Cellular neuroscienceHuman autoantibodies against early endosome antigen-1 enhance excitatory synaptic transmission
Section snippets
Antibodies
Human sera were obtained and stored as previously described (Selak et al 1999, Selak et al 2003). Control sera were randomly selected from a bank of 2000 blood donors or pooled from healthy volunteers. Control normal human immunoglobulin G (IgG) was purchased from Pierce (Rockford, IL, USA). For electrophysiological experiments, antibodies were purified on a protein G sepharose column (Sigma, Madrid, Spain). The bound antibodies were eluted in 100 mM glycine buffer pH 2.5, pH immediately
Human autoantibodies specifically target EEA1 in human and murine tissue
The human anti-EEA1 serum was from a 48 year old Caucasian female. The serum was selected by rigorous preliminary screening out of more than 30 EEA1-specific human sera and the presence of anti-EEA1 antibodies was confirmed by immunoprecipitation of 35S-labeled in vitro translation product of EEA1 cDNA (Selak et al., 1999). To investigate the specificity of the patient’s autoantibodies for EEA1 in more detail, we analyzed the co-localization pattern between human anti-EEA1 serum and mouse
Discussion
Our studies show that the autoantibodies isolated from the index patient with a neurological dysfunction and that exclusively recognized the endosomal EEA1 protein, specifically enhance the excitatory synaptic transmission in the hippocampus. This effect was determined in CA3 pyramidal neurons, since the mossy fiber synapses are established close to the soma, where the dendrite is thick and diffusion of the antibody from the recording pipette was expected to be facilitated. Indeed, we got proof
Conclusion
In conclusion, exposure of neurons to human anti-EEA1 autoantibodies drastically and specifically affected excitatory synaptic transmission. EEA1 is likely an important component of endocytic mechanisms regulating glutamate receptor sorting, such that its blockade affects excitatory synaptic transmission irrespective of the receptor mediating the synaptic response. While these effects may account for the neural alterations described for patients bearing EEA1 autoantibodies, it is also possible
Acknowledgments
This work was supported by grants to J.L. from the MCYT (BFI2003-00161) and the European Union (QLG3-CT2001-00929). We thank Ms. M. Llinares for technical help and Dr. B. Rico for sharing her expertise on immunohistology. S.S. was a recipient of a fellowship from the Program of Foreign Doctors and Technologists in Spain (MCYT) and currently is an I3P Program CSIC Research Fellow.
References (63)
- et al.
The endosomal protein NEEP21 regulates AMPA receptor-mediated synaptic transmission and plasticity in the hippocampus
Mol Cell Neurosci
(2005) Intracellular trafficking of GABA(A) receptors
Life Sci
(2000)- et al.
Subunit-specific NMDA receptor trafficking to synapses
Neuron
(2002) - et al.
NMDA receptor-dependent activation of the small GTPase Rab5 drives the removal of synaptic AMPA receptors during hippocampal LTD
Neuron
(2005) - et al.
Interaction with newly synthesized and retained proteins in the endoplasmic reticulum suggests a chaperone function for human integral membrane protein IP90 (calnexin)
J Biol Chem
(1993) Reinsertion or degradation of AMPA receptors determined by activity-dependent endocytic sorting
Neuron
(2000)- et al.
Penetration and internalization of plasma proteins in the human spinal cord
J Neurol Sci
(1991) - et al.
Calcium-permeable AMPA receptor plasticity is mediated by subunit-specific interactions with PICK1 and NSF
Neuron
(2005) - et al.
Molecular and cellular permeability control at the blood-brain barrier
Brain Res Rev
(2001) - et al.
