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

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.

Leptin signaling in the dorsomedial hypothalamus couples breathing and metabolism in obesity.

Mismatch between CO2 production (Vco2) and respiration underlies the pathogenesis of obesity hypoventilation. Leptin-mediated CNS pathways stimulate both metabolism and breathing, but interactions between these functions remain elusive. We hypothesized that LEPRb+ neurons of the dorsomedial hypothalamus (DMH) regulate metabolism and breathing in obesity. In diet-induced obese LeprbCre mice, chemogenetic activation of LEPRb+ DMH neurons increases minute ventilation (Ve) during sleep, the hypercapnic ventilatory response, Vco2, and Ve/Vco2, indicating that breathing is stimulated out of proportion to metabolism. The effects of chemogenetic activation are abolished by a serotonin blocker. Optogenetic stimulation of the LEPRb+ DMH neurons evokes excitatory postsynaptic currents in downstream serotonergic neurons of the dorsal raphe (DR). Administration of retrograde AAV harboring Cre-dependent caspase to the DR deletes LEPRb+ DMH neurons and abolishes metabolic and respiratory responses to leptin. These findings indicate that LEPRb+ DMH neurons match breathing to metabolism through serotonergic pathways to prevent obesity-induced hypoventilation.

TRPM7 channels regulate breathing during sleep in obesity by acting peripherally in the carotid bodies.

Sleep-disordered breathing (SDB) affects over 50% of obese individuals. Exaggerated hypoxic chemoreflex is a cardinal trait of SDB in obesity. We have shown that leptin acts in the carotid bodies (CB) to augment chemoreflex and that leptin activates the transient receptor potential melastatin 7 (TRPM7) channel. However, the effect of leptin-TRPM7 signalling in CB on breathing and SDB has not been characterized in diet-induced obesity (DIO). We hypothesized that leptin acts via TRPM7 in the CB to increase chemoreflex leading to SDB in obesity. DIO mice were implanted with EEG/EMG electrodes and transfected with Leprb short hairpin RNA (shRNA) or Trpm7 shRNA vs. control shRNA in the CB area bilaterally. Mice underwent a full-polysomnography and metabolic studies at baseline and after transfection. Ventilatory responses to hypoxia and hypercapnia were assessed during wakefulness. Leprb and Trpm7 were upregulated and their promoters were demethylated in the CB of DIO mice. Leprb knockdown in the CB did not significantly affect ventilation. Trpm7 knockdown in the CB stimulated breathing during sleep in normoxia. These effects were not driven by changes in CB chemosensitivity or metabolism. Under sustained hypoxia, Trpm7 shRNA in the CB augmented ventilation during sleep, but decreased oxyhaemoglobin saturation. We conclude that the suppression of TRPM7 in the CB improved sleep-related hypoventilation and that the respiratory effects of CB TRPM7 channels in obesity are independent of leptin. TRPM7 signalling in the CB could be a therapeutic target for the treatment of obesity-related SDB. KEY POINTS: The leptin-TRPM7 axis in the carotid bodies may play an important role in the pathogenesis of sleep-disordered breathing. TRPM7 channels regulate breathing during sleep by acting peripherally in the carotid bodies. Suppression of TRPM7 signalling in the carotid bodies improves the obesity-induced hypoventilation in mice. Pharmacological blockade of TRPM7 channels in the carotid bodies could be a therapy for sleep-disordered breathing in obesity.

LKB1 is the gatekeeper of carotid body chemosensing and the hypoxic ventilatory response.

The hypoxic ventilatory response (HVR) is critical to breathing and thus oxygen supply to the body and is primarily mediated by the carotid bodies. Here we reveal that carotid body afferent discharge during hypoxia and hypercapnia is determined by the expression of Liver Kinase B1 (LKB1), the principal kinase that activates the AMP-activated protein kinase (AMPK) during metabolic stresses. Conversely, conditional deletion in catecholaminergic cells of AMPK had no effect on carotid body responses to hypoxia or hypercapnia. By contrast, the HVR was attenuated by LKB1 and AMPK deletion. However, in LKB1 knockouts hypoxia evoked hypoventilation, apnoea and Cheyne-Stokes-like breathing, while only hypoventilation and apnoea were observed after AMPK deletion. We therefore identify LKB1 as an essential regulator of carotid body chemosensing and uncover a divergence in dependency on LKB1 and AMPK between the carotid body on one hand and the HVR on the other.

