Severe Aortic Stenosis in Dialysis Patients

Background: Characteristics and prognosis of hemodialysis patients with severe aortic stenosis have not yet been well defined. Methods and Results: The CURRENT AS (contemporary outcomes after surgery and medical treatment in patients with severe aortic stenosis) registry, a Japanese multicenter registry, enrolled 3815 consecutive patients with severe aortic stenosis. There were 405 hemodialysis patients (initial aortic valve replacement [AVR] group: N=135 [33.3%], and conservative group: N=270) and 3410 nonhemodialysis patients (initial AVR group: N=1062 [31.1%], and conservative group: N=2348). The median follow‐up duration after the index echocardiography was 1361 days, with 90% follow‐up rate at 2 years. The cumulative 5‐year incidence of all‐cause death was significantly higher in hemodialysis patients than in nonhemodialysis patients in both the entire cohort (71% versus 40%, P<0.001) and in the initial AVR group (63.2% versus 17.9%, P<0.001). Among hemodialysis patients, the initial AVR group as compared with the conservative group was associated with significantly lower cumulative 5‐year incidences of all‐cause death (60.6% versus 75.5%, P<0.001) and sudden death (10.2% versus 31.7%, P<0.001). Nevertheless, the rate of aortic valve procedure–related death, which predominantly occurred within 6 months of the AVR procedure, was markedly higher in the hemodialysis patients than in the nonhemodialysis patients (21.2% and 2.3%, P<0.001). Conclusions: Among hemodialysis patients with severe aortic stenosis, the initial AVR strategy as compared with the conservative strategy was associated with significantly lower long‐term mortality risk, particularly the risk for sudden death, although the effect size for the survival benefit of the initial AVR strategy was smaller than that in the nonhemodialysis patients

Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities

Cre complementation with variable dimerizers for inducible expression in neurons

Cre complementation is a process of reconstitution of the activity of DNA recombinase by noncovalent association of multiple segments of Cre recombinase, which are enzymatically inactive by themselves. Cre complementation is potentially useful in restriction of Cre activity in a specific subset of cells, with temporal regulation, by limiting overlap in expression of Cre fragments. We analyzed the efficiency of Cre complementation using three different dimerizing modules in the context of non-neuronal cells and found differential Cre complementation efficiency. We further tested the efficiency of Cre complementation in primary hippocampal neurons derived from transgenic mice harboring a reporter gene flanked by loxP sites and confirmed differential activity of dimerization modules in Cre-dependent recombination of the transgene. These results suggest possible application of dimerizer-based Cre complementation in inducible expression/inactivation of target genes in a specific subset of neurons in the complex environment of nervous tissue in vivo

Afadin regulates puncta adherentia junction formation and presynaptic differentiation in hippocampal neurons.

The formation and remodeling of mossy fiber-CA3 pyramidal cell synapses in the stratum lucidum of the hippocampus are implicated in the cellular basis of learning and memory. Afadin and its binding cell adhesion molecules, nectin-1 and nectin-3, together with N-cadherin, are concentrated at puncta adherentia junctions (PAJs) in these synapses. Here, we investigated the roles of afadin in PAJ formation and presynaptic differentiation in mossy fiber-CA3 pyramidal cell synapses. At these synapses in the mice in which the afadin gene was conditionally inactivated before synaptogenesis by using nestin-Cre mice, the immunofluorescence signals for the PAJ components, nectin-1, nectin-3 and N-cadherin, disappeared almost completely, while those for the presynaptic components, VGLUT1 and bassoon, were markedly decreased. In addition, these signals were significantly decreased in cultured afadin-deficient hippocampal neurons. Furthermore, the interevent interval of miniature excitatory postsynaptic currents was prolonged in the cultured afadin-deficient hippocampal neurons compared with control neurons, indicating that presynaptic functions were suppressed or a number of synapse was reduced in the afadin-deficient neurons. Analyses of presynaptic vesicle recycling and paired recordings revealed that the cultured afadin-deficient neurons showed impaired presynaptic functions. These results indicate that afadin regulates both PAJ formation and presynaptic differentiation in most mossy fiber-CA3 pyramidal cell synapses, while in a considerable population of these neurons, afadin regulates only PAJ formation but not presynaptic differentiation

Genetic deletion of afadin causes hydrocephalus by destruction of adherens junctions in radial glial and ependymal cells in the midbrain.

