Heterogeneous distribution of mitochondria and succinate dehydrogenase activity in human airway smooth muscle cells.

Succinate dehydrogenase (SDH) is a key mitochondrial enzyme involved in the tricarboxylic acid cycle, where it facilitates the oxidation of succinate to fumarate, and is coupled to the reduction of ubiquinone in the electron transport chain as Complex II. Previously, we developed a confocal-based quantitative histochemical technique to determine the maximum velocity of the SDH reaction (SDHmax) in single cells and observed that SDHmax corresponds with mitochondrial volume density. In addition, mitochondrial volume and motility varied within different compartments of human airway smooth muscle (hASM) cells. Therefore, we hypothesize that the SDH activity varies relative to the intracellular mitochondrial volume within hASM cells. Using 3D confocal imaging of labeled mitochondria and a concentric shell method for analysis, we quantified mitochondrial volume density, mitochondrial complexity index, and SDHmax relative to the distance from the nuclear membrane. The mitochondria within individual hASM cells were more filamentous in the immediate perinuclear region and were more fragmented in the distal parts of the cell. Within each shell, SDHmax also corresponded to mitochondrial volume density, where both peaked in the perinuclear region and decreased in more distal parts of the cell. Additionally, when normalized to mitochondrial volume, SDHmax was lower in the perinuclear region when compared to the distal parts of the cell. In summary, our results demonstrate that SDHmax measures differences in SDH activity within different cellular compartments. Importantly, our data indicate that mitochondria within individual cells are morphologically heterogeneous, and their distribution varies substantially within different cellular compartments, with distinct functional properties.

Aging related decreases in NM myosin expression and contractility in a resistance vessel.

Introduction: Vasodilatation in response to NO is a fundamental response of the vasculature, and during aging, the vasculature is characterized by an increase in stiffness and decrease in sensitivity to NO mediated vasodilatation. Vascular tone is regulated by the activation of smooth muscle and nonmuscle (NM) myosin, which are regulated by the activities of myosin light chain kinase (MLCK) and MLC phosphatase. MLC phosphatase is a trimeric enzyme with a catalytic subunit, myosin targeting subunit (MYPT1) and 20 kDa subunit of unknown function. Alternative mRNA splicing produces LZ+/LZ- MYPT1 isoforms and the relative expression of LZ+/LZ- MYPT1 determines the sensitivity to NO mediated vasodilatation. This study tested the hypothesis that aging is associated with changes in LZ+ MYPT1 and NM myosin expression, which alter vascular reactivity. Methods: We determined MYPT1 and NM myosin expression, force and the sensitivity of both endothelial dependent and endothelial independent relaxation in tertiary mesenteric arteries of young (6mo) and elderly (24mo) Fischer344 rats. Results: The data demonstrate that aging is associated with a decrease in both the expression of NM myosin and force, but LZ+ MYPT expression and the sensitivity to both endothelial dependent and independent vasodilatation did not change. Further, smooth muscle cell hypertrophy increases the thickness of the medial layer of smooth muscle with aging. Discussion: The reduction of NM myosin may represent an aging associated compensatory mechanism to normalize the stiffness of resistance vessels in response to the increase in media thickness observed during aging.

Cervical spinal cord hemisection impacts sigh and the respiratory reset in male rats.

Cervical spinal cord injury impacts ventilatory and non-ventilatory functions of the diaphragm muscle (DIAm) and contributes to clinical morbidity and mortality in the afflicted population. Periodically, integrated brainstem neural circuit activity drives the DIAm to generate a markedly augmented effort or sigh-which plays an important role in preventing atelectasis and thus maintaining lung function. Across species, the general pattern of DIAm efforts during a normal sigh is variable in amplitude and the extent of post-sigh "apnea" (i.e., the post-sigh inter-breath interval). This post-sigh inter-breath interval acts as a respiratory reset, following the interruption of regular respiratory rhythm by sigh. We examined the impact of upper cervical (C2 ) spinal cord hemisection (C2 SH) on the transdiaphragmatic pressure (Pdi ) generated during sighs and the post-sigh respiratory reset in rats. Sighs were identified in Pdi traces by their characteristic biphasic pattern. We found that C2 SH results in a reduction of Pdi during both eupnea and sighs, and a decrease in the immediate post-sigh breath interval. These results are consistent with partial removal of descending excitatory synaptic inputs to phrenic motor neurons that results from C2 SH. Following cervical spinal cord injury, a reduction in the amplitude of Pdi during sighs may compromise the maintenance of normal lung function.

