Major Noradrenergic, Dopaminergic and Serotonergic Pathways in the CNS

6. 비생물학적인 stresses에 대응한 복원 연구논문 review

β-Amyloid Plaques

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Neurodegenerative diseases are a varied assortment of central nervous system disorders characterized by neuronal loss and intraneuronal accumulations of fibrillary materials. Abnormal protein-protein interactions may allow the precipitation of these proteins, forming intracellular and extracellular aggregates. These abnormal interactions may play a role in the dysfunction and death of neurons in several common neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD).

AD is characterized by loss of function and death of nerve cells in the brain leading to loss of cognitive function. The cause of nerve cell death is unknown but fibrillar β-amyloid senile plaques (SP) and intraneuronal tau-rich neurofibrillary tangles (NFT) are seen. SP form by the extracellular accumulation of amyloid beta (Aβ) peptide into amyloid deposits, with the Aβ42 form being most prominant. Proteolytic enzymes β-secretase and γ-secretase sequentially cleave the β-amyloid precursor protein (APP) into Aβ. The enzyme BACE (β-site APP cleaving enzyme) has been identified as β-secretase. NFT are made up of

aggregated hyperphosphorylated tau protein. Abnormal phosphorylation, possibly caused by mutations in the tau gene, may be one of the events leading to aggregation.

The Notch-γ-Secretase Pathway

Notch activation involves the proteolytic cleavage of the Notch ligand/receptor complex by g-secretase to release the Notch intracellular domain fragment (NICD) that translocates to the nucleus and

upregulates expression of Myc, Hes1, and other upregulates expression of Myc, Hes1, and other genes.¹ When the DAOY medulloblastoma cell line was transfected with NICD2 to make Notch

signaling constitutively active, the transfected cells produced more xenograft tumors than the non- transformed DAOY cells and increased the population of both CD133⁺ and side population stem-like cells in culture. In contrast, inhibition of g-secretase reduced the side population to 0.01%

of the total cell count and inhibited by 90% the ability of cells to colonize soft agar or to form tumor xenografts in immune-compromised mice.

NICD2 transfection protected the cells from the effects of g-secretase inhibition.² Thus, in some tumor types, the inhibition of Notch signaling can deplete a population of cells that are required for tumor initiation.

Second Messenger Systems Involved in Reward and Addiction

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With repeated psychostimulant administration, these are changes in dopaminergic and glutamatergic

transmission in the nucleus accumbens. The figure depicts the major second messenger systems in the nucleus accumbens activated by dopamine and glutamate that are

influenced by acute and/or repeated psychostimulant injections.

Limbic Reward System

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This diagram represents a sagittal section of the rat brain. Highlighted are the nuclei representing the limbic

structures of the basal forebrain including the amygdala, hippocampus, prefrontal cortex (PFC), nucleus accumbens (N. Acc.), ventral pallidum (VP) and ventral tegmental area (VTA). Dopaminergic neurons in the VTA modulate information flow through the limbic circuit via projections to the nucleus accumbens, amygdala, hippocampus, PFC and VP. Increased dopaminergic transmission in limbic nuclei, particularly the nucleus accumbens, underlies the reinforcing effect of virtually every abused drug. Note that psychostimulants increase dopaminergic

transmission in areas receiving projections from the VTA via interactions with dopamine transporters.

Major Noradrenergic, Dopaminergic and Serotonergic Pathways in the CNS

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Intracellular Signaling Pathways Activated by Endothelins

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Occupation of the endothelin A receptor (ET_AR) induces activation of phospholipase C (PLC) which is central to the generation of cellular responses to endothelin 1 (ET-1). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP₂) to form diacylglycerol (DAG) and D-myo-inositol-1,4,5-trisphosphate (IP₃). IP₃ regulates intracellular Ca²⁺ by binding to the IP₃ receptor on the endoplasmic reticulum (ER) and stimulating Ca²⁺ release from the ER stores. DAG binds to and activates protein kinase C (PKC). Increases in the concentration of intracellular Ca²⁺ is critical for eliciting responses to ETs. The ET_A receptor is linked to a Ca²⁺ channel (CaCh) in the plasma membrane that opens in response to receptor occupation by ET-1 or its agonists, thus further increasing the intracellular Ca²⁺content. ETs have also been shown to open nonselective cation channels (NsCh) or chloride channels (ClCh) to induce cellular depolarization by increasing in influx of Na⁺ and efflux of Cl^–, respectively. ETs also open potassium channels (KCh) which leads to the passive efflux of K⁺ and hyperpolarization that, in turn, inhibits the CaCh and Ca²⁺ influx.

