We conclude that subtle variations in mAb sequence greatly affect responses towards low-pH incubation and subsequent neutralization, and demonstrate how orthogonal biophysical methods distinguish between reversible and irreversible mAb aggregation pathways at early stages of acidic treatment.
The presence of αSN fibrils indisputably associates with the development of synucleinopathies. However, while certain fibril morphologies have been linked to downstream pathological phenotypes, others appear less harmful, leading to the concept of fibril strains, originally described in relation to prion disease. Indeed, the presence of fibrils does not associate directly with neurotoxicity. Rather, it has been suggested that the toxic compounds are soluble amyloidogenic oligomers, potentially co-existing with fibrils. Here, combining synchrotron radiation circular dichroism, transmission electron microscopy and binding assays on native plasma membrane sheets, we reveal distinct biological and biophysical differences between initial and matured fibrils, transformed within the timespan of few days. Immature fibrils are reservoirs of membrane-binding species, which in response to even gentle experimental changes release into solution in a reversible manner. In contrast, mature fibrils, albeit macroscopically indistinguishable from their less mature counterparts, are structurally robust, shielding the solution from the membrane active soluble species. We thus show that particular biological activity resides transiently with the fibrillating sample, distinct for one, but not the other, spontaneously formed fibril polymorph. These results shed new light on the principles of fibril polymorphism with consequent impact on future design of assays and therapeutic development.
Parkinson's disease is associated with fibril deposition in the diseased brain. Misfolding events of the intrinsically disordered synaptic protein α-synuclein are suggested to lead to the formation of transient oligomeric and cytotoxic species. The etiology of Parkinson's disease is further associated with mitochondrial dysfunction and formation of reactive oxygen species. Oxidative stress causes chemical modification of native α-synuclein, plausibly further influencing misfolding events. Here, we present evidence for the spontaneous formation of covalent di-tyrosine α-synuclein dimers in standard recombinant protein preparations, induced without extrinsic oxidative or nitrative agents. The dimers exhibit no secondary structure but advanced SAXS studies reveal an increased structural definition, resulting in a more hydrophobic micro-environment than the highly disordered monomer. Accordingly, monomers and dimers follow distinct fibrillation pathways.
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...
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