Ralf Kleene

Glycans and neural cell interactions.

Carbohydrate-carrying molecules in the nervous system have important roles during development, regeneration and synaptic plasticity. Carbohydrates mediate interactions between recognition molecules, thereby contributing to the formation of a complex molecular meshwork at the cell surface and in the extracellular matrix. The tremendous structural diversity of glycan chains allows for immense combinatorial possibilities that might underlie the fine-tuning of cell–cell and cell–matrix interactions.

Synapsin I Is an Oligomannose-Carrying Glycoprotein, Acts As an Oligomannose-Binding Lectin, and Promotes Neurite Outgrowth and Neuronal Survival When Released via Glia-Derived Exosomes

Oligomannosidic glycans play important roles in nervous system development and function. By performing a phage display screening with oligomannose-specific antibodies, we identified an oligomannose-mimicking peptide that was functionally active in modulating neurite outgrowth and neuron–astrocyte adhesion. Using the oligomannose-mimicking peptide in crosslinking experiments, synapsin I was identified as a novel oligomannose-binding protein in mouse brain. Further analyses not only verified that synapsin I is an oligomannose-binding lectin, but also indicated that it is a glycoprotein carrying oligomannose and Lewisx. We also found that synapsin I is expressed in glia-enriched cultures and is released from glial cells via exosomes. Incubation of glial-derived exosomes in the presence of high KCl concentrations or subjecting glial cell cultures to either oxygen/glucose deprivation or hydrogen peroxide resulted in release of synapsin I from exosomes. Application of synapsin I promoted neurite outgrowth from hippocampal neurons and increased survival of cortical neurons upon hydrogen peroxide treatment or oxygen/glucose deprivation. Coculture experiments using wild-type hippocampal neurons and wild-type or synapsin-deficient glial cells showed enhanced neurite outgrowth when synapsin was expressed by glial cells. Synapsin-induced neurite outgrowth was dependent on oligomannose on synapsin I and the neural cell adhesion molecule NCAM at the neuronal cell surface. The data indicate that, under conditions of high neuronal activity and/or oxidative stress, synapsin can be released from glial-derived exosomes and promotes neurite outgrowth and neuronal survival by modulating the interactions between glia and neurons.

Proteolytic cleavage of the neural cell adhesion molecule by ADAM17/TACE is involved in neurite outgrowth.

The transmembrane and multidomain neural cell adhesion molecule (NCAM) plays important functional roles in the developing and adult nervous system. NCAM is proteolytically processed and appears in soluble forms in the cerebrospinal fluid and in serum under normal and pathological conditions. In this report, we present evidence that the metalloprotease a disintegrin and a metalloprotease (ADAM)17/tumour necrosis factor α converting enzyme (TACE) cleaves the polysialylated as well as the non‐polysialylated transmembrane isoforms of NCAM, whereas the glycophosphatidylinositol‐linked isoform of NCAM is not proteolytically cleaved. A truncated, enzymatically inactive mutant of TACE did not result in release of the NCAM110 cleavage product. Proteolytic cleavage was enhanced by a calmodulin‐specific inhibitor and the actin‐destabilizing agents cytochalasin D and latrunculin B. In contrast, the microtubule‐stabilizing agent colchicine or microtubule‐destabilizing agent paclitaxel did not affect the release of the 110‐kDa fragment of NCAM. Neurite outgrowth from cerebellar microexplants was inhibited in the presence of the metalloprotease inhibitor GM 6001 on substrate‐coated NCAM, but not on poly‐l‐lysine. Upon transfection of hippocampal neurones with an enzymatically inactive mutant of TACE, NCAM‐stimulated neurite outgrowth was inhibited without affecting neurite outgrowth on poly‐l‐lysine, showing that proteolytic processing of NCAM by the metalloprotease TACE is involved in NCAM‐mediated neurite outgrowth.

O-glycosylation pattern of CD24 from mouse brain.

