Glial Sphingosine-Mediated Epigenetic Regulation Stabilizes Synaptic Function in Drosophila Models of Alzheimer's Disease

Abstract

Destabilization of neural activity caused by failures of homeostatic regulation has been hypothesized to drive the progression of Alzheimer's Disease (AD). However, the underpinning mechanisms that connect synaptic homeostasis and the disease etiology are yet to be fully understood. Here, we demonstrated that neuronal overexpression of amyloid β (Aβ) causes abnormal histone acetylation in peripheral glia and completely blocks presynaptic homeostatic potentiation (PHP) at the neuromuscular junction in Drosophila The synaptic deficits caused by Aβ overexpression in motoneurons are associated with motor function impairment at the adult stage. Moreover, we found that a sphingosine analog drug, Fingolimod, ameliorates synaptic homeostatic plasticity impairment, abnormal glial histone acetylation, and motor behavior defects in the Aβ models. We further demonstrated that perineurial glial sphingosine kinase 2 (Sk2) is not only required for PHP, but also plays a beneficial role in modulating PHP in the Aβ models. Glial overexpression of Sk2 rescues PHP, glial histone acetylation, and motor function deficits that are associated with Aβ in Drosophila Finally, we showed that glial overexpression of Sk2 restores PHP and glial histone acetylation in a genetic loss-of-function mutant of the Spt-Ada-Gcn5 Acetyltransferase complex, strongly suggesting that Sk2 modulates PHP through epigenetic regulation. Both male and female animals were used in the experiments and analyses in this study. Collectively, we provided genetic evidence demonstrating that abnormal glial epigenetic alterations in Aβ models in Drosophila are associated with the impairment of PHP and that the sphingosine signaling pathway displays protective activities in stabilizing synaptic physiology.SIGNIFICANCE STATEMENT Fingolimod, an oral drug to treat multiple sclerosis, is phosphorylated by sphingosine kinases to generate its active form. It is known that Fingolimod enhances the cognitive function in mouse models of Alzheimer's disease (AD), but the role of sphingosine kinases in AD is not clear. We bridge this knowledge gap by demonstrating the relationship between impaired homeostatic plasticity and AD. We show that sphingosine kinase 2 (Sk2) in glial cells is necessary for homeostatic plasticity and that glial Sk2-mediated epigenetic signaling has a protective role in synapse stabilization. Our findings demonstrate the potential of the glial sphingosine signaling as a key player in glia-neuron interactions during homeostatic plasticity, suggesting it could be a promising target for sustaining synaptic function in AD.

Keywords: Alzheimer's disease; epigenetic regulation; glia; histone acetylation; presynaptic homeostatic plasticity; sphingosine kinase.

Figure 1.

