The basis for memory loss in early Alzheimer's disease (AD) seems likely to involve synaptic damage caused by soluble A␤-derived oligomers (ADDLs). ADDLs have been shown to build up in the brain and CSF of AD patients and are known to interfere with mechanisms of synaptic plasticity, acting as gain-of-function ligands that attach to synapses. Because of the correlation between AD dementia and synaptic degeneration, we investigated here the ability of ADDLs to affect synapse composition, structure, and abundance. Using highly differentiated cultures of hippocampal neurons, a preferred model for studies of synapse cell biology, we found that ADDLs bound to neurons with specificity, attaching to presumed excitatory pyramidal neurons but not GABAergic neurons. Fractionation of ADDLs bound to forebrain synaptosomes showed association with postsynaptic density complexes containing NMDA receptors, consistent with observed attachment of ADDLs to dendritic spines. During binding to hippocampal neurons, ADDLs promoted a rapid decrease in membrane expression of memory-related receptors (NMDA and EphB2). Continued exposure resulted in abnormal spine morphology, with induction of long thin spines reminiscent of the morphology found in mental retardation, deafferentation, and prionoses. Ultimately, ADDLs caused a significant decrease in spine density. Synaptic deterioration, which was accompanied by decreased levels of the spine cytoskeletal protein drebrin, was blocked by the Alzheimer's therapeutic drug Namenda. The observed disruption of dendritic spines links ADDLs to a major facet of AD pathology, providing strong evidence that ADDLs in AD brain cause neuropil damage believed to underlie dementia.

SUMMARYHypoxia-inducible factor (HIF) α, which has three isoforms, is central to the continuous balancing of the supply and demand of oxygen throughout the body. HIF-α is a transcription factor that modulates a wide range of processes, including erythropoiesis, angiogenesis, and cellular metabolism. We describe a family with erythrocytosis and a mutation in the HIF2A gene, which encodes the HIF-2α protein. Our functional studies indicate that this mutation leads to stabilization of the HIF-2α protein and suggest that wild-type HIF-2α regulates erythropoietin production in adults.A widely recognized example of an oxygen-regulated pathway is the erythropoietin system, in which the kidney senses decreased tissue oxygenation and, in turn, produces erythropoietin, thereby increasing the red-cell mass. 1 Studies of the regulation of the EPO gene led to the discovery of HIF, which consists of a labile α subunit and a constitutively expressed β subunit. 2 Under normoxic conditions, the α subunit, which consists of three isoforms -HIF-1α, HIF-2α, and HIF-3α -is hydroxylated on two specific prolyl residues. 3 This hydroxylation targets HIF-α for degradation by the von Hippel-Lindau (VHL) tumor-suppressor protein. 4 Under hypoxic conditions, the hydroxylation is inhibited, thereby maintaining a stable HIF-α protein, which activates not only the EPO gene but also a broad range of other genes that orchestrate adaptation to hypoxia. 2,5Familial erythrocytosis provides an opportunity to study the oxygen-sensing mechanism. 6 This genetic disorder can be caused by a mutation in one of the proteins that hydroxylates HIF-α (prolyl hydroxylase domain protein 2 [PHD2]) 7,8 or by a mutation in the VHL protein. 9,10 These findings raise the question of whether there are mutations in HIF-α itself.The particular HIF isoform involved in erythropoietin regulation has been the subject of intensive investigation. Several results point to HIF-1α, 11-13 but mouse Hif-2α (also known as endothelial PAS domain protein 1 [Epas1] or Hif-1α-like factor [Hlf]) has been implicated as the principal regulator of erythropoietin in postembryonic mice. 14-17 We investigated the HIF2A gene in a family with erythrocytosis and found a missense mutation that impairs hydroxylation of the HIF-2α protein, thereby allowing both for maintenance of its stable conformation and for its induction of erythrocytosis.

During metastatic progression, circulating cancer cells become lodged within the microvasculature of end-organs, where a majority die from mechanical deformation. Though this phenomenon was first described over a half-century ago, the mechanisms enabling certain cells to survive this metastasis-suppressive barrier remain unknown. By applying whole-transcriptome RNA-sequencing (RNA-seq) technology to isogenic cancer cells of differing metastatic capacity, we identified a mutation encoding a truncated form of the pannexin-1 (PANX1) channel, PANX1 1–89, as recurrently enriched in highly metastatic breast cancer cells. PANX1 1–89 functions to permit metastatic cell survival during traumatic deformation in the microvasculature by augmenting ATP release from mechanosensitive PANX1 channels activated by membrane stretch. PANX1-mediated ATP release acts as an autocrine suppressor of deformation-induced apoptosis via P2y-purinergic receptors. Finally, small-molecule therapeutic inhibition of PANX1 channels is found to reduce the efficiency of breast cancer metastasis. These data suggest a molecular basis for metastatic cell survival upon microvasculature-induced biomechanical trauma.

