Journal: JCI Insight

Article Title: Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function

doi: 10.1172/jci.insight.173707

Figure Lengend Snippet: ( A – C ) Gene expression of Prkaa1 and Prkaa2 (counts per million mapped reads) across different mouse tissues. Box plots show interquartile range, median (line), and minimum and maximum (whiskers). ( A ) Hepatocytes ( n = 4) of mice favor Prkaa2 over Prkaa1 expression while ( B ) macrophages ( n = 8) of mice favor Prkaa1 over Prkaa2 expression. ( C ) Mouse retinas demonstrate appreciable expression levels of Prkaa1 and Prkaa2 ( n = 6). ( D and E ) In situ hybridization of wild-type retina sections confirmed expression of ( D ) Prkaa1 and ( E ) Prkaa2 , seen in magenta dots within the outer nuclear layer (red arrows), suggesting expression of both isoforms in rod photoreceptors. RGC, retinal ganglion cells. ( F ) Scatterplot of Prkaa1 and Prkaa2 expression profiles of mouse retina cell types from single-cell RNA-sequencing data. The cell types show appreciable Prkaa1 and Prkaa2 expression. The rod photoreceptor cluster (red arrow) shows roughly a 2-fold expression of Prkaa2 over Prkaa1 . ( G ) Scatterplot of PRKAA1 and PRKAA2 expression profiles of human retina cell types. The cell types show appreciable PRKAA1 and PRKAA2 expression. The rod photoreceptor cluster (red arrow) shows roughly a 3-fold expression of Prkaa2 over Prkaa1 . ( H ) Scatterplot of Prkaa1 and Prkaa2 expression profiles of mouse brain cell types. Most cell types in the mouse brain demonstrate appreciable expression of Prkaa1 and Prkaa2 . ( I ) Scatterplot of PRKAA1 and PRKAA2 expression profiles of human PBMC types. These cell types do not appreciably express PRKAA2, unlike the central nervous system tissues. ( J ) Waterfall graph depicting the expression ratios of both catalytic isoforms across mouse retina, human retina, mouse brain, and human PBMCs. The majority of neuronal cell types express the α2 isoform over α1 whereas immune cells overwhelmingly express the α1 isoform over α2. Scale bar: 100 μm; insets: 20× original magnification.

Article Snippet: IMPDH1 (Proteintech catalog 22092-1-AP) and PRKAA2 (R&D Systems catalog AF2850) were used for primary antibody incubation.

Techniques: Expressing, In Situ Hybridization, RNA Sequencing Assay

Journal: JCI Insight

Article Title: Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function

doi: 10.1172/jci.insight.173707

Figure Lengend Snippet: ( A and B ) Schematic representations of the protein sequences in the Prkaa1 -Rhod/-Rhod and Prkaa2 -Rhod/-Rhod models, respectively. Knockout domains are within the kinase domain to abrogate function but preserve overall expression of the proteins. ( A ) Schematic representation of the PRKAA1 and ( B ) PRKAA2 protein sequence. ( C ) Representative transmission electron microscopy images of magnification, 2,500×, of rod photoreceptors. (Left) Rod photoreceptors of control mice ( Prkaa2 fl/fl ) demonstrate similar features to those of Prkaa1 -Rhod/-Rhod with consistent membrane structure and organization. (Middle) Rod photoreceptors of Prkaa1 -Rhod/-Rhod demonstrate intact connections between the outer and inner segments with consistent laminar organization of the outer segment membranes. (Right) Rod photoreceptors of Prkaa2 -Rhod/-Rhod consistently demonstrate detachments between the outer and inner segments and occasional outer segment membrane dysmorphisms (red arrow). ( D ) Representative transmission electron microscopy images of magnification, 6,000×, of the outer segments of rod photoreceptors. (Left) Outer segments from control mice ( Prkaa2 fl/fl ) show consistent striations and laminar organization resembling Prkaa1 -Rhod/-Rhod . (Middle) Prkaa1 -Rhod/-Rhod outer segments demonstrated organized and laminar structure indicative of normal wild-type structure. (Right) Prkaa2 -Rhod/-Rhod outer segments exhibited disorganized membrane layers and occasional manifestation of granular debris (red arrows). ( E and F ) Scotopic electroretinography of Prkaa1 -Rhod/-Rhod and respective wild-type littermates. ( E ) Representative traces of 0 dB intensity flashes demonstrate similar waveforms between Prkaa1 fl/fl and Prkaa1 -Rhod/-Rhod ( n = 7). ( F ) Analyses of scotopic a (left) and scotopic b (right) amplitude measurements reveal no significant changes in Prkaa1 -Rhod/-Rhod . ( G and H ) Scotopic electroretinography of Prkaa2 -Rhod/-Rhod and respective wild-type littermates ( n = 7). ( G ) Representative traces of 0 dB intensity flashes show a diminutive waveform from Prkaa2 -Rhod/-Rhod . ( H ) Analyses of scotopic a (left) and scotopic b (right) amplitude measurements confirm significant attenuation in Prkaa2 -Rhod/-Rhod measurements (** P < 0.01, **** P < 0.0001 by 2-way ANOVA with post hoc Bonferroni’s multiple comparisons test). Values are mean ± SEM. Scale bars represent 2 μm. Representative images selected from 40 images for each group.

