SMPD4 regulates mitotic nuclear envelope dynamics and its loss causes microcephaly and diabetes

Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder with progressive congenital microcephaly and early death. SMPD4 encodes a sphingomyelinase that hydrolyses sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes (NPC). We refine the clinical phenotype of loss-of-function SMPD4 variants by describing five individuals from three unrelated families with longitudinal data due to prolonged survival. All individuals surviving beyond infancy developed insulin-dependent diabetes, besides presenting with a severe neurodevelopmental disorder and microcephaly, making diabetes one of the most frequent age-dependent non-cerebral abnormalities. We studied the function of SMPD4 at the cellular and organ levels. Knock-down of SMPD4 in human neural stem cells causes reduced proliferation rates and prolonged mitosis. Moreover, SMPD4 depletion results in abnormal nuclear envelope breakdown and reassembly during mitosis and decreased post-mitotic NPC insertion. Fibroblasts from affected individuals show deficient SMPD4-specific neutral sphingomyelinase activity, without changing (sub)cellular lipidome fractions, which suggests a local function of SMPD4 on the nuclear envelope. In embryonic mouse brain, knockdown of Smpd4 impairs cortical progenitor proliferation and induces premature differentiation by altering the balance between neurogenic and proliferative progenitor cell divisions. We hypothesize that, in individuals with SMPD4-related disease, nuclear envelope bending, which is needed to insert NPCs in the nuclear envelope, is impaired in the absence of SMPD4 and interferes with cerebral corticogenesis and survival of pancreatic beta cells.</p

The IFN-γ-Inducible GTPase, Irga6, Protects Mice against Toxoplasma gondii but Not against Plasmodium berghei and Some Other Intracellular Pathogens

Clearance of infection with intracellular pathogens in mice involves interferon-regulated GTPases of the IRG protein family. Experiments with mice genetically deficient in members of this family such as Irgm1(LRG-47), Irgm3(IGTP), and Irgd(IRG-47) has revealed a critical role in microbial clearance, especially for Toxoplasma gondii. The in vivo role of another member of this family, Irga6 (IIGP, IIGP1) has been studied in less detail. We investigated the susceptibility of two independently generated mouse strains deficient in Irga6 to in vivo infection with T. gondii, Mycobacterium tuberculosis, Leishmania mexicana, L. major, Listeria monocytogenes, Anaplasma phagocytophilum and Plasmodium berghei. Compared with wild-type mice, mice deficient in Irga6 showed increased susceptibility to oral and intraperitoneal infection with T. gondii but not to infection with the other organisms. Surprisingly, infection of Irga6-deficient mice with the related apicomplexan parasite, P. berghei, did not result in increased replication in the liver stage and no Irga6 (or any other IRG protein) was detected at the parasitophorous vacuole membrane in IFN-γ-induced wild-type cells infected with P. berghei in vitro. Susceptibility to infection with T. gondii was associated with increased mortality and reduced time to death, increased numbers of inflammatory foci in the brains and elevated parasite loads in brains of infected Irga6-deficient mice. In vitro, Irga6-deficient macrophages and fibroblasts stimulated with IFN-γ were defective in controlling parasite replication. Taken together, our results implicate Irga6 in the control of infection with T. gondii and further highlight the importance of the IRG system for resistance to this pathogen

Strain-Specific Association of Cytotoxic Activity and Virulence of Clinical Staphylococcus aureus Isolates

Staphylococcus aureus has been shown to invade and induce the death of various cell types. Here we investigate whether the cytotoxicity of intracellular S. aureus is a general feature or rather characteristic of individual S. aureus strains. The majority of 23 randomly collected clinical S. aureus isolates were killed inside keratinocytes and fibroblasts, indicating that the uptake of S. aureus represents an important mechanism of cell-autonomous host defense. However, seven independent S. aureus isolates survived intracellularly and induced significant cytotoxicity for their host cells. Subcloning analysis revealed that the ability or inability to kill host cells is a stable, apparently genetically determined trait of a given S. aureus isolate. We show that noncytotoxic strains but not cytotoxic strains colocalize with the lysosomal marker LAMP-1, suggesting that only cytotoxic strains escape degradation by the endolysosomal pathway. In a mouse septicemic model, cytotoxic S. aureus isolates produce significantly greater lethality (96%) compared to noncytotoxic strains (41%), which corresponds to 23-, 63-, and 30,000-fold increases of bacterial loads in the liver, spleen, and kidney, respectively. Finally, cytotoxic S. aureus strains produce clinically apparent arthritis in mice at a greater frequency than compared to noncytotoxic S. aureus strains. The results of our study unravel a previously unrecognized dichotomy of cytotoxic and noncytotoxic S. aureus isolates, which may play an important role in the dissemination of, and mortality induced by, S. aureus infection

