Patients with amyotrophic lateral sclerosis (ALS) frequently have a string of repeated DNA code in brain neurons, carrying hundreds to thousands of copies within the gene C9orf72. New research looks at what triggers these repeated sequences to eventually produce the toxic proteins that are associated with ALS, frontotemporal dementia (FTD), and other neurodegenerative diseases in puatients carrying the C9orf72 mutation, the most common cause of inherited ALS. The work finds that neuronal excitation and stress trigger the protein production in cells, and reveals that targeting this stress response with a known drug could reduce toxic protein production. Through a collaboration with co-lead investigator Davide Trotti, PhD, and co-author Piera Pasinelli, PhD, who leads the Jefferson Weinberg ALS Center, the team tested what causes the repeat DNA sequences within C9orf72 to activate and produce a toxic protein. Suspecting stressors as a possible trigger, the investigators tested a number of agents that cause neurons to turn on the stress responses. Indeed, many of these also initiated production of toxic protein.
To better understand cellular pathways that drive this phenomenon, the team examined the effect of activating a variety of unique or overlapping molecular pathways on the unconventional translation mechanism. Therefore, they employed a panel of compounds that increased cellular stress through pathways such as ER stress, oxidative stress, or excitotoxic stress. Endoplasmic stress response (ESR) was triggered with thapsigargin and tunicamycin; oxidative stress was induced with bromobenzene, paraquat, diamide and other with similar mechanism. The researchers also showed that neuronal over-excitation, similar to what happens during a seizure, also triggered the protein production. Indeed, drugs simulating neuronal excitotoxicity (glutamate, homocysteine, ecc.) had similar effects on cells. A common final mediator was shown to be calcium ions. Somehow, dependently on integrated signaling, sustained calcium currents activate a cellular stress response called integrated stress response (ISR), derived from protein unfolding (UPR) and protein synthesis shift.
This is not new, since neuroscientists have deep knowledge how intracellular calcium may drive two opposite outcomes: neuronal activity and stability at regular and transient concentrations; apoptotic cell death with higher and sustained concentrations. Using AMPA or NMDA receptor antagonists MK801 or NBQX, respectively, we demonstrated that abnormal c9orf72 protein levels could be reduced in neurons under conditions of excitotoxic stress. These results suggest that cellular influx of calcium may be a signal that stimulates the ISR and has important implications in age‐dependent development of ALS. ISR relies on the eIF2-alpha protein, a general regulator of cellular protein synthesis. Beyond this role, this protein activates the ATF-4 transcription factor, a nuclear protein involved in stress gene response. Blocking components of ISR with the drug trazodone impaired the pathological translation of the protein. Trazodone, which is approved for the treatment of depression, is known to act on parts of the integrated stress response.
Trazodone is an antidepressant in the serotonin antagonist and reuptake inhibitor class, which has additional anxiolytic and hypnotic effects. It has been shown to reduce the behavioural and psychological symptoms of dementia in Alzheimer’s disease and in FTD, but no studies have looked at the progression of neurodegeneration with trazodone treatment. A second compound identified having the same effect was dibenzoyl-methane, a natural compound found in licorice roots. Both drugs were markedly neuroprotective in two mouse models of neurodegeneration, using clinically relevant doses over a prolonged period of time, without systemic toxicity. Understading what triggers toxic proteins production helped scientists hone in on drugs that could block them in laboratory tests. But honing in on this over-arching cellular mechanism, offered the researchers a glimpse into specific methods that might block the neuron-damaging response. The researchers are now expanding their research to screen for other molecules that might work better than the drug trazodone.
The research was published this month in EMBO Molecular Medicine.
- edited by Dr. Gianfrancesco Cormaci, PhD, specialist in Cinical Biochemistry
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