The Impact of Astrocyte Mitochondrial ATP Production on Neuroprotection After Stroke James D. Lechleiter, Ph.D., Professor

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The Impact of Astrocyte Mitochondrial Production The Impact of

of Cellular & Structural Biology University of Texas Health Science

ischemic stroke Neuron Astrocytes National Stroke Association (www.

org) Astrocyte neuroprotection Working Hypothesis: Protection Can Be

1 The Impact of Astrocyte Mitochondrial Production The Impact of Astrocyte Mitochondrial Production 1 Dept. of Cellular & Structural Biology University of Texas Health Science Center at San Antonio San Antonio, TX, USA Schematic diagram of an ischemic stroke Neuron Astrocytes National Stroke Association ( Astrocyte neuroprotection Working Hypothesis: Protection Can Be Enhanced by Stimulating Ca+ Sensitive Astrocyte Mitochondrial Production PY 1 R IP 3 Mitochondrion Astrocyte F1-F ase Neuron ANT Dependent Neuroprotection 3 ER IP 3 R 1

The Impact of Astrocyte Mitochondrial Production PY 1 -R stimulation reduces

RB & MeADP 4 CD4 (area x intensity) 8 4 RB & MeADP 1 3 4 Days * *.4 1.8 3.

8 PY 1 -R stimulation reduces RB-induced TTC stained infarctions Day 5 Day 5 Day

MeADP 4h 1 MeADP h MeADP, 4h 1 days 5 RB & MeADP, 4h Optical imaging In vivo of

(9) Chapter 3, In Astrocyte pathophysiology in the nervous system; Eds.

Boston, MA, Springer Figure 7 Optical Imaging in vivo of the mouse parietal

multiphoton microscope positioning the 6x 1.

higher magnification of the stainless steel ring holder that is glued to the

2 The Impact of Astrocyte Mitochondrial Production PY 1 -R stimulation reduces photothrombotic (RB) infarcts Day Day 3 Day 4 3. RB & MeADP 4 CD4 (area x intensity) 8 4 RB & MeADP Days * * CD4 (intensity) CD4 (intensity) 1.8 PY 1 -R stimulation reduces RB-induced TTC stained infarctions Day 5 Day 5 Day 1 Day 5 RB & MeADP h 5 Day 1 Day 5 RB & MeADP h RB & MeADP 4h TTC Lesion (mm ) MeADP 4h 1 MeADP h MeADP, 4h 1 days 5 RB & MeADP, 4h Optical imaging In vivo of the mouse parietal cortex Watts, L.T. and Lechleiter, J.D. (9) Chapter 3, In Astrocyte pathophysiology in the nervous system; Eds. Phil Haydon and Vladimir Parpura. Boston, MA, Springer Figure 7 Optical Imaging in vivo of the mouse parietal cortex Left panel shows the objective inverter attached to a Zeiss LSM 51 multiphoton microscope positioning the 6x 1.1 NA water immersion objective above the mouse parietal cortex Right panel is a higher magnification of the stainless steel ring holder that is glued to the skull and immobilizes the brain The center ring has been filled with 3% agarose (Sigma type VII) and sealed from above with a glass coverslip (#) 6 This essentially eliminates motion artifacts due to breathing when the hole in the cranium is less than 1- mm in diameter The red heating pad is maintained at 37 o C

3 The Impact of Astrocyte Mitochondrial Production Cerebral ischemic stroke in living mouse 1 mm 1 mm 7 1x Blood Brain Barrier (BBB) disruption induced by RB photothrombosis vol. 5 p. e1441 Zheng et al. 1 PLoS One Pre-RB RB 1 min RB 1 min RB min RB-clot RB-clot 1 min RB-clot min RB-clot 3 min µm Day Post-Photothrombosis RB min RB min RB 3 min 8 1 µm Pre-photothrombosis 9 Zheng et al. 1 PLoS One vol. 5 p. e1441 3

e1441 Necrotic core is not anoxic clot 1

4 The Impact of Astrocyte Mitochondrial Production 4 hours post-photothrombosis 1 Zheng et al. 1 PLoS One vol. 5 p. e hours post-photothrombosis 11 Zheng et al. 1 PLoS One vol. 5 p. e1441 Necrotic core is not anoxic clot 1 Zheng et al. 1 PLoS One vol. 5 p. e1441 4

