α-Synuclein oligomers induce early axonal dysfunction in human iPSC-based models of synucleinopathies

Abstract

α-Synuclein (α-Syn) aggregation, proceeding from oligomers to fibrils, is one central hallmark of neurodegeneration in synucleinopathies. α-Syn oligomers are toxic by triggering neurodegenerative processes in in vitro and in vivo models. However, the precise contribution of α-Syn oligomers to neurite pathology in human neurons and the underlying mechanisms remain unclear. Here, we demonstrate the formation of oligomeric α-Syn intermediates and reduced axonal mitochondrial transport in human neurons derived from induced pluripotent stem cells (iPSC) from a Parkinson's disease patient carrying an α-Syn gene duplication. We further show that increased levels of α-Syn oligomers disrupt axonal integrity in human neurons. We apply an α-Syn oligomerization model by expressing α-Syn oligomer-forming mutants (E46K and E57K) and wild-type α-Syn in human iPSC-derived neurons. Pronounced α-Syn oligomerization led to impaired anterograde axonal transport of mitochondria, which can be restored by the inhibition of α-Syn oligomer formation. Furthermore, α-Syn oligomers were associated with a subcellular relocation of transport-regulating proteins Miro1, KLC1, and Tau as well as reduced ATP levels, underlying axonal transport deficits. Consequently, reduced axonal density and structural synaptic degeneration were observed in human neurons in the presence of high levels of α-Syn oligomers. Together, increased dosage of α-Syn resulting in α-Syn oligomerization causes axonal transport disruption and energy deficits, leading to synapse loss in human neurons. This study identifies α-Syn oligomers as the critical species triggering early axonal dysfunction in synucleinopathies.

Keywords: axonal transport; neurodegeneration; oligomers; synucleinopathies; α-synuclein.

Fig. 1.

Human iPSC-derived PD Dupl neurons reveal increased α-Syn aggregation and impaired axonal transport. (A) IPSC from a PD Dupl patient and Ctrls were differentiated into NPC and neurons and infected with a Mito-DsRed lentivirus. (B) Ctrls and Dupl neurons expressed neuronal marker β3-tubulin (Tubb3, green). (Scale bar: 100 µm.) (C) Elevated α-Syn protein expression in Dupl neurons (three NPC lines: 1.1 and 1.2 from the Dupl1 iPSC clone and 2.1 from the Dupl2 iPSC clone) compared with Ctrls (three iPSC clones from two individuals: 1.1 and 1.2 from Ctrl1 and 2.1 from Ctrl2) by Western blot (WB). (D) Neurons were differentiated in microfluidic devices for 20 d, and transport of Mito-DsRed–positive mitochondria was measured (Experimental setup). Representative kymographs of Ctrl and four Dupl lines: three NPC lines as in C and the additional NPC line 2.2 from the Dupl2 iPSC clone. (E) Less frequently moving mitochondria (mito) in Dupl neuronal lines compared with Ctrls. (F) No differences in movement directionality. (G) Significantly impaired velocities of anterograde and retrograde transport in Dupl neurons. At least 20 neurites per line were tracked. Results from three different iPSC clones from two control individuals are shown collectively as Ctrls in E–G. (H) Neurons were lysed sequentially, and the amount of α-Syn was determined in the detergent-free soluble fraction (S1), Triton X-100–soluble (S2), SDS-soluble (S3), and urea/SDS-soluble (P3) fractions (Left) by WB using an anti–α-Syn Ab Syn1 (Middle: representative Ctrl and Dupl lines). Increase of insoluble α-Syn (P3 fraction) in Dupl neurons (Middle and Right). mol., molecular; P, pellet. (I) Analysis of α-Syn species by sucrose density gradient centrifugation revealed the increased presence of α-Syn aggregation intermediates in Dupl neurons (representative Ctrl and Dupl lines; red boxes). Dot blots within one black box were from the same membrane. Fractions (Fr.) 1–11 and 12–22 of each sample (Ctrl or Dupl) were from different parts of the same membrane (separated by gray lines). Fr. 1–22 correspond to sucrose concentrations gradiently decreasing from 60 to 10%. **P ≤ 0.01; ****P ≤ 0.01.

