Nucleotide repeat expansions underlie a heterogeneous groups of neurological and neuromuscular disorders with various disease mechanisms depending on the gene function, structure and location of the expansion. Hexanucleotide repeat expansion in the C9orf72 gene is the most frequent inherited cause of both ALS and FTD. A leading hypothesis for disease mechanism is gain of toxicity from the expanded repeats, which are transcribed in both sense and antisense directions and give rise to distinct sets of intranuclear RNA foci. This could sequester certain RNA-binding proteins and lead to their loss of function. An alternative but not mutually exclusive mechanism is the aberrant accumulation of dipeptide repeat proteins produced by repeat-associated non-ATG (RAN) translation in all six reading frames (poly-GA, poly-GR, poly-PA, poly-PR and poly-PG) of both strands. A third hypothesis is the expansions cause gene silencing and lead to haploid insufficiency of C9orf72 protein. Till today, there is no established evidence supporting what is the main factor driving the disease pathogenesis. Our research goal is to use biochemical, proteomic, genomic and screening approaches to decipher the various mechanisms of repeat expansion mediated toxicity and advance therapeutic target development, especially by applying RNA Biology knowledge and technologies.
- Determine the molecular mechanisms of RAN translation
Repeat expansions do not follow the canonical rules of ATG-dependent translation initiation and can generate a series of aberrant repeat proteins in multiple reading frames. This phenomenon has been identified in several repeat expansion disorders, including myotonic dystrophy (CAG repeats), spinocerebellar ataxia type 8 (CAG repeats), Fragile X-associated tremor/ataxia syndrome (CGG repeats), Huntington’s disease (CAG repeats), and now C9orf72-ALS/FTD. Despite the evidence of dipeptide accumulation in patients and their potential toxicity on neurons, the mechanisms of how RAN translation occurs and is regulated remain largely unknown. Identification of specific modifiers of RAN translation will provide tools to distinguish the RNA- versus dipeptides-mediated toxicity and develop drug targets to reduce dipeptide production as a therapeutic strategy. Understanding the molecular mechanisms of RAN translation will also address an interesting basic biological question of normal non-canonical translation process that might have physiological functions in other settings. We aim to use multiple high-throughput screening approaches to identify genetic regulators and small molecule inhibitors of RAN translation. We are also interested to explore what stress and aging factors could influence this process therefore contribute to this late onset neurodegenerative disease.
- Develop novel programmable RNA-binding protein system to target RNA in vivo
RNAs undergo complicated post-transcriptional processing to carry on proper function, such as splicing, polyadenylation, nuclear export, subcellular trafficking, turnover, and translation. These steps are regulated by RNA-binding proteins (RBPs) and dysfunctions of RNA metabolism causes various human diseases. In vivo approaches to target the processing of endogenous RNA will not only provide new research tools to understand basic biology questions and disease mechanisms but also open new opportunities for therapy development. We are highly interested to adapt and modify the novel gene-targeting techniques to visualize and manipulate the stability, structure, trafficking, or translation of endogenous C9orf72 repeat-containing RNAs to better understand the dynamics of the RNA repeats and their toxic properties underlying the neurodegeneration process.
- Determine the cell type-specific and age-dependent RNA processing alterations induced by ALS-causative mutations in RNA-binding proteins and C9orf72 repeat expansion
Two key questions in understanding the pathogenic mechanisms of neurodegeneration diseases are what causes the selective degeneration of certain neuronal types from a widely expressed mutant gene and what genetic regulators of aging influence the late-onset disease. Neurons have extremely complex RNA metabolism and highly specialized RNA processing pathways, probably due to their highly complex morphologies and functions. We will couple high-throughput RNA sequencing techniques with the translating ribosome affinity purification (TRAP) methodology to understand normal RNA processing pathways and RBP functions in specific cell types, and determine how they are altered to induce damage specifically in vulnerable neurons and the contribution from neighboring glia cells during disease course by ALS-causative mutant genes.
- ALS-causative mutations in FUS/TLS confer both gain and loss of RNA-processing functions by altered association with SMN and U1-snRN
Mutations in FUS/TLS account for about 4% of inherited ALS and neuronal inclusion/aggregate of FUS/TLS is the pathological marker for 10% of FTD cases. FUS/TLS is an RNA-binding protein that is predominantly nuclear but also shuttles between nucleus and cytoplasm. Using quantitative mass spectrometry (SILAC), we discovered unbalanced protein interaction with mutant FUS/TLS in multiple steps of RNA processing. Combining with global analysis of RNA splicing, we found both loss of function of mutant FUS/TLS by decreased interaction with U1 snRNP (recognize 5’ splice site as a core component of spliceosome) and gain of toxicity by disrupting SMN function on snRNP assembly and Gem formation, a potentially convergent disease mechanism of RNA metabolism defects in ALS and SMA, two most prominent motor neuron degenerative diseases in adults and children respectively. (Nature Communications 2015)
- Translational profiling identifies a cascade of damage initiated in motor neurons and spreading to glia in mutant SOD1-mediated ALS.
Ubiquitous expression of ALS-causing mutations in superoxide dismutase 1 (SOD1) provoke non-cell autonomous, fatal paralytic disease in mice. To determine how mutant SOD1 damages different cell types, we applied the targeted Translating Ribosome Affinity Purification (TRAP) methodology combined with high-throughput sequencing to identify cell type-specific and age-dependent changes. We identified a feature of selective vulnerability of motor neurons to be expression of a remarkably low level of endoplasmic reticulum (ER) chaperones, with mutant SOD1 driving changes earliest in motor neurons at disease onset and later in oligodendrocytes after disease initiation. This suggests a temporal cascade of cell type selective damage to motor neurons and oligodendrocytes that is essential to disease initiation and propagation. (PNAS 2015)
- Mechanisms of C9orf72 hexanucleotide repeat expansion caused neurodegeneration in ALS and FTD
With the discovery that the most frequent inherited cause of both ALS and FTD is hexanucleotide expansion in the C9orf72 gene, a leading hypothesis for disease mechanism is RNA-mediated toxicity from the expanded repeats. We discovered that the repeats are transcribed in both sense and antisense directions, which give rise to distinct sets of intranuclear RNA foci and dipeptide repeat proteins produced by RAN translation in all six reading frames. (PNAS 2013) We have also established a trans-differentiation method to directly convert human skin fibroblasts into neurons to define the disease linked RNA signature and decipher the toxic contribution from sense versus antisense transcripts using antisense oligonucleotide (ASO) to reduce repeat-containing RNA from either strand.