Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

Ca²⁺ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca_V1.3 Ca²⁺ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca²⁺ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca_V1.3 Ca²⁺ channels, resulting in reduced synaptic Ca²⁺ influx. Using superresolution microscopy, we found that Ca²⁺ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca²⁺ current, whereas the apparent Ca²⁺ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca²⁺ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca²⁺ channels at IHC AZs and are required for normal hearing.Tens of Ca_V1.3 Ca²⁺ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1–4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5–7). The mechanisms governing the number of Ca²⁺ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca²⁺ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca²⁺ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1–RIM4). All isoforms contain a C-terminal C₂ domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca²⁺ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca_Vβ) subunits (12, 13) and pore-forming Ca_Vα subunits (14, 15). In addition, RIMs are indirectly linked to Ca²⁺ channels via RIM-binding protein (14, 16, 17). A regulation of biophysical channel properties has been demonstrated in heterologous expression systems for RIM1 (12) and RIM2 (13).A role of RIM1 and RIM2 in clustering Ca²⁺ channels at the AZ was demonstrated by analysis of RIM1/2-deficient presynaptic terminals of cultured hippocampal neurons (14), auditory neurons in slices (18), and Drosophila neuromuscular junction (19). Because α-RIMs also bind the vesicle-associated protein Ras-related in brain 3 (Rab3) via the N-terminal zinc finger domain (20), they are also good candidates for molecular coupling of Ca²⁺ channels and vesicles (18, 21, 22). Finally, a role of RIMs in priming of vesicles for fusion is the subject of intense research (18, 21–27). RIMs likely contribute to priming via disinhibiting Munc13 (26) and regulating vesicle tethering (27). Here, we studied the expression and function of RIM in IHCs. We combined molecular, morphologic, and physiologic approaches for the analysis of RIM2α knockout mice [RIM2α SKO (28); see Methods] and of hair cell-specific RIM1/2 knockout mice (RIM1/2 cDKO). We demonstrate that RIM2α and RIM2β are present at IHC AZs of hearing mice, positively regulate the number of synaptic Ca_V1.3 Ca²⁺ channels, and are required for normal hearing.     

Acceleration of conduction velocity linked to clustering of nodal components precedes myelination

