Role of the cysteine protease cathepsin S in neuropathic hyperalgesia.

Using a gene expression analysis approach we found that the mRNA encoding the lysosomal cysteine protease cathepsin S (CatS) was up-regulated in rat dorsal root ganglia (DRG) following peripheral nerve injury. CatS protein was expressed in infiltrating macrophages in DRG and near the site of injury. At both sites CatS expression progressively increased from day 3 to day 14 after injury. In naïve rats, intraplantar injection of activated rat recombinant (rr) CatS (0.3, 1 microg/rat) induced a mechanical hyperalgesia that developed within half-an-hour, diminished by 3h and was absent after 24h. Activated rrCathepsin B (CatB) and non-activated rrCatS injected intraplantarly at the same or higher doses than activated rrCatS had no effect on rat nociceptive thresholds. In nerve-injured rats, mechanical hyperalgesia, but not allodynia, was significantly reversed for up to 3h by systemic administration of a non-brain penetrant, irreversible CatS inhibitor (LHVS, 3-30 mg/kg s.c.). Depletion of peripheral macrophages by intravenous injection of liposome encapsulate clodronate (1ml, 5 mg/ml) partially reduced established mechanical hyperalgesia but not allodynia, and abolished the anti-hyperalgesic effect of LHVS. Our results demonstrate a pro-nociceptive effect of CatS and indicate that endogenous CatS released by peripheral macrophages contributes to the maintenance of neuropathic hyperalgesia following nerve injury.

siRNA relieves chronic neuropathic pain.

Double stranded, short interfering RNAs (siRNA) of 21-22 nt length initiate a sequence-specific, post-trancriptional gene silencing in animals and plants known as RNA interference (RNAi). Here we show that RNAi can block a pathophysiological pain response and provide relief from neuropathic pain in a rat disease model by down regulating an endogenous, neuronally expressed gene. Rats, intrathecally infused with a 21 nt siRNA perfectly complementary to the pain-related cation-channel P2X3, showed diminished pain responses compared to missense (MS) siRNA-treated and untreated controls in models of both agonist-evoked pain and chronic neuropathic pain. This form of delivery caused no adverse effects in any of the animals receiving P2X3 siRNA, MS siRNA or vehicle. Molecular analysis of tissues revealed that P2X3 mRNA expressed in dorsal root ganglia, and P2X3 protein translocated into the dorsal horn of the spinal cord, were significantly diminished. These observations open a path toward use of siRNA as a genetic tool for drug target validation in the mammalian central nervous system, as well as for proof of concept studies and as therapeutic agents in man.

Functional downregulation of P2X3 receptor subunit in rat sensory neurons reveals a significant role in chronic neuropathic and inflammatory pain.

The excitation of nociceptive sensory neurons by ATP released in injured tissue is believed to be mediated partly by P2X3 receptors. Although an analysis of P2X3 knock-out mice has revealed some deficits in nociceptive signaling, detailed analysis of the role of these receptors is hampered by the lack of potent specific pharmacological tools. Here we have used antisense oligonucleotides (ASOs) to downregulate P2X3 receptors to examine their role in models of chronic pain in the rat. ASOs and control missense oligonucleotides (180 microg/d) were administered intrathecally to naive rats for up to 7 d via a lumbar indwelling cannula attached to an osmotic minipump. Functional downregulation of the receptors was confirmed by alphabeta-methylene ATP injection into the hindpaw, which evoked significantly less mechanical hyperalgesia as early as 2 d after treatment with ASOs relative to controls. At this time point, P2X3 protein levels were significantly downregulated in lumbar L4 and L5 dorsal root ganglia. After 7 d of ASO treatment, P2X3 protein levels were reduced in the primary afferent terminals in the lumbar dorsal horn of the spinal cord. In models of neuropathic (partial sciatic ligation) and inflammatory (complete Freund's adjuvant) pain, inhibition of the development of mechanical hyperalgesia as well as significant reversal of established hyperalgesia were observed within 2 d of ASO treatment. The time course of the reversal of hyperalgesia is consistent with downregulation of P2X3 receptor protein and function. This study demonstrates the utility of ASO approaches for validating gene targets in in vivo pain models and provides evidence for a role of P2X3 receptors in the pathophysiology of chronic pain.

The ducky mutation in Cacna2d2 results in altered Purkinje cell morphology and is associated with the expression of a truncated alpha 2 delta-2 protein with abnormal function.

The mouse mutant ducky, a model for absence epilepsy, is characterized by spike-wave seizures and cerebellar ataxia. A mutation in Cacna2d2, the gene encoding the alpha 2 delta-2 voltage-dependent calcium channel accessory subunit, has been found to underlie the ducky phenotype. The alpha 2 delta-2 mRNA is strongly expressed in cerebellar Purkinje cells. We show that du/du mice have abnormalities in their Purkinje cell dendritic tree. The mutation in alpha 2 delta-2 results in the introduction of a premature stop codon and predicts the expression of a truncated protein encoded by the first three exons of Cacna2d2, followed by 8 novel amino acids. We show that both mRNA and protein corresponding to this predicted transcript are expressed in du/du cerebellum and present in Purkinje cells. Whereas the alpha 2 delta-2 subunit increased the peak current density of the Ca(V)2.1/beta(4) channel combination when co-expressed in vitro, co-expression with the truncated mutant alpha 2 delta-2 protein reduced current density, indicating that it may contribute to the du phenotype.