NPY Inhibition of Catecholamine Release

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Catecholamine synthesis and release are two separate but related processes that influence the level of catecholaminergic neurotransmission. Neuropeptide Y (NPY) is a 36 amino acid peptide that is highly homologous to peptide YY (PYY).

NPY exerts its various biological effects through at least six classes of receptors, designated Y₁, Y₂, Y₃, Y₄, Y₅, and Y₆. It has been demonstrated that NPY inhibits catecholamine synthesis via the Y₃ receptor subtype, in contrast to the Y₂ subtype that mediates inhibition of catecholamine release. Although inhibition of both synthesis and release by these receptor subtypes is via inhibition of Ca²⁺ entry, the two processes are associated with actions on distinct Ca²⁺ channel subtypes. Inhibition of L-type Ca²⁺ channels mediates inhibition of catecholamine synthesis, while inhibition of N-type Ca²⁺ channels inhibits catecholamine release.

Ascending Pain Pathway

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IL: intralaminar nucleus of the thalamus, VP: ventroposterior nucleus of the thalamus.

Modulation of Pain Transmission

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Modulation of pain transmission in the dorsal horn of the spinal cord, illustrating the interaction of small diameter pain afferents and large diameter mechanoreceptors (touch) afferents, as well as the influence of descending neurons on afferent pain pathways; 5HT: serotonin, NE: norepinephrine. (Adapted from Watson, C., Basic Human Neuroanatomy, An Introductory Atlas, 4th Ed., Little, Brown & Company, Boston, 1991).

Mechanism of Action of Selective

Monoaminergic Reuptake Inhibitors Figure A

During neurotransmission, monoamine neurotransmitters such as serotonin, dopamine or norepinephrine, are released into the synaptic cleft from storage vesicles present in the pre-synaptic neuron (A). The neurotransmitters then bind to their respective receptors on the post-synaptic neuron, thereby transferring the signal. The actions of the

neurotransmitters are terminated via their reuptake into the pre-synaptic neuron via specific transporter proteins following which the neurotransmitter is taken up into the vesicles for re-release. When a monoaminergic reuptake inhibitor is present (B), it binds to the monoamine transporter and blocks the reuptake of the neurotransmitter whose levels increase in the synaptic cleft thereby enhancing neurotransmission.

Mechanism of Action of Selective

Monoaminergic Reuptake Inhibitors Figure B

During neurotransmission, monoamine neurotransmitters such as serotonin, dopamine or norepinephrine, are released into the synaptic cleft from storage vesicles present in the pre-synaptic neuron (A). The neurotransmitters then bind to their respective receptors on the post-synaptic neuron, thereby transferring the signal. The actions of the

neurotransmitters are terminated via their reuptake into the pre-synaptic neuron via specific transporter proteins following which the neurotransmitter is taken up into the vesicles for re-release. When a monoaminergic reuptake inhibitor is present (B), it binds to the monoamine transporter and blocks the reuptake of the neurotransmitter whose levels increase in the synaptic cleft thereby enhancing neurotransmission.

Drug Activation of Dopaminergic Neurons

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The main mechanism responsible for the reinforcing properties of most abused drugs is the direct or indirect activation of dopaminergic neurons in the ventral tegmental area (VTA). This figure represents a dopaminergic neuron in the VTA as well as a GABAergic interneuron and a GABAergic projection neuron with a recurrent collateral. Whereas, nicotine increases the firing rate of dopaminergic neurons in the VTA by activating nicotinic acetylcholine receptors located on these cells, other drugs of abuse activate dopaminergic neurotransmission by inhibiting GABAergic transmission in the VTA. Stimulation of m opioid receptors, GABA_A receptors, or CB1 cannabinoid receptors on VTA GABAergic neurons reduces GABA transmission, which increases the firing rate of dopaminergic neurons via disinhibition.

