What do these people have in common

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What do these people have in common ?
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Lecture 13 Nov 12, 2008Parkinsons disease
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History of Parkinsons disease (PD)

• First described in 1817 by an English physician,
  James Parkinson, in An Essay on the Shaking
  Palsy.
• The famous French neurologist, Charcot, further
  described the syndrome in the late 1800s.

Epidemiology of PD
Parkinson's is a idiopathic (unknown cause) is
disease. The most common movement disorder
affecting 1-2 of the general population over
the age of 65 years. The 2nd most common
neurodegenerative disorder after AD.
Clinical features of PD
Three cardinal symptoms i) resting tremor, ii)
bradykinesia (generalized slowness of movements),
iii) muscle rigidity
Neuropathology of PD

• Eosinophilic, round intracytoplasmic inclusions
  called lewy bodies and Lewy neurites
• First described in 1912 by a German
  neuropathologist - Friedrich Lewy
• Inclusions particularly numerous in the
  substantia nigra pars compacta (SNpc)

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Neurochemistry of PD

• Late 1950s Dopamine (DA) present in mammalian
  brain, and the levels highest within the
  striatum.
• 1960, Ehringer and Hornykiewicz The levels of DA
  severely reduced in the striatum of PD patients.
• PD symptoms become manifest when about 50-60 of
  the DA-containing neurons in the substantia nigra
  and 70-80 of striatal DA are lost.

Levodopa Therapy of PD and limitations

• Late 1950s L-dihydroxyphenylalanine (L-DOPA
  levodopa), a precursor of DA that crosses BBB,
  could restore brain DA levels and motor functions
  in animals treated with catecholamine depleting
  drug (reserpine).
• First treatment attempts in PD patients with
  levodopa resulted in dramatic but short-term
  improvements took years before it become an
  established and succesfull treatment.
• Still today, levodopa cornerstone of PD
  treatment virtually all the patients benefit.
• Efficacy tends to decrease as the disease
  progresses. Chronic treatment associated with
  adverse events (motor fluctuations, dyskinesias
  and neuropsychiatric problems).

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Dopamine pathways in human brain
Substantia Nigra A dark band of gray matter deep
within the brain where cells manufacture the
neurotransmitter dopamine for movement control.
Degeneration of cells in this region may lead to
a neurologic movement disorder such as
Parkinson's disease.
Dopamine affects muscle contraction via the five
dopamine receptors D1, D2, D3, D4, and D5. The
receptors D2, D3 and D4 are inhibitory. The
receptors D1 and D5 are stimulatory. The combined
inhibitory effect of D2, D3 and D4 is more
powerful in total than the combined stimulatory
effect of D1 and D5. So the overall effect of
dopamine is to inhibit muscle contraction.
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Dopamine metabolism
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Drugs that increase dopamine levels
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L-DOPA therapy

• L-DOPA
• metabolic precursor of the dopamine that crosses
  BBB,
• converted by endogenous aromatic amino acid
  decarboxylase (AAD, DOPA-decarboxylase) to
  dopamine
• Stored in surviving nigrostriatal terminals
• Large doses are required, because much of the
  drug is decarboxylated to dopamine in the
  periphery, resulting in side effects that include
  nausea, vomiting, cardiac arrhythmias and
  hypotension.
• DDI
• Administered with a peripheral dopa-decarboxylase
  inhibitor (DDI) as Carbidopa, (does not cross
  BBB).
• prevents the formation of dopamine peripherally
• increase availability of L-DOPA to CNS and allows
  a lower dose of L-DOPA to be administered.

