Brain Ischemia 102

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How do I use the Ischemia 102 Flashcards?

Back 1

These flashcards are meant to be a continuation of the introduction to brain ischemia you worked through in the electronic syllabus. At first, work the cards like a textbook, just reading through them. After a week or three, you'll find that you'll start to transition to using the cards to quiz yourself, and the answers will come faster and easier. Keep reading papers and thinking about this stuff as you're working at the bench. Pretty soon you'll have your head wrapped around the fundamentals of the field.

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Please set your preferences to show cards in standard order.

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Okay!

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EXCITOTOXICITY

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Excitotoxicity describes a series of events that begins during ischemia and continues through reperfusion. Ischemia results in massive neuronal depolarization, release of excitatory neurotransmitters, and a huge "spike" in intraneuronal Ca2+ concentrations, triggering multiple cytopathological processes.

Front 4

Which receptors are relevant to glutamate-induced excitatory synaptic transmission?

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These are ionotropic receptors sensitive to NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) or kainate.

Front 5

Briefly describe the structure of the NMDA receptor.

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Pentameric NMDA receptors consists of the “fundamental” subunit NMDAR1 and modulatory subunits NDMAR2A-2D.

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NMDA receptors are permeable to ______.

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Ca2+, Na+, K+.

Front 7

What happens to NMDA receptors when the plasma membrane is depolarized?

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The NMDA receptors can be fully activated by depolarization and the concommitant abolition of receptor inhibition by Mg2+.

Front 8

How does NMDA activation contribute to post-ischemic neuronal death?

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By contributing to a “spike” in intracellular [Ca2+] which triggers excitotoxicity, and by permitting Na+ influx which contributes to neuronal edema.

Front 9

Describe the AMPA receptor.

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AMPARs are non-NMDA-type ionotropic transmembrane receptor for glutamate that mediate fast synaptic transmission in the central nervous system (CNS). Its name is derived from its ability to be activated by the artificial glutamate analog, AMPA. AMPARs are found in many parts of the brain and are the most commonly found receptor in the nervous system.AMPA receptors are composed of combinations of GluR1-GluR4 subunits, and are permeable to Na+ and K+.

Front 10

What is the permeability of the AMPA receptor to Ca2+?

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Depends! Most neurons express a GluR2 subunit that renders the receptor impervious to Ca2+ -- but SVNs do not.

Front 11

What is the key temporal difference between NMDA- and AMPA-mediated excitotoxicity?

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NMDA receptors require as little as 3 min of activation to trigger neuronal death, while AMPA receptros require more prolonged activation (>1h).

Front 12

Describe the neuronal Ca2+ gradient.

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Cytoplasmic free [Ca2+] is on the order of 100nM, whereas the extracellular [Ca2+] is on the order of 1-2mM – a gradient of 1 to 10,000.

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How are intraneuronal levels of Ca2+ maintained?

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1. ligand-operated receptor or voltage-gated Ca2+ channels, 2. release of Ca2+ from the ER through stimulation of IP3 receptors 3. release of Ca2+ from the mitochondria throu Na+-Ca2+ exchanger, 4. extrusion of Ca2+ through Ca2+-ATP’ase or via th Na+- Ca2+ exchanger on the plasmalemma, 5. binding to globular proteins and 6. sequestration in ER or mitochondria.

Front 14

What are the principle mechanisms by which Ca2+ overload contributes to neuronal death after ischemia?

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1. Calpain activation, 2. activation of phospholipases (which provide the kindling for generation of ROS upon reperfusion) 3. activation of Ca2+-dependent protein kinases, 4. activation of Ca2+-dependent endonucleases, 5. induction of mitochondrial stress

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CALPAINS

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Activation of calpain occurs during ischemia, and contributes greatly to neuronal damage during ischemia and reperfusion.

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What are calpains?

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A family of Ca2+-activated neutral cysteine proteases.

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What is the physiologic role of the calpains?

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The physiologic role of calpain seems to be in modulating neurite outgrowth and remodeling of synapses. Synaptic activity, of course, is coupled to calcium flux across the membrane, and so it's easy to see how a protease linked to calcium flux could be useful in the controlled deconstruction of synaptic architecture.

Front 18

What are the two major isoforms of calpain and where are they located?

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Calpain I (μ calpain) and calpain II (m-calpain). Calpain I predominates in neurons, while calpain II is more concentrated in glia.

Front 19

Upon activation, what is the first thing calpain cuts?

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Itself—calpain is activated via autoproteolysis.

Front 20

What are the important molecular weights for calpains?

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Calpains consist of an 80kDa catalytic subunit and a 30 kDa regulatory subunit.

Front 21

What are substrates for activated calpain?

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Fodrin, spectrin, Ca2+-ATP’ase, PKC, Bax(?), caspases, NFκB, certain eIFs, and others.

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T or F: blockade of calpain with specific inhibitors has been shown to protect neurons from glutamate toxicity.

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False.

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Is there any relationship between calpain and selective vulnerability?

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Perhaps. There is some evidence that neurons richest in calpain are those most vulnerable to brain insult.

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PHOSPHOLIPASES

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These are enzymes that split free fatty acids out of membrane phospholipids.

Front 25

Describe the activation of phospholipases after an ischemic brain insult.