Postnatal survival of phrenic motor neurons is promoted by BDNF/TrkB.FL signaling.

The number of motor neurons (MNs) declines precipitously during the final trimester before birth. Thereafter, the number of MNs remains relatively stable, with their connections to skeletal muscle dependent on neurotrophins, including brain-derived neurotrophic factor (BDNF) signaling through its high-affinity full-length tropomyosin-related kinase receptor subtype B (TrkB.FL) receptor. As a genetic knockout of BDNF leads to extensive MN loss and postnatal death within 1-2 days after birth, we tested the hypothesis that postnatal inhibition of BDNF/TrkB.FL signaling is important for postnatal phrenic MN (PhMN) survival. In the present study, we used a 1NMPP1-sensitive TrkBF616A mutant mouse to evaluate the effects of inhibition of TrkB kinase activity on phrenic MN (PhMN) numbers and diaphragm muscle (DIAm) fiber cross-sectional area (CSA). Pups were exposed to 1NMPP1 or vehicle (DMSO) from birth to 21 days old (weaning) via the mother's ingestion in the drinking water. Following weaning, the right phrenic nerve was exposed in the neck and the proximal end dipped in a rhodamine solution to retrogradely label PhMNs. After 24 h, the cervical spinal cord and DIAm were excised. Labeled PhMNs were imaged using confocal microscopy, whereas DIAm strips were frozen at ∼1.5× resting length, cryosectioned, and stained with hematoxylin and eosin to assess CSA. We observed an ∼34% reduction in PhMN numbers and increased primary dendrite numbers in 1NMPP1-treated TrkBF616A mice. The distribution of PhMN size (somal surface area) DIAm fiber cross-sectional areas did not differ. We conclude that survival of PhMNs during early postnatal development is sensitive to BDNF/TrkB.FL signaling.NEW & NOTEWORTHY During early postnatal development, BDNF/TrkB signaling promotes PhMN survival. Inhibition of BDNF/TrkB signaling in early postnatal development does not impact PhMN size. Inhibition of BDNF/TrkB signaling in early postnatal development does not impact the number or CSA of DIAm fibers.

Molecular mechanisms underlying TNFα-induced mitochondrial fragmentation in human airway smooth muscle cells.

Tumor necrosis factor α (TNFα), a proinflammatory cytokine, plays a significant role in mediating the effects of acute inflammation in response to allergens, pollutants, and respiratory infections. Previously, we showed that acute exposure to TNFα induces mitochondrial fragmentation in human airway smooth muscle (hASM) cells, which is associated with increased expression of dynamin-related protein 1 (DRP1). Phosphorylation of DRP1 at serine 616 (pDRP1S616) promotes its translocation and binding to the outer mitochondrial membrane (OMM) and mediates mitochondrial fragmentation. Previously, we reported that TNFα exposure triggers protein unfolding and triggers an endoplasmic reticulum (ER) stress response involving phosphorylation of inositol-requiring enzyme 1α (pIRE1α) at serine 724 (pIRE1αS724) and subsequent splicing of X-box binding protein 1 (XBP1s) in hASM cells. We hypothesize that TNFα-mediated activation of the pIRE1αS724/XBP1s ER stress pathway in hASM cells transcriptionally activates genes that encode kinases responsible for pDRP1S616 phosphorylation. Using 3-D confocal imaging of MitoTracker green-labeled mitochondria, we found that TNFα treatment for 6 h induces mitochondrial fragmentation in hASM cells. We also confirmed that 6 h TNFα treatment activates the pIRE1α/XBP1s ER stress pathway. Using in silico analysis and ChIP assay, we showed that CDK1 and CDK5, kinases involved in the phosphorylation of pDRP1S616, are transcriptionally targeted by XBP1s. TNFα treatment increased the binding affinity of XBP1s on the promoter regions of CDK1 and CDK5, and this was associated with an increase in pDRP1S616 and mitochondria fragmentation. This study reveals a new underlying molecular mechanism for TNFα-induced mitochondrial fragmentation in hASM cells.NEW & NOTEWORTHY Airway inflammation is increasing worldwide. Proinflammatory cytokines mediate an adaptive mechanism to overcome inflammation-induced cellular stress. Previously, we reported that TNFα mediates hASM cellular responses, leading to increased force and ATP consumption associated with increased O2 consumption, and oxidative stress. This study indicates that TNFα induces ER stress, which induces mitochondrial fragmentation via pIRE1αS724/XBP1s mediated CDK1/5 upregulation and pDRP1S616 phosphorylation. Mitochondrial fragmentation may promote hASM mitochondrial biogenesis to maintain healthy mitochondrial pool.

