Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNAREs.

Mammalian homologs of yeast Atg8 protein (mAtg8s) are important in autophagy, but their exact mode of action remains ill-defined. Syntaxin 17 (Stx17), a SNARE with major roles in autophagy, was recently shown to bind mAtg8s. Here, we identified LC3-interacting regions (LIRs) in several SNAREs that broaden the landscape of the mAtg8-SNARE interactions. We found that Syntaxin 16 (Stx16) and its cognate SNARE partners all have LIR motifs and bind mAtg8s. Knockout of Stx16 caused defects in lysosome biogenesis, whereas a Stx16 and Stx17 double knockout completely blocked autophagic flux and decreased mitophagy, pexophagy, xenophagy, and ribophagy. Mechanistic analyses revealed that mAtg8s and Stx16 control several properties of lysosomal compartments including their function as platforms for active mTOR. These findings reveal a broad direct interaction of mAtg8s with SNAREs with impact on membrane remodeling in eukaryotic cells and expand the roles of mAtg8s to lysosome biogenesis.

Genetic and Epigenetic Characteristics of Autosomal Dominant Pseudohypoparathyroidism Type 1B: Case Reports and Literature Review.

Autosomal dominant pseudohypoparathyroidism 1B (AD-PHP1B) is a rare endocrine and imprinted disorder. The objective of this study is to clarify the imprinted regulation of the guanine nucleotide binding-protein α-stimulating activity polypeptide 1 (GNAS) cluster in the occurrence and development of AD-PHP1B based on animal and clinical patient studies. The methylation-specific multiples ligation-dependent probe amplification (MS-MLPA) was conducted to detect the copy number variation in syntaxin-16 (STX16) gene and methylation status of the GNAS differentially methylated regions (DMRs). Long-range PCR was used to confirm deletion at STX16 gene. In the first family, DNA analysis of the proband and proband's mother revealed an isolated loss of methylation (LOM) at exon A/B and a 3.0 kb STX16 deletion. The patient's healthy grandmother had the 3.0 kb STX16 deletion but no epigenetic abnormality. The patient's healthy maternal aunt showed no genetic or epigenetic abnormality. In the second family, the analysis of long-range PCR revealed the 3.0 kb STX16 deletion for the proband but not her children. In this study, 3.0 kb STX16 deletion causes isolated LOM at exon A/B in two families, which is the most common genetic mutation of AD-PHP1B. The deletion involving NESP55 or AS or genomic rearrangements of GNAS can also result in AD-PHP1B, but it's rare. LOM at exon A/B DMR is prerequisite methylation defect of AD-PHP1B. STX16 and NESP55 directly control the imprinting at exon A/B, while AS controls the imprinting at exon A/B by regulating the transcriptional level of NESP55.

Excess of a Rassf1-targeting microRNA, miR-193a-3p, perturbs cell division fidelity.

BACKGROUND: Several microRNA (miRNA) molecules have emerged as important post-transcriptional regulators of tumour suppressor and oncogene expression. Ras association domain family member 1 (RASSF1) is a critical tumour suppressor that controls multiple aspects of cell proliferation such as cell cycle, cell division and apoptosis. The expression of RASSF1 is lost in a variety of cancers due to the promoter hypermethylation. METHODS: miR-193a-3p was identified as a RASSF1-targeting miRNA by a dual screening approach. In cultured human cancer cells, immunoblotting, qRT-PCR, luciferase reporter assays, time-lapse microscopy and immunofluorescence methods were used to study the effects of excess miR-193a-3p on RASSF1 expression and cell division. RESULTS: Here, we report a new miRNA-mediated mechanism that regulates RASSF1 expression: miR-193a-3p binds directly to RASSF1-3'UTR and represses the mRNA and protein expression. In human cancer cells, excess of miR-193a-3p causes polyploidy through impairment of the Rassf1-Syntaxin 16 signalling pathway that is needed for completion of cytokinesis. In the next cell cycle the miR-193a-3p-overexpressing cells exhibit multipolar mitotic spindles, mitotic delay and elevated frequency of cell death. CONCLUSIONS: Our results suggest that besides epigenetic regulation, altered expression of specific miRNAs may contribute to the loss of Rassf1 in cancer cells and cause cell division errors.

Subversion of NPC1 pathway of cholesterol transport by Anaplasma phagocytophilum.

