Aptamers in Neuroscience

Neurodegenerative diseases are incurable

and debilitating conditions that result in

progressive degeneration or death of nerve

cells. They include Alzheimer's disease and

other dementias, Parkinson's disease,

Huntington's disease, motor neurone disease,

Creutzfeldt-Jakob disease and multiple

sclerosis. Of these, the dementias are

responsible for the greatest burden of disease,

with Alzheimer’s disease representing over

60-70% of the cases (MRC, 2016). The

dementias are one of the toughest medical

and economic challenges facing our society

today. Around 850,000 people in the UK suffer

from dementia, costing the health care system

over £26 billion a year (MRC, 2016). The

incidence of dementia will continue to grow

as the population in the UK and Europe ages,

with the number of people affected predicted

to double within the next 20 years (MRC,

2016). With the prevalence of human

neurodegenerative disorders set to

significantly increase, there is a critical need

for alternatives methods to facilitate early

diagnosis and as potential therapeutics.

Aptamers can occupy this niche and act to

solve the underlying complications at an early

stage before clinical progression and

manifestation take place.

What is Neurodegeneration?

Neurodegenerative diseases are defined as

hereditary and sporadic conditions which are

characterised by progressive nervous system

dysfunction. These disorders are often

associated with atrophy of the affected central

or peripheral structures of the nervous

system. The degeneration of the central

nervous system (CNS) is characterised by

chronic progressive loss of the structure and

function of neuronal materials, resulting in

functional and mental impairments (Chen,

Zhang, and Huang, 2016). The clinical hallmark

of demential neurodegenerative diseases is

the progressive impairment of intellectual

functions. These diseases classically exhibit

the accumulation of fibrillar protein

aggregates as a consequence of protein

misfolding (Mueller, Schuff, and Weiner 2006).

By far the most frequent form of dementia is

Alzheimer’s disease (AD). Other forms, such as

vascular dementia (VD), Lewy body disease,

frontotemporal lobe dementia and HIV-

associated dementia are less frequent

(Mueller, Schuff, and Weiner 2006).

What are Aptamers?

Aptamers are short strands of nucleic acids

that serve to bind to a target with high affinity

and specificity and as such, are commonly

referred to as ‘synthetic antibodies’. Aptamer

targets include proteins, cells, and even small

molecules which are typically problematic to

antibodies. In theory, almost any target

molecule can be selected, with aptamers

previously having demonstrated high

recognition and binding capability to low

molecular mass substances (Osborne and

Ellington, 1997). As a result, aptamers serve as

useful tools for the identification, separation

and purification of molecules and have

become popular in the research arena.

Consequently, aptamers open new avenues in

neuroscience research with their ability to

interrupt protein accumulation and ultimately

disrupt the perceived underlying factors

involved in neurodegenerative diseases.

Neurodegenerative diseases

Alzheimer’s disease (AD) is the most common

cause of dementia and is characterised by

progressive loss of memory and other

cognitive functions (Robinson, Fernandes,

Husi, 2014). It is considered a major epidemic

worldwide, where currently more than 35

million people live with this disease. By 2050

it is estimated this figure will reach 115

million. Due to its high incidence rate (5.9–

10.8 cases per 1000 above the age of 75

years), most efforts to find an effective

treatment for dementias have focused on AD

(Robinson, Fernandes, and Husi, 2014).

AD is characterised by two major

abnormalities; abnormal extracellular amyloid

β-protein (Aβ) disposition and intracellular

neurofibril-lary tangle (NFT) formation, both

leading to neuronal degeneration (Veedu,

2016). As Aβ aggregation is toxic, it follows

that reducing Aβ production would be

beneficial. The generation of Aβ is triggered

by B-site amyloid precursor protein cleaving

enzyme-1 (BACE1). Thus, BACE1 is a

prospective target for interfering with Aβ

production and the treatment of AD (Fig. 1)

(Das et al. 2016). A DNA aptamer selected by

Liang et al. has been shown to bind to BACE1

with high affinity and good specificity,

exhibiting a distinct inhibitory effect on BACE1

activity in an AD cell model.

