“The : 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 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 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 are essential  Cilia immotility in components of cilia structure infertile males – 2 PCD genes get the other Afzelius (1976) to their correct location in cilia

Courtesy of Hannah Mitchison PCD genes LRRC50 2009 KTU 2009

α

Outer 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 Function

BBS1 11q13 Novel 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. 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 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 . (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 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 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