Ca2+ is a key factor in α-synuclein-induced neurotoxicity

Abstract

Aggregation of α-synuclein leads to the formation of oligomeric intermediates that can interact with membranes to form pores. However, it is unknown how this leads to cell toxicity in Parkinson's disease. We investigated the species-specific effects of α-synuclein on Ca(2+) signalling in primary neurons and astrocytes using live neuronal imaging and electrophysiology on artificial membranes. We demonstrate that α-synuclein induces an increase in basal intracellular Ca(2+) in its unfolded monomeric state as well as in its oligomeric state. Electrophysiology of artificial membranes demonstrated that α-synuclein monomers induce irregular ionic currents, whereas α-synuclein oligomers induce rare discrete channel formation events. Despite the ability of monomeric α-synuclein to affect Ca(2+) signalling, it is only the oligomeric form of α-synuclein that induces cell death. Oligomer-induced cell death was abolished by the exclusion of extracellular Ca(2+), which prevented the α-synuclein-induced Ca(2+) dysregulation. The findings of this study confirm that α-synuclein interacts with membranes to affect Ca(2+) signalling in a structure-specific manner and the oligomeric β-sheet-rich α-synuclein species ultimately leads to Ca(2+) dysregulation and Ca(2+)-dependent cell death.

Keywords: Ca2+ signalling; Neuronal death; Parkinson's disease; α-Synuclein.

Fig. 1.

Application of extracellular α-synuclein induces a cytosolic Ca²⁺ signal. (A) A characteristic single-molecule FRET histogram of the 29-h time-point oligomerisation was used in this study. There are both globular non-toxic (18%, E=0.41, blue line), and toxic β-sheet-containing oligomers (82%, E=0.62), red and black lines, present. (B) Application of 40 nM oligomeric α-synuclein (α-syn) induces a Ca²⁺ signal in an acute rat brain slice. (C,D) 40 nM of monomeric (C) or oligomeric (D) α-synuclein induced an elevation of cytosolic Ca²⁺ signal in primary neurons and astrocytes. Both forms of α-synuclein induced a rise in basal cytosolic Ca²⁺ as well as Ca²⁺ spikes. (E) Dose–response experiment demonstrating the number of cells responding at different concentrations of oligomeric α-synuclein. (F) Histogram demonstrating the proportion of cells exhibiting each pattern of Ca²⁺ signal. (G) The α-synuclein-induced Ca²⁺ signal was also observed in neurons derived from iPSCs. (H) Enriched oligomeric α-synuclein induced a rise in basal cytosolic Ca²⁺ as well as Ca²⁺ spikes. Results are mean±s.e.m. [n=98 for monomers; n=183 for oligomers (E,F)].

Fig. 2.

Identification of the source of the α-synuclein-induced Ca²⁺ signal. (A,B) Both monomeric and oligomeric Ca²⁺ signals could be prevented completely by removal of Ca²⁺ from the extracellular medium. (C) Inhibition of PLC by U73122 and inhibition of IP³ receptors by Xestospongin C had no effect on the α-synuclein-induced Ca²⁺ signal. (D,E) Depletion of the ER Ca²⁺ store using thapsigargin did not affect the monomeric or oligomeric-induced Ca²⁺ signal. (F) Representative image of single neuron following application of equimolar ratio of α-synuclein labelled with monomeric AF488 (green) and AF647 (red) demonstrating intracellular uptake of recombinant α-synuclein species. Scale bar: 10 μm.

Fig. 3.

Mn²⁺ quench assay confirms that α-synuclein-induced Ca²⁺ transients originate from a Ca²⁺ influx across plasmalemmal membrane. (A,B) Fura-2-loaded neurons showed typical [Ca²⁺]_c fluctuations in response to monomeric (A) or oligomeric (B) α-synuclein. With the addition of 40 µM Mn²⁺, each [Ca²⁺]_c transient was accompanied by a step quench of the 360-nm fura-2 signal, confirming that each transient reflects a pulsed influx of divalent cations seen in response to α-synuclein. (C) Application of monomeric α-synuclein in the absence of MnCl₂ did not induce any alteration in the fura-2 360-nm signal. (D) The α-synuclein-induced Ca²⁺ influx was independent of the presence of inhibitors of plasmalemmal channels [20 µM verapamil, 1 µM Nifedipine, 10 µM MK-801, 20 µM CNQX or 50 µM (S)-MCPG].

Fig. 4.

Monomeric α-synuclein leads to impairment of Ca²⁺ efflux. Application of 5 μM glutamate to cells pre-incubated with α-synuclein monomers (A) resulted in significantly delayed recovery of the cytosolic Ca²⁺ signal (over a period of 10 min) compared to control (recovery within 2 min, B). Results are mean±s.e.m. [n=91, control; n=68, monomers (B)]

Fig. 5.

Channel activity of α-synuclein on artificial membranes. (A) Oligomeric α-synuclein was added to the cis compartment of a bilayer cuvette to a final concentration of 100 nM. The membrane was suspended between symmetric HBSS supplemented with 1 mM CaCl₂. (B) Channels induced by 100 nM oligomer α-synuclein in lipid bilayers suspended between aqueous solution of 15 mM NaCl (cis) and 150 mM NaCl (trans), 2 mM CaCl₂, 10 mM Tris-HCl pH 7.4 (symmetric). Voltage of 150 mV was applied across BLM. (C) Current–voltage dependence of the channels presented in B. (D) Irregular currents induced by 100 nM of monomeric α-synuclein in BLMs. The membrane was suspended between symmetric HBSS supplemented with 1 mM CaCl₂.

Fig. 6.

Protective effect of Ca²⁺-free medium against α-synuclein-induced caspase-3 activation and cell death. (A,B,D) 40 nM oligomeric α-synuclein significantly activates the NucView 488 caspase 3 substrate in neurons and astrocytes. (C) Pre-incubation in Ca²⁺-free medium significantly reduced caspase 3 activation, shown as the time from addition of the peptide to activation of the substrate (i.e. increase of fluorescence in A). (D) Quantification of α-synuclein-induced apoptosis in Ca²⁺-containing and Ca²⁺-free medium. (E) Cell death was assessed using propidium iodide to label dead cells and Hoechst 33342 to label all cells. (F) Quantification of α-synuclein-induced cell death in normal and Ca²⁺-free medium. Results are mean±s.e.m. [n=69 (D); n=32 (norm); n=37 (Ca²⁺-free) (F)]. ***P<0.001; ns, not significant (Student's t-test).