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A complete understanding of RNA biology requires methods for tracking transcripts in vivo. Common strategies rely on fluorogenic probes that are limited in sensitivity, dynamic range, and depth of interrogation, owing to their need for excitation light and tissue autofluorescence. To overcome these challenges, we report a bioluminescent platform for serial imaging of RNAs. The RNA tags are engineered to recruit light-emitting luciferase fragments (termed RNA lanterns) upon transcription. Robust photon production is observed for RNA targets both in cells and in live animals. Importantly, only a single copy of the tag is necessary for sensitive detection, in sharp contrast to fluorescent platforms requiring multiple repeats. Overall, this work provides a foundational platform for visualizing RNA dynamics from the micro to the macro scale.

BACKGROUND: One-fifth of the global population is infected with soil-transmitted helminths (STH). Mass drug administration (MDA) with deworming medication is widely implemented to control morbidity associated with STH infections. However, surveillance of human infection prevalence by collecting individual stool samples is time-consuming, costly, often stigmatized, and logistically challenging. Current methods of STH detection are poorly sensitive, particularly in low-intensity and low-prevalence populations. METHODOLOGY/PRINCIPAL FINDINGS: We aimed to develop a sensitive and specific molecular method for detecting STH DNA in large volumes of soil (20 g) by conducting laboratory and proof of concept studies across field sites in Kenya, Benin, and India. We collected human stool (n = 669) and soil (n = 478) from 322 households across the three study sites. We developed protocols for DNA extraction from 20 g of soil and qPCR to detect Ascaris lumbricoides, Trichuris trichiura, Necator americanus, and Ancylostoma duodenale. Agreement between detection of STH via qPCR, digital droplet PCR (ddPCR), and microscopy-based methods was assessed using the Cohens Kappa statistic. Finally, we estimated associations between soil characteristics and detection of STH in soil by qPCR, as well as between STH detected in soil and STH detected in stool from matched households, adjusting for soil characteristics. The overall prevalence of STH in soil by qPCR was 31% for A. lumbricoides, 3% for T. trichiura, and 13% for any hookworm species. ddPCR and qPCR performed similarly. However, there was poor agreement between STH detected in soil by qPCR versus light microscopy. Microscopy underestimated the prevalence of A. lumbricoides and N. americanus and overestimated T. trichiura. Detection of an STH species in household soil was strongly associated with increased odds of a household member being infected with that same species. CONCLUSIONS/SIGNIFICANCE: Soil surveillance for STH has several benefits over stool-based surveillance, including lower cost and higher success rates for sample collection. Considering that delivery of MDA occurs at the community level, environmental surveillance using molecular methods could be a cost-effective alternate strategy for monitoring STH in these populations.

Stimulation paradigms that induce perforant path long-term potentiation (LTP) initiate phosphorylation of ERK1/2 and induce expression of a variety of immediate early genes (IEGs). These events are thought to be critical components of the mechanism for establishing the changes in synaptic efficacy that endure for hours or longer. Here we show that in mice, perforant path LTP can be induced using a standard protocol (repeated trains at 250 Hz), without accompanying increases in immunostaining for p-ERK1/2 or increased in expression of representative IEGs (Arc and c-fos). Signaling pathways capable of inducing ERK phosphorylation and IEG transcription are intact in mice because ERK phosphorylation differs strikingly in awake versus anesthetized mice, and IEG expression is strongly induced by electroconvulsive seizures. In pursuing the reasons for the lack of induction with LTP, we found that in rats, one of the stimulation paradigms used to induce perforant path LTP (trains at 250 Hz) also does not activate MAP kinase or induce IEG expression, despite the fact that the LTP induced by 250 Hz stimulation requires NMDA receptor activation and persists for hours. These findings indicate that there are different forms of perforant path LTP, one of which does not require MAP kinase activation or IEG induction. Moreover, these data demonstrate that different LTP induction paradigms do not have identical molecular consequences, which may account for certain discrepancies between previous studies.

Recent studies have reported new evidence consistent with the hypothesis that reactivating a memory by re-exposure to a training context destabilizes the memory and induces "reconsolidation." In the present experiments, rats' memory for inhibitory avoidance (IA) training was tested 6 h (Test 1), 2 d (Test 2), and 6 d (Test 3) after training. On Test 1 the rats were either removed from the shock compartment immediately after entry or retained in the shock context for 200 sec, and intrahippocampal infusions of the protein synthesis inhibitor anisomycin (75 microgram/side) were administered immediately after the test. Anisomycin infusions administered after Test 1 impaired IA performance on Test 2 in animals given the brief re-exposure, but impaired extinction in animals exposed to the context for 200 sec. Rats with anisomycin-induced retention impairment on Test 2 demonstrated spontaneous recovery of retention performance on Test 3, whereas rats showing extinction on Test 2 showed further extinction on Test 3. The findings indicate that post-retrieval administration of anisomycin impairs subsequent retention performance only in the absence of extinction and that this impairment is temporary. (Contains 3 figures.)

