A delay in vesicle endocytosis by a C-terminal fragment of N-cadherin enhances Aß synaptotoxicity.

Synaptotoxic Aß oligomers are thought to play a major role in the early pathology of Alzheimer´s disease (AD). However, the molecular mechanisms involved in Aß-induced synaptic dysfunction and synapse damage remain largely unclear. Previously, Aß synaptotoxicity has been reported to be enhanced by increased levels of a C-terminal fragment of the synaptic adhesion molecule N-cadherin that is generated by proteolytic shedding of the extracellular domains [1]. To address the molecular mechanisms involved in this process, we have now studied the functional synaptic changes induced by C-terminal fragments (CTF1) of synaptic adhesion proteins. We used synaptophysin-pHluorin (SypHy) fluorescence imaging to monitor synaptic vesicle exo- and endocytosis in cultures of mouse cortical neurons. We increased the levels of C-terminal fragments of synaptic adhesion proteins by pharmacologically inhibiting γ-secretase, which further degrades CTF1 fragments. We found that this intervention caused a delay in synaptic vesicle endocytosis. A similar effect was induced by overexpression of N-cadherin CTF1, but not by overexpression of Neurexin3ß CTF1. Based on these observations, we further studied whether directly modulating synaptic vesicle endocytosis enhances Aß synaptotoxicity. We pharmacologically induced a delayed synaptic vesicle endocytosis by a low concentration of the endocytosis inhibitor dynasore. This treatment enhanced synaptoxicity of Aß oligomers as indicated by a reduced frequency of miniature postsynaptic currents. In conclusion, we propose that delayed endocytosis results in prolonged exposure of synaptic vesicle membranes to the extracellular space, thus enabling enhanced vesicle membrane binding of Aß oligomers. This might in turn promote the endocytic uptake of toxic Aß oligomers and might thus play an important role in intracellular Aß-mediated synaptotoxicity in AD.

Repurposing drugs against Alzheimer's disease: can the anti-multiple sclerosis drug fingolimod (FTY720) effectively tackle inflammation processes in AD?

Therapeutic approaches providing effective medication for Alzheimer's disease (AD) patients after disease onset are urgently needed. Previous studies in AD mouse models and in humans suggested that physical exercise or changed lifestyle can delay AD-related synaptic and memory dysfunctions when treatment started in juvenile animals or in elderly humans before onset of disease symptoms. However, a pharmacological treatment that can reverse memory deficits in AD patients was thus far not identified. Importantly, AD disease-related dysfunctions have increasingly been associated with neuro-inflammatory mechanisms and searching for anti-inflammatory medication to treat AD seems promising. Like for other diseases, repurposing of FDA-approved drugs for treatment of AD is an ideally suited strategy to reduce the time to bring such medication into clinical practice. Of note, the sphingosine-1-phosphate analogue fingolimod (FTY720) was FDA-approved in 2010 for treatment of multiple sclerosis patients. It binds to the five different isoforms of Sphingosine-1-phosphate receptors (S1PRs) that are widely distributed across human organs. Interestingly, recent studies in five different mouse models of AD suggest that FTY720 treatment, even when starting after onset of AD symptoms, can reverse synaptic deficits and memory dysfunction in these AD mouse models. Furthermore, a very recent multi-omics study identified mutations in the sphingosine/ceramide pathway as a risk factor for sporadic AD, suggesting S1PRs as promising drug target in AD patients. Therefore, progressing with FDA-approved S1PR modulators into human clinical trials might pave the way for these potential disease modifying anti-AD drugs.

Proteomics of the dentate gyrus reveals semantic dementia specific molecular pathology.