Intracellular cholesterol amounts, distribution and traffic are tightly regulated to maintain the healthy eukaryotic cell function. However, how intracellular pathogens that require cholesterol, interact with the host cholesterol homeostasis and traffic is not well understood. Anaplasma phagocytophilum is an obligatory intracellular and cholesterol-robbing bacterium, which causes human granulocytic anaplasmosis. Here we found that a subset of cholesterol-binding membrane protein, Niemann-Pick type C1 (NPC1)-bearing vesicles devoid of lysosomal markers were upregulated in HL-60 cells infected with A. phagocytophilum, and trafficked to live bacterial inclusions. The NPC1 localization to A. phagocytophilum inclusions was abolished by low-density lipoprotein (LDL)-derived cholesterol traffic inhibitor U18666A. Studies using NPC1 siRNA and the cell line with cholesterol traffic defect demonstrated that the NPC1 function is required for bacterial cholesterol acquisition and infection. Furthermore, trans-Golgi network-specific soluble N-ethylmaleimide-sensitive factor attachment protein receptors, vesicle-associated membrane protein (VAMP4) and syntaxin 16, which are associated with NPC1 and LDL-derived cholesterol vesicular transport were recruited to A. phagocytophilum inclusions, and VAMP4 was required for bacteria infection. Taken together, A. phagocytophilum is the first example of a pathogen that subverts the NPC1 pathway of intracellular cholesterol transport and homeostasis for bacterial inclusion membrane biogenesis and cholesterol capture.

VAMP4 is required to maintain the ribbon structure of the Golgi apparatus.

The Golgi apparatus forms a twisted ribbon-like network in the juxtanuclear region of vertebrate cells. Vesicle-associated membrane protein 4 (VAMP4), a v-SNARE protein expressed exclusively in the vertebrate trans-Golgi network (TGN), plays a role in retrograde trafficking from the early endosome to the TGN, although its precise function within the Golgi apparatus remains unclear. To determine whether VAMP4 plays a functional role in maintaining the structure of the Golgi apparatus, we depleted VAMP4 gene expression using RNA interference technology. Depletion of VAMP4 from HeLa cells led to fragmentation of the Golgi ribbon. These fragments were not uniformly distributed throughout the cytoplasm, but remained in the juxtanuclear area. Electron microscopy and immunohistochemistry showed that in the absence of VAMP4, the length of the Golgi stack was shortened, but Golgi stacking was normal. Anterograde trafficking was not impaired in VAMP4-depleted cells, which contained intact microtubule arrays. Depletion of the cognate SNARE partners of VAMP4, syntaxin 6, syntaxin 16, and Vti1a also disrupted the Golgi ribbon structure. Our findings suggested that the maintenance of Golgi ribbon structure requires normal retrograde trafficking from the early endosome to the TGN, which is likely to be mediated by the formation of VAMP4-containing SNARE complexes.

Neuromuscular symptoms in a patient with familial pseudohypoparathyroidism type Ib diagnosed by methylation-specific multiplex ligation-dependent probe amplification.

Pseudohypoparathyroidism type Ib (PHP-Ib) is a rare genetic disorder characterized by hypocalcemia and hyperphosphatemia due to imprinting defects in the maternally derived GNAS allele. Patients with PHP-Ib are usually identified by tetany, convulsions, and/or muscle cramps, whereas a substantial fraction of patients remain asymptomatic and are identified by familial studies. Although previous studies on patients with primary hypoparathyroidism have indicated that hypocalcemia can be associated with various neuromuscular abnormalities, such clinical features have been rarely described in patients with PHP-Ib. Here, we report a 12-year-old male patient with familial PHP-Ib and unique neuromuscular symptoms. The patient presented with general fatigue, steppage gait, and myalgia. Physical examinations revealed muscular weakness and atrophies in the lower legs, a shortening of the bilateral Achilles' tendons and absence of deep tendon reflexes. Laboratory tests showed hypocalcemia, hyperphosphatemia, elevated serum intact PTH level, and impaired responses of urinary phosphate and cyclic AMP in an Ellsworth-Howard test, in addition to an elevated serum creatine kinase level. Clinical features of the patient were significantly improved after 1 month of treatment with alfacalcidol and calcium. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and subsequent PCR analyses identified a methylation defect at exon A/B of GNAS and a microdeletion involving exons 4-6 of the GNAS neighboring gene STX16 in the patient and in his asymptomatic brother. The results suggest that various neuromuscular features probably associated with hypocalcemia can be the first symptoms of PHP-Ib, and that MS-MLPA serves as a powerful tool for screening of GNAS abnormalities in patients with atypical manifestations.

