Fig 1: Expression of key enzymes in glycogenesis and glycogenolysis under stress conditions. (A) Protein levels of glycogen synthase (GYS1 and GYS2), glycogen branching enzyme (GBE1), glycogen phosphorylase (PYGB, PYGM, and PYGL), and glycogen debranching enzyme (AGL) in mice under stress. The relative optical density was calculated by dividing the density of the target band by that of the corresponding ß-ACTIN band (n = 3). (B) Quantified GP activities in SS mice (n = 8). (C) Immunofluorescent double staining of S100ß and PYGB, scale bar = 50 µm. (D) Quantification of PYGB-relative fluorescence intensity in the medial prefrontal cortex of SS mice. PYGB-relative fluorescence intensity calculated as percentage of fluorescence intensity in the colocalization area (denoted as S100ß and PYGB) divided by that in the S100ß+ area (n = 6). *p < 0.05, ***p < 0.001. CTRL, control mouse models; SS, stress-susceptible mouse models.
Fig 2: PYGB upregulation changes glutamate levels and energy metabolites. (A) Glutamate level in SS mice, non-stressed controls, or CSDS attacked PygbH11/H11 mice (n = 5). (B) ATP level in SS, non-stressed control, or CSDS attacked PygbH11/H11 mice (n = 4). (C) Lactate level in SS, non-stressed control, or CSDS attacked PygbH11/H11 mice (n = 5). (D) NADPH level in SS and non-stressed control mice (n = 4). (E) Pyruvate level in SS and non-stressed control mice (n = 4). *p < 0.05, ***p < 0.001. ATP, adenosine triphosphate; CTRL, control mouse models; SS, stress-susceptible mouse models.
Fig 3: Increased astrocytic PYGB ameliorates depression-like behavior. (A) PYGB upregulation in Pygb+/+ mice; schematic overview of the CRISPR-/Cas-mediated genome strategy for creating GFAP-specific Pygb knock-in mice. (B) Schema of the experimental design of Pygb knock-in mice. Experimental timeline of the 10-day CSDS protocol, behavioral tests, and medial prefrontal cortex tissue collection. (C) CSDS induced depression-like behaviors as assessed by the social interaction (n = 8), (D) tail suspension (n = 8), and (E) sucrose preference tests (n = 8). *p < 0.05, **p < 0.01, ***p < 0.001. CSDS: chronic social defeat stress. GFAP, glial fibrillary acidic protein; PYGB, glycogen phosphorylase; CTRL, control mouse models; SS, stress-susceptible mouse models.
Fig 4: PYGB overexpression in the medial prefrontal cortex decreases susceptibility to depression. (A) Timeline of AAV injection, behavioral tests, and immunohistochemistry. (B) Immunofluorescence staining to identify the astrocyte-specific localization of exogenous PYGB in mice. Scale bar = 20 µm (n = 10). (C) Western blot and quantification of PYGB and P-PYGB protein levels (n = 3). (D) Glycogen level and GP activity in astrocytic KI-Pygb mouse models (n = 7). (E) Depression-like behaviors assessed by social interaction and tail suspension (n = 8). *p < 0.05. AAV, adeno associated virus; PYGB, glycogen phosphorylase; CSDS, chronic social defeat stress mouse models; KI-Pygb, Pygb-knockin mouse models.
Fig 5: PYGB knockdown increases susceptibility to depression. (A) Timeline of AAV injection, chronic social defeat stress model establishment, behavioral tests, and immunohistochemistry. (B) Immunofluorescence staining was performed to identify the astrocyte-specific localization of exogenous PYGB in mice (n = 8). Scale bars = 20 µm. (C) Protein levels of PYGB and P-PYGB were analyzed via immunoblotting in the medial prefrontal cortex of Pygb-knockdown mice (n = 3). (D) Glycogen level and GP activity in astrocytic Pygb-knockdown mouse models (n = 5). (E) Pygb-knockdown mouse models were assessed by social interaction and tail suspension tests (n = 7). *p < 0.05, **p < 0.01. AAV, adeno associated virus; GP, glycogen phosphorylase; PYGB, glycogen phosphorylase; CTRL, control mouse models; KD-Pygb, Pygb-knockdown mouse models.
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