Hart G.W. Lidov

IKKβ/NF-κB Activation Causes Severe Muscle Wasting in Mice

Muscle wasting accompanies aging and pathological conditions ranging from cancer, cachexia, and diabetes to denervation and immobilization. We show that activation of NF-kappaB, through muscle-specific transgenic expression of activated IkappaB kinase beta (MIKK), causes profound muscle wasting that resembles clinical cachexia. In contrast, no overt phenotype was seen upon muscle-specific inhibition of NF-kappaB through expression of IkappaBalpha superrepressor (MISR). Muscle loss was due to accelerated protein breakdown through ubiquitin-dependent proteolysis. Expression of the E3 ligase MuRF1, a mediator of muscle atrophy, was increased in MIKK mice. Pharmacological or genetic inhibition of the IKKbeta/NF-kappaB/MuRF1 pathway reversed muscle atrophy. Denervation- and tumor-induced muscle loss were substantially reduced and survival rates improved by NF-kappaB inhibition in MISR mice, consistent with a critical role for NF-kappaB in the pathology of muscle wasting and establishing it as an important clinical target for the treatment of muscle atrophy.

ArticleIKKβ/NF-κB Activation Causes Severe Muscle Wasting in Mice

Muscle wasting accompanies aging and pathological conditions ranging from cancer, cachexia, and diabetes to denervation and immobilization. We show that activation of NF-kappaB, through muscle-specific transgenic expression of activated IkappaB kinase beta (MIKK), causes profound muscle wasting that resembles clinical cachexia. In contrast, no overt phenotype was seen upon muscle-specific inhibition of NF-kappaB through expression of IkappaBalpha superrepressor (MISR). Muscle loss was due to accelerated protein breakdown through ubiquitin-dependent proteolysis. Expression of the E3 ligase MuRF1, a mediator of muscle atrophy, was increased in MIKK mice. Pharmacological or genetic inhibition of the IKKbeta/NF-kappaB/MuRF1 pathway reversed muscle atrophy. Denervation- and tumor-induced muscle loss were substantially reduced and survival rates improved by NF-kappaB inhibition in MISR mice, consistent with a critical role for NF-kappaB in the pathology of muscle wasting and establishing it as an important clinical target for the treatment of muscle atrophy.

An immunohistochemical study of serotonin neuron development in the rat: ascending pathways and terminal fields.

The ontogeny of the serotonergic axonal projections may be divided into three periods: one of initial axon elongation (E12-E16), the development of selective pathways (E15-E19) and terminal field development (E19-E21). All serotonergic axons that enter the prosencephalon ascend in the medial forebrain bundle From this bundle fascicles of immunoreactive axons enter several well-defined fiber tracts: specifically, the fasciculus retroflexus, stria medullaris, external capsule, fornix, and supracallosal stria. Axons from these pathways form terminal arborizations in the thalamus, hypothalamus, basal and limbic forebrain, and cerebral cortex. Serotonergic axons appear to be guided by pre-existing non-serotonergic tracts in reaching targets in the forebrain. Innervation of the cerebral cortex is a prolonged process extending from E19 through PND21. Axons enter directly into the marginal and intermediate zones of the immature cortex, at the medial, frontal and lateral edges of the hemisphere, and subsequently spread tangentially to cover the hemispheres. Terminal ramifications then arise from the bilaminar axons and fill in the middle cortical layers. This growth pattern gives rise to tangential and radial gradients in innervation density. While the growth of serotonin axons across the forebrain appears to be a continuous, sequential process, the development of terminal innervation is highly heterogeneous, occurring at different times and at different rates from region to region. Serotonergic axons do not innervate immature, primarily proliferative neuronal populations. The delay in serotonin innervation of the suprachiasmatic nucleus, striatum, and middle cortical layers long after the axons have reached these structures suggests that the formation of serotonin axon terminals is dependent on maturation of other elements in local neuronal circuitry.

The serotonin innervation of the cerebral cortex in the rat--an immunohistochemical analysis.

