X-linked Inhibitor of Apoptosis (XIAP) deficiency predisposes people to pathogen-associated hyperinflammation. Upon XIAP loss, Toll-like receptor (TLR) ligation triggers RIPK3-caspase-8-mediated IL-1β activation and death in myeloid cells. How XIAP suppresses these events remains unclear. Here, we show that TLR-MyD88 causes the proteasomal degradation of the related IAP, cIAP1, and its adaptor, TRAF2, by inducing TNF and TNF Receptor 2 (TNFR2) signaling. Genetically, we define that myeloid-specific cIAP1 loss promotes TLR-induced RIPK3-caspase-8 and IL-1β activity in the absence of XIAP. Importantly, deletion of TNFR2 in XIAP-deficient cells limited TLR-MyD88-induced cIAP1-TRAF2 degradation, cell death, and IL-1β activation. In contrast to TLR-MyD88, TLR-TRIF-induced interferon (IFN)β inhibited cIAP1 loss and consequent cell death. These data reveal how, upon XIAP deficiency, a TLR-TNF-TNFR2 axis drives cIAP1-TRAF2 degradation to allow TLR or TNFR1 activation of RIPK3-caspase-8 and IL-1β. This mechanism may explain why XIAP-deficient patients can exhibit symptoms reminiscent of patients with activating inflammasome mutations.

SUMMARY Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of anti-viral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2 deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV) and herpes simplex virus 1 (HSV-1) show impaired production of anti-viral cytokines and – in the case of EMCV and HSV-1 – reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.

Highlights d RTK oncoproteins can form de novo membraneless cytoplasmic protein granules d RTK protein granules activate RAS in a lipid membraneindependent manner d Higher-order protein assembly is critical for oncogenic RAS/ MAPK signaling d Protein granules serve as a subcellular platform for organizing RTK signaling

The differentiation process in neurons is a complex phenomenon involving changes in electrophysiological properties as well as morphological features characterized by dendritic and axonal outgrowths, broadly termed neurite outgrowth. The capacity to elaborate processes has achieved its maximal complexity in neurons, which utilize dendritic arbors to regulate the number of synaptic contacts and thus define the frequency at which impulses spread and thereby establish regionally distinct functional outputs (1). The regulation of neurite outgrowth is tightly controlled because of its critical physiological function. There are many signals that control the growth and directionality of dendrites and axons (2, 3). Many intracellular signaling components that control neurite outgrowth have been studied (4). However, their connections into networks have not been fully defined. Several G protein-coupled receptors trigger neurite outgrowth. For example, D2 dopamine receptors regulate neurite outgrowth in cortical neurons (5). Serotonin-1 B receptors are known to enhance neurite outgrowth in thalamic neurons (6). These receptors are coupled to G␣ i/o proteins, and G␣ o , one of the most abundant proteins in the neuronal growth cones, can induce neurite outgrowth (7). These studies indicate that G␣ i/o -coupled receptors play an important role in controlling neurite outgrowth. However, the signaling pathways by which the G␣ i/o signals trigger neurite outgrowth have not been determined. Recently, we demonstrated that the CB1 cannabinoid receptor, which is coupled to G␣ i/o , mediates neurite outgrowth through activation of Rap1 by enhancing proteasomal degradation of Rap1GAPII (8, 9). In a previous study, we show that G␣ i/o activates the c-Src-Stat3 pathway in NIH-3T3 fibroblast cells leading to cell transformation (10). Because c-Src is also an abundant protein in neuronal growth cones, we wondered as to the relationship between Rap and c-Src in G␣ i/o neurite formation and outgrowth. In this study, we showed that CB1 receptor activation leads to the Rap-dependent phosphorylation of Src and Stat3, and this network plays a critical role in neurite outgrowth. The network includes the small GTPases, such as Ral and Rac, as well as protein kinases such as JNK 3 /p38 that are involved in this process. These studies showed, within a fully endogenous system, the presence of a two-armed complex network that contains multiple small GTPases converging on Stat3 to regulate neurite outgrowth. EXPERIMENTAL PROCEDURESCell Culture and Transfection-Neuro-2A cells were cultured in 44% Dulbecco's modified Eagle's medium, 44% F-12 medium, 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% glutamine. Transfection was performed when cells were 75% confluent using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The catalytic domain of Src was replaced by green fluorescent protein to create a dominant negative form of Src (provided by Dr. Pamela L. Schwartzberg, National Institutes of Health). A dominant neg...

