STAT1-cooperative DNA binding distinguishes type 1 from type 2 interferon signaling.

STAT1 is an indispensable component of a heterotrimer (ISGF3) and a STAT1 homodimer (GAF) that function as transcription regulators in type 1 and type 2 interferon signaling, respectively. To investigate the importance of STAT1-cooperative DNA binding, we generated gene-targeted mice expressing cooperativity-deficient STAT1 with alanine substituted for Phe77. Neither ISGF3 nor GAF bound DNA cooperatively in the STAT1F77A mouse strain, but type 1 and type 2 interferon responses were affected differently. Type 2 interferon-mediated transcription and antibacterial immunity essentially disappeared owing to defective promoter recruitment of GAF. In contrast, STAT1 recruitment to ISGF3 binding sites and type 1 interferon-dependent responses, including antiviral protection, remained intact. We conclude that STAT1 cooperativity is essential for its biological activity and underlies the cellular responses to type 2, but not type 1 interferon.

Elezanumab, a human anti-RGMa monoclonal antibody, promotes neuroprotection, neuroplasticity, and neurorecovery following a thoracic hemicompression spinal cord injury in non-human primates.

Spinal cord injury (SCI) is a devastating condition characterized by loss of function, secondary to damaged spinal neurons, disrupted axonal connections, and myelin loss. Spontaneous recovery is limited, and there are no approved pharmaceutical treatments to reduce ongoing damage or promote repair. Repulsive guidance molecule A (RGMa) is upregulated following injury to the central nervous system (CNS), where it is believed to induce neuronal apoptosis and inhibit axonal growth and remyelination. We evaluated elezanumab, a human anti-RGMa monoclonal antibody, in a novel, newly characterized non-human primate (NHP) hemicompression model of thoracic SCI. Systemic intravenous (IV) administration of elezanumab over 6 months was well tolerated and associated with significant improvements in locomotor function. Treatment of animals for 16 weeks with a continuous intrathecal infusion of elezanumab below the lesion was not efficacious. IV elezanumab improved microstructural integrity of extralesional tissue as reflected by higher fractional anisotropy and magnetization transfer ratios in treated vs. untreated animals. IV elezanumab also reduced SCI-induced increases in soluble RGMa in cerebrospinal fluid, and membrane bound RGMa rostral and caudal to the lesion. Anterograde tracing of the corticospinal tract (CST) from the contralesional motor cortex following 20 weeks of IV elezanumab revealed a significant increase in the density of CST fibers emerging from the ipsilesional CST into the medial/ventral gray matter. There was a significant sprouting of serotonergic (5-HT) fibers rostral to the injury and in the ventral horn of lower thoracic regions. These data demonstrate that 6 months of intermittent IV administration of elezanumab, beginning within 24 h after a thoracic SCI, promotes neuroprotection and neuroplasticity of key descending pathways involved in locomotion. These findings emphasize the mechanisms leading to improved recovery of neuromotor functions with elezanumab in acute SCI in NHPs.

SUMO conjugation of STAT1 protects cells from hyperresponsiveness to IFNγ.

The biologic effects of IFNγ are mediated by the transcription factor STAT1. The activity of STAT1 is inhibited by small ubiquitin-like modifier (SUMO) conjugation. This occurs both directly through decreasing STAT1 tyrosine phosphorylation and indirectly by facilitating STAT1 dephosphorylation consequential to increased STAT1 solubility because of suppressed paracrystal assembly. However, the physiologic implications of SUMO conjugation have remained unclear. Here, we used fibroblasts and bone marrow-derived macrophages (BMMs) from knockin mice expressing SUMO-free STAT1 to explore the consequences of STAT1 sumoylation for IFNγ signaling. Our experiments demonstrated buffer property of paracrystals for activated STAT1, such that SUMO-mediated paracrystal dispersal profoundly reduced phosphorylation of STAT1, which affected both the activating tyrosine 701 and the transcription-enhancing serine 727. Accordingly, the curtailed STAT1 activity in the nucleus caused by SUMO conjugation resulted in diminished transcription of IFNγ-responsive genes; and increased the IFNγ concentration more than 100-fold required to trigger lipopolysaccharide-induced cytotoxicity in bone marrow-derived macrophages. These experiments identify SUMO conjugation of STAT1 as a mechanism to permanently attenuate the IFNγ sensitivity of cells, which prevents hyperresponsiveness to this cytokine and its potentially self-destructive consequences. This sets the mode of SUMO-mediated inhibition apart from the other negative STAT regulators known to date.

