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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 26, 2005 as doi:10.1096/fj.05-4195fje. |
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Cellular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA
1Correspondence: Cellular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, CA 92037, USA. E-mail: Schubert{at}salk.edu
SPECIFIC AIMS
The aims of this research were to 1) determine whether increased disulfide bonding of GAPDH occurs in brain tissue from both Alzheimer’s disease (AD) patients and transgenic AD mice; 2) examine the effect of both Aß and oxidant treatment on disulfide bonding, intracellular location and solubility properties of GAPDH in both primary and immortalized neurons.
PRINCIPAL FINDINGS
1. Increased disulfide bonding of GAPDH in AD brains
Since AD pathology is associated with increased oxidative damage, we examined the degree of GAPDH disulfide bonding in AD digitonin-soluble brain extracts by 1-dimension nonreducing SDS-PAGE and Redox 2D-PAGE. We observed an increase in GAPDH intermolecular disulfide bonding in all AD brain extracts examined compared with age-matched controls. Addition of the reducing agent DTT converted disulfide-linked GAPDH to a monomeric form.
2. Detergent-insoluble disulfide-linked GAPDH is found in cortical brain extracts from AD patients and aged transgenic AD mice
We performed a sequential protein extraction procedure on AD patient and transgenic AD mouse cortical tissue under nonreducing conditions starting with buffers containing 1% Triton-X, followed by 2% SDS and 70% formic acid (detergent-insoluble fraction), to assess the degree of GAPDH insolubility. Analysis of detergent-insoluble fractions by nonreducing SDS-PAGE revealed greatly increased disulfide bonding of GAPDH in AD brain samples (Fig. 1
A) that roughly correlated with the level of Aß accumulation (Fig. 1B
). Transgenic (tg) mice overexpressing FAD-linked mutations of both the human APP and PS1 gene develop amyloid deposits over time. To test the hypothesis that Aß accumulation promotes the formation of insoluble disulfide-linked GAPDH, we examined detergent-insoluble extracts in 4- to 20-month-old transgenic AD and littermate control mice by nonreducing SDS-PAGE. Examination of GAPDH disulfide bonding in detergent-insoluble extracts in 4-month-old mice (Fig. 1C
) revealed no difference between control and transgenic animals. However, by 12 months of age increased high molecular weight (HMW) disulfide bonding of GAPDH was observed that became progressively more pronounced by 20 months only in transgenic animals (Fig. 1C
). In contrast, none of the detergent-insoluble brain extracts in control mice contained GAPDH disulfide-bonded isoforms at any age. Furthermore, the degree of disulfide-bonded GAPDH in tg AD mice correlated with the level of insoluble Aß accumulation (Fig. 1D
).
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3. Aß treatment promotes nuclear accumulation of disulfide-bonded GAPDH
GAPDH participates in multiple nonglycolytic functions, including a proapoptotic role in the nucleus. Since Aß treatment increases the intracellular concentration of ROS in cultured nerve cells, we examined the effect of Aß exposure on the intracellular location and disulfide bond status of GAPDH in primary rat cortical neurons and the mouse hippocampal cell line HT22. Aß treatment of primary (Fig. 2
A) and immortalized neurons resulted in the loss of a 120 kDa disulfide-linked isoform of GAPDH in the cytosol and concomitant appearance of the same isoform in the nucleus. Overall levels of the reduced monomeric form of GAPDH remained constant in both cytoplasmic and nuclear extracts regardless of Aß treatment or exposure time, suggesting that Aß induces preferential nuclear accumulation of disulfide-linked GAPDH.
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4. Aß promotes the accumulation of detergent-insoluble disulfide-bonded GAPDH in cortical neurons
We exposed nondividing rat primary cortical neurons to both high (10 µM) and low (1 µM) concentrations of Aß and examined the intracellular localization and solubility properties of nonreduced GAPDH. Five days of 10 µM Aß treatment resulted in a pronounced increase in disulfide bonding of GAPDH in detergent-insoluble postnuclear extracts that was not observed in control or 1 µM Aß-treated neurons (Fig. 2A
). Immunofluorescent analysis of control neurons cultured for 7 days revealed an increase in punctate GAPDH staining, particularly along neuritic projections. Neurons treated for 7 days with Aß displayed pronounced GAPDH staining in large aggregate-like structures found predominately around nuclei (Fig. 2B
).
