Lipopolysaccharide-responsive beige-like anchor is involved in regulating NF-κB activation in B cells.

Introduction: Lipopolysaccharide-responsive and beige-like anchor (LRBA) is a scaffolding protein that interacts with proteins such as CTLA-4 and PKA, the importance of which has been determined in various cell types, including T regulatory cells, B cells, and renal cells. LRBA deficiency is associated with an inborn error in immunity characterized by immunodeficiency and autoimmunity. In addition to defects in T regulatory cells, patients with LRBA deficiency also exhibit B cell defects, such as reduced cell number, low memory B cells, hypogammaglobulinemia, impaired B cell proliferation, and increased autophagy. Although Lrba-/- mice do not exhibit the immunodeficiency observed in humans, responses to B cell receptors (BCR) in B cells have not been explored. Therefore, a murine model is for elucidating the mechanism of Lrba mechanism in B cells. Aim: To compare and evaluate spleen-derived B cell responses to BCR crosslinking in C57BL6 Lrba-/- and Lrba+/+ mice. Materials and methods: Spleen-derived B cells were obtained from 8 to 12-week-old mice. Subpopulations were determined by immunostaining and flow cytometry. BCR crosslinking was assessed by the F(ab')2 anti-µ chain. Activation, proliferation and viability assays were performed using flow cytometry and protein phosphorylation was evaluated by immunoblotting. The nuclear localization of p65 was determined using confocal microscopy. Nur77 expression was evaluated by Western blot. Results: Lrba-/- B cells showed an activated phenotype and a decreased proportion of transitional 1 B cells, and both proliferation and survival were affected after BCR crosslinking in the Lrba-/- mice. The NF-κB pathway exhibited a basal activation status of several components, resulting in increased activation of p50, p65, and IκBα, basal p50 activation was reduced by the Plcγ2 inhibitor U73122. BCR crosslinking in Lrba-/ - B cells resulted in poor p50 phosphorylation and p65 nuclear localization. Increased levels of Nur77 were detected. Discussion: These results indicate the importance of Lrba in controlling NF-κB activation driven by BCR. Basal activation of NF-κB could impact cellular processes, such as, activation, differentiation, proliferation, and maintenance of B cells after antigen encounter.

Lrba participates in the differentiation of IgA+ B lymphocytes through TGFßR signaling.

Introduction: Lrba is a cytoplasmic protein involved in vesicular trafficking. Lrba-deficient (Lrba-/-) mice exhibit substantially higher levels of IgA in both serum and feces than wild-type (WT) mice. Transforming growth factor ß1 (TGFß1) and its receptors (TGFßR I and II) is essential for differentiating IgA+ B cells. Furthermore, increased IgA production suggests a potential connection between Lrba and the TGFßR signaling pathway in IgA production. However, the specific function of Lrba in B cell biology remains unknown. Aim: Given the increased IgA levels in Lrba-/- mice, the goal in this work was to explore the lymph organs where the switch to IgA occurs, and if TGFßR function is affected. Methods: Non-immunized Lrba-/- mice were compared with Lrba+/+ mice. IgA levels in the serum and feces, as well as during peripheral B cell development, were determined. IgA+ B cells and plasma cells were assessed in the small intestine and secondary lymphoid organs, such as the spleen, mesenteric lymph nodes, and Peyer's patches. The TGFßR signaling pathway was evaluated by determining the expression of TGFßR on B cells. Additionally, SMAD2 phosphorylation was measured under basal conditions and in response to recombinant TGFß. Finally, confocal microscopy was performed to investigate a possible interaction between Lrba and TGFßR in B cells. Results: Lrba-/- mice exhibited significantly higher levels of circulating IgA, IgA+ B, and plasma cells than in peripheral lymphoid organs those in WT mice. TGFßR expression on the membrane of B cells was similar in both Lrba-/- and Lrba+/+ mice. However, intracellular TGFßR expression was reduced in Lrba-/- mice. SMAD2 phosphorylation showed increased levels under basal conditions; stimulation with recombinant TGFß elicited a poorer response than in that in Lrba+/+ B cells. Finally, we found that Lrba colocalizes with TGFßR in B cells. Conclusion: Lrba is essential in controlling TGFßR signaling, subsequently regulating SMAD2 phosphorylation on B cells. This mechanism may explain the increased differentiation of IgA+ B cells and production of IgA-producing plasma cells.