High-density accumulation of voltage-gated sodium (Na_v) channels at nodes of Ranvier ensures rapid saltatory conduction along myelinated axons. To gain insight into mechanisms of node assembly in the CNS, we focused on early steps of nodal protein clustering. We show in hippocampal cultures that prenodes (i.e., clusters of Na_v channels colocalizing with the scaffold protein ankyrinG and nodal cell adhesion molecules) are detected before myelin deposition along axons. These clusters can be induced on purified neurons by addition of oligodendroglial-secreted factor(s), whereas ankyrinG silencing prevents their formation. The Na_v isoforms Na_v1.1, Na_v1.2, and Na_v1.6 are detected at prenodes, with Na_v1.6 progressively replacing Na_v1.2 over time in hippocampal neurons cultured with oligodendrocytes and astrocytes. However, the oligodendrocyte-secreted factor(s) can induce the clustering of Na_v1.1 and Na_v1.2 but not of Na_v1.6 on purified neurons. We observed that prenodes are restricted to GABAergic neurons, whereas clustering of nodal proteins only occurs concomitantly with myelin ensheathment on pyramidal neurons, implying separate mechanisms of assembly among different neuronal subpopulations. To address the functional significance of these early clusters, we used single-axon electrophysiological recordings in vitro and showed that prenode formation is sufficient to accelerate the speed of axonal conduction before myelination. Finally, we provide evidence that prenodal clusters are also detected in vivo before myelination, further strengthening their physiological relevance.Voltage-gated sodium (Na_v) channels are highly enriched at the axon initial segment (AIS) and the node of Ranvier, allowing generation and rapid propagation of action potentials by saltatory conduction in myelinated fibers. These axonal domains also contain cell adhesion molecules [e.g., neurofascin 186 (Nfasc186)] and the scaffolding proteins ankyrinG (AnkG) and βIV spectrin, which provide a potential link with the actin cytoskeleton (1). Flanking the nodes are the paranodes, where axoglial junctions between paranodal myelin loops and the axon are formed through interactions between axonal contactin-associated protein (Caspr)/contactin and glial Nfasc155 (2, 3). Although the mechanisms of nodal assembly are best characterized in the peripheral nervous system (4–9), less is known about the cellular and molecular mechanisms underlying node assembly in the CNS. ECM proteins, adhesion molecules, such as Nfasc186, and also, axoglial paranodal junctions have been shown to trigger CNS nodal clustering, although their respective roles remain uncertain (10–19). Moreover, axonal clustering of Na_v channels before myelin deposition and oligodendroglial contact has been shown to occur in retinal ganglion cell (RGC) cultures, where these clusters were induced by oligodendroglial-secreted factor(s) (20, 21).Here, we have investigated the cellular and molecular mechanisms underlying nodal protein assembly in hippocampal neuron cultures. We first showed that evenly spaced clusters of Na_v channels, colocalizing with Nfasc186 and AnkG, are detected along axons before myelination. Strikingly, this prenode assembly is restricted to GABAergic interneurons, suggesting the existence of different mechanisms of nodal assembly. The prenodal clustering can be induced on purified neurons by the addition of oligodendroglial-secreted factor(s) and also depends on intrinsic cues, such as AnkG. Furthermore, we also provide evidence that these clusters are detected in vivo before myelination on hippocampal tissue sections. Finally, to gain insight into their functional significance, we performed in vitro simultaneous somatic and axonal recordings and showed that the presence of prenodes increases the speed of action potential propagation along axons before myelination.     

Changes in Volumetric Hemodynamic Parameters Induced by Fluid Removal on Hemodialysis in Critically Ill Patients

Management of volume status is difficult in critically ill patients with renal failure. Volumetric hemodynamic indices are increasingly being used to guide fluid therapy in the intensive care unit (ICU), but are not established to monitor hemodialysis‐induced fluid removal in critically ill patients. Using volumetric hemodynamic monitoring, changes in extravascular lung water index (EVLWI) and intrathoracic blood volume index (ITBVI) were measured immediately before and after hemodialysis sessions in 35 ICU patients. Additional hemodynamic and oxygenation related parameters were recorded at the same time, and online relative blood volume (RBV) monitoring was performed during hemodialysis. EVLWI decreased significantly with fluid removal (median 10.0 vs. 9.6 mL/kg, P = 0.001), whereas ITBVI remained stable (median 1012 vs. 1029 mL/m², P = 0.402). Significant changes were also observed in stroke volume variation (median 12.0 vs. 13.0 %, P = 0.012), cardiac index (median 4.2 vs. 3.5 mL/min/m², P = 0.003), mean arterial pressure (median 77 vs. 85.5 mmHg, P = 0.006), norepinephrine dose (median 0.092 vs. 0.114 μg/kg per min, P = 0.043), and hemoglobin values (median 9.5 vs. 10.4 gm/dL, P = 0.036). RBV decreased by 7.8% (median); there was no correlation with either the volumetric measurements or the other hemodynamic parameters recorded. EVLWI reduction with dialysis reflects the removal of excess body fluid, whereas preservation of cardiac preload is indicated by ITBVI stability. Volumetric hemodynamic measurements provide additional information concerning fluid status and are thus potentially useful to guide fluid removal on hemodialysis in critically ill patients.     

