VWMD patient fibroblasts and astrocytes exhibit dysregulated ISR marker expression
Mutations in eIF2B suppress both global and stress-induced protein translation, in response to ER stress (5, 15). Given that eIF2B is ubiquitously expressed, we examined patient fibroblasts and iPSC-derived astrocytes for evidence of a dysfunctional ISR. Fibroblasts were reprogrammed into iPSCs from two VWMD patients, along with gender-matched relatives as non-disease controls (Figure S1). A previous study identified that white matter-derived astrocytes, generated using a ciliary neurotrophic factor (CNTF)-based differentiation protocol, showed a more vulnerable phenotype to stress, compared to grey matter astrocytes generated using fetal bovine serum (7). Consequently, we generated astrocytes from iPSCs using a CNTF-based method (Figure S2).
Responses to oxidative and endoplasmic reticulum (ER) stresses are all proposed to be affected in VWMD cell and animal-based models (15). Studies employing cells bearing different VWMD mutations have shown variability in the ISR (5), and primary astrocytes from mice bearing a homozygous R191H mutation in eIF2Bε did not exhibit the ISR in vitro (16). Thus, first we established stress conditions that could generate an exacerbated cellular phenotype in the VWMD patient cells compared to controls. We evaluated the dose-dependent effect of H2O2, as a mediator of oxidative stress, thapsigargin, as a mediator of ER stress, and MG132. MG132 is commonly utilised as a proteasomal inhibitor that also induces ER and oxidative stress via the unfolded protein response, all of which trigger the ISR (17, 18). There was a small but significant reduction in cell survival for VWMD fibroblasts and iPSC-derived astrocytes, compared to non-disease controls, under all three stressors (Figure S4A).
Disrupted ISR homeostasis can be assessed by measuring the expression levels of ISR-relevant markers. The control of eIF2α phosphorylation or dephosphorylation acts as a pivotal mechanism that regulates global protein synthesis in response to cell stress (19); GADD34 dephosphorylates eIF2α (20) while CHOP is activated by the ISR to promote apoptosis (21). The expression levels of these ISR-relevant proteins, p-eIF2α (normalised to eIF2α), GADD34, and CHOP, were compared in VWMD and control fibroblasts, and iPSC-derived astrocytes, under MG132 stress (Fig. 1; antibody characterisation and representative blots shown in Figures S3-4). VWMD lymphoblasts have previously been reported to show reduced GADD34 expression and increased levels of p-eIF2α, following thapsigargin-induced acute ER stress (5). Under MG132-induced stress, VWMD fibroblasts showed reduced upregulation of eIF2α phosphorylation and reduced GADD34 expression, and increases in CHOP expression, compared to controls (Fig. 1A-C). MG132-stressed VWMD astrocytes also showed reduced upregulation of eIF2α phosphorylation and increased CHOP, compared to controls (Fig. 1D-F). VWMD astrocytes exhibited increased GADD34 (Fig. 1E), consistent with glia in animal model studies (15) and the hypophosphorylation of eIF2 observed in white matter patient tissue (22).
Cytoprotective drug screen in VWMD patient cultures
Based on the established capacity of MG132 to induce oxidative stress, and exacerbate ISR disease phenotypes, we performed a first-pass drug screen for candidates able to protect against the effect of low dose MG132 in VWMD1 EIF2B5R113H/A403V patient fibroblasts, with cell viability assessed by resazurin reduction activity (Figure 2).
Following the initial drug screen, a panel of 20 compounds (Table S2) was selected for downstream evaluation in VWMD1 EIF2B5R113H/A403V fibroblasts and in both VWMD1 EIF2B5R113H/A403V and VWMD6 EIF2B2G200V/E213G patient-derived astrocyte cultures. The compounds selected for downstream assays were chosen based on their potential for clinical translation, including considerations of bioavailability, route of administration and potential side effects. The drug panel included 14 protective compounds from the screen (>1.5 × standard deviation of MG132-stressed controls), and a further six compounds with relevant modes of action for VWMD. The sigma-1 receptor pathway was recently identified in a drug screen to protect against mitochondrial dysfunction in a murine EIF2B5R132H/R132H model (11). Nominated drugs on the basis of relevant mode of action to VWMD, included guanabenz (23), ISRIB (24), sigma-1 receptor agonists, AVex-73 and amitriptyline (11), as well as the CNS cytoprotective compounds tauroursodeoxycholic acid and alkaloid berberine (25). Overall, the panel of compounds for further evaluation included glucocorticosteroids, bile acids, iron chelators, antioxidants, ISR modulators and sigma-1 receptor agonists. All candidates elicited cytoprotective effects against MG132-induced stress at varying concentrations in VWMD1 fibroblasts, with the exception of ISRIB. Ursodiol and its taurine derivative, tauroursodeoxycholic acid, showed similar cytoprotective efficacies. Anti-inflammatories were amongst the largest category of compounds that improved cell viability under proteasomal stress, with a high proportion of these being glucocorticosteroids. The recent demonstration of mitochondrial dysfunction and inefficient respiration in murine models of VWMD (11) has expanded the search for possible therapeutics to include mitochondrial protective compounds and antioxidants. The sigma-1 receptor is a chaperone protein in ER membranes that governs a range of cellular processes, including calcium homeostasis and reactive oxygen species accumulation (26). However, in our study, the cytoprotective effect of the sigma-1 receptor agonists, AVex-73 and amitriptyline, was limited at higher concentrations (≥5 µM; Figures 2, S5). Drugs assessed in iPSC-derived astrocytes, VWMD1 EIF2B5R113H/A403V and VWMD6 EIF2B2G200V/E213G cells yielded comparable results (Figure S5). The majority of the drugs that were protective in VWMD1 fibroblasts were also protective in VWMD1 and VMWD6 astrocytes, with the exception of amitriptyline, curcumin and budesonide (Figures 2, S5).
