Fig 2: PPP1R15B is mutated, upregulated and correctly localized in patient cells. (A) Sanger sequencing demonstrating confirmation of the c.1972G>A;p.R658C PPP1R15B variant (NM_032833.4) complimentary strand shown. (B) Immunodetection of PPP1R15B in control and patient lymphoblast cell lines demonstrated no overt changes in PPP1R15B localization. (C) Western blot (left) and real-time PCR analysis (right) on extracts from control and patient cells showing elevated levels of PPP1R15B transcript and protein.

Fig 3: Stress preconditioning of human ES-derived neurons impairs their ability to maintain p-eIF2a levels and SG assembly. (a) Characterization of differentiated human neurons used in the study. Day 40 cultures of neurons differentiated from hES cells express typical neuronal markers ßIII-tubulin, synaptophysin and neurofilament M, as well as a motor neuron marker ChAT. (b) G3BP1-positive SGs and increased level of p-eIF2a in human neurons after 70 min of exposure to SA. Left column shows general plane images of treated and untreated cultures. Representative images are shown. Arrowheads indicate cells with SGs. (c) Pretreatment with MG132 upregulates CReP protein and results in attenuated build-up of p-eIF2a following SA stress as compared with control neurons. Representative western blots and quantification of band intensities are shown (n=4, * and #P<0.05, asterisks indicate significant difference as compared with control cells). (d) Pretreatment with MG132 reduces the fraction of human neurons with SGs following SA stress. Fraction of neurons with G3BP1-positive SGs in was quantified (~400 neurons per condition, *P<0.05). (e) MG132 pretreatment decreases survival of human motor neurons during oxidative stress. MG132-pretreated and control cultures were treated with SA for 70 min and the number of cleaved caspase 3 (CC3)-positive cells was quantified from three independent experiments as described in Materials and methods section (#P<0.05, **P<0.01, asterisks indicate significant difference as compared with control cells). Representative images for all conditions are also shown. Motor neuron cultures were pretreated with 1 µ MG132 for 4 h in all panels. Scale bars, 200 µm for top panels in (a), left column in (b) and top panel in (e); and 10 µm for bottom panel in (a), right columns in (b) and bottom panel in (e)

Fig 4: Stress preconditioning with a proteasome inhibitor MG132 upregulates eIF2a phosphatase regulatory subunits and limits the ability of SH-SY5Y cells to maintain p-eIF2a levels and SG assembly during stress. (a) SG assembly during oxidative stress is impaired after MG132 pretreatment. Cells were pretreated with MG132 (1 µM for 4 h) or left untreated, and SA was added for 10, 20 or 30 min. SGs were visualized with anti-TIAR staining. Representative images of all conditions are shown, asterisks indicate cells without SGs. Scale bar, 10 µm. (b) MG132 pretreatment leads to reduced number of cells with mature SGs after 30 min of SA exposure (n=3; *P<0.05). In contrast, pretreatment with a GADD34 phosphatase subunit inhibitor guanabenz does not affect SG assembly during stress. Cells were pretreated with 1 µM MG132 for 4 h or 5 µM guanabenz for 24 h. HSP70 inhibitor pifithrin (5 µM) was added 30 min before SA addition. (c) MG132-pretreated and control cells display different kinetics of p-eIF2a build-up during stress, and CReP protein is upregulated by MG132. Cells were treated with MG132 and subsequently stressed with SA as described in (a). Representative blots and quantification of band intensities for p-eIF2a and CReP in MG132-pretreated as compared with control cells are shown (n=5 or 6, *P<0.05, **P<0.01). For CReP and p-eIF2a band analysis, 0 and 10-min band intensity in control cells were taken as 1, respectively. (d) MG132 upregulates GADD34 mRNA. Cells were treated with 1 µM MG132 for 4 h before RNA extraction for qRT-PCR analysis. CHOP mRNA levels were measured in parallel to confirm activated stress response in MG132-treated cells (n=5, *P<0.05, **P<0.01, ***P<0.001). (e) Pharmacological inhibition of GADD34 and to a lesser extent CReP knockdown reverse the effect of MG132 pretreatment on p-eIF2a levels during stress. Cells were transfected with CReP siRNA and 72 h post-transfection treated with 1 µM MG132 for 4 h followed by SA stress; or co-treated with MG132 and 50 µM guanabenz for 4 h followed by SA stress. Cells were analyzed 20 min after SA addition. Representative western blots are shown

Fig 5: Transgenic mouse model of tauopathy is characterized by elevated basal p-eIF2a levels and impaired SG assembly in the brain. (a) p-eIF2a immunoreactivity is increased in the cortex of 8-month-old Tau22 transgenic animals compared with age-matched wild type (WT) controls under normal conditions and after HS. AT8 antibody was used to visualize phospho-tau on sagittal brain sections. (b) Levels of p-eIF2a, CReP and GADD34 proteins are higher in the hippocampus (CA1 subfield) of Tau22 animals compared with age-matched WT control animals (WT ctrl) and are upregulated by HS. Representative western blots and band intensity quantitation are shown (* and #P=0.05, asterisks indicate significant difference as compared with naive WT animals). For all proteins, band intensity for WT animals after HS was taken as equal 1. (c) Decreased p-eIF2a intensity in the cytoplasm of hippocampal neurons of Tau22 mice after HS. Representative images of p-eIF2a staining are shown for the same animals as in a. Details of fluorescence intensity analysis are given in Materials and methods section (**P<0.01). (d-f) Efficient HS-induced SG assembly is observed in cortical and hippocampal neurons of WT mice but not in Tau22 animals. SGs (arrowheads) were visualized using anti-TIAR antibody (d) or RNA-FISH with a fluorescently labeled poly(dT) probe (e); in (d), co-staining with an anti-FUS antibody in was used to highlight nuclei. For quantification of SG-containing cells, >100 neurons were assessed in each of the studied regions in three HS-treated mice per genotype (***P<0.001) (f). Scale bars, (a) 200 µm; (c)20 µm; (d, e) and inset in (a) 10 µm