1	Neurodegenerative Diseases II Friedreich Ataxia Clinical presentation and genetics Initial studies of the disease-gene homologue Pumping Iron? Initial studies of the disease protein and the iron connection What’s the primary defect? Iron-sulfur clusters, mouse models Is this relevant to humans with the disorder? Iron-sulfur clusters, mitochondrial dysfunction, oxidative stress Is there anything we can do about it? Current therapeutic initiatives Mitochondria and aging Oxidative stress versus apoptosis Experimental therapeutics Drug screens and development
2	Friedreich Ataxia First Described by Friedreich in 1863 Autosomal Recessive Prevalence ~1/40,000 in Caucasians, with a carrier frequency of ~1/100 Signs and Symptoms Ataxia, dysarthria, areflexia, sensory loss, muscle weakness Skeletal deformities and cardiomyopathy in most patients Diabetes mellitus and/or impaired glucose tolerance in ~1/3 of patients Reduced visual acuity and hearing loss occasionally seen Onset and Progression Onset usually around puberty Progressive; most patients wheelchair-bound by late 20s Myocardial failure, most common cause of premature death
3	X25/FRDA Map and Alignment with ORFs from Lower Eukaryotes (From Campuzano, Montermini, et al., Science, 1996)
4	GAA Repeat Expansions Campuzano et al., Science, 1996 GAA repeat expansions in the first intron of FRDA gene Normal, 7-40 repeats; disease, 70-1800 repeats Ohshima et al., J. Biol. Chem., 1998 GAA repeats inhibit transcription in proportion to length GAA repeats may form triplex DNA structures Sakamoto et al., Mol. Cell, 1999 GAA repeats form “sticky DNA” which inhibits transcription
5	GAAs Form Triplex Structures and “Sticky DNA” (From Sakamoto et al., 1999)
6	Hydrogen Bonding in Triplex DNA
7	GAA Repeat Expansions GAA repeat expansions decrease, but do not eliminate, frataxin expression GAA repeat expansion sizes correlate with measures of disease severity
8	GAA Repeat Size and Age of Onset From Durr et al., NEJM, 1996
9	Alignment of Frataxin and the Yeast Frataxin Homologue (From Wilson and Roof, Nat. Genet., 1997)
10	Tetrad Analysis of the Yfh1 Knockout From Wilson and Roof, Nature Genetics, 1997
11	Localization of Yfh1p
12	From Wilson and Roof, 1997
13	"Pumping Iron" Pumping Iron?
14	Babcock et al., Science, 1997, Fig. 3A and 3B
15	Babcock et al., Science, 1997, Fig. 3C
16	Electron Transport Chain
17	Reactive Oxygen Species and Antioxidant Defense Mechanisms
18	Reactive Oxygen Species and Antioxidant Defense Mechanisms
19	Rotig et al., Nature Genetics, 1997, Table 1
20	Structures of Iron-Sulfur Clusters
21	Electron Transport Chain
22	Rotig et al., 1997, Fig. 1
23	Underlying Vicious Cycle in FRDA
24	Radisky et al., JBC, 1999, Fig. 4C
25	Possible Effects of Decreased Mitochondrial Iron Efflux
26	Babcock et al., Science, 1997, Fig. 2
27	Possible Effects of Decreased Mitochondrial Iron Efflux
28	Serum Iron and Ferritin in Friedreich’s Ataxia Wilson et al., 1998 Patients Ten patients from nine families Ages 11-49 years, with wide range of repeat expansions (280-1000) and disease severity Serum Ferritin Concentrations Six female patients: 17-67 ng/ml, mean 34 ng/ml (reference range 15-95 ng/ml, geometric mean 46 ng/ml) Four male patients: 75-174 ng/ml, mean 109 ng/ml (reference range 30-370 ng/ml, geometric mean 127 ng/ml) Serum Iron Concentrations 50-118 mg/dl, mean 86 mg/dl (reference range 40-175 mg/dl, mean 110 mg/dl) Conclusion Iron-chelation therapy problematic
29	Possible Effects of Decreased Mitochondrial Iron Efflux
30	"What’s the primary defect" What’s the primary defect? (Or: How do we know where a vicious cycle starts?)