BACE1 contains short cytoplasmic tail (B1-CT).

Rentmeister et al. identified an RNA aptamer

that binds specifically to the BT-CT tail without

interfering with the B1-CT regulated BACE1

transport which remarkably discriminates

binding regions within a 3 kDa peptide

(Rentmeister et al., 2006). This aptamer can

be used to further elucidate B1-CT function

without hindering cellular localisation or

biological activates (Veedu, 2016).

The clinical diagnosis of AD is often made

during the mild stage of the disease, with

consideration of a list of cognitive-behavioral

signs and symptoms (Robinson, Fernandes,

and Husi, 2014). The current approach is

based on the combination of cognitive and

psychiatric assessment, genetic profiling and

structural magnetic resonance imaging (sMRI).

sMRI is able to measure morpho-anatomical

changes of the brain, such as loss of neural

cells and axons and expansion of the CSF

space (Robinson, Fernandes, and Husi, 2014).

Aptamers have shown great promise and

advances in the imaging and diagnosis of AD.

Recently, multiple aptamers have been

developed to facilitate imaging of the Aβ

peptides. Farrar et al. developed a

fluorescently tagged anti- Aβ -aptamer, b55,

that can efficiently bind Aβ plaques in both

human AD brain tissue in vitro and in vivo

using mice (Farrar et al., 2014) Similarly, Babu

et al. developed a simple luminescence

aptamer-ruthenium complex that binds and

inhibits the formation of Aβ oligomers (Babu

et al., 2015).

Figure1. Aptamers against AD targets.

Parkinson’s disease (PD), the second most

common neurodegenerative disease after AD,

affects over 7 million people worldwide

(Veedu, 2016). To date, there is no recognised

cure. The pathology is characterised by loss of

dopaminergic neurons leading to decreased

production of dopamine, a neurotransmitter

that regulates movement and cognition. PD is

considered a multifactorial disorder that

results, in most cases, from the combined

effects of multiple risk and protective factors,

including genetic and environmental ones. PD

is typically characterised by the accumulation

of α-synuclein proteins into Lewy bodies and

by the loss of dopaminergic neurons within

parts of the midbrain (Veedu, 2016).

Predominantly, α-synuclein oligomers are

more toxic than monomers and fibrils and are

seen as the key targets for PD therapeutics

(Veedu, 2016).

Figure 2. Aptamers targeting α -Syn oligomers

for diagnosing and preventing onset of PD and

dopamine for diagnosing dopamine

concentrations (Qu et al., 2016).

Therapeutics

Through Aptamer Group’s therapeutics

division aptarx, aptamers can be developed as

lead molecules for therapeutic intervention

against a range of targets. There are several

avenues for exploitation of aptamers as

therapeutics; they may be used directly, form

part of a therapeutic chimera, serve as a

carrier for other agents or be used to

modulate their activity.

Numerous examples in the literature have

shown the efficacy of aptamers against several

important targets. Aptamers have been

developed to bind to α-synuclein monomers

(Tsukakoshi et al., 2010) or its oligomer

(Tsukakoshi et al., 2012). These aptamers

recognised β-sheet structure, the moiety

though which they can bind not only to α-

synuclein oligomer, but also Aβ oligomer. This

indicates that these aptamers could also

potentially be deployed as drugs treat PD and

AD (Fig.2) (Tsukakoshi et al., 2012).

Glial cell line-derived neurotrophic factor

(GDNF) which promotes survival and

differentiation of dopaminergic neurons in

vitro is known to signal through a receptor

tyrosine kinase called RET (Cerchia et al.

