The Fabry disease-associated lipid Lyso-Gb3 enhances voltage-gated calcium currents in sensory neurons and causes pain

Abstract

Fabry disease is an X-linked lysosomal storage disorder characterised by accumulation of glycosphingolipids, and accompanied by clinical manifestations, such as cardiac disorders, renal failure, pain and peripheral neuropathy. Globotriaosylsphingosine (lyso-Gb3), a deacylated form of globotriaosylceramide (Gb3), has emerged as a marker of Fabry disease. We investigated the link between Gb3, lyso-Gb3 and pain. Plantar administration of lyso-Gb3 or Gb3 caused mechanical allodynia in healthy mice. In vitro application of 100nM lyso-Gb3 caused uptake of extracellular calcium in 10% of sensory neurons expressing nociceptor markers, rising to 40% of neurons at 1μM, a concentration that may occur in Fabry disease patients. Peak current densities of voltage-dependent Ca(2+) channels were substantially enhanced by application of 1μM lyso-Gb3. These studies suggest a direct role for lyso-Gb3 in the sensitisation of peripheral nociceptive neurons that may provide an opportunity for therapeutic intervention in the treatment of Fabry disease-associated pain.

Keywords: Calcium imaging; Dorsal root ganglia; Fabry disease; Pain; Voltage-dependent Ca(2+) channels.

Fig. 1

Effects of lyso-Gb3 on pain sensitivity and intracellular Ca²⁺ levels in nociceptive DRG neurons. (A) Either Gb3 or lyso-Gb3 injected into mice hind paws significantly enhanced pain sensitivity to mechanical stimuli over several hours post injection (n = 6–9 per group). Sensitivity resolved to a normal level within 24 h. Repeated measures ANOVA revealed effects of time: F (6,126) = 7.551, P < 0.0001, and of Fabry lipids (F (21,126) = 3.089, P < 0.001) but no difference between the two lipids (effect of lipid type: F (2,126) = 6.179, P < 0.05). (B) Lyso-Gb3 evoked an increase in cytoplasmic Ca²⁺ levels in DRG neurons in a dose-dependent manner. The concentration of lyso-Gb3 producing changes in Ca²⁺ levels is plotted against the percentage of responsive DRG neurons. (C) Repetitive applications of lyso-Gb3 (1 μM), but not vehicle evoked transient increases in Ca²⁺ levels in DRG neurons. (D) Representative real time recordings of the changes in Fura-2 ratio acquired every 10 s in individual DRG neurons during bath applications of 1 μM Lyso-Gb3, 50 mM KCl and 1 μM capsaicin. (E) Statistical summary of the changes in Fura-2 ratio before and after application of 1 μM of lyso-Gb3 (n = 74). *P < 0.05 and **P < 0.0001.

Fig. 2

Identification of the DRG neuron population responsive to lyso-Gb3. (A) Distribution of cells according to diameter; lyso-Gb3 responders were all small-diameter cells, as were 89 % of capsaicin-responsive cells. The third bar of the histogram signifies that IB-4 positive cells were all small-diameter neurons. (B) Lyso-Gb3 responsive cells were all stimulated by 1 μM capsaicin, but only 17 % of these bound IB-4.​ (B) Other lyso-Gb3 responsive capsaicin-sensitive cells were IB-4 negative. (C) Statistical summary of the lyso-Gb3-induced changes in Fura-2 ratio in neuronal subgroups shows no significant difference between IB4-positive and IB4-negative DRG neurons. (D) Representative images showing the changes in Fura-2 fluorescence during application of lyso-Gb3, KCl or capsaicin in IB4-positive DRG neurons. Arrows point to the IB4-positive neurons. (E) Subpopulation of lyso-Gb3-responding neurons represents approximately 63% of capsaicin-sensitive DRG neurons, only 17% of those were positive for IB-4 binding. **P < 0.0001.

Fig. 3

Requirement for extracellular calcium in the Fura-2 response to lyso-GB3. (A) Representative real time traces showing intracellular Ca²⁺ levels following superfusion with lyso-Gb3 in either Ca²⁺ free or standard recording buffer containing 2 mM Ca²⁺. (B) Peak Fura-2AM 340/380 ratio between baseline and lyso-Gb3. Cells appear red in accordance with Ca²⁺ levels. (C) Representative fluorescent images showing changes in Ca²⁺ levels. Note: Data sampled every 10 s during calcium imaging. **P < 0.0001.

Fig. 4

Effects of lyso-Gb3 on Ca²⁺ influx and current density of voltage-activated Ca²⁺ channels in DRG neurons. (A) Representative traces of Ca²⁺ currents evoked in the same small-diameter DRG neuron by voltage steps from holding potential (V_h) –70 mV to test potential (V_t) from –60 mV to +50 mV in 5 mV increments before (top) or during (medium) application of 1 μM lyso-Gb3 via a perfusion system. Ca²⁺ currents (bottom) in the presence of lyso-Gb3 were completely inhibited by 50 μM CdCl₂. Inset illustrates the voltage-clamp protocol used to elicit currents. (B) Representative Ca²⁺ currents recorded at V_t 0 mV before (control) and after lyso-Gb3 application. (C) Average current–voltage relationships of Ca²⁺ currents before and during an application of 1 μM lyso-Gb3. The peak current amplitudes at each V_t were normalized to cell capacitance and expressed as the current density. (D) A statistical summary of the current densities at V_t 0 mV before and during lyso-Gb3 application. Changes in current density were normalized to control.. *P < 0.05 (paired t-test).