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. 2018 Feb 16:12:81.
doi: 10.3389/fnins.2018.00081. eCollection 2018.

Silencing of Glucocerebrosidase Gene in Drosophila Enhances the Aggregation of Parkinson's Disease Associated α-Synuclein Mutant A53T and Affects Locomotor Activity

Salema B Abul Khair  1 , Nisha R Dhanushkodi  1 , Mustafa T Ardah  1 , Wenfeng Chen  2 , Yufeng Yang  2 , M Emdadul Haque  1
Affiliations

Silencing of Glucocerebrosidase Gene in Drosophila Enhances the Aggregation of Parkinson's Disease Associated α-Synuclein Mutant A53T and Affects Locomotor Activity

Salema B Abul Khair et al. Front Neurosci. .

Abstract

Background: Mutations in glucocerebrosidase (GBA), a lysosomal enzyme are the most common genetic risk factor for developing Parkinson's disease (PD). We studied how reduced GCase activity affects α-synuclein (α-syn) and its mutants (A30P and A53T) aggregation, neurodegeneration, sleep and locomotor behavior in a fly model of PD. Methods: We developed drosophila with GBA gene knockdown (RNAi) (with reduced GCase activity) that simultaneously expresses either wildtype (WT) or mutants such as A30P or A53T α-syn. Western blot and confocal microscopy were performed to study the α-syn aggregation and neurodegeneration in these flies. We also studied the sleep and locomotor activity of those flies using Drosophila activity monitor (DAM) system. Results: Western blot analysis showed that GBA RNAi A53T α-syn flies (30 days old) had an increased level of Triton insoluble synuclein (that corresponds to α-syn aggregates) compared to corresponding A53T flies without GBA RNAi (control), while mRNA expression of α-syn remained unchanged. Confocal imaging of whole brain staining of 30 days old drosophila showed a statistically significant decrease in neuron numbers in PPL1 cluster in flies expressing α-syn WT, A30P and A53T in the presence GBA RNAi compared to corresponding control. Staining with conformation specific antibody for α-syn aggregates showed an increased number of neurons staining for α-syn aggregates in A53T fly brain with GBA RNAi compared to control A53T flies, thus confirming our protein analysis finding that under decreased GBA enzyme activity, mutant A53T aggregates more than the control A53T without GBA silencing. Sleep analysis revealed decreased total activity in GBA silenced flies expressing mutant A53T compared to both A53T control flies and GBA RNAi flies without synuclein expression. Conclusion: In A53T flies with reduced GCase activity, there is increased α-syn aggregation and dopamine (DA) neuronal loss. This study demonstrates that reduced GCase activity both in the context of heterozygous GBA1 mutation associated with PD and in old age, contribute to increased aggregation of mutant α-syn A53T and exacerbates the phenotype in a fly model of PD.

Keywords: Drosophila; GBA; Parkinson's disease; neurodegeneration; sleep behavior; synuclein.

