Characteristics of DJ-1 protein in Parkinson’s Disease (PD)

Parkinson‘s Disease

• Neurodegenerative disorder• Selective loss of dopamine (DA) neurons in the substantia

nigra• Symptoms: resting tremor, rigidity, and slowness of

movement.• Traditional treatment includes substituting for the lack of

DA, eg with L-Dopa. Problematic• About 1-2% of the population over 65 affected, of these

10% inherited.• 10 different loci have been identified to date whose

mutation appears connected to PD eg alpha-synuclein and parkin.

• PARK7 locus codes for DJ-1.

DJ-1• 189 AA protein belonging to the ThiJ/PfpI protease family

• homodimer

• implicated for example in male fertility, cancer etc.

• DJ-1 is widely expressed especially in subcortical regions, in both neurones and astrocytes.

• Mostly localises to the cytoplasm, but also to mitochondria, especially in the case of increased oxidative stress.

• In the presence of reactive oxygen species (ROS), the isoelectric point (pI) of DJ-1 becomes markedly more acidic.

• Sensitive to oxidative stress, mainly through Cysteine 106.

Crystal Structure of DJ-1

Monomers 1+2: green and purple

Cys-106: yellow

Leu-166: red

Mutations in DJ-1

• Mutations in the DJ-1 gene have been shown to cause early onset recessive autosomal early onset PD

• L166P point mutation prevents dimerization of DJ-1.• Other mutations also present, eg early truncation due to

wrong insertion of stop codon.• One possible cause for PD might be damage of

neurones through inability to deal with the ROS accumulating during the metabolism of DA.

• Overexpression of DJ-1 has neuroprotective properties against oxidative stress.

Aims of the project

• Aim is to further characterise DJ-1 and to examine putative differences between distribution and biochemistry of DJ-1 between PD and healthy brains,

• Investigate DJ-1’s neuroprotective effects in the healthy and mutated state.

• The project will be based on ongoing studies in the Bandopadyhay lab.

STUDYING THE BIOCHEMICAL NATURE OF DJ-1 IN HUMAN BRAIN

- Comparison of insoluble and soluble DJ-1 in different regions of the brain, eg cerebellum, cortex, striatum, substantia nigra of healthy and PD brains, as well as sub-cellular localisation, using 2D gel electrophoresis.

- In 2D gel electrophoresis proteins are separated according to molecular weight (size) and pI.

IMMUNOHISTOCHEMISTRY

- DJ-1 also present in glial inclusions in multiple system atrophy. - But to date it could not be shown that DJ-1 stains Lewy bodies, inclusions abundant in PD.

- Labelling brain slices of various regions, investigate whether DJ-1 can be localised to Lewy bodies after all.- Use antibody against DJ-1, make that antibody visible with dyes. - Attempt to co-immunoprecipitate DJ-1 and alpha-synuclein.

Example of Immunohistochemistry

CELL CULTURE WORK

• Using various toxins on cell culture in different concentrations. Eg: rotenone or paraquat

• Investigate how those toxins affect the sub-cellular localisation of DJ-1.

• Also see how mitochondria are affected by assaying citrate synthase activity.

References• Bandopadhyay et al (2003). The expression of DJ-1 (PARK7) in normal human

CNS and idiopathic Parkinson’s disease. Brain 127: 420-430.• Bonifati et al (2004). Linking DJ-1 to neurodegeneration offers novel insights for

understanding the pathogenesis of Parkinson’s Disease. J. Mol. Med. 82: 163-174.• Canet-Avilés et al (2004). The Parkinson's disease protein DJ-1 is neuroprotective

due to cysteine-sulfinic acid-driven mitochondrial localization. PNAS 101: 9103-9108.

• Choi et al (2006). Oxidative Damage of DJ-1 is linked to sporadic Parkinson and Alzheimer Diseases. J. Bio. Chem. 281: 10816-10824.

• Cookson (2005) The Biochemistry of Parkinson’s disease. Annu. Rev. Biochem 74: 24-52.

• Moore et al (2005). Molecular Pathophysiology of Parkinson’s Disease. Annu. Rev. Neurosci. 28: 57-87.

• Wilson et al (2003). The 1.1A resolution crystal structure of DJ-1, the protein mutated in autosomal recessive early onset Parkinson’s disease. PNAS 100: 9256-9261.

• Wood-Kaczmar et al (2006). Understanding the molecular causes of Parkinson’s Disease. Trends in Mol. Med. 388.