Respiratory and autonomic dysfunction in congenital central hypoventilation syndrome

Abstract

The developmental lineage of the PHOX2B-expressing neurons in the retrotrapezoid nucleus (RTN) has been extensively studied. These cells are thought to function as central respiratory chemoreceptors, i.e., the mechanism by which brain Pco2 regulates breathing. The molecular and cellular basis of central respiratory chemoreception is based on the detection of CO2 via intrinsic proton receptors (TASK-2, GPR4) as well as synaptic input from peripheral chemoreceptors and other brain regions. Murine models of congenital central hypoventilation syndrome designed with PHOX2B mutations have suggested RTN neuron agenesis. In this review, we examine, through human and experimental animal models, how a restricted number of neurons that express the transcription factor PHOX2B play a crucial role in the control of breathing and autonomic regulation.

Keywords: CCHS; breathing; chemoreflex; ventrolateral medulla.

Fig. 1.

Schematic view of the control of CO₂ homeostasis and the role of PHOX2B-expressing neurons. Retrotrapezoid nucleus (RTN) chemoreceptor neurons are activated by CO₂ via their intrinsic pH sensitivity and via inputs from the carotid bodies. RTN neurons target various components of the respiratory central pattern generator (rCPG) and are presumed to play a key role in breathing automaticity during anesthesia, sleep, and quiet waking. The carotid body may also influence the activity of the rCPG neurons through connections (not shown) that bypass the RTN (Stornetta et al. 2006; Takakura et al. 2006). RTN neurons are also inhibited by lung inflation via slowly adapting receptor (SAR) activation and by the fact that the effect of SARs is mediated by GABAergic pump cells located within the nucleus of the solitary tract (NTS) at area postrema level (Moreira et al. 2007). In mammals, PHOX2B expression is required for the development of the carotid bodies (1) (Dauger et al. 2003), NTS (2) (Stornetta et al. 2006), and RTN chemoreceptors neurons (3 and 4) (Moreira et al. 2007; Stornetta et al. 2006), as well as brain stem catecholaminergic neurons, enteric nervous system, sympathetic ganglionic (but not preganglionic) neurons, and the cranial parasympathetic system but is not required for the serotonergic system or the rCPG neurons (5) (Stornetta et al. 2006). 1, Illustration showing the expression of PHOX2B immunoreactive in the carotid body between E13.5 and E16.5 in mice. [Modified from Dauger et al. (2003).]. 2, Awake rats subjected to hypoxia and hypoxia-activated NTS neurons were identified by the presence of Fos-immunoreactive nuclei. The brain tissue was processed for simultaneous detection of FluorGold (FG; native blue fluorescence), PHOX2B immunoreactivity (Alexa 488 fluorescence; green), Fos immunoreactivity (Cy3; red). The photomicrograph taken within the commissural portion of the NTS shows multiple aqua-colored neurons (arrows) with RTN projections, activated by carotid body stimulation and expressing PHOX2B. The color coding for other combinations of markers is indicated. Scale bar, 50 μm. [Modified from Stornetta et al. (2006).] 3, Example of one RTN chemoreceptor neuron (arrows) labeled in vivo with biotinamide [PHOX2B immunoreactivity revealed with Alexa 488 (green) and biotinamide with Cy-3 (red); colocalization shown in yellow]. Scale bar, 50 μm. [Modified from Stornetta et al. (2006).] 4, Example of one RTN chemoreceptor neuron (arrows) labeled in vivo with biotinamide [PHOX2B immunoreactivity revealed with Cy-3 (red) and biotinamide with Alexa 488 (green); colocalization shown in yellow]. [Modified from Moreira et al. (2007).] 5, Biotinamide labeling of the Bötzinger complex. This sample was also reacted for PHOX2B immunoreactivity (Cy3; red) revealing that the recorded neuron (Alexa 488; green) was PHOX2B negative. Scale bar, 50 μm. [Modified from Stornetta et al. (2006).]

Fig. 2.

