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Cited by 196Mitochondrial Function and PathologyCancer, Hypoxia, and MetabolismNeuroscience and Neuropharmacology Research

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Evidence for the Mitochondrial Lactate Oxidation Complex in Rat Neurons: Demonstration of an Essential Component of Brain Lactate Shuttles

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Hashimoto, Takeshi, Hussien, Rajaa, Cho, Hyung-Sook, Kaufer, Daniela, Brooks, George A.·2008·PLoS ONE
DOI 10.1371/journal.pone.0002915PMC PMC2488371
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Abstract

To evaluate the presence of components of a putative Intracellular Lactate Shuttle (ILS) in neurons, we attempted to determine if monocarboxylate (e.g. lactate) transporter isoforms (MCT1 and -2) and lactate dehydrogenase (LDH) are coexpressed in neuronal mitochondria of rat brains. Immunohistochemical analyses of rat brain cross-sections showed MCT1, MCT2, and LDH to colocalize with the mitochondrial inner membrane marker cytochrome oxidase (COX) in cortical, hippocampal, and thalamic neurons. Immunoblotting after immunoprecipitation (IP) of mitochondria from brain homogenates supported the histochemical observations by demonstrating that COX coprecipitated MCT1, MCT2, and LDH. Additionally, using primary cultures from rat cortex and hippocampus as well as immunohistochemistry and immunocoprecipitation techniques, we demonstrated that MCT2 and LDH are coexpressed in mitochondria of cultured neurons. These findings can be interpreted to mean that, as in skeletal muscle, neurons contain a mitochondrial lactate oxidation complex (mLOC) that has the potential to facilitate both intracellular and cell-cell lactate shuttles in brain.

