Giampietro Schiavo

Novel axonal pathways in motor neurons

The molecular machinery responsible for the long-range crosstalk between peripheral targets and the neuronal cell body is poorly understood. In particular, specific vesicular routes are responsible for the transport of endogenous ligands and various signals in axons, and allow the entry in the central nervous system of pathogens¹. Our laboratory has recently exploited atoxic fragments of tetanus neurotoxin (TeNT Hc), a well-known virulence factor responsible for all clinical symptoms of tetanus, as a paradigm to clarify both the mechanism of axonal retrograde transport in motor neurons and the pathogenesis of this important human disease^2,3.

Previous work in our laboratory demonstrated that TeNT is transported from the periphery to the neuronal cell body in carriers displaying a neutral pH, which is kept constant during movement. Therefore, TeNT does not enter a classical endosomal pathway and escapes targeting to a degradative compartment, thus enabling its central delivery in an intact form. To date, the only identified marker of TeNT carriers is the low affinity NGF receptor p75^NTR, suggesting that TeNT share its axonal transport route with neurotrophins and their signalling complexes². Studying TeNT dynamics in motor neurons therefore provides an ideal opportunity to characterise organelles involved in neurotrophin signalling and transport in health and disease.

To unravel the composition of these axonal retrograde carriers, we have recently developed a novel magnetic isolation strategy, which has allowed us to purify the TeNT-positive compartment in motor neurons. Upon internalisation and transport, the carriers containing TeNT-coupled magnetic beads were purified and their composition was determined by proteomic analysis. Sequencing of selected bands from the enriched population of TeNT carriers enabled us to identify, among known signalling proteins, motor proteins and accessory factors, the small GTPase family member Rab7. Rab7 is an important regulator of vesicular traffic, which is involved in the control of transport from early to late endosomes and lysosomes.

Several lines of evidence indicate that Rab7 is localised on TeNT carriers and acts as a crucial regulator of retrograde traffic in mammalian motor neurons. Both endogenous and a Rab7-GFP chimera co-localise with TeNT Hc carriers in axons and soma of motor neurons. Furthermore, over-expression of dominant-negative versions of Rab7, but not of other small GTPases, inhibits axonal retrograde transport of TeNT Hc. These data provide novel insights on a neuronal function for Rab7 on a neutral, non-classical endosomal transport compartment.

We now aim to uncover downstream effectors of Rab7 and their role in the sorting and maturation of retrograde transport carriers. Furthermore, we are keen to explore molecular defects in this pathways leading to human pathologies, such as motor neuron disease and hereditary neuropathies^4,5. This strategy will allow the identification of novel components of the axonal retrograde transport machinery and facilitate the characterisation of other carriers involved in long-range communication with the central nervous system.

The Neuropathobiology laboratory
The Molecular Neuropathobiology Laboratory aims to provide an integrated approach to the study of membrane dynamics at the nerve terminal. We are developing novel strategies to dissect sorting and exocytosis at the synapse and to characterise the molecular interactions regulating these processes, both under physiological and pathological conditions. Our basic research on neuronal trafficking can be exploited to uncover differences between normal and malignant brain tissue and to provide novel therapies for intractable pain in cancer patients.

References

1. Deinhardt K and Schiavo G. Endocytosis and retrograde axonal traffic in motor neurons. Biochem Soc Symp 2005; 72: 139-150.
2. Lalli G and Schiavo G. Analysis of retrograde transport in motor neurons reveals common endocytic carriers for Tetanus Toxin and neurotrophin receptor p75^NTR. J Cell Biol 2002; 156: 233-40.
3. Lalli G, et al. Myosin Va and microtubule-based motors are required for fast axonal retrograde transport of tetanus toxin in motor neurons. J Cell Sci 2003; 116: 4639-50.
4. Hafezparast M, et al. Mutations in dynein link motor neuron degeneration to defects in retrograde transport. Science 2003; 300: 808-12.
5. Kieran D, et al. Inducing mutations in dynein delays disease progression in SOD-1^G93A mice, a model of ALS. J Cell Biol 2005; in press.