Knockdown of PHOX2B in the retrotrapezoid nucleus reduces the central CO2 chemoreflex in rats

Abstract

PHOX2B is a transcription factor essential for the development of different classes of neurons in the central and peripheral nervous system. Heterozygous mutations in the PHOX2B coding region are responsible for the occurrence of Congenital Central Hypoventilation Syndrome (CCHS), a rare neurological disorder characterised by inadequate chemosensitivity and life-threatening sleep-related hypoventilation. Animal studies suggest that chemoreflex defects are caused in part by the improper development or function of PHOX2B expressing neurons in the retrotrapezoid nucleus (RTN), a central hub for CO₂ chemosensitivity. Although the function of PHOX2B in rodents during development is well established, its role in the adult respiratory network remains unknown. In this study, we investigated whether reduction in PHOX2B expression in chemosensitive neuromedin-B (NMB) expressing neurons in the RTN altered respiratory function. Four weeks following local RTN injection of a lentiviral vector expressing the short hairpin RNA (shRNA) targeting Phox2b mRNA, a reduction of PHOX2B expression was observed in Nmb neurons compared to both naive rats and rats injected with the non-target shRNA. PHOX2B knockdown did not affect breathing in room air or under hypoxia, but ventilation was significantly impaired during hypercapnia. PHOX2B knockdown did not alter Nmb expression but it was associated with reduced expression of both Task2 and Gpr4, two CO₂/pH sensors in the RTN. We conclude that PHOX2B in the adult brain has an important role in CO₂ chemoreception and reduced PHOX2B expression in CCHS beyond the developmental period may contribute to the impaired central chemoreflex function.

Keywords: PHOX2B; chemoreception; neuroscience; plethysmography; rat; retrotrapezoid nucleus; shRNA.

Figure 1.. Respiratory data 2 weeks post viral PHOX2B shRNA injection.

(A) Breathing frequency (ƒ_R,), (B) tidal volume (V_T), (C) allometric minute ventilation (V_E ALLO), (D) oxygen consumption (VO₂ ALLO), (E) convective requirement ratio (V_E/VO₂ ALLO), (F) hypercapnic ventilatory response (HCVR absolute change in V_E ALLO vs. corresponding room air) of baseline (pre-surgery, grey filled box), naive (black n=8), non-target control shRNA (NT-shRNA, grey n=17) and PHOX2B shRNA (PHOX2B-shRNA, red n=17) rats 2 weeks post injection during room air, hypercapnia 5% and 7.2% CO₂. ƒ_R was equally reduced in all experimental groups compared to baseline but no treatment effect was observed (A). V_T was significantly impaired following RTN injection in PHOX2B-shRNA group compared to naive animals (p=0.022) and baseline (p<0.001) at 7.2% CO₂ (B). V_E ALLO was increased in naive rats compared to the other treatment groups (p=0.005) in room air (C). Boxplots: median, 1st – 3rd quartiles and 10th – 90th percentiles, outliers = dots, ‘+’ indicates arithmetic mean. Bonferroni post-hoc as indicated. Black*, different from naive; Grey*, different from NT-shRNA; Red*, different from PHOX2B- shRNA.

Figure 1—figure supplement 1.. Respiratory and anatomical data 4 weeks post large viral PHOX2B shRNA injection.

(A) Allometric V_E is equally impaired following RTN injection of non-target control (NT-shRNA n=6) and PHOX2B-shRNA (n=8) in 5% CO2 (p<0.001) and 7.2% CO2 (p<0.001). Boxplots: median, 1st – 3rd quartiles and 10th – 90th percentiles, outliers = dots, ‘+’ indicates arithmetic mean. Bonferroni post-hoc as indicated. Asterisks, different from baseline. (B) The number of Nmb⁺/PHOX2B⁺ cells comprising the RTN are reduced in NT-shRNA (n=5; p=0.002), and shRNA rats (n=5; p=0.001) as compared to naive controls (n=4; Asterisks, different from baseline). The number of Nmb⁺/PHOX2B^- cells was unchanged in both surgical treatments compared to baseline (One-way ANOVA p=0.8).

Figure 2.. PHOX2B and Nmb expression and total cell count within the RTN area in naive, NT-shRNA and PHOX2B-shRNA injected rats two weeks post viral shRNA injection.