Phosphorylation of the N-terminus of Syntaxin-16 controls interaction with mVps45 and GLUT4 trafficking in adipocytes.

The ability of insulin to stimulate glucose transport in muscle and fat cells is mediated by the regulated delivery of intracellular vesicles containing glucose transporter-4 (GLUT4) to the plasma membrane, a process known to be defective in disease such as Type 2 diabetes. In the absence of insulin, GLUT4 is sequestered in tubules and vesicles within the cytosol, collectively known as the GLUT4 storage compartment. A subset of these vesicles, known as the 'insulin responsive vesicles' are selectively delivered to the cell surface in response to insulin. We have previously identified Syntaxin16 (Sx16) and its cognate Sec1/Munc18 protein family member mVps45 as key regulatory proteins involved in the delivery of GLUT4 into insulin responsive vesicles. Here we show that mutation of a key residue within the Sx16 N-terminus involved in mVps45 binding, and the mutation of the Sx16 binding site in mVps45 both perturb GLUT4 sorting, consistent with an important role of the interaction of these two proteins in GLUT4 trafficking. We identify Threonine-7 (T7) as a site of phosphorylation of Sx16 in vitro. Mutation of T7 to D impairs Sx16 binding to mVps45 in vitro and overexpression of T7D significantly impaired insulin-stimulated glucose transport in adipocytes. We show that both AMP-activated protein kinase (AMPK) and its relative SIK2 phosphorylate this site. Our data suggest that Sx16 T7 is a potentially important regulatory site for GLUT4 trafficking in adipocytes.

Quantitative proteomic analysis of the adipocyte plasma membrane.

The adipocyte is a key regulator of mammalian metabolism. To advance our understanding of this important cell, we have used quantitative proteomics to define the protein composition of the adipocyte plasma membrane (PM) in the presence and absence of insulin. Using this approach, we have identified a high confidence list of 486 PM proteins, 52 of which potentially represent novel cell surface proteins, including a member of the adiponectin receptor family and an unusually high number of hydrolases with no known function. Several novel insulin-responsive proteins including the sodium/hydrogen exchanger, NHE6 and the collagens III and VI were also identified, and we provide evidence of PM-ER association suggestive of a unique functional association between these two organelles in the adipocyte. Together these studies provide a wealth of potential therapeutic targets for the manipulation of adipocyte function and a valuable resource for metabolic research and PM biology.

Syntaxin 16 binds to cystic fibrosis transmembrane conductance regulator and regulates its membrane trafficking in epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a key membrane protein in the complex network of epithelial ion transporters regulating epithelial permeability. Syntaxins are one of the major determinants in the intracellular trafficking and membrane targeting of secretory proteins. In the present study we demonstrate the biochemical and functional association between CFTR and syntaxin 16 (STX16) that mediates vesicle transport within the early/late endosomes and trans-Golgi network. Immunoprecipitation experiments in rat colon and T84 human colonic epithelial cells indicate that STX16 associates with CFTR. Further analyses using the domain-specific pulldown assay reveal that the helix domain of STX16 directly interacts with the N-terminal region of CFTR. Immunostainings in rat colon and T84 cells show that CFTR and STX16 highly co-localize at the apical and subapical regions of epithelial cells. Interestingly, CFTR-associated chloride current was reduced by the knockdown of STX16 expression in T84 cells. Surface biotinylation and recycling assays indicate that the reduction in CFTR chloride current is due to decreased CFTR expression on the plasma membrane. These results suggest that STX16 mediates recycling of CFTR and constitutes an important component of CFTR trafficking machinery in intestinal epithelial cells.

Common and distinct roles for the binding partners Rabenosyn-5 and Vps45 in the regulation of endocytic trafficking in mammalian cells.

