Dysregulation of locus coeruleus development in congenital central hypoventilation syndrome

Abstract

Human congenital central hypoventilation syndrome (CCHS), resulting from mutations in transcription factor PHOX2B, manifests with impaired responses to hypoxemia and hypercapnia especially during sleep. To identify brainstem structures developmentally affected in CCHS, we analyzed two postmortem neonatal-lethal cases with confirmed polyalanine repeat expansion (PARM) or Non-PARM (PHOX2B∆8) mutation of PHOX2B. Both human cases showed neuronal losses within the locus coeruleus (LC), which is important for central noradrenergic signaling. Using a conditionally active transgenic mouse model of the PHOX2B∆8 mutation, we found that early embryonic expression (<E10.5) caused failure of LC neuronal specification and perinatal respiratory lethality. In contrast, later onset (E11.5) of PHOX2B∆8 expression was not deleterious to LC development and perinatal respiratory lethality was rescued, despite failure of chemosensor retrotrapezoid nucleus formation. Our findings indicate that early-onset mutant PHOX2B expression inhibits LC neuronal development in CCHS. They further suggest that such mutations result in dysregulation of central noradrenergic signaling, and therefore, potential for early pharmacologic intervention in humans with CCHS.

Fig. 1

Genotype and respiratory physiological phenotypes of the proband and transgenic mouse model. a PHOX2B contains three exons and proband 8-nucleotide frameshift mutation in exon 3 is shown. Nucleotide number refers to nucleotide position of GenBank accession number CCDS 3463.1. Wild-type PHOX2B protein is 314 amino acids long; PHOX2B∆8 mutation results in loss of the 20-alanine repeat domain and generates a protein of 355 amino acids. b Polysomnographic recording from the proband while on Synchronized Intermittent Mandatory Ventilation (SIMV) and after switching to continuous positive airway pressure (CPAP). Note the rapid decrease in oxygen saturation (blue arrow) and increase in carbon dioxide (pink arrow) levels. Values of E_TCO₂ at each epoch shown. When challenged with persistent hypercarbia and hypoxemia during CPAP, the proband showed no increase in respiratory effort. Heart rate variability during the challenge was minimal. EKG electrocardiogram, SpO ₂ oxygen saturation, E _T CO ₂ end-tidal CO₂, Nasal nasal airflow, Chest chest wall movements from respiratory inductance plethysmography. CPAP pressure of 5 cm H₂O was used. SIMV rate was 45 breaths/min. “Early” refers to 45 s after switching to CPAP, “late” refers to 75 s after switching to CPAP. Time scale is shown. c Targeting construct of patient-specific mouse model. Human PHOX2B exon 3 containing patient-specific PHOX2B mutation (denoted in blue color) is inserted following unmodified, non-mutated mouse Phox2b exon 3 flanked by loxP sites, to allow conditional expression of mutant gene by cre recombinase. For detailed generation of transgenic mouse line see Figure S4. d Endogenous respiratory output. Integrated C4 inspiratory activity from E18.5 control and Hprt-cre, Phox2b∆8 mutant mice under baseline (left) and stimulated (1 μM substance P, right) conditions. Note lack of response in Phox2b∆8 mouse brainstem (n = 4, a representative recording shown)

Fig. 2

Brainstem pathology in CCHS probands. a Cartoon of human hindbrain at levels of pons (pink) and medulla (blue) is shown. b Dramatic losses were observed in NPARM PHOX2BΔ8 proband locus coeruleus (LC), dorsal median raphe (MnR), mesencephalic trigeminal nucleus (MesV) and dorsal motor nucleus of vagus (DMNV). Note lack of dopamine β-hydroxylase (DBH) expression in LC and tryptophan hydroxylase (TrypH) in dorsal MnR indicating defects in synthesis of noradrenaline and serotonin production. DMNV showed diminished cholinergic neurons, indicated by choline acetyltransferase (CHAT) expression. Asterisk indicates diminished MesV fibers originating from the nucleus in the proband. H&E hematoxylin and eosin. c Dramatic loss was observed in PARM PHOX2B 20/27 proband LC. Note a significant reduction of DBH expression