Sarcopenia of the longitudinal tongue muscles in rats.

The tongue is a muscular hydrostat, with lingual movements occurring during breathing, chewing, swallowing, vocalization, vomiting, coughing and grooming/sexual activities. In the elderly, reduced lingual dysfunction and weakness contribute to increased risks of obstructive sleep apnea and aspiration pneumonia. In Fischer 344 (F344) rats, a validated model of aging, hypoglossal motor neuron death is apparent, although there is no information regarding tongue strength. The intrinsic tongue muscles, the superior and inferior longitudinal, transversalis and verticalis exist in an interdigitated state. Recently, we established a method to measure the specific force of individual intrinsic tongue muscle, accounting for the tissue bulk that is not in the direction of uniaxial force. In the longitudinal muscles of 6- (n = 10), 18- (n = 9) and 24-month-old (n = 12) female and male F344 rats, we assessed specific force, fatigability, fiber type dependent cross-sectional area (CSA) and overall CSA. Muscle force and fatigue was assessed ex vivo using platinum plate simulation electrodes. Tongue muscles were frozen in melting isopentane, and transverse sections cut at 10 µm. Muscle fiber type was classified based on immunoreactivity to myosin heavy chain (MyHC) isoform antibodies. In H&E stained muscle, CSA and uniaxial muscle contributions to total tongue bulk was assessed. We observed a robust ∼30% loss of longitudinal specific force, with reductions in overall longitudinal muscle fiber CSA and specific atrophy of type IIx/IIb fibers. It will be important to investigate the mechanistic underpinnings of hypoglossal motor neuron death and tongue muscle weakness to eventually provide therapies for age-associated lingual dysfunctions.

Chemical Chaperone 4-PBA Mitigates Tumor Necrosis Factor Alpha-Induced Endoplasmic Reticulum Stress in Human Airway Smooth Muscle.

Airway inflammation and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) underlie the pathophysiology of respiratory diseases, including asthma. Previously, we showed that TNFα activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 spliced (XBP1s) endoplasmic reticulum (ER) stress pathway in human airway smooth muscle (hASM) cells. The ER stress pathway is activated by the accumulation of unfolded proteins in the ER. Accordingly, chemical chaperones such as 4-phenylbutyric acid (4-PBA) may reduce ER stress activation. In the present study, we hypothesized that chemical chaperone 4-PBA mitigates TNFα-induced ER stress in hASM cells. hASM cells were isolated from bronchiolar tissue obtained from five patients with no history of smoking or respiratory diseases. The hASM cells' phenotype was confirmed via the expression of alpha-smooth muscle actin and elongated morphology. hASM cells from the same patient sample were then separated into three 12 h treatment groups: (1) TNFα (20 ng/mL), (2) TNFα + 4-PBA (1 µM, 30 min pretreatment), and (3) untreated control. The expressions of total IRE1α and phosphorylated IRE1α (pIRE1αS724) were determined through Western blotting. The splicing of XBP1 mRNA was analyzed using RT-PCR. We found that TNFα induced an increase in pIRE1αS724 phosphorylation, which was mitigated by treatment with chemical chaperone 4-PBA. We also found that TNFα induced an increase in XBP1s mRNA, which was also mitigated by treatment with chemical chaperone 4-PBA. These results support our hypothesis and indicate that chemical chaperone 4-PBA treatment mitigates TNFα-induced ER stress in hASM cells.

Neuromotor control of spontaneous quiet breathing in awake rats evaluated by assessments of diaphragm EMG stationarity.