The G(alpha)o/i-coupled CB1 cannabionoid receptor induces neurite outgrowth in Neuro-2A cells. The mechanisms of signaling through G(alpha)o/i to induce neurite outgrowth were studied. The expression of G(alpha)o/i reduces the stability of its direct interactor protein, Rap1GAPII, by targeting it for ubiquitination and proteasomal degradation. This results in the activation of Rap1. G(alpha)o/i-induced activation of endogenous Rap1 in Neuro-2A cells is blocked by the proteasomal inhibitor lactacystin. G(alpha)o/i stimulates neurite outgrowth that is blocked by the expression of dominant negative Rap1. Expression of Rap1GAPII also blocks the G(alpha)o/i-induced neurite outgrowth and treatment with proteasomal inhibitors potentiates this inhibition. The endogenous G(alpha)o/i-coupled cannabinoid (CB1) receptor in Neuro-2A cells stimulates the degradation of Rap1GAPII; activation of Rap1 and treatment with pertussis toxin or lactacystin blocks these effects. The CB1 receptor-stimulated neurite outgrowth is blocked by treatment with pertussis toxin, small interfering RNA for Rap, lactacystin, and expression of Rap1GAPII. Thus, the G(alpha)o/i-coupled cannabinoid receptor, by regulating the proteasomal degradation of Rap1GAPII, activates Rap1 to induce neurite outgrowth.

Human corneal endothelial cells (HCEnCs) form a monolayer of hexagonal cells whose main function is to maintain corneal clarity by regulating corneal hydration. HCEnCs are derived from neural crest and are arrested in the post-mitotic state. Thus cell loss due to aging or corneal endothelial disorders leads to corneal edema and blindness–the leading indication for corneal transplantation. Here we show the existence of morphologically distinct subpopulations of HCEnCs that are interspersed among primary cells and exhibit enhanced self-renewal competence and lack of phenotypic signs of cellular senescence. Colonies of these uniform and hexagonal HCEnCs (HCEnC-21) were selectively isolated and demonstrated high proliferative potential that was dependent on endogenous upregulation of telomerase and cyclin D/CDK4. Further transduction of HCEnC-21 with telomerase yielded a highly proliferative corneal endothelial cell line (HCEnT-21T) that was devoid of oncogenic transformation and retained critical corneal endothelial cell characteristics and functionality. This study will significantly impact the fields of corneal cell biology and regenerative medicine.

Trans-synaptic degeneration could exacerbate neurodegeneration in multiple sclerosis (MS). We aimed to assess whether anterograde trans-synaptic degeneration could be identified in the primary visual pathway in vivo. Diffusion tensor imaging (DTI) was used to assess the optic radiations in 15 patients with previous optic nerve inflammation and 9 healthy volunteers. A probabilistic atlas of the optic radiations was created from healthy diffusion tractography data. Lengthwise profiles for DTI parameters (axial [λ(||) ], radial [λ(⟂) ] and mean diffusivity [MD], fractional anisotropy [FA] and the angle of deviation of the principal eigenvector [α]) were analyzed for patients and controls. Patients also underwent multifocal visual evoked potential (mfVEP) assessments to characterize the latency and amplitude of cortical potentials. Correlations were performed between mfVEP latency and amplitude in the left and right visual hemi-fields and DTI parameters in the contra-lateral optic radiations. Patients displayed a significant decrease in λ(||) within the body of both optic radiations, which significantly correlated with loss of mfVEP amplitude. Abnormal λ(⟂) and FA were detected bilaterally throughout the optic radiations in patients but the abnormality was not associated with amplitude reduction or latency prolongation of the mfVEP. An abnormal α value was observed in the left optic radiations of patients, and the α value in the body of the optic radiations also correlated with mfVEP amplitude loss. The assocation between bilateral DTI abnormalities within the optic radiations and loss of afferent electrical activity could indicate anterograde trans-synaptic degeneration occurs following optic neuritis.

The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs). Although previous work reveals important interconnections between autophagy and EVP-mediated secretion, our understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition, which mediates EVP-associated release of autophagic cargo receptors, including p62/SQSTM1. This secretion is highly regulated and dependent on multiple ATGs required for autophagosome formation, as well as the small GTPase Rab27a. Furthermore, disrupting autophagosome maturation, either via genetic inhibition of autophagosome-to-autolysosome fusion or expression of SARS-CoV-2 ORF3a, is sufficient to induce EVP secretion of autophagy cargo receptors. Finally, ATG-dependent EVP secretion buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Thus, we propose secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis during endolysosomal dysfunction or impaired autophagosome maturation.