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.
The paper by Indrajit Das and colleagues supports accumulating evidence that regulating the phosphorylation or dephosphorylation of eukaryotic initiation factor 2α (eIF2α) is a potential therapeutic approach for treating neurodegenerative diseases. Regulation of phosphorylation of this translation initiation factor is central to the integrated stress response (ISR) and reduces protein synthesis under conditions of cellular stress. The authors designed Sephin1, an inhibitor of eIF2α dephosphorylation based on Guanabenz, a compound that inhibits the phosphatase subunit PPP1R15B. Like its predecessor, Sephin1 is selective for the stress-induced eIF2α phosphorylation, but it lacks side effects caused by interaction with the α2-adrenergic receptor. This paper raises the debate whether for neurodegenerative diseases can be treated by blocking the phosphorylation of eIF2α through the inhibition of eIF2α kinases such as PERK, or prolonging it by blocking phosphatases as in the current study. There is now substantial evidence that the approaches stimulate or reduce protein synthesis during stress, respectively (see Oct 2013 news).
The control of eIF2α phosphorylation and dephosphorylation is very complex. It is important to increase understanding of how eIF2α is controlled in physiological conditions, but the real questions (and answers) lie in human pathology. Data is accumulating that the ISR is involved in many human neurodegenerative conditions. Although the key players of the ISR are involved in human pathology, it is unclear how. Is the ISR blocked, over-activated, or under aberrant control? Is this process similar or different among diseases? The study by Das et al. nicely addresses the potential of eIF2α as a therapeutic target for neurodegeneration and shows that therapeutic side effects can be reduced by more specific approaches. For translation to human therapy, better understanding of how eIF2α is affected and controlled in different neurodegenerative diseases is required.
Whether eIF2α phosphorylation is detrimental or beneficial is now very well understood.
A transient phosphorylation of eIF2α is beneficial, but a persistent phosphorylation of eIF2α is detrimental. This is why mammals have evolved two eIF2α phosphatases: to avoid persistent phosphorylation of eIF2αand its deleterious effects. Because Sephin1 is a selective inhibitor of R15A, but not the related R15B, it is safe and doesn't cause deleterious side effects.
This is in full agreement with genetic investigations of this pathway, carefully carried out in the past 15 years, mostly in the labs of David Ron and Randy Kaufman. Mice lacking R15A are viable and appear largely normal. Likewise, as we show, pharmacological inhibition of R15A is safe.
To illustrate this mechanism in simple terms, it might be useful to compare the sophisticated adaptive eIF2α pathway to a diet and a hunger strike. A diet will be beneficial to someone who is overweight, but a prolonged hunger strike will inevitably be fatal.
We completely agree that the elegant design of the Sephin compound explains the lack of side effects. We thought it relevant to point out to readers that inhibition of eIF2α phosphorylation via genetic deletion or pharmacological inhibition of PERK protects when persistent eIF2α phosphorylation is observed, as in mouse models of prion disease and Aβ deposition (Ma et al., 2013; Moreno et al., 2012; Moreno et al., 2013). True, such interventions have too many side effects for clinical application, but these proof-of concept studies raise hope for more subtle interventions to inhibit phospho-eIF2α-mediated translational attenuation, such as ISRIB ( Sekine et al., 2015; Sidrauski et al., 2015; and Apr 2015 news).
In contrast, the opposite (prolonged eIF2α phosphorylation) is effected by Sephin and protective in ALS and CMT models as shown in the current paper. These paradoxical observations may, for example, relate to the stage of the disease process and the level of eIF2α phosphorylation at the start of treatment.
References:
Ma T, Trinh MA, Wexler AJ, Bourbon C, Gatti E, Pierre P, Cavener DR, Klann E.
Suppression of eIF2α kinases alleviates Alzheimer's disease-related plasticity and memory deficits.
Nat Neurosci. 2013 Sep;16(9):1299-305.
PubMed.
Moreno JA, Radford H, Peretti D, Steinert JR, Verity N, Martin MG, Halliday M, Morgan J, Dinsdale D, Ortori CA, Barrett DA, Tsaytler P, Bertolotti A, Willis AE, Bushell M, Mallucci GR.
Sustained translational repression by eIF2α-P mediates prion neurodegeneration.
Nature. 2012 May 24;485(7399):507-11.
PubMed.
Moreno JA, Halliday M, Molloy C, Radford H, Verity N, Axten JM, Ortori CA, Willis AE, Fischer PM, Barrett DA, Mallucci GR.
Oral treatment targeting the unfolded protein response prevents neurodegeneration and clinical disease in prion-infected mice.
