The failure of axons to regenerate is a major obstacle for functional recovery after central nervous system (CNS) injury. Removing extracellular inhibitory molecules results in limited axon regeneration in vivo. To test for the role of intrinsic impediments to axon regrowth, we analyzed cell growth control genes using a virus-assisted in vivo conditional knockout approach. Deletion of PTEN (phosphatase and tensin homolog), a negative regulator of the mammalian target of rapamycin (mTOR) pathway, in adult retinal ganglion cells (RGCs) promotes robust axon regeneration after optic nerve injury. In wild-type adult mice, the mTOR activity was suppressed and new protein synthesis was impaired in axotomized RGCs, which may contribute to the regeneration failure. Reactivating this pathway by conditional knockout of tuberous sclerosis complex 1, another negative regulator of the mTOR pathway, also leads to axon regeneration. Thus, our results suggest the manipulation of intrinsic growth control pathways as a therapeutic approach to promote axon regeneration after CNS injury.Axons do not regenerate after injury in the adult mammalian central nervous system (CNS), a phenomenon attributed to two properties of the adult CNS, the inhibitory extrinsic environment and a diminished intrinsic regenerative capacity of mature CNS neurons (1-4). Neutralization of the extracellular molecules identified as axon regrowth inhibitors allows only a limited degree of axon regeneration in vivo (5-7). Therefore, intrinsic mechanisms are likely to be important in controlling the process of axon regeneration. A hint about possible mechanisms of neuronal regenerative ability comes from the evolutionarily conserved molecular pathways that control cellular growth and size. For most cell types, specific mechanisms are necessary to prevent cellular overgrowth upon the completion of development (8). Because many of these molecules are often expressed in postmitotic mature neurons, we hypothesized that they may contribute to the diminished regenerative ability in adult CNS neurons.To circumvent the problem that germline knockout of individual cell growth control genes often results in compromised viability in mice, we designed a strategy based on intravitreal injection of adeno-associated viruses expressing Cre (AAV-Cre) in adult mice. This procedure resulted in the expression of Cre in more than 90% of retinal ganglion cells (RGCs) and few other non-RGC cells, as indicated in two reporter lines ( fig. S1, A and B). We thus injected AAV-Cre into the vitreous body of different adult floxed mice, including Rb f/f (9), P53 f/f (9),

Proteins of the bromodomain and extra-terminal (BET) family are epigenetics "readers" and promising therapeutic targets for cancer and other human diseases. We describe herein a structure-guided design of [1,4]oxazepines as a new class of BET inhibitors and our subsequent design, synthesis, and evaluation of proteolysis-targeting chimeric (PROTAC) small-molecule BET degraders. Our efforts have led to the discovery of extremely potent BET degraders, exemplified by QCA570, which effectively induces degradation of BET proteins and inhibits cell growth in human acute leukemia cell lines even at low picomolar concentrations. QCA570 achieves complete and durable tumor regression in leukemia xenograft models in mice at well-tolerated dose-schedules. QCA570 is the most potent and efficacious BET degrader reported to date.

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SUMMARY Loss of retinal ganglion cells (RGCs) accounts for visual function deficits after optic nerve injury, but how axonal insults leading to neuronal death remains elusive. By using an optic nerve crush model which results in the death of the majority of RGCs, we demonstrate that axotomy induces differential activation of distinct pathways of the unfolded protein response (UPR) in axotomized RGCs. Optic nerve injury provokes a sustained CCAAT/enhancer binding protein homologous protein (CHOP) up-regulation, and deletion of CHOP promotes RGC survival. In contrast, IRE/XBP-1 is only transiently activated, and forced XBP-1 activation dramatically protects RGCs from axon injury-induced death. Importantly, such differential activations of CHOP and XBP-1 and their distinct effects on neuronal cell death are also observed in RGCs with other types of axonal insults, such as vincristine treatment and intraocular pressure (IOP) elevation, suggesting a new protective strategy for neurodegeneration associated with axonal damage.