In several invertebrate organisms, the Sec1p/Munc18-like protein Vps45 interacts with the divalent Rab4/Rab5 effector, Rabenosyn-5 and carries out multiple functions in the endocytic/secretory pathways. In mammalian cells, Vps45 and Rabenosyn-5 also interact, but the molecular characterization of this binding, and the functional relationship between these two proteins has not been well defined. Here we identify a novel sequence within Rabenosyn-5 required for its interaction with Vps45. We demonstrate that hVps45-depletion decreases expression of Rabenosyn-5, likely resulting from Rabenosyn-5 degradation through the proteasomal pathway. Furthermore, we demonstrate that similar to Rabenosyn-5-depletion, hVps45-depletion causes impaired recycling of beta1 integrins, and a subsequent delay in human fibroblast cell migration on fibronectin-coated plates. Moreover, beta1 integrin recycling could be rescued by reintroduction of siRNA-resistant wild-type Rabenosyn-5, but not a mutant deficient in Vps45 binding. However, unlike Rabenosyn-5-depletion, which induces Golgi fragmentation and decreased recruitment of sorting nexin retromer subunits to the Golgi, hVps45-depletion induces Golgi condensation and accumulation of retromer subunits in the vicinity of the Golgi. In part, these phenomena could be attributed to reduced Syntaxin16 expression and altered localization of both Syntaxin16 and Syntaxin6 upon Vps45-depletion. Overall, these findings implicate hVps45 and Rabenosyn-5 in post early endosome transport, and we propose that their interaction serves as a nexus to promote bidirectional transport along the endosome-to-recycling compartment and endosome-to-Golgi axes.

Retrograde and Anterograde Transport of Lat-Vesicles during the Immunological Synapse Formation: Defining the Finely-Tuned Mechanism.

LAT is an important player of the signaling cascade induced by TCR activation. This adapter molecule is present at the plasma membrane of T lymphocytes and more abundantly in intracellular compartments. Upon T cell activation the intracellular pool of LAT is recruited to the immune synapse (IS). We previously described two pathways controlling LAT trafficking: retrograde transport from endosomes to the TGN, and anterograde traffic from the Golgi to the IS. We address the specific role of four proteins, the GTPase Rab6, the t-SNARE syntaxin-16, the v-SNARE VAMP7 and the golgin GMAP210, in each pathway. Using different methods (endocytosis and Golgi trap assays, confocal and TIRF microscopy, TCR-signalosome pull down) we show that syntaxin-16 is regulating the retrograde transport of LAT whereas VAMP7 is regulating the anterograde transport. Moreover, GMAP210 and Rab6, known to contribute to both pathways, are in our cellular context, specifically and respectively, involved in anterograde and retrograde transport of LAT. Altogether, our data describe how retrograde and anterograde pathways coordinate LAT enrichment at the IS and point to the Golgi as a central hub for the polarized recruitment of LAT to the IS. The role that this finely-tuned transport of signaling molecules plays in T-cell activation is discussed.

Syntaxin 16: unraveling cellular physiology through a ubiquitous SNARE molecule.

Syntaxin 16 (Syx16) is member of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family of molecules that functions in membrane fusion in eukaryotic cells. A rather ubiquitously expressed, tail-anchored membrane protein localized mainly at the trans-Golgi network (TGN), it mediates primarily retrograde endosomal-TGN transport. In spite of its ubiquitous expression, Syx16 has specific and interesting roles in the physiology of specialized cells, including Glut4 dynamics, dendritic outgrowth-related membrane traffic, and cytokinesis. We discussed these physiological functions of Syx16 in the light of what is known of its subcellular localization, vesicular trafficking pathways involved, cognate SNARE partners and other interacting proteins. Further, we speculate on some possible pathophysiological roles of Syx16.

Vps54 regulates Drosophila neuromuscular junction development and interacts genetically with Rab7 to control composition of the postsynaptic density.

Vps54 is a subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to the trans-Golgi network (TGN). In the wobbler mouse, a model for human motor neuron (MN) disease, reduction in the levels of Vps54 causes neurodegeneration. However, it is unclear how disruption of the GARP complex leads to MN dysfunction. To better understand the role of Vps54 in MNs, we have disrupted expression of the Vps54 ortholog in Drosophila and examined the impact on the larval neuromuscular junction (NMJ). Surprisingly, we show that both null mutants and MN-specific knockdown of Vps54 leads to NMJ overgrowth. Reduction of Vps54 partially disrupts localization of the t-SNARE, Syntaxin-16, to the TGN but has no visible impact on endosomal pools. MN-specific knockdown of Vps54 in MNs combined with overexpression of the small GTPases Rab5, Rab7, or Rab11 suppresses the Vps54 NMJ phenotype. Conversely, knockdown of Vps54 combined with overexpression of dominant negative Rab7 causes NMJ and behavioral abnormalities including a decrease in postsynaptic Dlg and GluRIIB levels without any effect on GluRIIA. Taken together, these data suggest that Vps54 controls larval MN axon development and postsynaptic density composition through a mechanism that requires Rab7.

