Hironao Saegusa

Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N‐type Ca2+ channel

The importance of voltage‐dependent Ca2+ channels (VDCCs) in pain transmission has been noticed gradually, as several VDCC blockers have been shown to be effective in inhibiting this process. In particular, the N‐type VDCC has attracted attention, because inhibitors of this channel are effective in various aspects of pain‐related phenomena. To understand the genuine contribution of the N‐type VDCC to the pain transmission system, we generated mice deficient in this channel by gene targeting. We report here that mice lacking N‐type VDCCs show suppressed responses to a painful stimulus that induces inflammation and show markedly reduced symptoms of neuropathic pain, which is caused by nerve injury and is known to be difficult to treat by currently available therapeutic methods. This finding clearly demonstrates that the N‐type VDCC is essential for development of neuropathic pain and, therefore, controlling the activity of this channel can be of great importance for the management of neuropathic pain.

Effects of ablation of N- and R-type Ca2+ channels on pain transmission

Recently several mutant mouse lines lacking neuronal voltage-dependent Ca(2+) channels (VDCCs) have been established by the use of gene targeting in embryonic stem cells. Pain-related behaviors in Ca(v)2.2 (alpha(1B)) and Ca(v)2.3 (alpha(1E)) knockout mice were studied to gain further insight into the mechanism of pain transmission, where VDCCs are thought to play important roles. We review here the data from these recent studies. Ca(v)2.3-/- mice showed normal responses to acute painful stimuli, and reduced responses to the somatic inflammatory pain stimuli. Ca(v)2.3+/- mice exhibited reduced symptoms of visceral inflammatory pain. Ca(v)2.3-/- mice showed abnormal behavior related to the descending antinociceptive mechanism activated by the intraperitoneal injection of acetic acid. Ca(v)2.2-/- mice showed variable acute nociceptive responses depending on the mutant lines. However, all the lines of Ca(v)2.2-/- mice exhibited reduced responses in the phase 2 of the formalin test, suggesting a suppression of inflammatory pain. Furthermore Ca(v)2.2-/- mice showed markedly reduced neuropathic pain symptoms after spinal nerve ligation. Impaired antinociception, similar to that seen in the Ca(v)2.3-/- mice, was also observed in the Ca(v)2.2-/- mice. Therefore, it is suggested that these mutant mice could provide novel models to delineate the nociceptive and antinociceptive mechanisms.

Properties of human Cav2.1 channel with a spinocerebellar ataxia type 6 mutation expressed in Purkinje cells

Spinocerebellar ataxia type 6 (SCA6) is caused by polyglutamine expansion in P/Q-type Ca2+ channels (Ca(v)2.1) and is characterized by predominant degeneration of cerebellar Purkinje cells. To characterize the Ca(v)2.1 channel with an SCA6 mutation in cerebellar Purkinje cells, we have generated knock-in mouse models that express human Ca(v)2.1 with 28 polyglutamine repeats (disease range) and with 13 polyglutamine repeats (normal range). Patch-clamp recordings of the Purkinje cells from homozygous control or SCA6 knock-in mice revealed a non-inactivating current that is highly sensitive to a spider toxin omega-Agatoxin IVA, indicating that the human Ca(v)2.1 expressed in Purkinje cells exhibits typical P-type properties in contrast to the previous data showing Q-type properties, when it was expressed in cultured cell lines. Furthermore, the voltage dependence of activation and inactivation and current density were not different between SCA6 and control, though these properties were altered in previous reports using non-neuronal cells as expression systems. Therefore, our results do not support the notion that the alteration of the channel properties may underlie the pathogenic mechanism of SCA6.

Cav2.3 (α1E) Ca2+ channel participates in the control of sperm function

To know the function of the Ca2+ channel containing α12.3 (α1E) subunit (Cav2.3 channel) in spermatozoa, we analyzed Ca2+ transients and sperm motility using a mouse strain lacking Cav2.3 channel. The averaged rising rates of Ca2+ transients induced by α‐D‐mannose–bovine serum albumin in the head region of Cav2.3−/− sperm were significantly lower than those of Cav2.3+/+ sperm. A computer‐assisted sperm motility assay revealed that straight‐line velocity and linearity were greater in Cav2.3−/− sperm than those in Cav2.3+/+ sperm. These results suggest that the Cav2.3 channel plays some roles in Ca2+ transients and the control of flagellar movement.