Article Snippet: IMPDH1 (Proteintech catalog 22092-1-AP) and PRKAA2 (R&D Systems catalog AF2850) were used for primary antibody incubation.

Techniques: Knock-Out, Expressing, Sequencing, Transmission Assay, Electron Microscopy, Control, Membrane

Journal: JCI Insight

Article Title: Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function

doi: 10.1172/jci.insight.173707

Figure Lengend Snippet: ( A – H ) Metabolomics analyses of different phospho-purines in Prkaa2 -Rhod/-Rhod retinas using LC-MS/MS ( n = 9). ( A ) Prkaa2 -Rhod/-Rhod retinas demonstrated near-significant increase in cGMP levels, a critical regulator of the dark current ( P = 0.056 by Welch’s t test). ( B and C ) GMP and GDP levels were not significantly changed in Prkaa2 -Rhod/-Rhod . ( D ) GTP levels were significantly increased in Prkaa2 -Rhod/-Rhod (** P < 0.01 by Welch’s t test). ( E ) Levels of IMP, a precursor to GMP, were not significantly changed in Prkaa2 -Rhod/-Rhod . ( F and G ) AMP and ADP levels were not significantly changed in Prkaa2 -Rhod/-Rhod . ( H ) ATP levels were significantly increased in Prkaa2 -Rhod/-Rhod (** P < 0.01 by Welch’s t test). ( I and J ) Extracellular flux analyses by retina Seahorse of Prkaa2 -Rhod/-Rhod . ( I ) Oxygen consumption rate as a measure of oxidative phosphorylative flux was not significantly changed in Prkaa2 -Rhod/-Rhod ( n = 7). ( J ) Extracellular acidification rate as a measure of glycolytic flux was significantly increased ( P < 0.0001 by 2-way ANOVA, comparing the wild-type group to the knockout group as a whole). Glycolysis (* P < 0.05, ** P < 0.01 by post hoc Bonferroni’s multiple comparisons test) and glycolytic capacity (*** P < 0.001 by post hoc Bonferroni’s multiple comparisons test) were significantly upregulated in Prkaa2 -Rhod/-Rhod ( n = 8). ( K ) Excreted retina lactate levels were significantly increased in Prkaa2 -Rhod/-Rhod , supporting the increased glycolysis phenotype ( n = 7, * P < 0.05 by Welch’s t test). Values are mean ± SEM.

Article Snippet: IMPDH1 (Proteintech catalog 22092-1-AP) and PRKAA2 (R&D Systems catalog AF2850) were used for primary antibody incubation.