Sci Transl Med. 2013 Oct 9;5(206):206ra138.
PubMed.
Sekine Y, Zyryanova A, Crespillo-Casado A, Fischer PM, Harding HP, Ron D.
Stress responses. Mutations in a translation initiation factor identify the target of a memory-enhancing compound.
Science. 2015 May 29;348(6238):1027-30. Epub 2015 Apr 9
PubMed.
Sidrauski C, McGeachy AM, Ingolia NT, Walter P.
The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly.
Elife. 2015 Feb 26;4
PubMed.
This beautiful paper by Indrajit Das and colleagues led by Anne Bertolotti describes Sephin1, a compound modified from Guanabenz that selectively inhibits the stress-inducible phosphatase isoform PPP1R15A, but not the constitutive PPP1R15B (Das et al., 2015). In conjunction with their previous report (Tsaytler et al., 2011), they have provided significant evidence that targeting R15A phosphatase activity could be relevant to restore cell proteostasis.
Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 at serine 51 (eIF2α-P) attenuates global protein synthesis rates in a variety of stressful conditions, including ER stress and accumulation of misfolded proteins. It is widely acknowledged that a transient and physiological elevation in eIF2α-P restores proteostasis, while a more chronic eIF2α-P rise leads to cell death. Nonetheless, it is possible that extending protein synthesis inhibition might help cells recover proteostasis in the face of abnormal deposition of misfolded proteins.
In this regard, the Bertolotti group has identified that maintaining higher eIF2α-P levels by inhibiting R15A phosphatase recovers functional deficits in two animal models of neurodegenerative disease (Charcot-Marie-Tooth 1B and ALS). Their results identify an elegant mechanism that specifically modulates eIF2α-P under stress, preserving normal eIF2α activity in conditions not affected by stress. This is indeed mirrored in normal behavior and physiology in non-diseased mice treated with Sephin1.
Protein misfolding is an especially relevant issue for neurodegenerative diseases, given that many of them relate to abnormal protein deposition (e.g., Alzheimer’s, Parkinson, ALS, etc.). Thus, much effort has been recently made to understand how modulating proteostasis could serve as a target for neurological disease treatment. In fact, the past few years have been considerably rich in terms of insightful reports exploring the role of eIF2α-P in brain disorders. Now, the discovery of Sephin1 might raise the idea that this compound could be used for other neurodegenerative diseases.
I wish to highlight that, at least in Alzheimer’s and prion disease models, prolonged eIF2α-P appears to lead to the observed behavioral impairment (Moreno et al., 2012; Lourenco et al., 2013; Ma et al., 2013; Moreno et al., 2013). As a proof of concept, strategies that reduce brain eIF2α-P levels in AD and prion mice improve behavioral phenotypes (Lourenco et al., 2013; Ma et al., 2013; Moreno et al., 2013; Halliday et al., 2015). We have further described that a mechanism involving pro-inflammatory signaling, stress kinase activation, and integrated stress response leads to increased eIF2α-P with deleterious consequences to memory in AD mice (Lourenco et al., 2013). Studies from the Eric Klann (Ma et al., 2013) and Ulrich Hengst (Baleriola et al., 2014) labs also support that excessive ISR/eIF2α-P/ATF4 signaling could be detrimental in AD. In a TDP-43 model of ALS, suppression of eIF2α-P also results in improved phenotype in Drosophila (Kim et al., 2013).
I believe that the combination of the exciting results by Bertolotti and colleagues with the aforementioned reports strengthens the notion that neuronal proteostasis obeys an hormetic (U-shaped) pattern, in which either too low or too high rates of protein synthesis prevent cells from properly responding to insults. Although Das and colleagues still haven’t plunged deeper into mechanistic aspects, their observations are of clear importance for two reasons: They (1) identified a novel and apparently specific eIF2α phosphatase inhibitor and (2) contributed original data that support a role for proteostasis modulation in neurological disease, with potential translational impact in the future.
Now it remains to be determined whether Sephin1 could be effective in animal models of diverse neurological conditions and, in order to fully establish the benefits triggered by Sephin1, it is essential that potential on- and off-target mechanisms be elucidated.
References:
Baleriola J, Walker CA, Jean YY, Crary JF, Troy CM, Nagy PL, Hengst U.
Axonally synthesized ATF4 transmits a neurodegenerative signal across brain regions.
Cell. 2014 Aug 28;158(5):1159-72.
PubMed.
Halliday M, Radford H, Sekine Y, Moreno J, Verity N, le Quesne J, Ortori CA, Barrett DA, Fromont C, Fischer PM, Harding HP, Ron D, Mallucci GR.
Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity.
Cell Death Dis. 2015 Mar 5;6:e1672.
PubMed.
Kim HJ, Raphael AR, LaDow ES, McGurk L, Weber RA, Trojanowski JQ, Lee VM, Finkbeiner S, Gitler AD, Bonini NM.
Therapeutic modulation of eIF2α phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models.
Nat Genet. 2014 Feb;46(2):152-60. Epub 2013 Dec 15
PubMed.
Lourenco MV, Clarke JR, Frozza RL, Bomfim TR, Forny-Germano L, Batista AF, Sathler LB, Brito-Moreira J, Amaral OB, Silva CA, Freitas-Correa L, Espírito-Santo S, Campello-Costa P, Houzel JC, Klein WL, Holscher C, Carvalheira JB, Silva AM, Velloso LA, Munoz DP, Ferreira ST, De Felice FG.
TNF-α mediates PKR-dependent memory impairment and brain IRS-1 inhibition induced by Alzheimer's β-amyloid oligomers in mice and monkeys.
Cell Metab. 2013 Dec 3;18(6):831-43.
PubMed.
Ma T, Trinh MA, Wexler AJ, Bourbon C, Gatti E, Pierre P, Cavener DR, Klann E.
Suppression of eIF2α kinases alleviates Alzheimer's disease-related plasticity and memory deficits.
Nat Neurosci. 2013 Sep;16(9):1299-305.
PubMed.
Moreno JA, Radford H, Peretti D, Steinert JR, Verity N, Martin MG, Halliday M, Morgan J, Dinsdale D, Ortori CA, Barrett DA, Tsaytler P, Bertolotti A, Willis AE, Bushell M, Mallucci GR.
Sustained translational repression by eIF2α-P mediates prion neurodegeneration.
Nature. 2012 May 24;485(7399):507-11.
PubMed.
Moreno JA, Halliday M, Molloy C, Radford H, Verity N, Axten JM, Ortori CA, Willis AE, Fischer PM, Barrett DA, Mallucci GR.
Oral treatment targeting the unfolded protein response prevents neurodegeneration and clinical disease in prion-infected mice.
Sci Transl Med. 2013 Oct 9;5(206):206ra138.
PubMed.
Tsaytler P, Harding HP, Ron D, Bertolotti A.
Selective inhibition of a regulatory subunit of protein phosphatase 1 restores proteostasis.
Science. 2011 Apr 1;332(6025):91-4. Epub 2011 Mar 3
PubMed.
Targeting stress-responsive signaling pathways that regulate cellular protein homeostasis (or proteostasis) is a promising strategy to potentially ameliorate pathologic protein misfolding associated with etiologically-diverse diseases including Alzheimer’s disease, Parkinson’s disease, and familial Amyotrophic Lateral Sclerosis (ALS) (Powers et al., 2009; Hetz et al., 2013). Despite this promise, few small molecules are available that target the activity of these stress-responsive signaling pathways. Furthermore, the small molecules that do are often non-selective and/or hampered by negative toxicity profiles, limiting the translation of this approach to intervene in human disease.
In this manuscript, Das et al. identify a new small-molecule derivative of the α2 adrenergic receptor agonist guanabenz, called Sephin1, that acts as a modulator of stress-responsive signaling induced downstream of eIF2α phosphorylation. EIF2α phosphorylation is induced in response to diverse cellular insults, including ER stress (Ron and Walter, 2007). Phosphorylation of eIF2α leads to translation attenuation that reduces the load of newly synthesized, unfolded proteins as an initial response to cellular ER stress. This translation attenuation promotes cellular proteostasis by freeing chaperones and folding enzymes to protect the established proteome and prevent the proteotoxic accumulation of misfolded proteins. Phosphorylated eIF2α also promotes the activation of a network of transcription factors that induce expression of stress-responsive genes, including cellular proteostasis genes (e.g., redox enzymes), the pro-apoptotic transcription factor CHOP, and the eIF2α phosphatase regulatory subunit GADD34/PPP1R15A. GADD34 binds Protein Phosphatase 1 (PP1) to dephosphorylate eIF2α and restore translational integrity in a well-established negative feedback loop of eIF2α signaling (Ron and Walter, 2007). The transient translation attenuation and increased expression of cellular proteostasis factors promote cellular survival in response to stress. Alternatively, pro-apoptotic signaling mediated downstream of eIF2α phosphorylation, predominantly through CHOP, promotes apoptosis in response to severe or chronic stress. As such, eIF2α phosphorylation is a critical determinant in dictating cell fate in the context of many diverse neurodegenerative disorders (Hetz and Mollereau, 2014).
