A mating-induced reproductive gene promotes Anopheles tolerance to Plasmodium falciparum infection.

Anopheles mosquitoes have transmitted Plasmodium parasites for millions of years, yet it remains unclear whether they suffer fitness costs to infection. Here we report that the fecundity of virgin and mated females of two important vectors-Anopheles gambiae and Anopheles stephensi-is not affected by infection with Plasmodium falciparum, demonstrating that these human malaria parasites do not inflict this reproductive cost on their natural mosquito hosts. Additionally, parasite development is not impacted by mating status. However, in field studies using different P. falciparum isolates in Anopheles coluzzii, we find that Mating-Induced Stimulator of Oogenesis (MISO), a female reproductive gene strongly induced after mating by the sexual transfer of the steroid hormone 20-hydroxyecdysone (20E), protects females from incurring fecundity costs to infection. MISO-silenced females produce fewer eggs as they become increasingly infected with P. falciparum, while parasite development is not impacted by this gene silencing. Interestingly, previous work had shown that sexual transfer of 20E has specifically evolved in Cellia species of the Anopheles genus, driving the co-adaptation of MISO. Our data therefore suggest that evolution of male-female sexual interactions may have promoted Anopheles tolerance to P. falciparum infection in the Cellia subgenus, which comprises the most important malaria vectors.

MicroRNA-dependent genetic networks during neural development.

The development of the structurally and functionally diverse mammalian nervous system requires the integration of numerous levels of gene regulation. Accumulating evidence suggests that microRNAs are key mediators of genetic networks during neural development. Importantly, microRNAs are found to regulate both feedback and feedforward loops during neural development leading to large changes in gene expression. These repressive interactions provide an additional mechanism that facilitates the establishment of complexity within the nervous system. Here, we review studies that have enabled the identification of microRNAs enriched in the brain and discuss the way that genetic networks in neural development depend on microRNAs.

Regulation of airway and alveolar epithelial cell apoptosis by p53-Induced plasminogen activator inhibitor-1 during cigarette smoke exposure injury.

Increased expression of tumor suppressor protein p53 and of plasminogen activator inhibitor (PAI)-1 is associated with cigarette smoke (CS) exposure-induced lung epithelial injury. p53 induces PAI-1 through mRNA stabilization in lung epithelial cells. However, it is unclear how this process affects lung epithelial damage. Here, we show that CS induces p53 and PAI-1 expression and apoptosis in cultured Beas2B and primary alveolar type (AT)II cells. CS exposure augmented binding of p53 protein with PAI-1 mRNA. Inhibition of p53 from binding to PAI-1 mRNA through expression of p53-binding 70 nt PAI-1 mRNA 3'UTR sequences suppressed CS-induced PAI-1 expression. Treatment of Beas2B cells with caveolin-1 scaffolding domain peptide (CSP) suppressed p53 expression and p53-PAI-1 mRNA interaction. These changes were associated with parallel inhibition of CS-induced PAI-1 expression and apoptosis in Beas2B cells. Wild-type mice exposed to passive CS likewise show augmented p53 and PAI-1 with parallel induction of ATII cell apoptosis, whereas mice deficient for p53 or PAI-1 expression resisted apoptosis of ATII cells. CSP suppressed CS-induced ATII cell apoptosis in wild-type mice and abrogated p53-PAI-1 mRNA interaction with parallel inhibition of p53 and PAI-1 expression. The protection against ATII cell apoptosis by CSP involves inhibition of passive CS-induced proapoptotic Bax and Bak expression and restoration of the prosurvival proteins Bcl-X(L). These observations demonstrate that inhibition of p53 binding to PAI-1 mRNA 3'UTR attenuates CS-induced ATII cell apoptosis. This presents a novel link between p53-mediated PAI-1 expression and CS-induced ATII cell apoptosis.

Regulation of urokinase expression at the posttranscription level by lung epithelial cells.

Urokinase-type plasminogen activator (uPA) is expressed by lung epithelial cells and regulates fibrin turnover and epithelial cell viability. PMA, LPS, and TNF-alpha, as well as uPA itself, induce uPA expression in lung epithelial cells. PMA, LPS, and TNF-alpha induce uPA expression through increased synthesis as well as stabilization of uPA mRNA, while uPA increases its own expression solely through uPA mRNA stabilization. The mechanism by which lung epithelial cells regulate uPA expression at the level of mRNA stability is unclear. To elucidate this process, we sought to characterize protein-uPA mRNA interactions that regulate uPA expression. Regulation of uPA at the level of mRNA stability involves the interaction of a ~40 kDa cytoplasmic-nuclear shuttling protein with a 66 nt uPA mRNA 3'UTR sequence. We purified the uPA mRNA 3'UTR binding protein and identified it as ribonucleotide reductase M2 (RRM2). We expressed recombinant RRM2 and confirmed its interaction with a specific 66 nt uPA 3'UTR sequence. Immunoprecipitation of cell lysates with anti-RRM2 antibody and RT-PCR for uPA mRNA confirmed that RRM2 binds to uPA mRNA. Treatment of Beas2B cells with uPA or LPS attenuated RRM2-endogenous uPA mRNA interactions, while overexpression of RRM2 inhibited uPA protein and mRNA expression through destabilization of uPA mRNA. LPS exposure of lung epithelial cells translocates RRM2 from the cytoplasm to the nucleus in a time-dependent manner, leading to stabilization of uPA mRNA. This newly recognized pathway could influence uPA expression and a broad range of uPA-dependent functions in lung epithelial cells in the context of lung inflammation and repair.

