Hexanucleotide repeat expansion in a non-coding region of C9orf72 was recently identified to be the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). The leading hypothesis for the disease mechanism is gain of toxicity from the expanded repeats, with two non- mutually exclusive mechanisms: 1) RNA foci formed by hexanucleotide repeats that could sequester RNA binding proteins and disrupt RNA processing; and 2) accumulation of dipeptide repeat proteins (DPRs) produced by repeat-associated non-ATG translation (RAN translation). Furthermore, the repeats are transcribed in both sense and antisense directions. How exactly or to what extent these different products contribute to disease is not established. In this project, propose to combine new genomic, biochemistry and cellular and mouse modeling tools to determine the molecular mechanism of disease pathogenesis and identify candidate targets for therapeutic development. I will define a C9orf72 repeat expansion-dependent RNA signature in human neurons derived from a large number of patient fibroblasts, including alterations in both RNA expression and alternative splicing using genome-wide sequencing approaches. I will determine whether decreasing sense and/or antisense repeat- containing transcripts by antisense oligonucleotide (ASO) treatment reverses the RNA signature in C9orf72 patient neurons. I will also decipher the functional contribution from repeat-containing RNAs and RAN translation-encoded poly-dipeptides by genome engineering control cells with individual potentially toxic product. I will identify specific RNA-binding protein(s) associated with either sense or antisense hexanucleotide repeats in intact cells using an in vivo RNA tagging system, and determine whether loss of function of any of these contributes to the RNA signature in C9orf72 neurons. I will then decipher the damaging pathways in neurons by manipulating the candidate gene changes, perturbed pathways and RNA-binding proteins. Finally, I will determine whether the repeat expansion in glial cells have toxic effects on neurons through a non-cell autonomous mechanism by combining mouse modeling for identification of cell type-specific, age- and repeat length-dependent RNA changes caused by C9orf72 repeat expansion in vivo, and co-culturing of iPSC- differentiated neurons with astrocytes or oligodendrocytes. Overall, I believe that my proposal has the potential to test several key hypotheses regarding C9orf72-mediated pathogenesis of ALS/FTD and identify a disease- dependent molecular signature that enables the development of therapeutic strategies.

Public Health Relevance

Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease, is a devastating disease in which progressive degeneration of motor neurons leads to muscle weakness and fatal paralysis. It is increasingly recognized to have clinical and pathological overlaps with frontotemporal dementia (FTD). Mutation in the C9orf72 gene has recently been identified to be the most frequent genetic cause of the two diseases. The proposed work will use multiple modern molecular approaches to determine disease mechanisms and identify targets for therapeutic interventions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Career Transition Award (K99)
Project #
1K99NS091538-01
Application #
8869891
Study Section
NST-2 Subcommittee (NST)
Program Officer
Gubitz, Amelie
Project Start
2015-04-01
Project End
2017-03-31
Budget Start
2015-04-01
Budget End
2016-03-31
Support Year
1
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Ludwig Institute for Cancer Research Ltd
Department
Type
DUNS #
627922248
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Ditsworth, Dara; Maldonado, Marcus; McAlonis-Downes, Melissa et al. (2017) Mutant TDP-43 within motor neurons drives disease onset but not progression in amyotrophic lateral sclerosis. Acta Neuropathol 133:907-922
Jiang, Jie; Zhu, Qiang; Gendron, Tania F et al. (2016) Gain of Toxicity from ALS/FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs. Neuron 90:535-50
Jovi?i?, Ana; Mertens, Jerome; Boeynaems, Steven et al. (2015) Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat Neurosci 18:1226-9
Sun, Shuying; Sun, Ying; Ling, Shuo-Chien et al. (2015) Translational profiling identifies a cascade of damage initiated in motor neurons and spreading to glia in mutant SOD1-mediated ALS. Proc Natl Acad Sci U S A 112:E6993-7002