Neurons synthesize glycogen under basal conditions. (A) Composition of neurons and astrocytes after culture for 5 days in vitro (DIV) in neurobasal medium with antimitotics. The presence of neurons and astrocytes using neuron- (β-III tubulin, TUJ1, middle panel, up) and astrocyte- (GFAP, middle panel, down) specific antibodies from three independent experiments is shown. Nuclei were stained with Hoechst 33342, and images were acquired with a confocal microscope using a × 10 objective. Scale bar=40 μm. The pictures correspond to representative images of three independent experiments. (B) Glucose incorporation into glycogen. Neurons obtained from WT mice were cultured for 5 days in neurobasal medium with antimitotics, and then incubated in 25 mmol/L Glucose containing neurobasal in the presence of [¹⁴C]-Glucose for the indicated times. After this incubation period, glycogen was extracted and the incorporation of [¹⁴C]-Glucose was measured. *P<0.05, **P<0.01 versus 0.5 hour. Data represent the mean±s.e.m. (n=4). (C) Neurons were obtained from wild-type (WT) and brain-specific muscle glycogen synthase (MGS) knockout (KO) mice and tested for the expression of MGS by western blot and (D) glycogen content after 5 DIV cultured in neurobasal medium with antimitotics. Glycogen levels represent the mean±s.e.m. (n=8). Actin was used as a loading control for the western blot. (E) Detection by immunofluorescence of MGS (red, left panel) and neuron-specific marker TUJ1 (gray, right panel) in WT neurons. Cells were seeded in coverslips and fixed with paraformaldehyde after 5 DIV. Nuclei were stained with Hoechst 33342, and images were acquired with a confocal microscope using a × 63 objective. Scale bar=10 μm. The pictures correspond to representative images of four independent experiments.

Neurons have the machinery necessary to degrade glycogen. Neurons were cultured for 5 days in Neurobasal with antimitotics. At 5 days in vitro (DIV5) medium was replaced with fresh neurobasal. (A) Glycogen content after exposure to hypoxia. At DIV5, cells were exposed to 1% O₂ for 4 hours and then rapidly frozen. Glycogen content was determined in control (Normoxia, exposed to environmental 21% O₂) and treated neurons (Hypoxia, exposed to 1% O₂) and represents the mean±s.e.m. (n=7). ***P<0.001 versus Normoxia. (B) Immunofluorescence analysis with muscle glycogen synthase (MGS) (red, left panel) and TUJ1 (gray, right panel) in control neurons and after hypoxic insult. At DIV5, cells were incubated in 1% O₂ for 4 hours and then fixed with paraformaldehyde. Cell nuclei were stained with Hoechst 33342. All images were acquired with a confocal microscope using a × 63 objective. Scale bar=10 μm. The images correspond to representative images of four independent experiments. (C) Glycogen content after hypoxic exposure of neurons overexpressing a constitutively active form of Glycogen Synthase (Ad MGS-9A). At DIV3, neuronal cultures were infected for 12 hours with adenovirus encoding MGS-9A. At DIV5, medium was replaced, and cells were subsequently exposed to 1% O₂ for 4 hours. The plates were rapidly frozen and analyzed for glycogen content. No inf=non-infected cells. Data represent the mean±s.e.m. (n=5). ***P<0.001 versus Normoxia Ad MGS-9A. (D) Glycogen (red, left panel) and MGS-9A (green, middle panel) immunocytochemistry of primary cultures of neurons infected with Ad MGS-9A and exposed to hypoxia for 4 hours under the same experimental conditions as (C). Cell nuclei were stained with Hoechst 33342. Images were acquired with an epifluorescent microscope using a × 40 objective. Scale bar=20 μm. The pictures correspond to representative images of five independent experiments. (E) Glycogen content in a Drosophila melanogaster line overexpressing human MGS specifically in neurons. Flies were exposed to 0.6% O₂ for 1 hour and then immediately frozen in liquid nitrogen. Heads were dissected on ice while still frozen, and glycogen content was determined from five heads per sample. The line elav> w1118 (w1118) was used as a control. Data represent the mean±s.e.m. (n=6). *P<0.05 versus w118 Normoxia and ***P<0.001 versus MGS Normoxia. All flies were tested at 7 days of age. (F) Neuronal glycogen degradation in vivo. Cerebella from eight-week-old control mice (Ctrl) and mice overexpressing a constitutively active form of glycogen synthase specifically in Purkinje neurons (MGS-9A^Pcp2) were isolated and rapidly frozen. In another subset of mice, the same groups were perfused intracardiacally with saline for 5 minutes before cerebella isolation to induce glycogen degradation. The glycogen content of these cerebella was measured. Ctrl animals correspond to MGS-9A^Pcp2 littermates lacking MGS-9A expression. Glycogen content represents the mean±s.e.m. (n=10). ***P<0.001 versus Ctrl—Saline and **P<0.01 versus MGS-9A^Pcp2—Saline.

