Axonal transport, protein trafficking and neurological disease
Lead Research Organisation:
King's College London
Department Name: Neuroscience
Abstract
There are currently no effective treatments for motor neuron disease/amyotrophic lateral sclerosis. Indeed, the mechanisms by which motor neurons die in ALS are not even properly understood. However, one of the earliest defects seen in models of the disease is disruption to movement of proteins through the cell by a process termed ?axonal transport?. Defective axonal transport is also seen in several other neurodegenerative diseases. This programme of studies is therefore to gain insight into the mechanisms by which axonal transport is damaged in ALS. We will disseminate our findings to the public in a variety of ways. Firstly, both Chris Miller and Chris Shaw regularly speak to lay audiences of patients and carers on their research at Motor Neurone Disease Association meetings. Chris Shaw has also communicated research findings and strategies on ALS to the public via radio and television broadcasts. In addition, the IOP hosts Open Days (which have been funded by the MRC) in which members of the public, through a series of talks and hands-on research presentations, are exposed to the wide range of neurodegenerative diseases-related research projects that are currently underway in the IOP. Finally, our findings are listed on the IOP WEB pages and receive exposure through the IOP press office.
Technical Summary
The hypothesis that underlies this proposal is that defects in axonal transport and protein trafficking are part of the pathogenic process in three familial forms of amyotrophic lateral sclerosis (ALS). These familial forms are those caused by mutations in the genes encoding copper/zinc superoxide dismutase-1 (SOD1), ALS2/Alsin and vesicle-associated membrane protein-associated protein-B (VAPB). The aim of the project is to understand how these different genetic insults induce defects in neuronal protein transport. The primary objectives are:-
1) To identify p38 stress-activated kinase phosphorylation sites in neurofilament proteins and to determine how this influences axonal transport of neurofilaments. p38 is activated in ALS, phosphorylates neurofilaments which in turn is known to be a regulator of neurofilament transport. This will be achieved by sequencing neurofilament proteins isolated from neuronal cell bodies in which we have activated p38 by transfection of dominantly-active MKK3. MKK3 is a direct upstream activator of p38. Identified sites will then be mutated to preclude or to mimic permanent phosphorylation and the transport properties of these mutants analysed by monitoring movement of green fluorescent protein (GFP)-tagged variants in transfected cultured neurons.
2) To determine whether ALS mutant SOD1 induces changes in phosphorylation of kinesin and dynein family proteins. These are phosphoproteins and there is emerging evidence that they may be substrates for p38. This will involve proteomic analyses of motor proteins isolated from cells and tissues expressing wild-type or mutant SOD1.
3) To determine how loss of ALS2 influences both fast and slow axonal transport by monitoring movement of GFP-tagged cargoes in living neurons derived from wild-type and ALS2 knockout mice.
4) To gain insight into the mechanisms by which ALS2 may signal to regulate axonal transport and how phosphorylation of ALS2 influences any such signalling.
5) To characterise new transgenic mouse models expressing ALS mutant VAPB that we have recently generated.
6) To determine whether ALS mutant VAPB damages axonal transport and if so, to gain insight into the mechanisms that underlie this effect.
The studies will thus provide information on the mechanisms underlying defective axonal transport in ALS and may reveal new therapeutic targets for this disorder. Since disruption to axonal transport is seen in other neurodegenerative diseases, these results are likely to be informative about pathogenic processes in a number of these disorders.
1) To identify p38 stress-activated kinase phosphorylation sites in neurofilament proteins and to determine how this influences axonal transport of neurofilaments. p38 is activated in ALS, phosphorylates neurofilaments which in turn is known to be a regulator of neurofilament transport. This will be achieved by sequencing neurofilament proteins isolated from neuronal cell bodies in which we have activated p38 by transfection of dominantly-active MKK3. MKK3 is a direct upstream activator of p38. Identified sites will then be mutated to preclude or to mimic permanent phosphorylation and the transport properties of these mutants analysed by monitoring movement of green fluorescent protein (GFP)-tagged variants in transfected cultured neurons.
2) To determine whether ALS mutant SOD1 induces changes in phosphorylation of kinesin and dynein family proteins. These are phosphoproteins and there is emerging evidence that they may be substrates for p38. This will involve proteomic analyses of motor proteins isolated from cells and tissues expressing wild-type or mutant SOD1.
3) To determine how loss of ALS2 influences both fast and slow axonal transport by monitoring movement of GFP-tagged cargoes in living neurons derived from wild-type and ALS2 knockout mice.
4) To gain insight into the mechanisms by which ALS2 may signal to regulate axonal transport and how phosphorylation of ALS2 influences any such signalling.
5) To characterise new transgenic mouse models expressing ALS mutant VAPB that we have recently generated.
6) To determine whether ALS mutant VAPB damages axonal transport and if so, to gain insight into the mechanisms that underlie this effect.
The studies will thus provide information on the mechanisms underlying defective axonal transport in ALS and may reveal new therapeutic targets for this disorder. Since disruption to axonal transport is seen in other neurodegenerative diseases, these results are likely to be informative about pathogenic processes in a number of these disorders.