Differential Expression of miR-4520a Associated With Pyrin Mutations in Familial Mediterranean Fever (FMF).

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute, and self-limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF, but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR-4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR-4520a. Although the relative expression levels of miR-4520a were variable among FMF patients, the statistical expression of miR-4520a was different between FMF mutation carriers and controls (P = 0.0061), indicating an association between miR-4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR-4520a expression levels (P = 0.00545). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR-4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results suggest a role of deregulated autophagy in the pathogenesis of FMF. J. Cell. Physiol. 232: 1326-1336, 2017. © 2016 Wiley Periodicals, Inc.

The phosphoinositide phosphatase MTM-1 regulates apoptotic cell corpse clearance through CED-5-CED-12 in C. elegans.

Multicellular organisms use programmed cell death to eliminate unwanted or potentially harmful cells. Improper cell corpse removal can lead to autoimmune diseases. The development of interventional therapies that increase engulfment activity could represent an attractive approach to treat such diseases. Here, we describe mtm-1, the Caenorhabditis elegans homolog of human myotubularin 1, as a potential negative regulator of apoptotic cell corpse clearance. Loss of mtm-1 function leads to substantially reduced numbers of persistent cell corpses in engulfment mutants, which is a result of a restoration of engulfment function rather than of impaired or delayed programmed cell death. Epistatic analyses place mtm-1 upstream of the ternary GEF complex, which consists of ced-2, ced-5 and ced-12, and parallel to mig-2. Over-activation of engulfment results in the removal of viable cells that have been brought to the verge of death under limiting caspase activity. In addition, mtm-1 also promotes phagosome maturation in the hermaphrodite gonad, potentially through CED-1 receptor recycling. Finally, we show that the CED-12 PH domain can bind to PtdIns(3,5)P(2) (one target of MTM-1 phosphatase activity), suggesting that MTM-1 might regulate CED-12 recruitment to the plasma membrane.

A conserved role for SNX9-family members in the regulation of phagosome maturation during engulfment of apoptotic cells.

Clearance of apoptotic cells is of key importance during development, tissue homeostasis and wound healing in multi-cellular animals. Genetic studies in the nematode Caenorhabditis elegans have identified a set of genes involved in the early steps of cell clearance, in particular the recognition and internalization of apoptotic cells. A pathway that orchestrates the maturation of phagosomes containing ingested apoptotic cells in the worm has recently been described. However, many steps in this pathway remain elusive. Here we show that the C. elegans SNX9-family member LST-4 (lateral signaling target) and its closest mammalian orthologue SNX33 play an evolutionary conserved role during apoptotic cell corpse clearance. In lst-4 deficient worms, internalized apoptotic cells accumulated within non-acidified, DYN-1-positive but RAB-5-negative phagosomes. Genetically, we show that LST-4 functions at the same step as DYN-1 during corpse removal, upstream of the GTPase RAB-5. We further show that mammalian SNX33 rescue C. elegans lst-4 mutants and that overexpression of truncated SNX33 fragments interfered with phagosome maturation in a mammalian cell system. Taken together, our genetic and cell biological analyses suggest that LST-4 is recruited through a combined activity of DYN-1 and VPS-34 to the early phagosome membrane, where it cooperates with DYN-1 to promote recruitment/retention of RAB-5 on the early phagosomal membrane during cell corpse clearance. The functional conservation between LST-4 and SNX33 indicate that these early steps of apoptotic phagosome maturation are likely conserved through evolution.

A pathway for phagosome maturation during engulfment of apoptotic cells.

Removal of apoptotic cells is critical for the physiological well-being of the organism and defects in corpse removal have been linked to disease states. Genes regulating corpse recognition and internalization have been identified, but few molecules involved in the processing of internalized corpses are known. Through a combination of targeted and unbiased reverse genetic screens in Caenorhabditis elegans, and studies in mammalian cells, we have identified genes required for maturation of apoptotic-cell-containing phagosomes. We have further ordered these candidates, which include the GTPases RAB-5 and RAB-7 and the HOPS complex, into a coherent linear pathway for the maturation of apoptotic cells within phagosomes. In depth analysis of two additional candidate genes, the phosphatidylinositol 3 kinase (PI(3)K) vps-34 (A001762) and dyn-1/dynamin, showed an accumulation of internalized, but undegraded, corpses within abnormal Rab5-negative phagosomes. We ordered these candidates in our pathway, with DYN-1 functioning upstream of VPS-34 in the recruitment and/or retention of RAB-5 to the phagosome. Finally, we have also identified a previously undescribed biochemical complex containing Vps34, dynamin and Rab5(GDP), thus providing a mechanism for Rab5 recruitment to the nascent phagosome.

C. elegans GLA-3 is a novel component of the MAP kinase MPK-1 signaling pathway required for germ cell survival.

During oocyte development in Caenorhabditis elegans, approximately half of all developing germ cells undergo apoptosis. While this process is evolutionarily conserved from worms to humans, the regulators of germ cell death are still largely unknown. In a genetic screen for novel genes involved in germline apoptosis in Caenorhabditis elegans, we identified and cloned gla-3. Loss of gla-3 function results in increased germline apoptosis and reduced brood size due to defective pachytene exit from meiosis I. gla-3 encodes a TIS11-like zinc-finger-containing protein that is expressed in the germline, from the L4 larval stage to adulthood. Biochemical evidence and genetic epistasis analysis revealed that GLA-3 participates in the MAPK signaling cascade and directly interacts with the C. elegans MAPK MPK-1, an essential meiotic regulator. Our results show that GLA-3 is a new component of the MAPK cascade that controls meiotic progression and apoptosis in the C. elegans germline and functions as a negative regulator of the MAPK signaling pathway during vulval development and in muscle cells.