Blockages in nerve-cell protein ‘factory’ implicated in neurodegenerative disease

To make an arm or a leg move, neurons – up to one meter in length – are required to send the brain’s signal to the limb through cell extensions called axons. Individuals with early- onset HSP begin to lose signaling to their legs during infancy and are often never able to walk. Advances in genome sequencing have linked more than 70 genes to HSP development, but their individual contributions to the disease state have never been clear.

“The main approach people have used in the past is to overexpress a mutant protein in a generic immortalized cell line, and then see the impact on cellular physiology. We took a different strategy by first engineering the mutation into human stem cells and then differentiating those cells into human neurons,” said Anjon Audhya, PhD, professor of biomolecular chemistry and senior author on the study. “The major implication of this work is that we found a result you could never identify using a standard cell line.”

Audhya and his team first used CRISPR-mediated gene editing in induced pluripotent stem cells (iPSCs) to introduce a TFG mutation found in hereditary spastic paraplegia patients. Then, they differentiated the stem cells to develop into cortical neurons, the same type of neurons affected in these patients.

By tracking TFG protein in normal compared to mutant cells, they found that both types of cells produced the same amount of TFG. The mutant TFG, however, failed to accumulate normally at one of the cell’s major protein-processing centers, known as the endoplasmic reticulum, or ER. This defect impaired protein trafficking from the ER, elevating a stress response in the neurons. When the researchers performed a cell “stress test” of sorts, the mutant cells showed elevated levels of ER stress.

“If a cell experiences constitutive insult to the ER, where they cannot secrete proteins efficiently, then that stress over months and years can lead to neuronal death,” Audhya said. “And we showed that cells harboring mutations in TFG are specifically impaired in this secretory pathway.”

Audhya and his team further examined axon outgrowth and morphology in the neurons. They found that while axon length was unaffected, the axons between cells failed to connect with each other in neurons expressing mutated TFG. An additional experiment showed that an adhesion protein required to bundle axons together was present at high levels on the surface of normal neurons, but dramatically reduced from the surface of mutant axons.

“Nerve conduction speed depends on the bundling of these axons, and if it’s reduced, then that translates into an inability to walk normally,” Audhya said. “And it could be that these other genes implicated in HSP cause other molecules not to be secreted appropriately by also affecting endoplasmic reticulum function.”

By identifying cell characteristics of the TFG mutant neurons, Audhya said his team will next screen for new drug therapies to treat the disease by monitoring how those potential drugs affect stress levels and axon bundling. Moreover, the commonalities between HSP and other neurodegenerative diseases such as ALS or Parkinson’s may now allow researchers to better screen for therapies for those diseases as well.

“People in the field broadly think that accumulated stress in the ER is an underlying cause of many neurodegenerative diseases,” Audhya said. “However, many of the clinical trials using drugs to target this stress haven’t been very successful, mainly because of the limitations of currently available small molecules. And that is one of the bases for why we want to go after small-molecule screening: to identify better drug candidates for many diseases in which constitutive ER stress is implicated.”