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The Ca2+ spike induces translocation of phospholipase A2 (cPLA2) to the plasma membrae, where it catalyzes the formation of FFAs, particularly arachidonate (AA) from glycerophospholipids.

Front 26

Cite some evidence for the participation of cPLA2 in ischemic neuronal death.

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Activation of cPLA2 has been documented in vitro after glutabmate stress. Furthermore, cPLA2(-/-) animals are resistant to ischemic injury. Direct application of cPLA2 induces neuronal injury and death in vitro.

Front 27

Besides AA, list some of the potentially neurotoxic products of cPLA2 activation during ischemia and reperfusion.

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Prostaglandins, leukotrienes, ROS, lysophospholipids, and platelet activating factor.

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Ca2+ - dependent kinases

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They're Ca2+-dependent. And they're kinases.

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CaMKII

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Calcium-Calmodulin Kinase II.

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What is CaMK II?

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This is a subset of the Ca2+ /calmodulin-dependent protein kinases, or CAMKs. These are serine-threonine kinases modulated by the Ca2+ /calmodulin complex. CaMKII are are multifunctional CaM kinases which play a role in many processes, such as neurotransmitter secretion, transcription factor regulation, and glycogen metabolism. Between 1% and 2% of the proteins in the brain are CaM kinase II.

Front 31

Discuss calmodulin.

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Calmodulin (CaM) is a calcium-binding protein expressed in all eukaryotic cells. It can bind to and regulate a number of different protein targets, thereby affecting many different cellular functions. CaM mediates processes such as inflammation, metabolism, apoptosis, muscle contraction, intracellular movement, short-term and long-term memory, nerve growth and the immune response. CaM is expressed in many cell types and can have different subcellular locations, including the cytoplasm, within organelles, or associated with the plasma or organelle membranes. Many of the proteins that CaM binds are unable to bind calcium themselves, and as such use CaM as a calcium sensor and signal transducer. CaM can also make use of the calcium stores in the endoplasmic reticulum, and the sarcoplasmic reticulum. CaM undergoes a conformational change upon binding to calcium, which enables it to bind to specific proteins for a specific response. CaM can bind up to four calcium ions, and can undergo post-translational modifications, such as phosphorylation, acetylation, methylation and proteolytic cleavage, each of which can potentially modulate its actions.

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Discuss the autoregulation and activation of the CaMKs.

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The CaM kinases consist of an N-terminal catalytic domain, a regulatory domain, and an association domain. In the absence of Ca2+/calmodulin, the catalytic domain is autoinhibited by the regulatory domain, which contains a pseudosubstrate sequence. Several CaM kinases aggregate into a homooligomer or heterooligomer. Upon activation by Ca2+/calmodulin, the activated CaM kinases autophosphorylate each other in an intermolecular reaction. This has two effects: (1) an increase in affinity for the calmodulin complex, prolonging the time the kinase is active. (2) Continued activation of the phosphorylated kinase complex even after the calmodulin complex has dissociated from the kinase complex, which prolongs the active state even more.

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What is the effect of glutamate on CaMKII?

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Adminsitration of glutamate results in Ca2+-dependent inhibition or activation of CaMKII in corcial and hippcampal neurons.

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What is the role of CaMKII in ischemic neuronal death?

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Homozygous knockout mice lacking the alpha subunit of CaMKII are more sensitive to hypoxic/ischemic insults. CaMKII inhibitors such as KN62 decrease neuronal death.

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MAPK

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The Mitogen-Activated Protein Kinases.

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What are MAPKs?

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Mitogen-activated protein (MAP) kinases are serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens) and regulate various cellular activities, such as gene expression, mitosis, differentiation, and cell survival/apoptosis.

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What are some of the major members of the MAPK family?

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Extracellular signal-regulated kinases (ERKs) 1 and 2; c-JUN N-terminal kinase (JNK); stress-activated protein kinase (SAPK), and p38, among others.

Front 38

Discuss the relevance of MAPKs in neuronal ischemia.

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Excitotoxins can activate major memebers of the MAPK family. SAPK and p38 both act as mediators of apoptosis. ERK inhibitors are known to decrease infarct volumes.

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ENDONUCLEASES

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As their name suggests, these are enzymes that cleave DNA.

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Describe how endonucleases get their freak on in ischemia/reperfusion.

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Intraneuronal Ca2+ overload activates Ca2+/Mg2+ dependent nucleases. Increaed acidity can activate DNAase II.

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Describe the classic DNA cleavage pattern manifested by activation of endonucleases.

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These enzymes cleave internucleosomal DNA, resulting in in characteristic fragments in multiples of 200 bp. This results in the classic DNA "ladder" on gel electrophoresis.

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True or false: administration of excitotoxic neurotransmitters to neuronal cultures or brain results in DNA laddering.

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True.

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True or false: DNA laddering is produced even in the absence of caspases.

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True.

Front 44

How do caspaces induce DNA laddering?

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Caspases can cleave ICAD, the Inhibitor of CAD. CAD is the Caspase-Activated DNAase. When ICAD is cleaved, CAD is released, and a nuclear localization signal brings CAD into the nucleus, where it cleaves DNA into nucleosomal fragments.