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Monoamine oxidase B (MAO-B) inhibition
L-DOPA is converted in the brain to dopamine.
COMT (Catechol-O-methyltransferase) inhibitors
block the methylation in the periphery 3-O-MD
(3-O-methyl DOPA), thereby increasing the L-DOPA
delivery to the brain. MAO inhibitors reduce the
metabolism of dopamine. AAD (aromatic L-amino
acid decarboxylase) DOPAC, 3,4-dihydroxyphenylace
tic acid 3MT, 3-methoxytyramine.
Selegilines (selective MAO-B inhibitor)
decreases the metabolism of dopamine by
preventing inter-neuronal degradation. Inhibition
of MAO-B slows the breakdown of dopamine in the
striatum. Drawback of selegiline is its
metabolism to ()-amphetamine and
()-methamphetamine. Rasagiline is a potent,
selective, irreversible inhibitor of MAO-B, and,
in contrast to selegiline, has no amphetamine
metabolites. Indeed, its major metabolite,
1(R)-aminoindan, has shown protective activity in
disease models relevant to PD. Schapira A, Nat
Rev Drug Discov 20054625.
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DA synthesis in striatum by gene delivery
TH is the rate-limiting enzyme that converts
tyrosine to L-DOPA, AADC (aromatic L-amino acid
decarboxylase) converts L-DOPA to DA. GCH (GTP
cyclohydrolase 1) is the rate-limiting enzyme for
the synthesis of an essential co-factor of TH,
which is tetrahydrobiopterin (BH4). Mixture of
the three recombinant adeno-associated virus
(rAAV) vectors expressing TH, AADC, and GCH was
stereotaxically injected into the unilateral
putamen. Co-expression of these three enzymes in
the putamen led to a marked improvement
persisted. TH-immunoreactive (TH-IR), AADC-IR,
and GCH-IR cells were present ingt90 of the
putamen. Okada T J Neurol 2002249 (supp 2)36.
Reversal of dyskinesias by continuous delivery of
DOPA
In vivo gene transfer of DA-synthetic enzyme
tyrosine hydroxylase and GCH1 using rAVV vectors
can provide constant source of DOPA production
locally in the striatum. Carlsson T Brain 128559
(2005)
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Animal models of Parkinsons Disease

• 6-hydroxydopamine (6-OHDA) is taken up by the
  dopamine transporter, then generates free
  radicals.
• Rotenone is a direct inhibitor of complex I,
  which also leads to free-radical generation.
• MPTP is converted by monoamine oxidase B (MAOB)
  to MPP, which is taken up by the dopamine
  transporter. MPP inhibits mitochondrial complex
  I, generating free radicals. Chronic MPTP
  administration may produce a-synuclein
  aggregation. MPP uptake by the vesicular
  monoamine transporter reduces toxicity.
• MPTP and rotenone increase the expression of
  a-synuclein and, in the latter case, leads to the
  formation of Lewy bodies.

Beal MF. Nat Rev Neurosci 20012325
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Lewy body dementia Parkinsons disease dementia
cortical Lewy bodies with the ubiquitin stain
Lewy Body abnormal aggregates of protein that
develop inside nerve cells. A Lewy body is
composed of the protein a associated with other
proteins such as ubiquitin, neurofilament
protein, and aB crystallin. Linked to PD.
Patients with diffuse Lewy body disease have
dementia and hallucinations.

• Lewy bodies are a-synuclein cytoplasmic
  inclusions
• Lewy body dementia (LBD) is a progressive brain
  disease and the second leading cause of
  degenerative dementia in the elderly.
• Clinical name dementia with Lewy bodies (DLB).
• Over 50 of Parkinsons disease patients develop
  Parkinsons disease dementia (PDD).
• Early 1900s Friederich H. Lewy, while
  researching PD, discovered abnormal protein
  deposits that disrupt the brain's normal
  functioning.
• Degeneration in substantia nigra as would be seen
  in Parkinson's disease.
• Normally the substantia nigra is populated by
  nerve cells which contain a dark-brown pigment
  called neuromelanin and also make dopamine.
• In both PD and LBD these cells die. Substantia
  nigra appears abnormally pale in comparison to
  normal. Remaining nerve cells contain abnormal
  structures called Lewy bodies, a pathological
  hallmark of the disease process.

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Parkinson's disease-associated genes
Abou-Sleiman et al. Nat. Rev. Neurosci. 20067207
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Emerging pathways in genetic Parkinson's disease
Potential role of ceramide metabolism in Lewy
body disease Genes associated with Lewy body
inclusions and their role potential role in
ceramide metabolism

• Several of the genes involved in disparate Lewy
  body diseases impinge on ceramide metabolism and
  this may be a common theme for pathogenesis.

Neurodegeneration with brain iron accumulation-1
(NBIA-1) leucine-rich repeat kinase 2 (LRRK2) are
a common cause of PD
Bras J FEBS J 20082755767.
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Radial glia cells as dopaminergic progenitors

• Radial glial cells
• A pivotal cell type in the developing CNS
  involved in key developmental processes,
  (patterning and neuronal migration) and
  precursors during neurogenesis
• ability to generate neurons and astrocytes
• First neuronal precursors only later shifts
  towards generation of astrocytes
• Cells isolated from E14 cortex half gave neuronal
  clones and rest gave astroglia, while E18 gave
  mostly astrocytes.
• This suggests as development proceeds radial glia
  differentiation becomes more restricted
• Arise early in development from neuroepithelial
  cells (a subtype of stem cells)
• Lead to the production of dopaminergic neurons in
  the midbrain.

