Integrated stress response inhibition prolongs the lifespan of a Pelizaeus-Merzbacher disease mouse model by increasing oligodendrocyte survival

Domain: neuroscience
Published: 2025-12-26
Editorial outcome: minor_revision

Abstract
The leukodystrophy Pelizaeus-Merzbacher disease (PMD) is caused by myelin protein proteolipid protein gene (PLP1) mutations. PMD is characterized by oligodendrocyte death and CNS hypomyelination; thus, increasing oligodendrocyte survival and enhancing myelination could provide therapeutic benefit. Here, we use the PMD mouse model Jimpy to determine the impact of the integrated stress response (ISR) on the oligodendrocyte response to mutant PLP expression. Male Jimpy animals in which the ISR-triggering eukaryotic initiation factor (eIF) 2α kinase, protein kinase-like endoplasmic reticulum kinase (PERK), is inactivated have an extended lifespan that correlates with increased oligodendrocyte survival and enhanced CNS myelination. Inactivation of downstream components of the ISR pathway, in contrast, does not rescue oligodendrocytes or myelin. Phosphorylated eIF2α inhibits the exchange factor eIF2B, resulting in diminished protein synthesis. Treatment with small molecule eIF2B activators 2BAct and ISRIB increases oligodendrocyte survival, CNS myelination, and doubled the Jimpy lifespan. These results suggest that ISR modulation could provide therapeutic benefit to PMD patients.

Introduction
Leukodystrophies represent a class of genetic disorders that result in abnormal central nervous system (CNS) myelin, which is produced by oligodendrocytes 1. Leukodystrophies are relatively rare, approximately one in 7500 births, but many lead to severe clinical symptoms resulting in childhood mortality 2. Pelizaeus-Merzbacher Disease (PMD) is a hypomyelinating X-linked leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene encoding the highly expressed CNS protein, proteolipid protein (PLP), which is exclusively expressed by myelinating cells 3. Oligodendrocyte apoptosis is thought to be the primary cause of myelin abnormalities in PMD patients. Although stem cell approaches designed to replace mutant oligodendrocytes have shown promise 4,5, and antisense oligonucleotides against the PLP1 transcript dramatically increase the lifespan of PMD mouse models 6, there are currently no approved therapies for PMD 7.

The severity of PMD varies based on the type of mutation 8. Most PMD cases are caused by duplications of the entire PLP1 gene locus, resulting in increased PLP expression 9. Loss-of-function PLP1 alleles result in relatively mild clinical symptoms, whereas missense and frameshift mutations lead to severe developmental abnormalities and early lethality 10. PLP1 point mutations that result in non-conservative amino acid substitutions lead to more severe clinical outcomes than conservative substitutions 10. PLP is a highly conserved integral membrane protein that passes the lipid bilayer multiple times 11. A subset of the more severe PLP1 mutations has been shown to disrupt PLP transport through the secretory pathway, resulting in accumulation in the endoplasmic reticulum (ER). This cytotoxic buildup gradually kills oligodendrocytes, ultimately causing axonal swelling and neurodegeneration 12. PLP accumulation in the ER activates the unfolded protein response (UPR) arm of the integrated stress response (ISR) 13. The ISR is a protective response to various cellular stresses that activates one of four eIF2α kinases, including the PKR-like ER kinase (PERK) 14. Phosphorylation of the translation initiation factor eIF2α results in the attenuation of protein synthesis and cellular protection 15. The PERK pathway is rapidly activated by ER stress in an effort to restore protein homeostasis in the secretory pathway. In addition to the PERK pathway, ER stress also activates the inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) arms of the UPR to ameliorate proteotoxicity by enhancing the transcription of chaperone genes and degradation of misfolded or aggregated polypeptides 16. Ultimately, if the cellular stress cannot be resolved, a maladaptive response is activated that results in the apoptotic elimination of the stressed cells 17.

A number of animal models with Plp1 mutations have proven useful in the study of PMD disease pathogenesis 18. These include the shaking pup canine model, the myelin-deficient rat model, as well as a number of mouse models of varying severity 19. The Jimpy mouse model is the result of a 74 base pair deletion in the Plp1 gene that causes a frameshift in the open reading frame, which changes the carboxy terminus 11,20. Although the Plp1 mutation of Jimpy mice is not an authentic mutation of a human disease allele, these mice recapitulate the cellular, molecular and neurologic features seen in severe “connatal” forms of human PMD 21. Jimpy mice, which exhibit tremors and seizures, and die at about postnatal day 21, have dramatically reduced numbers of oligodendrocytes and lack CNS myelin 22,23. The PERK arm of the UPR/ISR has been shown to be activated in Jimpy oligodendrocytes 24.

Previously, we have shown that oligodendrocytes in the presence of an inflammatory environment, in vitro and in vivo, also activate the PERK arm of the UPR/ISR 25,26. In this protective state, a subset of mRNA transcripts that encode cytoprotective proteins is preferentially translated. Our studies and those of others have demonstrated that an enhanced or prolonged stress response, using either genetic or pharmacologic approaches, provides increased protection to oligodendrocytes in the presence of inflammation 27–29. Here, we examined whether manipulating the ISR results in an altered oligodendroglial response to mutant PLP expression in Jimpy mice. We found that inhibition of the ISR in Jimpy oligodendrocytes, either genetically or pharmacologically, increased oligodendrocyte survival, enhanced CNS myelination, and prolonged lifespan.

Results
PERK inactivation increases Jimpy oligodendrocyte survival, CNS myelination, and lifespan
PERK is one of four stress-sensing kinases that trigger the UPR arm of the ISR by phosphorylating eIF2α 30. To determine the potential role that this pathway plays in modulating the response of oligodendrocytes against the ER stress caused by the Jimpy Plp1 mutation, we crossed Jimpy mice, which normally die at around postnatal day 21 (PND21) with Perk heterozygous mice. Jimpy males that were heterozygous for the Perk null allele (Jimpy; Perk+/−) displayed a significantly longer lifespan than Jimpy males on a WT background (Fig. 1a, b). Homozygous Perk null mice die shortly after birth 31; so to further examine the role of the PERK pathway in oligodendrocytes in response to chronic ER stress, we crossed female mice that carry the Jimpy mutation with mice that contain a conditional (floxed) Perk allele 32. These mice were crossed to Olig2-Cre mice, which express Cre throughout the oligodendrocyte lineage 33. Previously, we have shown that PERK is dispensable for normal oligodendrocyte development and CNS myelination 34,35. The Jimpy; Perkfl/fl; Olig2-Cre animals lived ~2 weeks longer than the Jimpy; Perkfl/fl mice (Fig. 1a, b), suggesting that PERK activity is detrimental to Jimpy mice. Despite their prolonged lifespan, the Jimpy; Perkfl/fl; Olig2-Cre animals displayed similar tremor and seizure symptoms as Jimpy control animals.

To further assess the impact of Perk inactivation on Jimpy oligodendrocytes, we examined these mice histologically at PND 19, which is close to the end of the expected lifespan of Jimpy controls. Antibodies against aspartoacylase (ASPA, mature oligodendrocytes) 36, breast carcinoma-amplified sequence 1 (BCAS1, differentiating oligodendrocytes) 37, and Olig2 (oligodendrocyte lineage cells) 38 were used to characterize the oligodendrocyte lineage in the Jimpy mice. Jimpy; Perkfl/fl mice had a significant lower density of ASPA immunoreactive (+) mature oligodendrocytes in the corpus callosum compared to wild-type (WT) controls (Fig. 1c, d). However, the oligodendrocyte-specific Perk mutant Jimpy animals (Jimpy; Perkfl/fl; Olig2-Cre) had a substantially higher density of ASPA+ oligodendrocytes than control Jimpy mice (Jimpy; Perkfl/fl). A similar change was found in newly differentiating oligodendrocytes (BCAS1+Olig2+)(Supplementary Fig. 1a, b), whereas the total number of Olig2+ oligodendrocyte lineage cells remained unchanged between Jimpy control and Perk mutant Jimpy animals (Supplementary Fig. 1a, c). Notably, the number of apoptotic oligodendrocyte lineage cells labeled by caspase 3 (Casp3+Olig2+) was significantly reduced in Perk mutant Jimpy mice (Jimpy; Perkfl/fl; Olig2-Cre)(Fig. 1e, f). Together, these data provide further support that PERK deletion in oligodendroglia partially ameliorates the loss of mature and newly differentiating Jimpy oligodendrocytes.

Jimpy mutant mice display severe CNS hypomyelination 39, which is reflected by minimal myelin basic protein (MBP) immunoreactivity in the corpus callosum of control Jimpy mice (Jimpy; Perkfl/fl)(Fig. 1c). In contrast, oligodendrocyte-specific Perk mutant Jimpy animals (Jimpy; Perkfl/fl; Olig2-Cre) exhibited higher MBP immunoreactivity, suggestive of increased myelination (Fig. 1c). Consistent with this possibility, at the electron microscopic (EM) level, myelin was absent in the Jimpy mice on a Perkfl/fl background. In contrast, myelinated sheaths were observed in Jimpy mice lacking Perk (Jimpy; Perkfl/fl; Olig2-Cre), though reaching only ~10% of the axons myelinated in WT mice (3 × 105/mm2) (Fig. 2a, b). Consistently, a large number of degenerating axons labeled by the DegenoTag antibody 40, which specifically recognizes a neoepitope in the neurofilament light chain that becomes exposed during axonal degeneration 39 and demyelination 41, were present in the brains of Jimpy; Perkfl/fl mice, while a significant reduction in Degenotag+ axons was observed in Jimpy; Perkfl/fl; Olig2-Cre animals (Fig. 2c, d). Moreover, Western blots revealed that a significant increase in myelin-associated glycoprotein (MAG) and MBP in the oligodendrocyte-specific Perk KO Jimpy animals compared to control Jimpy mice (Fig. 2e–g). Similarly, real time quantitative PCR (RT-qPCR) studies showed a comparable partial rescue of Mbp and Mag mRNA levels when Perk is deleted on the Jimpy background (Fig. 2h). Together, these data indicate that inhibition of the PERK pathway overcomes some of the detrimental effects of mutant PLP expression in Jimpy mice, allowing partial CNS myelination.

PERK inactivation increases Jimpy OPC proliferation and reduces astrogliosis
Oligodendrocyte precursor cells (OPCs) are distributed throughout the CNS in a uniform, tiled manner where they account for ~5% of all cells 42. OPCs represent the only mitotic stage in the oligodendrocyte lineage and have the capacity to replenish lost oligodendrocytes while maintaining OPC numbers 42,43. To determine whether the increased survival of oligodendrocytes in Jimpy mice with a diminished ISR response affects the OPC population, we quantified OPC density in the corpus callosum of Jimpy oligodendrocyte-specific Perk mutants (Fig. 3), through immunostaining with anti-platelet-derived growth factor receptor alpha (PDGFRα). There was no significant difference in OPC density between the groups at PND19 (Fig. 3a, b). OPCs exhibit homeostasis and proliferate to maintain a constant density when their numbers are reduced. To determine if the inability to form oligodendrocytes in Jimpy mice leads to persistent recruitment of OPCs and enhanced homeostatic proliferation, we assessed OPC proliferation using immunostaining against Ki67. The density of proliferating OPCs (PDGFRα+Ki67+ cells) in the Jimpy samples (24/mm2) was about 69% lower than WT controls (77/mm2) (Fig. 3a, c). This reduction was partially rescued in the oligodendrocyte-specific Perk mutant Jimpy animals, with the density of the double positive cells increasing to 51/mm2, reaching 67% of the mitotic OPCs (PDGFRα+Ki67+) observed in WT animals. When normalized to the total number of PDGFRα+ OPCs, the percentage of proliferating OPCs was not significantly different from controls, indicating that OPCs in Jimpy oligodendrocyte-specific Perk mutants remain highly mitotically active (Fig. 3d). On the WT background, the oligodendrocyte-specific Perk mutation (Perkfl/fl; Olig2-Cre) does not change the populations of OPCs (Supplementary Fig. 2), suggesting its effects are specific to the context of PLP deficiency. These results suggest that the rate of OPC proliferation (and homeostatic replacement) is reduced in Jimpy mice and that this change is partially rescued when Perk is deleted.