5 The Impact of Astrocyte Mitochondrial Production production in astrocytes Glucose Extracellular Cytoplasm Glycogen Glycolysis lactate LDH Glutamine Pyruvate Na + K + MCU Pyruvate ase Na + PDH (Ca ADP ) PC GS K + ANT Acetyl-CoA () () H + Oxaloacetate V F1-F Citrate ase Malate Glutamate ADP Isocitrate NADH GLUD1 H O Fumarate ICDH (ADP) a-ketoglutarate H + IV a-ketoglutarate O FADH II H + III NAD + Succinate a-kgdh II Succinyl-CoA 13 H + I NADH Ischemic production in astrocytes Glucose Extracellular Glycogen Glutamate Cytoplasm Glycolysis lactate Na + LDH Glutamine Pyruvate Na + K + MCU Na Pyruvate + ase PDH (Ca ADP + ) PC GS K + ANT Acetyl-CoA () () H + Oxaloacetate V F1-F Citrate ase Malate Glutamate ADP Isocitrate NADH GLUD1 H Fumarate ICDH O (ADP) a-ketoglutarate H + IV a-ketoglutarate O FADH II H + III NAD + Succinate a-kgdh II Succinyl-CoA 14 H + I NADH production in neurons Glucose Extracellular Cytoplasm Glycolysis lactate LDH Glutamine Pyruvate Na + K + MCU Pyruvate ase Na PDH (Ca ADP + ) PAG K + ANT X + Acetyl-CoA () H + Oxaloacetate V F1-F Citrate ase Malate Glutamate ADP Isocitrate NADH GLUD1 H Fumarate ICDH O (ADP) a-ketoglutarate H + IV a-ketoglutarate O FADH II H + III NAD + Succinate a-kgdh II Succinyl-CoA CyPD mptp 15 H + I NADH 5

post-photothrombosis (PY 1 R agonist ) 17

6 The Impact of Astrocyte Mitochondrial Production Pre-photothrombosis 16 4 hours post-photothrombosis (PY 1 R agonist ) hours post-photothrombosis (PY 1 R agonist ) Lesion Area (µm x1 5 ) 1 RB & Blood Vessel Diameter (µm) 8 4 MeADP 1 Days 1 Days 18 6

7 The Impact of Astrocyte Mitochondrial Production Pre-photothrombosis 19 4 hours post-photothrombosis (PY 1 R agonist added) 48 hours post-photothrombosis (4 hours Post PY 1 R agonist ) Lesion Area (µm x1 5 ) 1 RB & 1 Days 1 7

The Impact of Astrocyte Mitochondrial Production Astrocyte

Stimulating Sensitive Astrocyte Mitochondrial Production PY 1 R

Neuroprotection ER IP 3 R The astrocyte-specific mitochondrial

Mitochondrial toxin MCT4 isoform in astrocytes can transport

hrs) Can block specifically mitochondrial metabolism in

fluoroacetate + ) 3 Zheng et al. 1 PLoS One vol. 5 p.

on Dox off (8 days) Number of Mitochondria 1 Dox on Functional

8 The Impact of Astrocyte Mitochondrial Production Astrocyte neuroprotection Working Hypothesis: Protection can be Enhanced by Stimulating Sensitive Astrocyte Mitochondrial Production PY 1 R IP 3 Mitochondrion Astrocyte F1-F ase Neuron ANT Dependent Neuroprotection ER IP 3 R The astrocyte-specific mitochondrial toxin Fluoroacetate blocks PY 1 R protection Fluoroacetate: Mitochondrial toxin MCT4 isoform in astrocytes can transport acetate, the MCT in neurons cannot Pre-clot ( hrs) Pre-clot ( hrs) Can block specifically mitochondrial metabolism in astrocytes Post-clot Post-clot (4 hrs, fluoroacetate) (4 hrs, fluoroacetate + ) 3 Zheng et al. 1 PLoS One vol. 5 p. e1441 µm Dox off-regulated expression of mtecori decreases ΔΨ Dox on Dox off (8 days) Number of Mitochondria 1 Dox on Functional Mitochondria Dysfunctional Mitochondria Dox off 4 1 µm 1 µm GFAP-tTA-mtEcoRI Astrocytes ΔΨ = -6Log(F mit/f cyt) 8

