Transcript ABSTRACT - University of Colorado

PATHOLOGIC AGGREGATION OF THE BRAIN PROTEIN -SYNUCLEIN CAUSES CELL
DEATH IN PARKINSON AND ALZHEIMER DISEASE, Wenbo Zhou, PhD and Curt R. Freed, MD
Division of Clinical Pharmacology and Toxicology, Department of Medicine and the Neuroscience Program, University of
Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262, USA
The deposition of -synuclein and other cell proteins in Lewy bodies in midbrain dopamine neurons is a
pathological hallmark of Parkinson disease. In Alzheimer disease, a fragment of -synuclein is found in
the plaques deposited in brain. In vitro, oxidation and nitration of -synuclein leads to the formation of
dimers, polymers and fibrils through di-tyrosine cross-linking, suggesting that the cross-linking process
can seed and initiate protein precipitation. To determine if enhanced dimer formation can accelerate
protein aggregation and increase neuronal toxicity, we have substituted cysteine (C) for tyrosine (Y) at
positions 39, 125, 133, 136 in human wild-type -synuclein, and in A53T and A30P mutant -synuclein.
Overexpression of Mutant -Synuclein Causes
Rat DA Neurons Death in Culture
Overexpression of Human -Synuclein Leads to
Human DA Neuron Death in Culture
To reduce the likelihood of cross-linking, phenylalanine (F) was substituted for tyrosine at the same sites.
We examined aggregate formation and neurotoxic effects of these constructs in a rat dopaminergic cell line
(N27 cells) by transient transfection. Results showed that expression of Y39C or Y125C mutant proteins
led to large intracellular inclusions. Both proteins produced more cell death compared to wild type human
-synuclein. Overexpression of Y133C, Y136C and all four Y to F mutations did not generate inclusions
and were not more cytotoxic than wild type control. Under oxidizing conditions in vitro, recombinant
Y39C or Y125C proteins showed more abundant dimer and polymer formation than wild type synuclein. We conclude that increased dimer formation can accelerate protein aggregation and neuronal
toxicity of -synuclein.

Parkinson’s disease (PD) is pathologically characterized by the loss
of dopamine (DA) neuron and the presence of intracytoplasmic
Lewy bodies (LB) in DA neurons. -Synuclein is the major
component of LB.
Alzheimer’s disease (AD) is pathologically defined by the
widespread amyloid plaques and the intracellular neurofilament
tangles. In addition to the A, amyloid plaques also contain a
fragment of -synuclein (called NAC). Furthermore, patients with
AD frequently contain LB and Lewy neurites (LN).
Test if Structural Modifications of -Synuclein Change Protein Aggregate Property and Cellular Toxicity
 -Synuclein forms dityrosine dimer under nitrative / oxidative stress
 Dimer could be served as seed for oligomer and fibril formation
 Substitute tyrosine for cysteine or phenylalanine to see if it increases or decreases protein
aggregations and cellular toxicity
LBs in PD brain
LN in PD brain
LN in AD brain
-Synuclein Mutations Cause PD
• -Synuclein is a small, soluble, neuronal protein
• Normally present in presynaptic region associated with vesicle membrane
• Two point mutations A53T and A30P cause earlier onset PD
• -Synuclein protein is natively unfolded, can form fibrillar aggregates in vitro
A30P
1
Conclusions
•Overexpression of mutant human -synuclein kills dopamine neuron,
which may initiate the pathogenesis of Parkinson’s disease and related
Lewy Body diseases.
•Substitute of Y39C and Y125C increases the protein aggregations
and cellular toxicity, possible through the accelerated dimer and
oligomer formation.
A53T
140aa
•The tendency of human -synuclein to form aggregates under
oxidative stress and/or other conditions, suggesting finding the ways
to slow down the protein aggregate process can lead to the
development of novel treatment for PD and other related diseases.