLUSION We identified MIF as a PARP-1–dependent AIF-associated nuclease that is required for parthanatos. In response to oxidative stress or DNA damage, PARP-1 activation triggers AIF release from the mitochondria. AIF then recruits MIF to the nucleus where MIF cleaves genomic DNA into large fragments and causes cell death. Depletion of MIF, disruption of AIF and MIF interaction, or blocking MIF nuclease activity inhibited chromatinolysis and parthanatos. Targeting MIF nuclease activity may offer an important therapeutic opportunity for a variety of disorders with excessive PARP-1 activation. Stressors lead to DNA damage, PARP-1 activation, and PAR formation. PAR facilitates the release of AIF from mitochondria where it binds MIF. This complex translocates to the nucleus to bind DNA; the result is DNA fragmentation and cell death. Interference with this cascade by preventing the formation of the AIF-MIF complex or by a nuclease-deficient MIF prevents DNA fragmentation and promotes cell survival. Inhibition or genetic deletion of poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is protective against toxic insults in many organ systems. The molecular mechanisms underlying PARP-1–dependent cell death involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis. We identified macrophage migration inhibitory factor (MIF) as a PARP-1–dependent AIF-associated nuclease (PAAN). AIF was required for recruitment of MIF to the nucleus, where MIF cleaves genomic DNA into large fragments. Depletion of MIF, disruption of the AIF-MIF interaction, or mutation of glutamic acid at position 22 in the catalytic nuclease domain blocked MIF nuclease activity and inhibited chromatinolysis, cell death induced by glutamate excitotoxicity, and focal stroke. Inhibition of MIF’s nuclease activity is a potential therapeutic target for diseases caused by excessive PARP-1 activation.

Botch Promotes Neurogenesis by Antagonizing Notch

Regulation of self-renewal and differentiation of neural stem cells is still poorly understood. Here we investigate the role of a developmentally expressed protein, Botch, which blocks Notch, in neocortical development. Downregulation of Botch in vivo leads to cellular retention in the ventricular and subventricular zones, whereas overexpression of Botch drives neural stem cells into the intermediate zone and cortical plate. In vitro neurosphere and differentiation assays indicate that Botch regulates neurogenesis by promoting neuronal differentiation. Botch prevents cell surface presentation of Notch by inhibiting the S1 furin-like cleavage of Notch, maintaining Notch in the immature full-length form. Understanding the function of Botch expands our knowledge regarding both the regulation of Notch signaling and the complex signaling mediating neuronal development.

Advances in Neuronal Cell Death 2007

In a stroke lesion, there is a core of necrotic cell death surrounded by a zone of tissue at risk, termed the penumbra. In the penumbra, there is usually less severe tissue damage. Programmed cell death (PCD) pathways have been documented to be present in the penumbra. While many studies have used the term apoptosis as equivalent to PCD, the absence of complete biochemical and morphological characteristics of apoptosis, coupled with the ineffectiveness of caspase inhibitors, indicates that there are other important cell death pathways activated during stroke. A few caspase-independent cell death pathways have been identified and there are likely more to be described. Stroke injury leads to the activation of a cell death program, dependent on poly(ADP-ribose) polymerase-1 (PARP1) activation1 and culminating in apoptosis inducing factor (AIF) mediated cell death.2 Poly(ADP-ribose) (PAR) polymer is the death signal in this pathway and Thanatos is the Greek personification of death and mortality, hence, the name parthanatos. In this form of cell death that occurs in many different organ systems during ischemia-reperfusion injury, PAR polymer translocates from the nucleus to the cytoplasm and mitochondria. This leads to release of AIF from the mitochondria, an effect that is inhibited by poly(ADP-ribose) glycohydrolase (PARG), an enzyme that degrades PAR polymer.3 After cerebral focal ischemia reperfusion injury, mice with reduced PARG levels had higher infarct volumes than controls. On the other hand, mice overexpressing PARG had significantly lower infarct volumes. Combined, the current data indicate that PAR is a powerful death signal. What is not yet understood are the mechanisms of PAR translocation out of the nucleus and PAR-mediated AIF release from the mitochondria. Parthanatos is caspase-independent and is biochemically and morphologically distinct from apoptosis.4 Regulation of PAR signaling may be a useful therapeutic intervention to reduce stroke-mediated damage. Additional …

