respiratory and autonomic dysfunction in congenital ... · with phox2b mutations have suggested rtn...

Review

CALL FOR PAPERS Neurological Disease and Autonomic Dysfunction

Respiratory and autonomic dysfunction in congenital central hypoventilationsyndrome

Thiago S. Moreira,1 Ana C. Takakura,2 Catherine Czeisler,3 and Jose J. Otero3

1Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil;2Department of Pharmacology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil; and 3The OhioState University, College of Medicine, Department of Pathology, Division of Neuropathology, Columbus, Ohio

Submitted 8 January 2016; accepted in final form 25 May 2016

Moreira TS, Takakura AC, Czeisler C, Otero JJ. Respiratory and autonomicdysfunction in congenital central hypoventilation syndrome. J Neurophysiol 116:742–752, 2016. First published May 25, 2016; doi:10.1152/jn.00026.2016.—Thedevelopmental lineage of the PHOX2B-expressing neurons in the retrotrapezoidnucleus (RTN) has been extensively studied. These cells are thought to function ascentral respiratory chemoreceptors, i.e., the mechanism by which brain PCO2

regulates breathing. The molecular and cellular basis of central respiratory chemo-reception is based on the detection of CO2 via intrinsic proton receptors (TASK-2,GPR4) as well as synaptic input from peripheral chemoreceptors and other brainregions. Murine models of congenital central hypoventilation syndrome designedwith PHOX2B mutations have suggested RTN neuron agenesis. In this review, weexamine, through human and experimental animal models, how a restricted numberof neurons that express the transcription factor PHOX2B play a crucial role in thecontrol of breathing and autonomic regulation.

chemoreflex; CCHS; ventrolateral medulla; breathing

CEREBRAL AUTONOMIC NEURON DYSFUNCTION would be expected tolead to neurological disease. Such is the case with dysfunctionof cells in the retrotrapezoid nucleus (RTN). Seminal experi-ments underscore the importance that RTN neurons play inneurophysiological homeostasis. In 2003, Amiel and col-leagues published the first evidence that polyalanine expan-sions of the paired-like homeobox gene PHOX2B cause centralcongenital hypoventilation syndrome (CCHS) (Amiel et al.2003; Weese-Mayer et al. 2005). CCHS patients suffer lowerventilatory responses to hypercapnia and hypoxia comparedwith normal controls (Weese-Mayer et al. 2005). The proposedmechanism centers on a selective lack of central chemosensoryintegration rather than respiratory central pattern generator(rCPG) dysfunction (Ikeda et al. 2015; Ruffault et al. 2015;Stornetta et al. 2006). In these patients, breathing may beadequate during wakeful states with ventilation increasingnormally during volitional stimulation or exercise (Diedrich etal. 2007). However, when asleep, CCHS patients experiencelife-threatening hypoventilation or complete apnea. In short,the symptoms of CCHS support the existence of neurons thatselectively mediate the chemical drive to breathe and suggestthat these neurons are especially important during sleep.

In rodents, PHOX2B expression is required for the develop-ment of the nucleus of the solitary tract (NTS), brain stemcatecholaminergic neurons, carotid bodies, enteric nervoussystem, sympathetic ganglionic (but not preganglionic) neu-rons, and the cranial parasympathetic system but is not re-quired for the development of the serotonergic system (Fig. 1)(Ikeda et al. 2015; Ruffault et al. 2015). PHOX2B expressionpersists in most brain stem structures after birth (Dauger et al.2003), including the hypercapnia-sensitive neurons of the RTNand the NTS neurons that mediate the peripheral chemoreflex(Stornetta et al. 2006; Takakura et al. 2014). In short, anuninterrupted neuronal chain involved in chemosensory inte-gration express PHOX2B, whereas the rCPG itself lacksPHOX2B expression. This expression pattern clearly fits withthe symptoms of CCHS (normal day-time breathing but greatlyreduced peripheral and central chemoreflexes). Furthermore,from a basic science standpoint, the presence of PHOX2B inthe RTN and its absence from the rCPG provides an importantclue that the respiratory network’s pH sensitivity relies heavilyon specialized neurons that drive the rCPG, not on the rCPGitself (Guyenet and Bayliss 2015). Indeed, the presence ofPHOX2B-positive RTN neurons underscores a compellingdisease-based rationale to elucidate their roles in breathing.

Central respiratory chemoreception mechanisms. Recentliterature describes at least four prevalent theories on the celltypes responsible for mediating central respiratory chemore-

Address for reprint requests and other correspondence: T. S. Moreira, Dept.of Physiology and Biophysics, Institute of Biomedical Science, Univ. of SãoPaulo, Av. Prof Lineu Prestes, 1524, 05508-900, São Paulo, SP, Brazil (e-mail:[email protected]).

J Neurophysiol 116: 742–752, 2016.First published May 25, 2016; doi:10.1152/jn.00026.2016.

