Exercise‐linked improvement in age‐associated loss of balance is associated with increased vestibular input to motor neurons

Age‐associated loss of muscle function is exacerbated by a concomitant reduction in balance, leading to gait abnormalities and falls. Even though balance defects can be mitigated by exercise, the underlying neural mechanisms are unknown. We now have investigated components of the proprioceptive and vestibular systems in specific motor neuron pools in sedentary and trained old mice, respectively. We observed a strong age‐linked deterioration in both circuits, with a mitigating effect of exercise on vestibular synapse numbers on motor neurons, closely associated with an improvement in gait and balance in old mice. Our results thus describe how the proprioceptive and vestibular systems are modulated by age and exercise, and how these changes affect their input to motor neurons. These findings not only make a strong case for exercise‐based interventions in elderly individuals to improve balance, but could also lead to targeted therapeutic interventions aimed at the respective neuronal circuitry.     

Wild‐type FLT3 and FLT3 ITD exhibit similar ligand‐induced internalization characteristics

Class III receptor tyrosine kinases control the development of hematopoietic stem cells. Constitutive activation of FLT3 by internal tandem duplications (ITD) in the juxtamembrane domain has been causally linked to acute myeloid leukaemia. Oncogenic FLT3 ITD is partially retained in compartments of the biosynthetic route and aberrantly activates STAT5, thereby promoting cellular transformation. The pool of FLT3 ITD molecules in the plasma membrane efficiently activates RAS and AKT, which is likewise essential for cell transformation. Little is known about features and mechanisms of FLT3 ligand (FL)‐dependent internalization of surface‐bound FLT3 or FLT3 ITD. We have addressed this issue by internalization experiments using human RS4‐11 and MV4‐11 cells with endogenous wild‐type FLT3 or FLT3 ITD expression, respectively, and surface biotinylation. Further, FLT3 wild‐type, or FLT3 ITD‐GFP hybrid proteins were stably expressed and characterized in 32D cells, and internalization and stability were assessed by flow cytometry, imaging flow cytometry, and immunoblotting. FL‐stimulated surface‐exposed FLT3 WT or FLT3 ITD protein showed similar endocytosis and degradation characteristics. Kinase inactivation by mutation or FLT3 inhibitor treatment strongly promoted FLT3 ITD surface localization, and attenuated but did not abrogate FL‐induced internalization. Experiments with the dynamin inhibitor dynasore suggest that active FLT3 as well as FLT3 ITD is largely endocytosed via clathrin‐dependent endocytosis. Internalization of kinase‐inactivated molecules occurred through a different yet unidentified mechanism. Our data demonstrate that FLT3 WT and constitutively active FLT3 ITD receptor follow, despite very different biogenesis kinetics, similar internalization and degradation routes.     

Microfibril Orientation Dominates the Microelastic Properties of Human Bone Tissue at the Lamellar Length Scale

The elastic properties of bone tissue determine the biomechanical behavior of bone at the organ level. It is now widely accepted that the nanoscale structure of bone plays an important role to determine the elastic properties at the tissue level. Hence, in addition to the mineral density, the structure and organization of the mineral nanoparticles and of the collagen microfibrils appear as potential key factors governing the elasticity. Many studies exist on the role of the organization of collagen microfibril and mineral nanocrystals in strongly remodeled bone. However, there is no direct experimental proof to support the theoretical calculations. Here, we provide such evidence through a novel approach combining several high resolution imaging techniques: scanning acoustic microscopy, quantitative scanning small-Angle X-ray scattering imaging and synchrotron radiation computed microtomography. We find that the periodic modulations of elasticity across osteonal bone are essentially determined by the orientation of the mineral nanoparticles and to a lesser extent only by the particle size and density. Based on the strong correlation between the orientation of the mineral nanoparticles and the collagen molecules, we conclude that the microfibril orientation is the main determinant of the observed undulations of microelastic properties in regions of constant mineralization in osteonal lamellar bone. This multimodal approach could be applied to a much broader range of fibrous biological materials for the purpose of biomimetic technologies.     

