PD is the second most common chronic degenerative disease of the central nervous system after Alzheimer's disease [1]. It is characterized by the aging-dependent loss of dopamine (DA) neurons in the substantia nigra parenchyma and a decrease in dopamine levels [2]. In the early stages of Parkinson's disease, patients may experience pathological changes in the dorsal motor nucleus of the vagus nerve in the medulla oblongata and the anterior olfactory nucleus of the olfactory bulb in the presymptomatic stage, followed by changes in the locus coeruleus neurons and dopaminergic neurons in the pons. Therefore, olfactory and taste disorders may be early clinical features of Parkinson's disease. In the later stages, the pathology spreads to the amygdala, basal forebrain, and medial temporal lobe structures. The neocortex is affected in the final stages of the disease [3]. Since the occurrence of PD will seriously affect the patient's quality of life and increase the economic burden on society and the family, early prevention and diagnosis and treatment are particularly important. Studies have shown that the α-syn level in the cerebrospinal fluid of PD patients is decreased, the Aβ42 level is decreased, and the NF-L level is increased [4]. Taking advantage of these characteristics, this project aims to achieve early intervention by conducting early screening of patients with non-motor symptoms of PD but no motor symptoms of PD.

Neurofilament light polypeptide (NF-L) is a protein integral to the structure and function of neurons, particularly within the axons, where it forms part of the neurofilament network. In biology, neurofilaments act as a type of intermediate filament, providing mechanical stability to neurons and supporting the maintenance of axon diameter, which is crucial for efficient nerve signal conduction. The light chain, NF-L, serves as a foundation for neurofilament assembly, binding with medium and heavy neurofilament chains to form larger filaments that extend throughout the neuron.

One of the key biological properties of NF-L is its stability, which allows it to persist in the axonal structure for extended periods. This makes it highly valuable in research, particularly in the context of neurodegenerative diseases. For instance, elevated levels of NF-L in cerebrospinal fluid or blood can be an indicator of axonal damage, making it a reliable biomarker for diseases like amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Parkinson’s disease, and Alzheimer’s disease. Researchers use NF-L measurements to monitor disease progression and evaluate the effectiveness of treatments in clinical trials.

Moreover, NF-L plays a role in the biology of neural development and repair. It is involved in forming and maintaining the neuronal cytoskeleton, particularly in neurons with long axons, where structural integrity is essential for proper functioning. The size and density of the neurofilament network, which NF-L helps establish, directly influence the speed of electrical impulses along axons.

In experimental models, NF-L is used to study neurodegeneration and the mechanisms underlying axonal injury. Researchers investigate how mutations in the NF-L gene or disruptions in neurofilament assembly lead to neurodegenerative diseases, providing insight into potential therapeutic targets. Additionally, because NF-L is a stable and easily detectable protein, its levels in bodily fluids are increasingly used in clinical diagnostics as non-invasive indicators of neuronal health.

PCR was used to amplify the NF-L gene, and its length was 1059 bp. A band with the same size as the target gene appeared in Figure 1A, indicating that the target gene was successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was performed to obtain the recombinant plasmid pET28a(+)-NF-L.

Plasma Amyloid-β (Aβ42) is a peptide closely linked to the pathology of Alzheimer’s disease and plays a significant role in both research and clinical diagnostics. It is derived from the cleavage of amyloid precursor protein (APP), and while Aβ42 is produced naturally in the brain and circulates in the bloodstream, its biological relevance becomes particularly pronounced in the context of neurodegenerative diseases.

The biology of Aβ42 is characterized by its high tendency to misfold and aggregate, forming amyloid plaques in the brain. These plaques are a hallmark of Alzheimer's disease and are thought to disrupt neural communication and trigger inflammatory responses that contribute to neurodegeneration. What makes Aβ42 especially relevant in Alzheimer's research is that, compared to other amyloid-β isoforms, it is more prone to aggregation due to its hydrophobic nature and longer peptide sequence.