RNA Aptamers Recognizing Murine CCL17 Inhibit T Cell Chemotaxis and Reduce Contact Hypersensitivity In Vivo

1. Download high-res image (227KB)
2. Download full-size image

     

Granzyme B and perforin: constitutive expression in human polymorphonuclear neutrophils

Polymorphonuclear neutrophils (PMNs) produce an abundance of bactericidal and cytotoxic molecules consistent with their role as first-line defense against bacterial infection. PMNs, however, also cause efficient cellular cytotoxicity when targeted through Fc receptors to appropriate antibody-coated target cells. Although this so-called antibody-dependent cellular cytotoxicity (ADCC) was described many years ago, the mechanism of killing is still elusive. We now have found that PMNs contain perforin and granzyme B, the 2 molecules known as the cytotoxic entity of natural killer cells and of cytotoxic T lymphocytes as well. Lysates of PMNs were lytic for chicken erythrocytes in a time-, temperature-, and Ca(2+)-dependent manner. Moreover, apoptosis of Jurkat cells was induced, consistent with the observation that the PMN lysates contain enzymatically active granzyme B. Taken together, our data provide evidence for the presence of perforin and granzyme B within the cytotoxic arsenal of PMNs.     

Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria

F₁F_o-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology.F₁F_o-ATP synthases are ubiquitous, highly conserved energy-converting membrane protein complexes. ATP synthases produce ATP from ADP and inorganic phosphate (P_i) by rotary catalysis (1, 2) using the energy stored in a transmembrane electrochemical gradient. The ∼600-kDa monomer of the mitochondrial ATP synthase is composed of a soluble F₁ subcomplex and a membrane-bound F_o subcomplex (3). The main components of the F₁ subcomplex are the (αβ)₃ hexamer and the central stalk (4). The F_o subcomplex includes a rotor ring of 8–15 hydrophobic c subunits (5), the peripheral stalk, and several small hydrophobic stator subunits. Protons flowing through the membrane part of the F_o subcomplex drive the rotation of the c-ring (6–9). The central stalk transmits the torque generated by c-ring rotation to the catalytic head of the F₁ subcomplex, where it induces conformational changes of the α and β subunits that result in phosphate bond formation and the generation of ATP. The catalytic (αβ)₃ hexamer is held stationary relative to the membrane region by the peripheral stalk (10, 11). Several high-resolution structures of the F₁/rotor ring complexes have been solved by X-ray crystallography (12–16), and the structure of the complete assembly has been determined by cryoelectron microscopy (cryo-EM) (10, 17–20).In mitochondria, the ATP synthase forms dimers in the inner membrane. In fungi, plants, and metazoans, the dimers are V-shaped and associate into rows along the highly curved ridges of lamellar cristae (19–22). F_o subcomplexes of the two monomers in the dimer interact in the lipid bilayer via a number of hydrophobic stator subunits (20, 23–25). Coarse-grained molecular dynamics simulations have suggested that the V-shape of the ATP synthase dimers induces local membrane curvature, which in turn drives the association of ATP synthase dimers into rows (20). The exact role of the dimer rows is unclear, however rows of ATP synthase dimers have been proposed to promote the formation of lamellar cristae in yeast (20, 26).So far, all rows of ATP synthase dimers observed by electron cryotomography have been more or less straight (19–22, 27). However, an earlier deep-etch freeze-fracture study of mitochondria from the ciliate Paramecium multimicronucleatum revealed double rows of interdigitating 10-nm particles on helical tubular cristae (28). These particles were interpreted as ATP synthases, which, if correct, would suggest that the mitochondrial ATP synthase can assemble into rows that differ significantly from the standard geometry found in lamellar cristae (19, 21, 22).To investigate the helical rows in more detail, we performed electron cryotomography of isolated mitochondrial membranes from Paramecium tetraurelia. Using subtomogram averaging, we show that these helical rows do indeed consist of ATP synthase molecules, as suggested by Allen et al. (28). However, unlike the V-shaped dimers of metazoans, the ATP synthase of this species forms U-shaped dimers, which have new and unusual structural features. When assembled into the helical rows, the ATP synthase monomers interdigitate, whereas the U-shaped dimers align side by side. Thus, rows of ATP synthase dimers seem to be a universal feature of all mitochondria. We propose that the particular shape of the P. tetraurelia ATP synthase dimer induces its assembly into helical rows, which in turn cause the formation of the helical tubular cristae of ciliates.     

