Insights into the mechanism of oligodendrocyte protection and remyelination enhancement by the integrated stress response.

central nervous system (CNS) inflammation triggers activation of the integrated stress response (ISR). We previously reported that prolonging the ISR protects remyelinating oligodendrocytes and promotes remyelination in the presence of inflammation. However, the exact mechanisms through which this occurs remain unknown. Here, we investigated whether the ISR modulator Sephin1 in combination with the oligodendrocyte differentiation enhancing reagent bazedoxifene (BZA) is able to accelerate remyelination under inflammation, and the underlying mechanisms mediating this pathway. We find that the combined treatment of Sephin1 and BZA is sufficient to accelerate early-stage remyelination in mice with ectopic IFN-γ expression in the CNS. IFN-γ, which is a critical inflammatory cytokine in multiple sclerosis (MS), inhibits oligodendrocyte precursor cell (OPC) differentiation in culture and triggers a mild ISR. Mechanistically, we further show that BZA promotes OPC differentiation in the presence of IFN-γ, while Sephin1 enhances the IFN-γ-induced ISR by reducing protein synthesis and increasing RNA stress granule formation in differentiating oligodendrocytes. Finally, pharmacological suppression of the ISR blocks stress granule formation in vitro and partially lessens the beneficial effect of Sephin1 on disease progression in a mouse model of MS, experimental autoimmune encephalitis (EAE). Overall, our findings uncover distinct mechanisms of action of BZA and Sephin1 on oligodendrocyte lineage cells under inflammatory stress, suggesting that a combination therapy may effectively promote restoring neuronal function in MS patients.

Insights into the mechanism of oligodendrocyte protection and remyelination enhancement by the integrated stress response.

CNS inflammation triggers activation of the integrated stress response (ISR). We previously reported that prolonging the ISR protects remyelinating oligodendrocytes and promotes remyelination in the presence of inflammation (Chen et al., eLife , 2021). However, the exact mechanisms through which this occurs remain unknown. Here, we investigated whether the ISR modulator Sephin1 in combination with the oligodendrocyte differentiation enhancing reagent bazedoxifene (BZA) is able to accelerate remyelination under inflammation, and the underlying mechanisms mediating this pathway. We find that the combined treatment of Sephin1 and BZA is sufficient to accelerate early-stage remyelination in mice with ectopic IFN-γ expression in the CNS. IFN-γ, which is a critical inflammatory cytokine in multiple sclerosis (MS), inhibits oligodendrocyte precursor cell (OPC) differentiation in culture and triggers a mild ISR. Mechanistically, we further show that BZA promotes OPC differentiation in the presence of IFN-γ, while Sephin1 enhances the IFN-γ-induced ISR by reducing protein synthesis and increasing RNA stress granule formation in differentiating oligodendrocytes. Finally, the ISR suppressor 2BAct is able to partially lessen the beneficial effect of Sephin1 on disease progression, in an MS mouse model of experimental autoimmune encephalitis (EAE). Overall, our findings uncover distinct mechanisms of action of BZA and Sephin1 on oligodendrocyte lineage cells under inflammatory stress, suggesting that a combination therapy may effectively promote restoring neuronal function in MS patients.

Prolonging the integrated stress response enhances CNS remyelination in an inflammatory environment.

The inflammatory environment of demyelinated lesions in multiple sclerosis (MS) patients contributes to remyelination failure. Inflammation activates a cytoprotective pathway, the integrated stress response (ISR), but it remains unclear whether enhancing the ISR can improve remyelination in an inflammatory environment. To examine this possibility, the remyelination stage of experimental autoimmune encephalomyelitis (EAE), as well as a mouse model that incorporates cuprizone-induced demyelination along with CNS delivery of the proinflammatory cytokine IFN-γ were used here. We demonstrate that either genetic or pharmacological ISR enhancement significantly increased the number of remyelinating oligodendrocytes and remyelinated axons in the inflammatory lesions. Moreover, the combined treatment of the ISR modulator Sephin1 with the oligodendrocyte differentiation enhancing reagent bazedoxifene increased myelin thickness of remyelinated axons to pre-lesion levels. Taken together, our findings indicate that prolonging the ISR protects remyelinating oligodendrocytes and promotes remyelination in the presence of inflammation, suggesting that ISR enhancement may provide reparative benefit to MS patients.

m6A mRNA Methylation Is Essential for Oligodendrocyte Maturation and CNS Myelination.