Sephin1 affects eIF2α phosphorylation stress-signaling by inhibiting the GADD34/PP1 phosphatase complex involved in the negative feedback loop. Importantly, Sephin1 does not inhibit the constitutive eIF2α phosphatase regulatory subunit CREP/PPP1R15B. This selectivity for GADD34/PP1 is important, because the addition of Sephin1 does not influence eIF2α phosphorylation in the absence of stress. The consequence of the selective inhibition afforded by Sephin1 is a delay in the translational recovery following the stress insult, extending the translational attenuation afforded by eIF2α phosphorylation. Interestingly, Sephin1 also reduces the translation of the pro-apoptotic transcription factor CHOP, which could attenuate downstream apoptotic signaling.
Guanabenz also selectively inhibits GADD34/PP1 to promote cellular proteostasis in response to stress (Tsaytler et al., 2011), but the toxicity profile of this molecule (owing to its α2 adrenergic receptor activity) has largely precluded its use in protein-misfolding diseases. A critically important attribute of Sephin1 is that this molecule separates the GADD34 inhibitory activity of guanabenz from its α2 adrenergic receptor activity. Sephin1 shows no α2 adrenergic receptor activity in vitro, but still potently inhibits GADD34. Similarly, Sephin1 showed no detrimental phenotypes in mice that would reflect potent α2 adrenergic receptor activity. Furthermore, Sephin1 retained the desirable guanabenz bioavailabilty in the nervous system, suggesting that Sephin1 offers significant promise to ameliorate pathologic defects associated with eIF2α phosphorylation involved in protein-misfolding diseases.
The potency of Sephin1 for GADD34 inhibition combined with its desirable bioavailability profile provides a unique opportunity to demonstrate the potential for this therapeutic strategy to intervene in protein misfolding diseases. Das et al. demonstrated this potential in two etiologically distinct diseases. In a mouse model of Charcot-Marie Tooth 1B, which expresses a destabilizing mutant of myelin protein zero, the addition of Sephin 1 rescued myelination, attenuated neuronal expression of the pro-apoptotic transcription factor CHOP, and restored motor coordination. Then, in a mouse model of ALS that expresses the aggregation-prone G93A mutant of SOD1, Sephin1 restored weight gain, rescued motor coordination deficiencies, decreased SOD1G93A aggregates, and attenuated neuronal expression of ER stress markers, including CHOP. Thus, Sephin1-dependent inhibition of GADD34 was able to attenuate pathologic phenotypes and promote neuronal function in these two distinct neurodegenerative disorders.
The capacity for Sephin1 to attenuate degenerative phenotypes in distinct protein-misfolding diseases is extremely exciting, as it offers proof of principle that we can intervene in diverse protein misfolding diseases by targeting the activity of stress-responsive signaling pathways. The ability to separate the α2-adrenergic receptor activity of guanabenz from the GADD34/PP1 inhibitor activity through medicinal chemistry is a significant step toward translating this approach to the clinic to intervene in human neurodegenerative disorders including Charcot-Marie Tooth Type 1B and familial ALS. Furthermore, since stress-signaling downstream of eIF2α phosphorylation implicated in the pathophysiology of many other neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease (Hetz and Mollereau, 2014), this study suggests that targeting GADD34/PP1 activity using Sephin1 could potentially provide a broadly-applicable strategy to intervene in many distinct neurodegenerative disorders.
References:
Hetz C, Chevet E, Harding HP.
Targeting the unfolded protein response in disease.
Nat Rev Drug Discov. 2013 Sep;12(9):703-19.
PubMed.
Hetz C, Mollereau B.
Disturbance of endoplasmic reticulum proteostasis in neurodegenerative diseases.
Nat Rev Neurosci. 2014 Apr;15(4):233-49. Epub 2014 Mar 12
PubMed.
Powers ET, Morimoto RI, Dillin A, Kelly JW, Balch WE.
Biological and chemical approaches to diseases of proteostasis deficiency.
Annu Rev Biochem. 2009;78:959-91.
PubMed.
Ron D, Walter P.
Signal integration in the endoplasmic reticulum unfolded protein response.
Nat Rev Mol Cell Biol. 2007 Jul;8(7):519-29.
PubMed.
Tsaytler P, Harding HP, Ron D, Bertolotti A.
Selective inhibition of a regulatory subunit of protein phosphatase 1 restores proteostasis.
Science. 2011 Apr 1;332(6025):91-4. Epub 2011 Mar 3
PubMed.
