N-cadherin prodomain processing regulates synaptogenesis.

Classical cadherins, which are adhesion molecules functioning at the CNS synapse, are synthesized as adhesively inactive precursor proteins in the endoplasmic reticulum (ER). Signal sequence and prodomain cleavage in the ER and Golgi apparatus, respectively, activates their adhesive properties. Here, we provide the first evidence for sorting of nonadhesive precursor N-cadherin (ProN) to the neuronal surface, where it coexists with adhesively competent mature N-cadherin (N-cad), generating a spectrum of adhesive strengths. In cultured hippocampal neurons, a high ProN/N-cad ratio downregulates synapse formation. Neurons expressing genetically engineered uncleavable ProN make markedly fewer synapses. The synapse number can be rescued to normality by depleting surface ProN levels through prodomain cleavage by an exogenous protease. Finally, prodomain processing is developmentally regulated in the rat hippocampus. We conclude that it is the ProN/N-cad ratio and not mature N-cad alone that is critical for regulation of adhesion during synaptogenesis.

Cellular response to micropatterned growth promoting and inhibitory substrates.

BACKGROUND: Normal development and the response to injury both require cell growth, migration and morphological remodeling, guided by a complex local landscape of permissive and inhibitory cues. A standard approach for studying by such cues is to culture cells on uniform substrates containing known concentrations of these molecules, however this method fails to represent the molecular complexity of the natural growth environment. RESULTS: To mimic the local complexity of environmental conditions in vitro, we used a contact micropatterning technique to examine cell growth and differentiation on patterned substrates printed with the commonly studied growth permissive and inhibitory substrates, poly-L-lysine (PLL) and myelin, respectively. We show that micropatterning of PLL can be used to direct adherence and axonal outgrowth of hippocampal and cortical neurons as well as other cells with diverse morphologies like Oli-neu oligodendrocyte progenitor cell lines and fibroblast-like COS7 cells in culture. Surprisingly, COS7 cells exhibited a preference for low concentration (1 pg/mL) PLL zones over adjacent zones printed with high concentrations (1 mg/mL). We demonstrate that micropatterning is also useful for studying factors that inhibit growth as it can direct cells to grow along straight lines that are easy to quantify. Furthermore, we provide the first demonstration of microcontact printing of myelin-associated proteins and show that they impair process outgrowth from Oli-neu oligodendrocyte precursor cells. CONCLUSION: We conclude that microcontact printing is an efficient and reproducible method for patterning proteins and brain-derived myelin on glass surfaces in order to study the effects of the microenvironment on cell growth and morphogenesis.

Lipidome and proteome map of myelin membranes.

To understand the molecular anatomy of myelin membranes, we performed a large-scale, liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS)-based lipidome and proteome screen on freshly purified human and murine myelin fractions. We identified more than 700 lipid moieties and above 1,000 proteins in the two species, including 284 common lipids and 257 common proteins. This study establishes the first comprehensive map of myelin membrane components in human and mice. Although this study demonstrates many similarities between human and murine myelin, several components have been identified exclusively in each species. Future quantitative validation studies focused on interspecies differences will authenticate the myelin membrane anatomy. The combined lipidome and proteome map presented here can nevertheless be used as a reference library for myelin health and disease.

Implication of perturbed axoglial apparatus in early pediatric multiple sclerosis.

Cerebrospinal fluid samples collected from children during initial presentation of central nervous system inflammation, who may or may not subsequently be diagnosed as having multiple sclerosis (MS), were subjected to large-scale proteomics screening. Unexpectedly, major compact myelin membrane proteins typically implicated in MS were not detected. However, multiple molecules that localize to the node of Ranvier and the surrounding axoglial apparatus membrane were implicated, indicating perturbed axon-glial interactions in those children destined for diagnosis of MS.

Septin 7: actin cross-organization is required for axonal association of Schwann cells.

Myelin sheaths present two distinct domains: compacted myelin spirals and flanking non-compacted cytoplasmic channels, where lipid and protein segregation is established by unknown mechanisms. Septins, a conserved family of membrane and cytoskeletal interacting GTPases, form intracellular diffusion barriers during cell division and neurite extension and are expressed in myelinating cells. Septins, particularly septin 7 (Sept7), the central constituent of septin polymers, are associated with the cytoplasmic channels of myelinating cells. Here we show that Schwann cells deprived of Sept7 fail to wrap around axons from dorsal root ganglion neurons and exhibit disorganization of the actin cytoskeleton. Likewise, Sept7 distribution is dependent on microfilament but not microtubule organization.