Semantic dementia (SD) is a clinical subtype of frontotemporal dementia consistent with the neuropathological diagnosis frontotemporal lobar degeneration (FTLD) TDP type C, with characteristic round TDP-43 protein inclusions in the dentate gyrus. Despite this striking clinicopathological concordance, the pathogenic mechanisms are largely unexplained forestalling the development of targeted therapeutics. To address this, we carried out laser capture microdissection of the dentate gyrus of 15 SD patients and 17 non-demented controls, and assessed relative protein abundance changes by label-free quantitative mass spectrometry. To identify SD specific proteins, we compared our results to eight other FTLD and Alzheimer's disease (AD) proteomic datasets of cortical brain tissue, parallel with functional enrichment analyses and protein-protein interactions (PPI). Of the total 5,354 quantified proteins, 151 showed differential abundance in SD patients (adjusted P-value < 0.01). Seventy-nine proteins were considered potentially SD specific as these were not detected, or demonstrated insignificant or opposite change in FTLD/AD. Functional enrichment indicated an overrepresentation of pathways related to the immune response, metabolic processes, and cell-junction assembly. PPI analysis highlighted a cluster of interacting proteins associated with adherens junction and cadherin binding, the cadherin-catenin complex. Multiple proteins in this complex showed significant upregulation in SD, including ß-catenin (CTNNB1), γ-catenin (JUP), and N-cadherin (CDH2), which were not observed in other neurodegenerative proteomic studies, and hence may resemble SD specific involvement. A trend of upregulation of all three proteins was observed by immunoblotting of whole hippocampus tissue, albeit only significant for N-cadherin. In summary, we discovered a specific increase of cell adhesion proteins in SD constituting the cadherin-catenin complex at the synaptic membrane, essential for synaptic signaling. Although further investigation and validation are warranted, we anticipate that these findings will help unravel the disease processes underlying SD.

Hemisynapse Formation Between Target Astrocytes and Cortical Neuron Axons in vitro.

One of the most fundamental organizing principles in the mammalian brain is that neurons do not establish synapses with the other major cell type, the astrocytes. However, induced synapse formation between neurons and astrocytes appears conceivable, because astrocytes are well known to express functional ionotropic glutamate receptors. Here, we attempted to trigger synapse formation between co-cultured neurons and astrocytes by overexpressing the strongly synaptogenic adhesion protein LRRTM2 in astrocytes physically contacted by cortical axons. Interestingly, control experiments with immature cortical astrocytes without any overexpression resulted in the induction of synaptic vesicle clustering in contacting axons (hemisynapse formation). This synaptogenic activity correlated with the endogenous expression of the synaptogenic protein Neuroligin1. Hemisynapse formation was further enhanced upon overexpression of LRRTM2 in cortical astrocytes. In contrast, cerebellar astrocytes required overexpression of LRRTM2 for induction of synaptic vesicle clustering in contacting axons. We further addressed, whether hemisynapse formation was accompanied by the appearance of fully functional glutamatergic synapses. We therefore attempted to record AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) in innervated astrocytes using the whole-cell patch-clamp technique. Despite the endogenous expression of the AMPA receptor subunits GluA2 and to a lesser extent GluA1, we did not reliably observe spontaneous AMPA mEPSCs. In conclusion, overexpression of the synaptogenic protein LRRTM2 induced hemisynapse formation between co-cultured neurons and astrocytes. However, the formation of fully functional synapses appeared to require additional factors critical for nano-alignment of presynaptic vesicles and postsynaptic receptors.

Transsynaptic N-Cadherin Adhesion Complexes Control Presynaptic Vesicle and Bulk Endocytosis at Physiological Temperature.

