A Plasma-based Apolipoprotein and p-tau Elisa Assay to detect Alzheimer’s and differentiate from other dementia types for the NHS (ISSN 2753-8176 (online))

1. Ana Pedro

1.Gwyntwr1386 Pharmacy, Regus Chester Business Park, Heronsway, Chester, CH49QR, UK.

info@gwyntwr1386.com

A - Please select the innovation sector

In-vitro diagnostic

B- Please provide a brief description of the innovation including relevant disease area(s) and target patient and service user population(s)

Apolipoproteins are “any of various proteins that combine with a lipid to form a lipoprotein, such as HDL and LDL”, or “The protein components of lipoproteins, which remain after the lipids to which the proteins are bound have been removed”(1). Apolipoproteins are reported to be linked to many neurodegenerative disorders. APOE plays a role in many brain disorders, including Alzheimer’s disease, mild cognitive impairment, multiple sclerosis, traumatic brain injury, and maybe in more some others like Parkinson’s disease (PD) (2). We found out in a recent publication (3) that apoliproteins E (APOE) (total) and L1 (APOL1) are present in the plasma of autistic children and their parents but not in the plasma of their normal siblings. APOE4 is also associated with Alzheimer’s disease and other neurodegenerative disorders, and is expressed at low levels in brain and cerebrospinal fluid. Cultured astrocytes and neurons expressing APOE4 show reduced cholesterol and phospholipid secretion, decreased lipid-binding capacity, and increased intracellular degradation. Domain interaction, in which arginine-61 interacts ionically with glutamic acid-255, and a less stable conformation than APOE2 are supposed to be responsible for this dysfunction. Blocking this domain interaction by gene targeting (replacing arginine-61 with threonine) or by small-molecule structure correctors can increase CNS APOE4 levels and its lipid-binding capacity and decreases its intracellular degradation. Furthermore, plasma levels of APOE vary with APOE genotype (APOE2>APOE4) (7,8). In healthy controls, APOE2 is more abundant in plasma then APO4 (9). Total APOE is also less abundant in APOE4 carriers than in noncarriers and this gradient is also seen in cognitively impaired individuals. In cognitive normal individuals, mean plasma APOE is ≈5.7 mg/dL and total cerebrospinal fluid (CSF) APOE is 0.7 mg/dL (range,≈0.6–0.9 mg/dL; highest in APOE2 and lowest in APOE4 carriers). In plasma, this gradient reflects decreased LDL receptor binding of APOE2 (and thus higher plasma levels) and the preference of APOE4 for VLDL, due to its unique structural features (accelerates hepatic clearance and results in lower levels). In the brain, the lower APOE4 levels and thus lower levels of cholesterol transport could affect cholesterol homeostasis and neuronal plasticity (10). By other side, very recent proteomic analysis revealed that APOL1 is involved in lipid metabolism and transport, as a potential CSF biomarker for frontotemporal dementia (FTLD).Higher APOL1 immunoreactivity associated with neuronal and glia cells was observed in the frontal cortices of FTLD cases compared to controls (p<0.001) (11). Moreover, apolipoproteins that are involved in cholesterol removal from the cells and transport excess cholesterol to bile seem to have a protective role in PD such as APOA1. Also, elevated levels of APOD in glial cells of substancia nigra can be associated with PD (2).

Many of these neurological disorders show either similar symptoms or very characteristic symptoms (Table 1). In most of these pathologies APOE2/4 is involved (Table 2), exception is FTLD. Some of these disorders present interesting combinations of APOs such as AD (APOE4, APOA1), MCI (APOA1, APOA2, APOH, APOE, APOB/APOA1 ratio, APOE4, APOC3, APOB, all in plasma) and PD (APOA1, APOD in plasma) (Tables 1 and 2).

The performance of different biomarkers to identify AD cases, predict cognitive decline in pre-clinical AD cases, and differentiate AD cases from other dementia was analysed (Pais et al., 2023): combination of plasma A42/40 ratio, age, and APOE status showed the best accuracy in diagnosing brain amyloidosis with a liquid chromatography–mass spectrometry (LC–MS) assay. Plasma p-tau217 has shown the best accuracy in distinguishing A-PET+ from A-PET– even in cognitively unimpaired individuals in AD by using methods such as Lumipulse G or Simoa or MSD (Pais et al., 2023). Also, recent studies have been showing the best results for plasma p-tau231 and p-tau217 using Simoa, MSD, or MS based methods.

In this study we will look after ascertaining first: which is the best plasma biomarker of AD pathology which distinguishes AD from other dementia types and predicts cognitive decline in pre-clinical AD cases? Secondly, we will look after developing an accurate cost-effective plasma ELISA test which will allow us to answer our first aim questions.

C - References

1. Elliot et al (2010).Apolipoproteins in the brain: implications for neurological and psychiatric disorders. Clin Lipidol. 2010 August 1; 51(4): 555–573. doi:10.2217/CLP.10.37.

