Study 4 - Early Bio-marker for Parkinson’s Disease ... An update

Out of each of the studies I have planned, trying to utilise the data and information that I have learnt from the other studies together in such a way that we may highlight particular patterns that are linked with early cognitive or motor declines that can be potentially used as an early bio-marker is the hardest to draw to a conclusion that seems to make sense.

My current thinking is that we can't simply analyse the proteomic results, find particular protein aberrations, then analyse a younger sample ie 30 year olds, to see if the same proteins are showing any form of change or impairment. The problem is that the sample may be taken randomly from a younger person who may never go on to develop any form of neurodegenerative disease.

So how to overcome this dead end issue. We know from other studies that mitochondrial disease is definitely linked to other diseases such as coronary, vascular disease, diabetes, schizophrenia etc. which also have a higher correlation of being linked as comorbid diseases with Parkinson's disease.

By the time someone presents with Parkinson's they have already lost around 80% of their dopaminergic neurons, and research has indicated that this damage has been occuring over a period of upto 15 years. So if we can we identify a younger sample from individuals already showing signs of these other comorbid diseases and take our samples from this cohort, then this may increase the probability that some of the cohort will go on to develop Parkinson's in the future. An alternative tho this would be to run a cognitive battery of tests over the younger cohort and use samples that show cognitive changes compared to the test norm. 

Understanding Membrane Permeabilization ...

My initial understanding of how permeabilization worked was that when the permeabilizing agent of recombinant cytolysin protein was injected to create large pores in the leukocytes outer membrane, we would see a normal OXPHOS blueprint from each of the working complexes and a flatline for the impaired complex.

However on reflection, once the leukocytes outer membrane was permeabilized and the cells contents leaked out into the cytosol, there would be no further substrates of any type left in the cell to fuel any of the enzyme complexes CI-CIV.

What I now expect to see is that once the leukocytes membrane is permeabilized, and a specific substrate is injected, the only enzyme complex that will be activated to process the substrate and produce ATP, will be the complex that can oxidise the substrate injected into the cell.

As an example, by selectively injecting succinate we can test complex II’s ability to produce ATP.  Any increase or change in OCR will indicate production of ATP indicating that Complex II is working normally. However if there is no OCR consumption, then Complex II has been identified as impaired suggesting that CII, may be the cause of the PD mitochondrial bioenergetic impairment. This then gives us a target for more specific clinical and pharmaceutical investigation.

Study 1 - Bioenergetic changes ...

The first study will look at analysing changes in bioenergetic levels between Parkinson disease participants (PD)and healthy controls (HC) across differing age groups and by gender.

Initially in this study the levels of mitochondrial oxidative respiration will be measure using a Seahorse XFe-96 to simultaneously measure the oxygen consumption rate (OCR) and changes in glycosylation via the Extracellular Acidification Rate (ECAR).

Both oxygen and glucose are necessary in the production of ATP, so by measuring the OCR & ECAR we can estimate the ATP level of production. By measuring OCR & ECAR for healthy individual over the two age groups (40-50 & 60-70 yrs.), we can set up a benchmark to identify how mitochondrial bioenergetic levels change as individuals age.

These levels can be then used as a benchmark for comparison to the same measures in Parkinson disease participants as they age and also between gender.

Proteomic analysis shows PD mitochondria are upregulated

Starting to make some progress on understanding how proteomic analysis may work. The attached article has some interesting points:

1. proteomic results for upregulated Mitochondria, oxidative stress, and energy metabolism in SNpC
2. proteomic results for upregulated Inflammation, phagocytosis, cell signalling in blood plasma
3. “… Proteomic analysis of SN region of human brain demonstrated upregulation of mitochondrial complex III, ATP synthase D, complexin I, profilin, L-type calcium channel δ-subunit, and fatty acid-binding protein in PD patients in comparison to control subjects. These results provide the evidence of disrupted mitochondrial and antioxidant function in PD (Basso et al. 2004). Two other proteomic studies utilizing SN region of PD patients also confirmed the involvement of mitochondrial dysfunction, cytoskeleton impairment, and oxidative stress in PD pathogenesis (Kitsou et al. 2008; Licker et al. 2012)…”
4. “… mitochondria are the most studied organelle for understanding PD pathogenesis and are known to play a central role in PD pathology. A number of proteins related to disease such as PINK1, DJ-1, and parkin are either localized inside or interact with mitochondria, and death of dopaminergic neurons is caused due to induction of oxidative stress and apoptotic pathways involving mitochondria (Nicotra and Parvez 2002; Dixit et al. 2013). However, only a single proteomic study has been conducted on the mitochondrial fraction of the SN region of the PD brain, which demonstrated that 119 out of 842 identified proteins were significantly different in their relative abundance in comparison to age-matched controls…”

Each of these points seem to suggest that proteomic analysis of PD patients found changes to mitochondrial proteins were upregulated in all cases and in the case of oxidative stress and inflammation the proteins were also up[regulated.

Dixit, A., Mehta, R., & Singh, A. K. (2019). Proteomics in Human Parkinson's Disease: Present Scenario and Future Directions. Cellular & Molecular Neurobiology, 39(7), 901-915.