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Aberrant Rab11-Dependent Trafficking of the Neuronal Glutamate Transporter EAAC1 Causes Oxidative Stress and Cell Death in Huntington's Disease

Li, Xueyi, Valencia, Antonio, Sapp, Ellen, Masso, Nicholas, Alexander, Jonathan, Reeves, Patrick, Kegel, Kimberly B., Aronin, Neil, DiFiglia, Marian·2010·The Journal of Neuroscience
DOI 10.1523/jneurosci.5865-09.2010
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Abstract

Oxidative stress contributes to neurodegeneration in Huntington's disease (HD). However, the origins of oxidative stress in HD remain unclear. Studies in HD transgenic models suggest involvement of mitochondrial dysfunction, which would lead to overproduction of reactive oxygen species (ROS). Impaired mitochondria complexes occur in late stages of HD but not in presymptomatic or early-stage HD patients. Thus, other mechanisms may account for the earliest source of oxidative stress caused by endogenous mutant huntingtin. Here, we report that decreased levels of a major intracellular antioxidant glutathione coincide with accumulation of ROS in primary HD neurons prepared from embryos of HD knock-in mice (HD 140Q/140Q ), which have human huntingtin exon 1 with 140 CAG repeats inserted into the endogenous mouse huntingtin gene. Uptake of extracellular cysteine through the glutamate/cysteine transporter EAAC1 is required for de novo synthesis of glutathione in neurons. We found that, compared with wild-type neurons, HD neurons had lower cell surface levels of EAAC1 and were deficient in taking up cysteine. Constitutive trafficking of EAAC1 from recycling endosomes relies on Rab11 activity, which is defective in the brain of HD 140Q/140Q mice. Enhancement of Rab11 activity by expression of a dominant-active Rab11 mutant in primary HD neurons ameliorated the deficit in cysteine uptake, increased levels of intracellular glutathione, normalized clearance of ROS, and improved neuronal survival. Our data support a novel mechanism for oxidative stress in HD: Rab11 dysfunction slows trafficking of EAAC1 to the cell surface and impairs cysteine uptake, thereby leading to deficient synthesis of glutathione.