Neurobeachin regulates hematopoietic progenitor differentiation and survival by modulating Notch activity.

ABSTRACT: Hematopoietic stem cells (HSCs) can generate all blood cells. This ability is exploited in HSC transplantation (HSCT) to treat hematologic disease. A clear understanding of the molecular mechanisms that regulate HSCT is necessary to continue improving transplant protocols. We identified the Beige and Chediak-Higashi domain-containing protein (BDCP), Neurobeachin (NBEA), as a putative regulator of HSCT. Here, we demonstrated that NBEA and related BDCPs, including LPS Responsive Beige-Like Anchor Protein (LRBA), Neurobeachin Like 1 (NBEAL1) and Lysosomal Trafficking Regulator (LYST), are required during HSCT to efficiently reconstitute the hematopoietic system of lethally irradiated mice. Nbea knockdown in mouse HSCs induced apoptosis and a differentiation block after transplantation. Nbea deficiency in hematopoietic progenitor cells perturbed the expression of genes implicated in vesicle trafficking and led to changes in NOTCH receptor localization. This resulted in perturbation of the NOTCH transcriptional program, which is required for efficient HSC engraftment. In summary, our findings reveal a novel role for NBEA in the control of NOTCH receptor turnover in hematopoietic cells and supports a model in which BDCP-regulated vesicle trafficking is required for efficient HSCT.

LRBA, a BEACH protein mutated in human immune deficiency, is widely expressed in epithelia, exocrine and endocrine glands, and neurons.

Mutations in LRBA, a BEACH domain protein, cause severe immune deficiency in humans. LRBA is expressed in many tissues and organs according to biochemical analysis, but little is known about its cellular and subcellular localization, and its deficiency phenotype outside the immune system. By LacZ histochemistry of Lrba gene-trap mice, we performed a comprehensive survey of LRBA expression in numerous tissues, detecting it in many if not all epithelia, in exocrine and endocrine cells, and in subpopulations of neurons. Immunofluorescence microscopy of the exocrine and endocrine pancreas, salivary glands, and intestinal segments, confirmed these patterns of cellular expression and provided information on the subcellular localizations of the LRBA protein. Immuno-electron microscopy demonstrated that in neurons and endocrine cells, which co-express LRBA and its closest relative, neurobeachin, both proteins display partial association with endomembranes in complementary, rather than overlapping, subcellular distributions. Prominent manifestations of human LRBA deficiency, such as inflammatory bowel disease or endocrinopathies, are believed to be primarily due to immune dysregulation. However, as essentially all affected tissues also express LRBA, it is possible that LRBA deficiency enhances their vulnerability and contributes to the pathogenesis.

Remodeling of the postsynaptic proteome in male mice and marmosets during synapse development.

Postsynaptic proteins play crucial roles in synaptic function and plasticity. During brain development, alterations in synaptic number, shape, and stability occur, known as synapse maturation. However, the postsynaptic protein composition changes during development are not fully understood. Here, we show the trajectory of the postsynaptic proteome in developing male mice and common marmosets. Proteomic analysis of mice at 2, 3, 6, and 12 weeks of age shows that proteins involved in synaptogenesis are differentially expressed during this period. Analysis of published transcriptome datasets shows that the changes in postsynaptic protein composition in the mouse brain after 2 weeks of age correlate with gene expression changes. Proteomic analysis of marmosets at 0, 2, 3, 6, and 24 months of age show that the changes in the marmoset brain can be categorized into two parts: the first 2 months and after that. The changes observed in the first 2 months are similar to those in the mouse brain between 2 and 12 weeks of age. The changes observed in marmoset after 2 months old include differential expression of synaptogenesis-related molecules, which hardly overlap with that in mice. Our results provide a comprehensive proteomic resource that underlies developmental synapse maturation in rodents and primates.

Palmdelphin Regulates Nuclear Resilience to Mechanical Stress in the Endothelium.

