“The Ciliopathies: Recent advances" Phil Beales [email protected] Molecular Medicine Unit, Institute of Child Health and Great Ormond Street Hospital for Children, University College London Aims I. Structure and function of cilia II. Advances in ciliopathy research III. Potential for therapy Antony van Leeuwenhoek (1632-1723) "And though I must have seen quite 20 of these little animals on their long tails alongside one another very gently moving, with outstretched bodies and straightened-out tails; yet in an instant, as it were, they pulled their bodies and their tails together, and no sooner had they contracted their bodies and tails, than they began to stick their tails out again very leisurely, and stayed thus some time continuing their gentle motion: which sight I found mightily diverting.“ Letter to Royal Society of London - December 25, 1702 What is the evidence for cilia in disease? Fluid movement Vestigial remnants Sensory role: – Polycystic kidneys – Photoreceptors – L-R assymetry Signalling Cilia and signalling Hedgehog limbs defects Adapted from Goetz and Anderson Nat Rev Genet. 2010 What is the evidence for cilia in disease? Fluid movement Vestigial remnants Sensory role: – Polycystic kidneys – Photoreceptors – L-R assymetry Signalling Cystic renal disease Wildtype Orpk/Polaris Pazour G J et al. J Cell Biol 2000;151:709-718 Ciliated sensory cells Left-right asymmetry Courtesy N. Hirokawa Situs anomalies What is a ciliopathy? I. Motile (9+2) – Kartagener syndrome (PCD)– (e.g. DNAH5, DNAI1, DNAH11, TXNDC3 ) II. Non-motile (9+0) – Alstrom syndrome – Bardet-Biedl syndrome – Jeune syndrome – Joubert syndrome – Meckel syndrome – Nephronophthisis (NPHP)/Senior-Loken – Ellis van Creveld – Oral Facial Digital Type 1 (OFD1) (X-linked) – Polycystic kidney disease (ARPKD/ADPKD) Primary Ciliary Dyskinesia Baillie first described cilia beating situs inversus in 1793 Cilia lining the airways provide a motile surface to clear away bacteria from our Kartagener (1933) - airways triad - sinusitis, bronchiectasis, and situs inversus 7 PCD genes are essential Cilia immotility in components of cilia structure infertile males – 2 PCD genes get the other Afzelius (1976) proteins to their correct location in cilia Courtesy of Hannah Mitchison PCD genes LRRC50 2009 KTU 2009 α Outer dynein arm DC1 IC1 (Chlamydomonas) 8 6 DC3 DNAI2 DNAI1 TXNDC3 DNAH5 DNAH11 RSPH9, RSPH4A 2008 1999 2007 2002 2002 2009 9 genes causing 17-38% disease Courtesy of Hannah Mitchison Bardet-Biedl Syndrome . Retinal Degeneration . Cognitive impairment . Obesity . Polydactyly . Hypogenitalism/genital malformation . Renal dysfunction/malformation BBS genes Locus Chromosome Function BBS1 11q13 Novel gene BBS2 16q21 Novel gene BBS3/ARL6 3p13 ARL6 BBS4 15q23 TPRs/OGT BBS5 2q31 Novel gene BBS6/MKKS 20p12 Chaperone BBS7 4q27 Novel gene BBS8 14q32.1 PilF/TPRs BBS9 7p14 PTH-B1 BBS10 12q21 Chaperone BBS11/TRIM32 9q33.1 TRIM32 BBS12 4q27 Chaperone BBS13+ - MKS1, MKS3, Cep290 BBS8 first clue to cilia dysfunction TTC8 TTC8 TTC8 Ansley et al Nature 2003 C. elegans Adult worm has 959 cells 302 are neuronal 60 of these are ciliated Genes for osmolarity and chemotaxis are expressed exclusively in these 60 ciliated cells osm-1, osm-5, osm-6, che-2 bbs8 BBS8 Ac. tubulin Merge + DAPI Ciliary Interactome Meckel BBS7 BBS1 BBS2 