Investigating the Mechanism of Pathology in Parkinson’s Disease

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Parkinson’s disease (PD) is an age-related neurodegenerative disorder that affects almost 1 million people in the United States. PD is associated with motor symptoms including tremors and stiffness that affect balance and coordination. Symptoms appear as dopaminergic neurons are lost from the midbrain. Neuronal death appears to be due to the accumulation of toxic aggregates, called Lewy bodies, of the protein alpha-synuclein in neurons.

In recent work, Anandhan et al investigated the role of a transcription factor, NRF2, in alpha-synuclein driven pathology. NRF2 is known to regulate the cellular stress response and lack of NRF2 has been shown to exacerbate PD pathology, but the precise roll of NRF2 in alpha-synuclein driven pathology is unknown. The authors created mice that overexpress human alpha-synuclein as a model of PD. They then knocked out the gene coding for NRF2 so study the effect of the loss of NRF2 on behavioral tests, neuron loss in several brain regions, and phosphorylation and oligomerization of alpha-synuclein.

The research compared 4 groups of mice:

  1. Expressing human alpha-synuclein, expressing Nrf2 (ha-Syn+/Nrf2+)
  2. Expressing human alpha-synuclein, Nrf2 knockout (ha-Syn+/Nrf2-)
  3. Not expressing human alpha-synuclein, expressing Nrf2 (ha-Syn-/Nrf2+)
  4. Not expressing human alpha-synuclein, Nrf2 knockout (ha-Syn-/Nrf2-)
immunoblot
Figure 1. Generation of a novel humanized α-Syn/NRF2 mouse model of PD. (A) Mice overexpressing human wild-type α-Syn (hα-Syn+) were initially cross bred with Nrf2 knockout (Nrf2-/-) mice to result in hα-Syn+/Nrf2+/- and hα-Syn-/Nrf2+/- mouse strains. The hα-Syn+/Nrf2+/- mice were further crossed to finally generate four genotypes: hα-Syn+/Nrf2+/+, hα-Syn+/Nrf2-/-, hα-Syn-/Nrf2+/+ and hα-Syn-/Nrf2-/- strains. Mouse genotypes were confirmed by PCR (B and C) of tail DNA and Western blotting using Azure c600 (D).

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According to the study, lysates were boiled, sonicated, and resolved by SDS-PAGE. Membranes were subjected to appropriate antibodies at 4°C for overnight before being incubated with anti-mouse or anti-rabbit horseradish peroxidase (HRP) conjugated secondary antibodies from Sigma Aldrich and imaged using the Azure Biosystems c600 imager (Figure 1D).

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The study found increased alpha-synuclein phosphorylation and oligomerization in the mice expressing human alpha-synuclein and lacking Nrf2. These mice also lost tyrosine hydroxylase–expressing dopaminergic neurons in the substantia nigra and had behavioral defects consistent with early-state PD. The authors conclude that loss of NRF2 drives development of alpha-synuclein related PD pathogenesis via effects on oxidative stress, proteostasis, inflammation, and cell death. They suggest activating NRF2 might present a way to delay onset or progression of PD.

The Azure c600 Imager provides multiple imaging capabilities including chemiluminescence, multi-channel fluorescence, two-channel NIR fluorescence, and white light imaging of blots, gels, Petri dishes, and more. Learn more about the Azure Imaging line and how Azure can support your research by clicking here.

 

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