At mammalian glutamatergic synapses, most basic elements of synaptic transmission have been shown to be modulated by specific transsynaptic adhesion complexes. However, although crucial for synapse homeostasis, a physiological regulation of synaptic vesicle endocytosis by adhesion molecules has not been firmly established. The homophilic adhesion protein N-cadherin is localized at the peri-active zone, where the highly temperature-dependent endocytosis of vesicles occurs. Here, we demonstrate an important modulatory role of N-cadherin in endocytosis at near physiological temperature by synaptophysin-pHluorin imaging. Different modes of endocytosis including bulk endocytosis were dependent on N-cadherin expression and function. N-cadherin modulation might be mediated by actin filaments because actin polymerization ameliorated the knockout-induced endocytosis defect. Using super-resolution imaging, we found strong recruitment of N-cadherin to glutamatergic synapses upon massive vesicle release, which might in turn enhance vesicle endocytosis. This provides a novel, adhesion protein-mediated mechanism for efficient coupling of exo- and endocytosis.

Anti-Inflammatory Treatment with FTY720 Starting after Onset of Symptoms Reverses Synaptic Deficits in an AD Mouse Model.

Therapeutic approaches providing effective medication for Alzheimer's disease (AD) patients after disease onset are urgently needed. Previous studies in AD mouse models suggested that physical exercise or changed lifestyle can delay AD-related synaptic and memory dysfunctions when treatment started in juvenile animals long before onset of disease symptoms, while a pharmacological treatment that can reverse synaptic and memory deficits in AD mice was thus far not identified. Repurposing food and drug administration (FDA)-approved drugs for treatment of AD is a promising way to reduce the time to bring such medication into clinical practice. The sphingosine-1 phosphate analog fingolimod (FTY720) was approved recently for treatment of multiple sclerosis patients. Here, we addressed whether fingolimod rescues AD-related synaptic deficits and memory dysfunction in an amyloid precursor protein/presenilin-1 (APP/PS1) AD mouse model when medication starts after onset of symptoms (at five months). Male mice received intraperitoneal injections of fingolimod for one to two months starting at five to six months. This treatment rescued spine density as well as long-term potentiation in hippocampal cornu ammonis-1 (CA1) pyramidal neurons, that were both impaired in untreated APP/PS1 animals at six to seven months of age. Immunohistochemical analysis with markers of microgliosis (ionized calcium-binding adapter molecule 1; Iba1) and astrogliosis (glial fibrillary acid protein; GFAP) revealed that our fingolimod treatment regime strongly down regulated neuroinflammation in the hippocampus and neocortex of this AD model. These effects were accompanied by a moderate reduction of Aß accumulation in hippocampus and neocortex. Our results suggest that fingolimod, when applied after onset of disease symptoms in an APP/PS1 mouse model, rescues synaptic pathology that is believed to underlie memory deficits in AD mice, and that this beneficial effect is mediated via anti-neuroinflammatory actions of the drug on microglia and astrocytes.

Golgi-Cox impregnation combined with fluorescence staining of amyloid plaques reveals local spine loss in an Alzheimer mouse model.

BACKGROUND: Spine loss is a hallmark of Alzheimer´s and other neurodegenerative diseases, and testing candidate therapeutic drugs needs quantitative analysis of dendritic spine densities. Golgi-Cox impregnation of neurons is a classical method to visualize dendritic spines in diseased brains. Importantly, at early disease stages spine loss occurs locally in the vicinity of amyloid plaques, and concomitant fluorescence labeling of amyloid plaques is required to detect local spine damage. NEW METHOD: Because Golgi-Cox impregnation is done on unsectioned brains, whereas fluorescence staining is performed on sectioned material, the combination is technically challenging. We have now developed a novel combination of Golgi-Cox impregnation with methoxy-X04 fluorescence labeling of plaques that is performed on unsectioned brains. RESULTS: We used this new combination method to quantify dendritic spine densities in mouse hippocampal CA1 pyramidal neurons. Comparison of neurons from wildtype and APP/PS1 mice revealed local spine loss in the vicinity of amyloid plaques in both male and female APP/PS1 mice. COMPARISON WITH EXISTING METHOD: Golgi-Cox impregnation of neurons combined with methoxy-X04 staining of amyloid plaques is a highly reliable, easy-to-use method for permanent visualization of spines as compared to the technically more sophisticated and less stable fluorescence imaging of spines. CONCLUSION: Our novel combination method will be highly useful for testing potential therapeutic drugs in Alzheimer mouse models.

