Global expansion of tropical cyclone precipitation footprint.

Precipitation from tropical cyclones (TCs) can cause massive damage from inland floods and is becoming more intense under a warming climate. However, knowledge gaps still exist in changes of spatial patterns in heavy TC precipitation. Here we define a metric, DIST30, as the mean radial distance from centers of clustered heavy rainfall cells (> 30 mm/3 h) to TC center, representing the footprint of heavy TC precipitation. There is significant global increase in DIST30 at a rate of 0.34 km/year. Increases of DIST30 cover 59.87% of total TC impact areas, with growth especially strong in the Western North Pacific, Northern Atlantic, and Southern Pacific. The XGBoost machine learning model showed that monthly DIST30 variability is majorly controlled by TC maximum wind speed, location, sea surface temperature, vertical wind shear, and total water column vapor. TC poleward migration in the Northern Hemisphere contributes substantially to the DIST30 upward trend globally.

Inactivity-induced phrenic motor facilitation requires PKCζ activity within phrenic motor neurons.

Prolonged inhibition of respiratory neural activity elicits a long-lasting increase in phrenic nerve amplitude once respiratory neural activity is restored. Such long-lasting facilitation represents a form of respiratory motor plasticity known as inactivity-induced phrenic motor facilitation (iPMF). Although facilitation also occurs in inspiratory intercostal nerve activity after diminished respiratory neural activity (iIMF), it is of shorter duration. Atypical PKC activity in the cervical spinal cord is necessary for iPMF and iIMF, but the site and specific isoform of the relevant atypical PKC are unknown. Here, we used RNA interference to test the hypothesis that the zeta atypical PKC isoform (PKCζ) within phrenic motor neurons is necessary for iPMF but PKCζ within intercostal motor neurons is unnecessary for transient iIMF. Intrapleural injections of siRNAs targeting PKCζ (siPKCζ) to knock down PKCζ mRNA within phrenic and intercostal motor neurons were made in rats. Control rats received a nontargeting siRNA (NTsi) or an active siRNA pool targeting a novel PKC isoform, PKCθ (siPKCθ), which is required for other forms of respiratory motor plasticity. Phrenic nerve burst amplitude and external intercostal (T2) electromyographic (EMG) activity were measured in anesthetized and mechanically ventilated rats exposed to 30 min of respiratory neural inactivity (i.e., neural apnea) created by modest hypocapnia (20 min) or a similar recording duration without neural apnea (time control). Phrenic burst amplitude was increased in rats treated with NTsi (68 ± 10% baseline) and siPKCθ (57 ± 8% baseline) 60 min after neural apnea vs. time control rats (-3 ± 3% baseline), demonstrating iPMF. In contrast, intrapleural siPKCζ virtually abolished iPMF (5 ± 4% baseline). iIMF was transient in all groups exposed to neural apnea; however, intrapleural siPKCζ attenuated iIMF 5 min after neural apnea (50 ± 21% baseline) vs. NTsi (97 ± 22% baseline) and siPKCθ (103 ± 20% baseline). Neural inactivity elevated the phrenic, but not intercostal, responses to hypercapnia, an effect that was blocked by siPKCζ. We conclude that PKCζ within phrenic motor neurons is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for, transient iIMF.NEW & NOTEWORTHY We report important new findings concerning the mechanisms regulating a form of spinal neuroplasticity elicited by prolonged inhibition of respiratory neural activity, inactivity-induced phrenic motor facilitation (iPMF). We demonstrate that the atypical PKC isoform PKCζ within phrenic motor neurons is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for, transient inspiratory intercostal facilitation. Our findings are novel and advance our understanding of mechanisms contributing to phrenic motor plasticity.

Prolonged intermittent hypoxia differentially regulates phrenic motor neuron serotonin receptor expression in rats following chronic cervical spinal cord injury.

Low-dose (< 2 h/day), acute intermittent hypoxia (AIH) elicits multiple forms of serotonin-dependent phrenic motor plasticity and is emerging as a promising therapeutic strategy to restore respiratory and non-respiratory motor function after spinal cord injury (SCI). In contrast, high-dose (> 8 h/day), chronic intermittent hypoxia (CIH) undermines some forms of serotonin-dependent phrenic motor plasticity and elicits pathology. CIH is a hallmark of sleep disordered breathing, which is highly prevalent in individuals with cervical SCI. Interestingly, AIH and CIH preconditioning differentially impact phrenic motor plasticity. Although mechanisms of AIH-induced plasticity in the phrenic motor system are well-described in naïve rats, we know little concerning how these mechanisms are affected by chronic SCI or intermittent hypoxia preconditioning. Thus, in a rat model of chronic, incomplete cervical SCI (lateral spinal hemisection at C2 (C2Hx), we assessed serotonin type 2A, 2B and 7 receptor expression in and near phrenic motor neurons and compared: 1) intact vs. chronically injured rats; and 2) the impact of preconditioning with varied "doses" of intermittent hypoxia (IH). While there were no effects of chronic injury or intermittent hypoxia alone, CIH affected multiple receptors in rats with chronic C2Hx. Specifically, CIH preconditioning (8 h/day; 28 days) increased serotonin 2A and 7 receptor expression exclusively in rats with chronic C2Hx. Understanding the complex, context-specific interactions between chronic SCI and CIH and how this ultimately impacts phrenic motor plasticity is important as we leverage AIH-induced motor plasticity to restore breathing and other non-respiratory motor functions in people with chronic SCI.