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Figure 1 Immunohistochemical images of rat brain cross-sections demonstrating mitochondrial MCT1 in thalamic neurons. When signals from probes for the lactate/pyruvate transporter MCT1 (A, green) and mitochondrial cytochrome oxidase, COX (B, red) were merged with those of the neuronal marker MAP2 (C, blue), superposition of the signals (D, yellow/white) showed colocalization of MCT1 and components of the mitochondrial reticulum in neurons (white arrows). Scale bar = 20 µm.
Figure 1 Immunohistochemical images of rat brain cross-sections demonstrating mitochondrial MCT1 in thalamic neurons. When signals from probes for the lactate/pyruvate transporter MCT1 (A, green) and mitochondrial cytochrome oxidase, COX (B, red) were merged with those of the neuronal marker MAP2 (C, blue), superposition of the signals (D, yellow/white) showed colocalization of MCT1 and components of the mitochondrial reticulum in neurons (white arrows). Scale bar = 20 µm.CC BY · PubMed Central
Figure 2 Immunohistochemical images of rat brain cross-sections demonstrating mitochondrial MCT2 in thalamic neurons. When signals from probes for MCT2 (A, green), and mitochondrial COX (B, red) were merged with those of the neuronal marker MAP2 (C, blue), superposition of the signals (D, yellow/white) showed colocalization of MCT2 and components of the mitochondrial reticulum in neurons (white arrows). Scale bar = 20 µm.
Figure 2 Immunohistochemical images of rat brain cross-sections demonstrating mitochondrial MCT2 in thalamic neurons. When signals from probes for MCT2 (A, green), and mitochondrial COX (B, red) were merged with those of the neuronal marker MAP2 (C, blue), superposition of the signals (D, yellow/white) showed colocalization of MCT2 and components of the mitochondrial reticulum in neurons (white arrows). Scale bar = 20 µm.CC BY · PubMed Central
Figure 3 Immunohistochemical images of rat brain cross-sections demonstrating mitochondrial MCT2 in hippocampal neurons. Superposition of signals from probes for MCT2 (A, green), COX (B, red) and MAP2 (C, blue) showed clear colocalization (D, yellow/white) in neurons. Scale bar = 10 µm.
Figure 3 Immunohistochemical images of rat brain cross-sections demonstrating mitochondrial MCT2 in hippocampal neurons. Superposition of signals from probes for MCT2 (A, green), COX (B, red) and MAP2 (C, blue) showed clear colocalization (D, yellow/white) in neurons. Scale bar = 10 µm.CC BY · PubMed Central
Figure 4 Immunohistochemical images of rat brain cross-sections showing area-specific expression of LDH-M (A–C) and LDH-H (D–F). In hippocampus (A and D), signals for LDH-H predominate (D, arrows) whereas a signal for LDH-M is not evident (A). On the other hand, in cortex (B and E), signals for LDH-M predominate (B, arrows), but a signal for LDH-H is not evident (E). In thalamus (C and F), signals for LDH-H predominate (F, arrows), but a signal for LDH-M is also apparent (C, arrow). Scale bar = 200 µm.
Figure 4 Immunohistochemical images of rat brain cross-sections showing area-specific expression of LDH-M (A–C) and LDH-H (D–F). In hippocampus (A and D), signals for LDH-H predominate (D, arrows) whereas a signal for LDH-M is not evident (A). On the other hand, in cortex (B and E), signals for LDH-M predominate (B, arrows), but a signal for LDH-H is not evident (E). In thalamus (C and F), signals for LDH-H predominate (F, arrows), but a signal for LDH-M is also apparent (C, arrow). Scale bar = 200 µm.CC BY · PubMed Central
Figure 5 Immunohistochemical images of rat brain demonstrating mitochondrial LDH-M in cortical neurons. When signals from probes for LDH-M (B, red) and the mitochondrial protein VDAC (A, green) were merged with those for the neuronal marker MAP2 (C, blue), superposition of the signals (D, yellow/white) showed colocalization of LDH-M and components of the mitochondrial reticulum. Scale bar = 20 µm.
Figure 5 Immunohistochemical images of rat brain demonstrating mitochondrial LDH-M in cortical neurons. When signals from probes for LDH-M (B, red) and the mitochondrial protein VDAC (A, green) were merged with those for the neuronal marker MAP2 (C, blue), superposition of the signals (D, yellow/white) showed colocalization of LDH-M and components of the mitochondrial reticulum. Scale bar = 20 µm.CC BY · PubMed Central
Figure 6 Immunohistochemical images of rat brain cross-sections demonstrating mitochondrial LDH-H in hippocampal neurons. Superposition of signals from probes for VDAC (A, green), LDH-H (B, red) and MAP2 (C, blue) showed extensive colocalization throughout the neuron (D, yellow/white). Scale bar = 20 µm.
Figure 6 Immunohistochemical images of rat brain cross-sections demonstrating mitochondrial LDH-H in hippocampal neurons. Superposition of signals from probes for VDAC (A, green), LDH-H (B, red) and MAP2 (C, blue) showed extensive colocalization throughout the neuron (D, yellow/white). Scale bar = 20 µm.CC BY · PubMed Central
Figure 7 Antibodies to COX and normal IgG (nIgG) were used to develop immunoprecipitates (IP) from Tween 20 (1%)-solubilized mitochondrial fractions of brain tissue. IP proteins were subsequently probed with antibodies to MCT1, MCT2, LDH, and β1-Na+-K+-ATPase. MCT1 and MCT2 were coprecipitated with COX, and LDH was slightly, but significantly coprecipitated with COX as compared to nIgG (no protein was coprecipitated with nIgG). The plasma membrane marker β1-Na+-K+-ATPase was not coprecipitated with either COX or nIgG. Sup, supernatant after immunoprecipitation in lysates of mitochondrial fractions.
Figure 7 Antibodies to COX and normal IgG (nIgG) were used to develop immunoprecipitates (IP) from Tween 20 (1%)-solubilized mitochondrial fractions of brain tissue. IP proteins were subsequently probed with antibodies to MCT1, MCT2, LDH, and β1-Na+-K+-ATPase. MCT1 and MCT2 were coprecipitated with COX, and LDH was slightly, but significantly coprecipitated with COX as compared to nIgG (no protein was coprecipitated with nIgG). The plasma membrane marker β1-Na+-K+-ATPase was not coprecipitated with either COX or nIgG. Sup, supernatant after immunoprecipitation in lysates of mitochondrial fractions.CC BY · PubMed Central
