Amyloid polymorphisms constitute distinct clouds of conformational variants in different etiological subtypes of Alzheimer's disease

The molecular architecture of amyloids formed in vivo can be interrogated using luminescent conjugated oligothiophenes (LCOs), a unique class of amyloid dyes. When bound to amyloid, LCOs yield fluorescence emission spectra that reflect the 3D structure of the protein aggregates. Given that synthetic amyloid-β peptide (Aβ) has been shown to adopt distinct structural conformations with different biological activities, we asked whether Aβ can assume structurally and functionally distinct conformations within the brain. To this end, we analyzed the LCO-stained cores of β-amyloid plaques in postmortem tissue sections from frontal, temporal, and occipital neocortices in 40 cases of familial Alzheimer's disease (AD) or sporadic (idiopathic) AD (sAD). The spectral attributes of LCO-bound plaques varied markedly in the brain, but the mean spectral properties of the amyloid cores were generally similar in all three cortical regions of individual patients. Remarkably, the LCO amyloid spectra differed significantly among some of the familial and sAD subtypes, and between typical patients with sAD and those with posterior cortical atrophy AD. Neither the amount of Aβ nor its protease resistance correlated with LCO spectral properties. LCO spectral amyloid phenotypes could be partially conveyed to Aβ plaques induced by experimental transmission in a mouse model. These findings indicate that polymorphic Aβ-amyloid deposits within the brain cluster as clouds of conformational variants in different AD cases. Heterogeneity in the molecular architecture of pathogenic Aβ among individuals and in etiologically distinct subtypes of AD justifies further studies to assess putative links between Aβ conformation and clinical phenotype

Deletion of SERF2 in mice delays embryonic development and alters amyloid deposit structure in the brain

In age-related neurodegenerative diseases, like Alzheimer's and Parkinson's, disease-specific proteins become aggregation-prone and form amyloid-like deposits. Depletion of SERF proteins ameliorates this toxic process in worm and human cell models for diseases. Whether SERF modifies amyloid pathology in mammalian brain, however, has remained unknown. Here, we generated conditional Serf2 knockout mice and found that full-body deletion of Serf2 delayed embryonic development, causing premature birth and perinatal lethality. Brain-specific Serf2 knockout mice, on the other hand, were viable, and showed no major behavioral or cognitive abnormalities. In a mouse model for amyloid-β aggregation, brain depletion of Serf2 altered the binding of structure-specific amyloid dyes, previously used to distinguish amyloid polymorphisms in the human brain. These results suggest that Serf2 depletion changed the structure of amyloid deposits, which was further supported by scanning transmission electron microscopy, but further study will be required to confirm this observation. Altogether, our data reveal the pleiotropic functions of SERF2 in embryonic development and in the brain and support the existence of modifying factors of amyloid deposition in mammalian brain, which offer possibilities for polymorphism-based interventions. </p

Deletion of SERF2 in mice delays embryonic development and alters amyloid deposit structure in the brain

In age-related neurodegenerative diseases, like Alzheimer's and Parkinson's, disease-specific proteins become aggregation-prone and form amyloid-like deposits. Depletion of SERF proteins ameliorates this toxic process in worm and human cell models for diseases. Whether SERF modifies amyloid pathology in mammalian brain, however, has remained unknown. Here, we generated conditional Serf2 knockout mice and found that full-body deletion of Serf2 delayed embryonic development, causing premature birth and perinatal lethality. Brain-specific Serf2 knockout mice, on the other hand, were viable, and showed no major behavioral or cognitive abnormalities. In a mouse model for amyloid-β aggregation, brain depletion of Serf2 altered the binding of structure-specific amyloid dyes, previously used to distinguish amyloid polymorphisms in the human brain. These results suggest that Serf2 depletion changed the structure of amyloid deposits, which was further supported by scanning transmission electron microscopy, but further study will be required to confirm this observation. Altogether, our data reveal the pleiotropic functions of SERF2 in embryonic development and in the brain and support the existence of modifying factors of amyloid deposition in mammalian brain, which offer possibilities for polymorphism-based interventions

Alzheimer’s disease and the β-amyloid peptide: Aβ conformers and mechanisms of spreading

Alzheimer’ s disease (AD) is a progressive neurodegenerative condition characterized by an amnestic memory impairment and behavioral changes. According to the amyloid cascade hypothesis postulated by Hardy in 1992, the accumulation and subsequent aggregation of the β-amyloid protein (Aβ) is the initial trigger of the disease. Overwhelming research has recently shown that the Aβ peptide shares similarities with the prion protein - a protein that once aggregated transmits its misfolded shape onto physiological forms of the same protein and thereby becomes self-propagating. The overall objective of this doctoral dissertation was to further investigate the prion-like characteristics of the Aβ protein. Three studies were performed analyzing different aggregate conformations and the resistance of plaque polymorphism towards degradation as well as the characterization of new mouse models to study the spatiotemporal progression of protein aggregation throughout the brain. The first study analyzed the structural features of amyloid plaque cores in postmortem tissue of a heterogenous cohort of more than 40 AD patients displaying both sporadic as well as familiar forms of the disease. Evaluation of the structural plaque core features was performed using a combination of conformational sensitive amyloid dyes. The results indicate that amyloid conformations vary among patient subgroups and reveal important insight into the heterogeneity of plaque morphologies in postmortem end stage AD brain. The second study investigated the durability of plaque polymorphism in transgenic mouse brain. It emphasizes the prolonged durability of plaque polymorphism over serial transmission and the importance of the Aβ42 peptide in the propagation of strain-like morphologies. In the third study, two new mouse models overexpressing murine Aβ were characterized and their ability to study the propagation of Aβ seeds within axonally connected areas was evaluated