PHP is disrupted in Drosophila Aβ models. A, Schematic of PHP at the wild-type NMJ in Drosophila (left panel). Pharmacological inhibition of postsynaptic glutamate receptors induces a compensatory increase of presynaptic transmitter release, which offsets the reduction in mEPSP amplitude and recovers the excitation (EPSP) to the baseline value. Glial epigenetic signaling is required for PHP. It remains to be elucidated whether PHP is affected in Drosophila models of AD (right panel). B, Representative EPSP and mEPSP traces in wt (black) and overexpression of Aβ42 (OK371-Gal4>UAS-human-Aβ42, red) in motoneurons, in the absence (–PhTX) and presence (+PhTX) of philanthotoxin. C, mEPSP amplitudes (open bars) and presynaptic release (quantal content, filled bars). Data for each genotype are presented as the percentage change in PhTX compared with the same genotype recorded in the absence of PhTX. Genotypes and sample sizes are as follows: wt (–PhTX, n = 12; +PhTX, n = 9); OK371-Gal4>UAS-human-Aβ42 (Aβ42; –PhTX, n = 10; +PhTX, n = 10); OK371-Gal4>UAS-human-Aβ42^Ar (Aβ42^Ar; –PhTX, n = 13; +PhTX, n = 13); OK371-Gal4>UAS-APP^{Ar, Sw} (APP^{Ar, Sw}; –PhTX, n = 10; +PhTX, n = 10); and OK371-Gal4>UAS-MAPT (MAPT; –PhTX, n = 11; +PhTX, n = 13). Mean ± SEM; ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test were used. Non-normalized raw data were used for statistical tests. D–F, Non-normalized values for genotypes as in C: average mEPSP amplitude (D), EPSP amplitude (E), and presynaptic release (quantal content; F) in the absence (open) and presence (filled bars) of PhTX. Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. The levels of significance when comparing baseline values in the mutants to those in the wild type are labeled at the top in the graph. G, Representative EPSC traces wt (black) and overexpression of Aβ42 (OK371-Gal4>UAS-human-Aβ42, red) in motoneurons, in the absence (–PhTX) and the presence (+PhTX) of philanthotoxin. H, mEPSP amplitudes (open bars) and presynaptic release (apparent quantal content, filled bars). Data for each genotype are presented as the percentage change in PhTX compared with the same genotype recorded in the absence of PhTX. Genotypes and sample sizes are as follows: wt (–PhTX, n = 12; +PhTX, n = 9) and OK371-Gal4>UAS-human-Aβ42 (Aβ42; –PhTX, n = 11; +PhTX, n = 17). Mean ± SEM; ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. Non-normalized raw data were used for statistical tests. I–K, Non-normalized values for genotypes as in H: average mEPSP amplitude (I), EPSC amplitude (J), and presynaptic release (apparent quantal content; K) in the absence (open) and presence (filled bars) of PhTX. Mean ± SEM; ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. L, Representative confocal images of the NMJ immunolabeled with anti-Brp (green, presynaptic), anti-Dlg (red, postsynaptic), and the neuronal membrane (anti-HRP; blue) in wt, OK371-Gal4>UAS-human-Aβ42 (Aβ42), OK371-Gal4>UAS-human-Aβ42^Ar (Aβ42^Ar), and OK371-Gal4>UAS-APP^{Ar, Sw} (APP^{Ar, Sw}). M, L, The total number of presynaptic Brp puncta, total Dlg area, Brp density (Brp number/Dlg area), total number of synaptic boutons at muscle 6/7 in abdominal segment 2 for indicated genotypes (M) as in L. Sample sizes are as follows: wt, n = 4; Aβ42, n = 8; Aβ42^Ar, n = 4; APP^{Ar, Sw}, n = 8. Mean ± SEM; *p < 0.05, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. N, Schematic to show climbing behavioral analysis. The percentage of adult flies (day 25) that climb above 5, 10, 15, and 20 cm lines in 1 min are quantified. O, Quantification of the number of animals that climb above 5, 10, 15, and 20 cm lines in 1 min. Data for each genotype are normalized to the total number of animals used in each trial. Genotypes and sample sizes are as follows: wt (n = 12 trials), OK371-Gal4>UAS-human-Aβ42 (Aβ42, n = 12 trials), OK371-Gal4>UAS-human-Aβ42^Ar (Aβ42^Ar, n = 12 trials). Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test.

Figure 2.

