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

The final objective is to review the evidence from each of the five experimental studies to investigate the efficacy of using leukocytes to sense early deviations in bioenergetic levels and changes in metabolic stress associated with mitochondrial dysfunction. This capability would make leukocytes a potentially sensitive early biomarker for therapeutic and clinical intervention in neurodegenerative progression of PD (Braganza, Annarapu, & Shiva, 2020; Kramer, Ravi, Chacko, Johnson, & Darley-Usmar, 2014).

Statistical Analysis of Leukocyte Early Bio-marker

Correlation analysis will be used to review OCR and ECAR levels between PD and HC to establish high level relationships for any bioenergetic dysfunctions/impairment. Any changes detected will be tested for significance using between and within sample student t testing. Where patterns of significant change have been observed, multivariate analysis such as hierarchical and stepwise multiple regression will be used to factor out confounding patient data (i.e. BMI, weight etc.) and to investigate the veracity and accuracy of using age related mitochondrial changes in PD patient leukocytes as a predictor of predisposition for early development of PD or for increasing changes in the progression of existing PD symptomatology.

Study 3 - Proteomic Differential Pathway and Abundance Analysis ...

Changes in Protein Abundance by Age and Gender

Study 3 will support the signalling pathway analysis from study 2, by using a proteomic analysis of leukocytes across age and gender matched groups, to investigate differences in HC and PD participant protein abundance and gene expression along a specific signalling pathway.
Abnormal Protein Expression and Neurodegenerative Progression

A key manifestation of neurodegenerative progression in PD pathogenesis is the abnormal expression and aggregation of proteins through transcriptional and translational responses to biological perturbations, resulting in post-translational modifications (Wetie, Woods, & Darie, 2014), altered protein–protein interactions, protein degradation and functional changes to the proteome (Dixit, Mehta, & Singh, 2019; Li, Ganz, & Smit, 2019), in disease progression (Rubinsztein, 2006). Impaired protein expression has been consistently associated with cellular damage, mitochondrial bioenergetic dysfunction, metabolic impairment, increased oxidative stress, elevated proinflammatory responses and increased apoptosis linked to neurodegenerative progression in PD (Dixit et al., 2019; Jiang et al., 2019; Kitsou et al., 2008; Licker, Kovari, Hochstrasser, & Burkhard, 2009). Identification of specific protein changes along an impaired bioenergetic signalling pathway offers the possibility of developing targeted pharmacological and clinical solutions to slowing and treating PD.

Proposed Procedures

Identification of changes in protein expression and abundance will use liquid chromatography (LC) to fractionate blood samples into similar protein groups (Zhang et al., 2012) then mass spectrometry (MS) will fragment each of these long chain proteins into smaller peptides (Chin et al., 2008; Zhang et al., 2012). The molecular weight of the proteins present in the sample will then be calculated and compared against pre-established databases for identification. Microarray/RNA-Sequencing will be used to compare changes in specific leukocyte derived pathway mean differential gene expression levels between HC and PD participants (Ihnatova & Budinska, 2015). Microarray/RNA-Seq data will be analysed using an R statistic routine within Bioconductor v3.2 software.

Proteomic Analysis Workflow. The workflow diagram shows PD and HC blood samples are collected and initially lysed to release cellular content into the cytosol. The sample is then centrifuged with the resulting supernatant submitted to a liquid chromatograph where the proteins are ionised and fractionated into similar protein groups. These protein groups are then passed through a mass spectrometer which fragments the long chain proteins into smaller protein peptides to enable the calculation of the molecular weight of each peptide. Software then compares the calculated molecular weight to a predefined protein database to identify the proteins present in the sample. Adapted from Plasma Proteome Profiling to detect and avoid sample-related biases in biomarker studies, by Philipp E Geyer. et.al., 2019. EMBO Molecular Medicine, 11(11), e10427. doi:10.15252/emmm.201910427.
Proteomic Differential Pathway Analysis

Differential protein comparisons between PD and HC participants by molecular weight (Zhang et al., 2012), and along mitochondria signalling pathways identified as impaired should reveal alterations in PD protein expression and abundance indicating the potential location of the mechanisms driving mitochondrial and protein impairment in PD. Understanding the changes in gene expression, protein alteration and the mitochondria mechanisms that are impaired will offer significant opportunities to target new clinical and pharmacological solutions in treating PD.


Proteomic Differential Pathway Analysis. Figure simulates an anticipated result for a proteomic differential pathway analysis comparing gene expressions between Parkinson disease participants and healthy controls along a specified pathway. (A) Differentially expressed OXPHOS protein heat map, (B) Protein-protein interaction analysis of the significantly differentially expressed proteins, and (c) Network map of impaired mitochondrial proteins. Adapted from A dose-dependent perturbation in cardiac energy metabolism is linked to radiation-induced ischemic heart disease in Mayak nuclear workers, by Azimzadeh, O., et.al. (2017), Oncotarget, 8(6), 9067-9078.

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 2 - Impaired Bioenergetic Signalling Pathways ...

Having identified bioenergetic impairment in PD patients, study 2 will analyse how the mitochondria signalling pathways involved in energy production are dysfunctional. Different substrates are oxidised by different enzyme complexes along metabolic pathways to generate ATP. 