Differential Properties of the Synaptogenic Activities of the Neurexin Ligands Neuroligin1 and LRRTM2.

Synaptic cell adhesion molecules are well established to exhibit synaptogenic activity when overexpressed in target cells, indicating that they are involved in formation and functional maturation of synapses. The postsynaptic adhesion proteins Neuroligin1 and LRRTM2 both induce synaptic vesicle clusters in presynaptic axons in vitro by transsynaptically interacting with neurexins. In neurons, this is accompanied by the induction of glutamatergic, but not GABAergic synapses. Although the synaptogenic activity of Neuroligin1 has been well characterized, the properties of the synaptogenic activities of other synaptic adhesion molecules are largely unknown. In this paper, we now compared characteristics of the synaptogenic activities of Neuroligin1 and LRRTM2 upon overexpression in cultured mouse cortical neurons. Individual cortical neurons were transfected with Neuroligin1 and LRRTM2 expression plasmids, respectively, and synaptic vesicle clustering in contacting axons was examined by immunostaining for the vesicle membrane protein VAMP2. In immature neurons at 6-7 days in vitro (DIV) both Neuroligin1 and LRRTM2 exhibited strong synaptogenic activity. However, upon further neuronal differentiation only LRRTM2 retained significant synaptogenic activity at 12-13 DIV. A similar differential developmental maturation of the synaptogenic activities of Neuroligin1 and LRRTM2 was observed for the induction of glutamatergic synapses, which were detected by co-immunostaining for VGLUT1 and Homer1. Most interestingly, the synaptogenic activity of Neuroligin1 was strongly dependent on the expression and function of the synaptic adhesion molecule N-cadherin in immature neurons. In contrast, the synaptogenic activity of LRRTM2 was independent of N-cadherin expression and function in both immature (6-7 DIV) and more mature neurons (14-15 DIV). Taken together, our results with overexpression in cultured cortical neurons revealed striking differences in the properties of the synaptogenic activities of Neuroligin1 and LRRTM2, although both transsynaptically interact with presynaptic neurexins.

Astrocyte lineage cells are essential for functional neuronal differentiation and synapse maturation in human iPSC-derived neural networks.

Human astrocytes differ dramatically in cell morphology and gene expression from murine astrocytes. The latter are well known to be of major importance in the formation of neuronal networks by promoting synapse maturation. However, whether human astrocyte lineage cells have a similar role in network formation has not been firmly established. Here, we investigated the impact of human astrocyte lineage cells on the functional maturation of neural networks that were derived from human induced pluripotent stem cells (hiPSCs). Initial in vitro differentiation of hiPSC-derived neural progenitor cells and immature neurons (glia+ cultures) resulted in spontaneously active neural networks as indicated by synchronous neuronal Ca2+ transients. Depleting proliferating neural progenitors from these cultures by short-term antimitotic treatment resulted in strongly astrocyte lineage cell-depleted neuronal networks (glia- cultures). Strikingly, in contrast to glia+ cultures, glia- cultures did not exhibit spontaneous network activity. Detailed analysis of the morphological and electrophysiological properties of neurons by patch clamp recordings revealed reduced dendritic arborization in glia- cultures. In addition, a reduced action potential frequency upon current injection in pyramidal-like neurons was observed, whereas the electrical excitability of multipolar neurons was unaltered. Furthermore, we found a reduced dendritic density of PSD95-positive excitatory synapses, and more immature properties of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) miniature excitatory postsynaptic currents (mEPSCs) in glia- cultures, suggesting that the maturation of glutamatergic synapses depends on the presence of hiPSC-derived astrocyte lineage cells. Intriguingly, addition of the astrocyte-derived synapse maturation inducer cholesterol increased the dendritic density of PSD95-positive excitatory synapses in glia- cultures.

