Alzheimer's Disease (AD) currently affects around 50 million individuals worldwide, with a significant and growing burden in developing countries. Already 60% of people with dementia live in low- and middle-income countries, but by 2050 this will rise to 71%. The fastest growth in the elderly population is taking place in China, India, and their south Asian and western Pacific neighbours. Research shows that most people currently living with dementia have not received a formal diagnosis. In high income countries, only 20-50% of dementia cases are recognised and documented in primary care. This 'treatment gap' is certainly much greater in low- and middle-income countries, with one study in India suggesting 90% remain undiagnosed
This neurodegenerative disease severely affects the quality of life of patients and leads to severe impairments in mental and motor functions. Not only does it affect the patient's quality of life, but it also poses a growing emotional and economic burden on the caregivers. A meta-analysis reported that caregivers of people with dementia were significantly more likely to experience depression and anxiety than non-caregivers. Dementia caregivers also indicate more depressive symptoms than non-dementia caregivers.
The mechanism, progression and materialisation of the disease remain elusive. However, central to the pathology of Alzheimer's disease is the Amyloid Cascade Hypothesis. This hypothesis describes the formation of Amyloid β (Aβ) plaques as one of the potential causes for disease progression. In tandem to Amyloid β, the intracellular misfolding and the deposition of Tau proteins has also been shown to happen in the course of the disease.
Tau is an essential microtubule-associated protein that stabilises microtubules in neurons and facilitates neuronal transport. In AD and related tauopathies, Tau proteins undergo extensive post-translational modifications, such as hyperphosphorylation and truncation. These modifications predispose Tau proteins to lose their physiological functions, this leads to rise of many neurodegenerative diseases termed as 'Tauopathies'. These misfolded and highly aberrantly modified Taus form aggregates, resulting in protofibrillary helices and neurofibrillary tangles. Unfortunately, these misfolded proteins often evade or overwhelm the cell's Unfolded Protein Response, contributing to increased oxidative stress and triggering apoptotic pathways, which are crucial in the progression of dementia.
Previously, it was thought that the Tau pathway was orthogonal to the Amyloid cascade, however recent evidence suggests that there is a heavy interconnection between both.
One of the biggest problems with treating neurodegenerative diseases are issues of diagnosis, by the time visible symptoms show up, it is already too late. But recent research has shown some promise. Several biomarkers been proposed for Alzheimer's, we base our project around one of those biomarkers - 231 Threonine in Tau protein.
Phosphorylation in the 231 Threonine site of Tau is one of the most robust biomarkers in the detection of preclinical Alzheimer's disease. It has been established that the levels of p231 Tau react most promptly to the rapid changes in A β pathology, making it a valuable protein of interest for diagnostic and therapeutic studies.
Aptamers are short, single-stranded oligonucleotides which are chemically affine to a particular substrate with high specificity. Aptamers are sometimes loosely referred to as 'chemical antibodies'. Aptamers show some sort of advantages when compared to antibodies, most importantly the ease of production and the relatively low cost involved in the same. Aptamers are also not immunogenic and non-toxic; hence it suits perfectly in applications involving diagnostics or therapeutics.
Aptamers are generated via a process of SELEX (systematic evolution of ligands by exponential enrichment). This process can be loosely divided into two parts - amplification of existing pool of oligonucleotides via PCR (polymerase chain reaction), followed by applying a bias in the form of the target substrate, only the affine oligonucleotides remain bound to the target, while the unbound oligonucleotides get filtered away. The selection pressure increases with every round, and hence after 10-15 rounds the nucleotide pool is enriched with the most affine and specific aptamers for the target.
Certain aptamers have already been established for p231 site; we proceed to use the same aptamers as our beginner pool to start our process of SELEX. We expect evolving the affinity of our aptamers over randomly phosphorylated Tau-441 using GSK3β, having at least the 231 Threonine site phosphorylated. However, to reduce the scope of uncertainty, we decided to model the pathological Tau using 'phosphomimetics'. In this method, the phosphorylated substrate is substituted by a structurally similar acidic amino acid. By this, we can mimic the physical structure of the phosphorylated compound (which is what the aptamer binding mechanism depends on). However, we still reckon that the aptamers for the p231 Tau might not be completely compatible with our modifications, hence we also plan to run SELEX on them to generate a final pool of specific, affine aptamer for our investigations.
The second stage of our project would begin with tagging the generated aptamers to a E3-ligase system, which would help in the polyubiquitination of the bound p231-Tau, leading to the ultimate degradation of the protein assisted by the Ubiquitin-Proteosome Machinery.
The basic idea is to use click chemistry to 'click' our affine aptamer via a ligand and linker to our E3-ligase machinery (VHL, which is responsible for polyubiquitination). We have planned to introduce an alkynyl triphosphate in one of the bases of the aptamer, this would undergo a well reported ‘click’ addition with the azide moiety of our linker-ligand to finally form our Aptamer-PROTAC conjugate. We plan to use a diethylene glycol as our linker as it ensures enhanced solubility in the cell and makes the conjugate less susceptible to destabilising interactions.
Once our aptamers our generated, along with our work with Targeted Protein Delivery, we wish to parallely check out some ideas.
Diagnosis: By conjugating our aptamer with a light sensitive molecule, we hope to achieve for a tool which can help in diagnosis. Since reports have shown that p231 Tau can play a very important role in preclinical diagnosis of Alzheimer's, we would hope to develop this into an assay that can streamline the development of further diagnosis tools on CSF/plasma samples. This will help in a better understanding of the disease in a person in conjunction with other tests.
We are not claiming to develop a 'cure' or 'therapeutic' for AD, we are just planning to explore the limits of Aptamer technology with the hopes that it can be one day modelled successfully for a therapeutic application. While research on cell lines and mouse models have shown that TPD can be a very promising potential treatment strategy, it is NOT established via human trials. Hence, the possible side effects of such a strategy are not well understood. We are only wishing to expand the global scientific knowledge.
We hope to inspire future researchers to use our idea as a template for tackling other proteinopathies and also to expand and build on our core idea.