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ception. These theories attribute chemosensation to 1) special-ized respiratory chemoreceptors, 2) broad-spectrum chemore-ceptors, 3) ubiquitous chemoreceptors, and 4) pH-insensitiveneurons (Guyenet 2008; Guyenet et al. 2010; Guyenet and

Bayliss 2015; Nattie 2011). In the present review, we focus onthe role of specialized respiratory chemoreceptors. The che-mosensitivity process refers to the intrinsic pH sensitivity ofbrain cells including neurons, glia, and blood vessels, whereas

Fig. 1. Schematic view of the control of CO2 homeostasis and the role of PHOX2B-expressing neurons. Retrotrapezoid nucleus (RTN) chemoreceptor neuronsare activated by CO2 via their intrinsic pH sensitivity and via inputs from the carotid bodies. RTN neurons target various components of the respiratory centralpattern generator (rCPG) and are presumed to play a key role in breathing automaticity during anesthesia, sleep, and quiet waking. The carotid body may alsoinfluence the activity of the rCPG neurons through connections (not shown) that bypass the RTN (Stornetta et al. 2006; Takakura et al. 2006). RTN neurons arealso inhibited by lung inflation via slowly adapting receptor (SAR) activation and by the fact that the effect of SARs is mediated by GABAergic pump cellslocated within the nucleus of the solitary tract (NTS) at area postrema level (Moreira et al. 2007). In mammals, PHOX2B expression is required for thedevelopment 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 thecranial parasympathetic system but is not required for the serotonergic system or the rCPG neurons (5) (Stornetta et al. 2006). 1, Illustration showing theexpression of PHOX2B immunoreactive in the carotid body between E13.5 and E16.5 in mice. [Modified from Dauger et al. (2003).]. 2, Awake rats subjectedto hypoxia and hypoxia-activated NTS neurons were identified by the presence of Fos-immunoreactive nuclei. The brain tissue was processed for simultaneousdetection of FluorGold (FG; native blue fluorescence), PHOX2B immunoreactivity (Alexa 488 fluorescence; green), Fos immunoreactivity (Cy3; red). Thephotomicrograph taken within the commissural portion of the NTS shows multiple aqua-colored neurons (arrows) with RTN projections, activated by carotidbody stimulation and expressing PHOX2B. The color coding for other combinations of markers is indicated. Scale bar, 50 �m. [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 �m. [Modified from Stornetta et al. (2006).] 4, Example of one RTNchemoreceptor 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 forPHOX2B immunoreactivity (Cy3; red) revealing that the recorded neuron (Alexa 488; green) was PHOX2B negative. Scale bar, 50 �m. [Modified from Stornettaet al. (2006).]

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respiratory chemoreception, commonly called central chemo-reception, is the process by which central nervous system(CNS) PCO2 drives respiration (Feldman et al. 2003; Guyenet etal. 2010; Guyenet and Bayliss 2015; Nattie 2011). Thus respi-ratory chemoreception refers both to a subset of chemosensi-tive cells, the respiratory chemoreceptors, and to their relation-ship to a specific neuronal network, the breathing network.Intracellular pH changes mediate neuronal pH sensitivity invitro (Kumar et al. 2015; Mulkey et al. 2004; Ritucci et al.2005; Wang et al. 2013), presumably attributed to the simul-taneous effects of pH on multiple ion channels (Kumar et al.2015; Putnam et al. 2004). The correct identification of rele-vant central chemoreceptors is complicated by the fact thatbrain stem neurons very commonly respond to extracellularfluid acidification in vitro and that, equally common, some pHresponses persist after blockade of action potential-dependenttransmitter release with tetrodotoxin (Bayliss et al. 2001;Kumar et al. 2015; Mulkey et al. 2004; Wang et al. 2013). Suchresults have led others to theorize that all rCPG neurons couldbe equally sensitive to pH and to conclude that the entirerespiratory network possesses central respiratory chemosensi-tivity. That CCHS patients show a virtual loss of centralrespiratory chemoreception without impairment of daytimebreathing challenges the view that the entire rCPG showschemosensitive properties. An alternative view to which wesubscribe is that the widespread pH sensitivity of rCPG neu-rons observed in vitro does not explain central respiratorychemoreception. Rather, central chemoreception relies on spe-cialized neurons (Loeschcke 1982; Mitchell et al. 1963) thatare responsible for the highly sensitive respiratory outputresulting from brain pH changes (Eldridge et al. 1984). Wepropose that RTN neurons exemplify such cells and that others(locus coeruleus, medullary raphe, and the NTS) are yet to befound, presumably within brain stem regions where focalacidification in vivo activates breathing (Biancardi et al. 2008;Dean et al. 1989; Feldman et al. 2003; Guyenet et al. 2010;Guyenet and Bayliss 2015; Nattie 2011; Richerson 2004).

Chemoreceptors of the RTN. The respiratory stimulant ef-fects of ventral medullary surface (VMS) acidification and thedepressant effects caused by VMS cooling or topical applica-tion of inhibitory neurotransmitters lead to the hypothesis thatrespiratory chemoreception occurs near the VMS (Loeschcke1982; Millhorn 1986; Millhorn and Eldridge 1986; Mitchell etal. 1963). The most notable chemosensitive region of the VMSis located roughly ventral to the facial motor nucleus andoverlaps the region that was defined in 1989 as the RTN (Smithet al. 1989). Smith and colleagues defined the RTN as extend-ing from the caudal end of the trapezoid body to the Bötzingercomplex level of the ventral respiratory column (VRC) andfrom the lateral edge of the pyramids to the trigeminal tract(Smith et al. 2013). Classical lesion/stimulation experimentssupport the notion that an excitatory drive to the breathingpattern generators derives from RTN neurons (Nattie 2011;Nattie and Li 2000; Takakura et al. 2013). Furthermore, RTNacidification in vivo increases breathing, whereas inhibition ofthe same area with drugs or neuronal lesions reduces breathingand central chemosensitivity (Nattie 2011; Nattie and Li 2000;Takakura et al. 2008, 2013, 2014). Finally, Fos induction in theRTN occurs when animals or brain stem preparations areexposed to hypercapnia (Fortuna et al. 2009; Sato et al. 1992);these data suggest hypercapnia directly activates RTN cells. In

