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International audience; Cumulative evidence has shown that a delicate balance between serine proteases and their inhibitors is crucial for normal functioning of several biological pathways. The importance of proteases and their inhibitors is well documented in several human diseases. Among them, the best documented are hemophilia B, a genetic deficiency of the serine protease coagulation factor IX and serpinophathies. Alpha-1-antitrypsin deficiency (MIM 107400), is associated with early-onset emphysema and liver disease, while hereditary angioedema (HANE; MIM 106100) is caused by mutations in the C1 inhibitor, a serpin involved in the regulation of the complement cascade. Recently, two human genetic diseases of the central nervous system have been related to mutations in components of extracellular proteolytic systems. Here, we review the recent advances in this field.

Summary Endocytosis of amyloid-β precursor protein (APP) is thought to represent the major source of substrate for the production of the amyloidogenic Aβ peptide by the β-secretase BACE1. The irreversible nature of proteolytic cleavage implies the existence of an efficient replenishment route for APP from its sites of synthesis to the cell surface. We recently found that APP exits the trans-Golgi network in intimate association with calsyntenin-1, a transmembrane cargo-docking protein for Kinesin-1-mediated vesicular transport. Here we characterized the function of calsyntenin-1 in neuronal APP transport using selective immunoisolation of intracellular trafficking organelles, immunocytochemistry, live-imaging, and RNAi. We found that APP is co-transported with calsyntenin-1 along axons to early endosomes in the central region of growth cones in carriers that exclude the α-secretase ADAM10. Intriguingly, calsyntenin-1/APP organelles contained BACE1, suggesting premature cleavage of APP along its anterograde path. However, we found that APP contained in calsyntenin-1/APP organelles was stable. We further analyzed vesicular trafficking of APP in cultured hippocampal neurons, in which calsyntenin-1 was reduced by RNAi. We found a markedly increased co-localization of APP and ADAM10 in axons and growth cones, along with increased proteolytic processing of APP and Aβ secretion in these neurons. This suggested that the reduced capacity for calsyntenin-1-dependent APP transport resulted in mis-sorting of APP into additional axonal carriers and, therefore, the premature encounter of unprotected APP with its ectodomain proteases. In combination, our results characterize calsyntenin-1/APP organelles as carriers for sheltered anterograde axonal transport of APP.

A key feature at excitatory synapses is the remodelling of dendritic spines, which in conjunction with receptor trafficking modifies the efficacy of neurotransmission. Here we investigated whether activation of cholinergic receptors, which can modulate synaptic plasticity, also mediates changes in dendritic spine structure. Using confocal time-lapse microscopy in mouse slice cultures we found that brief activation of muscarinic receptors induced the emergence of fine filopodia from spine heads in all CA1 pyramidal cells examined. This response was widespread occurring in 48% of imaged spines, appeared within minutes, was reversible, and was blocked by atropine. Electron microscopic analyses showed that the spine head filopodia (SHFs) extend along the presynaptic bouton. In addition, the decay time of miniature EPSCs was longer after application of the muscarinic acetylcholine receptor agonist methacholine (MCh). Both morphological and electrophysiological changes were reduced by preventing microtubule polymerization with nocodazole. This extension of SHFs during cholinergic receptor activation represents a novel structural form of subspine plasticity that may regulate synaptic properties by fine-tuning interactions between presynaptic boutons and dendritic spines.

Gray matter pathology is increasingly recognized as an important feature of multiple sclerosis (MS), but the nature of the immune response that targets the gray matter is poorly understood. Starting with a proteomics approach, we identified contactin-2/transiently expressed axonal glycoprotein 1 (TAG-1) as a candidate autoantigen recognized by both autoantibodies and T helper (Th) 1/Th17 T cells in MS patients. Contactin-2 and its rat homologue, TAG-1, are expressed by various neuronal populations and sequestered in the juxtaparanodal domain of myelinated axons both at the axonal and myelin sides. The pathogenic significance of these autoimmune responses was then explored in experimental autoimmune encephalitis models in the rat. Adoptive transfer of TAG-1–specific T cells induced encephalitis characterized by a preferential inflammation of gray matter of the spinal cord and cortex. Cotransfer of TAG-1–specific T cells with a myelin oligodendrocyte glycoprotein-specific mAb generated focal perivascular demyelinating lesions in the cortex and extensive demyelination in spinal cord gray and white matter. This study identifies contactin-2 as an autoantigen targeted by T cells and autoantibodies in MS. Our findings suggest that a contactin-2–specific T-cell response contributes to the development of gray matter pathology.

Kinesin motors are required for the export of membranous cargo from the trans-Golgi network (TGN), yet information about how kinesins are recruited to forming transport intermediates is sparse. Here we show that the Kinesin-1 docking protein calsyntenin-1 localizes to the TGN in vivo and directly and specifically recruits Kinesin-1 to Golgi/TGN membranes as well as to dynamic post-Golgi carriers. Overexpression of various calsyntenin chimeras and kinesin light chain 1 (KLC1) at high levels caused the formation of aberrant membrane stacks at the endoplasmic reticulum (ER) or the Golgi, disrupted overall Golgi structure and blocked exit of calsyntenin from the TGN. Intriguingly, this blockade of calsyntenin exit strongly and selectively impeded TGN exit of amyloid precursor protein (APP). Using live cell microscopy we found that calsyntenins exit the TGN in Kinesin-1-decorated tubular structures which may serve as carriers for calsyntenin-1-mediated post-TGN transport of APP. Abrogation of this pathway via virus-mediated knockdown of calsyntenin-1 expression in primary cultured neurons caused a marked elevation of APP C-terminal fragments. Together, these results indicate a role for calsyntenin-1 in Kinesin-1-dependent TGN exit and post-Golgi transport of APP-containing organelles and further suggest that distinct intracellular routes may exhibit different capacities for proteolytic processing of APP.