Alkalosis-induced hypoventilation in cystic fibrosis: The importance of efficient renal adaptation.

The lungs and kidneys are pivotal organs in the regulation of body acid-base homeostasis. In cystic fibrosis (CF), the impaired renal ability to excrete an excess amount of HCO3- into the urine leads to metabolic alkalosis [P. Berg et al., J. Am. Soc. Nephrol. 31, 1711-1727 (2020); F. Al-Ghimlas, M. E. Faughnan, E. Tullis, Open Respir. Med. J. 6, 59-62 (2012)]. This is caused by defective HCO3- secretion in the ß-intercalated cells of the collecting duct that requires both the cystic fibrosis transmembrane conductance regulator (CFTR) and pendrin for normal function [P. Berg et al., J. Am. Soc. Nephrol. 31, 1711-1727 (2020)]. We studied the ventilatory consequences of acute oral base loading in normal, pendrin knockout (KO), and CFTR KO mice. In wild-type mice, oral base loading induced a dose-dependent metabolic alkalosis, fast urinary removal of base, and a moderate base load did not perturb ventilation. In contrast, CFTR and pendrin KO mice, which are unable to rapidly excrete excess base into the urine, developed a marked and transient depression of ventilation when subjected to the same base load. Therefore, swift renal base elimination in response to an acute oral base load is a necessary physiological function to avoid ventilatory depression. The transient urinary alkalization in the postprandial state is suggested to have evolved for proactive avoidance of hypoventilation. In CF, metabolic alkalosis may contribute to the commonly reduced lung function via a suppression of ventilatory drive.

Cerebral Autoregulation during Orthostatic Challenge in Congenital Central Hypoventilation Syndrome.

Rationale: Congenital central hypoventilation syndrome (CCHS) is a rare autonomic disorder with altered regulation of breathing, heart rate (HR), and blood pressure (BP). Aberrant cerebral oxygenation in response to hypercapnia/hypoxia in CCHS raises the concern that altered cerebral autoregulation may contribute to CCHS-related, variably impaired neurodevelopment. Objectives: To evaluate cerebral autoregulation in response to orthostatic challenge in CCHS cases versus controls. Methods: CCHS and age- and sex-matched control subjects were studied with head-up tilt (HUT) testing to induce orthostatic stress. Fifty CCHS and 100 control HUT recordings were included. HR, BP, and cerebral oxygen saturation (regional oxygen saturation) were continuously monitored. The cerebral oximetry index (COx), a real-time measure of cerebral autoregulation based on these measures, was calculated. Measurements and Main Results: HUT resulted in a greater mean BP decrease from baseline in CCHS versus controls (11% vs. 6%; P < 0.05) and a diminished increase in HR in CCHS versus controls (11% vs. 18%; P < 0.01) in the 5 minutes after tilt-up. Despite a similar COx at baseline, orthostatic provocation within 5 minutes of tilt-up caused a 50% greater increase in COx (P < 0.01) and a 29% increase in minutes of impaired autoregulation (P < 0.02) in CCHS versus controls (4.0 vs. 3.1 min). Conclusions: Cerebral autoregulatory mechanisms appear to be intact in CCHS, but the greater hypotension observed in CCHS consequent to orthostatic provocation is associated with greater values of COx/impaired autoregulation when BP is below the lower limits of autoregulation. Effects of repeated orthostatic challenges in everyday living in CCHS necessitate further study to determine their influence on neurodevelopmental disease burden.

Gavage approach to oxygen supplementation with oxygen therapeutic Ox66™ in a hypoventilation rodent model of respiratory distress.