31	Underlying Vicious Cycle in FRDA
32	Yfh1p and Fe-S Cluster Assembly Lill, 1999 Mutations affecting Fe-S cluster assembly in yeast cause the same phenotypes as mutations in Yfh1 Muhlenhoff et al., 2002 Yfh1p depletion associated with decreased Fe-S cluster assembly, in vivo and in vitro using mitochondrial extracts
33	Yfh1p and Fe-S Cluster Assembly Muhlenhoff, 2003
34	Yfh1p and Fe-S Cluster Assembly Gerber et al., 2003 Yfh1p interacts directly with Nfs1p and Isu1p
35	Mouse Models of Friedreich’s Ataxia Complete knockout (Cossee et al., 2000) Embryonic lethality with no evidence of iron accumulation Conditional gene-deletion (Puccio et al., 2001) MCK cre-lox to delete gene in skeletal and cardiac muscle Hypertrophic cardiomyopathy Average life expectancy of 76 days NSE cre-lox to delete gene in heart, brain, liver, kidney Hypertrophic cardiomyopathy Progressive ataxia, loss of proprioception Average life expectancy of 24 days Mitochondrial iron accumulation follows decreased iron-sulfur-cluster enzyme activities
36	Development of Mouse Models (Puccio et al., Nat. Genet., 2001)
37	Mitochondrial Iron Accumulation in MCK cre-lox FRDA Mice (Puccio et al., Nat. Genet., 2001)
38	Extra-mitochondrial Fe-S Cluster Assembly Lill and Muhlenhoff, 2005
39	Underlying Vicious Cycle in FRDA
40	So is there evidence of an iron-sulfur-cluster defect, mitochondrial dysfunction, mitochondrial iron accumulation, and oxidative stress in humans with FRDA?
41	IRP1 and Iron Homeostasis
42	REMSA of IRP binding activity in normal and FRDA fibroblasts Cells were grown in the presence of desferrioxamine (Des) or fully saturated human transferrin (hTf)
43	IRP1 and Iron Homeostasis
44	Lodi et al., PNAS, 1999 Deficit of mitochondrial ATP production in FRDA skeletal muscle in vivo by ³¹P-MRS
45	Lodi et al., PNAS, 1999 Deficit of mitochondrial ATP production in FRDA skeletal muscle in vivo by ³¹P-MRS
46	Lodi et al., PNAS, 1999 Deficit of mitochondrial ATP production in FRDA skeletal muscle in vivo by ³¹P-MRS
47	Near-Infrared Spectroscopy A. Normal Control B. Cytochrome C Oxidase Deficiency
48	Lynch et al., 2002 Prolonged deoxygenation recovery time in FRDA skeletal muscle in vivo by NIRS
49	Delatycki et al., Ann. Neurol., 1999 Increased mitochondrial iron in primary FRDA fibroblasts
50	Schultz et al., Neurol., 2000 Increased urinary 8OH2’dG in FRDA
51	Ian Blair’s Take “Nobody can measure 8-oxo-dGuo in urine with any confidence. It is not even clear how it could be in urine. Base excision repair (through hOGG1, which is known to remove 8-oxo-Gua from DNA) will release the free base rather than the mononucleoside. In a model of oxidative stress urinary 8-oxo-dGuo measurements were worthless.”
