NK cells in antibody-mediated rejection - Key effector cells in microvascular graft damage.

Antibody-mediated rejection (ABMR) stands as the major limitation to long-term transplant outcome. The immunologic understanding of ABMR continues to progress and has identified natural killer (NK) cells as key effector cells promoting and coordinating the immune attack on the graft microvascular endothelium. This review discusses the current concepts outlining the different ways that allow for NK cell recognition of graft endothelial cells which includes antibody-dependent as well as independent processes.

An in vitro model of antibody-mediated injury to glomerular endothelial cells: Upregulation of MHC class II and adhesion molecules.

Chronic active antibody-mediated rejection is a major cause of allograft failure in kidney transplantation. Microvascular inflammation and transplant glomerulopathy are defining pathologic features of chronic active antibody-mediated rejection and are associated with allograft failure. However, the mechanisms of leukocyte infiltration and glomerular endothelial cell injury remain unclear. We hypothesized MHC class II ligation on glomerular endothelial cells (GEnC) would result in upregulation of adhesion molecules and production of chemoattractants. A model of endothelial cell activation in the presence of antibodies to MHC classes I and II was used to determine the expression of adhesion molecules and chemokines. Murine GEnC were activated with IFNγ, which upregulated gene expression of ß2-microglobulin (MHC class I), ICAM1, VCAM1, CCL2, CCL5, and IL-6. IFNγ stimulation of GEnC increased surface expression of MHC class I, MHC class II, ICAM1, and VCAM1. Incubation with antibodies directed at MHC class I or class II did not further enhance adhesion molecule expression. Multispectral imaging flow cytometry and confocal microscopy demonstrated MHC molecules co-localized with the adhesion molecules ICAM1 and VCAM1 on the GEnC surface. GEnC secretion of chemoattractants, CCL2 and CCL5, was increased by IFNγ stimulation. CCL2 production was further enhanced by incubation with sensitized plasma. Endothelial activation induces de novo expression of MHC class II molecules and increases surface expression of MHC class I, ICAM1 and VCAM1, which are all co-localized together. Maintaining the integrity and functionality of the glomerular endothelium is necessary to ensure survival of the allograft. IFNγ stimulation of GEnC propagates an inflammatory response with production of chemokines and co-localization of MHC and adhesion molecules on the GEnC surface, contributing to endothelial cell function as antigen presenting cells and an active player in allograft injury.

Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms.

Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5-10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.

Generalization of the prion hypothesis to other neurodegenerative diseases: an imperfect fit.

Protein misfolding diseases have been classically understood as diffuse errors in protein folding, with misfolded protein arising autonomously throughout a tissue due to a pathologic stressor. The field of prion science has provided an alternative mechanism whereby a seed of pathologically misfolded protein, arising exogenously or through a rare endogenous structural fluctuation, yields a template to catalyze misfolding of the native protein. The misfolded protein may then spread intercellularly to communicate the misfold to adjacent areas and ultimately infect a whole tissue. Mounting evidence implicates a prion-like process in the propagation of several neurodegenerative diseases, including Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis, and the tauopathies. However, the parallels between the events observed in these conditions and those in prion disease are often incomplete. The aim of this review was to examine the current state of knowledge concerning the mechanisms of protein misfolding and aggregation for neurodegeneration-associated proteins. In addition, possible methods of intercellular spread are described that focus on the hypothesis that released microvesicles function as misfolded protein delivery vehicles, and the therapeutic options enabled by viewing these diseases from the prion perspective.

Intermolecular transmission of superoxide dismutase 1 misfolding in living cells.

Human wild-type superoxide dismutase-1 (wtSOD1) is known to coaggregate with mutant SOD1 in familial amyotrophic lateral sclerosis (FALS), in double transgenic models of FALS, and in cell culture systems, but the structural determinants of this process are unclear. Here we molecularly dissect the effects of intracellular and cell-free obligately misfolded SOD1 mutant proteins on natively structured wild-type SOD1. Expression of the enzymatically inactive, natural familial ALS SOD1 mutations G127X and G85R in human mesenchymal and neural cell lines induces misfolding of wild-type natively structured SOD1, as indicated by: acquisition of immunoreactivity with SOD1 misfolding-specific monoclonal antibodies; markedly enhanced protease sensitivity suggestive of structural loosening; and nonnative disulfide-linked oligomer and multimer formation. Expression of G127X and G85R in mouse cell lines did not induce misfolding of murine wtSOD1, and a species restriction element for human wtSOD1 conversion was mapped to a region of sequence divergence in loop II and ß-strand 3 of the SOD1 ß-barrel (residues 24-36), then further refined surprisingly to a single tryptophan residue at codon 32 (W32) in human SOD1. Time course experiments enabled by W32 restriction revealed that G127X and misfolded wtSOD1 can induce misfolding of cell-endogenous wtSOD1. Finally, aggregated recombinant G127X is capable of inducing misfolding and protease sensitivity of recombinant human wtSOD1 in a cell-free system containing reducing and chelating agents; cell-free wtSOD1 conversion was also restricted by W32. These observations demonstrate that misfolded SOD1 can induce misfolding of natively structured wtSOD1 in a physiological intracellular milieu, consistent with a direct protein-protein interaction.