CNS hypomyelination in PMD is often accompanied with neuroinflammation, which has been suggested to play a detrimental role in disease progression 44. Enhanced microglial activation (IBA1+) and astrogliosis (GFAP+) were observed in control Jimpy mice (Supplementary Fig. 3). Similar numbers of hypertrophic microglia were observed in the Jimpy mice with the Perk mutation (Supplementary Fig. 3a, c); however, the number of GFAP+ astrocytes was significantly reduced in Jimpy mice lacking Perk in oligodendrocytes (Supplementary Fig. 3b, d). Together, these results suggest that Perk deletion in oligodendroglia correlates with enhanced OPC differentiation in Jimpy mice and reduced astrocytic reactivity, thereby partially alleviating the neuroinflammatory pathology.

Downstream ISR inactivation fails to correct Jimpy oligodendrocyte deficits
GADD34 and CHOP are downstream ISR target genes, and RT-qPCR analysis revealed a significant upregulation of Gadd34 and Chop expression in the CNS of Jimpy mice (Supplementary Fig. 4a). GADD34 is the regulated co-factor of the protein phosphatase 1 (PP1) that dephosphorylates p-eIF2α to return stressed cells to proteostasis 45, and its increased expression upon stress contributes to the termination of the ISR. Previously, we have shown that oligodendrocytes from Gadd34 mutant mice, which display a prolonged ISR response, are more resistant to the presence of IFN-γ in vitro and in vivo 46. We also have found that Gadd34 mutant mice display an ameliorated experimental autoimmune encephalomyelitis (EAE) disease course that correlates with reduced oligodendrocyte death 27. To determine if a prolonged ISR response impacts the viability of Jimpy oligodendrocytes, we crossed the Jimpy mutation onto a homozygous mutant Gadd34 background (Gadd34ΔC/ΔC). The lifespan of Jimpy;Gadd34 mutants (Jimpy; Gadd34ΔC/ΔC) was indistinguishable from that of Jimpy; Gadd heterozygous (Supplementary Fig. 4b, c). There was also no difference in the density of CC1+ oligodendrocytes at PND19 in Jimpy mice with the Gadd34 mutation (Supplementary Fig. 4d, e), in contrast to the observed protection of oligodendrocytes upon prolongation of the ISR in inflammation-mediated demyelination models 27,46.

CHOP is a transcription factor that is activated in response to ER stress and other stressors that induce the ISR. CHOP has been shown to contribute to apoptosis that occurs in response to prolonged, unmitigated ER stress 47. To examine the impact of CHOP in Jimpy oligodendrocytes, we crossed the Jimpy mutation onto a homozygous Chop null background 48. The overall lifespan of the Jimpy mutants was slightly increased by the Chop mutation (Supplementary Fig. 4b). Although consistent, the average survival increased by less than 2 days in Jimpy; Chop−/− mutants (22.4 days) over that of Jimpy; Chop heterozygous (20.7 days) (Supplementary Fig. 4c). However, there was no increase in the density of oligodendrocytes in Jimpy mice when CHOP was deleted (Supplementary Fig. 4d, e).

Pharmacological ISR attenuation increases Jimpy myelination and lifespan
Phosphorylated eIF2α, which occurs as a result of PERK activation, interacts with and inhibits the guanine exchange factor eIF2B, resulting in diminished protein synthesis, and increased expression of the transcription factor ATF4 and its target genes 49. Small molecule activators of eIF2B (such as ISRIB and 2BAct) have the ability to rescue protein synthesis and attenuate the ISR by allosterically antagonizing phosphorylated eIF2α, thus desensitizing cells from stress 50,51. We next evaluated whether pharmacological ISR attenuation with 2BAct impacts the lifespan of Jimpy mutant mice and viability of Jimpy oligodendrocytes. Jimpy female carrier mice were mated with WT males and fed chow containing 100 μg/mg of 2BAct, which has been shown to rescue eIF2B activity, attenuating the ISR 52,53. Following birth, the dams and pups were continuously maintained on the 2BAct diet. Jimpy males on the control diet displayed the tremoring phenotype typical of the mutant animals and lived an average of 18.1 days. In contrast, Jimpy mice on the 2BAct diet, while still presenting tremor, survived approximately twice as long as Jimpy mice on the control diet (Fig. 4a, b). This increased survival was associated with an increased number of ASPA+ oligodendrocytes in the white matter area of corpus callosum, cerebellum and brainstem of Jimpy + 2BAct mice at PND19 (Fig. 4c, e, and Supplementary Fig. 5a, b, e, f). Consistently, myelinated axons were absent from these regions in Jimpy mice, but detectable in Jimpy mice on the 2BAct diet (Fig. 4d, f, and Supplementary Fig. 5c, d, g, h). Furthermore, myelin protein gene expression was significantly increased at both PND14 and PND19 in the Jimpy mice treated with 2BAct (Supplementary Fig. 6). In contrast to the Jimpy; Perkfl/fl; Olig2-Cre mice, no differences in the density of PDGFRα+ or NG2+ OPCs, nor OPC proliferation, as determined by Ki67 staining and EdU incorporation, were observed in the 2BAct-treated Jimpy animals at PND 19 (Fig. 5).

RNAseq pathways: Perk rescues myelination whereas 2BAct targets inflammation
To gain insight into the molecular mechanisms underlying the effect of oligodendrocyte-specific Perk mutants on Jimpy mice, we performed bulk RNAseq analysis of brain samples from Perkfl/fl, Perkfl/fl;Olig2-Cre, Jimpy; Perkfl/fl mice and Jimpy; Perkfl/fl; Olig2-Cre mice at PND19. There were 893 significantly upregulated genes and 629 significantly downregulated genes in Jimpy mice on the Perkfl/fl background (Jimpy; Perkfl/fl) compared to Perkfl/fl mice, and the introduction of Olig2-driven PERK knockout into Jimpy mice elicited significant upregulation of 112 genes and significant downregulation of 22 genes as compared to Jimpy mice (Jimpy; Perkfl/fl) (Fig. 6a, b, and Supplementary Data 1). Notably, several myelin sheath-related genes (Mbp, Plp1, Mag, Mog and Mobp) as well as genes related to oligodendrocyte terminal differentiation (Klk6, Opalin, and Ermn) were downregulated in Jimpy mice (Jimpy; Perkfl/fl) but rescued by the oligodendrocyte-specific PERK knockout in Jimpy Perkfl/fl; Olig2-Cre mice (Fig. 6c). Indeed, gene set enrichment analyzes indicated that the top downregulated gene sets in Jimpy mice (Jimpy; Perkfl/fl) compared to Perkfl/fl control mice included those related to oligodendrocytes, glial cell differentiation, and cholesterol biosynthesis, and these changes were rescued by the oligodendrocyte-specific PERK knockout (Fig. 6d, e, and Supplementary Data 2). Notably, we did not observe significant changes in the ER stress pathways and UPR response in Jimpy mice, perhaps because so few oligodendrocytes remain at this stage. The oligodendrocyte-specific PERK knockout Jimpy mice, however, showed upregulated gene sets involved in ER protein folding. This includes several target genes from the ATF6 pathway (Hspa5 and Hsp90b1) and the IRE1 pathway (Hspa5 and P4hb), the other arms of the UPR (Fig. 6f). In line with these findings, increased immunofluorescent intensities of ATF6, GRP78 (Hspa5) and GRP94 (Hsp90b1) were observed in Olig2+ cells of Jimpy mice lacking Perk in oligodendrocytes, with GRP78 and GRP94 reaching significance (Supplementary Fig. 7a–c, e–g). To assess ISR pathway activation, we queried the expression of a panel of 95 ISR target genes identified by Clustering by Inferred Co-Expression (referred to as ISRCLIC) 53. We found that the ISR CLIC gene set was significantly upregulated in Jimpy; Perkfl/fl mice compared to control mice and downregulated by PERK knockout in Jimpy Perkfl/fl; Olig2-Cre mice (Fig. 6e). Consistently, the volcano plots of ISR CLIC genes showed that the ISR gene Trib3 was upregulated in Jimpy; Perkfl/fl mice compared to Perkfl/fl control mice and downregulated in Jimpy Perkfl/fl; Olig2-Cre mice. The significant changes were also reflected in immunostaining (Supplementary Fig. 7d, h). In contrast, Atf3 was upregulated in Jimpy; Perkfl/fl mice and further increased in Jimpy Perkfl/fl; Olig2-Cre mice (Fig. 6g, h, and Supplementary Data 3). The upregulation of ATF3 has been associated with inflammation that is not always dependent on ISR induction 53.

We also characterized the effect of pharmacologically inhibiting the ISR in Jimpy mice through RNAseq analysis of brain samples from WT and Jimpy mice on control or 2BAct-supplemented diets at PND19. We identified 107 significantly upregulated genes and 315 significantly downregulated genes in Jimpy mice on control diet vs WT mice on control diet (Fig. 7a, and Supplementary Data 4). When Jimpy mice were on the 2BAct diet, 91 genes were significantly upregulated, and 98 genes significantly downregulated compared to control diet (Fig. 7b, and Supplementary Data 4). Though several myelin-related oligodendrocyte terminal differentiation genes were significantly rescued by 2BAct diet in Jimpy mice, the extent of these fold changes was relatively small compared to the rescue observed in oligodendrocyte-specific PERK mutant Jimpy mice (Figs. 6c and 7c). Notably, gene set enrichment analysis indicated that 2BAct significantly reversed inflammatory responses affected by the Jimpy mutation, including immature neutrophils and interferon gamma and interferon alpha pathways (Fig. 7d, e, and Supplementary Data 5). Consistent with these findings, immunostaining of the corpus callosum showed that 2BAct significantly reduced microglial activation at PND19, although no remarkable change was observed in astrocytes of Jimpy mice (Supplementary Fig. 8). However, unlike the oligodendrocyte-specific PERK mutant, 2BAct did not rescue the gene sets associated with oligodendrocytes, glial cell differentiation, or cholesterol homeostasis, (Figs. 6d, e, and 7d, e). Like the PERK mutant, 2BAct significantly downregulated the ISR CLIC gene set (Fig. 7e–g, and Supplementary Data 6).

