Development of the hair bundle and mechanotransduction

This review focuses on the cellular and molecular mechanisms underlying the development of the sensory hair bundle, an apical specialisation of the hair cell that is essential for mechanotransduction. The structure, function and development of the hair bundle is described, with an emphasis on the properties and possible roles played by the different link types that interconnect the individual elements of the hair bundle - the multiple stereocilia and the single kinocilium. Studies of mouse and zebrafish mutants have revealed that several classes of molecule are required for the genesis and maintenance of hair-bundle structure. These include cell surface molecules that are associated with the different hair-bundle links, along with myosin motors, scaffolding proteins and an actin cross-linker. Finally we consider how differences in the form and shape of hair bundles within and between different sensory organs are generated.     

The relationship between plasma potassium concentration and muscle torque during recovery following intense exercise

The present study investigated the relationship between plasma potassium ion concentration ([K⁺]) and skeletal muscle torque during three different 15-min recovery periods after fatigue induced by four 30-s sprints. Four males and one female completed the multiple sprint exercise on three separate days; recovery was passive, i.e. no cycling exercise (PRec), active cycling at 30% peak oxygen consumption O_2peak (30% Rec) and active cycling at 60% O_2peak (60% Rec). Plasma [K⁺] was measured from blood sampled from an antecubital vein of subjects at rest and at 0, 3, 5, 10 and 15 min into each recovery. Isokinetic leg strength was measured at rest and at 1, 6, 11 and 16 min during each recovery. Following the exhaustive sprints, [K⁺] increased significantly from an average mean (SEM) resting value of 3.81 (0.07) mmol · l⁻¹ to 4.48 (0.19) mmol · l⁻¹ (P < 0.01). In all recovery conditions, plasma [K⁺] returned to resting levels within 3 min following the fourth sprint. However, in the two active recovery conditions plasma [K⁺] increased over the remainder of the recovery periods to 4.36 (0.12) mmol · l⁻¹ in the 30% Rec condition and 4.62 (0.12) mmol · l⁻¹ in the 60% Rec condition, the latter being significantly higher than the former (P < 0.01). The maximum torque measured following the sprints decreased significantly, on average, to 61.1 (8.36)% of peak levels (P < 0.01). After 15 min of recovery, maximum torque was highest in the 30% Rec condition at 92.13 (3.06)% of peak levels (P < 0.01), compared to 85.23 (3.64)% and 85.71 (0.82)% for the PRec and 60% Rec conditions, respectively. In contrast to the significant differences in plasma [K⁺] across all three recovery conditions, muscle torque recovery was significantly different in only the 30% Rec condition. In summary, recovery of peak levels of muscle torque following fatiguing exercise does not appear to follow changes in plasma [K⁺]. Accepted: 18 October 1996     

Tubular epithelial C1orf54 mediates protection and recovery from acute kidney injury

Acute kidney injury (AKI) incidence among hospitalized patients is increasing steadily. Despite progress in prevention strategies and support measures, AKI remains correlated with high mortality, particularly among ICU patients, and no effective AKI therapy exists. Here, we investigated the function in kidney ischaemia‐reperfusion injury (IRI) of C1orf54, a newly identified protein encoded by an open reading frame on chromosome 1. C1orf54 expression was high in kidney and low in heart, liver, spleen, lung and skeletal muscle in healthy mice, and in the kidney, C1orf54 was expressed in tubular epithelial cells (TECs), but not in glomeruli. C1orf54 expression was markedly decreased on Day 1 after kidney IRI and then gradually recovered to baseline levels by Day 7. Notably, relative to wild‐type mice, C1orf54‐knockout mice exhibited impaired TEC proliferation and delayed recovery after kidney IRI, which led to deteriorated renal function and increased mortality. Conversely, adenovirus‐mediated C1orf54 overexpression promoted TEC proliferation and ameliorated kidney pathology, which resulted in accelerated renal repair and improved renal function. Mechanistically, C1orf54 was found to promote TEC proliferation through PI3K/AKT signalling. Thus, C1orf54 holds considerable potential as a therapeutic target in kidney IRI.     