addition, pathological evidence obtained from polyalanine re-peat mutation (PARM) CCHS animal models supports thenotion that central respiratory chemosensitivity relies on rela-tively few specialized neurons, such as the chemoreceptorneurons of the RTN (Dubreuil et al. 2008; Ramanantsoa et al.2011). In this genetic disease, central chemosensitivity isessentially absent; severe hypoventilation occurs during sleepbut relatively adequate breathing persists during waking, andsome degree of exercise-induced hyperventilation remains, atleast in the milder cases (Amiel et al. 2003, 2009; Carroll et al.2010; Gozal, 1998; Shea et al. 1993). The severity of thesymptoms increases according to the number of extra alanineresidues in the mutated polyalanine track of PHOX2B (Carrollet al. 2010). The respiratory symptoms of the disease suggestthat the central respiratory controller located in the pre-Bötz-inger complex of CCHS patients is largely intact but that thebrain of these individuals lacks neurons that are either uniquelyspecialized in detecting CO2 or are specialized in funneling theexcitatory influence of CO2 to the respiratory controller. Themouse models of the disease suggest that these missing neu-rons could be the RTN neurons (Dubreuil et al. 2008; Goridiset al. 2010; Nobuta et al. 2015; Ramanantsoa et al. 2011).

Chemoreceptor neurons in the RTN during development andin adult mammals. Lineage-tracing experiments suggest thatthe RTN originates from an Egr-2-dependent embryonic do-main (rhombomeres 3/5) and coexpresses Atoh-1 and PHOX2Bduring late embryogenesis (Dubreuil et al. 2009; Ramanantsoaet al. 2011). Proper RTN development depends on the afore-mentioned transcription factors (Huang et al. 2012; Ruffault etal. 2015). Perinatally, RTN neurons seem to retain many oftheir embryonic characteristics, i.e., their pacemaker proper-ties, pH modulation, and ability to activate the pre-Bötzingercomplex (Fortin and Thoby-Brisson 2009; Onimaru et al. 2008,2014; Onimaru and Homma 2003; Thoby-Brisson et al. 2009).In adult rats, RTN neurons function as central chemoreceptorsby controlling multiple aspects of breathing including fre-quency, depth of inspiration, active expiration, and airwaypatency (Abbott et al. 2013; Guyenet and Bayliss 2015; Silvaet al. 2016; Takakura et al. 2014). Several key pieces ofevidence support the role of RTN neurons as central chemo-receptors. First, these neurons respond with a massive increasein activity to hypercapnia in vivo (Mulkey et al. 2004;Takakura et al. 2006). The discharge activity rate of RTNchemoreceptors neurons varies by an average of �3.0 Hz for a1% change in arterial PCO2 (PaCO2

) (Guyenet et al. 2005).These cells are silent at low levels of CO2, and most of theRTN cells have a threshold (CO2 level above which the cellsare active) that is below that of the respiratory output (Guyenetet al. 2005; Takakura et al. 2006). In addition to their extremepH sensitivity, it is also important to point out that RTNneurons in vivo presumably rely on a combination of synapticdrives (peripheral chemoreceptors, raphe, hypothalamic neu-rons, and astrocytes) and intrinsic pH sensitivity (Barna et al.2014; Guyenet and Bayliss 2015; Nattie and Li 2002; Takakuraet al. 2014). We believe that such integrative characteristics areexpected from neurons (central chemoreceptors) that drive therespiratory system.

In summary, multiple studies performed on RTN neuronssupport a role in central chemosensation and include 1) theirintrinsic acid sensitivity (Kumar et al. 2015; Wang et al. 2013)and 2) their integration of excitatory inputs from the peripheral

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chemoreceptors, raphe, and hypothalamus and from neighbor-ing astrocytes that modulate respiratory drive (Barna et al.2014; Fortuna et al. 2009; Moreira et al. 2015; Nattie 2011;Richerson 2004; Takakura et al. 2006; Wenker et al. 2010).

CCHS: insights into pathophysiology from clinical, molec-ular, and genetic studies. CCHS was first described by modernmedicine in 1970 (Mellins et al. 1970) and is classicallycharacterized as a genetically inherited disorder with alveolarhypoventilation and monotonous respiratory rates despite ab-normal PaCO2

and pH concentrations, especially during non-rapid eye movement (NREM) sleep (Patwari et al. 2010).CCHS patients fail to arouse from sleep despite progressivehypercapnia or hypoxemia. Amiel et al. (2003) first describedthat mutations in the homeobox transcription factor PHOX2Bcause CCHS. The inheritance is considered overall most con-sistent with autosomal dominant inheritance pattern. However,cases of parental mosaic PHOX2B status or incomplete pen-etrance have characterized some families (Trochet et al. 2008).The mechanisms of variable penetrance are unknown but havebeen postulated to be due to other modifier mutations that mayoccur concurrently in CCHS patients. For example, mutationsin HASH1 lead to worsening noradrenergic phenotypes inexperimental animals with CCHS mutations (Amiel et al.2003; de Pontual et al. 2003). Furthermore, in addition toHASH1, other pertinent co-mutations found in some CCHSpatients include RET, GFRA1, and PHOX2A (Sasaki et al.2003). Family members of CCHS patients have a higherincidence of autonomic nervous system dysfunction,Hirschprung’s disease, neuroblastoma, and sudden infant deathsyndrome (Berry-Kravis et al. 2006; Devriendt et al. 2000;Weese-Mayer et al. 1993).