In addition, we analyzed the differentially expressed genes in Jimpy mice at earlier timepoints, PND7 and PND14, on 2BAct-medicated versus control diets (Supplementary Fig. 9). These analyzes indicate that there are more significantly changed genes at PND7 (n=735 in Jimpy control vs WT control diet; n=2056 in Jimpy 2BAct vs Jimpy control diet) and PND14 (n=5558 in Jimpy control vs WT control diet; n=5217 in Jimpy 2BAct vs Jimpy control diet) compared to PND19 (Supplementary Fig. 9a–d, and Supplementary Data 4). At PND7, cholesterol biogenesis, as well as hallmark cell cycle-related gene sets, such as E2F transcription factor target sets and G2M checkpoint genes were upregulated in 2BAct-treated Jimpy mice as compared to control diet (Supplementary Fig. 9e, g, and Supplementary Data 5). At PND14, the most significantly upregulated gene sets in Jimpy mice included development-related sets such as MYC targets and gene sets related to ion channel activities. Treatment with 2BAct significantly attenuated the expression of these gene sets in Jimpy mice (Supplementary Fig. 9f, h, and Supplementary Data 5). Notably, and in contrast with PND19, the ISR CLIC genes as a set did not show significant enrichment in Jimpy mice over WT mice on a control diet at PND7 and PND14 (Fig. 7e), nor was the signature significantly altered by 2BAct diet in Jimpy mice. However, many individual ISR genes that were significantly up- or downregulated in Jimpy mice versus WT mice on control diets were significantly reversed in Jimpy mice on the 2BAct-supplemented diet (Supplementary Fig. 10a–f, and Supplementary Data 6).

Together, the RNAseq data demonstrated that both oligodendrocyte-specific Perk inactivation and 2BAct can attenuate the upregulated ISR pathways in Jimpy mice at PND19. In addition, the Perk mutation rescued the gene sets related to oligodendrocyte markers, glial cell differentiation, and cholesterol homeostasis, while 2BAct partially attenuated these markers but mainly reversed inflammation related responses in PND19 Jimpy mice.

ISR inhibition promotes Jimpy oligodendrocyte maturation in vitro
To further assess the impact of cell autonomous eIF2B activation on Jimpy oligodendrocytes, we examined oligodendrocyte differentiation in vitro in the presence ISRIB, which is a 2BAct analog that has a longer half-life in cell culture media 54. OPCs were isolated from postnatal day 5 to 7 Jimpy pups and, after expansion in proliferation media supplemented with PDGFA, were placed in differentiation media containing either vehicle or increasing concentrations of ISRIB (1 μM, 2.5 μM or 5 μM). After 48 h (day 2), Jimpy oligodendrocytes in either control or ISRIB-containing differentiation media expressed MBP and extended branched processes (Supplementary Fig. 11a). However, by 72 h (day 3), few MBP+ oligodendrocytes were present in the Jimpy control cultures, whereas the ISRIB treated cells displayed numerous MBP+ cells that continued to mature over the next 2 days of culture (Fig. 8, and Supplementary Fig. 11a). The ISRIB rescue of the Jimpy oligodendrocytes occurred in a dose-dependent manner with the most significant changes occurring at a concentration of 5 μM ISRIB (Fig. 8b, c). At day 4 and day 5 in differentiation media, Jimpy cultures treated with 5 μM ISRIB contained over 50% of the number of MBP+ oligodendrocytes observed in the WT cultures (Fig. 8a–c). Notably, ISRIB did not alter the density of oligodendrocytes in WT cultures (Supplementary Fig. 11b, c). These data demonstrate a cell-autonomous impact of ISR inhibition on oligodendrocyte differentiation and survival.

Methods
Mouse lines and survival study
All animal experiments were executed in accordance with the National Institutes of Health guidelines for the Care and Use of Laboratory Animals and with the approval of the Institutional Animal Care Committee of Northwestern University Feinberg School of Medicine (IS00027307). Heterozygous Jimpy (Plp1jp) female mice were kindly provided by Paul Tesar, and the Perk heterozygous, Chop null and Gadd34ΔC/ΔC mice were a generous gift of David Ron. The Perkfl/fl mice were obtained from Douglas Cavener, and the Olig2-Cre mice were a gift from David Rowitch. Jimpy female mice were crossed with either Perk+/−, Perkfl/fl, Chop−/− or Gadd34ΔC/ΔC to generate Jimpy;Perk+/−, Jimpy;Perkfl/fl, Jimpy;Chop−/−, and Jimpy;Gadd34ΔC/ΔC mice. Jimpy;Perkfl/fl mice were then crossed with Olig2-Cre mice to produce Jimpy;Perkfl/fl; Olig2-Cre, in which the Olig2 promoter driven Cre recombinase inactivates Perk specifically in oligodendrocytes. All mice were housed in temperature-controlled specific pathogen–free facilities under 12-h light–dark cycles in the Center for Comparative Medicine at Northwestern University. Only male mice were used in the experiments. The survival study endpoint was defined by the signs of moribundity, including but not limited to lateral recumbency or immobility, lack of response to stimulation, inability to eat or drink and hypothermia 71.

Drugs
eIF2B activators 2BAct and ISRIB were provided by Calico Life Sciences LLC. Pregnant female Jimpy mice were exposed to 100 mg/kg of 2BAct in chow, with exposure continuing for both the dams and pups after birth. Identical animal chow without 2BAct was used as control. ISRIB was first dissolved in DMSO, and final concentrations of working solution in cell culture medium were 1 μM, 2.5 μM and 5 μM.

OPC isolation and cell culture
OPCs were isolated and purified following an immunopanning method 72. Briefly, mouse brain cortices from 5 to 7 Jimpy or WT pups were collected and digested with papain (#9001734, Worthington) at 37°C. Cells were triturated, suspended, and sequentially immunopanned on plates coated with Ran-2, GalC, and O4 antibodies from a hybridoma supernatant. OPCs adhering to the anti-O4 coated plate were trypsinized and seeded on poly-D-Lysine-coated plates in growth media supplemented with PDGFRA (#100-13A, PeproTech) for proliferation. After expansion, cells were plated into differentiating medium supplemented with T3 (#T6397, Sigma) in the presence or absence of ISRIB.

Immunofluorescence and immunocytochemistry
Male mice were anaesthetized by 1.2% avertin and transcardially perfused first with 0.9% NaCI followed by 4% paraformaldehyde (PFA) (#158127, Sigma). Brains were harvested, post-fixed with 4% PFA overnight and embedded in O.C.T compound (#4853, Sakura Finetek). Sagittal cerebral frozen sections were prepared in a series of 10 μm on a cryostat. Tissue sections or fixed cell culture were incubated with primary antibodies against ASPA (1:1000; #GTX13389, Genetex), MBP (1:50; #ab24567, Abcam), PDGFRα (1:50; #AF1062, R&D), Ki67 (1:100; #ab15580, Abcam), CC1 (1:100; OP80, Millipore), NG2 (1:100, Dwight Bergles’s house antibody), Olig2 (1:500; #AB9610, Millipore), NF-L-DegenoTag (1:1000; #MCA-6H63, Encor Biotech), IBA1 (1:500, #81728-1RR, Proteintech), GFAP (1:200, #AB5541, Millipore), cleaved caspase 3 (1:400, #9661S, Cell signaling), BCAS1 (1:1000, #445003, Synaptic Systems), ATF6a (1:50, #sc-166659, Santa Cruz), GRP94 (1:200, #10979-1-AP, Proteintech), Bip (1:100, #sc-166490, Santa Cruz), and TRIB3 (1:50, #A01414, Boster Bio) followed by appropriate species-specific fluorescent labeled secondary antibodies mounting and cover-slipping with HardSet™ Antifade Mounting Medium with DAPI (#H-1500-10, VECTASHIELD). The sections were scanned with a fluorescent research slide scanner (VS200, Evident). For tissue section staining, the number of immunopositive cells from at least five serial brain sections was quantified in each animal. For oligodendrocyte culture staining, numbers of MBP+ cells were counted in multiple image fields of culture from at least 3 different animals for each condition. The quantification was performed on Image J analysis software (NIH Image software) and Halo image analysis software (Indica Labs). The representative images were acquired under a Zeiss LSM880 confocal microscope.

EdU labeling
Labeling of dividing OPCs was carried out by intraperitoneal injection of EdU (5-ethynyal-2-deoxyuridine, #900584, Sigma) into Jimpy mice at 5 mg/kg body weight on PND 17 and PND18. Brain tissues were harvested on PND19. EdU staining was performed free-floating by treating the 35 μm of frozen section at room temperature with the EdU Click-it reaction kit (#C10340, Molecular Probes) and the Alexa fluor 647 dye (#A20006, ThermoFisher) for 30 min after the standard immunofluorescence.

Electron microscopy (EM)
Male mice were anaesthetized by 1.2% avertin and transcardially perfused with EM fixative made of 2.5% glutaraldehyde (#00710-5, Polysciences), 4% PFA in 0.1 M sodium cacodylate (#11652, EMS) buffer at pH 7.4. After 2 weeks of fixation at 4°C, brains were dissected, washed with 0.1 M sodium cacodylate and then postfixed in 1% osmium tetroxide (#19152, EMS) in 0.1 M sodium cacodylate. Sections were then dehydrated in a series of graded ethanol followed by incubation in propylene oxide (PO, #20401, EMS). Samples were then infiltrated with increasing ratio of PO:Epon-812 resin mixture (1:1 and 1:2, 2 h each), following by immersion of 100% Epon-812 resin mixture overnight. Epon 812 resin mixture was prepared using EMBed-812 (#14900, EMS), DDSA (#13710, EMS), NMA (#19000, EMS), and BDMA (#11400, EMS). Tissue were embedded in fresh Epon-812 resin mixture and polymerized at 60°C for 48 h. Semi-thin sections (1 μM thickness) were stained with toluidine blue, and then samples were ultrathin (60–90 nM thickness) sectioned on a Leica EM UC7 ultramicrotome. Grids containing ultrathin sections were examined on a FEI Tecnai Spirit G2 transmission electron microscope. Axon counts were performed using Image J and averaged from 10 images (area per image = 240 μm2) in each mouse.

Western-blot
Male mice were anaesthetized by 1.2% avertin and transcardially perfused with ice-cold PBS and then homogenized in cold RIPA buffer (#R0278, Sigma) containing Halt Protease and Phosphatase Inhibitor Cocktail (#PI78441, Thermo Fisher Scientific) with a motorized homogenizer. Lysates were centrifuged at 13,000 × g for 20 min at 4°C. A total 30 μg of protein lysates was separated by SDS-PAGE (#4561095, Bio-Rad) and transferred to a nitrocellulose membrane (Fig. 2e). The blots were incubated with the following primary antibodies: anti-MBP (1:1000; #808403, BioLegend), anti-MAG (1:1000; #346200, Invitrogen), and anti-β-actin (1:2000; # A2066, Sigma-Aldrich). The blots were then incubated with an HRP-conjugated secondary antibodies (1:5000; Invitrogen) and the signals were detected by chemiluminescence on a Bio-Rad ChemiDocTM Imager. The bands were analyzed using Image Lab Software (Bio-Rad).

RT-qPCR
Total RNA was isolated from brain tissue samples of PND19 mice using AurumTM total RNA fatty and fibrous tissue kit (#7326830, Bio-Rad). Reverse transcription was performed using the iScript cDNA synthesis kit (#1708891, Bio-Rad). Quantitative real-time reverse transcription PCR was performed on a CFX96 RT-PCR detection system using SYBR Green technology (#1725271, Bio-Rad). The relative gene expression levels were normalized to that of the housekeeping gene Gapdh. The primer sequences of the target genes were as follows: Mbp-f: TGTCACAATGTTCTTGAAGAAATGG, Mbp-r: TGTCACAATGTTCTTGAAGAAATGG; Mag-f: CTGCTCTGTGGGGCTGACAG, Mag-r: AGGTACAGGCTCTTGGCAACTG; Cnp-f: CTCTACTTTGGCTGGTT CCTGAC, Cnp-r: GCTTCTCCTTGGGTTCATCTCC; Gapdh-f: TGTGTCCGTCG TGGATCTGA; Gapdh-r: TTGCTGTTGAAGTCGCAGGA.