Model of stem cell lineage from embryogenesis to
adulthood. Stem cells are likely contained within
the neuroepithelial --gt radial glia --gtastrocyte
lineage. Radial glia (blue) not only give rise to
neurons (red) but also serve as the scaffolding
for their migration. SVZ and SGZ (subgranular
zone) astrocytes (blue) are stem cells in the
adult brain and give rise to neurons (red) via an
intermediate progenitor (green). Oligodendrocytes
may arise from radial glia during embryogenesis
and from astrocytes in the adult either directly
or via an intermediate progenitor (not shown in
schema).
Malatesta Development 1275253(2000) Bonilla S
(2008) Glia, 56, 809 (2008) Doetsch F. Nat
Neurosci 200361127
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Radial glia cells as dopaminergic progenitors

Cells labeled with BrdU in utero differentiated
into TH DA neurons (asterisks G, H) GFP/ TH DA
neurons were seen in both in clusters or isolated
(L-Q).
Bonilla S (2008) Glia, 56, 809 (2008)
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n-3 PUFA diet prevented MPTP-induced loss of TH
labeled nigral cells
Vehicle
n-3 PUFA diet
Control
Tyrosine hydroxylase (TH) neuronal counts
revealed a neuroprotective effect in animals fed
the diet containing high concentrations of n-3
PUFAs (D) compared to the control diet (C).
Bousquet M, FASEB J 2008221213
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n-3 PUFA diet prevented MPTP-induced loss of
NURR1 expression and dopamine transporter (DAT)
Nuclear receptor related 1 (NURR1) protein is a
member of the nuclear receptor family and plays a
key role in the maintenance of the dopaminergic
system of the brain. n-3 PUFAs protect against
MPTP-induced decrease of Nurr1 nigral
expression. Nurr1 mRNA levels in the SNpc
(Substantia nigra pars compacta) show partial
restoration of Nurr1 mRNA levels MPTP-treated
animals fed the high n-3 PUFA diet. Dopamine
transporter (DAT) transporting dopamine from the
synapse into a neuron. High n-3 PUFA diet
preserved DAT of MPTP treated mice.
vehicles
control
PUFA diet
Bousquet M, FASEB J 2008221213
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DHA reduces L-DOPA mediated dyskinesia in PD
Dyskinesia refers to involuntary movements,
similar to a tic or chorea. Time courses of the
development of the maximum dyskinetic scores
after L-dopa treatment in de novo MPTP monkeys.
In the context of Parkinson's disease,
dyskinesias are often the result of chronic
levodopa (L-DOPA) therapy or levodopa failure
syndrome. These motor fluctuations occur in more
than half of PD patients after 5 to 10 years of
levodopa therapy, with the percentage of affected
patients increasing over time.
Samadi P. Ann Neurol 200659282.
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Possible routes of DHA protection in PD

• DHA shown to
• increase glutathione reductase activity
• Decrease accumulation of oxidized proteins, lipid
  peroxides, and ROS
• MPTP toxicity involved increased ROS and damage
  to mitochondrial DNA
• Chronic n-3 PUFA might decrease MPTP ? to MPP
• N-PUFA reduces MAO-B activity in the brain

Bousquet M FASEB J 2008221213
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Generation of oxidative cholesterol metabolites
in vivo
Cholesterol (1) can be oxidized to form the
cholesterol 5,6-secosterol (2). Transient Schiff
base formation (box) between the aldehyde
(circled) functionality of (2) and amine groups
on proteins catalyzes the conversion of (2) into
(3) in vitro and in vivo, and therefore we refer
to the metabolites collectively as (3). Oxidation
of (3) yields (4) bearing a carboxylic acid.
Basco et al Nat. Chem Biol 2249 (2006)
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Cholesterol oxidation products a-synucleinopathy

• Mature a-synuclein fibrils were formed in
  reactions containing (4) by 48 h.
• In the presence of (2/3), protofibrils and short
  fibrils were detected after 48 h, which were
  transformed into mature fibrils by 96 h

