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CDKL5 deficiency disorder (CDD) is a rare, X-linked, developmental and epileptic encephalopathy characterised by severe global developmental impairment and seizures that can begin in the first few months after birth and are often treatment refractory. Ganaxolone, an investigational neuroactive steroid, reduced seizure frequency in an open-label, phase 2 trial that included patients with CDD. We aimed to further assess the efficacy and safety of ganaxolone in patients with CDD-associated refractory epilepsy. In the double-blind phase of this randomised, placebo-controlled, phase 3 trial, done at 39 outpatient clinics in eight countries (Australia, France, Israel, Italy, Poland, Russia, the UK, and the USA), patients were eligible if they were aged 2–21 years with a pathogenic or probably pathogenic CDKL5 variant and at least 16 major motor seizures (defined as bilateral tonic, generalised tonic-clonic, bilateral clonic, atonic, or focal to bilateral tonic-clonic) per 28 days in each 4-week period of an 8-week historical period. After a 6-week prospective baseline period, patients were randomly assigned (1:1) via an interactive web response system to receive either enteral adjunctive ganaxolone or matching enteral adjunctive placebo (maximum dose 63 mg/kg per day for patients weighing ≤28 kg or 1800 mg/day for patients weighing >28 kg) for 17 weeks. Patients, caregivers, investigators (including those analysing data), trial staff, and the sponsor (other than the investigational product manager) were masked to treatment allocation. The primary efficacy endpoint was percentage change in median 28-day major motor seizure frequency from the baseline period to the 17-week double-blind phase and was analysed (using a Wilcoxon-rank sum test) in all patients who received at least one dose of trial treatment and for whom baseline data were available. Safety (compared descriptively across groups) was analysed in all patients who received at least one dose of trial treatment. This study is registered with ClinicalTrials.gov, NCT03572933, and the open-label extension phase is ongoing. Between June 25, 2018, and July 2, 2020, 114 patients were screened for eligibility, of whom 101 (median age 6 years [IQR 3 to 10]) were randomly assigned to receive either ganaxolone (n=50) or placebo (n=51). All patients received at least one dose of a study drug, but seizure frequency for one patient in the ganaxolone group was not recorded at baseline and so the primary endpoint was analysed in a population of 100 patients. There was a median percentage change in 28-day major motor seizure frequency of –30·7% (IQR –49·5 to –1·9) in the ganaxolone group and of –6·9% (–24·1 to 39·7) in the placebo group (p=0·0036). The Hodges–Lehmann estimate of median difference in responses to ganaxolone versus placebo was –27·1% (95% CI –47·9 to – 9·6). Treatment-emergent adverse events occurred in 43 (86%) of 50 patients in the ganaxolone group and in 45 (88%) of 51 patients in the placebo group. Somnolence, pyrexia, and upper respiratory tract infections occurred in at least 10% of patients in the ganaxolone group and more frequently than in the placebo group. Serious adverse events occurred in six (12%) patients in the ganaxolone group and in five (10%) patients in the placebo group. Two (4%) patients in the ganaxolone group and four (8%) patients in the placebo group discontinued the trial. There were no deaths in the double-blind phase. Ganaxolone significantly reduced the frequency of CDD-associated seizures compared with placebo and was generally well tolerated. Results from what is, to our knowledge, the first controlled trial in CDD suggest a potential treatment benefit for ganaxolone. Long-term treatment is being assessed in the ongoing open-label extension phase of this trial. Marinus Pharmaceuticals.

