Neto2 Assembles with Kainate Receptors in DRG Neurons during Development and Modulates Neurite Outgrowth in Adult Sensory Neurons

Abstract

Peripheral sensory neurons in the dorsal root ganglia (DRG) are the initial transducers of sensory stimuli, including painful stimuli, from the periphery to central sensory and pain-processing centers. Small- to medium-diameter non-peptidergic neurons in the neonatal DRG express functional kainate receptors (KARs), one of three subfamilies of ionotropic glutamate receptors, as well as the putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2). Neto2 alters recombinant KAR function markedly but has yet to be confirmed as an auxiliary subunit that assembles with and alters the function of endogenous KARs. KARs in neonatal DRG require the GluK1 subunit as a necessary constituent, but it is unclear to what extent other KAR subunits contribute to the function and proposed roles of KARs in sensory ganglia, which include promotion of neurite outgrowth and modulation of glutamate release at the DRG-dorsal horn synapse. In addition, KARs containing the GluK1 subunit are implicated in modes of persistent but not acute pain signaling. We show here that the Neto2 protein is highly expressed in neonatal DRG and modifies KAR gating in DRG neurons in a developmentally regulated fashion in mice. Although normally at very low levels in adult DRG neurons, Neto2 protein expression can be upregulated via MEK/ERK signaling and after sciatic nerve crush and Neto2^-/- neurons from adult mice have stunted neurite outgrowth. These data confirm that Neto2 is a bona fide KAR auxiliary subunit that is an important constituent of KARs early in sensory neuron development and suggest that Neto2 assembly is critical to KAR modulation of DRG neuron process outgrowth.SIGNIFICANCE STATEMENT Pain-transducing peripheral sensory neurons of the dorsal root ganglia (DRG) express kainate receptors (KARs), a subfamily of glutamate receptors that modulate neurite outgrowth and regulate glutamate release at the DRG-dorsal horn synapse. The putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2) is also expressed in DRG. We show here that it is a developmentally downregulated but dynamic component of KARs in these neurons, that it contributes to regulated neurite regrowth in adult neurons, and that it is increased in adult mice after nerve injury. Our data confirm Neto2 as a KAR auxiliary subunit and expand our knowledge of the molecular composition of KARs in nociceptive neurons, a key piece in understanding the mechanistic contribution of KAR signaling to pain-processing circuits.

Keywords: auxiliary subunit; axon outgrowth; ionotropic glutamate receptor; nociceptor; pain; voltage-clamp.

Figure 1.

Neto2 is highly expressed in neonatal DRG and downregulated over development. a, In situ hybridization in a P4 spinal section with a probe directed against Neto2 transcripts. DRG are outlined with the dotted lines and denoted by arrowheads. b, Neto2 protein is detected by Western blot in wild-type but not Neto2^−/− DRG homogenate. c, Representative Western blot showing Neto2 protein expression from acutely homogenized P2, P5, P10, P14, and adult DRG. d, Densitometry quantification of Neto2 expression normalized to actin represented as percentage of Neto2 expression at P2. e, Representative current traces from neonatal and adult wild-type DRG neurons. Gray bar indicates glutamate (10 mm) application. Currents are reproduced from Figure 2a (neonatal) and Figure 3a (adult) for comparison. f, Quantification of mean weighted tau of glutamate-evoked desensitization for neonatal and adult DRG neurons. g, Quantification of peak current amplitude for neonatal and adult DRG neurons. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2.

Neto2 assembles into functional GluK1/GluK5-containing KARs in neonatal DRG neurons. a, Representative current traces from wild-type (also presented in Fig. 1e), Neto2^−/−, and GluK5^−/− neonatal DRG neurons. Gray bar indicates glutamate (10 mm) application. b, Proportion of small- and medium-diameter cells patched that expressed KAR-mediated current is represented in the filled bars. Empty bars are the proportion of KAR-negative cells patched for each genotype. c, Quantification of mean weighted tau of glutamate-evoked desensitization for wild-type, Neto1^−/−, Neto2^−/−, and GluK5^−/− neonatal DRG neurons. d, Cumulative probability histogram of individual cell desensitization rates for wild-type, Neto1^−/−, Neto2^−/−, and GluK5^−/− neonatal DRG neurons. e, Quantification of peak glutamate-evoked current amplitude for wild-type, Neto1^−/−, Neto2^−/−, and GluK5^−/− neonatal DRG neurons. f, Cumulative probability histogram of individual cell peak current amplitudes for wild-type, Neto1^−/−, Neto2^−/−, and GluK5^−/− neonatal DRG neurons. **p < 0.01; ***p < 0.001. Error bars in column graphs indicate SEM.

Figure 3.

