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 (Wld(S) 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.

Keywords: Axonal transport; Aβ42; HTTExon1; Secretion; α-Synuclein.

Fig. 1

Characterization of α-syn, Aβ42 and HTTExon1 fibrils. a MALDI-TOF mass spectra of, from left to right, Alexa-555 labeled α-synuclein (MH+ 14458.7), Alexa-555 labeled Aβ42 (MH+ 4644.7) and Alexa-555 labeled HTTExon1 (MH + 14,373.8). The number of Alexa 555 molecules covalently bound is indicated. b Electron micrographs of the fibrillar forms of α-synuclein, Aβ42 and HTTExon1 used throughout this study. The scale bar represents 200 nm. c Calibration curves for Alexa-555 labeled α-synuclein, Alexa-555 labeled Aβ42 and Alexa-555 labeled HTTExon1 within the concentration rang of 1 pM–20 µM

Fig. 2

Axonal transport of fibrils in microfluidic devices. Quantity (p.moles) of fibrillar α-syn, Aβ42 and HTTExon1 transported by axons in 24 h in the anterograde and retrograde directions. Primary embryonic cortical neurons were grown in microfluidic devices. Fibrils (1 µM monomer) were added to the soma or axon compartments. Fluorescence was measured in the opposite compartment after 24 h of incubation. a–d Anterograde transport. e–h Retrograde transport. a, e Schematics of the experimental design. Fibrils are depicted as coin piles. b–h Ordinates give the number of picomoles of fibrils transported per microfluidic device in 24 h. Note that the ordinate values are different for the three kinds of fibrils but that, for a given fibril, they are the same for anterograde and retrograde transport. M fibrils spontaneously released in the medium. L fibrils released after lysis by detergents of the axons or soma for, respectively the anterograde and the retrograde transport. n number of microfluidic devices used for the measurement

Fig. 3

Internalization of fibrils by neuron soma. Primary embryonic cortical neurons were grown in 8 well LabTek slides at low density. Alexa488-labeled fibrils (1 µM monomer) were added and incubated for 1, 3, 5, and 7 h. At each time point the fibrils were removed; the cells were washed with PBS; 0.1 % Trypan Blue in PBS was added and pictures (phase contrast and fluorescence) of tiled fields of the cultures were recorded. a Quantification of the  % of neurons that internalized fibrils was determined for a minimum of 150 neurons. Four independent experiments were performed. b, c Representative bright field and fluorescent images of primary cortical neurons 5 h after exposure to α-syn fibrils or control medium

Fig. 4

Transport of the fibrillar forms of Ure2p and Sup35p yeast prions by mouse neurons. The experiment was performed as for α-syn, Aβ42 and HTTExon1 fibrils (Fig. 2). Ure2p fibrils were obtained as described [49]. Sup35p fibrils were obtained as described [25]

Fig. 5

Quantity of α-syn, Aβ42 and HTTExon1 released in the medium in 24 h of anterograde transport by axons from C57BL/6wt (WT), C57BL/6Wld^S (Wlds/s) and C57BL/6Sarm1^−/− (Sarm1^−/−) mice. Primary embryonic cortical neurons from mice of the 3 genotypes were grown in microfluidic devices. Fibrils were added to the soma compartments. The fluorescence of the medium in the axon compartments was measured after 24 h of incubation. n number of microfluidic devices used for the measurement. n.s Not statistically significant. d, e Staining with the SMI-31 and SMI-32 monoclonal antibodies of the soma and axon of Wld^S neurons in microfluidic devices after 24 h of transport of α-syn fibrils. Similar results were obtained with WT and Sarm1^−/− neurons and with Aβ42 and HTTExon1 fibrils. SMI-32 did not detect un-phosphorylated neurofilaments, a sign of axonal damage. A positive control for the SMI-32 antibody was obtained by treating the soma with staurosporin to induce apoptosis (not shown)