Abstract The cell adhesion molecule CD24 is a highly glycosylated glycoprotein that plays important roles in the central nervous system, the immune system and in tumor biology. Since CD24 comprises only a short protein core of approximately 30 amino acids and low conservation among species, it has been proposed that the functions of CD24 are mediated by its glycosylation pattern. Our present study provides evidence that interaction of CD24 with the cell adhesion molecule L1 is mediated by O-linked glycans carrying α2,3-linked sialic acid. Furthermore, de-N-glycosylated CD24 was shown to promote or inhibit neurite outgrowth of cerebellar neurons or dorsal root ganglion neurons, respectively, to the same extent as untreated CD24. Therefore, this study is focused on the structural elucidation of the chemically released, permethylated CD24 O-glycans by electrospray ionization ion trap mass spectrometry. Our analyses revealed the occurrence of a diverse mixture of mucin-type and O-mannosyl glycans carrying, in part, functionally relevant epitopes, such as 3-linked sialic acid, disialyl motifs, LeX, sialyl-LeX or HNK-1 units. Hence, our data provide the basis for further studies on the contribution of carbohydrate determinants to CD24-mediated biological activities.

Elevated levels of neural recognition molecule L1 in the cerebrospinal fluid of patients with Alzheimer disease and other dementia syndromes

In this study we surveyed a total of 218 cerebrospinal fluid (CSF) samples from patients with different neurological diseases including Alzheimer disease, non-Alzheimer forms of dementia, other neurodegenerative diseases without dementia and normal controls to quantitate by capture ELISA the concentrations of the immunoglobulin superfamily adhesion molecules L1 and NCAM, and characterized by immunoblot analysis the molecular forms of L1 and NCAM. We found a significant increase of L1 and a strong tendency for increase of the soluble fragments of NCAM in the CSF of Alzheimer patients compared to the normal control group. The proteolytic fragments of L1, but not NCAM were also elevated in patients with vascular dementia and dementia of mixed type. Higher L1 concentrations were observed irrespective of age and gender. NCAM concentrations were independent of gender, but positively correlated with age and, surprisingly, also with incidence of multiple sclerosis. Thus, there was an influence of Alzheimer and non-Alzheimer dementias and neurodegeneration on L1, whereas age and neurodegeneration influenced NCAM concentrations. These observations point to an abnormal processing and/or shedding of L1 and NCAM in dementia-related neurodegeneration and age, respectively, reflecting changes in adhesion molecule-related cell interactions.

Cross-comparison of protein recognition of sialic acid diversity on two novel sialoglycan microarrays.

Background: Various glycan microarrays are currently widely used, but systematic cross-comparisons are lacking. Results: We compare and contrast two sialoglycan microarrays using a variety of sialic acid-binding proteins. Conclusion: Diverse array formats can strengthen the quality of information, but differences between arrays may be observed. Significance: Glycan arrays with similar glycan structures cannot be simply assumed to give similar results. DNA and protein arrays are commonly accepted as powerful exploratory tools in research. This has mainly been achieved by the establishment of proper guidelines for quality control, allowing cross-comparison between different array platforms. As a natural extension, glycan microarrays were subsequently developed, and recent advances using such arrays have greatly enhanced our understanding of protein-glycan recognition in nature. However, although it is assumed that biologically significant protein-glycan binding is robustly detected by glycan microarrays, there are wide variations in the methods used to produce, present, couple, and detect glycans, and systematic cross-comparisons are lacking. We address these issues by comparing two arrays that together represent the marked diversity of sialic acid modifications, linkages, and underlying glycans in nature, including some identical motifs. We compare and contrast binding interactions with various known and novel plant, vertebrate, and viral sialic acid-recognizing proteins and present a technical advance for assessing specificity using mild periodate oxidation of the sialic acid chain. These data demonstrate both the diversity of sialic acids and the analytical power of glycan arrays, showing that different presentations in different formats provide useful and complementary interpretations of glycan-binding protein specificity. They also highlight important challenges and questions for the future of glycan array technology and suggest that glycan arrays with similar glycan structures cannot be simply assumed to give similar results.

Prion Protein Regulates Glutamate-Dependent Lactate Transport of Astrocytes

Prion-related protein (PrP) is a neural cell adhesion molecule involved in neurite outgrowth, neuronal survival, and synaptic function. In search of novel binding partners for PrP, we identified the α2/β2-Na+/K+-ATPase and showed that this astroglial ATPase interacts directly with the immunoglobulin superfamily adhesion molecule basigin. In cultured astrocytes, PrP is involved in regulating lactate transport via the astroglial monocarboxylate transporter 1 (MCT1) and in conjunction with α2/β2-ATPase and basigin. Lactate transport via MCT1 is glutamate dependent and regulated by glutamate receptor 2 (GluR2)-containing AMPA receptors with which PrP interacts. The functional interplay between PrP, GluR2, α2/β2-ATPase, basigin, and MCT1 in regulating lactate transport of astrocytes may be functional in the metabolic cross talk between astrocytes and neurons, most likely under stress.

