Pumilio1 haploinsufficiency leads to SCA1-like neurodegeneration by increasing wild-type Ataxin1 levels.

Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative proteinopathy, in which a mutant protein (in this case, ATAXIN1) accumulates in neurons and exerts toxicity; in SCA1, this process causes progressive deterioration of motor coordination. Seeking to understand how post-translational modification of ATAXIN1 levels influences disease, we discovered that the RNA-binding protein PUMILIO1 (PUM1) not only directly regulates ATAXIN1 but also plays an unexpectedly important role in neuronal function. Loss of Pum1 caused progressive motor dysfunction and SCA1-like neurodegeneration with motor impairment, primarily by increasing Ataxin1 levels. Breeding Pum1(+/-) mice to SCA1 mice (Atxn1(154Q/+)) exacerbated disease progression, whereas breeding them to Atxn1(+/-) mice normalized Ataxin1 levels and largely rescued the Pum1(+/-) phenotype. Thus, both increased wild-type ATAXIN1 levels and PUM1 haploinsufficiency could contribute to human neurodegeneration. These results demonstrate the importance of studying post-transcriptional regulation of disease-driving proteins to reveal factors underlying neurodegenerative disease.

Alcohol impairs recognition and uptake of Mycobacterium tuberculosis by suppressing toll-like receptor 2 expression.

BACKGROUND: Alcohol impairs pulmonary innate immune function and is associated with an increased risk of tuberculosis (TB). Toll-like receptor 2 (TLR2) is a pattern recognition receptor on alveolar macrophages that recognizes Mycobacterium tuberculosis (Mtb). The expression of TLR2 depends, in part, on granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling. Given our prior work demonstrating the suppression of GM-CSF signaling following chronic alcohol ingestion, we hypothesized that alcohol impairs TLR2 expression via the suppression of GM-CSF and thereby reduces the ability of the macrophage to recognize and phagocytose Mtb. METHODS: Primary alveolar macrophages were isolated from control-fed and alcohol-fed rats. Prior to cell isolation, some alcohol-fed rats were treated with intranasal GM-CSF and then endotracheally inoculated with an attenuated strain of Mtb. Primary macrophages were then isolated and immunofluorescence was used to determine phagocytic efficiency and TLR2 expression in the presence and absence of GM-CSF treatment and phagocytic efficiency in the presence and absence of TLR2 neutralization. RESULTS: TLR2 expression and phagocytosis of Mtb were significantly lower in the alveolar macrophages of alcohol-fed rats than control-fed rats. In parallel, blocking TLR2 signaling recapitulated this decreased phagocytosis of Mtb. In contrast, intranasal GM-CSF treatment restored TLR2 expression and Mtb phagocytosis in the alveolar macrophages of alcohol-fed rats to levels comparable to those of control-fed rats. CONCLUSIONS: Chronic alcohol ingestion reduces TLR2 protein expression and phagocytosis of Mtb, likely due to impaired GM-CSF signaling. GM-CSF restores membrane-bound TLR2 expression and phagocytic function.

RAS-MAPK-MSK1 pathway modulates ataxin 1 protein levels and toxicity in SCA1.

Many neurodegenerative disorders, such as Alzheimer's, Parkinson's and polyglutamine diseases, share a common pathogenic mechanism: the abnormal accumulation of disease-causing proteins, due to either the mutant protein's resistance to degradation or overexpression of the wild-type protein. We have developed a strategy to identify therapeutic entry points for such neurodegenerative disorders by screening for genetic networks that influence the levels of disease-driving proteins. We applied this approach, which integrates parallel cell-based and Drosophila genetic screens, to spinocerebellar ataxia type 1 (SCA1), a disease caused by expansion of a polyglutamine tract in ataxin 1 (ATXN1). Our approach revealed that downregulation of several components of the RAS-MAPK-MSK1 pathway decreases ATXN1 levels and suppresses neurodegeneration in Drosophila and mice. Importantly, pharmacological inhibitors of components of this pathway also decrease ATXN1 levels, suggesting that these components represent new therapeutic targets in mitigating SCA1. Collectively, these data reveal new therapeutic entry points for SCA1 and provide a proof-of-principle for tackling other classes of intractable neurodegenerative diseases.

Extensive cryptic splicing upon loss of RBM17 and TDP43 in neurodegeneration models.

