Rab11 modulates α-synuclein-mediated defects in synaptic transmission and behaviour

Carlo Breda 2 Marie L. Nugent 1 2 Jasper G. Estranero 2 Charalambos P. Kyriacou 2 Tiago F. Outeiro 0 3 Joern R. Steinert 1 Flaviano Giorgini 2 0 Department of NeuroDegeneration and Restorative Research, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Goettingen , Go ttingen , Germany 1 MRC Toxicology Unit, University of Leicester , Lancaster Road, Leicester LE1 9HN , UK 2 Department of Genetics, University of Leicester , University Road, Leicester LE1 7RH , UK 3 Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa , Lisboa , Portugal A central pathological hallmark of Parkinson's disease (PD) is the presence of proteinaceous depositions known as Lewy bodies, which consist largely of the protein a-synuclein (aSyn). Mutations, multiplications and polymorphisms in the gene encoding aSyn are associated with familial forms of PD and susceptibility to idiopathic PD. Alterations in aSyn impair neuronal vesicle formation/transport, and likely contribute to PD pathogenesis by neuronal dysfunction and degeneration. aSyn is functionally associated with several Rab family GTPases, which perform various roles in vesicle trafficking. Here, we explore the role of the endosomal recycling factor Rab11 in the pathogenesis of PD using Drosophila models of aSyn toxicity. We find that aSyn induces synaptic potentiation at the larval neuromuscular junction by increasing synaptic vesicle (SV) size, and that these alterations are reversed by Rab11 overexpression. Furthermore, Rab11 decreases aSyn aggregation and ameliorates several aSyn-dependent phenotypes in both larvae and adult fruit flies, including locomotor activity, degeneration of dopaminergic neurons and shortened lifespan. This work emphasizes the importance of Rab11 in the modulation of SV size and consequent enhancement of synaptic function. Our results suggest that targeting Rab11 activity could have a therapeutic value in PD. - Parkinsons disease (PD) is the second most common neurodegenerative disorder and affects 4% of the population over 80 years of age (1,2). Neuropathologically, this disorder is characterized by the presence of Lewy bodies (LBs) and Lewy neurites in dopaminergic neurons located in the substantia nigra pars compacta, and the loss of neurons in this region (3). The main protein component of LBs is a-synuclein (aSyn) (4). Point mutations and multiplications in the gene encoding aSyn lead to autosomal dominant versions of PD. In addition, genome-wide association studies suggest that polymorphisms in this gene increase susceptibility to sporadic forms of this disease (5). Although the precise cellular role of aSyn is still ambiguous, aSyn is located at the pre-synaptic termini (6) and has been implicated in modulating neurotransmitter release (7). Moreover, in pathological conditions, aSyn has been suggested to cause defects in several vesicle trafficking pathways (8). Rab proteins are highly conserved small GTPases that orchestrate vesicle trafficking within a cellwith vesicle formation, movement, tethering and targeting controlled by specific Rabs (9). These proteins fluctuate between their target membranes and the cytosol dependent on their GTP-bound (active) or GDP-bound (inactive) status (10). Mutations in Rab genes have been associated with diverse neurological diseases (11). aSyn has been found to interact with several Rabs (12). Furthermore, aSyn disease-related pathology in model systems such as impaired endoplasmic reticulum (ER) Golgi vesicle trafficking and loss of dopaminergic neurons is reversed by the overexpression of several Rabs, including Rab1, Rab3A and Rab8A (13 15). On the other hand, Rab11 has not been previously implicated in PD, although it has been linked to other neurodegenerative disorders (16 18). Rab11 is involved in the trafficking of vesicles between the recycling endosome and plasma membrane (19) and the trans-Golgi network (20). Rab11 also sequesters plasma membrane receptors and directs them to the recycling pathway, which plays a role in cell migration (21) and long-term potentiation (22). The importance of Rab11 in the context of neurodegenerative disorders is emphasized by studies of Huntingtons (HD) and Alzheimers (AD) diseases. Indeed, Rab11 is functionally perturbed in several models of HD (23 26), and inhibition of Rab11 activity impairs vesicle formation from recycling endosomes in HD patient fibroblasts (24). Rab11 abrogates loss of dendritic spines in a primary neuronal model of HD, suggesting that Rab11 may play a critical early role in the synaptic dysfunction observed in HD (26). Rab11 overexpression also ameliorates synaptic dysfunction and neurodegeneration in a Drosophila model of HD (26,27). Regarding AD, direct interactions between Rab11 and the hydrophobic loops of presenilin 1 and 2 have been observed (28). Furthermore, oestrogen treatment has been found to divert Rab11 to the trans-Golgi network, thereby decreasing b-amyloid (Ab) generation by promoting the budding of amyloid precursor protein-containing vesicles (29), while blockage of Ab trafficking through Rab11 recycling vesicles enhances cellular Ab accumulation (30). Due to these many links between Rab11 and neurodegenerative disease, we hypothesized that Rab11 could play a role in modulation of PD pathogenesis. Here, we employ Drosophila melanogaster models of aSyn toxicityand a panel of electrophysiological, immunohistochemical, genetic and behavioural analysesto investigate the mechanistic role and therapeutic potential of Rab11 in PD. In a related recent study, we also demonstrated that Rab11 interacts with and modulates aSyn aggregation and secretion (31). Rab11 normalizes aSyn-dependent potentiation of synaptic transmission at the Drosophila larval neuromuscular junction Expression of aSyn in flies yields several PD-relevant phenotypes, including formation of LBs, dopaminergic neuron loss and locomotor impairments (32). Here, we employed the GAL4/UAS system (33) to drive aSyn expression in specific tissues using two independent fly models carrying UAS-aSyn transgenes [Model 1 from (34) and Model 2 from (35); see Materials and Methods]. As we previously established that aSyn oligomers enhance basal synaptic transmission in rat hippocampal slices (36), we assessed whether the electrophysiological parameters of the neuromuscular junction (NMJ) in aSyn-expressing larvae mirrored these effects. Indeed, panneuronal expression of aSyn via the elavGAL4 driver (elav. aSyn) in Model 1 strongly increased miniature excitatory junction potential (EJP) amplitudes (mEJP) from 1.17 + 0.05 to 1.45 + 0.09 mV (P , 0.05; Fig. 1A), with a similar pattern observed in Model 2, though this failed to reach statistical significance using ANOVA (Fig. 1B). More subtle effects on mEJP amplitudes in both models became apparent when analyzing mEJP distributions with the more sensitive Kolmogorov Smirnov test (KS test; Fig. 1C and D; Mo (...truncated)