Fingolimod rescues PHP deficits in Drosophila Aβ models. A, Representative EPSP and mEPSP traces in wt (black) and overexpression of Aβ42^Ar (OK371-Gal4>UAS-human-Aβ42^Ar, red) in the motoneuron, in the absence (–PhTX) and presence (+PhTX) of philanthotoxin. Data from Fingolimod (FTY720) treated and untreated conditions for each genotype are shown. B, mEPSP amplitudes (open bars) and presynaptic release (quantal content, filled bars) with and without FTY720 (300 µm) treatment. Data for each genotype are presented as the percentage change in PhTX compared with the same genotype recorded in the absence of PhTX. Genotypes and sample sizes are as follows: wt (–PhTX, n = 12; +PhTX, n = 9), wild type with FTY720 (wt FTY720; –PhTX, n = 10; +PhTX, n = 10), OK371-Gal4>UAS-human-Aβ42 (Aβ42; –PhTX, n = 10; +PhTX, n = 10), OK371-Gal4>UAS-human-Aβ42 with FTY720 (Aβ42 FTY720; –PhTX, n = 11; +PhTX, n = 11), OK371-Gal4>UAS-human-Aβ42^Ar (Aβ42^Ar; –PhTX, n = 13; +PhTX, n = 13), OK371-Gal4>UAS-human-Aβ42^Ar with FTY720 (Aβ42^Ar FTY720; –PhTX, n = 10; +PhTX, n = 9), OK371-Gal4>UAS-APP^{Ar, Sw} (APP^{Ar, Sw}; –PhTX, n = 10; +PhTX, n = 10), and OK371-Gal4>UAS-APP^{Ar, Sw} with FTY720 (APP^{Ar, Sw} FTY720; –PhTX, n = 11; +PhTX, n = 10). Mean ± SEM; ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. C, mEPSP amplitudes (open bars) and presynaptic release (quantal content, filled bars) with different concentrations of FTY720 treatment. Data for each genotype are presented as the percentage change in PhTX compared with the same genotype recorded in the absence of PhTX. Genotypes and sample sizes are as follows: wt (–PhTX, n = 12; +PhTX, n = 9), wild type with 300 µm FTY720 (wt+300 µM FTY720; –PhTX, n = 10; +PhTX, n = 10), OK371-Gal4>UAS-human-Aβ42^Ar (Aβ42^Ar; –PhTX, n = 13; +PhTX, n = 13), OK371-Gal4>UAS-human-Aβ42^Ar with 100 µm FTY720 (Aβ42^Ar+100µM FTY720; –PhTX, n = 12; +PhTX, n = 10), OK371-Gal4>UAS-human-Aβ42^Ar with 300 µm FTY720 (Aβ42^Ar+300µM FTY720; –PhTX, n = 10; +PhTX, n = 9), OK371-Gal4>UAS-human-Aβ42^Ar with 500 µm FTY720 (Aβ42^Ar+500µM FTY720; –PhTX, n = 9; +PhTX, n = 9). Mean ± SEM; **p < 0.01, ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. D–F, Non-normalized data for synaptic transmission in the Aβ models treated with different concentrations of FTY720. The average mEPSP amplitude (D), EPSP amplitude (E), and quantal content (F) are presented as in C. Mean ± SEM; **p < 0.01, ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. G, Representative confocal images of the NMJ immunolabeled with anti-Brp (green, presynaptic), anti-Dlg (red, postsynaptic), and the neuronal membrane (anti-HRP; blue) in wt treated with 300 µm FTY720 (wt+FTY720), OK371-Gal4>UAS-human-Aβ42^Ar (Aβ42^Ar), and OK371-Gal4>UAS-human-Aβ42^Ar treated with 300 µm FTY720 (Aβ42^Ar+FTY720). H, Quantification of the total number of presynaptic Brp puncta, total Dlg area, Brp density (Brp number/Dlg area), total number of synaptic boutons at muscle 6/7 in abdominal segment 2 for indicated genotypes as in C. Sample sizes are as follows: wt, n = 4; wt+FTY720, n = 4; Aβ42, n = 8; Aβ42+FTY720, n = 6; Aβ42^Ar, n = 4; Aβ42^Ar+FTY720, n = 4; APP^{Ar, Sw}, n = 8; APP^{Ar, Sw}+FTY720, n = 8; mean ± SEM; *p < 0.05, **p < 0.01; N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test.

Figure 3.

Fingolimod treatment rescues glial H3K9ac defects, motor function, and eye degeneration in Drosophila Aβ models. A, Representative confocal images of acetylated H3K9 (H3K9ac) in glial nuclei on the peripheral nerves. Glial nuclei (DAPI, green), acetylated H3K9 (H3K9ac, red), and neuronal membrane (HRP, blue) are shown for wt, OK371-Gal4>UAS-human-Aβ42^Ar (Aβ42^Ar), and OK371-Gal4>UAS-APP^{Ar, Sw} (APP^{Ar, Sw}) with (+FTY720) or without (–FTY720) treatment. B, Quantification of the average H3K9ac fluorescence intensity within glial nuclei. Genotypes and sample sizes are as follows: wt (n = 269 nuclei; n = 4 animals); wild type with FTY720 (wt+FTY720, n = 199 nuclei; n = 3 animals); OK371-Gal4>UAS-human-Aβ42 (Aβ42; n = 122 nuclei; n = 4 animals); OK371-Gal4>UAS-human-Aβ42 with FTY720 (Aβ42 FTY720; n = 198 nuclei; n = 4 animals); OK371-Gal4>UAS-human-Aβ42^Ar (Aβ42^Ar; n = 160 nuclei; n = 4 animals); OK371-Gal4>UAS-human-Aβ42^Ar with FTY720 (Aβ42^Ar FTY720; n = 246 nuclei; n = 4 animals); OK371-Gal4>UAS-APP^{Ar, Sw} (APP^{Ar, Sw}; n = 234 nuclei; n = 4 animals); and OK371-Gal4>UAS-APP^{Ar, Sw} with FTY720 (APP^{Ar, Sw} FTY720; n = 210 nuclei; n = 3 animals). Mean ± SEM; **p < 0.01, ***p < 0.001; N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test (Kruskal–Wallis score = 374.0, df = 7). C, Schematic to show climbing behavioral analysis with and without FTY720 treatment. The percentage of adult flies (day 25) that climb above 5, 10, 15, and 20 cm lines in 1 min are quantified. D, Quantification of the percentage of animals that climb above 5, 10, 15, and 20 cm lines in 1 min in the climbing assay. Genotypes and sample sizes are as follows: wt (n = 12 trials), wild type with FTY720 (wt+FTY720; n = 12 trials), OK371-Gal4>UAS-human-Aβ42^Ar (Aβ42^Ar; n = 12 trials), and OK371-Gal4>UAS-human-Aβ42^Ar with FTY720 (Aβ42^Ar+FTY720; n = 12 trials). Mean ± SEM; *p < 0.05, **p < 0.01, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. E, Cumulative percentage of flies with various degrees of eye degeneration (0–4: none to severe degeneration; see Materials and Methods for details). Cumulative percentage of flies with more than 0 (≥0) to more than 4 (≥4) degree of degeneration is shown. Genotypes and sample sizes are as follows: GMR-Gal4>UAS-human-Aβ42^Ar (–FTY720, n = 15; +FTY720, n = 16) and GMR-Gal4>UAS-human-APP^{Ar, Sw} (–FTY720, n = 15; +FTY720, n = 14).

