Elena Kartvelishvily

Neuron-derived D-serine release provides a novel means to activate N-methyl-D-aspartate receptors.

d-Serine is a coagonist of N-methyl-d-aspartate (NMDA) receptors that occurs at high levels in the brain. Biosynthesis of d-serine is carried out by serine racemase, which converts l-to d-serine. d-Serine has been demonstrated to occur in glial cells, leading to the proposal that astrocytes are the only source of d-serine. We now report significant amounts of serine racemase and d-serine in primary neuronal cultures and neurons in vivo. Several neuronal culture types expressed serine racemase, and d-serine synthesis was comparable with that in glial cultures. Immunohistochemical staining of brain sections with new antibodies revealed the presence of serine racemase and d-serine in neurons. Cortical neurons expressing serine racemase also expressed the NR2a subunit in situ. Neuron-derived d-serine contributes to NMDA receptor activation in cortical neuronal cultures. Degradation of endogenous d-serine by addition of the recombinant enzyme d-serine deaminase diminished NMDA-elicited excitotoxicity. Release of neuronal d-serine was mediated by ionotropic glutamate receptor agonists such as NMDA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate. Removal of either external Ca2+ or Na+ blocked d-serine release. Release of d-serine was mostly through a cytosolic route because it was insensitive to bafilomycin A1, a potent inhibitor of vesicular neurotransmitter uptake. d-Serine was also not transported into purified synaptic vesicles under conditions optimal for the uptake of known transmitters. Our results suggest that neurons are a major source of d-serine. Glutamate-induced neuronal d-serine release provides a novel mechanism for activating NMDA receptors by an autocrine or paracrine way.

D-Serine Is the Dominant Endogenous Coagonist for NMDA Receptor Neurotoxicity in Organotypic Hippocampal Slices

d-serine occurs at high levels in the brain, where it is an endogenous coagonist at the “glycine site” of NMDA receptors. However, d-serine action has not been previously compared with that of endogenous glycine, and the relative importance of the two coagonists remains unclear. We now investigated the efficiencies of the two coagonists in mediating NMDA receptor neurotoxicity in organotypic hippocampal slices. Removal of endogenous d-serine from slices was achieved by pretreating the tissue with recombinant d-serine deaminase enzyme. This enzyme is several orders of magnitude more efficient than previous methods to remove d-serine. We report that complete removal of d-serine virtually abolished NMDA-elicited neurotoxicity but did not protect against kainate. Although levels of glycine were 10-fold higher than d-serine, endogenous glycine was ineffective in mediating NMDA receptor neurotoxicity. The effect of endogenous glycine could be observed only after simultaneous removal of endogenous d-serine and blockage of the glycine transporter GlyT1. Our data indicate that d-serine is the dominant coagonist for NMDA receptor-elicited neurotoxicity, mediating all cell death elicited by NMDA in organotypic slices. The results suggest an essential role for this unusual d-amino acid, with implications for the mechanism of neuronal death in the nervous system.

Microbiota Diurnal Rhythmicity Programs Host Transcriptome Oscillations

The intestinal microbiota undergoes diurnal compositional and functional oscillations that affect metabolic homeostasis, but the mechanisms by which the rhythmic microbiota influences host circadian activity remain elusive. Using integrated multi-omics and imaging approaches, we demonstrate that the gut microbiota features oscillating biogeographical localization and metabolome patterns that determine the rhythmic exposure of the intestinal epithelium to different bacterial species and their metabolites over the course of a day. This diurnal microbial behavior drives, in turn, the global programming of the host circadian transcriptional, epigenetic, and metabolite oscillations. Surprisingly, disruption of homeostatic microbiome rhythmicity not only abrogates normal chromatin and transcriptional oscillations of the host, but also incites genome-wide de novo oscillations in both intestine and liver, thereby impacting diurnal fluctuations of host physiology and disease susceptibility. As such, the rhythmic biogeography and metabolome of the intestinal microbiota regulates the temporal organization and functional outcome of host transcriptional and epigenetic programs.

