The choroid plexus links innate immunity to CSF dysregulation in hydrocephalus.

The choroid plexus (ChP) is the blood-cerebrospinal fluid (CSF) barrier and the primary source of CSF. Acquired hydrocephalus, caused by brain infection or hemorrhage, lacks drug treatments due to obscure pathobiology. Our integrated, multi-omic investigation of post-infectious hydrocephalus (PIH) and post-hemorrhagic hydrocephalus (PHH) models revealed that lipopolysaccharide and blood breakdown products trigger highly similar TLR4-dependent immune responses at the ChP-CSF interface. The resulting CSF "cytokine storm", elicited from peripherally derived and border-associated ChP macrophages, causes increased CSF production from ChP epithelial cells via phospho-activation of the TNF-receptor-associated kinase SPAK, which serves as a regulatory scaffold of a multi-ion transporter protein complex. Genetic or pharmacological immunomodulation prevents PIH and PHH by antagonizing SPAK-dependent CSF hypersecretion. These results reveal the ChP as a dynamic, cellularly heterogeneous tissue with highly regulated immune-secretory capacity, expand our understanding of ChP immune-epithelial cell cross talk, and reframe PIH and PHH as related neuroimmune disorders vulnerable to small molecule pharmacotherapy.

Genetic variants in UNC93B1 predispose to childhood-onset systemic lupus erythematosus.

Rare genetic variants in toll-like receptor 7 (TLR7) are known to cause lupus in humans and mice. UNC93B1 is a transmembrane protein that regulates TLR7 localization into endosomes. In the present study, we identify two new variants in UNC93B1 (T314A, located proximally to the TLR7 transmembrane domain, and V117L) in a cohort of east Asian patients with childhood-onset systemic lupus erythematosus. The V117L variant was associated with increased expression of type I interferons and NF-κB-dependent cytokines in patient plasma and immortalized B cells. THP-1 cells expressing the variant UNC93B1 alleles exhibited exaggerated responses to stimulation of TLR7/-8, but not TLR3 or TLR9, which could be inhibited by targeting the downstream signaling molecules, IRAK1/-4. Heterozygous mice expressing the orthologous Unc93b1V117L variant developed a spontaneous lupus-like disease that was more severe in homozygotes and again hyperresponsive to TLR7 stimulation. Together, this work formally identifies genetic variants in UNC93B1 that can predispose to childhood-onset systemic lupus erythematosus.

G-CSF drives autoinflammation in APLAID.

Missense mutations in PLCG2 can cause autoinflammation with phospholipase C gamma 2-associated antibody deficiency and immune dysregulation (APLAID). Here, we generated a mouse model carrying an APLAID mutation (p.Ser707Tyr) and found that inflammatory infiltrates in the skin and lungs were only partially ameliorated by removing inflammasome function via the deletion of caspase-1. Also, deleting interleukin-6 or tumor necrosis factor did not fully prevent APLAID mutant mice from autoinflammation. Overall, these findings are in accordance with the poor response individuals with APLAID have to treatments that block interleukin-1, JAK1/2 or tumor necrosis factor. Cytokine analysis revealed increased granulocyte colony-stimulating factor (G-CSF) levels as the most distinct feature in mice and individuals with APLAID. Remarkably, treatment with a G-CSF antibody completely reversed established disease in APLAID mice. Furthermore, excessive myelopoiesis was normalized and lymphocyte numbers rebounded. APLAID mice were also fully rescued by bone marrow transplantation from healthy donors, associated with reduced G-CSF production, predominantly from non-hematopoietic cells. In summary, we identify APLAID as a G-CSF-driven autoinflammatory disease, for which targeted therapy is feasible.

TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS.