SummaryThe synaptic serine protease neurotrypsin is essential for cognitive function, as its deficiency in humans results in severe mental retardation. Recently, we demonstrated the activity-dependent release of neurotrypsin from presynaptic terminals and proteolytical cleavage of agrin at the synapse. Here we show that the activity-dependent formation of dendritic filopodia is abolished in hippocampal neurons from neurotrypsin-deficient mice. Administration of the neurotrypsin-dependent 22 kDa fragment of agrin rescues the filopodial response. Detailed analyses indicated that presynaptic action potential firing is necessary for the release of neurotrypsin, whereas postsynaptic NMDA receptor activation is necessary for the neurotrypsin-dependent cleavage of agrin. This contingency characterizes the neurotrypsin-agrin system as a coincidence detector of pre- and postsynaptic activation. As the resulting dendritic filopodia are thought to represent precursors of synapses, the neurotrypsin-dependent cleavage of agrin at the synapse may be instrumental for a Hebbian organization and remodeling of synaptic circuits in the CNS.

We have isolated and characterised the gene encoding the chicken axonal cell adhesion molecule axonin-1. This gene comprises 23 exons distributed over approximately 40 kb. Each of the six immunoglobulin-like domains and the four fibronectin-type-III-like domains of axonin-1 is encoded by two exons. The introns between two domains are exclusively phase I. Their exon/intron borders correspond to the domain borders of the protein, suggesting that the gene of axonin-1 had been generated by exon shuffling. Three transcripts with a length of 4.3 kb, 5 kb, and 8 kb are found, and we provide evidence that they result from alternative use of polyadenylation signals. In situ hybridization revealed co-localisation of these transcripts in time and space in the developing chicken retina. Several identical transcription initiation sites were found in retina, brain, and cerebellum by RNase protection assay and anchored polymerase chain reaction. By transfection of HeLa cells, rat PC-12 phaeochromocytoma cells, and chicken embryonic fibroblasts with serially truncated segments of the 5'-flanking region linked to a luciferase reporter gene, we have found that the sequence from -91 to +56 relative to the transcription initiation site is sufficient to promote efficient gene expression. Tissue-specific expression of the axonin-1 gene seems to be regulated in part by sequences more than 1 kb upstream of the transcription initiation site. As revealed by computer analysis, the sequence immediately upstream of exon 1 contains an AP-2 binding site, a tumor phorbol-ester-responsive element, and a homeodomain protein binding site, but no canonical TATA box. A second AP-2 binding site and a homeodomain protein binding site are located within exon 1.

Extracellular proteolysis plays an essential role in synaptic remodeling that is indispensable for cognitive function. The extracellular serine protease neurotrypsin was implicated in cognitive function because humans lacking a functional form of neurotrypsin suffer from severe mental retardation. By immunoelectron microscopy neurotrypsin has been localized to presynaptic terminals suggesting a local proteolytic function after its synaptic release. Here we studied axonal trafficking and synaptic exocytosis of neurotrypsin by live imaging of hippocampal neurons expressing neurotrypsin fused with enhanced green fluorescent protein or its pH sensitive variant superecliptic pHluorin. In differentiated neurons we identified neurotrypsin in mobile transport vesicles along axons and in both an intracellular and an extracellular pool at synapses. Short depolarization triggered rapid synaptic exocytosis of neurotrypsin. Once externalized neurotrypsin lingered at its synaptic release site for several minutes before it disappeared. Cell depolarization also enhanced synaptic capture of intracellular neurotrypsin transport vesicles and elevated synaptic activity increased both number and motility of mobile axonal neurotrypsin vesicles. We further observed trading of neurotrypsin vesicles between adjacent synapses. These activities may support the replenishment of neurotrypsin after activity induced synaptic exocytosis. Together the activity dependent recruitment of neurotrypsin to synapses and its exocytosis and transient persistence at its synaptic release site argue for a spatially and temporally restricted proteolytic action at the synapse. Thereby neurotrypsin may play a role in activity dependent remodeling of the synaptic circuitry that is key to adaptive synaptic changes in the context of cognitive functions such as learning and memory. Copyright © 2008 Society for Neuroscience.

Calsyntenins are members of the cadherin superfamily of cell adhesion molecules. They are present in postsynaptic membranes of excitatory neurons and in vesicles in transit to neuronal growth cones. In the current study, calsyntenin-1 (CST-1) and calsyntenin-3 (CST-3) were identified by mass spectrometric analysis (LC-MS/MS) of integral membrane proteins from highly enriched secretory granule preparations from bovine anterior pituitary gland. Immunofluorescence microscopy on thin frozen sections of rat pituitary revealed that CST-1 was present only in gonadotropes where it colocalized with follicle-stimulating hormone in secretory granules. In contrast, CST-3 was present not only in gonadotrope secretory granules but also in those of somatotropes and thyrotropes. Neither protein was detected in mammatropes. In addition, CST-1 was also localized to the glucagon-containing secretory granules of α cells in the pancreatic islets of Langerhans. Results indicate that calsyntenins function outside the nervous system and potentially are modulators of endocrine function. (J Histochem Cytochem 56:381–388, 2008)

The balance between tissue-type plasminogen activator (tPA) and one of its inhibitors, neuroserpin, has crucial roles in the central nervous system, including the control of neuronal migration, neuronal plasticity and neuronal death. In the present study, we demonstrate that the activation of the transforming growth factor-β (TGFβ)-related BMPR-IB (also known as BMPR1B and Alk6)- and Smad5-dependent signalling pathways controls neuroserpin transcription. Accordingly, we demonstrate for the first time that anti-Mullerian hormone (AMH), a member of the TGFβ family, promotes the expression of neuroserpin in cultured neurons but not in astrocytes. The relevance of these findings is confirmed by the presence of both AMH and AMH type-II receptor (AMHR-II) in brain tissues, and is supported by the observation of reduced levels of neuroserpin in the brain of AMHR-II-deficient mice. Interestingly, as previously demonstrated for neuroserpin, AMH protects neurons against N-methyl-D-aspartate (NMDA)-mediated excitotoxicity both in vitro and in vivo. This study demonstrates the existence of an AMH-dependent signalling pathway in the brain leading to an overexpression of the serine-protease inhibitor, neuroserpin, and neuronal survival.

Familial encephalopathy with neuroserpin inclusion bodies is a recently described neurodegenerative disease that is responsible for progressive myoclonic epilepsy or presenile dementia. In a French family with the S52R mutation of the neuroserpin gene, progressive myoclonic epilepsy was associated with a frontal syndrome. The typical cerebral inclusions (Collins bodies) were abundant in the frontal cortex and in the head of the caudate nucleus but spared the cerebellum.