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54	Emond et al., Neurol., 2000 Increased plasma malondialdehyde in FRDA
55	"Is there anything we can..." Is there anything we can do about it? Current therapeutic initiatives
56	Electron Transport Chain
57	Coenzyme Q (CoQ)
58	Idebenone for Friedreich’s Ataxia Mouse data Slightly delayed onset of cardiomyopathy and slightly prolonged lifespan Preliminary clinical trial data in humans Decreased cardiac hypertrophy after six months at 5 mg/kg/day
59	Clinical Trials of Idebenone for FRDA Mariotti et al., 2003 Idebenone 5 mg/kg/day 29 patients with heart-wall thicknesses ≥ 12 mm Double-blind, placebo controlled Decrease in heart-wall thicknesses after 6 months No change in cardiac ejection fraction or ICARS
60	Idebenone for Friedreich Ataxia The Good News May benefit heart and skeletal muscle The Bad News Statistically significant evidence of efficacy for ataxia lacking Imprecision of ataxia scales used in current trials Insufficient penetration of idebenone into neurons Intrinsic differences between neurons and muscle cells
61	What if you mess with mitochondria generally?
62	Mitochondrial Mutator Mouse Trifunovice et al., Nature 429: 417-423, 2004
63	Mitochondrial Mutator Mouse Trifunovice et al., Nature 429: 417-423, 2004
64	Mitochondrial Mutator Mouse Trifunovice et al., Nature 429: 417-423, 2004
65	Mitochondrial Mutator Mouse Kujoth et al., Science 309: 481-484, 2005
66	Mitochondrial Mutator Mouse Kujoth et al., Science 309: 481-484, 2005
67	Electron Transport Chain
68	Mitochondrial Mutator Mouse Kujoth et al., Science 309: 481-484, 2005
69	Apoptosis: Mitochondrial Pathway
70	Mitochondrial Mutator Mouse Kujoth et al., Science 309: 481-484, 2005
71	Mitochondrial Mutator Mouse Kujoth et al., Science 309: 481-484, 2005
72	Mitochondrial Mutator Mouse Kujoth et al., Science 309: 481-484, 2005
73	Mitochondrial Mutator Mouse Kujoth et al., Science 309: 481-484, 2005
74	From Wilson and Roof, Nature Genetics, 1997
75	Is there a connection between mitochondrial dysfuction and protein misfolding? (Or, Why does overexpression of Parkin reverse the phenotypes of Pink1 mutations?)
76	Gidalevitz et al., Science 311: 1471-1474, 2006
77	“Research is the art of the fundable” Steven Spitalnik
78	Yeast Model of Friedreich Ataxia for Drug Testing Turn off Yfh1 with inducible/repressible promoter Plate in 96-well plates in respiration-only medium Follow mitochondrial function and growth Tetrazolium dye reduction ATP content Absorbance at 630 nm Compounds versus carrier control
79	HTS Validation Signal-to-Background Ratio S/B = M_signal/M_background Should be > 3 Z’ Factor Z’ = 1 - 3 X (SD_signal + SD_background)/(M_signal - M_background) Should be > 0.4
80	Two of 30 compounds from Galileo Pharmaceuticals improved tetrazolium dye reduction in yeast lacking Yfh1 Confirmation of tetrazolium dye reduction assay S/B > 2 in growth assay (shaking tubes, Coulter counter) S/B > 3 with Cell-Titer-Glo assay for ATP content
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84	Drug Screening Using Primary FRDA Fibroblasts
85	From Lum et al., Cell 116: 121, 2004
86	From Lum et al., Cell 116: 121, 2004
87	Ataxia Scales Trial Purpose To develop sensitive, precise, and quantifiable measures of ataxia and ataxia progression Design Annual assessment Nine-hole pegboard test 25-foot timed walk Low-contrast vision test Participating Centers University of Pennsylvania (David Lynch), University of Iowa (Henry Paulson), University of Mississippi (Sub Subramony), University of California at Los Angeles (Susan Perlman), Emory University (George Wilmot), University of Minnesota (Christopher Gomez), and the University of Texas Medical Branch in Galveston (Tee Ashizawa)
88	Acknowledgements Laboratory Studies David Roof Rob Oldt Xiaohong Witmer Sizhen Gao Kevin Leclerc Susan Yoon Lioba Lobmayr Zhongwei Xu Claudia Provenzano David Brooks Grazia Cotticelli Clinical Studies David Lynch Jennifer Farmer Gwen Lech Kurt Fischbeck Paul Taylor Nick DiProspero William Bank Britton Chance Laura Balcer
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