Schematic Representation of the GABA

Receptor

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Representation of the GABA_B receptor heterodimer which is composed of two, seven transmembrane-spanning units that are linked by their carboxyl-termini. The apparent ligand binding domains are on the extracellular surface of R1 and R2. Activation of the heterodimer modulates adenyl cyclase activity via G-proteins that, in addition, influence K⁺ and Ca²⁺ conductances.

Oxidative Stress

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Oxidative stress is imposed on cells as a result of one of three factors: 1) an increase in oxidant generation, 2) a decrease in antioxidant protection, or 3) a failure to repair oxidative damage. Cell damage is induced by reactive oxygen species (ROS). ROS are either free radicals, reactive anions containing oxygen atoms, or molecules containing oxygen atoms that can either

produce free radicals or are chemically activated by them. Examples are hydroxyl radical, superoxide, hydrogen peroxide, and peroxynitrite. The main source of ROS in vivo is aerobic respiration, although ROS are also produced by peroxisomal ß-

oxidation of fatty acids, microsomal cytochrome P450 metabolism of xenobiotic compounds, stimulation of phagocytosis by pathogens or lipopolysaccharides, arginine metabolism, and tissue specific enzymes. Under normal conditions, ROS are cleared from the cell by the action of superoxide dismutase (SOD), catalase, or glutathione (GSH) peroxidase. The main damage to cells results from the ROS-induced alteration of macromolecules such as polyunsaturated fatty acids in membrane lipids, essential proteins, and DNA. Additionally, oxidative stress and ROS have been implicated in disease states, such as Alzheimer’s disease, Parkinson’s disease, cancer, and aging.

Neuronal Nitric Oxide Synthase (nNOS)

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Three isoforms of nitric oxide synthase (NOS) have been identified. All are homodimers with subunits of 130-160 kDa. All have binding sites for NADPH, FAD, and FMN near the carboxyl terminus (the reductase domain), and binding sites for tetrahydrobiopterin (BH₄) and heme near the amino terminus (the oxygenase domain). The reductase and oxygenase domains are linked by a calmodulin (CaM) binding site. Occupation of this site

facilitates electron transfer from the cofactors in the reductase domain to heme during nitric oxide production. NOS heme during nitric oxide production. NOS catalyzes the conversion of arginine to citrulline and nitric oxide (NO). Neuronal nitric oxide synthase (nNOS, bNOS, cNOS, Type I) is associated with the post-

synaptic density protein (PSD-95) in the neuronal membrane. In response to increased intracellular Ca²⁺, nNOS interacts with CaM. The Ca²⁺-CaM

complex, in combination with BH₄, binds to nNOS and induces its translocation from the plasma membrane to the cytoplasm. The dephosphorylation of nNOS by calcineurin initiates the production NO. NO activates guanylyl cyclase (GC) and activates the various cGMP-regulated signaling pathways.

nNOS is in activated by phosphorylation by protein kinase A (PKA) or protein kinase C (PKC).

Epithelial Nitric Oxide Synthase (eNOS)

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Nitric Oxide (NO) produced in the endothelial cells is involved in vasorelaxation, platelet aggregation, and mechanisms of cardiovascular homeostasis. Endothelial nitric oxide synthase (eNOS, cNOS, Type III) is constitutively expressed in endothelial and other cell types. Myristoylation and palmitoylation maintain the localization of eNOS to caveolae in the plasma membrane of resting cells where it is bound to caveolin. eNOS is inactive in the membrane-bound state. Activation of endothelial

acetylcholine receptors activate phospholipase C (PLC) that catalyzes the production of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (PIP₂). The IP₃-induced increase in intracellular Ca²⁺ activates calmodulin that binds to eNOS, which dissociates from caveolin and translocates to the cytoplasm. Phosphorylation of eNOS by protein kinase A (PKA) inactivates the enzyme, which then relocates to the membrane caveoli.