RNAseq and analysis
To analyze the effect of Perk mutation on Jimpy mice, mouse brains from four different mouse lines (Perkfl/fl, Perkfl/fl;Olig2-Cre, Jimpy; Perkfl/fl, Jimpy; Perkfl/fl; Olig2-Cre) were harvested at PND19. To analyze the 2BAct effect on Jimpy mice, mouse brains from four groups of mice (WT control diet, WT 2BAct, Jimpy control diet, and Jimpy 2BAct) were harvested at PND 7, PND 14 and PND 19. RNA was extracted from brain tissues, and the stranded total RNA-seq was conducted in the Northwestern University NUSeq Core Facility. Briefly, total RNA examples were checked for quality on Agilent Bioanalyzer 2100 and quantity with Qubit fluorometer. The Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit was used to prepare sequencing libraries. The kit procedure was performed without modifications. This procedure includes rRNA depletion, remaining RNA purification and fragmentation, cDNA synthesis, 3′ end adenylation, Illumina adapter ligation, library PCR amplification and validation. lllumina HiSeq 4000 Sequencer was used to sequence the libraries with the production of single-end, 50 bp reads. The quality of DNA reads, in fastq format, was evaluated using FastQC. Adapters were trimmed, and reads of poor quality or aligning to rRNA sequences were filtered. The cleaned reads were aligned to the Mus musculus genome (mm10) using STAR 73. Read counts for each gene were calculated using htseq-count 74 in conjunction with a gene annotation file for mm10 obtained from UCSC (University of California Santa Cruz; http://genome.ucsc.edu). For differential gene expression analysis, we used the limma-voom pipeline 75 to normalize data, define contrasts, and to fit a linear model. edgeR’s “filterByExpr” function was used to determine genes with sufficiently large counts to retain for statistical analysis, and the “calcNormFactors” function was used to calculate TMM scaling factors for each sample. Observational-level weights were computed using limma’s “voomWithQualityWeights” function to prepare data for linear modeling using the “lmFit” and “eBayes” functions. Differential expression results were used as input for the “seas” function (with parameters: methods=‘camera’, feature.max.padj = 0.05, feature.min.logFC = 0.5, inter.gene.cor = 0.01, feature.bias=“size”) in the sparrow R package 76 to test for enrichment of Hallmark, C2, and C5 gene sets from the Molecular Signatures Database 77.