Summary of molecular studies of PHOX2B mutation function. A: luciferase assay of PHOX2A promoter (top) and dopamine β-hydroxylase (DBH; bottom). Y-axis shows relative fluorescence units normalized to Renilla luciferase. Both PHOX2B and PHOX2B^Δ8 show activation of this promoter. For the DBH studies, both PHOX2B and PHOX2A cDNA constructs can transactivate the DBH promoter. Note that cotransfection of PHOX2A+PHOX2B as well as PHOX2A+PHOX2B^Δ8 shows synergistic activity relative to PHOX2A or PHOX2B alone. This synergy is not noted with PHOX2B-polyalanine (PA). B: immunofluorescent analyses of PHOX2B constructs expressed in 293FT cells. The transfected construct is delineated at top right and a color code for immunofluorescent staining at bottom left of each panel. Cells were transfected with PHOX2B, PHOX2B-PA, or PHOX2B^Δ8 cDNA and immunostained using NH₂-terminal (N-TERM) or COOH-terminal (C-TERM; which cannot detect PHOX2B^Δ8) PHOX2B antibodies (Santa Cruz Biotechnology), c-Myc epitope tag antibody (MYC; in frame with PHOX2B and PHOX2B-PA reading frame but not in frame with PHOX2B^Δ8 reading frame). Cells are counterstained with DAPI in blue, and colocalization of both antigens is indicated in yellow. Note that PHOX2B-PA shows significant cytoplasmic localization (B3 and B4), whereas PHOX2B and PHOX2B^Δ8 show nuclear localization. These findings support the notion that PARM PHOX2B mutations lead to accumulation in the cytoplasm. C: proximity ligation assay [PLA; see Supplemental Material (available for this article online at the Journal of Neurophysiology website) for epitopes and antibodies used] determines the proximity of 2 epitopes. Epitopes need to lie within 70 nm for a signal to be elicited and thus represent a way to identify histologically whether 2 proteins interact. PLA signal is red, and cells are counterstained with DAPI in blue. The transfected constructs in which PLA reaction is performed are shown at top right of each panel. PLA demonstrates nuclear localization of interactions between PHOX2A and PHOX2B (C1), PHOX2A and PHOX2B^Δ8 (C3), and PHOX2B and PHOX2B^Δ8 (C5). In contrast, significant cytoplasmic localization is noted between PHOX2A and PHOX2B-PA (C2) and PHOX2B-PHOX2B-PA (C4) interactions. C6 shows interactions of PHOX2B^Δ8 with a separate PHOX2B^Δ8 protein, indicating that NPARM mutations can homodimerize.

Fig. 3.

Genetic design of CCHS-type mutations in mouse experimental models. A: the gene structure of PHOX2B, located on mouse chromosome 5, is composed of 3 exons. B: knockin mice were generated with an extra 7 alanines to generate the PHOX2B^20/Ki-27 genotype. The neomycin resistance cassette was removed by interbreeding chimeric mice with PGK-cre mice. Experiments were performed with neomycin cassette-intact and neomycin cassette-ablated mice to ensure that the neomycin cassette did not affect the phenotype. C: humanized PHOX2B^20/27 conditional knockout mouse design. mE3 is flanked by loxP sites. The neomycin cassette was inserted between mE3 and the loxP site 5′ to mE3 and was removed by interbreeding founders. Located 3′ to the mE3 site is a humanized E3 with the additional 7 alanines and a human 3′-UTR. NPARM knockin models for 5 nucleotide deletions (D) and 8 nucleotide deletions (E) were done on mE3. Note that in D and E, the neomycin sites are located 3′ to the mutated E3 and were removed by in vitro Cre expression. F: a humanized conditional knockin strategy for NPARM analysis. mE3 is flanked by loxP sites. The neomycin cassette flanked by Frt sites was located between mE3 and the 3′ loxP site; this neomycin cassette was removed by interbreeding with flp-expressing mice. The humanized PHOX2B^Δ8 construct contains the human 3′-UTR and GFP after an internal ribosome entry site (IRES) sequence. After Cre-mediated recombination, proteins are generated from this locus: PHOX2B^Δ8 and green fluorescent protein (GFP). ALA, alanine; mE3, mouse exon 3.

Fig. 4.

Pedigrees of 2 PHOX2B mutation confirmed CCHS probands reported in literature with histopathological findings. A: family 1, NPARM. Note that the NPARM proband has a half-sibling deceased at 4 mo of age due to sudden infant death syndrome (with co-sleeping). B: family 2, PARM. The clinical history is complicated by a complex maternal obstetrical history, including 2 pregnancies not taken to term for unknown reasons. The PARM proband suffered preterm birth and was initially diagnosed with apnea of prematurity. This child could not become ventilator independent at ∼32–33 wk corrected postgestational age, an age at which central nervous system respiratory center maturation occurs. Squares represent males, circles represent females, triangles represent pregnancies not taken to term, and diagonal lines indicate deceased.