Microglia regulate motor neuron plasticity via reciprocal fractalkine/adenosine signaling.

Microglia are innate CNS immune cells that play key roles in supporting key CNS functions including brain plasticity. We now report a previously unknown role for microglia in regulating neuroplasticity within spinal phrenic motor neurons, the neurons driving diaphragm contractions and breathing. We demonstrate that microglia regulate phrenic long-term facilitation (pLTF), a form of respiratory memory lasting hours after repetitive exposures to brief periods of low oxygen (acute intermittent hypoxia; AIH) via neuronal/microglial fractalkine signaling. AIH-induced pLTF is regulated by the balance between competing intracellular signaling cascades initiated by serotonin vs adenosine, respectively. Although brainstem raphe neurons release the relevant serotonin, the cellular source of adenosine is unknown. We tested a model in which hypoxia initiates fractalkine signaling between phrenic motor neurons and nearby microglia that triggers extracellular adenosine accumulation. With moderate AIH, phrenic motor neuron adenosine 2A receptor activation undermines serotonin-dominant pLTF; in contrast, severe AIH drives pLTF by a unique, adenosine-dominant mechanism. Phrenic motor neuron fractalkine knockdown, cervical spinal fractalkine receptor inhibition on nearby microglia, and microglial depletion enhance serotonin-dominant pLTF with moderate AIH but suppress adenosine-dominant pLTF with severe AIH. Thus, microglia play novel functions in the healthy spinal cord, regulating hypoxia-induced neuroplasticity within the motor neurons responsible for breathing.

Cardiorespiratory Responses to Acute Intermittent Hypoxia in Humans With Chronic Spinal Cord Injury.

Brief exposure to repeated episodes of low inspired oxygen, or acute intermittent hypoxia (AIH), is a promising therapeutic modality to improve motor function after chronic, incomplete spinal cord injury (SCI). Although therapeutic AIH is under extensive investigation in persons with SCI, limited data are available concerning cardiorespiratory responses during and after AIH exposure despite implications for AIH safety and tolerability. Thus, we recorded immediate (during treatment) and enduring (up to 30 min post-treatment) cardiorespiratory responses to AIH in 19 participants with chronic SCI (>1 year post-injury; injury levels C1 to T6; American Spinal Injury Association Impairment Scale A to D; mean age = 33.8 ± 14.1 years; 18 males). Participants completed a single AIH (15, 60-sec episodes, inspired O2 ≈ 10%; 90-sec intervals breathing room air) and Sham (inspired O2 ≈ 21%) treatment, in random order. During hypoxic episodes: (1) arterial oxyhemoglobin saturation decreased to 82.1 ± 2.9% (p < 0.001); (2) minute ventilation increased 3.83 ± 2.29 L/min (p = 0.008); and (3) heart rate increased 4.77 ± 6.82 bpm (p = 0.010). Considerable variability in cardiorespiratory responses was found among subjects; some individuals exhibited large hypoxic ventilatory responses (≥0.20 L/min/%, n = 11), whereas others responded minimally (<0.20 L/min/%, n = 8). Apneas occurred frequently during AIH and/or Sham protocols in multiple participants. All participants completed AIH treatment without difficulty. No significant changes in ventilation, heart rate, or arterial blood pressure were found 30 min post-AIH p > 0.05). In conclusion, therapeutic AIH is well tolerated, elicits variable chemoreflex activation, and does not cause persistent changes in cardiorespiratory control/function 30 min post-treatment in persons with chronic SCI.

Inaugural Review Prize 2023: The exercise hyperpnoea dilemma: A 21st-century perspective.

During mild or moderate exercise, alveolar ventilation increases in direct proportion to metabolic rate, regulating arterial CO2 pressure near resting levels. Mechanisms giving rise to the hyperpnoea of exercise are unsettled despite over a century of investigation. In the past three decades, neuroscience has advanced tremendously, raising optimism that the 'exercise hyperpnoea dilemma' can finally be solved. In this review, new perspectives are offered in the hope of stimulating original ideas based on modern neuroscience methods and current understanding. We first describe the ventilatory control system and the challenge exercise places upon blood-gas regulation. We highlight relevant system properties, including feedforward, feedback and adaptive (i.e., plasticity) control of breathing. We then elaborate a seldom explored hypothesis that the exercise ventilatory response continuously adapts (learns and relearns) throughout life and ponder if the memory 'engram' encoding the feedforward exercise ventilatory stimulus could reside within the cerebellum. Our hypotheses are based on accumulating evidence supporting the cerebellum's role in motor learning and the numerous direct and indirect projections from deep cerebellar nuclei to brainstem respiratory neurons. We propose that cerebellar learning may be obligatory for the accurate and adjustable exercise hyperpnoea capable of tracking changes in life conditions/experiences, and that learning arises from specific cerebellar microcircuits that can be interrogated using powerful techniques such as optogenetics and chemogenetics. Although this review is speculative, we consider it essential to reframe our perspective if we are to solve the till-now intractable exercise hyperpnoea dilemma.