Atomic force microscopy reveals important differences in axonal resistance to injury.

Axonal degeneration after traumatic brain injury and nerve compression is considered a common underlying cause of temporary as well as permanent disability. Because a proper functioning of neural network requires phase coherence of all components, even subtle changes in circuitry may lead to network failure. However, it is still not possible to determine which axons will recover or degenerate after injury. Several groups have studied the pressure threshold for axonal injury within a nerve, but difficulty accessing the injured region; insufficient imaging methods and the extremely small dimensions involved have prevented the evaluation of the response of individual axons to injury. We combined microfluidics with atomic force microscopy and in vivo imaging to estimate the threshold force required to 1), uncouple axonal transport without impairing axonal survival, and 2), compromise axonal survival in both individual and bundled axons. We found that rat hippocampal axons completely recover axonal transport with no detectable axonal loss when compressed with pressures up to 65 ± 30 Pa for 10 min, while dorsal root ganglia axons can resist to pressures up to 540 ± 220 Pa. We investigated the reasons for the differential susceptibility of hippocampal and DRG axons to mechanical injury and estimated the elasticity of live axons. We found that dorsal root ganglia axons have a 20% lower elastic modulus than hippocampal axons. Our results emphasize the importance of the integrity of the axonal cytoskeleton in deciding the axonal fate after damage and open up new avenues to improve injury diagnosis and to identify ways to protect axons.

Misalignment of PLP/DM20 transmembrane domains determines protein misfolding in Pelizaeus-Merzbacher disease.

A large number of genetic diseases have been associated with truncated or misfolded membrane proteins trapped in the endoplasmic reticulum (ER). In the ER, they activate the unfolded protein response, which can trigger cell death. Hence, a better understanding of protein misfolding features might help in developing novel therapies. Here, we have studied the molecular basis of Pelizaeus-Merzbacher disease, a leukodystrophy defined by mutations of the PLP1 gene and ER retention of two encoded tetraspan myelin proteins, PLP and DM20. In mouse oligodendroglial cells, mutant isoforms of PLP/DM20 with fewer than all four transmembrane (TM) domains are fully ER retained. Surprisingly, a truncated PLP with only two N-terminal TM domains shows normal cell-surface expression when coexpressed with a second truncated PLP harboring the two C-terminal TM domains. This striking ability to properly self-align the TM domains is disease relevant, as shown for the smaller splice isoform DM20. Here, the increased length of TM domain 3 allows for compensation of the effect of several PLP1 point mutations that impose a conformational constraint onto the adjacent extracellular loop region. We conclude that an important determinant in the quality control of polytopic membrane proteins is the free alignment of their TM domains.

A role for Nr-CAM in the patterning of binocular visual pathways.

Retinal ganglion cell (RGC) axons diverge within the optic chiasm to project to opposite sides of the brain. In mouse, contralateral RGCs are distributed throughout the retina, whereas ipsilateral RGCs are restricted to the ventrotemporal crescent (VTC). While repulsive guidance mechanisms play a major role in the formation of the ipsilateral projection, little is known about the contribution of growth-promoting interactions to the formation of binocular visual projections. Here, we show that the cell adhesion molecule Nr-CAM is expressed by RGCs that project contralaterally and is critical for the guidance of late-born RGCs within the VTC. Blocking Nr-CAM function causes an increase in the size of the ipsilateral projection and reduces neurite outgrowth on chiasm cells in an age- and region-specific manner. Finally, we demonstrate that EphB1/ephrin-B2-mediated repulsion and Nr-CAM-mediated attraction comprise distinct molecular programs that each contributes to the proper formation of binocular visual pathways.

Rapid assembly of functional presynaptic boutons triggered by adhesive contacts.

CNS synapse assembly typically follows after stable contacts between "appropriate" axonal and dendritic membranes are made. We show that presynaptic boutons selectively form de novo following neuronal fiber adhesion to beads coated with poly-d-lysine (PDL), an artificial cationic polypeptide. As demonstrated by atomic force and live confocal microscopy, functional presynaptic boutons self-assemble as rapidly as 1 h after bead contact, and are found to contain a variety of proteins characteristic of presynaptic endings. Interestingly, presynaptic compartment assembly does not depend on the presence of a biological postsynaptic membrane surface. Rather, heparan sulfate proteoglycans, including syndecan-2, as well as others possibly adsorbed onto the bead matrix or expressed on the axon surface, are required for assembly to proceed by a mechanism dependent on the dynamic reorganization of F-actin. Our results indicate that certain (but not all) nonspecific cationic molecules like PDL, with presumably electrostatically mediated adhesive properties, can effectively bypass cognate and natural postsynaptic ligands to trigger presynaptic assembly in the absence of specific target recognition. In contrast, we find that postsynaptic compartment assembly depends on the prior presence of a mature presynaptic ending.