Neurons degrade glycogen using brain glycogen phosphorylase (GP). (A) Neurons were cultured in neurobasal medium with antimitotics, and were infected at 3 days in vitro (DIV3) with adenovirus encoding a constitutively active form of Glycogen Synthase (Ad MGS-9A) for 12 hours. At 5 days in vitro (DIV5), medium was replaced with fresh neurobasal, and neurons were incubated with inhibitors of GP (DAB), autophagy (Chloroquine) and acid alpha-1,4-glucosidase (Acarbose), and immediately exposed to 1% O₂ for 4 hours. Glycogen content was determined biochemically and represents the mean±s.e.m. (n=4). ***P<0.001 versus control without inhibitor inside the normoxic or hypoxic group. (B) Glycogen detection by immunocytochemistry in neurons treated with adenovirus and exposed to hypoxia under the same experimental conditions as (A). All images were acquired with an epifluorescent microscope using a × 40 objective. Scale bar=20 μm. The pictures correspond to representative images of four independent experiments. (C) Brain- and muscle-specific isoform levels of GP (brain GP and muscle GP, respectively) in neurons exposed to hypoxia. Neurons obtained from WT mice were cultured for 5 days in neurobasal medium with antimitotics. At DIV5 medium was replaced with fresh neurobasal, and cells were exposed to 1% O₂ for 4 hours. GFAP, an astroglial marker, was used to rule out possible contamination of astrocytes in the neuronal extracts, and total brain homogenate (B) was loaded as a positive control for brain, muscle GP, and GFAP. The image corresponds to a representative western blot (WB) of six independent experiments. For quantification, see in . (D) Brain GP intracellular detection (red, left panel) by immunofluorescence in primary neuronal cultures exposed to 1% O₂ for 4 hours as described in (C). TUJ1 (gray, right panel) and Hoechst 33342 (blue) are used to stain neurons and nuclei, respectively. All images were acquired with an epifluorescent microscope using a × 40 objective. Scale bar=20 μm. The pictures correspond to representative images of three independent experiments. (E) GP activity ratio and (F) GP activity in the presence of AMP of neurons exposed to 1% O₂ under the same conditions as in (C). The enzymatic activity is expressed as GP activity ratio, an adimensional value that results from dividing GP activity in the presence of caffeine (which measures phosphorylated, and thus active, GP) through GP activity in the presence of AMP (which is dependent in the case of brain GP, of the total amount of enzyme). Data represent the mean±s.e.m. (n=3). *P<0.05 versus Normoxia. (G) Representative images of brain GP distribution in hippocampal (CA2) slices from adult mice. Forty-week-old mice were perfused with paraformaldehyde, and brains were frozen. Slices were stained for the brain GP isoform (red, left panel, see white arrows), GFAP (gray, upper middle panels a–c) and Parvalbumin (green, lower middle panels d–f), markers specific for neurons and astroglia, respectively. Nuclei (blue) were stained with Hoechst 3342. Images were obtained with a confocal microscope using a × 63 objective. Scale bar=10 μm. The pictures correspond to representative images of three independent mice. (H) 3D and surface reconstruction of brain GP (red) and Parvalbumin (green)-positive neurons of the confocal images shown in (G). The reconstruction of the sections corresponding to the z axis shows colocalization of GP and Parvalbumin, giving rise to a yellow spot.