Intracellular protein deposition due to aggregation caused by conformational alteration is the hallmark of a number of neurodegenerative disorders, including Parkinson's disease, tauopathies, Huntington's disease, and familial encephalopathy with neuroserpin inclusion bodies. The latter is an autosomal dominant disorder caused by point mutations in neuroserpin resulting in its destabilization. Mutant neuroserpin polymerizes and forms intracellular aggregates that eventually lead to neurodegeneration. We generated genetically modified mice expressing the late-onset S49P-Syracuse or the early-onset S52R-Portland mutation of neuroserpin in central nervous system neurons. Mice exhibited morphological, biochemical, and clinical features resembling those found in the human disease. Analysis of brains revealed large intraneuronal inclusions composed exclusively of mutant neuroserpin, accumulating long before the development of clinical symptoms in a time-dependent manner. Clinical symptoms and amount of neuroserpin inclusions correlated with the predicted instability of the protein. The presence of inclusion bodies in subclinical mice indicates that in humans the prevalence of the disease could be higher than anticipated. In addition to shedding light on the pathophysiology of the human disorder, these mice provide an excellent model to study mechanisms of neurodegeneration or establish novel therapies for familial encephalopathy with neuroserpin inclusion bodies and other neurodegenerative diseases with intracellular protein deposition.

Considering its brain-specific expression, neuroserpin (NS), a potent inhibitor of tissue-type plasminogen activator (tPA), might be a good therapeutic target to limit the pro-excitotoxic effects of tPA within the cerebral parenchyma, without affecting the benefit from thrombolysis in stroke patients. Here, we aimed at determining the mechanisms of action responsible for the previously reported neuroprotective activity of NS in rodent experimental cerebral ischemia. First, we show in vivo that exogenous NS protects the cortex and the striatum against NMDA-induced injury. Then, the cellular mechanisms of this neuroprotection were investigated in primary cultures of cortical neurons. We show that NS fails to prevent serum deprivation-induced apoptotic neuronal death, while it selectively prevents NMDA- but not AMPA-induced excitotoxicity. This beneficial effect is associated to a decrease in NMDA receptor-mediated intracellular calcium influx. Altogether, these data suggest that an overexpression of neuroserpin in the brain parenchyma might limit the deleterious effect of tPA on NMDA receptor-mediated neuronal death, which occurs following experimental ischemia.

Neuroserpin is a neural serpin that inhibits the extracellular protease tissue-type plasminogen activator (tPA). We have generated neuroserpin-deficient mice which are viable and healthy. Zymographic analysis of neuroserpin-deficient brain showed unaltered tPA activity, suggesting that other inhibitors contribute to the regulation of tPA and may compensate for the defect. Analysis of explorative behavior revealed selective reduction of locomotor activity in novel environments, an anxiety-like response on the O-maze, and a neophobic response to novel objects. Mice overexpressing neuroserpin under the control of the Thy1.2 promoter are known to have a reduced brain tPA activity. They showed reduced center exploration in the open-field test and, like neuroserpin-deficient mice, a neophobic phenotype in the novel object test. Our results implicate neuroserpin in the regulation of emotional behavior through a mechanism that is at least in part independent of tPA activity. They are the first evidence for a role of protease inhibitors in mood regulation.

The neuronal serine protease inhibitor neuroserpin is widely expressed in the developing and adult brain. In the neocortex, neuroserpin is displayed particularly during the period of synaptic specification and refinement, indicating a role as modulator of extracellular proteolytic processes. The synaptic connections of the visual system of the mouse are shaped during early postnatal life by an activity-dependent process. We have studied the expression of the neuronal serine protease inhibitor neuroserpin in the primary visual cortex of mice from birth until the end of the critical period by means of reverse transcription polymerase chain reaction and in situ hybridization. The localization and the level of expression were constant throughout this period. Monocular deprivation with an eyelid sutured induced a decrease in neuroserpin expression in neurons of area 17 after 1 week of deprivation, the decrease being more pronounced on the side contralateral to the closed eye. The expression of neuroserpin in the visual cortex during the critical period and its decrease in parallel to the refinement of synaptic contacts after visual deprivation suggests a regulative role of neuroserpin on these processes.

Calsyntenin 1 is a transmembrane cargo docking protein important for kinesin 1 mediated fast transport of membrane bound organelles that exhibits peak expression levels at postnatal day 7. However its neuronal function during postnatal development remains unknown. We generated a knock out mouse to characterize calsyntenin 1 function in juvenile mice. In the absence of calsyntenin 1 synaptic transmission was depressed. To address the mechanism evoked EPSPs were analyzed revealing a greater proportion of synaptic GluN2B subunit containing receptors typical for less mature synapses. This imbalance was due to a disruption in calsyntenin 1 mediated dendritic transport of NMDA receptor subunits. As a consequence of increased expression of GluN2B subunits NMDA receptor dependent LTP was enhanced at Schaffer collateral CA1 pyramidal cell synapses. Interestingly these defects were accompanied by a decrease in dendritic arborization and increased proportions of immature filopodia like dendritic protrusions at the expense of thin type dendritic spines in CA1 pyramidal cells. Thus these results highlight a key role for calsyntenin 1 in the transport of NMDA receptors to synaptic targets which is necessary for the maturation of neuronal circuits during early development. © 2014 the authors.

TAG-1 is a member of the immunoglobulin superfamily of cell adhesion molecules thought to play important roles in neuronal differentiation and the establishment of connectivity during brain development. Because these are processes also affected by hypothyroidism, we studied the effects of thyroid hormone deprivation and administration on TAG-1 expression in the developing rat brain. By in situ hybridization, immunohistochemistry and Western blotting we found that TAG-1 RNA and protein levels are upregulated in the hypothyroid brain. From embryonic day 20 to postnatal day (P) 15, elevated TAG-1 RNA was found in several areas including the cerebral cortex, hippocampus and olfactory bulb. In agreement with this, TAG-1 protein was overexpressed in the major fibre tracts arising from these structures, including the corpus callosum, anterior and hippocampal commissures and lateral olfactory tract. A similar overexpression of TAG-1 by hypothyroidism was detected in the cerebellum, but starting only at P15. In all cases, elevation of TAG-1 RNA and protein expression could be reversed by thyroid hormone treatment. These results show that the deregulation of TAG-1 might contribute to the alterations caused by the lack of thyroid hormone during brain development.