Using mouse optic nerve (ON) crush as a CNS injury model, we and others have found that activation of the mammalian target of rapamycin complex 1 (mTORC1) in mature retinal ganglion cells by deletion of the negative regulators, phosphatase and tensin homolog (PTEN) and tuberous sclerosis 1, promotes ON regeneration. mTORC1 activation inhibits eukaryotic translation initiation factor 4E-binding protein (4E-BP) and activates ribosomal protein S6 kinase 1 (S6K1), both of which stimulate translation. We reasoned that mTORC1’s regeneration-promoting effects might be separable from its deleterious effects by differential manipulation of its downstream effectors. Here we show that S6K1 activation, but not 4E-BP inhibition, is sufficient to promote axon regeneration. However, inhibition of 4E-BP is required for PTEN deletion-induced axon regeneration. Both activation and inhibition of S6K1 decrease the effect of PTEN deletion on axon regeneration, implicating a dual role of S6K1 in regulating axon growth.

Injured mature CNS axons do not regenerate in mammals. Deletion of PTEN, the negative regulator of PI3K, induces CNS axon regeneration through the activation of PI3K-mTOR signaling. We have conducted an extensive molecular dissection of the cross-regulating mechanisms in axon regeneration that involve the downstream effectors of PI3K, AKT and the two mTOR complexes (mTORC1 and mTORC2). We found that the predominant AKT isoform in CNS, AKT3, induces much more robust axon regeneration than AKT1 and that activation of mTORC1 and inhibition of GSK3β are two critical parallel pathways for AKT-induced axon regeneration. Surprisingly, phosphorylation of T308 and S473 of AKT play opposite roles in GSK3β phosphorylation and inhibition, by which mTORC2 and pAKT-S473 negatively regulate axon regeneration. Thus, our study revealed a complex neuron-intrinsic balancing mechanism involving AKT as the nodal point of PI3K, mTORC1/2 and GSK3β that coordinates both positive and negative cues to regulate adult CNS axon regeneration.DOI: http://dx.doi.org/10.7554/eLife.14908.001

Interleukin-10 is a predominantly anti-inflammatory cytokine that inhibits macrophage and dendritic cell function, but can acquire proinflammatory activity during immune responses. We investigated whether type I IFNs, which are elevated during infections and in autoimmune diseases, modulate the activity of IL-10. Priming of primary human macrophages with low concentrations of IFN-α diminished the ability of IL-10 to suppress TNF-α production. IFN-α conferred a proinflammatory gain of function on IL-10, leading to IL-10 activation of expression of IFN-γ-inducible, STAT1-dependent genes such as IFN regulatory factor 1, IFN-γ-inducible protein-10 (CXCL10), and monokine induced by IFN-γ (CXCL9). IFN-α priming resulted in greatly enhanced STAT1 activation in response to IL-10, and STAT1 was required for IL-10 activation of IFN-γ-inducible protein-10 and monokine induced by IFN-γ expression in IFN-α-primed cells. In control, unprimed cells, IL-10 activation of STAT1 was suppressed by constitutive activity of protein kinase C and Src homology 2 domain-containing phosphatase 1. These results demonstrate that type I IFNs regulate the balance between IL-10 anti- and proinflammatory activity, and provide insight into molecular mechanisms that regulate IL-10 function. Gain of IL-10 proinflammatory functions may contribute to its pathogenic role in autoimmune diseases characterized by elevated type I IFN levels, such as systemic lupus erythematosus.

Axon injury is an early event in neurodegenerative diseases that often leads to retrograde neuronal cell death and progressive permanent loss of vital neuronal functions. The connection of these two obviously sequential degenerative events, however, is elusive. Deciphering the upstream signals that trigger the neurodegeneration cascades in both neuronal soma and axon would be a key step toward developing the effective neuroprotectants that are greatly needed in the clinic. We showed previously that optic nerve injury-induced neuronal endoplasmic reticulum (ER) stress plays an important role in retinal ganglion cell (RGC) death. Using two in vivo mouse models of optic neuropathies (traumatic optic nerve injury and glaucoma) and adeno-associated virus-mediated RGC-specific gene targeting, we now show that differential manipulation of unfolded protein response pathways in opposite directions-inhibition of eukaryotic translation initiation factor 2␣-C/EBP homologous protein and activation of X-box binding protein 1-promotes both RGC axons and somata survival and preserves visual function. Our results indicate that axon injury-induced neuronal ER stress plays an important role in both axon degeneration and neuron soma death. Neuronal ER stress is therefore a promising therapeutic target for glaucoma and potentially other types of neurodegeneration.