Comments
VU University Medical Center
VU University Medical Center
The paper by Indrajit Das and colleagues supports accumulating evidence that regulating the phosphorylation or dephosphorylation of eukaryotic initiation factor 2α (eIF2α) is a potential therapeutic approach for treating neurodegenerative diseases. Regulation of phosphorylation of this translation initiation factor is central to the integrated stress response (ISR) and reduces protein synthesis under conditions of cellular stress. The authors designed Sephin1, an inhibitor of eIF2α dephosphorylation based on Guanabenz, a compound that inhibits the phosphatase subunit PPP1R15B. Like its predecessor, Sephin1 is selective for the stress-induced eIF2α phosphorylation, but it lacks side effects caused by interaction with the α2-adrenergic receptor. This paper raises the debate whether for neurodegenerative diseases can be treated by blocking the phosphorylation of eIF2α through the inhibition of eIF2α kinases such as PERK, or prolonging it by blocking phosphatases as in the current study. There is now substantial evidence that the approaches stimulate or reduce protein synthesis during stress, respectively (see Oct 2013 news).
The control of eIF2α phosphorylation and dephosphorylation is very complex. It is important to increase understanding of how eIF2α is controlled in physiological conditions, but the real questions (and answers) lie in human pathology. Data is accumulating that the ISR is involved in many human neurodegenerative conditions. Although the key players of the ISR are involved in human pathology, it is unclear how. Is the ISR blocked, over-activated, or under aberrant control? Is this process similar or different among diseases? The study by Das et al. nicely addresses the potential of eIF2α as a therapeutic target for neurodegeneration and shows that therapeutic side effects can be reduced by more specific approaches. For translation to human therapy, better understanding of how eIF2α is affected and controlled in different neurodegenerative diseases is required.
MRC LMB
In response to Hoozemans and Scheper's comments:
Whether eIF2α phosphorylation is detrimental or beneficial is now very well understood.
A transient phosphorylation of eIF2α is beneficial, but a persistent phosphorylation of eIF2α is detrimental. This is why mammals have evolved two eIF2α phosphatases: to avoid persistent phosphorylation of eIF2αand its deleterious effects. Because Sephin1 is a selective inhibitor of R15A, but not the related R15B, it is safe and doesn't cause deleterious side effects.
This is in full agreement with genetic investigations of this pathway, carefully carried out in the past 15 years, mostly in the labs of David Ron and Randy Kaufman. Mice lacking R15A are viable and appear largely normal. Likewise, as we show, pharmacological inhibition of R15A is safe.
To illustrate this mechanism in simple terms, it might be useful to compare the sophisticated adaptive eIF2α pathway to a diet and a hunger strike. A diet will be beneficial to someone who is overweight, but a prolonged hunger strike will inevitably be fatal.
I hope this helps!
View all comments by Anne BertolottiVU University Medical Center
VU University Medical Center
We completely agree that the elegant design of the Sephin compound explains the lack of side effects. We thought it relevant to point out to readers that inhibition of eIF2α phosphorylation via genetic deletion or pharmacological inhibition of PERK protects when persistent eIF2α phosphorylation is observed, as in mouse models of prion disease and Aβ deposition (Ma et al., 2013; Moreno et al., 2012; Moreno et al., 2013). True, such interventions have too many side effects for clinical application, but these proof-of concept studies raise hope for more subtle interventions to inhibit phospho-eIF2α-mediated translational attenuation, such as ISRIB ( Sekine et al., 2015; Sidrauski et al., 2015; and Apr 2015 news).
In contrast, the opposite (prolonged eIF2α phosphorylation) is effected by Sephin and protective in ALS and CMT models as shown in the current paper. These paradoxical observations may, for example, relate to the stage of the disease process and the level of eIF2α phosphorylation at the start of treatment.
References:
Ma T, Trinh MA, Wexler AJ, Bourbon C, Gatti E, Pierre P, Cavener DR, Klann E. Suppression of eIF2α kinases alleviates Alzheimer's disease-related plasticity and memory deficits. Nat Neurosci. 2013 Sep;16(9):1299-305. PubMed.
Moreno JA, Radford H, Peretti D, Steinert JR, Verity N, Martin MG, Halliday M, Morgan J, Dinsdale D, Ortori CA, Barrett DA, Tsaytler P, Bertolotti A, Willis AE, Bushell M, Mallucci GR. Sustained translational repression by eIF2α-P mediates prion neurodegeneration. Nature. 2012 May 24;485(7399):507-11. PubMed.
