Signaling network alterations in mitochondrial disease

Xiaoyan Robert BaoMootha lab

Massachusetts General Hospital

• Most common inborn error of metabolism (~1/5000 live births)• Genetic heterogeneity (~75 known nuclear disease-causing genes; maternally

inherited mtDNA syndromes)• Clinical heterogeneity: epilepsy, stroke-like episodes, vision loss, hearing loss, GI

dismotility, lactic acidosis, hepatopathy• Diagnosis remains difficult, and no effective treatments exist

myopathy subacuteneurodegeneration

Mitochondrial diseases

Described in 1993 in Saguenay Lac-Saint-Jean (SLSJ)Incidence ~ 1/2063 live births (1979-1990)Carrier rate ~ 1/23 in SLSJ

Morin et al Am J Hum Genet 1993

An example of such a disease: Leigh Syndrome French Canadian Variant

Clinical FeaturesBrainstem gliosis, necrosisMild developmental delayAtaxiaReye-like syndromeFacial dysmorphismMortality due to acidotic crisesDeath between 6 mos and 12 yrs of age

Biochemical FeaturesCytochrome c oxidase deficiency

RNA1,600,000 mRNA measures

DNA2,000,000 nucleotides

Protein85,000 peptides

LRPPRC

Combining genomic information to discover the disease gene

Lee et al Am J Hum Genet 2001Mootha et al PNAS 2003

H2N COOH

Exon 9

C1277STOP R* Human RCGAIAEQTPILLLFLLRNSRKQGK

Mouse RCGAIAEQSSLLSVFCLRTSQKPKK

Exon 35

Physiological measurements1. O2 Consumption

2. Acid production

1. qRT-PCR

2. Western Blot

Confirmation of knockdown

Disease specific assay

1. Western Blot for respiratory chain proteins

shRNA

“Disease” cells

Can we model disease in a dish?

Gohil et al J Biol Chem 2010

Systematically defining pathways that are altered

Gohil et al J Biol Chem 2010

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ETHE1

V. Gohil, unpublished

RNAi against other OXPHOS disease genes

Shaham et al PNAS 2010

Chemical perturbation of OXPHOS function

Metabolic profiling of cell culture media

• We have constructed different types of cellular models of mitochondrial disease: RNAi, dominant negative, chemical

• In initial studies, we see robust transcriptional and metabolite changes in response to these genetic or chemical lesions.

• We hypothesize that some of these transcriptional/metabolic changes are mediated by kinase signaling events

• Such signaling events may contribute to disease pathogenesis.

• We look forward to working with the LINCS centers to identify kinase signaling events that drive these changes, using mass spectrometry (Broad) and imaging based (HMS) methods

Potential collaborations with LINCS centers

Acknowledgments

Vamsi MoothaCasey Belcher-TimmeVishal GohilOlga GoldbergerRoland NilssonOded ShahamNancy Slate

Katherine Sims

David E. RootBiao Luo

Clary ClishArvind RamanathanAmanda Souza

NIH/NIDDK R01DK081457