The molecular mechanisms that govern the maturation of oligodendrocyte lineage cells remain unclear. Emerging studies have shown that N6-methyladenosine (m6A), the most common internal RNA modification of mammalian mRNA, plays a critical role in various developmental processes. Here, we demonstrate that oligodendrocyte lineage progression is accompanied by dynamic changes in m6A modification on numerous transcripts. In vivo conditional inactivation of an essential m6A writer component, METTL14, results in decreased oligodendrocyte numbers and CNS hypomyelination, although oligodendrocyte precursor cell (OPC) numbers are normal. In vitro Mettl14 ablation disrupts postmitotic oligodendrocyte maturation and has distinct effects on OPC and oligodendrocyte transcriptomes. Moreover, the loss of Mettl14 in oligodendrocyte lineage cells causes aberrant splicing of myriad RNA transcripts, including those that encode the essential paranodal component neurofascin 155 (NF155). Together, our findings indicate that dynamic RNA methylation plays an important regulatory role in oligodendrocyte development and CNS myelination.

Targeted Complement Inhibition at Synapses Prevents Microglial Synaptic Engulfment and Synapse Loss in Demyelinating Disease.

Multiple sclerosis (MS) is a demyelinating, autoimmune disease of the central nervous system. While work has focused on myelin and axon loss in MS, less is known about mechanisms underlying synaptic changes. Using postmortem human MS tissue, a preclinical nonhuman primate model of MS, and two rodent models of demyelinating disease, we investigated synapse changes in the visual system. Similar to other neurodegenerative diseases, microglial synaptic engulfment and profound synapse loss were observed. In mice, synapse loss occurred independently of local demyelination and neuronal degeneration but coincided with gliosis and increased complement component C3, but not C1q, at synapses. Viral overexpression of the complement inhibitor Crry at C3-bound synapses decreased microglial engulfment of synapses and protected visual function. These results indicate that microglia eliminate synapses through the alternative complement cascade in demyelinating disease and identify a strategy to prevent synapse loss that may be broadly applicable to other neurodegenerative diseases. VIDEO ABSTRACT.

The UPR-PERK pathway is not a promising therapeutic target for mutant SOD1-induced ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, characterized by motor neuron death in the brain and spinal cord. Mutations in the Cu/Zn superoxide dismutase (SOD1) gene account for ~20% of all familial ALS forms, corresponding to 1%-2% of all ALS cases. One of the suggested mechanisms by which mutant SOD1 (mtSOD1) exerts its toxic effects involves intracellular accumulation of abnormal mtSOD1 aggregates, which trigger endoplasmic reticulum (ER) stress and activate its adaptive signal transduction pathways, including the unfolded protein response (UPR). PERK, an eIF2α kinase, is central to the UPR and is the most rapidly activated pathway in response to ER stress. Previous reports using mtSOD1 transgenic mice indicated that genetic or pharmacological enhancement of the UPR-PERK pathway may be effective in treating ALS. We investigated the response to PERK haploinsufficiency, and the response to deficiency of its downstream effectors GADD34 and CHOP, in five distinct lines of mtSOD1 mice. We demonstrate that, in contrast to a previously published study, PERK haploinsufficiency has no effect on disease in all mtSOD1 lines examined. We also show that deficiency of GADD34, which enhances the UPR by prolonging the phosphorylation of eIF2α, does not ameliorate disease in these mtSOD1 mouse lines. Finally, we demonstrate that genetic ablation of CHOP transcription factor, which is known to be pro-apoptotic, does not ameliorate disease in mtSOD1 mice. Cumulatively, our studies reveal that neither genetic inhibition of the UPR via ablation of PERK, nor genetic UPR enhancement via ablation of GADD34, is beneficial for mtSOD1-induced motor neuron disease. Therefore, the PERK pathway is not a likely target for therapeutic intervention in mtSOD1-induced ALS.

Sephin1, which prolongs the integrated stress response, is a promising therapeutic for multiple sclerosis.

