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. 2021 Feb 5;9(1):22.
doi: 10.1186/s40478-021-01123-8.

Non-cell autonomous astrocyte-mediated neuronal toxicity in prion diseases

Rajesh Kushwaha  1   2 , Anshuman Sinha  1   2 , Natallia Makarava  1   2 , Kara Molesworth  1   2 , Ilia V Baskakov  3   4
Affiliations

Non-cell autonomous astrocyte-mediated neuronal toxicity in prion diseases

Rajesh Kushwaha et al. Acta Neuropathol Commun. .

Abstract

Under normal conditions, astrocytes perform a number of important physiological functions centered around neuronal support and synapse maintenance. In neurodegenerative diseases including Alzheimer's, Parkinson's and prion diseases, astrocytes acquire reactive phenotypes, which are sustained throughout the disease progression. It is not known whether in the reactive states associated with prion diseases, astrocytes lose their ability to perform physiological functions and whether the reactive states are neurotoxic or, on the contrary, neuroprotective. The current work addresses these questions by testing the effects of reactive astrocytes isolated from prion-infected C57BL/6J mice on primary neuronal cultures. We found that astrocytes isolated at the clinical stage of the disease exhibited reactive, pro-inflammatory phenotype, which also showed downregulation of genes involved in neurogenic and synaptogenic functions. In astrocyte-neuron co-cultures, astrocytes from prion-infected animals impaired neuronal growth, dendritic spine development and synapse maturation. Toward examining the role of factors secreted by reactive astrocytes, astrocyte-conditioned media was found to have detrimental effects on neuronal viability and synaptogenic functions via impairing synapse integrity, and by reducing spine size and density. Reactive microglia isolated from prion-infected animals were found to induce phenotypic changes in primary astrocytes reminiscent to those observed in prion-infected mice. In particular, astrocytes cultured with reactive microglia-conditioned media displayed hypertrophic morphology and a downregulation of genes involved in neurogenic and synaptogenic functions. In summary, the current study provided experimental support toward the non-cell autonomous mechanisms behind neurotoxicity in prion diseases and demonstrated that the astrocyte reactive phenotype associated with prion diseases is synaptotoxic.

Keywords: Astrocytes; Microglia; Neuroinflammation; Prion diseases; Prions; Synaptic toxicity.

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Conflict of interest statement

The authors declare that they have no competing interest.

