Deletion of the phosphatase INPP5E in the murine retina impairs photoreceptor axoneme formation and prevents disc morphogenesis

Abstract

INPP5E, also known as pharbin, is a ubiquitously expressed phosphatidylinositol polyphosphate 5-phosphatase that is typically located in the primary cilia and modulates the phosphoinositide composition of membranes. Mutations to or loss of INPP5E is associated with ciliary dysfunction. INPP5E missense mutations of the phosphatase catalytic domain cause Joubert syndrome in humans—a syndromic ciliopathy affecting multiple tissues including the brain, liver, kidney, and retina. In contrast to other primary cilia, photoreceptor INPP5E is prominently expressed in the inner segment and connecting cilium and absent in the outer segment, which is a modified primary cilium dedicated to phototransduction. To investigate how loss of INPP5e causes retina degeneration, we generated mice with a retina-specific KO ( Inpp5e F/F ;Six3Cre, abbreviated as ret Inpp5e −/− ). These mice exhibit a rapidly progressing rod–cone degeneration resembling Leber congenital amaurosis that is nearly completed by postnatal day 21 (P21) in the central retina. Mutant cone outer segments contain vesicles instead of discs as early as P8. Although P10 mutant outer segments contain structural and phototransduction proteins, axonemal structure and disc membranes fail to form. Connecting cilia of ret Inpp5e −/− rods display accumulation of intraflagellar transport particles A and B at their distal ends, suggesting disrupted intraflagellar transport. Although INPP5E ablation may not prevent delivery of outer segment–specific proteins by means of the photoreceptor secretory pathway, its absence prevents the assembly of axonemal and disc components. Herein, we suggest a model for INPP5E–Leber congenital amaurosis, proposing how deletion of INPP5E may interrupt axoneme extension and disc membrane elaboration.

Multiple Ciliary Localization Signals Control INPP5E Ciliary Targeting

“…The complexity and redundancy in INPP5E ciliary targeting suggest this is a very important process, subject to fine regulation. This is consistent with the surprisingly wide range of functions INPP5E plays at the cilium, controlling among others their lipid and protein composition, assembly and disassembly, exovesicle release, and signaling 7,8,12,[32][33][34][35][36][37][38][39][40][41][42][43][44][45] . By providing a much broader view of the…”

“…INPP5E plays multiple important roles at the cilium. Among others, these roles include regulation of: (i) ciliary phosphoinositide levels, (ii) ciliary protein composition, (iii) ciliary Hedgehog and PI3K signaling, (iv) ciliary ectovesicle release, (v) ciliary stability, and (vi) ciliogenesis 7,8,12,[32][33][34][35][36][37][38][39][40][41][42][43][44][45] . Although most of its functions are ciliary, INPP5E also plays extraciliary roles, like promoting autophagosomelysosome fusion during autophagy 46 .…”

Deletion of CEP164 in mouse photoreceptors post-ciliogenesis interrupts ciliary intraflagellar transport (IFT)

“…CEP164 forms a complex with PDEδ (chaperone for prenylated protein), INPP5E (a farnesylated inositol polyphosphate 5 phosphatase) and ARL13B (GEF of ARL3) to traffic prenylated INPP5E to primary cilia in cell culture [22]. However, INPP5E of wildtype mouse photoreceptors localizes to the IS, is excluded from the OS, and is apparently independent of PDEδ [65]. Absence of INPP5E in the OS nullifies experiments to show that ARL13b, PDEδ or CEP164 act as OS chaperones for INPP5E.…”

“…Absence of INPP5E in the OS nullifies experiments to show that ARL13b, PDEδ or CEP164 act as OS chaperones for INPP5E. In ret Inpp5e -/- photoreceptors, CC and short rudimentary OS were established, but axonemal structure with discs failed to form [65]. ARL13B localizes to WT OS, but in ret Arl13b -/- [66] and ret Cep164 -/- photoreceptors ( Fig.…”

Superresolution Microscopy Reveals Distinct Phosphoinositide Subdomains Within the Cilia Transition Zone

“…Perhaps INPP5E transiently passes through the transition zone en route to the axoneme and in the process hydrolyses the PIs, however, such transient localization is not easily detected in fixed cells. Consistent with this contention, in a small subset of fixed cells, low level transition zone INPP5E has been observed (Dyson et al, 2017), and in photoreceptors that have a highly modified cilium, INPP5E localizes to the connecting cilium which corresponds to the transition zone (Sharif et al, 2021). Alternatively, it is possible that standard immunofluorescence (IF) techniques used to date do not accurately preserve and represent the correct INPP5E localization.…”