Glial α-synuclein promotes neurodegeneration characterized by a distinct transcriptional program in vivo

Abstract

α-Synucleinopathies are neurodegenerative diseases that are characterized pathologically by α-synuclein inclusions in neurons and glia. The pathologic contribution of glial α-synuclein in these diseases is not well understood. Glial α-synuclein may be of particular importance in multiple system atrophy (MSA), which is defined pathologically by glial cytoplasmic α-synuclein inclusions. We have previously described Drosophila models of neuronal α-synucleinopathy, which recapitulate key features of the human disorders. We have now expanded our model to express human α-synuclein in glia. We demonstrate that expression of α-synuclein in glia alone results in α-synuclein aggregation, death of dopaminergic neurons, impaired locomotor function, and autonomic dysfunction. Furthermore, co-expression of α-synuclein in both neurons and glia worsens these phenotypes as compared to expression of α-synuclein in neurons alone. We identify unique transcriptomic signatures induced by glial as opposed to neuronal α-synuclein. These results suggest that glial α-synuclein may contribute to the burden of pathology in the α-synucleinopathies through a cell type-specific transcriptional program. This new Drosophila model system enables further mechanistic studies dissecting the contribution of glial and neuronal α-synuclein in vivo, potentially shedding light on mechanisms of disease that are especially relevant in MSA but also the α-synucleinopathies more broadly.

Keywords: Drosophila; Parkinson's disease; glia; multiple system atrophy; α-Synuclein.

FIGURE 1

Glial α-synuclein impairs locomotion. Flies were subjected to a gentle tapping stimulus followed by a 15-s delay. The percentage of flies still in motion (% locomotion) following the delay was recorded and averaged over six technical replicates. Symbols above the “Glia” and “Both” curves represent statistically significant difference compared to the “Control” and “Neurons” curves, respectively, at a given time point. Slope of the line was determined by linear regression analysis and was also globally statistically significantly different between the four conditions. *p < .05, **p < .01, ***p < .005. n = minimum of 60 flies per genotype per time point (six biological replicates of 10 flies each)

FIGURE 2

Glial α-synuclein causes constipation. Flies were aged to 10 days of life, transferred to blue colored food for 24 hr, then returned to regular food. (a) Photographs were taken at 0, 2, and 4 hr after return to regular food. (b) The % of blue to total fecal matter was counted on an hourly basis for 8 hr after return to regular food (left). Area under the curve is statistically significantly different between conditions as measured by one-way ANOVA (right). **p < .01, ****p < .001. n = minimum six biological replicates of 10 flies each

FIGURE 3

Glial α-synuclein causes neurodegeneration. (a) Optic lobe sections stained with hematoxylin demonstrating vacuolization, an indicator of neurodegeneration. Glial α-synuclein caused infrequent large vacuoles (arrowhead) whereas neuronal α-synuclein caused frequent small vacuoles (arrows). Scale bar = 100 μm. (b) Representative anterior medulla sections stained with DAPI (blue) and tyrosine hydroxylase antibody (red, mouse, 1:200, Immunostar) to indicate dopaminergic neurons. Scale bar = 5 μm. (c) Quantification of total neurons from hematoxylin stained slides of anterior medulla (not shown), n = 6 replicates per genotype. (d) Quantification of dopaminergic neurons from anterior medulla, n = 6 replicates per genotype. *p < .05, **p < .01, ***p < .005, ****p < .001, determined with one-way ANOVA

FIGURE 4

α-Synuclein aggregates in neurons and glia. (a) Immunofluoresence for DAPI (blue), elav (red, mouse 1:5, DSHB), and α-synuclein (green, rabbit 1:1000) by confocal microscopy (3 μm scale). Arrows indicate α-synuclein inclusions in neurons. (b) Immunofluoresence for DAPI (blue), repo (red, mouse 1:5, DSHB), and α-synuclein (green, rabbit 1:1000) by confocal microscopy (3 μm scale). Arrows indicate α-synuclein inclusions in glia. (c) Quantification of total aggregates from optic lobe cortex, n = 5–six flies per genotype. (d) Representative immunofluorescence for tyrosine hydroxylase (red, mouse, 1:200, Immunostar), α-synuclein (green, rat, 1:10,000, Biolegend), and DAPI. Scale bar = 5 μm. Inclusions are quantified in the right panel

FIGURE 5

Transcriptional changes induced by α-synuclein depend on its cellular context. Bulk RNA-Seq from whole brains was performed on 10-day old flies. (a) Volcano plots demonstrating transcript expression changes. Colored dots (and numbers) represent statistically significant (p_adjust < .05 after correction for multiple comparisons) differentially expressed transcripts with ≥|1| log2fold change. (b) Venn diagram demonstrating little overlap between differentially expressed genes induced by glial and neuronal α-synuclein. (c) Venn diagram demonstrating significant similarity in differentially expressed genes with both glial and neuronal α-synuclein compared to neuronal α-synuclein alone. (d) When both glial and neuronal α-synuclein are present a common set of transcripts are further downregulated as compared to neuronal α-synuclein alone

FIGURE 6

Transcriptional changes induced by glial α-synuclein include upregulation of proteolysis and cell surface receptor signaling. Bulk RNA-Seq from whole brains was performed on 10-day old flies. (a) Gene ontology analysis for upregulated transcripts demonstrates enrichment of the terms “proteolysis” and “cell surface receptor signaling”. (b) Hierarchical clustering of proteolysis and cell surface receptor signaling related transcripts

FIGURE 7

Confirmation of transcriptional changes induced by neuronal α-synuclein. (a) Gene ontology analysis. *Other reproduction-related terms beyond “post-mating behavior” were also enriched (Data file S2). (b) qRT-PCR for male and lipid-related genes. Values in Neuron and Both are normalized to Control. n = 2–3 biological replicates. (c) Visualization of selected Acp and Sfp gene expression in single cell transcriptome atlas. The dot plot represents expression. For both Sfp77f and Acp53c14b, there is a high and low expressing population, indicated by dots that are the same color but different intensity. (d) Hierarchical clustering of lipid related genes. All genes were significantly differentially expressed with adjusted p-value < .05

FIGURE 8

Fatty acid metabolism and cytoskeletal genes downregulated by glial and neuronal α-synuclein. (a) Hierarchical clustering of fatty acid metabolism and peroxisome related genes. (b) Hierarchical clustering of cytoskeletal related genes. The top six that cluster together are those that contribute to the GO terms “myofibril assembly” and “muscle alpha-actinin binding,” whereas the lower four contribute to the GO term “sperm flagellum assembly”

FIGURE 9

Drosophila RNAseq identifies conserved targets and essential pathways in α-synucleinopathy pathogenesis. Mammalian orthologs of Drosophila genes were identified using DRSC Integrative Ortholog Prediction Tool. Orthologs have a ranked score from 1 to 14 indicating the degree of conservation (with 14 being the best). Orthologs that have been previously reported in MSA transcriptomic studies or in human α-synucleinopathy genome wide association studies (GWAS) or whole exome sequencing (WES) studies are shown. The type of evidence is indicated by the color of the circle. Human genetics, human α-synucleinopathy GWAS or WES; Human expr, expression is changed in human MSA patients; Model org expr, expression is changed in a mouse model of MSA; Cell culture expr, expression is changed in a rat oligodendrocyte model of MSA. Orthologs fall into nine pathways of known relevance to human α-synucleinopathies