A proteome map of axoglial specializations isolated and purified from human central nervous system.

Compact myelin, the paranode, and the juxtaparanode are discrete domains that are formed on myelinated axons. In humans, neurological disorders associated with loss of myelin, including Multiple Sclerosis, often also result in disassembly of the node of Ranvier. Despite the importance of these domains in the proper functioning of the CNS, their molecular composition and assembly mechanism remains largely unknown. We therefore performed a large-scale proteomics MudPIT screen for the identification of proteins in human myelin and axogliasomal fractions. We identified over 1,000 proteins in these fractions. Since even minor perturbations in neuron-glial interactions can uncouple the glial support of axons, the proteome map presented here can be used as a reference library for "myelin health" and disease states, including white matter disorders such as leukodystrophies and multiple sclerosis.

Spatiotemporal distribution and function of N-cadherin in postnatal Schwann cells: A matter of adhesion?

During embryonic development of the peripheral nervous system (PNS), the adhesion molecule neuronal cadherin (N-cadherin) is expressed by Schwann cell precursors and associated with axonal growth cones. N-cadherin expression levels decrease as precursors differentiate into Schwann cells. In this study, we investigated the distribution of N-cadherin in the developing postnatal and adult rat peripheral nervous system. N-cadherin was found primarily in ensheathing glia throughout development, concentrated at neuron-glial or glial-glial contacts of the sciatic nerve, dorsal root ganglia (DRG), and myenteric plexi. In the sciatic nerve, N-cadherin decreases with age and progress of myelination. In adult animals, N-cadherin was found exclusively in nonmyelinating Schwann cells. The distribution of N-cadherin in developing E17 DRG primary cultures is similar to what was observed in vivo. Functional studies of N-cadherin in these cultures, using the antagonist peptide INPISGQ, show a disruption of the attachment between Schwann cells, but no interference in the initial or long-term contact between Schwann cells and axons. We suggest that N-cadherin acts primarily in the adhesion between glial cells during postnatal development. It may form adherents/junctions between nonmyelinating glia, which contribute to the stable tubular structure encapsulating thin caliber axons and thus stabilize the nerve structure as a whole.

N-cadherin mediates interaction between precursor cells in the subventricular zone and regulates further differentiation.

Neurogenesis and cell differentiation in the brain continues throughout life. In the subventricular zone and rostral migratory stream, precursor cells contact each other. Cell-cell interactions mediated via adhesion molecules are no doubt involved in establishing and maintaining the neurogenic ability of these cells. Here, we demonstrate that N-cadherin plays important roles in forming cell clusters and in regulating cell differentiation. N-cadherin is abundantly expressed in chain migrating cells in the subventricular zone and rostral migratory stream but is down-regulated after cells exit these regions. We also show that neurosphere formation is inhibited via suppression of N-cadherin function and that N-cadherin expression is decreased after induction of neurosphere differentiation. Furthermore, we demonstrate that functional blockade of N-cadherin can enhance glial cell differentiation in explant cultures of precursors from the subventricular zone.

Cadherin activity is required for activity-induced spine remodeling.

Neural activity induces the remodeling of pre- and postsynaptic membranes, which maintain their apposition through cell adhesion molecules. Among them, N-cadherin is redistributed, undergoes activity-dependent conformational changes, and is required for synaptic plasticity. Here, we show that depolarization induces the enlargement of the width of spine head, and that cadherin activity is essential for this synaptic rearrangement. Dendritic spines visualized with green fluorescent protein in hippocampal neurons showed an expansion by the activation of AMPA receptor, so that the synaptic apposition zone may be expanded. N-cadherin-venus fusion protein laterally dispersed along the expanding spine head. Overexpression of dominant-negative forms of N-cadherin resulted in the abrogation of the spine expansion. Inhibition of actin polymerization with cytochalasin D abolished the spine expansion. Together, our data suggest that cadherin-based adhesion machinery coupled with the actin-cytoskeleton is critical for the remodeling of synaptic apposition zone.

TMEM10 Promotes Oligodendrocyte Differentiation and is Expressed by Oligodendrocytes in Human Remyelinating Multiple Sclerosis Plaques.

