Brucella-driven host N-glycome remodeling controls infection.

Many powerful methods have been employed to elucidate the global transcriptomic, proteomic, or metabolic responses to pathogen-infected host cells. However, the host glycome responses to bacterial infection remain largely unexplored, and hence, our understanding of the molecular mechanisms by which bacterial pathogens manipulate the host glycome to favor infection remains incomplete. Here, we address this gap by performing a systematic analysis of the host glycome during infection by the bacterial pathogen Brucella spp. that cause brucellosis. We discover, surprisingly, that a Brucella effector protein (EP) Rhg1 induces global reprogramming of the host cell N-glycome by interacting with components of the oligosaccharide transferase complex that controls N-linked protein glycosylation, and Rhg1 regulates Brucella replication and tissue colonization in a mouse model of brucellosis, demonstrating that Brucella exploits the EP Rhg1 to reprogram the host N-glycome and promote bacterial intracellular parasitism, thereby providing a paradigm for bacterial control of host cell infection.

The early macrophage response to pathogens requires dynamic regulation of the nuclear paraspeckle.

To ensure a robust immune response to pathogens without risking immunopathology, the kinetics and amplitude of inflammatory gene expression in macrophages need to be exquisitely well controlled. There is a growing appreciation for stress-responsive membraneless organelles (MLOs) regulating various steps of eukaryotic gene expression in response to extrinsic cues. Here, we implicate the nuclear paraspeckle, a highly ordered biomolecular condensate that nucleates on the Neat1 lncRNA, in tuning innate immune gene expression in murine macrophages. In response to a variety of innate agonists, macrophage paraspeckles rapidly aggregate (0.5 h poststimulation) and disaggregate (2 h poststimulation). Paraspeckle maintenance and aggregation require active transcription and MAPK signaling, whereas paraspeckle disaggregation requires degradation of Neat1 via the nuclear RNA exosome. In response to lipopolysaccharide treatment, Neat1 KO macrophages fail to properly express a large cohort of proinflammatory cytokines, chemokines, and antimicrobial mediators. Consequently, Neat1 KO macrophages cannot control replication of Salmonella enterica serovar Typhimurium or vesicular stomatitis virus. These findings highlight a prominent role for MLOs in orchestrating the macrophage response to pathogens and support a model whereby dynamic assembly and disassembly of paraspeckles reorganizes the nuclear landscape to enable inflammatory gene expression following innate stimuli.

Serine/arginine-rich splicing factor 7 promotes the type I interferon response by activating Irf7 transcription.

Tight regulation of macrophage immune gene expression is required to fight infection without risking harmful inflammation. The contribution of RNA-binding proteins (RBPs) to shaping the macrophage response to pathogens remains poorly understood. Transcriptomic analysis reveals that a member of the serine/arginine-rich (SR) family of mRNA processing factors, SRSF7, is required for optimal expression of a cohort of interferon-stimulated genes in macrophages. Using genetic and biochemical assays, we discover that in addition to its canonical role in regulating alternative splicing, SRSF7 drives transcription of interferon regulatory transcription factor 7 (IRF7) to promote antiviral immunity. At the Irf7 promoter, SRSF7 maximizes STAT1 transcription factor binding and RNA polymerase II elongation via cooperation with the H4K20me1 histone methyltransferase KMT5a (SET8). These studies define a role for an SR protein in activating transcription and reveal an RBP-chromatin network that orchestrates macrophage antiviral gene expression.

Serine arginine-rich splicing factor (SRSF7) cooperates with the histone methyltransferase KMT5a to promote the type I interferon response via transcriptional activation of IRF7.