Release activity-dependent control of vesicle endocytosis by the synaptic adhesion molecule N-cadherin.

At synapses in the mammalian brain, continuous information transfer requires the long-term maintenance of homeostatic coupling between exo- and endocytosis of synaptic vesicles. Because classical endocytosis is orders of magnitude slower than the millisecond-range exocytosis of vesicles, high frequency vesicle fusion could potentially compromise structural stability of synapses. However, the molecular mechanisms mediating the tight coupling of exo- and endocytosis are largely unknown. Here, we investigated the role of the transsynaptic adhesion molecules N-cadherin and Neuroligin1 in regulating vesicle exo- and endocytosis by using activity-induced FM4-64 staining and by using synaptophysin-pHluorin fluorescence imaging. The synaptic adhesion molecules N-cadherin and Neuroligin1 had distinct impacts on exo- and endocytosis at mature cortical synapses. Expression of Neuroligin1 enhanced vesicle release in a N-cadherin-dependent way. Most intriguingly, expression of N-cadherin enhanced both vesicle exo- and endocytosis. Further detailed analysis of N-cadherin knockout neurons revealed that the boosting of endocytosis by N-cadherin was largely dependent on preceding high levels of vesicle release activity. In summary, regulation of vesicle endocytosis was mediated at the molecular level by N-cadherin in a release activity-dependent manner. Because of its endocytosis enhancing function, N-cadherin might play an important role in the coupling of vesicle exo- and endocytosis.

Amyloid-ß dimers in the absence of plaque pathology impair learning and synaptic plasticity.

Despite amyloid plaques, consisting of insoluble, aggregated amyloid-ß peptides, being a defining feature of Alzheimer's disease, their significance has been challenged due to controversial findings regarding the correlation of cognitive impairment in Alzheimer's disease with plaque load. The amyloid cascade hypothesis defines soluble amyloid-ß oligomers, consisting of multiple amyloid-ß monomers, as precursors of insoluble amyloid-ß plaques. Dissecting the biological effects of single amyloid-ß oligomers, for example of amyloid-ß dimers, an abundant amyloid-ß oligomer associated with clinical progression of Alzheimer's disease, has been difficult due to the inability to control the kinetics of amyloid-ß multimerization. For investigating the biological effects of amyloid-ß dimers, we stabilized amyloid-ß dimers by an intermolecular disulphide bridge via a cysteine mutation in the amyloid-ß peptide (Aß-S8C) of the amyloid precursor protein. This construct was expressed as a recombinant protein in cells and in a novel transgenic mouse, termed tgDimer mouse. This mouse formed constant levels of highly synaptotoxic soluble amyloid-ß dimers, but not monomers, amyloid-ß plaques or insoluble amyloid-ß during its lifespan. Accordingly, neither signs of neuroinflammation, tau hyperphosphorylation or cell death were observed. Nevertheless, these tgDimer mice did exhibit deficits in hippocampal long-term potentiation and age-related impairments in learning and memory, similar to what was observed in classical Alzheimer's disease mouse models. Although the amyloid-ß dimers were unable to initiate the formation of insoluble amyloid-ß aggregates in tgDimer mice, after crossbreeding tgDimer mice with the CRND8 mouse, an amyloid-ß plaque generating mouse model, Aß-S8C dimers were sequestered into amyloid-ß plaques, suggesting that amyloid-ß plaques incorporate neurotoxic amyloid-ß dimers that by themselves are unable to self-assemble. Our results suggest that within the fine interplay between different amyloid-ß species, amyloid-ß dimer neurotoxic signalling, in the absence of amyloid-ß plaque pathology, may be involved in causing early deficits in synaptic plasticity, learning and memory that accompany Alzheimer's disease.

Y-P30 promotes axonal growth by stabilizing growth cones.