In the United States, CCHS clinical diagnostic and manage-ment guidelines are rendered by the American Thoracic Soci-ety (ATS). Specifically, in patients with alveolar hypoventila-tion, a documented PHOX2B mutation represents the sine quanon for CCHS diagnosis (Weese-Mayer et al. 2010). PHOX2B(human chromosome 4p12) has a 20-alanine stretch in exon 3.CCHS-causing mutations include 1) polyalanine repeat muta-tions (PARM) in the 20-alanine repeat of exon 3 resulting inpolyalanine tracts ranging from 25–35 alanines, or 2) non-polyalanine repeat mutations (NPARM) resulting in exon 3deletions that cause frameshift mutations, missense mutations,or nonsense mutations (Weese-Mayer et al. 2003). Of note,PHOX2B mutations resulting in loss of alanines manifest asPHOX2B20/15 genotypes; these mutations exist in 1–1.5% ofthe general population and are not diagnostic of CCHS even ifthe patient suffers hypoventilation (Weese-Mayer et al. 2003).ATS guidelines recommend biannual and then annual in-hospital comprehensive evaluations during awake and asleepstates so that ventilatory needs during all stages of sleep and awide range of activity can be determined. These comprehen-sive evaluations include 1) 72-h Holter monitoring, 2) echo-cardiogram, 3) autonomic nervous system (ANS) evaluationacross other organs, and 4) neurocognitive evaluation. Theseguidelines represent an attempt to decrease acute episodes ofhypoventilation. However, the extent to which long-term au-tonomic dysfunction affects other outcomes, including neuro-cognitive state or other quality of life measures, is unclear.

CCHS is a rare genetic disorder, yet its exact incidence andprevalence is unknown. Using the definition originally pro-posed by Trang et al. (2005), the French CCHS working group

estimated an incidence of 1 in 200,000 births. Furthermore,incidence may vary with ethnicity, with some studies demon-strating up to 90% of their affected CCHS population beingCaucasian (Weese-Mayer et al. 2010). To date, no study hassuggested a gender bias in CCHS. Accurate epidemiologicalCCHS data utilizing ATS diagnostic criteria have not beengenerated throughout the world. Further confounding accuratestatistics, surveys have shown loose adherence to ATS guide-lines in North America (Amin et al. 2013). In summary, CCHSrepresents a rare genetic disorder without gender bias, withmost experts believing that the incidence will increase if theATS diagnostic criteria become widely implemented.

CCHS patients suffer a spectrum of severity. Continuousventilatory support is required in 35% of PARM patients and67% of NPARM patients (Weese-Mayer et al. 2010). Forexample, patients with a PHOX2B20/25 phenotype never need24-h ventilatory support. In contrast, patients with PHOX2B20/28-

20/33 typically require continuous ventilatory support and patientswith PHOX2B20/26 show variable awake needs depending onactivity (Berry-Kravis et al. 2006; Matera et al. 2004; Weese-Mayer et al. 2003). Patients with PHOX2B20/25 have the mildestproblems, may present only after exposure to respiratory depres-sants or severe respiratory infection, and are typically managedwith nocturnal ventilatory support (Antic et al. 2006; Repetto et al.2009; Weese-Mayer et al. 2005). Other ANS manifestationscorrelate with PHOX2B genetic aberrations. For example, studiesmeasuring cardiac asystole in CCHS patients demonstrate nosinus pauses of �3 s in patients with PHOX2B20/25, whereas thisis found in 19% and 83% of patients with PHOX2B20/26 andPHOX2B20/27, respectively. Hirschprung’s disease occurs in 19%of PARM patients but in 80% of NPARM patients; neural cresttumors are found in 1% of PARM patients and 41% ofNPARM patients. In summary, when PARM clinical pheno-types are compared, polyalanine expansion positively corre-lates with disease severity. Furthermore, although alveolarhypoventilation and neural crest tumors are clinically criticalevents, other sequelae of long-term autonomic dysfunction areunknown and are not addressed in current clinical managementguidelines.

The molecular mechanisms by which PHOX2B mutationscause dysfunction are not entirely known. Molecular studies donot indicate that these PHOX2B mutations yield simple “dom-inant negative” proteins. Bachetti et al. (2005) demonstratedthat polyalanine expansions cause aggregate formation andreduced transcriptional activities. These findings are widelyreproducible and are illustrated in Fig. 2A. The PHOX2Apromoter, a direct target of PHOX2B, can be tested in aluciferase transcriptional assay. A PHOX2B frameshift muta-tion (PHOX2B�8) characterized by an eight-nucleotide dele-tion found in a CCHS case (Otero et al. 2011) shows robustactivation of the PHOX2A promoter, whereas a polyalanineexpansion mutation shows no such activation. The dopamine�-hydroxylase promoter, a direct target of both PHOX2B andPHOX2A proteins, can be activated by PHOX2B andPHOX2A. Of note, synergistic activation can be achieved bycotransfection of PHOX2A � PHOX2B or PHOX2A �PHOX2B�8. Overexpression of PHOX2B-polyalanine(PHOX2B-PA) in 293FT cells results in localization of thistranscription factor in the cytoplasm, at times as a discoidaggregate (Fig. 2, B3 and B4); PHOX2B and PHOX2B�8 showonly nuclear localization in such assays. As determined by

