Feeding of ambroxol ameliorates neurodevelopmental defects and ER stress in the mutated hGBA induced <i>Drosophila</i> eye.

<p>Ambroxol can recover morphological defects and decrease ER stress in transgenic flies. (A) Less fluorescence emitted by the eye imaginal discs of hGBA^RecNciI transgenic combinations treated with, than without 1 mM Ambroxol. (B) Values generated by different transgenic combinations at fixed quantities of fluorescence intensity (n = 12–43 eye imaginal discs of third instar larvae per transgenic combination). Error bars represent SE. *Significant difference compared with controls (all without Ambroxol) (***P<0.001; Student's t test). (C) Ambroxol (1 mM) decreases expression levels of dBiP mRNA in the heads of hGBA^RecNciI transgenic combinations (n  =  about 30 fly heads per transgenic combination). Internal control was dRpL32. Error bars represent SE. (D) Eye phenotypes of hGBA^RecNciI transgenic combinations incubated without or with 1 mM Ambroxol. Size and shape of ocelli were uniform, and layout uniformity was more similar to that of normal fly eyes treated with 1 mM Ambroxol. (E) Size histograms of ocelli in hGBA^RecNciI transgenic combinations treated with or without 1 mM Ambroxol. (n = 6–10 flies per transgenic combination; about 400 ocelli each). Dispersion analysis showed significant differences from hGBA^RecNciI transgenic combinations treated with and without 1 mM Ambroxol (F = 2.07–3.35; P<0.001; Levene's test).</p

Generation of transgenic flies carrying hGBA variants.

<p>(A) Sequence of hGBA. Blue and red fonts show R120W and RecNciI mutations, respectively. (B) Expression levels of hGBA mRNA confirmed by quantitative RT-PCR (n  =  about 30 fly heads per transgenic combination) with dRpL32 as internal control. Error bars represent SE. (C) Levels of hGBA protein confirmed by Western blotting (n  =  about 100 fly heads per transgenic combination). Total amounts of hGBA protein were decreased in hGBA^R120W, and significantly decreased in hGBA^RecNciI transgenic combinations, compared with hGBA^WT transgenic combination.</p

Neurodevelopmental defects in the <i>Drosophila</i> eye caused by expression of hGBA carrying the RecNciI mutation.

<p>We investigated the effects of overexpression to mutated hGBAs in fly eyes. (A) Phenotype of eyes overexpressing hGBA^WT transgenic combination do not significantly differ from those of GMR control. Phenotype of eyes overexpressing hGBA^R120W transgenic combinations occasionally differed in terms of morphology in some flies compared with control. Eye morphology is obviously affected in hGBA^RecNciI transgenic combinations compared with control. (B) Size histograms of ocelli in transgenic combinations (n = 3–5 flies each, about 100 ocelli each). Dispersion analysis showed obvious differences in variance of the sizes of ocelli between the hGBA^RecNciI transgenic combinations and the GMR control (F = 29.50–37.19; P<0.001; Levene's test).</p

Endoplasmic reticulum (ER) stress detected in the mutated hGBA induced <i>Drosophila</i> eye.

<p>We used xbp1-EGFP as an ER stress marker in which EGFP is expressed in frame only after ER stress <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069147#pone.0069147-Ryoo1" target="_blank">[31]</a>. (A) Weak fluorescence is generated in eye imaginal discs expressing the hGBA^WT construct. The eye imaginal discs of hGBA^R120W transgenic combinations emitted more fluorescence than that of hGBA^WT transgenic combination. The eye imaginal discs of hGBA^RecNciI transgenic combinations emitted the most intense fluorescence. (B) Values generated by different transgenic combinations with fixed quantities of fluorescence intensity (n = 7–15 eye imaginal discs from third instar larvae per transgenic combination). Error bars represent SE. *Significant difference compared with values from GMR control (***P<0.001; Student's t test). (C) Endoplasmic reticulum stress marker gene, dBiP (major ER chaperone) mRNA expression in hGBA^R120W and hGBA^RecNciI transgenic combinations was upregulated (n  =  about 30 fly heads per transgenic combination). Internal control was dRpL32. Error bars represent SE. *Significant difference compared with GMR control (*P<0.05; Student's t test). (D) High levels of hGBAs are expressed in whole bodies of heat-shocked flies. Expression levels of dBiP mRNA of hGBA^R120W and hGBA^RecNciI transgenic combinations were also upregulated (n  =  about 30 flies per transgenic combination). Internal control was dRpL32. Error bars represent SE. *Significant difference compared with hs control (*P<0.05; **P<0.01; ***P<0.001; Student's t test).</p

Primer sequences for Quantitative RT-PCR.

<p>Human GBA primers were designed at Universal Probe Library Assay Design Center (Roche Applied Science).</p><p>Primers for dBiP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069147#pone.0069147-Plongthongkum1" target="_blank">[32]</a> and dRpL32 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069147#pone.0069147-Takehana1" target="_blank">[35]</a> were as described in respective citations.</p