Aptamer Applications in Neuroscience Part 2

Another neurotransmitter, serotonin, plays a role in neuromodulation activities affecting sleep, aggression, appetite, and sexual activity. It is produced in different body parts, such as the brain, spinal cord, platelets, and intestine [81–84]. 

Serotonin is a neurotransmitter that plays an important role in human physical and mental health. In the human body, serotonin regulates mood, controls appetite and sleep, and promotes feelings of physical pleasure and well-being. However, serotonin also plays an important role in affecting memory.

Research has found that serotonin can promote memory enhancement by regulating signaling between neurons. When people are positively stimulated, the body releases serotonin, which helps strengthen connections between neurons and improves memory efficiency. Serotonin can also make people more creative, improve mental agility, and help drive people to learn and think faster.

In addition, serotonin can help reduce symptoms of anxiety and depression, mood problems that can negatively impact people's concentration and memory. A happy mood and mental state can often promote people's recognition and learning ability of new things.

To sum up, serotonin plays an indispensable role in our physical and mental health learning, and memory. We can increase the release of serotonin and [improve memory] by changing our eating habits, amount of exercise, learning methods, etc., cultivating a positive and healthy mentality, and constantly challenging ourselves to continuously promote the enhancement of memory. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche deserticola can also promote the growth and repair of nerve cells, thus enhancing the connectivity and function of neural networks. These effects can help improve memory, learning, and thinking speed, and may also prevent the development of cognitive dysfunction and neurodegenerative diseases.

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Imbalances of peripheral serotonin levels have been linked to several disease conditions, including hypertension, kidney disease, and depression. Thus, using serotonin to predict the disease state and to initiate proper treatment has clinical importance. 

A plasmonic assay for serotonin was developed with an aptamer–AuNP conjugate for which aggregation is altered in the presence of serotonin. The change in particle aggregation can be measured as a shift in the peak wavelength of absorption. 

The assay showed a specific response to serotonin and gave no statistically significant signal with its metabolites, 5-hydroxyindoleacetic acid (5-HIAA), epinephrine, or norepinephrine when each was tested at 1 µM. 

Fetal bovine serum, as a mimic of human serum, also initiated no signal [85]. Recently, implantable aptamer–field-effect transistor (FET) neuroprobes have been developed for monitoring serotonin levels in brain tissue. Ultrathin In2O3 surfaces of nanoscale FETs were modified with aptamers and these neuroprobes enabled fM serotonin detection limits in vivo with minimal biofouling [86]. 

With additional neuro probes, spatiotemporal recordings of neural activity will be possible so brain functions can be understood at a much deeper level. Epinephrine is a catecholamine neurotransmitter with a wide range of physiological functions for which abnormal levels may cause health problems like arrhythmia, myocardial infarction, blood pressure increase, and pulmonary edema. 

It is important to quantify circulating epinephrine for observing these related disease states. With this aim, a colorimetric detection method, based on the interaction of epinephrine with aptamer functionalized Au-NP, was developed. The system is less expensive and more specific than other techniques for quantifying epinephrine, such as by liquid chromatography, and spectrophotometry. 

Also, a detection limit of 0.9 nM was determined by UV-visible spectroscopy, which is the lowest detection limit recorded for epinephrine using any colorimetric method. Analogs of epinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC), tryptophan, ascorbic acid, dopamine, tyrosine, and homovanillic acid, showed very little or no response, which indicates a high specificity of the aptasensor [87]. 

A change in the level of a single neurotransmitter can indicate any of several diseases, which makes it highly unlikely that a disease diagnosis can be achieved based on the measurement of a single neurotransmitter. Therefore, the ability of most aptasensors to be integrated into a multiplex platform will result in diagnostic tools better suited to clinics and with the potential to support more accurate disease identifications.

2.3. Biomarker Detection
A biomarker is any substance, structure, or process in the body that either results in an outcome or indicates the presence of a specific disease. Many biomarkers are substances that can be measured in biofluids such as the blood, saliva, and urine. 

The main problem of biomarker characterization for neurological diseases is the blood-brain barrier (BBB) through which many molecules, including many proteins, cannot pass. Although proteins are the dominant biomarkers for certain diseases, small peptides, such as neuropeptide Y or fragments of proteins, are more likely to pass through the BBB and be detected in the circulation. 