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proximity ligation assay (Fig. 2C), both PHOX2B-PA andPHOX2B�8 are capable of binding to PHOX2A and PHOX2B;of note, PHOX2B-PA interaction can occur in nucleus andcytoplasm. In summary, although both NPARM and PARMmutations cause alveolar hypoventilation, these proteins likelyinduce the defects through distinct molecular mechanisms ashas been postulated by Wu et al. (2009). These distinct mo-lecular mechanisms may mediate the varying clinical pheno-types observed between PARM and NPARM patients.

Experimental models of CCHS and comparison to humanpathology. CCHS pathobiology research is unique in thatmurine experimental models of the disease were generatedbefore the extent of human anatomic pathology was fully

understood. This was because, in addition to CCHS being ahighly rare disease, patients with CCHS usually die in child-hood or late adulthood. It is therefore difficult for health careproviders to discuss consents to medical autopsies in thisdisease because of the sensitivity required for a normal griev-ing process. Nevertheless, animal models have yielded signif-icant insights. One of the first clues that the embryonic loss ofRTN neurons leads to breathing abnormalities came fromstudies of ATOH1 knockout mice. Such ATOH1�/� mice showablation of the ATOH1 locus and die due to breathing problemsshortly after birth (Ben-Arie et al. 1997). Indeed, localizationof RTN neurons in the ventrolateral medulla and the RTN’sventral respiratory column connections requires ATOH1

C PROXIMITY LIGATION PLA 2A:2B PLA 2A:PA PLA 2A: 8

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Fig. 2. Summary of molecular studies of PHOX2B mutation function. A: luciferase assay of PHOX2A promoter (top) and dopamine �-hydroxylase (DBH;bottom). Y-axis shows relative fluorescence units normalized to Renilla luciferase. Both PHOX2B and PHOX2B�8 show activation of this promoter. For the DBHstudies, both PHOX2B and PHOX2A cDNA constructs can transactivate the DBH promoter. Note that cotransfection of PHOX2A�PHOX2B as well asPHOX2A�PHOX2B�8 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 forimmunofluorescent staining at bottom left of each panel. Cells were transfected with PHOX2B, PHOX2B-PA, or PHOX2B�8 cDNA and immunostained usingNH2-terminal (N-TERM) or COOH-terminal (C-TERM; which cannot detect PHOX2B�8) PHOX2B antibodies (Santa Cruz Biotechnology), c-Myc epitope tagantibody (MYC; in frame with PHOX2B and PHOX2B-PA reading frame but not in frame with PHOX2B�8 reading frame). Cells are counterstained with DAPIin blue, and colocalization of both antigens is indicated in yellow. Note that PHOX2B-PA shows significant cytoplasmic localization (B3 and B4), whereasPHOX2B and PHOX2B�8 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 andantibodies 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 identifyhistologically whether 2 proteins interact. PLA signal is red, and cells are counterstained with DAPI in blue. The transfected constructs in which PLA reactionis performed are shown at top right of each panel. PLA demonstrates nuclear localization of interactions between PHOX2A and PHOX2B (C1), PHOX2A andPHOX2B�8 (C3), and PHOX2B and PHOX2B�8 (C5). In contrast, significant cytoplasmic localization is noted between PHOX2A and PHOX2B-PA (C2) andPHOX2B-PHOX2B-PA (C4) interactions. C6 shows interactions of PHOX2B�8 with a separate PHOX2B�8 protein, indicating that NPARM mutations canhomodimerize.

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(Huang et al. 2012). The age of CCHS transgenic mousebiology emerged with the publication of a seminal manuscriptby Dubreuil et al. (2008) whereby they achieved a knockin tothe PHOX2B locus of a modified exon 3 harboring a 7-alanineexpansion (Fig. 3B). This results in embryonic developmentwith all cells showing the PHOX2B20/KI-27 genotype, which isthe most common PHOX2B mutation in CCHS patients. Theseanimals had unique breathing abnormalities. Specifically, 3 of18 animals showed intermittent gasping at room air, with theremainder showing a continuum of rhythmic breathing abnor-malities ranging from normal (albeit slower) rhythmic breath-ing rates to intermittent interruption by apneic episodes. How-ever, fully penetrant in all of the PHOX2B20/KI-27 animals is alack of responsiveness to hypercapnia. These animals showloss of RTN neurons and precursor populations by embryonicday 15.5 (E15.5), with other areas, including the locus coer-uleus, A5, and carotid bodies, showing no significant develop-mental pathology.

