Peripherally targeted analgesia via AAV-mediated sensory neuron-specific inhibition of multiple pronociceptive sodium channels in rat

Abstract

Here, we present evidence showing Piezo1 expression in the primary sensory neurons (PSNs) and non-neuronal cells of rat peripheral nervous system. Using a knockdown/knockout validated antibody, we detected Piezo1 immunoreactivity (IR) in ~80% of PSNs of rat dorsal root ganglia (DRG) with higher IR density in the small- and medium-sized neurons, and within axons extending to both central presynaptic terminals innervating to the spinal dorsal horn and peripheral cutaneous sensory terminals in the skin. Piezo-IR was clearly identified in DRG perineuronal glia, including satellite glial cells (SGCs) and non-myelinating Schwann cells; in sciatic nerve Schwann cells surrounding the axons and cutaneous afferent endings; and in skin epidermal Merkel cells and melanocytes. Neuronal and non-neuronal Piezo1 channels were functional, since various cells (dissociated PSNs and SGCs from DRGs, isolated Schwann cells, and primary human melanocytes) exhibited a robust response to Piezo1 agonist Yoda1 by an increase of intracellular Ca2+ concentration ([Ca2+]i). These responses were abolished by Piezo1 antagonist GsMTx4. Immunoblots showed elevated Piezo1 protein in DRG proximal to peripheral nerve injury-induced painful neuropathy, while PSNs and SGCs from rats with neuropathic pain showed greater Yuda1-evoked elevation of [Ca2+]i and an increased frequency of cells responding to Yoda1, compared to controls. Ipsilateral sciatic nerve application of GsMTx4 alleviated mechanical hypersensitivity following nerve injury. Overall, our data show that Piezo1 is widely expressed by the neuronal and non-neuronal cells in the peripheral sensory pathways and that painful nerve injury is associated with activation of Piezo1 in PSNs and peripheral glia cells.

Disordered but effective: short linear motifs as gene therapy targets for hyperexcitability disorders

“…The study by Shin et al ( 27 ) provides an example of an analgesic gene therapy approach that reduces proexcitatory TTX-S sodium currents in peripheral sensory neurons by roughly 50%, which also adds to our foundational knowledge of the role of specific SLiMs within cytoplasmic regions of Na V channels in regulating channel’s biophysical properties, delivery, and stability at the plasma membrane. Although the physiological role of the IDD regions of Na V channels are not well understood and the mechanism underlying the inhibitory effects of Na V iPA1 on the TTX-S channels remain to be elucidated, targeting of protein-protein interacting sites may offer an innovative strategy for dissecting structure-function models of Na V channels.…”

“…Further, since Na V iPA1 acts on all of the neuronal TTX-S Na V channels, this approach might be used to treat other excitability disorders, for example epilepsy. It remains to be seen whether a clinical version of the Na V iPA1 will prove to be an effective analgesic in humans, especially if it avoids adverse autonomic and motor effects, but the Shin et al ( 27 ) preclinical studies provide a first step.…”

“…In a well-designed study in this issue, Shin and colleagues ( 27 ) used in silico analysis of intrinsically disordered domains (IDD) in cytoplasmic sequences of Na V 1.7 channel and identified a polyampholytic interfering peptide aptamer (which they called Na V iPA1) that corresponded to a sequence in the proximal end of L1 ( Figure 1A ). The Na V iPA1 sequence carries a short linear polybasic motif and potentially phosphorylatable serine residues, which are conserved in TTX-S but not TTX-R channels.…”

“…The Shin et al study ( 27 ) leaves some questions unanswered and also suggests new opportunities. It is not known whether the SLiMs in the Na V iPA1 exert their effects as a decoy via an adapter molecule or via an intramolecular interaction; and the lack of high-resolution atomic structures of cytoplasmic regions of Na V channels published to date limits our ability to predict and test potential intramolecular interacting sequences that might influence channel trafficking.…”