2005). A nuclease resistant 2'-

fluoropyrimidine RNA aptamer was identified

exhibiting high affinity and specificity to the

extracellular domain of RET monomers,

inhibiting its dimerization and signalling

(Cerchia et al., 2005). Despite not having

direct therapeutic use, these aptamers can be

utilised to investigate important and major

signalling components.

Multiple Sclerosis (MS) is a chronic

inflammatory and demyelinating disorder of

the nervous system. Currently, there is no

cure for MS and the available medications

only shorten the duration of attacks to slow

the progression of the disease (Veedu, 2016).

Remyelination is a naturally occuring process

in the body to restore damaged myelin

sheaths after an MS attach. However this

restoration process often leads to only

incomplete recovery (Veedu, 2016).

Rozenblum et al. identified a 40-nucleotide

DNA aptamer which exhibits affinity towards

murine myelin and binds to multiple myelin

components in vitro (Rozenblaum et al.,

2014). Through intraperitoneal (IP) injection in

mice, improved distribution and uptake in CNS

tissue was demonstrated. Furthermore, the

aptamer promoted remyelination of CNS

lesions in mice infected by Theiler virus

(Nastasijevic et al., 2012). This aptamer could

therefore prove valuable in the body for

recovery following an MS attack and could

palliate MS symptoms.

In the field of MS diagnostics, a 2'-F modified

RNA aptamer was selected to bind specifically

to proteolytic autoantibodies against myelin

basic proteins (Vorobjeva et al., 2014). This

high affinity aptamer was conjugated with a

Ca (2+) regulated photo protein and used in a

bioluminescent microplate assay to detect

these antibodies. This technique is not only

sensitive but also simple and fast,

demonstrating the potential to be used as a

specific laboratory test for diagnostic MS

(Vorobjeva et al., 2014).

Variant Creutzfeldt - Jakob diseases (CJD) is a

degenerative neurological disorder and a form

of spongiform encephalopathy that is

invariably fatal. The accumulation of

abnormally folded insoluble prion protein

(PrP) especially in the brain is a characteristic

of CJD. PrP replicates in the host by

promoting the misfolding of native proteins of

the diseased conformation (Veedu, 2016).

Several aptamers were used to stop the

conversion of normal to abnormal PrPs.

Proske et al. developed a modified RNA

aptamer against PrP, targeting a domain

thought to be important in the conversion of

PrP from its normal to abnormal confirmation

(Proske et al. 2002). They were able to show a

reduction in misfolded PrP levels with the

administration of this aptamer into the

medium of prion-infected neuroblastoma cells

(Proske et al. 2002).

Murakami et al. identified an RNA aptamer

consisting of only 12 nucelotides that binds to

a normal cellular form of bovine PrP with high

affinity. The aptamer also exhibits antiprion

activity in mouse neural cells by binding and

stabilising the normal form of PrP, thus

blocking its conversion to abnormal PrP

(Murakami et al. 2008). These advances could

be the first steps to successful prophylaxis of

CJD.

Brain tumour like glioblastoma (GBM) is the

most lethal form of malignant tumour in

adults (Wilson et al., 2014). GBM is

characterised by rapid growth and is highly

invasive; its capacity to spread into critical

neurological areas within the brain well

documented. Recently, Kang et al. developed

two aptamers with high affinity and specificity

against GBM cells and displaying no

nonspecific binding to normal astraglial cells

or normal brain tissue. Kim et al. also

developed aptamers to tumour initiating cells

(TIC), with dissociation constants (Kd) in the

pM to nM range. These aptamers select and

internalise into GBM cells that self-renew,

proliferate, and initiate tumours (Kim et al.,

2013).

PEG–PLGA nanoparticles incorporating a DNA

aptamer targeting nucleolin, a molecule highly

expressed in the plasma membrane of both

cancer cells and tumour endothelium,

enhanced the anti-proliferative effects of

paclitaxel against C6 glioma cells in vitro

(Srikanth and Kessler, 2012). These aptamer

nanoparticles reduced C6 glioma xenograft

volumes in nude mice over 3 fold, and

prolonged survival of animals with C6

intracranial gliomas compared to treatment

with either paclitaxel alone, or paclitaxel

loaded into undecorated nanoparticles

(Srikanth and Kessler, 2012). Importantly, the

aptamer-decorated nanoparticles showed

greater efficacy than undecorated particles

highlighting the value of targeted delivery.