KARs in acutely plated adult DRG do not contain Neto2 but increase Neto2 incorporation over time in culture. a, Representative current traces from wild-type, Neto2^−/−, and GluK5^−/− adult DRG neurons are shown for both early and late recording time points: 3–12 h and 12–22 h in culture, respectively. b, Proportion of small- and medium-diameter cells patched that expressed KAR-mediated current is represented in the filled bars. Empty bars are the proportion of KAR-negative cells patched for each genotype. c, Quantification of mean weighted tau of glutamate-evoked desensitization for wild-type, Neto1^−/−, Neto2^−/−, and GluK5^−/− adult DRG neurons. Data are grouped by whether the cell was recorded at 3–12 h or 12–22 h after the cultures were plated. d, Cumulative probability histogram of individual cell desensitization rates for wild-type, Neto1^−/−, Neto2^−/−, and GluK5^−/− adult DRG neurons. e, Quantification of peak glutamate-evoked current amplitudes for wild-type, Neto1^−/−, Neto2^−/−, and GluK5^−/− adult DRG neurons. Data are grouped by whether the cell was recorded 3–12 h or 12–22 h after the cultures were plated. f, Cumulative probability histogram of individual cell peak current amplitudes for wild-type, Neto1^−/−, Neto2^−/−, and GluK5^−/− adult DRG neurons. g, Wild-type and Neto2^−/− data from d represented as desensitization rate versus time-in-culture to demonstrate visually the correlations reported in the text. h, Wild-type and Neto2^−/− data from e represented as peak current amplitude versus time-in-culture to demonstrate visually the correlations reported in the text. ***p < 0.001. Error bars in column graphs indicate SEM.

Figure 4.

Neto2 is upregulated in older DRG cultures and ERK activation is required for Neto2 upregulation in adult DRG neurons. a, Representative Western blot of Neto2 expression in young wild-type DRG cultures at 4 and 24 h. b, Densitometry quantification of Neto2 expression normalized to actin and represented as the percentage of P5 culture at 24 h. c, Representative Western blot of Neto2 expression in adult wild-type DRG cultures at 4 and 24 h in untreated cultures and at 24 h in cultures treated with the ERK1/2 inhibitor U0126 (50 μm), the PI3K inhibitor wortmannin (10 nm), and the Akt inhibitor triciribine (20 μm). d, Densitometry quantification of Neto2 expression normalized to actin and represented as percentage of control culture at 24 h. Error bars indicate SEM. *p < 0.05; ***p < 0.001. con, Untreated control cultures; U0, cultures treated with U0126; wor, cultures treated with wortmannin; tric, cultures treated with triciribine.

Figure 5.

Neto^−/− mice show normal formalin pain behaviors and normal formalin-induced inflammatory heat hypersensitivity. a, Neto1^−/− and Neto2^−/− mouse spontaneous pain behavior after formalin injection compared with their wild-type littermates. Measured time spent on nocifensive behaviors was pooled into 5 min bins. Saline-injected animals did not show nocifensive behaviors over the 60 min after injection and these data are omitted from graphs for the purpose of clarity. b, Neto1^−/− and Neto2^−/− mouse thermal hypersensitivity compared with their wild-type littermates. Hypersensitivity was measured by Hargreaves test 3 h after formalin injection and is represented as the fold change from the withdrawal latency measured before formalin injection. Error bars indicate SEM. *p < 0.05; ***p < 0.001.

Figure 6.

Neto2^−/− DRG neurons from adult mice show stunted neurite outgrowth and maturation in culture compared with wild-type neurons. a, Representative images of wild-type and Neto2^−/− neurons from each maturation stage. Stage 2 and 3 inverted images show the full axon arbor for each cell and the area within the blue boxes is magnified to demonstrate branching. Black scale bars, 100 μm; white scale bars, 50 μm. b, Percentage of wild-type and Neto2^−/− cells at each maturation stage (see Materials and Methods for details). c, Length of the longest neurite measured from each cell in wild-type and Neto2^−/− adult neurons. Cells were electroporated with eGFP and fixed after 24 h in culture. Data are grouped by the cell's maturation stage. d, Number of branch points measured on each cell in wild-type and Neto2^−/− adult neurons. Data are grouped by the cell's maturation stage. e, Sholl analysis was performed on axon arbor tracings of wild-type and Neto2^−/− adult neurons at 5 μm intervals from the soma center. Sholl data are grouped by maturation stage for presentation and analysis. Error bars on column graphs indicate SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7.

Sciatic nerve crush upregulates Neto2 in vivo. a, Representative Western blot showing Neto2 and actin expression in homogenized DRG and sciatic nerve tissue taken 3, 7, or 10 d post-sciatic nerve crush (dp-SNC). Tissue was taken ipsilateral to the crushed nerve (Ipsil.), contralateral to the crushed nerve (Contral.), or ipsilateral to an exposed surgical sham nerve (Sham). b, Densitometry quantification of Neto2 normalized to actin. Within each time point, Neto2 expression in Sham tissue was set to 100%. Error bars indicate SEM. *p < 0.05.