Further studies were conducted to gain insight into the mode of action of drugs with therapeutic potential for VWMD (Figures 2, S5), including AVex-73, berberine, deflazacort, guanabenz, ISRIB, ursodiol and zileuton. Assays to investigate drug mechanisms were performed with VWMD1 EIF2B5R113H/A403V patient astrocytes, given that the majority of VWMD-causing mutations affect the eIF2Bε subunit (3).
Effect of drug candidates on cell death in VWMD iPSC-astrocytes
To determine whether candidate drugs reduced the proportion of bulk cell death, the membrane permeabilisation of treated astrocyte cultures was evaluated. In MG132-stressed VWMD1 astrocytes there were no significant changes in the percentage of membrane-permeabilised cells, with the exception of ISRIB increasing the proportion of membrane-permeabilised cells (Figure S6). To test whether the drugs caused a reduction in mitochondrial apoptosis, the BAX:BCL2 ratio was evaluated. The ratio of BAX/BCL2 is used to assess the levels of Bax (proapoptotic) and Bcl-2 (antiapoptotic) expression. There was no detectable change in BAX:BCL2 ratio caused by any of the drug candidates, with the exception of an increase in BAX:BCL2 caused by ISRIB (Figure S6).
Effect of candidate drugs on ISR-relevant proteins p-eIF2α, GADD34 and CHOP
Candidate drugs were assessed for their effect on the ISR under MG132 stress in astrocytes. The expression levels of the ISR-relevant proteins, p-eIF2α (normalised to eIF2α), GADD34 and CHOP, were evaluated following candidate drug treatment (Figure 3A-C). ISRIB markedly increased p-eIF2α and decreased GADD34 and CHOP expression under MG132 stress conditions. AVex-73, berberine and deflazacort significantly decreased CHOP expression in the presence of MG132.
Previous cell stress and neurodegeneration studies have established ISR-modulating roles for guanabenz and ISRIB (27, 28). Guanabenz is considered to exert cytoprotective effects by inhibiting the activity of GADD34 to recruit eIF2 phosphatases, thus prolonging translation inhibition and avoiding the added stress of resuming protein synthesis (27). However, at concentrations of guanabenz that induced a cytoprotective effect (5 µM), we did not observe a significant impact on expression of any ISR markers in astrocytes. Conversely, ISRIB-mediated cytoprotection of astrocytes (1.25 µM) was associated with increased eIF2α phosphorylation, and downregulation of GADD34 and CHOP.
Effect of candidate drugs on indicators of mitochondrial function
The eIF2B mutations in murine models have been shown to decrease mitochondrial membrane potential and impair mitochondrial complex I function, resulting in a compensatory increase in mitochondrial abundance (9). Furthermore, mutations in eIF2B genes impair mitochondrial function during oxidative stress conditions in VWMD murine fibroblasts and astrocytes (11). VWMD1 and VWMD6 patient iPSC-derived astrocytes demonstrated reduced mitochondrial membrane potential in both the presence and absence of MG132, suggesting reduced mitochondrial membrane potential is a cellular phenotype of VWMD patient cells, even under basal conditions (Figure S8).
The candidate drugs were investigated for their ability to protect against oxidative stress and improve mitochondrial membrane potential. VWMD1 EIF2B5R113H/A403V patient astrocytes treated with ursodiol or zileuton led to decreased generation of reactive oxygen species, in both the presence and absence of MG132 stress (Figure 4A). This correlates with the purported radical scavenging activity of the 5-lipoxygenase antagonist zileuton (29). Ursodiol also increased the relative mitochondrial membrane potential of VWMD1 EIF2B5R113H/A403V astrocytes under both unstressed and stressed conditions (Figure 4B), consistent with a loss of mitochondrial membrane potential as a cellular phenotype of VWMD astrocytes. Ursodiol reduced oxidative stress in both control and VWMD astrocytes (Figure 4C) and improved the mitochondrial membrane potential phenotype of VWMD astrocytes, bringing the levels of TMRE to those of control cells (Figure 4D-G). Together, these data suggest that ursodiol may promote mitochondrial function and reduce oxidative stress in VWMD astrocytes.