In terms of usage, Aβ42 has become a critical focus in the study of Alzheimer’s disease, both as a target for therapeutic intervention and as a biomarker for early detection. Plasma Aβ42 levels, along with Aβ40, are often measured to assess the amyloid burden in the brain. Although the exact levels in the blood don’t directly correspond to the amount in the brain, changes in the plasma Aβ42/Aβ40 ratio can offer insights into Alzheimer’s pathology before clinical symptoms manifest. As such, Aβ42 has become an essential biomarker in preclinical studies, helping researchers develop diagnostic tools and track the progression of Alzheimer's in clinical trials.

In addition to its role as a biomarker, therapeutic strategies have focused on reducing Aβ42 production or preventing its aggregation. Approaches like monoclonal antibodies, small molecule inhibitors, and immunotherapies aim to lower Aβ42 levels or enhance its clearance from the brain to halt or slow disease progression. These strategies highlight the dual role of Aβ42 in both diagnostics and treatment research.

The biology of Aβ42 extends beyond Alzheimer’s, as it also plays a role in general amyloid-related research, where understanding its aggregation properties and interactions with other proteins informs studies on other neurodegenerative conditions. Overall, Aβ42 remains central to unraveling the complexities of Alzheimer's and developing both preventative and therapeutic strategies.

The Aβ42 gene was amplified by PCR, and its length was 789 bp. As shown in Figure 1A, a band with the same size as the target gene appeared, indicating that the target gene was successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was performed to obtain the recombinant plasmid pET28a(+)-Aβ42.

Alpha-synuclein (α-Syn) is a crucial protein in neurodegenerative research, particularly for its role in Parkinson’s disease and related disorders known as synucleinopathies. Biologically, α-Syn is abundant in neurons, especially at presynaptic terminals, where it is thought to regulate synaptic vesicle trafficking and neurotransmitter release. Although normally soluble and unstructured, α-Syn can misfold and aggregate into insoluble fibrils under pathological conditions. These fibrils form Lewy bodies, which are the hallmark of Parkinson’s disease and other neurodegenerative diseases.

Research on α-Syn focuses on its propensity to misfold and how these misfolded proteins spread between neurons, contributing to the progression of disease. It is hypothesized that α-Syn misfolding follows a prion-like mechanism, where abnormal forms of the protein can induce normal α-Syn to misfold, propagating toxic aggregates across brain regions. This mechanism has become a central focus in understanding disease progression and is being studied for potential therapeutic interventions.

In terms of clinical application, α-Syn is a major target for biomarker development. Misfolded α-Syn can be detected in cerebrospinal fluid or blood, offering a potential tool for early diagnosis of Parkinson’s disease before motor symptoms appear. It is also a therapeutic target, with various strategies in development aimed at reducing α-Syn aggregation, clearing misfolded proteins, or preventing their spread. These include immunotherapies, small molecule inhibitors, and gene therapies aimed at reducing α-Syn production or enhancing its clearance.

Overall, the biology of α-Syn highlights its central role in neurodegeneration, from its normal function in synaptic regulation to its pathological role in disease progression, making it a key protein in both basic neuroscience research and therapeutic development.

PCR was used to amplify the α-syn gene, and its length was 522 bp. As shown in Figure 1A, a band with the same size as the target gene appeared, indicating that the target gene was successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was performed to obtain the recombinant plasmid pET28a(+)-α-syn.

In our iGEM project focused on early screening for Parkinson’s disease using three biomarkers and an ELISA kit, our contributions at least two key areas:

The fact that our team successfully expressed the three target proteins—likely α-synuclein (α-Syn), Amyloid-β (Aβ42), and Neurofilament light polypeptide (NF-L)—is a significant achievement. This demonstrates that the fundamental step of producing the biomarkers necessary for Parkinson's early detection has been accomplished. It lays the groundwork for the development of an ELISA kit, as protein expression is a critical prerequisite for the production of antibodies and the establishment of diagnostic tools.

our approach to utilizing three specific biomarkers for Parkinson's early screening is a novel and impactful contribution. Combining these proteins in an ELISA kit for early detection can provide a more comprehensive diagnostic tool, potentially allowing for earlier and more accurate diagnosis compared to current methods. This innovative approach addresses a significant gap in the early diagnosis of neurodegenerative diseases, which is crucial for improving patient outcomes. Even though the ELISA test itself has not been fully completed, our work on identifying and expressing these biomarkers sets a solid foundation for future experiments and potential clinical applications.