Intracellular Sort- and Transportsystems in HSP - in vitro and in vivo models for testing the role of Atlastin and KIF5A
Dr. med. Clemens Neusch Zentrum Neurologische Medizin der Georg-August-Universität Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Tel.: +49-(0)551-3912838, Email: firstname.lastname@example.org
PD Dr. rer. nat. Frank Kirchhoff Neurogenetik, Max-Planck-Institut für experimentelle Medizin, Göttingen, Tel.: +49-(0)551-3899770, Email: email@example.com
The hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of neurodegenerative diseases characterized by progressive spasticity in the lower limbs without (‘pure’) or with (‘complicated’) additional neurological symptoms. Thirty different loci (SPG 1-30) have been mapped so far, and eleven responsible genes have been identified.
The physiological role and pathophysiological impact of these proteins on disease onset and progression has only partially being elucidated. Some of the underlying mutated proteins are involved in signal transduction pathways, others in cell migration, myelination or belong to the chaperone family. Additionally and importantly, defects in the intracellular transport machinery are being discussed.
This project aims to understand the subcellular expression and physiological role and of two proteins that when mutated, cause clinically ‘pure’ HSP: KIF5A and Atlastin. Mutated KIF5A is most likely involved in defective axonal transport as the protein is thought to belong to the group of microtubule-associated proteins. Atlastin is expressed in close association with the Golgi apparatus and may impair intracellular sorting, trafficking and/or membrane transport.
Both proteins are therefore part of the highly dynamic intracellular transport machinery of the neuronal cell. Point mutations in these proteins may lead to impairment of these dynamic processes, either from the ER to the cell membrane (atlastin) or in fast anterograde axonal transport of vesicles (KIF5A).
In this project, we propose the following approach to understand the function of KIF5A and atlastin:
- Generation of KIF5A/EGFP and Atlastin/EGFP fusion proteins
- Overexpression of wild-type fusion proteins in heterologous expression systems and in primary neuronal cultures
- Determination of the subcellular expression and
- functional analysis using conventional epifluorescence or laser scanning microscopy.
In a second step we will insert known human mutations and analyse mutated versus wild-type expression of both fusion proteins in vitro. Here, our analysis will focus on putative changes within the described transport systems: anterograde axonal transport and Golgi-trafficking. We will study (i) changes of structural proteins, e. g. neurofilaments, a- and b-tubulin, or the Golgi apparatus by Western blot analysis and (ii) functional consequences of the inserted mutations on transport mechanisms by time-lapse microscopy.
The aim of this project is to help understanding the (i) physiological role of the two proteins in intracellular transport system (a probable underlying cause of axonal and neuronal degeneration in HSP) and (ii) the cellular and intracellular consequences of the expression of known KIF5A and atlastin mutations for neuronal cell integrity and transport systems.