Myosin light chain kinase mediates intestinal barrier disruption following burn injury

Background

Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC) phosphorylation mediated by MLC kinase (MLCK) is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction.

Methodology/Principal Findings

Male balb/c mice were assigned randomly to either sham burn (control) or 30% total body surface area (TBSA) full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg), an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC)-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression.

Conclusions/Significance

The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury.     

Discharge patterns of chicken cochlear ganglion neurons following kanamycin-induced hair cell loss and regeneration

Hair cells in the basal, high frequency region (>1100 Hz) of the chicken cochlea were destroyed with kanamycin (400 mg/kg/d × 10 d) and allowed to regenerate. Afterwards, single unit recordings were made from cochlear ganglion neurons at various times post-treatment. During the first few weeks post-treatment, only neurons with low characteristic frequencies (<1100 Hz) responded to sound. Despite the fact that the low frequency region of the cochlea was not destroyed, neurons with low characteristic frequencies had elevated thresholds, abnormally broad U-shaped or W-shaped tuning curves and low spontaneous discharge rates. At 2 days post-treatment, the spontaneous discharge rates of some acoustically unresponsive units fluctuated in a rhythmical manner. As recovery time increased, thresholds decreased, tuning curves narrowed and developed a symmetrical V-shape, spontaneous rate increased and neurons with higher characteristic frequencies began to respond to sound. In addition, the proportion of interspike interval histograms with regularly spaced peaks increased. These improvements progressed along a low-to-high characteristic frequency gradient. By 10–20 weeks post-treatment, the thresholds and tuning curves of neurons with characteristic frequencies below 2000 Hz were within normal limits; however, the spontaneous discharge rates of the neurons were still significantly lower than those from normal animals.Abbreviations KM kanamycin - BrdU bromodeoxyuridine - CF characteristic frequency - CAP compound action potential - ISI interspike interval     

Rapid carbon loss and slow recovery following permafrost thaw in boreal peatlands

Permafrost peatlands store one‐third of the total carbon (C) in the atmosphere and are increasingly vulnerable to thaw as high‐latitude temperatures warm. Large uncertainties remain about C dynamics following permafrost thaw in boreal peatlands. We used a chronosequence approach to measure C stocks in forested permafrost plateaus (forest) and thawed permafrost bogs, ranging in thaw age from young (<10 years) to old (>100 years) from two interior Alaska chronosequences. Permafrost originally aggraded simultaneously with peat accumulation (syngenetic permafrost) at both sites. We found that upon thaw, C loss of the forest peat C is equivalent to ~30% of the initial forest C stock and is directly proportional to the prethaw C stocks. Our model results indicate that permafrost thaw turned these peatlands into net C sources to the atmosphere for a decade following thaw, after which post‐thaw bog peat accumulation returned sites to net C sinks. It can take multiple centuries to millennia for a site to recover its prethaw C stocks; the amount of time needed for them to regain their prethaw C stocks is governed by the amount of C that accumulated prior to thaw. Consequently, these findings show that older peatlands will take longer to recover prethaw C stocks, whereas younger peatlands will exceed prethaw stocks in a matter of centuries. We conclude that the loss of sporadic and discontinuous permafrost by 2100 could result in a loss of up to 24 Pg of deep C from permafrost peatlands.     

Eps8 regulates hair bundle length and functional maturation of mammalian auditory hair cells

Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells.     

Two-state model for outer hair cell stiffness and motility

With discovery of the protein prestin and the gathering evidence linking it to outer hair cell electromotility, the working mechanism of outer hair cells is becoming clearer. Recent experiments have established the voltage-dependent stiffness of outer hair cells and given an insight into the nature of variation of stiffness with respect to voltage. These and earlier experiments are used to analyze and develop models of outer hair cell response. In this article, recent modeling efforts have been reconciled and placed into a common mechanics-based framework. The constitutive models are analyzed with regard to their capability to replicate experimental results. We extend the area motor model to include elastic constants dependent on motor state. The modified model successfully captures stiffness variations of outer hair cells and capacitance changes with respect to voltage.     

