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Mutant Cu/Zn superoxide dismutase (SOD1) causes mitochondrial alterations that contribute to motor neuron demise in amyotrophic lateral sclerosis (ALS). When mitochondria are damaged, cells activate mitochondria quality control (MQC) mechanisms leading to mitophagy. Here, we show that in the spinal cord of G93A mutant SOD1 transgenic mice (SOD1-G93A mice), the autophagy receptor p62 is recruited to mitochondria and mitophagy is activated. Furthermore, the mitochondrial ubiquitin ligase Parkin and mitochondrial dynamics proteins, such as Miro1, and Mfn2, which are ubiquitinated by Parkin, and the mitochondrial biogenesis regulator PGC1 alpha are depleted. Unexpectedly, Parkin genetic ablation delays disease progression and prolongs survival in SOD1-G93A mice, as it slows down motor neuron loss and muscle denervation and attenuates the depletion of mitochondrial dynamics proteins and PGC1 alpha. Our results indicate that Parkin is a disease modifier in ALS, because chronic Parkin-mediated MQC activation depletes mitochondrial dynamics-related proteins, inhibits mitochondrial biogenesis, and worsens mitochondrial dysfunction.

Mdn1 is an essential AAA (ATPase associated with various activities) protein that removes assembly factors from distinct precursors of the ribosomal 60S subunit. However, Mdn1's large size (similar to 5,000 amino acid [aa]) and its limited homology to other well-studied proteins have restricted our understanding of its remodeling function. Here, we present structures for S. pombe Mdn1 in the presence of AMPPNP at up to similar to 4 angstrom or ATP plus Rbin-1, a chemical inhibitor, at similar to 8 angstrom resolution. These data reveal that Mdn1's MIDAS domain is tethered to its ring-shaped AAA domain through an similar to 20 nm long structured linker and a flexible similar to 500 aa Asp/Glu-rich motif. We find that the MIDAS domain, which also binds other ribosome-assembly factors, docks onto the AAA ring in a nucleotide state-specific manner. Together, our findings reveal how conformational changes in the AAA ring can be directly transmitted to the MIDAS domain and thereby drive the targeted release of assembly factors from ribosomal 60S-subunit precursors.

Chronic inflammatory diseases are characterized by recurrent inflammatory attacks in the tissues mediated by autoreactive T cells. Identity and functional programming of CD8(+) T cells at the target site of inflammation still remain elusive. One key question is whether, in these antigen-rich environments, chronic stimulation leads to CD8(+) T cell exhaustion comparable to what is observed in infectious disease contexts. In the synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients, a model of chronic inflammation, an overrepresentation of PD-1(+)CD8(+) T cells was found. Gene expression profiling, gene set enrichment analysis, functional studies, and extracellular flux analysis identified PD-1(+)CD8(+ )T cells as metabolically active effectors, with no sign of exhaustion. Furthermore, PD-1(+)CD8(+) T cells were enriched for a tissue-resident memory (Trm) cell transcriptional profile and demonstrated increased clonal expansion compared with the PD-1(-) counterpart, suggesting antigen-driven expansion of locally adapted cells. Interestingly, this subset was also found increased in target tissues in other human chronic inflammatory diseases. These data indicate that local chronic inflammation drives the induction and expansion of CD8(+) T cells endowed with potential detrimental properties. Together, these findings lay the basis for investigation of PD-1-expressing CD8(+) T cell targeting strategies in human chronic inflammatory diseases.

PurposeCARD9 deficiency is an inborn error of immunity that predisposes otherwise healthy humans to mucocutaneous and invasive fungal infections, mostly caused by Candida, but also by dermatophytes, Aspergillus, and other fungi. Phaeohyphomycosis are an emerging group of fungal infections caused by dematiaceous fungi (phaeohyphomycetes) and are being increasingly identified in patients with CARD9 deficiency. The Corynespora genus belongs to phaeohyphomycetes and only one adult patient with CARD9 deficiency has been reported to suffer from invasive disease caused by C. cassiicola. We identified a Colombian child with an early-onset, deep, and destructive mucocutaneous infection due to C. cassiicola and we searched for mutations in CARD9.MethodsWe reviewed the medical records and immunological findings in the patient. Microbiologic tests and biopsies were performed. Whole-exome sequencing (WES) was made and Sanger sequencing was used to confirm the CARD9 mutations in the patient and her family. Finally, CARD9 protein expression was evaluated in peripheral blood mononuclear cells (PBMC) by western blotting.ResultsThe patient was affected by a large, indurated, foul-smelling, and verrucous ulcerated lesion on the left side of the face with extensive necrosis and crusting, due to a C. cassiicola infectious disease. WES led to the identification of compound heterozygous mutations in the patient consisting of the previously reported p.Q289* nonsense (c.865C > T, exon 6) mutation, and a novel deletion (c.23_29del; p.Asp8Alafs10*) leading to a frameshift and a premature stop codon in exon 2. CARD9 protein expression was absent in peripheral blood mononuclear cells from the patient.ConclusionWe describe here compound heterozygous loss-of-expression mutations in CARD9 leading to severe deep and destructive mucocutaneous phaeohyphomycosis due to C. cassiicola in a Colombian child.