This paper explores the potential of rAAV2-retro to deliver gene modifying cargoes to the cells of origin of multiple pathways that are interrupted by spinal cord injury (SCI), summarizing data from previous studies and new data from additional experiments. rAAV-retro exhibits uniquely robust and reliable long-distance retrograde transport from pre-terminal axons and synapses back to neuronal bodies. Previous studies have documented that various AAV-based genetic modifications can enable axon regeneration after SCI, but these have targeted the cells of origin of one pathway at a time. In contrast, rAAV-retro can simultaneously transduce large numbers of neurons of origin of multiple spinal pathways with single injections into the spinal cord. Our initial studies use RosatdTomato and double transgenic PTENf/f; RosatdTomato mice in which transfection with rAAV-retro/Cre deletes PTEN and activates tdT expression in the same neurons. Injections of rAAV-retro/Cre into the cervical, thoracic and lumbar spinal cord led to topographically specific retrograde transduction in cortical motoneurons and neurons in subcortical regions that give rise to different spinal pathways. Our results confirm and extend previous studies indicating selective transduction of neurons that terminate at the level of the injection with minimal retrograde transduction of axons in transit to lower levels. We document feasibility of using rAAV-retro expressing shRNA against PTEN along with a GFP reporter (rAAV-retro-shPTEN/GFP) to effectively knock down PTEN in multiple populations of neurons, which can be used in any species. Some limitations and caveats of currently available rAAV-retros are discussed. Together, our results support the potential applications of rAAV-retro for AAV-based gene-modifications for SCI.

The highly lethal brain cancer glioblastoma (GBM) poses a daunting challenge because the blood-brain barrier renders potentially druggable amplified or mutated oncoproteins relatively inaccessible. Here, we identify sphingomyelin phosphodiesterase 1 (SMPD1), an enzyme that regulates the conversion of sphingomyelin to ceramide, as an actionable drug target in GBM. We show that the highly brain-penetrant antidepressant fluoxetine potently inhibits SMPD1 activity, killing GBMs, through inhibition of epidermal growth factor receptor (EGFR) signaling and via activation of lysosomal stress. Combining fluoxetine with temozolomide, a standard of care for GBM, causes massive increases in GBM cell death and complete tumor regression in mice. Incorporation of real-world evidence from electronic medical records from insurance databases reveals significantly increased survival in GBM patients treated with fluoxetine, which was not seen in patients treated with other selective serotonin reuptake inhibitor (SSRI) antidepressants. These results nominate the repurposing of fluoxetine as a potentially safe and promising therapy for patients with GBM and suggest prospective randomized clinical trials.

Injury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in murine dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of translation in vitro and in vivo Inflammatory cues promote local translation of Arc in the skin. Arc-deficient male mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles (EVs), we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found that the excessive thermal responses and vasodilation observed in Arc defective mice are rescued by injection of Arc-containing EVs into the skin. Our findings suggest that activity-dependent production of Arc in afferent fibers regulates neurogenic inflammation potentially through intercellular signaling.SIGNIFICANCE STATEMENT Nociceptors play prominent roles in pain and inflammation. We examined rapid changes in the landscape of nascent translation in cultured dorsal root ganglia (DRGs) treated with a combination of inflammatory mediators using ribosome profiling. We identified several hundred transcripts subject to rapid preferential translation. Among them is the immediate early gene (IEG) Arc. We provide evidence that Arc is translated in afferent fibers in the skin. Arc-deficient mice display several signs of exaggerated inflammation which is normalized on injection of Arc containing extracellular vesicles (EVs). Our work suggests that noxious cues can trigger Arc production by nociceptors which in turn constrains neurogenic inflammation in the skin.

Rostro-caudal specificity of corticospinal tract (CST) projections from different areas of the cortex was assessed by retrograde labeling with fluorogold and retrograde transfection following retro-AAV/Cre injection into the spinal cord of tdT reporter mice. Injections at C5 led to retrograde labeling of neurons throughout forelimb area of the sensorimotor cortex and a region in the dorsolateral cortex near the barrel field (S2). Injections at L2 led to retrograde labeling of neurons in the posterior sensorimotor cortex (hindlimb area) but not the dorsolateral cortex. With injections of biotinylated dextran amine (BDA) into the main sensorimotor cortex (forelimb region), labeled axons terminated selectively at cervical levels. With BDA injections into caudal sensorimotor cortex (hindlimb region), labeled axons passed through cervical levels without sending collaterals into the gray matter and then elaborated terminal arbors at thoracic sacral levels. With BDA injections into the dorsolateral cortex near the barrel field, labeled axons terminated at high cervical levels. Axons from medial sensorimotor cortex terminated primarily in intermediate laminae and axons from lateral sensorimotor cortex terminated primarily in laminae III-V of the dorsal horn. One of the descending pathways seen in rats (the ventral CST) was not observed in most mice.

