Project Details
Projekt
Mechanisms of presynaptic dysfunction under acute metabolic stress
Applicant
Professor Dr. Jürgen Klingauf
Subject Area
Molecular Biology and Physiology of Neurons and Glial Cells
Term
since 2018
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 394431587
Pre- and postsynaptic mechanisms consume up to 55% of the total ATP used on action potentials. Therefore, we systematically analyzed during the first funding period the relative ATP demand of action potential generation and presynaptic processes such as exocytosis, release site clearance, Ca2+ clearance, endocytosis and SV reformation/refilling. We found that chemical interference with ATP production does not have an immediate effect on the excitability of neurons and that that ATP depletion manifested earliest on the process of compensatory endocytosis. However, surprisingly we found that ATP depletion causes an immediate shutdown of the sodium-proton-antiporter 1 (NHE1-transporter) leading to a block of cytosolic re-alkalinization upon stimulation-induced acidification. As the slow endocytosis phenotype upon ATP depletion can be mimicked by pharmacological block of NHE1 function, we hypothesize that slowed endocytosis is only a secondary consequence of ATP depletion while block of NHE1 transporters is the primary effect leading to pronounced and sustained intracellular acidification. This way, Na+ toxicity is avoided by block of NHE1 mediated Na+ influx, but at the expense of intracellular pH and accurate synaptic vesicle recycling.Therefore, we aim in the second funding period to rescue the pH-phenotype and decipher the “real” ATP dependence of exo/endocytosis. For this purpose, we will co-express the light-driven proton pump Arch3 together with various optical reporters of presynaptic activity and re-analyze the ATP dependence of the individual steps of presynaptic exo/endocytosis under conditions of stabilized cytosolic pH. In addition, we will screen for NHE1 mutants with abolished ATP sensitivity to prevent sustained acidification upon ATP depletion. In a second part of the project, we will focus on the clinical relevance of NHE1 transporters in ischemia. We will establish a flow chamber, which allows to mimic ischemic conditions induced by reduced oxygen supply. The degree of ATP depletion for different experimental paradigms will be quantitated using a FRET-based ATP sensor. In combination with optical reporters of presynaptic activity and cytosolic pH, we will analyze the effects of ischemia and reperfusion on presynaptic function, cytosolic pH and NHE1 activity. Once this approach is established, we will expand our cellular model system to iPSC-derived human neurons, which are already routinely used in our lab.
DFG Programme
Research Units
