| dc.description.abstract | It is commonly recognised that mislocalisation and aggregation of Transactive response
DNA- binding protein-43 (TDP-43) occurs in over 97% of amyotrophic lateral sclerosis (ALS)
patients and around 50% of fronto-temporal dementia (FTD) patients. However, despite the
commonality of this molecular phenotype, its precise role in disease onset and progression
remains elusive. Although TDP-43 is an attractive target for therapeutic intervention and
diagnostic testing, it has thus far not been possible to exploit either of these approaches.
A core problem remains the lack of efficient detection and targeting probes for TDP-
43. A large variety of micrometre-scale TDP-43 inclusions have been described, however,
smaller protein assemblies have not been studied in detail. Although traditional ensembleaveraging
biochemical techniques fail to deliver information on single nanometre-scale
aggregates, single-molecule and super-resolution (SR) methods can be used to circumvent
these issues, as has been demonstrated in other neurodegenerative diseases involving
protein aggregation. To date, the number of studies employing these techniques to examine
TDP-43 is extremely limited. Highly specific and sensitive imaging probes are required for
single-molecule measurements and would allow counting and characterisation of individual
aggregates. Precise localisation of aggregates in tissue and the cellular milieu could enhance
the understanding of TDP-43 relevance in a disease context. Meanwhile, separation of
disease- relevant aggregates from physiological TDP-43 in biofluids could assist in the
establishment of a reliable biomarker.
Aptamers are artificial oligonucleotides capable of binding to specific molecular targets and
are attractive alternatives to antibodies with regards to biomolecule labelling. In this study,
aptamers targeting TDP-43’s RNA recognition motifs (RRM1-2) were designed via the in silico
algorithm, catRAPID, and validated in vitro. The highest affinity candidate aptamer, Apt-1, was
labelled for fluorescent imaging and used to track the aggregation of a TDP-43 construct. Apt-
1 was capable of generating diffraction limited fluorescence images, as well as SR images at
a resolution of 20 nm via Aptamer DNA-points accumulation in nanoscale topography (ADPAINT)
and a newly developed technique, dubbed Aptamer-PAINT.
To establish if Apt-1 could be used for imaging in biological samples of a complex composition,
a comparative study of TDP-43 detection using gold-standard immunohistochemistry (IHC)
and Apt-1 fluorescence imaging, was carried out. Despite low-agreement between both
techniques, Apt-1 was capable of separating ALS cases from healthy controls based on
the number of detected TDP-43 aggregates. Furthermore, Aptamer-PAINT SR images of
aggregates could be acquired in tissue with a similar resolution to that achieved in vitro.
TDP-43 can be detected in human biofluids, such as serum and cerebrospinal fluid (CSF). SR
imaging of CSF samples using Apt-1 was carried out to establish if the number of detected
species in samples from ALS patients with TDP-43 proteinopathy was higher than in ALS
disease controls. Additionally, size characterisation of the detected species was used to
determine differences in the relative distribution of aggregate populations.
Overall, the work presented in this thesis demonstrates the development of a new TDP-43
imaging probe capable of characterising aggregate species individually in variety of in vitro
and patient-relevant samples. Although these studies are exploratory, they form the basis of
establishing a new class of detection probes in the field of ALS and FTD research, and show
promise in the development of diagnostic tools. In this manner it is hoped that the developed
aptamers will aid researchers and clinicians in understanding the mechanisms of TDP-43
pathology and assist in diagnosing patients. | en |
