shRNA-Based Screen Identifies Endocytic Recycling Pathway Components That Act as Genetic Modifiers of Alpha-Synuclein Aggregation, Secretion and Toxicity

Abstract

Alpha-Synuclein (aSyn) misfolding and aggregation is common in several neurodegenerative diseases, including Parkinson’s disease and dementia with Lewy bodies, which are known as synucleinopathies. Accumulating evidence suggests that secretion and cell-to-cell trafficking of pathological forms of aSyn may explain the typical patterns of disease progression. However, the molecular mechanisms controlling aSyn aggregation and spreading of pathology are still elusive. In order to obtain unbiased information about the molecular regulators of aSyn oligomerization, we performed a microscopy-based large-scale RNAi screen in living cells. Interestingly, we identified nine Rab GTPase and kinase genes that modulated aSyn aggregation, toxicity and levels. From those, Rab8b, Rab11a, Rab13 and Slp5 were able to promote the clearance of aSyn inclusions and rescue aSyn induced toxicity. Furthermore, we found that endocytic recycling and secretion of aSyn was enhanced upon Rab11a and Rab13 expression in cells accumulating aSyn inclusions. Overall, our study resulted in the identification of new molecular players involved in the aggregation, toxicity, and secretion of aSyn, opening novel avenues for our understanding of the molecular basis of synucleinopathies.

Vesicular Dysfunction and the Pathogenesis of Parkinson’s Disease: Clues From Genetic Studies

“…These proteins have been shown to play a part in various neurodegenerative diseases through their role in cellular functions including but not limited to endocytosis, vesicle trafficking, vesicle docking, ciliogenesis and interactions in the trans-Golgi network (Schimmöller et al, 1998;Rodman and Wandinger-Ness, 2000;Seabra et al, 2002). While the Rab family encompasses roughly 50 proteins with variable functions across the vesicular pathway, 10 have been shown to have a possible relationship to PD either directly or indirectly (Gonçalves et al, 2016;Steger et al, 2016;Chung et al, 2017). The myriad of functions attributed to the Rab proteins and the varied hypothesized routs to pathogenesis in PD can be summarized as the following three pathways: endocytic function and lysosomal stress, ciliogenesis and sonic hedgehog signaling and, lastly the trans-Golgi network and endoplasmic reticulum (ER) stress.…”

“…It is important to note that RAB39b is neuron specific and plays a role in synapse formation and maintenance (Giannandrea et al, 2010). A ShRNA-based screen identified several Rab proteins from endocytic recycling pathway acted as genetic modifiers of α-synuclein secretion, aggregation and toxicity (Gonçalves et al, 2016). Examination of Rab proteins and vesicle recycling components in the context of PD are warranted which might open up novel avenues for therapeutic intervention.…”

Rab GTPases: The Key Players in the Molecular Pathway of Parkinson’s Disease

“…Using a Drosophila model, Breda et al (2015) revealed that overexpression of Rab11 colocalizing with α-synuclein in intracellular inclusions could significantly reverse the synaptic potentiation at the neuromuscular junction due to the increase of synaptic vesicle size. Through a shRNA-based screen, some Rabs (Rab8b, Rab11a, and Rab13) have been identified as modulators of α-Synuclein in living cells and reduced the toxicity induced by aggregated α-synuclein via secretion enhancement (Goncalves et al, 2016). Rab8, playing a role in the post-Golgi trafficking, binded with the C terminal of α-synuclein, which was confirmed by the nuclear magnetic resonance spectroscopy.…”

The Molecular Physiopathogenesis of Islet Amyloidosis

“…Several independent studies reported the involvement of various endocytotic pathways in uptake of the extracellular Aβ and α-Syn in various cell and tissue types (Goncalves et al 2016; Volpicelli-Daley et al 2014; Hansen et al 2011). Over dozen of neuronal receptors, such as glutamate and acetylcholine receptors, integrins and soluble receptors like apolipoprotein E were implicated in the uptake of extracellular Aβ in neuronal cells (Lai and McLaurin 2010).…”

“…Further studies by Yu et al, (2010), demonstrated that oligomeric Aβ 42 neuronal toxicity and intracellular levels remained unchanged following downregulation of clathrin’s expression and function, indicating the importance of clathrin-independent endocytotic pathways in Aβ turnover and toxicity, similar to hA (Trikha and Jeremic 2011, 2013; Yu et al 2010). Analogous to uptake mechanisms of nontoxic Aβ monomers, the primary mechanism of extracellular α-Syn uptake is clathrin-mediated endocytosis (Ben Gedalya et al 2009; Goncalves et al 2016; Sung et al 2001; Trikha and Jeremic 2011, 2013). This is in good agreement with hA monomer uptake studies showing macropinocytosis (within the first few hours of internalization) and clathrin-dependent endocytosis [at later stage (>12 hours)] as main internalization routes for hA in pancreatic β–cells (Trikha and Jeremic 2013).…”