The neural cell adhesion molecules axonin-1 and NgCAM have been genetically engineered and covalently immobilized on glass and silicon oxide surfaces in their correct orientation. Surfaces treated with these adhesion molecules were used as substrates for culturing dorsal root ganglion neurons. The cleft between the neuron cell membrane and the surface was determined using fluorescence interference contrast (FLIC) microscopy. For comparison, cell--material distances on laminin, RGDC, polylysine and amino-terminated surfaces were measured. When the neurons grow on axonin-1 the cell--surface distance is at a minimum (37 nm) probably because the glycocalyx hinders a closer contact. A selective treatment of extracellular electrodes with axonin-1 could be used to improve the cell-material contact and thus increase extracellularly recorded signals.

We have determined the crystal structure of the ligand binding fragment of the neural cell adhesion molecule axonin-1/TAG-1 comprising the first four immunoglobulin (Ig) domains. The overall structure of axonin-1Ig1–4 is U-shaped due to contacts between domains 1 and 4 and domains 2 and 3. In the crystals, these molecules are aligned in a string with adjacent molecules oriented in an anti-parallel fashion and their C termini perpendicular to the string. This arrangement suggests that cell adhesion by homophilic axonin-1 interaction occurs by the formation of a linear zipper-like array in which the axonin-1 molecules are alternately provided by the two apposed membranes. In accordance with this model, mutations in a loop critical for the formation of the zipper resulted in the loss of the homophilic binding capacity of axonin-1.

Conformational diseases result from cellular dysfunctions induced by aberrant aggregation of proteins. They are caused either by excess of secretion of a normal protein, or more frequently, by mutation in a protein, as in prion diseases. Recently, one of them, familial encephalopathy with neuroserpin inclusion bodies (FENIB, OMIM #604218), an autosomal dominant dementia, was recognized. It is caused by mutations in PI12 (proteinase inhibitor 12, SERPINI1 or neuroserpin, OMIM #602445), a neuron-specific serine proteinase inhibitor (serpin).1,2 Neuroserpin was first identified in culture medium of chicken axons, then in human neurons.3 It plays roles in synapses and vessel permeability, and is known to be associated with learning, memory, and behavior. It belongs to the serpin super-family, members of which display at least 30% amino acid sequence homology with their archetype, alpha-1-antitrypsin. Mutated neuroserpin progressively polymerizes in neuronal endoplasmic reticulum, inducing cognitive impairment and sometimes myoclonic epilepsy. Neuropathology is characterized by neuronal intra-cytoplasmic rounded inclusions, homogeneously pale to intensely pink with eosin, and PAS positive after diastase treatment (Collins bodies). Four different mutations have been described in six families affected by FENIB: two in exon 2 (S49P and S52R) and two in exon 9 (H338R and G392E). Known mutations in serpins are localized in the mobile regions of the molecule (exon 2 and 9). They result in proteins …

Designed networks of neurons are potentially very useful to investigate neural activities. Using photolithography microgrooves suited in size for single neurons have been produced on glass chips. Two conducting gold lanes ending in each microgroove allow extracelluar stimulation of the neurons and recording of their activity. A cell adhesive surface was created by functionalization of glass with the adhesion peptide RGDC. In addition, in order to optimize the contact of the neuronal cell membrane to the electrode surface axonin-1, a specific neural adhesion protein was used. A recombinant form of axonin-1 was produced and immobilized in a correct orientation on protected gold surfaces through a C-terminal cysteine residue. Neurite outgrowth of neurons cultured on chips derivatized with RGDC or axonin-1 were compared. The developed materials and methods represent a first step towards establishing designed functionalized glass surfaces for neurophysiological investigations.

Neuroserpin is a novel serine protease inhibitor of the serpin family. It has been reported as a 55-kDa glycoprotein that is secreted from the axons of cultured central and peripheral nervous system neurons. In situ hybridization and Northern blot analyses at different developmental stages of the chicken revealed that neuroserpin is predominantly expressed in the nervous system and that most cells expressing neuroserpin can be qualified as bona fide neurons. We have isolated the full-length cDNA for human neuroserpin from a fetal retina cDNA library. The open reading frame of the cDNA of human neuroserpin, like that of its chicken counterpart, encodes a protein of 410 amino acids. The human and the chicken neuroserpin exhibit an amino acid sequence identity of 80%. Northern blot analysis of human organs demonstrated predominant expression of neuroserpin in the brain. By fluorescence in situ hybridization the human neuroserpin gene (HGMW-approved symbol PI12) was mapped to region q26 of chromosome 3.

By homologous recombination, a first-generation adenovirus-based gene transfer vector, AdCMVax-1, was constructed as a means of manipulating the expression level of the axonal cell adhesion molecule axonin-1 in neurons and glial cells. AdCMVax-1 harbours the entire coding region of the chicken axonin-1 cDNA under the transcriptional control of the Cytomegalovirus enhancer/promoter in the early-region 1 of the viral genome. Characterization of AdCMVax-1 in vitro revealed highly efficient gene transfer and expression of recombinant axonin-1 in neurons and glial cells of dissociated rat dorsal root ganglia. Similar to its native counterpart, virus-derived axonin-1 was detected on the cell body, neurites, and growth cones of transduced neurons, occurred in a secreted and membrane-associated form, and could be cleaved from the membrane with phosphatidylinositol-specific phospholipase C. Functional characterization of recombinant axonin-1 revealed the same binding properties as previously reported for native axonin-1 isolated from the vitreous fluid of chicken embryos. In vivo gene transfer was studied by stereotactic injection of AdCMVax-1 in the dentate gyrus of the hippocampus and the facial nucleus in the brainstem of adult Wistar rats and revealed high level expression of recombinant axonin-1 in a subset of hippocampal neurons and motor neurons in the facial nucleus.

