Phosphodiesterase Inhibitors Revert Axonal Dystrophy in Friedreich's Ataxia Mouse Model

Abstract

Friedreich's ataxia (FRDA) is a neurodegenerative disorder caused by an unstable GAA repeat expansion within intron 1 of the FXN gene and characterized by peripheral neuropathy. A major feature of FRDA is frataxin deficiency with the loss of large sensory neurons of the dorsal root ganglia (DRG), namely proprioceptive neurons, undergoing dying-back neurodegeneration with progression to posterior columns of the spinal cord and cerebellar ataxia. We used isolated DRGs from a YG8R FRDA mouse model and C57BL/6J control mice for a proteomic study and a primary culture of sensory neurons from DRG to test novel pharmacological strategies. We found a decreased expression of electron transport chain (ETC) proteins, the oxidative phosphorylation (OXPHOS) system and antioxidant enzymes, confirming a clear impairment in mitochondrial function and an oxidative stress-prone phenotype. The proteomic profile also showed a decreased expression in Ca²⁺ signaling related proteins and G protein-coupled receptors (GPCRs). These receptors modulate intracellular cAMP/cGMP and Ca²⁺ levels. Treatment of frataxin-deficient sensory neurons with phosphodiesterase (PDE) inhibitors was able to restore improper cytosolic Ca²⁺ levels and revert the axonal dystrophy found in DRG neurons of YG8R mice. In conclusion, the present study shows the effectiveness of PDE inhibitors against axonal degeneration of sensory neurons in YG8R mice. Our findings indicate that PDE inhibitors may become a future FRDA pharmacological treatment.

Keywords: Ca2+ signaling; FRDA; G protein-coupled receptor (GPCR); PDE inhibitors; axonal degeneration.

Fig. 1

Protein profile differential expression in DRG from frataxin-deficient mouse YG8R versus C57BL/6J control. (A) Representative 2D-DIGE blot of DRG protein extraction. The spots showing significant differences in protein levels between cases and controls are labeled. (B) 15 spots were identified as different between YG8R versus C57BL/6J, with ratio varying between − 1.33 and − 4.19. (C) Representation of molecular function of differential proteins expressed in YG8R mice versus C57BL/6J control by Gene Ontology (GO) database with PANTHER classification system. 44.30% proteins have catalytic activity (GO:0003824), 35.20% are binding proteins (GO:0005488), 22.80% proteins have structural molecule activity (GO:0005198), 8.20% proteins have enzyme regulator activity (GO:0030234), 5.70% have a receptor activity (GO:0004872), 2.70% are nucleic acid binding transcription factor activity (GO:0001071), 2.50% have translation regulator activity (GO:0045182), 0.70% have protein binding transcription factor activity (GO:0000988), and 0.20% have antioxidant activity (GO:0016209)

Fig. 2

cAMP measurements and PKA and CREB phosphorylation. (A) DRG tissues of YG8R mice and C57BL6/J were analyzed with cAMP enzyme immunoassay kit (Cayman Chemical Company). There was a significant variation in YG8R mice versus C57BL6/J. (B) Western blot analysis shows that the phosphorylation of PKA and CREB proteins were similar in YG8R and C57BL6J mice. Western blot results were quantified for each lane using Fujifilm’s Multi-Gauge Software. The ratio between phosphorylated and total forms was calculated and represented in (C) p-CREB/CREB and (D) p-PKA/PKA

Fig. 3

In vivo measurement of cytosolic Ca²⁺ in sensory neurons of YG8R mouse model. (A) Quantification of Fluo-8 AM fluorescence corresponding with intracellular Ca²⁺ levels by confocal microscopy. Final values were expressed as a ratio of the YG8R basal and the graph represents the mean ± S.E.M. of three experimental repeats (N = 3) with a total of 92, 135, 130, 131, and 108 measured neurons corresponding with C57BL/6J basal, YG8R basal, YG8R treated with nicardipine, YG8R treated with sildenafil, and YG8R treated with rolipram. One-way ANOVA (genotype); the results did not show statistically significant differences. (B) Microscopy images of Fluo-8 AM (green) and MitoTracker fluorescence (red) in primary culture of DRG of FRDA mouse model. Arrowheads show neuronal bodies and arrows show axonal spheroids with calcium and mitochondria retained. 40×, confocal microscopy. Scale 50 μm

Fig. 4

Treatment with PDE inhibitors recovers mitochondrial morphology in frataxin-deficient neurons. (A) Pattern of neuritic and mitochondrial network by immunodetection of β-tubulin III (green) and MitoTracker fluorescence (red) in primary culture of DRG from YG8R mouse. Arrowheads show neuronal bodies and arrows show axonal spheroids with mitochondria retained in YG8R mice sensory neurons that are absent in YG8R mice treated with PDE inhibitors. 40×, confocal microscopy. Scale 50 μm. (B–F) Quantification of mitochondrial network descriptors in proximal axon: number of mitochondria per 100 μm of neurite (B), percentage of axonal area occupied by mitochondria (C), mitochondrial elongation index (D), mitochondrial interconnectivity (E), and mitochondrial swelling (F) are expressed as mean ± S.E.M. of three experimental repeats (N = 3) with a total of 185, 188, 25, 213, and 193 measured neurons corresponding with C57BL/6J basal, YG8R basal, YG8R nicardipine, YG8R sildenafil, and YG8R rolipram. One-way ANOVA followed by Bonferroni post hoc test to determine the significance of values between different experimental groups. Significant P values: *P < 0.05, **P < 0.01, and ***P < 0.001 were considered. (G) Mitochondrial swelling expressed as cumulative distribution was analyzed using the Kolmogorov–Smirnov test

Fig. 5

Presumptive mechanism by which PDE inhibitors recover axonal dystrophy in FRDA neurons. According to previous reports (see references), frataxin-deficient neurons show (1.) a decrease in mitochondrial Fe-S proteins; (2.) a reduced mitochondrial membrane potential; (3.) a failure in mitochondrial biogenesis; (4.) a defect in Ca²⁺ buffering by mitochondria; (5.) high cytosolic Ca²⁺ levels; and (6.) axonal dystrophy. Treatment of neurons with PDE inhibitors recovers cytosolic Ca²⁺ to normal levels and repair axonal morphology (shown in this work). A possible recovery of Ca²⁺ influx activity due to restoration of mitochondrial function and mitochondrial biogenesis after PDE inhibitor treatments could explain our findings. A summary of previous reports is listed in black, data obtained in this work are shown in blue, and possible mechanisms explaining the action of PDE inhibitors are listed in red