Week 7B: Parkinson's Disease: Basics, Apoptosis, Vulnerability SNc Flashcards
HC 40, 41, 42
HC40: Symptoms Parkinson’s Disease
Involuntary tremulous motion, lessened muscle power (muscles stiffen), in parts not in action
> Shaking when resting: resting tremor
> propensity to bend trunk forward
> Slowness in movement (bradykinesia)
> difficulty walking
TRAP in Parkinson’s
-Tremor
-Rigidity
-Akinesia (slow (bradykinesia), to no movement (akinesia)
-Postural instability
Lewy bodies
alpha-synuclein aggregates in the pigmented neurons of the substantia nigra (SN)
SN neurons contain the pigment…
melanin
Basal ganglia
Subcortical nuclei responsible for primary motor control
Stimuli for movement path in brain
Cortex > travels to putamen and globus palidus segments to the thalamus
- SNc plays effect in stimulating striatum
SNr and SNc
SNr: substantia nigra pars reticula
SNc: substantia nigra pars compacta
Cortico-striatal pathways of basal ganglia (only the names, pathway details in other flashcard)
-Direct pathway: stimulates voluntary movements
-Indirect pathway: inhibits voluntary movement
Direct pathway cortico-striatal of basal ganglia
Decision is made in the cortex
Excitatory pathway
Cortex: Glutamate (Glu) >+ Striatum
SNc: Dopamine (DA) >+ Striatum neurons with D1 receptor (Gs)
Striatum: GABA >- GPi (Globus palidus int.) & SNr
GPi, SNr: GABA >- Thalamus
Thalamus: Glu >+ Cortical (for movement)
» So, the GPi and SNr are inhibited so that Thalamus is active
Indirect excitatory pathway via SNc
Cortex: Glu >+ Striatum
SNc: DA >- Striatum neurons with D2 receptor (Gi)
Striatum: GABA >- GPe (Globus palidus ext.)
GPe: GABA >- STN (subthalamic nucleus)
STN: Glu >+ GPi, SNr
GPi, SNr: GABA >- Thalamus
Thalamus: Glu >+ cortical (movement)
» So, the striatum inhibits the GPe which promotes inhibition of Thalamus via STN and GPi and SNr. > excitatory by blocking inhibition
SNc contain … neurons
Dopaminergic neruons
Defect which neurons in Parkinson’s disease (PD)?
Dopamine neurons in SNc
What kind of receptor is the DA receptor?
Trimeric G protein coupled receptor
Dopamine biosynthesis
Tyrosine is substrate
Tyrosine > L-DOPA (tyrosine hydroxylase)
L-DOPA > Dopamine (DOPA decarboxylase) (remove carboxyl group, from amino acid structure to amine only)
> monoamine neurotransmitter dopamine
Where does the dopamine synthesis for SNc occur?
In the cells themselves > in the cytosol
Dopamine biosynthesis pathway also used to make the hormones / neurotransmitter
Noradrenline and then adrenaline
Dopamine breakdown
Dopamine (DA) > DOPAL (dihydroxyphenylacetaldehyde) + NH3 (MAO-B)
(oxidative deamination)
DOPAL > DOPAX (dihydroxyphenyl acetic acid) (ALDH)
MAO-B characteristics
Monoamine oxidase B (MAO-B)
> Flavoprotein that catalyzes oxidative deamination
> oxidative: removal electrons, oxidation
> Flavoprotein is stuck with the electrons > cannot be donates to ETC (not in mitochondrium, just like VLCFA oxidation in peroxisomes)
> use molecular oxygen to accept electrons (high affinity for it)
> create hydrogen peroxide (addition two protons as well)
Loss dopamine neurons over time
SNc dopamine neurons are lost naturally over time due to aging
> but in PD: quicker loss DA neurons, quicker reaching threshold amount of neurons for symptoms
> amount of SNc neurons born with varies
What if DOPAL is not converted quickly after made?
It is an aldehyde: toxic > can create oxidative damage
> reactive
Treatment strategies PD (to prevent loss DA)
Stimulate synthesis or inhibit breakdown
> DA has longer half life if MAO-B inhibited
Prodromal stage symptoms of PD
-Loss of smell
Current most used treatment for PD
Levodopa (L-DOPA supplement)
> L-DOPA can be transported past blood brain barrier because it is an amino acid and amino acid transporter LAT-1 > DA is not! no transport!
