Anti-clarin-1 AAV-delivered ribozyme induced apoptosis in the mouse cochlea.
Usher syndrome type 3 is caused by mutations in the USH3A gene, which encodes the protein clarin-1. Clarin-1 is a member of the tetraspanin superfamily (TM4SF) of transmembrane proteins, expressed in the organ of Corti and spiral ganglion cells of the mouse ear. We have examined whether the AAV-mediated anti-clarin ribozyme delivery causes apoptotic cell death in vivo in the organ of Corti. We used an AAV-2 vector delivered hammerhead ribozyme, AAV-CBA-Rz, which specifically recognizes and cleaves wild type mouse clarin-1 mRNA. Cochleae of CD-1 mice were injected either with 1mul of the AAV-CBA-Rz, or control AAV vectors containing the green fluorescent protein (GFP) marker gene (AAV-CBA-GFP). Additional controls were performed with saline only. At one-week and one-month post-injection, the animals were sacrificed and the cochleae were studied by histology and fluorescence imaging. Mice injected with AAV-CBA-GFP displayed GFP reporter expression of varying fluorescence intensity throughout the length of the cochlea in the outer and inner hair cells and stria vascularis, and to a lesser extent, in vestibular epithelial cells. GFP expression was not detectable in the spiral ganglion. The pro-apoptotic effect of AAV-CBA-delivered anti-clarin-1 ribozymes was evaluated by TUNEL-staining. We observed in the AAV-CBA-Rz, AAV-CBA-GFP and saline control groups apoptotic nuclei in the outer and inner hair cells and in the stria vascularis one week after the microinjection. The vestibular epithelium was also observed to contain apoptotic cells. No TUNEL-positive spiral ganglion neurons were detected. After one-month post-injection, the AAV-CBA-Rz-injected group had significantly more apoptotic outer and inner hair cells and cells of the stria vascularis than the AAV-CBA-GFP group. In this study, we demonstrate that AAV-CBA mediated clarin-1 ribozyme may induce apoptosis of the cochlear hair cells and cells of the stria vascularis. Surprisingly, we did not observe apoptosis in spiral ganglion cells, which should also be susceptible to clarin-1 mRNA cleavage. This result may be due to the injection technique, the promoter used, or tropism of the AAV serotype 2 viral vector. These results suggest the role of apoptosis in the progression of USH3A hearing loss warrants further evaluation.
Aarnisalo
A A
AA
Department Otorhinolaryngology, University of Helsinki, Helsinki, Finland. antti.aarnisalo@hus.fi
Pietola
L
L
Joensuu
J
J
Isosomppi
J
J
Aarnisalo
P
P
Dinculescu
A
A
Lewin
A S
AS
Flannery
J
J
Hauswirth
W W
WW
Sankila
E-M
EM
Jero
J
J
D000818:Animals; D017209:Apoptosis; D003051:Cochlea / Q000378:metabolism / Q000473:pathology*; D000229:Dependovirus / Q000235:genetics*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006198:Hair Cells, Auditory / Q000378:metabolism / Q000473:pathology; D020287:In Situ Nick-End Labeling; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013316:Stria Vascularis / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D052245:Usher Syndromes / Q000235:genetics / Q000378:metabolism / Q000473:pathology*
C512668:Clrn1 protein, mouse; D008565:Membrane Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme; D049452:Green Fluorescent Proteins