Mutations in CLN3 cause juvenile neuronal ceroid lipofuscinoses (JNCL) or Batten disease. Mutations result in rapid visual failure, seizures, progressive cognitive and psychomotor decline, and premature death. We previously generated cln3 knockdown zebrafish using antisense morpholinos (cln3 MO), which emulate key disease aspects (Wager et al, 2016).
Here we report the generation of a stable, adult viable, cln3 mutant zebrafish line (cln3^ex2Δ5bp), a 5 bp deletion in exon 2 predicted to cause a loss-of-function (null) allele. Compared to wild-type siblings the homozygous mutant displays significant impairments in behavioural, visual and mitochondrial assays from 1 to 5 dpf, as well as retinal structure and visual function in adults. We have used this cln3 null mutant to confirm that the cln3 morpholino does indeed target cln3 and does not have off-target effects, by showing that the cln3 morpholino does not cause phenotypes when injected into cln3 null mutants. We aim to have completed rescue experiments by the time of the meeting, further confirming the specificity of the cln3 morpholino. We also expect to report on lipidomics, including GPD and BMP measurements, at 5 dpf in both cln3 MO and our cln3 mutant.
We also report that cln3 MO larvae show significantly impaired motor behaviour and mitochondrial function compared to wild-type siblings. Treatment with three candidate drugs and nutritional supplements (RVC1/Pregnenolone, RVC4, RVC5) demonstrates therapeutic rescue, with RVC4 producing the strongest and most consistent improvements in phenotype and mitochondrial activity. These findings demonstrate the validity and usefulness of our zebrafish models for studying CLN3 disease and comparing potential therapeutics.