Syntaxin 16's Newly Deciphered Roles in Autophagy.

Syntaxin 16, a Qa-SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor), is involved in a number of membrane-trafficking activities, particularly transport processes at the trans-Golgi network (TGN). Recent works have now implicated syntaxin 16 in the autophagy process. In fact, syntaxin 16 appears to have dual roles, firstly in facilitating the transport of ATG9a-containing vesicles to growing autophagosomes, and secondly in autolysosome formation. The former involves a putative SNARE complex between syntaxin 16, VAMP7 and SNAP-47. The latter occurs via syntaxin 16's recruitment by Atg8/LC3/GABARAP family proteins to autophagosomes and endo-lysosomes, where syntaxin 16 may act in a manner that bears functional redundancy with the canonical autophagosome Qa-SNARE syntaxin 17. Here, I discuss these recent findings and speculate on the mechanistic aspects of syntaxin 16's newly found role in autophagy.

Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation.

The adapter molecule linker for activation of T cells (LAT) orchestrates the formation of signalosomes upon T cell receptor (TCR) stimulation. LAT is present in different intracellular pools and is dynamically recruited to the immune synapse upon stimulation. However, the intracellular traffic of LAT and its function in T lymphocyte activation are ill defined. We show herein that LAT, once internalized, transits through the Golgi-trans-Golgi network (TGN), where it is repolarized to the immune synapse. This retrograde transport of LAT depends on the small GTPase Rab6 and the target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) Syntaxin-16, two regulators of the endosome-to-Golgi/TGN retrograde transport. We also show in vitro in Syntaxin-16- or Rab6-silenced human cells and in vivo in CD4+ T lymphocytes of the Rab6 knockout mouse that this retrograde traffic controls TCR stimulation. These results establish that the retrograde traffic of LAT from the plasma membrane to the Golgi-TGN controls the polarized delivery of LAT at the immune synapse and T lymphocyte activation.

Analysis of Multiple Families With Single Individuals Affected by Pseudohypoparathyroidism Type Ib (PHP1B) Reveals Only One Novel Maternally Inherited GNAS Deletion.

Proximal tubular resistance to parathyroid hormone (PTH) resulting in hypocalcemia and hyperphosphatemia are preeminent abnormalities in pseudohypoparathyroidism type Ib (PHP1B), but resistance toward other hormones as well as variable features of Albright's Hereditary Osteodystrophy (AHO) can occur also. Genomic DNA from PHP1B patients shows epigenetic changes at one or multiple differentially methylated regions (DMRs) within GNAS, the gene encoding Gαs and splice variants thereof. In the autosomal dominant disease variant, these methylation abnormalities are caused by deletions in STX16 or GNAS on the maternal allele. The molecular defect(s) leading to sporadic PHP1B (sporPHP1B) remains in most cases unknown and we therefore analyzed 60 sporPHP1B patients and available family members by microsatellite markers, single nucleotide polymorphisms (SNPs), multiplex ligation-dependent probe amplification (MLPA), and methylation-specific MLPA (MS-MLPA). All investigated cases revealed broad GNAS methylation changes, but no evidence for inheritance of two paternal chromosome 20q alleles. Some patients with partial epigenetic modifications in DNA from peripheral blood cells showed more complete GNAS methylation changes when testing their immortalized lymphoblastoid cells. Analysis of siblings and children of sporPHP1B patients provided no evidence for an abnormal mineral ion regulation and no changes in GNAS methylation. Only one patient revealed, based on MLPA and microsatellite analyses, evidence for an allelic loss, which resulted in the discovery of two adjacent, maternally inherited deletions (37,597 and 1427 bp, respectively) that remove the area between GNAS antisense exons 3 and 5, including exon NESP. Our findings thus emphasize that the region comprising antisense exons 3 and 4 is required for establishing all maternal GNAS methylation imprints. The genetic defect(s) leading in sporPHP1B to epigenetic GNAS changes and thus PTH-resistance remains unknown, but it seems unlikely that this disease variant is caused by heterozygous inherited or de novo mutations involving GNAS.

Drosophila SNAP-29 is an essential SNARE that binds multiple proteins involved in membrane traffic.