Figure 4.

Sk2 is necessary for the rapid induction and long-term expression of PHP. A, Schematic to show sphingosine kinase-mediated phosphorylation of sphingosine and FTY720 in wild type and Sk2 mutant. B, Superimposed AlphaFold predicted structures of human SPHK2 (blue) and Drosophila Sk2 (orange). rmsd and TM-score are shown. C, Representative EPSP and mEPSP traces in wt (black), Sk1 (green), and Sk2 (blue), in the absence (–PhTX) and the presence (+PhTX) of philanthotoxin. D–G, Normalized data (D) for mEPSP amplitudes (open bars) and presynaptic release (QC; filled bars) and non-normalized data of mEPSP amplitudes (E), EPSP amplitudes (F), and quantal content (G) for wt (–PhTX, n = 48; +PhTX, n = 21), Sk1 (–PhTX, n = 22; +PhTX, n = 13), and Sk2 (–PhTX, n = 27; +PhTX, n = 16). Data for each genotype are presented as the percentage change in PhTX compared with the same genotype recorded in the absence of PhTX in D. Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. H, Representative EPSC traces in wt (black) and Sk2 mutant (Sk2, blue), in the absence (–PhTX) and the presence (+PhTX) of philanthotoxin. I–L, Normalized data (I) for mEPSP amplitudes (open bars) and presynaptic release (QC; filled bars) and non-normalized data of mEPSP amplitudes (J), EPSC amplitudes (K), and apparent quantal content (L) for wt (–PhTX, n = 12; +PhTX, n = 9) and Sk2 mutant (Sk2; –PhTX, n = 10; +PhTX, n = 11). Data for each genotype are presented as the percentage change in PhTX compared with the same genotype recorded in the absence of PhTX in I. Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. M, Representative EPSP and mEPSP traces in the wt (gray), GluRIIA mutant (GluRIIA, black), Sk2 (light purple), and GluRIIA;Sk2 double mutant (GluRIIA;Sk2). N–Q, Normalized data (N) for mEPSP amplitudes (open bars) and presynaptic release (QC; filled bars) and non-normalized data of mEPSP amplitudes (O), EPSP amplitudes (P), and quantal content (Q) for wt (n = 28), GluRIIA mutant (GluRIIA, n = 18), Sk2 (n = 10), and GluRIIA;Sk2 double mutant (GluRIIA;Sk2, n = 10). Mean ± SEM; *p < 0.05, ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test.

Figure 5.

