α-Synuclein Radiotracer Development and In Vivo Imaging: Recent Advancements and New Perspectives

α-Synucleinopathies including idiopathic Parkinson's disease, dementia with Lewy bodies and multiple systems atrophy share overlapping symptoms and pathological hallmarks. Selective neurodegeneration and Lewy pathology are the main hallmarks of α-synucleinopathies. Currently, there is no imaging biomarker suitable for a definitive early diagnosis of α-synucleinopathies. Although dopaminergic deficits detected with single-photon emission computed tomography (SPECT) and positron emission tomography (PET) radiotracers can support clinical diagnosis by confirming the presence of dopaminergic neurodegeneration, dopaminergic imaging cannot visualize the preceding disease process, nor distinguish α-synucleinopathies from tauopathies with dopaminergic neurodegeneration, especially at early symptomatic disease stage when clinical presentation is often overlapping. Aggregated α-synuclein (αSyn) could be a suitable imaging biomarker in α-synucleinopathies, because αSyn aggregation and therefore, Lewy pathology is evidently an early driver of α-synucleinopathies pathogenesis. Additionally, several antibodies and small molecule compounds targeting aggregated αSyn are in development for therapy. However, there is no way to directly measure if or how much they lower the levels of aggregated αSyn in the brain. There is clearly a paramount diagnostic and therapeutic unmet medical need. To date, aggregated αSyn and Lewy pathology inclusion bodies cannot be assessed ante-mortem with SPECT or PET imaging because of the suboptimal binding characteristics and/or physicochemical properties of current radiotracers. The aim of this narrative review is to highlight the suitability of aggregated αSyn as an imaging biomarker in α-synucleinopathies, the current limitations with and lessons learned from αSyn radiotracer development, and finally to propose antibody-based ligands for imaging αSyn aggregates as a complementary tool rather than an alternative to small molecule ligands. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.</p

Probing Amyloid-beta protein structure and dynamics with a selective antibody

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. The AD brain is characterized by significant neuronal loss and accumulation of insoluble fibrillar amyloid-β protein (Aβ) plaques and tau protein neurofibrillary tangles in the brain. However, over the last decade, many studies have shown that the neurodegenerative effect of Aβ may in fact be caused by various soluble oligomeric forms as opposed to the insoluble fibrils. Furthermore, the data suggest that a pre-fibrillar aggregated form, termed protofibrils, mediates direct neurotoxicity, and triggers a robust neuroinflammatory response. Antibodies targeting the various conformation of Aβ are important therapeutic agents to prevent the progression of AD. We have generated conformationally-selective monoclonal antibody St. Louis (mAbSL) that selectively targets Aβ42 protofibrils compared to Aβ42 monomers and fibrils. The development aspects of these antibodies include the cloning of HC and LC variable fragments into the plasmid vector, transfection of the plasmids into 293 F cells, collection of the supernatant and purification using Protein A or protein G affinity chromatography. Sequencing of the heavy and light chain variable regions for multiple antibodies identified sequence characteristics that may impart conformational selectivity to the antibodies. Thus, I have successfully developed, expressed, and characterized these conformationally selective antibodies using various ELISA formats. Exploration of Aβ42 aggregation in the presence a selective (mAbSL 113) and a non-selective antibody (mAb Ab 513) using spectroscopic and microscopic techniques is quintessential to looking at the effect of these antibodies on Aβ42 monomer aggregation and protofibril dynamics. It yielded a unique inhibitory mechanism on Aβ42 monomer aggregation offered by mAbSL antibodies. Aβ42 protofibril dynamics were prominently altered in the presence of mAbSL 113 with an insoluble complex formation by the antibody at low sub-stoichiometric molar ratios. We focused on accurately determining the conformational epitope of our developed antibodies on Aβ42 protofibrils. The conformational epitope on Aβ42 protofibril was detected using a monoclonal antibody in various experimental formats like antibody competition ELISA, HDX-MS, and FPOP analysis. Our findings demonstrated new insights into monoclonal antibodies that target AD progression

Imaging Alzheimer’s Disease Beta-amyloid Pathology in Transgenic Mouse Models Using Positron Emission Tomography

