The protective effect of naringenin in REEP1 drosophila model of hereditary spastic paraplegia is mediated by induction of ER-phagy

Defects in endoplasmic reticulum (ER) membrane shaping and interactions to other organelles seem to be one of the crucial mechanisms underlying Hereditary Spastic Paraplegia (HSP), a complex genetic disorder characterized by the axonal degeneration of corticospinal tracts. The structural organization of this complex organelle is created and maintained thanks to a continuous process of membrane remodeling, governed by homotypic fusion events, tubulation and curvature rearrangements as well as cytoskeletal transport (Goyal and Blackstone, 2013; Chen et al., 2013) and autophagy. In parallel to the ER remodeling process, lipid metabolism is another important emerging cellular aspect of HSP mechanism. The importance of the LDs role in HSP mechanism is highlighted by recent evidences that proteins as seipin/spg17, erlin2/spg18, atlastin/spg3a, spartan/spg20, REEP1/spg31 and spastin/spg4 localize or affect the LDs turnover in cells (Belzil and Rouleau, 2012; Papadopoulos et al., 2015; Tan et al., 2014). In spite of these evidences, the role of ER, its relationship with the lipid pathway, and the mechanism implicated in generating neuronal disorder in HSP still remain unknown. Here we report the analysis of a Drosophila melanogaster model for an autosomal dominant form of HSP caused by mutations in the SPG31 gene. SPG31 codifies for REEP1, a transmembrane protein belonging to the TB2/Dp1/HVA22 family. REEP1 interaction with atlastin-1 (SPG3A) and spastin (SPG4), the other two major HSP linked proteins, has been demonstrated to modify ER architecture in vitro. Indeed, REEP1 is required to confer stress resistance against the accumulation of unfolded proteins (UPR) induced by tunicamycin, thapsigargin and 1,4 dithiothreitol (DTT) (Appocher et al. 2014). We manipulated the expression of Drosophila REEP1 by using loss of function and gain of function alleles and analyzed the ER morphology, ER stress response, mitochondria defects, LDs biogenesis and the activation of autophagic pathway. The absence of D-REEP1 caused ER and mitochondria elongation, decreased LDs biogenesis and attenuated the UPR response. The attenuation of the adaptive UPR response sustained by activation of the ATF4 and the decline of unspliced and spliced levels of XBP1 is a condition reported in prolonged ER stress. We compared the UPR response of REEP1 loss of function with control flies after the chronic treatment with DTT and thapsigargin and found a similar UPR response. However, the autophagic flux was activated in control flies upon ER stress induction and absent in flies lacking REEP1 with or without ER stressor administration. Conversely, the upregulation of REEP1 caused ER and mitochondria fragmentation and the activation of autophagic machinery, suggesting the involvement of REEP1 in controlling ER functionality and morphology by the autophagic flux. Finally, we treated control and REEP1 loss of function flies with Naringenin, one of the compounds selected in a screen targeted to induce LDs biogenesis in Drosophila nervous system and muscle tissues. Administration of naringenin activated the autophagic flux in control flies as well as in flies lacking REEP1 and rescued the ER, LDs and mitochondria defects of REEP1 loss function alleles. There is substantial evidence on the involvement of autophagy and chronic ER stress in many diseases, including neurodegeneration (Hetz et al. 2009) and it is known that UPR accumulation can induce autophagy but the mechanisms and the proteins involved in these processes are still unknown. The characterization of a new player in modulating the ER homeostasis by autophagic degradation is of particular relevance to comprehend the maintenance and the functionality of neuronal cells as well as the neurodegeneration process.