Optimization of RT-QuIC Assay Duration for Screening Chronic Wasting Disease in White-Tailed Deer.

Real-time quaking-induced conversion (RT-QuIC) assays have become a common tool to detect chronic wasting disease (CWD) and are very sensitive provided the assay duration is sufficient. However, a prolonged assay duration may lead to non-specific signal amplification. The wide range of pre-defined assay durations in current RT-QuIC applications presents a need for methods to optimize the RT-QuIC assay. In this study, receiver operating characteristic (ROC) analysis was applied to optimize the assay duration for CWD screening in obex and retropharyngeal lymph node (RLN) tissue specimens. Two different fluorescence thresholds were used: a fixed threshold based on background fluorescence (Tstdev) and a max-point ratio (maximum/background fluorescence) threshold (TMPR) to determine CWD positivity. The optimal assay duration was 33 h for obex and 30 h for RLN based on Tstdev, and 29 h for obex and 32 h for RLN based on TMPR. The optimized assay durations were then evaluated for screening CWD in white-tailed deer from an affected farm. At a replicate level, using the optimized assay durations with TStdev and TMPR, the level of agreement with enzyme-linked immunosorbent assay (ELISA) was significantly higher (p < 0.05) than that when using a 40 h assay duration. These findings demonstrate that the optimization of assay duration via a ROC analysis can improve RT-QuIC assays for screening CWD in white-tailed deer.

A Research Protocol to Study the Priming Effects of Breathing Low Oxygen on Enhancing Training-Related Gains in Walking Function for Persons With Spinal Cord Injury: The BO2ST Trial.

Brief episodes of low oxygen breathing (therapeutic acute intermittent hypoxia; tAIH) may serve as an effective plasticity-promoting primer to enhance the effects of transcutaneous spinal stimulation-enhanced walking therapy (WALKtSTIM) in persons with chronic (>1 year) spinal cord injury (SCI). Pre-clinical studies in rodents with SCI show that tAIH and WALKtSTIM therapies harness complementary mechanisms of plasticity to maximize walking recovery. Here, we present a multi-site clinical trial protocol designed to examine the influence of tAIH + WALKtSTIM on walking recovery in persons with chronic SCI. We hypothesize that daily (eight sessions, 2 weeks) tAIH + WALKtSTIM will elicit faster, more persistent improvements in walking recovery than either treatment alone. To test our hypothesis, we are conducting a placebo-controlled clinical trial on 60 SCI participants who randomly receive one of three interventions: tAIH + WALKtSTIM; Placebo + WALKtSTIM; and tAIH + WALKtSHAM. Participants receive daily tAIH (fifteen 90-sec episodes at 10% O2 with 60-sec intervals at 21% O2) or daily placebo (fifteen 90-sec episodes at 21% O2 with 60-sec intervals at 21% O2) before a 45-min session of WALKtSTIM or WALKtSHAM. Our primary outcome measures assess walking speed (10-Meter Walk Test), endurance (6-Minute Walk Test), and balance (Timed Up and Go Test). For safety, we also measure pain levels, spasticity, sleep behavior, cognition, and rates of systemic hypertension and autonomic dysreflexia. Assessments occur before, during, and after sessions, as well as at 1, 4, and 8 weeks post-intervention. Results from this study extend our understanding of the functional benefits of tAIH priming by investigating its capacity to boost the neuromodulatory effects of transcutaneous spinal stimulation on restoring walking after SCI. Given that there is no known cure for SCI and no single treatment is sufficient to overcome walking deficits, there is a critical need for combinatorial treatments that accelerate and anchor walking gains in persons with lifelong SCI. Trial Registration: ClinicalTrials.gov, NCT05563103.

Progressive tauopathy disrupts breathing stability and chemoreflexes during presumptive sleep in mice.