Multiple sclerosis is a chronic autoimmune demyelinating disorder of the CNS. Immune-mediated oligodendrocyte cell loss contributes to multiple sclerosis pathogenesis, such that oligodendrocyte-protective strategies represent a promising therapeutic approach. The integrated stress response, which is an innate cellular protective signalling pathway, reduces the cytotoxic impact of inflammation on oligodendrocytes. This response is initiated by phosphorylation of eIF2α to diminish global protein translation and selectively allow for the synthesis of protective proteins. The integrated stress response is terminated by dephosphorylation of eIF2α. The small molecule Sephin1 inhibits eIF2α dephosphorylation, thereby prolonging the protective response. Herein, we tested the effectiveness of Sephin1 in shielding oligodendrocytes against inflammatory stress. We confirmed that Sephin1 prolonged eIF2α phosphorylation in stressed primary oligodendrocyte cultures. Moreover, by using a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis, we demonstrated that Sephin1 delayed the onset of clinical symptoms, which correlated with a prolonged integrated stress response, reduced oligodendrocyte and axon loss, as well as diminished T cell presence in the CNS. Sephin1 is reportedly a selective inhibitor of GADD34 (PPP1R15A), which is a stress-induced regulatory subunit of protein phosphatase 1 complex that dephosphorylates eIF2α. Consistent with this possibility, GADD34 mutant mice presented with a similar ameliorated experimental autoimmune encephalomyelitis phenotype as Sephin1-treated mice, and Sephin1 did not provide additional therapeutic benefit to the GADD34 mutant animals. Results presented from the adoptive transfer of encephalitogenic T cells between wild-type and GADD34 mutant mice further indicate that the beneficial effects of Sephin1 are mediated through a direct protective effect on the CNS. Of particular therapeutic relevance, Sephin1 provided additive therapeutic benefit when combined with the first line multiple sclerosis drug, interferon ß. Together, our results suggest that a neuroprotective treatment based on the enhancement of the integrated stress response would likely have significant therapeutic value for multiple sclerosis patients.

The integrated stress response in hypoxia-induced diffuse white matter injury.

Currently no treatments exist for preterm infants with diffuse white matter injury (DWMI) caused by hypoxia. Due to improved care of preterm neonates and increased recognition by advanced imaging techniques, the prevalence of DWMI is increasing. A better understanding of the pathophysiology of DWMI is therefore of critical importance. The integrated stress response (ISR), a conserved eukaryotic response to myriad stressors including hypoxia, may play a role in hypoxia-induced DWMI and may represent a novel target for much needed therapies. In this study we utilize in vitro and in vivo hypoxic models of DWMI to investigate whether the ISR is involved in DWMI. We demonstrate that hypoxia activates the ISR in primary mouse oligodendrocyte precursor cells (OPCs) in vitro and that genetically inhibiting the ISR in differentiating OPCs increases their susceptibility to in vitro hypoxia. We also show that a well-established in vivo mild chronic hypoxia (MCH) mouse model and a new severe acute hypoxia (SAH) mouse model of DWMI activates the initial step of the ISR. Nonetheless, genetic inhibition of the ISR has no detectable effect on either MCH or SAH-induced DWMI. In addition, we demonstrate that genetic enhancement of the ISR does not ameliorate MCH or SAH-induced DWMI. These studies suggest that while the ISR protects OPCs from hypoxia in vitro, it does not appear to play a major role in either MCH or SAH-induced DWMI and is therefore not a likely target for therapies aimed at improving neurological outcome in preterm neonates with hypoxia-induced DWMI.SIGNIFICANCE STATEMENTDiffuse white matter injury (DWMI) caused by hypoxia is a leading cause of neurological deficits following premature birth. An increased understanding of the pathogenesis of this disease is critical. The integrated stress response (ISR) is activated by hypoxia and protects oligodendrocyte lineage cells in other disease models. This has led to an interest in the potential role of the ISR in DWMI. Here we examine the ISR in hypoxia-induced DWMI and show that while the ISR protects oligodendrocyte lineage cells from hypoxia in vitro, genetic inhibition or enhancement of the ISR has no effect on hypoxia-induced DWMI in vivo suggesting that the ISR does not play a major role in, and is not a likely therapeutic target for, DWMI.