Metabolites are another important group of biomarkers for neurological disorders. NeoVentures Biotechnology Inc. has developed an aptamer selection approach called FRELEX, which allows the selection of aptamers based on competition between a short oligonucleotide attached to a surface and the target. Like for cell-SELEX and other forms of structure-switching SELEX, this protocol can be performed with a sample containing a mixture of components without prior knowledge of the target (s) to which aptamers will be selected. 

With these forms of SELEX, counter selections are performed with particular subsets of components to drive the selection of aptamers that fulfill the desired specificities. Using FRELEX, aptamers were selected against serum from transgenic mice that overexpressed the human tau protein. 

To drive aptamer selection toward human tau and the consequences of tau overexpression, serum from wild-type mice was used in the counter selection. The group identified the enriched aptamer sequences using NGS and characterized certain aptamers for late-stage transgenic mice and wild-type mice by comparing their relative abundances. 

They hypothesized that the difference in abundance reflects the respective concentration of the epitopes (increased in response to tau overexpression) with which the aptamers can interact [29].

3. Diagnostic and Therapeutic Applications

Neurodegenerative diseases (NDs) involve the degeneration of neurons in the brain that results in the loss of structures and functions of the central nervous system. ND can be caused by aging, genetic, and environmental factors. 

Dementia is one of the most common age-related NDs as is Alzheimer's disease (AD). Parkinson's disease (PD) can be due to genetic mutations and/or environmental toxins. Amyotrophic lateral sclerosis (ALS), Multiple Sclerosis (MS), Huntington's disease (HD), and prion diseases are other common NDs with genetic links. 

Unfortunately, neither effective cures nor early detection strategies are available for these diseases. Like the widely used antibodies, aptamers have become attractive agents to apply to developing novel biosensors for early diagnosis of NDs and cure of these diseases [88].

3.1. Alzheimer's Disease

As an age-related progressive brain disorder resulting in mental deficiencies, AD is the most common form of dementia. Its pathology is characterized by the aggregation of amyloid-β (Aβ) derived from the amyloid precursor protein (APP) and initiated in the brain region of the hippocampus. 

Although scientists hypothesized that Aβ-induced neurotoxicity was correlated with insoluble Aβ plaques (AβP) and fibrils (AβF), recent evidence indicates that soluble Aβ oligomers (AβO) are also associated with AD onset.
Therefore, AβO has been identified as an attractive biomarker for early diagnosis of AD, and Aβ and tau are considered significant therapeutic targets for treating AD [89].

3.1.1. AD Diagnostics

Early described aptamers against AβO had low affinities and specificities, but later studies were more successful in identifying aptamers that selectively recognized fibrils of a 40-residue form of Aβ (Aβ40) but not Aβ40 polymers [90]. 

However, the aptamers lacked high specificity for Aβ40 fibrils over the fibrils of several other amyloidogenic proteins that were tested. A DNA aptamer selected to interact with α-synuclein (α-syn) oligomer with 68-nM affinity was also shown to bind AβO with 25-nM affinity [91]. In 2019, using the same aptamers obtained by Tsukakoshi et al., a label-free electrochemical aptasensor was developed for more specific recognition of AβO [92]. 

The aptamer self-assembles on gold rod electrodes via thiol (-S) interaction and the system has a detection limit of 30 ppm, determined by EIS. This was the first aptasensor successfully used to monitor Aβ protein aggregation based on EIS, which was possible because of the aptamer's high selectivity among Aβ species. With easy fabrication and effective regeneration, this aptasensor might be a promising diagnostic tool for the early detection of AD and for demonstrating Aβ protein accumulation [92]. 

An aptasensor to detect Aβ oligomers was developed using the α-syn DNA aptamer [91] in complex with methylene blue (MB) and attached to nanoporous anodic alumina. The high absorption coefficient of MB for white light resulted in a low intensity of reflected white light. 

In the presence of Aβ oligomers, the aptamer/MB complex dissociated and the increase in reflected light was detected by interferometric reflectance spectroscopy (IRS) [93]. The aptasensor had a detection limit of 8 pM and a good response to the Aβ oligomers, in the concentration range from 0.5 to 50 nM. Neurofibrillary tangles (NFTs) are pathological hallmarks of AD. 