Abstract The serotonergic innervation of the cerebral cortex in the rat has been studied by immunohistochemistry employing an antibody directed against the neurotransmitter, serotonin. The dorsal raphe, median raphe and B9 cell groups contain intensely labelled neuronal perikarya. Bundles of large diameter axons suggestive of fibers of passage are observed in successive sections as they ascend through the midbrain tegmentum, medial forebrain bundle, diagonal band and supracallosal stria en route to the cortex. In addition, a lateral pathway to the cerebral cortex traversing the ansa peduncularis is visualized. All regions of the cerebral cortex appear to be innervated by serotonergic axons which have a distinctive morphology: they are fine (0.1–0.5 μm), varicose, and extremely convoluted. Serotonergic axons of passage are thicker and comparatively straight. Throughout the lateral neocortex, as well as in the anterior cingulate cortex, serotonergic axons form a densely arborizing plexus through all cortical layers. Contrary to earlier reports, based on histofluorescence, describing a sparse innervation of the cortex with most of the fibers found in the molecular layer, the present study reveals that the innervation is relatively uniform across all cortical layers. In most of the cortex the density of serotonin-containing axons exceeds that of noradrenergic fibers. A distinctive and different pattern of serotonin innervation is found in the posterior cingulate cortex (cytoarchitectonic field RSg): the serotonergic axons are restricted largely to lamina I and III. A restricted laminar pattern also characterizes the innervation of the hippocampus; dense axonal plexuses occur in the outer rim of the dentate hilus and in the stratum lacunosum-moleculare. The serotonergic afferents to the cortex appear to have at least two different modes of distribution, a relatively uniform pattern in the anterior cingulate and the lateral neocortex and a restricted, laminar pattern in the posterior cingulate and the hippocampus. The density and extent of the serotonin innervation is such that the raphe neurons may contact every cell in the cortex. The widespread arborization of serotonin axons contrasts with the spatially restricted termination of thalamic afferents. The distribution of serotonin-containing fibers also differs substantially from the terminal patterns of noradrenergic and dopaminergic fibers. The differences in axonal morphology and distribution amongst the monoamine afferents reflect differences in their contributions to cortical circuitry. The present findings indicate that the serotonin-containing neurons may exert a profound and global, but not necessarily uniform, influence upon cortical function.

Distinctive patterns of microRNA expression in primary muscular disorders

The primary muscle disorders are a diverse group of diseases caused by various defective structural proteins, abnormal signaling molecules, enzymes and proteins involved in posttranslational modifications, and other mechanisms. Although there is increasing clarification of the primary aberrant cellular processes responsible for these conditions, the decisive factors involved in the secondary pathogenic cascades are still mainly obscure. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, we searched for miRNAs regulated during the degenerative process of muscle to gain insight into the specific regulation of genes that are disrupted in pathological muscle conditions. We describe 185 miRNAs that are up- or down-regulated in 10 major muscular disorders in humans [Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophies types 2A and 2B, Miyoshi myopathy, nemaline myopathy, polymyositis, dermatomyositis, and inclusion body myositis]. Although five miRNAs were found to be consistently regulated in almost all samples analyzed, pointing to possible involvement of a common regulatory mechanism, others were dysregulated only in one disease and not at all in the other disorders. Functional correlation between the predicted targets of these miRNAs and mRNA expression demonstrated tight posttranscriptional regulation at the mRNA level in DMD and Miyoshi myopathy. Together with direct mRNA–miRNA predicted interactions demonstrated in DMD, some of which are involved in known secondary response functions and others that are involved in muscle regeneration, these findings suggest an important role of miRNAs in specific physiological pathways underlying the disease pathology.

Immunohistochemical study of the development of serotonergic neurons in the rat CNS.