Oligodendrocyte precursor cells (OPCs) differentiate during postnatal development into myelin-forming oligodendrocytes, in a process distinguished by substantial changes in morphology and the onset of myelin gene expression. A mammalian-specific CNS myelin gene, tmem10, also called Opalin, encodes a type 1 transmembrane protein that is highly upregulated during early stages of OPC differentiation; however, a function for TMEM10 has not yet been identified. Here, consistent with previous studies, we detect TMEM10 protein in mouse brain beginning at ~P10 and show that protein levels continue to increase as oligodendrocytes differentiate and myelinate axons in vivo. We show that constitutive TMEM10 overexpression in the Oli-neu oligodendroglial cell line promotes the expression of the myelin-associated genes MAG, CNP and CGT, whereas TMEM10 knock down in primary OPCs reduces CNP mRNA expression and decreases the percentage of MBP-positive oligodendrocytes that differentiate in vitro. Ectopic TMEM10 expression evokes an increase in process extension and branching, and blocking endogenous TMEM10 expression results in oligodendrocytes with abnormal cell morphology. These findings may have implications for human demyelinating disorders, as oligodendrocytes expressing TMEM10 are detected in human remyelinating multiple sclerosis lesions. Together, our findings provide evidence that TMEM10 promotes oligodendrocyte terminal differentiation and may represent a novel target to promote remyelination in demyelinating disorders.

Structure of the neural (N-) cadherin prodomain reveals a cadherin extracellular domain-like fold without adhesive characteristics.

Classical cadherins mediate cell-cell adhesion through calcium-dependent homophilic interactions and are activated through cleavage of a prosequence in the late Golgi. We present here the first three-dimensional structure of a classical cadherin prosequence, solved by NMR. The prototypic prosequence of N-cadherin consists of an Ig-like domain and an unstructured C-terminal region. The folded part of the prosequence-termed prodomain-has a striking structural resemblance to cadherin "adhesive" domains that could not have been predicted from the amino acid sequence due to low sequence similarities. Our detailed structural and evolutionary analysis revealed that prodomains are distant relatives of cadherin "adhesive" domains but lack all the features known to be important for cadherin-cadherin interactions. The presence of an additional "nonadhesive" domain seems to make it impossible to engage homophilic interactions between cadherins that are necessary to activate adhesion, thus explaining the inactive state of prodomain-bearing cadherins.

Gamma-protocadherins are targeted to subsets of synapses and intracellular organelles in neurons.

The clustered protocadherins (Pcdhs) comprise >50 putative synaptic recognition molecules that are related to classical cadherins and highly expressed in the nervous system. Pcdhs are organized into three gene clusters (alpha, beta, and gamma). Within the alpha and gamma clusters, three exons encode the cytoplasmic domain for each Pcdh, making these domains identical within a cluster. Using an antibody to the Pcdh-gamma constant cytoplasmic domain, we find that all interneurons in cultured hippocampal neurons express high levels of Pcdh-gamma(s) in a nonsynaptic distribution. In contrast, only 48% of pyramidal-like cells expressed appreciable levels of these molecules. In these cells, Pcdh-gamma(s) were associated with a subset of excitatory synapses in which they may mediate presynaptic to postsynaptic recognition in concert with classical cadherins. Immunogold localization in hippocampal tissue showed Pcdh-gamma(s) at some synapses, in nonsynaptic plasma membranes, and in axonal and dendritic tubulovesicular structures, indicating that they may be exchanged among synapses and intracellular compartments. Our results show that although Pcdh-gamma(s) can be synaptic molecules, synapses form lacking Pcdh-gamma(s). Thus, Pcdh-gamma(s) and their relatives may be late additions to the classical cadherin-based synaptic adhesive scaffold; their presence in intracellular compartments suggests a role in modifying synaptic physiology or stability.

N-cadherin relocalizes from the periphery to the center of the synapse after transient synaptic stimulation in hippocampal neurons.

