Carbonic anhydrase-8 regulates inflammatory pain by inhibiting the ITPR1-cytosolic free calcium pathway

Abstract

Calcium dysregulation is causally linked with various forms of neuropathology including seizure disorders, multiple sclerosis, Huntington's disease, Alzheimer's, spinal cerebellar ataxia (SCA) and chronic pain. Carbonic anhydrase-8 (Car8) is an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1), which regulates intracellular calcium release fundamental to critical cellular functions including neuronal excitability, neurite outgrowth, neurotransmitter release, mitochondrial energy production and cell fate. In this report we test the hypothesis that Car8 regulation of ITPR1 and cytoplasmic free calcium release is critical to nociception and pain behaviors. We show Car8 null mutant mice (MT) exhibit mechanical allodynia and thermal hyperalgesia. Dorsal root ganglia (DRG) from MT also demonstrate increased steady-state ITPR1 phosphorylation (pITPR1) and cytoplasmic free calcium release. Overexpression of Car8 wildtype protein in MT nociceptors complements Car8 deficiency, down regulates pITPR1 and abolishes thermal and mechanical hypersensitivity. We also show that Car8 nociceptor overexpression alleviates chronic inflammatory pain. Finally, inflammation results in downregulation of DRG Car8 that is associated with increased pITPR1 expression relative to ITPR1, suggesting a possible mechanism of acute hypersensitivity. Our findings indicate Car8 regulates the ITPR1-cytosolic free calcium pathway that is critical to nociception, inflammatory pain and possibly other neuropathological states. Car8 and ITPR1 represent new therapeutic targets for chronic pain.

Fig 1. Car8 deficiency alters nociception by inducing hypersensitivity.

Nociception was tested in background C57BLKS/J (WT) mice (white bars), C57BLKS mice heterozygous for a 19 Bp deletion in exon 8 of the Car8 gene (Car8 wdl+/−)(HET) (grey bars); and C57BLKS mice homozygous for this deletion (Car8 wdl−/−)(MT) (black bars). (A) The “up-down” method (see Methods for details) was used to measure mechanical responses by probing the plantar aspect of the hindpaw with von Frey filaments and determining the paw withdrawal threshold (grams). (B) Thermal withdrawal response latencies were measured to radiant heat (75 units) applied to the plantar aspect of the hind paw (seconds). (N = 12; ** P<0.01; *** P<0.001; one way ANOVA)

Fig 2. DRG Car8 expression WT and MT animals.

(Fig. 2A-D) Immunoreactivity for anti-Car8 (Fig. 2A, D, green), anti-NF200 (Fig. 2B, red), and anti-Car8 with anti-NF200 (Fig. 2C) antibodies, respectively. The merged image (Fig. 2C) is from A and B. Immunohistochemistry demonstrates Car8 is expressed in the WT DRG (Fig. 2A) but little or none in the MT DRG (Fig. 2D). Percentage of different size neurons of Car8-containing neurons (measuring neuronal somata with visible nuclei) in the WT DRG (Fig. 2E). Small: <300 μm²; Medium: 300–700 μm²; Large: >700 μm². RT-PCR demonstrates Car8 mRNA in DRG tissues (Fig. 2F). No template (TEMP) was used as a negative control (CTRL). The cerebellum was used as positive control. N = 4. Scale bar: 100 μm.

Fig 3. DRG ITPR1, pITPR1 and steady-state cytoplasmic free calcium in WT and MT mice.

Immunohistochemistry data show there are no significant differences in ITPR1 DRG expression between WT and MT mice (Fig. 3C, D); pITPR1 was higher in MT DRG as compared to WT DRG (Fig. 3A, B, G). Western blot data also show an increase of pITPR1 in DRG from MT mice as compared to WT mice (ratio MT/WT pITPR1 = 3.0)(Fig. 3H). Vinculin was used as a loading control. Calcium image analyses from cultured DRG cells also demonstrate free calcium concentration is higher in MT DRG as compared to WT DRG (Fig. 3E, F, G) N = 4. Scale bar: 100 μm (Fig. 3A-D), 30 μm (Fig. 3E-F). (*** P<0.001; Student’s t-test or one-way ANOVA.)

Fig 4. Inflammation decreases DRG Car8 increasing pITPR1.

Carrageenan was injected (30 μl 1% carrageenan) into the left hind palm. Analyses of DRG immunohistochemistry of WT rats (Fig. 4A-L); and western blot of rat DRG (Fig. 4N, 4O) show Car8 expression is clearly reduced from 6 h to 48 h (4A-D, M, N) and pITPR1 expression significantly increased from 6h to 48 h (Fig. 4E-H, M, O), while ITPR1 levels remained unchanged (Fig. 4I-M). N = 4. Scale bar: 100 μm. (* P<0.1; ** P<0.01; *** P<0.001; Student’s t-test or one-way ANOVA.)

