Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Abstract

Use of the in vivo alpha-synuclein preformed fibril (α-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson's disease synucleinopathy and nigrostriatal degeneration. The standardization of α-syn PFF generation and in vivo application is critical in order to ensure consistent, robust α-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric α-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of α-syn PFFs into rats or mice. Starting with monomeric α-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed α-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated α-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively. Video LinkThe video component of this article can be found at https://www.jove.com/video/59758/ 1,7,8,9 . Inclusions share similar properties to Lewy bodies: containing α-syn phosphorylated at serine 129 (pSyn), ubiquitin, and p62; possess amyloid quaternary structures as shown with positive thioflavin staining; and are resistant to proteinase K digestion 1,3,5,7,8,9,10,11,12 . PFF exposure leads to α-syn inclusion formation in primary and some immortalized neurons in culture, as well as mice, rats, and non-human primates in vivo 1,2,3,4,5,6,7,8,9,13 . It is important to note that PFFs will not lead to α-syn inclusion formation in all cell culture models and some cultured neurons will seed better than others.Another important feature of the in vivo α-syn PFF model is the distinct sequential pathological phases that emerge over several months. In rodents, following intrastriatal injection, α-syn inclusion formation generally peaks within the SNpc and many cortical regions within 1-2 months. This aggregation peak is followed by nigrostriatal degeneration ≈2-4 months later 1,3,5 . These distinct pathological stages provide researchers the platform with which to study and develop strategies that 1) decrease α-syn aggregation, 2) clear already formed α-syn inclusions, and/or 3) prevent subsequent neurodegeneration. The PFF mod...

Modeling Parkinson’s Disease With the Alpha-Synuclein Protein

“…PFFs are generated in vitro from recombinant a-Syn monomers. Subsequently, the aggregation of monomers into fibrils is induced and the fibrils are sonicated to generate short fibrils w h i c h a f t e r i n j e c t i o n , t r i g g e r t h e a g g r e g a t i o n , hyperphosphorylation and ubiquitination of endogenous a-Syn (Patterson et al, 2019). Intracerebral injection of a-Syn PFFs has been extended to rodents, both mice (Luk et al, 2012a;Masuda-Suzukake et al, 2013;Karampetsou et al, 2017;Milanese et al, 2018;Okuzumi et al, 2018;Patterson et al, 2019) and rats (Paumier et al, 2015;Harms et al, 2017;Thakur et al, 2017;Duffy et al, 2018b;Durante et al, 2019) and non-human primates (Shimozawa et al, 2017;Chu et al, 2019).…”

“…Subsequently, the aggregation of monomers into fibrils is induced and the fibrils are sonicated to generate short fibrils w h i c h a f t e r i n j e c t i o n , t r i g g e r t h e a g g r e g a t i o n , hyperphosphorylation and ubiquitination of endogenous a-Syn (Patterson et al, 2019). Intracerebral injection of a-Syn PFFs has been extended to rodents, both mice (Luk et al, 2012a;Masuda-Suzukake et al, 2013;Karampetsou et al, 2017;Milanese et al, 2018;Okuzumi et al, 2018;Patterson et al, 2019) and rats (Paumier et al, 2015;Harms et al, 2017;Thakur et al, 2017;Duffy et al, 2018b;Durante et al, 2019) and non-human primates (Shimozawa et al, 2017;Chu et al, 2019). PFFs have been mainly injected in the striatum (Luk et al, 2012a;Paumier et al, 2015;Karampetsou et al, 2017;Shimozawa et al, 2017;Duffy et al, 2018b;Milanese et al, 2018;Okuzumi et al, 2018;Chu et al, 2019;Durante et al, 2019;Patterson et al, 2019), the substantia nigra (Masuda-Suzukake et al, 2013;Harms et al, 2017) and the cortex (Luk et al, 2012b).…”

“…Similarly, the inoculation of purified brain extracts from PD patients or transgenic mice containing pathological a-Syn has been performed in rodents and non-human primates (Luk et al, 2012b;Recasens et al, 2014). Most of these models have succeeded in producing the accumulation and aggregation of phosphorylated a-Syn (pa-Syn), the progressive degeneration of dopaminergic neurons, a significant reduction in striatal dopamine levels, neuroinflammation, and the development of motor deficits (Recasens et al, 2014;Paumier et al, 2015;Karampetsou et al, 2017;Shimozawa et al, 2017;Patterson et al, 2019). It is well described that intracerebral inoculation of PFFs leads to the formation of pa-Syn-immunoreactive aggregates that are distributed both in the soma and neuronal processes.…”

Developmental exposure to the organochlorine pesticide dieldrin causes male-specific exacerbation of α-synuclein-preformed fibril-induced toxicity and motor deficits

“…Fibrils were generated using wild-type, full-length, recombinant mouse α-syn monomers, as previously described. 38,44,[48][49][50][51] Quality control was performed on full length fibrils to confirm fibril formation (by transmission electron microscopy), amyloid content (by thioflavin T assay), a shift to insoluble species compared to monomers (by sedimentation assay), and low bacterial contamination (<0.5 endotoxin units mg −1 of total protein via a Limulus amebocyte lysate assay). On the day of surgery, PFFs were thawed to room temperature, diluted to 2 μg/μl in Dulbecco's phosphate buffered saline (PBS) (Gibco), and sonicated at room temperature using an ultrasonic homogenizer (Q125 Sonicator; Qsonica, Newtown, CT. Sonication was performed for 1 minute, using 1 second pulses with 1 second between pulses and amplitude set at 30%.…”

A simple, versatile and robust centrifugation-based filtration protocol for the isolation and quantification of α-synuclein monomers, oligomers and fibrils: towards improving experimental reproducibility in α-synuclein research

“…In response to the high variability in aSyn fibril preparations and concerns about reproducibility, there is an urgent need to share the best practices, to develop reproducible protocols and methods for generating aSyn fibrils, and to follow and simple methods for quality control, characterization and validation of such preparations (Polinski et al 2018;Patterson et al 2019).…”