The formation of NFTs is initiated in the hippocampus and the extent of NFT formation is associated with the severity of dementia in AD. Hyperphosphorylation of the microtubule-associated protein, tau promotes its ability to recruit and organize normal tau into filaments, which become the NFTs. Thus, hyperphosphorylation of tau is proposed as one of the main causes of AD. 

In addition to its normal roles in promoting microtubule assembly and stabilizing the assembled microtubules, tau was observed by non-equilibrium capillary electrophoresis (CE) to bind three randomly selected ssDNA oligonucleotides, one of which, ssDNA1, bound to tau381 with high affinity (Kd = 190 nM) [94]. 

An aptamer/antibody sandwich assay was developed using ssDNA1 to detect femtomolar concentrations of tau protein in human plasma by surface plasmon resonance (SPR) [95]. 

In another study, DNA aptamers were selected against the entire tau protein of 441 amino acids (tau441) by a rapid selection technique based on capillary electrophoresis partitioning with three selection rounds completed in a single day. Five aptamers chosen from the high throughput sequencing results were evaluated by SPR for their tau recognition ability. 

The analytical potential of the aptamer with higher affinity was demonstrated by a homogeneous-phase fluorescence anisotropy assay. This high-affinity aptamer bound tau441 protein and the shorter isoforms, tau352, tau381, and tau383 with detection limits of 28 nM, 6.3 nM, 3.2 nM, and 22 nM, respectively, in this assay [96].

3.1.2. AD Therapeutics

As one of the potential therapies, RNA aptamers were selected to inhibit Aβ40 aggregation. During selection, an Aβ oligomer was conjugated to gold nanoparticles (Aβ-AuNPs) and two aptamers were identified as aptamer candidates after SELEX. 

The Kids of the 10–20 nM. These aptamers inhibited Aβ plaque and fibril formation as revealed by transmission electron microscopy and an Aβ40 enzyme-linked immunosorbent assay (ELISA) [97]. 

Alcohol dehydrogenase (ABAD) decoy peptide (DP), which interacts with Aβ, antagonizes the cytotoxicity of the Aβ peptide. The ABAD-DP sequence was inserted between two thiols that form a disulfide bond in thioredoxin (TRX) to create the TRX1-ABAD-DP-TRX2 peptide aptamer. Its stable expression in NIH 3T3 cells using adeno-associated viruses was verified with immunofluorescent staining. 

The expressed aptamer could bind the Aβ peptide and improve cell viability. This investigation confirmed the effective inhibition of the cytotoxic effect of Aβ peptide by peptide aptamers [98]. Tau proteins normally associate with and stabilize microtubules, but their hyperphosphorylation can result in protein aggregates termed "tauopathy", which is toxic. 

DNA aptamer Tau-1, which was selected against the human tau441, inhibits Tau1 oligomerization in vitro [99]. This inhibition was also validated in cell culture studies using HEK293 cells [100]. Beta-secretase (BACE1) has a role in Aβ production, and inhibition of BACE1 expression is lethal in mice. Treatment for AD might involve BACE1 modulation. 

However, its large active site makes BACE1 a challenging target for small molecule inhibitors. To circumvent this, nucleic acid aptamers might be novel tools to inhibit BACE1 activity. This enzyme has a cytoplasmic tail, B1-CT, serving as a docking site for proteins such as the copper chaperone for superoxide dismutase-1 (CCS) and ADP ribosylation factor-binding (GGA1) protein. 

The physiological role of the B1-CT is largely unknown. RNA aptamers targeting the B1-CT bound the membrane-proximal half of the C terminus of BACE1 prevented CCS recruitment while allowing GGA1 binding to BACE1 and regulating BACE1 transport to recycling endosomes [101]. 

To specifically target BACE1, the DNA aptamers, BI1 and BI2, were generated against BACE1, which inhibited BACE1 but neither alpha- nor gamma-secretase. Using a stably transfected HEK293 cell line, they expressed the amyloid protein precursor (APP) and, using an in-vitro fluorescence resonance energy transfer (FRET) assay, they showed that Aβ level was reduced, and cellular deficiency was rescued in a primary cultured neuronal cell line [102]. The aptamer efficiency was further improved by the addition of cholesteryl tetra ethylene glycol (TEG).