Accumulation of beta-amyloid (Aβ) in the brain is known to have an important role in the complex chain of pathological events leading to Alzheimer’s disease (AD). Based on current knowledge, Aβ is also seen as an interesting target for novel disease modifying therapies. Non-invasive and quantitative imaging of transgenic mouse models of AD by positron emission tomography (PET) would represent an ideal translational approach for evaluation of novel Aβ targeted therapeutics in vivo already during the preclinical phase of drug development. The aim of this thesis was to evaluate the suitability of Aβ targeted PET tracers, 11C-PIB and 18F-flutemetamol, for small animal PET imaging and for longitudinal follow-up of β-amyloidosis in three AD mouse models, i.e. transgenic APP23, Tg2576 and APPswe-PS1dE9 mice. In addition, the effect of novel functionalized Aβ targeted nanoliposomes, known as mApoE-PA-LIPs, were investigated in APP23 mice exploiting longitudinal 11C-PIB PET imaging. A temporal increase in tracer retention reflecting increased Aβ-deposition could be detected in vivo only in the APP23 mouse model. Both tracers specifically bound Aβ in mouse brain sections, however, the higher non-specific binding of [18F]flutemetamol to the white matter structures limited its sensitivity in comparison to 11C-PIB. In the APP23 model, the mApoE-PA-LIP treatment showed a trend to reduce the increase in the 11C-PIB binding ratios as compared to the saline-treated group; in addition, the binding ratios correlated well with the histologically-assessed amyloid load, further validating the method. In summary, these results demonstrate that 11C-PIB binding is a valid biomarker of Aβ deposition in the APP23 mouse – an animal model that expresses abundant, large and congophilic Aβ deposits. However, the sensitivity of the method is not sufficient for use in animal models with lower plaque loads and different plaque morphologies, nor does it seem capable of detecting early pathological changes in young AD mice.Siirretty Doriast

Imaging glucose metabolism, neuroinflammation, and cannabinoid receptor 1 in transgenic mouse models of Alzheimer’s disease

The pathophysiological cascade leading to Alzheimer’s disease is characterized by the accumulation of destructive β-amyloid in the brain. Convincing evidence has also shown that cerebral energy hypometabolism and an overproduction of translocator protein during neuroinflammation, as well as deficits in the endocannabinoid system, play major roles in progression of the disease. Monitoring temporal changes inside the diseased brain with noninvasive positron emission tomography (PET) would be a unique translational tool, bridging the gap between disease models and patients and aiding in the discovery of disease-modifying therapies against Alzheimer’s disease. The aim of this thesis was to evaluate the translational feasibility of cerebral glucose metabolism targeting PET tracer 18F-FDG in APPswe-PSIdE9, Tg2576, and APP/PS1-21 mouse models of Alzheimer’s disease. In addition, this thesis aimed to examine the suitability of neuroinflammation-specific protein targeting tracer 18F-DPA-714 for longitudinal follow-up in aging APP/PS1-21 mice and whether it correlates with changes in glucose metabolism. Furthermore, the translational applicability of 18F-FMPEP-d2 was evaluated as a tool to assist in preclinical research targeting cannabinoid receptor 1 (CB1R) in wild-type and APP/PS1-21 mice. Of the tested models, APP/PS1-21 mice demonstrated the most aggressive β-amyloid pathology. Furthermore, repeated PET scans with 18F-FDG and 18F-DPA-714 detected progressive glucose hypometabolism and neuroinflammation in the APP/PS1-21 model as the mice aged. However in the APPswe-PSIdE9 and Tg2576 mouse models, only a weak or non-altered glucose metabolism was observed. 18F-FMPEP-d2 was able to reveal altered CB1R availability when aging APP/PS1- 21 mice were followed with repeated PET scans. This thesis work demonstrated that Alzheimer’s disease mouse models differ in terms of amyloidosis and cerebral glucose metabolism, which creates challenges when comparing the research results between the models. The feasibility of 18F-FDG small animal PET depends on the chosen disease model and environmental factors. In the APP/PS1-21 model, longitudinal 18FFMPEP- d2 and 18F-DPA-714 PET scans were able to demonstrate pathological features related to Alzheimer´s disease, which were confirmed by ex vivo examinations.Aivojen energia-aineenvaihdunnan, tulehduksen ja tyypin 1 kannabinoidireseptorin kuvantaminen Alzheimerin taudin muuntogeenisissä hiirimalleissa Alzheimerin taudin keskeisimmät aivomuutokset ovat sakkautuvien β-amyloidipeptidien muodostuminen plakeiksi, aivojen heikentynyt energia-aineenvaihdunta, tulehduksen lisääntyminen ja endokannabinoidijärjestelmässä tapahtuvat muutokset, jotka lopulta johtavat hermosolujen vaurioitumiseen ja tyypillisten kognitiivisten häiriöiden ilmentymiseen. Aivomuutoksia on mahdollista seurata elävässä tutkittavassa kajoamattoman positroniemissiotomografia (PET)-kuvantamisen avulla. Muuntogeenisten Alzheimerin taudin eläinmallien PET-kuvantaminen antaa ainutlaatuisen mahdollisuuden selvittää sairauden monimutkaisia patologisia tapahtumia ja seurata uusien lääkeaineiden vaikutusta ja turvallisuutta. Tämän tutkimuksen tavoitteena oli arvioida aivojen glukoosiaineenvaihduntaa mallintavan 18FFDG-merkkiaineen soveltuvuutta muuntogeenisten APPswe-PSIdE9, Tg2576 ja APP/PS1-21 hiirimallien pieneläinPET-kuvantamiseen. Toisena tavoitteena oli arvioida tulehdusproteiiniin sitoutuvan PET-merkkiaineen, 18F-DPA-714, soveltuvuutta aivoissa etenevän tulehduksen seuraamiseen muuntogeenisessä APP/PS1-21 hiirimallissa. Kolmantena tavoitteena oli tutkia tyypin 1 kannabinoidireseptori-PET-merkkiaineen, 18F-FMPEP-d2, soveltuvuutta pieneläinkuvantamiseen villityypin hiirillä ja Alzheimerin taudin reseptorimuutosten seuraamiseen APP/PS1-21 hiirimallilla. APP/PS1-21 hiirimallin β-amyloidipatologia eteni muita malleja nopeammin. Lisäksi hiirimallin aivojen glukoosiaineenvaihduntaa mallintavan merkkiaineen kertymä heikentyi ja tulehdusproteiiniin sitoutuvan merkkiaineen määrä kasvoi, kun hiiriä kuvattiin toistuvasti PETmenetelmällä. Vastaavasti APPswe-PSIdE9 ja Tg2576 hiirimalleilla havaittiin vain lievää tai olematonta glukoosiaineenvaihdunnan heikkenemistä. 18F-FMPEP-d2 PET-tutkimukset osoittivat alentunutta merkkiainekertymää APP/PS1-21 hiirimallissa verrattuna terveisiin eläimiin, ja soveltuvuutta tuleviin pieneläinkuvantamistutkimuksiin. Tutkimustulokset osoittivat, että muuntogeeniset eläinmallit eroavat merkittävästi toisistaan, mikä asettaa haasteita tutkimustulosten vertaamiseen mallien kesken. Aivojen 18F-FDG-kertymä vaihtelee tautimallin ja ympäristötekijöiden mukaan, mikä tuo rajoitteita pieneläinkuvantamisen toteuttamiseen. Sekä 18F-DPA-714- ja 18F-FMPEP-d2-merkkiaineet pystyivät osoittamaan Alzheimerin taudille tyypillisiä aivomuutoksia APP/PS1-21 hiirissä, mitkä voitiin varmentaa ex vivo menetelmin hiirten aivoleikkeistä

