Cell biological studies using high content, high resolution bio imaging and metabolic profiling of individual cells offer in depth insights into adaptive and plastic mechanisms of neurons maintaining their health or coping with disease conditions. Yet, these analyses are just beginning to account for the stunning neuronal complexity, with multiple compartments of very different functions and metabolic demands, namely axons, somata, dendrites or synapses. In this project, we aim at accommodating a joint cell biological and metabolic analysis, addressing neuronal compartment-specific characteristics of a key homeostatic process termed autophagy.
Autophagy - self-eating - of cell contents serves to remove damaged or aged macromolecules such as proteins or lipids and entire malfunctioning organelles. The material is lysed and recycled, so that energy as well as building blocks are returned to the cellular metabolism. The cellular waste is packaged into double-walled vesicles termed autophagosomes, which in neurons arise at ER outposts in axon terminals with the help of the actin cytoskeleton. Subsequently, via extensively regulated intracellular transport along microtubules in the axon, autophagosomes fuse with lysosomes near the neuronal soma, ensuring recycling or deposition of their cargo. Thus autophagy represents a true recycling mechanism allowing neurons to maintain or adjust their composition according to their needs.
This compartment-specific autophagic flux progression as well as its consequences for the cell’s metabolism and homeostasis are by far understudied. Moreover, both, impaired formation or destruction of autophagic vesicles are considered among the key mechanisms driving the progression of severe neurodegenerative diseases
To tackle cellular transport in cultured neuronal compartments microfluidic chambers developed in TP1 will be used for stereotypic outgrowth of axons through an array of microgrooves from cultured primary neurons derived from mouse and zebrafish tissue. This structured alignment of neurons enables soma, axon shaft and axon terminal specific analysis by high-resolution microscopy as well as compartment-selective interference with dynamic cell biological processes.
Representative of neurodegenerative disease, spinocerebellar ataxia type13 (SCA13) (described in more detail in TP6), and nephrocerebellar syndrome caused by the whamm gene will be studied. Following expression of Kcnc3 channel variants or mutation of the whamm gene, subcellular structures such as certain vesicles involved in autophagy and the microtubule skeleton responsible for axonal transport are visualized using fluorescent marker proteins. Establishment of a virus-based method with high transduction efficiency in the microfluidic chamber allows simultaneous expression of Kcnc3 channel vatiants, Whamm mutations, and/or markers for modeling transport and degradation processes. Thus, cell biological analyses, such as monitoring microtubule dynamics, can be characterized in terms of autophagic membrane transport in neuronal compartments by time-lapse microscopy. Targeted manipulation of the various cellular compartments, which are individually addressable in the microfluidic chambers, will be used to determine whether autophagic vesicle turnover is promoted or attenuated, and whether destruction of axons and dendrites and neurodegeneration are enhanced or alleviated. The main findings will be confirmed in vivo using an established SCA13 disease model in zebrafish and a new model for nephrocerebellar syndrome in mice.
Prof. Dr. Theresia Stradal
Helmholtz Centre for Infection Research (HZI)
Phone: +49 531 6181 2900
Prof. Dr. Reinhard Köster
Zelluläre und Molekulare Neurobiologie
Tel: +49 531 391 3230
Fax: +49 531 391 8178