Rationale: Although sleep apnea occurs in over 50% of individuals with Alzheimer's Disease (AD) or related tauopathies, little is known concerning the potential role of tauopathy in the pathogenesis of sleep apnea. Here, we tested the hypotheses that, during presumptive sleep, a murine model of tauopathy (rTg4510) exhibits: 1) increased breathing instability; 2) impaired chemoreflex function; and 3) exacerbation of these effects with tauopathy progression. Methods: rTg4510 mice initially develop robust tauopathy in the hippocampus and cortex, and eventually progresses to the brainstem. Type I and II post-sigh apnea, Type III (spontaneous) apnea, sigh, and hypopnea incidence were measured in young adult (5-6 months; n = 10-14/group) and aged (13-15 months; n = 22-24/group) non-transgenic (nTg), monogenic control tetracycline transactivator, and bigenic rTg4510 mice using whole-body plethysmography during presumptive sleep (i.e., eyes closed, curled/laying posture, stable breathing for >200 breaths) while breathing room air (21% O2). Peripheral and central chemoreceptor sensitivity were assessed with transient exposures (5 min) to hyperoxia (100% O2) or hypercapnia (3% and 5% CO2 in 21% O2), respectively. Results: We report significant increases in Type I, II, and III apneas (all p < 0.001), sighs (p = 0.002) and hypopneas (p < 0.001) in aged rTg4510 mice, but only Type III apneas in young adult rTg4510 mice (p < 0.001) versus age-matched nTg controls. Aged rTg4510 mice exhibited profound chemoreflex impairment versus age matched nTg and tTA mice. In rTg4510 mice, breathing frequency, tidal volume and minute ventilation were not affected by hyperoxic or hypercapnic challenges, in striking contrast to controls. Histological examination revealed hyperphosphorylated tau in brainstem regions involved in the control of breathing (e.g., pons, medullary respiratory column, retrotrapezoid nucleus) in aged rTg4510 mice. Neither breathing instability nor hyperphosphorylated tau in brainstem tissues were observed in young adult rTg4510 mice. Conclusion: Older rTg4510 mice exhibit profound impairment in the neural control of breathing, with greater breathing instability and near absence of oxygen and carbon-dioxide chemoreflexes. Breathing impairments paralleled tauopathy progression into brainstem regions that control breathing. These findings are consistent with the idea that tauopathy per se undermines chemoreflexes and promotes breathing instability during sleep.

Magnitude and Mechanism of Phrenic Long-term Facilitation Shift Between Daily Rest Versus Active Phase.

Plasticity is a fundamental property of the neural system controlling breathing. One key example of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a persistent increase in phrenic nerve activity elicited by acute intermittent hypoxia (AIH). pLTF can arise from distinct cell signaling cascades initiated by serotonin versus adenosine receptor activation, respectively, and interact via powerful cross-talk inhibition. Here, we demonstrate that the daily rest/active phase and the duration of hypoxic episodes within an AIH protocol have profound impact on the magnitude and mechanism of pLTF due to shifts in serotonin/adenosine balance. Using the historical "standard" AIH protocol (3, 5-min moderate hypoxic episodes), we demonstrate that pLTF magnitude is unaffected by exposure in the midactive versus midrest phase, yet the mechanism driving pLTF shifts from serotonin-dominant (midrest) to adenosine-dominant (midactive). This mechanistic "flip" results from combined influences of hypoxia-evoked adenosine release and daily fluctuations in basal spinal adenosine. Since AIH evokes less adenosine with shorter (15, 1-min) hypoxic episodes, midrest pLTF is amplified due to diminished adenosine constraint on serotonin-driven plasticity; in contrast, elevated background adenosine during the midactive phase suppresses serotonin-dominant pLTF. These findings demonstrate the importance of the serotonin/adenosine balance in regulating the amplitude and mechanism of AIH-induced pLTF. Since AIH is emerging as a promising therapeutic modality to restore respiratory and nonrespiratory movements in people with spinal cord injury or ALS, knowledge of how time-of-day and hypoxic episode duration impact the serotonin/adenosine balance and the magnitude and mechanism of pLTF has profound biological, experimental, and translational implications.

APOE4, Age, and Sex Regulate Respiratory Plasticity Elicited by Acute Intermittent Hypercapnic-Hypoxia.

