Background & Aims
Fabry disease (FD) is an X-linked rare genetic condition due to variants in the alpha-galactosidase A (GLA) gene, which encodes a lysosomal enzyme responsible for glycosphingolipid degradation. When GLA activity is reduced, the accumulation of globotriaosylceramide (Gb3) occurs in various tissues. Neuropathic pain is one of the earliest symptoms in FD and Gb3 accumulation in primary sensory neurons (DRG) has been proposed as the mechanism behind this neuropathy. Emerging research has hinted at a connection between intracellular calcium signaling and FD-associated symptoms. When DRG were exposed to Gb3, it resulted in a temporary increase in cytosolic Ca2+ levels (1). Additionally, the Ca2+-activated potassium channel KCa 3.1 has been proposed to be involved in the pathogenesis of FD (2).
The study aimed to investigate the calcium homeostasis in dorsal root ganglion (DRG) neurons of the FD knock-out (KO) mouse model and identify molecular mechanisms involved in FD neuropathic pain.
Methods
We used the FD knock-out male ?-Gal A (-/0) murine model (3). Specifically, 8-12 weeks and 1-year-old male ?-Gal A (-/0, KO) and relative wild-type ?-Gal A (+/0, WT) were used. All the experiments were performed on isolated DRG neurons at 2 days in vitro. To investigate calcium dynamics, we performed ratiometric calcium imaging experiments using a microfluorimetry setup and the Fura-2 AM probe. The same system was applied for pH measurements, using the fluorescence probe BCECF-AM.
We performed western-blot experiments and immunofluorescence analysis coupled with confocal microscopy to assess protein expression levels. Electrophysiology/patch clamp analysis in the whole cell configuration was used to record action potentials in DRG neurons.
Results
We first investigated the electrophysiological properties of single action potentials elicited in KO DRG. We observed a significant reduction in the threshold potential and the afterhyperpolarization peak, suggesting an increased neuronal excitability compared to WT animals. On the other hand, the AP peak was strongly reduced at both ages. Despite we noted a downregulation of the KCa 3.1, perfusing KO DRG with a specific activator did not alter the single action potential profile.
Next, we assessed resting calcium levels in KO DRG and observed a significant increase in KO cells, both in young and old animals. Analyzing the mechanisms behind this calcium dysregulation, we found a reduction in plasma membrane Ca2+ ATPase activity without relevant changes in protein expression. The reduced activity correlated with a time-dependent decline in intracellular pH, which regulates pump activity.
Furthermore, we evaluated the mitochondrial calcium contribution in DRG neurons and observed impaired mitochondrial buffer capacity in KO DRG. These alterations were accompanied by increased autophagic markers and mitochondria/lysosome colocalization, suggesting an alteration in mitophagic flux and mitochondrial quality control.
Conclusions
In this study, we propose that calcium dysregulation in DRG neurons from the FD mouse model constitutes an early event in the disease mechanisms. Previous reports have indicated enhanced apoptosis in these neurons (4). The observed calcium overload may be associated with the neurodegenerative phenotype in these neurons, where Gb3 deposition is secondary to lysosomal dysfunction. Furthermore, we suggest that reduced plasma membrane Ca2+ ATPase activity and compromised mitochondrial buffer capacity contribute to calcium dysregulation, possibly affecting both synaptic transmission at the spinal level and neuronal survival. In this context, manipulating calcium signaling and restoring mitochondrial fitness could represent valuable therapeutic approaches for treating this neuropathic pain condition.
References
1)Choi L, Vernon J, Kopach O, Minett MS, Mills K, Clayton PT, Meert T, Wood JN. The Fabry disease-associated lipid Lyso-Gb3 enhances voltage-gated calcium currents in sensory neurons and causes pain. Neurosci Lett, 2015.
2)Choi JY, Shin MY, Suh SH, Park S. Lyso-globotriaosylceramide downregulates KCa3.1 channel expression to inhibit collagen synthesis in fibroblasts. Biochem Biophys Res Commun, 2015.
3)Formaggio F, Rimondini R, Delprete C, Scalia L, Merlo Pich E, Liguori R, Nicoletti F, Caprini M. L-Acetylcarnitine causes analgesia in mice modeling Fabry disease by up-regulating type-2 metabotropic glutamate receptors. Mol Pain, 2022
4)Hofmann L, Hose D, Grießhammer A, Blum R, Döring F, Dib-Hajj S, Waxman S, Sommer C, Wischmeyer E, Üçeyler N. Characterization of small fiber pathology in a mouse model of Fabry disease. Elife, 2018.
Presenting Author
Francesco Formaggio
Poster Authors
Topics
- Models: Chronic Pain - Neuropathic