Basco et al Nat. Chem Biol 2249 (2006)
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cPLA2 null mice are resistant to MPTP toxicity
TH-immunostained sections of substantia nigra
MPTP-treated cPLA2 wt mice show marked neuronal
loss within the medial segment of the substantia
nigra pars compacta (SNpc) compared to
PBS-treated and cPLA2 null mice
Effects of MPTP on striatal dopamine, DOPAC, and
HVA levels in cPLA2wt, cPLA2/-, and
cPLA2-/-mice.
p lt 0.05, p lt 0.01, p lt0.001 compared with
PBS p lt 0.05 compared with MPTP-treated control
cPLA2 wt and heterozygous cPLA2/-mice.
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cPLA2 null mice are resistant to

• MPTP toxicity (PD model) (Klivenyi et al, J
  Neurochem 1998)
• Stroke (Bonventre et al, 1997 Nature)
• MS-EAE (Marusic, J Expt Med 2005)
• AD (Sanchez-Mejia RO, Nat Neurosci 2008)

cPLA2 has been implicated in

• Spinal cord Injury (Liu, Ann Neurol 2006)
• Schizophrenia (Law, Mol Psychiatry 2006)
• Bipolar disorders (Rao JS, Mol Psychiatry 2008)

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Iron and oxidative stress in PD
Neuromelanin is a byproduct of the synthesis of
monoamine neurotransmitters for which the
pigmented neurons are the only source. The loss
of pigmented neurons from specific nuclei is seen
in a variety of neurodegenerative diseases. In
Parkinson's disease there is massive loss of
dopamine producing pigmented neurons in the
substantia nigra.
Zecca et al Nat Rev Neurosci 20045863
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Iron and oxidative stress in PD

• Dopamine (DA) required for neuronal signaling is
  vesicle bound and redox inert.
• when DA is released from the vesicle into the
  cytoplasm it is able to coordinate Fe and undergo
  redox reactions that result in the formation of
  neuromelanin (NM) and reactive oxygen species
  (ROS).
• Neuromelanin will also coordinate Fe and produce
  ROS.
• The equilibrium between vesicle bound DA and
  cytoplasmic DA is regulated by a-synuclein
  mutations in this protein shift the dopamine
  equilibrium in favor of the cytoplasm.
• In the presence of Fe and under conditions of
  oxidative stress a-synuclein will aggregate and
  form deposits. MPP, 1-methyl-4-phenyl pyridine.

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The protective and toxic role of neuromelanin (NM)

• The synthesis of NM removes excess cytosolic
  catecholamines that are not accumulated by
  synaptic vesicles, thus preventing the consequent
  damage. In this example DA, synthesized from
  tyrosine by TH and AADC, is normally taken up
  into vesicles by the vesicular monoamine
  transporter VMAT2. Excess DA can interact with
  Fe3 to form quinones and semiquinones. If these
  react with cysteine to form cysteinyl-dopamine,
  which can be further converted into NM,
  neurodegeneration can be avoided.
• Exogenous metals can be released in reactive and
  potentially toxic forms in the cytosol toxic
  metals such as Cd and Hg can also reach the
  neuronal cytosol following environmental
  exposure. Neuromelanin chelates toxic metals in
  the cytosol, forming stable complexes. Such
  chelation blocks toxic effect of metals,
  including Fenton's reaction and enzyme
  inactivation.
• Following a neuronal damage (due to an
  environmental or genetic factor), the released NM
  induces microglial activation, with production of
  the neurotoxic (TNF-a, IL-6 and NO, which damage
  other neurons. These degenerating neurons will
  release NM, then establishing a chronic condition
  of neuroinflammation and neurodegeneration. Zecca
  LTrends Neurosci 200326578.

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Iron, oxidative stress and neuromelanin (NM) in PD
Iron histochemistry with modified Perls' staining
of human substantia nigra (a) and locus coeruleus
(b) from a normal male subject aged 88. NM in
dopaminergic neurons of the substantia nigra and
noradrenaline neurons of the locus coeruleus are
seen as brown granules, and iron deposits are
stained blue. Neuromelanin-containing neurons in
both the substantia nigra (large arrows in a) and
locus coeruleus (large arrows in b) do not stain
blue for iron.
Zecca et al Nat Rev Neurosci 20045863
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Diagnostic methods for Parkinsons
Disease Sydney-based Diagnosing dopamine cell
loss  (DEDCeL) study 
In PD many of the NM-containing pigmented brain
cells which control normal movement die,
releasing their NM pigment into the brain. The
pigment is removed from the brain by cells of the
immune system into the blood and that these
immune cells make a new protein, an antibody, as
a response to being exposed to the released
pigment. The test measures this new protein in a
small blood sample.  If high blood levels of this
new protein are found this suggests that the
NM-containing brain cells are actively dying.
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END OF LECTURE