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Figure 1 Reduction of GSH levels precedes neuronal death in HD. Neuron-enriched primary cultures were used for MTT conversion, which positively correlates with survival, to determine cell viability at DIV8 and DIV11 as described in Materials and Methods. Four separate experiments were performed using neurons prepared from embryos of four WT and four HD pregnant mice. In each experiment, MTT conversion was performed in each of a six-well culture plate containing three wells of WT neurons and three wells of HD neurons. The average production of formazaninabsorbanceunitsintheWTneuroncultureswasusedforcalculatingthepercentage of survival in HD neuron cultures ( n = 4 experiments; mean ± SD; Student's t test: ns, no significance; * p < 0.00001).
Figure 1 Reduction of GSH levels precedes neuronal death in HD. Neuron-enriched primary cultures were used for MTT conversion, which positively correlates with survival, to determine cell viability at DIV8 and DIV11 as described in Materials and Methods. Four separate experiments were performed using neurons prepared from embryos of four WT and four HD pregnant mice. In each experiment, MTT conversion was performed in each of a six-well culture plate containing three wells of WT neurons and three wells of HD neurons. The average production of formazaninabsorbanceunitsintheWTneuroncultureswasusedforcalculatingthepercentage of survival in HD neuron cultures ( n = 4 experiments; mean ± SD; Student's t test: ns, no significance; * p < 0.00001).CC-BY-NC-SA · From the PDF
Figure 2 Elevation of ROS levels in HD neurons. To detect reactive oxygen species, live cortical neurons were incubated with carboxy-DCFDA at 37°C for 30 min at DIV8. After washes in PBS, live neurons were imaged using a confocal microscope as described in Materials and Methods. Shown are examples of confocal images of live neurons containing DCF fluorescence. Scale bar, 25 𝛍 m.
Figure 2 Elevation of ROS levels in HD neurons. To detect reactive oxygen species, live cortical neurons were incubated with carboxy-DCFDA at 37°C for 30 min at DIV8. After washes in PBS, live neurons were imaged using a confocal microscope as described in Materials and Methods. Shown are examples of confocal images of live neurons containing DCF fluorescence. Scale bar, 25 𝛍 m.CC-BY-NC-SA · From the PDF
Figure 3 Deficient uptake of cysteine and reduced cell surface levels of EAAC1 in HD neurons. A , At DIV8, primary cortical neurons in six-well culture plates were used for performing L-[ 14 C]cysteine uptake at room temperature for 30 s with L-[ 14 C]cysteine concentration of 0.1, 1, 5, 10, and 50 𝛍 M, respectively, and harvested for preparation of cell lysates. The amount of L-[ 14 C]cysteine in cell lysates for each cysteine concentration was determined by scintillation count in triplicate and represented as mean cpm per milligram per second with SD (two-tailed Student's t test, * p < 0.01). WT neurons were prepared from three pregnantmice,andHDneuronswerepreparedfromthreepregnantmice. B , Biotinylation of cell surface proteins of neurons in a 60 mmculture dish was conducted as in Materials and Methods. Biotinylated cell surface proteins and the corresponding unbiotinylated intracellular proteins were analyzed by SDS-PAGE and Western blot with indicated antibodies. Shown is one blot analysis of three experiments. The broad migration of EAAC1 signal, which is also found in previous studies (Grewer et al., 2000), may be attributable to glycosylation. Biotinylated EAAC1 at the cell surface (arrow) is detected at a longer exposure of Western blot film (middle panel).
Figure 3 Deficient uptake of cysteine and reduced cell surface levels of EAAC1 in HD neurons. A , At DIV8, primary cortical neurons in six-well culture plates were used for performing L-[ 14 C]cysteine uptake at room temperature for 30 s with L-[ 14 C]cysteine concentration of 0.1, 1, 5, 10, and 50 𝛍 M, respectively, and harvested for preparation of cell lysates. The amount of L-[ 14 C]cysteine in cell lysates for each cysteine concentration was determined by scintillation count in triplicate and represented as mean cpm per milligram per second with SD (two-tailed Student's t test, * p < 0.01). WT neurons were prepared from three pregnantmice,andHDneuronswerepreparedfromthreepregnantmice. B , Biotinylation of cell surface proteins of neurons in a 60 mmculture dish was conducted as in Materials and Methods. Biotinylated cell surface proteins and the corresponding unbiotinylated intracellular proteins were analyzed by SDS-PAGE and Western blot with indicated antibodies. Shown is one blot analysis of three experiments. The broad migration of EAAC1 signal, which is also found in previous studies (Grewer et al., 2000), may be attributable to glycosylation. Biotinylated EAAC1 at the cell surface (arrow) is detected at a longer exposure of Western blot film (middle panel).CC-BY-NC-SA · From the PDF
Figure 4 EAAC1 traffics through Rab11-positive endosomes and accumulates in the cytoplasm of HD neurons. EAAC1 and Rab11 colocalize in WT and HD neurons. Primary cortical neurons prepared from WT and HD 140Q/140Q mouse embryos were fixed and processed for immunolabeling with antibodies against EAAC1 (green) and Rab11 (red). The merged signals show areas of colocalization in yellow. Shown are confocal images of one neuron from a WT culture and one neuron from a HD culture. The arrowheadpointstoaccumulationofimmunoreactiveEAAC1inthesomaofanHD 140Q/140Q neuron.Theboxedregionscontainpart of a neurite and are enlarged below. The arrows in the merged photos indicate loci along the neurite where there is colabeling of EAAC1 and Rab11 (yellow).
