Expression of an antioxidative enzyme in the normal and pakinsonian brain presented by Simone Abercrombie Bio 475

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Expression of an antioxidative enzyme in the
normal and pakinsonian brainpresented by Simone
AbercrombieBio 475

• van Muiswinkel F.L., R. A. I. de Vos, J.M. Bol,
  G. Andringa, E.H. Jansen Steur, D. Ross, D.
  Siegel and B. Drukarch

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Parkinsons disease (PD)

• Neurodegenerative disorder
• Death of dopaminergic neurons which contain
  neuromelanin.

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Loss of dopaminergic neurons cause

• Gliosis- Scars that are produced by enlargement
  of Astrocyte processes. When a portion of the CNS
  is damaged (Neuron or Axon), Astrocyte processes
  enlarge and replace the damaged tissue. This
  process is referred to as Gliosis. Scar-sclerosis
• Accumuulation of proteins called Lewy bodies and
  Lewy neurites

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od.jpg
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on/Figures/MS_gliosis_draw.jpg
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Normal function of dopamine
Dopamine

• The cells in this area need a proper balance of
  dopamine, a neurotransmitter, for proper motor
  function.
• Loss of dopamine causes the nerve cells of the
  striatum to fire out of control, leaving patients
  unable to control their movements in a normal
  manner.
• Parkinson's patients have a loss of 80 percent or
  more of dopamine-producing cells in the
  substantia nigra.

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Dopamine
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M/brain_images/DAmet.gif
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Pathway of neurotransmitter Dopamine
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Dopaminergic terminal
Prototypic dopaminergic terminal with cycle of
synthesis, storage, release and removal of
dopamine. (Cooper, Bloom Roth, 1996)
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Major neural pathways in normal and Parkinsonian
basal ganglia, (Vermeulen, 1994) . The thickness
of the arrows represents the strength of the
signal.
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Pathogenic factors

• Genetic factors (familial PD)
• Environmental factors (sporatic)
• Oxidative metabolism of dopamine-A chemical
  process which leads to leads to the
• Formation of hydrogen peroxide (H2O2). This leads
  to the formation of highly reactive hydroxyl
  radicals that can cause cell damage.
• In PD, levels of reduced glutathione (clears
  H2O2) are decreased. Thus, loss of protection
  against formation of free radicals. Iron is
  increased in the substantia nigra (brain) and
  serve as a source of donor electrons, thereby
  promoting the formation of free radicals.
• Oxidative stress-A condition in which antioxidant
  levels are lower than normal. Antioxidant levels
  are usually measured in blood plasma.

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Free radicals in depth

• They are highly unstable because they seek other
  compounds and break bonds between stable
  compounds causing a chain reaction.
• Free radicals may come from environmental
  pollution, radiation, cigarette smoke, chemicals,
  and herbicides

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Genetics

• Inheriting mutated genes are linked to the same
  genes that are altered sporadically by
  environmental factors and toxins.
• alpha-synuclein-This gene was mutated in families
  with familial PD
• Parkin-encoded into a protein which functions to
  help cells break down and recycle proteins
• DJ-1-normally helps control gene activity and
  protect cells from oxidative stress
• PINK1-when it is mutated it increases
  vulnerability to cellular stress
• LRRK2-linked to late onset of familial PD and a
  small percentage in sporadic PD

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Aging

• In some individuals, the normal, age-related
  wearing away of dopamine-producing neurons
  accelerates.
• This theory is supported by the fact that the
  loss of antioxidative protective mechanisms is
  associated with both Parkinson's disease and
  increasing age.

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Expression of NAD(P)H Quinone oxidoreductase in
the normal and Parkinsonian substantia nigra
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NAD(P)HQuinone oxidoreductase (NQO1)

• detoxication enzyme
• Has antioxidative properties
• catalyses the two-electron reduction of DAQs
  (electron deficient, highly reactive) into
  DAhydroquinone, a relatively redox stable entity.

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Rationale

• Examine the cellular expression of NQO1 in the
  brain of a large series of idiopathic(no known
  cause) PD patients and age-matched controls
• Data on the cellular localization of NQO1 in the
  Parkinsonian SNpc is lacking
• Investigate the potential role of NQO1 in the
  pathogenesis of PD

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Methods

• Tissue obtained at autopsy
• Fixed by immersion in buffered formaldehide
• Embedded in paraffin
• Stained
• Treated with H2O2 in ethanol
• Undergo microwave irradation to achieve antigen
  retrieval.
• NQO1 immunoreactivity was detected by using
  anti-NQO1 antibodies raised against human NQO1
  proteins.
• As a positive control for NQO1, non-small cell
  lung cancer and small cell lung cancer tissue was
  used with the same procedure.