Pretargeted Imaging beyond the Blood-Brain Barrier-Utopia or Feasible?

Pretargeting is a promising nuclear imaging technique that allows for the usage of antibodies (Abs) with enhanced imaging contrast and reduced patient radiation burden. It is based on bioorthogonal chemistry with the tetrazine ligation-a reaction between trans-cyclooctenes (TCOs) and tetrazines (Tzs)-currently being the most popular reaction due to its high selectivity and reactivity. As Abs can be designed to bind specifically to currently 'undruggable' targets such as protein isoforms or oligomers, which play a crucial role in neurodegenerative diseases, pretargeted imaging beyond the BBB is highly sought after, but has not been achieved yet. A challenge in this respect is that large molecules such as Abs show poor brain uptake. Uptake can be increased by receptor mediated transcytosis; however, it is largely unknown if the achieved brain concentrations are sufficient for pretargeted imaging. In this study, we investigated whether the required concentrations are feasible to reach. As a model Ab, we used the bispecific anti-amyloid beta (A beta) anti-transferrin receptor (TfR) Ab 3D6scFv8D3 and conjugated it to a different amount of TCOs per Ab and tested different concentrations in vitro. With this model in hand, we estimated the minimum required TCO concentration to achieve a suitable contrast between the high and low binding regions. The estimation was carried out using pretargeted autoradiography on brain sections of an Alzheimer's disease mouse model. Biodistribution studies in wild-type (WT) mice were used to correlate how different TCO/Ab ratios alter the brain uptake. Pretargeted autoradiography showed that increasing the number of TCOs as well as increasing the TCO-Ab concentration increased the imaging contrast. A minimum brain concentration of TCOs for pretargeting purposes was determined to be 10.7 pmol/g in vitro. Biodistribution studies in WT mice showed a brain uptake of 1.1% ID/g using TCO-3D6scFv8D3 with 6.8 TCO/Ab. According to our estimations using the optimal parameters, pretargeted imaging beyond the BBB is not a utopia. Necessary brain TCO concentrations can be reached and are in the same order of magnitude as required to achieve sufficient contrast. This work gives a first estimate that pretargeted imaging is indeed possible with antibodies. This could allow the imaging of currently 'undruggable' targets and therefore be crucial to monitor (e.g., therapies for intractable neurodegenerative diseases)

Pretargeted Imaging Beyond the Blood-Brain-Barrier

Pretargeting is a powerful nuclear imaging strategy to achieve enhanced imaging contrast for nanomedicines. It reduces the radiation burden to healthy tissue. Pretargeting is based on bioorthogonal chemistry. The most attractive reaction for this purpose is currently the tetrazine ligation, which occurs between trans-cyclooctene (TCO) tags and tetrazines (Tzs). Pretargeted imaging beyond the blood-brain-barrier (BBB) has not been reported thus far. In this study, we developed Tz imaging agents that are capable to ligate in vivo to targets beyond the BBB. We chose to develop 18F-labeled Tzs as they can be applied to positron emission tomography (PET) - the most powerful molecular imaging technology. Fluorine-18 is an ideal radionuclide for PET due to its almost ideal decay properties. Fluorine-18 also allows - as a non-metal radionuclide - to develop Tzs with physicochemical properties enabling passive brain diffusion. In order to develop these imaging agents, we applied a rational drug design approach. This approach was based on estimated and experimental determined parameters such as the BBB score, pretargeted autoradiography contrast, in vivo input and washout curves as well as on metabolism studies. From initially 18 developed structures, five Tzs were selected to be tested on their in vivo click performance. Whereas all selected structures clicked in vivo into the brain, [18F]18 displayed the most favorable characteristics with respect to brain pretargeting. [18F]18 is our lead compound for future pretargeted imaging studies based on BBB-penetrant monoclonal antibodies. Pretargeting beyond the BBB will allow us to image targets beyond the BBB that are currently not imageable. For example, soluble protein isoforms could be imaged. These proteins are valuable drug targets for several neurodegenerative diseases and can currently not be imaged. Imaging would allow for diagnosis of these diseases, identifying responders from non-responders or to monitor treatment. Consequently, imaging will provide valuable information to accelerate drug development and greatly benefit patient care