Adherens junctions (AJs) play a role in mechanically connecting adjacent cells to maintain tissue structure, particularly in epithelial cells. The major cell-cell adhesion molecules at AJs are cadherins and nectins. Afadin binds to both nectins and α-catenin and recruits the cadherin-β-catenin complex to the nectin-based cell-cell adhesion site to form AJs. To explore the role of afadin in radial glial and ependymal cells in the brain, we generated mice carrying a nestin-Cre-mediated conditional knockout (cKO) of the afadin gene. Newborn afadin-cKO mice developed hydrocephalus and died neonatally. The afadin-cKO brain displayed enlarged lateral ventricles and cerebral aqueduct, resulting from stenosis of the caudal end of the cerebral aqueduct and obliteration of the ventral part of the third ventricle. Afadin deficiency further caused the loss of ependymal cells from the ventricular and aqueductal surfaces. During development, radial glial cells, which terminally differentiate into ependymal cells, scattered from the ventricular zone and were replaced by neurons that eventually covered the ventricular and aqueductal surfaces of the afadin-cKO midbrain. Moreover, the denuded ependymal cells were only occasionally observed in the third ventricle and the cerebral aqueduct of the afadin-cKO midbrain. Afadin was co-localized with nectin-1 and N-cadherin at AJs of radial glial and ependymal cells in the control midbrain, but these proteins were not concentrated at AJs in the afadin-cKO midbrain. Thus, the defects in the afadin-cKO midbrain most likely resulted from the destruction of AJs, because AJs in the midbrain were already established before afadin was genetically deleted. These results indicate that afadin is essential for the maintenance of AJs in radial glial and ependymal cells in the midbrain and is required for normal morphogenesis of the cerebral aqueduct and ventral third ventricle in the midbrain

Impaired presynaptic functions in cultured afadin-deficient hippocampal neurons.

<p>(A, B) Labeling of functional synapses in cultured live hippocampal neurons. (A) Recycling synaptic vesicles labeled by incubation of cultured live hippocampal neurons at 14 DIV with the synaptotagmin I luminal domain Ab. Scale bars, 1 µm. (B) Quantification of the total integrated intensity of the internalized synaptotagmin I Ab. Sixty punctae for each genotype were analyzed. (C–E) Modulation of presynaptic vesicular release by afadin in cultured hippocampal neurons. (C) Examples of postsynaptic responses evoked by paired action potentials in a presynaptic cell with a 50-ms interval in control and <i>afadin</i> cKO neurons. Superimposed images for 15 recordings of unitary EPSC (top), average unitary EPSC (middle), and action potentials (bottom). A vertical scale bar denotes 20 pA for the top and middle rows and 5 mV for the bottom row; a horizontal scale bar denotes 20 ms. (D) The average unitary amplitudes (n = 10 and 12 in control and <i>afadin</i> cKO neurons, respectively). (E) paired-pulse ratios (n = 8 and 11 in control and <i>afadin</i> cKO neurons, respectively). Error bars, s.e.m. Control, <i>afadin</i>^+/f; cKO, <i>afadin</i>^f/f;nestin-Cre.</p

Conditional ablation of <i>afadin</i> in the brain.

<p>(A) Expression levels of various synaptic components in P14 forebrains. The indicated synaptic proteins were analyzed by Western blotting. Twenty µg of protein lysates were loaded in each lane. (B) Expression pattern of afadin in the CA3 stratum lucidum at P14. Hippocampal sections were stained with the l-afadin Ab (red) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue). The signal for afadin in nuclei was likely non-specific because it was not abolished by genetic ablation of <i>afadin</i>. The results shown are representative of three independent experiments. SR, stratum radiatum; SL, stratum lucidum; SP, stratum pyramidale. Control, <i>afadin</i>^+/f; cKO, <i>afadin</i>^f/f;nestin-Cre. Scale bars, 25 µm.</p

Differences in AMPA and Kainate Receptor Interactomes Facilitate Identification of AMPA Receptor Auxiliary Subunit GSG1L

AMPA receptor (AMPA-R) complexes consist of channel-forming subunits, GluA1-4, and auxiliary proteins, including TARPs, CNIHs, synDIG1, and CKAMP44, which can modulate AMPA-R function in specific ways. The combinatorial effects of four GluA subunits binding to various auxiliary subunits amplify the functional diversity of AMPA-Rs. The significance and magnitude of molecular diversity, however, remain elusive. To gain insight into the molecular complexity of AMPA and kainate receptors, we compared the proteins that copurify with each receptor type in the rat brain. This interactome study identified the majority of known interacting proteins and, more importantly, provides candidates for additional studies. We validate the claudin homolog GSG1L as a newly identified binding protein and unique modulator of AMPA-R gating, as determined by detailed molecular, cellular, electrophysiological, and biochemical experiments. GSG1L extends the functional variety of AMPA-R complexes, and further investigation of other candidates may reveal additional complexity of ionotropic glutamate receptor function