Amytrophic lateral sclerosis (ALS) is a fatal neurodegenrative disease characterised by selective degeneration and death of motor neurons. The architecture of neurons makes them dependent upon the proper transport of protein and organelle cargoes, especially through axons (axonal transport). Indeed, disruption to axonal transport is a very early, pathological event in ALS. A proline to serine substitution at position 56 in the vesicle-associated membrane protein-associated protein B (VAPB; VAPBP56S) causes some dominantly inherited familial forms of motor neurone disease including ALS type-8. How VAPBP56S causes ALS is not properly understood. In this thesis, I investigated the effect of VAPBP56S on axonal transport of mitochondria in primary rat cortical neurons and primarybmouse motor neurons. Using time-lapse microscopy, I showed that expression of VAPBP56S but not wild-type VAPB in neurons selectively disrupts anterograde axonal transport of mitochondria. Anterograde axonal transport of mitochondria is mediated by the microtubule-based molecular motor kinesin-1. Attachment of kinesin-1 to mitochondria involves the outer mitchondrial membrane protein Rho GTPase-1 (Miro1) which acts as a sensor for cytosolic calcium levels ([Ca2]c); elevated [Ca2+]c disrupts mitochondrial transport via an effect on Miro1. To gain insight into the mechanism underlying the VAPBP56S effect on mitochondrial transport, I monitored [Ca2+]c levels in VAPBP56S expressing primary rat cortical neurons. Expression of VAPBP56S but not VAPB increased resting [Ca2+]c in these cells. Moreover, the amounts of tubulin but not kinesin-1 that were associated with Miro1 were reduced in VAPBP56S compared to VAPB transfected HEK293 cells. Also, expression of a Ca2+ insensitive mutant of Miro1 rescued defective mitochondrial axonal transport and restored the amounts of tubulin associated with the Miro1/kinesin-1 complex to normal in VAPBP56S expressing HEK293 cells. Thus VAPBP56S may perturb axonal tranport of mitochondria by disrupting Ca2+ homeostasis and affecting the interaction of Miro1/kinesin-1 with tubulin.
Investigating the effect of amyotrophic lateral sclerosis-associated mutant vesicle-associated membrane protein B on axonal transport
Morotz, G. M. (Author). 1 Jan 2013
Student thesis: Doctoral Thesis › Doctor of Philosophy