(A) Schematic and representative image of a transverse brainstem section at the level of the RTN (–11.5 mm distance from Bregma) showing the area of investigation containing RTN neurons. (B) Expression of Phox2b mRNA (red), PHOX2B protein (white) and Nmb mRNA (green) in RTN Nmb⁺/PHOX2B⁺ and Nmb⁺/PHOX2B^- neurons in naive (left), NT-shRNA (middle), and PHOX2B-shRNA (right) rats (magnified view inserts below). Arrowheads indicate absence of PHOX2B protein. Scale bar = 400 μm (top figures), 150 μm (inserts below). (C) The number of total cells (Nmb⁺/PHOX2B⁺ plus Nmb⁺/PHOX2B^-) comprising the RTN were reduced in PHOX2B-shRNA rats (n=4) as compared to naive (n=4) and NT-shRNA (n=4), and in NT-shRNA rats as compared to naive rats (black^#, One-way ANOVA, p<0.001). The number of Nmb⁺/PHOX2B⁺ cells were reduced in PHOX2B-shRNA rats as compared to naive and NT-shRNA (blue*, One-way ANOVA, p<0 0.001). The number of Nmb⁺/PHOX2B^- cells was unchanged across groups. (D,E) Rostral-caudal distribution (distance from the caudal tip of the facial nucleus, 7Mn) of Nmb⁺/PHOX2B⁺ (D) and Nmb⁺/PHOX2B^- (E) neurons along the RTN.

Figure 3.. PHOX2B and Nmb expression and total cell count within the RTN area in naive, NT-shRNA and PHOX2B-shRNA injected rats 4 weeks post viral shRNA injection.

(A) Schematic and representative image of a transverse brainstem section at the level of the RTN (–11.5 mm distance from Bregma) showing the area of investigation containing RTN neurons. (B) Expression of Phox2b mRNA (red), PHOX2B protein (white) and Nmb mRNA (green) in naive (left), NT-shRNA (middle), and PHOX2B-shRNA (right) rats (magnified view insert). Arrowheads indicate absence of PHOX2B protein. Scale bar = 400 μm (top figures), 150 μm (inserts below). (C) The number of total cells (Nmb⁺/PHOX2B⁺ + Nmb⁺/PHOX2B^-) comprising the RTN were reduced in PHOX2B-shRNA rats (n=6) as compared to naive (n=4) (Black^#, One-way ANOVA, p=0.0087) but not to NT-shRNA (n=10). The number of Nmb⁺/PHOX2B⁺ cells were reduced in PHOX2B-shRNA rats as compared to naive and NT-shRNA (Blue*, one-way ANOVA, p<0.001). The number of Nmb⁺/PHOX2B^- cells were increased in PHOX2B-shRNA rats as compared to both naive and NT-shRNA (Green*, one-way ANOVA p<0.001). (D,E) Rostral-caudal distribution (distance from the caudal tip of the facial nucleus, 7Mn) of Nmb⁺/PHOX2B⁺ (D) and Nmb⁺/PHOX2B^- (E) neurons along the RTN.

Figure 4.. Respiratory data following 4 weeks post viral shRNA injection.

(A) Breathing frequency (ƒ_R,), (B) tidal volume (V_T), (C) allometric minute ventilation (V_E ALLO), (D) convective requirement ratio (V_E/VO₂ ALLO), (E) hypercapnic ventilatory response (HCVR, absolute change in V_E ALLO vs. corresponding room air), (F) HCVR at baseline, week 2 and week 4 post-viral injections in naive (black n=8), non-target control shRNA (NT-shRNA, grey n=10) and PHOX2B-shRNA (PHOX2B-shRNA, red n=6). ƒ_R was equally impaired in all experimental group compared to baseline but no treatment effect was observed (A). V_T was significantly impaired following RTN injection in PHOX2B-shRNA group compared to baseline pre-surgery (p<0.001), naive rats (p<0.001), and NT-shRNA rats (p=0.002) at 7.2% CO₂. (B). V_E ALLO was impaired in PHOX2B-shRNA rats during exposure to hypercapnia (7.2% CO₂) compared to baseline (p=0.0025), naive rats (p=0.007), and NT-shRNA rats (p=0.002). (C). V_E/VO₂ ALLO was reduced in PHOX2B-shRNA animals compared to NT-shRNA rats both at 5% (p=0.023) and 7.2% (p=0.004) CO₂ (D). HCVR during 7.2% CO₂ was lower in PHOX2B-shRNA rats compared to baseline (p=0.007), naive rats (p=0.001), and NT-shRNA rats (p=0.016) (E). Boxplots: median, 1st – 3rd quartiles and 10th – 90th percentiles, outliers = dots, ‘+’ indicates arithmetic mean. Bonferroni post-hoc as indicated. HCVR was significantly impaired only in PHOX2B-shRNA rats 4 weeks post-surgery (One-way ANOVA p=0.007) (F). Black*, different from naive; Grey*, different from NT-shRNA; Red*, different from PHOX2B- shRNA.