Adenomatous polyposis coli regulates radial axonal sorting and myelination in the PNS.

The tumor suppressor protein adenomatous polyposis coli (APC) is multifunctional - it participates in the canonical Wnt/ß-catenin signal transduction pathway as well as modulating cytoskeleton function. Although APC is expressed by Schwann cells, the role that it plays in these cells and in the myelination of the peripheral nervous system (PNS) is unknown. Therefore, we used the Cre-lox approach to generate a mouse model in which APC expression is specifically eliminated from Schwann cells. These mice display hindlimb weakness and impaired axonal conduction in sciatic nerves. Detailed morphological analyses revealed that APC loss delays radial axonal sorting and PNS myelination. Furthermore, APC loss delays Schwann cell differentiation in vivo, which correlates with persistent activation of the Wnt signaling pathway and results in perturbed extension of Schwann cell processes and disrupted lamellipodia formation. In addition, APC-deficient Schwann cells display a transient diminution of proliferative capacity. Our data indicate that APC is required by Schwann cells for their timely differentiation to mature, myelinating cells and plays a crucial role in radial axonal sorting and PNS myelination.

Pharmaceutical integrated stress response enhancement protects oligodendrocytes and provides a potential multiple sclerosis therapeutic.

Oligodendrocyte death contributes to the pathogenesis of the inflammatory demyelinating disease multiple sclerosis (MS). Nevertheless, current MS therapies are mainly immunomodulatory and have demonstrated limited ability to inhibit MS progression. Protection of oligodendrocytes is therefore a desirable strategy for alleviating disease. Here we demonstrate that enhancement of the integrated stress response using the FDA-approved drug guanabenz increases oligodendrocyte survival in culture and prevents hypomyelination in cerebellar explants in the presence of interferon-γ, a pro-inflammatory cytokine implicated in MS pathogenesis. In vivo, guanabenz treatment protects against oligodendrocyte loss caused by CNS-specific expression of interferon-γ. In a mouse model of MS, experimental autoimmune encephalomyelitis, guanabenz alleviates clinical symptoms, which correlates with increased oligodendrocyte survival and diminished CNS CD4+ T cell accumulation. Moreover, guanabenz ameliorates relapse in relapsing-remitting experimental autoimmune encephalomyelitis. Our results provide support for a MS therapy that enhances the integrated stress response to protect oligodendrocytes against the inflammatory CNS environment.

miR-9 is an essential oncogenic microRNA specifically overexpressed in mixed lineage leukemia-rearranged leukemia.

MicroRNAs (miRNAs), small noncoding RNAs that regulate target gene mRNAs, are known to contribute to pathogenesis of cancers. Acute myeloid leukemia (AML) is a group of heterogeneous hematopoietic malignancies with various chromosomal and/or molecular abnormalities. AML with chromosomal translocations involving the mixed lineage leukemia (MLL) gene are usually associated with poor survival. In the present study, through a large-scale, genomewide miRNA expression assay, we show that microRNA-9 (miR-9) is the most specifically up-regulated miRNA in MLL-rearranged AML compared with both normal control and non-MLL-rearranged AML. We demonstrate that miR-9 is a direct target of MLL fusion proteins and can be significantly up-regulated in expression by the latter in human and mouse hematopoietic stem/progenitor cells. Depletion of endogenous miR-9 expression by an appropriate antagomiR can significantly inhibit cell growth/viability and promote apoptosis in human MLL-rearranged AML cells, and the opposite is true when expression of miR-9 is forced. Blocking endogenous miR-9 function by anti-miRNA sponge can significantly inhibit, whereas forced expression of miR-9 can significantly promote, MLL fusion-induced immortalization/transformation of normal mouse bone marrow progenitor cells in vitro. Furthermore, forced expression of miR-9 can significantly promote MLL fusion-mediated leukemogenesis in vivo. In addition, a group of putative target genes of miR-9 exhibited a significant inverse correlation of expression with miR-9 in a series of leukemia sample sets, suggesting that they are potential targets of miR-9 in MLL-rearranged AML. Collectively, our data demonstrate that miR-9 is a critical oncomiR in MLL-rearranged AML and can serve as a potential therapeutic target to treat this dismal disease.