BBS6 BBS4 BBS9 JS PCM1 BBS8 BBS5 AS YTH Proteome NPHP GM reduction in ant temporal lobes, olfactory bulbs Hippocampal volume Smo, Ift88 and Fantom knockouts – cilia required for postnatal neural stem cell proliferation via the Sonic Hedgehog pathway Alström syndrome Cone-rod dystrophy Early onset obesity Type 2 diabetes mellitus Neuro-sensory hearing loss Cardiomyopathy Short Male hypogonadism AR ALMS1 Adult Alström cardiomyopathy - fibrotic process causing impairment of both ventricles Serial cardiac MRI advocated Cause of fibrosis? Alström and hearing loss Alms1 localises to basal body of cochlear hair cells Alms1 required for normal cell pattern likely accounting for hearing loss Jagger et al. 2010 HMG (AOP) 102 likely ciliopathies 14 proven Jeune syndrome (asphyxiating thoracic dystrophy) . Rare autosomal recessive chondrodysplasia . Often leads to death in infancy - severely constricted thoracic cage (short ribs) and respiratory insufficiency . Metaphyseal abnormalities/short long bones . Liver (fibrocystic), pancreatic (fibrosis) involvement . Post-axial polydactyly . Renal cysts . Retinal degeneration . Multiple loci mapped (15q13, 3q25) JATD-01 JATD-02 JATD-03 wt H105Q L549del wt L549del A701P A701P wt Tetrahymena thermophila GFPift80 wild-type ift80 depletion Acetylated tubulin (cilia) Courtesy Mark Winey Centrin (basal bodies) Blacque et al. Frontiers in Bioscience 13, 2633-2652 IFT46 IFT88 IFT80 IFT172 IFT140 IFT52 IFT122 Adapted from Blacque et al. Frontiers in Bioscience 13, 2633-2652 American Journal of Human Genetics 84, 706–711, 2009 American Journal of Human Genetics 84, 542–549, 2009 Polycystic kidney disease Autosomal recessive PKD (ARPKD) – 1 in 10,000 Autosomal dominant PKD (ADPKD) – 1 in 800 mTOR signalling in PKD mTOR inhibitors reduce cyst formation mTORC1 activity contributes to ADPKD PC1 suppresses mTOR activity TSC proteins regulate ciliary length (but no evidence for vv) Latest human trials - rapamycin 3 placebo-controlled trials Different outcomes SIRENA (Sirolimus) study – inhibits cyst growth and >parenchymal volume Swiss ADPKD study – no change in TKV or eGFR Everolimus study – slowed increase in TKV May stabilise renal function if given early Treating ciliopathies Gene therapy – disadvantage in multisystem disorders Exon skipping – gentamicin/PTC124 Pharmacological interventions a. Pathway targetted approach b. Library screen to rescue individual phenotypes – Small molecules – prolonged development period – Approved drugs – safety, toxicity, side-effects Zebrafish kidney Drummond et al 1998 Zebrafish morphants develop pronephric cysts Rapamycin reduces renal cysts & recovers function control bbs4 MO bbs6 MO bbs6 MO + Rapa bbs8 MO bbs8 MO + Rapa ift80 MO ift80 MO + Rapa Rescue of kidney cyst Size of kidney 200000 180000 160000 140000 120000 100000 80000 Kidney surface area (um2) 60000 40000 20000 0 control ift80 MO ift80 MO ift80 MO bbs6 MO 0nM bbs6 MO bbs8 MO 0nM bbs8 MO 0nM Rapa 0nM Rapa 10nM Rapa 100nM Rapa Rapa 100nM Rapa Rapa 100nM Rapa (n=8) (n=22) (n=20) (n=12) (n=10) (n=4) (n=11) (n=8) Tobin and Beales Ped Nephr 2006 Ciliopathies Summary Ciliopathies – diverse overlapping conditions Collectively common Share fundamental cellular defects but specific differences Many more ...... Therapeutic approaches – reprofiling promising.