LUPAS Luminescent Polymers for in vivo Imaging of Amyloid Signatures A research project for innovative diagnostics for neurodegenerative disorders Funded by the European Union under the 7 th Framework Programme Alzheimer- diseased human brain with amyloid plaque visualised by LUPAS technology Project objectives Alzheimer s disease, prion disorders and many other age- related neurodegenerative disorders are referred to as conformation or misfolding disorders. Significant evidence suggests that the accumulation of soluble and/or insoluble protein aggregates is central to their pathogenesis. We propose to develop novel agents and methods for diagnostic imaging that will rely on reporter molecules based on luminescent conjugated polymers, LCPs. The LCP molecules target the pathogenic protein aggregates with high selectivity and specificity. By the development of associated hyper spectral imaging and multiphoton imaging techniques, which have been conceptually demonstrated by us, the imaging agents - LCPs - will not only improve the quality of diagnosis of neurodegenerative diseases, but also be of great advantage for 1
monitoring and understanding the disease progression. Secondly, LCPs can be properly adapted for conventional imaging configurations, such as magnetic resonance imaging (MRI) that are being used for routine clinical diagnosis within the health care system, thus paving the way to clinical therapy and drug development in the near future. Moreover, we envision that the imaging agents developed within LUPAS have potential to be used also for prevention of protein aggregation and could thereby facilitate treatment of neurodegenerative disorders. Consortium The LUPAS partners are assembled from a wide range of areas ranging from experts within organic synthetic chemistry, synthetic nano- chemistry, amyloid structure, prion disease Alzheimer s disease, magnetic resonance imaging, multi- photon physics and hyper spectral imaging. This group form the critical mass of competences needed to reach towards the project s ambitious goals. The partner organisations in the LUPAS consortium are: Linköping University (Sweden), Université Claude Bernard Lyon 1 (France), University of Tübingen (Germany), Norwegian University of Science and Technology (Norway), Zürich University Hospital (Switzerland), Charité - Universitätsmedizin Berlin (Germany), Applied Spectral Imaging (Israel) and Genovis AB (Sweden). Results LUPAS is a 3 year funded research project and have now reached its mid- term point at 18 months. The work towards the projects scientific and technical goals has progressed well during this first period and significant achievements have been made. By combining the diversity of scientific knowledge available within the consortium, progress towards the development of multimodal amyloid ligands for non- invasive imaging with the possibility to visualize the dynamics and biochemical activity of pathological processes of Alzheimer s disease (AD) and prion diseases in real- time from the molecular level to the organ full body scale has been achieved. In addition, novel molecular insight regarding the pathological hallmarks of these diseases and the specific chemical requirements for an optimal amyloid ligand has been obtained. One of the major tasks within LUPAS is to develop novel luminescent conjugated polythiophenes (LCPs) that can be utilized as tools for selective identification of protein deposits, the pathological hallmarks in AD and prion diseases. From a library of LCPs, we have identified a couple of LCPs with distinct chemical functionalisations that can be employed for spectral assignment of protein deposits in transgenic mouse models for AD and prion diseases (Figure 1). Furthermore, these LCPs can also be used for in vivo imaging of protein deposits in the brain of living animals. Hence, LCP scaffolds that are selective amyloid ligands and capable of crossing the blood brain barrier has already been developed within the consortium. These LCPs can also be synthesized in gram scale amounts and are now being implemented within a variety of subprojects within LUPAS. 2
Figure 1: LCP staining of protein deposits in brain tissue from transgenic mouse models for AD and prion diseases. a) Brain tissue from an age matched control mouse. b) Brain tissue from a mouse with AD- like pathology. The LCP stained protein plaque deposits are clearly seen in green colour. c) Brain tissue from a mouse infected with a distinct prion strain. The LCP stained prion deposits are seen in yellow- red colour. To achieve a total characterization of the LCP spectral profiles towards different types of protein deposits, we generated different amyloid molecular targets (AMTs) in vitro and screened the LCP library towards these targets. Interestingly, the LCPs were able to identify a variety of AMTs and several LCP candidates show augmented shifts of excitation and/or emission wavelengths when bound to different amyloid targets in solution and/or in microscopy either in vitro or from ex vivo tissue samples. Hence, several LCPs could be utilized for spectral discrimination of amyloid structures both from the same protein (PrP, A) as well as for discrimination of different amyloid within the same sample (Aß, tau). In addition, we have shown that pre- fibrillar aggregates of PrP, and Aß (oligomers) that goes undetectable by conventional amyloid ligands are readily detectable by LCPs. The ability of LCPs to detect heterogeneous population of