Inducible Nitric Oxide Synthase (iNOS)

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Macrophages are important for early immune responses to invading microorganisms, and the production of nitric oxide (NO) is central to this function. NO is generated by inducible nitric function. NO is generated by inducible nitric oxide synthase (iNOS, macNOS, Type II NOS) following exposure to certain cytokines, such as interferon-γ (IFN-γ). The IFN-γ receptor signals through the Janus kinase (JAK) family and signal transducers and activators of transcription (STAT) proteins. Receptor occupation and dimerization induces the phosphorylation of associated STATs. Activated STATs dimerize and translocate to the nucleus where they increase expression of the transcription factor, IRF-1, that, in turn, binds to specific DNA elements in the iNOS gene promoter region to increase iNOS gene expression. iNOS is a soluble enzyme that, unlike eNOS and nNOS, does not require elevated intracellular Ca²⁺ levels for activation.

Nitric Oxide Metabolism

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Nitric oxide (NO) is as a major signaling molecule in neurons and in the immune system, either acting within the cell in which it is produced or by penetrating cell membranes to affect adjacent cells. Nitric oxide is generated from arginine by the action of nitric oxide synthase (NOS). NO has a half-life of only a few seconds in vivo . However, since it is soluble in both aqueous and lipid media, it readily diffuses through the cytoplasm and

plasma membranes. NO has effects on neuronal transmission as well as on synaptic plasticity in the central nervous system. In the vasculature, NO central nervous system. In the vasculature, NO reacts with iron in the active site of the enzyme guanylyl cyclase (GC), stimulating it to produce the intracellular mediator cyclic GMP (cGMP), that in turn enhances the release of neurotransmitters resulting in smooth muscle relaxation and vasodilation. NO may also be involved in the regulation of protein activity through S-

nitrosylation. In the extracellular milieu, NO reacts with oxygen and water to form nitrates and

nitrites. NO toxicity is linked to its ability to combine with superoxide anions

(O^2–) to form peroxynitrite (ONOO^–), an oxidizing free radical that can cause DNA fragmentation and lipid oxidation. In the mitochondria, ONOO^–acts on the respiratory chain (I-IV) complex and manganese superoxide dismutase (MnSOD), to generate superoxide anions and hydrogen peroxide (H₂O₂), respectively.

Proteasome/Ubiquitination

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Attachment of ubiquitin to proteins targets them for proteolytic degradation by a complex cellular structure, the proteasome. Degradation of proteins by proteasomes removes denatured,

damaged or improperly translated

proteins from cells and regulates the level of proteins such as cyclins and some transcription factors. E1 and E2 enzymes prepare the ubiquitin chains that are then attached to proteins by the E3 enzyme.

The core proteasome (20S proteasome) consists of four rings each with 14 subunits stacked on top of each other subunits stacked on top of each other that are responsible for the proteolytic activity of the proteasome. The PA700 regulatory complex is stacked on the ends of the cylindrical core to form a 26S proteasome. Proteins that are tagged with ubiquitin are recognized and bound by the regulatory subunits, then unfolded in an ATP-dependent manner, and inserted into the core particle, where proteases degrade the protein, releasing small peptides and releasing the ubiquitin intact. The PA28 regulatory complex is an alternative regulatory complex that

appears to play a role in antigen

processing for presentation of peptides to immune cells in the major

histocompatibility complex I (MHC I) complex.

Formation of Activated 20S Proteasome

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Intracellular Proteolytic Systems Recognize and Destroy misfolded or damaged proteins, unassembled

polypeptide chains, and short-lived regulatory proteins.

There are a number of mechanisms of protein degradation within cells. Two systems that play an important role in proteolysis resulting from cell stress are calpain proteases and the ubiquitin-proteasome pathway.

The calpains are a family of heterodimeric, calcium- activated cysteine proteases. They are composed of a common 30 kDa subunit and an isoform specific, catalytic 80 kDa subunit. The large subunit of Calpain I catalytic 80 kDa subunit. The large subunit of Calpain I m-isoform) is activated by micromolar calcium levels, while Calpain II (m-isoform) requires millimolar levels of calcium for activation. Cell stress or injury can lead to sustained elevation of intracellular calcium levels, causing sustained activation of calpains. The common, late-stage of the cell death pathway induced by

excitotoxic compounds in the nervous system involves calpain-mediated proteolysis.

