Cell Metabolism and Cell Death in Neurodegenerative Disease · 2011. 1. 25. · Cell death is a normal process during brain development. It is mostly abnormal in the adult brain.

Cell Metabolism and Cell Deathin Neurodegenerative Disease

Neurology Clerkship

Dr. Ranney MizeCell Biology and AnatomyEmail: [email protected]

• Subcellular organelles in neurons• Protein Synthesis and Degradation• Mitochondria and Oxidative Metabolism• Cell Death: Necrosis and Apoptosis• Pathways leading to cell death• Parkinson’s: Mitochondrial dysfunction+• Alzheimer’s: Protein aggregation+

Cell Biology and Death of Neurons

• Alzheimer’s Disease: abnormal protein cleavage producing toxic protein aggregation + dysfunction of mitochondria

• Parkinson’s Disease: gene mutations leading to mitochondrial dysfunction + protein aggregation

• ALS: mutation in superoxide dismutase (SOD1) gene leading to mitochondrial dysfunction, oxidative stress, and protein aggregation

• Huntington’s Disease: huntingtin gene mutation > polyglutamine repeats > mitochondrial dysfunction, oxidative stress, protein aggregation

• Stroke, Epilepsy: glutamate excitotoxicity yielding mitochondrial dysfunction + oxidative stress, apoptosis.

Neurodegenerative Disorders

PresenterPresentation NotesFocus of this lecture will be on protein aggregation, mitochondrial dysfunction, and oxidative stress.

Intracellular Organelles in the Neuron

Dendrites

Cell Body

Dendrites

Axon

Synapse

PresenterPresentation NotesNeuronal morphology varies enormously, with different shapes, sizes (5-50 um), axon projections. Inhibitory vs. excitatory. Dendritic trees are the receptive zones; axons transmit signal to other cells. Nonetheless, soma contents are similar to other cells, including nucleus (gnee transcription), rough ER (protein synthesis), Golgi (transport, routing of proteins), mitochondria (energy source). Other organelles are polyribosomes and smooth ER. Rought ER, mitochondria, are also found in dendrites.

1. Rough Endoplasmic Reticulum (rER): stacks of membrane dotted with ribosomes. Function: synthesis of integral membrane proteins. mRNA transcripts bind to ribosomes which translate the mRNA blueprint in order to assemble proteins from amino acid sequences.2. Mitochondria: membrane bound “kidney” shaped organelles. Function: cellular respiration where converted proteins, fats, sugars (glucose) and oxygen enter the Krebs cycle to produce reactions that yield adenosine triphosphate (ATP). ATP is released into the cytosol as the energy source of the cell.3. Smooth Endoplasmic Reticulum (sER): membrane without ribosomes. Function: varied, including regulation of intracellular concentrations of Ca++.

Subcellular Organelles of the Neuron

PresenterPresentation NotesSeveral of the neuron’s organelles are players in the cascade of cell death that occurs after brain injury and disease. Rough ER is a site where proteins are mis-expressed and also a site for failed intracellular Ca++ storage.

rER: Protein Synthesis(integral membrane proteins)

DNA Transcription

Protein Degradation(ubiquitin-proteosome sys)

Amino acids

Proteolysis occurs to break peptide bonds

via proteases

requiresenergy

rER

Disruption =Protein

Aggregation

translocatedthru pore

Regulatory proteinthat targets proteins to

the proteosome

Protein complexwith a core

(Bear et al, 2007)

ActivationConjugationRecognition (ligase)

chaperone

PresenterPresentation NotesInitial protein synthesis from mRNA code occurs in free polysomes. N terminus then allows signal recognition particle (SRP) to bind to its receptor located on the ER membrane. Protein is then translocated thru an aquaquious pore (TRAM).Ubiquitination refers to the post-translational modification of a protein by the covalent attachment (via an isopeptide bond) of one or more ubiquitin monomers. The most prominent function of ubiquitin is labeling proteins for proteasomal degradation. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that carry out such reactions are called proteases. Proteasomes are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins, first to peptides, then to constituent amino acids,

Mitochondrial Oxidative Metabolism

I II III IV Velectron chain protein complexes

NADH

Cyt c

(electron transport chain)

Glucose

respiration-energy

ATP

mtDNA

complex I

complex III

NADHreducing agent > antioxidants. Free radicals incrif blocked.