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True or false: excitotoxins produce DNA ladders primarily via caspase activation and CAD release.

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False. Maybe.

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NFκB

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NFκB appears to play an important role in neuronal survival and death.

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What is NFκB?

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NFκB is a transcription factor that is activated by cytokines. NFκB activation has been documented to enhance neuronal survival by inhibiting apoptosis. Conversely, inhibition of NFκB makes cells more sensitive to apoptosis. On the other hand, some investigators have reported that NFκB contributes to excitotoxicity. These results are not without controversy.

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________is a "super-repressor" of NFκB function.

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IκB-alpha protein.

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OXIDATIVE STRESS

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Oxidative stress is an important component of the cytopathology of brain ischemia.

Front 50

Define "oxidative stress."

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Simply put, oxidative stress is the excess accumulation of pro-oxidants over antioxidants. In chemical terms, oxidative stress is a large increase (becoming less negative) in the cellular reduction potential, or a large decrease in the reducing capacity of the cellular redox couples, such as glutathione or NADH. Put yet another way, oxidative stress is caused by an imbalance between the production of reactive oxygen and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage.

Front 51

What percentage of inspired 02 is ultimately consumed by the brain?

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As much as 20%.

Front 52

How does ischemia and reperfusion result in oxidative stress.

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Multiple pathways are involved. Excitotoxic calcium dumping activates phospholipases during ischemia. The resultant FFAs are metabolized upon reperfusion, with production of superoxide, which generates secondary free radicals such as peroxynitrite. Calcium may activate other prooxidant enzyme systems, such as xanthine oxidase and nitric oxide synthase. Mitochondrial stress from Ca2+ accumulation and hypoxia results in increased "leakage" of superoxide as well. Transition metals such as Fe2+, Cu2+, and Zn2+ may contribute to generation of free radicals.

Front 53

How is superoxide "leakage" from mitochondria usually removed?

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MnSOD usually clears superoxide to H202. Glutathione peroxidase is another important antioxidant system in neurons.

Front 54

Summarize the processes by which mitochondria contribute to oxidative stress after ischemia.

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Excitotoxicity leads to accumulation of calcium in the mitochondria and collapse of the mitochondrial membrane potential. This stops the flow of electrons, leading to production of superoxide. Mitochondrial dysfunction leads to a transient spike in NADH, followed by a gradual loss of adenine dinucleotides, which has an effect on the NAD+/NADH redox pair. ROS further inhibit the electron transport system.

Front 55

How does xanthine oxidase get activated?

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Glutamate administration results in activation of xanthine oxidase via calcium-dependent proteases. Activated calpain xanthine dehydrogenase to xanthine oxidase.

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What does xanthine oxidase do?

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Catalyzes the oxidation of xanthine and hypoxanthine to uric acid. This reaction produces superoxide as a by-product.

Front 57

True or false: transition metals such as copper and iron are not particularly abundant in the brain.

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False. These species are abundant in brain (0.1-0.5mM.

Front 58

How do transition metals lead to ROS production?

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Iron-catalyzed or copper-catalyzed Haber-Weiss reactions that generate hydroxyls from superoxide and peroxide.

Front 59

Describe the Haber-Weiss reaction.

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Haber-Weiss reaction generates •OH (hydroxyl radicals) from H2O2 (hydrogen peroxide) and superoxide (•O2-). This reaction can occur in cells and is therefore a possible source for oxidative stress. The reaction is very slow, but is catalyzed by iron. The first step of the catalytic cycle involves reduction of ferric ion to ferrous: Fe3+ + •O2− → Fe2+ + O2 The second step is the Fenton reaction: Fe2+ + H2O2 → Fe3+ + OH− + •OH Net reaction:•O2- + H2O2 → •OH + HO- + O2 The reaction is named after Fritz Haber and his student Joseph Weiss.

Front 60

What happens to iron during ischemia?

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During ischemia, Fe2+ is released from iron-binding proteins, to participate in Haber-Weiss and Fenton reactions.

Front 61

What is the role of Zn2+ in cerebral ischemia?

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Zn2+ mediates death of neuronal and nonneuronal cells in ischemia and other neurologic injury patterns.

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Where is Zn2+ stored and how is it mobilized in the central nervous system?

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Zn2+ is stored in the presynaptic vesicles of glutaminergic neurons, released with glutamate in an activity-dependent manner, and translocated into adjacent neurons.

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How does Zn2+ enter into target neurons?

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Through voltage-gated calcium channels (VGCC), NMDA or AMPA/kainate glutamate receptors permeable Ca2+, Na+/Ca2+ exchanger, or Zn2+ transportes.

Front 64

How does Zn2+ mediate the production of ROS?

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Appears to work in part through activation of cyclooxygenases and PKC.

Front 65

What are the three isoforms of NOS? What does NOS do?

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iNOS, eNOS and nNOS. NOS catalyzes the conversion of arginine to NO and citrulline.

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nNOS and eNOS are both _________-dependent NOS isoforms.

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Ca2+/calmodulin-dependent, meaning they are both activated by increased Ca2+.

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How is iNOS activated?

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Inducible NOS is increased in astrocytes and microglia by cytokines such as IL-1, IL-2, IF-gamma, TNF, etc.