BACKGROUND: PALMD (palmdelphin) belongs to the family of paralemmin proteins implicated in cytoskeletal regulation. Single nucleotide polymorphisms in the PALMD locus that result in reduced expression are strong risk factors for development of calcific aortic valve stenosis and predict severity of the disease. METHODS: Immunodetection and public database screening showed dominant expression of PALMD in endothelial cells (ECs) in brain and cardiovascular tissues including aortic valves. Mass spectrometry, coimmunoprecipitation, and immunofluorescent staining allowed identification of PALMD partners. The consequence of loss of PALMD expression was assessed in small interferring RNA-treated EC cultures, knockout mice, and human valve samples. RNA sequencing of ECs and transcript arrays on valve samples from an aortic valve study cohort including patients with the single nucleotide polymorphism rs7543130 informed about gene regulatory changes. RESULTS: ECs express the cytosolic PALMD-KKVI splice variant, which associated with RANGAP1 (RAN GTP hydrolyase activating protein 1). RANGAP1 regulates the activity of the GTPase RAN and thereby nucleocytoplasmic shuttling via XPO1 (Exportin1). Reduced PALMD expression resulted in subcellular relocalization of RANGAP1 and XPO1, and nuclear arrest of the XPO1 cargoes p53 and p21. This indicates an important role for PALMD in nucleocytoplasmic transport and consequently in gene regulation because of the effect on localization of transcriptional regulators. Changes in EC responsiveness on loss of PALMD expression included failure to form a perinuclear actin cap when exposed to flow, indicating lack of protection against mechanical stress. Loss of the actin cap correlated with misalignment of the nuclear long axis relative to the cell body, observed in PALMD-deficient ECs, Palmd-/- mouse aorta, and human aortic valve samples derived from patients with calcific aortic valve stenosis. In agreement with these changes in EC behavior, gene ontology analysis showed enrichment of nuclear- and cytoskeleton-related terms in PALMD-silenced ECs. CONCLUSIONS: We identify RANGAP1 as a PALMD partner in ECs. Disrupting the PALMD/RANGAP1 complex alters the subcellular localization of RANGAP1 and XPO1, and leads to nuclear arrest of the XPO1 cargoes p53 and p21, accompanied by gene regulatory changes and loss of actin-dependent nuclear resilience. Combined, these consequences of reduced PALMD expression provide a mechanistic underpinning for PALMD's contribution to calcific aortic valve stenosis pathology.

Neocortical Expansion Due to Increased Proliferation of Basal Progenitors Is Linked to Changes in Their Morphology.

The evolutionary expansion of the mammalian neocortex (Ncx) is thought to be linked to increased proliferative capacity of basal progenitors (BPs) and their neurogenic capacity. Here, by quantifying BP morphology in the developing Ncx of mouse, ferret, and human, we show that increased BP proliferative capacity is linked to an increase in BP process number. We identify human membrane-bound PALMDELPHIN (PALMD-Caax) as an underlying factor, and we show that it drives BP process growth and proliferation when expressed in developing mouse and ferret Ncx. Conversely, CRISPR/Cas9-mediated disruption of PALMD or its binding partner ADDUCIN-γ in fetal human Ncx reduces BP process numbers and proliferation. We further show that PALMD-induced processes enable BPs to receive pro-proliferative integrin-dependent signals. These findings provide a link between BP morphology and proliferation, suggesting that changes in BP morphology may have contributed to the evolutionary expansion of the Ncx.

Molecular Dissection of Neurobeachin Function at Excitatory Synapses.

Spines are small protrusions from dendrites where most excitatory synapses reside. Changes in number, shape, and size of dendritic spines often reflect changes of neural activity in entire circuits or at individual synapses, making spines key structures of synaptic plasticity. Neurobeachin is a multidomain protein with roles in spine formation, postsynaptic neurotransmitter receptor targeting and actin distribution. However, the contributions of individual domains of Neurobeachin to these functions is poorly understood. Here, we used mostly live cell imaging and patch-clamp electrophysiology to monitor morphology and function of spinous synapses in primary hippocampal neurons. We demonstrate that a recombinant full-length Neurobeachin from humans can restore mushroom spine density and excitatory postsynaptic currents in neurons of Neurobeachin-deficient mice. We then probed the role of individual domains of Neurobeachin by comparing them to the full-length molecule in rescue experiments of knockout neurons. We show that the combined PH-BEACH domain complex is highly localized in spine heads, and that it is sufficient to restore normal spine density and surface targeting of postsynaptic AMPA receptors. In addition, we report that the Armadillo domain facilitates the formation of filopodia, long dendritic protrusions which often precede the development of mature spines, whereas the PKA-binding site appears as a negative regulator of filopodial extension. Thus, our results indicate that individual domains of Neurobeachin sustain important and specific roles in the regulation of spinous synapses. Since heterozygous mutations in Neurobeachin occur in autistic patients, the results will also improve our understanding of pathomechanism in neuropsychiatric disorders associated with impairments of spine function.