Mutations in interleukin-10 receptor (IL-10R) genes are one cause of very early-onset inflammatory bowel disease with perianal lesions, which can be cured by hematopoietic stem cell transplantation. Using a functional test, which assesses responsiveness of peripheral monocytes to IL-10, we identified three unrelated Portuguese patients carrying two novel IL-10RB mutations. In the three patients, sequencing of genomic DNA identified the same large deletion of exon 3 which precluded protein expression. This mutation was homozygous in two patients born from consanguineous families and heterozygous in the third patient born from unrelated parents. Microsatellite analysis of the IL1ORBgenomic region revealed a common haplotype in the three Portuguese families pointing to a founder deletion inherited from a common ancestor 400 years ago. In the third patient, surface expression of IL-10R was normal but signaling in response to IL-10 was impaired. Complementary DNA sequencing and next-generation sequencing of IL10RB locus with custom-made probes revealed a 6 Kb duplication encompassing the exon approximate to 6 which leads to a frameshift mutation and a loss of the TYK2-interacting Box 2 motif. Altogether, we describe two novel copy number variations in IL10RB, one with founder effect and one preserving cell surface expression but abolishing signaling.

MicroRNA-206 (miR-206) has demonstrated tumor suppressive effects in a variety of cancers. Numerous studies have identified aberrantly expressed targets of miR-206 that contribute to tumor progression and metastasis, however, the broader gene-networks and pathways regulated by miR-206 remain poorly defined. Here, we have ectopically expressed miR-206 in lung adenocarcinoma cell lines and tumors to identify differentially expressed genes, and study the effects on tumor growth and metastasis. In H1299 tumor xenograft assays, stable expression of miR-206 suppressed both tumor growth and metastasis in mice. Profiling of xenograft tumors using small RNA sequencing and a targeted panel of tumor progression and metastasis-related genes revealed a network of genes involved in TGF-beta signalling that were regulated by miR-206. Among these were the TGFB1 ligand, as well as direct transcriptional targets of Smad3. Other differentially expressed genes included components of the extracellular matrix involved in TGF-beta activation and signalling, including Thrombospondin-1, which is responsible for the activation of latent TGF-beta in the stroma. In cultured lung adenocarcinoma cells treated with recombinant TGF-beta, ectopic expression of miR-206 impaired canonical signalling, and expression of TGF-beta target genes linked to epithelial-mesenchymal transition. This was due at least in part to the suppression of Smad3 protein levels in lung adenocarcinoma cells with ectopic miR-206 expression. Together, these findings indicate that miR-206 can suppress tumor progression and metastasis by limiting autocrine production of TGF-beta, and highlight the potential utility of TGF-beta inhibitors for the treatment of lung adenocarcinomas.

HIV-1 Env, a trimer of gp120-gp41 heterodimers, mediates membrane fusion after binding host receptor CD4. Receptor binding displaces V1V2 loops from Env's apex, allowing coreceptor binding and opening Env to enable gp41-mediated fusion. We present 3.54 angstrom and 4.06 angstrom cryoelectron microscopy structures of partially open soluble native-like Env trimers (SOSIPs) bound to CD4. One structure, a complex with a coreceptor-mimicking antibody that binds both CD4 and gp120, stabilizes the displaced V1V2 and reveals its structure. Comparing partially and fully open Envs with closed Envs shows that gp41 re-arrangements are independent of the CD4-induced rearrangements that result in V1V2 displacement and formation of a 4-stranded bridging sheet. These findings suggest ordered conformational changes before coreceptor binding: (1) gp120 opening inducing side-chain rearrangements and a compact gp41 central helix conformation, and (2) 4-stranded bridging-sheet formation and V1V2 displacement. These analyses illuminate potential receptor-induced Env changes and inform design of therapeutics disrupting viral entry.

A network of a few hundred neurons in the Drosophila central complex carries an estimate of the fly's heading in the world, akin to the mammalian head-direction system. Here we describe how anatomically defined neuronal classes in this network are poised to implement specific sub-processes for building and updating this population-level heading signal. The computations we describe in the fly central complex strongly resemble those posited to exist in the mammalian brain, in computational models for building head-direction signals. By linking circuit anatomy to navigational physiology, the Drosophila central complex should provide a detailed example of how a heading signal is built.

Steroid-resistant nephrotic syndrome (SRNS) almost invariably progresses to end-stage renal disease. Although more than 50 monogenic causes of SRNS have been described, a large proportion of SRNS remains unexplained, Recently, it was discovered that mutations of NUP93 and NUP205, encoding 2 proteins of the inner ring subunit of the nuclear pore complex (NPC), cause SRNS. Here, we describe mutations in genes encoding 4 components of the outer rings of the NPC, namely NUP107, NUP85, NUP133, and NUP160, in 13 families with SRNS. Using coimmunoprecipitation experiments, we showed that certain pathogenic alleles weakened the interaction between neighboring NPC subunits. We demonstrated that morpholino knockdown of nup107, nup85, or nup133 in Xenopus disrupted glomerulogenesis. Re-expression of WT mRNA, but not of mRNA reflecting mutations from SRNS patients, mitigated this phenotype. We furthermore found that CRISPR/Cas9 knockout of NUP107, NUP85, or NUP133 in podocytes activated Cdc42, an important effector of SRNS pathogenesis. CRISPR/Cas9 knockout of nup107 or nup85 in zebrafish caused developmental anomalies and early lethality. In contrast, an in-frame mutation of nup107 did not affect survival, thus mimicking the allelic effects seen in humans. In conclusion, we discovered here that mutations in 4 genes encoding components of the outer ring subunits of the NPC cause SRNS and thereby provide further evidence that specific hypomorphic mutations in these essential genes cause a distinct, organ-specific phenotype.