Neural cell adhesion molecules of the immunoglobulin/fibronectin type III family on axons have been implicated in promotion of neurite outgrowth, fasciculation, and the mediation of specific cell adhesion. The present study demonstrates that two of these molecules on dorsal root ganglion neurons are associated with distinct protein kinases, axonin-1 with the src-related nonreceptor tyrosine kinase fyn and NgCAM with a casein kinase II-related activity and a serine/ threonine kinase related to S6 kinase. When neurites grew without contacts involving axonin-1 and NgCAM, strong fyn kinase activity was associated with axonin-1, whereas the NgCAM-associated kinase activities were low. Clustering of axonin-1 with NgCAM induced by the formation of cell-cell contacts correlated with a reduction of the axonin-1-associated fyn activity and an increased phosphorylation of NgCAM by the associated casein kinase II-related activity. Thus, axonin-1 and NgCAM trigger distinctive intracellular signals during in vitro differentiation depending on their state of association.

We have identified and chromatographically purified an axonally secreted glycoprotein of CNS and PNS neurons. Several peptides derived from it were microsequenced. Based on these sequences, a fragment of the corresponding cDNA was amplified and used as a probe to isolate a full length cDNA from a chicken brain cDNA library. Because the deduced amino acid sequence qualified the protein as a novel member of the serpin family of serine protease inhibitors, we called it neuroserpin. Analysis of the primary structural features further characterized neuroserpin as a heparin-independent, functional inhibitor of a trypsin-like serine protease. In situ hybridization revealed a predominantly neuronal expression during the late stages of neurogenesis and in the adult brain in regions which exhibit synaptic plasticity. Thus, neuroserpin might function as an axonally secreted regulator of the local extracellular proteolysis involved in the reorganization of the synaptic connectivity during development and synapse plasticity in the adult.

The human TAX-1 gene (HGMW-approved symbol TAX1) is located on chromosome 1 (1q32.1) and encodes the neuronal cell adhesion molecule TAG-1/axonin-1. The gene product, termed TAG-1 in the rat and axonin-1 in the chicken, is composed of six immunoglobulin (Ig)-like and four fibronectin type III (FNIII)-like domains. It is found predominantly on the axons of particular nerve fiber tracts during neural development, and it has been demonstrated to function as a potent substratum for neurite outgrowth in vitro. Here we report the cloning and structural characterization of the TAX-1 gene. The transcribed region of the TAX-1 gene extends over about 40 kb. Like its chicken homologue, the human TAX-1 gene consists of 23 exons. Two GT/CA microsatellites were localized in the first intron; a polymorphism was found for one of them. Reporter gene analysis with serially truncated fragments of the 5'-flanking region indicated that a 164-bp fragment located immediately upstream of the putative transcription initiation site was sufficient to function as a basal promoter.

Asarta alpicolella Lebensraum: Ich kenne die Art nur von einem Lebensraum am Fusse des Glacier de Tsanfleuron (Abb. 4) im Wallis, 2380 m, 26.07. 2008. Hier kommt die Art in den alpinen Schutt- und Ger��llfluren mit wenig Vegetation vor. 1800��� 2400 m, zum Teil in den gletschernahen Gebieten, die vor 50 Jahren noch vergletschert waren. Flugzeit: Asarta alpicolella ist eine Art, die kurz nach der Schneeschmelze erscheint und deshalb lokal grosse Unterschiede in der Flugzeit aufweist. Funddaten: 18. Mai bis 25. August. Verbreitung: Die Art ist sehr lokal in den Franz��sischen und Schweizer Alpen verbreitet. Wallis: Glacier de Tsanfleuron (2008, 2009 und 2010). Varenalp, Ende Juni 1906 durch Rougemont beobachtet. Graub��nden: Il Fuorn, 1800 m, 18.05. 1927 (Thomann); Alp Murt��r, 2400 m, 25.08. 1926 (Thomann); Munt Baselgia 2400 ��� 2500m, 15.07. 1951 (Thomann); Ofenpass 2100m, 0 4.06. 2010, 23.05.2011; Zernez, Val Laschadura, 2100m, 23.05. 2011 (J��rg Schmid). In Frankreich aus den Departementen Hautes- Alpes und Alpes-de-Haute-Provence gemeldet., Published as part of Peter Sonderegger, 2012, Asartodes monspesulalis (Duponchel, 1834), Asarta aethiopella (Duponchel, 1837) und Asarta alpicolella (Zeller, 1839) (Lepidoptera: Pyralidae) aus der Schweiz, pp. 35-45 in Entomo Helvetica 5 on page 43, DOI: 10.5281/zenodo.269121

The quantification of synapses in neuronal cultures is essential in studies of the molecular mechanisms underlying synaptogenesis and synaptic plasticity. Conventional counting of synapses based on morphological or immunocytochemical criteria is extremely work-intensive. We developed a fully automated method which quantifies synaptic elements and complete synapses based on immunocytochemistry. Pre- and postsynaptic elements are detected by their corresponding fluorescence signals and their proximity to dendrites. Synapses are defined as the combination of a pre- and postsynaptic element within a given distance. The analysis is performed in three dimensions and all parameters required for quantification can be easily adjusted by a graphical user interface. The integrated batch processing enables the analysis of large datasets without any further user interaction and is therefore efficient and timesaving. The potential of this method was demonstrated by an extensive quantification of synapses in neuronal cultures from DIV 7 to DIV 21. The method can be applied to all datasets containing a pre- and postsynaptic labeling plus a dendritic or cell surface marker.