Statistical analysis
GraphPad Prism 10 was used to perform statistical analyzes. All results were expressed as mean±SEM. Statistical significance for these data was determined by Log-rank (Mantel–Cox) test, unpaired t-tests/Welch test, one way analysis of variance (ANOVA) followed by Turkey post hoc test for multiple group experiments, two-way ANOVA with Sidak’s multiple comparison test. Significance levels were set at p < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, Chen et al. investigate the impact of ISR inhibition on ameliorating Pelizaeus-Merzbacher disease (PMD) using the Jimpy mouse model. By employing both pharmacological (2Bact) and genetic (PERK conditional KO) ISR inhibition, they demonstrate that blunting the ISR extends lifespan and reverses oligodendrocyte cell death and demyelination characteristic of PMD. The authors also performed a detailed gene expression analysis to identify differentially expressed genes in Jimpy mice compared to controls and ISR-OFF animals. The manuscript is well-written, the experiments are clearly described, and the data convincingly support the authors’ conclusions. Therefore, I support the publication of this manuscript in Nature Communications. However, the manuscript would benefit from addressing the following points: 1. Involvement of GADD34 and CHOP in PMD Progression: The authors assess the roles of GADD34 and CHOP by measuring viability and oligodendrocyte density in Jimpy/GADD34ΔC/ΔC and Jimpy/CHOP-/- mice, concluding that their inactivation does not affect oligodendrocyte deficits. However, whether the ISR feedback loop is fully functional in Jimpy mice remains unclear. What are the expression levels of GADD34 and CHOP in Jimpy oligodendrocytes? Is ISR signaling at its maximum in these cells, or could depletion of CReP further impact oligodendrocyte viability? Clarification on these points would strengthen the findings. 2. PLP1 Levels in ISR-OFF Jimpy Mice: PMD is a gain-of-function disease in which the misfolded PLP1 protein accumulates in the endoplasmic reticulum. Since ISR inhibition enhances protein synthesis, it would be expected to increase the accumulation of the toxic PLP1 protein. Despite this, Jimpy mice show an improved phenotype when ISR signaling is ablated. The authors should clarify the impact of ISR inhibition on PLP1 levels in Jimpy mice. This data would help reconcile the apparent paradox between PLP1 accumulation and phenotypic improvement. 3. Cell-autonomous Efficacy of ISR Inhibition: The authors show pharmacological ISR inhibition's cell-autonomous efficacy in primary cultured oligodendrocytes (Figure 8A). Including untreated wild-type cells in the panel and comparing Jimpy-treated oligodendrocytes to wild-type cells would provide valuable context and make the findings more robust. This additional comparison would help to illustrate the relative efficacy of the treatment better. 4. Functional Testing in Mice: The manuscript would benefit from functional tests in the mice, such as assessing locomotion in Jimpy mice treated with 2Bact. Including such behavioral or motor function data would provide more comprehensive evidence of ISR inhibition's therapeutic potential and further strengthen the findings.", "reviewer_2": "The paper from Y. Chen, R. Kunjamma et al. is addressing the role of integrated stress response (ISR) in a mouse model of leukodystrophy Pelizaeus-Merzbacher disease (PMD). Authors have combined a well-characterized PMD model (Jimpy mice) with a PERKcKO line (Olig2-cre;Perk-flox) or used pharmacological inhibitors (2BAct or ISRIB) to study the impact of ISR inactivation on oligodendroglial cell lineage, myelination and lifespan. By analyzing bulk RNA-Sequencing data, the authors have highlighted the differentially regulated pathways, after ISR inactivation, during development (post-natal day 7, P14, P19). Currently, there are no efficient treatment for PMD, a really disabling disease. Based on the authors previous work showing a protective effect of ISR manipulation in demyelinating models and the current clinical trial for another leukodystrophy, this work tests an interesting and promising hypothesis. However, at the moment, the manuscript misses some essential data to confirm their results. And unfortunately, the overall gain from the ISR inhibition tends to be limited in their models. - In their first model, the authors cross the Jimpy line with a PERKcKO line (Olig2-cre;Perk-flox). They mention that “PERK is dispensable for normal oligodendrocyte development and CNS myelination” (l.139). However, in their previous work, the Perk-flox has been crossed with a Cnp-cre line, which is targeting a later stage of the oligodendroglial lineage. It is important to add in the current manuscript the Olig2-cre;Perk-flox line as control, for all IHC quantifications and RNA-Sequencing. Is Perk ablation in OLIG2 cells affecting the oligodendroglial cell lineage (OPC and OL population, survival and proliferation of OPC, myelination)? In the Cnp-cre;Perk-flox line, the authors noticed that an increased susceptibility to inflammation. Is inflammation changed in their current Olig2-cre;Perk-flox line? - What is the efficiency of the recombination in the (Jimpy-)PERKcKO line? Could it explain the low effect in this model (25% of OLs, only 10% of myelinating axons)? - Currently, the characterization of oligodendroglial cell lineage by IHC is limited in the Jimpy-PERKcKO line. The quantification of the whole oligodendroglial population (OLIG2) could help distinguish which stage of the lineage is affected (whole population vs. OPC or OL). Authors should add survival and death quantification if they want to support their claims. Jimpy-PERKcKO mice tend to present a decrease (trend) in the number of PDGFRa+ OPC (Fig.3b). Could this explain the relative increase of Ki67+ cells, to maintain OPC homeostatic number (Fig.3c)? What is the percentage of PDGFRa+ and Ki67+ (among the PDGFRa+ cells)? Are these numbers different in the control PERKcKO only? - In conclusion, the authors states “the rate of OPC differentiation” (l.192), but they have quantified PDGFRa and Ki67 stainings here. Do they mean “differentiation”? Additional BCAS1 staining could highlight the number of differentiating OLs, and better show if the switch from cell proliferation to differentiation is impacted. - On the WB (Fig.2c), the quantification of MBP is surprising (50%) compared to the representative blot where MBP signal is really light. Could the authors explain their quantification process? - At the transcriptomic level (qPCR and RNA-Seq) and IHC level (ASPA staining), the pharmacological approach tends to have an earlier effect (P14) and lower effect. Mice lifespan is extended as mush as in the Jimpy-PERKcKO line. Did the authors notice any impact on other cell types, especially on inflammation, which could indirectly affect the oligodendroglial lineage? In l.328, the authors mention that ISRIB has a longer half-life. Did the authors test it in vivo? Could it be more efficient? The proliferation and OPC quantifications reveal a trend, but further points are needed (Fig.5). Authors should increase their n (n=2 for some points), as their data also show strong variability (especially for WT+2BAct samples). - The puncted MBP stainings (Fig.1c and Fig.4c) can not be associated with “increased” myelination (l.159), as further noted by the EM results on Fig.2a. So additional EM for the pharmacological approach is needed. - The impact of ISRIB on OPC proliferation in vitro needs to be quantified to complete Supp.Fig.5 (especially because OPC density in vitro strongly affects their differentiation capacities). Could the authors add the dots for their biological replicates on Fig.8b? Depending on the variability of the in vitro assay, the authors should add 1 or 2 biological replicates. - While extensive, the RNA-Sequencing data do not add much to the current story. Due to the bulk approach, part of the differential expression observed are resulting of different cell population representation in the tissues, as shown by IHC (less oligodendroglial cells for example). As observed previously (by qPCR), the pharmacological approach tends to have a lower effect and earlier effect (P14 and not P19) at the transcriptomic level. Because of the global impact of the drug, adding a cell-specific sequencing could lead to more interesting findings. RNA-sequencing also highlight inflammatory changes between the cKO and pharmacological approaches. Is it confirmed by IHC on tissues? - Over the manuscript, some titles are overselling: “increased oligodendrocyte survival” (l.126) “rescues oligodendrocytes death” when no death assessment has been performed (Fig.1 legend), “rescues myelin loss” of only 10% (Fig.2 legend), “protect Jimpy oligodendrocytes” while only 1/8 of oligodendrocytes compared to controls are detected (l.223), “rescues oligodendrocyte death and demyelination” without death assessment neither EM quantification (Fig.4 legend). Minor points: - The Suppl. Fig. 2 showing the “rescued downregulated” genes (l. 258-262) is not really readable. A similar graph as Fig. 6 would be more useful. - Is the PNS affected in the Jimpy model? To what extend? Is the ISR also involved in Schwann cells? It could be briefly discussed in the discussion, to justify the importance of the pharmacological global approach.", "reviewer_3": "In this study, Chen et al. report that oligodendrocyte condition knockout (cKO) of PERK in Jimpy mutant mice improves oligodendrocyte survival and extends animal lifespan. They observed increased myelination within the corpus callosum, along with elevated levels of OPC proliferation and production of myelin protein components MBP, MAG at the protein and RNA levels. Modest improvements of oligodendrocyte survival were observed with eIF2B activation drug treatment in Jimpy mice, but not with KO of CHOP or GADD34, downstream components of the integrated stress response (ISR). Bulk RNAseq highlights differential gene expression in Jimpy; Perkfl/fl; Olig2-Cre vs Jimpy;Perkfl/fl cKO brains. This is an interesting study, but the following points should be addressed. Major points: 1. It was unclear whether PERK is upregulated in Jimpy and cKO mice OLs compared to WT/controls so immunohistochemistry (IHC) is needed or other references provided. 2. Jimpy pathology includes inflammation, ROS production, astrogliosis, and neuronal loss, so have the authors assessed this compared to Jimpy PERK-cKO mice? 3. The study shows that PERK inactivation increases the proliferation of oligodendrocyte progenitor cells (OPCs) in vivo. The authors suggest that in this context, OPC proliferation is dependent on their capacity to differentiate to maintain a constant OPC density (homeostatic replacement). It is unclear whether increased OPC proliferation is intrinsically driven by PERK mutation. The authors should discuss this point. 4. It remains unclear from this manuscript whether 2BAct extends the lifespan of Jimpy mice by reducing inflammation, increasing myelination, or both. Specifically: a. The 2BAct diet improved the survival of Jimpy and increased oligodendrocytes (OLs) in the corpus callosum. How about other brain regions? It should be relatively easy to show impact in cerebellum or brainstem. Indeed, brainstem is of particular interest because better myelination might help explain improved survival, so this analysis is recommended. b. Because 2BAct enters via the mother's diet, it is not specific to OLs. the authors should confirm by IHC that the drug reduces PERK pathway activity in oligodendrocytes. Indeed, there is no clear evidence that 2BAct improves Jimpy mice survival by promoting myelination, similar to the prior comment further studies to document improved myelination in brainstem or other brain regions is recommended. c. The authors show in Figure 7 that 2BAct treatment reduces the inflammatory response in Jimpy mice at the transcriptional level. How about measures of gliosis by IHC (e.g., Iba1, GFAP in 2BAct-treated Jimpy mice compared to vehicle? 5. RNA sequencing was performed on total brain lysate, which limits conclusions on OPCs/OLs. To validate RNAseq findings, the authors should use ISH or IHC to confirm upregulation of genes involved in ER protein folding (e.g., Hspa5, Hsp90b1, P4hb) or protein markers (e.g., IRE1, ATF6, TRIB3, AKT) in OPCs or OLs. Alternatively, single-cell RNA sequencing would provide this detail. Minor points: 6. Is Jimpy a mouse model of Pelizaeus-Merzbacher disease (PMD)? It is a severe mutation of PLP but does it relate to a PMD patient-specific mutation? Please provide a reference or qualify characterization of Jimpy as a model. 7. Line 182: Instead of \"there was no appreciable difference,\" rephrasing a less subjective statement is needed e.g., \"no significant differences were observed.\" 8. For all figures: The authors should indicate \"n.s.\" (not significant) where applicable to clarify the statistical relevance of the data.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point by point response to the reviewers’ comments. Dear reviewers, We want to express our sincere gratitude for the time and effort that you took to review our manuscript. Your comments have been invaluable in improving the quality of our work. Please find our point-by-point response below. Reviewer(s)' Comments to Author: Reviewer: 1 1. Involvement of GADD34 and CHOP in PMD Progression: The authors assess the roles of GADD34 and CHOP by measuring viability and oligodendrocyte density in Jimpy/GADD34ΔC/ΔC and Jimpy/CHOP-/- mice, concluding that their inactivation does not affect oligodendrocyte deficits. However, whether the ISR feedback loop is fully functional in Jimpy mice remains unclear. What are the expression levels of GADD34 and CHOP in Jimpy oligodendrocytes? Is ISR signaling at its maximum in these cells, or could depletion of CReP further impact oligodendrocyte viability? Clarification on these points would strengthen the findings. Response: We thank reviewer for giving us this opportunity to clarify these points. We have now included the mRNA expression results for GADD34 and CHOP in our revised manuscript in Suppl. Figure 4a. The expression levels of both GADD34 and CHOP were significantly increased in Jimpy mice compared to WT. CReP is a constitutive regulatory subunit of protein phosphatase 1. Depletion of CReP leads to enhanced eIF2a phosphorylation and a prolonged ISR. Given that inactivating PERK and thereby inhibiting the ISR is beneficial, we believe that depletion of CReP would most likely exacerbate the Jimpy phenotype. 