Neurobeachin and the Kinesin KIF21B Are Critical for Endocytic Recycling of NMDA Receptors and Regulate Social Behavior.

Autism spectrum disorders (ASDs) are associated with mutations affecting synaptic components, including GluN2B-NMDA receptors (NMDARs) and neurobeachin (NBEA). NBEA participates in biosynthetic pathways to regulate synapse receptor targeting, synaptic function, cognition, and social behavior. However, the role of NBEA-mediated transport in specific trafficking routes is unclear. Here, we highlight an additional function for NBEA in the local delivery and surface re-insertion of synaptic receptors in mouse neurons. NBEA dynamically interacts with Rab4-positive recycling endosomes, transiently enters spines in an activity-dependent manner, and regulates GluN2B-NMDAR recycling. Furthermore, we show that the microtubule growth inhibitor kinesin KIF21B constrains NBEA dynamics and is present in the NBEA-recycling endosome-NMDAR complex. Notably, Kif21b knockout decreases NMDAR surface expression and alters social behavior in mice, consistent with reported social deficits in Nbea mutants. The influence of NBEA-KIF21B interactions on GluN2B-NMDAR local recycling may be relevant to mechanisms underlying ASD etiology.

Mutations outside the N-terminal part of RBCK1 may cause polyglucosan body myopathy with immunological dysfunction: expanding the genotype-phenotype spectrum.

A subset of patients with polyglucosan body myopathy was found to have underlying mutations in the RBCK1 gene. Affected patients may display diverse symptoms ranging from skeletal muscular weakness, cardiomyopathy to chronic autoinflammation and immunodeficiency. It was suggested that the exact localization of the mutation within the gene might be responsible for the specific phenotype, with N-terminal mutations causing severe immunological dysfunction and mutations in the middle or C-terminal part leading to a myopathy phenotype. We report the clinical, immunological and genetic findings of two unrelated individuals suffering from a childhood-onset RBCK1-asscociated disease caused by the same homozygous truncating mutation (NM_031229.2:c.896_899del, p.Glu299Valfs*46) in the middle part of the RBCK1 gene. Our patients suffered from a myopathy with cardiac involvement, but in contrast to previous reports on mutations in this part of the gene, also displayed signs of autoinflammation and immunodeficiency. Our report suggests that RBCK1 mutations at locations that were previously thought to lack immunological features may also present with immunological dysfunction later in the disease course. This notably broadens the genotype-phenotype correlation of RBCK1-related polyglucosan body myopathy.

The BEACH protein LRBA is required for hair bundle maintenance in cochlear hair cells and for hearing.

Lipopolysaccharide-responsive beige-like anchor protein (LRBA) belongs to the enigmatic class of BEACH domain-containing proteins, which have been attributed various cellular functions, typically involving intracellular protein and membrane transport processes. Here, we show that LRBA deficiency in mice leads to progressive sensorineural hearing loss. In LRBA knockout mice, inner and outer hair cell stereociliary bundles initially develop normally, but then partially degenerate during the second postnatal week. LRBA deficiency is associated with a reduced abundance of radixin and Nherf2, two adaptor proteins, which are important for the mechanical stability of the basal taper region of stereocilia. Our data suggest that due to the loss of structural integrity of the central parts of the hair bundle, the hair cell receptor potential is reduced, resulting in a loss of cochlear sensitivity and functional loss of the fraction of spiral ganglion neurons with low spontaneous firing rates. Clinical data obtained from two human patients with protein-truncating nonsense or frameshift mutations suggest that LRBA deficiency may likewise cause syndromic sensorineural hearing impairment in humans, albeit less severe than in our mouse model.

The BEACH Protein LRBA Promotes the Localization of the Heterotrimeric G-protein Golf to Olfactory Cilia.