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Figures

Figure 1
Figure 1
Targeted silencing of drosophila GBA gene using RNAi (dG2/dG2). (A) Eye expression shows phenotypic effect in male and female flies maintained at 25° and 29°C carrying two copies of dG2 (GBA RNAi) with GMR-Gal4 driver compared to two copies of GMR-Gal4 alone. (B) RT-PCR results showing that there is a significant downregulation of GBA in fly lysates of dG1/dG1 and dG2/dG2 (two different clones targeting CG13414, fly homolog of GBA1) under control of GMR-Gal4 driver compared to corresponding GMR-Gal4 control and the corresponding graph shown in (C). On doing other preliminary screenings, we decided to continue this study with dG2/dG2. (D) GCase enzyme activity assay was performed to confirm silencing of GBA enzyme in flies with two copies of dG2 in the presence of two copies of GMR-Gal4 driver. The enzyme activity is expressed as percentage of control. Graph represents mean ± S. E. M for 3 independent experiments. Differences in means compared by Students t-test *** represents p < 0.0001.
Figure 2
Figure 2
Absence of transcriptional changes in α-syn expression in dG2/dG2 RNAi flies. (A) RT-PCR confirms knockdown of GBA1 mRNA in flies with dG2/dG2 (in presence/absence of α-syn-WT, A30P, A53T) with single copy of GMR-Gal4 driver compared to respective control flies. This confirms significant transcriptional downregulation of GBA even in the absence of eye phenotype (in flies with single copy of GMR). Corresponding densitometric analysis of RT-PCR was done by normalizing controls to 100% and experimental flies (dG2/dG2 with/without α-syn) are compared against their respective controls. (B) The effect of GBA silencing on α-syn protein expression, immunoblotting was done by whole tissue lysis extraction method. Further densitometric analysis revealed no change in α-syn protein level in flies with dG2/dG2 expressing α-syn (WT, A30P, and A53T) under GMR-Gal4 drive compared to control flies expressing α-syn (WT, A30P, and A53T) alone. Corresponding densitometric graph where controls (GMR with/without α-syn) normalized to 100% is shown. (C) RT-PCR results show no changes in the transcriptional level of α-syn during GBA silencing. In densitometric analysis of α-syn RT-PCR, controls (GMR with/without α-syn) are normalized to 100% to compare experimental flies (dG2/dG2 with/without α-syn) against their respective controls. Graph represents mean ± S.E.M. for 3 independent experiments. Differences in means were compared by one-way ANOVA followed by the Newman-Keuls Multiple Comparison post hoc test. *** represents p < 0.0001.
Figure 3
Figure 3
Triton-soluble and triton-insoluble α-syn level in dG2/dG2 RNAi flies expressing WT, A30P, and A53T synuclein. Western blot of triton-soluble and insoluble fraction of 0 day (A) and 30 day (B) old fly head lysates probed for α-syn showed increase in triton-insoluble α-syn levels in dG2/dG2 A53Tmutant flies compared to corresponding A53T control flies at 30days. In the corresponding densitometric analysis, controls (GMR with/without α-syn) are normalized to 100% and experimental flies (dG2/dG2 with/without α-syn) are compared against their respective controls. Triton-Insoluble α-syn was also normalized to corresponding triton-soluble α-syn. Graph represents mean ± S.E.M. for 3 independent experiments. Differences in means were compared by one-way ANOVA followed by the Newman-Keuls Multiple Comparison post hoc test. * represents p < 0.05.
Figure 4
Figure 4
Increased neurodegeneration in dG2/dG2 flies expressing WT, A30P, and A53T synuclein. (A) Representative image of a projected Z-series of 30 day-old control fly brain with TH-Gal4 driver stained with anti-Tyrosine Hydroxylase (TH) to identify dopaminergic (DA) neurons. DA neurons within each cluster are indicated by labels. (B) Relative number of DA neurons within the PPL1 cluster of 30 day-old dG2/dG2 RNAi flies under control of TH-Gal4 driver expressing WT, A30P, A53T synuclein (n = 16), compared to age-matched corresponding control flies without dG2/dG2 RNAi (n = 16). There was also significant decrease in number of DA neurons in flies expressing α-syn (WT/A30P/A53T) compared to control TH/+ flies. Differences in means were compared by one-way ANOVA followed by the Newman-Keuls Multiple Comparison post hoc test. ** represents p < 0.001, *p < 0.05. (C) Representative image of projected Z-series of PPL1 cluster in 30 day-old fly brain with under control of TH-Gal4 driver co-stained with anti-TH (green) and anti-syn antibody (red) confirms expression of α-syn in DA neurons in different genotypes. (D) Representative immunofluorescent staining of 30 day-old fly brain expressing WT, A30P, and A53T synuclein (TH-Gal4 UAS transgenes) with/without GBA silencing stained with conformational antibody (anti-α-synuclein filament antibody) for α-syn aggregates.
Figure 5
Figure 5
Decreased activity of dG2/dG2 flies in negative geotaxis assay. (A) Negative geotaxis assay revealed a decreased movement in dG2/dG2; A53/TH compared to A53T/TH and dG2/dG2 control flies at Day0. (B) Climbing Index of old flies (20 and 30 day) normalized to 0 day young flies for each genotype showed a significant decline in activity with age in case of control dG2/dG2 flies. The dG2/dG2 A53T flies did not show prominent decline compared to dG2/dG2 control because the movement is severely compromised in these flies even at day 0. Data represents Mean + S. E. M of 3 independent experiments. Multiple comparisons between means were done by one-way ANOVA followed by Bonferroni correction. Comparison was done between TH/+ vs. dG2/dG2;TH/+, dG2/dG2 vs. dG2/dG2;WT/TH, dG2/dG2; A30P/TH and dG2/dG2;A53T and WT/TH vs. dG2/dG2;WT/TH and A30P vs. dG2/dG2;A30P/TH and A53T vs. dG2/dG2;A53T/TH). *** represents p < 0.0001.
Figure 6
Figure 6
Sleep Behavior in dG2/dG2 GBA RNAi flies expressing wildtype and mutant α-synuclein. Sleep behavior in young flies (0–3days) with and without dG2/dG2 silencing, expressing wildtype (WT), A30P and A53T mutant synuclein using TH-Gal4 is represented as total length of sleep, fragmentation in sleep indicated by sleep bout number, and the average length of sleep in 12 h light (Day) and 12 h dark (Night) cycles (A,B). Activity index represents fly activity level during the wake periods in day, night and in total (C). GBA silenced A53T old flies showed pathogenic symptoms such as increased daytime sleep, with an increased number of bouts (D) and a decreased night sleep with decreased night bout length (E). These flies also show marked reduction in wake activity during day, night and in total (F). Bars represent mean values of at least three independent experiments ± the standard error of the mean from 16 flies that were individually recorded in each experiment conducted using drosophila activity monitor. Differences in means were compared by one-way ANOVA followed by the Newman-Keuls Multiple Comparison post hoc test (between TH/+ vs. dG2/dG2;TH/+, dG2/dG2 vs. dG2/dG2;WT/TH, dG2/dG2; A30P/TH and dG2/dG2;A53T and WT/TH vs. dG2/dG2;WT/TH and A30P vs. dG2/dG2;A30P/TH and A53T vs. dG2/dG2;A53T/TH). *** represents p < 0.0001, **p < 0.001, and *p < 0.05.
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