The neuromotor control of the diaphragm muscle (DIAm) is dynamic. The activity of the DIAm can be recorded via electromyography (EMG), which represents the temporal summation of motor unit action potentials. Our goal in the present study was to investigate DIAm neuromotor control during quiet spontaneous breathing (eupnea) in awake rats by evaluating DIAm EMG at specific temporal locations defined by motor unit recruitment and derecruitment. We evaluated the nonstationarity of DIAm EMG activity to identify DIAm motor unit recruitment and derecruitment durations. Combined with assessments of root mean square (RMS) and sum of squares (SS) EMG, the durations of these phases provide physiological information about the temporal aspects of motor control. During eupnea in awake rats (n = 10), the duration of motor unit recruitment comprised 61 ± 19 ms of the onset-to-peak duration (214 ± 62 ms) of the DIAm RMS EMG. The peak-to-offset duration of DIAm EMG activity was 453 ± 96 ms, with a terminating period of derecruitment of 161 ± 44 ms. The burst duration was 673 ± 128 ms. Both the RMS EMG amplitude and the SS EMG were higher at the completion of motor unit recruitment than at the start of motor unit derecruitment, suggesting that offset discharge rates were lower than onset discharge rates. Our analyses provide novel insights into the time domain aspects of DIAm neuromotor control and allow indirect estimates of the contribution of recruitment and frequency to RMS EMG amplitude during eupnea in awake rats.NEW & NOTEWORTHY We characterized three phases of neuromotor control-motor unit recruitment, sustained activity, and derecruitment-based on statistical assessments of stationarity of the diaphragm muscle (DIAm) EMG activity in awake rats. Our findings may allow indirect estimates of the contribution of motor unit recruitment and frequency coding toward generating force and provide novel insights about the temporal aspects of DIAm neuromotor control and descending respiratory drive in unanesthetized animals.

Chloroquine impairs maximal transdiaphragmatic pressure generation in old mice.

Aging results in increased neuromuscular transmission failure and denervation of the diaphragm muscle, as well as decreased force generation across a range of motor behaviors. Increased risk for respiratory complications in old age is a major health problem. Aging impairs autophagy, a tightly regulated multistep process responsible for clearing misfolded or aggregated proteins and damaged organelles. In motor neurons, aging-related autophagy impairment may contribute to deficits in neurotransmission, subsequent muscle atrophy, and loss of muscle force. Chloroquine is commonly used to inhibit autophagy. We hypothesized that chloroquine decreases transdiaphragmatic pressure (Pdi) in mice. Old mice (16-28 mo old; n = 26) were randomly allocated to receive intraperitoneal chloroquine (50 mg/kg) or vehicle 4 h before measuring Pdi during eupnea, hypoxia (10% O2)-hypercapnia (5% CO2) exposure, spontaneous deep breaths ("sighs"), and maximal activation elicited by bilateral phrenic nerve stimulation (Pdimax). Pdi amplitude and ventilatory parameters across experimental groups and behaviors were evaluated using a mixed linear model. There were no differences in Pdi amplitude across treatments during eupnea (∼8 cm H2O), hypoxia-hypercapnia (∼10 cm H2O), or sigh (∼36 cm H2O), consistent with prior studies documenting a lack of aging effects on ventilatory behaviors. In vehicle and chloroquine-treated mice, average Pdimax was 61 and 46 cm H2O, respectively. Chloroquine decreased Pdimax by 24% compared to vehicle (P < 0.05). There were no sex or age effects on Pdi in older mice. The observed decrease in Pdimax suggests aging-related susceptibility to impairments in autophagy, consistent with the effects of chloroquine on this important homeostatic process.NEW & NOTEWORTHY Recent findings suggest that autophagy plays a role in the development of aging-related neuromuscular dysfunction; however, the contribution of autophagy impairment to the maintenance of diaphragm force generation in old age is unknown. This study shows that in old mice, chloroquine administration decreases maximal transdiaphragmatic pressure generation. These chloroquine effects suggest a susceptibility to impairments in autophagy in old age.

Cell-Based Measurement of Mitochondrial Function in Human Airway Smooth Muscle Cells.