Front 68

Discuss the activation of nNOS and its contribution to neuronal injury.

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nNOS is activated by increased Ca2+ via Ca2+/calmodulin and generates NO, which can diffuse freely across membranes. NO can combine with superoxide to form the highly reactive species peroxynitrite, which causes nitration of DNA and protein as well as oxidation of DNA, lipids and proteins. ONOO may activate PARP synthetase, leading to depletion of NADH and inhibition of mitochondrial respiration.

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ONOO eventually breaks down into________.

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Hydroxyl radical and nitrogen dioxide.

Front 70

What is the role of eNOS?

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eNOS is produced in endothelial cells and mediates vasodilation. This improves perfusion and limits microthrombosis, and is neuroprotective.

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What is the role iNOS?

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iNOS is increased in glial cells and neutrophils over several days after reperfusion. The NO produced by iNOS diffuses across membranes and increases NO-mediated damage in the ischemic area.

Front 72

Summarize the effect of the various NOS isoforms on neuroprotection in ischemia.

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eNOS - neuroprotective in ischemia iNOS - neurotoxic in ischemia nNOS - neurotoxic in ischemia

Front 73

Describe the metabolism of arachidonic acid.

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Arachidonate is produced during ischemia secondary to excitotoxic induction of PLA2. Upon reperfusion, AA is converted into eicosanoids by cyclooxygenase and lipoxygenase, with concominant production of superoxide.

Front 74

What is the role of COX-2 in brain ischemia and reperfusion?

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COX-2 activity appears to be enhanced in post-ischemic brain, and to depend on activation of NMDA receptors in neurons. COX-2 inhibition appears to ameliorate ischemic injury.

Front 75

Discuss how ROS may act as mediators of apoptotic or oncotic cell death.

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We know that ROS are produced in the process of apoptotic cell death, and that ROS scavengers prevent apoptosis. Administration of prooxidants can trigger apoptotic processes. ROS are both products and instigators of mitochondrial stress and may well be involved in the egress of cytochrome c that activates intrinsic apoptosis.

Front 76

Discuss the limitation of antioxidant therapy for ischemic injury.

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Results of trials have been disappointing. Trilizad mesylate, a lipid peroxidation inhibitor, and ebselen, a selenoorganic, failed to improve outcome in stroke patients. NXY-059, a PBN-based spin-trap, seemed to show an effect in the SAINT trial, but failed in the SAINT-2 trial, although this may be due to on-the-shelf "decay" of the compound during a delay before study. In the final anaylysis, we may find that ROS therapy is necessary but not sufficient for treatment because of the contribution of other complex pathways.

Front 77

APOPTOSIS

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Apoptosis, also called program cell death, probably plays a role in ischemic neuronal death, although it does not generally display classic phenotypes in this system.

Front 78

What are the major genetic homologies between apoptosis in C. elegans and mammalian systems?

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Ced 3 is a caspase homolog. Ced 4 is the Apaf-1 homolog. Ced 9 is the Bcl family homolog.

Front 79

True or false: inhibitors of caspases or Bcl-2 overexpression attenuates neuronal death after both global and focal ischemia.

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True.

Front 80

What are factors that have been hypothesized to trigger apoptosis after an ischemic injury?

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pH, calcium flux, Fas receptor activation, oxidative stress, mitochondrial energy failure, altered gene expression, calpain activation (with cross-talk to apoptotic processes), MAPK signalling processes, decreased growth factor "tone," HIF1-P53 activation, and others. Bottom line: we just don't know yet.

Front 81

Discuss postulated mechanisms of pH-mediated activation of apoptosis.

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Intracellular neuronal acidosis could have an effect on ΔΨm, leading (through an unspecified mechanism) to egress of cytochrome c. Intracellular acidosis has been shown to to cause apoptosis in cultured neurons.

Front 82

What role do growth factors play in the hypoxia-apoptosis hypothesis?

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Growth factors (and certain members of Bcl-2 family) prevent cytosolic acidification (Furlong et al, Reynolds et al).

Front 83

What is the role of calcium imbalance in triggering apoptosis?

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Accumulation of Ca2+ in neuronal cytoplasm is sufficient to trigger apoptosis. Potential mediators include calpain, Ca2+-induced mitochondrial dysfunction, phospholipase activation, and Ca2+-dependent endonucleases. and DNAase II.

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What is the Fas receptor?

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The death receptor Fas (CD95 or APO-1) is a member of the tumor necrosis factor (TNF) receptor superfamily. This receptor is activated by the Fas ligand, either by autocrine or paracrine activation.

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Describe the activation process of the Fas receptor.

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Ligand binding brings about association of the intracellular part of the Fas receptor with procaspase-8 with the Death Domain FADD. This leads to procaspase activation and a caspase cascade, a process called extrinsic apoptosis.

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What is the role of Fas in ischeic neuronal death?

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Neuronal cells that have been deprived of trophic factors activate extrinsic apoptosis in a Fas-dependent manner. Expression of Fas and Fas ligand has been shown to increase in neuronal tissue after ischemia. In both focal and global ischemia, recruitment of Fas ligand to the recptor has been demonstrated in both cerebral cortex and hippocampus. Mice deficient in Fas are relatively resistant to ischemic insult.

