Age-related Loss in Cell’s Control Over Protein Levels Tied to Clumping

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by Steve Bryson, PhD |

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The loss of chemical modifications in the regulatory sequence of the TARDBP gene, called DNA methylation, seems to contribute to increased levels and clumping of the TDP-43 protein in the motor cortex of people with amyotrophic lateral sclerosis (ALS), a study found.

As methylation in this gene — which prevents cellular machinery from reading this gene and making proteins from it — decreases with age, the study’s findings begin to explain why older age is a major risk factor for ALS.

Age-related demethylation of the TDP-43 autoregulatory region in the human motor cortex” was published in the journal Nature Communications Biology.

In over 95% of sporadic ALS cases, the TDP-43 protein forms abnormal clumps in motor neurons, the nerve cells that control muscle movements. This aggregation leads to the loss of motor neurons in a brain region known as the motor cortex, and to disease symptoms.

Aging is a risk factor for the loss of motor neurons and ALS development, and researchers think there may be a link between age and higher levels of TDP-43. However, the exact mechanism linking this protein, aging, and the impact on motor neurons is still unknown.

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The amount of TDP-43 is strictly controlled by how its messenger RNA — mRNA, the molecule that carries instructions from the TARDBP gene to make the TDP-43 protein — is processed, via a mechanism called splicing.

Splicing essentially determines which parts of the gene are retained to be read by the protein-making machinery. In the case of TARDBP, if too much of the TDP-43 protein is already in the nucleus, cells change the splicing process to produce an mRNA molecule that is degraded and won’t lead to protein production, helping to keep TDP-43 levels in check.

Since aging is associated with the loss of DNA methylation — the biological process by which methyl groups are added to DNA to suppress gene activity — researchers based at the Niigata University in Japan wondered if DNA methylation of the TARDBP gene changes with age.

They also examined whether a change in methylation status could alter the splicing mechanism of TDP-43 mRNA to increase TDP-43 production in motor neurons, eventually leading to aggregation and disease.

“It’s been a big mystery why TDP-43 selectively accumulates in the motor cortex as we get older in ALS. TDP-43 accumulates when its amount increases,” Osamu Onodera, MD, PhD, director of the Brain Research Institute at Niigata University and the study’s lead author, said in a press release.

“However, TDP-43 itself is expressed in all cells, and the amount of TDP-43 in a cell is maintained at a strictly constant level through splicing,” Onodera added.

To find out, the researchers selectively demethylated the TARDBP gene to determine if it affected TDP-43 expression and to investigate the DNA methylation status in brain tissue collected post-mortem from seven ALS patients and eight individuals without brain disease serving as a control group.

“Many neurological diseases, including ALS, have ‘aging’ as a risk factor. DNA methylation modifications, which shape tissue specificity, are also affected by aging, which in turn affects splicing,” Onodera said. “This is why we focused on methylation at the regulatory site of the TDP-43 gene.”

The team demonstrated that demethylation suppressed the proper splicing of TARDBP mRNA, with a 1.85-fold increase in unspliced mRNA. In brain samples, the amount of DNA methylation in the cerebral cortex and cerebellum did not differ between ALS patients and controls.

In the brain’s motor cortex, however, samples from controls showed lesser methylation in TARDBP with older age. People in their 50s had about 80% DNA methylation, which dropped to nearly 50% in individuals in their 80s. Among those more elderly, methylation was “extremely low,” the researchers noted.

No correlation between methylation and age was evident among ALS patients. But their methylation levels were about 65% of those normally found at age 45, and similar to levels seen in people in their 60s and 70s.

Further experiments showed that TARDBP expression tended to increase when DNA was demethylated in samples from the motor cortex, but this association was only significant in control group samples.

Researchers also measured TDP-43 protein levels in motor cortex tissue. Although there was no association between the DNA methylation levels and normally folded TDP-43 protein, less methylation was linked to more aggregated TDP-43.

Finally, they investigated the relationship between TARDBP methylation in the motor cortex and the clinical features of ALS patients. A significant correlation was found between lesser methylation in the TARDBP gene and the age of ALS onset, but not with disease duration.

“Thus, the unique profile of TARDBP … DNA methylation in the motor cortex may contribute to susceptibility to TDP-43 pathology [disease] during brain aging in individuals with ALS,” the researchers wrote.

“Regulation of methylation in the regulatory region of TDP-43 may provide a new therapeutic approach for ALS,” Onodera said. “Tissue-specific, age-related methylation changes in disease-associated genes may play a role in other neurodegenerative diseases as well.”