Tight regulation of macrophage immune gene expression is required to fight infection without risking harmful inflammation. The contribution of RNA binding proteins (RBPs) to shaping the macrophage response to pathogens remains poorly understood. Transcriptomic analysis revealed that a member of the serine/arginine-rich (SR) family of mRNA processing factors, SRSF7, is required for optimal expression of a cohort of interferon stimulated genes (ISGs) in macrophages. Using genetic and biochemical assays, we discovered that in addition to its canonical role in regulating alternative splicing, SRSF7 drives transcription of interferon regulatory transcription factor 7 (IRF7) to promote antiviral immunity. At the Irf7 promoter, SRSF7 maximizes STAT1 transcription factor binding and RNA polymerase II elongation via cooperation with the H4K20me1 histone methyltransferase KMT5a (SET8). These studies define an unorthodox role for an SR protein in activating transcription and reveal an unappreciated RNA binding protein-chromatin network that orchestrates macrophage antiviral gene expression.

Mitochondrial reactive oxygen species: double agents in Mycobacterium tuberculosis infection.

In addition to housing the major energy-producing pathways in cells, mitochondria are active players in innate immune responses. One critical way mitochondria fulfill this role is by releasing damage-associated molecular patterns (mtDAMPs) that are recognized by innate sensors to activate pathways including, but not limited to, cytokine expression, selective autophagy, and cell death. Mitochondrial reactive oxygen species (mtROS) is a multifunctional mtDAMP linked to pro- and antimicrobial immune outcomes. Formed as a by-product of energy generation, mtROS links mitochondrial metabolism with downstream innate immune responses. As a result, altered cellular metabolism can change mtROS levels and impact downstream antimicrobial responses in a variety of ways. MtROS has emerged as a particularly important mediator of pathogenesis during infection with Mycobacterium tuberculosis (Mtb), an intracellular bacterial pathogen that continues to pose a significant threat to global public health. Here, we will summarize how Mtb modulates mtROS levels in infected macrophages and how mtROS dictates Mtb infection outcomes by controlling inflammation, lipid peroxidation, and cell death. We propose that mtROS may serve as a biomarker to predict tuberculosis patient outcomes and/or a target for host-directed therapeutics.

Gasdermins gone wild: new roles for GSDMs in regulating cellular homeostasis.

Since their discovery, members of the gasdermin (GSDM) family of proteins have been firmly established as executors of pyroptosis, with the N-terminal fragment of most GSDMs capable of forming pores in the plasma membrane. More recent findings suggest that some GSDMs can drive additional cell death pathways, such as apoptosis and necroptosis, through mechanisms independent of plasma membrane perforation. There is also emerging evidence that by associating with cellular compartments such as mitochondria, peroxisomes, endosomes, and the nucleus, GSDMs regulate cell death-independent aspects of cellular homeostasis. Here, we review the diversity of GSDM function across several cell types and explore how various cellular stresses can promote relocalization - and thus refunctionalization - of GSDMs.

SRSF6 balances mitochondrial-driven innate immune outcomes through alternative splicing of BAX.

To mount a protective response to infection while preventing hyperinflammation, gene expression in innate immune cells must be tightly regulated. Despite the importance of pre-mRNA splicing in shaping the proteome, its role in balancing immune outcomes remains understudied. Transcriptomic analysis of murine macrophage cell lines identified Serine/Arginine Rich Splicing factor 6 (SRSF6) as a gatekeeper of mitochondrial homeostasis. SRSF6-dependent orchestration of mitochondrial health is directed in large part by alternative splicing of the pro-apoptosis pore-forming protein BAX. Loss of SRSF6 promotes accumulation of BAX-κ, a variant that sensitizes macrophages to undergo cell death and triggers upregulation of interferon stimulated genes through cGAS sensing of cytosolic mitochondrial DNA. Upon pathogen sensing, macrophages regulate SRSF6 expression to control the liberation of immunogenic mtDNA and adjust the threshold for entry into programmed cell death. This work defines BAX alternative splicing by SRSF6 as a critical node not only in mitochondrial homeostasis but also in the macrophage's response to pathogens.

Mitochondrial ROS promotes susceptibility to infection via gasdermin D-mediated necroptosis.