Acute respiratory distress syndrome (ARDS) features pulmonary dysfunction capable of causing life-threatening hypoxaemia. Ventilation and hyperoxic therapies force oxygen through dysfunctional alveoli but risk exacerbating damage. Ox66™ is an ingestible, solid-state oxygen product designed for oxygen supplementation. Eighteen anaesthetized, ventilated rats were subjected to a 40% reduction in tidal volume to produce a hypoventilatory simulation of the hypoxia in ARDS (HV-ARDS). After 60 min, animals were randomized to receive either normal saline (Saline; volume control) or Ox66™ gavage. Cardiovascular function and blood oximetry/chemistry were measured alongside interstitial oxygenation (PISFO2) of the peripheral spinotrapezius muscle. HV-ARDS reduced mean arterial pressure by ∼20% and PISFO2 by ∼35% for both groups. Ox66™ gavage treatment at 60 min improved PISFO2 over Saline (p < .0001), restoring baseline values, however, the effect was temporary. A second bolus at 120 min repeated the OX66™ PISFO2 response, which remained elevated over Saline (p < .01) until study end and was supported by systemic parameters of lactate, PaO2, SO2, and base deficit. Saline remained hypotensive, whereas Ox66™ became normotensive. Vasoconstriction was observed in the Saline, but not Ox66™ group. Supplemental oxygenation through Ox66™ gavage increased peripheral tissue oxygenation, warranting further study for disorders featuring dysfunction of pulmonary perfusion like ARDS.

In vivo evidence for the cellular basis of central hypoventilation of Rett syndrome and pharmacological correction in the rat model.

Rett syndrome (RTT) is a neurodevelopmental disorder caused mostly by mutations in the MECP2 gene. RTT patients show periodical hypoventilation attacks. The breathing disorder contributing to the high incidence of sudden death is thought to be due to depressed central inspiratory (I) activity via unknown cellular processes. Demonstration of such processes may lead to targets for pharmacological control of the RTT-type hypoventilation. We performed in vivo recordings from medullary respiratory neurons on the RTT rat model. To our surprise, both I and expiratory (E) neurons in the ventral respiratory column (VRC) increased their firing activity in Mecp2-null rats with severe hypoventilation. These I neurons including E-I phase-spanning and other I neurons remained active during apneas. Consistent with enhanced central I drive, ectopic phrenic discharges during expiration as well as apnea were observed in the Mecp2-null rats. Considering the increased I neuronal firing and ectopic phrenic activity, the RTT-type hypoventilation does not seem to be caused by depression in central I activity, neither reduced medullary I premotor output. This as well as excessive E neuronal firing as shown in our previous studies suggests inadequate synaptic inhibition for phase transition. We found that the abnormal respiratory neuronal firing, ectopic phrenic discharge as well as RTT-type hypoventilation all can be corrected by enhancing GABAergic inhibition. More strikingly, Mecp2-null rats reaching humane endpoints with severe hypoventilation can be rescued by GABAergic augmentation. Thus, defective GABAergic inhibition among respiratory neurons is likely to play a role in the RTT-type hypoventilation, which can be effectively controlled with pharmacological agents.

Breath holding as an example of extreme hypoventilation: experimental testing of a new model describing alveolar gas pathways.

NEW FINDINGS: What is the central question of this study? We modelled the alveolar pathway during breath holding on the hypothesis that it follows a hypoventilation loop on the O2 -CO2 diagram. What is the main finding and its importance? Validation of the model was possible within the range of alveolar gas compositions compatible with consciousness. Within this range, the experimental data were compatible with the proposed model. The model and its characteristics might allow predictions of alveolar gas composition whenever the alveolar ventilation goes to zero; for example, static and dynamic breath holding at the surface or during ventilation/intubation failure in anaesthesia. ABSTRACT: According to the hypothesis that alveolar partial pressures of O2 and CO2 during breath holding (BH) should vary following a hypoventilation loop, we modelled the alveolar gas pathways during BH on the O2 -CO2 diagram and tested it experimentally during ambient air and pure oxygen breathing. In air, the model was constructed using the inspired and alveolar partial pressures of O2 ( PIO2 and PAO2 , respectively) and CO2 ( PICO2 and PACO2 , respectively) and the steady-state values of the pre-BH respiratory exchange ratio (RER). In pure oxygen, the model respected the constraint of PACO2=-PAO2+PIO2 . To test this, 12 subjects performed several BHs of increasing duration and one maximal BH at rest and during exercise (30 W cycling supine), while breathing air or pure oxygen. We measured gas flows, PAO2 and PACO2 before and at the end of all BHs. Measured data were fitted through the model. In air, PIO2  = 150 ± 1 mmHg and PICO2  = 0.3 ± 0.0 mmHg, both at rest and at 30 W. Before BH, steady-state RER was 0.83 ± 0.16 at rest and 0.77 ± 0.14 at 30 W; PAO2  = 107 ± 7 mmHg at rest and 102 ± 8 mmHg at 30 W; and PACO2  = 36 ± 4 mmHg at rest and 38 ± 3 mmHg at 30 W. By model fitting, we computed the RER during the early phase of BH: 0.10 [95% confidence interval (95% CI) = 0.08-0.12] at rest and 0.13 (95% CI = 0.11-0.15) at 30 W. In oxygen, model fitting provided PIO2 : 692 (95% CI = 688-696) mmHg at rest and 693 (95% CI = 689-698) mmHg at 30 W. The experimental data are compatible with the proposed model, within its physiological range.

