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

Reviewer #1 (Public Review):

Summary:
This important study investigated the role of the PHOX2B transcription factor in neurons in the key brainstem chemosensory structure, the retrotrapezoid nucleus (RTN), for maintaining proper CO2 chemoreflex responses of breathing in the adult rat in vivo. PHOX2B has an important transcriptional role in neuronal survival and/or function, and mutations of PHOX2B severely impair the development and function of the autonomic nervous system and RTN, resulting in the developmental genetic disease congenital central hypoventilation syndrome (CCHS) in neonates, where the RTN may not form and is functionally impaired. The function of the wild-type PHOX2B protein in adult RTN neurons that continue to express PHOX2B is not fully understood. By utilizing a viral PHOX2B-shRNA approach for knockdown of PHOX2B specifically in RTN neurons, the authors' solid results show impaired ventilatory responses to elevated inspired CO2, measured by whole-body plethysmography in freely behaving adult rats, that develop progressively over a four-week period in vivo, indicating effects on RTN neuron transcriptional activity and associated blunting of the CO2 ventilatory response. The RTN neuronal mRNA expression data presented suggests the impaired hypercapnic ventilatory response is possibly due to the decreased expression of key proton sensors in the RTN. This study will be of interest to neuroscientists studying respiratory neurobiology as well as the neurodevelopmental control of motor behavior.

Strengths:
1. The authors used a shRNA viral approach to progressively knock down the PHOX2B protein, specifically in RTN neurons to determine whether PHOX2B is necessary for the survival and/or chemosensory function of adult RTN neurons in vivo.

2. To determine the extent of PHOX2B knockdown in RTN neurons, the authors combined RNAScope® and immunohistochemistry assays to quantify the subpopulation of RTN neurons expressing PHOX2B and neuromedin B (Nmb), which has been proposed to be key chemosensory neurons in the RTN.

3. The authors demonstrate that knockdown efficiency is time-dependent, with a progressive decrease in the number of Nmb-expressing RTN neurons that co-express PHOX2B over a four-week period.

4. Their results convincingly show hypoventilation particularly in 7.2% CO2 only for PHOX2B-shRNA RTN-injected rats after four weeks as compared to naïve and non-PHOX2B-shRNA targeted (NT-shRNA) RTN injected rats, suggesting a specific impairment of chemosensitive properties in RTN neurons with PHOX2B knockdown.

5. Analysis of the association between PHOX2B knockdown in RTN neurons and the attenuation of the hypercapnic ventilatory response (HCVR), by evaluating the correlation between the number of Nmb+/PHOX2B+ or Nmb+/PHOX2B- cells in the RTN and the resulting HCVR, showed a significant correlation between HCVR and number of Nmb+/PHOX2B+ and Nmb+/PHOX2B- cells, suggesting that the number of PHOX2B-expressing cells in the RTN is a predictor of the chemoreflex response and the reduction of PHOX2B protein impairs the CO2-chemoreflex.

6. The data presented indicate that PHOX2B knockdown not only causes a reduction in the HCVR but also a reduction in the expression of Gpr4 and Task2 mRNAs, suggesting that PHOX2B knockdown affects RTN neurons transcriptional activity and decreases the CO2 response, possibly by reducing the expression of key proton sensors in the RTN.

7. Results of this study show that independent of the role of PHOX2B during development, PHOX2B is still required to maintain proper CO2 chemoreflex responses in the adult brain, and its reduction in CCHS may contribute to the respiratory impairment in this disorder.

Weaknesses:
1. The authors found a significant decrease in the total number of Nmb+ RTN neurons (i.e., Nmb+/PHOX2B+ plus Nmb+/ PHOX2B-) in NT-shRNA rats at two weeks post viral injection, and also at the four-week period where the impairment of the chemosensory function of the RTN became significant, suggesting some inherent cell death possibly due to off-target toxic effects associated with shRNA procedures that may affect the experimental results.

2. The tissue sampling procedures for quantifying numbers of cells expressing proteins/mRNAs throughout the extended RTN region bilaterally have not been completely validated to accurately represent the full expression patterns in the RTN under experimental conditions.

