PARAPLEGIA ENDOSOMES · Spastic paraplegia genes and endosomal signaling in Drosophila

Spastic paraplegia genes and endosomal signaling in Drosophila

Hereditary spastic paraplegias (HSPs) are a group of neurological disorders characterised by retrograde degeneration of long nerve fibres in the corticospinal tracts and posterior columns. Existing treatments are limited to management of spasticity and the disease mechanisms are poorly understood. Over 45 Spastic paraplegia gene (SPG) loci and 20 causative genes have been identified (refer to Dion et al. 2009). While these encode a wide range of proteins, the largest single class are intracellular membrane proteins with a variety of sub-cellular localisations, principally endosomal or Endoplasmic reticulum (ER) (Blackstone et al. 2011). SPG3A/atlastin, SPG4/spastin and SPG31/REEP1, the most commonly mutated proteins in autosomal dominant HSP, as well as their wider family members, have a common feature of ER localisation and a hairpin-loop domain that can insert in membranes and increase their curvature (refer to Voeltz et al., 2006; Hu et al., 2008, Hu et al., 2009; Orso et al., 2009 and Park et al., 2010), suggesting common in vivo functions. Indeed, these domains can bind to each other to form oligomeric complexes, which can also contain a fourth class of hairpin loop proteins, the so-called reticulon, as was noted by Voeltz et al., 2006 and Hu et al., 2009. These HSP proteins contribute to ER topology in a number of different ways: 1. reticulon and reep proteins share a partly redundant role in the formation of tubular ER and by induction of curvature at the edges of sheet ER (refer to Voeltz et al., 2006 and Shibata et al., 2010); 2. atlastin is a GTPase that can catalyse membrane fusion. In case its function is impaired there are fewer connections between ER tubules arguing for a role in fusing ER tubules into a network ( refer to Hu et al., 2009; Orso et al., 2009 and Bian et al., 2011); 3. reep proteins, possibly assisted by the Microtubule (MT) severing activity of spastin, mediate the alignment of ER tubules in parallel with MTs, as mentioned by Evans et al. (2005), Roll-Mecak and Vale (2005), Sanderson et al. (2006) and Park et al. (2010). In order to understand how abnormality of these gene functions could give rise to axonopathy, we characterised the effects of loss of reticulon in drosophila. Rtnl1, the drosophila ortholog of reticulon one to four in humans, was required for tubular ER formation, as Rtnl1 knockdown expanded ER sheets and reduced tubular ER. Rtnl1, which was widely expressed in nervous tissue and co-localised with MT markers in axons, was also found to be required for the maintenance of ER and MT cytoskeleton in long axons, e.g. in Rtnl1 knockdown larvae, in which motor axons in posterior segments disrupted staining of ER and MT markers compared to control while anterior segments of the same axons did not. In addition, loss of Rtnl1 from motor neurons resulted in fewer mitochondria within the boutons of posterior, but not anterior, neuromuscular junctions. This did not appear to be trafficking defect, as mitochondrial number and distribution did not change from control in either anterior or posterior segments Rtnl1 knockdown motor neuron axons. Mitochondria and the ER had extensive contact points (refer to Friedman et al., 2011). Finally, we found that loss of Rtnl1 adult flies had locomotor deficits similar to those described in spastin or atlastin mutants, as noted by Sherwood et al., 2004 and Lee et al., 2008. There was no evidence that these behavioural phenotypes were relevant to the spasticity and weakness that occurred in the lower limbs of human patients with HSP. However, it was suggested that ER shaping proteins were required for the maintenance of motor axons in drosophila. The hairpin loop protein reticulon, along with reep, atlastin and spastin, was required for ER network formation. Disruption of the ER network by loss of any of these proteins resulted in degeneration of long axons as observed in HSP. We proposed that drosophila loss of Rtnl1 provided a novel model for the investigation of tubular ER defects in vivo.

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