Sk2 functions in perineurial glia for PHP. A, Representative EPSP and mEPSP traces in the absence (–PhTX) and the presence (+PhTX) of philanthotoxin. Genotypes are as follows: wt (black), OK371-Gal4>UAS-Sk2 RNAi (Motoneuron Sk2 RNAi, orange), BG57-Gal4>UAS-Sk2 RNAi (Muscle Sk2 RNAi, red), and NP6293-Gal4>UAS-Sk2 RNAi (Glial Sk2 RNAi, blue). B–E, Normalized data (B) for mEPSP amplitudes (open bars) and presynaptic release (QC; filled bars), and non-normalized data of mEPSP amplitudes (C), EPSP amplitudes (D), and quantal content (E) for wt (–PhTX, n = 32, +PhTX, n = 17), NP6293-Gal4/+ (Glial Gal4/+; –PhTX, n = 10; +PhTX, n = 9), OK371-Gal4>UAS-Sk2 RNAi #1 (Motoneuron Sk2 RNAi #1; –PhTX, n = 13; +PhTX, n = 11), BG57-Gal4>UAS-Sk2 RNAi (Muscle Sk2 RNAi #1; –PhTX, n = 10; +PhTX, n = 10), NP6293-Gal4>UAS-Sk2 RNAi (Glial Sk2 RNAi #1; –PhTX, n = 19; +PhTX, n = 13), and NP6293-Gal4>UAS-Sk2 RNAi #2 (Glial Sk2 RNAi #2; –PhTX, n = 10; +PhTX, n = 8). Data for each genotype are presented as the percentage change in PhTX compared with the same genotype recorded in the absence of PhTX in B. Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. F–I, Normalized data (F) for mEPSP amplitudes (open bars) and presynaptic release (QC; filled bars), and non-normalized data of mEPSP amplitudes (G), EPSP amplitudes (H), and quantal content (I) for wt (–PhTX, n = 48; +PhTX, n = 24), Sk2 (–PhTX, n = 27; +PhTX, n = 17), and NP6293-Gal4>UAS-Sk2;Sk2 (Sk2;Glial rescue; –PhTX, n = 11; +PhTX, n = 10). Data for each genotype are presented as the percentage change in PhTX compared with the same genotype recorded in the absence of PhTX in F. Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. J, Perineurial glia cell clusters are shown in t-SNE (t-distributed stochastic neighbor embedding) plots using a published scRNA-seq dataset from Drosophila adult brains (57,000 cells; as published in Davie et al., 2018; GSE107451). The perineurial glia cluster is highlighted in red (left panel). Individual perineurial glia expressing Sk1 (green) or Sk2 (blue) are shown on the right. K, L, The total number of perineurial glia expressing Sk1 (green) and Sk2 (blue; K), and the average expression level of Sk1 (green) and Sk2 (blue; L) in perineurial glia. CPM, Counts per million.

Figure 6.