2. PLP1 Levels in ISR-OFF Jimpy Mice: PMD is a gain-of-function disease in which the misfolded PLP1 protein accumulates in the endoplasmic reticulum. Since ISR inhibition enhances protein synthesis, it would be expected to increase the accumulation of the toxic PLP1 protein. Despite this, Jimpy mice show an improved phenotype when ISR signaling is ablated. The authors should clarify the impact of ISR inhibition on PLP1 levels in Jimpy mice. This data would help reconcile the apparent paradox between PLP1 accumulation and phenotypic improvement. Response: We thank reviewer for raising this critical point. We agree that ISR inhibition should increase protein synthesis, including mutant PLP1. However, we believe that the benefit of ISR inhibition comes not from modulating synthesis of the toxic protein itself, but from alleviating the maladaptive consequences of chronic ISR activation. In Jimpy mice, we propose that sustained and excessive ISR activation becomes maladaptive, leading to the apoptosis of oligodendrocytes. In addition, a recent study using a vanishing white matter disease model indicates that the sustained ISR leads to dysfunction of cholesterol synthesis, affecting oligodendrocyte maturation (Lin K et al., JCI insight., 2025). By inhibiting the ISR, we mitigate this programmed cell death pathway and rescue cholesterol synthesis, thereby preserving oligodendrocyte survival and maturation. While ISR inhibition may permit continued synthesis of mutant PLP1, preserving a larger pool of viable oligodendrocytes provides a critical therapeutic window. We have revised the discussion to address this paradox (Page 14, line 412-422). 3. Cell-autonomous Efficacy of ISR Inhibition: The authors show pharmacological ISR inhibition's cell-autonomous efficacy in primary cultured oligodendrocytes (Figure 8A). Including untreated wild-type cells in the panel and comparing Jimpy-treated oligodendrocytes to wild-type cells would provide valuable context and make the findings more robust. This additional comparison would help to illustrate the relative efficacy of the treatment better. Response: We thank reviewer for this suggestion. We have now included the WT cultures in revised Fig. 8 and Suppl Fig. 11. We found that primary Jimpy cultures treated with 5 μM ISRIB displayed greater than 50% of the MBP+ oligodendrocytes observed in WT culture (Fig. 8b). ISRIB did not alter the density of oligodendrocytes in WT cultures (Suppl Fig. 11b,c). 4. Functional Testing in Mice: The manuscript would benefit from functional tests in the mice, such as assessing locomotion in Jimpy mice treated with 2Bact. Including such behavioral or motor function data would provide more comprehensive evidence of ISR inhibition's therapeutic potential and further strengthen the findings. Response: We thank the reviewer for this suggestion to include quantitative motor assessment data. In response we have tried several different approaches. The treated Jimpy mice do not visually appear different than untreated Jimpy mice in their motor defects. We tried videotaping the mice in slow motion in an effort to detect differences in tremor frequency, but we were unable to identify any differences using this approach. Furthermore, we attempted to quantify the tremor using an acoustic startle response assay, but this assay was also insufficiently sensitive to detect differences. Lastly, we weighed the mice daily under the premise that reduced tremor might correlate with increased weight gain, but we did not detect differences. Unfortunately, the premature death of untreated Jimpy mice (~PND 21) presents a challenge to most motor assays. Therefore, the prolonged survival of the 2BAct treated and Perk mutant Jimpy mice (Fig. 4a,b) represents the only functional improvement that we have been able to document. Nevertheless, we believe that increased survival is a critically important parameter for outcome. Reviewer: 2 1. In their first model, the authors cross the Jimpy line with a PERKcKO line (Olig2-cre;Perk-flox). They mention that “PERK is dispensable for normal oligodendrocyte development and CNS myelination” (l.139). However, in their previous work, the Perk-flox has been crossed with a Cnp-cre line, which is targeting a later stage of the oligodendroglial lineage. It is important to add in the current manuscript the Olig2-cre;Perk-flox line as control, for all IHC quantifications and RNA-Sequencing. Is Perk ablation in OLIG2 cells affecting the oligodendroglial cell lineage (OPC and OL population, survival and proliferation of OPC, myelination)? In the Cnp-cre;Perk-flox line, the authors noticed that an increased susceptibility to inflammation. Is inflammation changed in their current Olig2-cre;Perk-flox line? Response: We thank the reviewer for their comments and suggestions. Our lab previously reported that both the Cnp-cre; Perk flox line (Hussien et al., Glia, 2014) and the Olig2-cre; Perk flox line (Clayton et al., J. Neurosci., 2017) have no effect on developmental myelination, suggesting that PERK activity is not required for oligodendrocyte development or CNS myelination; As such, we did not include the Olig2-cre line as a separate control. In the revised manuscript, we showed that Perk ablation in Olig2 expressing cells in Jimpy mice significantly increased the number of ASPA+ mature OLs and newly formed OLs labeled by BCSA1 without altering the total number of Olig2+ lineage cells (Fig. 2c,d; Suppl Fig. 1). We also observed an increased portion of proliferating OPCs (Ki67+Pdgfra+), although the overall reduction of OPCs is not significant (Fig. 3). Furthermore, our EM analysis, along with analyses of myelin-related proteins (Fig. 2a,b, e-h), indicate an increase in myelinated axons and enhanced myelin-related proteins. Inflammation is changed in the Olig2-cre; Perk-flox line. In the revised manuscript, we observed a significant reduction of GFAP+ astrocytes in Jimpy mice lacking Perk in oligodendrocytes, although the hypertrophic active microglia remained unchanged (Suppl Fig. 3). 2. What is the efficiency of the recombination in the (Jimpy-)PERKcKO line? Could it explain the low effect in this model (25% of OLs, only 10% of myelinating axons)? Response: We thank reviewer for giving us the opportunity to clarify this point. The Olig2-Cre line is a robust driver of LoxP recombination. We have previously reported that recombination efficiency of Olig2-Cre line is as high as 79.6 ± 11.6%, as determined in the Olig2-Cre; ROSA26-YFP report line (Clayton et al., J. Neurosci., 2017). Therefore, the moderate rescue observed in the Jimpy model is unlikely due to inefficient recombination. We believe that the ability of the PERK deletion to rescue oligodendrocytes and myelination is limited due to the profound severity of the Jimpy phenotype. 3. Currently, the characterization of oligodendroglial cell lineage by IHC is limited in the Jimpy-PERKcKO line. The quantification of the whole oligodendroglial population (OLIG2) could help distinguish which stage of the lineage is affected (whole population vs. OPC or OL). Authors should add survival and death quantification if they want to support their claims. Jimpy-PERKcKO mice tend to present a decrease (trend) in the number of PDGFRa+ OPC (Fig.3b). Could this explain the relative increase of Ki67+ cells, to maintain OPC homeostatic number (Fig.3c)? What is the percentage of PDGFRa+ and Ki67+ (among the PDGFRa+ cells). Are these numbers different in the control PERKcKO only? Response: We thank reviewer for this comment. In the revised manuscript, as suggested, we characterized the whole oligodendrocyte lineage cells as well as cell death. We found that Olig2+ oligodendrocyte lineage cells remained unchanged between Jimpy control and Perk mutant Jimpy animals (Suppl Fig. 1a,c). Notably, the number of apoptotic oligodendrocyte lineage cells labeled by caspase 3 (Casp3+Olig2+) was significantly reduced in Perk mutant Jimpy mice (Jimpy; Perkfl/fl; Olig2-Cre) (Fig. 1e,f). We agree with reviewer that there is a decreased trend in the number of Pdgfra+ OPCs with a significant increase of Ki67+Pdgfra+ cells. When normalized to the total PDGFRa+ population, the percentage of proliferating OPCs was significantly increased and restored to WT levels, indicating that OPCs in Jimpy oligodendrocyte-specific Perk mutants remain highly mitotically active despite their reduced overall abundance (Fig. 3d). On the WT background, the oligodendrocyte-specific Perk mutation (Perkfl/fl; Olig2-Cre) does not change the total number of OPCs or the number of proliferating oligodendrocytes (Suppl Fig. 2), suggesting its effects are specific to the context of the PLP mutation. 4. In conclusion, the authors states “the rate of OPC differentiation” (l.192), but they have quantified PDGFRa and stainings here. Do they mean “differentiation”? Additional BCAS1 staining could highlight the number of differentiating OLs, and better show if the switch from cell proliferation to differentiation is impacted. Response: We apologize for being imprecise in our original submission. We now change the rate of OPC “differentiation” to “proliferation”. Moreover, in response to the reviewer’s suggestion, we have also included BCAS1 staining: we found that Perk ablation in Olig2 expressing Jimpy cells significantly increased the number of newly differentiated oligodendrocytes labeled by BCSA1 (Suppl Fig. 1a,c). 5. On the WB (Fig.2c), the quantification of MBP is surprising (50%) compared to the representative blot where MBP signal is really light. Could the authors explain their quantification process? Response: In response to the reviewer’s insightful comment, we have redone the quantification of the MBP western-blots, and the new graph is now included in the revised manuscript (Fig. 2g). Indeed, the amount of MBP in the mutant, as suggested by the reviewer, is less than we originally reported. 6. At the transcriptomic level (qPCR and RNA-Seq) and IHC level (ASPA staining), the pharmacological approach tends to have an earlier effect (P14) and lower effect. Mice lifespan is extended as much as in the Jimpy-PERKcKO line. Did the authors notice any impact on other cell types, especially on inflammation, which could indirectly affect the oligodendroglial lineage? In l.328, the authors mention that ISRIB has a longer half-life. Did the authors test it in vivo? Could it be more efficient? The proliferation and OPC quantifications reveal a trend, but further points are needed (Fig.5). Authors should increase their n (n=2 for some points), as their data also show strong variability (especially for WT+2BAct samples). Response: We thank reviewer for these comments. In the revised manuscript, we further analyzed neuroinflammation. Our results indicated that 2BAct significantly reduced microglial activation, though astrogliosis was not altered at PND19 (Suppl Fig. 8). As we state in the manuscript (line 362), 2BAct may provide benefit by reducing inflammation. We did not test ISRIB in vivo because it has been reported to have poor in vivo pharmacokinetics. In fact, 2BAct was developed to improve the in vivo pharmacokinetics of ISRIB (Wong YL, et al., eLife, 2019). 2BAct is a highly selective eIF2B activator that efficiently crosses the BBB and is well-suited for dietary formulation enabling long-term dosing beginning at pregnancy in our experimental design. ISRIB, paradoxically, has a much longer half-life in tissue culture media, which is why we chose it for our in vitro studies. In the revised manuscripts, we analyzed additional samples, increasing the group size to n=4~5 per group, which increased statistical power. With these additional samples, we still did not observe a significant difference on the number of Pdgfra+ OPCs between Jimpy mice on control diet and those on 2BAct diet. OPC proliferation, as determined by Ki67, was reduced in Jimpy mice compared to WT, but this reduction was not altered by 2BAct (Fig. 5a,c,d). 7. The puncted MBP stainings (Fig.1c and Fig.4c) can not be associated with “increased” myelination (l.159), as further noted by the EM results on Fig.2a. So additional EM for the pharmacological approach is needed. Response: We thank reviewer for giving us the opportunity to address this critical point. In the revised manuscript, we included EM of corpus callosum (Fig.2a,b), brainstem and cerebellum for the 2BAct experiment (Suppl Fig.5c,d,g,h). We found that myelinated axons were absent in Jimpy mice, but detectable in Jimpy mice on the 2BAct diet. These data confirm that both the Perk mutation as well as 2BAct increase oligodendrocyte survival and CNS myelination in Jimpy mice. Nevertheless, as the reviewer suggests, it is not clear that the MBP staining in these animals is entirely due to increased myelination, especially since we detect faint MBP staining in the Jimpy animals, which do not have detectable myelin on EM. Although the source of the MBP immunoreactivity is unclear, one possibility is that it is derived from microglia. These cells have been shown capable of engulfing MBP mRNA during development and in demyelinating disorders (Li et al., Neuron 2019 101:207-223; Schrimer et al., Nature 2019 573:75-82). We will explore this possibility in future studies, and we now mention in the discussion the possibility that exogenous MBP transcripts are translated in the Jimpy microglia. 8. The impact of ISRIB on OPC proliferation in vitro needs to be quantified to complete Supp.Fig.5 (especially because OPC density in vitro strongly affects their differentiation capacities). Could the authors add the dots for their biological replicates on Fig.8b? Depending on the variability of the in vitro assay, the authors should add 1 or 2 biological replicates. Response: We thank reviewer for these helpful suggestions. It is unlikely that ISRIB has an impact on OPC proliferation since in our experimental paradigm the drug is not added to the culture media until the cells are switched to differentiation media. We have updated the Figure 8b format to the scatter dot plots to reflect the biological replicates (Fig. 8b). The analyses now include four biological replicates for each condition. 9. While extensive, the RNA-Sequencing data do not add much to the current story. Due to the bulk approach, part of the differential expression observed are resulting of different cell population representation in the tissues, as shown by IHC (less oligodendroglial cells for example). As observed previously (by qPCR), the pharmacological approach tends to have a lower effect and earlier effect (P14 and not P19) at the transcriptomic level. Because of the global impact of the drug, adding a cell-specific sequencing could lead to more interesting findings. RNA-sequencing also highlight inflammatory changes between the cKO and pharmacological approaches. Is it confirmed by IHC on tissues? Response: We thank reviewer for the thoughtful comments. We agree that a cell-specific transcriptomic study could lead to additional depth and identify more cell-specific changes; however, we believe that this approach, which is costly and prolonged, is beyond the scope of the current work. We see this as an important direction for future studies and have included mention of this in the Discussion part in the revised manuscript. In the current work, our focus was to establish the overall impact of modulating the ISR on the Jimpy phenotype. The bulk RNA-sequencing allowed us to capture these global transcriptional changes, including inflammatory signatures. In the revised manuscript, we analyzed the neuroinflammation by IHC, and we observed that 2BAct reduced microglial activation but had minimal impact on astrogliosis (Supp Fig. 8). 