Although mutations in mitochondrial-associated genes are linked to inflammation and susceptibility to infection, their mechanistic contributions to immune outcomes remain ill-defined. We discovered that the disease-associated gain-of-function allele Lrrk2G2019S (leucine-rich repeat kinase 2) perturbs mitochondrial homeostasis and reprograms cell death pathways in macrophages. When the inflammasome is activated in Lrrk2G2019S macrophages, elevated mitochondrial ROS (mtROS) directs association of the pore-forming protein gasdermin D (GSDMD) to mitochondrial membranes. Mitochondrial GSDMD pore formation then releases mtROS, promoting a switch to RIPK1/RIPK3/MLKL-dependent necroptosis. Consistent with enhanced necroptosis, infection of Lrrk2G2019S mice with Mycobacterium tuberculosis elicits hyperinflammation and severe immunopathology. Our findings suggest a pivotal role for GSDMD as an executer of multiple cell death pathways and demonstrate that mitochondrial dysfunction can direct immune outcomes via cell death modality switching. This work provides insights into how LRRK2 mutations manifest or exacerbate human diseases and identifies GSDMD-dependent necroptosis as a potential target to limit Lrrk2G2019S-mediated immunopathology.

Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense.

The phagocytosis and destruction of pathogens in lysosomes constitute central elements of innate immune defense. Here, we show that Brucella, the causative agent of brucellosis, the most prevalent bacterial zoonosis globally, subverts this immune defense pathway by activating regulated IRE1α-dependent decay (RIDD) of Bloc1s1 mRNA encoding BLOS1, a protein that promotes endosome-lysosome fusion. RIDD-deficient cells and mice harboring a RIDD-incompetent variant of IRE1α were resistant to infection. Inactivation of the Bloc1s1 gene impaired the ability to assemble BLOC-1-related complex (BORC), resulting in differential recruitment of BORC-related lysosome trafficking components, perinuclear trafficking of Brucella-containing vacuoles (BCVs), and enhanced susceptibility to infection. The RIDD-resistant Bloc1s1 variant maintains the integrity of BORC and a higher-level association of BORC-related components that promote centrifugal lysosome trafficking, resulting in enhanced BCV peripheral trafficking and lysosomal destruction, and resistance to infection. These findings demonstrate that host RIDD activity on BLOS1 regulates Brucella intracellular parasitism by disrupting BORC-directed lysosomal trafficking. Notably, coronavirus murine hepatitis virus also subverted the RIDD-BLOS1 axis to promote intracellular replication. Our work establishes BLOS1 as a novel immune defense factor whose activity is hijacked by diverse pathogens.

PRMT5 Promotes Symmetric Dimethylation of RNA Processing Proteins and Modulates Activated T Cell Alternative Splicing and Ca2+/NFAT Signaling.

Protein arginine methyltransferase (PRMT) 5 is the type 2 methyltransferase catalyzing symmetric dimethylation of arginine. PRMT5 inhibition or deletion in CD4 Th cells reduces TCR engagement-induced IL-2 production and Th cell expansion and confers protection against experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. However, the mechanisms by which PRMT5 modulates Th cell proliferation are still not completely understood, and neither are the methylation targets in T cells. In this manuscript, we uncover the role of PRMT5 on alternative splicing in activated mouse T cells and identify several targets of PRMT5 symmetric dimethylation involved in splicing. In addition, we find a possible link between PRMT5-mediated alternative splicing of transient receptor potential cation channel subfamily M member 4 (Trpm4) and TCR/NFAT signaling/IL-2 production. This understanding may guide development of drugs targeting these processes to benefit patients with T cell-mediated diseases.

The opportunistic intracellular bacterial pathogen Rhodococcus equi elicits type I interferon by engaging cytosolic DNA sensing in macrophages.