Cytoplasmic accumulation of TDP-43 is a disease hallmark for many cases of amyotrophic lateral sclerosis (ALS), associated with a neuroinflammatory cytokine profile related to upregulation of nuclear factor κB (NF-κB) and type I interferon (IFN) pathways. Here we show that this inflammation is driven by the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) when TDP-43 invades mitochondria and releases DNA via the permeability transition pore. Pharmacologic inhibition or genetic deletion of cGAS and its downstream signaling partner STING prevents upregulation of NF-κB and type I IFN induced by TDP-43 in induced pluripotent stem cell (iPSC)-derived motor neurons and in TDP-43 mutant mice. Finally, we document elevated levels of the specific cGAS signaling metabolite cGAMP in spinal cord samples from patients, which may be a biomarker of mtDNA release and cGAS/STING activation in ALS. Our results identify mtDNA release and cGAS/STING activation as critical determinants of TDP-43-associated pathology and demonstrate the potential for targeting this pathway in ALS.

Small Molecule Targets TMED9 and Promotes Lysosomal Degradation to Reverse Proteinopathy.

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.

Germline NLRP1 Mutations Cause Skin Inflammatory and Cancer Susceptibility Syndromes via Inflammasome Activation.

Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition.

RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis.

Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.

Recording gene expression order in DNA by CRISPR addition of retron barcodes.

Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this through engineered RNA barcodes, based on prokaryotic retrons1, that are reverse transcribed into DNA and integrated into the genome using the CRISPR-Cas system2. The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record through simple, logical rules rather than relying on pretrained classifiers or post hoc inferential methods. For disambiguation in the field, we will refer to this system as a Retro-Cascorder.

Caspase-8-driven apoptotic and pyroptotic crosstalk causes cell death and IL-1ß release in X-linked inhibitor of apoptosis (XIAP) deficiency.

Genetic lesions in X-linked inhibitor of apoptosis (XIAP) pre-dispose humans to cell death-associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency-associated inflammatory bowel disease display increased inflammatory IL-1ß maturation as well as cell death-associated caspase-8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase-8-driven cell death and bioactive IL-1ß release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase-8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase-1, -3, -7, -11 and BID), while caspase-8 can still cause cell death in the absence of both GSDMD and GSDME when caspase-3 and caspase-7 are present. Neither caspase-3 and caspase-7-mediated activation of the pannexin-1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase-1 and IL-1ß maturation downstream of XIAP inhibition and caspase-8 activation, even though the pannexin-1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, reveal how XIAP loss can co-opt dual cell death programs, and uncover strategies for targeting the cell death and inflammatory pathways that result from XIAP deficiency.

The transcriptional regulators IRF4, BATF and IL-33 orchestrate development and maintenance of adipose tissue-resident regulatory T cells.

Foxp3(+) regulatory T (Treg) cells in visceral adipose tissue (VAT-Treg cells) are functionally specialized tissue-resident cells that prevent obesity-associated inflammation and preserve insulin sensitivity and glucose tolerance. Their development depends on the transcription factor PPAR-γ; however, the environmental cues required for their differentiation are unknown. Here we show that interleukin 33 (IL-33) signaling through the IL-33 receptor ST2 and myeloid differentiation factor MyD88 is essential for development and maintenance of VAT-Treg cells and sustains their transcriptional signature. Furthermore, the transcriptional regulators BATF and IRF4 were necessary for VAT-Treg differentiation through direct regulation of ST2 and PPAR-γ expression. IL-33 administration induced vigorous population expansion of VAT-Treg cells, which tightly correlated with improvements in metabolic parameters in obese mice. Human omental adipose tissue Treg cells also showed high ST2 expression, suggesting an evolutionarily conserved requirement for IL-33 in VAT-Treg cell homeostasis.

Pathogenic variants in autism gene KATNAL2 cause hydrocephalus and disrupt neuronal connectivity by impairing ciliary microtubule dynamics.