Although abnormal breathing was not a primary outcome oftheir work, additional insights in CCHS pathology were ob-tained from transgenic mice generated by Nagashimada et al.(2012), who focused their analyses on NPARM of PHOX2B.These mice were constructed to have deletions of 5 nucleotidesand 8 nucleotides in PHOX2B exon 3 (Fig. 3D for PHOX2B20/

Ki-�5 and Fig. 3E for PHOX2B20/Ki-�8). Both genotypes showsignificant defects in enteric nervous system development and

other peripheral nervous system structures. Interestingly, bothgenotypes also show loss of PHOX2B-positive neurons in theventrolateral medulla, implying RTN neuron hypoplasia. How-ever, these mice differed in their effects on the 7th cranialnerve nucleus, which was completely ablated in thePHOX2B20/Ki-�8 animal yet intact in the PHOX2B20/Ki-�5. De-livery by caesarean section at E18.5 showed that pups fromboth genotypes suffer from persistent cyanosis, which suggeststhat these mice had trouble initiating breathing at room air.

The above knockin models of PARM and NPARM CCHSsuffer the caveats that the experimental mice show significantdefects at birth, which precludes the ability to propagate thesemice. Several idiosyncrasies about the PHOX2B locus requirespecial consideration for the generation of conditional experi-mental models of PARM CCHS (Ramanantsoa et al. 2011) andNPARM CCHS (Nobuta et al. 2015). The primary structures ofmouse and human PHOX2B protein are identical, yet signifi-cant differences exist at the genomic level. This is important toconsider, because frameshift mutations, such as thePHOX2B20/Ki-�8, would generate murine C-terminal fragmentscompletely different from the human disease if the deletionwere engineered in mouse exon 3. Additional differences in-clude differences in the 3=-untranslated region (UTR) betweenmice and humans. Luckily, the 3= splice acceptor site forexon 3 is 100% identical between mouse and human genes,which facilitated the genetic design of conditional, human-

Fig. 3. Genetic design of CCHS-type mutations in mouse experimental models. A: the gene structure of PHOX2B, located on mouse chromosome 5, is composedof 3 exons. B: knockin mice were generated with an extra 7 alanines to generate the PHOX2B20/Ki-27 genotype. The neomycin resistance cassette was removedby interbreeding chimeric mice with PGK-cre mice. Experiments were performed with neomycin cassette-intact and neomycin cassette-ablated mice to ensurethat the neomycin cassette did not affect the phenotype. C: humanized PHOX2B20/27 conditional knockout mouse design. mE3 is flanked by loxP sites. Theneomycin 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 humanizedE3 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 onmE3. 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 knockinstrategy 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; thisneomycin cassette was removed by interbreeding with flp-expressing mice. The humanized PHOX2B�8 construct contains the human 3=-UTR and GFP after aninternal ribosome entry site (IRES) sequence. After Cre-mediated recombination, proteins are generated from this locus: PHOX2B�8 and green fluorescentprotein (GFP). ALA, alanine; mE3, mouse exon 3.

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ized PHOX2B PARM (Fig. 3C, hereafter referred to asPHOX2B20/E3-cKi-27) and NPARM (Fig. 3F, hereafter re-ferred to as PHOX2B20/E3-cKi-�8-ires-GFP) experimental mod-els. These studies were then able to express the mousemutants either in germline with PGK-cre (Lallemand et al.1998) or HPRT-cre mice (Tang et al. 2002), in the nervoussystem with Brn4-cre (Ahn et al. 2001) and BLBP1-cre mice(Hegedus et al. 2007), or in rhombomeres 3 and 5, whichgive rise to RTN neurons (Ramanantsoa et al. 2011), withEgr2-cre mice (Voiculescu et al. 2000).

The most instructive information on respiratory physiology canbe obtained from the PARM PHOX2B20/E3-cKi-27 studies. BothPGK-cre, PHOX2B20/E3-cKi-27 and Brn4-cre, PHOX2B20/E3-cKi-27

showed findings similar to the PHOX2B20/KI-27 genotype, includ-ing perinatal lethality. To focus the pathological misexpression inRTN neurons, PHOX2B20/E3-cKi-27 mice were then bred to Egr2-cre, which induces Cre-mediated recombination in rhombomeres3/5. These mice suffer a massive loss of RTN neurons withnormal phrenic nerve root activity, indicating that downstreamcomponents of the RTN neural network are intact. Marked lack ofchemosensation occurs up until postnatal day 9; these mice alsoshow reduced mean ventilation in normoxia and a mild metabolicacidosis. Switching the animals to 100% oxygen leads to deepbreaths interrupted by prolonged apneas, underscoring that themain defect in respiratory drive is in the CO2 detection cells.

The above-described findings in PHOX2B20/E3-cKi-27 micedemonstrate a clear link between chemosensation andPHOX2B mutations at a functional and neuroanatomical level.However, complicating the translation of these findings intohumans is the fact that the anatomical location of the humanRTN remains elusive. One study, based on PHOX2B immu-nohistochemistry, suggested that the human RTN is locatedbetween the 7th cranial nerve and the superior olivary nuclearcomplex (Rudzinski and Kapur 2010); however, this interpre-tation is fraught with caveats. Specifically, the RTN is princi-pally defined functionally, and PHOX2B immunohistochemis-try shows diffuse immunoreactivity in the brain stem. There-fore, immunohistochemistry-based analyses are insufficient todetermine with certainty RTN-like phenotypes in human brainstem sections. In addition, PHOX2B immunohistochemistry isnotoriously inconsistent in human postmortem brain stem ap-plications. Thus the hypothesis that the human RTN is locatedbetween the 7th cranial nerve and superior olivary nuclearcomplex cannot be accepted or rejected using immunohisto-chemical approaches. Further understanding of the human neu-ropathology of CCHS is thus required determine the extent towhich human clinical phenotypes are caused by RTNdysfunction.