Sphingolipids constitute a heterogeneous lipid category that is involved in many key cellular functions. For high-throughput analyses of sphingolipids, tandem mass spectrometry (MS/MS) is the method of choice, offering sufficient sensitivity, structural information, and quantitative precision for detecting hundreds to thousands of species simultaneously. While glycerolipids and phospholipids are predominantly non-hydroxylated, sphingolipids are typically dihydroxylated. However, species containing one or three hydroxylation sites can be detected frequently. This variability in the number of hydroxylation sites on the sphingolipid long-chain base and the fatty acyl moiety produces many more isobaric species and fragments than for other lipid categories. Due to this complexity, the automated annotation of sphingolipid species is challenging, and incorrect annotations are common. In this study, we present an extension of the Lipid Data Analyzer (LDA) "decision rule set" concept that considers the structural characteristics that are specific for this lipid category. To address the challenges inherent to automated annotation of sphingolipid structures from MS/MS data, we first developed decision rule sets using spectra from authentic standards and then tested the applicability on biological samples including murine brain and human plasma. A benchmark test based on the murine brain samples revealed a highly improved annotation quality as measured by sensitivity and reliability. The results of this benchmark test combined with the easy extensibility of the software to other (sphingo)lipid classes and the capability to detect and correctly annotate novel sphingolipid species make LDA broadly applicable to automated sphingolipid analysis, especially in high-throughput settings.

Patients infected with influenza are at high risk of secondary bacterial infection, which is a major proximate cause of morbidity and mortality. We have shown that in mice, prior infection with influenza results in increased inflammation and mortality upon Staphylococcus aureus infection, recapitulating the human disease. Lipidomic profiling of the lungs of superinfected mice revealed an increase in CYP450 metabolites during lethal superinfection. These lipids are endogenous ligands for the nuclear receptor PPARα, and we demonstrate that Ppara-/- mice are less susceptible to superinfection than wild-type mice. PPARα is an inhibitor of NFκB activation, and transcriptional profiling of cells isolated by bronchoalveolar lavage confirmed that influenza infection inhibits NFκB, thereby dampening proinflammatory and prosurvival signals. Furthermore, network analysis indicated an increase in necrotic cell death in the lungs of superinfected mice compared to mice infected with S. aureus alone. Consistent with this, we observed reduced NFκB-mediated inflammation and cell survival signaling in cells isolated from the lungs of superinfected mice. The kinase RIPK3 is required to induce necrotic cell death and is strongly induced in cells isolated from the lungs of superinfected mice compared to mice infected with S. aureus alone. Genetic and pharmacological perturbations demonstrated that PPARα mediates RIPK3-dependent necroptosis and that this pathway plays a central role in mortality following superinfection. Thus, we have identified a molecular circuit in which infection with influenza induces CYP450 metabolites that activate PPARα, leading to increased necrotic cell death in the lung which correlates with the excess mortality observed in superinfection.

Multiple sclerosis (MS) is a chronic, inflammatory autoimmune disease that affects the central nervous system (CNS) for which there is no cure. In MS, encephalitogenic T cells infiltrate the CNS causing demyelination and neuroinflammation; however, little is known about the role of regulatory T cells (Tregs) in CNS tissue repair. Transplantation of neural stem and progenitor cells (NSCs and NPCs) is a promising therapeutic strategy to promote repair through cell replacement, although recent findings suggest transplanted NSCs also instruct endogenous repair mechanisms. We have recently described that dampened neuroinflammation and increased remyelination is correlated with emergence of Tregs following human NPC transplantation in a murine viral model of immune-mediated demyelination. In the current study we utilized the prototypic murine autoimmune model of demyelination experimental autoimmune encephalomyelitis (EAE) to test the efficacy of hNSC transplantation. Eight-week-old, male EAE mice receiving an intraspinal transplant of hNSCs during the chronic phase of disease displayed remyelination, dampened neuroinflammation, and an increase in CNS CD4+CD25+FoxP3+ regulatory T cells (Tregs). Importantly, ablation of Tregs abrogated histopathological improvement. Tregs are essential for maintenance of T cell homeostasis and prevention of autoimmunity, and an emerging role for Tregs in maintenance of tissue homeostasis through interactions with stem and progenitor cells has recently been suggested. The data presented here provide direct evidence for collaboration between CNS Tregs and hNSCs promoting remyelination.