Fig 5. Overexpression of V5-Car8^WT in vitro inhibits forskolin-induced ITPR1-phosphorylation.

Western blotting analyses demonstrate that expression of V5-Car8^WT is significantly higher than that of V5-Car8^MT in mouse-derived N2A cultures (Fig. 5A). Real-time PCR data show that there is no significant difference between V5-Car8 ^WT and V5-Car8 ^MT mRNA expression levels from N2A cultures (Fig. 5B). Western blotting analyses of pITPR1 demonstrate that forskolin increases ITPR1 activation intensity in N2A cultures in a dose-dependent manner (Fig. 5C). Overexpression of V5-Car8^WT protein using the AAV2-V5-Car8 ^WT vector reduced forskolin-induced pITPR1 increases in N2A cultures (Fig. 5D). Overexpression of V5-Car8^MT protein using the AAV2-V5-Car8 ^WT vector failed to reduce pITPR1 activation by forskolin in N2A cultures (Fig. 5D). N = 6 from 2 independent cultures in triplicate. (* P<0.05; ***P<0.001; Student’s t-test or one-way ANOVA.)

Fig 6. Overexpression of V5-Car8^WT in vitro inhibits ATP-induced cytoplasmic free calcium increases.

Calcium imaging data show overexpression of V5-Car8^WT protein reduced 1μM ATP-induced cytoplasmic free calcium increases in HEK293 cultures (Fig. 6A-C, G), when compared to no vector (S1A–F Fig.) and the V5-Car8 ^MT vector (Fig. 6D-F, G). (N = 6 from 2 independent cultures in triplicate. * P<0.05; *** P<0.001; one-way ANOVA).

Fig 7. Gene transfer of V5-Car8^WT regulates nociception and produces analgesia in MT mice.

Sciatic nerve injections of AAV8-V5-Car8 ^WT virus (1.5μl, 1.29E+14 genome copies /mL) and AAV8-V5-Car8 ^MT virus (1.5μl, 1.61E+14 genome copies /mL) were used in MT mice. (7A) The “up-down” method (see Methods for details) was used to measure mechanical responses by probing the plantar aspect of the hindpaw with von Frey filaments and determining the paw withdrawal threshold (grams). (7B) Thermal withdrawal response latencies were measured to radiant heat (70 units) applied to the plantar aspect of the hind paw (seconds). AAV8-V5-Car8 ^WT increased both basal mechanical thresholds (Fig. 7A) and thermal latencies (Fig. 7B), starting on day 7 after injection and lasting more than 28 days. Sciatic nerve injections of AAV8-V5-Car8 ^MT failed to affect nociception (Fig. 7A, B) over the 28 d. (N = 8. * P<0.1; ** P<0.01; *** P< 0.001; Student t-test and two-way repeated measure ANOVA.)

Fig 8. Gene transfer of V5-Car8^WT produces analgesia and anti-hyperalgesia in a carrageenan subacute inflammatory pain model in WT mice.

Sciatic nerve injections of AAV8-V5-Car8 ^WT virus (1.5μl, 1.29E+14 genome copies /mL) increase basal mechanical thresholds (Fig. 8A) and thermal latencies (Fig. 8C) by day 13, before carrageenan injection. Sciatic nerve injections of AAV8-V5-Car8 ^MT virus (1.5μl, 1.61E+14 genome copies /mL) failed to alter basal mechanical thresholds (Fig. 8B) and thermal latencies (Fig. 8D) by day 13, before carrageenan injection. After carrageenan injections, the AAV8-V5-Car8 ^WT virus group showed a reduction in mechanical thresholds (Fig. 8A) and thermal latencies (Fig. 8C) on days 14 and 16 when compared to day 13; but these did not differ from baseline. After carrageenan injections, the AAV8-V5-Car8 ^MT virus group showed a reduction in mechanical thresholds (Fig. 8B) and thermal latencies (Fig. 8D) well below day 13 and baseline. (N = 8. * P<0.1; ** P<0.01; *** P<0.001 by one-way ANOVA.)

Fig 9. Gene transfer of V5-Car8^WT produces thermal anti-hyperalgesia in a complete Freund’s adjuvant (CFA) chronic inflammatory pain model in WT mice.

Chronic inflammatory pain produced by injection of 30 μl 1% CFA in the left hind palm on minus day 2, and day 9. CFA induces thermal hyperalgesia starting on minus d1. Sciatic nerve injections of AAV8-V5-Car8 ^WT virus (1.5μl, 1.29E+14 genome copies /mL) on day zero increased basal latencies (Fig. 9A) by day 13. In contrast, sciatic nerve injections of AAV8-V5-Car8 ^MT virus (1.5μl, 1.61E+14 genome copies /mL) failed to alter basal thermal latencies (Fig. 9B) at any time. (N = 8. *** P<0.001 by one-way ANOVA.)