Neural Cell Adhesion Molecule Modulates Dopaminergic Signaling and Behavior by Regulating Dopamine D2 Receptor Internalization

The dopaminergic system plays an important role in the etiology of schizophrenia, and most antipsychotic drugs exert their functions by blocking dopamine D2 receptors (D2Rs). Since the signaling strength mediated by D2Rs is regulated by internalization and degradation processes, it is crucial to identify molecules that modulate D2R localization at the cell surface. Here, we show that the neural cell adhesion molecule (NCAM) promotes D2R internalization/desensitization and subsequent degradation via direct interaction with a short peptide in the third intracellular loop of the D2R. NCAM deficiency in mice leads to increased numbers of D2Rs at the cell surface and augmented D2R signaling as a result of impaired D2R internalization. Furthermore, NCAM-deficient mice show higher sensitivity to the psychostimulant apomorphine and exaggerated activity of dopamine-related locomotor behavior. These results demonstrate that, in addition to its classical function in cell adhesion, NCAM is involved in regulating the trafficking of the neurotransmitter receptor D2R as well as receptor-mediated signaling and behavior, thus implicating NCAM as modulator of the dopaminergic system and a potential pharmacological target for dopamine-related neurological and psychiatric disorders.

NCAM-Induced Neurite Outgrowth Depends on Binding of Calmodulin to NCAM and on Nuclear Import of NCAM and fak Fragments

The neural cell adhesion molecule NCAM plays important functional roles not only during nervous system development, but also in the adult after injury and in synaptic plasticity. Homophilic binding of NCAM triggers intracellular signaling events resulting in cellular responses such as neurite outgrowth that require NCAM palmitoylation-dependent raft localization and activation of the nonreceptor tyrosine kinases fyn and fak. In this study, we show that stimulation of NCAM by a function-triggering NCAM antibody results in proteolytic processing of NCAM and fak. The C-terminal fragment of NCAM, consisting of the intracellular domain, the transmembrane domain, and a stub of the extracellular domain, and the N-terminal fragment of fak are imported into the nucleus. NCAM-stimulated fak activation, generation, and nuclear import of NCAM and fak fragments as well as neurite outgrowth are abolished by mutation of the calmodulin binding motif in the intracellular domain of NCAM that is responsible for the calcium-dependent binding of calmodulin to NCAM. This mutation interferes neither with NCAM cell surface expression, palmitoylation, and raft localization nor with fyn activation. The way by which the transmembrane NCAM fragment reaches the nucleus in a calmodulin- and calcium-dependent manner is by endocytotic transport via the endoplasmic reticulum and the cytoplasm. The generation and nuclear import of NCAM and phosphorylated fak fragments resulting from NCAM stimulation may represent a signal pathway activating cellular responses in parallel or in association with classical kinase- and phosphorylation-dependent signaling cascades.

Lewisx and α2,3-Sialyl Glycans and Their Receptors TAG-1, Contactin, and L1 Mediate CD24-Dependent Neurite Outgrowth

Although carbohydrates have been implicated in cell interactions in the nervous system, the molecular bases of their functions have remained largely obscure. Here, we show that promotion or inhibition of neurite outgrowth of cerebellar or dorsal root ganglion neurons, respectively, induced by the mucin-type adhesion molecule CD24 depends on α2,3-linked sialic acid and Lewisx present on glia-specific CD24 glycoforms. α2,3-Sialyl residues of CD24 bind to a structural motif in the first fibronectin type III domain of the adhesion molecule L1. Following the observation that the adhesion molecules TAG-1 and Contactin show sequence homologies with fucose-specific lectins, we obtained evidence that TAG-1 and Contactin mediate Lewisx-dependent CD24-induced effects on neurite outgrowth. Thus, L1, TAG-1, and Contactin function as lectin-like neuronal receptors. Their cis interactions with neighboring adhesion molecules, e.g., Caspr1 and Caspr2, and with their triggered signal transduction pathways elicit cell type-specific promotion or inhibition of neurite outgrowth induced by glial CD24 in a glycan-dependent trans interaction.