Prof. Dr. Hilmar BadingResearch Foci

It is well known that NMDAR-mediated neurotoxicity is dependent on its subcellular localization, where the synaptic NMDAR promotes survival signals while the extrasynaptic NMDAR activation leads to cell death (Hardingham et al., 2002; Bading, 2017). Recently, we have unraveled the molecular basis of extrasynaptic NMDAR-mediated toxicity by discovering the NMDAR/TRPM4 death complex (Yan et al., 2020). TRPM4 is a ubiquitously expressed monovalent channel protein that is only localized outside of synapses; therefore, the N/T death complex localized extrasynaptically and explained why the locaition of NMDAR is so crucial for its function and neuronal fate. Now we are particularly interested in the following questions:

1. What is the physiological function of the NMDAR/TRPM4 complex?
2. Can extrasynaptic NMDARs act like synaptic NMDARs, when they are disassociated from TRPM4?
3. Can we use N/T interface inhibitors as a new therapy for neurodegenerative diseases?

Related bio-techniques: Cloning, qRT-PCR, western blotting, rAAV production, calcium imaging, mitochondrial-related live-cell imaging, immunochemistry, surgery & behavior tests in animals.

NMDA receptors mediate multiple forms of plasticity important for learning and memory. They are also critical for both acquired neuroprotection and excitotoxicity in acute and chronic pathological conditions of the brain, such as stroke, traumatic brain injury and many neurodegenerative diseases. NMDA receptor function is critical for learning and memory as well as neurotoxicity yet their function is biophysically complex and is dynamically modulated. NMDA receptors are receptor associated non-selective cation channels permeable to Na⁺, K⁺, and Ca²⁺ ions. Both presynaptic glutamate release and postsynaptic depolarization (Mg²⁺ unblock) are required for their activation and they undergo desensitization in a time, voltage and subunit dependent fashion relative to the presence of glycine, Ca²⁺ and Zn²⁺. Synaptic and extrasynaptic receptor function can be directly quantified with whole cell patch clamp electrophysiology in acute brain slices (see Figure) where their localisation, binding partners and developmentally regulated subunit composition can be studied in a near- in vivo context. Similarly, synaptic NMDA receptor dependent activation of nuclear calcium signals critical for transcription-dependent plasticity and learning in the hippocampus can be directly quantified with nuclear-localised recombinant calcium indicators in such slices. These methods can directly investigate alterations in NMDA receptor function following molecular or pharmacological manipulation (eg. selected protein knockdown or small molecule testing) or in distinct genetic backgrounds (eg. human vs mouse).

For more information on this project, please contact Dr. Peter Bengtson (bengtson@nbio.uni-heidelberg.de).

Excitotoxic cell death and strategies to prevent it have primarily been studied in mouse or rat neurons and, thus, rodents are the major source for our understanding of the molecular and cellular mechanisms of neurodegeneration and pro-survival programs. We have recently established that similar to rodent neurons, human induced pluripotent stem cell (iPSC)-derived neurons are sensitive to NMDAR-mediated excitotoxicity. Our aim now is to thoroughly characterize NMDAR signaling in human neurons to provide a human cell-based experimental platform for investigating neuroprotective strategies that target NMDAR activity and function. We take two approaches. We model human forebrain with iPSC-derived spheroids and culture monolayers of human excitatory neurons induced form iPSCs by overexpression of the transcription factor NGN2 (iNeurons). In addition to offering human cell-based models for studying NMDAR signaling and neurodegenerative processes, these cell culture systems can be used to validate the therapeutic potential of neuroprotective gene products and drug candidates.

Methods: iPS cell culture and molecular biology approaches, including rAAV and lentiviral tools and CRISPR-Cas9

For more information on this project, please contact Dr. Priit Pruunsild (pruunsild@nbio.uni-heidelberg.de).

Synaptic activity is supported by an extensive and complex network of cell signaling events among which calcium signaling plays important regulatory functions. At the postsynaptic level, the activation of NMDA receptors leads to local and transient increases in Ca²⁺ concentration. High levels of synaptic activity might induce calcium entry that no longer is confined to dendritic spines, but flows into the dendritic shaft where the Ca²⁺ transients can reach the mitochondria, thus affecting the local metabolism.

Extracellular Ca²⁺ can also enter the neurons through voltage gated calcium channels during action potential firing and through store operated calcium entry channels. The latter channels are important to replenish and maintain the endoplasmic reticulum (ER) Ca²⁺ levels which are relevant to the proper working of the neuronal Ca²⁺ signaling.

To further understand the functions of the dynamic compartmentalization of the Ca²⁺ signaling at the level of plasma membrane, endoplasmic reticulum, mitochondria and their contacts, we are using neuronal primary cultures and hippocampal organotypic cultures as models. The main techniques we are using include cell culture, fluorescence microscopy (confocal, spinning disk), image analysis and genetically encoded Ca²⁺ indicators directed to different subcellular locations including mitochondrial matrix, mitochondrial outer membrane, ER membrane and plasma membrane.

In the figure, the high affinity Ca²⁺ indicator GCaMP6f is targeted to the outer mitochondrial membrane in hippocampal neurons. The mitochondria does not maintain homogeneous Ca²⁺ levels across the network, but locally regulates surface Ca²⁺ concentration.

For more information on this project, please contact Dr. Omar Ramírez (ramirez@nbio.uni-heidelberg.de).