Etiology and pathogenesis of sarcope- nia, the progressive decline in skeletal muscle mass and strength that occurs with aging, are still poorly under- stood. We recently found that overexpression of the neural serine protease neurotrypsin in motoneurons resulted in the degeneration of their neuromuscular junctions (NMJ) within days. Therefore, we wondered whether neurotrypsin-dependent NMJ degeneration also affected the structure and function of the skeletal muscles. Using histological and functional analyses of neurotrypsin-overexpressing and neurotrypsin-defi- cient mice, we found that overexpression of neurotryp- sin in motoneurons installed the full sarcopenia pheno- type in young adult mice. Characteristic muscular alterations included a reduced number of muscle fi- bers, increased heterogeneity of fiber thickness, more centralized nuclei, fiber-type grouping, and an in- creased proportion of type I fibers. As in age-depen- dent sarcopenia, excessive fragmentation of the NMJ accompanied the muscular alterations. These results suggested the destabilization of the NMJ through pro- teolytic cleavage of agrin at the onset of a pathogenic pathway ending in sarcopenia. Studies of neurotrypsin- deficient and agrin-overexpressing mice revealed that old-age sarcopenia also develops without neurotrypsin and is not prevented by elevated levels of agrin. Our results define neurotrypsin- and age-dependent sar- copenia as the common final outcome of 2 etiologically distinct entities.—Butikofer, L., Zurlinden, A., Bolliger, M. F., Kunz, B., Sonderegger, P. Destabilization of the neuromuscular junction by proteolytic cleavage of agrin results in precocious sarcopenia. FASEB J. 25, 000 - 000 (2011). www.fasebj.org

Protein expression patterns of morphologically different cloned capillary endothelial cells from porcine and murine brain cortices were examined. Type I cells, grown in medium containing heparin and endothelial cell growth factor (ECGF), exhibited a polygonal, cobblestone appearance and appeared to replicate the cells of the blood-brain barrier endothelium. Type II cells, grown in medium without heparin and ECGF, were elongated and appeared to replicate capillaries in central nervous system tissue. Cells of both phenotypes stained positive by the specific endothelial cell marker Bandeiraea simplicifolia lectin. The expression of alpha smooth-muscle actin (mRNA and protein) was taken as a marker for type II cells. By use of 2-D gel images and the GELLAB II system, a data base was created revealing that two proteins (90 kDa, pI 5.1, and 35 kDa, pI 5.7) were exclusively expressed in type I cells. Furthermore, the synergistic action of ECGF and heparin in respect to the phenotypic determination of cerebral endothelial cells was demonstrated.

The C-terminal 95 kDa fragment of some isoforms of vertebrate agrins is sufficient to induce clustering of acetylcholine receptors but despite two decades of intense agrin research very little is known about the function of the other isoforms and the function of the larger, N-terminal part of agrins that is common to all isoforms. Since the N-terminal part of agrins contains several follistatin-domains, a domain type that is frequently implicated in binding TGFbetas, we have explored the interaction of the N-terminal part of rat agrin (Agrin-Nterm) with members of the TGFbeta family using surface plasmon resonance spectroscopy and reporter assays. Here we show that agrin binds BMP2, BMP4 and TGFbeta1 with relatively high affinity, the K(D) values of the interactions calculated from SPR experiments fall in the 10(-8) M-10(-7) M range. In reporter assays Agrin-Nterm inhibited the activities of BMP2 and BMP4, half maximal inhibition being achieved at approximately 5x10(-7) M. Paradoxically, in the case of TGFbeta1 Agrin N-term caused a slight increase in activity in reporter assays. Our finding that agrin binds members of the TGFbeta family may have important implications for the role of these growth factors in the regulation of synaptogenesis as well as for the role of agrin isoforms that are unable to induce clustering of acetylcholine receptors. We suggest that binding of these TGFbeta family members to agrin may have a dual function: agrin may serve as a reservoir for these growth factors and may also inhibit their growth promoting activity. Based on analysis of the evolutionary history of agrin we suggest that agrin's growth factor binding function is more ancient than its involvement in acetylcholine receptor clustering.

Kinesin motors play crucial roles in the delivery of membranous cargo to its destination and thus for the establishment and maintenance of cellular polarization. Recently, calsyntenin-1 was identified as a cargo-docking protein for Kinesin-1-mediated axonal transport of tubulovesicular organelles along axons of central nervous system neurons. To further define the function of calsyntenin-1, we immunoisolated calsyntenin-1 organelles from murine brain homogenates and determined their proteome by MS. We found that calsyntenin-1 organelles are endowed with components of the endosomal trafficking machinery and contained the β-amyloid precursor protein (APP). Detailed biochemical analyses of calsyntenin-1 immunoisolates in conjunction with immunocytochemical colocalization studies with cultured hippocampal neurons, using endosomal marker proteins for distinct subcompartments of the endosomal pathways, indicated that neuronal axons contain at least two distinct, nonoverlapping calsyntenin-1-containing transport packages: one characterized as early-endosomal, APP positive, the other as recycling-endosomal, APP negative. We postulate that calsyntenin-1 acts as a general mediator of anterograde axonal transportation of endosomal vesicles. In this role, calsyntenin-1 may actively contribute to axonal growth and pathfinding in the developing as well as to the maintenance of neuronal polarity in the adult nervous system; further, it may actively contribute to the stabilization of APP during its anterograde axonal trajectory.

Recent studies have deepened our understanding of multiple mechanisms by which extracellular matrix (ECM) molecules regulate various aspects of synaptic plasticity and have strengthened a link between the ECM and learning and memory. New findings also support the view that the ECM is important for homeostatic processes, such as scaling of synaptic responses, metaplasticity and stabilization of synaptic connectivity. Activity-dependent modification of the ECM affects the formation of dendritic filopodia and the growth of dendritic spines. Thus, the ECM has a dual role as a promoter of structural and functional plasticity and as a degradable stabilizer of neural microcircuits. Both of these aspects are likely to be important for mental health.

During the initial stage of neuromuscular junction (NMJ) formation, nerve-derived agrin cooperates with muscle-autonomous mechanisms in the organization and stabilization of a plaque-like postsynaptic specialization at the site of nerve–muscle contact. Subsequent NMJ maturation to the characteristic pretzel-like appearance requires extensive structural reorganization. We found that the progress of plaque-to-pretzel maturation is regulated by agrin. Excessive cleavage of agrin via transgenic overexpression of an agrin-cleaving protease, neurotrypsin, in motoneurons resulted in excessive reorganizational activity of the NMJs, leading to rapid dispersal of the synaptic specialization. By contrast, expression of cleavage-resistant agrin in motoneurons slowed down NMJ remodeling and delayed NMJ maturation. Neurotrypsin, which is the sole agrin-cleaving protease in the CNS, was excluded as the physiological agrin-cleaving protease at the NMJ, because NMJ maturation was normal in neurotrypsin-deficient mice. Together, our analyses characterize agrin cleavage at its proteolytic α- and β-sites by an as-yet-unspecified protease as a regulatory access for relieving the agrin-dependent constraint on endplate reorganization during NMJ maturation.