Nocturnal hypoventilation in Down syndrome children with or without sleep apnea.

BACKGROUND: There is a high prevalence of obstructive sleep apnea (OSA) in children with Down syndrome (DS), sometimes associated with alveolar hypoventilation. OBJECTIVE: To compare transcutaneous partial pressure of carbon dioxide (PtcCO2 ) and pulse oximetry (SpO2 ) in children with DS and in control children with OSA. PATIENTS AND METHODS: This retrospective case-control study involved children followed in Trousseau Hospital (Paris) Sleep Center. Polysomnography (PSG) recordings and clinical files of children with DS were reviewed to identify clinical signs of OSA and comorbidities associated with DS. Controls were children who presented with OSA of ENT origin without other comorbidities (exceptions: two overweight, one obese, and three with well-controlled asthma). DS subjects and controls were matched for age and apnea hypopnea index. RESULTS: There were 28 children in each group. Mean PtcCO2 during sleep was significantly higher in patients with DS compared to controls (44 mm Hg vs 42 mm Hg, P = .001). Five (21%) patients with DS met the American Academy of Sleep medicine criteria for hypoventilation, compared to one (4%) in the control group. The mean PtcO2 during sleep was significantly lower in patients with DS (77 mm Hg vs 82 mm Hg, P = .003). CONCLUSIONS: This is the first study to compare nocturnal gas exchange in children with DS to a control group of children with similar OSA. Our data demonstrate that children with DS have increased PtcCO2 regardless of the presence of OSA and its severity. This may be due to respiratory muscle hypotonia and/or ventilatory control alteration in patients with DS.

Physiological adaptations to repeated sprint training in hypoxia induced by voluntary hypoventilation at low lung volume.

PURPOSE: This study investigated the effects of repeated-sprint (RS) training in hypoxia induced by voluntary hypoventilation at low lung volume (RSH-VHL) on physiological adaptations, RS ability (RSA) and anaerobic performance. METHODS: Over a 3-week period, eighteen well-trained cyclists completed six RS sessions in cycling either with RSH-VHL or with normal conditions (RSN). Before (Pre) and after (Post) the training period, the subjects performed an RSA test (10 × 6-s all-out cycling sprints) during which oxygen uptake [Formula: see text] and the change in both muscle deoxyhaemoglobin (Δ[HHb]) and total haemoglobin (Δ[THb]) were measured. A 30-s Wingate test was also performed and maximal blood lactate concentration ([La]max) was assessed. RESULTS: At Post compared to Pre, the mean power output during both the RSA and the Wingate tests was improved in RSH-VHL (846 ± 98 vs 911 ± 117 W and 723 ± 112 vs 768 ± 123 W, p < 0.05) but not in RSN (834 ± 52 vs 852 ± 69 W, p = 0.2; 710 ± 63 vs 713 ± 72 W, p = 0.68). The average [Formula: see text] recorded during the RSA test was significantly higher in RSH-VHL at Post but did not change in RSN. No change occurred for Δ[THb] whereas Δ[HHb] increased to the same extent in both groups. [Lamax] after the Wingate test was higher in RSH-VHL at Post (13.9 ± 2.8 vs 16.1 ± 3.2 mmol L-1, p < 0.01) and tended to decrease in RSN (p = 0.1). CONCLUSIONS: This study showed that RSH-VHL could bring benefits to both RSA and anaerobic performance through increases in oxygen delivery and glycolytic contribution. On the other hand, no additional effect was observed for the indices of muscle blood volume and O2 extraction.

Mutation in LBX1/Lbx1 precludes transcription factor cooperativity and causes congenital hypoventilation in humans and mice.