Enlargement of the cerebrospinal fluid (CSF)-filled brain ventricles (cerebral ventriculomegaly), the cardinal feature of congenital hydrocephalus (CH), is increasingly recognized among patients with autism spectrum disorders (ASD). KATNAL2, a member of Katanin family microtubule-severing ATPases, is a known ASD risk gene, but its roles in human brain development remain unclear. Here, we show that nonsense truncation of Katnal2 (Katnal2Δ17) in mice results in classic ciliopathy phenotypes, including impaired spermatogenesis and cerebral ventriculomegaly. In both humans and mice, KATNAL2 is highly expressed in ciliated radial glia of the fetal ventricular-subventricular zone as well as in their postnatal ependymal and neuronal progeny. The ventriculomegaly observed in Katnal2Δ17 mice is associated with disrupted primary cilia and ependymal planar cell polarity that results in impaired cilia-generated CSF flow. Further, prefrontal pyramidal neurons in ventriculomegalic Katnal2Δ17 mice exhibit decreased excitatory drive and reduced high-frequency firing. Consistent with these findings in mice, we identified rare, damaging heterozygous germline variants in KATNAL2 in five unrelated patients with neurosurgically treated CH and comorbid ASD or other neurodevelopmental disorders. Mice engineered with the orthologous ASD-associated KATNAL2 F244L missense variant recapitulated the ventriculomegaly found in human patients. Together, these data suggest KATNAL2 pathogenic variants alter intraventricular CSF homeostasis and parenchymal neuronal connectivity by disrupting microtubule dynamics in fetal radial glia and their postnatal ependymal and neuronal descendants. The results identify a molecular mechanism underlying the development of ventriculomegaly in a genetic subset of patients with ASD and may explain persistence of neurodevelopmental phenotypes in some patients with CH despite neurosurgical CSF shunting.

Simultaneous multi-site editing of individual genomes using retron arrays.

During recent years, the use of libraries-scale genomic manipulations scaffolded on CRISPR guide RNAs have been transformative. However, these existing approaches are typically multiplexed across genomes. Unfortunately, building cells with multiple, nonadjacent precise mutations remains a laborious cycle of editing, isolating an edited cell and editing again. The use of bacterial retrons can overcome this limitation. Retrons are genetic systems composed of a reverse transcriptase and a noncoding RNA that contains an multicopy single-stranded DNA, which is reverse transcribed to produce multiple copies of single-stranded DNA. Here we describe a technology-termed a multitron-for precisely modifying multiple sites on a single genome simultaneously using retron arrays, in which multiple donor-encoding DNAs are produced from a single transcript. The multitron architecture is compatible with both recombineering in prokaryotic cells and CRISPR editing in eukaryotic cells. We demonstrate applications for this approach in molecular recording, genetic element minimization and metabolic engineering.

SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition.

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral 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 antiviral 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.

Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease.

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 cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. 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 Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/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 Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ 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.

Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway.

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca2+-ATPase (SERCA) in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca2+ sensitivity of active SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca2+ affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows transitions in the SERCA enzymatic cycle associated with Ca2+ binding, altering transport Ca2+ dependence without actually affecting the equilibrium binding affinity of the Ca2+-coordinating sites. Here, we consider another hypothesis, that measurements of Ca2+ binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca2+ binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca2+ affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca2+ affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays versus Ca2+ binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca2+-binding sites. We propose this pathway is disrupted by PLB binding. Thus, PLB reduces the equilibrium Ca2+ affinity of SERCA by interrupting allosteric activation of the pump by ATP.

Role of ecto-5'-nucleotidase in bladder function.