Cellular mitochondrial function can be assessed using high-resolution respirometry that measures the O2 consumption rate (OCR) across a number of cells. However, a direct measurement of cellular mitochondrial function provides valuable information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, which participates in both the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) as Complex II. In this study, we determine the maximum velocity of the SDH reaction (SDHmax) in individual human airway smooth muscle (hASM) cells. To measure SDHmax, hASM cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT, reaction indicator). As the reaction proceeded, the change in optical density (OD) due to the reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) was measured using a confocal microscope with the pathlength for light absorbance tightly controlled. SDHmax was determined during the linear period of the SDH reaction and expressed as mmol fumarate/liter of cell/min. We determine that this technique is rigorous and reproducible, and reliable for the measurement of mitochondrial function in individual cells.

Size-dependent differences in mitochondrial volume density in phrenic motor neurons.

Neuromotor control of diaphragm muscle (DIAm) motor units is dependent on an orderly size-dependent recruitment of phrenic motor neurons (PhMNs). Slow (type S) and fast, fatigue resistant (type FR) DIAm motor units, which are frequently recruited to sustain ventilation, comprise smaller PhMNs that innervate type I and IIa DIAm fibers. More fatigable fast (type FF) motor units, which are infrequently recruited for higher force, expulsive behaviors, comprise larger PhMNs that innervate more type IIx/IIb DIAm fibers. We hypothesize that due to the more frequent activation and thus higher energy demand of type S and FR motor units, the mitochondrial volume density (MVD) of smaller PhMNs is greater compared with larger PhMNs. In eight adult (6 mo old) Fischer 344 rats, PhMNs were identified via intrapleural injection of Alexa488-conjugated cholera toxin B (CTB). Following retrograde CTB labeling, mitochondria in PhMNs were labeled by transdural infusion of MitoTracker Red. PhMNs and mitochondria were imaged using multichannel confocal microscopy using a ×60 oil objective. Following optical sectioning and three-dimensional (3-D) rendering, PhMNs and mitochondria were analyzed volumetrically using Nikon Elements software. Analysis of MVD in somal and dendritic compartments was stratified by PhMN somal surface area. Smaller PhMNs (likely S and FR units) had greater somal MVDs compared with larger PhMNs (likely FF units). By contrast, proximal dendrites or larger PhMNs had higher MVD compared with dendrites of smaller PhMNs. We conclude that more active smaller PhMNs have a higher mitochondrial volume density to support their higher energy demand in sustaining ventilation.NEW & NOTEWORTHY Type S and FR motor units, comprising smaller phrenic motor neurons (PhMNs) are regularly activated to perform indefatigable ventilatory requirements. By contrast, type FF motor units, comprising larger PhMNs, are infrequently activated to perform expulsive straining and airway defense maneuvers. This difference in activation history is mirrored in the mitochondrial volume density (MVD), with smaller PhMNs having higher MVD than larger PhMNs. In proximal dendrites, this trend was reversed, with larger PhMNs having higher MVD than smaller PhMNs, likely due to the maintenance requirements for the larger dendritic arbor of FF PhMNs.

Electrophysiological effects of BDNF and TrkB signaling at type-identified diaphragm neuromuscular junctions.

Previous studies show that synaptic quantal release decreases during repetitive stimulation, i.e., synaptic depression. Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). We hypothesized that BDNF mitigates synaptic depression at the neuromuscular junction and that the effect is more pronounced at type IIx and/or IIb fibers compared to type I or IIa fibers given the more rapid reduction in docked synaptic vesicles with repetitive stimulation. Rat phrenic nerve-diaphragm muscle preparations were used to determine the effect of BDNF on synaptic quantal release during repetitive stimulation at 50 Hz. An ∼40% decline in quantal release was observed during each 330-ms duration train of nerve stimulation (intratrain synaptic depression), and this intratrain decline was observed across repetitive trains (20 trains at 1/s repeated every 5 min for 30 min for 6 sets). BDNF treatment significantly enhanced quantal release at all fiber types (P < 0.001). BDNF treatment did not change release probability within a stimulation set but enhanced synaptic vesicle replenishment between sets. In agreement, synaptic vesicle cycling (measured using FM4-64 fluorescence uptake) was increased following BDNF [or neurotrophin-4 (NT-4)] treatment (∼40%; P < 0.05). Conversely, inhibiting BDNF/TrkB signaling with the tyrosine kinase inhibitor K252a and TrkB-IgG (which quenches endogenous BDNF or NT-4) decreased FM4-64 uptake (∼34% across fiber types; P < 0.05). The effects of BDNF were generally similar across all fiber types. We conclude that BDNF/TrkB signaling acutely enhances presynaptic quantal release and thereby may serve to mitigate synaptic depression and maintain neuromuscular transmission during repetitive activation.NEW & NOTEWORTHY Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). Rat phrenic nerve-diaphragm muscle preparations were used to determine the rapid effect of BDNF on synaptic quantal release during repetitive stimulation. BDNF treatment significantly enhanced quantal release at all fiber types. BDNF increased synaptic vesicle cycling (measured using FM4-64 fluorescence uptake); conversely, inhibiting BDNF/TrkB signaling decreased FM4-64 uptake.