2. Emamzadeh F (2017).Role of Apolipoproteins and α-Synuclein in Parkinson’s Disease.J Mol Neurosci (2017) 62:344–355

3. Pedro A (2022). The role of apoliproteins APOE and APOL1 in Autism DOI:10.13140/RG.2.2.23055.05286, ISSN 2753-8176 (online)

4.Hu et al. (2018).APOE hypermethylation is associated with autism spectrum disorder in a Chinese population. EXPERIMENTAL AND THERAPEUTIC MEDICINE 15: 4749-4754, 2018

5.Elliot et al (2010). Apolipoproteins in the brain: implications for neurological and psychiatric disorders. Clin Lipidol. 2010 August 1; 51(4): 555–573.

6. Giunco CT, de Oliveira AB, Carvalho-Salles AB, Souza DS, Silva AE, da Rocha SS and Fett-Conte AC: Association between APOE polymorphisms and predisposition for autism. Psychiatr Genet 19: 338, 2009.

7.Utermann G. Genetic polymorphism of apolipoprotein E – impact on plasma lipoprotein metabolism. In: Crepaldi G, Tiengo A, Baggio G, eds. Diabetes, Obesity and Hyperlipidemias – III. Amsterdam: Elsevier Science Publishers; 1985:1–28

8. Rasmussen KL, Tybjaerg-Hansen A, Nordestgaard BG, Frikke-Schmidt R. Plasma levels of apolipoprotein E and risk of dementia in the general population. Ann Neurol. 2015;77:301–311. doi: 10.1002/ana.24326.

9. Gupta VB, Wilson AC, Burnham S, et al; AIBL Research Group. Follow-up plasma apolipoprotein E levels in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL) cohort. Alzheimers Res Ther. 2015;7:16. doi: 10.1186/s13195-015-0105-6.

10. Mahley R. Central Nervous System Lipoproteins: ApoE and Regulation of Cholesterol Metabolism. Arterioscler Thromb Vasc Biol, 2016 Jul;36(7):1305-15

11.Hok-A-Hin et al. Apolipoprotein L1 is increased in frontotemporal lobar degeneration post-mortem brain but not in ante-mortem cerebrospinal fluid. Neurobiol Dis 2022 Oct 1;172:105813.

R. Kim, J.S Jun, H.J Kim, B.S Jeon. Apolipoprotein E ε4 and risk of freezing of gait in early Parkinson’s disease [abstract]. Mov Disord. 2020; 35 (suppl 1). https://www.mdsabstracts.org/abstract/apolipoprotein-e-%ce%b54-and-risk-of-freezing-of-gait-in-early-parkinsons-disease/

D- Please provide a brief description of the unmet need or health care problem you are looking to address and how you think your innovation will solve or improve on the current standard of care. Please include the potential benefits you’ve identified for patients and service users i.e sustainability, cost savings, supporting underserved populations, addressing health inequalities/barriers etc.

A combination of plasma A42/40 ratio, age, and APOE status showed the best accuracy in diagnosing brain amyloidosis with a liquid chromatography–mass spectrometry (LC–MS) assay for AD. Plasma p-tau217 has shown the best accuracy in distinguishing A-PET+ from A-PET– even in cognitively unimpaired individuals in AD by using methods such as Lumipulse G or Simoa or MSD (Pais et al., 2023).

We aim to develop for the NHS an accurate plasma-based apolipoproteins ELISA assay, which helps us to distinguish between different type of dementias and AD and AD progression which would be more cost-effective if compared with the tests mentioned above.

E- Please provide a brief description of the research proposed, including any research questions you are looking to answer and what you aim to get out of the research. Please include any questions you would like to ask potential collaborators from the research network. What evidence or data have you generated so far and can the findings/results or publication be shared with researchers in the NIHR network?

1. Collect 100 control plasma samples + patient plasma samples (tables 1 and 2)

2. Validate biomarkers. 3 methods: LC-MS, ELISA, MSD. Biomarkers to analyze: Aß40, Aß42, APOE, p-tau 217, p-tau 231, APOA1, APOA2, APOH, APOB, APOC3, APOL1, APOD

3. Ana Pedro will analyse the data and develop the commercial ELISA assay, MHRA applications, commercialize, clinical phase (Gwyntwr1386 Pharmacy)

- Second, studies should follow standard operating procedures to control for pre-analytical and analytical biases, investigating test re-test variability of different laboratory methods

- studies need to investigate sex differences and include diverse populations

- First, studies should include head-to-head comparisons of different analytical methods

F- Methods

For this project I will need plasma samples from patients with Alzheimer’s, MCI, PD, FTLD, vascular dementia, mixed dementia, Lewy bodies (tables 1 and 2). I will also need plasma samples from healthy volunteers.