Rhodococcus equi is a major cause of foal pneumonia and an opportunistic pathogen in immunocompromised humans. While alveolar macrophages constitute the primary replicative niche for R. equi, little is known about how intracellular R. equi is sensed by macrophages. Here, we discovered that in addition to previously characterized pro-inflammatory cytokines (e.g., Tnfa, Il6, Il1b), macrophages infected with R. equi induce a robust type I IFN response, including Ifnb and interferon-stimulated genes (ISGs), similar to the evolutionarily related pathogen, Mycobacterium tuberculosis. Follow up studies using a combination of mammalian and bacterial genetics demonstrated that induction of this type I IFN expression program is largely dependent on the cGAS/STING/TBK1 axis of the cytosolic DNA sensing pathway, suggesting that R. equi perturbs the phagosomal membrane and causes DNA release into the cytosol following phagocytosis. Consistent with this, we found that a population of ~12% of R. equi phagosomes recruits the galectin-3,-8 and -9 danger receptors. Interestingly, neither phagosomal damage nor induction of type I IFN require the R. equi's virulence-associated plasmid. Importantly, R. equi infection of both mice and foals stimulates ISG expression, in organs (mice) and circulating monocytes (foals). By demonstrating that R. equi activates cytosolic DNA sensing in macrophages and elicits type I IFN responses in animal models, our work provides novel insights into how R. equi engages the innate immune system and furthers our understanding how this zoonotic pathogen causes inflammation and disease.

Galectin-8 Senses Phagosomal Damage and Recruits Selective Autophagy Adapter TAX1BP1 To Control Mycobacterium tuberculosis Infection in Macrophages.

Mycobacterium tuberculosis (Mtb) causes one of the deadliest infectious diseases worldwide. Upon infection, Mtb is phagocytosed by macrophages and uses its virulence-associated ESX-1 secretion system to modulate the host cell. We showed previously that the ESX-1 secretion system perturbs the Mtb-containing phagosome, and a population (∼30%) of intracellular Mtb is tagged with ubiquitin and targeted to selective autophagy. However, our understanding of how macrophages sense and respond to damaged Mtb-containing phagosomes remains incomplete. Here, we demonstrate that several cytosolic glycan-binding proteins called galectins recognize Mtb-containing phagosomes; in macrophage cell lines and in primary macrophages, galectin-3, -8, and -9 are all recruited to the same Mtb population that colocalizes with selective autophagy markers (ubiquitin, p62, and LC3). To test whether galectins are required for controlling Mtb replication in macrophages, we generated CRISPR/Cas9 knockouts and found that galectin-8-/- and galectin-3/8/9-/- macrophages were similarly defective in targeting Mtb to selective autophagy and controlling replication. This suggests galectin-8 plays a unique role in anti-Mtb autophagy. In investigating galectin-8's role, we identified a novel and specific interaction between galectin-8 and the selective autophagy adapter TAX1BP1 and found that this galectin-8/TAX1BP1 interaction was necessary for macrophages to efficiently target Mtb to selective autophagy. Remarkably, overexpressing galectin-8 increased targeting of Mtb to autophagy and limited Mtb replication. Taken together, these data demonstrate that while several galectins are capable of recognizing damaged Mtb-containing phagosomes, galectin-8 plays a privileged role in recruiting downstream autophagy machinery and may represent a promising target for host-directed tuberculosis therapies. IMPORTANCE Mycobacterium tuberculosis (Mtb) infects one-quarter of the global population and causes one of the deadliest infectious diseases worldwide. Macrophages are the first line of defense against Mtb infection and are typically incredibly efficient at destroying intracellular pathogens, but Mtb has evolved to survive and replicate in this harsh environment. Previous work has found that a portion of intracellular Mtb bacilli damage their phagosomes, leaving them vulnerable to detection by the host and delivery to an antibacterial pathway called selective autophagy. Here, we show that in macrophages, galectin-8 recognizes damaged Mtb-containing phagosomes and targets Mtb to selective autophagy; we found that galectin-8, unlike other highly similar and closely related galectins, is required for targeting and controlling Mtb in macrophages. The specific role for galectin-8 appears to stem from its interaction with TAX1BP1, a selective autophagy adapter protein. Interestingly, overexpressing galectin-8 helps macrophages target and control Mtb, highlighting the importance of galectin-8 in the innate immune response to Mtb.

Global Transcriptomics Uncovers Distinct Contributions From Splicing Regulatory Proteins to the Macrophage Innate Immune Response.