10. Over the manuscript, some titles are overselling: “increased oligodendrocyte survival” (l.126) “rescues oligodendrocytes death” when no death assessment has been performed (Fig.1 legend), “rescues myelin loss” of only 10% (Fig.2 legend), “protect Jimpy oligodendrocytes” while only 1/8 of oligodendrocytes compared to controls are detected (l.223), “rescues oligodendrocyte death and demyelination” without death assessment neither EM quantification (Fig.4 legend). Response: We thank reviewing for pointing out these statements. In the revised manuscript, we analyzed cell death using antibodies against the apoptotic marker caspase 3. Our results showed that the number of apoptotic oligodendrocyte lineage cells labeled by caspase 3 (Casp3+Olig2+) was significantly reduced in Perk mutant Jimpy mice (Jimpy; Perkfl/fl; Olig2-Cre) (Fig. 1e,f). The new evidence provides support for the statements “increased oligodendrocyte survival” (now l.120) “rescues oligodendrocytes death” (Fig.1 legend). In addition, we have revised, as suggested by the reviewer, the wording of the sub-headings to avoid overstating our findings. We changed “rescues myelin loss” to “partially rescue myelin loss” (Fig. 2 legend); “protects Jimpy oligodendrocytes” to “increases Jimpy myelination” (original l.223 , now l.252); and “rescues oligodendrocyte death and demyelination” to “partially rescues oligodendrocyte death and demyelination” (Fig.4 legend). Importantly, we added new EM quantification data demonstrating that Jimpy mice on the 2BAct diet presented with detectable myelinated axons (Fig. 4d,f). Minor points: 11. The Suppl. Fig. 2 showing the “rescued downregulated” genes (l. 258-262) is not really readable. A similar graph as Fig. 6 would be more useful. Response: We thank reviewer for this suggestion. In the revised manuscript, we have reformatted the graph in original Supple Fig. 2 in a style similar to Fig. 6, and this is now presented as Suppl Fig. 6. 12. Is the PNS affected in the Jimpy model? To what extend? Is the ISR also involved in Schwann cells? It could be briefly discussed in the discussion, to justify the importance of the pharmacological global approach. Response: We thank reviewer for raising this important point. Peripheral nerve function is generally normal in patients with PMD (Carbern et al., Neuron, 1997). Jimpy mice primarily present with severe CNS hypomyelination without apparent PNS involvement, and Schwann cells do not undergo cell death in this model (Wright et al., Neurochem Res, 2006). This is likely because although Schwann cells express the PLP1 gene, the amount of PLP protein in peripheral nerve is minimal compared to the CNS. We have now included this in the Discussion part of the revised manuscript. Reviewer: 3 & 4 Major points: 1. It was unclear whether PERK is upregulated in Jimpy and cKO mice OLs compared to WT/controls so immunohistochemistry (IHC) is needed or other references provided. Response: We thank reviewer for this comment. There is a profound loss of oligodendrocytes in Jimpy mice. It is not feasible to assess PERK expression in oligodendrocyte by IHC. Notably, prior studies have demonstrated that PERK is expressed in oligodendrocytes and activated by ER stress (Lin et al., J Clin Invest, 2007; Lin et al., J Neurosci, 2013). 2. Jimpy pathology includes inflammation, ROS production, astrogliosis, and neuronal loss, so have the authors assessed this compared to Jimpy PERK-cKO mice? Response: We thank reviewer for this comment. In the revised manuscript, we extensively analyzed the Jimpy pathology, including microglial aviation, astrogliosis, and axon degeneration. Our results showed that microglial activation (IBA1+) and astrogliosis (GFAP+) were present in control Jimpy mice (Jimpy; Perkfl/fl). Astrogliosis, not microgliosis, was significantly reduced in Jimpy mice lacking Perk in oligodendrocytes (Suppl Fig. 3). In addition, a large number of damaged axons, as revealed by the DegenoTag antibody, were present in the brains of Jimpy; Perkfl/flmice, while a significant reduction was observed in Jimpy; Perkfl/fl; Olig2-Cre mice (Fig. 2c,d), indicating that oligodendrocyte protection had a beneficial impact on axons. 3. The study shows that PERK inactivation increases the proliferation of oligodendrocyte progenitor cells (OPCs) in vivo. The authors suggest that in this context, OPC proliferation is dependent on their capacity to differentiate to maintain a constant OPC density (homeostatic replacement). It is unclear whether increased OPC proliferation is intrinsically driven by PERK mutation. The authors should discuss this point. Response: We thank reviewer for the comments and suggestions. Our lab previously showed that oligodendrocyte-specific Perk deletion has no effect on developmental myelination using the Olig2-cre; Perk flox line, suggesting that PERK activity is not required for oligodendrocyte development or myelination (Hussien, et al., Glia, 2014; Clayton et al., J. Neurosci., 2017). Moreover, in our revised manuscript, we showed that, on the WT background, the oligodendrocyte-specific Perk mutation (Perkfl/fl; Olig2-Cre) does not change the total number of OPCs or the number of proliferating oligodendrocytes (Suppl Fig. 2). Our previous and current findings suggest that the increased OPC proliferation observed in the current study is unlikely to be intrinsically driven by the PERK mutation. This clarification and the reference have been incorporated into the revised manuscript. 4. It remains unclear from this manuscript whether 2BAct extends the lifespan of Jimpy mice by reducing inflammation, increasing myelination, or both. Specifically: a. The 2BAct diet improved the survival of Jimpy and increased oligodendrocytes (OLs) in the corpus callosum. How about other brain regions? It should be relatively easy to show impact in cerebellum or brainstem. Indeed, brainstem is of particular interest because better myelination might help explain improved survival, so this analysis is recommended. Response: We thank reviewer for this helpful comment. In the revised manuscript, we analyzed the impact of 2BAct on the cerebellum and brainstem. Similar to the corpus callosum, the cerebellum and brainstem from Jimpy mice on the 2BAct diet have increased numbers of oligodendrocytes as well as myelinated axons compared to Jimpy control (Suppl Fig. 5). b. Because 2BAct enters via the mother's diet, it is not specific to OLs. the authors should confirm by IHC that the drug reduces PERK pathway activity in oligodendrocytes. Indeed, there is no clear evidence that 2BAct improves Jimpy mice survival by promoting myelination, similar to the prior comment further studies to document improved myelination in brainstem or other brain regions is recommended. Response: We thank the reviewer for this comment. We have now examined myelin by EM in the corpus callosum, cerebellum, and brainstem of 2BAct-treated animals (Fig. 4d,f; Suppl Fig. 5c,d,g,h). Increased myelin content is observed in the 2BAct treated mice in all CNS regions examined. c. The authors show in Figure 7 that 2BAct treatment reduces the inflammatory response in Jimpy mice at the transcriptional level. How about measures of gliosis by IHC (e.g., Iba1, GFAP in 2BAct-treated Jimpy mice compared to vehicle? Response: We thank review for this suggestion. In the revised manuscript, we analyzed the inflammation response. Our immunostaining of corpus callosum showed that 2BAct significantly reduced microglial activation of Jimpy mice at PND19, although no remarkable change was observed in astrocytes at this timepoint (Suppl Fig. 8). 5. RNA sequencing was performed on total brain lysate, which limits conclusions on OPCs/OLs. To validate RNAseq findings, the authors should use ISH or IHC to confirm upregulation of genes involved in ER protein folding (e.g., Hspa5, Hsp90b1, P4hb) or protein markers (e.g., IRE1, ATF6, TRIB3, AKT) in OPCs or OLs. Alternatively, single-cell RNA sequencing would provide this detail. Response: We thank review for this insightful comment. To validate the RNA sequencing results, in the revised manuscript, we performed IHC and confirmed the changes in ER stress related protein in oligodendrocytes, including Hspa5 (BIP/GRP78), Hsp90b1 (GRP94), ATF6 and TRIB3 (Suppl Fig. 7). These results support the transcriptomic data. Although single-cell RNA sequencing would provide additional resolution, our IHC validation provides important cell specific confirmation of the RNAseq results. Minor points: 6. Is Jimpy a mouse model of Pelizaeus-Merzbacher disease (PMD)? It is a severe mutation of PLP but does it relate to a PMD patient-specific mutation? Please provide a reference or qualify characterization of Jimpy as a model. Response: We thank reviewer for giving us the opportunity to clarify this point. The Jimpy mouse is a model for severe PMD, but it is not an authentic patient-specific human mutation. Instead, it expresses an abnormal PLP1 protein and recapitulates the cellular, molecular and neurologic features seen in severe \"connatal\" forms of human PMD, including near-total absence of myelin in the CNS, extensive oligodendrocyte loss and premature death (Nave, et al., The Neuroscientist ,1996). This clarification and the reference have been incorporated into the revised manuscript. 7. Line 182: Instead of \"there was no appreciable difference,\" rephrasing a less subjective statement is needed e.g., \"no significant differences were observed.\" Response: We thank the reviewer for this comment. We have replaced “appreciable” to “significant” in the revised manuscript. 8. For all figures: The authors should indicate \"n.s.\" (not significant) where applicable to clarify the statistical relevance of the data. Response: We thank the reviewer for this suggestion. We have updated all figures to indicate “n.s.” (not significant) where applicable to clarify the statistical relevance of the data. Integrated stress response inhibition prolongs the lifespan of a Pelizaeus-Merzbacher disease mouse model by increasing oligodendrocyte survival Author’s Response Reviewers’ new comments are highlighted in blue and our point-by-point response is highlighted in red. REVIEWERS' COMMENTS Reviewer #1 (Remarks to the Author): The revised manuscript satisfactorily addresses all my major points, having incorporated additional experimental data (GADD34 and CHOP mRNA measurements) and necessary narrative corrections. The study's focus on inhibiting the ISR in PMD is highly relevant. Given that the inhibitor used (2Bact) is safe and already in clinical trials, this research constitutes a critical step toward translational studies for PMD. I strongly recommend publication. Response: We thank the reviewer for their supportive comments. Reviewer #2 (Remarks to the Author): 1. In their first model, the authors cross the Jimpy line with a PERKcKO line (Olig2-cre;Perk-flox). They mention that “PERK is dispensable for normal oligodendrocyte development and CNS myelination” (l.139). However, in their previous work, the Perk-flox has been crossed with a Cnp-cre line, which is targeting a later stage of the oligodendroglial lineage. It is important to add in the current manuscript the Olig2-cre;Perk-flox line as control, for all IHC quantifications and RNA-Sequencing. Is Perk ablation in OLIG2 cells affecting the oligodendroglial cell lineage (OPC and OL population, survival and proliferation of OPC, myelination)? In the Cnp-cre;Perk-flox line, the authors noticed that an increased susceptibility to inflammation. Is inflammation changed in their current Olig2-cre;Perk-flox line? Response: We thank the reviewer for their comments and suggestions. Our lab previously reported that both the Cnp-cre; Perk flox line (Hussien et al., Glia, 2014) and the Olig2-cre; Perk flox line (Clayton et al., J. Neurosci., 2017) have no effect on developmental myelination, suggesting that PERK activity is not required for oligodendrocyte development or CNS myelination; As such, we did not include the Olig2-cre line as a separate control. In the revised manuscript, we showed that Perk ablation in Olig2 expressing cells in Jimpy mice significantly increased the number of ASPA+ mature OLs and newly formed OLs labeled by BCSA1 without altering the total number of Olig2+ lineage cells (Fig. 2c,d; Suppl Fig. 1). We also observed an increased portion of proliferating OPCs (Ki67+Pdgfra+), although the overall reduction of OPCs is not significant (Fig. 3). Furthermore, our EM analysis, along with analyses of myelin-related proteins (Fig. 2a,b, e-h), indicate an increase in myelinated axons and enhanced myelin-related proteins. Inflammation is changed in the Olig2-cre; Perk-flox line. In the revised manuscript, we observed a significant reduction of GFAP+ astrocytes in Jimpy mice lacking Perk in oligodendrocytes, although the hypertrophic active microglia remained unchanged (Suppl Fig. 3). The inclusion of these controls is appreciated. Regarding the PDGFRa+/Ki67+ quantification, the reduction in OPCs is noted as not significant. However, the Perk-flox data points appear to separate into two groups (n=2 high PDGFRa+/Ki67+ and n=2 low PDGFRa+/Ki67+). Could there be intrinsic differences within these Perk-flox mice, or would increasing the sample size help clarify and strengthen the statistical outcome? Response: We thank reviewer for this comment. After reviewing the litter source, genotyping information, staining/imaging batches, we did not find biological or technical variables to distinguish the two clusters within the PERK flox mice. We increased the sample size with n=8 of Perkfl/fl mice and n=6 of Perk fl/fl; Olig2-cre mice and no significant difference was detected in the density/percentage of PDGFRa+/Ki67+ cell between these two groups. The reason for the variability within in the Perkfl/fl group remains unclear. Therefore, the intrinsic difference within PERK flox mice cannot be excluded. The new quantification data is now included in the supplementary file (Supplementary figure 2b,c,d). 2. What is the efficiency of the recombination in the (Jimpy-)PERKcKO line? Could it explain the low effect in this model (25% of OLs, only 10% of myelinating axons)? Response: We thank reviewer for giving us the opportunity to clarify this point. The Olig2-Cre line is a robust driver of LoxP recombination. We have previously reported that recombination efficiency of Olig2-Cre line is as high as 79.6 ± 11.6%, as determined in the Olig2-Cre; ROSA26-YFP report line (Clayton et al., J. Neurosci., 2017). Therefore, the moderate rescue observed in the Jimpy model is unlikely due to inefficient recombination. We believe that the ability of the PERK deletion to rescue oligodendrocytes and myelination is limited due to the profound severity of the Jimpy phenotype. Thank you for providing these details and the additional reference. 