Stochastic Loss of Silencing of the Imprinted Ndn/NDN Allele,in a Mouse Model and Humans with Prader-Willi Syndrome,Has Functional Consequences

Genomic imprinting is a process that causes genes to be expressed from one allele only according to parental origin, the other allele being silent. Diseases can arise when the normally active alleles are not expressed. In this context, low level of expression of the normally silent alleles has been considered as genetic noise although such expression has never been further studied. Prader-Willi Syndrome (PWS) is a neurodevelopmental disease involving imprinted genes, including NDN, which are only expressed from the paternally inherited allele, with the maternally inherited allele silent. We present the first in-depth study of the low expression of a normally silent imprinted allele, in pathological context. Using a variety of qualitative and quantitative approaches and comparing wild-type, heterozygous and homozygous mice deleted for Ndn, we show that, in absence of the paternal Ndn allele, the maternal Ndn allele is expressed at an extremely low level with a high degree of non-genetic heterogeneity. The level of this expression is sex-dependent and shows transgenerational epigenetic inheritance. In about 50% of mutant mice, this expression reduces birth lethality and severity of the breathing deficiency, correlated with a reduction in the loss of serotonergic neurons. In wild-type brains, the maternal Ndn allele is never expressed. However, using several mouse models, we reveal a competition between non-imprinted Ndn promoters which results in monoallelic (paternal or maternal) Ndn expression, suggesting that Ndn allelic exclusion occurs in the absence of imprinting regulation. Importantly, specific expression of the maternal NDN allele is also detected in post-mortem brain samples of PWS individuals. Our data reveal an unexpected epigenetic flexibility of PWS imprinted genes that could be exploited to reactivate the functional but dormant maternal alleles in PWS. Overall our results reveal high non-genetic heterogeneity between genetically identical individuals that might underlie the variability of the phenotype.     

Validation of a patient-specific one-dimensional model of the systemic arterial tree

The aim of this study is to develop and validate a patient-specific distributed model of the systemic arterial tree. This model is built using geometric and hemodynamic data measured on a specific person and validated with noninvasive measurements of flow and pressure on the same person, providing thus a patient-specific model and validation. The systemic arterial tree geometry was obtained from MR angiographic measurements. A nonlinear viscoelastic constitutive law for the arterial wall is considered. Arterial wall distensibility is based on literature data and adapted to match the wave propagation velocity of the main arteries of the specific subject, which were estimated by pressure waves traveling time. The intimal shear stress is modeled using the Witzig-Womersley theory. Blood pressure is measured using applanation tonometry and flow rate using transcranial ultrasound and phase-contrast-MRI. The model predicts pressure and flow waveforms in good qualitative and quantitative agreement with the in vivo measurements, in terms of wave shape and specific wave features. Comparison with a generic one-dimensional model shows that the patient-specific model better predicts pressure and flow at specific arterial sites. These results obtained let us conclude that a patient-specific one-dimensional model of the arterial tree is able to predict well pressure and flow waveforms in the main systemic circulation, whereas this is not always the case for a generic one-dimensional model.     

Electroporation-aided DNA immunization generates polyclonal antibodies against the native conformation of human endothelin B receptor

Endothelin B receptor (ET(B)R) is a G protein-coupled receptor (GPCR) specific for endothelin peptides (including endothelin-1, ET1), which mediates a variety of key physiological functions in normal tissues, such as modulation of vasomotor tone, tissue differentiation, or cell proliferation. Moreover, ET(B)R, overexpressed in various cancer cells including melanoma, has been implicated in the growth and progression of tumors, as well as in controlling T cell homing to tumors. To gather information on receptor structure and function, antibodies are generally considered choice molecular probes, but generation of such reagents against the native conformation of GPCRs is a real technical challenge. Here, we show that electroporation-aided genetic immunization, coupled to cardiotoxin pretreatment, is a simple and very efficient method to raise large amounts of polyclonal antibodies highly specific for native human ET(B)R (hET(B)R), as assessed by both flow cytometry analysis of different stably transfected cell lines and a new and rapid cell-based enzyme-linked immunosorbent assay that we also describe. The antibodies recognized two major epitopes on hET(B)R, mapped within the N-terminal extracellular domain. They were used to reveal hET(B)R on membranes of three different human melanoma cell lines, by flow cytometry and confocal microscopy, a method that we show is more relevant than mRNA polymerase chain reaction in assessing receptor expression. In addition, ET-1 partially competed with antibodies for receptor binding. The strategy described here, thus, efficiently generated new immunological tools to further analyze the role of ET(B)R under both normal and pathological conditions, including cancers. Above all, it can now be used to raise monoclonal antibodies against hET(B)R and, more generally, against GPCRs that constitute, by far, the largest reservoir of potential pharmacological targets.