Permeabilizing the leukocyte plasma membrane causing large pores on the outer membrane surface, allows the cell contents to leak out into the cytoplasm. It is then possible to selectively control the type, amount and timing of particular substrates delivered into the cell for oxidation by the mitochondria during OXPHOS and ATP production. 

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.

Research Studies slowly coming together ...

Overall the focus of my PhD research will be on changes in mitochondrial bioenergetics and how the lack of available energy in the form of adenosine triophosphate (ATP) is linked to a wide variety of disease pathologies. 

Specific Studies : 

1. Impact of mitochondrial bioenergetic dysfunction/failure on aged Parkinson’s Disease patients
2. Identify impaired bioenergetic signalling pathways in PD 
3. Proteomic analysis of specific pathway changes in protein abundance
4. Investigate the viability of Leukocytes as a bio-marker of Parkinson’s Disease

Confirmation Presentation ...

Beginning work on my presentation. The talk will need to be around 25 minutes and cover most aspects of my research methods, expectations, potential issues and overall project timing. Like every talk you have to put together, you end up with more information than you are allowed to present. I am only up to study 1 and already I have 25 slides.

I think the way to proceed is to get all the information I want for each study and then at the end trim the number back by deleting and combining information. 

Laura Bassi Scholarship

Drafting up an application for the Laura Bassi Scholarship which was established by Editing Press in 2018 with the aim of providing editorial assistance to postgraduates and junior academics whose research focuses on neglected topics of study, broadly construed, within their disciplines. 

The scholarships are open to every discipline and are awarded three times per year: December, April, and August. The value of the scholarships are remitted through editorial assistance for a PhD at $2,500.

I do not think there is any cash involved, just the equivalent dollar value in the editing press assistance with getting my PhD thesis professionally edited.

Update - 11th July: Turns out both Sheila & Philippe had misread the application, thinking it was for $24,000 pa paid out as $8,000 three times a year. When I contacted them to discuss, the decision was made to not apply at this time as we cannot see any value in getting Editing Press involved in reviewing each of my articles that will constitute each of the key PhD chapters.

How many patients are significant ?

In order to calculate an effective sample size that will yield a significant result I have set up Gpower using the Linear Multiple Regression: Fixed model single regression co-efficient with the following parameters:

1. Confidence interval of 95%
2. Critical value of 0.05%
3. Power of the test at 80%, and 
4. Large Effect size of 0.80
5. Number of predictor variables set at 1

The analysis results in a proposed sample size of 10 which seems close to the 10 sample we are expecting to get access to from the hospital.

Proteomic Protein Targeting ....

Yesterday (1st July 2020), I had a meeting with Rohan Lowe over in Proteomics to outline the planned OXPHOS experiments and to discuss how study 1 looks to confirm mitochondrial impairment and study 2 attempts to tracks each of the ETC complexes to identify the signalling pathway associated with the source of the impairment. From this initial outline we then talked about the practicalities of using leukocytes, setting up protocols and the types of proteomic methods that might be appropriate ie label free, protein mapping, depletion etc.

Whilst it was a short high level discussion three interesting concepts that came out of the discussion:

1. Rohan commented that our Seahorse experiments and data will generate a mass of information to help them with the design of our experiments and that if all goes according to plan the inter-related nature of studies 1 & 2 will be a great source of meta data that will be very useful in defining the proteomic experiments.
   
   
2. When we discussed protein mapping, what seem to offer the greatest opportunity (at least at this early stage), was if we know from study 2 which pathway is impaired i.e. the use of pyruvate reveals poor OXPHOS indicating the TCA cycle and Complex I are the potential source of the impairment, then we can look to identify specific proteins involved in that process and investigate their abundance. In a similar way, if we look at another neurodegenerative disease such as Alzheimer’s or Huntington’s and find proteomic analysis has identified a specific protein as a problem, then an alternative for us is to bring the study 2 pathway analysis together with the other neurodegenerative proteomic analysis and ask if that problem protein in Alzheimer’s is also a problem in PD and is it on our identified impaired pathway.
   
   
3. We discussed how a sample run might work and he suggested we meet with them early ie when we are lining up the samples and before we begin the Seahorse experiments to ensure we can define a collection protocol that meets the quality criteria for preparing the OXPHOX and proteomic samples at exactly the same time so the meta data will be comparable.
   

I think I need to begin looking at proteomic research on neurodegenerative disease where specific proteins have been identified as a problem i.e. missing, low abundance, mutated etc. so that we can see if asking the question “ if it is a problem in that disease, is it also a problem in PD ?”

How are we going so far ....

I was reflection on how we are going as a PhD "team" and whilst I am enjoying the research and writing the papers, I cannot help lamenting the impact Corona is taking on all those casual conversations I am missing, where parts of ideas are exchanged and discussed to form even better leads to follow up. 

Sheila has such an enormous wealth of knowledge, that I know it may seem selfish, but sometimes I feel I am really missing out on learning something new and unexpected. So I am looking forward to the end of Corona and getting back to normal so we can get those discussion and ideas flowing.

Designing and researching the confirmation paper was interesting but I feel it would have been a significantly more enriching experience if there had of been lots of conversation along the way.

 Just a thought.