Though the symptoms of neurodegenerative disorders vary, the molecular mechanisms are often eerily similar: misfolded proteins activate the unfolded protein response. Stress granules that sequester RNAs and RNA-binding proteins may form. Prolonged silencing of translation of many proteins may occur and lead to neuronal loss.
A crucial nexus in this stress granule assembly and translational block is phosphorylation of the signaling molecule eIF2α, but it’s been tricky to identify safe, soluble medications that would prevent its effects. Now, a research team led by University of Cambridge’s Giovanna Mallucci in England identifies drugs that work downstream of eIF2α phosphorylation, including the antidepressant trazodone, that could alleviate the translational block in multiple forms of neurodegeneration. What’s more, these drugs protected neurons and prevented symptoms in mouse models of frontotemporal dementia (FTD) and prion infection, the researchers reported on April 19 in Brain.
Could it work for ALS? “It’s a pathway that should be examined,” said the University of Pennsylvania’s Nancy Bonini in Philadelphia, who did not participate in the study.
From 1,040 to Two
NNancy Bonini suspected in 2013 that the phosphorylation of eIF2α, which triggers stress granule formation, may play a role in ALS. The translation regulator popped up in a genetic screen in yeast for proteins that mediate TDP-43 toxicity. And, inhibition of the eIF2α kinase known as PERK (protein kinase RNA-like endoplasmic reticulum kinase) improved the mobility of a Drosophila model of ALS, and also protected cultured rat cortical neurons from cytoplasmic TDP-43-mediated toxicity. The results suggested that blocking PERK could be a potential therapeutic approach for the disease (see Dec 2013 news; Kim et al., 2013).
Across the pond, Giovanna Mallucci and colleagues knew the unfolded protein response (UPR),is overactivated in Alzheimer’s and other neurodegenerative disorders, presumably leading to PERK-mediated eIF2α phosphorylation and potentially a shut down of general protein synthesis. So, her team turned to a similar treatment strategy as Bonini, to restore translation in hopes of treating these diseases. Mallucci’s team reported beginning in 2013 that blocking PERK restored translation and prevented neuronal loss in the brain of mouse models of prion infection and Tau-linked FTD (Moreno et al., 2013; Radford et al., 2015).
However, the current drugs aren’t suitable for therapeutic use. GSK2606414, which Bonini and Mallucci’s team used in their studies, is so effective in blocking PERK that it’s toxic to the pancreas. And another, ISRIB, which Mallucci’s team later turned to (Halliday et al., 2015), is insoluble and therefore unsuitable to be administered to people.
To look for alternative therapeutic approaches, first author Mark Halliday and colleagues mined a library of 1,040 compounds from the National Institute of Neurological Disorders and Stroke, of which 75% are approved by the Food and Drug Administration. They started in nematodes, in which UPR-activating tunicamycin stalls larval development, and discovered 20 UPR down-regulators that allowed development to continue.
Next, the researchers wanted to narrow their list to drugs that specifically relieve the PERK-mediated translational block, not other branches of the UPR. Plus, they wanted compounds that weren’t too effective, like toxic GSK2606414. Therefore, they screened stressed CHO cells for drugs that partially restored PERK-activated translation of CHOP. So doing, they shaved the pool down to five molecules.
Of these, they focused on trazodone and dibenzoylmethane (DBM). The other three hits were unsuitable, due to the inability to cross the blood-brain barrier, unwanted side effects, or toxicity to the mouse models the researchers wanted to work with next.
They evaluated both trazodone and DBM in mouse models of two neurodegenerative diseases. In a model of prion infection, trazodone or DBM prevented the appearance of key clinical features of the disease, including motor signs such as limb-dragging, in most mice tested.
In a mouse model of Tau-based FTD, five-month-old mice showed signs of improved memory by being able to identify novel objects, compared to untreated controls. Protein synthesis reached at least nearly 75% of normal rates in either model treated with either medication.
Both medications were well-tolerated, and showed no signs of toxicity to the pancreas, suggesting they could be safely administered. The researchers are now planning a small pilot trial of trazodone in people with Alzheimer’s disease, Halliday said.
What about ALS?
“One of the most exciting features of our approach is that it is predicted to be effective against any neurodegenerative disorder in which the unfolded protein response is overactivated,” according to Halliday.