Moreno JA, Halliday M, Molloy C, Radford H, Verity N, Axten JM, Ortori CA, Willis AE, Fischer PM, Barrett DA, Mallucci GR. Oral treatment targeting the unfolded protein response prevents neurodegeneration and clinical disease in prion-infected mice. Sci Transl Med. 2013 Oct 9;5(206):206ra138. PubMed.
Sekine Y, Zyryanova A, Crespillo-Casado A, Fischer PM, Harding HP, Ron D. Stress responses. Mutations in a translation initiation factor identify the target of a memory-enhancing compound. Science. 2015 May 29;348(6238):1027-30. Epub 2015 Apr 9 PubMed.
Sidrauski C, McGeachy AM, Ingolia NT, Walter P. The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly. Elife. 2015 Feb 26;4 PubMed.
View all comments by Jeroen HoozemansFederal University of Rio de Janeiro
This beautiful paper by Indrajit Das and colleagues led by Anne Bertolotti describes Sephin1, a compound modified from Guanabenz that selectively inhibits the stress-inducible phosphatase isoform PPP1R15A, but not the constitutive PPP1R15B (Das et al., 2015). In conjunction with their previous report (Tsaytler et al., 2011), they have provided significant evidence that targeting R15A phosphatase activity could be relevant to restore cell proteostasis.
Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 at serine 51 (eIF2α-P) attenuates global protein synthesis rates in a variety of stressful conditions, including ER stress and accumulation of misfolded proteins. It is widely acknowledged that a transient and physiological elevation in eIF2α-P restores proteostasis, while a more chronic eIF2α-P rise leads to cell death. Nonetheless, it is possible that extending protein synthesis inhibition might help cells recover proteostasis in the face of abnormal deposition of misfolded proteins.
In this regard, the Bertolotti group has identified that maintaining higher eIF2α-P levels by inhibiting R15A phosphatase recovers functional deficits in two animal models of neurodegenerative disease (Charcot-Marie-Tooth 1B and ALS). Their results identify an elegant mechanism that specifically modulates eIF2α-P under stress, preserving normal eIF2α activity in conditions not affected by stress. This is indeed mirrored in normal behavior and physiology in non-diseased mice treated with Sephin1.
Protein misfolding is an especially relevant issue for neurodegenerative diseases, given that many of them relate to abnormal protein deposition (e.g., Alzheimer’s, Parkinson, ALS, etc.). Thus, much effort has been recently made to understand how modulating proteostasis could serve as a target for neurological disease treatment. In fact, the past few years have been considerably rich in terms of insightful reports exploring the role of eIF2α-P in brain disorders. Now, the discovery of Sephin1 might raise the idea that this compound could be used for other neurodegenerative diseases.
I wish to highlight that, at least in Alzheimer’s and prion disease models, prolonged eIF2α-P appears to lead to the observed behavioral impairment (Moreno et al., 2012; Lourenco et al., 2013; Ma et al., 2013; Moreno et al., 2013). As a proof of concept, strategies that reduce brain eIF2α-P levels in AD and prion mice improve behavioral phenotypes (Lourenco et al., 2013; Ma et al., 2013; Moreno et al., 2013; Halliday et al., 2015). We have further described that a mechanism involving pro-inflammatory signaling, stress kinase activation, and integrated stress response leads to increased eIF2α-P with deleterious consequences to memory in AD mice (Lourenco et al., 2013). Studies from the Eric Klann (Ma et al., 2013) and Ulrich Hengst (Baleriola et al., 2014) labs also support that excessive ISR/eIF2α-P/ATF4 signaling could be detrimental in AD. In a TDP-43 model of ALS, suppression of eIF2α-P also results in improved phenotype in Drosophila (Kim et al., 2013).
I believe that the combination of the exciting results by Bertolotti and colleagues with the aforementioned reports strengthens the notion that neuronal proteostasis obeys an hormetic (U-shaped) pattern, in which either too low or too high rates of protein synthesis prevent cells from properly responding to insults. Although Das and colleagues still haven’t plunged deeper into mechanistic aspects, their observations are of clear importance for two reasons: They (1) identified a novel and apparently specific eIF2α phosphatase inhibitor and (2) contributed original data that support a role for proteostasis modulation in neurological disease, with potential translational impact in the future.
Now it remains to be determined whether Sephin1 could be effective in animal models of diverse neurological conditions and, in order to fully establish the benefits triggered by Sephin1, it is essential that potential on- and off-target mechanisms be elucidated.
References:
Baleriola J, Walker CA, Jean YY, Crary JF, Troy CM, Nagy PL, Hengst U. Axonally synthesized ATF4 transmits a neurodegenerative signal across brain regions. Cell. 2014 Aug 28;158(5):1159-72. PubMed.