Mitochondrial uncoupling protein-2 mediates steatotic liver injury following ischemia/reperfusion

Steatotic livers are not used for transplantation because they have a reduced tolerance for ischemic events with reduced ATP levels and greater levels of cellular necrosis, which ultimately result in total organ failure. Mitochondrial uncoupling protein-2 (UCP2) is highly expressed in steatotic livers and may be responsible for liver sensitivity to ischemia through mitochondrial and ATP regulation. To test this hypothesis, experiments were conducted in lean and steatotic (ob/ob), wild-type, and UCP2 knock-out mice subjected to total warm hepatic ischemi-a/reperfusion. Although ob/ob UCP2 knock-out mice and ob/ob mice have a similar initial phenotype, ob/ob UCP2 knock-out animal survival was 83% when compared with 30% in ob/ob mice 24 h after reperfusion. Serum alanine aminotransferase concentrations and hepatocellular necrosis were decreased in the ob/ob UCP2 knock-out mice when compared with ob/ob mice subjected to ischemia. Liver ATP levels were increased in the ob/ob UCP2 knock-out animals after reperfusion when compared with the ob/ob mice but remained below the concentrations from lean livers. Lipid peroxidation (thiobarbituric acid-reactive substances) increased after reperfusion most significantly in the steatotic groups, but the increase was not affected by UCP2 deficiency. These results reveal that UCP2 expression is a critical factor, which sensitizes steatotic livers to ischemic injury, regulating liver ATP levels after ischemia and reperfusion.     

Multipotent hair follicle stem cells promote repair of spinal cord injury and recovery of walking function

The mouse hair follicle is an easily accessible source of actively growing, pluripotent adult stem cells. C57BL transgenic mice, labeled with the fluorescent protein GFP, afforded follicle stem cells whose fate could be followed when transferred to recipient animals. These cells appear to be relatively undifferentiated since they are positive for the stem cell markers nestin and CD34 but negative for the keratinocyte marker keratin 15. These hair follicle stem cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Implanting hair follicle stem cells into the gap region of severed sciatic or tibial nerves greatly enhanced the rate of nerve regeneration and restoration of nerve function. The transplanted follicle cells transdifferentiated mostly into Schwann cells, which are known to support neuron regrowth. The treated mice regained the ability to walk essentially normally. In the present study, we severed the thoracic spinal chord of C57BL/6 immunocompetent mice and transplanted GFP-expressing hair follicle stem cells to the injury site. Most of the transplanted cells also differentiated into Schwann cells that apparently facilitated repair of the severed spinal cord. The rejoined spinal cord reestablished extensive hind-limb locomotor performance. These results suggest that hair follicle stem cells can promote the recovery of spinal cord injury. Thus, hair follicle stem cells provide an effective accessible, autologous source of stem cells for the promising treatment of peripheral nerve and spinal cord injury.     

The BEACH protein LRBA is required for hair bundle maintenance in cochlear hair cells and for hearing

Lipopolysaccharide‐responsive beige‐like anchor protein (LRBA) belongs to the enigmatic class of BEACH domain‐containing proteins, which have been attributed various cellular functions, typically involving intracellular protein and membrane transport processes. Here, we show that LRBA deficiency in mice leads to progressive sensorineural hearing loss. In LRBA knockout mice, inner and outer hair cell stereociliary bundles initially develop normally, but then partially degenerate during the second postnatal week. LRBA deficiency is associated with a reduced abundance of radixin and Nherf2, two adaptor proteins, which are important for the mechanical stability of the basal taper region of stereocilia. Our data suggest that due to the loss of structural integrity of the central parts of the hair bundle, the hair cell receptor potential is reduced, resulting in a loss of cochlear sensitivity and functional loss of the fraction of spiral ganglion neurons with low spontaneous firing rates. Clinical data obtained from two human patients with protein‐truncating nonsense or frameshift mutations suggest that LRBA deficiency may likewise cause syndromic sensorineural hearing impairment in humans, albeit less severe than in our mouse model.     