The enzyme RIPK1 functions through its RHIM domain to prevent ZBP1-mediated activation of RIPK3–MLKL-dependent necroptosis, thus preventing perinatal lethality and skin inflammation in adult mice. RIPK1 inhibition of inflammation Manolis Pasparakis and colleagues report that receptor-interacting protein kinase 1 (RIPK1) functions via its RIP homotypic interaction motif (RHIM) to prevent skin inflammation in mice by inhibiting activation of RIPK3–MLKL-dependent necroptosis mediated by Z-DNA binding protein 1 (ZBP1; also known as DAI). The finding that ZBP1 is a critical mediator of inflammation beyond its previously known role in antiviral defence suggests that ZBP1 might be involved in the pathogenesis of necroptosis-associated inflammatory diseases. Receptor-interacting protein kinase 1 (RIPK1) regulates cell death and inflammation through kinase-dependent and -independent functions 1 , 2 , 3 , 4 , 5 , 6 , 7 . RIPK1 kinase activity induces caspase-8-dependent apoptosis and RIPK3 and mixed lineage kinase like (MLKL)-dependent necroptosis 8 , 9 , 10 , 11 , 12 , 13 . In addition, RIPK1 inhibits apoptosis and necroptosis through kinase-independent functions, which are important for late embryonic development and the prevention of inflammation in epithelial barriers 14 , 15 , 16 , 17 , 18 . The mechanism by which RIPK1 counteracts RIPK3–MLKL-mediated necroptosis has remained unknown. Here we show that RIPK1 prevents skin inflammation by inhibiting activation of RIPK3–MLKL-dependent necroptosis mediated by Z-DNA binding protein 1 (ZBP1, also known as DAI or DLM1). ZBP1 deficiency inhibited keratinocyte necroptosis and skin inflammation in mice with epidermis-specific RIPK1 knockout. Moreover, mutation of the conserved RIP homotypic interaction motif (RHIM) of endogenous mouse RIPK1 (RIPK1 mRHIM ) caused perinatal lethality that was prevented by RIPK3, MLKL or ZBP1 deficiency. Furthermore, mice expressing only RIPK1 mRHIM in keratinocytes developed skin inflammation that was abrogated by MLKL or ZBP1 deficiency. Mechanistically, ZBP1 interacted strongly with phosphorylated RIPK3 in cells expressing RIPK1 mRHIM , suggesting that the RIPK1 RHIM prevents ZBP1 from binding and activating RIPK3. Collectively, these results show that RIPK1 prevents perinatal death as well as skin inflammation in adult mice by inhibiting ZBP1-induced necroptosis. Furthermore, these findings identify ZBP1 as a critical mediator of inflammation beyond its previously known role in antiviral defence and suggest that ZBP1 might be implicated in the pathogenesis of necroptosis-associated inflammatory diseases.

NEK7, a member of the NIMA-related kinase family, is identified as a regulator of NLRP3 inflammasome oligomerization and activation; NEK7 functions downstream of potassium efflux in a manner that is independent of its kinase activity. NEK7 mediates NLRP3 inflammasome activation The NLRP3 inflammasome, a critical component of the innate immune system, has been linked to multiple acquired and inherited diseases. However, the molecular mechanism that leads to NLRP3 oligomerization and activation remains elusive. Here Gabriel Núñez and colleagues identify a member of the family of NIM related kinases (NEK7) as a regulator of NLRP3 inflammasome oligomerization and activation. NEK7 functions downstream of potassium efflux in a manner that is independent of its kinase activity. Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases 1 . So far, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor protein ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-18 proteins 2 , 3 . The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases 4 , 5 as well as cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations 6 , 7 . Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by many stimuli 8 , 9 . Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. Here we report the identification of NEK7, a member of the family of mammalian NIMA-related kinases (NEK proteins) 10 , as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of NEK7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasomes. NLRP3-activating stimuli promoted the NLRP3–NEK7 interaction in a process that was dependent on potassium efflux. NLRP3 associated with the catalytic domain of NEK7, but the catalytic activity of NEK7 was shown to be dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3–NEK7 complex, which, along with ASC oligomerization and ASC speck formation, was abrogated in the absence of NEK7. NEK7 was required for macrophages containing the CAPS-associated NLRP3(R258W) activating mutation to activate caspase-1. Mouse chimaeras reconstituted with wild-type, Nek7 −/− or Nlrp3 −/− haematopoietic cells showed that NEK7 was required for NLRP3 inflammasome activation in vivo . These studies demonstrate that NEK7 is an essential protein that acts downstream of potassium efflux to mediate NLRP3 inflammasome assembly and activation.

In the absence of RIPK1, ZBP1 engages RIPK3 in a RHIM-dependent manner and acts as a critical activator of RIPK3/MLKL-dependent necroptosis. RIPK1 regulation of necroptosis Receptor-interacting protein kinase 1 (RIPK1) is known to act as a brake on apoptosis and necroptosis during development. Here Kim Newton et al . report that, in the absence of RIPK1, the adapter protein ZBP1 (also known as DAI) engages RIPK3 in a RIP homotypic interaction motif (RHIM)-dependent manner and acts as a critical activator of RIPK3–MLKL-dependent necroptosis. This finding, together with related analyses, suggests an unexpected role for ZBP1 in triggering necroptosis in the perinatal period. Receptor-interacting protein kinase 1 (RIPK1) promotes cell survival—mice lacking RIPK1 die perinatally, exhibiting aberrant caspase-8-dependent apoptosis and mixed lineage kinase-like (MLKL)-dependent necroptosis 1 , 2 , 3 . However, mice expressing catalytically inactive RIPK1 are viable 2 , 4 , 5 , and an ill-defined pro-survival function for the RIPK1 scaffold has therefore been proposed. Here we show that the RIP homotypic interaction motif (RHIM) in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 (Z-DNA binding protein 1; also known as DAI or DLM1) from activating RIPK3 upstream of MLKL. Ripk1 RHIM/RHIM mice that expressed mutant RIPK1 with critical RHIM residues IQIG mutated to AAAA died around birth and exhibited RIPK3 autophosphorylation on Thr231 and Ser232, which is a hallmark of necroptosis 6 , in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1 RHIM/RHIM mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses 7 , 8 but previously had no defined role in development, also prevented perinatal lethality in Ripk1 RHIM/RHIM mice. Consistent with the RHIM of RIPK1 functioning as a brake that prevents ZBP1 from engaging the RIPK3 RHIM, ZBP1 interacted with RIPK3 in Ripk1 RHIM/RHIM Mlkl −/− macrophages, but not in wild-type, Mlkl −/− or Ripk1 RHIM/RHIM Ripk3 RHIM/RHIM macrophages. Collectively, these findings indicate that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.