Figure 5.. PHOX2B knockdown in RTN neurons does not alter TH or Nmb expression but impairs the hypercapnic ventilatory response.

(A) PHOX2B protein (white) and TH (red) expression in C1 neurons of nave (left), NT-shRNA (middle), and PHOX2B-shRNA (right) rats. Magnified view at the bottom. Arrowheads indicate the colocalization of PHOX2B and TH protein in C1 neurones cells. Scale bar = 400 μm (top figures), 150 μm (bottom figures). (B) No differences were observed in the rostral-caudal distribution of TH⁺/PHOX2B⁺ catecholaminergic C1 neurons cells caudal to the RTN between naive (black, n=4), NT-shRNA (grey, n=10) and PHOX2B-shRNA (red, n=6) rats. (C) PHOX2B protein (white) and Nmb mRNA (green) expression in NT-shRNA rat at the level of NTS and RTN regions. Magnified view on the left. Arrowheads indicate cells with Nmb mRNA expression. Scale bar = 400 μm (right figures), 150 μm (left figures). (D) Quantification of single cells Nmb mRNA fluorescence intensity along the rostro-caudal extension of RTN in naive (black, n=4), NT-shRNA (grey, n=10) and PHOX2B-shRNA (red, n=6) rat calculated as average ratio between RTN and NTS cells showed no difference between treatment groups. Data are shown as average cell fluorescence value at different rostro-caudal levels. (E-F) X-Y plot of HCVR during 7.2% CO₂ exposure relative to the number of Nmb⁺/PHOX2B⁺ (E, slope is different from ‘0’ at p<0.001; r²=0.739) and Nmb⁺/PHOX2B^- (F, p<0.001 for difference between slopes; r²=0.482) in the RTN.

Figure 6.. Gpr4 and Task2 mRNA expression within the RTN area in naive, and PHOX2B-shRNA injected rats 4 weeks post viral shRNA injection.

(A, D) Nmb (green), Gpr4, Task2 mRNA (red) and PHOX2B protein (grey) expression in RTN neurons in naive (top) and PHOX2B-shRNA (bottom) rats. Scale bar = 150 μm. Arrowheads indicate colocalization of Gpr4 (A) and Task2 (D) with Nmb⁺/PHOX2B⁺ neurons. Asterisks indicate colocalization of Gpr4 (A) and Task2 (D) with Nmb⁺/PHOX2B^- neurons. (B, E) Quantification of single cells Gpr4 and Task2 mRNA fluorescence intensity along the rostro-caudal extension of RTN in naive (black), NT-shRNA (grey) and PHOX2B-shRNA (red) rats. Black*, different from naive, p<0.001. Grey*, different from NT-shRNA, p<0.001 (C, F) Quantification of single cells Gpr4 and Task2 mRNA fluorescence staining intensity along the rostro-caudal extension of RTN in Nmb⁺/PHOX2B⁺ (red filled dot) and Nmb⁺/PHOX2B^- (red empty dot) neurons in PHOX2B-shRNA rats (C, p=0.022); (F, p=0.029). Data are shown as average cell fluorescence value at different rostro-caudal levels of the RTN. Mean corrected total cell fluorescence (CTCF) value ± SEM combined (naive, n=4; NT-shRNA, n=10; PHOX2B-shRNA, n=6) (see Materials and methods for detail). One-way ANOVA repeated measures.