Pathogen sensing via pattern recognition receptors triggers massive reprogramming of macrophage gene expression. While the signaling cascades and transcription factors that activate these responses are well-known, the role of post-transcriptional RNA processing in modulating innate immune gene expression remains understudied. Given their crucial role in regulating pre-mRNA splicing and other RNA processing steps, we hypothesized that members of the SR/hnRNP protein families regulate innate immune gene expression in distinct ways. We analyzed steady state gene expression and alternatively spliced isoform production in ten SR/hnRNP knockdown RAW 264.7 macrophage-like cell lines following infection with the bacterial pathogen Salmonella enterica serovar Typhimurium (Salmonella). We identified thousands of transcripts whose abundance is increased or decreased by SR/hnRNP knockdown in macrophages. Notably, we observed that SR and hnRNP proteins influence expression of different genes in uninfected versus Salmonella-infected macrophages, suggesting functionalization of these proteins upon pathogen sensing. Likewise, we found that knockdown of SR/hnRNPs promoted differential isoform usage (DIU) for thousands of macrophage transcripts and that these alternative splicing changes were distinct in uninfected and Salmonella-infected macrophages. Finally, having observed a surprising degree of similarity between the differentially expressed genes (DEGs) and DIUs in hnRNP K and U knockdown macrophages, we found that hnRNP K and U knockdown macrophages are both more restrictive to Vesicular Stomatitis Virus (VSV), while hnRNP K knockdown macrophages are more permissive to Salmonella Typhimurium. Based on these findings, we conclude that many innate immune genes evolved to rely on one or more SR/hnRNPs to ensure the proper magnitude of their induction, supporting a model wherein pre-mRNA splicing is critical for regulating innate immune gene expression and controlling infection outcomes in macrophages ex vivo.

Interactions between fungal hyaluronic acid and host CD44 promote internalization by recruiting host autophagy proteins to forming phagosomes.

Phagocytosis and autophagy play critical roles in immune defense. The human fungal pathogen Cryptococcus neoformans (Cn) subverts host autophagy-initiation complex (AIC)-related proteins, to promote its phagocytosis and intracellular parasitism of host cells. The mechanisms by which the pathogen engages host AIC-related proteins remain obscure. Here, we show that the recruitment of host AIC proteins to forming phagosomes is dependent upon the activity of CD44, a host cell surface receptor that engages fungal hyaluronic acid (HA). This interaction elevates intracellular Ca2+ concentrations and activates CaMKKß and its downstream target AMPKα, which results in activation of ULK1 and the recruitment of AIC components. Moreover, we demonstrate that HA-coated beads efficiently recruit AIC components to phagosomes and CD44 interacts with AIC components. Taken together, these findings show that fungal HA plays a critical role in directing the internalization and productive intracellular membrane trafficking of a fungal pathogen of global importance.

Mitochondria: Powering the Innate Immune Response to Mycobacterium tuberculosis Infection.

Within the last decade, we have learned that damaged mitochondria activate many of the same innate immune pathways that evolved to sense and respond to intracellular pathogens. These shared responses include cytosolic nucleic acid sensing and type I interferon (IFN) expression, inflammasome activation that leads to pyroptosis, and selective autophagy (called mitophagy when mitochondria are the cargo). Because mitochondria were once bacteria, parallels between how cells respond to mitochondrial and bacterial ligands are not altogether surprising. However, the potential for cross talk or synergy between bacterium- and mitochondrion-driven innate immune responses during infection remains poorly understood. This interplay is particularly striking, and intriguing, in the context of infection with the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb). Multiple studies point to a role for Mtb infection and/or specific Mtb virulence factors in disrupting the mitochondrial network in macrophages, leading to metabolic changes and triggering potent innate immune responses. Research from our laboratories and others argues that mutations in mitochondrial genes can exacerbate mycobacterial disease severity by hyperactivating innate responses or activating them at the wrong time. Indeed, growing evidence supports a model whereby different mitochondrial defects or mutations alter Mtb infection outcomes in distinct ways. By synthesizing the current literature in this minireview, we hope to gain insight into the molecular mechanisms driving, and consequences of, mitochondrion-dependent immune polarization so that we might better predict tuberculosis patient outcomes and develop host-directed therapeutics designed to correct these imbalances.