A-to-G Hypermutation in the Genome of Lymphocytic Choriomeningitis Virus

The interferon-inducible adenosine deaminase that acts on double-stranded RNA (ADAR1-L) has been proposed to be one of the antiviral effector proteins within the complex innate immune response. Here, the potential role of ADAR1-L in the innate immune response to lymphocytic choriomeningitis virus (LCMV), a widely used virus model, was studied. Infection with LCMV clearly upregulated ADAR1-L expression and activity. The editing activity of ADAR1-L on an RNA substrate was not inhibited by LCMV replication. Accordingly, an adenosine-to-guanosine (A-to-G) and uracil-to-cytidine (U-to-C) hypermutation pattern was found in the LCMV genomic RNA in infected cell lines and in mice. In addition, two hypermutated clones with a high level of A-to-G or U-to-C mutations within a short stretch of the viral genome were isolated. Analysis of the functionality of viral glycoprotein revealed that A-to-G- and U-to-C-mutated LCMV genomes coded for nonfunctional glycoprotein at a surprisingly high frequency. Approximately half the GP clones with an amino acid mutation lacked functionality. These results suggest that ADAR1-L-induced mutations in the viral RNA lead to a loss of viral protein function and reduced viral infectivity. This study therefore provides strong support for the contribution of ADAR1-L to the innate antiviral immune response

Reexamination of the Role of Ubiquitin-Like Modifier ISG15 in the Phenotype of UBP43-Deficient Mice

UBP43/USP18 was described as a specific protease that removes conjugated ubiquitin-like modifier ISG15 from target proteins. The severe phenotype of UBP43(−/−) mice characterized by premature death, brain cell injury, and deregulated STAT1 signaling was ascribed to an enhanced conjugation of ISG15. In contrast, no phenotypic changes were detected in ISG15(−/−) mice. To verify the role of ISG15 in the phenotype of UBP43(−/−) mice, we employed mice deficient for both ISG15 and UBP43. Here, we show that the phenotype of UBP43(−/−) mice was not rescued by the absence of ISG15, as evident from unchanged mortality, neurological symptoms, and occurrence of hydrocephalus. Also, the reported hypersensitivity of UBP43(−/−) mice to an interferon inducer, poly(I · C), was ISG15 independent. Furthermore, no evidence for a role of ISG15 in the modulation of STAT1 signaling or in the resistance against lymphocytic choriomeningitis virus and vesicular stomatitis virus was found. Presented results clearly demonstrate that the phenotypic alterations of UBP43(−/−) mice are not caused by the lack of ISG15 deconjugation and must be due to another, non-ISG15-mediated molecular mechanism

ISG15, an Interferon-Stimulated Ubiquitin-Like Protein, Is Not Essential for STAT1 Signaling and Responses against Vesicular Stomatitis and Lymphocytic Choriomeningitis Virus

ISG15 is an interferon-induced ubiquitin-like modifier which can be conjugated to distinct, but largely unknown, proteins. ISG15 has been implicated in a variety of biological activities, which encompass antiviral defense, immune responses, and pregnancy. Mice lacking UBP43 (USP18), the ISG15-deconjugating enzyme, develop a severe phenotype with brain injuries and lethal hypersensitivity to poly(I:C). It has been reported that an augmented conjugation of ISG15 in the absence of UBP43 induces prolonged STAT1 phosphorylation and that the ISG15 conjugation plays an important role in the regulation of JAK/STAT and interferon signaling (O. A. Malakhova, M. Yan, M. P. Malakhov, Y. Yuan, K. J. Ritchie, K. I. Kim, L. F. Peterson, K. Shuai, and D. E. Zhang, Genes Dev. 17:455-460, 2003). Here, we report that ISG15(−/−) mice are viable and fertile and display no obvious abnormalities. Lack of ISG15 did not affect the development and composition of the main cellular compartments of the immune system. The interferon-induced antiviral state and immune responses directed against vesicular stomatitis virus and lymphocytic choriomeningitis virus were not significantly altered in the absence of ISG15. Furthermore, interferon- or endotoxin-induced STAT1 tyrosine-phosphorylation, as well as expression of typical STAT1 target genes, remained unaffected by the lack of ISG15. Thus, ISG15 is dispensable for STAT1 and interferon signaling

Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease

The immune cells of the central nervous system (CNS) comprise parenchymal microglia and at the CNS border regions meningeal, perivascular, and choroid plexus macrophages (collectively called CNS-associated macrophages, CAMs). While previous work has shown that microglial properties depend on environmental signals from the commensal microbiota, the effects of microbiota on CAMs are unknown. By combining several microbiota manipulation approaches, genetic mouse models, and single-cell RNA-sequencing, we have characterized CNS myeloid cell composition and function. Under steady-state conditions, the transcriptional profiles and numbers of choroid plexus macrophages were found to be tightly regulated by complex microbiota. In contrast, perivascular and meningeal macrophages were affected to a lesser extent. An acute perturbation through viral infection evoked an attenuated immune response of all CAMs in germ-free mice. We further assessed CAMs in a more chronic pathological state in 5xFAD mice, a model for Alzheimer's disease, and found enhanced amyloid beta uptake exclusively by perivascular macrophages in germ-free 5xFAD mice. Our results aid the understanding of distinct microbiota-CNS macrophage interactions during homeostasis and disease, which could potentially be targeted therapeutically