The 30-amino acid peptide Y-P30, generated from the N-terminus of the human dermcidin precursor protein, has been found to promote neuronal survival, cell migration and neurite outgrowth by enhancing the interaction of pleiotrophin and syndecan-3. We now show that Y-P30 activates Src kinase and extracellular signal-regulated kinase (ERK). Y-P30 promotes axonal growth of mouse embryonic stem cell-derived neurons, embryonic mouse spinal cord motoneurons, perinatal rat retinal neurons, and rat cortical neurons. Y-P30-mediated axon growth was dependent on heparan sulfate chains. Y-P30 decreased the proportion of collapsing/degenerating growth cones of cortical axons in an Src and ERK-dependent manner. Y-P30 increased for 90 min in axonal growth cones the level of Tyr418-phosphorylated Src kinase and the amount of F-actin, and transiently the level of Tyr-phosphorylated ERK. Levels of total Src kinase, actin, GAP-43, cortactin and the glutamate receptor subunit GluN2B were not altered. When exposed to semaphorin-3a, Y-P30 protected a significant fraction of growth cones of cortical neurons from collapse. These results suggest that Y-P30 promotes axonal growth via Src- and ERK-dependent mechanisms which stabilize growth cones and confer resistance to collapsing factors.

mESC-based in vitro differentiation models to study vascular response and functionality following genotoxic insults.

Because of high exposure to systemic noxae, vascular endothelial cells (EC) have to ensure distinct damage defense and regenerative mechanisms to guarantee vascular health. For meaningful toxicological drug assessments employing embryonic stem cell (ESC)-based in vitro models, functional competence of differentiated progeny and detailed knowledge regarding damage defense mechanisms are essential. Here, mouse ESCs (mESC) were differentiated into functionally competent vascular cells (EC and smooth muscle cells [SMC]). mESC, EC, and SMC were comparatively analyzed regarding DNA repair and DNA damage response (DDR). Differentiation was accompanied by both congruent and unique alterations in repair and DDR characteristics. EC and SMC shared the downregulation of genes involved cell cycle regulation and repair of DNA double-strand breaks (DSBs) and mismatches, whereas genes associated with nucleotide excision repair (NER), apoptosis, and autophagy were upregulated when compared with mESC. Expression of genes involved in base excision repair (BER) was particularly low in SMC. IR-induced formation of DSBs, as detected by nuclear γH2AX foci formation, was most efficient in SMC, the repair of DSBs was fastest in EC. Together with substantial differences in IR-induced phosphorylation of p53, Chk1, and Kap1, the data demonstrate complex alterations in DDR capacity going along with the loss of pluripotency and gain of EC- and SMC-specific functions. Notably, IR exposure of early vascular progenitors did not impair differentiation into functionally competent EC and SMC. Summarizing, mESC-based vascular differentiation models are informative to study the impact of environmental stressors on differentiation and function of vascular cells.

Embryonic stem cells stably expressing BDNF-GFP exhibit a BDNF-release-dependent enhancement of neuronal differentiation.

Brain-derived neurotrophic factor (BDNF) is known to be a crucial regulator of neuronal survival and synaptic plasticity in the mammalian brain. Furthermore, BDNF positively influences differentiation of embryonic neural precursors, as well as that of neural stem cells from adult neurogenic niches. To study the impact of cell-released BDNF on neural differentiation of embryonic stem cells (ESCs), which represent an attractive source for cell transplantation studies, we have generated mouse ESC clones overexpressing BDNF-GFP by use of knock-in technology. After neural differentiation in vitro, we observed that ESC clones overexpressing BDNF-GFP gave rise to an increased number of neurons as compared to control ESCs. Neurons derived from BDNF-GFP-expressing ESCs harbored a more complex dendritic morphology and differentiated into the GABAergic lineage more than controls. Moreover, we show that ESC-derived neurons released BDNF-GFP in an activity-dependent manner and displayed similar electrophysiological properties as cortical neurons. Thus, our study describes the generation of ESCs stably overexpressing BDNF-GFP, which are ideally suited to investigate the ameliorating effects of BDNF in cell transplantation studies of various neuropathological conditions.