Techniques: Liquid Chromatography with Mass Spectroscopy, Knock-Out

Journal: JCI Insight

Article Title: Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function

doi: 10.1172/jci.insight.173707

Figure Lengend Snippet: ( A ) Schematic representation of the workflow to isolate rod photoreceptors to use for phosphoproteomics analysis. Six retinas were dissected per sample and first dissociated using papain digestion. CD73-PE and Rhodopsin-biotin antibodies were used to tag rod photoreceptor inner and outer segments and were isolated using immunomagnetic beads. The cells were then lysed and processed by LC-MS/MS using an unbiased phosphoproteomics pipeline and analyzed. ( B and C ) Rod photoreceptors from Prkaa1 -Rhod/-Rhod and Prkaa2 -Rhod/-Rhod were processed for phosphoproteomic analyses ( n = 4). 1.75 fold-change and 0.01 P value cutoffs with < 1% false discovery rate were used to determine significant changes. ( B ) Prkaa1 -Rhod/-Rhod analysis revealed 2 downregulated targets, which were unrelated to rod photoreceptor function. ( C ) Prkaa2 -Rhod/-Rhod analysis revealed 45 downregulated targets, including species related to the phototransduction cascade and photoreceptor function. ( D ) A selection of downregulated phosphoproteins from the Prkaa2 -Rhod/-Rhod phosphoproteomics data set were plotted on a heatmap to visualize the spread of individual samples. Samples with higher z scores are visualized as a deeper red color while samples with lower z scores are visualized as a deeper blue color. Each row represents a phospho-site of the denoted protein, while each column represents either a sample from Prkaa2 fl/fl (WT) or Prkaa2 -Rhod/-Rhod (KO). ( E and F ) Tandem mass tag (TMT) signal-to-noise ratios of IMPDH1-S416 and IMPDH2-S416 from individual Prkaa2 fl/fl (WT) and Prkaa2 -Rhod/-Rhod (KO) samples. KO samples are colored as pink dots while WT samples are colored as blue dots. In both IMPDH1-S416 and IMPDH2-S416 measurements, the KO samples overall present lower signal-to-noise ratios than WT samples.

Article Snippet: IMPDH1 (Proteintech catalog 22092-1-AP) and PRKAA2 (R&D Systems catalog AF2850) were used for primary antibody incubation.

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Selection

Journal: JCI Insight

Article Title: Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function

doi: 10.1172/jci.insight.173707

Figure Lengend Snippet: ( A ) Schematic representation of IMPDH function in rod photoreceptors according to light exposure. Previous work has shown GTP allosterically inhibits IMPDH in dark-adapted conditions while IMPDH is active to produce GMP in light-adapted conditions. ( B ) Ambient light–adapted retinas from Prkaa2 -Rhod/-Rhod were assessed for IMPDH activity ( n = 4). No significant changes were detected. ( C ) Dark-adapted retinas from Prkaa2 -Rhod/-Rhod showed significantly increased IMPDH activity ( n = 6; ** P < 0.01 by 2-way ANOVA). ( D ) Ambient light–adapted wild-type retinas were assessed for IMPDH activity after treatment with 5 mM metformin, an AMPK activator. Metformin is a slower activator of AMPK, and metformin treatment inhibits IMPDH activity compared with that of control retinas ( n = 6, P < 0.0001 by 2-way ANOVA). ( E ) Dark-adapted wild-type retinas were assessed for IMPDH activity after treatment with 40 μM compound C, an AMPK inhibitor. Lysates treated with compound C exhibited significantly higher IMPDH activity compared with controls ( n = 4, ** P < 0.01 by 2-way ANOVA). ( F and G ) Recombinant human AMPK (α2β1γ1) was incubated with recombinant human ( F ) IMPDH1 or ( G ) IMPDH2 to assess the effect of AMPK on IMPDH activity. Both IMPDH1 and IMPDH2 when incubated with AMPK had significantly attenuated IMPDH activity compared with IMPDH incubated alone ( n = 4, P < 0.0001 by 2-way ANOVA). ( H ) Schematic workflow of the co-immunoprecipitation protocol of IMPDH1. Six dark-adapted retinas from wild-type mice were dissected and lysed in extraction buffer. Dynabeads coated with IMPDH1 antibody were added to the suspension and allowed to bind to IMPDH1. Attached IMPDH1 along with bound proteins were isolated. The resulting eluant was then used for Western blots to detect IMPDH1 and bound protein. ( I ) The results of Western blot detection of co-immunoprecipitation samples are depicted. Clear bands representing IMPDH1 along with PRKAA2 demonstrate PRKAA2 was bound to IMPDH1 in wild-type retinas. Values are mean ± SEM.

Article Snippet: IMPDH1 (Proteintech catalog 22092-1-AP) and PRKAA2 (R&D Systems catalog AF2850) were used for primary antibody incubation.