Cyt c = heme protein >apoptosis if released

( O-2, NO) ( ATP)lipid

peroxidation

PresenterPresentation NotesThe pathway for production of ATP is mitochondrial respiration. After glycolysis, the pyruvate product is taken into the mitochondia, oxidatively decarboxylated to acetyl coenzyme-A and further oxidized in the TCA (tricarboxylic acid) cycle. This cycle deposits energy in reduced coenzymes which transfer that energy through what is called the electron transport chain. Energy given to the electrons of the reduced coenzyme NADH by the TCA cycle is transferred in small steps in the inner membrane of the mitochondrion through a chain of five protein complexes. These small oxidation steps accomplish the conversion of ADP to the energy currency molecule ATP. The protein complex ATP synthase makes use of a mitochondrial membrane potential to accomplish the phosphorylation of ADP to ATP Acetyl coenzyme A is an important molecule in metabolism, used to convey carbon atoms within the acetyl group to the citric acid cycle to be oxidized for energy production. NADH = nicotinamide adenine dinucleotide hydrogenase = antioxidant.In metabolism, NAD+ is involved in redox reactions, carrying electrons from one reaction to another. The coenzyme is, therefore, found in two forms in cells: NAD+ is an oxidizing agent – it accepts electrons from other molecules and becomes reduced. This reaction forms NADH, which can then be used as a reducing agent to donate electrons. Complex I = (NADH-ubiquinone reductase), complex III (cytochrome oxidase) and mitochondrial nitric oxide synthase (mtNOS).Mitochondrial complex I (NADH-UQ reductase) catalyzes electron transfer from NADH to ubiquinone and is the molecular pathway to connect the tricarboxylic acid cycle and NADH with the mitochondrial respiratory chain. Complex I is a supra-molecular protein complex of 850 kDa composed of about 40 polypeptide units. peroxynitrite (ONOO-).

Cell death is a normal process during brain development.It is mostly abnormal in the adult brain. Neurons die during acute trauma and chronic disease as well as in aging.There are two basic types of cell death: necrotic cell death and programmed cell death.Necrotic cell death occurs most commonly when the brain is damaged by impact injury, subarachnoid hemorrhage, ischemic stroke, seizures, and other sudden insults.Programmed cell death (apoptosis) occurs more commonly in chronic progressive diseases such as Alzheimer’s and Parkinson’s. However, both necrosis and apoptosis can occur in both acute and chronic brain injury. Understanding the mechanisms underlying necrosis and apoptosis has contributed to treatment protocols for these diseases.

How Do Cells Die

Cells Undergoing Necrosis

1. Necrosis; from Greek for “death”.2. It is a non-programmed cell death

occurring due to injury or disease= abnormal, inappropriate.

3. Cell bodies initially swell up leadingto rupture of the cell membrane.

4. Nucleolus breaks up and nucleusbecomes multi-nucleolated.

5. Significant cytoplasmic vacuolizationoccurs with breakdown of organelles.

6. Cell membrane rupture causes releaseof internal contents of the cytoplasm.

7. This debris produces an inflammatory auto-immune response in brain.

8. Necrosis is triggered in part by endogenous release of intracellularCa++ produced by ion failure.

(From Squire et al, 2008)

vacuoles

multi-nucleolatednucleus

darkened chromatin

swelling, membrane rupture

http://www.imbb.forth.gr/worms/worms/nrn1174-f1.jpg�

Cells Undergoing Apoptosis

1. Apoptosis; Gr. for “leaves dropping”.2. It is one form of programmed cell death

(PCD) termed type 1.= normal, appropriate in development.

3. Cell bodies “round up”, shrink in size.4. Nuclear chromatin condenses, nuclei

become pyknotic (shrink).5. DNA fragmentation occurs, mediated by

proteases.6. Eventually the cytoplasm and nucleus

break up, blebs form on membrane andbud off to form apoptotic bodies.

7. Residues are phagocytized by glial cells, macrophages.

8. Triggered by activation of cell-suicidecysteine proteases called caspases.

(From Squire et al, 2008)

blebs

condensed chromatin

shrinking cell

apoptotic body

Cell Death Summary

blebs

apoptotic bodiescondensed chromatin

dense nucleusApoptosis

macrophage, lysosomedegradation

membranerupture

somatic swellingvacuolization

phagocytosis

(From Squire et al, 2008)

Necrosis

‘Normal’ genetic programoccurring in single cells

Occurs In Multiple Cells > toxic content release which causes brain inflammation

Toxic releaseof debris

• Cell injury (trauma, ischemia, toxins, hypoglycemia) leads to glutamate excitotoxity and significant Ca++ influx into a damaged cell (stroke, status epilepticus)

• Oxidative stress- ROS generation also lead to release of Ca++ from intracellular stores in the endoplasmic reticulum.