The respiratory rhythm is generated by the preBötzinger complex in the medulla oblongata, and is modulated by neurons in the retrotrapezoid nucleus (RTN), which are essential for accelerating respiration in response to high CO2 Here we identify a LBX1 frameshift (LBX1FS ) mutation in patients with congenital central hypoventilation. The mutation alters the C-terminal but not the DNA-binding domain of LBX1 Mice with the analogous mutation recapitulate the breathing deficits found in humans. Furthermore, the mutation only interferes with a small subset of Lbx1 functions, and in particular with development of RTN neurons that coexpress Lbx1 and Phox2b. Genome-wide analyses in a cell culture model show that Lbx1FS and wild-type Lbx1 proteins are mostly bound to similar sites, but that Lbx1FS is unable to cooperate with Phox2b. Thus, our analyses on Lbx1FS (dys)function reveals an unusual pathomechanism; that is, a mutation that selectively interferes with the ability of Lbx1 to cooperate with Phox2b, and thus impairs the development of a small subpopulation of neurons essential for respiratory control.

Diagnostic accuracy of simple tools in monitoring patients with chronic hypoventilation treated with non-invasive ventilation; a prospective cross-sectional study.

OBJECTIVES: To evaluate the sensitivity and specificity of a screening test panel for nocturnal hypoventilation (NH) and other sleep related respiratory events during monitoring of patients with chronic hypercapnic respiratory failure (CRF) treated with NIV. METHODS: We performed a prospective study at Oslo University Hospital. Eligible for inclusion were consecutive adults with CRF due to neuromuscular diseases or chest wall disorders treated with NIV scheduled for a follow-up visit. All patients underwent the screening test panel (clinical evaluation, daytime arterial blood gas (ABG), nocturnal pulse oximetry (SpO2) and data from ventilator software) and the reference tests; sleep polygraphy and nocturnal transcutaneous CO2. RESULTS: Of 67 patients included, NH was confirmed in 23-50 according to the 3 definitions used for NH, apnea-hypopnea index (AHIpolygraphy) ≥ 10 was confirmed in 16 and patient-ventilator asynchrony (PVA) ≥ 10% of total recording time in 14. Sensitivity of the combined screening test panel for NH was 87% (95% confidence interval 66-97), 84% (66-95) and 80% (66-90), for abnormal AHIpolygraphy 91% (59-100) and for PVA 71% (42-92). Sensitivity for NH of SpO2 was 48% (27-69), 39% (22-58) and 38% (24-53) and of daytime ABG 74% (52-90), 74% (55-88) and 68% (53-80). Sensitivity and specificity of AHIsoftware for AHIpolygraphy ≥ 10 was 93% (68-100) and 92% (81-98) respectively. DISCUSSION: In patients treated with long term NIV, screening test panel, nocturnal SpO2 and daytime ABG all failed to accurately detect NH, underlining the importance of nocturnal monitoring of CO2. AHIsoftware accurately identified obstructive events and can be used to modify NIV settings. TRIAL REGISTRATION: N° NCT01845233.

DCTN1 F52L mutation case of Perry syndrome with progressive supranuclear palsy-like tauopathy.

INTRODUCTION: Perry syndrome is a rapidly progressive, autosomal dominant parkinsonism characterized by central hypoventilation, depression and severe weight loss. To date, eight DCTN1 mutations have been identified associated with Perry syndrome. A novel F52L DCTN1 mutation case of Perry syndrome is characterized by late-onset parkinsonism and frontotemporal atrophy. METHODS: A Japanese woman suffered from slowly progressing parkinsonism since age 48. At age 59, she developed central hypoventilation, and required breathing assistance. Gene analysis identified a p.F52L mutation in DCTN1 and she was diagnosed with Perry syndrome. She died of aspiration pneumonia at age 74. RESULTS: Postmortem examination revealed severe neuronal loss in the substantia nigra and the putamen. Immunohistochemistry for DCTN1 revealed many abnormal aggregates, mainly in neurons in the brainstem and basal ganglia. Additionally, numerous abnormal phosphorylated tau deposits including neurofibrillary tangles, tuft-shaped astrocytes and coiled bodies were observed mainly in the basal ganglia, brainstem and cerebellum. These correspond with the neuropathologic criteria for progressive supranuclear palsy. Colocalization of DCTN1 and tau were occasionally seen. Colocalization of phosphorylated α-synuclein and DCTN1 were also observed in Lewy body-like structures in oculomotor nuclei. Phosphorylated TARDBP-positive neuronal cytoplasmic inclusions were few. CONCLUSION: In conjunction with long disease duration and aging, our findings suggest that the F52L DCTN1 mutation may evoke severe tauopathy and moderate α-synucleinopathy.