3. Currently, the characterization of oligodendroglial cell lineage by IHC is limited in the Jimpy-PERKcKO line. The quantification of the whole oligodendroglial population (OLIG2) could help distinguish which stage of the lineage is affected (whole population vs. OPC or OL). Authors should add survival and death quantification if they want to support their claims. Jimpy-PERKcKO mice tend to present a decrease (trend) in the number of PDGFRa+ OPC (Fig.3b). Could this explain the relative increase of Ki67+ cells, to maintain OPC homeostatic number (Fig.3c)? What is the percentage of PDGFRa+ and Ki67+ (among the PDGFRa+ cells). Are these numbers different in the control PERKcKO only? Response: We thank reviewer for this comment. In the revised manuscript, as suggested, we characterized the whole oligodendrocyte lineage cells as well as cell death. We found that Olig2+ oligodendrocyte lineage cells remained unchanged between Jimpy control and Perk mutant Jimpy animals (Suppl Fig. 1a,c). Notably, the number of apoptotic oligodendrocyte lineage cells labeled by caspase 3 (Casp3+Olig2+) was significantly reduced in Perk mutant Jimpy mice (Jimpy; Perkfl/fl; Olig2-Cre) (Fig. 1e,f). We agree with reviewer that there is a decreased trend in the number of Pdgfra+ OPCs with a significant increase of Ki67+Pdgfra+ cells. When normalized to the total PDGFRa+ population, the percentage of proliferating OPCs was significantly increased and restored to WT levels, indicating that OPCs in Jimpy oligodendrocyte-specific Perk mutants remain highly mitotically active despite their reduced overall abundance (Fig. 3d). On the WT background, the oligodendrocyte-specific Perk mutation (Perkfl/fl; Olig2-Cre) does not change the total number of OPCs or the number of proliferating oligodendrocytes (Suppl Fig. 2), suggesting its effects are specific to the context of the PLP mutation. The improved characterization of the oligodendroglial lineage is informative and significantly strengthens the study. 4. In conclusion, the authors states “the rate of OPC differentiation” (l.192), but they have quantified PDGFRa and stainings here. Do they mean “differentiation”? Additional BCAS1 staining could highlight the number of differentiating OLs, and better show if the switch from cell proliferation to differentiation is impacted. Response: We apologize for being imprecise in our original submission. We now change the rate of OPC “differentiation” to “proliferation”. Moreover, in response to the reviewer’s suggestion, we have also included BCAS1 staining: we found that Perk ablation in Olig2 expressing Jimpy cells significantly increased the number of newly differentiated oligodendrocytes labeled by BCSA1 (Suppl Fig. 1a,c). The improved characterization of the oligodendroglial lineage is informative and significantly strengthens the study. 5. On the WB (Fig.2c), the quantification of MBP is surprising (50%) compared to the representative blot where MBP signal is really light. Could the authors explain their quantification process? Response: In response to the reviewer’s insightful comment, we have redone the quantification of the MBP western-blots, and the new graph is now included in the revised manuscript (Fig. 2g). Indeed, the amount of MBP in the mutant, as suggested by the reviewer, is less than we originally reported. The revised western blot presentation is clearer, and the representative bands now correspond well with the quantification. 6. At the transcriptomic level (qPCR and RNA-Seq) and IHC level (ASPA staining), the pharmacological approach tends to have an earlier effect (P14) and lower effect. Mice lifespan is extended as much as in the Jimpy-PERKcKO line. Did the authors notice any impact on other cell types, especially on inflammation, which could indirectly affect the oligodendroglial lineage? In l.328, the authors mention that ISRIB has a longer half-life. Did the authors test it in vivo? Could it be more efficient? The proliferation and OPC quantifications reveal a trend, but further points are needed (Fig.5). Authors should increase their n (n=2 for some points), as their data also show strong variability (especially for WT+2BAct samples). Response: We thank reviewer for these comments. In the revised manuscript, we further analyzed neuroinflammation. Our results indicated that 2BAct significantly reduced microglial activation, though astrogliosis was not altered at PND19 (Suppl Fig. 8). As we state in the manuscript (line 362), 2BAct may provide benefit by reducing inflammation. We did not test ISRIB in vivo because it has been reported to have poor in vivo pharmacokinetics. In fact, 2BAct was developed to improve the in vivo pharmacokinetics of ISRIB (Wong YL, et al., eLife, 2019). 2BAct is a highly selective eIF2B activator that efficiently crosses the BBB and is well-suited for dietary formulation enabling long-term dosing beginning at pregnancy in our experimental design. ISRIB, paradoxically, has a much longer half-life in tissue culture media, which is why we chose it for our in vitro studies. In the revised manuscripts, we analyzed additional samples, increasing the group size to n=4~5 per group, which increased statistical power. With these additional samples, we still did not observe a significant difference on the number of Pdgfra+ OPCs between Jimpy mice on control diet and those on 2BAct diet. OPC proliferation, as determined by Ki67, was reduced in Jimpy mice compared to WT, but this reduction was not altered by 2BAct (Fig. 5a,c,d). Increasing the sample size has strengthened the robustness of the results. Notably, the Jimpy + CTRL group shows a higher proportion of PDGFRa+/Ki67+ cells (35–40%, Supp. Fig. 5d) compared to Jimpy-Perk (20%, Fig. 3). These levels are similar to those in Jimpy-Olig2cre-Perkflox mice (35–40%, Supp. Fig. 5d), which could explain why the Jimpy + 2BAct group shows no additional effect (35–40%, Supp. Fig. 5d). Could the authors clarify what the CTRL condition corresponds to? Is it diet plus DMSO? Response: We thank reviewer for giving us the opportunity to clarify this point. CTRL is the control diet, identically formulated to the 2BACT chow but without adding 2BAct. The clarification of the CTRL condition is included now in the methods section of the revised manuscript. 7. The puncted MBP stainings (Fig.1c and Fig.4c) can not be associated with “increased” myelination (l.159), as further noted by the EM results on Fig.2a. So additional EM for the pharmacological approach is needed. Response: We thank reviewer for giving us the opportunity to address this critical point. In the revised manuscript, we included EM of corpus callosum (Fig.2a,b), brainstem and cerebellum for the 2BAct experiment (Suppl Fig.5c,d,g,h). We found that myelinated axons were absent in Jimpy mice, but detectable in Jimpy mice on the 2BAct diet. These data confirm that both the Perk mutation as well as 2BAct increase oligodendrocyte survival and CNS myelination in Jimpy mice. Nevertheless, as the reviewer suggests, it is not clear that the MBP staining in these animals is entirely due to increased myelination, especially since we detect faint MBP staining in the Jimpy animals, which do not have detectable myelin on EM. Although the source of the MBP immunoreactivity is unclear, one possibility is that it is derived from microglia. These cells have been shown capable of engulfing MBP mRNA during development and in demyelinating disorders (Li et al., Neuron 2019 101:207-223; Schrimer et al., Nature 2019 573:75-82). We will explore this possibility in future studies, and we now mention in the discussion the possibility that exogenous MBP transcripts are translated in the Jimpy microglia. The inclusion of EM data is appreciated, as it clearly reveals myelinated axons, albeit in limited numbers. It would be interesting to determine whether the myelination rate continues to increase over time (up to P35, when Jimpy-Perk animals typically die) or if it plateaus. Response: We thank the reviewer for this comment. We agree that it would be informative to evaluate the persistence of myelination over time. However, this longitudinal analysis is beyond the scope the current study. The goal of the current study was to compare the difference between Jimpy mice and Jimpy mice treated with 2BAct. Because Jimpy mice typically do not survive much beyond P21, it is not feasible to perform a longer-term comparison. The myelination rate mediated by 2BAct over a longer term can be further investigated in the future studies. 8. The impact of ISRIB on OPC proliferation in vitro needs to be quantified to complete Supp.Fig.5 (especially because OPC density in vitro strongly affects their differentiation capacities). Could the authors add the dots for their biological replicates on Fig.8b? Depending on the variability of the in vitro assay, the authors should add 1 or 2 biological replicates. Response: We thank reviewer for these helpful suggestions. It is unlikely that ISRIB has an impact on OPC proliferation since in our experimental paradigm the drug is not added to the culture media until the cells are switched to differentiation media. We have updated the Figure 8b format to the scatter dot plots to reflect the biological replicates (Fig. 8b). The analyses now include four biological replicates for each condition. Thank you for clarifying the timing of drug addition. The inclusion of additional biological replicates is appreciated. 9. While extensive, the RNA-Sequencing data do not add much to the current story. Due to the bulk approach, part of the differential expression observed are resulting of different cell population representation in the tissues, as shown by IHC (less oligodendroglial cells for example). As observed previously (by qPCR), the pharmacological approach tends to have a lower effect and earlier effect (P14 and not P19) at the transcriptomic level. Because of the global impact of the drug, adding a cell-specific sequencing could lead to more interesting findings. RNA-sequencing also highlight inflammatory changes between the cKO and pharmacological approaches. Is it confirmed by IHC on tissues? Response: We thank reviewer for the thoughtful comments. We agree that a cell-specific transcriptomic study could lead to additional depth and identify more cell-specific changes; however, we believe that this approach, which is costly and prolonged, is beyond the scope of the current work. We see this as an important direction for future studies and have included mention of this in the Discussion part in the revised manuscript. In the current work, our focus was to establish the overall impact of modulating the ISR on the Jimpy phenotype. The bulk RNA-sequencing allowed us to capture these global transcriptional changes, including inflammatory signatures. In the revised manuscript, we analyzed the neuroinflammation by IHC, and we observed that 2BAct reduced microglial activation but had minimal impact on astrogliosis (Supp Fig. 8). Comments noted. Quantification of ATF6, GRP78, GRP94, and TRIB3 would be helpful to complement Supp. Fig. 7, as intensity differences are difficult to assess solely from representative IHC images. Response: In response to the reviewer’s comment, we have quantified the mean fluorescence intensity (MFI) of ATF6, GRP78, GRP94, and TRIB3 in Olig2+ oligodendroglia cells (Supp Fig. 7). Our results showed that, increased immunofluorescent intensities of ATF6, GRP78 and GRP94 were observed in Olig2+ cells of Jimpy mice lacking Perk in oligodendrocytes, with GRP78 and GRP94 reaching significance (Suppl Fig. 7a-c,e-g). In addition, TRIB3 intensity was significantly increased in Jimpy; Perkfl/fl mice compared to WT control and reduced in Jimpy Perkfl/fl; Olig2-Cre mice (Suppl Fig. 7d,h). 10. Over the manuscript, some titles are overselling: “increased oligodendrocyte survival” (l.126) “rescues oligodendrocytes death” when no death assessment has been performed (Fig.1 legend), “rescues myelin loss” of only 10% (Fig.2 legend), “protect Jimpy oligodendrocytes” while only 1/8 of oligodendrocytes compared to controls are detected (l.223), “rescues oligodendrocyte death and demyelination” without death assessment neither EM quantification (Fig.4 legend). Response: We thank reviewing for pointing out these statements. In the revised manuscript, we analyzed cell death using antibodies against the apoptotic marker caspase 3. Our results showed that the number of apoptotic oligodendrocyte lineage cells labeled by caspase 3 (Casp3+Olig2+) was significantly reduced in Perk mutant Jimpy mice (Jimpy; Perkfl/fl; Olig2-Cre) (Fig. 1e,f). The new evidence provides support for the statements “increased oligodendrocyte survival” (now l.120) “rescues oligodendrocytes death” (Fig.1 legend). In addition, we have revised, as suggested by the reviewer, the wording of the sub-headings to avoid overstating our findings. We changed “rescues myelin loss” to “partially rescue myelin loss” (Fig. 2 legend); “protects Jimpy oligodendrocytes” to “increases Jimpy myelination” (original l.223 , now l.252); and “rescues oligodendrocyte death and demyelination” to “partially rescues oligodendrocyte death and demyelination” (Fig.4 legend). Importantly, we added new EM quantification data demonstrating that Jimpy mice on the 2BAct diet presented with detectable myelinated axons (Fig. 4d,f). The revisions are appropriate, and the updated text now aligns more accurately with the presented results. Minor points: 11. The Suppl. Fig. 2 showing the “rescued downregulated” genes (l. 258-262) is not really readable. A similar graph as Fig. 6 would be more useful. Response: We thank reviewer for this suggestion. In the revised manuscript, we have reformatted the graph in original Supple Fig. 2 in a style similar to Fig. 6, and this is now presented as Suppl Fig. 6. 12. Is the PNS affected in the Jimpy model? To what extend? Is the ISR also involved in Schwann cells? It could be briefly discussed in the discussion, to justify the importance of the pharmacological global approach. Response: We thank reviewer for raising this important point. Peripheral nerve function is generally normal in patients with PMD (Carbern et al., Neuron, 1997). Jimpy mice primarily present with severe CNS hypomyelination without apparent PNS involvement, and Schwann cells do not undergo cell death in this model (Wright et al., Neurochem Res, 2006). This is likely because although Schwann cells express the PLP1 gene, the amount of PLP protein in peripheral nerve is minimal compared to the CNS. We have now included this in the Discussion part of the revised manuscript. Thank you for the additional explanations and clarifications. Reviewer #4 (Remarks to the Author): I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}]

Decision letter