PBX3 is an important cofactor of HOXA9 in leukemogenesis.

Although PBX proteins are known to increase DNA-binding/transcriptional activity of HOX proteins through their direct binding, the functional importance of their interaction in leukemogenesis is unclear.We recently reported that overexpression of a 4-homeobox-gene signature (ie, PBX3/HOXA7/HOXA9/HOXA11) is an independent predictor of poor survival in patients with cytogenetically abnormal acute myeloid leukemia (CA-AML). Here we show that it is PBX3, but not PBX1 or PBX2, that is consistently coexpressed with HOXA9 in various subtypes of CA-AML, particularly MLL-rearranged AML, and thus appears as a potential pathologic cofactor of HOXA9 in CA-AML. We then show that depletion of endogenous Pbx3 expression by shRNA significantly inhibits MLL-fusion-mediated cell transformation, and coexpressed PBX3 exhibits a significantly synergistic effect with HOXA9 in promoting cell transformation in vitro and leukemogenesis in vivo. Furthermore, as a proof of concept, we show that a small peptide, namely HXR9, which was developed to specifically disrupt the interactions between HOX and PBX proteins, can selectively kill leukemic cells with overexpression of HOXA/PBX3 genes. Collectively, our data suggest that PBX3 is a critical cofactor of HOXA9 in leukemogenesis, and targeting their interaction is a feasible strategy to treat presently therapy resistant CA-AML (eg, MLL-rearranged leukemia) in which HOXA/PBX3 genes are overexpressed.

MiR-495 is a tumor-suppressor microRNA down-regulated in MLL-rearranged leukemia.

Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic malignancies with variable response to treatment. AMLs bearing MLL (mixed lineage leukemia) rearrangements are associated with intermediate or poor survival. MicroRNAs (miRNAs), a class of small noncoding RNAs, have been postulated to be important gene expression regulators virtually in all biological processes, including leukemogenesis. Through a large-scale, genome-wide miRNA expression profiling assay of 85 human AML and 15 normal control samples, we show that among 48 miRNAs that are significantly differentially expressed between MLL- and non-MLL-rearranged AML samples, only one (miR-495) is expressed at a lower level in MLL-rearranged AML than in non-MLL-rearranged AML; meanwhile, miR-495 is also significantly down-regulated in MLL-rearranged AML samples compared with normal control samples. Through in vitro colony-forming/replating assays and in vivo bone marrow transplantation studies, we show that forced expression of miR-495 significantly inhibits MLL-fusion-mediated cell transformation in vitro and leukemogenesis in vivo. In human leukemic cells carrying MLL rearrangements, ectopic expression of miR-495 greatly inhibits cell viability and increases cell apoptosis. Furthermore, our studies demonstrate that PBX3 and MEIS1 are two direct target genes of miR-495, and forced expression of either of them can reverse the effects of miR-495 overexpression on inhibiting cell viability and promoting apoptosis of human MLL-rearranged leukemic cells. Thus, our data indicate that miR-495 likely functions as a tumor suppressor in AML with MLL rearrangements by targeting essential leukemia-related genes.

Blockade of miR-150 maturation by MLL-fusion/MYC/LIN-28 is required for MLL-associated leukemia.

Expression of microRNAs (miRNAs) is under stringent regulation at both transcriptional and posttranscriptional levels. Disturbance at either level could cause dysregulation of miRNAs. Here, we show that MLL fusion proteins negatively regulate production of miR-150, an miRNA widely repressed in acute leukemia, by blocking miR-150 precursors from being processed to mature miRNAs through MYC/LIN28 functional axis. Forced expression of miR-150 dramatically inhibited leukemic cell growth and delayed MLL-fusion-mediated leukemogenesis, likely through targeting FLT3 and MYB and thereby interfering with the HOXA9/MEIS1/FLT3/MYB signaling network, which in turn caused downregulation of MYC/LIN28. Collectively, we revealed a MLL-fusion/MYC/LIN28⊣miR-150⊣FLT3/MYB/HOXA9/MEIS1 signaling circuit underlying the pathogenesis of leukemia, where miR-150 functions as a pivotal gatekeeper and its repression is required for leukemogenesis.