Neutralizing RGMa with Elezanumab Promotes Cerebroprotection and Recovery in Rabbit Middle Cerebral Artery Occlusion.

Repulsive guidance molecule A (RGMa) is an inhibitor of neuronal growth and survival which is upregulated in the damaged central nervous system following acute spinal cord injury (SCI), traumatic brain injury, acute ischemic stroke (AIS), and other neuropathological conditions. Neutralization of RGMa is neuroprotective and promotes neuroplasticity in several preclinical models of neurodegeneration and injury including multiple sclerosis, AIS, and SCI. Given the limitations of current treatments for AIS due to narrow time windows to intervention (TTI), and restrictive patient selection criteria, there is significant unmet need for therapeutic agents that enable tissue survival and repair following acute ischemic damage for a broader population of stroke patients. In this preclinical study, we evaluated whether elezanumab, a human anti-RGMa monoclonal antibody, could improve neuromotor function and modulate neuroinflammatory cell activation following AIS with delayed intervention times up to 24 h using a rabbit embolic permanent middle cerebral artery occlusion model (pMCAO). In two replicate 28-day pMCAO studies, weekly intravenous infusions of elezanumab, over a range of doses and TTIs of 6 and 24 h after stroke, significantly improved neuromotor function in both pMCAO studies when first administered 6 h after stroke. All elezanumab treatment groups, including the 24 h TTI group, had significantly less neuroinflammation as assessed by microglial and astrocyte activation. The novel mechanism of action and potential for expanding TTI in human AIS make elezanumab distinct from current acute reperfusion therapies, and support evaluation in clinical trials of acute CNS damage to determine optimal dose and TTI in humans. A: Ramified/resting astrocytes and microglia in a normal, uninjured rabbit brain. B: Rabbit pMCAO brain illustrating lesion on right side of brain (red), surrounded by penumbra (pink) during acute phase post stroke, with minimal injury to left brain hemisphere. Penumbra characterized by activated astrocytes and microglia (region in crosshair within circle), with upregulation of free and bound RGMa. C: Elezanumab binds to both free and bound RGMa, preventing full activation of astrocytes and microglia. D: Elezanumab is efficacious in rabbit pMCAO with a 4 × larger TTI window vs. tPA (6 vs. 1.5 h, respectively). In human AIS, tPA is approved for a TTI of 3-4.5 h. Elezanumab is currently being evaluated in a clinical Ph2 study of AIS to determine the optimal dose and TTI (NCT04309474).

Cytokine-induced paracrystals prolong the activity of signal transducers and activators of transcription (STAT) and provide a model for the regulation of protein solubility by small ubiquitin-like modifier (SUMO).

The biological effects of cytokines are mediated by STAT proteins, a family of dimeric transcription factors. In order to elicit transcriptional activity, the STATs require activation by phosphorylation of a single tyrosine residue. Our experiments revealed that fully tyrosine-phosphorylated STAT dimers polymerize via Tyr(P)-Src homology 2 domain interactions and assemble into paracrystalline arrays in the nucleus of cytokine-stimulated cells. Paracrystals are demonstrated to be dynamic reservoirs that protect STATs from dephosphorylation. Activated STAT3 forms such paracrystals in acute phase liver cells. Activated STAT1, in contrast, does not normally form paracrystals. By reversing the abilities of STAT1 and STAT3 to be sumoylated, we show that this is due to the unique ability of STAT1 among the STATs to conjugate to small ubiquitin-like modifier (SUMO). Sumoylation had one direct effect; it obstructed proximal tyrosine phosphorylation, which led to semiphosphorylated STAT dimers. These competed with their fully phosphorylated counterparts and interfered with their polymerization into paracrystals. Consequently, sumoylation, by preventing paracrystal formation, profoundly curtailed signal duration and reporter gene activation in response to cytokine stimulation of cells. The study thus identifies polymerization of activated STAT transcription factors as a positive regulatory mechanism in cytokine signaling. It provides a unifying explanation for the different subnuclear distributions of STAT transcription factors and reconciles the conflicting results as to the role of SUMO modification in STAT1 functioning. We present a generally applicable system in which protein solubility is maintained by a disproportionately small SUMO-modified fraction, whereby modification by SUMO partially prevents formation of polymerization interfaces, thus generating competitive polymerization inhibitors.