A bispecific Tribody PET radioligand for visualization of amyloid-beta protofibrils - a new concept for neuroimaging

Antibodies are highly specific for their target molecules, but their poor brain penetrance has restricted their use as PET ligands for imaging of targets within the CNS. The aim of this study was to develop an antibody-based radioligand, using the Tribody(TM) format, for PET imaging of soluble amyloid-beta (All) protofibrils, which are suggested to cause neurodegeneration in Alzheimer's disease. Antibodies, even when expressed in smaller engineered formats, are large molecules that do not enter the brain in sufficient amounts for imaging purposes. Hence, their transport across the blood-brain barrier (BBB) needs to be facilitated, for example through interaction with the transferrin receptor (TfR). Thus, a Fab fragment of the TfR antibody 8D3 was fused with two single chain variable fragments (scFv) of the A beta protofibril selective antibody mAb158. Five Tribody proteins (A1-A5) were generated with different linkers between the Fab-8D3 and scFv-158. All proteins bound to TfR and All protofibrils in vitro. Three of the proteins (A1-A3) were radiolabeled with iodine-125 and studied ex vivo in wild-type (wt) and transgenic mice overexpressing human All. The systemic pharmacokinetics were similar with half-lives in blood of around 9 h for all three ligands. Brain concentrations at 2 h were around 1% of the injected dose per gram brain tissue, which is similar to what is observed for small molecular radioligands and at least 10-fold higher than antibodies in general. At 72 h, transgenic mice showed higher concentrations of radioactivity in the brain than wt mice (12, 15- and 16-fold for Al, A2 and A3 respectively), except in the cerebellum, an area largely devoid of A beta pathology. A3 was then labelled with iodine-124 for in vivo positron emission tomography (PET) imaging. Brain concentrations were quantified in six different regions showing a clear distinction both quantitatively and visually between wt and transgenic mice and a good correlation with A beta pathology. We have thus produced a recombinant, bispecific protein, actively transported into the brain, for PET imaging within the CNS. In a longer perspective, this technique may enable imaging of other proteins involved in neurodegenerative diseases for which imaging agents are completely lacking today