BEACH domain proteins are involved in membrane protein traffic and human diseases, but their molecular mechanisms are not understood. The BEACH protein LRBA has been implicated in immune response and cell proliferation, and human LRBA mutations cause severe immune deficiency. Here, we report a first functional and molecular phenotype outside the immune system of LRBA-knockout mice: compromised olfaction, manifesting in reduced electro-olfactogram response amplitude, impaired food-finding efficiency, and smaller olfactory bulbs. LRBA is prominently expressed in olfactory and vomeronasal chemosensory neurons of wild-type mice. Olfactory impairment in the LRBA-KO is explained by markedly reduced concentrations (20-40% of wild-type levels) of all three subunits αolf, ß1 and γ13 of the olfactory heterotrimeric G-protein, Golf, in the sensory cilia of olfactory neurons. In contrast, cilia morphology and the concentrations of many other proteins of olfactory cilia are not or only slightly affected. LRBA is also highly expressed in photoreceptor cells, another cell type with a specialized sensory cilium and heterotrimeric G-protein-based signalling; however, visual function appeared unimpaired by the LRBA-KO. To our knowledge, this is the first observation that a BEACH protein is required for the efficient subcellular localization of a lipid-anchored protein, and of a ciliary protein.

Immunological phenotype of the murine Lrba knockout.

Biallelic mutations in the human lipopolysaccharide responsive beige-like anchor (LRBA) gene lead to a primary immunodeficiency known as LRBA deficiency, characterized by a broad range of clinical manifestations including autoimmunity, organomegaly, hypogammaglobulinemia and recurrent infections. Considering the phenotypic heterogeneity in patients and the severity of the disease, our aim was to assess the role of LRBA in immune cells and to understand the underlying pathomechanisms through the study of a Lrba knockout (Lrba-/-) mouse model. LRBA-deficient mice did not show severe clinical or immunological signs of disease, either at steady state under specific-pathogen-free conditions, after vaccination with T-dependent and T-independent antigens, or in the context of acute infections with lymphocytic choriomeningitis virus (LCMV) or Salmonella Typhimurium. Although Lrba-/- mice were able to produce normal serum immunoglobulin M (IgM) and IgG and to mount a specific immune response after immunization, they showed elevated serum and secretory basal IgA levels. LRBA was dispensable for B- and T-cell development, as well as for in vitro B-cell proliferation, survival, isotype switching and plasmablast differentiation. Interestingly, Lrba-/- mice displayed decreased cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) expression by regulatory T cells and activated conventional CD4+ and CD8+ T lymphocytes, reduced frequency of peritoneal B-1a cells along with diminished interleukin-10 production and increased percentages of T follicular helper cells in Peyer's patches, but without developing overt signs of autoimmunity. Our findings expand the role of LRBA in immune regulatory mechanisms previously reported in patients, and suggest a novel role in IgA production that is crucial for the protection of mucosal surfaces and gut-associated immune tolerance.

Glycogen pathways in disease: new developments in a classical field of medical genetics.

Glycogen is the storage form of glucose in animal cells. Its degradation can rapidly provide fuel for energy production (particularly important in muscle), or replenish blood glucose during fasting by the liver. Genetic defects of glycogen metabolism give rise to glycogen storage diseases (GSDs), manifesting histologically in abnormal quantity or quality of glycogen in the cells. GSDs can be caused by defects of proteins participating in the synthesis or degradation of glycogen itself, in the glycolytic degradation of glucose phosphates in muscle and erythrocytes, in the release of glucose from liver and kidney into the bloodstream, in the clearance of glycogen from lysosomes (all, "primary GSDs"), or in the control of these pathways ("secondary GSDs"). Most genes responsible for classical, primary GSDs have probably been identified, and future progress in understanding the biochemical and genetic defects underlying unsolved disorders presenting with glycogen storage abnormalities will perhaps be predominantly in the field of secondary GSDs.

Severe neurodegeneration with impaired autophagy mechanism triggered by ostm1 deficiency.

