Pathogenic chaperone-like RNA induces congophilic aggregates and facilitates neurodegeneration in Drosophila

Abstract

Protein aggregation is a hallmark of many neurodegenerative diseases. RNA chaperones have been suggested to play a role in protein misfolding and aggregation. Noncoding, highly structured RNA recently has been demonstrated to facilitate transformation of recombinant and cellular prion protein into proteinase K-resistant, congophilic, insoluble aggregates and to generate cytotoxic oligomers in vitro. Transgenic Drosophila melanogaster strains were developed to express highly structured RNA under control of a heat shock promoter. Expression of a specific construct strongly perturbed fly behavior, caused significant decline in learning and memory retention of adult males, and was coincident with the formation of intracellular congophilic aggregates in the brain and other tissues of adult and larval stages. Additionally, neuronal cell pathology of adult flies was similar to that observed in human Parkinson's and Alzheimer's disease. This novel model demonstrates that expression of a specific highly structured RNA alone is sufficient to trigger neurodegeneration, possibly through chaperone-like facilitation of protein misfolding and aggregation.

Fig 1.

Heat shock–inducible expression of RQ and RQT RNA in flies. Upper panel: Northern blot hybridization of ³²P-labeled RQ construct DNA probe with total RNA isolated from adult flies of transgenic strains. RNA from uninduced flies is compared with RNA from hs-induced flies at 0, 5, and 15 hours after treatment for 30 minutes at 37°C. Lane 1: RQ2 flies, no hs; lane 2: RQT flies, no hs; lane 3: RQ2 flies immediately after hs; lane 4: RQT flies immediately after hs; lane 5: RQ2 flies after 5 hours of recovery from hs; lane 6: RQT flies after 5 hours of recovery from hs; lane 7: RQ2 flies after 15 hours of recovery from hs; lane 8: RQT flies after 15 hours of recovery from hs. Lower panel: ethidium bromide–stained gel before transfer to indicate even loading of RNA samples

Fig 2.

Congophilic aggregate formation in salivary glands of larvae. Congo red staining accompanied by Mayer's Hematoxylin counterstaining to see the nuclei in blue, of salivary glands was performed on Canton-S (A) uninduced and (B) induced larvae and RQ2 (C) uninduced and (D) induced. Salivary glands from induced larvae were collected at 1 hour after hs at 37°C for 30 minutes

Fig 3.

Congophilic aggregates in the brains of RQ2 flies and neural pathology. (A) Frontal fly brain sections at the level of central complex from RQ2 transgenic flies stained with Congo red (CR): (A) uninduced; (B) hs-treated, arrows indicate congophilic aggregates. Brain sections at the level of the optic lobes from RQ2 transgenic adult flies stained with CR: (C) uninduced; (D) hs-treated, arrows indicate congophilic aggregates. Frontal protocerebral cellular mass of RQ2 transgenic adult flies: (E) uninduced; (F) hs-treated, arrows indicate Giemsa-positive pycnomorphic cells with high level of basophily; and (G ) hs-treated individual, arrows indicate dying nerve cells. The Giemsa-positive, needle-like structures are seen near the dying cells. Magnification: 10 × 100, immersion

Fig 4.

Learning indices (LI) in wild type and transgenic strains. Abscissa: time after training (min); ordinate: LI. The sample size for each time point was 20 males. * = LI after heat shock significantly differ from LI at 25°C (one-sided randomization test, α_R < 0.05); & = LI significantly lower than that of Canton-S strain under similar conditions (one-sided randomization test, α_R < 0.05); @ = LI significantly lower than that of RQT157 strain under similar conditions (one-sided randomization test, α_R < 0.05); # = LI 3 hours after training significantly differ from LI 0 hours after training (one-sided randomization test, α_R < 0.05); ^ = LI after HS1 or HS2 significantly differ from LI after HS (one-sided randomization test, α_R < 0.05)

Fig 5.

Lifespan of RQ2 males hs-induced at stage of third instar. Heat shock treatment of RQ2 males at the stage of third instar larvae decreased the lifespan of adult flies