Journal: Nature Metabolism

Article Title: Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions

doi: 10.1038/s42255-023-00838-3

Figure Lengend Snippet: a , Experimental design to validate dysregulated TCA cycle enzymes identified by MitoTag proteomics in EAE. b – j , Immunofluorescence analysis of Idh3a ( b – d ), Mdh2 ( e – g ) and Idh2 ( h – j ) in spinal cord neuronal somata (top) and axons (bottom) of control (left) or of acute EAE (right) Thy1 -mitoRFP mice (TCA cycle enzymes, green; RFP, red for neuronal mitochondria in somata or neurofilament (NF) for axonal staining, respectively). Graphs ( d , g , j ) show area occupancy (soma, P = 0.5009 for Idh3a, 0.1371 for Mdh2 and 0.9907 for Idh2; axon, P = 0.053 for Idh3a, 0.0357 for Mdh2 and 0.5615 for Idh2), mean fluorescence intensities (MFIs) (soma, P = 0.0007 for Idh3a, 0.0003 for Mdh2 and 0.0317 for Idh2; axon, P = 0.00006 for Idh3a, 0.0357 for Mdh2 and 0.0299 for Idh2) and their product, integrated density (soma, P = 0.0046 for Idh3a, 0.0286 for Mdh2 and 0.265 for Idh2; axon, P = 0.0079 for Idh3a, 0.0023 for Mdh2 and 0.0653 for Idh2), in EAE neuronal somata and axons normalized to the mean of control (mean ± s.e.m.; compared per animal by two-tailed, unpaired Student’s t -test or Mann–Whitney U -test where normal distribution could not be confirmed from four control and four EAE mice for Idh3a and Mdh2; five control and five EAE mice for Idh2. Scale bar, 25 μm ( h ) applies also to b , e and their details; 10 μm ( i ), applies also to c and f . * P < 0.05; ** P < 0.01, *** P < 0.005; **** P < 0.001; NS, not significant. See source data for individual data points and further statistical parameters. Illustration created with BioRender.

Article Snippet: Primary antibodies were incubated overnight at 4 °C at a dilution of 1:400 (Mdh2, Novus Biologicals, NBP1-32259; Idh3a, Novus Biologicals, NBP1-32396; Idh2, Thermo Scientific, 702713; NeuN, clone A60, Sigma, MAB377) or 1:1,000 (RFP, Novus Biologicals, NBP1-97371 and GFP, Abcam, ab13970).

Techniques: Immunofluorescence, Control, Staining, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature Metabolism

Article Title: Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions

doi: 10.1038/s42255-023-00838-3

Figure Lengend Snippet: a , Overview of an MS lesion in human cortex, with areas of NAWM and the lesion site marked and magnified in insets. Myelin (Luxol fast blue, LFB, blue; periodic acid–Schiff, PAS, purple) and macrophages (CD68, black) are labeled. b , c , Immunofluorescence analysis of Idh3a in cortical axons in NAWM (left) or lesion areas (right; Idh3a, green; NF, red). d , e , Immunofluorescence analysis of Mdh2 in cortical axons in NAWM (left) or lesion areas (right; Mdh2, green; NF, red). f , g , Immunofluorescence analysis of Idh2 in cortical axons in NAWM (left) or lesion areas (right; Idh2, green; NF, red). Graphs ( c , e , g show area occupancy ( P = 0.0251 for Idh3a, 0.2995 for Mdh2 and 0.1599 for Idh2), MFIs ( P = 0.0156 for Idh3a, 0.0099 for Mdh2 and 0.5144 for Idh2) and their product, integrated density ( P = 0.0075 for Idh3a, 0.0606 for Mdh2 and 0.6875 for Idh2), in pairs of NAWM and lesion areas from seven cases, as listed in Extended Data Table and compared by two-tailed, paired t -test or Wilcoxon matched-pairs signed-rank test where normal distribution could not be confirmed. Scale bars, 1,000 μm ( a ); 50 μm in inset; 25 μm ( f ), also applies to b and d . * P < 0.05; ** P < 0.01; NS, not significant. See source data for individual data points and further statistical parameters.

