Figure 4. Intracellular domain (ICD) of Crb is required for Aβ42 mediated neurodegeneration.

(A) A cartoon depicting full length type I transmembrane Crb protein and various truncated constructs used in this study. The full length Crb protein consists of an extracellular domain (ECD), transmembrane domain (TM), and a short cytoplasmic intracellular domain (ICD), which consists of the juxtamembrane Ferm-binding motif (JM) and PDZ-binding motif (PBM) domains [28]. GMR-Gal4 driver was used for the misexpression studies in the differentiating photoreceptor neurons [22]. (B–D) Adult eyes of (B) Wild-Type, (C) GMR>Aβ42 (GMR enhancer driving overexpression of human Aβ42 in the developing neural retina), and (D) GMR>Crb (FL) are shown as controls. (A, E–F) Misexpression of (E) Crb^intra alone, comprising of fully intact ICD, shows a severe phenotype with a small scab on the head cuticle in the adult eye, which is similar to the (F) GMR>Aβ42+Crb^intra adult eye. (G) In the GMR>Aβ42+Crb^intra eye disc big gaps and holes between photoreceptors of the ommatidia are seen, Dlg (white) marks the membrane and provide an outline of the imaginal disc and pan neural marker Elav [52] marks the photoreceptors. (A, H–P) In the three other Crb constructs, one of the two domains (JM and PBM) of the ICD is either missing or both of them are missing. (H–P) When Crb is missing either (H–J) PBM, or (K–M) JM, or (N–P) both the PBM and JM domain of the ICD, the GMR>Aβ42 neurodegenerative phenotype is restored significantly with the adult eye having a larger size, higher number of ommatidia, and interommatidial bristles. Furthermore, the Elav staining in the eye-imaginal discs shows more organized photoreceptors in comparison to the GMR>Aβ42 eye imaginal disc. (H, K, N) The controls (H) GMR>Crb^{intra ΔPBM}, (K) GMR>Crb^{intra ΔJM}, and (N) GMR>Crb^{intra ΔPBM ΔJM} showed adult eye phenotypes that are significantly closer to the wild-type. (I, J) When the PBM domain (GMR>Aβ42+Crb^{intra ΔPBM}) is missing, (I) the adult eye and (J) the eye imaginal disc showed significant rescue in comparison to the GMR>Aβ42 phenotype. (L, M) When the JM domain (GMR>Aβ42+Crb^{intra ΔJM}) is missing, (L) the adult eye and (M) the eye-imaginal disc showed significant rescue in comparison to the GMR>Aβ42 phenotype. (O, P) Finally, when both PBM and JM domains of the ICD are missing (GMR>Aβ42+Crb^{intra ΔPBM ΔJM}), a significant rescue was seen in (O) the adult eye and (P) the eye imaginal disc in comparison to the GMR>Aβ42 phenotype.