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Discuss the process of intrinsic apoptosis.

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The critical event in intrinsic apoptosis is the release of cytochrome c from intermembranous space in the mitochondria. Assembly of Bax pores on the outer mitochondrial membrane appears to be a necessary event for the release of cytochrome c, but it is unclear whether Bax pore formation is sufficient. Once released into the cytoplasm, cytochrome c binds to the ced 4 analog Apaf-1, which causes Apaf-1 to undergo a conformational change and expose its CARD death domain. Through a protein-protein interaction, the cytochrome c-Apaf-1 complex recruits a CARD domain on procaspase 9, resulting in its activation. The cytochrome c-Apaf-1-caspase 9 complex is called the "apoptosome." The apoptosome cleaves downstreame executor caspases, leading to a caspase cascade and apoptotic cell death.

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How do MAPKs participate in the execution of ischemic neuronal apoptosis?

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Among the MAPKs, JNK and p38 are known to be activated and execute apoptotic death of neurons during trophic factor withdrawl or exposure to pro-apoptogenic factors. P38 inhibitors attenuate neuronal death after ischemia. Furthermore, JNK and p38 appear to inhibit antiapoptotic Bcl-2 family proteins, promoting intinsic apoptotis via release of cytochrome c.

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What are the Bcl-2 family proteins?

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These are ced-9 homologs, and comprise a family of proteins that undergo complex protein-protein interactions. Some members of this family, such as Bcl-2, Bcl-XL, and Mcl, are anti-apoptotic, while others (BH3-only members) such as Bad, Bid, Bim, Bax and Bak, are pro-apoptotic. The relative abundance of pro-apoptotic and anti-apoptotic Bcl-2 family proteins, and their complex interplay with each other, has a profound impact on whehter the cell survives or succumbs to apoptotic processes.

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Describe the struture of Bcl-2 family proteins.

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The members of the Bcl-2 family share one or more of the four characteristic domains of homology entitled the Bcl-2 homology (BH) domains (named BH1, BH2, BH3 and BH4). The anti-apoptotic Bcl-2 proteins, such as Bcl-2 and Bcl-xL, conserve all four BH domains. The BH domains also serve to subdivide the pro-apoptotic Bcl-2 proteins into those with several BH domains (e.g. Bax and Bak) or those proteins that have only the BH3 domain (e.g. Bid, Bim and Bad). The Bcl-2 family has a general structure that consists of a hydrophobic helix surrounded by amphipathic helices. Many members of the family have transmembrane domains. The site of action for the Bcl-2 family is mostly on the outer mitochondrial membrane. Within the mitochondria are apoptogenic factors (cytochrome c, Smac/DIABLO, Omi) that if released activate the executioners of apoptosis, the caspases. Depending on their function, once activated, Bcl-2 proteins either promote the release of these factors, or keep them sequestered in the mitochondria. Whereas the activated pro-apoptotic Bak and/or Bax would form MAC and mediate the release of cytochrome c, the anti-apoptotic Bcl-2 would block it, possibly through inhibition of Bax and/or Bak.

Front 91

Who is Bcl-2?

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Bcl-2 derives its name from B-cell lymphoma 2, as it is the second member of a range of proteins initially described as a reciprocal gene translocation in chromosomes 14 and 18 in follicular lymphomas. This protein and its close relatives, Bcl XL, Mcl, maintain all of the BH3 domains, and work (at least in part) by "sequestering" pro-apoptotic proteins like Bax and Bak.

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Who is Bax?

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Bax is the Bcl-2 Associated X Protein, contain the BH1, BH2 and BH3 and transmembrane domains. In healthy mammalian cells, the majority of Bax is found in the cytosol, but upon initiation of apoptotic signaling, Bax undergoes a conformation shift, and inserts into organelle membranes, primarily the outer mitochondrial membrane.[2] Bax is believed to interact with, and induce the opening of the mitochondrial voltage-dependent anion channel, VDAC. Alternatively, growing evidence suggest that activated Bax and/or Bak form an oligomeric pore, MAC in the outer membrane. This results in the the release of cytochrome c and other pro-apoptotic factors from the mitochondria, often referred to as mitochondrial outer membrane permeabilization, leading to activation of caspases. This defines a direct role for Bax in mitochondrial outer membrane permeabilization, a role common to the Bcl-2 proteins containing the BH1, BH2 and BH3 domains.

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What is a key transcriptional factor in the upregulation of Bax expression?

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The expression of BAX is upregulated by the tumor suppressor protein p53, and Bax has been shown to be involved in p53-mediated apoptosis. The p53 protein is a transcription factor that, when activated as part of the cell's response to stress, regulates many downstream target genes, including BAX. However, p53 also has a transcription-independent role in apoptosis. In particular, p53 interacts with Bax, promoting Bax activation and the insertion of Bax into the mitochondrial membrane.

Front 94

Who is Bak?

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The Bcl-2 homologous Antagonist/Killer (BAK) protein is a pro-apoptotic member of the Bcl-2 gene family which is involved in initiating apoptosis. BH1, BH2 and BH3 and transmembrane domains are all present in Bak. Bak appears in some in vitro systems to form heterooligomers with Bax to form pores in the mitochondrial membrane.