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Demographic information, clinical status, and
neuropathologial characteristics of contol and PD
cases
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Hoehn and Yahr stages III-V

• 3. Stage Three
• Significant slowing of body movements Early
  impairment of equilibrium on walking or standing
  Generalized dysfunction that is moderately severe
  
• 4. Stage Four
• Severe symptoms Can still walk to a limited
  extent no longer able to live alone .
• 5. Stage Five
• Cachectic stage Cannot stand or walk.
  Requires constant nursing care.

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DLB

• Dimentia with lewy bodies

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Braak stages 1-IVdistribution of
neurofibrillary-Accumulation of twisted protein
fragments inside nerve cells.

• 1-2 Characterized by an either mild or severe
  alteration of the transentorhinal layer Pre-alpha
  
• 3-4 The two forms of limbic stages (stages
  III-IV) are marked by a conspicuous affection of
  layer Pre-alpha in both transentorhinal region
  and proper entorhinal cortex. In addition, there
  was mild involvement of the first Ammon's horn
  sector.

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Results

• Specificity of NQO1 immunocytochemistry
• Expression of NQO1 in the control SNpc
• Expressio of NQO1 in the parkinsonian SNpc

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Specificity of NQO1 immunocytochemistry

• Tissue staining staining with antibodies raised
  against NQO1 protein shows expression of NQO1 in
  normal respiratory tissue and non-small cell lung
  cancer (NSCLC).
• No expression in small cell lung cancer (SCLC),
  or surrounding tissue.

A) SCLC (asterisk) tissue with normal respiratory
epithelium (brown) (B and C) non-NSCLC (double
asterisks) tissue with scattered coal deposits
and normal respiratory epithelium (C, brown) (D)
NSCLC tissue immunostained for NQO1 with AEC.
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Expression of NQO1 in the control substantia
nigra, pars compacta (SNpc)

• NQO1 immunoreactivity was detected in three major
  cell types melanized dopaminergic neurons,
  astrocytes, and vascular endothelium.
• NQO1 staining was not detected when antibodies
  were omitted or replaced by C100 control
  hybridoma supernatant (data not shown).
• Cytoplasmatic staining of NQO1 was observed in
  neuromelanin containing dopaminergic neurons
• A, star points out the neuromelanin pigment
• B, astroglial cells
• C, vascular endothelium
• D shows a NQO1-immunopositive astrocyte in a
  Parkinsoninan SNpc.

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Expression of NQO1 and glial markers in the
normal SNpc. contd

• Case 3 control
• Most of the NQO1-immunopositive glial cells were
  astrocytes(A-C).
• Arrows in A and B indicate NQO1 immunopositive
  astrocytes.

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Expression of NQO1 in PD

• CASE 22 BD show high-power magnifications of A.
  
• Reactive astrocytes are indicated by arrows
• Arrowheads indicate NQO1-immunonegative Lewy
  bodies.
• C-D shows reactive astrogliosis and NQO1
  immunopositive neurons.

Note further examination on the basis of early,
intermediate or end-stage revealed NQO1 response
dissapeared when the loss of neurons has advanced.
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Expression of NQO1 in the Parkinsonian substantia
nigra, pars compacta

• CASE 21 A-C (intermediate stage)
• CASE 24 D-F (end stage)
• Intermediate-stage PD is characterized by the
  presence of marked gliosis and intense
  NQO1-immunostaining of astroglial cells (arrows)
• End-stage PD NQO1 expression is limited to
  vascular endothelial cells (double arrow).

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• Discussion

• Expression of NQO1 was restricted to the SNpc.
• NQO1 is present in the form of reactive fibrous
  astrocytes, pigmented neurons, or a combination
  of both.
• The increase in cellular expression of NQO1
  occours when degeneration of neurons is actively
  taking place, not in the end stages (figure
  5A-C).
• NQO1 reactivity coincides with the presence of
  activated (phagocytic) microglial cells (figure 5
  B C).
• At end stage when degeneration of neuron is
  almost complete, NQO1 was absent.
• The increase in NQO1 expression in the
  Parkinsonian SNpc indicates a possible
  correlation between the expression of NQO1 and
  ongoing degeneration of dopaminergic neurons.

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Conclusion

• NQO1 detoxication enzymes are potential targets
  for neuroprotective theraputic strategies for PD

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References

• References
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