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Role of spastic paraplegia genes and BMP signaling in regulating axonal microtubules and transport

Periodic Report Summary - HSP AXONS DROSOPHILA (Role of spastic paraplegia genes and BMP signaling in regulating axonal microtubules and transport)

The hereditary spastic paraplegias (HSPs) are a diverse group of neurological disorders that show progressive spasticity of the lower extremities. To understand the function of one membrane-associated HSP protein, NIPA1/SPG6, the host lab studied its Drosophila ortholog, spichthyin (Spict) and showed that Spict and its human ortholog SPG6 are novel antagonists of BMP signalling, which regulate traffic of BMP receptors. How can this influence axonal pathology? Overexpression of Spict caused reduction of the MT cytoskeleton in axons, and loss of BMP signalling had the same effect, accompanied by impaired axonal transport and reduced speed of transport of synaptotagmin-containing vesicles. The aim of this proposal was to determine mechanisms by which BMP signalling can regulate MT assembly and stability. The identification of such mechanisms would allow a better understanding of both BMP signalling and MT regulation in both healthy and disease states.

To determine which branch(es) of the BMP signalling pathway mediate MT regulation, I started to develop Drosophila lines to generate larvae deficient for different members of either the canonical Mad pathway, the LIMK pathway (non-canonical), or both. I also developed lines to allow expression of different BMP components in BMP mutant backgrounds, to test the rescue of MT loss by either muscular or neuronal expression of BMP components. I also initiated the construction of stocks combining a BMP mutation (tkv, gbb, mad, med, limk) and either a Tubulin-GFP construct or an EB1-GFP (or -YFP) construct to allow live imaging of GFP-tagged MTs and MT-interacting proteins. Unfortunately, although BMP mutant stocks showed other phenotypes expected for BMP mutations (including NMJ undergrowth), I had serious troubles to reproduce the initial phenotype of less acetylated tubulin staining in axons, in different BMP mutants (tkv, gbb, mad, med, limk). Trying a number of modifications to larval fixation and staining conditions did not improve this. While another group did report effects of BMP mutants on axonal microtubules, and I did indeed observe synaptic vesicle accumulation along motorneuron axons, in spite of my efforts I could not find a robust and reproducible MT phenotype on which I could have worked confidently. I therefore discontinued this work.

I then joined a project developed by Sophie Zaessinger in the lab, on the role of known regulators of BMP signalling as NMJ growth regulators. The Smad anchor for receptor activation (Sara) protein was chosen to test its involvement in BMP signalling in a situation where it is not required for its recently characterised roles in segregation of signalling components. In Drosophila wing disc epithelia, Sara can also either stimulate or inhibit BMP signalling, by acting as an adaptor for other components of the pathway. However, nonspecific background anti-Sara staining prevented us from seeing endogenous Sara at the NMJ, and we expressed a Sara-GFP construct either in muscles or in motoneurons. In muscles, Sara-GFP was detected in the perinuclear area, but not at the postsynaptic NMJ. In motoneurons, Sara-GFP could be detected as dots in cell bodies, and predominantly in the small boutons (Is) at the NMJ. We saw undergrowth of the NMJ in sara mutants, largely confined to Is boutons. Those data suggest that Sara could have a specific role in growth regulation in some but not all motorneurons. To determine whether Sara was affecting the BMP transcriptional response in neurons, we determined levels of Trio protein (a transcriptional target of BMP signalling) in Sara mutant larvae. Our preliminary experiment showed a decrease in Trio protein levels, as it is the case for BMP mutants. Therefore, Sara may be a new positive regulator of NMJ growth, through regulation of BMP signalling. Analysis of NMJ growth and Trio levels, using another sara mutant Drosophila line, are currently being analysed in the lab.