Phosphorylation of pyruvate dehydrogenase inversely associates with neuronal activity

Abstract

For decades, the expression of immediate early genes (IEGs) such as FOS has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity. Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected activity decreases across the brain, which were induced by a wide range of factors including general anesthesia, chemogenetic inhibition, sensory experiences, and natural behaviors. Thus, as an inverse activity marker (IAM) in vivo, pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors.

Keywords: immediate early genes; inhibition; inverse activity marker; neuronal activity.

Figure 1.. Activity-dependent phosphoproteomic screen identifies phospho-PDH in vitro. (See also Figure S1)

(A) Schematic of the activity-dependent phosphoproteomic screen. Cultured cortical neurons infected with AAV9-Channelrhodopsin 2 (ChR2) were stimulated with blue light at different frequencies. Cells were harvested after stimulation for biochemical analysis. (B-C) Validation of optical spiking in cultured cortical neurons expressing ChR2. (B) Representative light-evoked traces using cell-attached recording at the indicated light pulse frequencies and each pulse durations (40 pulses, λ = 470 nm, 5 mW/mm). (C) Quantification of light-evoked spike fidelity in (B). N = 21 neurons per condition. (D) Gene expression changes of immediate early genes after light stimulation in cultured cortical neurons expressing ChR2. (N = 3 biological replicates for control, 0.5 Hz, 10 Hz, N = 2 for KCl). (E) Workflow of phosphoproteomic analysis. Cells stimulated with different frequencies were lysed followed by tandem mass tag (TMT)-based labeling. TMT-labeled peptides were then enriched for phosphopeptides. (F) TMT-based phosphopeptide quantification revealed pPDH was significantly enriched in the 0.5 Hz stimulated group compared to 10 Hz stimulation group. (7,504 plotted phosphopeptides, cutoff fold change = 2, p = 0.05). (G) Schematic overview of glucose metabolism regulated by pPDH. All values are mean ± s.e.m. Statistics determined by two-tailed unpaired t test in (D). * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2.. pPDH is inversely correlated with neuronal activity in vitro. (See also Figure S2)

(A-C) The effects of different cellular stimuli on pPDH. (A) Cultured cortical neurons were applied with light (0.1 or 5 Hz), BDNF (50 ng/mL), Forskolin (FSK,10 μM) or KCl (50 mM) for 10 minutes. Cells were lysed at the end of stimulation (marked by red triangles) for immunoblot analysis (B), and pPDH levels were quantified in (C). N = 3 replicates. (D-F) The effect of photostimulation duration on pPDH. (D) Cultured cortical neurons were stimulated with light (5 Hz) for indicated durations. Whole-cell lysates were analyzed by immunoblotting (E), and pPDH levels were quantified in (F). N = 4 replicates. (G-I) The effect of post-stimulation recovery time on pPDH. Cultured cortical neurons stimulated with light (5 Hz, 10 minutes) were given recovery times as indicated (G) before whole-cell lysis and immunoblot analysis (H). pPDH levels were quantified in (I). N = 3 replicates. (J-L) The effect of pharmacological inhibition on pPDH. Cultured cortical neurons were treated with an inhibitor cocktail (1 μM TTX, 50 μM AP5 and 10 μM CNQX) for indicated time (J) before whole-cell lysis and immunoblot analysis (K). pPDH levels were quantified in (L). N = 4 replicates. Cultured neurons in A-I were pre-incubated with inhibitor cocktails (50 μM AP5, 10 μM CNQX and 100 μM Picrotoxin) overnight before stimulation. All values are mean ± s.e.m. The statistical significance between the treated group and the control group was determined using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. BDNF: brain-derived neurotrophic factor; TTX: tetrodotoxin; AP5: D-(−)-2-Amino-5-phosphonopentanoic acid; CNQX: 6-Cyano-7-nitroquinoxaline-2,3-dione disodium.

Figure 3.. pPDH inversely correlates with neural activity in general anesthesia. (See also Figure S3)

(A) General anesthesia paradigm. Mice were exposed to isoflurane, and brain samples were collected at indicated time points (orange triangles) for immunohistochemistry analysis. (B-C) pPDH labeling in the whole brain after 2 hours of general anesthesia. (B) Representative images of brain coronal sections at indicated bregma positions. (C) Representative zoomed-in images of pPDH and FOS labeling in different brain regions. N = 4 animals per condition. Scale bar: 500 μm in (B) and 50 μm in (C). M2: secondary motor cortex; LH: lateral hypothalamic area; BLA: basolateral amygdaloid nucleus, anterior part; VMH: ventromedial hypothalamic nucleus.

Figure 4.. pPDH inversely correlates with neural activity in chemogenetic inhibition and visual stimulation. (See also Figure S4)