The first human neuropathological reports of CCHS began in1997, before the discovery of PHOX2B mutations; these pa-tients were classified as CCHS due to clinical criteria of thetime (Cutz et al. 1997). These patients showed severe loss ofthe carotid bodies and a decrease in dense core vesicles ofglomus cells, with an increase in the number of sustentacularcells estimated at up to a twofold increase above normal. In thelung, these patients showed increased frequency and size ofneuroepithelial bodies. These findings shed light on the findingthat CCHS patients suffer reduced respiratory drive in thesetting of hypoxia, which was not identified in murine PARMexperimental models. Radiographic studies have shown con-currence of other brain stem malformations, including Chiari

type I malformations, in CCHS patients (Bachetti et al. 2006).Kumar et al. (2008) reported diffuse neuropathology in CCHSusing in vivo imaging modalities. Specifically, they measuredradial diffusivity and axial diffusivity, measures of myelinalterations and axonal injury, respectively. These patientsshowed increased axial diffusivity in the lateral medulla, andclusters of injured areas were noted from the dorsal midbrainthrough the basal pons. Tomcyz et al. (2010) evaluated histo-pathologically a case suspicious for CCHS (without a confir-matory PHOX2B mutation) and found significantly decreasedtyrosine hydroxylase staining in the locus coeruleus and cor-tical malformations. These cortical malformations included adevelopmentally inappropriate operculum, small superior tem-poral gyrus, an abnormally split posterior temporal gyrus,hyperconvoluted gyri, fused sulci, and focal abnormal lamina-tions in cortical layers I–IV.

The only histopathological findings of CCHS with con-firmed PHOX2B mutations was reported by Nobuta et al.(2015). This data set included two CCHS patients, one PARMand one NPARM (Fig. 4 for simplified pedigrees). TheNPARM patient suffered an 8-nucleotide deletion in PHOX2Bexon 3, whereas the PARM patient showed a PHOX2B20/27

phenotype. The NPARM patient’s pathology was significantfor markedly diffuse abnormalities. These included signifi-cantly atrophic locus coeruleus, decreases in the dorsal motor

Age: 4 months.Diagnosis: Sudden Infant Death Syndrome.

Age: 6 weeks (term delivery).Diagnosis: Congenital Central HypoventilationSyndrome.

A Family 1, NPARM

B Family 2, PARM

Age: 41 weeks corrected post-conception age.Diagnosis: 1. Congenital Central HypoventilationSyndrome 2. Grade II/III intraventricular hemorrhage 3. Premature birth (27 5/7 weeks)

Fig. 4. Pedigrees of 2 PHOX2B mutation confirmed CCHS probands reportedin literature with histopathological findings. A: family 1, NPARM. Note thatthe NPARM proband has a half-sibling deceased at 4 mo of age due to suddeninfant death syndrome (with co-sleeping). B: family 2, PARM. The clinicalhistory is complicated by a complex maternal obstetrical history, including 2pregnancies not taken to term for unknown reasons. The PARM probandsuffered preterm birth and was initially diagnosed with apnea of prematurity.This child could not become ventilator independent at �32–33 wk correctedpostgestational age, an age at which central nervous system respiratory centermaturation occurs. Squares represent males, circles represent females, trianglesrepresent pregnancies not taken to term, and diagonal lines indicate deceased.

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nucleus of the vagus nerve, absent mesencephalic trigeminalnucleus, decreased serotonergic median raphe neurons, de-creased dopamine �-hydroxylase-positive adrenal medullaryneurons, and marked colonic aganglionosis characterized byloss of enteric ganglia from 10 cm distal to the ligament ofTreitz to anus. Of note, the intestinal aganglionosis showedpathological features distinct from classical Hirschprung’s dis-ease. Specifically, the NPARM proband was remarkable forloss of both chromogranin-positive neurons and S100 protein-positive Schwann cells. In contrast, classical Hirschprung’spathology manifests histologically as loss of neurons withnormal to increased S100 protein-positive peripheral nerves.Unfortunately, the NPARM patient’s carotid body was notsampled. The PARM patient’s histopathology was also char-acterized by marked reduction in dopamine �-hydroxylase-positive neurons of the locus coeruleus. Other comorbiditiesfound in the PARM patient included marked hypoxic-ischemicencephalopathy, likely due to the patient’s premature birth. ThePARM patient showed an intact dorsal motor nucleus of thevagus nerve and a median raphe. However, diffuse brain stemastrogliosis was noted in the PARM patient with significantaccentuation in the median raphe. The PARM patient’s adrenalgland, intestine, and the carotid body were not sampled. In bothpatients, an intact facial nerve nucleus was identified.