The ubiquitin-proteasome pathway functions widely in intracellular protein turnover. It plays a central role in degradation of short-lived and regulatory proteins important in a variety of basic cellular processes,

including regulation of the cell cycle, modulation of cell surface receptors and ion channels, and antigen

processing and presentation. The pathway employs an enzymatic cascade by which multiple ubiquitin

molecules are covalently attached to the protein substrate. The polyubiquitin modification marks the

Ubiquitin-Proteasome Pathway

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Intracellular proteolytic systems recognize and destroy misfolded or damaged proteins, unassembled polypeptide chains, and short-lived regulatory proteins. There are several mechanisms for protein degradation within cells. Two systems that play important roles in proteolysis resulting from cell stress are the calpain proteases and the ubiquitin- calpain proteases and the ubiquitin- proteasome pathway. The ubiquitin- proteasome pathway functions widely in intracellular protein turnover. It plays a central role in degradation of short-lived and regulatory proteins important in a variety of basic cellular processes, including regulation of the cell cycle, modulation of cell surface receptors and ion channels, and antigen processing and presentation.

The pathway employs an enzymatic cascade by which multiple ubiquitin molecules are covalently attached to the protein substrate. The

polyubiquitin modification marks the protein for destruction and directs it to the 26S proteasome complex for degradation.

The Mitogen-activated Protein Kinase (MAPK) Cascades

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Several MAPK cascades have been identified in mammalian cells, including the extracellular signal- related kinase pathways (ERK1/2, ERK5) and the stress activated kinase pathways (JNK/SAPK, p38 MAPK). These pathways are linked to many G protein-linked cell surface receptors and receptor protein-linked cell surface receptors and receptor tyrosine kinases. Thus, most cytokines, growth factors, hormones, and neurotransmitters can selectively activate these cascades via receptor activation of intracellular second messengers. All MAPK pathways operate through sequential phosphorylation events to phosphorylate

transcription factors and regulate gene expression.

They can also phosphorylate cytosolic targets to regulate intracellular events. These cascades are implicated in the regulation of cellular proliferation, differentiation, development, cell cycle, and

transmission of oncogenic signals.

Courtesy of Rony Seger, Ph.D., Dept. Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.

Akt Signaling

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The serine/threonine protein kinase Akt/PKB is the cellular homologue of the viral oncogene v-Akt and is activated by various growth and survival factors. In mammals, there are three known isoforms of the Akt kinase, Akt1, Akt2, and Akt3. Many cell surface receptors induce the production of second messengers that activate phosphoinositide 3-kinase (PI3K). Akt is located downstream of PI3K and, therefore, functions as part of a wortmannin-sensitive signaling, pathway. PI3K generates phosphorylated phosphatidylinositides (PI- 3,4-P₂and PI-3,4,5-P₃) in the cell membrane that bind to the amino-terminal pleckstrin homology (PH) domain of Akt. PI-3,4-P₂and PI-3,4,5-P₃also activate phosphoinositide-dependent kinase (PDK) which phosphorylates Thr³⁰⁸of membrane-bound Akt. Ser⁴⁷³ is phosphorylated by integrin-linked kinase (ILK). Activated Akt promotes cell survival through two distinct pathways: 1) Akt inhibits apoptosis by phosphorylating the Bad component of the Bad/Bcl-X_Lcomplex. Phosphorylated Bad binds to 14-3-3 causing

dissociation of the Bad/Bcl-X_Lcomplex and allowing cell survival. 2) Akt activates IKK-α which ultimately leads to NF-κβ activation and cell survival.

Signaling Pathways Activated by VEGF

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VEGF regulates several endothelial cell functions, including proliferation, differentiation, permeability, vascular tone, and the production of vasoactive molecules. Upon ligand binding, the receptor tyrosines are phosphorylated, allowing the receptor to associate with and activate a range of signaling molecules, including phosphatidylinositol 3-kinase (PI3K), Shc, Grb2, and the phosphatases SHP-1 and SHP-2. VEGF receptor activation can induce activation of the MAPK cascade via Raf stimulation leading to gene expression and cell proliferation, activation of PI3K leading to PKB activation and cell survival, activation of PLC-g leading to cell proliferation, vasopermeability, and angiogenesis.