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Loss of endoplasmic reticulum shaping protein Reticulon-like 1 protein in Drosophila melanogaster to study locomotor deficits in Hereditary Spastic Paraplegia



Hereditary Spastic Paraplegia (HSP) is a genetic neurodegenerative disorder characterized principally as degeneration of the longest motor axons. Clinically, it is commonly seen as progressive stiffness of the lower limbs. Pathologically, it is characterized by retrograde degeneration of axons. Endoplasmic reticulum (ER) shaping proteins have recently been associated with HSP. We studied the loss of locomotor ability created by the loss of an endoplasmic reticulum protein, Reticulon, which interacts with mutated proteins in HSP. We tested changes in climbing ability (negative geotaxis) of Drosophila melanogaster caused by Reticulon-like 1 (Rtnl1) protein gene mutations and RNAi silencing created by the UAS-Gal4 driver. Twenty four-hours old flies were placed in a 10 cm glass cylinder in sets of 10 flies, with 3 replicates per fly line. The total number of flies climbing to the top of the glass cylinder within 15 seconds was recorded every 3 days for 4 weeks. RNAi control flies were homozygous for just the UAS-Gal4 driver, while experimental flies were homozygous for the driver and RNAi genes. RNAi silenced Rtnl1 confirmation was done by RNA extraction, cDNA synthesis of Rtnl1 transcripts of control and silenced samples followed by PCR amplification and agarose gel electrophoresis. Differences in expression were first normalized among fly lines using the ribosomal protein Rp49. Videos of mutant and RNAi silenced flies were watched and total flies climbed recorded. The average of 3 trials was taken for each fly line (i.e., mutant and RNAi). The averages of each day for the same fly lines were combined into a single average and plotted against age of flies. Percentage of flies climbing per day was analyzed by 2-way ANOVA testing. Homozygous mutant flies did not climb as well as wild type flies only on 1 day, day 21 (P<0.01, n =3). Homozygous RNAi silenced flies exhibited different results. Control flies showed significantly less climbing ability than RNAi silenced flies on days 8 to 19 (P<0.001, n=3) from control flies. This project demonstrated that there is a difference in climbing abilities due to changes in expression of Rtnl1- a molecular interactor of HSP proteins. The inability of the control RNAi flies to perform better at climbing could be due to deleterious effects as a consequence of having just the UAS-Gal4 driver inserted without an RNAi interfering gene. In conclusion, we demonstrated locomotor changes associated to the Rtnl1 levels.


hereditary spastic paraplegia , neurodegenerative disorders, drosophila melanogaster