Glial-specific expression of Sk2 rescues PHP, H3K9ac, and motor deficits in Drosophila Aβ models. A, Representative EPSP and mEPSP traces in the absence (–PhTX) and the presence (+PhTX) of philanthotoxin in wt, wt with FTY720 (wt+FTY720), Sk2 mutant (Sk2), and Sk2 mutant with FTY720 (Sk2+FTY720). B, mEPSP amplitudes (open bars) and presynaptic release (QC; filled bars) in genotypes as in A. Data for each genotype are presented as the percentage change in PhTX compared with the same genotype recorded in the absence of PhTX. Sample sizes are as follows: wt (–PhTX, n = 28; +PhTX, n = 15), wild type with FTY720 (wt FTY720; –PhTX, n = 10; +PhTX, n = 10), Sk2 mutant (Sk2; –PhTX, n = 17; +PhTX, n = 13), and Sk2 mutant with FTY720 (Sk2 FTY720; –PhTX, n = 10; +PhTX, n = 10). Mean ± SEM; ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. C, mEPSP amplitudes (open bars) and presynaptic release (quantal content, filled bars) for glial-specific overexpression of Sk1 and Sk2 in the wild-type background. Data for each genotype are presented as the percentage change in PhTX compared with the same genotype recorded in the absence of PhTX. Genotypes and sample sizes: wt (–PhTX, n = 28; +PhTX, n = 15), NP6293-Gal4>UAS-Sk1 (Glial UAS-Sk1; –PhTX, n = 12; +PhTX, n = 10), NP6293-Gal4>UAS-Sk2 (Glial UAS-Sk2; –PhTX, n = 11; +PhTX, n = 11). Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. D, Non-normalized data of mEPSP amplitudes (left panel), EPSP amplitudes (middle panel), and quantal content (right panel) for genotypes shown in A and B. Mean ± SEM; *p < 0.05, ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. E, Non-normalized data of mEPSP amplitudes (left panel), EPSP amplitudes (middle panel), and quantal content (right panel) for genotypes shown in C. Mean ± SEM; ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. F, Schematic to show neuronal overexpression of Aβ42 and glial overexpression of Sk2 at the same synapse. G, Representative EPSP and mEPSP traces in the absence (–PhTX) and the presence (+PhTX) of philanthotoxin in heterozygous nSyb-QF2/+ (Neural QF/+), nSyb-QF2>QUAS-human-Aβ42 (Neural Aβ42), and nSyb-QF2>QUAS-human-Aβ42;NP6293-Gal4>UAS-Sk2 (Neural Aβ42+Glial Sk2 rescue). H, mEPSP amplitudes (open bars) and presynaptic release (QC; filled bars) for wt (–PhTX, n = 32; +PhTX, n = 15), heterozygous nSyb-QF2/+ (Neural QF/+; –PhTX, n = 11; +PhTX, n = 9), nSyb-QF2>QUAS-human-Aβ42 (Neural hAβ42; –PhTX, n = 12; +PhTX, n = 14), nSyb-QF2>QUAS-human-Aβ42;UAS-Sk2/+ (Neural hAβ42;UAS-Sk2/+; –PhTX, n = 10; +PhTX, n = 8), and nSyb-QF2>QUAS-human-Aβ42;NP6293-Gal4>UAS-Sk2 (Neural hAβ42;Glial Sk2; –PhTX, n = 11; +PhTX, n = 9). Data for each genotype are presented as the percentage change in PhTX compared with the same genotype recorded in the absence of PhTX. Mean ± SEM; **p < 0.01, ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. I–K, Non-normalized data of mEPSP amplitudes (I), EPSP amplitudes (J), and quantal content (K) for genotypes presented in H. Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. L, Representative confocal images of acetylated H3K9 (H3K9ac) in peripheral glial nuclei on the peripheral nerves. Peripheral glial nuclei (DAPI, green), acetylated H3K9 (H3K9ac, red), and neuronal membrane (HRP, blue) are shown for the wt, nSyb-QF2>QUAS-human-Aβ42 (Neural QF>QUAS-hAβ42), nSyb-QF2>QUAS-human-Aβ42;NP6293-Gal4/+ (Neural QF>QUAS-hAβ42;Glial-Gal4/+), and nSyb-QF2>QUAS-human-Aβ42;NP6293-Gal4>UAS-Sk2 (Neural QF>QUAS-hAβ42;Glial-Gal4>UAS-Sk2). M, Average H3K9ac fluorescence intensity within peripheral glial nuclei. Genotypes are shown as in L. Sample sizes are as follows: wt: n = 429 nuclei; n = 5 animals; Neural QF>QUAS-hAβ42: n = 429 nuclei; n = 5 animals; Neural QF>QUAS-hAβ42;Glial-Gal4/+: n = 192 nuclei; n = 6 animals; Neural QF>QUAS-hAβ42;Glial-Gal4>UAS-Sk2: n = 276 nuclei; n = 6 animals. Mean ± SEM; ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test (Kruskal–Wallis score = 98.25; df = 3). N, The number of adult animals (day 23–25) that climb above 5, 10, 15, and 20 cm lines in 1 min in the climbing assay. Data for each genotype are normalized to the total number of animals used in each trial. Genotypes and sample sizes: wt, n = 21 trials; Neural QF>QUAS-hAβ42, n = 59 trials, Neural QF>QUAS-hAβ42;NP6293-Gal4/+, n = 56 trials, Neural QF>QUAS-hAβ42;NP6293-Gal4>UAS-Sk2, n = 47 trials. Mean ± SEM; ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. O, The total numbers of astrocyte, microglia, OPC, and oligodendrocyte that express SPHK1 (green) and SPHK2 (blue) using a published human scRNA-seq dataset (Hodge et al., 2019). P, Quantification of the average expression level of human SPHK1 (green) and SPHK2 (blue) in astrocyte, microglia, OPC, and oligodendrocyte using a published human scRNA-seq dataset (Hodge et al., 2019). CPM, Counts per million. Q, The mean expression level of SPHK1 and SPHK2 in nonpathological (no patho) and pathological (patho; AD) from human brain samples as published in Mathys et al. (2019).

Figure 7.

