Acid sphingomyelinase - a regulator of canonical transient receptor potential channel 6 (TRPC6) activity.

Recent investigations propose the acid sphingomyelinase (ASM)/ceramide system as a novel target for antidepressant action. ASM catalyzes the breakdown of the abundant membrane lipid sphingomyelin to the lipid messenger ceramide. This ASM-induced lipid modification induces a local shift in membrane properties, which influences receptor clustering and downstream signaling. Canonical transient receptor potential channels 6 (TRPC6) are non-selective cation channels located in the cell membrane that play an important role in dendritic growth, synaptic plasticity and cognition in the brain. They can be activated by hyperforin, an ingredient of the herbal remedy St. John's wort for treatment of depression disorders. Because of their role in the context of major depression, we investigated the crosstalk between the ASM/ceramide system and TRPC6 ion channels in a pheochromocytoma cell line 12 neuronal cell model (PC12 rat pheochromocytoma cell line). Ca2+ imaging experiments indicated that hyperforin-induced Ca2+ influx through TRPC6 channels is modulated by ASM activity. While antidepressants, known as functional inhibitors of ASM activity, reduced TRPC6-mediated Ca2+ influx, extracellular application of bacterial sphingomyelinase rebalanced TRPC6 activity in a concentration-related way. This effect was confirmed in whole-cell patch clamp electrophysiology recordings. Lipidomic analyses revealed a decrease in very long chain ceramide/sphingomyelin molar ratio after ASM inhibition, which was connected with changes in the abundance of TRPC6 channels in flotillin-1-positive lipid rafts as visualized by western blotting. Our data provide evidence that the ASM/ceramide system regulates TRPC6 channels likely by controlling their recruitment to specific lipid subdomains and thereby fine-tuning their physical properties.

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.

CHL1 is a selective organizer of the presynaptic machinery chaperoning the SNARE complex.

Proteins constituting the presynaptic machinery of vesicle release undergo substantial conformational changes during the process of exocytosis. While changes in the conformation make proteins vulnerable to aggregation and degradation, little is known about synaptic chaperones which counteract these processes. We show that the cell adhesion molecule CHL1 directly interacts with and regulates the activity of the synaptic chaperones Hsc70, CSP and alphaSGT. CHL1, Hsc70, CSP and alphaSGT form predominantly CHL1/Hsc70/alphaSGT and CHL1/CSP complexes in synapses. Among the various complexes formed by CHL1, Hsc70, CSP and alphaSGT, SNAP25 and VAMP2 induce chaperone activity only in CHL1/Hsc70/alphaSGT and CHL1/CSP complexes, respectively, indicating a remarkable selectivity of a presynaptic chaperone activity for proteins of the exocytotic machinery. In mice with genetic ablation of CHL1, chaperone activity in synapses is reduced and the machinery for synaptic vesicle exocytosis and, in particular, the SNARE complex is unable to sustain prolonged synaptic activity. Thus, we reveal a novel role for a cell adhesion molecule in selective activation of the presynaptic chaperone machinery.

The adhesion molecule CHL1 regulates uncoating of clathrin-coated synaptic vesicles.

In searching for binding partners of the intracellular domain of the immunoglobulin superfamily adhesion molecule CHL1, we identified the clathrin-uncoating ATPase Hsc70. CHL1 gene ablation resulted in reduced targeting of Hsc70 to the synaptic plasma membrane and synaptic vesicles, suggesting CHL1 as a synapse-targeting cue for Hsc70. CHL1 accumulates in presynaptic membranes and, in response to synapse activation, is targeted to synaptic vesicles by endocytosis. CHL1 deficiency or disruption of the CHL1/Hsc70 complex results in accumulation of abnormally high levels of clathrin-coated synaptic vesicles with a reduced ability to release clathrin. Generation of new clathrin-coated synaptic vesicles in an activity-dependent manner is inhibited when the CHL1/Hsc70 complex is disrupted, resulting in impaired uptake and release of FM dyes in synaptic boutons. Abnormalities in clathrin-dependent synaptic vesicle recycling may thus underlie brain malfunctions in humans and mice that carry mutations in the CHL1 gene.