Although these in vivo and postmortem analyses of CCHSpatients provide insight into the pathological processes af-flicted by CCHS patients, the nature of these does not allowone to incisively test hypotheses regarding which pathologicalprocesses represent developmental syndrome versus a devel-opmental sequence. For these reasons, we developed a human-ized NPARM murine experimental model based on theNPARM patients described by Nobuta et al. (2015). In thisexperiment, we used Hprt-cre interbreeding to evaluate theneuropathology of mice showing early recombination of thePHOX2B20/E3-cKi-�8-ires-GFP locus and compared these findingswith recombination in later stages of CNS development withBLBP1-cre mice. An added benefit of this design is that cellsthat express the PHOX2B�8 protein also show concurrentcytoplasmic GFP expression. We found that HPRT-cre,PHOX2B20/E3-cKi-�8-ires-GFP mice were not born at expectedMendelian ratios, and those that were died soon after birth.These mice showed intestinal aganglionosis and loss of theRTN and facial nerve nucleus. Of particular relevance was thatin HPRT-cre, PHOX2B20/E3-cKi-�8-ires-GFP mice, several NeuN-positive neurons colabeled for GFP and PHOX2B in an areaanatomically appropriate for the locus coeruleus, but thesecells did not show significant tyrosine hydroxylase staining.Recombining the PHOX2B20/E3-cKi-�8-ires-GFP in later neuraldevelopment by interbreeding with Blbp-cre mice showedpreserved tyrosine hydroxylase-positive locus coeruleus neu-rons. However, the RTN and the facial nerve nucleus ofBlbp-cre, PHOX2B20/E3-cKi-�8-ires-GFP mice were both essen-tially absent. Blbp-cre, PHOX2B20/E3-cKi-�8-ires-GFP mice wereviable at birth and maintained spontaneous breathing. Al-though respiratory physiology was not a primary endpoint ofthe NPARM PHOX2B20/E3-cKi-�8-ires-GFP studies, these studiesunderscore the requirement of noradrenergic neuron functionfor appropriate respiration at birth. In summary, locus coer-uleus pathology has been reported in two CCHS patients withconfirmed PHOX2B mutations, in one case suspicious forCCHS, and in a humanized NPARM model.

Conclusion. In the brain, we have a well-organized networkto maintain one of the most important systems to life, therespiratory system. One important task of the neural control ofbreathing is to maintain within normal ranges, most of the time,the levels of CO2 and pH in the body (Dempsey and Smith2014; Guyenet and Bayliss 2015; Smith et al. 2015). Centralrespiratory chemoreception is a mechanism by which verysmall changes in tissue CO2/H� produce very large changes inbreathing. One region that is involved in central respiratorychemoreception is the retrotrapezoid nucleus (RTN) (Guyenetet al. 2010; Guyenet and Bayliss 2015; Nattie 2011). Thisregion is located in the ventrolateral surface of the medullawith neurons that have a genetic lineage (derived from neuronsthat express PHOX2B, Atoh-1, and Egr-2) and express differ-ent phenotypes (PHOX2B, NK1 receptors, VGlut2, TASK-2,GPR4, and galanin but not GABA, glycine, acetylcholine, orcatecholamines) (Kumar et al. 2015; Lazarenko et al. 2009;Ruffault et al. 2015; Takakura et al. 2008, 2014; Wang et al.2013). In this concise review, we discuss, on the basis ofclinical and genetic studies in human respiratory disease andevidence from neurophysiology and experimental animal mod-els, how a restricted number of neurons, located in the VMS,expressing and depending on the PHOX2B transcription factorcould be essential in the control of breathing and autonomicregulation. The neurons in the VMS named RTN chemorecep-tors neurons are considered the principal central respiratorychemoreceptors (Guyenet and Bayliss 2015). Such knowledgewill certainly enable new approaches (genetics, physiology,pharmacology, and biochemistry) to elucidate how the breath-ing networks are assembled and configured in normal andpathological conditions. Elucidating the mechanisms by whichthese neural networks regulate respiratory control represents afertile area for future investigation. Furthermore, current un-answered questions regarding clinical presentation of CCHSinclude 1) determining the genetic mechanisms to CCHS’sknown variable penetrance, 2) developing uniform diagnosticand patient management guidelines to improve prognosis andquality of life for patients with CCHS, and 3) determining theextent to which long-term suboptimal management of ventila-tor drive contributes to other neurocognitive/neuropsychiatricmanifestations of the disease.

GRANTS

This work was supported by São Paulo Research Foundation (FAPESP)Grants 2014/22406-1 (to A. C. Takakura), 2009/54888-7 (to T. S. Moreira),and 2013/10573-8 (to T. S. Moreira), Conselho Nacional de DesenvolvimentoCientífico e Tecnológico (CNPq) Grants 471744/2011-5 (to A. C. Takakura),471263/2013-3 (to A. C. Takakura), and 471283/2012-6 (to T. S. Moreira), andCNPq Fellowships 305533/2012-6 (to T. S. Moreira) and 301651/2013-2 (toA. C. Takakura). J. J. Otero acknowledges support from National Institutes ofHealth (NIH) Grant 1R21EB017539-01A1. J. J. Otero and C. Czeisler weresupported by NIH Grant R011HL132355-01A1.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

T.S.M., A.C.T., C.C., and J.J.O. conception and design of research; T.S.M.,A.C.T., C.C., and J.J.O. performed experiments; T.S.M., A.C.T., C.C., andJ.J.O. analyzed data; T.S.M., A.C.T., C.C., and J.J.O. interpreted results ofexperiments; T.S.M., A.C.T., C.C., and J.J.O. prepared figures; T.S.M.,

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A.C.T., C.C., and J.J.O. drafted manuscript; T.S.M., A.C.T., C.C., and J.J.O.edited and revised manuscript; T.S.M., A.C.T., and J.J.O. approved finalversion of manuscript.

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