Impact of human CA8 on thermal antinociception in relation to morphine equivalence in mice

Recently, we showed that murine dorsal root ganglion (DRG) Car8 expression is a cis-regulated eQTL that determines analgesic responses. In this report, we show that transduction through sciatic nerve injection of DRG with human wild-type carbonic anhydrase-8 using adeno-associated virus viral particles (AAV8-V5-CA8WT) produces analgesia in naive male C57BL/6J mice and antihyperalgesia after carrageenan treatment. A peak mean increase of about 4 s in thermal hindpaw withdrawal latency equaled increases in thermal withdrawal latency produced by 10 mg/kg intraperitoneal morphine in these mice. Allometric conversion of this intraperitoneal morphine dose in mice equals an oral morphine dose of about 146 mg in a 60-kg adult. Our work quantifies for the first time analgesia and antihyperalgesia in an inflammatory pain model after DRG transduction by CA8 gene therapy

Human carbonic anhydrase-8 AAV8 gene therapy inhibits nerve growth factor signaling producing prolonged analgesia and anti-hyperalgesia in mice

Carbonic anhydrase-8 (Car8; murine gene symbol) is an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1), which regulates neuronal intracellular calcium release. We previously reported that wild-type Car8 overexpression corrects the baseline allodynia and hyperalgesia associated with calcium dysregulation in the waddle (wdl) mouse due to a 19 bp deletion in exon 8 of the Car8 gene. In this report, we provide preliminary evidence that overexpression of the human wild-type ortholog of Car8 (CA8 ), but not the reported CA8 S100P loss-of-function mutation (CA8 ), inhibits nerve growth factor (NGF)-induced phosphorylation of ITPR1, TrkA (NGF high-affinity receptor), and ITPR1-mediated cytoplasmic free calcium release in vitro. In addition, we show that gene transfer using AAV8-V5-CA8 viral particles via sciatic nerve injection demonstrates retrograde transport to dorsal root ganglia (DRG) producing prolonged V5-CA8 expression, pITPR1 and pTrkA inhibition, and profound analgesia and anti-hyperalgesia in male C57BL/6J mice. AAV8-V5-CA8 -mediated overexpression prevented and treated allodynia and hyperalgesia associated with chronic neuropathic pain produced by the spinal nerve ligation (SNL) model. These AAV8-V5-CA8 data provide a proof-of-concept for precision medicine through targeted gene therapy of NGF-responsive somatosensory neurons as a long-acting local analgesic able to prevent and treat chronic neuropathic pain through regulating TrkA signaling, ITPR1 activation, and intracellular free calcium release by ITPR1

Car8 dorsal root ganglion expression and genetic regulation of analgesic responses are associated with a cis-eQTL in mice

Carbonic anhydrase-8 (Car8 mouse gene symbol) is devoid of enzymatic activity, but instead functions as an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1) to regulate this intracellular calcium release channel important in synaptic functions and neuronal excitability. Causative mutations in ITPR1 and carbonic anhydrase-8 in mice and humans are associated with certain subtypes of spinal cerebellar ataxia (SCA). SCA mice are genetically deficient in dorsal root ganglia (DRG) Car8 expression and display mechanical and thermal hypersensitivity and susceptibility to subacute and chronic inflammatory pain behaviors. In this report, we show that DRG Car8 expression is variable across 25 naïve-inbred strains of mice, and this cis-regulated eQTL (association between rs27660559, rs27706398, and rs27688767 and DRG Car8 expression; P < 1 × 10 ) is correlated with nociceptive responses in mice. Next, we hypothesized that increasing DRG Car8 gene expression would inhibit intracellular calcium release required for morphine antinociception and might correlate with antinociceptive sensitivity of morphine and perhaps other analgesic agents. We show that mean DRG Car8 gene expression is directly related to the dose of morphine or clonidine needed to provide a half-maximal analgesic response (r = 0.93, P < 0.00002; r = 0.83, P < 0.0008, respectively), suggesting that greater DRG Car8 expression increases analgesic requirements. Finally, we show that morphine induces intracellular free calcium release using Fura 2 calcium imaging in a dose-dependent manner; V5-Car8 overexpression in NBL cells inhibits morphine-induced calcium increase. These findings highlight the 'morphine paradox' whereby morphine provides antinociception by increasing intracellular free calcium, while Car8 and other antinociceptive agents work by decreasing intracellular free calcium. This is the first study demonstrating that biologic variability associated with this cis-eQTL may contribute to differing analgesic responses through altered regulation of ITPR1-dependent calcium release in mice

