TP6 - Physiological signatures of neurons and compartments under homeostatic and pathogenic conditions
Investigating the cellular and molecular mechanisms underlying the emergence and progression of neurodegenerative diseases lay the foundation for the rationale design of therapeutic approaches. Yet our current understanding of differences between neuronal homeostasis and pathogenic processes occurring in affected patients is limited. What is needed is an investigation on the level of neuronal compartments in order to reveal the interplay of neuronal structures dedicated to signal reception (dendrites), processing (soma) and signal release (axon).
Spinocerebellar Ataxia Type 13 (SCA13) is a neurodegenerative disease severely affecting the principal neuron of the cerebellar cortex, the Purkinje cells as well as cortical and hippocampal neurons. This monogenetic autosomal dominant disease is caused by a missense mutation inside a potassium channel. Incorporation of one mutated subunit into the multimeric channel results in electrophysiological impairment of the entire channel and altered electrophysiological properties of the affected neurons. SCA13 patients suffer from temporal lobe epilepsy, hippocampal sclerosis and cerebellar atrophy. Physiological, genetic and cell biological studies have shown that neurodegeneration is not only due to altered electrophysiological properties of affected neurons, but also caused by impaired trafficking of the mutated channel subunit. As many cell biological processes in neurons are activity-regulated both pathological mechanisms could be interdependent.
Compartmented microfluidic chambers established in TP1 and used for cell biological analysis in TP1 and TP5 of cultured neurons with structured dendrite and axon outgrowth guided by microgrooves will be further equipped with microelectrode arrays (MEA) for compartment selective electrophysiological recording and stimulation. Furthermore, the MEA can also be used for high-throughput drug screening as it is applicable to any cell type (including fibroblast HEK293 cells expressing selected proteins of interest). Readout is based on fluorescence imaging and stimulation (e.g. optogenetics) and recording of electrical local field potentials. Whole cell patch clamp electrophysiological analyses within the micro chamber will complement data sets obtained in a cell type and cell type compartment specific way.
This setup will be employed in TP6 for investigating the activity dependence of microtubule dynamics and autophagic flux in wildtype and SCA13 neurons studied in TP5. Autophagic vesicles are known to form at axon terminals and are retrogradely transported along microtubules. During this process autophagosomes mature and eventually fuse near the soma with lysosomes. Such transport processes as well as microtubule dynamics are likely influenced by neuronal activity to deliver and recycle cellular content on demand.
To relate these findings to physiological and cell biological changes in human cells, SCA13-associated mutations will be introduced by genome editing into human fibroblasts. Subsequently, these cells will be reprogrammed to induced pluripotent stem cell lines followed by their in vitro differentiation into kcnc3-expression neuronal cell types such as cortical neurons. Electrophysiological analysis of differentiated neurons will confirm SCA13-associated physiological alterations, while fluorescent protein reporters will serve to validate that cell biologically impaired transport processes are conserved between SCA13-cells in different vertebrate classes such as fish, rodents and humans. Furthermore, human iPSC-derived SCA13-neurons and isogenic wildtype controls will be used for metabolic profiling performed within TP3.
These studies will provide a comprehensive understanding of the physiological, cell biological and metabolic consequences of SCA13 progression in neurons and will reveal promising entry points as well as limitations for therapeutic approaches.
Prof. Dr. Christine Klein Institute of Neurogenetics and Section of Clinical and Molecular Neurogenetics Department of Neurology University of Lübeck Ratzeburger Allee 160 23538 Lübeck Germany Phone: +49-451-2903351 Fax: +49-451-2903353 Email: firstname.lastname@example.org