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Frieg, B.

Publications and source records attributed to Frieg, B..

9 recordsLinked to original sources

Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

The existence of -synuclein conformational strains provides a potential explanation for the clinical and pathological differences among synucleinopathies such as Parkinsons disease and multiple system atrophy. However, how distinct -synuclein strains are formed in vivo remains unknown. Here, we examined whether unique strains of self-propagating -synuclein aggregates can arise within a consistent molecular environment. Unexpectedly, we observed conformational heterogeneity between individual preparations of -synuclein pre-formed fibrils (PFFs) generated by polymerizing recombinant wild-type or A53T-mutant human -synuclein under identical conditions. Moreover, we found that -synuclein aggregates formed spontaneously in the brains of a transgenic synucleinopathy mouse model were conformationally diverse, leading to the identification of three distinct disease subtypes. Propagation of putative PFF- and brain-derived -synuclein strains in mice initiated several distinct synucleinopathies, characterized by differences in disease onset times, cerebral -synuclein deposition patterns, and the conformational attributes of -synuclein aggregates. The conformational diversity of -synuclein aggregates across PFF preparations and between the brains of individual transgenic mice demonstrates that -synuclein can spontaneously form multiple self-propagating strains within an identical environment both in vitro and in vivo. This suggests that stochastic misfolding into distinct aggregate structures drives the emergence of -synuclein strains and implies that the intrinsic variability of common synucleinopathy research tools must be considered when designing and interpreting experiments.

neuroscience↗

The clinical drug candidate anle138b binds predominantly to the central cavity in lipidic Aβ fibrils and modulates fibril formation.

Alzheimers disease is a specific neurodegenerative disorder, distinct from normal aging, with a growing unmet medical need. It is characterized by the accumulation of amyloid plaques in the brain, primarily consisting of amyloid beta (A{beta}) fibrils. Therapeutic antibodies can slow down the disease, but are associated with potential severe side effects, motivating the development of small molecules to halt disease progression. This study investigates the interaction between the clinical drug candidate small molecule anle138b and lipidic A{beta}40 fibrils of type 1 (L1). L1 fibrils were previously shown to closely resemble fibrils from Alzheimers patients18. Using high-resolution structural biology techniques, including cryo-electron microscopy (cryo-EM), nuclear magnetic resonance (NMR) spectroscopy enhanced by dynamic nuclear polarization (DNP), and molecular dynamics (MD) simulations, we find that anle138b selectively binds to a cavity within the fibril. This structural insight provides a deeper understanding of a potential drug-binding mechanism at the atomic level and may inform the development of novel therapies and diagnostic approaches. In addition, anle138b reduces fibril formation in the presence of lipids by approximately 75%. This may suggest a mechanistic connection to its previously reported activity in animal models of Alzheimers disease15.

biochemistry↗

Structural insight into binding of novel PET tracer MODAG-005 to lipidic ????-Synuclein fibrils

Accumulation of -synuclein (Syn) aggregates in the human brain is the major hallmark of synucleinopathies such as Parkinsons Disease, Multiple System Atrophy, and Dementia with Lewy bodies. Positron Emission Tomography (PET) plays a vital role in diagnosing these diseases and monitoring their progression by enabling the non-invasive and sensitive detection of Syn aggregates in the brain. However, developing PET tracers with specific target binding, as well as identifying the binding site and complex structure present significant challenges. Here, we investigated the interaction between lipidic Syn aggregates and MODAG-005, a new Syn PET tracer candidate, using nuclear magnetic resonance spectroscopy, cryogenic electron microscopy and molecular dynamics (MD) simulations. Two binding sites of MODAG-005 were found, one on the surface and one in a tubular cavity of the fibril, of which the occupancies were found to depend on the preparation protocols. The cavity binding site is thermodynamically more stable than the external binding site and is the only site occupied when MODAG-005 is applied with liposome as carriers to the aggregates. This is corroborated by MD simulations in which stable interactions between MODAG-005 and glycine containing backbone motifs of the tubular cavity of the aggregates are observed.

biochemistry↗

Cryo-EM structures of lipidic fibrils of amyloid-β (1-40)

Alzheimers disease (AD) is a progressive and incurable neurodegenerative disease characterized by the extracellular deposition of amyloid plaques. Investigation into the composition of these plaques revealed a high amount of amyloid-{beta} (A{beta}) fibrils and a high concentration of lipids, suggesting that fibril-lipid interactions may also be relevant for the pathogenesis of AD. Therefore, we grew A{beta}40 fibrils in the presence of lipid vesicles and determined their structure by cryo-electron microscopy (cryo-EM) to high resolution. The fold of the major polymorph is similar to the structure of brain-seeded fibrils reported previously. The majority of the lipids are bound to the fibrils as we show by cryo-EM and NMR spectroscopy. This apparent lipid extraction from vesicles observed here in vitro provides structural insights into potentially disease-relevant fibril-lipid interactions.

