a–f Identification of biallelic RNF170 mutations in four families and functional characterization. a Pedigree of the family in which genome sequencing identified a homozygous splice region mutation in RNF170 segregating with the disease. b Confirmation of the intronic variant c.396+3A>G in genomic DNA. c Gel electrophoresis and d consecutive Sanger sequencing confirmed the sole expression of a shorter transcript lacking exon 5 (wildtype transcript: 395bp; aberrant transcript: 321bp). e Quantitative real-time PCR from blood and fibroblast derived cDNA from individual A.4 demonstrated significantly reduced RNF170 expression in comparison with three control samples (Wilcoxon rank sum test, two-sided); f No residual RNF170 expression could be detected in patient fibroblasts. Note the unspecific band in the RNF170 western blot as well as the specific 25 kDa band corresponding to RNF170, that is abolished upon knockout of RNF170 in SH-SY5Y cells. g Pedigree of family B and h variant confirmation by Sanger sequencing. i Pedigree of family C and segregation in the family. j The deletion was confirmed by visual analysis of split reads in the IGV browser. k, l In addition, primers were designed flanking the breakpoints as well as the deletion. m Subsequent Sanger sequencing of the breakpoint fragment was used to further characterize the variant. n The frameshift variant segregating in family D could be confirmed by o Sanger sequencing

Loss of RNF170 results in decreased degradation of IP3R-3 in patient fibroblasts. a Immunoblot analysis of IP3R-3 in fibroblasts derived from individuals A.4 and C.4 shows increased expression levels in comparison with five controls (Co1, Co2, Co3, Co4, Co5). Western blots from a representative experiment are shown. b Semiquantitative immunoblot analysis indicates significantly increased (Tukey–Kramer HSD, t-sided) IP3R-3 expression. In the quantile blot, boxes indicate the 1st and 3rd quartile and median (center line); whiskers depict the 1st/3rd quartile ± 1.5* interquartile range). c, d IP3R-3 was activated by bradykinin stimulation of fibroblasts to trigger RNF170-dependent IP3R-3 degradation by the proteasomal system. IP3R-3 levels were assessed at baseline as well as 30 and 60 mins after stimulation. Physiological IP3R-3 reduction was observed in all three control cell lines (Co1, Co2, Co3), whereas levels were unaltered in patient-derived fibroblasts (derived from patients A.4 and C.4) (full-factorial repeated measures analysis; means and standard deviations are shown for each data point)

Effect of RNF170 mutations on IP3R-1 degradation and abundance in neuronal cells. a, b Immunoblot analysis of IP3R-1 in wildtype and knockout SH-SY5Y cells (SH-SY5Y(RNF170^wt)/n = 14 biologically independent samples; SH-SY5Y(RNF170^ko)/n = 14 biologically independent samples) and after re-expression of RNF170 in a knockout background (SH-SY5Y(RNF170^ko(wt-HA))/n = 6 biologically independent samples). SH-SY5Y(RNF170^ko) cells demonstrate significant accumulation of IP3R-1 (ko: mean 0.437 ± 0.133; wt: mean 0.247 ± 0.043) that can be rescued by re-expression of RNF170 (rescue: mean 0.318 ± 0.066; Tukey–Kramer HSD, two-sided). In the quantile blot, boxes indicate the 1st and 3rd quartile and median (center line); whiskers depict the 1st/3rd quartile ± 1.5* interquartile range. c, d IP3R-1 was activated by carbachol stimulation in neuronal SH-SY5Y cells, including wt and CRISPR/Cas9 generated RNF170 knockout cell lines (SH-SY5Y(RNF170^wt), SH-SY5Y(RNF170^ko) as well as SH-SY5Y cells stably expressing wildtype HA-tagged RNF170 in a knockout background (SH-SY5Y(RNF170^ko(wt-HA)). IP3R-1 levels were quantified by western blot at baseline (t = 0 h) and 2 h/4 h after stimulation. Neither the effect of the genotype on IP3R-1 degradation (wt vs. ko: p = 0.1806; repeated measures full-factorial analysis) nor the interaction between genotype and time was significant (wt vs. ko, genotype*time: p = 0.1956; repeated measures full-factorial analysis). Nine independent biological replicates were examined per genotype. Means and standard deviations are shown for each data point. e Expression of episomally expressed HA-tagged RNF170 was analyzed by immunoblot in SH-SY5Y cells

MO knockdown of rnf170 results in morphological abnormalities, impaired neurogenesis, and motoneuron defects. a Representative images showing the morphology of live zebrafish embryos at 48 hpf injected with two different splice-blocking rnf170 antisense MOs. rnf170 morphants are characterized by a shortened body axis, micropthalmia (arrows), microcephaly (brackets), and alterations in pigmentation (arrowheads). Scale bar represents 500 µm. b Staining for the axonal marker acetylated tubulin at 48 hpf indicates impaired neurogenesis as shown by reduced neuronal density and migration (brackets) in the developing hindbrain of rnf170 morphants, compared with control embryos. Asterisks indicate the position of the eye, scale bar represents 50 µm. c Dorsal flatmount images of acetyated tubulin stained embryos at 48 hpf showing loss of migrating axons across the intertectal commissure (arrow and asterisks), reduction of arborization in the tectum (Te), and thickening of the tracts of the habenular commissure (THC) and tracts of the posterior commissure (TPC) (arrowheads). Scale bar 200 µm. The eye, trigeminal glia (Tg) and hindbrain glia (Hg) are given as further landmarks. d Aberrant eye and brain development was observed in wax sections of rnf170 morphants at 4 dpf stained with H&E. Reduction of cranial width (brackets) and ventricular cavities was apparent (arrowheads). Scale bar represents 100 µm

Overexpression of mutant RNF170 in zebrafish. a, b Overexpression of wt but not mutant RNF170 results in morphological abnormalities in zebrafish larvae. Representative images showing normal, mild, moderate, and severe morphology phenotypes in live zebrafish at 48 hpf. Overexpression of wt RNF170 results in more severe phenotypes when compared with mock injected controls (MIC). Overexpression of truncated RNF170 as well as mutant RNF170 harboring the mutation c.304T>C, which was identified in family B, does not result in morphological abnormalities implying that the missense mutation results in a loss of protein function. Scale bar represents 400 μm. c, d Overexpression of wt but not mutant RNF170 results in shortened body axis and smaller eye area. Representative images of MIC (n = 18) zebrafish larvae in comparison with overexpression of wt RNF170 (n = 16) as well as mutants (c.304T>C, n = 26; and truncated RNA, n = 22) at 48 hpf. Only overexpression of wt RNF170 but not mutant RNF170 (both c.304T>C and truncated RNA) result in reduced body length and eye area in comparison with MIC embryos further delineating a loss of function effect of the mutation c.304T>C (one-way ANOVA with Tukey’s multiple comparison test)

RNF170-dependent degradation of activated IP3R and genetic disorders affecting this pathway. Upon activation of the IP3R with IP3, calcium is released from the ER into the cytoplasm. This triggers the association of IP3R with the ERLIN1/2 complex leading to the ubiquitination of IP3R by the E3 ubiquitin ligase RNF170, resulting in the proteasomal degradation of IP3R. Mutations in all genes encoding components of this pathway are known to cause hereditary neurologic disorders, especially spastic paraplegia and spinocerebellar ataxia

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