Fig. 3

Brain pathology in Hprt-cre, Phox2b∆8 mouse. Top Cartoon of mouse hindbrain at levels of rostral (pink) and caudal (blue) hindbrain is shown. a–d The Hprt-cre, Phox2b∆8 mutant mouse showed profoundly abnormal differentiation of LC, characterized by absent expression of tyrosine hydroxylase (TH). Additional abnormalities were diminished MesV (Brn3) and DMNV Choline Acetyltransferase (CHAT) neurons. In contrast to NPARM PHOX2BΔ8 proband (see Fig. 2b), the dorsal MnR in Hprt-cre, Phox2b∆8 mouse showed non-significant reduction in counts of TrypH and 5HT cells compared with controls (n = 3). V, 4th ventricle. Scale bar unit µm

Fig. 4

Noradrenergic neurons are affected by stage-specific activation of PHOX2B∆8. a Left panels the specification of noradrenergic neurons in LC appears by E10.5 in control, which express TH in addition to Phox2b. In Hprt-cre fate-mapped early-onset mice, the TH expression is diminished despite available Phox2b+ precursors. Note expression of PHOX2B∆8 protein (GFP+) in the same cells. Blbp-cre fate-mapped late-onset mice showed normal TH expressions and PHOX2B∆8 is not expressed. Right panels by E11.5, cre-activation in late-onset mice commences in noradrenergic neurons, shown by GFP+ (n = 3–4). Note that mouse E10.5 and E11.5 are roughly equivalent to human gestational age week 6 and week 7, respectively. b Central noradrenergic neurons affected by early-onset Hprt-cre, Phox2b∆8 were pervasive at E18.5 C-section including hindbrain nuclei LC and A1 and forebrain projection to periventricular nucleus of the hypothalamus. In the late-onset Blbp-cre, Phox2b∆8, noradrenergic neurons were intact in all areas at P0 (n = 3)

Fig. 5

Central chemosensor RTN and CNVII are dispensable for perinatal respiratory regulation. a–c Respiratory phenotype and central chemosensor of a control, b early-onset Hprt-cre, Phox2b∆8, and c late-onset Blbp-cre, Phox2b∆8 mice. Whereas Hprt-cre, Phox2b∆8 mice failed to show any respiratory effort, were cyanotic (dark violet skin tone), and died in the immediate perinatal period, Blbp-cre, Phox2b∆8 mice showed spontaneous respiration and were pink (n = 7–11 from at least 3 litters/genotype). Both Hprt-cre, Phox2b∆8 and Blbp-cre, Phox2b∆8 mice lacked the central chemosensor RTN (Phox2b+/Islet−, and NK1R+/Phox2b+) and CNVII (Phox2b+/Islet+), despite continuous respiration in Blbp-cre, Phox2b∆8 and lack of respiration in Hprt-cre, Phox2b∆8 (n = 3). d Quantification of RTN (Phox2b+/Islet−) and CNVII (Phox2b+/Islet+) showed significant losses of both nuclei at E18.5 (n = 3). e When mice were harvested at E18.5, no Hprt-cre, Phox2b∆8 mice showed continuous respiration in contrast to Blbp-cre, Phox2b∆8 and control mice (n = 7–11 from at least 3 litters/genotype). f Quantification of breathing rate at birth showed spontaneous/continuous breathing in Blbp-cre, Phox2b∆8 mice albeit at reduced frequency. Respiratory rate ranged 20–132 breaths/min depending on time post-birth, normalized to control within the same litter (n = 3–5). N/A not assessed due to absence of respiration

Fig. 6

Abnormal migration of facial nucleus CNVII in Hprt-cre, Phox2b∆8 mouse. a Neurons of CNVII migrate caudally from their origin in rhombomere (r)4 toward r6 followed by lateral migration toward ventral surface. Horizontal sections at E11.5 to visualize trans-rhombomere migration showed in Hprt-cre, Phox2b∆8 mouse, precocious termination of rostral-to-caudal migration (note accumulation of Phox2b+ Islet1+ cells at r4–5), and abnormal lateral migration within r4–5 (arrowheads) (V 4th ventricle, A anterior, P posterior, L lateral, M medial). b Top cartoon of mouse hindbrain levels of rostral (R) and caudal (C) at E14 is shown. At E13.5, stalled migration of CNVII/RTN, detected by Islet1 and Phox2b antibodies, was found at rostral levels (denoted by solid arrowheads) of hindbrain than normally found at caudal level (denoted by empty arrowheads) in control littermates. c The same pattern described in b was observed at E15.5, implicating permanent migration defect of CNVII. The number of putative Phox2b+ Islet1+ CNVII cells found in the mutants declined from 37.6 to 16.2 % of WT at E13.5 and E15.5, respectively. Scale bar unit µm