Article Snippet: Primary antibodies were incubated overnight at 4 °C at a dilution of 1:400 (Mdh2, Novus Biologicals, NBP1-32259; Idh3a, Novus Biologicals, NBP1-32396; Idh2, Thermo Scientific, 702713; NeuN, clone A60, Sigma, MAB377) or 1:1,000 (RFP, Novus Biologicals, NBP1-97371 and GFP, Abcam, ab13970).

Techniques: Labeling, Immunofluorescence, Two Tailed Test

Journal: Nature Metabolism

Article Title: Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions

doi: 10.1038/s42255-023-00838-3

Figure Lengend Snippet: ( a ) Schematic of experiment, analysis of same data sets as shown in Fig. . ( b ) Confocal images of spinal cord sections of a Thy1 -PercevalHR mouse (green) that was injected with rAAV.hSyn:Idh3a-tdTomato (tdTomato, tdTom, red). Bottom row shows immunostainings for Idh3a, with details highlighting, left, a tdTomato-negative (tdTom − ) and, right, a tdTomato-positive (tdTom + ) neuron with Idh3a overexpression. ( c ) Expression level of Idh3a in tdTomato-negative (tdTom − ) and tdTomato-positive (tdTom + ) neurons (mean ± s.e.m.; n = 266 tdTom − neurons and 803 tdTom + neurons using two-tailed, Mann-Whitney test, p < 0.001). Scale bar: 25 μm (top) and 10 μm (bottom) in b. ****, p < 0.001. See source data for individual data points and further statistical parameters. Illustration created with BioRender.

Article Snippet: Primary antibodies were incubated overnight at 4 °C at a dilution of 1:400 (Mdh2, Novus Biologicals, NBP1-32259; Idh3a, Novus Biologicals, NBP1-32396; Idh2, Thermo Scientific, 702713; NeuN, clone A60, Sigma, MAB377) or 1:1,000 (RFP, Novus Biologicals, NBP1-97371 and GFP, Abcam, ab13970).

Techniques: Injection, Over Expression, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Metabolism

Article Title: Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions

doi: 10.1038/s42255-023-00838-3

Figure Lengend Snippet: a , Experimental design to measure [ATP/ADP] axon in acute EAE in Thy1 -PercevalHR mice that virally overexpressed Idh3a or a control protein (Cre recombinase) together with tdTomato in a subset of axons. b , Maximum intensity projections of in vivo multi-photon image stacks of spinal cord axons in Idh3a-overexpressing Thy1 -PercevalHR mice. Grayscale LUT of tdTomato (left). Ratiometric [ATP/ADP] axon LUT (λ ex ratio 950 nm/840 nm) (right). Details below show image pairs of tdTomato-negative (tdTom − , left) and tdTomato-positive (tdTom + , right) normal-appearing, swollen and fragmented axons (tdTom − has dashed outlines) in acute EAE. c , Comparison of [ATP/ADP] axon in tdTom + and tdTom − axons (plotted as λ ex ratio 950 nm/840 nm, normalized to control axon mean indicated as the black line; values above 1.5 are lined up on the ‘≥1.5’ dashed line). [ATP/ADP] axon of single tdTom − (gray) and tdTom + (orange) axons in Idh3a-overexpressing EAE mice (left). Lesion-specific paired analysis of mean [ATP/ADP] axon in tdTom − (gray) and tdTom + (orange) axon populations of the three morphological stages (right). d , Same analysis as c , but in a mouse cohort overexpressing a control protein (Cre recombinase). Mean ± s.e.m. Comparison of 176 tdTom − axons versus 153 tdTom + axons in 12 lesions from four mice in c ; 214 tdTom − axons versus 159 tdTom + axons in 11 lesions from three mice in d using a two-tailed, unpaired Student’s t -test ( c , d , left graphs) and a paired t -test ( c , d , right graphs). Scale bar, 25 μm ( b ). ** P < 0.01; *** P < 0.005; **** P < 0.001. See source data for individual data points and further statistical parameters. Illustration created with BioRender.

Article Snippet: Primary antibodies were incubated overnight at 4 °C at a dilution of 1:400 (Mdh2, Novus Biologicals, NBP1-32259; Idh3a, Novus Biologicals, NBP1-32396; Idh2, Thermo Scientific, 702713; NeuN, clone A60, Sigma, MAB377) or 1:1,000 (RFP, Novus Biologicals, NBP1-97371 and GFP, Abcam, ab13970).