In this study the development of serotonergic (5-HT) neurons is followed from their initiation of transmitter synthesis until the establishment of an essentially mature morphology. We have used the new and sensitive technique of 5-HT immunocytochemistry to visualize the precise features of this process. The great stability of this method, and the feasibility of counter-staining tissue sections permits the visualization of dendritic processes and axon terminals, as well as perikarya, and facilitates the localization of these structures with respect to non-5-HT components of the neuropil. Serial transverse and sagittal sections of rat fetuses on embryonic days (ED) 13, 14, 15, 17, 19 and 21, and postnatal rats on days 1, 3, 4 and 10 were examined. A detailed photomicrographic map showing the locations of 5-HT neurons at all prenatal stages is provided. The development of 5-HT neurons is evaluated in terms of their cellular morphology, particularly dendritic architecture, the relationship of these cells to the development of the surrounding brainstem, and the morphology and packing density of the 5-HT nuclei. From these considerations a model is proposed of the pattern of cell migration within the nuclei that give rise to the ascending 5-HT projections. At E14 a relatively simple configuration of bilateral superior (rostral) and inferior (caudal) 5-HT cell groups is present. In the period extending from E14 to E19 several subgroupings of these cells develop, presumably as the result of differential cell migration. Based on the predominant dendritic orientation of these cells it is possible to reconstruct their probable migratory paths. At E19 the 5-HT neurons are distributed in groups that are similar to those seen in the adult. In the time from E19 until the end of the first postnatal week there is rapid growth of 5-HT dendrites and a marked decrease in cellular packing density. These alterations shape the nuclear aggregates into the form seen in the adult. The development of the 5-HT cell groups is discussed in the context of known features of neurogenesis, migration, and axonal projections of the raphe and medial reticular nuclei of the brainstem. The possibility is raised that the decrease in cellular packing density in the 5-HT nuclei may reflect the appearance of the non-5-HT components of the raphe nuclei.

Long-term persistence of donor nuclei in a Duchenne muscular dystrophy patient receiving bone marrow transplantation

Duchenne muscular dystrophy (DMD) is a severe progressive muscle-wasting disorder caused by mutations in the dystrophin gene. Studies have shown that bone marrow cells transplanted into lethally irradiated mdx mice, the mouse model of DMD, can become part of skeletal muscle myofibers. Whether human marrow cells also have this ability is unknown. Here we report the analysis of muscle biopsies from a DMD patient (DMD-BMT1) who received bone marrow transplantation at age 1 year for X-linked severe combined immune deficiency and who was diagnosed with DMD at age 12 years. Analysis of muscle biopsies from DMD-BMT1 revealed the presence of donor nuclei within a small number of muscle myofibers (0.5-0.9%). The majority of the myofibers produce a truncated, in-frame isoform of dystrophin lacking exons 44 and 45 (not wild-type). The presence of bone marrow-derived donor nuclei in the muscle of this patient documents the ability of exogenous human bone marrow cells to fuse into skeletal muscle and persist up to 13 years after transplantation.

Exome sequencing identifies BRAF mutations in papillary craniopharyngiomas

Craniopharyngiomas are epithelial tumors that typically arise in the suprasellar region of the brain. Patients experience substantial clinical sequelae from both extension of the tumors and therapeutic interventions that damage the optic chiasm, the pituitary stalk and the hypothalamic area. Using whole-exome sequencing, we identified mutations in CTNNB1 (β-catenin) in nearly all adamantinomatous craniopharyngiomas examined (11/12, 92%) and recurrent mutations in BRAF (resulting in p.Val600Glu) in all papillary craniopharyngiomas (3/3, 100%). Targeted genotyping revealed BRAF p.Val600Glu in 95% of papillary craniopharyngiomas (36 of 39 tumors) and mutation of CTNNB1 in 96% of adamantinomatous craniopharyngiomas (51 of 53 tumors). The CTNNB1 and BRAF mutations were clonal in each tumor subtype, and we detected no other recurrent mutations or genomic aberrations in either subtype. Adamantinomatous and papillary craniopharyngiomas harbor mutations that are mutually exclusive and clonal. These findings have important implications for the diagnosis and treatment of these neoplasms.