Figure 4 EAAC1 traffics through Rab11-positive endosomes and accumulates in the cytoplasm of HD neurons. EAAC1 and Rab11 colocalize in WT and HD neurons. Primary cortical neurons prepared from WT and HD 140Q/140Q mouse embryos were fixed and processed for immunolabeling with antibodies against EAAC1 (green) and Rab11 (red). The merged signals show areas of colocalization in yellow. Shown are confocal images of one neuron from a WT culture and one neuron from a HD culture. The arrowheadpointstoaccumulationofimmunoreactiveEAAC1inthesomaofanHD 140Q/140Q neuron.Theboxedregionscontainpart of a neurite and are enlarged below. The arrows in the merged photos indicate loci along the neurite where there is colabeling of EAAC1 and Rab11 (yellow).CC-BY-NC-SA · From the PDF
Image 5
CC-BY-NC-SA · From the PDF
Figure 6 Expression of Rab11Q70L enhances clearance of ROS in HD neurons. Primary cortical neurons were infected at DIV4 with lentivirus expressing EGFP alone or lentivirus expressing Rab11Q70L/EGFP. At DIV8, virus-infected primary neurons were treat with DCFDA to assess ROSlevels as described in Materials and Methods. Cells expressing EGFP alone (not shown here) had low fluorescent signals that were of comparable levels in WT and HD neurons. Thus, EGFP signals did not interfere with our quantitative analysis of DCF signals. A , Shown are examples of confocal images of DCF-labeled neurons. B , Bar graph shows quantification of intensity of fluorescence generated by carboxy-DCFDA after reaction with ROS and EGFP in the soma. Mean fluorescence intensity with SD was graphed ( n = 53WT-EGFP,95WT-Rab11Q70L,46HD-EGFP, and 84 HD-Rab11Q70L; one-way ANOVA: ns, no significance; * p < 0.01, ** p < 0.001). Note that there is no significance (ns) between WT-Rab11Q70L and HD-Rab11Q70L. Neurons were from two sets of cultures from embryos of two WT and two HD pregnant mice.
Figure 6 Expression of Rab11Q70L enhances clearance of ROS in HD neurons. Primary cortical neurons were infected at DIV4 with lentivirus expressing EGFP alone or lentivirus expressing Rab11Q70L/EGFP. At DIV8, virus-infected primary neurons were treat with DCFDA to assess ROSlevels as described in Materials and Methods. Cells expressing EGFP alone (not shown here) had low fluorescent signals that were of comparable levels in WT and HD neurons. Thus, EGFP signals did not interfere with our quantitative analysis of DCF signals. A , Shown are examples of confocal images of DCF-labeled neurons. B , Bar graph shows quantification of intensity of fluorescence generated by carboxy-DCFDA after reaction with ROS and EGFP in the soma. Mean fluorescence intensity with SD was graphed ( n = 53WT-EGFP,95WT-Rab11Q70L,46HD-EGFP, and 84 HD-Rab11Q70L; one-way ANOVA: ns, no significance; * p < 0.01, ** p < 0.001). Note that there is no significance (ns) between WT-Rab11Q70L and HD-Rab11Q70L. Neurons were from two sets of cultures from embryos of two WT and two HD pregnant mice.CC-BY-NC-SA · From the PDF
Figure 7 Expression of Rab11Q70L promotes neuronal survival. Primary cortical neurons at DIV4 were untreated or infected with lentivirus expressing EGFP alone or lentivirus bicistronically expressing EGFP and Rab11Q70L. At DIV11, virus-treated primary neurons were used for the MTT assay to determine cell viability as in Figure 1. The average production of formazan in absorbance units from four cultures of WT neurons with infection of lenti-EGFP was used for calculating the percentage of the survival rate of WT-Rab11Q70L, HD-EGFP, and HD-Rab11Q70L neurons. The bar graph shows mean percentage with SD of surviving HD neurons relative to WT neurons infected with lenti-EGFP ( n = 4; one-way ANOVA: ns, no significance; * p < 0.001).
Figure 7 Expression of Rab11Q70L promotes neuronal survival. Primary cortical neurons at DIV4 were untreated or infected with lentivirus expressing EGFP alone or lentivirus bicistronically expressing EGFP and Rab11Q70L. At DIV11, virus-treated primary neurons were used for the MTT assay to determine cell viability as in Figure 1. The average production of formazan in absorbance units from four cultures of WT neurons with infection of lenti-EGFP was used for calculating the percentage of the survival rate of WT-Rab11Q70L, HD-EGFP, and HD-Rab11Q70L neurons. The bar graph shows mean percentage with SD of surviving HD neurons relative to WT neurons infected with lenti-EGFP ( n = 4; one-way ANOVA: ns, no significance; * p < 0.001).CC-BY-NC-SA · From the PDF

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Metadata

Authors
Li, X., Valencia, A., Sapp, E., Masso, N., Alexander, J., Reeves, P., Kegel, K. B., Aronin, N., & DiFiglia, M.
Venue
The Journal of Neuroscience
Publisher
Society for Neuroscience
Published
2010-03-31
Volume
30
Issue
13
Pages
4552-4561
ISSN
  • 0270-6474
  • 1529-2401
DOI
10.1523/jneurosci.5865-09.2010
Cited by
147
References
4
Language
en
Licence
creativecommons.org/licenses/by-nc-sa/4.0/ (vor, 183 d)

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