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Expression of an antioxidative enzyme in the normal and pakinsonian brain presented by Simone Abercrombie Bio 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. Parkinson’s disease (PD). Neurodegenerative disorder
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Expression of an antioxidative enzyme in the normal and pakinsonian brainpresented bySimone 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
Parkinson’s disease (PD) • Neurodegenerative disorder • Death of dopaminergic neurons which contain neuromelanin.
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 http://www.nottingham.ac.uk/pathology/images/lewybod.jpg http://missinglink.ucsf.edu/lm/ids_104_Demyelination/Figures/MS_gliosis_draw.jpg
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.
Dopaminergic terminal Prototypic dopaminergic terminal with cycle of synthesis, storage, release and removal of dopamine. (Cooper, Bloom & Roth, 1996)
Major neural pathways in normal and Parkinsonian basal ganglia, (Vermeulen, 1994) . The thickness of the arrows represents the strength of the signal.
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.
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
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
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.
Expression of NAD(P)H: Quinone oxidoreductase in the normal and Parkinsonian substantia nigra
NAD(P)H:Quinone 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.
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
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.
Demographic information, clinical status, and neuropathologial characteristics of contol and PD cases
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.
DLB • Dimentia with lewy bodies
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. Braak stages 1-IVdistribution of neurofibrillary-Accumulation of twisted protein fragments inside nerve cells.
Results • Specificity of NQO1 immunocytochemistry • Expression of NQO1 in the control SNpc • Expressio of NQO1 in the parkinsonian SNpc
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.
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.
Expression of NQO1 and glial markers in the normal SNpc. …cont’d • Case 3: control • Most of the NQO1-immunopositive glial cells were astrocytes(A-C). • Arrows in A and B indicate NQO1 immunopositive astrocytes.
Expression of NQO1 in PD • CASE 22: B–D 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.
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).
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. Discussion
Conclusion • NQO1 detoxication enzymes are potential targets for neuroprotective theraputic strategies for PD
References References: • Beyer R.E, J. Segura-Aguilar, S. Di Bernado, M. Cavazzoni, R. Fato, and D. Fiorentini. 1997. The two-electron quinone reductase DT-diaphorase generates and maintains the antioxidant (reduced) form of coenzyme Q in membranes. Molecular aspects of medicine. 18:15–23. • Cadenas E. 1995. Antioxidant and prooxidant functions of DT-diaphorase in quinone metabolism. Biochemical Pharmacology. 49:127–140. • Drukarch B. and F.L. van Muiswinkel. 2000. Drug treatment of Parkinson’s disease: time for phase II. Biochemical Pharmacology. 59:1023–1031. • Drukarch B. and F.L. van Muiswinkel. 2001. Neuroprotection for Parkinsons’s disease: a new approach for a new millennium. Expert opinion on investigational drugs. 10:1855–1868. • Harada S., C. Fujii, A. Hayashi and N. Ohkoshi. 2001. An association between idiopathic Parkinson’s disease and polymorphisms of phase II detoxification enzymes: glutathione S-transferase M1 and quinone oxidoreductase 1 and 2. Biochemical and biophysical research communications. 288:887–892. • Segura-Aguilar J. and C. Lind. 1989. On the mechanism of the Mn3+-induced neurotoxicity of dopamine: prevention of quinone-derived oxygen toxicity by DT diaphorase and superoxide dismutase. Chemico-biological interactions. 72:309–324. • van Muiswinkel F.L., R. I. de Vos, J.M. Bol, G. Andringa, E.H. Jansen-Steur, D. Ross, D. Siegel and B. Drukarch. 2003. Expression of NAD(P)H:quinone oxidoreductase in the normal and Parkinsonian substantia nigra. Neurobiology of Aging. 25:1253-1262.