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Phosphorylation-dependent subcellular localization of the small heat shock proteins HspB1/Hsp25 and HspB5/αB-crystallin in cultured hippocampal neurons

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Abstract

The so-called stress response involving upregulation of heat shock proteins (Hsps) is a powerful mechanism of cells to deal with harmful conditions to which they are exposed throughout life, such as hyperthermia, hypoxia or oxidative stress. To gain more information about the molecular targets by which HspB1 (Hsp25) and HspB5 (αB-crystallin) might exert their neuroprotective effect we investigated the subcellular localization of unphosphorylated and phosphorylated HspB1 and B5 in neurons by immunocytochemistry and subcellular fractionation. In cultured hippocampal neurons, the unphosphorylated forms of both Hsps were localized in the perikaryon and nucleus, whereas the phosphorylated forms were recruited into neuronal processes. pHspB1-Ser15 and -Ser 86 were found within dendrites with a punctate distribution pattern partially colocalizing with the synaptic marker vGlut-1. pHspB5-Ser19 and -Ser45 localized to axons and dendrites with a filamentous-like staining pattern, whereas pHspB5-Ser59 was found in dendrites, especially along the plasma membrane and in spines. Biochemical analysis, i.e. subcellular fractionation of rat brain with subsequent Western blotting supported these localizations. These data show that in neurons HspB1 and B5 may have various molecular interaction partners at synapses, within dendrites and axons and that this interaction is likely to be regulated by phosphorylation. Stress-induced phosphorylation of HspB1 and B5 may lead to binding of these Hsps to their targets at synapses and neuronal processes which might provide one important mechanism of how they exert their neuroprotective effect.

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Acknowledgments

We thank Bianca Mekle and Silke Zschemisch for their excellent technical assistance and the International Graduate School in Molecular Medicine (University of Ulm, Germany) of which Thomas Schmidt is a member.

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  1. Institute of Anatomy and Cell Biology, University of Ulm, Albert-Einstein-Allee 11, 89081, Ulm, Germany

    Thomas Schmidt, Britta Bartelt-Kirbach & Nikola Golenhofen

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Correspondence to Nikola Golenhofen.

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Supplementary figure 1: Cultured hippocampal neurons immunolabelled for phospho-HspB1 and phospho-HspB5 as indicated (a-e) without and (f-j) with preabsorption of the antibodies with the respective immunogen. Note strong reduction or absence of immunosignals in panels f-j compared to panels a-e demonstrating specificity of the phospho-specific-Hsp-antibodies. Bar: 20 μm (PDF 558 kb)

418_2012_964_MOESM2_ESM.pdf

Supplementary figure 2: Quantification of band intensities of Western blots for HspB1, B5 and their phospho-forms of subcellular fractionations of rat brains as shown for a representative experiment in figure 5. HspB1 was significantly enriched in the nuclear fraction but also present in all other fractions investigated. pHspB1-Ser86 showed a similar subcellular distribution whereas pHspB1-S15 was enriched in the membrane fraction, synaptosomes and synaptic junctions and was detectable in little amount in the postsynaptic densities. HspB5 was enriched in the myelin, membrane fraction and postsynaptic densities The immunoblots using the phospho-specific antibodies against HspB5 showed that HspB5 in the myelin fraction was strongly phosphorylated at all three phosphorylation sites. Regarding the synaptic fractions pHspB5-Ser 19 was not present in any of these fractions analyzed. pHspB5-Ser45 and -Ser59 were detectable in low amounts in synaptosomes and synaptic membranes and to a much higher amount in postsynaptic densities. This hints to a postsynaptic localization of these two phospho forms of HspB5. n = 3, * p < 0.05 versus homogenate (PDF 109 kb)

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Schmidt, T., Bartelt-Kirbach, B. & Golenhofen, N. Phosphorylation-dependent subcellular localization of the small heat shock proteins HspB1/Hsp25 and HspB5/αB-crystallin in cultured hippocampal neurons. Histochem Cell Biol 138, 407–418 (2012). https://doi.org/10.1007/s00418-012-0964-x

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