Antidepressant action of ketamine via mTOR is mediated by inhibition of nitrergic Rheb degradation

As traditional antidepressants act only after weeks/months, the discovery that ketamine, an antagonist of glutamate/N-methyl-d-aspartate (NMDA) receptors, elicits antidepressant actions in hours has been transformative. Its mechanism of action has been elusive, though enhanced mammalian target of rapamycin (mTOR) signaling is a major feature. We report a novel signaling pathway wherein NMDA receptor activation stimulates generation of nitric oxide (NO), which S-nitrosylates glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nitrosylated GAPDH complexes with the ubiquitin-E3-ligase Siah1 and Rheb, a small G protein that activates mTOR. Siah1 degrades Rheb leading to reduced mTOR signaling, while ketamine, conversely, stabilizes Rheb that enhances mTOR signaling. Drugs selectively targeting components of this pathway may offer novel approaches to the treatment of depression.

Inositol polyphosphate multikinase is a transcriptional coactivator required for immediate early gene induction

Significance The induction of immediate early genes (IEGs) by neural stimuli underlies much of the plasticity of brain function, but regulatory mechanisms have been obscure. Inositol polyphosphate multikinase (IPMK) is a notably pleiotropic enzyme that displays inositol phosphate kinase activity and phosphatidylinositol kinase activity and exhibits physiologically noncatalytic actions such as stabilizing the mammalian target of rapamycin complex 1 complex. We report that IPMK is required for IEG induction by neural activation and neurotrophic stimuli. We have elucidated the molecular mechanisms responsible for IPMK influences; namely, that it enhances the transcriptional coactivation ability of Creb-binding protein (CBP). This epigenetic regulation of IEGs may have both neural and nonneural implications, as IPMK and CBP are broadly expressed in a variety of tissues. Profound induction of immediate early genes (IEGs) by neural activation is a critical determinant for plasticity in the brain, but intervening molecular signals are not well characterized. We demonstrate that inositol polyphosphate multikinase (IPMK) acts noncatalytically as a transcriptional coactivator to mediate induction of numerous IEGs. IEG induction by electroconvulsive stimulation is virtually abolished in the brains of IPMK-deleted mice, which also display deficits in spatial memory. Neural activity stimulates binding of IPMK to the histone acetyltransferase CBP and enhances its recruitment to IEG promoters. Interestingly, IPMK regulation of CBP recruitment and IEG induction does not require its catalytic activities. Dominant-negative constructs, which prevent IPMK-CBP binding, substantially decrease IEG induction. As IPMK is ubiquitously expressed, its epigenetic regulation of IEGs may influence diverse nonneural and neural biologic processes.

Cocaine elicits autophagic cytotoxicity via a nitric oxide-GAPDH signaling cascade

Significance Cocaine is one of the most abused drugs in modern society, with overdoses that are frequently lethal. Molecular mechanisms underlying its toxic actions have been obscure. The present study demonstrates that cocaine’s cellular toxicity involves a signaling cascade that utilizes the gasotransmitter nitric oxide, which leads to autophagy, a cellular modification that can cause cell death. Thus, manipulations that impair nitric oxide signaling and autophagy diminish cytotoxic actions of cocaine. By contrast, alterations of apoptosis and other nonautophagic modes of cell death are ineffective. Therapies directed toward the autophagic process may be beneficial in treating cocaine neurotoxicity. Cocaine exerts its behavioral stimulant effects by facilitating synaptic actions of neurotransmitters such as dopamine and serotonin. It is also neurotoxic and broadly cytotoxic, leading to overdose deaths. We demonstrate that the cytotoxic actions of cocaine reflect selective enhancement of autophagy, a process that physiologically degrades metabolites and cellular organelles, and that uncontrolled autophagy can also lead to cell death. In brain cultures, cocaine markedly increases levels of LC3-II and depletes p62, both actions characteristic of autophagy. By contrast, cocaine fails to stimulate cell death processes reflecting parthanatos, monitored by cleavage of poly(ADP ribose)polymerase-1 (PARP-1), or necroptosis, assessed by levels of phosphorylated mixed lineage kinase domain-like protein. Pharmacologic inhibition of autophagy protects neurons against cocaine-induced cell death. On the other hand, inhibition of parthanatos, necroptosis, or apoptosis did not change cocaine cytotoxicity. Depletion of ATG5 or beclin-1, major mediators of autophagy, prevents cocaine-induced cell death. By contrast, depleting caspase-3, whose cleavage reflects apoptosis, fails to alter cocaine cytotoxicity, and cocaine does not alter caspase-3 cleavage. Moreover, depleting PARP-1 or RIPK1, key mediators of parthanatos and necroptosis, respectively, did not prevent cocaine-induced cell death. Autophagic actions of cocaine are mediated by the nitric oxide-glyceraldehyde-3-phosphate dehydrogenase signaling pathway. Thus, cocaine-associated autophagy is abolished by depleting GAPDH via shRNA; by the drug CGP3466B, which prevents GAPDH nitrosylation; and by mutating cysteine-150 of GAPDH, its site of nitrosylation. Treatments that selectively influence cocaine-associated autophagy may afford therapeutic benefit.

