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Aberrant lysosomal carbohydrate storage accompanies endocytic defects and neurodegeneration in Drosophila benchwarmer.

Dermaut B, Norga KK, Kania A, Verstreken P, Pan H, Zhou Y, Callaerts P, Bellen HJ - J. Cell Biol. (2005)

Bottom Line: Here, we report that loss of Drosophila benchwarmer (bnch), a predicted lysosomal sugar carrier, leads to carbohydrate storage in yolk spheres during oogenesis and results in widespread accumulation of enlarged lysosomal and late endosomal inclusions.Finally, we find that loss of bnch strongly enhances tau neurotoxicity in a dose-dependent manner.We hypothesize that, in bnch, defective lysosomal carbohydrate efflux leads to endocytic defects with functional consequences in synaptic strength, neuronal viability, and tau neurotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
Lysosomal storage is the most common cause of neurodegenerative brain disease in preadulthood. However, the underlying cellular mechanisms that lead to neuronal dysfunction are unknown. Here, we report that loss of Drosophila benchwarmer (bnch), a predicted lysosomal sugar carrier, leads to carbohydrate storage in yolk spheres during oogenesis and results in widespread accumulation of enlarged lysosomal and late endosomal inclusions. At the bnch larval neuromuscular junction, we observe similar inclusions and find defects in synaptic vesicle recycling at the level of endocytosis. In addition, loss of bnch slows endosome-to-lysosome trafficking in larval garland cells. In adult bnch flies, we observe age-dependent synaptic dysfunction and neuronal degeneration. Finally, we find that loss of bnch strongly enhances tau neurotoxicity in a dose-dependent manner. We hypothesize that, in bnch, defective lysosomal carbohydrate efflux leads to endocytic defects with functional consequences in synaptic strength, neuronal viability, and tau neurotoxicity.

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Abnormal membranous structures and presynaptic endocytic defects at the bnch NMJ. (A–D) Ultrastructure of yw (A, control), yw; bnchE14.1/bnchP (B) and yw; bnch11F5/bnchΔ2B (C and D) NMJ boutons at rest. Bars represent 0.5 μm. Membranous inclusions in bnch mutant boutons range from smaller (0.2 μm) vesicular structures with a single limiting membrane (C, arrow) to larger multilamellar bodies (0.3–1.2 μm) (B and D, arrows) that are absent in controls (A). (E and F) EJP recordings in 1 mM Ca2+ from muscle 6 in yw (control), yw; bnch11F5/bnchΔ2B, and bnch11F5/bnchΔ2B third instar larvae. Quantification of EJP amplitudes shows no significant differences between bnch mutants and controls (E). (F) High frequency (10 Hz) stimulation during 10 min reveals a gradual rundown of the EJP amplitudes in bnch mutants (green and yellow) but not controls (blue). (G and H) FM1-43 dye loading and unloading experiments in yw (control) and yw; bnch11F5/bnchΔ2B third instar larval NMJs preparations. FM1-43 dye was loaded during 5 min 30 Hz stimulation in 1.5 mM Ca2+ and 5 min rest (G, a and b). Unloading of the RRP of synaptic vesicles was achieved by 90 mM K+ stimulation during 5 min (G, c and d). Note the subtle decrease in FM1-43 uptake in bnch mutants (G, b) compared with controls (G, a). Unloading of the RRP is similar in bnch mutants (G, d) and controls (G, c). Quantification of these results is shown (H).
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fig5: Abnormal membranous structures and presynaptic endocytic defects at the bnch NMJ. (A–D) Ultrastructure of yw (A, control), yw; bnchE14.1/bnchP (B) and yw; bnch11F5/bnchΔ2B (C and D) NMJ boutons at rest. Bars represent 0.5 μm. Membranous inclusions in bnch mutant boutons range from smaller (0.2 μm) vesicular structures with a single limiting membrane (C, arrow) to larger multilamellar bodies (0.3–1.2 μm) (B and D, arrows) that are absent in controls (A). (E and F) EJP recordings in 1 mM Ca2+ from muscle 6 in yw (control), yw; bnch11F5/bnchΔ2B, and bnch11F5/bnchΔ2B third instar larvae. Quantification of EJP amplitudes shows no significant differences between bnch mutants and controls (E). (F) High frequency (10 Hz) stimulation during 10 min reveals a gradual rundown of the EJP amplitudes in bnch mutants (green and yellow) but not controls (blue). (G and H) FM1-43 dye loading and unloading experiments in yw (control) and yw; bnch11F5/bnchΔ2B third instar larval NMJs preparations. FM1-43 dye was loaded during 5 min 30 Hz stimulation in 1.5 mM Ca2+ and 5 min rest (G, a and b). Unloading of the RRP of synaptic vesicles was achieved by 90 mM K+ stimulation during 5 min (G, c and d). Note the subtle decrease in FM1-43 uptake in bnch mutants (G, b) compared with controls (G, a). Unloading of the RRP is similar in bnch mutants (G, d) and controls (G, c). Quantification of these results is shown (H).

Mentions: At the larval neuromuscular junction (NMJ) of bnch mutants, however, Sweeney and Davis (2002) reported an expanded presynaptic vesicular compartment that is acidic as evidenced by Lysotracker staining. Interestingly, at the larval NMJ we observe abnormal ultrastructural membrane compartments in the cytoplasm of bnch mutant boutons (Fig. 5, B–D), but not in wild-type controls (Fig. 5 A). Given their size and absence in wild-type boutons, these structures likely represent the reported presynaptic acidic compartments (Sweeney and Davis, 2002). These multilamellar structures range in size from 0.3 to 2 μm and resemble the multilayered lysosomal membrane structures present in the visual system.