Molecular Mechanisms Underlying TNFα-Induced Mitochondrial Biogenesis in Human Airway Smooth Muscle.

Proinflammatory cytokines such as TNFα mediate airway inflammation. Previously, we showed that TNFα increases mitochondrial biogenesis in human ASM (hASM) cells, which is associated with increased PGC1α expression. We hypothesized that TNFα induces CREB and ATF1 phosphorylation (pCREBS133 and pATF1S63), which transcriptionally co-activate PGC1α expression. Primary hASM cells were dissociated from bronchiolar tissue obtained from patients undergoing lung resection, cultured (one-three passages), and then differentiated by serum deprivation (48 h). hASM cells from the same patient were divided into two groups: TNFα (20 ng/mL) treated for 6 h and untreated controls. Mitochondria were labeled using MitoTracker green and imaged using 3D confocal microscopy to determine mitochondrial volume density. Mitochondrial biogenesis was assessed based on relative mitochondrial DNA (mtDNA) copy number determined by quantitative real-time PCR (qPCR). Gene and/or protein expression of pCREBS133, pATF1S63, PCG1α, and downstream signaling molecules (NRFs, TFAM) that regulate transcription and replication of the mitochondrial genome, were determined by qPCR and/or Western blot. TNFα increased mitochondrial volume density and mitochondrial biogenesis in hASM cells, which was associated with an increase in pCREBS133, pATF1S63 and PCG1α expression, with downstream transcriptional activation of NRF1, NRF2, and TFAM. We conclude that TNFα increases mitochondrial volume density in hASM cells via a pCREBS133/pATF1S63/PCG1α-mediated pathway.

Chemogenetic inhibition of TrkB signalling reduces phrenic motor neuron survival and size.

Brain derived neurotrophic factor (BDNF) signalling through its high-affinity tropomyosin receptor kinase B (TrkB) is known to have potent effects on motor neuron survival and morphology during development and in neurodegenerative diseases. Here, we employed a novel 1NMPP1 sensitive TrkBF616 rat model to evaluate the effect of 14 days inhibition of TrkB signalling on phrenic motor neurons (PhMNs). Adult female and male TrkBF616 rats were divided into 1NMPP1 or vehicle treated groups. Three days prior to treatment, PhMNs in both groups were initially labeled via intrapleural injection of Alexa-Fluor-647 cholera toxin B (CTB). After 11 days of treatment, retrograde axonal uptake/transport was assessed by secondary labeling of PhMNs by intrapleural injection of Alexa-Fluor-488 CTB. After 14 days of treatment, the spinal cord was excised 100 µm thick spinal sections containing PhMNs were imaged using two-channel confocal microscopy. TrkB inhibition reduced the total number of PhMNs by ∼16 %, reduced the mean PhMN somal surface areas by ∼25 %, impaired CTB uptake 2.5-fold and reduced the estimated PhMN dendritic surface area by ∼38 %. We conclude that inhibition of TrkB signalling alone in adult TrkBF616 rats is sufficient to lead to PhMN loss, morphological degeneration and deficits in retrograde axonal uptake/transport.

CTB-targeted protocells enhance ability of lanthionine ketenamine analogs to induce autophagy in motor neuron-like cells.