Front 95

Who are Bik and Bim?

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These are BH3-only proteins which also contain a transmembrane domains. Bim in particular may be required for Bax oligomerization.

Front 96

Who is Bad?

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Bad is a BH3-only protein that is normally phosphorylated on two residues. When phosphorylated by Akt, it is sequestered in the cytosol by 14-3-3 proteins. When it is dephosphorylated (as during apoptosis), it can interfere with the anti-apoptotic activity of Bcl-2. Work in our lab with Bad failed to uncover any significant change in its phosphorylation state after transient global brain ischemia.

Front 97

What is MAC?

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Aka "MAC the knife," the Mitochondrial Apoptosis-Induced Channel, is an early marker of the onset of apoptosis. This ion channel is formed on the outer mitochondrial membrane in response to certain apoptotic stimuli. MAC activity is detected by patch clamping mitochondria from apoptotic cells at the time of cytochrome c release. Members of the Bcl-2 protein family regulate apoptosis by controlling the formation of MAC: the pro-apoptotic members Bax and/or Bak form MAC, whereas the anti-apoptotic members like Bcl-2 or Bcl-xL prevent MAC formation. Once formed, MAC mediates the release of cytochrome c to the cytosol, triggering the commitment step of the mitochondrial apoptotic cascade.

Front 98

GROWTH FACTOR SIGNALING AND APOPTOSIS

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Growth factor signal transduction is an important mechanism for countering cell death processes.

Front 99

What are growth factors?

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The term growth factor refers to a naturally occurring protein capable of stimulating cellular proliferation and cellular differentiation. Growth factors are important for regulating a variety of cellular processes. From the point of view of iscemia and reperfusion, growth factors are important because they are strong antagonists of apoptotic processes.

Front 100

Differentiate between growth factors and cytokines.

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Growth factor is sometimes used interchangeably among scientists with the term cytokine. Historically, cytokines were associated with hematopoietic (blood forming) cells and immune system cells. While growth factor implies a positive effect on cell division, cytokine is a neutral term with respect to whether a molecule affects proliferation. In this sense, some cytokines can be growth factors, such as G-CSF and GM-CSF. However, some cytokines have an inhibitory effect on cell growth or proliferation. Yet others, such as Fas ligand are used as "death" signals; they cause target cells to undergo programmed cell death or apoptosis.

Front 101

Examples of growth factors.

Back 101

Individual growth factor proteins tend to occur as members of larger families of structurally and evolutionarily related proteins. There are dozens and dozens of growth factor families such as insulin, IGF-1 (insulin-like growth factor-1)TGF-beta (transforming growth factor-beta), BMP (bone morphogenic protein), neurotrophins (NGF, BDNF, and NT3), fibroblast growth factor (FGF), and so on.

Front 102

Growth factor receptors.

Back 102

Growth factor receptors are characterized by extracellular peptide hormone binding domains and intracellular tyrosine kinase domains. For most growth factors, binding to the receptor induces homo- or heterodimerization of the receptor, with autophosphorylation of the tyrosine kinase domain. Proteins contains SH-2 domains bind to the phosphotyrosine residues, and this binding transduces the growth factor signals, which have diverse effects on metabolism, survival and gene expression.

Front 103

The insulin receptor.

Back 103

The insulin receptor is a permanent homotetramer; binding does not induce dimerization. Like other growth factor receptors, binding induces autophosphorylation at tyrosine residues. However, unlike other gf receptors, the insulin receptor does not induce much binding of SH2 proteins at its own phosphotyrosine residues. Instead, the tyrosine kinase function of the insulin receptor phosphorylates tyrosine residues on a protein called the insulin receptor substrate-1, or IRS-1 protein. SH2 proteins then bind to the phosphotyrosines on IRS-1.

Front 104

What is the role of insulin signaling in brain ischemia?

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Insulin was initially tried in the setting of experimental stroke with the idea of ameliorating injury by controlling glucose. It subsequently became apparent that insulin's neuron-sparing effect was independent of hypoglycemia. Since then, it has been shown that insulin has salutary effects on protein synthesis in brain reperfusion. Insulin and other growth factors have also been shown to induce activation of Akt, an anti-apoptotic signaling "clearinghouse" that also effects diverse other elements of cellular metabolism; and to also effect Bcl-2 family interactions and prevent release of cytochrome c from mitochondria.

Front 105

THE PI3K-AKT SIGNALING SYSTEM

Back 105

This signaling system, which is activated by insulin and other growth factors, has important survival signaling properties.

Front 106

Discuss PI3K and its activation.

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Phosphoinositide 3-kinases (PI 3-kinases or PI3Ks) are a family of related enzymes that are capable of phosphorylating the 3 position hydroxyl group of the inositol ring of phosphatidylinositol (PtdIns). PI3Ks interact with the IRS (Insulin receptor substrate) in order to regulate cellular processes through a series of phosphorylation events. The various 3-phosphorylated phosphoinositides that are produced by PI 3-kinases (PtdIns3P, PtdIns(3,4)P2, PtdIns(3,5)P2 and PtdIns(3,4,5)P3) function in a mechanism by which an assorted group of signalling proteins, containing PX domain, pleckstrin homology domains (PH domains), FYVE domains and other phosphoinositide-binding domains, are recruited to various cellular membranes. PI 3-kinases have been linked to an extraordinarily diverse group of cellular functions, including cell growth, proliferation, differentiation, motility, survival and intracellular trafficking. Many of these functions relate to the ablilty of class I PI 3-kinases to activate protein kinase B (PKB, aka Akt).