Deletion of the Hippo pathway component Salvador in mouse hearts with established ischaemic heart failure after myocardial infarction induces a reparative genetic program with increased scar border vascularity, reduced fibrosis, and recovery of pumping function. Salvador deletion reverses heart failure Previous work has shown that interfering with Hippo signalling during myocardial injury improves heart function in mice. Clinical outcomes of acute myocardial infarction in humans have improved as a result of better emergency care, but chronic heart failure, whereby the heart tissue undergoes pathological remodelling, remains a leading cause of death. James Martin and colleagues now show that the failing heart has a previously unrecognized capacity for repair. They show that blocking Hippo signalling can rescue established heart failure in mice. Deletion of the Hippo pathway component Salvador (Salv) or virus-mediated delivery of Salv short hairpin RNA when ischaemic heart failure is established can improve heart function in mice. The authors attribute the effect to the induction of a reparative genetic program, including increased expression of stress response genes and proliferative genes and preservation of mitochondrial quality control. Mammalian organs vary widely in regenerative capacity. Poorly regenerative organs, such as the heart are particularly vulnerable to organ failure. Once established, heart failure commonly results in mortality 1 . The Hippo pathway, a kinase cascade that prevents adult cardiomyocyte proliferation and regeneration 2 , is upregulated in human heart failure. Here we show that deletion of the Hippo pathway component Salvador (Salv) in mouse hearts with established ischaemic heart failure after myocardial infarction induces a reparative genetic program with increased scar border vascularity, reduced fibrosis, and recovery of pumping function compared with controls. Using translating ribosomal affinity purification, we isolate cardiomyocyte-specific translating messenger RNA. Hippo-deficient cardiomyocytes have increased expression of proliferative genes and stress response genes, such as the mitochondrial quality control gene, Park2 . Genetic studies indicate that Park2 is essential for heart repair, suggesting a requirement for mitochondrial quality control in regenerating myocardium. Gene therapy with a virus encoding Salv short hairpin RNA improves heart function when delivered at the time of infarct or after ischaemic heart failure following myocardial infarction was established. Our findings indicate that the failing heart has a previously unrecognized reparative capacity involving more than cardiomyocyte renewal.

RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage 1 – 7 . The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis 8 . Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy—a condition we term ‘cleavage-resistant RIPK1-induced autoinflammatory syndrome’. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1 D325A mutant mouse strain. Whereas Ripk1 −/− mice died postnatally from systemic inflammation, Ripk1 D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3 , but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1 D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1 D325A/D325A and Ripk1 D325A /+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1 D325A /+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life. Heterozygous mutateons in the caspase-8 cleavage site of RIPK1 cause a range of autoinflammatory symptoms in humans, and caspase-8 cleavage of RIPK1 in a mouse model limits TNF-induced cell death and inflammation.

Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia–reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia–reperfusion injury model, the high-dose TNF model, and in A20 −/− mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia–reperfusion injury model and no benefit in A20 −/− mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease.

The phytohormone auxin controls many processes in plants, at least in part through its regulation of cell expansion 1 . The acid growth hypothesis has been proposed to explain auxin-stimulated cell expansion for five decades, but the mechanism that underlies auxin-induced cell-wall acidification is poorly characterized. Auxin induces the phosphorylation and activation of the plasma membrane H + -ATPase that pumps protons into the apoplast 2 , yet how auxin activates its phosphorylation remains unclear. Here we show that the transmembrane kinase (TMK) auxin-signalling proteins interact with plasma membrane H + -ATPases, inducing their phosphorylation, and thereby promoting cell-wall acidification and hypocotyl cell elongation in Arabidopsis . Auxin induced interactions between TMKs and H + -ATPases in the plasma membrane within seconds, as well as TMK-dependent phosphorylation of the penultimate threonine residue on the H+-ATPases. Our genetic, biochemical and molecular evidence demonstrates that TMKs directly phosphorylate plasma membrane H + -ATPase and are required for auxin-induced H + -ATPase activation, apoplastic acidification and cell expansion. Thus, our findings reveal a crucial connection between auxin and plasma membrane H + -ATPase activation in regulating apoplastic pH changes and cell expansion through TMK-based cell surface auxin signalling. Auxin induces transmembrane-kinase-dependent activation of H + -ATPase in the plasma membrane through phosphorylation of its penultimate threonine residue, promoting apoplastic acidification and hypocotyl cell elongation in Arabidopsis .