Techniques: Control, In Vivo, Comparison, Two Tailed Test

Journal: Nature Metabolism

Article Title: Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions

doi: 10.1038/s42255-023-00838-3

Figure Lengend Snippet: ( a ) Experimental design for [ATP/ADP] axon measurements in chronic EAE in Thy1 - PercevalHR mice virally overexpressing Idh3a or a control protein (Cre recombinase) and tdTomato in a subset of axons. ( b ) In vivo multi-photon image projections of chronically Idh3a-overexpressing Thy1 -PercevalHR spinal axons. Left: Grayscale LUT of tdTomato. Right: Ratiometric [ATP/ADP] axon LUT (λ ex ratio 950 nm/840 nm). Details: Image pairs of tdTomato-negative (tdTom − ; left) and -positive (tdTom + ; right) normal-appearing, swollen, and fragmented axons. ( c ) Comparison of [ATP/ADP] axon in tdTom − and tdTom + axons (λ ex ratio 950 nm/840 nm, normalized to control axon mean indicated by black line; values above 1.5 lined up on the “≥1.5” dashed line). Left: [ATP/ADP] axon of single tdTom − (gray) and tdTom + (orange) axons. Right: Lesion-specific paired analysis of mean [ATP/ADP] axon in tdTom − (gray) and tdTom + (orange) axon populations of the three morphological stages. Mean ± s.e.m. Comparison of 130 tdTom − versus 115 tdTom + axons in 12 lesions from four mice in c using two-tailed, unpaired Student’s t-test or Mann-Whitney test where normal distribution could not be confirmed (c, left graphs; p = 7 × 10 -6 , 9.9 × 10 -5 and 3.8 × 10 -5 for stages 0, 1 and 2) and a paired t-test (right graph; 0.001, 0.0273 and 0.0547 for stages 0, 1 and 2.) ( d ) Paired analysis of the frequency of stage 0, 1 and 2 in tdTom − (gray) and tdTom + (orange) axon populations. Mean ± s.e.m. Comparison of 551 tdTom − versus 539 tdTom + axons in 38 lesions from six mice in d using two-tailed, paired Student’s t-test or Wilcoxon test where normal distribution could not be confirmed (p = 0.2108, 0.743 and 0.0575 for stage 0, 1 and 2). Scale bars: 25 μm in b. *, p < 0.05; ***, p < 0.005; ****, p < 0.001. See source data for individual data points and further statistical parameters. Illustration created with BioRender.

Article Snippet: Primary antibodies were incubated overnight at 4 °C at a dilution of 1:400 (Mdh2, Novus Biologicals, NBP1-32259; Idh3a, Novus Biologicals, NBP1-32396; Idh2, Thermo Scientific, 702713; NeuN, clone A60, Sigma, MAB377) or 1:1,000 (RFP, Novus Biologicals, NBP1-97371 and GFP, Abcam, ab13970).

Techniques: Control, In Vivo, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Adenylate cyclase 10 promotes brown adipose tissue thermogenesis

doi: 10.1016/j.isci.2025.111833

Figure Lengend Snippet:

Article Snippet: Idh3a (NM_029573) Mouse Tagged ORF Clone , Origene , MR205632.

Techniques: Recombinant, Transfection, cAMP Assay, Activity Assay, Software

Journal: CNS & neurological disorders drug targets

Article Title: Alteration of Isocitrate Dehydrogenase Following Acute Ischemic Injury as a Means to Improve Cellular Energetic Status in Neuroadaptation

doi:

Figure Lengend Snippet: Primary neuronal cultures were exposed to 5 min of OGD on three consecutive days. Whole cell extracts were harvested 24 h following the third PC session. Fifteen μg of total protein was separated on 4–12% Bis-Tris gels, transferred onto Hybond PVDF membranes, and probed with antibodies specific for IDH3 and HSC70 (loading control). (A) A decrease in IDH3α expression was observed after 3 days of PC (n=3) and quantified (B). Neurons were photo-documented prior to PC (C), 24h after 1 day of PC (D), 24 h after 2 days of PC (E), and 24 h following day 1, 2, and 3 of PC (F) in order to demonstrate that there was no change in cell viability as a result of three consecutive days of PC. These images are in keeping with our previous use of this model [14].