Survival from tumours of the central nervous system in Danish children: Is survival related to family circumstances?

Little is known about social inequalities in childhood cancer survival. We investigated the impact of family circumstances on survival from paediatric central nervous system (CNS) tumours in a nationwide, register‐based cohort of Danish children. All children born between 1973 and 2006 and diagnosed with a CNS tumour before the age of 20 years (N = 1,261) were followed until 10 years from diagnosis. Using Cox proportional hazards models, the impact of various family characteristics on overall survival was estimated. Hazard ratios (HRs) for all CNS tumours combined did not show strong associations between survival and any family characteristic. Analyses by CNS tumour subtypes showed reduced survival for children with glioma when living outside of Copenhagen (HR 1.55; CI 1.03; 2.35). For embryonal CNS tumours, the number of full siblings was associated with worse survival (HR for having 3+ siblings 3.60; CI 1.52; 8.53) and a trend of better survival was observed for children with parents of younger age at child's diagnosis and poorer survival of children with parents of older age. Despite free and uniform access to health care services, some family circumstances appear to affect survival from specific CNS tumour types in Danish children. Further research is warranted to gain a more comprehensive understanding of the impact of family factors on childhood cancer survival in other populations and to elaborate underlying mechanisms and pathways of those survival inequalities observed.     

Ebola Virus IgG Seroprevalence in Southern Mali

Mali had 2 reported introductions of Ebola virus (EBOV) during the 2013–2016 West Africa epidemic. Previously, no evidence for EBOV circulation was reported in Mali. We performed an EBOV serosurvey study in southern Mali. We found low seroprevalence in the population, indicating local exposure to EBOV or closely related ebola viruses.     

The spatial pattern of coronary capillaries in patients with dilated, ischemic, or inflammatory cardiomyopathy.

INTRODUCTION: The goal of the present study was to compare the pattern of coronary capillaries in healthy participants and in patients with end-stage heart failure due to idiopathic dilated cardiomyopathy (DCM), ischemic cardiomyopathy (ICM), or inflammatory cardiomyopathy (InfCM). METHODS: Capillary patterns were studied in histological sections from resected hearts from patients with DCM (n=5), ICM (n=5), or InfCM (n=5) and compared with donor hearts showing no signs of cardiac disease (n=3). Patterns were characterized by the distribution of Voronoi polygon areas, A, associated with the centers of capillary profiles, nearest-neighbor distances, d, intercapillary distances (ICD), as well as by means of the pair correlation function g(r). The coefficient of variation of A, CV, was used to characterize capillary patterns as regular, random, or clustered. RESULTS: CV increased from 30.5% (control) to 33.8% (DCM), 36.6% (ICM), and to 40.3% (InfCM). d was minimal in the control group (16.5+/-4.3 microm) and increased to 18.1+/-5.2 microm in the DCM group and to 20.9+/-6.8 and 20.6+/-6.6 microm in the ICM and InfCM groups, respectively. ICD increased from 25.6+/-7.9 (control) to 28.5+/-9.2 (DCM), 34.4+/-12.2 (ICM), and 33.6+/-12.0 microm (InfCM). In all groups, g(r) was markedly different from random points; in the control and DCM group, g(r) showed a weak but distinct first maximum, characteristic for short-range order in a point pattern. CONCLUSIONS: Our data suggest that, for the patients studied, (1) the pattern of coronary capillaries is not purely random, (2) the regularity of the pattern diminishes from the control to DCM, ICM, and InfCM, and (3) ICD increases, whereas capillary density (CD) decreases, from control to DCM, ICM, and InfCM.