Protein and Familial Aggregation

Familial aggregation had been recognised as a

prominent characteristic of many

neurodegenerative disorders decades before

the underlying molecular genetic or

biochemical properties were known (Bertram

and Tanzi, 2005). It was often the

identification of specific, disease-segregating

mutations in previously unknown genes that

directed the attention to certain proteins and

pathways now considered crucial in the

pathogenesis of these diseases. These include

mutations in the Aβ precursor protein, causing

AD, in α-synuclein, causing PD, or in

microtubule-associated protein tau, causing

frontotemporal dementia (FTD) with

Parkinsonism (Bertram and Tanzi, 2005).

Tau is a cytosolic protein that functions in the

assembly and stabilisation of axonal

microtubule networks. Its oligomerisation may

be the rate-limiting step of insoluble

aggregate formation; a neuropathological

hallmark of Alzheimer’s disease (AD) and a

number of other tauopathies (Kim et al.,

2016). Kim et al. identified RNA aptamers that

target human tau and significantly inhibited

the oligomerization propensity of tau both in

vitro and in cultured cell models of tauopathy,

without affecting the half-life of tau.

Tauopathy model cells treated with the

aptamers were less sensitised to proteotoxic

stress induced by tau overexpression (Kim et

al., 2016).

Figure 3. Tau assembly with RNA aptamer in

protecting cells under stresses from

pathogenic tau oligomerization (Kim et al.,

2016).

Moreover, the tau aptamers significantly

alleviated synthetic tau oligomer-mediated

neurotoxicity and dendritic spine loss in

primary hippocampal neurons. This

demonstrated that delaying tau assembly with

RNA aptamers (Fig.3) is an effective strategy

for protecting cells under various

neurodegenerative stresses originating from

pathogenic tau oligomerisation (Kim et al.,

2016).

The Blood - Brain Barrier

Aptamers have emerged as an exciting and

promising new means of treating neurological

disease, with the potential to fundamentally

change the way we approach CNS-targeted

therapeutics. With their ability to penetrate

the blood–brain barrier (BBB), aptamers can

target specific cell or signalling systems,

respond to endogenous stimuli, act as vehicles

for gene delivery, or as a matrix to promote

axon elongation and support cell survival.

The BBB regulates brain homeostasis and the

transport of endogenous and exogenous

compounds by controlling their selective and

specific uptake, efflux, and metabolism in the

brain (De Boer and Gaillard, 2007). Brain

capillary endothelial cells, pericytes, astrocytic

foot processes and nerve endings terminating

on the capillary surface constitute the BBB

(Pardridge et al., 2013)

The unique structure of the BBB hinders many

therapies directed at brain pathologies.

Several non-invasive strategies have been

proposed to overcome this problem.

This includes delivery through (Cheng et al.,

2013):

Nasal mucosa,

Osmotic opening of the BBB,

Nanoparticle coating,

Transporter vectors,

Viral vectors

Given the chemical and physical attributes of

aptamers, it is unlikely that they enter the

brain via paracellular aqueous routes or

transcellular lipophilic pathways. However,

aptamers may enter via adsorptive-mediated

transcytosis, channel and/or receptors for

uptake or fluid-phase pinocytosis (Hanss et al.,

1998). Recent work suggests that a

quadruplex-forming DNA aptamer binds to

nucleolin via macropinocytosis. Cheng et al.

identified an aptamer that can enter brain

endothelia cells under physiological

conditions, and in vivo, into the brain

parenchyma.

Figure 4: Schematic representation of the blood-brain barrier and aptamers toward targeted

therapy of neurological diseases (Veedu, 2016).