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

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

Carbonic Anhydrase-8 Regulates Inflammatory Pain by Inhibiting the ITPR1-Cytosolic Free Calcium Pathway

<div><p>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.</p></div

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

<p>Sciatic nerve injections of <i>AAV8-V5-Car8</i>^<i>WT</i> virus (1.5μl, 1.29E+14 genome copies /mL) and <i>AAV8-V5-Car8</i>^<i>MT</i> virus (1.5μl, 1.61E+14 genome copies /mL) were used in MT mice. (7A) The “up-down” method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#sec002" target="_blank">Methods</a> 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). <i>AAV8-V5-Car8</i>^<i>WT</i> increased both basal mechanical thresholds (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g007" target="_blank">Fig. 7A</a>) and thermal latencies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g007" target="_blank">Fig. 7B</a>), starting on day 7 after injection and lasting more than 28 days. Sciatic nerve injections of <i>AAV8-V5-Car8</i>^<i>MT</i> failed to affect nociception (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g007" target="_blank">Fig. 7A, B</a>) over the 28 d. (N = 8. * P<0.1; ** P<0.01; *** P< 0.001; Student <i>t</i>-test and two-way repeated measure ANOVA.)</p

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

<p>Sciatic nerve injections of <i>AAV8-V5-Car8</i>^<i>WT</i> virus (1.5μl, 1.29E+14 genome copies /mL) increase basal mechanical thresholds (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g008" target="_blank">Fig. 8A</a>) and thermal latencies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g008" target="_blank">Fig. 8C</a>) by day 13, before carrageenan injection. Sciatic nerve injections of <i>AAV8-V5-Car8</i>^<i>MT</i> virus (1.5μl, 1.61E+14 genome copies /mL) failed to alter basal mechanical thresholds (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g008" target="_blank">Fig. 8B</a>) and thermal latencies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g008" target="_blank">Fig. 8D</a>) by day 13, before carrageenan injection. After carrageenan injections, the <i>AAV8-V5-Car8</i>^<i>WT</i> virus group showed a reduction in mechanical thresholds (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g008" target="_blank">Fig. 8A</a>) and thermal latencies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g008" target="_blank">Fig. 8C</a>) on days 14 and 16 when compared to day 13; but these did not differ from baseline. After carrageenan injections, the <i>AAV8-V5-Car8</i>^<i>MT</i> virus group showed a reduction in mechanical thresholds (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g008" target="_blank">Fig. 8B</a>) and thermal latencies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g008" target="_blank">Fig. 8D</a>) well below day 13 and baseline. (N = 8. * P<0.1; ** P<0.01; *** P<0.001 by one-way ANOVA.)</p

Car8 deficiency alters nociception by inducing hypersensitivity.

<p>Nociception was tested in background C57BLKS/J (WT) mice (white bars), C57BLKS mice heterozygous for a 19 Bp deletion in exon 8 of the <i>Car8</i> gene (<i>Car8 wdl+/−</i>)(HET) (grey bars); and C57BLKS mice homozygous for this deletion <i>(Car8 wdl−/−</i>)(MT) (black bars). (A) The “up-down” method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#sec002" target="_blank">Methods</a> 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)</p

DRG Car8 expression WT and MT animals.

<p>(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g002" target="_blank">Fig. 2A-D</a>) Immunoreactivity for anti-Car8 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g002" target="_blank">Fig. 2A, D</a>, green), anti-NF200 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g002" target="_blank">Fig. 2B</a>, red), and anti-Car8 with anti-NF200 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g002" target="_blank">Fig. 2C</a>) antibodies, respectively. The merged image (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g002" target="_blank">Fig. 2C</a>) is from A and B. Immunohistochemistry demonstrates Car8 is expressed in the WT DRG (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g002" target="_blank">Fig. 2A</a>) but little or none in the MT DRG (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g002" target="_blank">Fig. 2D</a>). Percentage of different size neurons of Car8-containing neurons (measuring neuronal somata with visible nuclei) in the WT DRG (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g002" target="_blank">Fig. 2E</a>). Small: <300 μm²; Medium: 300–700 μm²; Large: >700 μm². RT-PCR demonstrates <i>Car8</i> mRNA in DRG tissues (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118273#pone.0118273.g002" target="_blank">Fig. 2F</a>). No template (TEMP) was used as a negative control (CTRL). The cerebellum was used as positive control. N = 4. Scale bar: 100 μm.</p