Evidence for abnormal forward trafficking of AMPA receptors in frontal cortex of elderly patients with schizophrenia

Abstract

Several lines of evidence point to alterations of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor trafficking in schizophrenia. Multiple proteins, including synapse-associated protein 97 (SAP97), glutamate receptor-interacting protein 1 (GRIP1), and N-ethylmaleimide sensitive factor (NSF), facilitate the forward trafficking of AMPA receptors toward the synapse. Once localized to the synapse, AMPA receptors are trafficked in a complex endosomal system. We hypothesized that alterations in the expression of these proteins and alterations in the subcellular localization of AMPA receptors in endosomes may contribute to the pathophysiology of schizophrenia. Accordingly, we measured protein expression of SAP97, GRIP1, and NSF in the dorsolateral prefrontal cortex and found an increase in the expression of SAP97 and GRIP1 in schizophrenia. To determine the subcellular localization of AMPA receptor subunits, we developed a technique to isolate early endosomes from post-mortem tissue. We found increased GluR1 receptor subunit protein in early endosomes in subjects with schizophrenia. Together, these data suggest that there is an alteration of forward trafficking of AMPA receptors as well as changes in the subcellular localization of an AMPA receptor subunit in schizophrenia.

Figure 1

Western blot analysis of AMPA-interacting proteins ((a)—GRIP1, (b)—NSF, (c)—SAP97, and (d)—EEA1) in total brain homogenate normalized to β-tubulin. ^*p<0.05.

Figure 2

Flow chart of EEA1 immunoisolation protocol with non-precleared and precleared tissue. Western blotting was used to determine protein expression of EEA1 and PSD95. Nonspecific binding of PSD95 in EEA1 immunoisolation is present with non-precleared tissue (non-precleared, IM, pellet lane). EEA1 immunoisolation with precleared tissue has markedly reduced expression of PSD95 but maintains EEA1 expression (precleared, IM, pellet lane). PSD95 sticks nonspecifically to beads (preclear bead lane). IM, immunoisolation; – control, negative control.

Figure 3

Electron micrograph of early endosome immunoisolation. Early endosomes were isolated using magnetic beads and EEA1 capture antibody and imaged using electron microscopy. The panel on the right is an enlarged view of the area indicated by the arrow on the left.

Figure 4

Characterization of early endosome isolation by western blot analysis. Proteins not expressed in early endosomes (IM—pellet) include those found in the postsynaptic density (PSD95), endoplasmic reticulum (GRP78/BiP), astrocytes (glutamine synthetase), and late endosomes (Rab7). Expression of the AMPA receptor subunit (GluR2) is present in early endosomes (IM—pellet). IM, immunoisolation; – control, negative control; EEA1, early endosome antigen 1; PSD95, postsynaptic density 95; GRP78/BiP, glucose-regulated protein 78/binding immunoglobulin protein; GluR2, ionotropic glutamate receptor 2.

Figure 5

Western blot analysis of AMPA receptor subunits GluR1 (a), GluR2 (b), and GluR3 (c) in early endosomes normalized to EEA1 levels. Data are expressed as the ratio of subunit relative to isolated endosome expression. ^*p<0.05.

Figure 6

Analysis of antipsychotic effects on GluR1 expression relative to expression of isolated early endosomes. Post hoc analysis of expression of GluR1 relative to expression of isolated early endosomes. Patients on medication at time of death (On Rx). Patients off medication for ⩾6 weeks at time of death (Off Rx). ^*p<0.05.

Figure 7

AMPA receptor-interacting protein expression in early endosomes. The expression of the AMPA receptor-interacting proteins, NSF (a) and SAP97 (b), relative to expression of EEA1 was detected by western blot after enrichment of early endosomes.