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Jul 3
2018

Packard scientists close in on how small, toxic protein causes ALS

ALS Headlines, Packard Center News
Work by Packard scientists has revealed how toxic peptides produced by the C9orf72 repeat expansion interfere with the cellular stress response and the cell’s protein-synthesizing machinery.

A new mouse model of the most common genetic cause of ALS and frontotemporal dementia (FTD) has provided new insights into the molecular mechanisms of the disease. Work by Packard scientists Leonard Petrucelli, a neuroscientist at the Mayo Clinic, Aaron Gitler, a geneticist at Stanford University, and led by Yong-Jie Zhang, also a neuroscientist at Mayo, has revealed how toxic peptides produced by the C9orf72 repeat expansion interfere with the cellular stress response and the cell’s protein-synthesizing machinery. The study, published today in Nature Medicine, may provide new drug targets for ALS and FTD, the authors say.

As soon as researchers discovered the C9orf72 mutation, scientists from Packard and around the world have worked to figure out how the repeat expansion (in which six DNA bases are repeated hundreds or thousands of times in the middle of the C9orf72 gene) leads to disease. The repetitive DNA sequence confuses the machinery that transcribes DNA to RNA and then translates it into protein. This produces large amounts of abnormal mRNA and small, toxic proteins called dipeptide repeats (DPRs). Although all five different types of DPRs are toxic, some cause more damage than others. A DPR protein known as Poly(GR) is one of the most harmful. Previous studies showed that Poly(GR) harms a neuron’s ability to create energy and to shuttle important proteins and molecules between the nucleus and the cytoplasm.

Work on these mechanisms have been done in cultured cells and fruit flies, not live mammalian models that would provide more insight into what was actually happening in people with ALS and FTD. This led Zhang, Petrucelli, Gitler, and colleagues to build a mouse model of Poly(GR) toxicity. They created a virus vector to insert the Poly(GR) dipeptide attached to a green fluorescent tag into mouse neurons, which they directly injected into the brains of newborn mouse pups. As the mice aged, they began to develop behavioral and physiological signs of ALS/FTD. By six months, the mice had lost cells in their hippocampus and showed signs of cortical thinning. Behavioral changes paralleled the neurodegeneration; motor and locomotive abilities had deteriorated significantly in three- and six-month-old mice.

Microscopy studies revealed that Poly(GR) were located near ribosomes, which translate mRNA into protein. This showed that Poly(GR) could interfere with ribosomal functioning, a hypothesis supported by further experiments showing upregulation of ribosomal genes, perhaps in response to obstructions from Poly(GR). This decrease in ribosomal activity is also a sign of the cellular stress response. Other indications include the formation of stress granules, dense aggregates of RNA and protein that form and disassemble as the cell enters and exits stress. Although cells in the Poly(GR) mice formed the same number of stress granules as control mice, these stress granules did not dissolve after the stress had passed. Being in a prolonged state of stress also promotes the assembly of Poly(GR). As the protein accumulates and aggregates, however, it can also switch off the cellular processes that facilitate its own synthesis.

The researchers say that reducing levels of Poly(GR) and/or reducing its ability to interfere with ribosomes and stress granule disassembly may provide useful drug targets in future experiments.

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