Figure 8 Immunohistochemical images demonstrating mitochondrial MCT2 in a cultured neuron from rat hippocampus. Superposition of signals from probes for MCT2 (A, green), COX (B, red), and MAP2 (C, blue) showed extensive colocalization (D, white) in a cultured neuron. Scale bar = 10 µm.
Figure 8 Immunohistochemical images demonstrating mitochondrial MCT2 in a cultured neuron from rat hippocampus. Superposition of signals from probes for MCT2 (A, green), COX (B, red), and MAP2 (C, blue) showed extensive colocalization (D, white) in a cultured neuron. Scale bar = 10 µm.CC BY · PubMed Central
Figure 9 Immunohistochemical images demonstrating mitochondrial LDH in neurons cultured from rat hippocampus and cortex. Superposition of signals from probes for LDH (A, green), COX (B, red) and MAP2 (C, blue) showed clear colocalization (D, yellow/white) in neuron cells. Scale bar = 10 µm.
Figure 9 Immunohistochemical images demonstrating mitochondrial LDH in neurons cultured from rat hippocampus and cortex. Superposition of signals from probes for LDH (A, green), COX (B, red) and MAP2 (C, blue) showed clear colocalization (D, yellow/white) in neuron cells. Scale bar = 10 µm.CC BY · PubMed Central
Figure 10 Immunoblots of mitochondrial fractions (MI) from primary neuronal cultures developed from rat cortex and hippocampus. Whole homogenates (WH) of the same cells were loaded as positive controls. Mitochondria were immunoblotted using plasma membrane markers for β1-Na+-K+-ATPase and GLUT1 as well as the cytosol marker F-actin. Mitochondrial fractions (MI) showed the presence of β1-Na+-K+-ATPase. However, while prominent in whole cell homogenates (WH), the other plasma membrane marker Glut1 and the cytosol marker F-actin were not apparent.
Figure 10 Immunoblots of mitochondrial fractions (MI) from primary neuronal cultures developed from rat cortex and hippocampus. Whole homogenates (WH) of the same cells were loaded as positive controls. Mitochondria were immunoblotted using plasma membrane markers for β1-Na+-K+-ATPase and GLUT1 as well as the cytosol marker F-actin. Mitochondrial fractions (MI) showed the presence of β1-Na+-K+-ATPase. However, while prominent in whole cell homogenates (WH), the other plasma membrane marker Glut1 and the cytosol marker F-actin were not apparent.CC BY · PubMed Central
Figure 11 To probe for an association between mitochondrial cytochrome oxidase (COX) and lactate dehydrogenase (LDH), antibodies to COX and normal IgG (nIgG) were used to develop immunoprecipitates (IP) from Tween 20 (0.3%)-solubilized mitochondrial fractions of cortex and hippocampus primary cultures. Results were compared to those obtained on the supernatant remaining after immunoprecipitation (Sup). COX and nIgG immunoprecipitates were subsequently probed with an antibody that recognized all LDH isoforms. Mitochondrial fractions (MI) were also probed for LDH and results are presented as a positive control. LDH was coprecipitated with COX as compared to nIgG (no protein was coprecipitated with nIgG) in mitochondrial fractions derived from cortical as well as hippocampal areas of rat primary cultures. When the inhibitor to astrocyte development Ara-C was not used, COX coprecipitated LDH in mitochondrial fractions from cortical and hippocampal areas of rat primary cultures. Similar results obtained with and without Ara-C (the lack of Ara-C facilitates development of astrocytes among as neurons in cultures) indicate that astrocytes also contain mLDH.
Figure 11 To probe for an association between mitochondrial cytochrome oxidase (COX) and lactate dehydrogenase (LDH), antibodies to COX and normal IgG (nIgG) were used to develop immunoprecipitates (IP) from Tween 20 (0.3%)-solubilized mitochondrial fractions of cortex and hippocampus primary cultures. Results were compared to those obtained on the supernatant remaining after immunoprecipitation (Sup). COX and nIgG immunoprecipitates were subsequently probed with an antibody that recognized all LDH isoforms. Mitochondrial fractions (MI) were also probed for LDH and results are presented as a positive control. LDH was coprecipitated with COX as compared to nIgG (no protein was coprecipitated with nIgG) in mitochondrial fractions derived from cortical as well as hippocampal areas of rat primary cultures. When the inhibitor to astrocyte development Ara-C was not used, COX coprecipitated LDH in mitochondrial fractions from cortical and hippocampal areas of rat primary cultures. Similar results obtained with and without Ara-C (the lack of Ara-C facilitates development of astrocytes among as neurons in cultures) indicate that astrocytes also contain mLDH.CC BY · PubMed Central

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Metadata

Authors
Hashimoto, T., Hussien, R., Cho, H.-S., Kaufer, D., & Brooks, G. A.
Affiliations
University of California, Berkeley (US)
Corresponding author
Brooks, George A.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2008-08-13
Volume
3
Issue
8
Pages
e2915
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0002915
PMC
PMC2488371
OpenAlex
W1988714133
Cited by
196
References
43
Citations by year
2026: 10 · 2025: 9 · 2024: 8 · 2023: 14 · 2022: 9 · 2021: 10 · 2020: 14 · 2019: 7 · 2018: 14 · 2017: 15 · 2016: 13 · 2015: 5 · 2014: 11 · 2013: 17 · 2012: 13
FWCI
3.56
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • eScholarship (California Digital Library) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
Funders
National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR050459)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
lactate dehydrogenase, monocarboxylate transporters, MCT1, MCT2, neuronal mitochondria, cytochrome c oxidase, cortical neurons, hippocampal neurons, thalamic neurons
MeSH terms
Animals, Brain, Female, L-Lactate Dehydrogenase, Lactates, Mesencephalon, Mitochondria, Neuroglia, Neurons, Oxidation-Reduction, Rats, Wistar, Monocarboxylic Acid Transporters, Rats

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