Wolbachia cifB induces cytoplasmic incompatibility in the malaria mosquito vector.

Wolbachia, a maternally inherited intracellular bacterial species, can manipulate host insect reproduction by cytoplasmic incompatibility (CI), which results in embryo lethality in crosses between infected males and uninfected females. CI is encoded by two prophage genes, cifA and cifB. Wolbachia, coupled with the sterile insect technique, has been used in field trials to control populations of the dengue vector Aedes albopictus, but CI-inducing strains are not known to infect the malaria vector Anopheles gambiae. Here we show that cifA and cifB can induce conditional sterility in the malaria vector An. gambiae. We used transgenic expression of these Wolbachia-derived genes in the An. gambiae germline to show that cifB is sufficient to cause embryonic lethality and that cifB-induced sterility is rescued by cifA expression in females. When we co-expressed cifA and cifB in male mosquitoes, the CI phenotype was attenuated. In female mosquitoes, cifB impaired fertility, which was overcome by co-expression of cifA. Our findings pave the way towards using CI to control malaria mosquito vectors.

Deconstructing Stepwise Fate Conversion of Human Fibroblasts to Neurons by MicroRNAs.

Cell-fate conversion generally requires reprogramming effectors to both introduce fate programs of the target cell type and erase the identity of starting cell population. Here, we reveal insights into the activity of microRNAs miR-9/9∗ and miR-124 (miR-9/9∗-124) as reprogramming agents that orchestrate direct conversion of human fibroblasts into motor neurons by first eradicating fibroblast identity and promoting uniform transition to a neuronal state in sequence. We identify KLF-family transcription factors as direct target genes for miR-9/9∗-124 and show their repression is critical for erasing fibroblast fate. Subsequent gain of neuronal identity requires upregulation of a small nuclear RNA, RN7SK, which induces accessibilities of chromatin regions and neuronal gene activation to push cells to a neuronal state. Our study defines deterministic components in the microRNA-mediated reprogramming cascade.

MicroRNAs Induce a Permissive Chromatin Environment that Enables Neuronal Subtype-Specific Reprogramming of Adult Human Fibroblasts.

Directed reprogramming of human fibroblasts into fully differentiated neurons requires massive changes in epigenetic and transcriptional states. Induction of a chromatin environment permissive for acquiring neuronal subtype identity is therefore a major barrier to fate conversion. Here we show that the brain-enriched miRNAs miR-9/9∗ and miR-124 (miR-9/9∗-124) trigger reconfiguration of chromatin accessibility, DNA methylation, and mRNA expression to induce a default neuronal state. miR-9/9∗-124-induced neurons (miNs) are functionally excitable and uncommitted toward specific subtypes but possess open chromatin at neuronal subtype-specific loci, suggesting that such identity can be imparted by additional lineage-specific transcription factors. Consistently, we show that ISL1 and LHX3 selectively drive conversion to a highly homogeneous population of human spinal cord motor neurons. This study shows that modular synergism between miRNAs and neuronal subtype-specific transcription factors can drive lineage-specific neuronal reprogramming, providing a general platform for high-efficiency generation of distinct subtypes of human neurons.

MicroRNA-based conversion of human fibroblasts into striatal medium spiny neurons.

The ability to generate human neurons of specific subtypes of clinical importance offers experimental platforms that may be instrumental for disease modeling. We recently published a study demonstrating the use of neuronal microRNAs (miRNAs) and transcription factors to directly convert human fibroblasts to a highly enriched population of striatal medium spiny neurons (MSNs), a neuronal subpopulation that has a crucial role in motor control and harbors selective susceptibility to cell death in Huntington's disease. Here we describe a stepwise protocol for the generation of MSNs by direct neuronal conversion of human fibroblasts in 30 d. We provide descriptions of cellular behaviors during reprogramming and crucial steps involved in gene delivery, cell adhesion and culturing conditions that promote cell survival. Our protocol offers a unique approach to combine microRNAs and transcription factors to guide the neuronal conversion of human fibroblasts toward a specific neuronal subtype.