Axonal transport and secretion of fibrillar forms of α-synuclein, Aβ42 peptide and HTTExon 1

Abstract

Accruing evidence suggests that prion-like behavior of fibrillar forms of α-synuclein, β-amyloid peptide and mutant huntingtin are responsible for the spread of the lesions that characterize Parkinson disease, Alzheimer disease and Huntington disease, respectively. It is unknown whether these distinct protein assemblies are transported within and between neurons by similar or distinct mechanisms. It is also unclear if neuronal death or injury is required for neuron-to-neuron transfer. To address these questions, we used mouse primary cortical neurons grown in microfluidic devices to measure the amounts of α-synuclein, Aβ42 and HTTExon1 fibrils transported by axons in both directions (anterograde and retrograde), as well as to examine the mechanism of their release from axons after anterograde transport. We observed that the three fibrils were transported in both anterograde and retrograde directions but with strikingly different efficiencies. The amount of Aβ42 fibrils transported was ten times higher than that of the other two fibrils. HTTExon1 was efficiently transported in the retrograde direction but only marginally in the anterograde direction. Finally, using neurons from two distinct mutant mouse strains whose axons are highly resistant to neurodegeneration (WldS and Sarm1−/−), we found that the three different fibrils were secreted by axons after anterograde transport, in the absence of axonal lysis, indicating that trans-neuronal spread can occur in intact healthy neurons. In summary, fibrils of α-synuclein, Aβ42 and HTTExon1 are all transported in axons but in directions and amounts that are specific of each fibril. After anterograde transport, the three fibrils were secreted in the medium in the absence of axon lysis. Continuous secretion could play an important role in the spread of pathology between neurons but may be amenable to pharmacological intervention.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1538-0) contains supplementary material, which is available to authorized users.

Selective imaging of internalized proteopathic α-synuclein seeds in primary neurons reveals mechanistic insight into transmission of synucleinopathies

“…), indicating either photophysical quenching of GFP fluorescence (at pH Ͻ6.0) (24) in progressively acidifying compartments or the beginning of proteolytic lysosomal degradation of mSyn-GFP. This observation is consistent with previous studies using traditional biochemical approaches or microfluidic chamber imaging systems, (15,25) with additional advantages of quantitative whole-culture imaging with both spatial and temporal resolution of intracellular puncta. By 2 days pTd, few objects are detectable, indicating either robust degradation or pH-dependent fluorescence quenching of intracellular mSyn-GFP seeds.…”

“…Brief perturbation of lysosomes leads to an increase in detectable mSyn-GFP puncta 24 h post-transduction ␣-Syn pffs have been shown to at least partially localize to lysosomes in the hours following transduction in model cell lines and neurons (14,15,30). In this context, our results indicate that the majority of the intracellular pffs are localized in the endocytic pathway shortly after transduction (Fig.…”

“…Our studies indicate that proteopathic seeds remain in lysosomes for days, providing an extended window for direct cell-to-cell transfer if such mechanisms occur in tissue. Direct release of exogenous pffs from neurons has been demonstrated following treatment in microfluidic culture systems (15). Anterograde and retrograde axonal trafficking of endocytosed ␣-syn pffs has been observed with the kinetics of slow component b of axonal transport (14).…”

“…Despite strong evidence gleaned from these models implicating a causal relationship between exposure of neurons to insoluble ␣-syn aggregates and the subsequent development of inclusions bearing pathological hallmarks of synucleinopathies, the specific processes underlying cell-to-cell transmission of proteopathic seeds and resulting intracellular events leading to the development of pathological aggregates remain poorly understood. Whereas recent advances using flow cytometry or microfluidic culture chambers have been employed to study internalization and intracellular trafficking of amyloidogenic species, respectively, there exists a dearth of quantitative methods capable of unambiguous and spatially resolved discernment of intracellular from extracellular misfolded proteopathic species in a primary neuronal culture (11)(12)(13)(14)(15). As such, the study of the uptake and intracellular processing of proteopathic ␣-syn seeds, as well as their subsequent interaction with endogenous ␣-syn in the seeding of intracellular aggregates, has been impeded.…”

The Effect of Fragmented Pathogenic α-Synuclein Seeds on Prion-like Propagation

“…It is plausible that smaller structures would be more easily incorporated into cells by endocytosis. There is evidence that ␣-syn fibrous structures interact with cellular membranes, can move between cells, and are secreted from axons (38,39). Additionally, Pieri et al (40) reported that ␣-syn fibrils are more cytotoxic than oligomeric species.…”