Synaptic expression of TAR-DNA-binding protein 43 in the mouse spinal cord determined using super-resolution microscopy

Funding: This work was supported by Motor Neurone Disease (MND) Association UK (Miles/Apr18/863-791), Chief Scientist Office, RS Macdonald Charitable Trust, ALS CURE Project, the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (695568 SYNNOVATE), Simons Foundation Autism Research Initiative (529085), and the Wellcome Trust (Technology Development grant 202932).Amyotrophic Lateral Sclerosis (ALS) is characterised by a loss of motor neurons in the brain and spinal cord that is preceded by early-stage changes in synapses that may be associated with TAR-DNA-Binding Protein 43 (TDP-43) pathology. Cellular inclusions of hyperphosphorylated TDP-43 (pTDP-43) are a key hallmark of neurodegenerative diseases such ALS. However, there has been little characterisation of the synaptic expression of TDP-43 inside subpopulations of spinal cord synapses. This study utilises a range of high-resolution and super-resolution microscopy techniques with immunolabelling, as well as an aptamer-based TDP-43 labelling strategy visualised with single-molecule localisation microscopy, to characterise and quantify the presence of pTDP-43 in populations of excitatory synapses near where motor neurons reside in the lateral ventral horn of the mouse lumbar spinal cord. We observe that TDP-43 is expressed in approximately half of spinal cord synapses as nanoscale clusters. Synaptic TDP-43 clusters are found most abundantly at synapses associated with VGLUT1-positive presynaptic terminals, compared to VGLUT2-associated synapses. Our nanoscopy techniques showed no difference in the subsynaptic expression of pTDP-43 in the ALS mouse model, SOD1G93a, compared to healthy controls, despite prominent structural deficits in VGLUT1-associated synapses in SOD1G93a mice. This research characterizes the basic synaptic expression of TDP-43 with nanoscale precision and provides a framework with which to investigate the potential relationship between TDP-43 pathology and synaptic pathology in neurodegenerative diseases.Publisher PDFPeer reviewe

pTDP-43 aggregates accumulate in non-central nervous system tissues prior to symptom onset in amyotrophic lateral sclerosis : a case series linking archival surgical biopsies with clinical phenotypic data

Acknowledgements The authors would like to thank the staff at the NHS Lothian BioResource (Vishad Patel and Craig Marshall) and the NHS Grampian biorepository (Joan Wilson) and the staff and corefunded resources of the imaging and histology core facility at the Institute of Medical Sciences (Gillian Milne, Lucinda Wight, and Debbie Wilkinson). This study was funded by the Pathological Society/Jean Shanks Foundation (JSPS CLSG 202002 to JMG and JO’S), The Royal Society (RGS\R1\221396 to JMG) and the Wellcome Trust (108890/Z/15/Z to OR). Funders had no role in study design, data collection, data analyses, interpretation, or writing the manuscriptPeer reviewedPostprin

Synaptic expression of TAR-DNA-binding protein 43 in the mouse spinal cord determined using super-resolution microscopy

Amyotrophic Lateral Sclerosis (ALS) is characterised by a loss of motor neurons in the brain and spinal cord that is preceded by early-stage changes in synapses that may be associated with TAR-DNA-Binding Protein 43 (TDP-43) pathology. Cellular inclusions of hyperphosphorylated TDP-43 (pTDP-43) are a key hallmark of neurodegenerative diseases such ALS. However, there has been little characterisation of the synaptic expression of TDP-43 inside subpopulations of spinal cord synapses. This study utilises a range of high-resolution and super-resolution microscopy techniques with immunolabelling, as well as an aptamer-based TDP-43 labelling strategy visualised with single-molecule localisation microscopy, to characterise and quantify the presence of pTDP-43 in populations of excitatory synapses near where motor neurons reside in the lateral ventral horn of the mouse lumbar spinal cord. We observe that TDP-43 is expressed in approximately half of spinal cord synapses as nanoscale clusters. Synaptic TDP-43 clusters are found most abundantly at synapses associated with VGLUT1-positive presynaptic terminals, compared to VGLUT2-associated synapses. Our nanoscopy techniques showed no difference in the subsynaptic expression of pTDP-43 in the ALS mouse model, SOD1G93a, compared to healthy controls, despite prominent structural deficits in VGLUT1-associated synapses in SOD1G93a mice. This research characterises the basic synaptic expression of TDP-43 with nanoscale precision and provides a framework with which to investigate the potential relationship between TDP-43 pathology and synaptic pathology in neurodegenerative diseases

Development of RNA aptamers as super-resolution imaging tools to study TDP-43 aggregation in ALS

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

Probing TDP-43 condensation using an in silico designed aptamer

We thank the “RNA Initiative” at IIT, all members of the M.H.H., A.P., and G.G.T. groups, and especially Fernando Cid Samper. M.H.H. wishes to thank UCB Biopharma and Dr Jim Love for providing funding for the instrument used to generate the super-resolution data in this manuscript. A.P. acknowledges funding from UK DRI (grant REI 3556) and AlzheimerUK (grant ARUK-PG2019B-020). O.K. was supported by a Scottish PhD Research & Innovation Network Traineeships in MND/MS. E.Z. received funding from the Newton fellowship scheme and the MINDED fellowship of the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 754490. K.J. was funded via the BBSRCEastBIO doctoral training program (BB/M010996/1). The research leading to these results was supported by European Research Council [RIBOMYLOME n. 309545 to G.G.T. and ASTRA n. 855923 to G.G.T.], H2020 [IASIS n. 727658 to G.G.T. and INFORE n. 825080 to G.G.T.] and MND [840-791 to G.G.T. and A.P.] projects. The authors would also like to acknowledge the help and support received during confocal images acquisition by the group of Giuseppe Vicidomini at the Molecular Microscopy and Spectroscopy Department of IIT.Peer reviewedPublisher PD