Each membrane fusion event along the secretory and endocytic pathways requires a specific set of SNAREs to assemble into a 4-helical coiled-coil, the so-called trans-SNARE complex. Although most SNAREs contribute one helix to the trans-SNARE complex, members of the SNAP-25 family contribute two helixes. We report the characterization of the Drosophila homologue of SNAP-29 (dSNAP-29), which is expressed throughout development. Unlike the other SNAP-25 like proteins in fruit fly (i.e., dSNAP-25 and dSNAP-24), which form SDS-resistant SNARE complexes with their cognate SNAREs, dSNAP-29 does not participate in any SDS-resistant complexes, despite its interaction with dsyntaxin1 and dsyntaxin16 in vitro. Immunofluorescence studies indicated that dSNAP-29 is distributed in various tissues, locating in small intracellular puncta and on the plasma membrane, where it associates with EH domain-containing proteins implicated in the endocytic pathway. Overexpression and RNAi studies suggested that dSNAP-29 mediates an essential process in Drosophila development.

GNAS -Related Loss-of-Function Disorders and the Role of Imprinting.

GNAS (guanine nucleotide-binding protein, α stimulating) is a complex imprinted locus coding, besides the α-stimulatory subunit of the G protein, the paternally (extra-large, antisense and A/B) and maternally (neuroendocrine secretory protein) transcripts. Heterozygous mutations in the coding sequence of GNAS produce dominant phenotypes (combination of resistances to hormones signaling through G-protein-coupled receptors, osteodystrophy and obesity) that depend on the parental origin of the mutated allele. Likewise, alterations in the methylation at promoters of GNAS transcripts, associated or not with deletions of imprinting control regions in the nearby STX16 gene or within GNAS, prompt resistance to parathormone when affecting the maternal allele. Therefore, imprinting of GNAS is the determining factor for the variability of the phenotype. Knowledge of the various phenotypes is necessary for genetic counseling as well as an appropriate therapeutic balance between regular follow-up, prevention of disease complications and iatrogeny.

Syntaxin 16 is a master recruitment factor for cytokinesis.

Recently it was shown that both recycling endosome and endosomal sorting complex required for transport (ESCRT) components are required for cytokinesis, in which they are believed to act in a sequential manner to bring about secondary ingression and abscission, respectively. However, it is not clear how either of these complexes is targeted to the midbody and whether their delivery is coordinated. The trafficking of membrane vesicles between different intracellular organelles involves the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. Although membrane traffic is known to play an important role in cytokinesis, the contribution and identity of intracellular SNAREs to cytokinesis remain unclear. Here we demonstrate that syntaxin 16 is a key regulator of cytokinesis, as it is required for recruitment of both recycling endosome-associated Exocyst and ESCRT machinery during late telophase, and therefore that these two distinct facets of cytokinesis are inextricably linked.

Partial sleep restriction activates immune response-related gene expression pathways: experimental and epidemiological studies in humans.

Epidemiological studies have shown that short or insufficient sleep is associated with increased risk for metabolic diseases and mortality. To elucidate mechanisms behind this connection, we aimed to identify genes and pathways affected by experimentally induced, partial sleep restriction and to verify their connection to insufficient sleep at population level. The experimental design simulated sleep restriction during a working week: sleep of healthy men (N = 9) was restricted to 4 h/night for five nights. The control subjects (N = 4) spent 8 h/night in bed. Leukocyte RNA expression was analyzed at baseline, after sleep restriction, and after recovery using whole genome microarrays complemented with pathway and transcription factor analysis. Expression levels of the ten most up-regulated and ten most down-regulated transcripts were correlated with subjective assessment of insufficient sleep in a population cohort (N = 472). Experimental sleep restriction altered the expression of 117 genes. Eight of the 25 most up-regulated transcripts were related to immune function. Accordingly, fifteen of the 25 most up-regulated Gene Ontology pathways were also related to immune function, including those for B cell activation, interleukin 8 production, and NF-κB signaling (P<0.005). Of the ten most up-regulated genes, expression of STX16 correlated negatively with self-reported insufficient sleep in a population sample, while three other genes showed tendency for positive correlation. Of the ten most down-regulated genes, TBX21 and LGR6 correlated negatively and TGFBR3 positively with insufficient sleep. Partial sleep restriction affects the regulation of signaling pathways related to the immune system. Some of these changes appear to be long-lasting and may at least partly explain how prolonged sleep restriction can contribute to inflammation-associated pathological states, such as cardiometabolic diseases.