T H~ mechanism of formation of connections between neurons by axons and dendrites is one of the crucial and interesting aspects of neural development. To reach their intended targets, extending axons depend on a complex pattern of guidance cues present in their local environment (Dodd and Jessell, 1988). Several distinct mechanisms have been postulated for such guidance functions. (a) Neurite growth-promoting proteins exposed either at the surface of neuronal or nonneuronal cells, or localized in the extracellular matrix (ECM) ~ provide pathways along which axons can preferentially elongate (Rathjen and Jessell, 1991; Grumet, 1991; Reichardt and Tomaselli, 1991; Takeichi, 1991). (b) Repulsive molecules may block axonal extension into certain territories; e.g., by contact inhibition of the mobility of growth cones. In spatial and temporal concert with molecules favorable to axon growth, such molecules may be involved in guidance (Caroni and Schwab, 1988; Stahl et al., 1990; Raper and Kapfhammer, 1990; Keynes et al., 1991; Chiquet-Ehrismann, 1991). (c) Soluble chemoattractants, released by intermediate or final cellular targets, might guide axons by the establishment of concentration gradients (Lumsden and Davies, 1986; Tessier-Lavigne et al., 1988; Heffner et al., 1990; Placzek et al., 1990). Axon growth on favorable substrata involves specific interactions with cell surface glycoproteins. Three major structural classes of axonal membrane proteins have been described so far to undergo specific interactions with neurite growth-promoting ECM or cell surface molecules, and thus, have been implicated as axonal receptors in the growthgenerating machinery: (a) integrins of the 13~ and/33 subfamilies (Reichardt and Tomaselli, 1991), (b) cadherins (Takeichi, 1991), and (c) axonal membrane proteins of the Ig superfamily (Rathjen and Jessell, 1991; Grumet, 1991; Hortsch and Goodman, 1991). Ig-like proteins concentrated on axons can be further classified by the expression of both Igand fibronectin type III (FNIII)-related repeats (referred to as Ig/FNIII-like proteins in the following), or Ig-like domains alone. According to the overall domain organization and primary sequence, the group of Ig/FNIII-like proteins in vertebrates can be further divided into subgroups (Fig. 1).

Axonin-1 is an axon-associated cell adhesion molecule with dualistic expression, one form being glycophosphatidylinositol-anchored to the axonal membrane, the other secreted from axons in a soluble form. When presented as a substratum for neuronal cultures it strongly promotes neurite outgrowth from chicken embryonic dorsal root ganglia neurons. In this study, the axon-associated cell adhesion molecule G4, which is identical with Ng-CAM and 8D9, and homologous or closely related to L1 of the mouse and NILE of the rat, was investigated with respect to a receptor function for axonin-1. Using fluorescent microspheres with covalently coupled axonin-1 or L1(G4) at their surface we showed that these proteins bind to each other. Within the sensitivity of this microsphere assay, no interaction of axonin-1 with itself could be detected. Axonin-1-coated microspheres also bound to the neurites of cultured dorsal root ganglia neurons. This interaction was exclusively mediated by L1(G4), as indicated by complete binding suppression by monovalent anti-L1(G4) antibodies. The interaction between neuritic L1(G4) and immobilized axonin-1 was found to mediate the promotion of neurite growth on axonin-1, as evidenced by the virtually complete arrest of neurite outgrowth in the presence of anti-L1(G4) antibodies. Convincing evidence has recently been presented that neurite growth on L1(8D9) is mediated by the homophilic binding of neuritic L1(G4) (1989. Neuron. 2: 1597-1603). Thus, both L1(G4)- and axonin-1-expressing axons may serve as "substrate pathways" for the guidance of following axons expressing L1(G4) into their target area. Conceivably, differences in the concentration of axonin-1 and L1(G4), and/or modulatory influences on their specific binding parameters in leading pathways and following axons could represent elements in the control of axonal pathway selection.

Axonin-1 is a neuronal glycoprotein occurring both as a membrane-bound and a secreted form. Membrane-bound axonin-1 is predominantly located in membranes of developing nerve fiber tracts and has recently been characterized as a cell adhesion molecule; the soluble form is secreted from axons and accumulates in the cerebrospinal fluid and the vitreous fluid of the eye. In the present study, we addressed the question as to whether secreted axonin-1 was released in a functionally competent form and we found that it strongly promotes neurite outgrowth when presented to neurons as an immobilized substratum. Neurite lengths elaborated by embryonic dorsal root ganglia neurons on axonin-1 were similar to those on the established neurite-promoting substrata L1 and laminin. Fab fragments of axonin-1 antibodies completely inhibited neurite growth on axonin-1, but not on other substrata. In soluble form, axonin-1 had an anti-adhesive effect, as revealed by perturbation of neurite fasciculation. In view of their structural similarity, we conclude that secreted and membrane-bound axonin-1 interact with the same growth-promoting neuritic receptor. The fact that secreted axonin-1 is functionally active, together with our previous findings that it is secreted from an internal cellular pool, suggests a functional dualism between membrane-bound and secreted axonin-1 at the site of secretion, which is most likely the growth cone. The secretion of adhesion molecules could represent a powerful and rapidly acting regulatory element of growth cone-neurite interactions in the control of neurite elongation, pathway selection, and possibly target recognition.

The synaptic serine protease neurotrypsin is considered to be essential for the establishment and maintenance of cognitive brain functions, because humans lacking functional neurotrypsin suffer from severe mental retardation. Neurotrypsin cleaves agrin at two homologous sites, liberating a 90-kDa and a C-terminal 22-kDa fragment from the N-terminal moiety of agrin. Morphological analyses indicate that neurotrypsin is contained in presynaptic terminals and externalized in association with synaptic activity, while agrin is localized to the extracellular space at or in the vicinity of the synapse. Here, we present a detailed biochemical analysis of neurotrypsin-mediated agrin cleavage in the murine brain. In brain homogenates, we found that neurotrypsin exclusively cleaves glycanated variants of agrin. Studies with isolated synaptosomes obtained by subcellular fractionation from brains of wild-type and neurotrypsin-overexpressing mice revealed that neurotrypsin-dependent cleavage of agrin was concentrated at synapses, where the most heavily glycanated variants of agrin predominate. Because agrin has been shown to play an important role in the formation and the maintenance of excitatory synapses in the central nervous system, its local cleavage at the synapse implicates the neurotrypsin/agrin system in the regulation of adaptive reorganizations of the synaptic circuitry in the context of cognitive functions, such as learning and memory.

