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. 2017 Nov 22:11:365.
doi: 10.3389/fncel.2017.00365. eCollection 2017.

Pannexin 1 Modulates Axonal Growth in Mouse Peripheral Nerves

Steven M Horton  1 , Carlos Luna Lopez  1 , Elisabeth Blevins  1   2 , Holly Howarth  3 , Jake Weisberg  1 , Valery I Shestopalov  4 , Helen P Makarenkova  5 , Sameer B Shah  1   2   3
Affiliations

Pannexin 1 Modulates Axonal Growth in Mouse Peripheral Nerves

Steven M Horton et al. Front Cell Neurosci. .

Abstract

The pannexin family of channels consists of three members-pannexin-1 (Panx1), pannexin-2 (Panx2), and pannexin-3 (Panx3) that enable the exchange of metabolites and signaling molecules between intracellular and extracellular compartments. Pannexin-mediated release of intracellular ATP into the extracellular space has been tied to a number of cellular activities, primarily through the activity of type P2 purinergic receptors. Previous work indicates that the opening of Panx1 channels and activation of purinergic receptors by extracellular ATP may cause inflammation and apoptosis. In the CNS (central nervous system) and PNS (peripheral nervous system), coupled pannexin, and P2 functions have been linked to peripheral sensitization (pain) pathways. Purinergic pathways are also essential for other critical processes in the PNS, including myelination and neurite outgrowth. However, whether such pathways are pannexin-dependent remains to be determined. In this study, we use a Panx1 knockout mouse model and pharmacological inhibitors of the Panx1 and the ATP-mediated signaling pathway to fill gaps in our understanding of Panx1 localization in peripheral nerves, roles for Panx1 in axonal outgrowth and myelination, and neurite extension. Our data show that Panx1 is localized to axonal, myelin, and vascular compartments of the peripheral nerves. Knockout of Panx1 gene significantly increased axonal caliber in vivo and axonal growth rate in cultured dorsal root ganglia (DRG) neurons. Furthermore, genetic knockout of Panx1 or inhibition of components of purinergic signaling, by treatment with probenecid and apyrase, resulted in denser axonal outgrowth from cultured DRG explants compared to untreated wild-types. Our findings suggest that Panx1 regulates axonal growth in the peripheral nervous system.

Keywords: axon; dorsal root ganglion; pannexin; peripheral nerve; purinergic signaling.

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Figures

Figure 1
Figure 1
Panx1 is expressed in axons. (A–I) Immunolabeled sections from wild-type sciatic nerves. (A–C) β3-tubulin (A) and Panx1 (B) colocalize (C). Insets in (B,C) show, respectively, enlargements of blood vessels and co-localizing Panx1 and β3-tubulin. (D–E) β3-tubulin (D) and Panx1 (E) colocalize over the entire cross-section of sciatic nerves, suggesting Panx1 localization to motor and sensory axons. (F–H) Serial sections show that β3-tubulin (F) and myelin basic protein (MBP) (G) colocalize (H). Nerve fibers are separated by laminin-delineated sheaths (I). (J) β3-tubulin immuno-labeling shows a characteristic punctate pattern within the nerve. β3-tubulin and Panx1 (K) co-localize (L) in cultured DRG explants from wild-type mice. Explant mass is indicated by an asterisk. (M) β3-tubulin labeling in Panx1 knockout nerves shows a characteristic punctate pattern, though axons appear to be larger. (N–Q) Axonal localization appears normal relative to myelin (N–P) and laminin (Q). (R–S) Panx1 labeling of Panx1 knockout nerves shows diffuse, non-specific labeling consistent with the truncated protein that results in functional knockout. (T) Quantification reveals that cross-sectional area is larger in PANx1−/− axons than wild-type. *Indicates significant difference. Bars: (A–C), 50 μm; (D–E), 100 μm; (F–I), 50 μm; (J–L), 100 μm; (M), 50 μm; (N–P), 25 μm.
Figure 2
Figure 2
Axonal outgrowth from Panx1−/− DRG explants is accelerated compared to that from wild-type explants. (A) Wild-type: Axons are shown in a fluorescence (β3-tubulin) image for Day 11. (B) Panx1−/−: Axons are shown in a fluorescence (β3-tubulin) image for Day 11. Bars: 50 μm. (C) Quantification of growth rates for axons of each genotype. Significance was assessed at each time point using Welch's t-test. *P < 0.01.
Figure 3
Figure 3
Disruption of purinergic signaling increases axonal density. (A–D) Samples of axonal outgrowth from (A) untreated wild-type, (B) untreated Panx1 knockout, (C) apyrase-treated wild-type, (D) probenecid-treated wild-type, and (E) 10Panx peptide-treated wild-type DRG explants. Insets show binarized images used to calculate axonal density. (F) Quantification of axonal density shows significant increases (*) in density in Panx1 knockout and apyrase, probenecid, and 10Panx-peptide-treated explants. Bars in brightfield images: 100 μm.
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