TRIM14 Is a Key Regulator of the Type I IFN Response during Mycobacterium tuberculosis Infection.

Tripartite motif-containing proteins (TRIMs) play a variety of recently described roles in innate immunity. Although many TRIMs regulate type I IFN expression following cytosolic nucleic acid sensing of viruses, their contribution to innate immune signaling and gene expression during bacterial infection remains largely unknown. Because Mycobacterium tuberculosis is an activator of cGAS-dependent cytosolic DNA sensing, we set out to investigate a role for TRIM proteins in regulating macrophage responses to M. tuberculosis In this study, we demonstrate that TRIM14, a noncanonical TRIM that lacks an E3 ubiquitin ligase RING domain, is a critical negative regulator of the type I IFN response in Mus musculus macrophages. We show that TRIM14 interacts with both cGAS and TBK1 and that macrophages lacking TRIM14 dramatically hyperinduce IFN stimulated gene (ISG) expression following M. tuberculosis infection, cytosolic nucleic acid transfection, and IFN-ß treatment. Consistent with a defect in resolution of the type I IFN response, Trim14 knockout macrophages have more phospho-Ser754 STAT3 relative to phospho-Ser727 and fail to upregulate the STAT3 target Socs3, which is required to turn off IFNAR signaling. These data support a model whereby TRIM14 acts as a scaffold between TBK1 and STAT3 to promote phosphorylation of STAT3 at Ser727 and resolve ISG expression. Remarkably, Trim14 knockout macrophages hyperinduce expression of antimicrobial genes like Nos2 and are significantly better than control cells at limiting M. tuberculosis replication. Collectively, these data reveal an unappreciated role for TRIM14 in resolving type I IFN responses and controlling M. tuberculosis infection.

LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis.

The Parkinson's disease (PD)-associated gene leucine-rich repeat kinase 2 (LRRK2) has been studied extensively in the brain. However, several studies have established that mutations in LRRK2 confer susceptibility to mycobacterial infection, suggesting LRRK2 also controls immunity. We demonstrate that loss of LRRK2 in macrophages induces elevated basal levels of type I interferon (IFN) and interferon stimulated genes (ISGs) and causes blunted interferon responses to mycobacterial pathogens and cytosolic nucleic acid agonists. Altered innate immune gene expression in Lrrk2 knockout (KO) macrophages is driven by a combination of mitochondrial stresses, including oxidative stress from low levels of purine metabolites and DRP1-dependent mitochondrial fragmentation. Together, these defects promote mtDNA leakage into the cytosol and chronic cGAS engagement. While Lrrk2 KO mice can control Mycobacterium tuberculosis (Mtb) replication, they have exacerbated inflammation and lower ISG expression in the lungs. These results demonstrate previously unappreciated consequences of LRRK2-dependent mitochondrial defects in controlling innate immune outcomes.

Parkinson's disease is a progressive nervous system disorder that causes tremors, slow movements, and stiff and inflexible muscles. The symptoms are caused by the loss of cells known as neurons in a specific part of the brain that helps to regulate how the body moves. Researchers have identified mutations in several genes that are associated with an increased risk of developing Parkinson's. The most common of these mutations occur in a gene called LRRK2. This gene produces a protein that has been shown to be important for maintaining cellular compartments known as mitochondria, which play a crucial role in generating energy. It remains unclear how these mutations lead to the death of neurons. Mutations in LRRK2 have also been shown to make individuals more susceptible to bacterial infections, suggesting that the protein that LRRK2 codes for may help our immune system. Weindel, Bell et al. set out to understand how this protein works in immune cells called macrophages, which 'eat' invading bacteria and produce type I interferons, molecules that promote immune responses. Mouse cells were used to measure the ability of normal macrophages and macrophages that lack the mouse equivalent to LRRK2 (referred to as Lrrk2 knockout macrophages) to make type I interferons. The experiments showed that the Lrrk2 knockout macrophages made type I interferons even when they were not infected with bacteria, suggesting they are subject to stress that triggers immune responses. It was possible to correct the behavior of the Lrrk2 knockout macrophages by repairing their mitochondria. When mice missing the gene equivalent to LRRK2 were infected with the bacterium that causes tuberculosis, they experienced more severe disease. The protein encoded by the LRRK2 gene is considered a potential target for therapies to treat Parkinson's disease, and several drugs that inhibit this protein are being tested in clinical trials. The findings of Weindel, Bell et al. suggest that these drugs may have unintended negative effects on a patient's ability to fight infection. This work also indicates that LRRK2 mutations may disrupt immune responses in the brain, where macrophage-like cells called microglia play a crucial role in maintaining healthy neurons. Future studies that examine how mutations in LRRK2 affect microglia may help us understand how Parkinson's disease develops.