Rationale: Acute intermittent hypoxia (AIH) shows promise for enhancing motor recovery in chronic spinal cord injuries and neurodegenerative diseases. However, human trials of AIH have reported significant variability in individual responses. Objectives: Identify individual factors (eg, genetics, age, and sex) that determine response magnitude of healthy adults to an optimized AIH protocol, acute intermittent hypercapnic-hypoxia (AIHH). Methods: In 17 healthy individuals (age = 27 ± 5 yr), associations between individual factors and changes in the magnitude of AIHH (15, 1-min O2 = 9.5%, CO2 = 5% episodes) induced changes in diaphragm motor-evoked potential (MEP) amplitude and inspiratory mouth occlusion pressures (P0.1) were evaluated. Single nucleotide polymorphisms (SNPs) in genes linked with mechanisms of AIH induced phrenic motor plasticity (BDNF, HTR2A, TPH2, MAOA, NTRK2) and neuronal plasticity (apolipoprotein E, APOE) were tested. Variations in AIHH induced plasticity with age and sex were also analyzed. Additional experiments in humanized (h)ApoE knock-in rats were performed to test causality. Results: AIHH-induced changes in diaphragm MEP amplitudes were lower in individuals heterozygous for APOE4 (i.e., APOE3/4) compared to individuals with other APOE genotypes (P = 0.048) and the other tested SNPs. Males exhibited a greater diaphragm MEP enhancement versus females, regardless of age (P = 0.004). Additionally, age was inversely related with change in P0.1 (P = 0.007). In hApoE4 knock-in rats, AIHH-induced phrenic motor plasticity was significantly lower than hApoE3 controls (P < 0.05). Conclusions: APOE4 genotype, sex, and age are important biological determinants of AIHH-induced respiratory motor plasticity in healthy adults. Addition to Knowledge Base: AIH is a novel rehabilitation strategy to induce functional recovery of respiratory and non-respiratory motor systems in people with chronic spinal cord injury and/or neurodegenerative disease. Figure 5 Since most AIH trials report considerable inter-individual variability in AIH outcomes, we investigated factors that potentially undermine the response to an optimized AIH protocol, AIHH, in healthy humans. We demonstrate that genetics (particularly the lipid transporter, APOE), age and sex are important biological determinants of AIHH-induced respiratory motor plasticity.

Cervical spinal hemisection effects on spinal tissue oxygenation and long-term facilitation of phrenic, renal and splanchnic sympathetic nerve activity.

HYPOTHESES: Moderate acute intermittent hypoxia (mAIH) elicits plasticity in both respiratory (phrenic long-term facilitation; pLTF) and sympathetic nerve activity (sympLTF) in rats. Although mAIH produces pLTF in normal rats, inconsistent results are reported after cervical spinal cord injury (cSCI), possibly due to greater spinal tissue hypoxia below the injury site. There are no reports concerning cSCI effects on sympLTF. Since mAIH is being explored as a therapeutic modality to restore respiratory and non-respiratory movements in humans with chronic SCI, both effects are important. To understand cSCI effects on mAIH-induced pLTF and sympLTF, partial or complete C2 spinal hemisections (C2Hx) were performed and, 2 weeks later, we assessed: 1) ipsilateral cervical spinal tissue oxygen tension; 2) ipsilateral & contralateral pLTF; and 3) ipsilateral sympLTF in splanchnic and renal sympathetic nerves. METHODS: Male Sprague-Dawley rats were studied intact, or after partial (single slice) or complete C2Hx (slice with ∼1 mm aspiration). Two weeks post-C2Hx, rats were anesthetized and prepared for recordings of bilateral phrenic nerve activity and spinal tissue oxygen pressure (PtO2). Splanchnic and renal sympathetic nerve activity was recorded in intact and complete C2Hx rats. RESULTS: Spinal PtO2 near phrenic motor neurons was decreased after C2Hx, an effect most prominent with complete vs. partial injuries; baseline PtO2 was positively correlated with mean arterial pressure. Complete C2Hx impaired ipsilateral but not contralateral pLTF; with partial C2Hx, ipsilateral pLTF was unaffected. In intact rats, mAIH elicited splanchnic and renal sympLTF. Complete C2Hx had minimal impact on baseline ipsilateral splanchnic or renal sympathetic nerve activity and renal, but not splanchnic, sympLTF remained intact. CONCLUSION: Greater tissue hypoxia likely impairs pLTF and splanchnic sympLTF post-C2Hx, although renal sympLTF remains intact. Increased sympathetic nerve activity post-mAIH may have therapeutic benefits in individuals living with chronic SCI since anticipated elevations in systemic blood pressure may mitigate hypotension characteristic of people living with SCI.

Increased spinal adenosine impairs phrenic long-term facilitation in aging rats.

Moderate acute intermittent hypoxia (mAIH) elicits a form of spinal, respiratory motor plasticity known as phrenic long-term facilitation (pLTF). In middle-aged male and geriatric female rats, mAIH-induced pLTF is attenuated through unknown mechanisms. In young adults, mAIH activates competing intracellular signaling cascades, initiated by serotonin 2 and adenosine 2A (A2A) receptors, respectively. Spinal A2A receptor inhibition enhances mAIH-induced pLTF, meaning, serotonin dominates, and adenosine constrains mAIH-induced plasticity in the daily rest phase. Thus, we hypothesized elevated basal adenosine levels in the ventral cervical spinal cord of aged rats shifts this balance, undermining mAIH-induced pLTF. A selective A2A receptor antagonist (MSX-3) or vehicle was delivered intrathecally at C4 in anesthetized young (3-6 mo) and aged (20-22 mo) Sprague-Dawley rats before mAIH (3,5-min episodes; arterial Po2 = 45-55 mmHg). In young males, spinal A2A receptor inhibition enhanced pLTF (119 ± 5%) vs. vehicle (55 ± 9%), consistent with prior reports. In old males, pLTF was reduced to 25 ± 11%, but A2A receptor inhibition increased pLTF to levels greater than in young males (186 ± 19%). Basal adenosine levels in ventral C3-C5 homogenates are elevated two- to threefold in old vs. young males. These findings advance our understanding of age as a biological variable in phrenic motor plasticity and will help guide translation of mAIH as a therapeutic modality to restore respiratory and nonrespiratory movements in older populations afflicted with clinical disorders that compromise movement.NEW & NOTEWORTHY Advanced age undermines respiratory motor plasticity, specifically phrenic long-term facilitation (pLTF) following moderate acute intermittent hypoxia (mAIH). We report that spinal adenosine increases in aged male rats, undermining mAIH-induced pLTF via adenosine 2A (A2A) receptor activation, an effect reversed by selective spinal adenosine 2A receptor inhibition. These findings advance our understanding of mechanisms that impair neuroplasticity, and the ability to compensate for the onset of lung or neural injury with age, and may guide efforts to harness mAIH as a treatment for clinical disorders that compromise breathing and other movements.