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What are important inhibitors of PI3K used in research?

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As wortmannin and LY294002 are broad inhibitors against PI 3-kinases and a number of unrelated proteins at higher concentrations they are too toxic to be used as therapeutics. A number of pharmaceutical companies have recently been working on PI 3-kinase isoform specific inhibitors including the class I PI 3-kinase, p110δ isoform specific inhibitors, IC486068 and IC87114, ICOS Corporation.

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Who is the major downstream effector of PI3K activation and how does PI3K turn it on?

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AKT is activated as a result of PI3-kinase activity, because AKT requires the formation of the PtdIns(3,4,5)P3 (or "PIP3") molecule in order to be translocated to the cell membrane. At PIP3, AKT is then phosphorylated by another kinase called PDK1, and is thereby activated. The "PI3-k/AKT" signaling pathway has been shown to be required for an extremely diverse array of cellular activities - most notably cellular proliferation and survival. In addition to AKT and PDK1, one other related serine threonine kinase is bound at the PIP3 molecule created as a resulte of PI3-kinase activity, SGK.

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Who is Akt?

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Akt1, also known as "Akt" or protein kinase B (PKB) is an important molecule in mammalian cellular signaling. It has a weight of

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What are the Akt isoforms?

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In humans, there are three genes in the "Akt family": Akt1, Akt2, and Akt3. These enzymes are members of the serine/threonine-specific protein kinase family. Akt1 is involved in cellular survival pathways, by inhibiting apoptotic processes. Akt1 is also able to induce protein synthesis pathways, and has been implicated as a major factor in many types of cancer. Akt2 is an important signaling molecule in the Insulin signaling pathway. It is required to induce glucose transport. Akt2 is more specific for the insulin receptor signaling pathway. The role of Akt3 is less clear, though it appears to be predominantly expressed in brain.

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MITOCHONDRIA

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Mitochondrial dysfunction is an area of increasing interest in brain ischemia research.

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What percentage of neuronal volume is occupied by mitochondria?

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25%

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What is the MPT?

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The Mitochondrial Permeability Transition pore is a channel in the inner membrane permeable to species less than 1,500 Da. Opening of the pore dissipates the proton gradient, depletes pyridine nucleotides from the matrix, leads to mitochondrial swelling, and results in mitochondrial energy failure, leading to cell death.

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What is the structure of the MPT?

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Though the exact structure of the MPTP is still unknown, several proteins probably come together to form the pore, including adenine nucleotide translocase (ANT), the mitochondrial inner membrane protein transporter (Tim), the protein transporter at the outer membrane (Tom), the outer membrane voltage-dependent anion channel (VDAC) and cyclophilin-D.

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What are factors that trigger MPT opening?

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Calcium, high inorganic phosphate (Pi), oxidative stress, and oxidized pyridine nucleotides.

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What is the most important trigger for MPT opening?

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Calcium.

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What factors inhibit MPT opening?

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Adenine nucleotides (ATP, ADP), magnesium, low intramitochondrial pH, and high membrane potential inhibit pore opening (Bernardi et al., 1992, 1999).

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For research purposes, what is the most important inhibitor of the MPT? How does it work?

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Cyclosporin A (CsA). Inhibits the MPT by binding to a mitochondrial cyclophilin, cyclophilin D.

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What are the most prevalent biological redox carriers? Why is this important from the point of view of mitochondrial dysfunction?

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Pyridine nucleotides are the most prevalent redox carriers in all organisms. In reperfusion, activated PARP hydrolyzes NAD and transfers the ADP-ribose to poly(ADP-ribose) on acceptor proteins, depleting cellular NAD in a situation where the lack of ATP already inhibits its synthesis. Once the [NAD] falls below a certain level, ATP production falls further. This vicious circle leads to energy failure and death.

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How does pyruvate help in the preceding circumstance?

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Pyruvate promotes the conversion of NAD+ to NADH—which can donate e- to Complex I, forestalling energy failure and perhaps breaking the vicious cycle.

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What appears to be the critical link between consumption of adenine nucleotides and apoptosis in this situation?

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PARP.

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Do mitochondria belong exclusively to the intrinsic or extrinsic pathway?

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Actually, both. Although mitochondrial dysfunction and release of cyto-c are classically associated with intrinsic apoptosis, ther eis evidence that they can be recruited into the extrinsic pathway by death receptor ligands such as FASl and TNF in some cells (including neurons).

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What are the two specific mechanisms by which apoptogenic proteins can be released from mitochondria?

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1. Selective OMM permeabilization by protein or lipid pores. 2. Rupture of the OMM.

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True or false: selective permeabilization of the OMM by Bcl-2 family proteins occurs in conjunction with MPT formation and loss of IMM integrity.

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False! Bax, Bak and a BH3-only protein like tBid are the minimum requirements for this process to occur.

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What happens to animals deficient in cyclophilin D in the setting of MCAO?

