Elsevier

Brain Research

Volume 765, Issue 1, 8 August 1997, Pages 74-80
Brain Research

Research report
Excitable membranes and synaptic transmission: postsynaptic mechanisms.: Localization of α-internexin in the postsynaptic density of the rat brain

https://doi.org/10.1016/S0006-8993(97)00492-7Get rights and content

Abstract

The synaptic localization of α-internexin, a brain-specific intermediate filament protein, was investigated immunohistochemically in the rat brain. The specificity of the antibody used in this study was confirmed by Western blotting and the antibody specifically reacted with α-internexin in the neurofilament preparation and in the postsynaptic density (PSD) fraction. The α-internexin immunoreactivity was distributed in neurons, especially in the somata and dendrites, throughout the cerebral cortex. Immunoelectron microscopic examination showed the immunoreactivity in the PSD, while neurofilament M was not in the PSD. Thus α-internexin and neurofilament M are differentially localized in neuronal cells. α-Internexin content in the PSD fraction was relatively high even before the period of synaptogenesis and the content in the fraction was unchanged between young and adult rats (2–6 weeks old). These results suggest a role of α-internexin for early development and organization of the PSD.

Introduction

The postsynaptic density (PSD) is a cytoskeletal specialization tightly apposed to the postsynaptic membranes of dendrites and spines 26, 30. The PSD is a major site for modulation of neuronal signal transduction and is believed to change its shape dynamically dependent on input signals to the synapses. Morphological changes in PSD may be one of the mechanisms responsible for long-term modulation of synaptic strength 1, 2. In addition to molecules that are directly required for the signal processing, the PSD contains various kinds of cytoskeletal proteins and cytoskeleton-associated molecules such as fodrin (calspectin), actin, α-actinin, tensin (actin capping protein), myosin, tubulin, microtubule-associated protein 2 (MAP2), τ-protein, 205 kDa MAP, gystrophin, N-CAM (cell adhesion molecule), and N-cadherin [30]. However, the precise molecular architecture and the mechanisms of dynamic changes in PSD morphology have not yet been determined. Certain types of as yet undetermined intermediate filaments may participate in the construction of the PSD because PSDs partially broken up accidentally or by sonication revealed 10 nm filaments as major constituents of PSD 3, 5. Major PSD protein (mPSDp), now known as the α subunit of Ca2+/calmodulin-dependent protein kinase II 9, 16, 18, is not a major cytoskeletal element of PSD [31].

α-Internexin was found in cytoskeletal extract from adult rat optic nerve and was purified in homogeneity by Pachter and Liem [24]. α-Internexin binds various intermediate filament proteins [24]and on electrophoresis migrates to a position immediately below the 68 kDa neurofilament L subunit protein 4, 14, 24, 33. This intermediate filament protein is expressed specifically in the brain, and its distribution in the adult central nervous system closely parallels those of neurofilaments L and M, but its expression precedes those of these neurofilaments 7, 14. These properties of α-internexin suggest fundamental roles for the organization of the intermediate filament-based cytoskeleton in the brain.

The observations described above prompted us to explore the distribution of α-internexin at synaptic sites. We report here the localization of this molecule in PSD of rat brain as detected by immunoelectron microscopy.

Section snippets

Reagents

Anti-α-internexin antibody (AB1515) was purchased from Chemicon International; anti-neurofilament 145 kDa subunit antibody (Ab-1, mouse) was from Oncogene Science; horseradish peroxidase-coupled goat anti-rabbit immunoglobulin was from Cappel; horseradish peroxidase-coupled anti-mouse and anti-rabbit immunoglobulin for immunohistochemistry were from Nichirei Laboratories; ECL Western blotting detection reagents were from Amersham; polyvinylidene difluoride (PVDF) membranes (Immobilon) were from

Identification of α-internexin in the PSD fraction

The anti-α-internexin antibody used in this study reacted specifically with the 63 kDa protein, α-internexin 4, 14, 24, 33, which migrated to a position immediately below neurofilament L in the neurofilament-enriched fraction on electrophoresis (Fig. 1). The antibody also selectively reacted with a 63 kDa band with a pI of around 6.0 identical to that of α-internexin [24], in the PSD fraction prepared from the rat forebrain (Fig. 2a). The 63 kDa immunoreactive protein was also detected in the

Discussion

We examined the localization of α-internexin, a brain-specific intermediate filament protein, in the rat brain. α-Internexin was detected specifically in the neurons and was localized in the somata and dendrites. Immunoelectron microscopic analysis demonstrated that α-internexin was localized in the spines and PSD, but not in the presynaptic terminals. Thus, α-internexin is a PSD constituent in vivo.

The occurrence of α-internexin in the PSD in vivo was in marked contrast to neurofilament M

Acknowledgements

This research was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture, the Ichiro Kanehara Foundation, and Toyota Physical and Chemical Research Institute.

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