Proteinopathies are a group of disorders associated with the misfolding, aggregation, and deposition of proteins, leading to loss of functionality and ultimately resulting in cell death. A lot of neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease are examples of proteinopathies. Understanding the mechanisms of the proteinopathies is challenging due to the underlying complexity of protein folding, the rich interwebbing of multiple important cellular pathways, and the unique pathophysiology of each disease. Proteins interact with various other macromolecules in the cell such as - other proteins, lipids, and nucleic acids. These interactions usually affect and influence the stability, localization, and function of a protein in its native state. Understanding these networks is essential to deduce which pathways are involved and get affected in proteinopathies. Proteinopathies also exhibit a broad abstract range of symptoms which seem to overlap with a range of other age related disorders. Disease progression, even amongst patients with the same diagnosis, seem to differ and vary a lot. This heterogeneity complicates the identification of common underlying mechanisms and hence effective therapeutic targets. Several animal models and cell cultures often fail to replicate the complexity and exactitude of human diseases accurately. While these models provide valuable insights and intuition, they may not capture the full spectrum of pathology seen in patients. Unfortunately, many proteinopathies show clinical symptoms only after extensive damage has already occurred, making early diagnosis potentially very challenging. The underlying pathology may already be well-established, hence leaving the clinician with limited therapeutic options.
Proteins are amazing macromolecules that fold into specific three-dimensional structures to perform their functions. However, factors such as mutations, environment induced stress, and aging can lead to misfolding, causing proteins to adopt non-functional conformations. These misfolded proteins can sometime aggregate into larger complexes, such as neurofibrillary tangles and protofibrillary helices which often do more harm than good for the cell. Sometimes, these complexes might self-catalyse the conversion of normal proteins into misfolded ones!
Aptamers are oligonucleotides, usually composed of DNA or RNA, that interact and bind to a specific target molecule with high affinity and specificity. There is functional similarly between antibodies and aptamers in the sense that they both bind specifically, but aptamers offer distinct advantages in terms of ease of production, high flexibility, and enhanced potential for application, making them very relavent in contemporary biomedical research. Aptamers form unique three-dimensional structures that facilitate specific interactions with a wide repertoire of targets, such as proteins, small molecules, and sometimes even entire cells! These interactions are due to the folding of the oligonucleotide into complex secondary and tertiary structures like hairpins, loops, and bulges, which allow aptamers to "lock" onto their targets with remarkable precision.
One of the greatest advantages aptamers have over traditional antibodies is ease of production and functionalisation. Aptamers can be synthesized in vitro using the principles of PCR (polymerase chain reaction) , making their production relatively inexpensive, massively scalable, and free from huge amount of variability. Additionally, aptamers are mostly non-immunogenic and non-toxic, which causes them to have very less side effects, hence they are highly amenable for diagnostic and therapeutic investigations.
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.
The specificity of aptamers have made them valuable tools in numerous research areas such as - diagnostics, therapeutics, and targeted drug delivery. For example, aptamers have been successfully developed to detect several biomarkers in diseases such as cancer, HIV, and cardiovascular conditions.
Recent research into aptamers has expanded to fields such as biosensing and regenerative medicine. Aptamer-based biosensors, known as aptasensors, have gained attention for their potential in point-of-care diagnostics due to their simplicity, rapid response, and cost-effectiveness. In regenerative medicine, aptamers are being investigated for their ability to guide stem cells to injured tissues, potentially facilitating tissue repair and regeneration. In addition, aptamers are now being employed in CRISPR/Cas9 systems to enhance gene editing specificity by acting as molecular guides to improve targeting accuracy.
Alzheimer's Disease (AD) currently affects around 50 million individuals worldwide, with a significant and growing burden in developing countries. Alzheimer's remains the fifth leading cause of death in Americans of the age 65 and above. 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. By 2030, over 10 million people in India are projected to be living with Alzheimer's or other forms of dementia, making it one of the fastest-growing regions for dementia cases. 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 leading 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. Studies show that caregivers in India face extreme challenges, with over 60% of caregivers reporting high levels of stress, depression, and anxiety due to the lack of formal caregiving support. The prevalence of dementia in rural areas of South Asia, including India, is underreported and often confused with "normal aging," leaving a huge gap in treatment and support.
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 with Amyloid β, the intracellular misfolding and 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, leading to a rise in 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. Current disease models suggest that Aβ (either as plaques or as non-fibrillar, soluble, oligomeric forms) initiates a pathophysiological cascade leading to tau misfolding and assembly that spreads throughout the cortex, ultimately resulting in neural system failure, neurodegeneration and cognitive decline.
One of the biggest problems with treating neurodegenerative diseases is the issue of diagnosis; by the time visible symptoms show up, it is already too late. But recent research has shown some promise. Several biomarkers have been proposed for Alzheimer's, and we base our project around one of those biomarkers - 231 Threonine in the Tau protein.
Phosphorylation at 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 rapid changes in Aβ pathology, making it a valuable protein of interest for diagnostic and therapeutic studies.
Furthermore, new diagnostic tools like blood-based biomarkers and PET imaging have shown significant potential in detecting early-stage AD. Blood tests measuring levels of plasma Aβ and phosphorylated Tau (pTau) are being developed as less invasive and more cost-effective diagnostic methods compared to cerebrospinal fluid (CSF) analysis. Additionally, positron emission tomography (PET) scans using radiotracers targeting Aβ and Tau aggregates have improved the accuracy of early AD diagnosis, offering potential for early therapeutic intervention.
Economically, the burden of dementia is projected to surpass $2 trillion by 2030 as the global population ages, especially in countries where healthcare systems are underdeveloped. This highlights the urgency for improving early detection and developing more effective treatments.
In 2018, Aptamers were raised on several peptide segments of Tau, one of them being Thr-231; we then proceed to use those 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.
PROTACs are bifunctional molecules having two critical components: one that binds to the target protein (which in our case is the Aptamer) and another that recruits an E3 ubiquitin ligase. Once the PROTAC binds to both the target protein and the E3 ligase, it brings them into close proximity, enabling the ligase to tag the protein with ubiquitin molecules. This ubiquitination process marks the protein for degradation by the proteasome, which we conjecture would help in the removal of the misfolded proteins.
The VHL E3 ligase system is a crucial component of the ubiquitin-proteasome pathway, which is responsible for tagging proteins for degradation. The von Hippel-Lindau (VHL) protein is a well-studied E3 ubiquitin ligase that forms part of a multi-subunit complex, specifically designed to target hypoxia-inducible factors (HIFs) for ubiquitination under normoxic (normal oxygen) conditions. However, its role has been widely adapted in research for controlled degradation of other target proteins, using innovative techniques like PROTACs (PROteolysis-TArgeting Chimeras).
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.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.