RNA splicing problems start early in nerve cell’s life, study indicates

Troubled process of protein production seen to precede Huntington's onset

Marisa Wexler, MS avatar

by Marisa Wexler, MS |

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An illustration of nerve cells and their axons.

Alternative splicing, a molecular process that’s crucial for genes being “read” to produce proteins, is dysregulated in Huntington’s disease from the early stages of neuronal development, a study indicates.

Notably, splicing changes were found to be dependent on the length of CAG repeats in the HTT gene — the cause of Huntington’s.

The study, “Widespread dysregulation of mRNA splicing implicates RNA processing in the development and progression of Huntington’s disease,” was published in eBioMedicine.

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HTT, other genes use alternative splicing to produce versions of a protein

Huntington’s is caused by excessive repeats of three nucleotides, or DNA’s building blocks — C, A, and G — in the HTT gene, driving the production of a mutant HTT protein (mHTT) that forms clumps and leads to nerve cell death.

A healthy HTT gene contains 10 to 35 CAG repeats. People with a higher number of repeats usually develop Huntington’s. The length of CAG repeats in the HTT gene has been shown to determine the age at which Huntington’s motor symptoms appear, with longer repeat expansions leading to earlier onset.

How mHTT drives the development of Huntington’s, however, remains poorly understood.

HTT and many other genes can generate different versions of a protein through a process called alternative splicing.

Much like in a recipe, adding or removing certain key ingredients — in this case, pieces of genetic information — from the gene’s messenger RNA (mRNA) can change the final protein. mRNA is the molecule derived from DNA that guides protein production.

Alternative splicing “is a crucial process in [nerve cell formation], brain development, and neuronal function, where neuron-specific splicing factors and RNA-binding proteins act together to regulate [alterative splicing] in genes associated with neuronal functions,” the researchers wrote.

Previous research showed that mHTT RNA and protein can trap proteins involved in RNA processing, “suggesting that splicing dysregulation partially contributes to HD [Huntington’s disease],” the researchers wrote.

Abnormal alternative splicing also has been reported in brain cells of people with Huntington’s, but these findings mainly come from deceased patients and represent changes relatively late in the disease process.

Alternative splicing events tied to CAG length in stem cells, mature neurons

To investigate alternative splicing changes in early Huntington’s, a team of researchers in Singapore and Canada conducted comprehensive analyses in an established cellular model of Huntington’s.

They analyzed three types of cells in this model: embryonic stem cells, which are made early in fetal development; neural precursor cells, which are more mature stem cells on a path to grow into nerve cells; and mature nerve cells, or neurons.

All these cell types had three genetic versions: one containing a normal CAG repeat number, one with 45 repeats that are associated with adult-onset Huntington’s, and another with 81 CAG repeats and associated with juvenile onset.

Through a battery of tests, the researchers identified hundreds to thousands of alternative splicing events associated with CAG length in each of the three cell types. Only 35 genes — about 1% of all differentially spliced genes — were found to be differentially spliced in all three types of cells relative to their healthy counterparts.

This suggests that excessive CAG repeats have different effects on RNA splicing in cells at different stages of development, the scientists noted.

Splicing changes at the mRNA level also were seen to lead to changes in the types of proteins being produced in these different types of cells.

Further analyses showed that many of the differentially spliced genes play important roles in neuronal development and function, mRNA splicing, and epigenetics — heritable changes in gene activity that do not involve DNA mutations.

‘Feedback loops’ of dysregulated splicing may underlie Huntington’s

According to the scientists, these findings suggest that there may be a feedback loop in Huntington’s, where early splicing problems lead to dysregulation of splicing-related genes, which in turn causes more splicing problems.

“We identified several molecular processes that are enriched in [alterative splicing] events and may represent feedback loops of RNA processing dysregulation,” the scientists wrote. “Although these molecular processes have been previously observed to be disrupted in Huntington’s disease, this study highlights mHTT-driven splicing dysregulation as a potential major contributing factor to multiple HD [underlying] mechanisms.”

Many of the alternative splicing events found in the cell models also were detectable in the brains of deceased Huntington’s patients and in mouse models, particularly in part of the brain called the striatum that is known to be deeply impacted in Huntington’s.

Specifically, about 13.4% of all differentially spliced genes in patients’ striatum showed the same alternative splicing event in at least one of the three cell maturation stages studied.

“Combined, these results indicate that dysregulation of splicing occurs at early stages, well before the onset of symptoms, and that there may be continued alterations to a subset of genes through to the end stages of the disease,” the researchers wrote.

“By disrupting biological processes crucial to early development of striatal neurons and other implicated cell types, mHTT could impair cell survival and function eventually leading to selective vulnerability and cell death,” they added.

The scientists stressed that, while these cell studies provided important data, additional work is needed to fully understand the role of splicing dysregulation in Huntington’s. To help support further research, the scientists have made their data available online for other researchers to use.