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Ultrastructural features of aberrant glial cells isolated from the spinal cord of paralytic rats expressing the amyotrophic lateral sclerosis-linked SOD1G93A mutation

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Abstract

In the rat model of amyotrophic lateral sclerosis expressing the G93A superoxide dismutase-1 mutation, motor neuron death and rapid paralysis progression are associated with the emergence of a population of aberrant glial cells (AbAs) that proliferate in the degenerating spinal cord. Targeting of AbAs with anti-neoplasic drugs reduced paralysis progression, suggesting a pathogenic potential contribution of these cells accelerating paralysis progression. In the present study, analyze the cellular and ultrastructural features of AbAs following their isolation and establishment in culture during several passages. We found that AbAs exhibit permanent loss of contact inhibition, absence of intermediate filaments and abundance of microtubules, together with an important production of extracellular matrix components. Remarkably, AbAs also exhibited exacerbated ER stress together with a significant abundance of lipid droplets, as well as autophagic and secretory vesicles, all characteristic features of cellular stress and inflammatory activation. Taken together, the present data show AbA cells as a unique aberrant phenotype for a glial cell that might explain their pathogenic and neurotoxic effects.

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Funding

This study was funded by the Program for the Development of Basic Sciences (PEDECIBA), a shared program from the University of the Republic (UDELAR) and the Ministry of Education and Culture (MEC). JMR, PDA and EI were granted Postgraduate Grants from the National Agency for Innovation and Research (ANII).

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Authors and Affiliations

  1. Instituto de Investigaciones Biológicas Clemente Estable, Ministry of Education and Culture (MEC), Av. Italia 3318, 11600, Montevideo, Uruguay

    Marcie Jiménez-Riani, Pablo Díaz-Amarilla, Eugenia Isasi & Silvia Olivera-Bravo

  2. Transmission Electron Microscopy Unit, School of Sciences, University of the Republic (UdelaR), Iguá 4225, 11400, Montevideo, Uruguay

    Marcie Jiménez-Riani & Gabriela Casanova

  3. Institute Pasteur Montevideo, Mataojo 2020, 11400, Montevideo, Uruguay

    Luis Barbeito

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  1. Marcie Jiménez-Riani
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  2. Pablo Díaz-Amarilla
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  3. Eugenia Isasi
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  4. Gabriela Casanova
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  5. Luis Barbeito
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  6. Silvia Olivera-Bravo
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Correspondence to Silvia Olivera-Bravo.

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Fig. S1

Intermediate filaments, mitochondria and ER in cultured astrocytes (a) Abundant and ordered gliofilaments (f) present in the cytoskeleton of cultured astrocytes processed simultaneously to AbAs. Some ER cisternae are also evident (white arrows). (b) Mitochondria (m) with characteristic features including elongated shapes and well delineated crests as well as gliofilaments (f) that are found in the vicinity of mitochondria and nuclei. (c) Mitochondrial morphology and distribution throughout the whole soma of living neonatal astrocytes were evidenced by incubation with the vital probe Mitotracker green. Note abundant elongated mitochondria widespread throughout the whole AbAs’cytoplasm. (d) Representative ER cisterna (white arrows) of clear content and delineated by abundant ribosomes in astrocytes. Free ribosomes (r) were also frequently observed either isolated or as polisomes. Mitochondria of typical appearance are also observed widespread in the whole image (black arrows). (f-g) Charts show the comparison of the length of mitochondrion main axis and the diameter of ER cross section between astrocytes (AST) and AbAs. Note that AbA cells have significantly shorter mitochondria as well as much more dilated ER cisternae with a diameter that is almost four times those of astrocytes. (*): statistical significant difference at p < 0.05. (JPEG 53 kb)

High resolution image (TIFF 15422 kb)

Fig. S2

ER stress and secretory activity in astrocytes and AbA cells (a) Signals of ATF6α, ATF4 and GRP78/BiP present in astrocytes. (b) Comparison between the mean gray value (MGV, intensity of the signal per area) of ATF6α, ATF4 and GRP78/BiP in AbAs (black columns) compared to astrocytes (white colums). Note the increases of up to 60, 50 and 100%, respectively. (c) Percentage of cells positive to the ER stress markers evidenced that the number of AbAs positive to each marker doubled that of astrocytes. (d-e) Western blottings show the levels of Secretogranin II (SgII) and Chromogranin A (Chr-A) in astrocytes and AbA cells. The semi-quantitative analysis shows significant higher expression in AbAs. (*) indicates statistical signification at p < 0.05. (JPEG 46 kb)

High resolution image (TIFF 15375 kb)

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Jiménez-Riani, M., Díaz-Amarilla, P., Isasi, E. et al. Ultrastructural features of aberrant glial cells isolated from the spinal cord of paralytic rats expressing the amyotrophic lateral sclerosis-linked SOD1G93A mutation. Cell Tissue Res 370, 391–401 (2017). https://doi.org/10.1007/s00441-017-2681-1

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