ALS seems to qualify: Post-mortem analyses of the spinal cords of people with sporadic disease indicate the UPR is upregulated, eIF2α is highly phosphorylated, and PERK and CHOP are also upregulated (reviewed in Matus et al., 2013). But the situation is complicated. ALS does not have one single cause. It can be associated with mutations in the gene SOD1 or, more commonly, a buildup of toxic TDP-43 protein in the cytoplasm. And, the impact of modulating the UPR depends on when the intervention is administered and on the nature of the disease (Vieira et al., 2015).
Roy Parker of the University of Colorado in Boulder said it’s possible that restoring general protein synthesis with trazodone or a similar medication could be beneficial in ALS. But it depends on whether translational inhibition plays an important role in ALS, and at what stage it’s important in the disease.
“One connection to ALS would be in [SOD1 ALS], where there’s pretty good evidence that SOD1 is misfolding and triggering an unfolded protein response which then leads to PERK activation,” he said. But, added Parker, whether modifying the eIF2α pathway is of benefit in SOD1 ALS, according to preclinical studies, remains an open question.
Treatment with guanabenz, which preserves eIF2α phosphorylation and thus the UPR, has been reported to either mitigate (Wang et al., 2014) or hasten symptoms in SOD1-G93A mice (see Aug 2015 news; Vieira et al., 2015). Treatment with salubrinal, which blocks eIF2a dephosphorylation, improved survival of SOD1-G93A mice (see Mar 2009 news; Boyce et al., 2005; Saxena et al., 2009). In other words, modulating eIF2α phosphorylation makes the disease better or worse, depending on who you ask.
Another key question, according to Parker, is when eIF2α phosphorylated and, potentially, protein synthesis shuts down: “Is it kind of at the very end, or is it the early stage?” he asked. It would be important to provide a treatment like trazodone when the PERK-eIF2α pathway is activated. Such a therapy may need to be administered very early in the disease, according to a report from the ALS Therapy Development Institute.(Vieira et al., 2015).
Therefore, whether modulation of the UPR in could be helpful in SOD1-linked ALS remains unclear. Halliday thinks trazodone would have the best chance in more common forms of ALS including sporadic disease, in which TDP-43 builds up in the cytoplasm. The sequestration of TDP-43 may lead to a block in the synthesis of at least some proteins (Coyne et al., 2014; Majumder et al., 2016). And, at increased levels, cytoplasmic TDP-43 may stick to polyribosomes –leading to a global shut down of protein synthesis suggesting that a strategy might be therapeutically beneficial (see February 2017 news; Russo et al., 2017).
A stress reliever?
Daryl Bosco of the University of Massachusetts Medical School in Worcester found Halliday’s results intriguing, and wondered how universal this treatment approach would turn out to be. It’s not known if translation inhibition plays an important role in ALS. But much attention recently has focused on stress granules, also triggered by eIF2α phosphorylation, and which also diminish translation. These granules might sequester TDP-43 in the cytoplasm and promote its aggregation in most ALS cases (Li et al., 2013).
The formation of stress granules and translational blockade are mediated by overlapping pathways, explained Halliday, suggesting that targeting one pathway would likely reduce activation of the other, too. ISRIB, in fact, not only reactivates translation but also cuts down on stress granule accumulation by triggering their disassembly (Sidrauski et al., 2015).
Stress granules are also in researchers’ direct target sights. Scientists recently reported that by knocking down a stress granule assembly regulator, ataxin-2, they could improve survival in a TDP-43 mouse model of ALS (see May 2017 news; Becker et al., 2017). “I think they’re both great approaches,” said Bonini, who was involved in early work on ataxin-2 and ALS. She and Bosco agreed that if translational repression does indeed occur in ALS model mice, trazodone or DBM would be worth trying.
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Damage control. When cellular proteins are misfolded, the cell activates an unfolded protein response kicked off by PERK, which phosphorylates eIF2a. The end result is a slowing or protein synthesis, leading to neurodegeneration. Different drugs act at different levels of the cascade. [Courtesy of Halliday et al., 2017, Brain.] Figure 2A scheme. https://academic.oup.com/brain/article-lookup/doi/10.1093/brain/awx074
Chop To It. Scientists identified potential drugs that restore general translation by screening for those that at least partially block PERK-dependent translation of the protein CHOP. [Courtesy of Halliday et al., 2017, Brain.]