9 The Impact of Astrocyte Mitochondrial Production PY 1 R-enhanced neuroprotection dependent on astrocyte mitochondria Disrupted Astrocyte Mitochondria Wildtype Mitochondria Pre-Photothrombosis (GFAP-tTA-EcoRI-GFP) Pre-Photothrombosis (GFAP-tTA-EcoRI-GFP) Pre-Photothrombosis (WIldtype mice) 4 hours Post-Photothrombosis 5 1 µm 4 hours Post-Photothrombosis (PY 1R agonist ) 6 mt-ecori RB & Lesion area 4 Days 1 RB & Wildtype 4 hours Post-Photothrombosis 4 hours post-clot Gradient Cell Swelling towards thrombosis 6 Pre-clot Post-clot (3 hours) Astrocytes (GFP) 7 Soma Area (1 µm ) 4 MeADP h * * * * Hours Cytotoxic Edema 9

10 The Impact of Astrocyte Mitochondrial Production Pre-clot Post-clot (3 hrs) Post-clot (4 hrs) 8 Pre-clot Post-clot (3 hrs) Post-clot (4 hrs) 9 Ischemia induces astrocyte death (via swelling?) Ischemic 16 min 19 min Normal tissue penumbra Core 195 min 5 min 3 1

R IP 3 Mitochondrion Astrocyte X F1-F ase Neuron ANT Dependent Neuroprotection 3 ER IP 3 R PY 1 R stimulation decreases whole brain swelling

11 The Impact of Astrocyte Mitochondrial Production Astrocyte swelling is reversible Post 8 hrs 8.5 hrs Normal tissue penumbra Core 8.75 hrs 9. hrs 31 Astrocyte neuroprotection Working Hypothesis: Protection can be Enhanced by Stimulating Sensitive Astrocyte Mitochondrial Production PY 1 R IP 3 Mitochondrion Astrocyte X F1-F ase Neuron ANT Dependent Neuroprotection 3 ER IP 3 R PY 1 R stimulation decreases whole brain swelling following TBI TBI TBI + 11 * * * * 3 day 1 day % of control hemisphere Cn TBI Me Cn TBI Me 33 1 day 3 day Watts et al. 13 J. Neurotrauma3,

4 μm 34 Soma area (μm ) 1 9 8 7 6 5 TB I

ANT Dependent Neuroprotection 35 ER IP 3

Sayre, PhD Yanan Chen & Deborah Holstein

12 The Impact of Astrocyte Mitochondrial Production PY 1 R stimulation decreases cell edema in the hippocampus following TBI Day TBI, Day1 Day + TBI +, Day1 CA3 4 μm 34 Soma area (μm ) TB I * * Post-injury 1 3(days) 7 Watts et al. 13 J. Neurotrauma3, Astrocyte neuroprotection Working Hypothesis: Protection can be Enhanced by Stimulating Sensitive Astrocyte Mitochondrial Production PY 1 R IP 3 Mitochondrion Astrocyte F1-F ase Neuron ANT Dependent Neuroprotection 35 ER IP 3 R Acknowledgements Lechleiter Lab (past & present): Wei Zheng, PhD Jun Wu, PhD Da-Ting Lin, PhD Lora Watts, PhD Naomi Sayre, PhD Yanan Chen & Deborah Holstein Collaborators: Christi Walter, Charles Keller and Murat H. Digicaylioglu 36 Thank you 1

13 The Impact of Astrocyte Mitochondrial Production 37 13

Lecture 10. Proton Gradient-dependent ATP Synthesis. Oxidative. Photo-Phosphorylation

Lecture 10. Proton Gradient-dependent ATP Synthesis. Oxidative. Photo-Phosphorylation Lecture 10 Proton Gradient-dependent ATP Synthesis Oxidative Phosphorylation Photo-Phosphorylation Model of the Electron Transport Chain (ETC) Glycerol-3-P Shuttle Outer Mitochondrial Membrane G3P DHAP