The molecular basis of the erythrocytosis group of red cell disorders is incompletely defined. Some cases are due to dysregulation of erythropoietin (Epo) synthesis. The hypoxia inducible transcription factor (HIF) tightly regulates Epo synthesis. HIF in turn is regulated through its ␣ subunit, which under normoxic conditions is hydroxylated on specific prolines and targeted for degradation by the von Hippel Lindau (VHL) protein. Several mutations in VHL have been reported in erythrocytosis, but only 1 mutation in the HIF prolyl hydroxylase PHD2 (prolyl hydroxylase domain protein 2) has been described. Here, we report a novel PHD2 mutation, Arg371His, which causes decreased HIF binding, HIF hydroxylase, and HIF inhibitory activities. In the tertiary structure of PHD2, Arg371 lies close to the previously described Pro317Arg mutation site. These findings substantiate PHD2 as a critical enzyme controlling HIF and therefore Epo in humans, and furthermore suggest the location of an active site groove in PHD2 that binds HIF. (Blood.

A classic physiologic response to hypoxia in humans is the up-regulation of the ERYTHROPOIETIN (EPO) gene, which is the central regulator of red blood cell mass. The EPO gene, in turn, is activated by hypoxia inducible factor (HIF). HIF is a transcription factor consisting of an ␣ subunit (HIF-␣) and a ␤ subunit (HIF-␤). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-␣ in a conserved LXXLAP motif (where underlining indicates the hydroxylacceptor proline). This provides a recognition motif for the von Hippel Lindau protein, a component of an E3 ubiquitin ligase complex that targets hydroxylated HIF-␣ for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-␣ and allowing it to activate the EPO gene. We previously identified and characterized an erythrocytosis-associated HIF2A mutation, G537W. More recently, we reported two additional erythrocytosis-associated HIF2A mutations, G537R and M535V. Here, we describe the functional characterization of these two mutants as well as a third novel erythrocytosisassociated mutation, P534L. These mutations affect residues C-terminal to the LXXLAP motif. We find that all result in impaired degradation and thus aberrant stabilization of HIF-2␣. However, each exhibits a distinct profile with respect to their effects on PHD2 binding and von Hippel Lindau interaction. These findings reinforce the importance of HIF-2␣ in human EPO regulation, demonstrate heterogeneity of functional defects arising from these mutations, and point to a critical role for residues C-terminal to the LXXLAP motif in HIF-␣.

Anatomy as the foundation of surgery is a concept no better exemplified than by the history of tracheal surgery. Incremental advancements in our understanding of the trachea's position, structure, blood supply and adjacent organs each allowed for stepwise improvements in the thoracic surgeon's ability to address upper airway disease. As such, the mastery of tracheal anatomy is fundamental to those clinicians responsible for treating such ailments. In this article, tracheal anatomy is reviewed and points critical to the thoracic surgeon are highlighted. The structure and location of the trachea, the blood supply to the trachea, and the trachea's spatial relationship to critical mediastinal organs are presented. This material provides the groundwork for understanding all aspects of tracheal surgery today.

No abstract

Human diseases have provided important insights into the function of fundamental physiological processes, and studying erythrocytosis has increased our understanding of the oxygen sensing pathway. In some cases this rare disorder arises specifically from dysregulation of the erythropoietin (Epo) axis. Epo gene transcription is under the control of a negative feedback mechanism involving the kidneys, which sense tissue hypoxia at the molecular level via the hypoxia inducible factor (HIF) transcription complex. Both subunits of the HIF complex, alpha and beta, are constitutively expressed but only the beta subunit is detectable in the presence of oxygen. Key prolines in the oxygen degradation domain of HIFalpha are hydroxylated by a family of prolyl hydroxylase enzymes, PHD1-3. Of these PHD2 has the most widespread tissue distribution. Upon hydroxylation of HIF, the von Hippel Lindau (VHL) protein, a component of an E3 ligase complex, promotes the ubiquitination of the alpha subunit. HIFalpha is then targeted to the proteasome and the transcription of HIF target genes is prevented. In hypoxia the activity of the PHD enzymes is low and the HIF complex assembles causing upregulation of genes such as Epo. Over the last ten years we have maintained a registry of individuals with erythrocytosis. To date 181 patients have been included with clinical details and consented DNA samples have been collected. There is a preponderance of males with ratio of 1.7 males to each female. The mean age of erythrocytosis individuals on the registry is 37 years. The majority of erythrocytosis patients have inappropriately normal (46%) or raised (26%) serum Epo levels as compared to PV, where the Epo level is often undetectable. Thus it can be inferred that dysregulation of the Epo axis via the oxygen sensing pathway would be a significant cause of erythrocytosis. Consequently, PHD2 and VHL have been investigated. We have now identified 2 different mutations, Pro317Arg and Arg371His, in PHD2. In the tertiary structure of PHD2, Pro317and Arg371 are close to essential iron binding residues and furthermore suggest the location of a HIF binding groove. The Arg200Trp VHL mutation, commonly described as the Chuvash mutation, has been detected in 8 Asian families and their large kindred. Two Western European individuals were found to be compound heterozygous for the Arg200Trp and either the novel Pro192Thr or Gly144Arg variants. Intriguingly 2 further individuals possess one wild type and one mutated allele, Arg200Trp or Leu188Val. Screening other genes of the oxygen sensing pathway has failed to reveal further defects in these individuals. In summary, defects in the oxygen sensing pathway are associated with the development of erythrocytosis. Mutations in VHL are more common than PHD2 and highlight the importance of these proteins in the maintenance of red cell homeostasis.