Asymmetric N-cadherin expression results in synapse dysfunction, synapse elimination, and axon retraction in cultured mouse neurons.

Synapse elimination and pruning of axon collaterals are crucial developmental events in the refinement of neuronal circuits. While a control of synapse formation by adhesion molecules is well established, the involvement of adhesion molecules in developmental synapse loss is poorly characterized. To investigate the consequences of mis-match expression of a homophilic synaptic adhesion molecule, we analysed an asymmetric, exclusively postsynaptic expression of N-cadherin. This was induced by transfecting individual neurons in cultures of N-cadherin knockout mouse neurons with a N-cadherin expression vector. 2 days after transfection, patch-clamp analysis of AMPA receptor-mediated miniature postsynaptic currents revealed an impaired synaptic function without a reduction in the number of presynaptic vesicle clusters. Long-term asymmetric expression of N-cadherin for 8 days subsequently led to synapse elimination as indicated by a loss of colocalization of presynaptic vesicles and postsynaptic PSD95 protein. We further studied long-term asymmetric N-cadherin expression by conditional, Cre-induced knockout of N-cadherin in individual neurons in cultures of N-cadherin expressing cortical mouse neurons. This resulted in a strong retraction of axonal processes in individual neurons that lacked N-cadherin protein. Moreover, an in vivo asymmetric expression of N-cadherin in the developmentally transient cortico-tectal projection was indicated by in-situ hybridization with layer V neurons lacking N-cadherin expression. Thus, mis-match expression of N-cadherin might contribute to selective synaptic connectivity.

C-terminal fragment of N-cadherin accelerates synapse destabilization by amyloid-ß.

The aetiology of Alzheimer's disease is thought to include functional impairment of synapses and synapse loss as crucial pathological events leading to cognitive dysfunction and memory loss. Oligomeric amyloid-ß peptides are well known to induce functional damage, destabilization and loss of brain synapses. However, the complex molecular mechanisms of amyloid-ß action resulting ultimately in synapse elimination are incompletely understood, thus limiting knowledge of potential therapeutic targets. Under physiological conditions, long-term synapse stability is mediated by trans-synaptically interacting adhesion molecules such as the homophilically binding N-cadherin/catenin complexes. In this study, we addressed whether inhibition of N-cadherin function affects amyloid-ß-induced synapse impairment. We found that blocking N-cadherin function, both by specific peptides interfering with homophilic binding and by expression of a dominant-negative, ectodomain-deleted N-cadherin mutant, resulted in a strong acceleration of the effect of amyloid-ß on synapse function in cultured cortical neurons. The frequency of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor-mediated miniature excitatory postsynaptic currents was reduced upon amyloid-ß application much earlier than observed in controls. We further hypothesized that ectodomain-shed, transmembrane C-terminal fragments that are generated during N-cadherin proteolytic processing might similarly enhance amyloid-ß-induced synapse damage. Indeed, expression of human N-cadherin C-terminal fragment 1 strongly accelerated amyloid-ß-triggered synapse impairment. Ectodomain-shed N-cadherin C-terminal fragment 1 is further proteolytically cleaved by γ-secretase. Therefore, both pharmacological inhibition of γ-secretase and expression of the dominant-negative presenilin 1 mutant L166P were used to increase the presence of endogeneous N-cadherin C-terminal fragment 1. Under these conditions, we again found a strong acceleration of amyloid-ß-induced synapse impairment, which could be compensated by over-expression of full-length N-cadherin. Intriguingly, western blot analysis of post-mortem brains from patients with Alzheimer's disease revealed an enhanced presence of N-cadherin C-terminal fragment 1. Thus, an inhibition of N-cadherin function by proteolytically generated N-cadherin C-terminal fragment 1 might play an important role in Alzheimer's disease progression by accelerating amyloid-ß-triggered synapse damage.