> DA cannot diffuse through: charges > water shell attached
> transporter for amino acids but not for monoamines
> after L-DOPA reaches neurons, conversion to DA by DOPA decarboxylase
Levodopa treatment is in combination with … because …
Carbidopa, which inhibits DOPA decarboxylase in the blood
> it is no danger, cannot pass blood brain barrier
> prevents conversion L-DOPA to DA in blood, then it cannot pass the blood brain barrier
Genetics of familial PD: name the heritance of the protein markers and their function and presentation
-alpha-synuclein: Dominant heritance, early onset, involved in synaptic vesicle release
-LRRK2: Dominant, mid-late onset, signaling protein for synaptic transmission
-Parkin: recessive, juvenile PD, E3 ubiquitin ligase
-PINK1: recessive, juvenile PD, targets malfunctioning mitochondria
-DJ-1: recessive, early onset, cytoplasmic sensor of oxidative stress
Alpha-synuclein function
Protein involved in docking of vesicles with for example dopamine at the PM
> binds directly to synaptobrevin (VAMP) and stimulates assembly synaptic vesicle complex
> found in Lewy Bodies: forms fibrils when defect
LRRK2 function and in PD
Leucine-rich repeat kinase-2
> large kinase with many protein-protein binding domains
> involved in docking vesicles via activating the small GTPase Rab
> in dominant mutation: hyperactive: too much Rab phosphorylated, toxicity
PINK1 and Parkin are involved in the …
and the global path
PINK1/Parkin dependent mitophagy of damaged mitochondria
> Mitochondrion does not work: no ETC, no proton gradient, no transmembrane potential (depolarization): sensed by PINK1 (damaged mitochondrion recognized)
> Pink1 phosphorylates mitochondrion and binds Parkin > polyubiquitination mitochondrion
> autophagy and fusion with lysosome
What happens with Pink1 when normal mitochondrial transmembrane potential
Degraded via ubiquitination proteasome pathway
> Tail of Pink1 goes through mitochondrial membranes and in the matrix, the tail is cleaved by proteases there > released for degradation
Effect depolarization mitochondrial membrane potential of Pink1
Tail of Pink1 cannot go through into mitochondrial matrix anymore
> tail not cleaved, Pink1 not released
> dimerization Pink1 on mitochondria
> active Pink1 > auto-phosphorylation for fully active
> Phosphorylate mitochondrion proteins
> and phosphorylate and activate Parkin
Pathway Pink1/Parkin-dependent mitophagy
-Proton gradient lost, depolarization transmembrane potential
-Pink1 dimerization and activation by auto-phosphorylation
-Pink1 phosphorylates and activates Parkin
-Parkin (E3 ubiquitin ligase) ubiquitinates mitochondrial proteins (mitochondrion decorated by unusual phosphorylation and ubiquitination)
-Binding mitophagy receptors (OPTN) to ubiquitinated mitochondrial proteins
-Binding phagophore via LC3 (to OPTN)
-Elongation phagophore and engulfment of mitochondrion
-Fusion with lysosome
-Degradation mitochondrion
What if there is a defect in one of the proteins of the Pink1/Parkin-dependent mitophagy
Damaged mitochondria not recycled
> not enough energy
> SNc neurons use a lot of energy: sensitive to energy loss, die first due to loss ATP synthesis
Safe fusion mitophagosome and lysosome
Double membrane bilayers around mitochondrion in phagosome
> fuses with lysosomal bilayer (single membrane)
> while fusion, mitochondrion still in one bilayer membrane.
DJ-1 function and in PD
Sensor oxidative stress: redox-sensitive chaperone-like activity, active in oxidative stress
> specific cleft with Cys-106: putative binding site
> oxidized at Cys-106: sensor oxidative stress
> -SH (inactive, reduced thiol form) > -S-OH (transient) > -S=O-OH (protective active, reversible) > S=O=O-OH (protective active, irreversible)
Environmental causes PD: effect Rotenone
Rotenone: fish killer and insecticide
> inhibitor of ETC Complex 1
> Binds competitively to the multiple ubiquinone (Q) binding sites
> energy deprivation, SNc cell death
Carbon monooxide (CO) is toxic because…
It can block cytochrome c oxidase (ETC Complex 4)
> chance to generate ROS and energy deprivation
Trichloroethylene effect
Increases risk PD
> cleaning solvent
> correlation: possibly toxic
HC41: Is there a cure to prevent SNc cell loss
No, progressive loss over time
Side effect levodopa treatment
Serotonin neurons affected (whole brain gets the L-DOPA)
> L-DOPA taken up by serotonin neurons and converted to DA by AADC (DOPA decarboxylase?)
> Release 5-HT (serotonin) with DA in same vesicles
> Less 5-HT signalling in synaptic cleft
-Increasing dose needed after time: can get toxic
Rate limiting enzyme in PD
Tyrosine hydroxylase, in DA synthesis
Men get PD more ofter than women. But after the menopause, they may have risk factors, why then?