3.2. Parkinson's Disease

Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting about seven million people globally. While being a progressive disease characterized by motor and nonmotor features, it has significant clinical impacts on patients due to their loss of mobility and muscle control. 

Loss of striatal dopaminergic neurons and neuronal loss in nondopaminergic areas characterize PD. Loss of neurons is generally associated with characteristic Lewy body formation. Thus, the presence of Lewy bodies containing α-syn oligomers in the brain is considered a possible therapeutic and diagnostic target for PD.

3.2.1. PD Diagnostics

The first aptamer, "M5-15", selected against α-syn lacked specificity for oligomers and could also bind to α-syn monomers [103]. DNA aptamers, then selected, were shown to interact with α-syn oligomers and not α-syn monomers but did bind Aβ1–40 oligomers. 

The investigators also explored the aptamer's specificity for α-syn oligomers over other proteins with structures that are predominantly β-sheet, such as the β-sheet propeller structure of pyrroloquinoline quinine glucose dehydrogenase (PQQH), and demonstrated that the aptamer did not bind PQQH [91]. A variety of aptasensors have been reported to be capable of quantifying α-syn oligomers. 

A colorimetric aptasensor, employing a DNA aptamer against the α-syn oligomer, was adsorbed to gold nanoparticles (AuNPs), which prevented salt-induced aggregation of the nanoparticles. Its binding to the α-syn oligomer prevents the absorption of aptamer onto the surface of AuNPs, which allows the aggregation of AuNPs to be triggered in high salt concentrations and results in a color change. 

This system was reported with a LoD of 10 nM. However, serum (even at 2%) contributed to absorbing compounds in the range of color change in the assay and interfered with the salt-dependent aggregation of the aptamer-Au-NPs. 

Aptasensors using EIS and SPR were more sensitive, with LoDs of 1 pM and 8 pM, respectively [104]. An EIS aptasensor was found highly specific for oligomers over monomers and not affected by the presence of serum [104]. An elaborate design for an electrochemical aptasensor to detect α-syn oligomer involved a hybrid formed by the complementary sequence to an α-syn oligomer aptamer linked to a gold surface through a thiol bond and included terminal deoxynucleotidyl transferase (TdT) and dTTP to extend the 30 ends of the aptamer and its complement with additional polyp. 

The extended ssDNA bound methylene blue and promoted its association with the gold surface, thereby creating an electrochemical signal. In addition, the system contained exonuclease I (Exo I), which is specific for ssDNA. When the aptamer was removed from the binary complex with its complement by its binding to the α-syn oligomer, the complementary strands were digested by Exo I, preventing the accumulation of methylene blue at the gold surface and thus decreasing the electrochemical signal. 

The detection limit was 10 PM (S/N = 3) and recovery of the signal in the presence of 10% and lower concentrations of serum was a very acceptable 95.3–107% [105]. However, its complexity and the incorporation of enzymes make this system less likely to be stable for storage and reproducible in the field. A combination of capillary electrophoresis-mass spectrometry (CE-MS) with solid phase extraction (SPE) to pre-concentrate the target with an aptamer-modified micro cartridge in a large volume of injected samples decreased the detection limit 100-fold compared to CE-MS systems. 

In this novel system, the sample is cleaned, and the volume is minimized for enhanced analysis by the use of a micro cartridge that selectively retains the target. An aptamer affinity (AA) sorbent in the cartridge was found to be a superior alternative to an antibody-based immunoaffinity (IA) sorbent as the background electrolytes required to rinse the system also denature the antibodies. 

The development of an AA-SPE-CE-MS was achieved by modifying the micro cartridge with the M5-15 DNA aptamer. The system demonstrated a LoD of 2.8 nM and had a linear detection range between 7 and 140 nM of α-syn in the blood sample. 

Also, identification of free and N-acetylated α-syn proteoforms was possible as a result of the highly accurate mass detection and resolving power of the MS. Finally, the micro cartridge was found stable even with acidic BGE and could be used for about 20 analyses with a low chance of erroneous quantification of the target [106].

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