Kinetics of α-synuclein prions preceding neuropathological inclusions in multiple system atrophy

Abstract

Multiple system atrophy (MSA), a progressive neurodegenerative disease characterized by autonomic dysfunction and motor impairment, is caused by the self-templated misfolding of the protein α-synuclein. With no treatment currently available, we sought to characterize the spread of α-synuclein in a transgenic mouse model of MSA prion propagation to support drug discovery programs for synucleinopathies. Brain homogenates from MSA patient samples or mouse-passaged MSA were inoculated either by standard freehand injection or stereotactically into TgM83 +/mice, which express human α-synuclein with the A53T mutation. Following disease onset, brains from the mice were tested for biologically active α-synuclein prions using a cell-based assay and examined for α-synuclein neuropathology. Inoculation studies using homogenates prepared from brain regions lacking detectable α-synuclein neuropathology transmitted neurological disease to mice. Terminal animals contained similar concentrations of α-synuclein prions; however, a time-course study where mice were terminated every five days through disease progression revealed that the kinetics of α-synuclein prion replication in the mice were variable. Stereotactic inoculation into the thalamus reduced variability in disease onset in the mice, although incubation times were consistent with standard inoculations. Using human samples with and without neuropathological lesions, we observed that α-synuclein prion formation precedes neuropathology in the brain, suggesting that disease in patients is not limited to brain regions containing neuropathological lesions.

Similar neuronal imprint and absence of cross-seeded partner fibrils in α-synuclein aggregates from MSA and Parkinson’s disease brains

“…In pafticular, using amygdala or putamen extracts for focusing on LBs or GCIs protein compositions, respectively, should help refining the present lists and identifying less represented candidates. Also, Sarkospin extraction and purification of synucleinopathy subject brain tissue yet devoid of any inclusion 36 , and the insoluble proteomes’ comparison to the ones we defined here is of high importance for understanding the formation and composition of IBs. The Sarkospin procedure we developed here allows testing the seeding ability 37 or the pathogenicity of aggregated proteins 38 , an asset to understand the role of the inclusions in the disease process.…”

Is Multiple System Atrophy a Prion-like Disorder?

“…Hemizygous TgM83 mice do not develop α-synucleinopathy spontaneously, allowing them to detect disease transmission following extended incubation periods of more than one year post-inoculation. Inoculation studies using homogenates from MSA brain regions lacking detectable αSyn pathology also transmitted neurological lesions to mice indicating that αSyn prion formation precedes neuropathology in the brain, suggesting that the lesions are not limited to affected brain regions [ 25 ]. This clearly indicates that αSyn fibrils, like prions, can neuroinvade the CNS after a single oral or intravenous challenge and cause neuropathology.…”

“…The pathogenic cascade leading to αSyn aggregation and the neurodegeneration of this oligodendroglioneuronal proteinopathy is poorly understood [ 10 , 11 ], but recent studies elucidated the early cellular dysfunction in MSA indicating both increased susceptibility to oxidative stress and disease-related translocation of αSyn to the cell nucleus [ 12 ], while others demonstrated mislocalization of myelin-associated oligodendrocyte basic protein (MOBP) and huntingtin protein 1 (HIP1) due to DNA methylation interacting with αSyn in the oligodendrocyte as a pathogenic way of MSA [ 13 ]. Converging evidence suggests a “prion-like” spreading of misfolded αSyn “strains” as a pathogenic key event [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ], while others suggested that MSA is a prion disease [ 26 , 27 , 28 , 29 ]. The prion hypothesis of human synucleinopathies and the question of whether αSyn is a prion or prion-like are a matter of continuous discussion [ 15 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ].…”

Potential of Microfluidics and Lab-on-Chip Platforms to Improve Understanding of “prion-like” Protein Assembly and Behavior

“…A second strategy is amplification of the pathogenic amyloid in the presence of its monomeric counterparts using biochemical reactions, such as the protein misfolding cyclic amplification (PMCA) method (Saborio et al, 2001;Soto et al, 2002), real-time quaking-induced conversion (RT-QuIC) assay (Wilham et al, 2010;Sano et al, 2018), and ASA assay (Colby et al, 2007). These methods have evolved and have been adapted for the reliable detection of the presence and aggregative stage of various amyloids (i.e., Orru et al, 2017;Saijo et al, 2019) in different biological samples (i.e., Fairfoul et al, 2016;Haley et al, 2016;Bongianni et al, 2017).…”

“…Indeed, cell-spread of amyloid seeds can act as a self-propagating template disrupting cell viability and leading to both the death of recipient cells and the progression of the neurodegenerative disorder (Ashe and Aguzzi, 2013;Aguzzi and Lakkaraju, 2016;Collinge, 2016;Holmes and Diamond, 2017). However, some criticisms appeared with this terminology [i.e., for tau (Polanco and Gotz, 2015), β-amyloid (Watts and Prusiner, 2018), and MSA-derived α-synuclein (Woerman et al, 2020)]. We suggest to the reader interested in this topic examining the reports of Castilla's and Requena's laboratories (i.e., Castilla and Requena, 2015;Erana et al, 2017) and the recent review of Wells et al (2019).…”