Aberrant lysosomal carbohydrate storage accompanies endocytic defects and neurodegeneration in Drosophila benchwarmer.

Dermaut B, Norga KK, Kania A, Verstreken P, Pan H, Zhou Y, Callaerts P, Bellen HJ - J. Cell Biol. (2005)

Abnormal membranous structures and presynaptic endocytic defects at the bnch NMJ. (A–D) Ultrastructure of yw (A, control), yw; bnchE14.1/bnchP (B) and yw; bnch11F5/bnchΔ2B (C and D) NMJ boutons at rest. Bars represent 0.5 μm. Membranous inclusions in bnch mutant boutons range from smaller (0.2 μm) vesicular structures with a single limiting membrane (C, arrow) to larger multilamellar bodies (0.3–1.2 μm) (B and D, arrows) that are absent in controls (A). (E and F) EJP recordings in 1 mM Ca2+ from muscle 6 in yw (control), yw; bnch11F5/bnchΔ2B, and bnch11F5/bnchΔ2B third instar larvae. Quantification of EJP amplitudes shows no significant differences between bnch mutants and controls (E). (F) High frequency (10 Hz) stimulation during 10 min reveals a gradual rundown of the EJP amplitudes in bnch mutants (green and yellow) but not controls (blue). (G and H) FM1-43 dye loading and unloading experiments in yw (control) and yw; bnch11F5/bnchΔ2B third instar larval NMJs preparations. FM1-43 dye was loaded during 5 min 30 Hz stimulation in 1.5 mM Ca2+ and 5 min rest (G, a and b). Unloading of the RRP of synaptic vesicles was achieved by 90 mM K+ stimulation during 5 min (G, c and d). Note the subtle decrease in FM1-43 uptake in bnch mutants (G, b) compared with controls (G, a). Unloading of the RRP is similar in bnch mutants (G, d) and controls (G, c). Quantification of these results is shown (H).
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fig5: Abnormal membranous structures and presynaptic endocytic defects at the bnch NMJ. (A–D) Ultrastructure of yw (A, control), yw; bnchE14.1/bnchP (B) and yw; bnch11F5/bnchΔ2B (C and D) NMJ boutons at rest. Bars represent 0.5 μm. Membranous inclusions in bnch mutant boutons range from smaller (0.2 μm) vesicular structures with a single limiting membrane (C, arrow) to larger multilamellar bodies (0.3–1.2 μm) (B and D, arrows) that are absent in controls (A). (E and F) EJP recordings in 1 mM Ca2+ from muscle 6 in yw (control), yw; bnch11F5/bnchΔ2B, and bnch11F5/bnchΔ2B third instar larvae. Quantification of EJP amplitudes shows no significant differences between bnch mutants and controls (E). (F) High frequency (10 Hz) stimulation during 10 min reveals a gradual rundown of the EJP amplitudes in bnch mutants (green and yellow) but not controls (blue). (G and H) FM1-43 dye loading and unloading experiments in yw (control) and yw; bnch11F5/bnchΔ2B third instar larval NMJs preparations. FM1-43 dye was loaded during 5 min 30 Hz stimulation in 1.5 mM Ca2+ and 5 min rest (G, a and b). Unloading of the RRP of synaptic vesicles was achieved by 90 mM K+ stimulation during 5 min (G, c and d). Note the subtle decrease in FM1-43 uptake in bnch mutants (G, b) compared with controls (G, a). Unloading of the RRP is similar in bnch mutants (G, d) and controls (G, c). Quantification of these results is shown (H).
Mentions: At the larval neuromuscular junction (NMJ) of bnch mutants, however, Sweeney and Davis (2002) reported an expanded presynaptic vesicular compartment that is acidic as evidenced by Lysotracker staining. Interestingly, at the larval NMJ we observe abnormal ultrastructural membrane compartments in the cytoplasm of bnch mutant boutons (Fig. 5, B–D), but not in wild-type controls (Fig. 5 A). Given their size and absence in wild-type boutons, these structures likely represent the reported presynaptic acidic compartments (Sweeney and Davis, 2002). These multilamellar structures range in size from 0.3 to 2 μm and resemble the multilayered lysosomal membrane structures present in the visual system.

Bottom Line: Here, we report that loss of Drosophila benchwarmer (bnch), a predicted lysosomal sugar carrier, leads to carbohydrate storage in yolk spheres during oogenesis and results in widespread accumulation of enlarged lysosomal and late endosomal inclusions.Finally, we find that loss of bnch strongly enhances tau neurotoxicity in a dose-dependent manner.We hypothesize that, in bnch, defective lysosomal carbohydrate efflux leads to endocytic defects with functional consequences in synaptic strength, neuronal viability, and tau neurotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
Lysosomal storage is the most common cause of neurodegenerative brain disease in preadulthood. However, the underlying cellular mechanisms that lead to neuronal dysfunction are unknown. Here, we report that loss of Drosophila benchwarmer (bnch), a predicted lysosomal sugar carrier, leads to carbohydrate storage in yolk spheres during oogenesis and results in widespread accumulation of enlarged lysosomal and late endosomal inclusions. At the bnch larval neuromuscular junction, we observe similar inclusions and find defects in synaptic vesicle recycling at the level of endocytosis. In addition, loss of bnch slows endosome-to-lysosome trafficking in larval garland cells. In adult bnch flies, we observe age-dependent synaptic dysfunction and neuronal degeneration. Finally, we find that loss of bnch strongly enhances tau neurotoxicity in a dose-dependent manner. We hypothesize that, in bnch, defective lysosomal carbohydrate efflux leads to endocytic defects with functional consequences in synaptic strength, neuronal viability, and tau neurotoxicity.

Show MeSH
Related in: MedlinePlus