(A-E) Chemogenetic inhibition paradigm. (A) Diagram of chemogenetic inhibition of substantia nigra (SNr). Vgat-Cre mice were injected with AAV-DIO-Gi-mCherry into the left SNr and AAV-DIO-mCherry into the right SNr. 1h after CNO administration, the mice were perfused and collected for brain samples. (B) Behavior validation of SNr inhibition. On two separate days, mice were injected with either vehicle or CNO and recorded 30 minutes post-injection. The cumulative number of turning rounds during the recording session was quantified. N=9 animals. Statistics determined by two-way ANOVA. (C) Representative images of AAV infection and pPDH staining in the left and right SNr from the same animal. (D) Quantification of total number of pPDH⁺ infected (mCherry⁺) cells in SNr. N = 8 animals. Statistics determined by two-tailed paired t test. (E) Quantification of pPDH staining intensity in mCherry⁺ cells. N = 8 animals. Statistics determined by two-tailed paired t test. (F-H) Visual stimulation paradigm. (F) Mice were kept in the dark for 48 hours before being exposed to visual stimuli (V. S.) for 4 hours and then returned to the dark immediately thereafter. Tissues were harvested at indicated timepoints (shown by orange triangles) for immunostaining. (G) Representative images and quantification of pPDH and FOS staining in the primary visual cortex (V1). Positively stained cells in layer 5 of V1 were quantified and normalized to 48-hour dark group. N = 6, 10, 7, 6 animals for the dark, V.S., 2h, 12h conditions respectively. The statistical significance between adjacent conditions were determined by one-way ANOVA with Šídák’s multiple comparisons test. (H) Representative images and quantification of pPDH and FOS staining in the lateral geniculate nucleus (LGN). Positively stained cells were quantified and normalized to 48-hour dark group. N = 4 animals for each condition. The statistical significance between adjacent conditions were determined by one-way ANOVA with Šídák’s multiple comparisons test. All values are mean ± s.e.m. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Scale bar, 50 μm. SNr: substantia nigra; V1: primary visual cortex; LGN: lateral geniculate nucleus.

Figure 5.. pPDH inversely correlates with neural activity in water deprivation models. (See also Figure S5)

(A-B) Water deprivation and drinking paradigm. (A) Mice were water deprived (W.D.) for 24 hours before given access to water. Brain samples were collected at indicated timepoints (orange triangles) for immunohistochemistry analysis. (B) Representative images of pPDH staining in the median preoptic nucleus (MEPO). Positively stained cells were quantified and normalized to the control group. N = 4 animals per group. The statistical significance between adjacent conditions were determined by one-way ANOVA with Šídák’s multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001. (C-G) Whole-brain activity analysis after water deprivation paradigm. (C) Workflow of the whole-brain activity screening with FOS antibody staining after water deprivation. N = 7 animals per condition. (D) Volcano plot illustrating the results of automated whole-brain FOS staining quantification following water deprivation. Brain regions demonstrating upregulated (marked with red dots) and downregulated (marked with green dots) FOS expression are highlighted. N = 7 animals per condition, left and right hemisphere quantified separately. Statistics determined by two-tailed t tests (cutoff: p < 0.05). (E) Quantification of pPDH intensity ratio between water-deprived and control groups in brain regions exhibiting increased FOS expression following water deprivation. Brain regions identified as having upregulated FOS expression from (D) were filtered to exclude regions associated with the blood-brain barrier or regions that were too small for accurate assessment (gray label in (D)). FOS fold change is color-coded on the left. Control: N = 8; W.D.: N = 7. Statistics determined by multiple two-sided t-tests corrected for multiple comparisons with the two-stage step-up (Benjamini, Krieger, and Yekutieli). *false discovery rate (FDR) < 10%. (F) Representative images of pPDH staining in indicated regions. Scale bar, 50 μm. AMB: nucleus ambiguous; PVH: paraventricular hypothalamic nucleus; MEPO: median preoptic nucleus; ADP: anterodorsal preoptic nucleus; VII: facial motor nucleus; SO: supraoptic nucleus; PSV: principal sensory nucleus of the trigeminal; TU: tuberal nucleus; TRS: triangular nucleus of septum; VTA: ventral tegmental area.

Figure 6.. pPDH inversely correlates with neural activity in food deprivation models. (See also Figure S6)

(A-E) Examination of arcuate nucleus (ARC) (inside blood brain barrier) in fasting-refeeding paradigm. (A) Schematics of fast-refeeding paradigm. Mice were food deprived for 16 hours before given access to food for 1 hour. Brain samples were collected at indicated timepoints (orange triangle) for immunohistochemistry analysis. (B) Quantification of total pPDH⁺ cells in ARC across all conditions. N = 7 mice per condition. (C) Quantification of total FOS⁺ cells in ARC across all conditions. N = 7 mice per condition. (D) Representative images showing pPDH staining and cell-type markers, AgRP (marked by AgRP-Ai9) and POMC (marked by β-endorphin staining) in the ARC under re-fed condition. Blood-brain barrier indicated by yellow dashed line. (E) Quantification of the percentage of pPDH⁺ cells in either AgRP⁺ or POMC⁺ cells across all conditions. N =5 animals per conditions for AgRP and N = 4 animals per conditions for POMC. (F-H) Analysis of lateral hypothalamic area (LH) activity in fasting-refeeding paradigm. (F) Representative images of pPDH and FOS staining in LH. Positively stained cells were quantified and normalized to the control group. N = 4 animals per group. (G) Analysis of cell types in the LH. Representative images showing cell-type markers and pPDH staining in LH under re-fed conditions. The percentage of indicated cell-type markers in total pPDH⁺ cells under re-fed conditions was quantified. Cell-type markers include orexin (marked by orexin staining, N = 7 animals), MCH (marked by MCH staining, N = 3), Vgat (marked by Vgat-Ai9 mice, N = 4). (H) Fiber photometry recording of Orexin⁺ neuron activity in LH during feeding. AAV-DIO-gCamp6f was injected into LH of Hcrt-Cre mice. A representative raw calcium trace from one animal and averaged calcium signals during feeding from all animals (N = 5) are shown. All values are mean ± s.e.m. in (B-G). Statistics determined by one-way ANOVA with Šídák’s multiple comparisons test for adjacent conditions in (B), (C), (F); 2-way ANOVA with Tukey’s multiple comparisons test in (E); one-way ANOVA with Tukey’s multiple comparisons test in (G). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Scale bar, 50 μm. MCH: melanin-concentrating hormone; LH: lateral hypothalamic area; ARC: arcuate nucleus.