Hypoxia induces muscle glycogen synthase (MGS) activation and glycogen synthesis, but no net accumulation. Neurons were cultured for 5 days in neurobasal medium with antimitotics. At 5 days in vitro (DIV5) medium was replaced with fresh neurobasal and hypoxia was induced. (A) Glycogen synthase (GS) Activity Ratio (GS activity measured in the absence of glucose-6-phosphate (Glc-6-P)/total GS activity, measured in the presence of Glc-6-P, left panel) and total GS activity (measured in the presence of Glc-6-P, right panel) in neuronal cultures exposed at DIV5 to 1% O₂ for 4 hours. The GS activity ratio is an estimation of the activation state of the enzyme, while total GS activity depends only on the total levels of GS in the cells. Data represent the mean±s.e.m. (n=6). ***P<0.001 versus normoxia. (B) Western blot analysis using antibodies against total (MGS) and phosphorylated 641 Serine MGS (pMGS) in neuronal cultures after the same treatment as (A). Actin was used as a loading control. The image corresponds to a representative western blot of seven independent experiments. For quantification see in (C) Glycogen content of neurons after hypoxia in the presence of the GP inhibitor DAB. At DIV5, neuronal cultures were incubated in the presence or absence of 0.1 mmol/L DAB for 1 hour and subsequently exposed to 1% O₂ for 4 hours. Cells were frozen, and glycogen was determined. Data represent the mean±s.e.m. (n=8). *P<0.05 of +DAB hypoxia versus +DAB normoxia. ***P<0.001 of +DAB hypoxia versus—DAB hypoxia. (D) Intracellular UDP Glucose (left panel) and Glc-6-P (right panel) levels of neurons obtained either from WT mice or brain-specific MGS knockout (KO) mice and exposed at DIV5 to 4 hours 1% O₂. Data represent the mean±s.e.m. (n=8). ***P<0.001 Normoxia WT versus Hypoxia WT of UDP Glucose and Glc-6-P, ***P<0.001 Normoxia WT vs Normoxia KO of UDP Glucose and *P<0.05 Hypoxia WT versus Hypoxia KO of Glc-6-P. (E) Glycogen content and (F) UDP Glucose levels in reoxygenated neurons. Neurons at DIV5 were exposed to 1% O₂ for 4 hours and then incubated at 21% O₂ (Reoxygenation) for the indicated times. Data represent the mean±s.e.m. (n=4). ***P<0.001 and *P<0.05 versus Control levels. (G) Schematic representation of glycogen turnover in normoxia and hypoxia. MGS and BGP are mostly inactive in normoxia (circles) and become activated in hypoxia (stars).

Glycogen synthase (GS) has a protective role in neurons under hypoxia. (A) Death fold change after increasing exposure to hypoxia in GS wild-type (WT) (black) and knockout (KO) (white) neurons. Neurons were isolated from brain-specific muscle glycogen synthase (MGS) WT or KO mice and cultured for 5 days with neurobasal medium with antimitotics. At 5 days in vitro (DIV5), medium was replaced by fresh neurobasal, and neurons were exposed for the indicated times to 1% O₂. Neurons were stained with two cell-permeable dyes, which differentially stained living and dead cells. The average number of dead cells was estimated in 10 fields from 5 to 8 independent mice, 900 to 1000 total cells. Data were normalized against 2 hours Normoxia for WT neurons, which corresponded to around 30% of cellular death. *P<0.05, **P<0.005, ***P<0.001 of Hypoxia KO versus Hypoxia WT. (B) ATP levels (arbitrary units) after increasing exposure to hypoxia in GS WT (black) and KO (white) neurons. Neurons were isolated from brain-specific MGS WT or KO mice, under the same experimental conditions as in (A), exposed for the indicated times to 1% O₂ and rapidly frozen in liquid nitrogen for the metabolite assay. Data represent the mean±s.e.m. (n=14). **P<0.005, ***P<0.001 of Normoxia KO versus Normoxia WT. (C) Drosophila GS (dGS) mRNA levels from whole adult heads in several Drosophila melanogaster transgenic lines. In these lines, the promoter elav drives the expression of UAS-GFP control (GFP) and two short hairpin RNAi constructs targeting dGS: UAS-sh dGS Gsi III (GSi III) or UAS-sh dGS NIG III (NIG III), using the UAS-GAL4 system. W1118 elav-driven control lines lacking any UAS insertion were used as an additional control (w1118). Data represent the mean±s.e.m. (n=4). *P<0.05 versus GFP. (D) Time to recovery after hypoxic exposure of the transgenic lines of Drosophila melanogaster used in (C) (control lines, GFP, black and w1118, gray; and the dGS knock down lines GSi III, green and NIG III, red). Groups of 16 flies were placed in a nylon-sealed tube and incubated in 0.6% O₂ for 1 hour, during which they entered a state of stupor. After 1 hour, flies were again exposed to environmental oxygen tension, and the time required to recover their motor functions was determined for each line. n=100 to 150 flies per group. P values were calculated against GFP using the Cox proportional hazard model. Genotypes of flies: GFP (elav-Gal4/+ UAS-GFP/+), w1118 (elav-Gal4/w[1118]), GSi III (elav-Gal4/+ UAS-dGS RNAi-III/+); NIG III (elav-Gal4/+ UAS-dGS RNAi-NIII/+); UAS-dGS RNAi lines were from VDRC (GSi III, v35136) and NIG (NIII, 6904R-3).