Phosphatase Inhibitor Promotes Protein Folding, Helps ALS Mice

Scientists have modified a medicine for high blood pressure into one that might tame misfolded protein diseases, they report in the April 10 Science. The new molecule, dubbed Sephin1, countered the effects of aggregating proteins in mouse models of amyotrophic lateral sclerosis and Charcot-Marie-Tooth disease. It might do so for other neurodegenerative disorders, speculated senior author Anne Bertolotti of the Medical Research Council Laboratory of Molecular Biology in Cambridge, England. Moreover, Sephin1 did this by selectively inhibiting dephosphorylation of a translation factor, something thought to be almost impossible because phosphatases have so many substrates.

Change for the Better.

Removing a chlorine from guanabenz generated Sephin1, which caused fewer side effects in mice. [Image courtesy of Science/AAAS.]

Protein phosphatase 1C (PP1c) dephosphorylates the eukaryotic translation initiation factor 2α subunit (eIF2α), among a plethora of other protein targets. As part of the unfolded protein response (UPR) in the endoplasmic reticulum, phosphorylated eIF2α temporarily shuts down translation, giving chaperones a chance to catch up on protein folding. The UPR often kicks in to counteract accumulation of misfolded proteins in neurodegenerative disease. Dephosphorylation of eIF2α turns off the UPR and allows normal translation to resume. PP1c removes the regulatory phosphate in conjunction with one of two accessory subunits that go by the complex monikers protein phosphatase 1, regulatory subunits 15A and 15B (PPP1R15A and PPP1R15B, or R15A and R15B for short). R15B is always around, but the cell only makes R15A during times of ER stress. By promoting eIF2α dephosphorylation, these subunits ensure the cell does not go too long without making proteins, which would be lethal.

In 2011, Bertolotti reported that the anti-hypertensive drug guanabenz binds to R15A, but not R15B, and inhibits eIF2α dephosphorylation. Guanabenz protected cells from misfolded proteins and tunicamycin, a drug that induces ER stress (Tsaytler et al., 2011). Other scientists have reported that guanabenz treatment slows down disease in mouse models of ALS (Wang et al., 2014; Jiang et al., 2014), multiple sclerosis (Way et al., 2015), and prion disease (Tribouillard-Tanvier et al., 2008).

Unfortunately, guanabenz has side effects such as drowsiness and lethargy, and even coma after overdose (Hall et al., 1985). This is due to activation of the α2-adrenergic receptor, causing a potent reduction in blood pressure. Guanabenz was designed and approved as an anti-hypertensive in 1982, but it fell out of use when better medications came along. Bertolotti and first author Indarjit Das sought to eliminate α2 binding, while preserving activity in the UPR.

From a panel of guanabenz derivatives, the authors zeroed in on one missing a chlorine (see image above). They christened it Sephin1, for “selective inhibitor of a holophosphatase.” Like guanabenz, Sephin1 only bound and inhibited R15A, not R15B. Sephin1 treatment rescued cells exposed to tunicamycin, but not cells missing R15A, confirming it worked via that subunit.

Maintaining this specificity for R15A was crucial, Bertolotti said. By subduing R15A, Sephin1 prolongs the phosphorylation of eIF2α in times of stress, but eventually R15B kicks in and dephosphorylates eIF2α, allowing translation to resume. In addition, Sephin1 did not interact with the α2-adrenergic receptor.

Those cell-culture experiments indicated that Sephin1 might avoid the undesirable side effects of guanabenz, which Das next tested by treating healthy 1-month-old mice with oral Sephin1, twice a day for a month. While guanabenz-treated mice toppled off a rotating rod due to lethargy, Sephin1-treated mice had no balance problems. The treated young mice gained weight as fast as untreated controls. Since eIF-2α plays a role in memory (see Apr 2015 news), Das also tested the mice for their ability to find a hidden platform in a water maze, and to associate a sound with a foot shock in fear-cue experiments. Treated mice had no problems with either. Bertolotti said the researchers have continued the Sephin1 treatment for as long as six months. “For those six months, we see no side effects whatsoever,” she said.

What about treating disorders of protein misfolding? The researchers tested Sephin1 in a mutant SOD1 mouse model of fast-progressing ALS. They selected this model, Bertolotti said, because there was already evidence from genetic studies that knocking down R15A protected a slower-progressing version of mSOD1 mice (Wang et al., 2014). Das treated the animals with daily Sephin1 or control vehicle solution for seven weeks, beginning at when they were 4 weeks old. The Sephin1 mice grew faster and balanced better on the rotating rod. “The SOD1 mice treated with Sephin1 behave almost like normal,” Bertolotti wrote in an email to Alzforum.

When examined a month later, the Sephin1-treated mice contained more motor neurons in the lumbar spinal cord than the untreated mSOD1 controls, again almost matching wild-type controls. The Sephin1 treatment greatly reduced the amount of insoluble, presumably misfolded, SOD1 in spinal cord extracts, though they had normal amounts of SOD1 protein overall. The Sephin1 mice also expressed fewer markers of ER stress. “It looks like we have improved [SOD1] folding ,” Bertolotti said.

Raymond Roos of the University of Chicago, who was not involved in the work, said he would have liked to see how Sephin1 affected survival. Given the effects on weight, motor neuron number, and SOD1 solubility, he said, “I would be surprised if there was not a significant survival effect.” Bertolotti told Alzforum that those studies, which require more animals, are now in the works.

Sephin1 also helps mice that mimic a form of Charcot-Marie-Tooth neuropathy. Might it have broader effects against misfolding of neurodegeneration-related proteins, such as Aβ or tau? “Since they show this striking protective effect, it is worth trying in other neurodegenerative diseases,” commented Yang Hu of Temple University School of Medicine in Philadelphia, who did not participate in the study. However, he cautioned that Sephin1 is far from a shoo-in. Researchers have not confirmed that ER stress due to protein aggregates is a generalized cause of neurodegeneration, he pointed out. Bertolotti suspects that Sephin1 or a similar molecule would benefit more diseases than just ALS and CMT, but probably not all neurodegenerative conditions.

Hu and Roos were not entirely convinced that prolonging eIF2α phosphorylation was the only mechanism at work, either. Roos pointed out that R15A also dampens inflammation (Mesman et al., 2014). Bertolotti said R15A probably affects inflammation because of its influence on eIF2α. She is convinced that Sephin1 has no other mechanism of action because it mimics R15A knockout.

Jeroen Hoozemans and Wiep Scheper of VU University Medical Center in Amsterdam added that some studies suggest that eIF2α phosphorylation would be detrimental, not beneficial. Inhibition of PERK, the kinase that phosphorylates and activates eIF2α, protected mice from prion disease (see Oct 2013 news). “The control of eIF2α phosphorylation and dephosphorylation is very complex,” they wrote in an email to Alzforum. “For translation to human neurodegeneration, better understanding of how eIF2α is affected and controlled in different neurodegenerative diseases is required.”

Reference:

Das I, Krzyzosiak A, Schneider K, Wrabetz L, D’Antonio M, Barry N, Sigurdardottir A, Bertolotti A. Preventing proteostasis diseases by selective inhibition of a phosphatase regulatory subunit. Science.2015 Apr 10;348(6231):239-42. PubMed.


To view commentaries, primary articles and linked stories, go to the original posting on Alzforum.org here.

Copyright © 1996–2017 Biomedical Research Forum, LLC. All Rights Reserved.

disease-als topic-preclinical
Share this:
Facebooktwittergoogle_plusmailFacebooktwittergoogle_plusmail