• DNA damage activates poly ADP-ribose polymerase which decreases pH and leads to acidosis.

• Increased Ca++ and acidosis produce calpain proteases which together with free radicals rupture the membrane of lysosomes which contain ‘digestive’ enzymes.

• Release of proteolytic enzymes cathepsin B and D from the lysosome degrade cell structure and metabolism, leading to necrotic cell death.

Signaling Cascades Leading to Necrosis

PresenterPresentation NotesPARP is composed of four domains of interest: the DNA binding domain, the cleavage domain, the auto-modification domain, and the catalytic domain. The DNA binding domain is composed of two zinc finger motifs. In the presence of damaged DNA (base pair excised), the DNA binding domain will bind the DNA and induce a conformational shift. It has been shown that this binding occurs independent of the other domains. This is integral in a programmed cell death model based on caspase cleavage inhibition of PARP. The auto-modification domain is responsible for releasing the protein from the DNA after catalysis. It too plays an integral role in cleavage induced inactivation.

Pathways to Neuronal Necrosis

IP3

ROS overproduction

Acidosis

Ca++ Ca++

Ca++

Ca++ Ca++

zinc

NMDA receptor

Na+

(cisteineprotease)

(aspartylprotease)

Na2+

AMPA receptor

Mg entrypH

Intracellularcalcium stores

Poly ADP-ribosepolymerase

“suicide bag”degrade cell

organelles

Glutamate Excitotoxicity(excessive depolarization)

• Bcl proteins regulate apoptosis by controlling mitochondrial outer membrane permeabilization, in particular by regulating release of cytochrome c.

• Anti-apoptotic Bcl-2 proteins in the mitochondrial membrane normally inhibit cytochrome c release.

• Pro-apoptotic Bcl proteins (Bax, Bid, Bad) translocate to the mitochondrial outer membrane and promote cytochrome c release during injury and disease.

• Upon release from mitochondria cytochrome c binds to APAF-1 and forms a complex with caspase 9 which is called an ‘apoptosome’.

• Caspase 9 activates caspase 3 leading to membrane blebbing, chromatin condensation, DNA fragmentation and apoptotic cell death.

Signaling Cascades In Apoptosis

PresenterPresentation NotesAPAF-1: Apoptotic protease activating factor 1. Bcl-2 = B cell lymphoma related proteins encoded by Bcl-2 gene.Cytochrome c = protein involved in apoptosis.

Intrinsic-Extrinsic Apoptotic Pathways

(From Bredesen et al, 2006)

BAX

BID

(caspase 3)

BID = BH3 interacting domain death agonistBAX = Bcl-2–associated pro-apoptotic X- protein

translocation

Tissue Necrosis Factor Receptor(TNFR)

Extrinsic PathwayIntrinsic Pathway

Mitochondrial MembranePermeabilization

PresenterPresentation NotesSMAC = second mitochondria-derived activator of caspases.APAF1 = Apoptotic protease activating factorBID = BH3 interacting domain death agonistBAX = Bcl-2–associated pro-apoptotic X- protein

• Normal protein degradation involves the ubiquitin-proteosome system for recycling, redistribution of protein. Ubiquitin guides the protein, the proteosome degrades it.

• The first pathological step blocking protein degradation is the formation of a misfolded protein.

• Once misfolded, the hydrophobic fragments that are normally buried inside the protein get exposed to the aqueous environment.

• This intermediate has a high tendency to aggregate and become stabilized because the proteosome is unable to degrade the protein.

• Incorporation of additional monomers to the protein gives rise to soluable oligomers, protofibrils, and then ‘mature’ protein aggregates which are insoluble.

Protein Aggregation Pathways

oligomers

fibrils

Proteolysis occurs to break peptide bonds

via proteases

Post-translationalmodification via

covalent bond

requiresenergy

Normal Protein Degradation Abnormal Formation of Aggregates

aggregates

Aβ 1-40B sheets

(cleavage or phosphorylation)

chaperone

• Progressive neurodegenerative disorder affecting 0.5 million Americans, about 1% of the pop. over 60.