Loss of Ostm1 leads to the most severe form of osteopetrosis in mice and humans. Because functional rescue of the osteopetrotic defect in these mice extended their lifespan from ∼3 weeks to 6 weeks, this unraveled a second essential role of Ostm1. We discovered that Ostm1 is highly expressed in the mouse brain in neurons, microglia, and astrocytes. At 3-4 weeks of age, mice with Ostm1 loss showed 3-10-fold stimulation of reactive gliosis, with an increased astrocyte cell population and microglia activation. This inflammatory response was associated with marked retinal photoreceptor degeneration and massive neuronal loss in the brain. Intracellular characterization of neurons revealed abnormal storage of carbohydrates, lipids, and ubiquitinated proteins, combined with marked accumulation of autophagosomes that causes frequent axonal swelling. Stimulation of autophagy was provided by specific markers and by significant down-regulation of the mammalian target of rapamycin signaling, identifying a cellular pathologic mechanism. A series of transgenic mouse lines specifically targeted to distinct central nervous system cell subpopulations determined that Ostm1 has a primary and autonomous role in neuronal homeostasis. Complete functional complementation demonstrated that the development of severe and rapid neurodegeneration in these mice is independent of the hematopoietic lineage and has clinical implications for treatment of osteopetrosis. Importantly, this study establishes a novel neurodegenerative mouse model critical for understanding the multistep pathogenic cascade of cellular autophagy disorders toward therapeutic strategy design.

Paralemmin-1 is expressed in lymphatic endothelial cells and modulates cell migration, cell maturation and tumor lymphangiogenesis.

The lymphatic system, the network of lymphatic vessels and lymphoid organs, maintains the body fluid balance and ensures the immunological surveillance of the body. In the adult organism, the de novo formation of lymphatic vessels is mainly observed in pathological conditions. In contrast to the molecular mechanisms governing the generation of the lymphatic vasculature during embryogenesis, the processes underlying pathological lymphangiogenesis are less well understood. A genome-wide screen comparing the transcriptome of tumor-derived lymphatic endothelial cells with that of blood vessel endothelial cells identified paralemmin-1 as a protein prominently expressed in lymphatic endothelial cells. Paralemmin-1 is a lipid-anchored membrane protein that in fibroblasts and neurons plays a role in the regulation of cell shape, plasma membrane dynamics and cell motility. Here, we show that paralemmin-1 is expressed in tumor-derived lymphatic endothelial cells as well as in lymphatic endothelial cells of normal, non-tumorigenic tissue. Paralemmin-1 represses cell migration and delays the formation of tube-like structures of lymphatic endothelial cells in vitro by modulating cell-substrate adhesion, filopodia formation and plasma membrane blebbing. While constitutive genetic ablation of paralemmin-1 expression in mice has no effect on the development and physiological function of the lymphatic system, the loss of paralemmin-1 impaired tumor-associated lymphangiogenesis. Together, these results newly identify paralemmin-1 as a protein highly expressed in lymphatic endothelial cells. Similar to its function in neurons, it may link the cytoskeleton to the plasma membrane and thereby modulate lymphatic endothelial cell adhesion, migration and lymphangiogenesis.

Neurobeachin regulates neurotransmitter receptor trafficking to synapses.

The surface density of neurotransmitter receptors at synapses is a key determinant of synaptic efficacy. Synaptic receptor accumulation is regulated by the transport, postsynaptic anchoring, and turnover of receptors, involving multiple trafficking, sorting, motor, and scaffold proteins. We found that neurons lacking the BEACH (beige-Chediak/Higashi) domain protein Neurobeachin (Nbea) had strongly reduced synaptic responses caused by a reduction in surface levels of glutamate and GABA(A) receptors. In the absence of Nbea, immature AMPA receptors accumulated early in the biosynthetic pathway, and mature N-methyl-d-aspartate, kainate, and GABA(A) receptors did not reach the synapse, whereas maturation and surface expression of other membrane proteins, synapse formation, and presynaptic function were unaffected. These data show that Nbea regulates synaptic transmission under basal conditions by targeting neurotransmitter receptors to synapses.

Evolution of the vertebrate paralemmin gene family: ancient origin of gene duplicates suggests distinct functions.

