The inherent flexibility of intrinsically disordered proteins (IDPs) makes it difficult to interpret experimental data using structural models. On the other hand, molecular dynamics simulations of IDPs often suffer from force-field inaccuracies, and long simulation times or enhanced sampling methods are needed to obtain converged ensembles. Here, we apply metainference and Bayesian/Maximum Entropy reweighting approaches to integrate prior knowledge of the system with experimental data, while also dealing with various sources of errors and the inherent conformational heterogeneity of IDPs. We have measured new SAXS data on the protein α-synuclein, and integrate this with simulations performed using different force fields. We find that if the force field gives rise to ensembles that are much more compact than what is implied by the SAXS data it is difficult to recover a reasonable ensemble. On the other hand, we show that when the simulated ensemble is reasonable, we can obtain an ensemble that is consistent with the SAXS data, but also with NMR diffusion and paramagnetic relaxation enhancement data.

Background: Parkinson’s disease (PD) is a neurodegenerative disorder associated with insoluble pathological aggregates of the protein α-synuclein. While PD is diagnosed by motor symptoms putatively due to aggregated α-synuclein-mediated damage to substantia nigra (SN) neurons, up to a decade before motor symptom appearance, patients exhibit sleep disorders (SDs). Therefore, we hypothesized that α-synuclein, which can be present in monomeric, fibril, and other forms, has deleterious cellular actions on sleep-control nuclei. Objective: We investigated whether native monomer and fibril forms of α-synuclein have effects on neuronal function, calcium dynamics, and cell-death-induction in two sleep-controlling nuclei: the laterodorsal tegmentum (LDT), and the pedunculopontine tegmentum (PPT), as well as the motor-controlling SN. Methods: Size exclusion chromatography, Thioflavin T emission, and circular dichroism spectroscopy were used to isolate structurally defined forms of recombinant, human α-synuclein. Neuronal and viability effects of characterized monomeric and fibril forms of α-synuclein were determined on LDT, PPT, and SN neurons using electrophysiology, calcium imaging, and neurotoxicity assays. Results: In LDT and PPT, both forms of α-synuclein induced excitation and increased calcium, and the monomeric form heightened putatively excitotoxic neuronal death, whereas, in the SN we saw inhibition, decreased intracellular calcium, and monomeric α-synuclein was not associated with heightened cell death. Conclusion: Nucleus-specific differential effects suggest mechanistic underpinnings of SDs’ prodromal appearance in PD. While speculative, we hypothesize that the monomeric form of α-synuclein compromises functionality of sleep-control neurons, leading to the presence of SDs decades prior to motor dysfunction.

Parkinson's disease, Multiple System Atrophy, and Lewy Body Dementia are incurable diseases called αsynucleinopathies as they are mechanistically linked to the protein, α-synuclein (α-syn). α-syn exists in different structural forms which have been linked to clinical disease distinctions. However, sleeping disorders (SDs) are common in the prodromal phase of all three α-synucleinopathies, which suggests that sleep-controlling neurons are affected by multiple forms of α-syn. To determine whether a structureindependent neuronal impact of α-syn exists, we compared and contrasted the cellular effect of three different α-syn forms on neurotransmitter-de ned cells of two sleep-controlling nuclei located in the brainstem: the Laterodorsal Tegmental nucleus and the Pedunculopontine Tegmental nucleus. We utilized size exclusion chromatography, uorescence spectroscopy, circular dichroism spectroscopy and transmission electron microscopy to precisely characterize timepoints in the α-syn aggregation process with three different dominating forms of this protein (monomeric, oligomeric and bril) and we conducted an in-depth investigation of the underlying neuronal mechanism behind cellular effects of the different forms of the protein using electrophysiology, multiple-cell calcium imaging, single-cell calcium imaging and live-location tracking with uorescently-tagged α-syn. Interestingly, α-syn altered membrane currents, enhanced ring, increased intracellular calcium and facilitated cell death in a structure-independent manner in sleep-controlling nuclei, and postsynaptic actions involved a G-protein-mediated mechanism.These data are novel as the sleep-controlling nuclei are the rst brain regions reported to be affected by αsyn in a structure-independent manner. These regions may represent highly important targets for future neuroprotective therapy to modify or delay disease progression in α-synucleinopathies.The most common SDs found in prodromal phases of α-synucleinopathies are: Rapid Eye Movement (REM) Sleep Behavior Disorder, which presents with abnormal motor activity during REM sleep when atonia should be prevalent [11][12][13][14][15][16] and Excessive Daytime Sleepiness which is characterized by low arousal levels during wakefulness [17, 18]. Both SDs involve altered activity of neurons in two brainstem nuclei that play a role in the reticular activating system and REM sleep control: the Laterodorsal Tegmental nucleus (LDT) and the Pedunculopontine Tegmental nucleus (PPT) [19,20]. Localized at the junction of the midbrain and pons in the brainstem, both of these nuclei are cytologically heterogeneous and are composed of acetylcholine-containing, glutamatergic and GABAergic neurons [20][21][22][23] with the cholinergic neurons being the most studied for the role they play in sleep behavior and alert arousal [19,24]. Interestingly, analyses of postmortem brain of patients with α-synucleinopathies showed extensive degeneration in these two brain nuclei of cholinergic neurons [25][26][27], which was associated with deposit...

The inherent flexibility of intrinsically disordered proteins (IDPs) makes it difficult to interpret experimental data using structural models. On the other hand, molecular dynamics simulations of IDPs often suffer from force-field inaccuracies, and long simulations times or enhanced sampling methods are needed to obtain converged ensembles. Here, we apply metainference and Bayesian/Maximum Entropy reweighting approaches to integrate prior knowledge of the system with experimental data, while also dealing with various sources of errors and the inherent conformational heterogeneity of IDPs. We have measured new SAXS data on the protein α-synuclein, and integrate this with simulations performed using different force fields. We find that if the force field gives rise to ensembles that are much more compact than what is implied by the SAXS data it is difficult to recover a reasonable ensemble. On the other hand, we show that when the simulated ensemble is reasonable, we can obtain an ensemble that is consistent with the SAXS data, but also with NMR diffusion and paramagnetic relaxation enhancement data

The intrinsically disordered protein α-synuclein (aSN) is, in its fibrillated state, the main component of Lewy bodies—hallmarks of Parkinson’s disease. Additional Lewy body components include glycosaminoglycans, including heparan sulfate proteoglycans. In humans, heparan sulfate has, in an age-dependent manner, shown increased levels of sulfation. Heparin, a highly sulfated glycosaminoglycan, is a relevant mimic for mature heparan sulfate and has been shown to influence aSN fibrillation. Here, we decompose the underlying properties of the interaction between heparin and aSN and the effect of heparin on fibrillation. Via the isolation of the first 61 residues of aSN, which lacked intrinsic fibrillation propensity, fibrillation could be induced by heparin, and access to the initial steps in fibrillation was possible. Here, structural changes with shifts from disorder via type I β-turns to β-sheets were revealed, correlating with an increase in the aSN1–61/heparin molar ratio. Fluorescence microscopy revealed that heparin and aSN1–61 co-exist in the final fibrils. We conclude that heparin can induce the fibrillation of aSN1–61, through binding to the N-terminal with an affinity that is higher in the truncated form of aSN. It does so by specifically modulating the structure of aSN via the formation of type I β-turn structures likely critical for triggering aSN fibrillation.