Alternatively activated (also known as M2) macrophages are involved in the repair of various types of organs. However, the contribution of M2 macrophages to cardiac repair after myocardial infarction (MI) remains to be fully characterized. Here, we identified CD206+F4/80+CD11b+ M2-like macrophages in the murine heart and demonstrated that this cell population predominantly increases in the infarct area and exhibits strengthened reparative abilities after MI. We evaluated mice lacking the kinase TRIB1 (Trib1-/-), which exhibit a selective depletion of M2 macrophages after MI. Compared with control animals, Trib1-/- mice had a catastrophic prognosis, with frequent cardiac rupture, as the result of markedly reduced collagen fibril formation in the infarct area due to impaired fibroblast activation. The decreased tissue repair observed in Trib1-/- mice was entirely rescued by an external supply of M2-like macrophages. Furthermore, IL-1α and osteopontin were suggested to be mediators of M2-like macrophage-induced fibroblast activation. In addition, IL-4 administration achieved a targeted increase in the number of M2-like macrophages and enhanced the post-MI prognosis of WT mice, corresponding with amplified fibroblast activation and formation of more supportive fibrous tissues in the infarcts. Together, these data demonstrate that M2-like macrophages critically determine the repair of infarcted adult murine heart by regulating fibroblast activation and suggest that IL-4 is a potential biological drug for treating MI.

Haematopoietic expression of the adaptor protein Trib1 is shown to be required for the presence of adipose-tissue-resident macrophages with an M2-like phenotype; Trib1 deficiency leads to aberrant expression of C/EBPα and impaired adipose tissue function. Trib1 protein role in macrophage function Macrophages are classified loosely into two types: M1 cells are immune cells active against microbial infection, and M2 cells have a broad spectrum of activities involving tissue repair, helminth infection, tumour progression and various metabolic disorders. This paper demonstrates that Tribbles homolog 1 (Trib1), an adaptor protein involved in protein degradation through interaction with COP1 ubiquitin ligase, is essential for the development of adipose-tissue-resident macrophages with an M2-like phenotype. Trib1 deficiency leads to aberrant expression of the transcription factor C/EBPα and impaired adipose tissue function. TRIB1 mutations have been implicated in metabolic disorders including atherosclerosis and hyperlipidaemia, and this work points to possible explanation of the relations between TRIB1 and metabolic disorders in humans. Macrophages consist of at least two subgroups, M1 and M2 (refs 1 , 2 , 3 ). Whereas M1 macrophages are proinflammatory and have a central role in host defence against bacterial and viral infections 4 , 5 , M2 macrophages are associated with responses to anti-inflammatory reactions, helminth infection, tissue remodelling, fibrosis and tumour progression 6 . Trib1 is an adaptor protein involved in protein degradation by interacting with COP1 ubiquitin ligase 7 . Genome-wide association studies in humans have implicated TRIB1 in lipid metabolism 8 , 9 , 10 . Here we show that Trib1 is critical for the differentiation of F4/80 + MR + tissue-resident macrophages—that share characteristics with M2 macrophages (which we term M2-like macrophages)—and eosinophils but not for the differentiation of M1 myeloid cells. Trib1 deficiency results in a severe reduction of M2-like macrophages in various organs, including bone marrow, spleen, lung and adipose tissues. Aberrant expression of C/EBPα in Trib1-deficient bone marrow cells is responsible for the defects in macrophage differentiation. Unexpectedly, mice lacking Trib1 in haematopoietic cells show diminished adipose tissue mass accompanied by evidence of increased lipolysis, even when fed a normal diet. Supplementation of M2-like macrophages rescues the pathophysiology, indicating that a lack of these macrophages is the cause of lipolysis. In response to a high-fat diet, mice lacking Trib1 in haematopoietic cells develop hypertriglyceridaemia and insulin resistance, together with increased proinflammatory cytokine gene induction. Collectively, these results demonstrate that Trib1 is critical for adipose tissue maintenance and suppression of metabolic disorders by controlling the differentiation of tissue-resident M2-like macrophages.