Fig. 2.

Increased α-Syn oligomerization and impaired anterograde axonal transport in iPSC-derived neurons expressing α-Syn mutants. (A) Control iPSC-derived neurons (20 d of differentiation) were infected with WTS or α-Syn mutants (E46K or E57K) and Mito-DsRed lentivirus. (B) An increase of insoluble α-Syn [Triton X-100–soluble (S2), SDS-soluble (S3), and urea/SDS-soluble (P3) fractions] was observed in E57K and E46K neurons. S1, a detergent-free soluble fraction. (C) Soluble α-Syn multimers were analyzed by size exclusion chromatography. Soluble α-Syn trimers (Tr) were significantly increased in E46K and E57K lysates compared with WTS. D, dimers; HMWO, higher-molecular-weight oligomers; M, monomers; T, tetramers. (D) Clustering (asterisks) and shortening of mitochondria in neurites with α-Syn oligomers (E46K, E57K) compared with elongated mitochondria in control neurons (Mock). (Scale bar: 500 nm.) (E) ATP levels were significantly reduced in neurons expressing the α-Syn mutants E46K and E57K. (F) Representative kymographs. (G) Less frequently moving mitochondria were found in all α-Syn overexpressing neurons and (H) can be restored by NPT100-18A. Positive deltas indicate an increase and negative deltas indicate a decrease of a value in NPT100-18A compared with DMSO (diluent control in all NPT100-18A experiments). (I) Neurons expressing α-Syn mutants (E46K and E57K) revealed a lower incidence of anterograde axonal transport, which was significantly increased by NPT100-18A (J). (K) Decreased maximal (max.) anterograde velocities in WTS, E46K, and E57K neurons, which were improved by NPT100-18A in α-Syn mutant neurons (Left). NPT100-18A reduced slow-moving (0.1 µm/s) and increased middle-speed (0.3 µm/s) anterograde mitochondria frequencies in WTS, E46K, and E57K neurons (Middle). Significant increase of fast-moving (>0.3 µm/s) anterograde mitochondria by NPT100-18A was observed in E46K and E57K neurons (Middle). No changes of retrograde axonal transport were detected independently of NPT100-18A usage (Right). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Data are presented as mean ± SD in H, J, and K and as mean ± SEM in C. Data from two independent iPSC clones from Ctrl1 are presented collectively in H, J, and K.

Fig. 3.

Spatial distribution and expression of kinesin adaptor proteins is profoundly altered in the presence of α-Syn oligomers. (A) Significantly decreased neurite-to-soma ratios of Miro1 were measured in WTS, E46K, and E57K neurons. (B) A significant increase of SNPH was found in E46K neurites. (C) Significantly decreased levels of KLC1 were found in neurites of oligomer-containing E46K and E57K neurons. (D) Significantly increased total Tau levels (green) especially in a perinuclear region (asterisks) and (E) pTau (red) accumulations (asterisks) in E46K and E57K neurons compared with Mock and WTS. Tau and pTau levels were normalized to the number of DAPI-positive viable cells. Data are presented as mean ± SEM. MFI, mean fluorescence intensity. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (Scale bars: 20 µm in A–C and 100 µm in D and E.)

Fig. 4.

Axonal and synaptic compartments are severely affected in the presence of increased levels of α-Syn oligomers. (A) β3-Tubulin (Tubb3) immunostaining reveals (B) impaired axonal fiber densities in neuronal cultures expressing mutant α-Syn (E46K and E57K) compared with Mock. (Scale bar: 100 µm.) Tubb3 fiber densities were normalized to numbers of neurons. *P ≤ 0.05. Data are shown as mean ± SD. (C) Ultrastructural analysis identifies irregular synaptic structures and lack of postsynaptic densities (arrows) in α-Syn mutant neuronal cultures (E46K and E57K). (Scale bars: 1 µm.) (D) A model of axonopathy induced by α-Syn oligomers in human neurons. α-Syn oligomers lead to mitochondrial morphological alterations and to impaired anterograde axonal transport, attributed to reduced abundancy of Miro1 and KLC1 within axons accompanied by increased levels of SNPH and Tau/pTau. These changes result in a profound degeneration of synapses.