Purinergic signaling plays an important role in regulating bladder contractility and voiding. Abnormal purinergic signaling is associated with lower urinary tract symptoms (LUTS). Ecto-5'-nucleotidase (NT5E) catalyzes dephosphorylation of extracellular AMP to adenosine, which in turn promotes adenosine-A2b receptor signaling to relax bladder smooth muscle (BSM). The functional importance of this mechanism was investigated using Nt5e knockout (Nt5eKO) mice. Increased voiding frequency of small voids revealed by voiding spot assay was corroborated by urodynamic studies showing shortened voiding intervals and decreased bladder compliance. Myography indicated reduced contractility of Nt5eKO BSM. These data support a role for NT5E in regulating bladder function through modulation of BSM contraction and relaxation. However, the abnormal bladder phenotype of Nt5eKO mice is much milder than we previously reported in A2b receptor knockout (A2bKO) mice, suggesting compensatory response(s) in Nt5eKO mouse bladder. To better understand this compensatory mechanism, we analyzed changes in purinergic and other receptors controlling BSM contraction and relaxation in the Nt5eKO bladder. We found that the relative abundance of muscarinic CHRM3 (cholinergic receptor muscarinic 3), purinergic P2X1, and A2b receptors was unchanged, whereas P2Y12 receptor was significantly downregulated, suggesting a negative feedback response to elevated ADP signaling. Further studies of additional ecto-nucleotidases indicated significant upregulation of the nonspecific urothelial alkaline phosphatase ALPL, which might mitigate the degree of voiding dysfunction by compensating for Nt5e deletion. These data suggest a mechanistic complexity of the purinergic signaling network in bladder and imply a paracrine mechanism in which urothelium-released ATP and its rapidly produced metabolites coordinately regulate BSM contraction and relaxation.

TRIM71 mutations cause a neurodevelopmental syndrome featuring ventriculomegaly and hydrocephalus.

Congenital hydrocephalus (CH), characterized by cerebral ventriculomegaly, is one of the most common reasons for pediatric brain surgery. Recent studies have implicated lin-41 (lineage variant 41)/TRIM71 (tripartite motif 71) as a candidate CH risk gene, however, TRIM71 variants have not been systematically examined in a large patient cohort or conclusively linked with an OMIM syndrome. Through cross-sectional analysis of the largest assembled cohort of patients with cerebral ventriculomegaly, including neurosurgically-treated CH (totaling 2,697 parent-proband trios and 8,091 total exomes), we identified 13 protein-altering de novo variants (DNVs) in TRIM71 in unrelated children exhibiting variable ventriculomegaly, CH, developmental delay, dysmorphic features, and other structural brain defects including corpus callosum dysgenesis and white matter hypoplasia. Eight unrelated patients were found to harbor arginine variants, including two recurrent missense DNVs, at homologous positions in RPXGV motifs of different NHL domains. Seven additional patients with rare, damaging, unphased or transmitted variants of uncertain significance were also identified. NHL-domain variants of TRIM71 exhibited impaired binding to the canonical TRIM71 target CDKN1A; other variants failed to direct the subcellular localization of TRIM71 to processing bodies. Single-cell transcriptomic analysis of human embryos revealed expression of TRIM71 in early first-trimester neural stem cells of the brain. These data show TRIM71 is essential for human brain morphogenesis and that TRIM71 mutations cause a novel neurodevelopmental syndrome featuring ventriculomegaly and CH.

A novel SMARCC1 BAFopathy implicates neural progenitor epigenetic dysregulation in human hydrocephalus.

