Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - TransSplicHD (Single-Cell Transcriptomics and Spliceosome analysis to uncover new mechanisms of neuronal vulnerability to Huntington’s Disease.)

Teaser

Huntington\'s disease (HD) is a hereditary, fatal neurodegenerative disorder, mainly affecting people in mid-life. Although more than 20 years have passed since the discovery of the disease-causing genetic mutation - an expanded DNA repeat at the tip of chromosome 4 -, the...

Summary

Huntington\'s disease (HD) is a hereditary, fatal neurodegenerative disorder, mainly affecting people in mid-life. Although more than 20 years have passed since the discovery of the disease-causing genetic mutation - an expanded DNA repeat at the tip of chromosome 4 -, the challenge is still to determine which pathways are directly responsible for the pathological process. Although recent efforts aimed to decrease mutant huntingtin protein show promising results, no treatments are available to the clinic to delay or to arrest the disease progression. In order to identify molecular mechanisms driving HD, in this project we investigated how RNA processing could be altered by the HD mutation, specifically in those neurons that are more sensitive to the disease. To answer this question we resourced to the use of “HD mice”, carefully replicating the human mutation, focusing on striatal brain regions that are most vulnerable to HD. We searched for changes presenting more severe alterations with increasing severity in the HD mutation – longer triplet expansion - in a manner that totally recapitulated the human HD mutation. Particularly, through an integrative effort to combine cutting edge methods to visualize, isolate and profile single neurons and analysis of “big, bioinformatic datasets”, we aimed to characterize the molecular sensitizers that render some subpopulation of neurons susceptible to HD mutation. Thanks to the HD genetics criteria utilized – mouse model faithfully replicating the HD mutation, selection of molecular phenotypes based on for severity of the mutation – and the pioneering approach applied, we have every expectation to have identified dysregulated pathways proximal to the mutation, thus likely to more relevant for the disease pathologic process. Thus, the outcome of this project will lead to the discovery of early pathways and networks of genes altered by the mutation and ultimately responsible for neuronal cell death to be targeted or protected in search of new, effective therapeutic treatments.

Work performed

Recent findings revealed that the process of alternative splicing (AS) - crucial for the establishment of a repertoire of protein coding isoforms extremely relevant for the proper physiological characteristics of the nervous system - might be compromised in HD.
Therefore, the goal of our research was to take advantage of genetically engineered Htt knock-in (KI) mouse models bearing normal or pathological Htt CAG repeat lengths to discover alterations in transcription and RNA processing that might reflect on the levels of expression as well as the composition of a repertoire of proteins, thus contributing to HD striatal vulnerability.
To discover alterations in linear splicing, we resourced to a publicly available dataset (Langfelder P. et al.,2016, Nature Neuroscience ) analyzing full transcriptomic profiling dataset for striatum, cortex and liver at different developmental time points, identifying and quantifying the total numbers of differential AS events. Our analysis demonstrated that specifically for the striatum - most vulnerable to HD degeneration - the total number of detected, differential AS events increased significantly with increasing CAG expansion. Genes presenting high number of AS events were enriched in ‘synapse’ and ‘cell junction’ pathways. Notably not all the AS events increased with CAG-expansion and age, but only a specific subtype of AS event - EXON SKIPPING (63.8 %) - revealed to be the most affected.
We then further focused at single-cell level by analyzing CAG-driven transcriptomic differences in striatal medium-sized spiny neurons of the direct and indirect pathways. In fact, cell specific differences in the vulnerability of the striatal neurons to the HD mutation have been previously reported. Particularly, D2 dopamine receptor (Drd2) expressing neurons appear to be the most vulnerable. Thus, we tested the hypothesis that 1. single neurons in the indirect pathway (Drd2) might have a peculiar RNA signature acting as disease sensitizers and/or 2. single neurons in the direct pathway (Drd1) might express genes or RNA isoforms that could be protective against mutant huntingtin toxicity.

In conclusion, our results support the idea that the AS machinery is responding to HD mutational process by altering linear splicing events at the genome-wide level. This knowledge, already presented at international meetings and congresses and part of upcoming scientific publication, is important to uncover new biological pathways sensible to the HD mutation and potentially responsible for striatal vulnerability. On the other hand, our studies will possibly pave the way to new trials of therapeutic intervention aimed to target spliceosomal alteration.

Final results

With the TranSplicHD project we aim to offer novel insight toward the understanding of HD pathogenesis mechanisms and putative innovative treatment for degeneration by i) using of accurate experimental models, ii) producing an unbiased genome-wide analysis and iii) developing Single-Cell Transcriptomics Analysis.
To study the HD pathogenic process, we capitalized on accurate genetic KI mouse models of HD, previously generated. These lines of mice accurately reproduce normal and disease-producing human HD alleles, thus providing an ideal system for investigating the consequences of HD mutation.
We produced an unbiased genome-wide analysis of the striatal, cortical transcriptome in WT and huntingtin-mutant animals that unveiled important, global pathways altered since the early stages of the pathological process. The use of RNAseq enabled the concomitant analysis of the levels of protein coding transcripts, non-coding RNAs and the detection of aberrantly spliced, processed isoforms crucial for the understanding of neuronal vulnerability in HD
Limited information exists about the transcriptional differences among single neurons in vivo and specifically into neuronal subtypes in the striatum. Through the analysis of single-neurons we will uncover differences at transcripts and exons level of genes that might be responsible for subtype neuronal vulnerability in HD. This part of the project, still ongoing, will be particularly important for the HD field and will drive new paths to therapeutic intervention or prevention to sensibly ameliorate HD patient life expectation.

In conclusion, the results of this proposal will represent a huge progress beyond the current state of the art in HD research highlighting for the first time i) whether and how transcription and AS is altered by the presence of mutant alleles in vivo, at genome-wide level; ii) how peculiarly transcription and AS of neuronal subpopulations respond to the HD mutational process. All these information will set the stage for follow-up studies aimed to specifically understand the impact of each transcriptional or spliceosomal alteration on the mouse HD phenotypes and human disease pathology. Moreover, the knowledge of pathways and genes specifically altered in affected brain districts or, even more finely, in single-neurons of the striatum, will pave the way to new trials of therapeutic intervention aimed to target transcriptional/spliceosomal alterations through specific drugs or genetic manipulation.

Website & more info

More info: https://www.cibio.unitn.it/184/neuroepigenetics-laboratory AND https.