Hypoventilation Therapy Alleviates Panic by Repeated Induction of Dyspnea.

BACKGROUND: Previous research has shown that hypoventilation therapy reduces panic symptoms in part by increasing basal partial pressure of carbon dioxide (PCO2) levels. We tested an additional pathway by which hypoventilation therapy could exert its therapeutic effects: through repeated interoceptive exposure to sensations of dyspnea. METHODS: A total of 35 patients with panic disorder were trained to perform exercises to raise their end-tidal PCO2 levels using a portable capnometry device. Anxiety, dyspnea, end-tidal PCO2, and respiratory rate were assessed during each exercise across 4 weeks of training. Mixed-model analysis examined whether within-exercise levels of dyspnea were predictive of reduction of panicogenic cognitions. RESULTS: As expected, within-exercise anxiety and respiratory rate decreased over time. Unexpectedly, PCO2 dropped significantly from the beginning to the end of exercise, with these drops becoming progressively smaller across weeks. Dyspnea increased and remained consistently above basal levels across weeks. As hypothesized, greater dyspnea was related to significantly lower panicogenic cognitions over time even after controlling for anxiety and PCO2. Additional exploratory analyses showed that within-exercise increases in dyspnea were related to within-exercise increases in anxiety but were not related to within-exercise increases in PCO2. CONCLUSIONS: In support of the interoceptive exposure model, we found that greater dyspnea during hypoventilation exercises resulted in lower panicogenic cognitions even after the effect of PCO2 was taken into account. The findings offer an additional important target in panic treatment.

Inhalational Anesthetics Induce Neuronal Protein Aggregation and Affect ER Trafficking.

Anesthetic agents have been implicated in the causation of neurological and cognitive deficits after surgery, the exacerbation of chronic neurodegenerative disease, and were recently reported to promote the onset of the neurologic respiratory disease Congenital Central Hypoventilation Syndrome (CCHS), related to misfolding of the transcription factor Phox2B. To study how anesthetic agents could affect neuronal function through alterations to protein folding, we created neuronal cell models emulating the graded disease severity of CCHS. We found that the gas anesthetic isoflurane and the opiate morphine potentiated aggregation and mislocalization of Phox2B variants, similar to that seen in CCHS, and observed transcript and protein level changes consistent with activation of the endoplasmic reticulum (ER) unfolded protein response. Attenuation of ER stress pathways did not result in a correction of Phox2B misfolding, indicating a primary effect of isoflurane on protein structure. We also observed that isoflurane hindered the folding and activity of proteins that rely heavily on ER function, like the CFTR channel. Our results show how anesthetic drugs can alter protein folding and induce ER stress, indicating a mechanism by which these agents may affect neuronal function after surgery.

Structural and functional differences in PHOX2B frameshift mutations underlie isolated or syndromic congenital central hypoventilation syndrome.

Heterozygous mutations in the PHOX2B gene are causative of congenital central hypoventilation syndrome (CCHS), a neurocristopathy characterized by defective autonomic control of breathing due to the impaired differentiation of neural crest cells. Among PHOX2B mutations, polyalanine (polyAla) expansions are almost exclusively associated with isolated CCHS, whereas frameshift variants, although less frequent, are often more severe than polyAla expansions and identified in syndromic CCHS. This article provides a complete review of all the frameshift mutations identified in cases of isolated and syndromic CCHS reported in the literature as well as those identified by us and not yet published. These were considered in terms of both their structure, whether the underlying indels induced frameshifts of either 1 or 2 steps ("frame 2" and "frame 3" mutations respectively), and clinical associations. Furthermore, we evaluated the structural and functional effects of one "frame 3" mutation identified in a patient with isolated CCHS, and one "frame 2" mutation identified in a patient with syndromic CCHS, also affected with Hirschsprung's disease and neuroblastoma. The data thus obtained confirm that the type of translational frame affects the severity of the transcriptional dysfunction and the predisposition to isolated or syndromic CCHS.