Estrogen has protective effects
Search biomarkers PD, why
Lewy bodies only seen in postmortem histology
> early diagnosis and halting progressive decline of DA neurons could benefit patient
Balance L-DOPA concentrations, what if too much and too little
Too much: dyskinesia
Too little: Akinesia/rigidity
Over time in PD progression: window of L-DOPA good concentration narrows
L-DOPA in dopamine neuron terminal
Conversion to DA
> release upon activation
> re-uptake leftovers through DAT
> D2 binding on presynaptic membrane > Gi trimeric protein activated > inhibits DA (negative feedback)
Prodromal symptom: REM-sleep behaviour disorder injury
Physical things done when in dreams: can cause injuries
The risk factors (genetic) of PD, like alpha-synuclein, LRRK2, Pink1, Parkin and DJ-1 are expressed where?
In many neurons, but they do cause PD
> also expression in cortex: leads to behavioural changes in PD, dependent on the expression programs of the genes
Length SNc neurons
Huge, 10 cm length in brain
> SN closer to midpart brain
> striatum more to the front, and the SN axons span across this distance.
> The SN neurons require immense amounts of energy because of this
> not enough ATP, first neurons to die
Are there environmental protective factors for PD?
Yes, like coffee and Ca2+ channel blockers
Observed in PD brains, which suggest a role of this cell death pathway in dopamine neurons
-Loss of cells
-Caspase 3 activity
-DNA cleavage
-Fragmented nucleus
> Apoptosis (intrinsic)
Survival midbrain dopamine neurons depend on the Bcl2 factor … and the cell death during PD development is driven by …
Mcl1, driven by BH3-only factors (pro-apoptotic)
Intrinsic apoptosis regulation
Bak/Bax form channels on mitochondria for cytochrome c release, activation Caspase-9 by bining complex with Apaf1 > activation Caspase-3 > cell death
-Bcl-2 likes inhibit Bak/Bax by binding them
-BH3-only factors, activated by cellular stress, bind and inhibit Bcl2-likes
Name important Bcl-2 likes and BH3-only factors
Bcl2-likes: Bcl-2, Bcl-xL, Mcl-1
BH3-only: Bid, Bim , Puma, Bad
Defect anti-apoptotic Bcl-2 like Bcl-2 factors leads to
Shift balance to pro-apoptotic Bcl-2 factors
Bcl-2 modulators and growth factors can shift balance back to anti-apoptotic Bcl-2 factors. Is this a cure for PD?
No, delay cells death, but mutation and defect is still there, just longer symptom free.
Apoptosome which can activate Casp-3 or 7 is made from?
Active Casp-9, activated when Cyt-C binds Procasp-9/Apaf1 complex
Which Bcl-2 factor defect leads to SNc dopamine neuron death
Mcl1: highly dependent on it
What is Pitx3 and what if KO
Homeodomain TF, activates a gene program
> mRNA Pitx3 specifically expressed in part which forms SNc dopaminergic neurons
> KO: absence SNc in adult
Transient GFP assay
GFP replacement for one allele for a gene, for example Pitx3
> show expression in which cells > discovered that it was in SNc.
If Mcl1 is functional inhibited, the CC3 and PI in cells are shown. CC3 is the cleaved caspase 3. Why is the PI marker also needed?
To see if the cell actually died (red DNA dye, dead cells, leaky membrane, PI goes through)
Function Mcl1 under basal conditions
Bind Bax to keep it in inactive state
Proximity ligation assay to measure Mcl1-Bax
Secondary antibodies coupled to oligonucleotides (PLA probes) > bind the proteins
> connector oligos ligate probes when in close proximity
> acts as primer for DNA polymerase > detection because oligos are coupled to fluorochromes
> spatial information on protein-protein interaction
Where does Mcl1 bind to Bax?
In the cytosol
Dopamine neurons are basally more vulnerable than non-dopaminergic cells, how is this seen in mice models?
Increased CC3 after Mcl1 functional inhibition
> Mcl1 functional inhibition also induces increased CC3 in SN of WT mice
> increased cell death in PD is not due to loss of one allele Pitx3 in GFP marking the cells
Loss Pitx3 (entirely) leads to
Increase CC3 in dopamine neurons
> increased Noxa mRNA (pro-apoptotic BH3-only factor) > overexpression Noxa leads to induction caspase activation
Which molecule can block Noxa
Bax inhibting peptide (BIP V5)
Mcl1 leads to survival dopamine neurons because it …
inhibis Noxa-dependent apoptosis
HC42: Which dopamine neurons are vulnerable in PD?
SNc, substantia nigra pars compacta
Mesodiencephalic (midbrain) DA neurons originate from
Ventral midbrain (VZ: ventricular zone)
Which enzyme marks DA neurons?