Higher hematocrit improves cerebral outcome after deep hypothermic circulatory arrest

BACKGROUND Various degrees of hemodilution are currently in clinical use during deep hypothermic circulatory arrest to counteract deleterious rheologic effects linked with brain injury by previous reports. MATERIAL AND METHODS Seventeen piglets were randomly assigned to three groups. Group I piglets (n = 7) received colloid and crystalloid prime (hematocrit < 10%), group II piglets (n = 5) received blood and crystalloid prime (hematocrit 20%), group III piglets (n = 5) received blood prime (hematocrit 30%). All groups underwent 60 minutes of deep hypothermic circulatory arrest at 15 degrees C with continuous magnetic resonance spectroscopy and near-infrared spectroscopy Neurologic recovery was evaluated for 4 days (neurologic deficit score 0, normal, to 500, brain death; overall performance category 1, normal, to 5, brain death). Neurohistologic score (0, normal, to 5+, necrosis) was assessed after the animals were euthanized on day 4. RESULTS Group I had significant loss of phosphocreatine and intracellular acidosis during early cooling (phosphocreatine in group I, 86.3% +/- 26.8%; group II, 117.3% +/- 8.6%; group III, 110.9% +/- 2.68%; p = 0.0008; intracellular pH in group I, 6.95 +/- 0.18; group II, 7.28 +/- 0.04; group III, 7.49 +/- 0.04; p = 0.0048). Final recovery was the same for all groups. Cytochrome aa3 was more reduced in group I during deep hypothermic circulatory arrest than in either of the other groups (group I, -43.6 +/- 2.6; group II, -16.0 +/- 5.2; group III, 1.3 +/= 3.1; p < 0.0001). Neurologic deficit score was best preserved in group III (p < 0.05 group II vs group III) on the first postoperative day, although this difference diminished with time and all animals were neurologically normal after 4 days. Histologic assessment was worst among group I in neocortex area (group I, 1.33 +/- 0.3; group II, 0.22 +/- 0.1; group III, 0.40 +/- 0.2, p < 0.05, group I vs group II; p = 0.0287, group I vs group III). CONCLUSION Extreme hemodilution during cardiopulmonary bypass may cause inadequate oxygen delivery during early cooling. The higher hematocrit with a blood prime is associated with improved cerebral recovery after deep hypothermic circulatory arrest.

The distribution of dystrophin in the murine central nervous system: An immunocytochemical study

A mild non-progressive cognitive defect is a feature of the fatal X-linked disease, Duchenne muscular dystrophy. Recent studies have identified the genetic defect and the resulting loss of the protein dystrophin, and shown that dystrophin messenger RNA and protein are present in normal brain tissue. We have performed western immunoblotting and fluorescence immunocytochemistry using a sensitive antibody made against a large fragment of the dystrophin molecule to study the regional, cellular and subcellular distribution of dystrophin in the mammalian brain. The brains of B10 (control) and mdx (dystrophin deficient null mutant) mouse brain were compared on a point-by-point basis to verify that only dystrophin and not autosomal dystrophin related protein or cross-reacting proteins were being identified. In addition three murine neurologic mutants, nervous, lurcher, and weaver, were studied to refine the localization of dystrophin. In western immunoblots, dystrophin is present in all regions of the brain and in greatest abundance in the cerebellum. Dystrophin, as demonstrated in immunofluorescence, is present in neurons, but not in glia or myelin, and forms punctate foci associated with the plasma membrane of perikarya and dendrites, but not axons. While dystrophin is abundant in cerebral cortical neurons and cerebellar Purkinje cells, it is absent from most subcortical neurons, the granule cells of fascia dentata, and cerebellar neurons other than Purkinje cells. The absence of dystrophin in the cerebellum of the Purkinje cell deficient mutants nervous and lurcher, and its presence in the granule cell deficient mutant weaver indicate that dystrophin is a component of Purkinje cells rather than closely apposed afferents to those cells. The distribution and localization of dystrophin suggests a role in organizing the plasma membrane, possibly as an anchor of the postsynaptic apparatus, a possible basis for the cognitive defect in Duchenne dystrophy.