Halliday M, Radford H, Sekine Y, Moreno J, Verity N, le Quesne J, Ortori CA, Barrett DA, Fromont C, Fischer PM, Harding HP, Ron D, Mallucci GR. Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity. Cell Death Dis. 2015 Mar 5;6:e1672. PubMed.
Kim HJ, Raphael AR, LaDow ES, McGurk L, Weber RA, Trojanowski JQ, Lee VM, Finkbeiner S, Gitler AD, Bonini NM. Therapeutic modulation of eIF2α phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models. Nat Genet. 2014 Feb;46(2):152-60. Epub 2013 Dec 15 PubMed.
Lourenco MV, Clarke JR, Frozza RL, Bomfim TR, Forny-Germano L, Batista AF, Sathler LB, Brito-Moreira J, Amaral OB, Silva CA, Freitas-Correa L, Espírito-Santo S, Campello-Costa P, Houzel JC, Klein WL, Holscher C, Carvalheira JB, Silva AM, Velloso LA, Munoz DP, Ferreira ST, De Felice FG. TNF-α mediates PKR-dependent memory impairment and brain IRS-1 inhibition induced by Alzheimer's β-amyloid oligomers in mice and monkeys. Cell Metab. 2013 Dec 3;18(6):831-43. PubMed.
Ma T, Trinh MA, Wexler AJ, Bourbon C, Gatti E, Pierre P, Cavener DR, Klann E. Suppression of eIF2α kinases alleviates Alzheimer's disease-related plasticity and memory deficits. Nat Neurosci. 2013 Sep;16(9):1299-305. PubMed.
Moreno JA, Radford H, Peretti D, Steinert JR, Verity N, Martin MG, Halliday M, Morgan J, Dinsdale D, Ortori CA, Barrett DA, Tsaytler P, Bertolotti A, Willis AE, Bushell M, Mallucci GR. Sustained translational repression by eIF2α-P mediates prion neurodegeneration. Nature. 2012 May 24;485(7399):507-11. PubMed.
Moreno JA, Halliday M, Molloy C, Radford H, Verity N, Axten JM, Ortori CA, Willis AE, Fischer PM, Barrett DA, Mallucci GR. Oral treatment targeting the unfolded protein response prevents neurodegeneration and clinical disease in prion-infected mice. Sci Transl Med. 2013 Oct 9;5(206):206ra138. PubMed.
Tsaytler P, Harding HP, Ron D, Bertolotti A. Selective inhibition of a regulatory subunit of protein phosphatase 1 restores proteostasis. Science. 2011 Apr 1;332(6025):91-4. Epub 2011 Mar 3 PubMed.
View all comments by Mychael LourencoThe Scripps Research Institute
Targeting stress-responsive signaling pathways that regulate cellular protein homeostasis (or proteostasis) is a promising strategy to potentially ameliorate pathologic protein misfolding associated with etiologically-diverse diseases including Alzheimer’s disease, Parkinson’s disease, and familial Amyotrophic Lateral Sclerosis (ALS) (Powers et al., 2009; Hetz et al., 2013). Despite this promise, few small molecules are available that target the activity of these stress-responsive signaling pathways. Furthermore, the small molecules that do are often non-selective and/or hampered by negative toxicity profiles, limiting the translation of this approach to intervene in human disease.
In this manuscript, Das et al. identify a new small-molecule derivative of the α2 adrenergic receptor agonist guanabenz, called Sephin1, that acts as a modulator of stress-responsive signaling induced downstream of eIF2α phosphorylation. EIF2α phosphorylation is induced in response to diverse cellular insults, including ER stress (Ron and Walter, 2007). Phosphorylation of eIF2α leads to translation attenuation that reduces the load of newly synthesized, unfolded proteins as an initial response to cellular ER stress. This translation attenuation promotes cellular proteostasis by freeing chaperones and folding enzymes to protect the established proteome and prevent the proteotoxic accumulation of misfolded proteins. Phosphorylated eIF2α also promotes the activation of a network of transcription factors that induce expression of stress-responsive genes, including cellular proteostasis genes (e.g., redox enzymes), the pro-apoptotic transcription factor CHOP, and the eIF2α phosphatase regulatory subunit GADD34/PPP1R15A. GADD34 binds Protein Phosphatase 1 (PP1) to dephosphorylate eIF2α and restore translational integrity in a well-established negative feedback loop of eIF2α signaling (Ron and Walter, 2007). The transient translation attenuation and increased expression of cellular proteostasis factors promote cellular survival in response to stress. Alternatively, pro-apoptotic signaling mediated downstream of eIF2α phosphorylation, predominantly through CHOP, promotes apoptosis in response to severe or chronic stress. As such, eIF2α phosphorylation is a critical determinant in dictating cell fate in the context of many diverse neurodegenerative disorders (Hetz and Mollereau, 2014).