A Secreted Disulfide Catalyst Controls Extracellular Matrix Composition and Function

Form and Function The contribution of disulfide bonding to oxidative protein folding and assembly, quality control, and stress responses in the endoplasmic reticulum (ER) are widely recognized. In contrast, catalysis of disulfide bond formation downstream of the ER is uncharted territory. QSOX, a Golgi-localized or secreted disulfide catalyst, was identified in the 1970s and was more recently shown to be upregulated in many cancers. However, the physiological importance of QSOX catalytic activity has been unclear. Ilani et al. (p. 74, published online 23 May) found that human QSOX1 is essential for incorporation of laminin into the extracellular matrix, with profound effects on the capability of the matrix to support integrin-mediated cell adhesion and migration. Laminin incorporation is promoted by a secreted enzyme, which is important for cell adhesion and migration. Disulfide bond formation in secretory proteins occurs primarily in the endoplasmic reticulum (ER), where multiple enzyme families catalyze cysteine cross-linking. Quiescin sulfhydryl oxidase 1 (QSOX1) is an atypical disulfide catalyst, localized to the Golgi apparatus or secreted from cells. We examined the physiological function for extracellular catalysis of de novo disulfide bond formation by QSOX1. QSOX1 activity was required for incorporation of laminin into the extracellular matrix (ECM) synthesized by fibroblasts, and ECM produced without QSOX1 was defective in supporting cell-matrix adhesion. We developed an inhibitory monoclonal antibody against QSOX1 that could modulate ECM properties and undermine cell migration.

A Metabolic Gene Cluster in the Wheat W1 and the Barley Cer-cqu Loci Determines β-Diketone Biosynthesis and Glaucousness

A metabolic gene cluster underlying the known glaucousness loci W1 in wheat and Cer-cqu in barley establishes a novel molecular pathway for β-diketone wax biosynthesis The glaucous appearance of wheat (Triticum aestivum) and barley (Hordeum vulgare) plants, that is the light bluish-gray look of flag leaf, stem, and spike surfaces, results from deposition of cuticular β-diketone wax on their surfaces; this phenotype is associated with high yield, especially under drought conditions. Despite extensive genetic and biochemical characterization, the molecular genetic basis underlying the biosynthesis of β-diketones remains unclear. Here, we discovered that the wheat W1 locus contains a metabolic gene cluster mediating β-diketone biosynthesis. The cluster comprises genes encoding proteins of several families including type-III polyketide synthases, hydrolases, and cytochrome P450s related to known fatty acid hydroxylases. The cluster region was identified in both genetic and physical maps of glaucous and glossy tetraploid wheat, demonstrating entirely different haplotypes in these accessions. Complementary evidence obtained through gene silencing in planta and heterologous expression in bacteria supports a model for a β-diketone biosynthesis pathway involving members of these three protein families. Mutations in homologous genes were identified in the barley eceriferum mutants defective in β-diketone biosynthesis, demonstrating a gene cluster also in the β-diketone biosynthesis Cer-cqu locus in barley. Hence, our findings open new opportunities to breed major cereal crops for surface features that impact yield and stress response.

Genetic deletion of Cadm4 results in myelin abnormalities resembling Charcot-Marie-Tooth neuropathy.

The interaction between myelinating Schwann cells and the axons they ensheath is mediated by cell adhesion molecules of the Cadm/Necl/SynCAM family. This family consists of four members: Cadm4/Necl4 and Cadm1/Necl2 are found in both glia and axons, whereas Cadm2/Necl3 and Cadm3/Necl1 are expressed by sensory and motor neurons. By generating mice lacking each of the Cadm genes, we now demonstrate that Cadm4 plays a role in the establishment of the myelin unit in the peripheral nervous system. Mice lacking Cadm4 (PGK-Cre/Cadm4fl/fl), but not Cadm1, Cadm2, or Cadm3, develop focal hypermyelination characterized by tomacula and myelin outfoldings, which are the hallmark of several Charcot-Marie-Tooth neuropathies. The absence of Cadm4 also resulted in abnormal axon–glial contact and redistribution of ion channels along the axon. These neuropathological features were also found in transgenic mice expressing a dominant-negative mutant of Cadm4 lacking its cytoplasmic domain in myelinating glia Tg(mbp-Cadm4dCT), as well as in mice lacking Cadm4 specifically in Schwann cells (DHH-Cre/Cadm4fl/fl). Consistent with these abnormalities, both PGK-Cre/Cadm4fl/fl and Tg(mbp-Cadm4dCT) mice exhibit impaired motor function and slower nerve conduction velocity. These findings indicate that Cadm4 regulates the growth of the myelin unit and the organization of the underlying axonal membrane.