Splicing regulation is an important step of post-transcriptional gene regulation. It is a highly dynamic process orchestrated by RNA-binding proteins (RBPs). RBP dysfunction and global splicing dysregulation have been implicated in many human diseases, but the in vivo functions of most RBPs and the splicing outcome upon their loss remain largely unexplored. Here we report that constitutive deletion of Rbm17, which encodes an RBP with a putative role in splicing, causes early embryonic lethality in mice and that its loss in Purkinje neurons leads to rapid degeneration. Transcriptome profiling of Rbm17-deficient and control neurons and subsequent splicing analyses using CrypSplice, a new computational method that we developed, revealed that more than half of RBM17-dependent splicing changes are cryptic. Importantly, RBM17 represses cryptic splicing of genes that likely contribute to motor coordination and cell survival. This finding prompted us to re-analyze published datasets from a recent report on TDP-43, an RBP implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as it was demonstrated that TDP-43 represses cryptic exon splicing to promote cell survival. We uncovered a large number of TDP-43-dependent splicing defects that were not previously discovered, revealing that TDP-43 extensively regulates cryptic splicing. Moreover, we found a significant overlap in genes that undergo both RBM17- and TDP-43-dependent cryptic splicing repression, many of which are associated with survival. We propose that repression of cryptic splicing by RBPs is critical for neuronal health and survival. CrypSplice is available at www.liuzlab.org/CrypSplice.

Red Blood Cell DNA Capture and Delivery Drives Host Responses During Polymicrobial Sepsis.

Red blood cells (RBCs), traditionally recognized for their role in transporting oxygen, play a pivotal role in the body's immune response by expressing TLR9 and scavenging excess host cell-free DNA. DNA capture by RBCs leads to accelerated RBC clearance and triggers inflammation. Whether RBCs can also acquire microbial DNA during infections is unknown. Murine RBCs acquire microbial DNA in vitro and bacterial-DNA-induced macrophage activation was augmented by WT but not TLR9-deleted RBCs. In a mouse model of polymicrobial sepsis, RBC-bound bacterial DNA was elevated in WT but not in erythroid TLR9-deleted mice. Plasma cytokine analysis revealed distinct sepsis endotypes, characterized by persistent hypothermia and hyperinflammation in the most severely affected subjects. RBC-TLR9 deletion attenuated plasma and tissue IL-6 production in the most severe endotype. Parallel findings in human subjects confirmed that RBCs from septic patients harbored more bacterial DNA compared to healthy individuals. Further analysis through 16S sequencing of RBC-bound DNA illustrated distinct microbial communities, with RBC-bound DNA composition correlating with plasma IL-6 in patients with sepsis. Collectively, these findings unveil RBCs as overlooked reservoirs and couriers of microbial DNA, capable of influencing host inflammatory responses in sepsis.

Immunologic alterations in the pancreatic cancer microenvironment of patients treated with neoadjuvant chemotherapy and radiotherapy.

Pancreatic ductal adenocarcinoma (PDAC) has dismal 5-year survival (<9%). We hypothesize that exposure of tumors to conventional therapies may preferentially modulate immune biomarkers in the tumor microenvironment in PDAC. PDAC patients who underwent upfront surgical resection or who received neoadjuvant FOLFIRINOX with or without neoadjuvant radiotherapy followed by surgical resection were selected for study. Total expression of immunologically relevant transcripts and spatially resolved expression of immunologically relevant proteins was quantitated using multiplexed methods (NanoString nCounter and GeoMX platforms). This analysis identified numerous differentially expressed transcripts associated with the type of neoadjuvant therapy received. Moreover, we identified significant alterations in the expression and/or spatial distribution of immunologically relevant proteins in different regions (tumor cell rich, immune cell rich, stromal cell rich) of the tumor microenvironment. These data provide insight into the immunological effects of clinically relevant neoadjuvant therapy for resectable/borderline-resectable PDAC by describing significant differences in the expression of key immunologic biomarkers within the PDAC microenvironment that were associated with the type of treatment patients received prior to surgical resection. This represents a comprehensive analysis of numerous biomarkers conducted on the PDAC microenvironment. This work may guide strategic new combination therapies for pancreatic cancer.

Induction Therapy in Localized Pancreatic Cancer.

OBJECTIVES: Pancreatic cancer (PDAC) with localized stage includes resectable (RPC), borderline resectable (BRPC), or locally advanced unresectable (LAPC). Standard of care for RPC is adjuvant chemotherapy. There are no prospective randomized trials for best treatment of BRPC and LAPC. We evaluate the impact of induction chemotherapy on localized PDAC. METHODS: Charts of PDAC patients treated at Emory University between 2009 and 2016 were reviewed. The primary end point was overall survival (OS). RESULTS: A total of 409 localized PDACs were identified. Resectability was prospectively determined at a multidisciplinary tumor conference. Median age was 67 years (range, 30-92 years), 49% were male, 66% were white, 171 had RPC, 131 had BRPC, and 107 had LAPC. Median OSs for RPC, BRPC, and LAPC were 19.5, 16.1, and 12.7 months, respectively. Type of chemotherapy and age were predictors of OS. Induction chemotherapy was used in 106 with BRPC (81%) and 74 with RPC (56.5%); patients with BRPC who received combination chemotherapy and resection had a median OS of 31.5 compared with 19.5 months in patients with RPC (P = 0.0049). Patients with LAPC had a median OS of 12.7 months. CONCLUSIONS: In patients with BRPC who undergo resection after induction treatment, the OS was significantly better than in patients with RPC. Neoadjuvant treatment should be considered for all localized PDACs.