Figures

Fig. 1
Fig. 1
Astrocytes isolated from 22L-infected mice exhibit a reactive phenotype. a Analysis of gene expression in 22L-PACs, normalized by the expression of the same genes in CT-PACs, using qRT-PCR. b Representative Western blots and densitometric analysis of GFAP expression normalized per expression of β-actin in CT-PACs and 22L-PACs. c Representative images of CT-PACs and 22L-PACs stained for GFAP, and morphometric analyses of cell area, perimeter and process number in astrocytes from CT-PACs and 22L-PACs. Insets show magnified images. d Analysis of expression of PAN-, A1- and A2-specific genes in 22L-PACs normalized by the expression levels in CT-PACs using qRT-PCR. e Representative images of co-immunostaining of CT-PACs and 22L-PACs for GFAP (green), LCN2 (red) and nuclei (DAPI, blue), and quantification of integrated fluorescence intensity of LCN2 in CT-PACs and 22L-PACs. f Representative Western blots and densitometric analysis of LCN2 expression normalized per expression of β-actin in CT-PACs and 22L-PACs. g Analysis of expression of pro-inflammatory genes in 22L-PACs normalized by the expression levels in CT-PACs using qRT-PCR. h Analysis of IL-6 concentration in media conditioned by CT-PACs and 22L-PACs. In panels a, d and g, Gapdh was used as a housekeeping gene. In panels a–h, data represent means ± SE, n = 3 independent cultures isolated from individual animals, ***p < 0.001, **p < 0.01, *p < 0.05, and ‘ns’ non-significant (two tailed, unpaired t test). Scale bar = 50 µm for panels c and e
Fig. 2
Fig. 2
22L-derived reactive astrocytes show impairment in supporting neuronal growth. Primary cortical neurons were plated on CT-PACs or 22L-PACs and co-cultured for 10–12 days. a Representative fluorescent images of neuron-astrocyte co-cultures co-immunostained for MAP2 (green) and GFAP (red), and quantification of neurite length. b Representative fluorescent images of cortical neuronal cells co-cultured with CT- PACs or 22L-PACs, and co-immunostained for pre- and post-synaptic markers synaptophysin (SYP, green) and PSD95 (red), respectively, and MAP2 (blue). Co-localization between SYP and PSD95 was analyzed for quantification of synapses in neuronal cells co-cultured with CT-PACs or 22L-PACs (yellow puncta). c Representative images of cortical neuronal cells co-cultured with CT-PACs or 22L-PACs, and co-immunostained with a spine marker Drebrin (red) and MAP2 (green). Quantification of spine size and density in neurons co-cultured with CT-PACs and 22L-PACs. In a–c, data represent mean ± SE, n = 3 independent experiments in which CT-PACs or 22L-PACs were isolated individual animals, 50 neurons (in a and b) and 40 neurons (in c) per conditions were analyzed, **p < 0.01 and *p < 0.05 (two tailed, unpaired t test with Welch's correction). Scale bars = 50 µm in a, 25 µm in b, and 10 µm in c
Fig. 3
Fig. 3
Deleterious effects of factors released by 22L-astrocytes on neuronal morphology, viability and expression of functional genes. a Analysis of expression of synaptogenic and neurotrophic genes in 22L-PACs normalized by the expression levels in CT-PACs using qRT-PCR. b Schematic illustration of experiments on treatment of mouse primary cortical neuronal cultures with astrocyte-conditioned media (ACM) collected from CT-PACs and 22L-PACs. c Representative images of primary neuronal cultures isolated from P1–P2 mice and co-immunostained for MAP2 (green), NeuN (red) and DAPI (blue). d Left: representative images of primary neuronal cultures treated with CT-ACM and 22L-ACM for 72 h and co-immunostained for MAP2 (green) and NeuN (red). Right: quantification of neuronal morphology using MAP2 fluorescence in primary neuronal cultures treated with CT-ACM and 22L-ACM for 72 h. e Analysis of expression of Syp, Syn2, Dlg4, Thbs2, Gria1 and Gria4 in primary neuronal cultures treated with 22L-ACM for 72 h and normalized by the expression levels in cultures treated with CT-ACM using qRT-PCR. f Cell viability in primary neuronal cultures assessed by MTT assay as a function of incubation time with CT-ACM or 22L-ACM. In panels a and e, Gapdh was used as housekeeping gene. In panels. a, d–f, data represent means ± SE, n = 3 independent astrocyte or microglia cultures isolated from individual animals, ***p < 0.001, **p < 0.01, *p < 0.05, and ‘ns’ non-significant (two tailed, unpaired student t test). Scale bar = 50 µm
Fig. 4
Fig. 4
Factors released by 22L-derived reactive astrocytes impairs synapse integrity and spine density. a Representative images of primary cortical neurons treated with CT-ACM or 22L-ACM for 72 h and co-immunostained for the pre- and post-synaptic markers synaptophysin (SYP, green) and PSD-95 (red), respectively, and MAP2 (blue). Arrows point at puncta of co-localization of synaptophysin and PSD-95. b Quantification of co-localized puncta in CT-ACM- and 22L-ACM-treated primary neurons. c Representative Western blots and densitometric analysis of synaptophysin (SYP) and PSD-95 expression normalized per expression of β-actin in neuronal cultures treated with CT-ACM or 22L-ACM. d Representative images of neuronal cultures treated with CT-ACM or 22L-ACM for 72 h and co-immunostained for a spine marker Drebrin (red) and MAP2 (green). Arrows point at the secondary dendritic spine branches. Quantification of spine size and density in primary neurons incubated with CT-ACM or 22L-ACM. In b–d, data represent mean ± SE, n = 3 independent experiments in which CT-ACM or 22L-ACM was collected from cultures established form individual animals, 50 neurons (in b) and 40 neurons (in d) were counted for each condition. *p < 0.05 (two tailed, unpaired student t test with Welch's correction). Scale bar = 25 µm for a and 10 µm for d panels
Fig. 5
Fig. 5
22L-derived reactive microglia induce a pro-inflammatory state in primary astrocytes. a Left: representative images of primary astrocyte cultures isolated from adult (200–300 days old) C57 Black mice, treated with CT-MCM or 22L-MCM for 72 h, and co-immunostained for GFAP (green) and nuclei (DAPI, blue). Right: morphometric analysis of astrocytes following treatment of primary cultures with CT-MCM or 22L-MCM and staining for GFAP. Data represent means ± SE, n = 3 independent experiments, **p < 0.01 and *p < 0.05 (two tailed, unpaired student t test with Welch's correction). Scale bar = 50 µm. b and c. Analysis of expression of PAN-, A1- and A2-specific genes (b) and synaptogenic genes (c) in primary astrocyte cultures following treatment with CT-MCM or 22L-MCM for 72 h using qRT-PCR. Gapdh was used as housekeeping gene. Data represent means ± SE, n = 3 independent experiments, i.e. astrocyte cultures isolated from individual animals was treated by CT-ACM or 22L-ACM collected from cultures also established form individual animals, ***p < 0.001, **p < 0.01 and *p < 0.05 and ‘ns’ non-significant (two tailed, unpaired student t test)
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