Impaired autophagy, a cellular digestion process that eliminates proteins and damaged organelles, has been implicated in neurodegenerative diseases, including motor neuron disorders. Motor neuron targeted upregulation of autophagy may serve as a promising therapeutic approach. Lanthionine ketenamine (LK), an amino acid metabolite found in mammalian brain tissue, activates autophagy in neuronal cell lines. We hypothesized that analogs of LK can be targeted to motor neurons using nanoparticles to improve autophagy flux. Using a mouse motor neuron-like hybrid cell line (NSC-34), we tested the effect of three different LK analogs on autophagy modulation, either alone or loaded in nanoparticles. For fluorescence visualization of autophagy flux, we used a mCherry-GFP-LC3 plasmid reporter. We also evaluated protein expression changes in LC3-II/LC3-I ratio obtained by western blot, as well as presence of autophagic vacuoles per cell obtained by electron microscopy. Delivering LK analogs with targeted nanoparticles significantly enhanced autophagy flux in differentiated motor neuron-like cells compared to LK analogs alone, suggesting the need of a delivery vehicle to enhance their efficacy. In conclusion, LK analogs loaded in nanoparticles targeting motor neurons constitute a promising treatment option to induce autophagy flux, which may serve to mitigate motor neuron degeneration/loss and preserve motor function in motor neuron disease.

Novel regenerative drug, SPG302 promotes functional recovery of diaphragm muscle activity after cervical spinal cord injury.

Spinal cord hemisection at C2 (C2 SH), sparing the dorsal column is widely used to investigate the effects of reduced phrenic motor neuron (PhMN) activation on diaphragm muscle (DIAm) function, with reduced DIAm activity on the injured side during eupnoea. Following C2 SH, recovery of DIAm EMG activity may occur spontaneously over subsequent days/weeks. Various strategies have been effective at improving the incidence and magnitude of DIAm recovery during eupnoea, but little is known about the effects of C2 SH on transdiaphragmatic pressure (Pdi ) during other ventilatory and non-ventilatory behaviours. We employ SPG302, a novel type of pegylated benzothiazole derivative, to assess whether enhancing synaptogenesis (i.e., enhancing spared local connections) will improve the incidence and the magnitude of recovery of DIAm EMG activity and Pdi function 14 days post-C2 SH. In anaesthetised Sprague-Dawley rats, DIAm EMG and Pdi were assessed during eupnoea, hypoxia/hypercapnia and airway occlusion prior to surgery (C2 SH or sham), immediately post-surgery and at 14 days post-surgery. In C2 SH rats, 14 days of DMSO (vehicle) or SPG302 treatments (i.p. injection) occurred. At the terminal experiment, maximum Pdi was evoked by bilateral phrenic nerve stimulation. We show that significant EMG and Pdi deficits are apparent in C2 SH compared with sham rats immediately after surgery. In C2 SH rats treated with SPG302, recovery of eupneic, hypoxia/hypercapnia and occlusion DIAm EMG was enhanced compared with vehicle rats after 14 days. Treatment with SPG302 also ameliorated Pdi deficits following C2 SH. In summary, SPG302 is an exciting new therapy to explore for use in spinal cord injuries. KEY POINTS: Despite advances in our understanding of the effects of cervical hemisection (C2 SH) on diaphragm muscle (DIAm) EMG activity, very little is understood about the impact of C2 SH on the gamut of ventilatory and non-ventilatory transdiaphragmatic pressures (Pdi ). Recovery of DIAm activity following C2 SH is improved using a variety of approaches, but very few pharmaceuticals have been shown to be effective. One way of improving DIAm recovery is to enhance the amount of latent local spared connections onto phrenic motor neurons. A novel pegylated benzothiazole derivative enhances synaptogenesis in a variety of neurodegenerative conditions. Here, using a novel therapeutic SPG302, we show that 14 days of treatment with SPG302 ameliorated DIAm EMG and Pdi deficits compared with vehicle controls. Our results show that SPG302 is a compound with very promising potential for use in improving functional outcomes post-spinal cord injury.

Aging affects the number and morphological heterogeneity of rat phrenic motor neurons and phrenic motor axons.

Diaphragm muscle (DIAm) motor units comprise a phrenic motor neuron (PhMN), the phrenic nerve and the muscle fibers innervated, with the size of PhMNs and axons characteristic of motor unit type. Smaller PhMNs and their axons comprise slow (type S) and fatigue-resistant (type FR) DIAm motor units, while larger PhMNs and their axons comprise more fatigable (type FF) motor units. With aging, we have shown a loss of larger PhMNs, consistent with selective atrophy of type IIx/IIb DIAm fibers and reduced maximum DIAm force. In the present study, we hypothesized that with aging there is a loss of larger myelinated phrenic α motor axons. Female and male young (6 months) and old (24 months) Fischer 344 rats were studied. PhMNs were retrogradely labeled by intrapleural injection of 488-conjugated CTB. The phrenic nerves were excised ~1 cm from the DIAm insertion and mounted in resin, and phrenic α motor axons were delineated based on size (i.e., >4 µm diameters). In older rats, the number of larger PhMNs and larger phrenic α motor axons were reduced. There were no differences in non-α axons. In addition, there was evidence of demyelination of larger phrenic α motor axons in older rats. Together, these findings are consistent with the selective age-related vulnerability of larger PhMNs and denervation of type FF motor units, which may underlie DIAm sarcopenia.