Tyrosine hydroxylase
Radial glia cells
Progenitor cells which produce neurons in cerebral cortex
> cell bodies (somata) reside in VZ
Development dopeminergic neurons in neural tube in WT signalling
Floorplate: DA neurons > Shh signalling strong there
> and little stripes into part baseplate as well
Molecular distinction DA neurons: subgroups and relevance.
-Specific vulnerabilities may reside in molecular differences between SNc and VTA
> SNc, substantia nigra pars compacta
> VTA: ventral tegmental area
Mutation Pitx3
Mutation in key TF in dopamine neuronal development leads to ablation SNc.
> in the basal plate
Which DA neurons contain Pitx3
All of them
Which important enzyme is activated by Pitx3
Aldh1a1 > Adh2 (aldehyde dehydrogenase)
> important in breakdown dopamine
Remove Pitx3, only SNc die, not VTA, why?
VTA neurons in floor plate, only SNc neurons in basal plate affected
Medial DA neurons (VTA) show … Adh2 expression and lateral neurons (SNc) show ..
VTA show restricted gene expression, Adh2 expression in SNc
> Ahd2 only expressed in subset of mdFA neurons
> Pitx3 regulates endogenous Ahd2 gene
> Ahd2 downregulated in Pitx3 -/- mdDA neurons
Pitx3 -/- in SNc
No Ahd2
> Accumulation DA and DOPAL (aldehyde) because deficient breakdown
> reactive aldehyde
> only expressed in lateral position thus frontal area
Retinoic acid production
-Retinol (Vitamin A) > retinaldehyde (retinol dehydrogenase)
-Retinaldehyde > (all-trans) retinoic acid (Retinal dehydrogenase
» Ahd2 required! aldehyde dehydrogenase can convert retinaldehyde to retinoic acid
Why is all-trans retinoic acid needed
Conversion undifferentiated mdDA neurons to differentiated mdDA neurons
Rescue SNc neurons when Pitx3 -/-
Supplement retinoic acid
Complication not enough retinoic acid
Spinal cord problems
SNc neurons need retinoic acid signalling to remain, what about VTA neurons
They do not require it that much
RA signalling in SNc neurons: RA dependent pathway
Pitx3 binds Nurr1
> complex upregulates Ahd2
> Retinaldehyde > RA (Adh2 catalysis)
> RA > RAR-beta
> Expression D2R (D2 receptor) and Th (tyrosine hydroxylase) by RAR-beta/RXR-beta complex (neclear receptor)
Pitx3 RA independent pathway
Pitx3/Nurr1
-Rostral mdDA
> Ahd2, Vmat2 (monoamine neurotransmitter transporter, like DA) , Dat (DAT, transporter re uptake), Th (tyrosine hydroxylase),
> inactivation Cck
Caudal mdDA
> Vmat2, Dat
Cross-inhibiting function Pitx3/Nurr1
Meet in the middle
> rostrolateral mdDA neuron: Adh2 subset: RA signalling, inactivation Cck and En1 by Pitx3
> causal mdDA neuron: Cck subset. No expression Ahd2 and RA signalling, activation Cck by En1. > no cross-inhibition
» specific molecular coding differences lead to vulnerability of differetn mDA subgroups
How many subgroups DA neurons
At least 12-14 > complex TF interplay
Genetic variance in … influences pesticide toxicity towards PD (like paraquat, not rotenone) > toxicity depend on transport by …
DAT
Turnover DA in the VTA is very …
Low
> less dependent on Adh2
Character SNc compared to VTA
Very high arborization > many branches made
> more energy needed to maintain this
> higher bioenenergetic: more DA made, transported and secreted: energy demanding
> small error in energy metabolism leads to big problem
Basal oxygen consumption SNc compared to VTA
Higher
ROS production SNc and VTA
More in SNc
> quicker problems when something is wrong
SNc have … axon length and amount of processes than VTA
Higher
Mitochondrion density SNc vs VTA
Higher is SNc, matching its higher ATP needs and content
Rotenone / paraquat induce blockade of …
Electron transport chain
> error in energy metabolism, SNc vulnerable
> higher survival VTA than SNc when giving those two.
Which important transporter, which is blocked by paraquat, is high expressed in SNc
DAT
SNc + Rotenone + Paraquat
SNc: energy dependent and dependent on DAT
> rotenone blocks Complex 1 and paraquat blocks DAT
> cell death
Reducing axon arborisation in SNc
Sema7a treatment reduces axonal outgrowth
> reduces overall metabolic activity in SNc neurons
> increases resilience of SNc neurons to energy metabolism error
> reduces OCR (oxygen consumption rate)
> the treatment has no significant effect in VTA neurons
The higher load of DA in SNc neurons induces…
Higher risk of toxic effects through aldehydes
Which molecular distinctions induce specific vulnerability of SNc
-Differential DAT expression (higher load DA)
-Differential RA dependence
-Axon arborisation is higher
