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.
Human induced pluripotent stem cell (iPSC)-derived neurons have been proposed to be a highly valuable cellular model for studying the pathomechanisms of Alzheimer's disease (AD). Studies employing patient-specific human iPSCs as models of familial and sporadic forms of AD described elevated levels of AD-related amyloid-β (Aβ). However, none of the present AD iPSC studies could recapitulate the synaptotoxic actions of Aβ, which are crucial early events in a cascade that eventually leads to vast brain degeneration. Here we established highly reproducible, human iPSC-derived cortical cultures as a cellular model to study the synaptotoxic effects of Aβ. We developed a highly efficient immunopurification procedure yielding immature neurons that express markers of deep layer cortical pyramidal neurons and GABAergic interneurons. Upon long-term cultivation, purified cells differentiated into mature neurons exhibiting the generation of action potentials and excitatory glutamatergic and inhibitory GABAergic synapses. Most interestingly, these iPSC-derived human neurons were strongly susceptible to the synaptotoxic actions of Aβ. Application of Aβ for 8 days led to a reduction in the overall FM4–64 and vGlut1 staining of vesicles in neurites, indicating a loss of vesicle clusters. A selective analysis of presynaptic vesicle clusters on dendrites did not reveal a significant change, thus suggesting that Aβ impaired axonal vesicle clusters. In addition, electrophysiological patch-clamp recordings of AMPA receptor-mediated miniature EPSCs revealed an Aβ-induced reduction in amplitudes, indicating an impairment of postsynaptic AMPA receptors. A loss of postsynaptic AMPA receptor clusters was confirmed by immunocytochemical stainings for GluA1. Incubation with Aβ for 8 days did not result in a significant loss of neurites or cell death. In summary, we describe a highly reproducible cellular AD model based on human iPSC-derived cortical neurons that enables the mechanistic analysis of Aβ-induced synaptic pathomechanisms and the development of novel therapeutic approaches.
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 αSGT. CHL1, Hsc70, CSP and αSGT form predominantly CHL1/Hsc70/αSGT and CHL1/CSP complexes in synapses. Among the various complexes formed by CHL1, Hsc70, CSP and αSGT, SNAP25 and VAMP2 induce chaperone activity only in CHL1/Hsc70/αSGT 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 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-β peptides are well known to induce functional damage, destabilization and loss of brain synapses. However, the complex molecular mechanisms of amyloid-β 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-β-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-β 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-β 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-β-induced synapse damage. Indeed, expression of human N-cadherin C-terminal fragment 1 strongly accelerated amyloid-β-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-β-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-β-triggered synapse damage.
More than the sum of its parts: Novel hybrid compounds consisting of an organic β‐sheet‐breaking moiety and a signaling, D‐enantiomeric Aβ‐recognizing peptide moiety have been designed (see picture). The compounds, which were chemically synthesized and characterized by several techniques, combine rational design and drug selection from libraries and inhibit Aβ oligomerization and Aβ‐induced synaptic pathology.
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.
A lzheimer's disease (AD) is the most prevalent neurodegenerative disorder with the amyloid-β-peptide (Aβ) playing a central role in its pathogenesis. 1 Aβ is generated as a 38À43 residue peptide from the amyloid precursor protein (APP) by the proteolytic activity of β-and γ-secretases. 2,3 Aβ assembles into oligomers of various sizes and Aβ plaques, a neuropathological hallmark of AD. 1 Unlike Aβ fibrils or plaques, only the presence of Aβ oligomers correlates with the cognitive status of AD. 4,5 Aβ oligomers cause synaptic pathology 1,6 in line with findings that loss of dendritic spines correlates to early morphological changes in AD patients. 7 Many different oligomeric Aβ species have been identified, 8À10 and neurotoxicity has been attributed to Aβ dimers, 6 Aβ dodecamers, 10,11 or others. A recent study from Shankar et al. 6 has provided solid evidence that natively secreted Aβ dimers purified from post-mortem brains of patients with clinical AD are the predominant species sufficient to cause synaptic pathology. To gain insight into structureÀfunction relationships of Aβ dimers, it is of paramount importance to generate or purify highly homogeneous, distinct species of Aβ oligomers. So far, detailed structural investigations of native Aβ oligomers have been hampered by their low abundance in available transgenic mouse models of AD or AD patient brains, and laborious purification procedures.Synthetic Aβ has been a convenient tool for studying Aβ protein biochemistry, since it can be generated to relatively high purity in large amounts and is therefore readily available for biophysical and structural studies of some aspects of Aβ. However, evidence has accumulated suggesting that synthetic Aβ oligomers are only insufficiently modeling naturally secreted Aβ oligomer functions, even if refolded and purified by laborious procedures. Several studies clearly indicate that the bioactivity of naturally secreted Aβ oligomers from transfected cells is similar to that of AD brain-derived Aβ oligomers, but more than hundred times stronger than that of synthetic Aβ oligomers, when long-term potentiation (LTP) effects are taken as readout. 12 The major difference between natively secreted and synthetic Aβ is that natively secreted Aβ is the result of a highly controlled and quality-checked, cofactor-assisted proteolytic process at low local concentrations, while bulk refolding of synthetic Aβ occurs without regulating cofactors at high local concentrations. Bulk refolding leads to conformational heterogenetiy of refolded Aβ oligomers correlating with different neurotoxicities. 13 Received: February 20, 2011 Accepted: March 11, 2011 ABSTRACT: Aβ oligomers play a key role in the pathophysiology of Alzheimer's disease. Research into structureÀfunction relationships of Aβ oligomers has been hampered by the lack of large amounts of homogeneous and stable material. Using computational chemistry, we designed conservative cysteine substitutions in Aβ aiming at accelerating and stabilizing assembly of Aβ dimers by an intermolecu...
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.
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