Reduced orexin immunoreactivity in Perry syndrome and multiple system atrophy.

INTRODUCTION: Orexin is a neuropeptide that plays a key role in maintaining a state of arousal, and possibly associates with sleep apnea syndrome (SAS). Reduced orexin immunoreactivity has been reported in various neurologic conditions such as narcolepsy, Alzheimer's disease, Lewy body disease and multiple system atrophy (MSA); however, there has been no report investigating orexin in Perry syndrome, a rare hereditary neurodegenerative disease characterized by four clinical cardinal signs (parkinsonism, depression/apathy, weight loss, and central hypoventilation). Perry syndrome patients frequently have sleep disturbances, including SAS and insomnia. METHODS: We evaluated orexin immunoreactivity in Perry syndrome. Using imaging analysis, we quantitatively assessed orexin immunoreactivity in the nucleus basalis of Meynert in three Perry syndrome cases, as well as five cases of frontotemporal lobar degeneration with motor neuron disease, five cases of MSA and five age-matched controls. For these cases, antemortem clinical information on sleep disturbances has been reviewed. RESULTS: In Perry syndrome and MSA, there was reduction of orexin immunoreactivity compared with controls (Perry syndrome: p = 0.020, MSA: p < 0.001). In contrast, FTLD-MND did not have significant reduction of orexin immunoreactivity. Two out of three cases of Perry syndrome had SAS confirmed by polysomnography. CONCLUSIONS: This is the first report assessing orexin immunoreactivity in Perry syndrome, and it showed significant reduction, similar to select neurodegenerative diseases, such as MSA. Further analysis with more cases will be needed to elucidate the specific mechanism of orexin loss in these disorders.

Reduced TDP-43 Expression Improves Neuronal Activities in a Drosophila Model of Perry Syndrome.

Parkinsonian Perry syndrome, involving mutations in the dynein motor component dynactin or p150Glued, is characterized by TDP-43 pathology in affected brain regions, including the substantia nigra. However, the molecular relationship between p150Glued and TDP-43 is largely unknown. Here, we report that a reduction in TDP-43 protein levels alleviates the synaptic defects of neurons expressing the Perry mutant p150G50R in Drosophila. Dopaminergic expression of p150G50R, which decreases dopamine release, disrupts motor ability and reduces the lifespan of Drosophila. p150G50R expression also causes aggregation of dense core vesicles (DCVs), which contain monoamines and neuropeptides, and disrupts the axonal flow of DCVs, thus decreasing synaptic strength. The above phenotypes associated with Perry syndrome are improved by the removal of a copy of Drosophila TDP-43 TBPH, thus suggesting that the stagnation of axonal transport by dynactin mutations promotes TDP-43 aggregation and interferes with the dynamics of DCVs and synaptic activities.

Expanded polyalanine tracts function as nuclear export signals and promote protein mislocalization via eEF1A1 factor.

Polyalanine (poly(A)) diseases are caused by the expansion of translated GCN triplet nucleotide sequences encoding poly(A) tracts in proteins. To date, nine human disorders have been found to be associated with poly(A) tract expansions, including congenital central hypoventilation syndrome and oculopharyngeal muscular dystrophy. Previous studies have demonstrated that unexpanded wild-type poly(A)-containing proteins localize to the cell nucleus, whereas expanded poly(A)-containing proteins primarily localize to the cytoplasm. Because most of these poly(A) disease proteins are transcription factors, this mislocalization causes cellular transcriptional dysregulation leading to cellular dysfunction. Correcting this faulty localization could potentially point to strategies to treat the aforementioned disorders, so there is a pressing need to identify the mechanisms underlying the mislocalization of expanded poly(A) protein. Here, we performed a glutathione S-transferase pulldown assay followed by mass spectrometry and identified eukaryotic translation elongation factor 1 α1 (eEF1A1) as an interacting partner with expanded poly(A)-containing proteins. Strikingly, knockdown of eEF1A1 expression partially corrected the mislocalization of the expanded poly(A) proteins in the cytoplasm and restored their functions in the nucleus. We further demonstrated that the expanded poly(A) domain itself can serve as a nuclear export signal. Taken together, this study demonstrates that eEF1A1 regulates the subcellular location of expanded poly(A) proteins and is therefore a potential therapeutic target for combating the pathogenesis of poly(A) diseases.