Human GAPDH Is a Target of Aspirin’s Primary Metabolite Salicylic Acid and Its Derivatives

The plant hormone salicylic acid (SA) controls several physiological processes and is a key regulator of multiple levels of plant immunity. To decipher the mechanisms through which SA’s multiple physiological effects are mediated, particularly in immunity, two high-throughput screens were developed to identify SA-binding proteins (SABPs). Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) from plants (Arabidopsis thaliana) was identified in these screens. Similar screens and subsequent analyses using SA analogs, in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology, established that human GAPDH (HsGAPDH) also binds SA. In addition to its central role in glycolysis, HsGAPDH participates in several pathological processes, including viral replication and neuronal cell death. The anti-Parkinson’s drug deprenyl has been shown to suppress nuclear translocation of HsGAPDH, an early step in cell death and the resulting cell death induced by the DNA alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine. Here, we demonstrate that SA, which is the primary metabolite of aspirin (acetyl SA) and is likely responsible for many of its pharmacological effects, also suppresses nuclear translocation of HsGAPDH and cell death. Analysis of two synthetic SA derivatives and two classes of compounds from the Chinese medicinal herb Glycyrrhiza foetida (licorice), glycyrrhizin and the SA-derivatives amorfrutins, revealed that they not only appear to bind HsGAPDH more tightly than SA, but also exhibit a greater ability to suppress translocation of HsGAPDH to the nucleus and cell death.

Huntington’s disease: Neural dysfunction linked to inositol polyphosphate multikinase

Significance Huntington’s disease (HD) is a progressive neurodegenerative disorder affecting the striatum. The striatal-enriched transcription factor COUP-TF-interacting protein 2 (Ctip2) is depleted in HD and has been identified as a putative transcription factor for the enzyme inositol polyphosphate multikinase (IPMK). IPMK displays soluble inositol phosphate kinase activity, lipid kinase activity, and several noncatalytic activities including its role as a transcriptional coactivator. We describe severe depletion in IPMK protein in HD patients and several animal and cell models of the disease. IPMK overexpression rescues the metabolic impairments in a cell model of HD. Furthermore, delivery of IPMK in a transgenic HD model improves pathological changes and motor performance. The Ctip2–IPMK–Akt signaling pathway provides a previously unidentified therapeutic target for HD. Huntington’s disease (HD) is a progressive neurodegenerative disease caused by a glutamine repeat expansion in mutant huntingtin (mHtt). Despite the known genetic cause of HD, the pathophysiology of this disease remains to be elucidated. Inositol polyphosphate multikinase (IPMK) is an enzyme that displays soluble inositol phosphate kinase activity, lipid kinase activity, and various noncatalytic interactions. We report a severe loss of IPMK in the striatum of HD patients and in several cellular and animal models of the disease. This depletion reflects mHtt-induced impairment of COUP-TF-interacting protein 2 (Ctip2), a striatal-enriched transcription factor for IPMK, as well as alterations in IPMK protein stability. IPMK overexpression reverses the metabolic activity deficit in a cell model of HD. IPMK depletion appears to mediate neural dysfunction, because intrastriatal delivery of IPMK abates the progression of motor abnormalities and rescues striatal pathology in transgenic murine models of HD.