The research goal of the Methner lab is neuronal cell death and how to prevent it. We study mechanisms of resistance against oxidative stress, the role of the calcium machinery in cell death and protection, and transcriptional effectors of cell survival in models of neurological disease. We employ a combination of molecular and cell biological, biochemical, and genetic approaches and use neurons and neuronal stem cells in primary culture. The Methner lab is part of the Focus program translational neuroscience (FTN) at the Johannes Gutenberg University Mainz / Germany.
We are currently focused on three major areas of research in basic research and one more clinically relevant project:
Calcium signaling in primary astrocytes shown by Fura2 imaging
Calcium ions are involved in a plethora of cellular functions including cell death and mitochondrial energy metabolism. Within the cell, Ca2+ is mainly stored in the endoplasmic reticulum (ER), where its concentration is controlled among others by anti-apoptotic proteins of the Bcl-2 family or the evolutionarily conserved cell death modulator Bax inhibitor-1 (BI-1). Ca2+ is released from the ER after activation of membrane receptors that generate the second messenger inositol 1,4,5-trisphosphate (IP3). IP3 binds and opens IP3 receptors 1-3 at the ER membrane triggering Ca2+-release from the ER lumen. The subsequent decrease in [Ca2+]ER induces a strong Ca2+ influx from the extracellular space, referred to as store-operated Ca2+ entry (SOCE), which is followed by removal of cytosolic Ca2+ and replenishment of luminal Ca2+ through sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). The Ca2+ sensor that conveys information about the Ca2+ load of the ER lumen to store-operated Ca2+ channels is stromal interaction molecule 1 (STIM1), a single transmembrane protein located in the ER membrane with a luminal EF hand and a cytosolic domain, which activates Ca2+ release-activated Ca2+ (CRAC) channels. Upon store depletion, STIM1 clusters into punctae near the plasma membrane and activates CRAC channels within the plasma membrane, mainly Orai1, resulting in a sustained Ca2+ influx and subsequent activation of downstream proteins. We are interested in the role of the Ca2+ machinery in oxidative and ER stress. We study the role of SOCE in neurodegenerative diseases within the EU-sponsored ERAnet RUS program in the collaborative research effort TargetSOCE.
The C terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore. Bultynck, G. et al. (2012). Journal of Biological Chemistry, 287(4), 2544–2557
The plasma membrane channel Orai1 mediates detrimental calcium influx caused by endogenous oxidative stress. Henke, N. et al. (2013). Cell Death and Disease, 4, e470.
Stromal Interaction Molecule 1 (STIM1) Is Involved in the Regulation of Mitochondrial Shape and Bioenergetics and Plays a Role in Oxidative Stress. Henke, N. et al. (2012). Journal of Biological Chemistry, 287(50), 42042–42052.
BI-1 protects cells from oxygen glucose deprivation by reducing the calcium content of the endoplasmic reticulum. Westphalen, B. C. et al. (2005). Cell Death and Differentiation, 12(3), 304–306.
Spinal motoneuron derived from human embryonic stem cells treated with the neuroprotective antibiotic Ceftriaxone
Oxidative stress plays a fundamental role in a wide variety of neurological diseases. Treating cells with glutamate, which inhibits cystine uptake through the glutamate/cystine antiporter system xc-, causes endogenous oxidative stress because, within the cell, cystine is rapidly converted to cysteine, the rate-limiting amino acid for GSH synthesis. Cystine deprivation therefore causes secondary GSH depletion and a programmed cell death by oxytosis or oxidative glutamate toxicity, which is clearly distinct from apoptosis, necrosis, and cell death associated with autophagy. A well-established model system for oxytosis is glutamate-induced cell death in the hippocampal cell line HT22, which has been extensively used by others and us to clarify the cascade leading to cell death and to identify antioxidant pathways and proteins. In this system, GSH depletion leads to an exponential increase in reactive oxygen species, which mostly originate from mitochondrial complex I activity, and is accompanied by a gradual disruption of the mitochondrial membrane potential. After approximately six hours of glutamate exposure, the lipid-oxidizing enzyme 12/15-lipoxygenase is activated and generates 12- and 15- hydroxyeicosatetraenoic acids that directly damage mitochondria, which is followed or accompanied by translocation of the pro-apoptotic BCL-2 family member BID (BH3-interacting domain death agonist) to mitochondria and release of apoptosis-inducing factor from mitochondria. We are particularly interested in the role of mitochondria and energy metabolism in the susceptibility to endogenous oxidative stress and use this model for the identification of neuroprotective substances and signal transduction pathways.
Effects of dimethyl fumarate on neuroprotection and immunomodulation. Albrecht, P. et al. (2012). Journal of Neuroinflammation, 9, 163.
Charcot-Marie-Tooth disease CMT4A: GDAP1 increases cellular glutathione and the mitochondrial membrane potential. Noack, R. et al. (2012). Human Molecular Genetics, 21(1), 150–162.
Induction of Nrf2 and xCT are involved in the action of the neuroprotective antibiotic ceftriaxone in vitro. Lewerenz, J. et al. (2009). Journal of Neurochemistry, 111(2), 332–343.
Neural stem cells are the self-renewing, multipotent cells that generate the main phenotypes of the nervous system. Adult neural stem cells are mainly located in the subventricular zone. We are interested in factors that mediate survival of neural stem cells against oxidative or ER stress. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. Previous work in the lab has focused on neural differentiation using a human neuronal progenitor cell line NTERA-2cl.D1, but we have now shifted to murine neurospheres and human glioblastoma-derived stem cell like cells. We have now established neuronal cultured from human induced pluripotent stem cells in the lab.
Neuronal differentiation of cultured human NTERA-2cl.D1 cells leads to increased expression of synapsins. Leypoldt F et al. (2002) Neuroscience Letters 324(1):37-40
Human septin 3 on chromosome 22q13.2 is upregulated by neuronal differentiation. Methner A et al. (2001) Biochemical and Biophysical Research Communications 83(1):48-
Identification of genes up-regulated by retinoic-acid-induced differentiation of the human neuronal precursor cell line NTERA-2 cl.D1. Leypoldt F et al. (2001) Journal of Neurochemistry 76(3):806-14
Biomarkers are necessary to evaluate the efficacy of potentially neuroprotective therapies in the clinic. To this end, we use the thickness of the retinal nerve fiber layer as readout for axonal destruction using Optical Coherence Tomography (OCT), a noncontact, noninvasive imaging technique capable to obtain high-resolution cross-sectional images of the retina. OCT is analogous to ultrasound B-scan imaging except that light rather than sound waves are used in order to obtain a much higher longitudinal resolution of approximately 10 µm in the retina. We have recently shown that OCT can measure axonal degeneration in Multiple Sclerosis and other neurological diseases.
Retinal Neurodegeneration in Wilson’s Disease Revealed by Spectral Domain Optical Coherence Tomography. Albrecht, P et al. (2012). PLoS ONE, 7(11), e49825.
Optical coherence tomography in parkinsonian syndromes. Albrecht, P et al. (2012). PLoS ONE, 7(4), e34891.
Degeneration of retinal layers in multiple sclerosis subtypes quantified by optical coherence tomography. Albrecht, P et al. (2012). Multiple Sclerosis Journal, 18(10), 1422–1429.