Blocking the R-type (Cav2.3) Ca2+ channel enhanced morphine analgesia and reduced morphine tolerance

Morphine is the drug of choice to treat intractable pain, although prolonged administration often causes undesirable side‐effects including analgesic tolerance. It is speculated that voltage‐dependent Ca2+ channels (VDCCs) play a key role in morphine analgesia and tolerance. To examine the subtype specificity of VDCCs in these processes, we analysed mice lacking N‐type (Cav2.2) or R‐type (Cav2.3) VDCCs. Systemic morphine administration or exposure to warm water swim‐stress, known to induce endogenous opioid release, resulted in greater analgesia in Cav2.3−/− mice than in controls. Moreover, Cav2.3−/− mice showed resistance to morphine tolerance. In contrast, Cav2.2−/− mice showed similar levels of analgesia and tolerance to control mice. Intracerebroventricular (i.c.v.) but not intrathecal (i.t.) administration of morphine reproduced the result of systemic morphine in Cav2.3−/− mice. Furthermore, i.c.v. administration of an R‐type channel blocker potentiated morphine analgesia in wild‐type mice. Thus, the inhibition of R‐type Ca2+ current could lead to high‐efficiency opioid therapy without tolerance.

Increased sensitivity to halothane but decreased sensitivity to propofol in mice lacking the N-type Ca2+ channel

Volatile anesthetics are known to depress excitatory synaptic transmission. Inhibition of voltage-dependent Ca2+ channels is speculated to underlie this mechanism, which remains to be clarified in vivo. We examined the sensitivity to halothane in mice lacking the N-type Ca2+ channel, a major contributor of presynaptic neurotransmitter release. Sensitivity to halothane was significantly increased in the knockout mice compared with the wild-type littermates. Halothane also depressed field excitatory postsynaptic potentials recorded from the Schaffer collateral-CA1 hippocampal synapses more greatly in the knockout mice. We further examined sleep time induced by injection of propofol, an intravenous anesthetic that mainly affects inhibitory synaptic transmission. In contrast, sensitivity to propofol was significantly decreased in the knockout mice. We suggest that inhibition of the N-type Ca2+ channel underlies mechanisms of halothane anesthesia but counteracts propofol anesthesia.

The carboxy-terminal tail region of human Cav2.1 (P/Q-type) channel is not an essential determinant for its subcellular localization in cultured neurones.

A recent report on the mechanism of synaptic targeting of Cav2.2 channel suggested that this process depends upon the presence of long C‐terminal tail and that protein interactions mediated by SH3‐binding and PDZ‐binding motifs in the tail region are important. To examine the possibility that C‐terminal tail of the Cav2.1 channel and the polyglutamine stretch therein are also involved in the mechanism for channel localization, we constructed several expression plasmids for human Cav2.1 channel tagged with enhanced green fluorescent protein (EGFP) and introduced them into mouse hippocampal neuronal culture. HC construct encodes short version of Cav2.1, and HS and HL encode Cav2.1 channel with a long C‐terminal tail, which contains polyglutamine tract of 13 (normal range) and 28 (SCA6 disease range) repeat units, respectively. Surprisingly, transfection with HC, HS, and HL gave essentially the same results: EGFP signal was observed in cell soma, dendrites, and the axon as well. Furthermore, mutation of the PDZ‐binding motif located at the C‐terminus of the long version of Cav2.1, by adding FLAG tag, did not affect the localization patterns of HS and HL as well. Therefore, the C‐terminal region is not indispensable for the subcellular localization of Cav2.1 channel, nor expansion of polyglutamine length affected the localization of the channel. Thus, it is possible that the localization mechanism of Cav2.1 channel is different from that of Cav2.2, though these channels share various structural and functional characteristics.