N-cadherin is a cell adhesion molecule which is enriched at synapses. Binding of N-cadherin molecules to each other across the synaptic cleft has been postulated to stabilize adhesion between the presynaptic bouton and the postsynaptic terminal. N-cadherin is also required for activity-induced changes at synapses, including hippocampal long term potentiation and activity-induced spine expansion and stabilization. We hypothesized that these activity-dependent changes might involve changes in N-cadherin localization within synapses. To determine whether synaptic activity changes the localization of N-cadherin, we used structured illumination microscopy, a super-resolution approach which overcomes the conventional resolution limits of light microscopy, to visualize the localization of N-cadherin within synapses of hippocampal neurons. We found that synaptic N-cadherin exhibits a spectrum of localization patterns, ranging from puncta at the periphery of the synapse adjacent to the active zone to an even distribution along the synaptic cleft. Furthermore, the N-cadherin localization pattern within synapses changes during KCl depolarization and after transient synaptic stimulation. During KCl depolarization, N-cadherin relocalizes away from the central region of the synaptic cleft to the periphery of the synapse. In contrast, after transient synaptic stimulation with KCl followed by a period of rest in normal media, fewer synapses have N-cadherin present as puncta at the periphery and more synapses have N-cadherin present more centrally and uniformly along the synapse compared to unstimulated cells. This indicates that transient synaptic stimulation modulates N-cadherin localization within the synapse. These results bring new information to the structural organization and activity-induced changes occurring at synapses, and suggest that N-cadherin relocalization may contribute to activity dependent changes at synapses.

Substrate Micropatterning as a New in Vitro Cell Culture System to Study Myelination.

Myelination is a highly regulated developmental process whereby oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system ensheathe axons with a multilayered concentric membrane. Axonal myelination increases the velocity of nerve impulse propagation. In this work, we present a novel in vitro system for coculturing primary dorsal root ganglia neurons along with myelinating cells on a highly restrictive and micropatterned substrate. In this new coculture system, neurons survive for several weeks, extending long axons on defined Matrigel tracks. On these axons, myelinating cells can achieve robust myelination, as demonstrated by the distribution of compact myelin and nodal markers. Under these conditions, neurites and associated myelinating cells are easily accessible for studies on the mechanisms of myelin formation and on the effects of axonal damage on the myelin sheath.

Opposite roles of furin and PC5A in N-cadherin processing.

We recently demonstrated that lack of Furin-processing of the N-cadherin precursor (proNCAD) in highly invasive melanoma and brain tumor cells results in the cell-surface expression of a nonadhesive protein favoring cell migration and invasion in vitro. Quantitative polymerase chain reaction analysis of malignant human brain tumor cells revealed that of all proprotein convertases (PCs) only the levels of Furin and PC5A are modulated, being inversely (Furin) or directly (PC5A) correlated with brain tumor invasive capacity. Intriguingly, the N-terminal sequence following the Furin-activated NCAD site (RQKR↓DW(161), mouse nomenclature) reveals a second putative PC-processing site (RIRSDR↓DK(189)) located in the first extracellular domain. Cleavage at this site would abolish the adhesive functions of NCAD because of the loss of the critical Trp(161). This was confirmed upon analysis of the fate of the endogenous prosegment of proNCAD in human malignant glioma cells expressing high levels of Furin and low levels of PC5A (U343) or high levels of PC5A and negligible Furin levels (U251). Cellular analyses revealed that Furin is the best activating convertase releasing an ~17-kDa prosegment, whereas PC5A is the major inactivating enzyme resulting in the secretion of an ~20-kDa product. Like expression of proNCAD at the cell surface, cleavage of the NCAD molecule at RIRSDR↓DK(189) renders the U251 cancer cells less adhesive to one another and more migratory. Our work modifies the present view on posttranslational processing and surface expression of classic cadherins and clarifies how NCAD possesses a range of adhesive potentials and plays a critical role in tumor progression.

14-3-3 proteins regulate a cell-intrinsic switch from sonic hedgehog-mediated commissural axon attraction to repulsion after midline crossing.

Axons must switch responsiveness to guidance cues during development for correct pathfinding. Sonic Hedgehog (Shh) attracts spinal cord commissural axons ventrally toward the floorplate. We show that after crossing the floorplate, commissural axons switch their response to Shh from attraction to repulsion, so that they are repelled anteriorly by a posterior-high/anterior-low Shh gradient along the longitudinal axis. This switch is recapitulated in vitro with dissociated commissural neurons as they age, indicating that the switch is intrinsic and time dependent. 14-3-3 protein inhibition converted Shh-mediated repulsion of aged dissociated neurons to attraction and prevented the correct anterior turn of postcrossing commissural axons in vivo, an effect mediated through PKA. Conversely, overexpression of 14-3-3 proteins was sufficient to drive the switch from Shh-mediated attraction to repulsion both in vitro and in vivo. Therefore, we identify a 14-3-3 protein-dependent mechanism for a cell-intrinsic temporal switch in the polarity of axon turning responses.