Heterozygosity for cervid S138N polymorphism results in subclinical CWD in gene-targeted mice and progressive inhibition of prion conversion.

Prions are proteinaceous infectious particles that replicate by structural conversion of the host-encoded cellular prion protein (PrPC), causing fatal neurodegenerative diseases in mammals. Species-specific amino acid substitutions (AAS) arising from single nucleotide polymorphisms within the prion protein gene (Prnp) modulate prion disease pathogenesis, and, in several instances, reduce susceptibility of homo- or heterozygous AAS carriers to prion infection. However, a mechanistic understanding of their protective effects against clinical disease is missing. We generated gene-targeted mouse infection models of chronic wasting disease (CWD), a highly contagious prion disease of cervids. These mice express wild-type deer or PrPC harboring the S138N substitution homo- or heterozygously, a polymorphism found exclusively in reindeer (Rangifer tarandus spp.) and fallow deer (Dama dama). The wild-type deer PrP-expressing model recapitulated CWD pathogenesis including fecal shedding. Encoding at least one 138N allele prevented clinical CWD, accumulation of protease-resistant PrP (PrPres) and abnormal PrP deposits in the brain tissue. However, prion seeding activity was detected in spleens, brains, and feces of these mice, suggesting subclinical infection accompanied by prion shedding. 138N-PrPC was less efficiently converted to PrPres in vitro than wild-type deer (138SS) PrPC. Heterozygous coexpression of wild-type deer and 138N-PrPC resulted in dominant-negative inhibition and progressively diminished prion conversion over serial rounds of protein misfolding cyclic amplification. Our study indicates that heterozygosity at a polymorphic Prnp codon can confer the highest protection against clinical CWD and highlights the potential role of subclinical carriers in CWD transmission.

Mild inflammation impairs acute intermittent hypoxia-induced phrenic long-term facilitation by a spinal adenosine-dependent mechanism.

Inflammation undermines neuroplasticity, including serotonin-dependent phrenic long-term facilitation (pLTF) following moderate acute intermittent hypoxia (mAIH: 3, 5-min episodes, arterial Po2: 40-50 mmHg; 5-min intervals). Mild inflammation elicited by a low dose of the TLR-4 receptor agonist, lipopolysaccharide (LPS; 100 µg/kg, ip), abolishes mAIH-induced pLTF by unknown mechanisms. In the central nervous system, neuroinflammation primes glia, triggering ATP release and extracellular adenosine accumulation. As spinal adenosine 2 A (A2A) receptor activation impairs mAIH-induced pLTF, we hypothesized that spinal adenosine accumulation and A2A receptor activation are necessary in the mechanism whereby LPS impairs pLTF. We report that 24 h after LPS injection in adult male Sprague Dawley rats: 1) adenosine levels increase in ventral spinal segments containing the phrenic motor nucleus (C3-C5; P = 0.010; n = 7/group) and 2) cervical spinal A2A receptor inhibition (MSX-3, 10 µM, 12 µL intrathecal) rescues mAIH-induced pLTF. In LPS vehicle-treated rats (saline, ip), MSX-3 enhanced pLTF versus controls (LPS: 110 ± 16% baseline; controls: 53 ± 6%; P = 0.002; n = 6/group). In LPS-treated rats, pLTF was abolished as expected (4 ± 6% baseline; n = 6), but intrathecal MSX-3 restored pLTF to levels equivalent to MSX-3-treated control rats (120 ± 14% baseline; P < 0.001; n = 6; vs. LPS controls with MSX-3: P = 0.539). Thus, inflammation abolishes mAIH-induced pLTF by a mechanism that requires increased spinal adenosine levels and A2A receptor activation. As repetitive mAIH is emerging as a treatment to improve breathing and nonrespiratory movements in people with spinal cord injury or ALS, A2A inhibition may offset undermining effects of neuroinflammation associated with these neuromuscular disorders.NEW & NOTEWORTHY Mild inflammation undermines motor plasticity elicited by mAIH. In a model of mAIH-induced respiratory motor plasticity (phrenic long-term facilitation; pLTF), we report that inflammation induced by low-dose lipopolysaccharide undermines mAIH-induced pLTF by a mechanism requiring increased cervical spinal adenosine and adenosine 2 A receptor activation. This finding advances the understanding of mechanisms impairing neuroplasticity, potentially undermining the ability to compensate for the onset of lung/neural injury or to harness mAIH as a therapeutic modality.