Splicing Defects of the Profilin Gene Alter Actin Dynamics in an S. pombe SMN Mutant.

Spinal muscular atrophy (SMA) is a devastating motor neuron disorder caused by mutations in the survival motor neuron (SMN) gene. It remains unclear how SMN deficiency leads to the loss of motor neurons. By screening Schizosaccharomyces pombe, we found that the growth defect of an SMN mutant can be alleviated by deletion of the actin-capping protein subunit gene acp1+. We show that SMN mutated cells have splicing defects in the profilin gene, which thus directly hinder actin cytoskeleton homeostasis including endocytosis and cytokinesis. We conclude that deletion of acp1+ in an SMN mutant background compensates for actin cytoskeleton alterations by restoring redistribution of actin monomers between different types of cellular actin networks. Our data reveal a direct correlation between an impaired function of SMN in snRNP assembly and defects in actin dynamics. They also point to important common features in the pathogenic mechanism of SMA and ALS.

The Splicing Factor hnRNP M Is a Critical Regulator of Innate Immune Gene Expression in Macrophages.

While transcriptional control of innate immune gene expression is well characterized, almost nothing is known about how pre-mRNA splicing decisions influence, or are influenced by, macrophage activation. Here, we demonstrate that the splicing factor hnRNP M is a critical repressor of innate immune gene expression and that its function is regulated by pathogen sensing cascades. Loss of hnRNP M led to hyperinduction of a unique regulon of inflammatory and antimicrobial genes following diverse innate immune stimuli. While mutating specific serines on hnRNP M had little effect on its ability to control pre-mRNA splicing or transcript levels of housekeeping genes in resting macrophages, it greatly impacted the protein's ability to dampen induction of specific innate immune transcripts following pathogen sensing. These data reveal a previously unappreciated role for pattern recognition receptor signaling in controlling splicing factor phosphorylation and establish pre-mRNA splicing as a critical regulatory node in defining innate immune outcomes.

Exploring the "Multiple-Hit Hypothesis" of Neurodegenerative Disease: Bacterial Infection Comes Up to Bat.

Despite major strides in personalized genomics, it remains poorly understood why neurodegenerative diseases occur in only a fraction of individuals with a genetic predisposition and conversely, why individuals with no genetic risk of a disorder develop one. Chronic diseases like Alzheimer's, Parkinson's, and Multiple sclerosis are speculated to result from a combination of genetic and environmental factors, a concept commonly referred to as the "multiple hit hypothesis." A number of bacterial infections have been linked to increased risk of neurodegeneration, and in some cases, clearance of bacterial pathogens has been correlated with amelioration of central nervous system (CNS) deficits. Additionally, mutations in several genes known to contribute to CNS disorders like Parkinson's Disease have repeatedly been implicated in susceptibility to intracellular bacterial infection. Recent data has begun to demonstrate roles for these genes (PARK2, PINK1, and LRRK2) in modulating innate immune outcomes, suggesting that immune dysregulation may play an even more important role in neurodegeneration than previously appreciated. This review will broadly explore the connections between bacterial infection, immune dysregulation, and CNS disorders. Understanding this interplay and how bacterial pathogenesis contributes to the "multiple-hit hypothesis" of neurodegeneration will be crucial to develop therapeutics to effectively treat both neurodegeneration and infection.