An increasing number of studies indicate that serine proteases play an important role in structural plasticity associated with learning and memory formation. Neurotrypsin is a multidomain serine protease located at the presynaptic terminal of neurons. It is thought to be crucial for cognitive brain functions. A deletion in the neurotrypsin gene causes severe mental retardation in humans. For a biochemical characterization we produced murine neurotrypsin recombinantly in a eukaryotic expression system using myeloma cells. From the culture medium we purified neurotrypsin using heparin hydrophobic interaction and immobilized metal affinity chromatography. For an enzymological characterization two fragments of agrin containing the natural cleavages sites of neurotrypsin were used as substrates. The highest catalytic activity of neurotrypsin was observed in the pH range between 7.0 and 8.5. Calcium ions were required for neurotrypsin activity and an ionic strength exceeding 500 mM decreased substrate cleavage. Site specific mutations of the amino acids flanking the scissile bonds showed that cleavage is highly specific and requires a basic amino acid preceded by a glutamate residue on the N terminal side of the scissile bond. This sequence requirement argues for a unique substrate binding pocket of neurotrypsin. This observation was further substantiated by the fact that almost all tested serine protease inhibitors except dichloroisocoumarin and PMSF did not affect neurotrypsin activity. (C) 2008 Elsevier Inc. All rights reserved.

The synaptic serine protease neurotrypsin is thought to be important for adaptive synaptic processes required for cognitive functions, because humans deficient in neurotrypsin suffer from severe mental retardation. In the present study, we describe the biochemical characterization of neurotrypsin and its so far unique substrate agrin. In cell culture experiment as well as in neurotrypsin-deficient mice, we showed that agrin cleavage depends on neurotrypsin and occurs at two conserved sites. Neurotrypsin and agrin were expressed recombinantly, purified, and assayed in vitro. A catalytic efficiency of 1.3 x 10(4) M(-1) x s(-1) was determined. Neurotrypsin activity was shown to depend on calcium with an optimal activity in the pH range of 7-8.5. Mutagenesis analysis of the amino acids flanking the scissile bonds showed that cleavage is highly specific due to the unique substrate recognition pocket of neurotrypsin at the active site. The C-terminal agrin fragment released after cleavage has recently been identified as an inactivating ligand of the Na+/K+-ATPase at CNS synapses, and its binding has been demonstrated to regulate presynaptic excitability. Therefore, dysregulation of agrin processing is a good candidate for a pathogenetic mechanism underlying mental retardation. In turn, these results may also shed light on mechanisms involved in cognitive functions.

Didelot et al . (Reports, 11 August 2006, p. 851) claimed that Drosophila Tequila (Teq) and human neurotrypsin are orthologs and concluded that deficient long-term memory after Teq inactivation indicates that neurotrypsin plays its essential role for human cognitive functions through a similar mechanism. Our analyses suggest that Teq and neurotrypsin are not orthologous, leading us to question their equivalent roles in higher brain function.

Human TAG-1 is a neural cell adhesion molecule that is crucial for the development of the nervous system during embryogenesis. It consists of six immunoglobulin-like and four fibronectin III-like domains and is anchored to the membrane by glycosylphosphatidylinositol. Herein we present the crystal structure of the four N-terminal immunoglobulin-like domains of TAG-1 (TAG-1(Ig1-4)), known to be important in heterophilic and homophilic macromolecular interactions. The contacts of neighboring molecules within the crystal were investigated. A comparison with the structure of the chicken ortholog resulted in an alternative mode for the molecular mechanism of homophilic TAG-1 interaction. This mode of TAG-1 homophilic interaction is based on dimer formation rather than formation of a molecular zipper as proposed for the chicken ortholog.

We identified a direct interaction between the neuronal transmembrane protein calsyntenin-1 and the light chain of Kinesin-1 (KLC1). GST pulldowns demonstrated that two highly conserved segments in the cytoplasmic domain of calsyntenin-1 mediate binding to the tetratricopeptide repeats of KLC1. A complex containing calsyntenin-1 and the Kinesin-1 motor was isolated from developing mouse brain and immunoelectron microscopy located calsyntenin-1 in association with tubulovesicular organelles in axonal fiber tracts. In primary neuronal cultures, calsyntenin-1–containing organelles were aligned along microtubules and partially colocalized with Kinesin-1. Using live imaging, we showed that these organelles are transported along axons with a velocity and processivity typical for fast axonal transport. Point mutations in the two kinesin-binding segments of calsyntenin-1 significantly reduced binding to KLC1 in vitro, and vesicles bearing mutated calsyntenin-1 exhibited a markedly altered anterograde axonal transport. In summary, our results indicate that calsyntenin-1 links a certain type of vesicular and tubulovesicular organelles to the Kinesin-1 motor.

Various studies suggest that proteolytic activity may be involved in a number of neurodegenerative disorders, including stroke and seizure. In this report, we examined the role of tryptic serine proteases, plasminogen activators (PAs), in the evolution of a neurodegenerative disease. Transgenic mice overexpressing an axonally secreted inhibitor of serine proteases (neuroserpin) were crossed with mice characterized by a “dying-back” motor neuron disease [progressive motor neuronopathy (pmn/pmn)]. Compared withpmn/pmnmice that showed an increase in PA activity, double mutant mice had decreased PA activity in sciatic nerves and spinal cord; their lifespan was increased by 50%, their motor behavior was stabilized, and histological analysis revealed increased numbers of myelinated axons and rescue of motoneuron number and size. This is the first report showing that a class of serine proteases (PAs) may be involved in the pathogenesis of a motor neuron disease and more specifically in axonal degeneration. Inhibiting serine proteases could offer a new strategy for delaying these disorders.