NSF ATPase and alpha-/beta-SNAPs disassemble the AMPA receptor-PICK1 complex
Neuron
(2002)
Rapid and differential regulation of AMPA and kainate receptors at hippocampal mossy fibre synapses by PICK1 and GRIP
Neuron
Sequential roles for phosphatidylinositol 3-phosphate and Rab5 in tethering and fusion of early endosomes via their interaction with EEA1
J Biol Chem
The FYVE domain of early endosome antigen 1 is required for both phosphatidylinositol 3-phosphate and Rab5 bindingCritical role of this dual interaction for endosomal localization
J Biol Chem
Pathogenic autoantibodies: Emerging insights into tissue injury
Immunol Lett
PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking
Neuron
Regulation of AMPA receptor-mediated synaptic transmission by clathrin-dependent receptor internalization
Neuron
Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13
Cell
Involvement of the endosomal autoantigen EEA1 in homotypic fusion of early endosomes
Curr Biol
EEA1, an early endosome-associated proteinEEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine “fingers” and contains a calmodulin-binding IQ motif
J Biol Chem
NSF binding to GluR2 regulates synaptic transmission
Neuron
The AMPA receptor GluR2 C terminus can mediate a reversible, ATP-dependent interaction with NSF and alpha- and beta-SNAPs
Neuron
Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis
Neuron
Kainate receptors presynaptically downregulate GABAergic inhibition in the rat hippocampus
Neuron
Selective membrane recruitment of EEA1 suggests a role in directional transport of clathrin-coated vesicles to early endosomes
J Biol Chem
Characterization of early endosome antigen 1 in neural tissues
Biochem Biophys Res Commun
Identification of the B-cell epitopes of the early endosome antigen 1 (EEA1)
Clin Immunol
PDZs and receptor/channel clustering: rounding up the latest suspects
Neuron
Regulation of AMPA receptors during synaptic plasticity
Trends Neurosci
Endosomal localization of the autoantigen EEA1 is mediated by a zinc-binding FYVE finger
J Biol Chem
Autoantibodies to a novel early endosome antigen-1
Clin Immunol Immunopathol
Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles
Cell
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2020, NeuroscienceCitation Excerpt :However, EEA1 protein level in the soma was unaltered (Ackermann et al., 2019). The increased EEA1 measured in our study could indeed emanate from the postsynaptic compartment, which is the previously demonstrated localization for EEA1 (Wilson et al., 2000; Selak et al., 2006; Lomash et al., 2015). The protein encoded by the EEA1 gene is known to interact with Rab5, N-ethymaleimide-sensitive factor (NSF) and Syntaxin 13 in order to regulate vesicle fusion at the early endosome after endocytosis (McBride et al., 1999; Lawe et al., 2002; Selak et al., 2006).
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2018, Cell ReportsCitation Excerpt :Neurons in the amygdala nuclei were identified by their firing patterns when they were depolarized by current passing through the patch pipette. In a few instances, the correlation between morphology and electrical responses was confirmed by filling the neurons with biocytin, which was then visualized with streptavidin (Selak et al., 2006). The perfusion solution was supplemented with picrotoxin (100 μM), D-2-amino-5-phosphonovalerate (D-APV) (50 μM), and LY303070 (25 μM) to isolate spontaneous KAR-mediated EPSCs.
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2009, NeuronCitation Excerpt :The identification of SNAP25 as a key element of this process should reveal important for this endeavor. Membrane extracts or subcellular fractions were prepared from the brain tissue of P21–P26 C57 mice, as previously described (Selak et al., 2006). Immunoprecipitations were performed by incubating membrane preparations with 50% protein A/G-Sepharose slurry (Amersham Biosciences) at 4°C to eliminate nonspecific binding.
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2009, NeuroscienceCitation Excerpt :Interestingly, Rab5 activation is involved in LTD-induced AMPAR endocytosis, but not in constitutive endocytosis of AMPAR (Brown et al., 2005). In this context it is interesting that infusion of auto-antibodies against early endosome antigen-1 (EEA1), an effector protein of Rab5, caused a run-up of EPSC, presumably due to impaired endocytosis of AMPAR (Selak et al., 2006). LTD also activates a parallel pathway dependent on Rap2 and the JNK pathway to control AMPAR internalization via GluR1 (Zhu et al., 2005).
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2006, NeuronCitation Excerpt :Blots were visualized by the chemiluminescence protocol, scanned, and quantified using ImageJ software. Live cultured hippocampal neurons were prepared using a protocol described elsewhere (Selak et al., 2006). Neurons were transfected at DIV10 with 1 μg of EGFP-tagged GluR6 wt, R6(R523K), or R6(K696C/E787C) using Lipofectamine 2000, according to the manufacturer's instructions.