protein aggregates and pre- fibrillar Aß aggregates is of great importance, as it is evident that significant morphological variation can exist between different protein deposits formed from the same peptide or protein and that pre- fibrillar states preceding the formation of well- defined amyloid fibrils are likely to play a critical role in the pathogenesis of protein aggregation diseases. Multiphoton imaging is a preferable technique for studying protein aggregation diseases in real time in transgenic mouse models and we have shown that the unique optical properties of LCPs make these dyes highly efficient for multiphoton in vivo imaging. Several LCPs cross the blood brain barrier and and specifically labels Aß amyloid plaques in the parenchyma as well as intraneuronal tau in transgenic mouse models with AD pathology. Hence, in vivo imaging of protein deposits in living mice was achieved using distinct LCPs and these LCP scaffolds are well tolerated to mice even during longer time periods and repetitive injections. The latter is of great importance, as the final goal of LUPAS is to achieve an LCP based agent for non- invasive clinical diagnostics of AD and prion diseases. From a diagnostic perspective, we have employed the LCPs on post- mortem tissue sections from patients with AD or prion disease. The LCP selectively stain the protein deposits in all of these brain sections and the major pathological hallmarks of AD, A plaques, neurofibrillary tangles and dystrophic neuritis can easily be identified and distinguished due to the colour emitted from the LCP bound to the different entities (Figure 2). Hence, the LCP technique shows great promise for being implemented as a complementary technique in routine clinical diagnostics of AD and prion diseases. 3
Figure 2: LCP staining of protein deposits in brain tissue from human patients with AD or Creutzfeldt- Jakob disease (CJD). a) LCP stained brain tissue from a patient with AD. The different pathological hallmarks, plaques (green colour), neurofibrillary tangles (yellow- red colour) and dystrophic A neuritis (yellow- red colour) can clearly be distinguished due to the colour from the LCP. b) LCP staining of brain tissue from a patient with variant CJD (v- CJD). The LCP stained prion deposits are clearly seen in yellow colour. c) LCP staining of brain tissue from a patient with sporadic CJD (s- CJD). The LCP stained prion deposits are clearly seen in pale- yellow colour. Optical probes are not the preferable agents for non- invasive imaging in humans, due to the limitations of multiphoton imaging. In this regard, the LUPAS consortium is aiming at develop novel multimodal LCPs that can be used for both optical imaging and magnetic resonance imaging (MRI). The latter is today a standard technique for imaging of pathological conditions in humans. Contrast agents based on magnetic nanoparticles (MNPs) hold great promise for MRI and within LUPAS we have synthesized a variety of nanoparticles for enhanced T1 and T2 relaxation dispersion potentially useful as contrast agents for MRI. In addition, the first prototype of a LCP- MNP conjugate can specifically target amyloid in tissue samples and in vitro. These hybrid molecules will be evaluated further during the next stage of LUPAS. 4
Conclusions and continued work plans In conclusion, we have shown that LCPs are excellent amyloid ligands that selectively targets protein deposits in AD and prion diseases. Furthermore, the LCPs can be used for real time optical imaging of protein deposits in transgenic mouse models and this technique can be utilized to gain novel molecular insights regarding the pathological events of these diseases. We have also synthesized a variety of MNPs that can be utilized as contrast agents for MRI. Our continued work plans within LUPAS aims at combining the LCPs with the MNPs to achieve excellent imaging agents for non- invasive clinical diagnostics of AD and prion diseases. Hence, during the next stage of LUPAS, we will aim towards proof- of- concept for MRI of protein deposits in animal models by a multimodal LCP/MNPs contrast agent. This phase of LUPAS will also require an increased focus on dissemination and IPR protection. Currently we are working on filing patent applications for promising LUPAS results. Expected final outcome There is a tremendous need for quantitative diagnostic methods for early detection and evaluation of neurodegenerative disorders, such as Alzheimer s disease and prion diseases. The need is underlined by he recent development of proposed therapeutical interventions targeting disease, so called disease modifiers, including immune therapy. Herein, quantitative physical outcome measures are urgently needed in terms of amyloid pathology within living subjects. Within the brief 3 year time frame of LUPAS, we will develop these tools for use in disease model systems (mouse models) in vivo and on histological ex vivo samples from humans. If successful, as it appears from the LUPAS mid- term report, the realistic prognosis is that it will take a few more years to validate this technology in the preclinical phase prior to going to the clinic. The LUPAS consortium strives for continuing the efforts towards these goals beyond the project time frame. Contacts Coordinator Prof. Per HammarstroÃàm, LinkoÃàping University, Sweden E- mail: perha@ifm.liu.se Dissemination manager Dr. Sarah Fredriksson, Genovis AB, Sweden E- mail: sarah.fredriksson@genovis.com Website www.lupas- amyloid.eu 5