ALS, a rare but devastating disorder also known as Lou Gehrig’s disease, attacks cells in the nervous system, causing progressive muscle weakness as neurons degrade over time. There is no cure. People with ALS eventually lose their strength and the ability to move their arms, legs and body. About a third of those with ALS also develop frontotemporal dementia (FTD), a destruction of neurons in the brain that causes profound personality changes and disabilities. The two diseases are similar in both pathology and genetics. FTD tends to strike people before Alzheimer’s disease, the most common type of dementia, occurs. Researchers have long known that for most people with ALS and FTD, deposits of a protein called TDP-43 build up in nerve cells. Accumulation of TDP-43 may be responsible for nerve cell death in ALS and FTD. Understanding exactly how and why this happens is the focus of Michigan Medicine Sami Barmada, MD, PhD, an assistant professor of neurology and his research team.
Their most recent preclinical findings, published at Cell Reports, locate a structure within the TDP-43 protein that is critical to this protein’s function and its ability to cause nerve cell death. In the process, they discovered a potential clue to stopping the destruction of nerve cells. Dr. Barmada said: “RNA’s main function is to translate the molecular instructions from DNA to build proteins. For RNA to be translated into proteins, it must be processed properly and must last long enough to undergo translation. TDP-43 helps regulate both RNA processing and stability. By manipulating the protein structure, we determined that RNA binding is critical for maintaining TDP-43 stability, function and toxicity in disease models. RNA is destabilized: Most affected RNAs have been involved in energy and protein production, two pathways that nerve cells need to survive, and we found an identical pattern in cells from ALS patients, suggesting that TDP-43 could to be responsible”.
So how does the accumulation of TDP-43 cause nerve cells to die? Suspecting that it involves TDP-43’s ability to bind RNA, Barmada and colleagues focused on manipulating this protein’s function. Since protein structure is critical to function, they wondered if they could alter the function of TDP-43 by altering its structure. By introducing specific mutations, they disrupted an interaction between two parts of the protein required for RNA binding, creating versions of TDP-43 that were unable to bind RNA. Unexpectedly, they found that when TDP-43 can’t bind RNA it is rapidly degraded, leading them to believe that these versions of TDP-43 would not be as lethal to nerve cells. To determine whether these engineered versions of TDP-43 are toxic to nerve cells, Barmada’s team used a method called automated microscopy. Here, thousands of cultured nerve cells are visualized over time using a fully program-controlled microscope. Additional programs analyze the data, determine when each cell dies, and compare different conditions using methods obtained from human clinical trials.
Dr. Barmada explained: “This is like a clinical trial in a dish, measuring the fate of each nerve cell as if it were a person. We saw that when we disrupted the structure, it destabilized the protein dramatically. We know in disease that if there’s too much TDP-43, the cells die, if the excess TDP-43 is degraded, like here, the cells are saved.” To further support their conclusions, the investigators collaborated with Asim Beg, PhD, Department of Pharmacology, to create a worm model of TDP-43 with the same structural change using CRISPR engineering. Worms expressing these versions of TDP-43 were identical to worms without any TDP-43, suggesting that the targeted structure mutations are essential for TDP-43 toxicity and function. Taken together, the results show that modifying the TDP-43 structure eliminates its ability to bind RNA and cause nerve cell death in ALS and FTD models. Targeting this facility opens up the potential to explore new therapies for ALS and FTD.
But there are the results of another research group, that of the St. Jude Children Research Hospital, which have “cracked” another related molecular mechanism. Mutations in the C9orf72 gene make it the most common genetic cause of ALS and FTD. The mutation leads to a dramatic increase in the number of short repetitive DNA sequences and leads to the formation of abnormal repetitive proteins of variable length. These proteins are referred to as dipeptide repeats (DPRs). Two of the formed DPRs contain the amino acid arginine and are particularly toxic to neurons. Until now, key details about the molecular mechanisms involved were uncertain. Dr. Richard Kriwacki, PhD, a member of the St. Jude Department of Structural Biology, and his team identified the nucleophosmin protein as a site of DPR toxicity, also demonstrating that this toxicity is length-dependent. Typically, a segment of the C9orf72 gene is repeated 20 to 30 times or less. However, those with ALS and FTD have hundreds or even thousands of repeats, which then cause DPR to form.
Previous research by Kriwacki and others reported that toxic DPRs disrupt the assembly and function of the nucleolus, the largest non-membrane organelle in cells. This study highlights how they disrupt nucleolar assembly. The study also shows that longer DPRs are dramatically more toxic to cells. The nucleolus resides in the nucleus and is where the cells’ protein factories (called ribosomes) are assembled. Membraneless organelles such as the nucleolus rely on a process called liquid-liquid phase separation to form and give cells flexibility to respond to changing conditions. The same process explains why oil forms droplets in water. Kriwacki and his colleagues showed that toxic DPRs disrupt cellular function by binding tightly to key regions of nucleophosmin, displacing other binding partners that help maintain nucleolus and ribosome assembly. The higher the concentration of toxic DPRs, the faster the nucleolus is altered.
Going forward, the researchers believe that DPR duration may have prognostic value for people diagnosed with ALS.
- By Dr. Gianfrancesco Cormaci, PhD, specialist in Clinical Biochemistry.
Scientific publications
White MR, Mitrea DM et al., Kriwacki RW. Mol Cell. 2019 Apr 3.
Yamamoto I et al. Yamaguchi M. Sci Rep. 2018 Jul 26; 8(1):11291.
Sharkey L et al. Proc Natl Acad Sci USA 2018; 115(44):10495-504 .
Weskamp K, Barmada SJ. Brain Res. 2018 Aug; 1693(Pt A):67-74.
Archbold HC et al., Barmada SJ. Sci Rep. 2018 Mar 15; 8(1):4606.