Paralemmin-1 is a protein implicated in plasma membrane dynamics, the development of filopodia, neurites and dendritic spines, as well as the invasiveness and metastatic potential of cancer cells. However, little is known about its mode of action, or about the biological functions of the other paralemmin isoforms: paralemmin-2, paralemmin-3 and palmdelphin. We describe here evolutionary analyses of the paralemmin gene family in a broad range of vertebrate species. Our results suggest that the four paralemmin isoform genes (PALM1, PALM2, PALM3 and PALMD) arose by quadruplication of an ancestral gene in the two early vertebrate genome duplications. Paralemmin-1 and palmdelphin were further duplicated in the teleost fish specific genome duplication. We identified a unique sequence motif common to all paralemmins, consisting of 11 highly conserved residues of which four are invariant. A single full-length paralemmin homolog with this motif was identified in the genome of the sea lamprey Petromyzon marinus and an isolated putative paralemmin motif could be detected in the genome of the lancelet Branchiostoma floridae. This allows us to conclude that the paralemmin gene family arose early and has been maintained throughout vertebrate evolution, suggesting functional diversification and specific biological roles of the paralemmin isoforms. The paralemmin genes have also maintained specific features of gene organisation and sequence. This includes the occurrence of closely linked downstream genes, initially identified as a readthrough fusion protein with mammalian paralemmin-2 (Palm2-AKAP2). We have found evidence for such an arrangement for paralemmin-1 and -2 in several vertebrate genomes, as well as for palmdelphin and paralemmin-3 in teleost fish genomes, and suggest the name paralemmin downstream genes (PDG) for this new gene family. Thus, our findings point to ancient roles for paralemmins and distinct biological functions of the gene duplicates.

Paralemmin-1 is over-expressed in estrogen-receptor positive breast cancers.

BACKGROUND: Paralemmin-1 is a phosphoprotein lipid-anchored to the cytoplasmic face of membranes where it functions in membrane dynamics, maintenance of cell shape, and process formation. Expression of paralemmin-1 and its major splice variant (Δ exon 8) as well as the extent of posttranslational modifications are tissue- and development-specific. Paralemmin-1 expression in normal breast and breast cancer tissue has not been described previously. RESULTS: Paralemmin-1 mRNA and protein expression was evaluated in ten breast cell lines, 26 primary tumors, and 10 reduction mammoplasty (RM) tissues using real time RT-PCR. Paralemmin-1 splice variants were assessed in tumor and RM tissues using a series of primers and RT-PCR. Paralemmin-1 protein expression was examined in cell lines using Western Blots and in 31 ductal carcinomas in situ, 65 infiltrating ductal carcinomas, and 40 RM tissues using immunohistochemistry. Paralemmin-1 mRNA levels were higher in breast cancers than in RM tissue and estrogen receptor (ER)-positive tumors had higher transcript levels than ER-negative tumors. The Δ exon 8 splice variant was detected more frequently in tumor than in RM tissues. Protein expression was consistent with mRNA results showing higher paralemmin-1 expression in ER-positive tumors. CONCLUSIONS: The differential expression of paralemmin-1 in a subset of breast cancers suggests the existence of variation in membrane dynamics that may be exploited to improve diagnosis or provide a therapeutic target.

Neurobeachin, a regulator of synaptic protein targeting, is associated with body fat mass and feeding behavior in mice and body-mass index in humans.

Neurobeachin (Nbea) regulates neuronal membrane protein trafficking and is required for the development and functioning of central and neuromuscular synapses. In homozygous knockout (KO) mice, Nbea deficiency causes perinatal death. Here, we report that heterozygous KO mice haploinsufficient for Nbea have higher body weight due to increased adipose tissue mass. In several feeding paradigms, heterozygous KO mice consumed more food than wild-type (WT) controls, and this consumption was primarily driven by calories rather than palatability. Expression analysis of feeding-related genes in the hypothalamus and brainstem with real-time PCR showed differential expression of a subset of neuropeptide or neuropeptide receptor mRNAs between WT and Nbea+/- mice in the sated state and in response to food deprivation, but not to feeding reward. In humans, we identified two intronic NBEA single-nucleotide polymorphisms (SNPs) that are significantly associated with body-mass index (BMI) in adult and juvenile cohorts. Overall, data obtained in mice and humans suggest that variation of Nbea abundance or activity critically affects body weight, presumably by influencing the activity of feeding-related neural circuits. Our study emphasizes the importance of neural mechanisms in body weight control and points out NBEA as a potential risk gene in human obesity.