Article Snippet: Antibodies were purchased as follows: IDH3α (Aviva Systems Biology, ARP42237_T100), heat shock conjugate 70 (HSC70) (Assay Designs, SPA-816), hypoxia inducible factor-1α (HIF1α) (Novus Biologicals, NB100-110); IDH1 (sc-49996), IDH2 (sc-55668) and α-KG (sc-49589) antibodies were purchased from Santa Cruz Biotechnology, Inc. All other immunoblot supplies, unless noted, were purchased from Bio-Rad Laboratories.

Techniques: Control, Expressing

Journal: CNS & neurological disorders drug targets

Article Title: Alteration of Isocitrate Dehydrogenase Following Acute Ischemic Injury as a Means to Improve Cellular Energetic Status in Neuroadaptation

doi:

Figure Lengend Snippet: HT-22 neural cells were exposed to 3 mM glutamate for 24 h. Whole cell extracts were harvested at 24 h and 10 μg of total protein was probed with antibodies specific for IDH1, IDH2, IDH3, α-KG, HIF1α, and HSC70. (A) IDH1, IDH2, IDH3, α-KG, and HIF1α showed no change in expression compared to control lysates (n=3). (B) Cell viability was assessed and quantified by MTT assay (n=3), showing this oxidative challenge was non-lethal (p>0.05). The data are expressed as a percent survival compared to sorbitol (“Total Kill”) and were post-hoc analyzed using a one-way ANOVA and Turkey’s multiple comparison post-hoc analysis. HT-22 cells were photo documented following treatment to visually assess cellular viability: both the control condition (C) and the 3mM glutamate condition (D) illustrate negligible changes in cellular viability while the sorbitol condition (E) exhibits appreciable cell death.

Article Snippet: Antibodies were purchased as follows: IDH3α (Aviva Systems Biology, ARP42237_T100), heat shock conjugate 70 (HSC70) (Assay Designs, SPA-816), hypoxia inducible factor-1α (HIF1α) (Novus Biologicals, NB100-110); IDH1 (sc-49996), IDH2 (sc-55668) and α-KG (sc-49589) antibodies were purchased from Santa Cruz Biotechnology, Inc. All other immunoblot supplies, unless noted, were purchased from Bio-Rad Laboratories.

Techniques: Expressing, Control, MTT Assay, Comparison

Journal: CNS & neurological disorders drug targets

Article Title: Alteration of Isocitrate Dehydrogenase Following Acute Ischemic Injury as a Means to Improve Cellular Energetic Status in Neuroadaptation

doi:

Figure Lengend Snippet: HT-22 cells were exposed to glucose-free medium substituted with 25 mM galactose for 72 h. Whole cell extracts were harvested at 72 h and 10 μg of protein was probed with antibodies specific for IDH1, IDH2, IDH3α, α-KG, HIF1α, and HSC70. (A) IDH1, IDH2, IDH3, and α-KG all demonstrated significant increases in expression compared to control lysates, while a decrease in HIF1α was observed (n=3). (B) At 72 h, the proliferative status of the 25 mM galactose condition compared to the 25 mM glucose control was assessed via MTT assays. Data are expressed as percent of control and were analyzed using a Student’s t-test (***p<0.0001). At 72 h, cells were fixed and further processed for immunocytochemistry. Ki67 (red) was used as a marker of mitotically active cells, β-tubulin (green) was used as a cytoarchitectural marker, and DAPI (blue) was utilized as a label of cell nuclei. (C) The control condition shows a predominance of Ki67 within the nuclei of the cells while (D) the 25 mM galactose condition has minimal Ki67 staining, demonstrating galactose challenge halts cell proliferation.

Article Snippet: Antibodies were purchased as follows: IDH3α (Aviva Systems Biology, ARP42237_T100), heat shock conjugate 70 (HSC70) (Assay Designs, SPA-816), hypoxia inducible factor-1α (HIF1α) (Novus Biologicals, NB100-110); IDH1 (sc-49996), IDH2 (sc-55668) and α-KG (sc-49589) antibodies were purchased from Santa Cruz Biotechnology, Inc. All other immunoblot supplies, unless noted, were purchased from Bio-Rad Laboratories.

Techniques: Expressing, Control, Immunocytochemistry, Marker, Staining