E-cadherin is required at GABAergic synapses in cultured cortical neurons.

Classical cadherins are cell adhesion molecules that are thought to contribute to the control of synapse formation, synaptic transmission, and synaptic plasticity. This is largely based on studies investigating the functions of N-cadherin at glutamatergic synapses, whereas other classical cadherins have hardly been examined at central synapses. We have now used a conditional knockout approach in cultured cortical neurons to address the role of E-cadherin mainly at inhibitory, GABAergic synapses. Cortical neurons were cultured from mouse fetuses carrying floxed E-cadherin alleles in homozygous configuration. E-cadherin knockout was induced in individual neurons by expression of an EGFP-Cre fusion protein. Immunocytochemical stainings for the vesicular GABA (VGAT) and glutamate (VGLUT1) transporters revealed a reduced density of dendritic GABAergic synapses in E-cadherin knockout neurons, whereas glutamatergic synapses were unaffected. Electrophysiological recordings of miniature and action potential-evoked, GABA(A) receptor-mediated postsynaptic currents confirmed an impairment of GABAergic synapses at the functional level. In summary, our immunocytochemical and electrophysiological analysis of E-cadherin knockout neurons suggested that E-cadherin signaling importantly contributes to the regulation of GABAergic synapses in cortical neurons.

Molecular engineering of a secreted, highly homogeneous, and neurotoxic aß dimer.

Aß oligomers play a key role in the pathophysiology of Alzheimer's disease. Research into structure-function relationships of Aß oligomers has been hampered by the lack of large amounts of homogeneous and stable material. Using computational chemistry, we designed conservative cysteine substitutions in Aß aiming at accelerating and stabilizing assembly of Aß dimers by an intermolecular disulfide bond without changing its folding. Molecular dynamics simulations suggested that mutants AßS8C and AßM35C exhibited structural properties similar to those of Aß wildtype dimers. Full length, mutant APP was stably expressed in transfected cell lines to study assembly of Aß oligomers in the physiological, secretory pathway and to avoid artifacts resulting from simultaneous in vitro oxidation and aggregation. Biochemical and neurophysiological analysis of supernatants indicated that AßS8C generated an exclusive, homogeneous, and neurotoxic dimer, whereas AßM35C assembled into dimers, tetramers, and higher oligomers. Thus, molecular engineering enabled generation of bioactive, homogeneous, and correctly processed Aß dimers in vivo.

Properties of glutamatergic synapses in immature layer Vb pyramidal neurons: coupling of pre- and postsynaptic maturational states.

Following initial contact formation, glutamatergic synapses in cortical neurons undergo pronounced functional maturation. These maturational events, occurring both pre- and postsynaptically, have been well described in the developing hippocampus. In this paper, we characterized glutamatergic synapses in immature layer Vb pyramidal neurons of the mouse somatosensory cortex during early postnatal development. At postnatal day 7, a significant subpopulation of glutamatergic synapses exhibited a low release probability that was accompanied by strong paired-pulse facilitation of AMPA EPSCs (paired-pulse ratio C > or = 2). Increasing extracellular Ca(2+) concentration increased release probability and led to paired-pulse depression. During further postnatal development, these functionally immature synapses disappeared. As shown pharmacologically,these synapses expressed postsynaptic NMDA receptors containing NR2B subunits, while NMDA receptors with NR2A subunits were lacking. Taken together, a low release probability presynaptically was coupled to postsynaptic NR2B signaling. This subpopulation of neocortical synapses thus differed from the majority of synapses in the developing hippocampus, where high release probability is coupled to NR2B signaling. The novel type of functionally immature glutamatergic synapse described here might play an important role in early developmental synapse elimination and in the activity-dependent refinement of the neocortical synaptic microcircuitry.