Brain derived neurotrophic factor/tropomyosin related kinase B signaling impacts diaphragm neuromuscular transmission in a novel rat chemogenetic model.

The neuromuscular junction (NMJ) mediates neural control of skeletal muscle fibers. Neurotrophic signaling, specifically brain derived neurotrophic factor (BDNF) acting through its high-affinity tropomyosin related kinase B (TrkB) receptor is known to improve neuromuscular transmission. BDNF/TrkB signaling also maintains the integrity of antero- and retrograde communication between the motor neuron soma, its distal axons and pre-synaptic terminals and influences neuromuscular transmission. In this study, we employed a novel rat chemogenetic mutation (TrkB F616), in which a 1-naphthylmethyl phosphoprotein phosphatase 1 (1NMPP1) sensitive knock-in allele allowed specific, rapid and sustained inhibition of TrkB kinase activity. In adult female and male TrkB F616 rats, treatment with either 1NMPP1 (TrkB kinase inhibition) or DMSO (vehicle) was administered in drinking water for 14 days. To assess the extent of neuromuscular transmission failure (NMTF), diaphragm muscle isometric force evoked by nerve stimulation at 40 Hz (330 ms duration trains repeated each s) was compared to isometric forces evoked by superimposed direct muscle stimulation (every 15 s). Chronic TrkB kinase inhibition (1NMPP1 group) markedly worsened NMTF compared to vehicle controls. Acute BDNF treatment did not rescue NMTF in the 1NMPP1 group. Chronic TrkB kinase inhibition did not affect the apposition of pre-synaptic terminals (labeled with synaptophysin) and post-synaptic endplates (labeled with α-Bungarotoxin) at diaphragm NMJs. We conclude that inhibition of BDNF/TrkB signaling in TrkB F616 rats disrupts diaphragm neuromuscular transmission in a similar manner to TrkB F616A mice, likely via a pre-synaptic mechanism independent of axonal branch point failure.

Functional recovery after accidental deep hypothermic cardiac arrest: Comparison of different cardiopulmonary bypass rewarming strategies.

Introduction: Using a porcine model of accidental immersion hypothermia and hypothermic cardiac arrest (HCA), the aim of the present study was to compare effects of different rewarming strategies on CPB on need for vascular fluid supply, level of cardiac restitution, and cerebral metabolism and pressures. Materials and Methods: Totally sixteen healthy, anesthetized castrated male pigs were immersion cooled to 20°C to induce HCA, maintained for 75 min and then randomized into two groups: 1) animals receiving CPB rewarming to 30°C followed by immersion rewarming to 36°C (CPB30, n = 8), or 2) animals receiving CPB rewarming to 36°C (CPB36, n = 8). Measurements of cerebral metabolism were collected using a microdialysis catheter. After rewarming to 36°C, surviving animals in both groups were further warmed by immersion to 38°C and observed for 2 h. Results: Survival rate at 2 h after rewarming was 5 out of 8 animals in the CPB30 group, and 8 out of 8 in the CPB36 group. All surviving animals displayed significant acute cardiac dysfunction irrespective of rewarming method. Differences between groups in CPB exposure time or rewarming rate created no differences in need for vascular volume supply, in variables of cerebral metabolism, or in cerebral pressures and blood flow. Conclusion: As 3 out of 8 animals did not survive weaning from CPB at 30°C, early weaning gave no advantages over weaning at 36°C. Further, in surviving animals, the results showed no differences between groups in the need for vascular volume replacement, nor any differences in cerebral blood flow or pressures. Most prominent, after weaning from CPB, was the existence of acute cardiac failure which was responsible for the inability to create an adequate perfusion irrespective of rewarming strategy.