5. Oxidant-induced disulfide bonding of GAPDH leads to a reduction in enzyme activity and punctate immunofluorescent staining in the cytoplasm of HT22 cells
We asked whether prooxidant conditions can directly affect disulfide bonding, activity, and intracellular localization of GAPDH. Treatment of HT22 cells for 20 min with H2O2 or diamide caused formation of HMW disulfide-bonded GAPDH multimers in the cytoplasm and nucleus, which persisted in the nucleus 24 h after removal of the oxidant. H2O2 exposure resulted in an 
94% drop in GAPDH activity compared with untreated cells while only a partial recovery in activity was observed after BME exposure. Diamide exposure caused a reduction in GAPDH activity (66%) that was restored after addition of BME. A similar pattern of enzyme inhibition was observed with purified rabbit GAPDH treated with H2O2 or diamide. Apparently disulfide bonding of GAPDH leads to varying degrees of enzyme inhibition depending on the type of oxidant exposure.
We next examined the intracellular location of GAPDH in H2O2-treated cells over 2 days by immunofluorescent staining. Untreated HT22 cells display both cytoplasmic and nuclear GAPDH staining patterns. After exposure to 0.1 mM H2O2 for 1 day, increased GAPDH staining was observed in the nucleus and punctuate staining of GAPDH was observed in the cytoplasm of treated cells (H2O2-treated: 17.7%±4.3% vs. control: 6.7%±2%). Increased apoptosis (H2O2-treated: 22.7%±4.1% vs. control: 1%±0.4%) was observed after 2 days of H2O2 treatment with GAPDH immunostaining overlapping with DAPI-stained apoptotic nuclei. These results are in accordance with other studies showing a correlation between nuclear accumulation of GAPDH and apoptosis.
CONCLUSIONS
A detergent-insoluble disulfide-linked form of GAPDH is found in brain tissue from AD patients and transgenic AD mice but is not present in control tissues (Fig. 1)
. Exposure of HT22 immortalized nerve cells or primary cortical neurons to Aß promoted the preferential accumulation of disulfide-bonded GAPDH in the nucleus (Fig. 2)
. Long-term exposure of cortical neurons to Aß resulted in the progressive formation of insoluble disulfide-linked GAPDH multimers and large aggregate-like structures in the cytoplasm that were not present in control cells (Fig. 2)
. Oxidant exposure readily induced disulfide bonding of GAPDH in both the cytoplasm and nucleus, led to a loss of activity, and promoted the formation of cytoplasmic aggregate-like inclusions and the accumulation of GAPDH in apoptotic nuclei. Collectively, these results indicate that oxidant- and/or Aß-induced disulfide bonding promotes nuclear accumulation and protein insolubility of GAPDH in a time-dependent manner.
Our observation that GAPDH is prone to disulfide-mediated aggregation is consistent with studies examining a link between GAPDH and neurodegenerative disorders. Nuclear aggregated GAPDH and colocalization of GAPDH with fragmented and/or condensed chromatin in neurons have been observed in post mortem AD brain tissue but not in control tissue. GAPDH immunoreactivity has also been detected in amyloid plaques and co-immunoprecipitates with abnormal forms of tau in AD brains. Specific protein-protein interactions have been detected between GAPDH and the ß-amyloid precursor protein, immobilized Aß peptide, and with several aggregate-prone proteins associated with polyglutamine repeat diseases. GAPDH colocalizes with 
-synuclein in Lewy bodies and binds to aggregated -synuclein in vitro. These data show the propensity of GAPDH to aggregate or interact with aggregate-prone proteins.
Based on the proapoptotic and protein aggregate association properties of GAPDH, we propose a model in which Aß or oxidant exposure leads to increased accumulation of a disulfide-bonded isoform of GAPDH in the nucleus that undergoes further disulfide bonding and becomes less soluble (Fig. 3
). Disulfide bonding of GAPDH acts as a seed to facilitate the misfolding of GAPDH leading to an insoluble conformation and ultimately conversion to HMW aggregates, which themselves may be cytotoxic or indirectly trigger apoptosis. During apoptosis, the insoluble form of GAPDH is released from the nucleus and accumulates in inclusion bodies in the cytoplasm. After cell death, the liberated GAPDH insoluble aggregates may then accumulate in extracellular plaques.
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A significant association of late-onset AD (LOAD) and polymorphic variation within GAPDH genes was recently found. A missense mutation within a GAPDH gene (p-GAPD) is associated with a later age of onset of AD. The enzymatic, aggregation, and proapoptotic properties of pGAPD are unknown. It is possible that different isoforms of GAPDH and polymorphic variation within various GAPDH genes may alter the disulfide bonding properties of the enzyme and affect aggregation.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4195fje;
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