Diagnosis

The diagnostic tool commonly used to assess

cognitive impairment in neurodegenerative

diseases is based on established clinical

practices. However, the differential diagnosis

between disorders can be difficult, especially

in early phases or atypical variants. This takes

on particular importance when it is still

possible to use an appropriate treatment.

To solve this problem, physicians need access

to an arsenal of diagnostic tests, such as

neurofunctional imaging, that enable higher

specificity in clinical assessment.

Through Aptamer Group’s diagnostics division

aptadx, aptamers can be developed under a

wider variety of conditions, simplifying

development in systems requiring complex

matrices. Aptamers are highly specific and can

be generated against a wide range of markers

of disease, making them an logical choice for

researchers and diagnosticians seeking

improved function and reliability.

Optical imaging is one form of diagnosis; a

cost - effective imaging method typically using

fluorescent or bioluminescent molecules.

However, optical imaging methods that rely

on the availability of either small molecule

reporters or genetically encoded florescent

proteins can be challenging and time

consuming to develop (Kong and Byun, 2013).

Aptamers have demonstrably addressed this

problem, examples having been engineered

and developed with high specificity and

sensitivity for use as optical imaging agents

(Kong and Byun, 2013). Farrar et al. developed

a fluorescently tagged anti-Aβ RNA aptamer

that binds Aβ plaques in both ex vivo human

AD brain tissue and in vivo APP/PS1 transgenic

mice. This work suggested RNA aptamers may

have complementary, and perhaps

advantageous, properties compared to

conventional optical imaging probes due to

their high binding affinity, ease of probe

development and ability to incorporate

multiple and multimodal imaging reporters.

Biomarker Discovery

Biomarkers are essential for performing early

diagnosis, monitoring neurodegenerative

disease progression, gauging responses to

therapies and stratifying neurodegenerative

diseases into their different subtypes (Lausted

et al., 2014). A wide range of molecular

markers are under investigation in tissues and

bio-fluids as well as through imaging.

Moreover, many are prominent proteins

present in cerebrospinal fluid. However, in

more frequently and easily collected fluids

such as plasma, these proteins show only a

modest correlation with disease and thus lack

the necessary sensitivity or specificity for

clinical use (Lausted et al., 2014).

Through the Aptamer Group biomarker

discovery division Aptasort, the Aptamer

Group holds the technology and necessary

expertise to identify novel biomarkers on cell

surfaces or in samples of biological fluids.

Our approach can also be used to develop

leads to faster identification and validation of

novel, important diseased targets for

conventional drug library screening.

Aptamers can play a prominent role in the

identification and discovery of novel disease

correlated biomarkers. Baird et al. used a

slow-flow-rate aptamer array to investigate

age dependant changes in the cerebrospinal

fluid proteome. Using this technology over

200 proteins were identified that could be

valuable biomarkers for the diagnosis and

treatment of neurological diseases (Baird et

al., 2012).

Similarly, Tenascin-C which is an extracellular

glycoprotein that is overexpressed on glioma

cells and therefore acts as a marker for brain

tumours, has also been targeted by aptamers

to bind to or facilitate imaging of glioma cells

(Ye et al., 2012). These aptamers are of high

value for diagnostics and the identification of

GBM biomarkers, molecular imaging, and

targeted drug delivery.

Aptamer Group

Aptamer Group takes a high-throughput

approach using liquid handling robotics and

dedicated researchers to identify aptamers

against novel and significant targets. We are

committed to finding the perfect aptamers to

your target and use a proprietary selection

technique to identify high affinity aptamers

with specificity in as short as 3 months.

Aptamer Group’s biomarker discovery,

diagnostic and therapeutic divisions aim to

conduct further research in the prevention

and treatment of neurodegenerative diseases.

Through our know-how and key

collaborations, we are able to facilitate the

development of aptamers as novel

therapeutics or diagnostic agents for your

target of interest.

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