Cilia dysfunction underlies a class of human diseases with variable penetrance in different organ systems. Across eukaryotes, intraflagellar transport (IFT) facilitates cilia biogenesis and cargo trafficking, but our understanding of mammalian IFT is insufficient. Here we perform live analysis of cilia ultrastructure, composition and cargo transport in native mammalian tissue using olfactory sensory neurons. Proximal and distal axonemes of these neurons show no bias towards IFT kinesin-2 choice, and Kif17 homodimer is dispensable for distal segment IFT. We identify Bardet–Biedl syndrome proteins (BBSome) as bona fide constituents of IFT in olfactory sensory neurons, and show that they exist in 1:1 stoichiometry with IFT particles. Conversely, subpopulations of peripheral membrane proteins, as well as transmembrane olfactory signalling pathway components, are capable of IFT but with significantly less frequency and/or duration. Our results yield a model for IFT and cargo trafficking in native mammalian cilia and may explain the penetrance of specific ciliopathy phenotypes in olfactory neurons.

Cilia are evolutionarily conserved microtubule-based organelles that are crucial for diverse biological functions, including motility, cell signaling and sensory perception1. In humans, alterations in the formation and function of cilia manifest clinically as ciliopathies, a growing class of pleiotropic genetic disorders2–4. Despite the substantial progress that has been made in identifying genes that cause ciliopathies, therapies for these disorders are not yet available to patients. Although mice with a hypomorphic mutation in the intraflagellar transport protein IFT88 (Ift88Tg737Rpw mice, also known as ORPK mice)5 have been well studied, the relevance of IFT88 mutations to human pathology is unknown. We show that a mutation in IFT88 causes a hitherto unknown human ciliopathy. In vivo complementation assays in zebrafish and mIMCD3 cells show the pathogenicity of this newly discovered allele. We further show that ORPK mice are functionally anosmic as a result of the loss of cilia on their olfactory sensory neurons (OSNs). Notably, adenoviral-mediated expression of IFT88 in mature, fully differentiated OSNs of ORPK mice is sufficient to restore ciliary structures and rescue olfactory function. These studies are the first to use in vivo therapeutic treatment to reestablish cilia in a mammalian ciliopathy. More broadly, our studies indicate that gene therapy is a viable option for cellular and functional rescue of the complex ciliary organelle in established differentiated cells.

In comparing purified mouse olfactory sensory neurons (OSNs) with neighboring cells, we identified 54 differentially expressed transcripts. One-third of the transcripts encode proteins with no known function, but the others have functions that correlate with challenges faced by OSNs. The OSNs expressed a diversity of signaling protein genes, including stomatin (Epb7.2), S100A5, Ddit3, Sirt2, CD81, Sdc2, Omp, and Ptpla. The elaboration of dendrites, cilia, and axons that places OSNs in contact with diverse cell types and signals presumably also requires large investments in cytoskeletal-associated proteins, lipid biosynthesis, and energy production. Several of the genes encode proteins that participate in these biological processes, including ATP5g3, Ndufa9, Sqrdl, Mdh1, Got1, beta-2 tubulin, Capza1, Bin3, Tom1, Acl6, and similar to O-MACS. Three transcripts had restricted expression patterns. Similar to O-MACS and Gstm2 had zonally restricted expression patterns in OSNs and sustentacular cells but not in Bowman's glands, suggesting that zonality can be differentially regulated by cell type. The mosaic expression pattern of S100A5 in approximately 70% of OSNs predicts that it is coexpressed with a subset of odorant receptors. We captured four abundant transcripts, Cyp2a4, similar to Cyp2g1, Gstm2, and Cbr2, that encode xenobiotic metabolizing enzymes expressed by sustentacular cells or Bowman's glands, reinforcing the interpretation that clearance of xenobiotic compounds is a major function of these cells. Within the olfactory epithelium, Cbr2 is a new anatomical marker for sustentacular cells. We also discovered that Reg3g is a marker for respiratory epithelium.