Fingolimod and glial-specific expression of Sk2 rescues PHP and H3K9ac in a SAGA complex mutant. A, Schematic to show histone acetylation is reduced in the ada2b (a SAGA component) mutant (bottom left) compared with wild type (top left). Sphingosine kinase-mediated phosphorylation of sphingosine and FTY720 can modulate histone acetylation (right panel). B, Representative EPSP and mEPSP traces in the absence (–PhTX) and the presence (+PhTX) of philanthotoxin in wt, ada2b mutant (ada2b), ada2b with FTY720 (ada2b+FTY720), and ada2b;NP6293-Gal4>UAS-Sk2 (ada2b;Glial UAS-Sk2). C–F, Normalized data (C) for mEPSP amplitudes (open bars) and presynaptic release (QC; filled bars), and non-normalized data of mEPSP amplitudes (D), EPSP amplitudes (E), and quantal content (F) for wt (–PhTX, n = 28; +PhTX, n = 15), ada2b (–PhTX, n = 10; +PhTX, n = 10), ada2b with FTY720 (ada2b+FTY720; –PhTX, n = 10; +PhTX, n = 9), and ada2b;NP6293-Gal4>UAS-Sk2 (ada2b;Glial UAS-Sk2; –PhTX, n = 9; +PhTX, n = 10). Data for each genotype are presented as the percentage change in PhTX compared with the same genotype recorded in the absence of PhTX in C. Mean ± SEM; **p < 0.01, ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. G, Representative confocal images of acetylated H3K9 (H3K9ac) in perineurial glial nuclei on the peripheral nerves. Perineurial glial nuclei (NP6293-Gal4>UAS-Redstinger.nls, green), acetylated H3K9 (H3K9ac, red), and neuronal membrane (HRP, blue) are shown for NP6293-Gal4>UAS-Redstinger.nls (wt), ada2b;NP6293-Gal4>UAS-Redstinger.nls (ada2b), and NP6293-Gal4>UAS-Redstinger.nls;ada2b with FTY720 (ada2b+FTY720). H, Average H3K9ac fluorescence intensity within perineurial glial nuclei (left panel) and all peripheral glial nuclei (right panel) for genotypes as in G. Sample sizes for perineurial glia: wt: n = 310 nuclei; n = 4 animals; ada2b: n = 203 nuclei; n = 3 animals; ada2b+FTY720: n = 98 nuclei; n = 3 animals. Kruskal–Wallis score = 281.6, df = 2. Sample sizes for all peripheral glia are as follows: wt: n = 439 nuclei; n = 4 animals; ada2b: n = 361 nuclei; n = 3 animals; ada2b+FTY720: n = 228 nuclei; n = 3 animals. Kruskal–Wallis score = 362.5, df = 2. Mean ± SEM; ***p < 0.001, N.S. not significant; Kruskal–Wallis test with post hoc Dunn's test. I, Representative confocal images of acetylated H3K9 (H3K9ac) in glial nuclei on the peripheral nerves. Glial nuclei (DAPI, green), acetylated H3K9 (H3K9ac, red), and neuronal membrane (HRP, blue) are shown for ada2b (ada2b) and ada2b;NP6293-Gal4>UAS-Sk2 (ada2b;Glial Sk2). J, Average H3K9ac fluorescence intensity within peripheral glial nuclei for genotypes as in I. Sample sizes are as follows: ada2b, n = 204 nuclei; n = 3 animals; ada2b;Glial-Gal4>UAS-Sk2; n = 427 nuclei; n = 3 animals. U score = 15815. Mean ± SEM; ***p < 0.001; Mann–Whitney U test.

Figure 8.

Glial sphingosine signaling modulates PHP in Drosophila Aβ models. Neuronal overexpression of Aβ results in decreased histone acetylation in peripheral glia, leading to a disruption of glial epigenome-dependent downstream signaling (right side of the figure). This aberrant histone acetylation and subsequent transcriptomic alterations in glia impair PHP, resulting in synapse destabilization. Conversely, glial Sk2 can activate either endogenous sphingosine or exogenous FTY720, which act as HDAC inhibitors, thereby restoring histone acetylation and homeostatic plasticity in Drosophila models of AD (left side of the figure). We propose that glial epigenome-mediated processes, including the synthesis and secretion of signaling factors, are required for glial–neuron interactions, thus enabling PHP and synapse stabilization.