BDNF-induced phrenic motor facilitation shifts from PKCθ to ERK dependence with mild systemic inflammation.

Moderate acute intermittent hypoxia (mAIH) elicits a form of phrenic motor plasticity known as phrenic long-term facilitation (pLTF), which requires spinal 5-HT2 receptor activation, ERK/MAP kinase signaling, and new brain-derived neurotrophic factor (BDNF) synthesis. New BDNF protein activates TrkB receptors that normally signal through PKCθ to elicit pLTF. Phrenic motor plasticity elicited by spinal drug administration (e.g., BDNF) is referred to by a more general term: phrenic motor facilitation (pMF). Although mild systemic inflammation elicited by a low lipopolysaccharide (LPS) dose (100 µg/kg; 24 h prior) undermines mAIH-induced pLTF upstream from BDNF protein synthesis, it augments pMF induced by spinal BDNF administration through unknown mechanisms. Here, we tested the hypothesis that mild inflammation shifts BDNF/TrkB signaling from PKCθ to alternative pathways that enhance pMF. We examined the role of three known signaling pathways associated with TrkB (MEK/ERK MAP kinase, PI3 kinase/Akt, and PKCθ) in BDNF-induced pMF in anesthetized, paralyzed, and ventilated Sprague Dawley rats 24 h post-LPS. Spinal PKCθ inhibitor (TIP) attenuated early BDNF-induced pMF (≤30 min), with minimal effect 60-90 min post-BDNF injection. In contrast, MEK inhibition (U0126) abolished BDNF-induced pMF at 60 and 90 min. PI3K/Akt inhibition (PI-828) had no effect on BDNF-induced pMF at any time. Thus, whereas BDNF-induced pMF is exclusively PKCθ-dependent in normal rats, MEK/ERK is recruited by neuroinflammation to sustain, and even augment downstream plasticity. Because AIH is being developed as a therapeutic modality to restore breathing in people living with multiple neurological disorders, it is important to understand how inflammation, a common comorbidity in many traumatic or degenerative central nervous system disorders, impacts phrenic motor plasticity.NEW & NOTEWORTHY We demonstrate that even mild systemic inflammation shifts signaling mechanisms giving rise to BDNF-induced phrenic motor plasticity. This finding has important experimental, biological, and translational implications, particularly since BDNF-dependent spinal plasticity is being translated to restore breathing and nonrespiratory movements in diverse clinical disorders, such as spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS).

APOE4, Age & Sex Regulate Respiratory Plasticity Elicited By Acute Intermittent Hypercapnic-Hypoxia.

Rationale: Acute intermittent hypoxia (AIH) is a promising strategy to induce functional motor recovery following chronic spinal cord injuries and neurodegenerative diseases. Although significant results are obtained, human AIH trials report considerable inter-individual response variability. Objectives: Identify individual factors ( e.g. , genetics, age, and sex) that determine response magnitude of healthy adults to an optimized AIH protocol, acute intermittent hypercapnic-hypoxia (AIHH). Methods: Associations of individual factors with the magnitude of AIHH (15, 1-min O 2 =9.5%, CO 2 =5% episodes) induced changes in diaphragm motor-evoked potential amplitude (MEP) and inspiratory mouth occlusion pressures (P 0.1 ) were evaluated in 17 healthy individuals (age=27±5 years) compared to Sham. Single nucleotide polymorphisms (SNPs) in genes linked with mechanisms of AIH induced phrenic motor plasticity ( BDNF, HTR 2A , TPH 2 , MAOA, NTRK 2 ) and neuronal plasticity (apolipoprotein E, APOE ) were tested. Variations in AIHH induced plasticity with age and sex were also analyzed. Additional experiments in humanized ( h ) ApoE knock-in rats were performed to test causality. Results: AIHH-induced changes in diaphragm MEP amplitudes were lower in individuals heterozygous for APOE 4 ( i.e., APOE 3/4 ) allele versus other APOE genotypes (p=0.048). No significant differences were observed between any other SNPs investigated, notably BDNFval/met ( all p>0.05 ). Males exhibited a greater diaphragm MEP enhancement versus females, regardless of age (p=0.004). Age was inversely related with change in P 0.1 within the limited age range studied (p=0.007). In hApoE 4 knock-in rats, AIHH-induced phrenic motor plasticity was significantly lower than hApoE 3 controls (p<0.05). Conclusions: APOE 4 genotype, sex and age are important biological determinants of AIHH-induced respiratory motor plasticity in healthy adults. ADDITION TO KNOWLEDGE BASE: Acute intermittent hypoxia (AIH) is a novel rehabilitation strategy to induce functional recovery of respiratory and non-respiratory motor systems in people with chronic spinal cord injury and/or neurodegenerative diseases. Since most AIH trials report considerable inter-individual variability in AIH outcomes, we investigated factors that potentially undermine the response to an optimized AIH protocol, acute intermittent hypercapnic-hypoxia (AIHH), in healthy humans. We demonstrate that genetics (particularly the lipid transporter, APOE ), age and sex are important biological determinants of AIHH-induced respiratory motor plasticity.