An overlapping reading frame in the PRNP gene encodes a novel polypeptide distinct from the prion protein.

The prion protein gene PRNP directs the synthesis of one of the most intensively studied mammalian proteins, the prion protein (PrP). Yet the physiological function of PrP has remained elusive and has created controversies in the literature. We found a downstream alternative translation initiation AUG codon surrounded by an optimal Kozak sequence in the +3 reading frame of PRNP. The corresponding alternative open reading frame encodes a polypeptide termed alternative prion protein (AltPrP) with a completely different amino acid sequence from PrP. We introduced a hemagglutinin (HA) tag in frame with AltPrP in PrP cDNAs from different species to test the expression of this novel polypeptide using anti-HA antibodies. AltPrP is constitutively coexpressed with human, bovine, sheep, and deer PrP. AltPrP is localized at the mitochondria and is up-regulated by endoplasmic reticulum stress and proteasomal inhibition. Generation of anti-AltPrP antibodies allowed us to test for endogenous expression of AltPrP in wild-type human cells expressing PrP. By transfecting cells with siRNA against PrP mRNA, we repressed expression of both PrP and AltPrP, confirming endogenous expression of AltPrP from PRNP. AltPrP was also detected in human brain homogenate, primary neurons, and peripheral blood mononuclear cells. These results demonstrate an unexpected function for PRNP, which, in addition to plasma membrane-anchored PrP, also encodes a second polypeptide termed AltPrP.

Evaluation of Prader-Willi Syndrome gene MAGEL2 in severe childhood-onset obesity.

MAGEL2 is one of the five genes inactivated in Prader-Willi Syndrome, a neurodevelopmental chromosome microdeletion disorder modified by genomic imprinting. By early childhood, individuals with Prader-Willi Syndrome exhibit hypothalamic dysfunction, including hyperphagia, and become obese in the absence of behavioral intervention. Murine Magel2 is highly expressed in the hypothalamus during development. We screened the MAGEL2 open reading frame for mutations in genomic DNA samples from hyperphagic but non-dysmorphic individuals with severe childhood-onset obesity. Although no mutations likely to affect gene function were identified, we identified three variant alleles. We conclude that severe childhood-onset obesity is not commonly caused by MAGEL2 mutations.

FOXC1 transcriptional regulatory activity is impaired by PBX1 in a filamin A-mediated manner.

FOXC1 mutations underlie Axenfeld-Rieger syndrome, an autosomal dominant disorder that is characterized by a spectrum of ocular and nonocular phenotypes and results in an increased susceptibility to glaucoma. Proteins interacting with FOXC1 were identified in human nonpigmented ciliary epithelial cells. Here we demonstrate that FOXC1 interacts with the actin-binding protein filamin A (FLNA). In A7 melanoma cells possessing elevated levels of nuclear FLNA, FOXC1 is unable to activate transcription and is partitioned to an HP1alpha, heterochromatin-rich region of the nucleus. This inhibition is mediated through an interaction between FOXC1 and the homeodomain protein PBX1a. In addition, we demonstrate that efficient nuclear and subnuclear localization of PBX1 is mediated by FLNA. Together, these data reveal a mechanism by which structural proteins such as FLNA can influence the activity of a developmentally and pathologically important transcription factor such as FOXC1. Given the resemblance of the skeletal phenotypes caused by FOXC1 loss-of-function mutations and FLNA gain-of-function mutations, this inhibitory activity of FLNA on FOXC1 may contribute to the pathogenesis of FLNA-linked skeletal disorders.

Essential role for the Prader-Willi syndrome protein necdin in axonal outgrowth.

Necdin and Magel2 are related proteins inactivated in Prader-Willi syndrome (PWS), a sporadic chromosomal deletion disorder. We demonstrate that necdin and Magel2 bind to and prevent proteasomal degradation of Fez1, a fasciculation and elongation protein implicated in axonal outgrowth and kinesin-mediated transport, and also bind to the Bardet-Biedl syndrome (BBS) protein BBS4 in co-transfected cells. The interactions among necdin, Magel2, Fez1 and BBS4 occur at or near centrosomes. Centrosomal or pericentriolar dysfunction has previously been implicated in BBS and may also be important in the features of PWS that overlap with BBS, such as learning disabilities, hypogonadism and obesity. Morphological abnormalities in axonal outgrowth and fasciculation manifest in several regions of the nervous system in necdin null mouse embryos, including axons of sympathetic, retinal ganglion cell, serotonergic and catecholaminergic neurons. These data demonstrate that necdin mediates intracellular processes essential for neurite outgrowth and that loss of necdin impinges on axonal outgrowth. We further suggest that loss of necdin contributes to the neurological phenotype of PWS, and raise the possibility that co-deletion of necdin and the related protein Magel2 may explain the lack of single gene mutations in PWS.