molecular biology↗

Cryo-EM Structures of Amyloid-β Fibrils from Alzheimer's Disease Mouse Models

The development of novel drugs for Alzheimers disease has proven difficult, with a high failure rate in clinical trials. Typically, transgenic mice displaying amyloid-{beta} peptide brain pathology are used to develop therapeutic options and to test their efficacy in preclinical studies. However, the properties of A{beta} in such mice have not been systematically compared to A{beta} from the patient brains. Here, we determined the structures of nine ex vivo A{beta} fibrils from six different mouse models by cryo-EM. We found novel A{beta} fibril structures in the APP/PS1, ARTE10, and tg-SwDI models, whereas the human familial type II fibril fold was found in the ARTE10, tg-APPSwe, and APP23 models. The tg-APPArcSwe mice showed an A{beta} fibril whose structure resembles the human sporadic type I fibril. These structural elucidations are key to the selection of adequate mouse models for the development of novel plaque-targeting therapeutics and PET imaging tracers. One Sentence SummaryCryo-EM structures of A{beta} fibrils extracted from brains of mouse models used for Alzheimers disease preclinical research are presented.

neuroscience↗

The 3D structure of lipidic fibrils of α-synuclein

-synuclein (Syn) is abundant in neurons, but its misfolding and abnormal fibrillization are associated with severe neurodegenerative diseases. Although interactions between Syn and phospholipid membranes are relevant during Syn fibril assembly, insights into the interactions of Syn fibrils with phospholipids have remained elusive. Here, we present six novel polymorphic atomic structures of Syn fibrils aggregated in the presence of phospholipids. The structures reveal that phospholipids favor a novel protofilament fold, mediate an unusual arrangement of protofilaments, and fill the central cavities between the protofilaments. These findings provide a structural rationale for fibril-induced lipid extraction, a mechanism likely to be involved in the development of -synucleinopathies.

molecular biology↗

Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.95 {+/-} 1.28 M. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

biophysics↗

α-Synuclein polymorphism determines oligodendroglial dysfunction

Synucleinopathies, such as Parkinsons disease (PD) and Multiple System Atrophy (MSA) are progressive and unremitting neurological diseases. For both PD and MSA, -synuclein fibril inclusions inside brain cells are neuropathological hallmarks. In addition, amplification of -synuclein fibrils from body fluids is a potential biomarker distinguishing PD from MSA. However, little is known about the structure of -synuclein fibrils amplified from human samples and its connection to -synuclein fibril structure in the human brain. Here we amplified -synuclein fibrils from PD and MSA brain tissue, characterized its seeding potential in oligodendroglia, and determined the 3D structures by cryo-electron microscopy. We show that the -synuclein fibrils from a MSA patient are more potent in recruiting the endogenous -synuclein and evoking a redistribution of TPPP/p25 protein in mouse primary oligoden-droglial cultures compared to those amplified from a PD patient. Cryo-electron microscopy shows that the PD- and MSA-amplified -synuclein fibrils share a similar protofilament fold but differ in their inter-protofilament interface. The structures of the brain-tissue amplified -synuclein fibrils are also similar to other in vitro and ex vivo -synuclein fibrils. Together with published data, our results suggest that Syn fibrils differ between PD and MSA in their quaternary arrangement and could further vary between different forms of PD and MSA.

neuroscience↗

Functional and structural characterization of interactions between opposite subunits in HCN pacemaker channels

Hyperpolarization-activated and cyclic nucleotide (HCN) modulated channels are tetrameric cation channels. In each of the four subunits, the intracellular cyclic nucleotide-binding domain (CNBD) is coupled to the transmembrane domain via a helical structure, the C-linker. High-resolution channel structures suggest that the C-linker enables functionally relevant interactions with the opposite subunit, which might be critical for coupling the conformational changes in the CNBD to the channel pore. We combined mutagenesis, patch-clamp technique, confocal patch-clamp fluorometry, and molecular dynamics simulations to show that residue K464 of the C-linker is essential for stabilizing the closed state of the mHCN2 channel by forming interactions with the opposite subunit. MD simulations revealed that both cAMP and K464E induce a rotation of the intracellular domain relative to the channel pore, weakening the autoinhibitory effect of the unoccupied CL-CNBD region. The adopted poses are in excellent agreement with structural results.

biophysics↗