Hydrocephalus, characterized by cerebral ventriculomegaly, is the most common disorder requiring brain surgery in children. Recent studies have implicated SMARCC1, a component of the BRG1-associated factor (BAF) chromatin remodelling complex, as a candidate congenital hydrocephalus gene. However, SMARCC1 variants have not been systematically examined in a large patient cohort or conclusively linked with a human syndrome. Moreover, congenital hydrocephalus-associated SMARCC1 variants have not been functionally validated or mechanistically studied in vivo. Here, we aimed to assess the prevalence of SMARCC1 variants in an expanded patient cohort, describe associated clinical and radiographic phenotypes, and assess the impact of Smarcc1 depletion in a novel Xenopus tropicalis model of congenital hydrocephalus. To do this, we performed a genetic association study using whole-exome sequencing from a cohort consisting of 2697 total ventriculomegalic trios, including patients with neurosurgically-treated congenital hydrocephalus, that total 8091 exomes collected over 7 years (2016-23). A comparison control cohort consisted of 1798 exomes from unaffected siblings of patients with autism spectrum disorder and their unaffected parents were sourced from the Simons Simplex Collection. Enrichment and impact on protein structure were assessed in identified variants. Effects on the human fetal brain transcriptome were examined with RNA-sequencing and Smarcc1 knockdowns were generated in Xenopus and studied using optical coherence tomography imaging, in situ hybridization and immunofluorescence. SMARCC1 surpassed genome-wide significance thresholds, yielding six rare, protein-altering de novo variants localized to highly conserved residues in key functional domains. Patients exhibited hydrocephalus with aqueductal stenosis; corpus callosum abnormalities, developmental delay, and cardiac defects were also common. Xenopus knockdowns recapitulated both aqueductal stenosis and cardiac defects and were rescued by wild-type but not patient-specific variant SMARCC1. Hydrocephalic SMARCC1-variant human fetal brain and Smarcc1-variant Xenopus brain exhibited a similarly altered expression of key genes linked to midgestational neurogenesis, including the transcription factors NEUROD2 and MAB21L2. These results suggest de novo variants in SMARCC1 cause a novel human BAFopathy we term 'SMARCC1-associated developmental dysgenesis syndrome', characterized by variable presence of cerebral ventriculomegaly, aqueductal stenosis, developmental delay and a variety of structural brain or cardiac defects. These data underscore the importance of SMARCC1 and the BAF chromatin remodelling complex for human brain morphogenesis and provide evidence for a 'neural stem cell' paradigm of congenital hydrocephalus pathogenesis. These results highlight utility of trio-based whole-exome sequencing for identifying pathogenic variants in sporadic congenital structural brain disorders and suggest whole-exome sequencing may be a valuable adjunct in clinical management of congenital hydrocephalus patients.

ADAR regulates APOL1 via A-to-I RNA editing by inhibition of MDA5 activation in a paradoxical biological circuit.

APOL1 risk variants are associated with increased risk of kidney disease in patients of African ancestry, but not all individuals with the APOL1 high-risk genotype develop kidney disease. As APOL1 gene expression correlates closely with the degree of kidney cell injury in both cell and animal models, the mechanisms regulating APOL1 expression may be critical determinants of risk allele penetrance. The APOL1 messenger RNA includes Alu elements at the 3' untranslated region that can form a double-stranded RNA structure (Alu-dsRNA) susceptible to posttranscriptional adenosine deaminase acting on RNA (ADAR)-mediated adenosine-to-inosine (A-to-I) editing, potentially impacting gene expression. We studied the effects of ADAR expression and A-to-I editing on APOL1 levels in podocytes, human kidney tissue, and a transgenic APOL1 mouse model. In interferon-γ (IFN-γ)-stimulated human podocytes, ADAR down-regulates APOL1 by preventing melanoma differentiation-associated protein 5 (MDA5) recognition of dsRNA and the subsequent type I interferon (IFN-I) response. Knockdown experiments showed that recognition of APOL1 messenger RNA itself is an important contributor to the MDA5-driven IFN-I response. Mathematical modeling suggests that the IFN-ADAR-APOL1 network functions as an incoherent feed-forward loop, a biological circuit capable of generating fast, transient responses to stimuli. Glomeruli from human kidney biopsies exhibited widespread editing of APOL1 Alu-dsRNA, while the transgenic mouse model closely replicated the edited sites in humans. APOL1 expression in mice was inversely correlated with Adar1 expression under IFN-γ stimuli, supporting the idea that ADAR regulates APOL1 levels in vivo. ADAR-mediated A-to-I editing is an important regulator of APOL1 expression that could impact both penetrance and severity of APOL1-associated kidney disease.