Tailchaser (Tlc): A new mouse mutation affecting hair bundle differentiation and hair cell survival

We have undertaken a phenotypic approach in the mouse to identifying molecules involved in inner ear function by N-ethyl-N-nitrosourea mutagenesis followed by screening for new dominant mutations affecting hearing or balance. The pathology and genetic mapping of the first of these new mutants, tailchaser (Tlc), is described here. Tlc/+ mutants display classic behavioural symptoms of a vestibular dysfunction, including head-shaking and circling. Behavioural testing of ageing mice revealed a gradual deterioration of both hearing and balance function, indicating that the pathology caused by the Tlc mutation is progressive, similar to many dominant nonsyndromic deafnesses in humans. Based on scanning electron microscopy (SEM) studies, Tlc clearly plays a developmental role in the hair cells of the cochlea since the stereocilia bundles fail to form the characteristic V-shape pattern around the time of birth. By young adult stages, Tlc/+ outer hair bundles are grossly disorganised although inner hair bundles appear relatively normal by SEM. Increased compound action potential thresholds revealed that the Tlc/+ cochlear hair cells were not functioning normally in young adults. Similar to inner hair cells, the hair bundles of the vestibular hair cells also do not appear grossly disordered. However, all types of hair cells in the Tlc/+ inner ear eventually degenerate, apparently regardless of the degree of organisation of their hair bundles. We have mapped the Tlc mutation to a 12 cM region of chromosome 2, between D2Mit164 and D2Mit423. Based on the mode of inheritance and map location, Tlc appears to be a novel mouse mutation affecting both hair cell survival and stereocilia bundle development.     

Functional recovery in a primate model of Parkinson's disease following motor cortex stimulation

A concept in Parkinson's disease postulates that motor cortex may pattern abnormal rhythmic activities in the basal ganglia, underlying the genesis of observed motor symptoms. We conducted a preclinical study of electrical interference in the primary motor cortex using a chronic MPTP primate model in which dopamine depletion was progressive and regularly documented using 18F-DOPA positron tomography. High-frequency motor cortex stimulation significantly reduced akinesia and bradykinesia. This behavioral benefit was associated with an increased metabolic activity in the supplementary motor area as assessed with 18-F-deoxyglucose PET, a normalization of mean firing rate in the internal globus pallidus (GPi) and the subthalamic nucleus (STN), and a reduction of synchronized oscillatory neuronal activities in these two structures. Motor cortex stimulation is a simple and safe procedure to modulate subthalamo-pallido-cortical loop and alleviate parkinsonian symptoms without requiring deep brain stereotactic surgery.     

Epidermal growth factor accelerates recovery of LLC-PK1 cells following oxidant injury

Summary In renal tubular epithelial cells, oxidant injury results in several metabolic alterations including ATP depletion, decreased Na⁺K⁺ ATPase activity, and altered intracellular sodium and potassium content. To investigate the recovery of LLC-PK1 cells following oxidant injury and to determine if recovery can be accelerated, we induced oxidant stress in LLC-PK1 cells with 500 μM hydrogen peroxide for 60 min. Identical cohorts of oxidant-stressed cells were incubated in recovery medium without epidermal growth factor (EGF) or recovery medium containing 25 ng EGF per ml. ATP levels, Na⁺K⁺ ATPase activity in whole cells, Na⁺K⁺ ATPase activity in disrupted cells, and intracellular sodium and potassium ion content were determined at 0, 5, 24, 48, and 72 h following oxidant injury in each cohort of cells. In oxidant-stressed cells recovering in medium without EGF, ATP levels, Na⁺K⁺ ATPase activity, and intracellular ion content improved but continued to remain substantially lower than control values at all time points following oxidant stress. In cells recovering in medium with EGF, ATP levels, Na⁺K⁺ ATPase activity, and the intracellular potassium-to-sodium ratio were significantly higher at nearly all time points than values in cells recovering in medium alone. In cells recovering with added EGF, Na⁺K⁺ ATPase activity had improved to control levels, whereas ATP levels and intracellular ion content approached control values by 72 h following oxidant stress. We conclude that oxidant-mediated ATP depletion, altered Na⁺K⁺ ATPase activity, and intracellular ion content remain depressed for several d following oxidant stress and that EGF accelerated recovery of LLC-PK1 cells from oxidant injury.