Sephin1 affects eIF2α phosphorylation stress-signaling by inhibiting the GADD34/PP1 phosphatase complex involved in the negative feedback loop. Importantly, Sephin1 does not inhibit the constitutive eIF2α phosphatase regulatory subunit CREP/PPP1R15B. This selectivity for GADD34/PP1 is important, because the addition of Sephin1 does not influence eIF2α phosphorylation in the absence of stress. The consequence of the selective inhibition afforded by Sephin1 is a delay in the translational recovery following the stress insult, extending the translational attenuation afforded by eIF2α phosphorylation. Interestingly, Sephin1 also reduces the translation of the pro-apoptotic transcription factor CHOP, which could attenuate downstream apoptotic signaling.
Guanabenz also selectively inhibits GADD34/PP1 to promote cellular proteostasis in response to stress (Tsaytler et al., 2011), but the toxicity profile of this molecule (owing to its α2 adrenergic receptor activity) has largely precluded its use in protein-misfolding diseases. A critically important attribute of Sephin1 is that this molecule separates the GADD34 inhibitory activity of guanabenz from its α2 adrenergic receptor activity. Sephin1 shows no α2 adrenergic receptor activity in vitro, but still potently inhibits GADD34. Similarly, Sephin1 showed no detrimental phenotypes in mice that would reflect potent α2 adrenergic receptor activity. Furthermore, Sephin1 retained the desirable guanabenz bioavailabilty in the nervous system, suggesting that Sephin1 offers significant promise to ameliorate pathologic defects associated with eIF2α phosphorylation involved in protein-misfolding diseases.
The potency of Sephin1 for GADD34 inhibition combined with its desirable bioavailability profile provides a unique opportunity to demonstrate the potential for this therapeutic strategy to intervene in protein misfolding diseases. Das et al. demonstrated this potential in two etiologically distinct diseases. In a mouse model of Charcot-Marie Tooth 1B, which expresses a destabilizing mutant of myelin protein zero, the addition of Sephin 1 rescued myelination, attenuated neuronal expression of the pro-apoptotic transcription factor CHOP, and restored motor coordination. Then, in a mouse model of ALS that expresses the aggregation-prone G93A mutant of SOD1, Sephin1 restored weight gain, rescued motor coordination deficiencies, decreased SOD1G93A aggregates, and attenuated neuronal expression of ER stress markers, including CHOP. Thus, Sephin1-dependent inhibition of GADD34 was able to attenuate pathologic phenotypes and promote neuronal function in these two distinct neurodegenerative disorders.
The capacity for Sephin1 to attenuate degenerative phenotypes in distinct protein-misfolding diseases is extremely exciting, as it offers proof of principle that we can intervene in diverse protein misfolding diseases by targeting the activity of stress-responsive signaling pathways. The ability to separate the α2-adrenergic receptor activity of guanabenz from the GADD34/PP1 inhibitor activity through medicinal chemistry is a significant step toward translating this approach to the clinic to intervene in human neurodegenerative disorders including Charcot-Marie Tooth Type 1B and familial ALS. Furthermore, since stress-signaling downstream of eIF2α phosphorylation implicated in the pathophysiology of many other neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease (Hetz and Mollereau, 2014), this study suggests that targeting GADD34/PP1 activity using Sephin1 could potentially provide a broadly-applicable strategy to intervene in many distinct neurodegenerative disorders.
References:
Hetz C, Chevet E, Harding HP. Targeting the unfolded protein response in disease. Nat Rev Drug Discov. 2013 Sep;12(9):703-19. PubMed.
Hetz C, Mollereau B. Disturbance of endoplasmic reticulum proteostasis in neurodegenerative diseases. Nat Rev Neurosci. 2014 Apr;15(4):233-49. Epub 2014 Mar 12 PubMed.
Powers ET, Morimoto RI, Dillin A, Kelly JW, Balch WE. Biological and chemical approaches to diseases of proteostasis deficiency. Annu Rev Biochem. 2009;78:959-91. PubMed.
Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol. 2007 Jul;8(7):519-29. PubMed.
Tsaytler P, Harding HP, Ron D, Bertolotti A. Selective inhibition of a regulatory subunit of protein phosphatase 1 restores proteostasis. Science. 2011 Apr 1;332(6025):91-4. Epub 2011 Mar 3 PubMed.
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