Biodegradation of bovine spongiform encephalopathy prions in compost.

To reduce the transmission risk of bovine spongiform encephalopathy prions (PrPBSE), specified risk materials (SRM) that can harbour PrPBSE are prevented from entering the feed and food chains. As composting is one approach to disposing of SRM, we investigated the inactivation of PrPBSE in lab-scale composters over 28 days and in bin composters over 106-120 days. Lab-scale composting was conducted using 45 kg of feedlot manure with and without chicken feathers. Based on protein misfolding cyclic amplification (PMCA), after 28 days of composting, PrPBSE seeding activity was reduced by 3-4 log10 with feathers and 3 log10 without. Bin composters were constructed using ~ 2200 kg feedlot manure and repeated in 2017 and 2018. PMCA results showed that seeding activity of PrPBSE was reduced by 1-2 log10 in the centre, but only by 1 log10 in the bottom of bin composters. Subsequent assessment by transgenic (Tgbov XV) mouse bioassay confirmed a similar reduction in PrPBSE infectivity. Enrichment for proteolytic microorganisms through the addition of feathers to compost could enhance PrPBSE degradation. In addition to temperature, other factors including varying concentrations of PrPBSE and the nature of proteolytic microbial populations may be responsible for differential degradation of PrPBSE during composting.

Intermittent Hypoxia Differentially Regulates Adenosine Receptors in Phrenic Motor Neurons with Spinal Cord Injury.

Cervical spinal cord injury (cSCI) impairs neural drive to the respiratory muscles, causing life- threatening complications such as respiratory insufficiency and diminished airway protection. Repetitive "low dose" acute intermittent hypoxia (AIH) is a promising strategy to restore motor function in people with chronic SCI. Conversely, "high dose" chronic intermittent hypoxia (CIH; ∼8 h/night), such as experienced during sleep apnea, causes pathology. Sleep apnea, spinal ischemia, hypoxia and neuroinflammation associated with cSCI increase extracellular adenosine concentrations and activate spinal adenosine receptors which in turn constrains the functional benefits of therapeutic AIH. Adenosine 1 and 2A receptors (A1, A2A) compete to determine net cAMP signaling and likely the tAIH efficacy with chronic cSCI. Since cSCI and intermittent hypoxia may regulate adenosine receptor expression in phrenic motor neurons, we tested the hypotheses that: 1) daily AIH (28 days) downregulates A2A and upregulates A1 receptor expression; 2) CIH (28 days) upregulates A2A and downregulates A1 receptor expression; and 3) cSCI alters the impact of CIH on adenosine receptor expression. Daily AIH had no effect on either adenosine receptor in intact or injured rats. However, CIH exerted complex effects depending on injury status. Whereas CIH increased A1 receptor expression in intact (not injured) rats, it increased A2A receptor expression in spinally injured (not intact) rats. The differential impact of CIH reinforces the concept that the injured spinal cord behaves in distinct ways from intact spinal cords, and that these differences should be considered in the design of experiments and/or new treatments for chronic cSCI.

Respiratory neuroplasticity: Mechanisms and translational implications of phrenic motor plasticity.

Widespread appreciation that neuroplasticity is an essential feature of the neural system controlling breathing has emerged only in recent years. In this chapter, we focus on respiratory motor plasticity, with emphasis on the phrenic motor system. First, we define related but distinct concepts: neuromodulation and neuroplasticity. We then focus on mechanisms underlying two well-studied models of phrenic motor plasticity: (1) phrenic long-term facilitation following brief exposure to acute intermittent hypoxia; and (2) phrenic motor facilitation after prolonged or recurrent bouts of diminished respiratory neural activity. Advances in our understanding of these novel and important forms of plasticity have been rapid and have already inspired translation in multiple respects: (1) development of novel therapeutic strategies to preserve/restore breathing function in humans with severe neurological disorders, such as spinal cord injury and amyotrophic lateral sclerosis; and (2) the discovery that similar plasticity also occurs in nonrespiratory motor systems. Indeed, the realization that similar plasticity occurs in respiratory and nonrespiratory motor neurons inspired clinical trials to restore leg/walking and hand/arm function in people living with chronic, incomplete spinal cord injury. Similar application may be possible to other clinical disorders that compromise respiratory and non-respiratory movements.