Identification of Tmem10/opalin as an oligodendrocyte enriched gene using expression profiling combined with genetic cell ablation

Oligodendrocytes form an insulating multilamellar structure of compact myelin around axons, which allows efficient and rapid propagation of action potentials. However, little is known about the molecular mechanisms operating at the onset of myelination and during maintenance of the myelin sheath in the adult. Here we use a genetic cell ablation approach combined with Affymetrix GeneChip microarrays to identify a number of oligodendrocyte‐enriched genes that may play a key role in myelination. One of the “oligogenes” we cloned using this approach is Tmem10/Opalin, which encodes for a novel transmembrane glycoprotein. In situ hybridization and RT‐PCR analysis revealed that Tmem10 is selectively expressed by oligodendrocytes and that its expression is induced during their differentiation. Developmental immunofluorescence analysis demonstrated that Tmem10 starts to be expressed in the white matter tracks of the cerebellum and the corpus callosum at the onset of myelination after the appearance of other myelin genes such as MBP. In contrast to the spinal cord and brain, Tmem10 was not detected in myelinating Schwann cells, indicating that it is a CNS‐specific myelin protein. In mature oligodendrocytes, Tmem10 was present at the cell soma and processes, as well as along myelinated internodes, where it was occasionally concentrated at the paranodes. In myelinating spinal cord cultures, Tmem10 was detected in MBP‐positive cellular processes that were aligned with underlying axons before myelination commenced. These results suggest a possible role of Tmem10 in oligodendrocyte differentiation and CNS myelination.

Three-dimensional localization of T-cell receptors in relation to microvilli using a combination of superresolution microscopies

Significance T lymphocytes play a central role in cell-mediated immunity. Their surfaces are covered by narrow and short protrusions called microvilli. It is not known whether there is a role for microvilli in the immune response. To shed light on this question, we probed the location of T-cell receptors (TCRs), the molecules that initiate the immune response of T cells, with respect to the 3D structure of microvilli. Superresolution optical microscopy showed that TCRs are highly concentrated on microvilli. Previous studies stressed the role of small clusters of TCRs in the immune process; our study provides a natural explanation as to how these clusters form. Leukocyte microvilli are flexible projections enriched with adhesion molecules. The role of these cellular projections in the ability of T cells to probe antigen-presenting cells has been elusive. In this study, we probe the spatial relation of microvilli and T-cell receptors (TCRs), the major molecules responsible for antigen recognition on the T-cell membrane. To this end, an effective and robust methodology for mapping membrane protein distribution in relation to the 3D surface structure of cells is introduced, based on two complementary superresolution microscopies. Strikingly, TCRs are found to be highly localized on microvilli, in both peripheral blood human T cells and differentiated effector T cells, and are barely found on the cell body. This is a decisive demonstration that different types of T cells universally localize their TCRs to microvilli, immediately pointing to these surface projections as effective sensors for antigenic moieties. This finding also suggests how previously reported membrane clusters might form, with microvilli serving as anchors for specific T-cell surface molecules.

Differential clustering of Caspr by oligodendrocytes and Schwann cells

Formation of the paranodal axoglial junction (PNJ) requires the presence of three cell adhesion molecules: the 155‐kDa isoform of neurofascin (NF155) on the glial membrane and a complex of Caspr and contactin found on the axolemma. Here we report that the clustering of Caspr along myelinated axons during development differs fundamentally between the central (CNS) and peripheral (PNS) nervous systems. In cultures of Schwann cells (SC) and dorsal root ganglion (DRG) neurons, membrane accumulation of Caspr was detected only after myelination. In contrast, in oligodendrocytes (OL)/DRG neurons cocultures, Caspr was clustered upon initial glial cell contact already before myelination had begun. Premyelination clustering of Caspr was detected in cultures of oligodendrocytes and retinal ganglion cells, motor neurons, and DRG neurons as well as in mixed cell cultures of rat forebrain and spinal cords. Cocultures of oligodendrocyte precursor cells isolated from contactin‐ or neurofascin‐deficient mice with wild‐type DRG neurons showed that clustering of Caspr at initial contact sites between OL processes and the axon requires glial expression of NF155 but not of contactin. These results demonstrate that the expression of membrane proteins along the axolemma is determined by the type of the contacting glial cells and is not an intrinsic characteristic of the axon.

Hyaluronan Nanoparticles Selectively Target Plaque-Associated Macrophages and Improve Plaque Stability in Atherosclerosis

Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe–/– mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received 89Zr-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe–/– mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP–immune cell interactions significantly decreased over the disease progression. 89Zr-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosis.