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. 2020 Sep 8;10(9):183.
doi: 10.3390/life10090183.

Extracellular Alpha-Synuclein Promotes a Neuroinhibitory Secretory Phenotype in Astrocytes

Bruno Di Marco Vieira  1 , Rowan A W Radford  2 , Junna Hayashi  1 , Emma D Eaton  3 , Ben Greenaway  1 , Mark Jambas  1 , Eugen B Petcu  4 , Roger S Chung  2   3 , Dean L Pountney  1
Affiliations

Extracellular Alpha-Synuclein Promotes a Neuroinhibitory Secretory Phenotype in Astrocytes

Bruno Di Marco Vieira et al. Life (Basel). .

Abstract

Multiple system atrophy (MSA) and dementia with Lewy bodies (DLB) are α-synucleinopathies that exhibit widespread astrogliosis as a component of the neuroinflammatory response. Munc18, a protein critical to vesicle exocytosis, was previously found to strongly mark morphologically activated astrocytes in brain tissue of MSA patients. Immunofluorescence of MSA, DLB and normal brain tissue sections was combined with cell culture and co-culture experiments to investigate the relationship between extracellular α-synuclein and the transition to a secretory astrocyte phenotype. Increased Munc18-positive vesicles were resolved in activated astrocytes in MSA and DLB tissue compared to controls, and they were also significantly upregulated in the human 1321N1 astrocytoma cell line upon treatment with α-synuclein, with parallel increases in GFAP expression and IL-6 secretion. In co-culture experiments, rat primary astrocytes pretreated with α-synuclein inhibited the growth of neurites of co-cultured primary rat neurons and upregulated chondroitin sulphate proteoglycan. Taken together, these results indicate that the secretory machinery is significantly upregulated in the astrocyte response to extracellular α-synuclein and may participate in the release of neuroinhibitory and proinflammatory factors in α-synucleinopathies.

Keywords: DLB; MSA; Munc18; astrocytes; astrogliosis.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Primary rat astrocyte increased expression of GFAP and CSPG upon α-syn treatments. (A–D) WT and mutant α-syn treatment and GFAP expression in primary rat astrocytes. GFAP expression in primary rat astrocytes was measured in controls and compared to different α-syn forms tested at three concentrations. (A) WT α-syn compared to vehicle-only control. (B) Mutant A53T α-syn compared to vehicle-only control. (C) Mutant A46K α-syn compared to vehicle-only control. (D) Mutant A30P α-syn compared to vehicle-only control. (E,F) CSPG immunofluorescence of rat primary astrocytes treated with exogenous mutant A53T α-syn and (F) vehicle-only control. (G) CSPG ELISA assays of primary rat astrocytes treated with exogenous WT α-syn, mutant A53T α-syn, vehicle-only control and IFN-γ positive control. **, p, 0.01; *, p, 0.05.
Figure 2
Figure 2
Human 1321N1 astrocytes treated with WT α-syn show increased GFAP expression, activated morphology and IL-6 secretion. (A,B) Vehicle-treated and α-syn-treated 1321N1 cells. (C) GFAP immunofluorescence mean cytoplasmic intensity. (D,E) DIC images of control and α-syn-treated 1321N1 cells. (F) IL-6 concentration in 1321N1 conditioned medium with and without α-syn treatment. Error bars represent ± SEM. **, p, 0.01; *, p, 0.05.
Figure 3
Figure 3
Munc18-positive vesicles in astrocytes in dementia with Lewy bodies (DLB), multiple system atrophy (MSA) and normal tissue. (A–C) Representative images of Munc18 (red) and α-syn (green) immunofluorescence in normal tissue (A), MSA (B) and DLB (C), showing high expression in MSA and DLB tissue compared to normal tissue (frontal cortex) and intense perinuclear punctate expression (normal arrow) in the vicinity of glial cytoplasmic inclusions (GCIs) (small arrows). (D,E) Enlarged views of MSA tissue. DAPI (blue) nuclear stain. (F,G) Example Munc18 (red) expression in MSA visual cortex, illustrating the intense punctate expression (thick arrow) in activated astrocytes observed in areas of high GCI (normal arrows) pathology. (H–J) Enlarged views of DLB tissue.
Figure 4
Figure 4
Munc18-positive vesicles increased in α-syn-treated 1321N1 cells. (A,B) Representative immunofluorescence of control vehicle-only and α-syn-treated 1321N1 cells at 3 h. (C) Enlarged area. (D,E) Representative image outputs from Columbus software showing an increase of Munc18-positive vesicles (in red) in the α-syn-treated group of cells at 3 h. Vesicle positivity is established based on inclusion criteria (cytoplasmic location and signal intensity). The nuclear region is not considered for analysis of Munc18. Red dots are Munc18 vesicles. The nuclear region is blue and cytoplasm is outlined in green. (F) Graph of means ± SEM of Munc18-positive vesicles between controls and α-syn treated groups at 3 h. A significant increase (1.7-fold) in the number of positively marked vesicles is observed in the treated group when compared to controls. (G,H) Representative immunofluorescence of control vehicle-only and α-syn-treated 1321N1 cells at 24 h. (I) Enlarged area. ***, p, 0.001.
Figure 5
Figure 5
Neuron and astrocyte co-culture show astrocyte activation and neuronal inhibition. (A,B) Co-culture of rat primary neurons and astrocytes without (A) and with α-syn A53T pretreatment of astrocytes (B). (C) Quantification of neurite outgrowth of neurons plated onto control, α-syn WT, α-syn A53T and IFNγ pretreated astrocytes. **, p, 0.01; *, p, 0.05.
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