Olfactory dysfunction is a pervasive but underappreciated health concern that affects personal safety and quality of life. Patients with olfactory dysfunctions have limited therapeutic options, particularly those involving congenital diseases. Bardet-Biedl syndrome (BBS) is one such disorder, where olfactory loss and other symptoms manifest from defective cilium morphology and/or function in various cell types/tissues. Olfactory sensory neurons (OSNs) of BBS mutant mice lack the capacity to build/maintain cilia, rendering the cells incapable of odor detection. Here we examined OSN cilium defects in Bbs1 mutant mice and assessed the utility of gene therapy to restore ciliation and function in young and adult mice. Bbs1 mutant mice possessed short residual OSN cilia in which BBSome protein trafficking and odorant detection were defective. Gene therapy with an adenovirus-delivered wild-type Bbs1 gene restored OSN ciliation, corrected BBSome cilium trafficking defects, and returned acute odor responses. Finally, using clinically approved AAV serotypes, we demonstrate, for the first time, the capacity of AAVs to restore ciliation and odor detection in OSNs of Bbs1 mutants. Together, our data demonstrate that OSN ciliogenesis can be promoted in differentiated cells of young and adult Bbs1 mutants and highlight the potential of gene therapy as a viable restorative treatment for congenital olfactory disorders.

Cilia are highly specialized microtubule-based organelles that have pivotal roles in numerous biological processes, including transducing sensory signals. Defects in cilia biogenesis and transport cause pleiotropic human ciliopathies. Mutations in over 30 different genes can lead to cilia defects, and complex interactions exist among ciliopathy-associated proteins. Mutations of the centrosomal protein 290 kDa (CEP290) lead to distinct clinical manifestations, including Leber congenital amaurosis (LCA), a hereditary cause of blindness due to photoreceptor degeneration. Mice homozygous for a mutant Cep290 allele (Cep290 rd16 mice) exhibit LCA-like early-onset retinal degeneration that is caused by an in-frame deletion in the CEP290 protein. Here, we show that the domain deleted in the protein encoded by the Cep290 rd16 allele directly interacts with another ciliopathy protein, MKKS. MKKS mutations identified in patients with the ciliopathy BardetBiedl syndrome disrupted this interaction. In zebrafish embryos, combined subminimal knockdown of mkks and cep290 produced sensory defects in the eye and inner ear. Intriguingly, combinations of Cep290 rd16 and Mkks ko alleles in mice led to improved ciliogenesis and sensory functions compared with those of either mutant alone. We propose that altered association of CEP290 and MKKS affects the integrity of multiprotein complexes at the cilia transition zone and basal body. Amelioration of the sensory phenotypes caused by specific mutations in one protein by removal of an interacting domain/protein suggests a possible novel approach for treating human ciliopathies.

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Recent work suggests an important role for the Ran importin system in cilia trafficking. At the onset of ciliogenesis, Ran GTP levels rise markedly at the centrosome. Altering Ran GTP levels by varying RanBP1 expression modulates cilia formation and trafficking.

The mechanisms selecting a single odorant receptor (OR) gene for expression in each olfactory sensory neuron (OSN) establish an OR expression pattern critical for odor discrimination. These mechanisms are largely unknown, but putative OR promoters contain homeodomain-like sites, implicating homeobox transcription factors such as Emx2. At embryonic day 18.5, expression of 49-76% of ORs was decreased in mice lacking Emx2, depending on the metric used. The decreases were due to fewer OSNs expressing each OR. Affected ORs showed changes that were disproportionately greater than the 42% reduction in mature neurons and similar decreases in unrelated olfactory neuron-enriched messenger RNAs in Emx2(-/-) mice. Both Class I and Class II ORs decreased, as did ORs expressed in both the dorsal and ventral regions of the epithelium. Conversely, 7% of Class II ORs tested were expressed more frequently, suggesting that some ORs are independent of Emx2. Emx2 helps stimulate transcription for many OR genes, which we hypothesize is through direct action at OR promoters, but Emx2 appears to have no significant role in regulating other aspects of OR gene expression, including the zonal patterns, OR gene cluster selection mechanisms, and singularity of OR gene choice.