PINK1 has the kind of name you’d want for every gene – short, succinct, and easy to remember. PINK1 is a gene pathway that when expressed, has certain protective features against Alzheimer pathologies. Its presence reduces beta-amyloid protein accumulation and mitochondrial dysfunction, making it an extremely efficient neuroprotectant. When I think of the PINK1 pathway, I imagine a giant, pink shield defending us against Alzheimer’s, because of how the pathway protects against the aforementioned Alzheimer pathologies. Ultimately, people care about this pathway because it harbors great potential as a solution to Alzheimer’s.
An important problem in Alzheimer’s is the prevalence of defective mitochondria and subsequent breakdown of the powerhouses of the cell. In the body, damaged mitochondria go on to increase production of reactive oxygen species, which leads to tissue damage and accelerated aging; however, PINK1 specifically protects the cell via a mechanism called mitophagy, where dysfunctional mitochondria are eaten up by internal vacuoles and broken down. Through this mechanism mediated by PINK1, the neuron can preserve a careful equilibrium to go on and live a happy, productive life. In order to experimentally prove that PINK1 was responsible for these effects, the researchers had to create a method to either turn on or turn off this specific gene. Thus, in this paper, I will focus on breaking down a single method: production of the rAAV2-PINK Construct for regulating PINK1 gene expression.
From the paper:
“Viral production and packaging using recombinant adeno-associated virus type 2 (rAAV2) encoding a protein of interest under control of the cytomegatovirus (CMV) promoter has been described in our previous study (Rappold et al., 2014). Briefly, human wild-type PINK1 and mutant PINK1-L347P (Cui et al., 2010) were subcloned into the pBSFBrmcs shuttle vector and then the modified pFBGR plasmid backbone containing EGFP to monitor the expression of PINK1 proteins after rAAV injection. Finally, rAAV2 packaging was performed as described previously (Rappold et al., 2014). Control experiments were performed with an AAV2-GFP construct.”
Initially, I was intrigued by this protocol because adeno-associated viruses are commonly used vectors for insertion of a gene of interest; however, despite being described so simply, there turned out to be an incredible amount of complexity to the protocol. Consequently, I labeled the terms of interest.
Terms to know:
- Recombinant: DNA and various genetic information created by combining multiple sources of DNA 
- Adeno-associated virus type 2: A non-pathogenic virus commonly used to help insert genes of interest into cells for expression. The virus is prevented from stably integrating its genome into the AAVS1 region of chromosome 19 of the host by removal of the rep and cap genes 
- Cytomegalovirus promoter: A common mammalian promoter normally used for general expression of a gene 
- Sub-cloned: A technique to move a piece of DNA from one vector to a different vector 
- Shuttle Vector: A piece of DNA that can be moved around from cell-to-cell that can exist in multiple species i.e. a plasmid from E. coli that can be inserted into humans 
- Plasmid Backbone: Empty plasmids that you can put your gene of interest on 
- PINK1-L347P: A mutant PINK1 variant that does not have a functional active site after swapping a L (leucine) for a P (proline) amino acid.
- Stereotactic Insertion: A minimally invasive surgery that uses tiny bone landmarks within the skull to position and direct cutting, injection, etc. 
After reading Rappold et al., 2014, I realized that the packaging step of the virus is not difficult to understand — rather, it utilizes large, complex words for many simple ideas. The most interesting step of the isolation they used required a cesium chloride gradient for removal of active viral particles
“Fractions with a refractive index of 1.374–1.370, corresponding to the position of viable viral particles, were collected and subsequently dialysed against PBS.” (Rappold et al., 2014)
In conclusion, the researchers wanted to overexpress both the normal PINK1 gene and a mutant isoform of the PINK1 gene, PINK1-L347P, in neuron cells to test their effects on beta-amyloid plaque formation. They accomplished this by messing with an adeno-associated viral vector, and through a multi-step process, expressed the genes on separate plasmid within neurons. In the future, I look forward to working with similar systems in research and although I think that this ubiquitous method of gene insertion may become antiquated in the coming years, I’m glad to add another method to my molecular toolbox.
- Fang Du, Qing Yu, Shijun Yan, Gang Hu, Lih-Fen Lue, Douglas G Walker, Long Wu, Shi Fang Yan, Kim Tieu, Shirley ShiDu Yan; PINK1 signalling rescues amyloid pathology and mitochondrial dysfunction in Alzheimer’s disease, Brain, , awx258, https://doi.org/10.1093/brain/awx258
- Rappold, P. M. et al. Drp1 inhibition attenuates neurotoxicity and dopamine release deficits in vivo. Nat. Commun.5:5244 doi: 10.1038/ncomms6244 (2014).