3. The inferences about RTN neuronal expression of NMB, GPR4, or TASK2 are based on changes in mRNA levels, so it remains speculation that the observed reduction in Gpr4 and Task2 mRNA translates to a reduction in the protein levels and associated reduction of RTN neuronal chemosensitive properties.

Reviewer #2 (Public Review):

Summary:
The authors used a short hairpin RNA technique strategy to elucidate the functional activity of neurons in the retrotrapezoid nucleus (RTN), a critical brainstem region for central chemoreception. Dysfunction in this area is associated with the neuropathology of congenital central hypoventilation syndrome (CCHS). The subsequent examination of these rats aimed to shed light on the intricate aspects of RTN and its implications for central chemoreception and disorders like CCHS in adults. They found that using the short hairpin RNA (shRNA) targeting Phox2b mRNA, a reduction of Phox2b expression was observed in Nmb neurons. In addition, Phox2b knockdown did not affect breathing in room air or under hypoxia, but the hypercapnia ventilatory response was significantly impaired. They concluded that Phox2b in the adult brain has an important role in CO2 chemoreception. They thought that their findings provided new evidence for mechanisms related to CCHS neuropathology. The conclusions of this paper are well supported by data, but careful discussion seems to be required for comparison with the results of various previous studies performed by different genetic strategies for the RTN neurons.

Strengths:
The most exciting aspect of this work is the modelling of the Phox2b knockdown in one element of the central neuronal circuit mediating respiratory reflexes, that is in the RTN. To date, mutations in the PHOX2B gene are commonly associated with most patients diagnosed with CCHS, a disease characterized by hypoventilation and absence of chemoreflexes, in the neonatal period, which in severe cases can lead to respiratory arrest during sleep. In the present study, the authors demonstrated that the role of Phox2b extends beyond the developmental period, and its reduction in CCHS may contribute to the respiratory impairment observed in this disorder.

Weaknesses:
Whereas the most exciting part of this work is the knockdown of the Phox2b in the RTN in adult rodents, the weakness of this study is the lack of a clear physiological, developmental, and anatomical distinction between this approach and similar studies already reported elsewhere (Ruffault et al., 2015, DOI: 10.7554/eLife.07051; Ramanantsoa et al., 2011, DOI: 10.1523/JNEUROSCI.1721-11.2011; Huang et al., 2017, DOI: 10.1016/j.neuron.2012.06.027; Hernandez-Miranda et al., 2018, DOI: 10.1073/pnas.1813520115; Ferreira et al., 2022 DOI: 10.7554/eLife.73130; Takakura et al., 2008 DOI: 10.1113/jphysiol.2008.153163; Basting et al., 2015 DOI: 10.1523/JNEUROSCI.2923-14.2015; Marina et al., 2010 DOI: 10.1523/JNEUROSCI.3141-10.2010). In addition, several conclusions presented in this work are not directly supported by the provided data.

Reviewer #3 (Public Review):

A brain region called the retrotrapezoid nucleus (RTN) regulates breathing in response to changes in CO2/H+, a process termed central chemoreception. A transcription factor called PHOX2B is important for RTN development and mutations in the PHOX2B gene result in a severe type of sleep apnea called Congenital Central Hypoventilation Syndrome. PHOX2B is also expressed throughout life, but its postmitotic functions remain unknown. This study shows that knockdown of PHOX2B in the RTN region in adult rats decreased expression of Task2 and Gpr4 in Nmb-expressing RTN chemoreceptors and this corresponded with a diminished ventilatory response to CO2 but did not impact baseline breathing or the hypoxic ventilatory response. These results provide novel insight regarding the postmitotic functions of PHOX2B in RTN neurons.

Main issues:

1. The experimental approach was not targeted to Nmb+ neurons and since other cells in the area also express Phox2b, conclusions should be tempered to focus on Phox2b expressing parafacial neurons NOT specifically RTN neurons

2. It is not clear whether PHOX2B is important for the transcription of pH sensing machinery, cell health, or both. If knockdown of PHOX2B knockdown results in loss of RTN neurons this is also expected to decrease Task2 and Gpr4 levels, albeit by a transcription-independent mechanism.