• Symptoms include tremor-at-rest, cogwheel rigidity, postural instability, festinating gate, slow movement(bradykinesia), flat affect, depression.

• Pathology includes loss of >70% of dopamine neurons in SN compacta and build-up of intracellular inclusions(Lewy bodies) composed of fibrillar α-synuclein.

• Mitochondrial dysfunction (complex I inhibition leading to oxidative stress) may be the initial step in killing DAcells. However, protein aggregation is also involved.

• Drug treatments are l-dopa, dopamine agonists, MAOinhibitors. Surgical approach is DBS, usually in the subthalamus.

Parkinson’s Disease

Parkinson’s DiseasePathology

pill-rolling tremor at rest

Improved blood flow afterSurgical pallidotomy

Loss of dopamine inParkinson’s brain

rigidityakinesia

Pre Post

MRI of pathology in

putamen-globus pallidus

festinatinggate

Lewy bodies

SN

abnormalposture

• Inhibition of complex I of the electron transport chain impairs mitochondrial respiration, including reduction in NADPH and other antioxidants.

• This leads to increased production of intra- and extra-mitochondrial Reactive Oxygen Species (free radicals such as superoxide anion, hydrogen peroxide, NO).

• ROS increases the soluble pool of cytochrome c in IMS as well as ROS in the extra-mitochondrial cytosol.

• Cytosolic ROS produces DNA fragmentation yielding transcriptional upregulation of pro-apoptotic Bax which translocates to the mitochondrial outer membrane.

• Bax then induces release of cytochrome c which triggers caspase activation and apoptotic cell death.

Mitochondrial Induced Cell Death Cascade in Parkinson’s

PresenterPresentation NotesThe molecular mechanisms involved in complex I inactivation include the synergistic inactivations produced by ONOO- mediated reactions, by reactions with free radical intermediates of lipid peroxidation and by amine-aldehyde adduction reactions. The increased rate of O2- generation promotes lipid peroxidation, protein oxidative damage and peroxynitrite (ONOO−) mediated protein nitration (as 3-nitrotyrosine) and nitrosylation (as –S–NO of thiol groups), in a process that lead neurons to apoptosis

Perier C et al. PNAS 2007;104:8161-8166

©2007 by National Academy of Sciences

Mitochondrial Dysfunction in Parkinson’s Cell Death

DNA fragmentation

MPTP

up-regulation

translocation

oxidative phosphorylation

(NADH)

increasedpermeabilization

transcription

(Bcl-2 interactingmediator)

PTP

Bax, Bim =Pro-apoptoticproteins

PresenterPresentation NotesFirst step is blocking complex I and hence downregulating NADH. This produces a build-up of reactive oxygen species (ROS). ROS increases cytochrome c from complex III in the intermembrane space (IMS). This cyt c may be released thru a permeability transition pore. Alternatively, cytoplasmic ROS can produce DNA fragmentation which leads to P53 mediated transcription of the pro-apoptotic Bcl-2 protein Bax. Bim then translocates Bax to the mitochondrion, which also permeabilizes the membrane, releases cyto c, activates the caspase reaction leading to apoptosis.MPTP – methyl phenyl tetra-hydropyridine. Proposed pathogenic scenario induced by complex I deficiency with MPTP in which DA neuronal death results from a self-amplifying cascade of deleterious events that start at the mitochondria with the alteration of oxidative phosphorylation and finish at the mitochondria with the activation of the programmed cell death machinery. In this scenario, p53 transcriptionally up-regulates Bax, whereas JNK promotes Bax mitochondrial translocation through transcriptional activation of the BH3-only protein Bim.MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a neurotoxin that causes permanent symptoms of Parkinson's disease. The pesticide rotenone has a similar effect.

Protein Aggregation in Parkinson’s Disease

protofibrils

ROS

cytochrome c

caspases

Gene MutationsProtein Misfolding

Abnormal ProteinAggregation

Reactive OxygenSpecies Production

Oxidative Stress

Cell Apoptosis

ATP

apoptosis

disrupts proteinguidance

Proteinstructure

X

gene mutationsE3 ubiquitin ligase

PresenterPresentation NotesLeucine rich repeat kinase-2 may also contribute to proteosomal dysfunction. It is a current hypothesis in the field that cytosolic oxidative stress (increased GSSG and H2O2 levels) favor the post-translational oxidative modification of α-synuclein and its aggregation.