Upregulation of casein kinase 1ε in dorsal root ganglia and spinal cord after mouse spinal nerve injury contributes to neuropathic pain

BackgroundNeuropathic pain is a complex chronic pain generated by damage to, or pathological changes in the somatosensory nervous system. Characteristic features of neuropathic pain are allodynia, hyperalgesia and spontaneous pain. Such abnormalities associated with neuropathic pain state remain to be a significant clinical problem. However, the neuronal mechanisms underlying the pathogenesis of neuropathic pain are complex and still poorly understood. Casein kinase 1 is a serine/threonine protein kinase and has been implicated in a wide range of signaling activities such as cell differentiation, proliferation, apoptosis, circadian rhythms and membrane transport. In mammals, the CK1 family consists of seven members (α, β, γ1, γ2, γ3, δ, and ε) with a highly conserved kinase domain and divergent amino- and carboxy-termini.ResultsPreliminary cDNA microarray analysis revealed that the expression of the casein kinase 1 epsilon (CK1ε) mRNA in the spinal cord of the neuropathic pain-resistant N- type Ca2+ channel deficient (Cav2.2-/-) mice was decreased by the spinal nerve injury. The same injury exerted no effects on the expression of CK1ε mRNA in the wild-type mice. Western blot analysis of the spinal cord identified the downregulation of CK1ε protein in the injured Cav2.2-/- mice, which is consistent with the data of microarray analysis. However, the expression of CK1ε protein was found to be up-regulated in the spinal cord of injured wild-type mice. Immunocytochemical analysis revealed that the spinal nerve injury changed the expression profiles of CK1ε protein in the dorsal root ganglion (DRG) and the spinal cord neurons. Both the percentage of CK1ε-positive neurons and the expression level of CK1ε protein were increased in DRG and the spinal cord of the neuropathic mice. These changes were reversed in the spinal cord of the injured Cav2.2-/- mice. Furthermore, intrathecal administration of a CK1 inhibitor IC261 produced marked anti-allodynic and anti-hyperalgesic effects on the neuropathic mice. In addition, primary afferent fiber-evoked spinal excitatory responses in the neuropathic mice were reduced by IC261.ConclusionsThese results suggest that CK1ε plays important physiological roles in neuropathic pain signaling. Therefore CK1ε is a useful target for analgesic drug development.

Role of Cav2.3 (α1E) Ca2+ channel in ischemic neuronal injury

We investigated the role of the Ca v 2.3 (α 1E ) channel in ischemic neuronal injury using Ca v 2.3 mutant mice. In focal ischemia model with a complete occlusion of the middle cerebral artery in vivo, infarct at 24 h was significantly larger in Ca v 2.3 mutant mice compared with that in wild-type controls. In vitro Ca 2+ imaging studies using hippocampal slices revealed that oxygen-glucose deprivation induced a [Ca 2+ ] i increase in the hippocampal CA1 region more vigorously in Ca v 2.3 mutant mice than in wild-type controls, and that tetrodotoxin or bicuculline application abolished the difference between the genotypes. These results suggest that the Ca v 2.3 channel plays a protective role in ischemic neuronal injury by a mechanism in which GABAergic neuronal actions are involved.

Deficit of heat shock transcription factor 1‐heat shock 70 kDa protein 1A axis determines the cell death vulnerability in a model of spinocerebellar ataxia type 6

Spinocerebellar ataxia type 6 (SCA6) is caused by a small expansion of polyglutamine (polyQ)‐encoding CAG repeat in Cav2.1 calcium channel gene. To gain insights into pathogenic mechanism of SCA6, we used HEK293 cells expressing fusion protein of enhanced green fluorescent protein and Cav2.1 carboxyl terminal fragment (EGFP‐Cav2.1CT) [L24 and S13 cells containing 24 polyQ (disease range) and 13 polyQ (normal range), respectively] and examined their responses to some stressors. When exposed to CdCl2, L24 cells showed lower viability than the control S13 cells and caspase‐dependent apoptosis was enhanced more in L24 cells. Localization of EGFP‐Cav2.1CT was almost confined to the nucleus, where it existed as speckle‐like structures. Interestingly, CdCl2 treatment resulted in disruption of more promyelocytic leukemia nuclear bodies (PML‐NBs) in L24 cells than in S13 cells and in cells where PML‐NBs were disrupted, aggregates of EGFP‐Cav2.1CT became larger. Furthermore, a large number of aggregates were formed in L24 cells than in S13 cells. Results of RNAi experiments indicated that HSPA1A determined the difference against CdCl2 toxicity. Furthermore, protein expression of heat shock transcription factor 1 (HSF1), which activates HSPA1A expression, was down‐regulated in L24 cells. Therefore, HSF1‐HSPA1A axis is critical for the vulnerability in L24 cells.