Techniques: Activity Assay, Control, Recombinant, Incubation, Immunoprecipitation, Extraction, Suspension, Isolation, Western Blot

Journal: JCI Insight

Article Title: Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function

doi: 10.1172/jci.insight.173707

Figure Lengend Snippet: ( A and B ) Representative electroretinography traces of Prkaa2 -Rhod/-Rhod mass rod recovery. A 0 dB flash with 800 ms interstimulus time (IST) is represented. ( A ) A trace from Prkaa2 fl/fl shows typical attenuated scotopic a and b waves in the test response indicated by the probe flash with 800 ms interstimulus time. ( B ) A trace from Prkaa2 -Rhod/-Rhod shows abnormally large scotopic a and b waves with 800 ms IST in the test response indicated by the probe flash. ( C ) Quantification of scotopic A and scotopic B rod recovery ( n = 7). Prkaa2 -Rhod/-Rhod mice show significantly faster rod recovery compared to wild-type littermates (*** P < 0.001, **** P < 0.0001 by post hoc Bonferroni’s multiple comparisons test; P < 0.0001 by 2-way ANOVA for both graphs). ( D and E ) Representative electroretinography traces of Prkaa2 -Rhod/-Rhod mass rod recovery from eyes treated with vehicle or mycophenolate mofetil. ( D ) Vehicle-treated eyes demonstrate similar waveform shapes as ( E ) mycophenolate-treated eyes from the probe flash. ( F ) Quantification of scotopic a and scotopic b rod recovery ( n = 5). Mycophenolate-treated eyes show significantly slower scotopic a and scotopic b rod recovery compared with vehicle-treated eyes ( †† P < 0.01 by post hoc Bonferroni’s multiple comparisons test; *** P < 0.001 by 2-way ANOVA). ( G ) Representative traces of full-field scotopic electroretinography from vehicle- and mycophenolate-treated eyes. Although the waveform shape is slightly altered, mycophenolate treatment improves scotopic a and scotopic b wave amplitudes compared with vehicle treatment. ( H ) Quantification of scotopic a and scotopic b wave amplitudes from full-field scotopic electroretinography ( n = 5). Mycophenolate-treated eyes had significantly improved scotopic a and scotopic b wave amplitudes compared with vehicle-treated eyes (** P < 0.01, *** P < 0.001 by 2-way ANOVA). Values are mean ± SEM.

Article Snippet: IMPDH1 (Proteintech catalog 22092-1-AP) and PRKAA2 (R&D Systems catalog AF2850) were used for primary antibody incubation.

Techniques:

Journal: Clinical immunology (Orlando, Fla.)

Article Title: Metabolic Reprogramming in Memory CD4 T Cell Responses of Old Adults

doi: 10.1016/j.clim.2019.07.003

Figure Lengend Snippet: (A) AMPK phosphorylation was compared by Peggy Sue western in day 2 activated CD4 memory T cells from 12 young and 12 older adults. Representative blot of signal intensities (left) and summary data (right). Comparison by unpaired one-tailed t-test. (B) Activated CD4 T memory cells were transfected with siRNA for AMPKα on day 1. Knockdown efficiency by qPCR (left) and CPT1a expression by Peggy Sue western (right) are shown on day 2 after activation (n=9). (C) SIRT1 expression in day 2 activated CD4 memory T cells were determined by Peggy Sue western. A representative blot of signal intensities from a young and an old adult (left) and the ratio of SIRT1 to actin from the samples shown in (A) are shown. (D) Activated CD4 T memory cells were transfected with siRNA for SIRT1 on day 1. Knockdown efficiency by Peggy Sue western (left) and CPT1a expression by qPCR (middle) and Peggy Sue western (right) are shown on day 2 after activation (n=6). Comparisons were done by paired one-tailed t-test.

Article Snippet: The high sensitivity protocol was applied by increasing the stacking loading time to 20 seconds, the sample loading time to 12 seconds, and the separation time to 45 min. Primary antibodies were as listed: CPTIa (Cell Signaling, Cat#: 12252), SIRT1 (Novus Biologicals, Cat#: NBP1–49540), AMPK (R&D, Cat#: MAB3197), phospho-AMPK (Cell Signaling, Cat#: 2535), Cytochrome c (Cell Signaling, Cat#: 11940).

Techniques: Western Blot, One-tailed Test, Transfection, Expressing, Activation Assay