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Decreased infarct volume, probably because of an effect on the MPT. (Remember, CsA, which inhibits cylophilin D, also decreases infarct size.)

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What is Apop-1 and what does it do?

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Apop-1 is a non-Bcl-2 protein that localizes to mitochondria after injury. It appears to induce apoptosis b a Bcl-2/Bax-indepenent mechanism that is cyclosporin-dependent, suggesting that it is interacting somehow interacting with the MPT.

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What is the morphological consequence of cytochrome c relea se from mitochondria?

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Intramitochondrial sturcture appears to undergo a dramatic remodeling upon release of cyto-c. This process appears to involve tBid.

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Summarize the electron transport chain.

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The mitochondrial electron transport chain removes electrons from an electron donor (NADH or FADH2) and passes them to a terminal electron acceptor (O2) via a series of redox reactions. These reactions are coupled to the creation of a proton gradient across the mitochondrial inner membrane. There are three proton pumps: I, III, and IV. The resulting transmembrane proton gradient is used to make ATP via ATP synthase.

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Complex I

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Aka NADH dehydrogenase, removes two electrons from NADH and transfers them to a lipid-soluble carrier, ubiquinone (Q). The reduced product, ubiquinol (QH2) is free to diffuse within the membrane. At the same time, Complex I moves four protons (H+) across the membrane, producing a proton gradient. Complex I is one of the main sites at which premature electron leakage to oxygen occurs, thus being one of main sites of production of a harmful free radical called superoxide..

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Describe the flow of electrons in Complex I

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The pathway of electrons occurs as follows: NADH is oxidized to NAD+, reducing Flavin mononucleotide to FMNH2 in one two-electron step. The next electron carrier is a Fe-S cluster, which can only accept one electron at a time to reduce the ferric ion into a ferrous ion. In a convenient manner, FMNH2 can be oxidized in only two one-electron steps, through a semiquinone intermediate. The electron thus travels from the FMNH2 to the Fe-S cluster, then from the Fe-S cluster to the oxidized Q to give the free-radical (semiquinone) form of Q. This happens again to reduce the semiquinone form to the ubiquinol form, QH2. During this process, four protons are translocated across the inner mitochondrial membrane, from the matrix to the intermembrane space. This creates a proton gradient that will be later used to generate ATP through oxidative phosphorylation

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Complex II

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Aka succinate dehydrogenase. Complex II is not a proton pump. It serves to funnel additional electrons into the quinone pool (Q) by removing electrons from succinate and transferring them (via FAD) to Q. Complex II consists of four protein subunits. Other electron donors (e.g., fatty acids and glycerol 3-phosphate) also funnel electrons into Q (via FAD), again without producing a proton gradient.

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Complex III

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Complex III (cytochrome bc1 complex) removes in a stepwise fashion two electrons from QH2 and transfers them to two molecules of cytochrome c, a water-soluble electron carrier located within the intermembrane space. At the same time, it moves two protons across the membrane, producing a proton gradient (in total 4 protons: 2 protons are translocated and 2 protons are released from ubiquinol). When electron transfer is hindered (by a high membrane potential, point mutations or respiratory inhibitors such as antimycin A), Complex III may leak electrons to oxygen resulting in the formation of superoxide, a highly toxic species, which is thought to contribute to the pathology of a number of diseases, including aging.

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Complex IV

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Complex IV (cytochrome c oxidase) removes four electrons from four molecules of cytochrome c and transfers them to molecular oxygen (O2), producing two molecules of water (H2O). At the same time, it moves four protons across the membrane, producing a proton gradient.

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Oxidative Phosphorylation

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The chemiosmotic coupling hypothesis, as proposed by Nobel Prize in Chemistry winner Peter D. Mitchell, explains that the electron transport chain and oxidative phosphorylation are coupled by a proton gradient across the inner mitochondrial membrane. The efflux of protons creates both a pH gradient and an electrochemical gradient. This proton gradient is used by the FOF1 ATP synthase complex to make ATP via oxidative phosphorylation. ATP synthase is sometimes regarded as complex V of the electron transport chain. The FO component of ATP synthase acts as an ion channel for return of protons back to mitochondrial matrix. During their return, the free energy produced during the generation of the oxidized forms of the electron carriers (NAD+ and FAD—literally from “burning” NADH and FADHH) is released. This energy is used to drive ATP synthesis, catalyzed by the F1 component of the complex.

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How does the excitotoxic calcium spike effect the mitochondria?

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The postischemic ytosolic calcium spike results in accumulation of calcium in the mitochondria. An electrophoretic uniporter driven by the negative membrane potential ΔΨm pulls the calcium into the mitochondria.

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What effect does the buildup of calcium in mitochondria have on oxidative phosphorylation?

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Accumulation of calcium in the matrix results in mitochondrial depolarization and inhibition of oxidative phosphorylation.

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True or false: inhibiting mitochondrial calcium uptake mitigates Ca2+-mediated excitotoxicity.

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True, of course.

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How does excess calcium in mitochondria induce mitochondrial dysfunction?

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Accumulation of calcium inhibits oxidative phosphorylation and leads to decreased ATP, which further impairs the ATP-dependent pumps the mito needs to maintain Ca2+ homeostasis.