Alzheimer’s Disease• Progressive neurodegenerative disease producing severe

cognitive decline, memory loss, behavioral disorders.• Familial early age Alzheimer’s is an autosomal-dominant

genetic abnormality in the coding of one or more proteins. Accounts for ~5% of cases. All others are sporadic.

• Pathology characterized by senile plaques of amyloid-βpeptide and neurofibrillary tangles consisting of hyperphosphorylated tau protein.

• Genes thought to be involved include amyloid precursor protein (APP) gene and those encoding presenilins 1-2. Presence of the apoE4 gene allele is also a risk factor.

• Acetylcholine and glutamate transmitter systems are also effected. FDA approved drugs target ACHe (Aricept, an inhibitor), or the NMDA receptor (Namenda, a antagonist)

• Ultimate neuronal loss occurs first in hippocampus, basal forebrain, temporal and prefrontal cortex, then broadly.

• In familial early-onset AD three gene mutations have been identified: amyloid precursor protein (APP) and genes (PS1-PS2) encoding presenilins 1 and 2.

• Deposits of amyloid β in extracellular space are produced by cleavage of transmembrane APP by β and γ secretases.Aβ-42 is likely the fragment causing amyloid plaques.

• The abnormal cleavage of APP is produced by a mutation in the presenilin gene. The resulting plaques disrupt cell function.

• Plaques also increase ROS which produces cell membrane damage and mitrochondrial oxidative stress leading to apoptosis.

• Neurofibrillary tangles (NFT) also accumulate inside damaged cells. NFT are paired helical filaments consisting of the hyper-phosphorylated microtubule-associated protein tau. These also damage cell function.

Protein Aggregation and Oxidative Stress in Alzheimer’s Disease

Assembly of Plaques and Tangles

Multi-protein complexwith active docking,

ATP binding sites

N-APP

Amyloid plaques

NeurofibrillaryTangles

Tau

APP Intracellularcytoplasmic domain

β cleavageenzyme

BACE = β-site of APP cleaving enzyme

Amyloid Plaque

Hyperphosphorylated Tau

Cell SignalingPathways in

Alzheimer’s Disease

Aβ Protein Aggregation(senile plaques)

Hyperphosphorylated Tau (neurofibrillary tangles)

Oxidative Stress

Apoptosis

T1 MRI ofAD brain

ROS Formation

Bax, CaspasesOxidative degradation

by free radicals

?

• Stroke = A “Brain Attack” defined as an “abrupt onset of a focal neurologic deficit”. 3rd leading cause of death inthe US, no. 1 cause of morbidity.

• Stroke is a vascular insult produced by either obstruction in a blood vessel (embolus, thrombus) or hemorrhage

(anuerysm, AVM). • Stroke causes ischemia (blood loss), hypoxia ( oxygen)

and infarct (cell death) leading to paralysis, impaired speech, loss of vision, etc.

• Tissue damage is due to glutamate excitotoxicity > ionicfailure, excess intracellular Ca+, oxidative stress, etc.

• Within the infarct, the core is largely necrotic while thesurrounding penumbra can be reperfused. Withoutreperfusion, apoptosis occurs in the penumbra.

Ischemic and Hemorrhagic Stroke

Core and Penumbra in StrokePET

Ischemic penumbra(perinfarct zone)

< 20% blood flowdepleted ATPno energy metabolismnecrosis

• Glutamate excitotoxicity: ion channel and reuptake failure produces glu overload > prolonged NMDA and AMPA stimulation. This is one source of Ca+ influx.

• Calcium Dysregulation: Ca+ is also released from mitochondria and ER stores and by activation of Na+/Ca+ exchanger.

• Oxidative Stress: overload of ROS oxidants from mitochondria (superoxide, hydrogen peroxide, NADPH oxidase, NO) > release of proteases and caspases leading to apoptosis.

• Spreading Depolarization: triggered by high levels of glu and K+ disrupts ionic gradients yielding massive depolarization of cells.

• Inflammation: release of cytokines (TNF, IL1β), prosto-glandins, produce severe inflammation in the affected area.

Detrimental Events in Stroke

Imaging the IschemicPenumbra In Stroke

Moskowitz et al 2010

Early

Late

DWI

PWI

Difference

A

B

C

D

H2O

Blood flow

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