Elsevier

Neuroscience

Volume 143, Issue 4, 28 December 2006, Pages 953-964
Neuroscience

Cellular neuroscience
Human autoantibodies against early endosome antigen-1 enhance excitatory synaptic transmission

https://doi.org/10.1016/j.neuroscience.2006.10.014Get rights and content

Abstract

Early endosome antigen 1 (EEA1), a peripheral membrane protein associated with the cytoplasmic face of early endosomes, controls vesicle fusion during endocytosis, as extensively studied in non-neuronal cells. In neurons, early endosomes are involved in recycling of synaptic vesicles and neurotransmitter receptors. Since certain patients bearing autoantibodies that target EEA1 develop neurological disease, we studied the subcellular distribution of EEA1 in neurons and the effect on neurotransmission of purified immunoglobulins from the serum of a patient bearing EEA1 autoantibodies. EEA1 was localized in the soma and in the postsynaptic nerve terminals. Electrophysiological recordings in hippocampal slices including purified EEA1 antibodies in the patch pipette solution, revealed a run-up of AMPA, N-methyl-d-aspartate and kainate receptor-mediated excitatory post-synaptic currents recorded from CA3 pyramidal neurons, which was absent in the recordings obtained in the presence of control human immunoglobulin G. Inclusion of human EEA1 antibodies had no effect on inhibitory post-synaptic responses. Recordings in the presence of a dominant-negative C-terminal EEA1 deletion mutant produced a similar effect as observed with human anti-EEA1 antibodies. This specific effect on the excitatory synaptic transmission may be due to the impairment of internalization of specific glutamate receptors and their subsequent accumulation in the synapse. These results may account for the neurological deficits observed in some patients developing EEA1 autoantibodies.

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)

  • H. Hirbec et al.

    Rapid and differential regulation of AMPA and kainate receptors at hippocampal mossy fibre synapses by PICK1 and GRIP

    Neuron

    (2003)
  • D.C. Lawe et al.

    Sequential roles for phosphatidylinositol 3-phosphate and Rab5 in tethering and fusion of early endosomes via their interaction with EEA1

    J Biol Chem

    (2002)
  • D.C. Lawe et al.

    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

    (2000)
  • P.L. Lim et al.

    Pathogenic autoantibodies: Emerging insights into tissue injury

    Immunol Lett

    (2006)
  • W. Lu et al.

    PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking

    Neuron

    (2005)
  • H.Y. Man et al.

    Regulation of AMPA receptor-mediated synaptic transmission by clathrin-dependent receptor internalization

    Neuron

    (2000)
  • H.M. McBride et al.

    Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13

    Cell

    (1999)
  • I.G. Mills et al.

    Involvement of the endosomal autoantigen EEA1 in homotypic fusion of early endosomes

    Curr Biol

    (1998)
  • F.T. Mu et al.

    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

    (1995)
  • A. Nishimune et al.

    NSF binding to GluR2 regulates synaptic transmission

    Neuron

    (1998)
  • P. Osten et al.

    The AMPA receptor GluR2 C terminus can mediate a reversible, ATP-dependent interaction with NSF and alpha- and beta-SNAPs

    Neuron

    (1998)
  • N. Ringstad et al.

    Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis

    Neuron

    (1999)
  • A. Rodríguez-Moreno et al.

    Kainate receptors presynaptically downregulate GABAergic inhibition in the rat hippocampus

    Neuron

    (1997)
  • M. Rubino et al.

    Selective membrane recruitment of EEA1 suggests a role in directional transport of clathrin-coated vesicles to early endosomes

    J Biol Chem

    (2000)
  • S. Selak et al.

    Characterization of early endosome antigen 1 in neural tissues

    Biochem Biophys Res Commun

    (2004)
  • S. Selak et al.

    Identification of the B-cell epitopes of the early endosome antigen 1 (EEA1)

    Clin Immunol

    (2003)
  • M. Sheng

    PDZs and receptor/channel clustering: rounding up the latest suspects

    Neuron

    (1996)
  • I. Song et al.

    Regulation of AMPA receptors during synaptic plasticity

    Trends Neurosci

    (2002)
  • H. Stenmark et al.

    Endosomal localization of the autoantigen EEA1 is mediated by a zinc-binding FYVE finger

    J Biol Chem

    (1996)
  • R.L. Waite et al.

    Autoantibodies to a novel early endosome antigen-1

    Clin Immunol Immunopathol

    (1998)
  • B. Wiedenmann et al.

    Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles

    Cell

    (1985)
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