Diwas Gautam, a sophomore neuroscience major at Duke University, analyzed and validated Translating Ribosome Affinity Purification (TRAP) data to identify a specific gene involved in pathways that lead to Huntington’s disease-like phenotypes.
Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by CAG trinucleotide repeat expansion in the Huntingtin gene (HTT). My summer at the Broad Institute was challenging but very enriching. My mentor pushed me to ask thoughtful scientific questions as well as develop the proper lab techniques to become a more independent and efficient young scientist. This experience has helped me gain the confidence to say that ‘I, too, can become a scientist.’The number of repeats in HTT is highly correlated with the age of symptom onset, and transcription dysregulation is one of the main phenotypes observed in patients and disease models. Striatal neurons, specifically Drd2-expressing neurons, are the most vulnerable to mutant HTT.
To understand how changes in gene expression progress in Drd2-expressing neurons as we increase CAG repeat, we carried out translating ribosome affinity purification (TRAP) from 3-month old HD knock-in-model mice with increasing CAG lengths of Q20,Q50, Q111, and Q170. TRAP allowed us to specifically identify translating mRNAs in a cell-type-specific manner with high sensitivity.
When comparing each dataset to the Q20 allele, we identified at least 675 mRNAs that were dysregulated, and 86 mRNAs were identified as differentially represented in all datasets. Pathway analysis identified synaptic genes as being the most affected at this 3-month time point. These data suggest that, as observed by others, synaptic changes occur early in HD and are driven by multiple proteins. Aside from synaptic genes, we identified a specific gene: Polo-like kinase 1 (Plk1). Plk1 is widely studied in cancer cells and we speculated it to be involved in multiple pathways leading to HD like phenotypes such as DNA damage response, protein aggregation, and cell death. Upon validating the role of this gene in cell culture using the knock-in striatal HdhQ111 and Q7 cell lines, we looked further into the pathways it’s involved in by artificially inhibiting Plk1. Our results suggest that inhibition of Plk1 in wild-type Q7 cells increases DNA damage. This finding tells us that Plk1 is playing a role in this pathway, but we would like to further study exactly how in the future by changing Plk1’s expression in wild type and mutant HTT cells.
Understanding the expression pattern and specific role of this in HD has the potential to open new directions for developing novel therapeutic methods.
Project: Identifying early markers of Huntington disease using cell type-specific translating ribosome affinity purification (TRAP)
Mentos: Martine Therrien, Picower Institute for Learning and Memory