A genetically modified minipig model for Alzheimer's disease with SORL1 haploinsufficiency.

The established causal genes in Alzheimer's disease (AD), APP, PSEN1, and PSEN2, are functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease's initial preclinical phase. Mutations in SORL1, encoding the endosome recycling receptor SORLA, are found in 2%-3% of individuals with early-onset AD, and SORL1 haploinsufficiency appears to be causal for AD. To test whether SORL1 can function as an AD causal gene, we use CRISPR-Cas9-based gene editing to develop a model of SORL1 haploinsufficiency in Göttingen minipigs, taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young adult minipigs is found to phenocopy the preclinical in vivo profile of AD observed with APP, PSEN1, and PSEN2, resulting in elevated levels of ß-amyloid (Aß) and tau preceding amyloid plaque formation and neurodegeneration, as observed in humans. Our study provides functional support for the theory that SORL1 haploinsufficiency leads to endosome cytopathology with biofluid hallmarks of autosomal dominant AD.

The SUMO2/3 specific E3 ligase ZNF451-1 regulates PML stability.

The small ubiquitin related modifier SUMO regulates protein functions to maintain cell homeostasis. SUMO attachment is executed by the hierarchical action of E1, E2 and E3 enzymes of which E3 ligases ensure substrate specificity. We recently identified the ZNF451 family as novel class of SUMO2/3 specific E3 ligases and characterized their function in SUMO chain formation. The founding member, ZNF451isoform1 (ZNF451-1) partially resides in PML bodies, nuclear structures organized by the promyelocytic leukemia gene product PML. As PML and diverse PML components are well known SUMO substrates the question arises whether ZNF451-1 is involved in their sumoylation. Here, we show that ZNF451-1 indeed functions as SUMO2/3 specific E3 ligase for PML and selected PML components in vitro. Mutational analysis indicates that substrate sumoylation employs an identical biochemical mechanism as we described for SUMO chain formation. In vivo, ZNF451-1 RNAi depletion leads to PML stabilization and an increased number of PML bodies. By contrast, PML degradation upon arsenic trioxide treatment is not ZNF451-1 dependent. Our data suggest a regulatory role of ZNF451-1 in fine-tuning physiological PML levels in a RNF4 cooperative manner in the mouse neuroblastoma N2a cell-line.

A new vertebrate SUMO enzyme family reveals insights into SUMO-chain assembly.

SUMO chains act as stress-induced degradation tags or repair factor-recruiting signals at DNA lesions. Although E1 activating, E2 conjugating and E3 ligating enzymes efficiently assemble SUMO chains, specific chain-elongation mechanisms are unknown. E4 elongases are specialized E3 ligases that extend a chain but are inefficient in the initial conjugation of the modifier. We identified ZNF451, a representative member of a new class of SUMO2 and SUMO3 (SUMO2/3)-specific enzymes that execute catalysis via a tandem SUMO-interaction motif (SIM) region. One SIM positions the donor SUMO while a second SIM binds SUMO on the back side of the E2 enzyme. This tandem-SIM region is sufficient to extend a back side-anchored SUMO chain (E4 elongase activity), whereas efficient chain initiation also requires a zinc-finger region to recruit the initial acceptor SUMO (E3 ligase activity). Finally, we describe four human proteins sharing E4 elongase activities and their function in stress-induced SUMO2/3 conjugation.

SUMO rules: regulatory concepts and their implication in neurologic functions.

Posttranslational modification of proteins by the small ubiquitin-like modifier (SUMO) is a potent regulator of various cellular events. Hundreds of substrates have been identified, many of them involved in vital processes like transcriptional regulation, signal transduction, protein degradation, cell cycle regulation, DNA repair, chromatin organization, and nuclear transport. In recent years, protein sumoylation increasingly attracted attention, as it could be linked to heart failure, cancer, and neurodegeneration. However, underlying mechanisms involving how modification by SUMO contributes to disease development are still scarce thus necessitating further research. This review aims to critically discuss currently available concepts of the SUMO pathway, thereby highlighting regulation in the healthy versus diseased organism, focusing on neurologic aspects. Better understanding of differential regulation in health and disease may finally allow to uncover pathogenic mechanisms and contribute to the development of disease-specific therapies.