G protein-coupled receptor 120 (GPR120) and PPARγ agonists each have insulin sensitizing effects. But whether these two pathways functionally interact and can be leveraged together to markedly improve insulin resistance has not been explored. Here, we show that treatment with the PPARγ agonist rosiglitazone (Rosi) plus the GPR120 agonist Compound A leads to additive effects to improve glucose tolerance and insulin sensitivity, but at lower doses of Rosi, thus avoiding its known side effects. Mechanistically, we show that GPR120 is a PPARγ target gene in adipocytes, while GPR120 augments PPARγ activity by inducing the endogenous ligand 15d-PGJ2 and by blocking ERK-mediated inhibition of PPARγ. Further, we used macrophage- (MKO) or adipocyte-specific GPR120 KO (AKO) mice to show that GRP120 has anti-inflammatory effects via macrophages while working with PPARγ in adipocytes to increase insulin sensitivity. These results raise the prospect of a safer way to increase insulin sensitization in the clinic.

Background & aimsChronic alcohol consumption is a leading risk factor for the development of hepatocellular carcinoma (HCC), which is associated with a marked increase in hepatic expression of pro-inflammatory IL-17A and its receptor IL-17RA.MethodsGenetic deletion and pharmacological blocking were used to characterize the role of IL-17A/IL-17RA signaling in the pathogenesis of HCC in mouse models and human specimens.ResultsWe demonstrate that the global deletion of the Il-17ra gene suppressed HCC in alcohol-fed diethylnitrosamine-challenged Il-17ra-/- and major urinary protein-urokinase-type plasminogen activator/Il-17ra-/- mice compared with wild-type mice. When the cell-specific role of IL-17RA signaling was examined, the development of HCC was decreased in both alcohol-fed Il-17raΔMΦ and Il-17raΔHep mice devoid of IL-17RA in myeloid cells and hepatocytes, but not in Il-17raΔHSC mice (deficient in IL-17RA in hepatic stellate cells). Deletion of Il-17ra in myeloid cells ameliorated tumorigenesis via suppression of pro-tumorigenic/inflammatory and pro-fibrogenic responses in alcohol-fed Il-17raΔMΦ mice. Remarkably, despite a normal inflammatory response, alcohol-fed Il-17raΔHep mice developed the fewest tumors (compared with Il-17raΔMΦ mice), with reduced steatosis and fibrosis. Steatotic IL-17RA-deficient hepatocytes downregulated the expression of Cxcl1 and other chemokines, exhibited a striking defect in tumor necrosis factor (TNF)/TNF receptor 1-dependent caspase-2-SREBP1/2-DHCR7-mediated cholesterol synthesis, and upregulated the production of antioxidant vitamin D3. The pharmacological blocking of IL-17A/Th-17 cells using anti-IL-12/IL-23 antibodies suppressed the progression of HCC (by 70%) in alcohol-fed mice, indicating that targeting IL-17 signaling might provide novel strategies for the treatment of alcohol-induced HCC.ConclusionsOverall, IL-17A is a tumor-promoting cytokine, which critically regulates alcohol-induced hepatic steatosis, inflammation, fibrosis, and HCC.Lay summaryIL-17A is a tumor-promoting cytokine, which critically regulates inflammatory responses in macrophages (Kupffer cells and bone-marrow-derived monocytes) and cholesterol synthesis in steatotic hepatocytes in an experimental model of alcohol-induced HCC. Therefore, IL-17A may be a potential therapeutic target for patients with alcohol-induced HCC.

Genetic deletion or knockdown of PTEN enables regeneration of CNS axons, enhances sprouting of intact axons after injury, and induces de novo growth of uninjured adult neurons. It is unknown, however how PTEN deletion in mature neurons alters neuronal physiology. As a first step to address this question, we used immunocytochemistry for activity-dependent markers to assess consequences of PTEN knockdown in cortical neurons and granule cells of the dentate gyrus. In adult rats that received unilateral intra-cortical injections of AAV expressing shRNA against PTEN, immunostaining for c-fos under resting conditions (home cage, HC) and after 1 h of exploration of a novel enriched environment (EE) revealed no hot spots of c-fos expression that would suggest abnormal activity. Counts revealed similar numbers of c-fos positive neurons in the area of PTEN deletion vs. homologous areas in the contralateral cortex in the HC and similar induction of c-fos with EE. However, IEG induction in response to high frequency stimulation (HFS) of the cortex was attenuated in areas of PTEN deletion. In rats with AAVshRNA-mediated PTEN deletion in the dentate gyrus, induction of the IEGs c-fos and Arc with HFS of the perforant path was abrogated in areas of PTEN deletion. Immunostaining using phosphospecific antibodies for phospho-S6 (a downstream marker for mTOR activation) and phospho-ERK1/2 revealed abrogation of S6 phosphorylation in PTEN-deleted areas but preserved activation of phosphorylation of ERK1/2. SIGNIFICANCE STATEMENT: Deletion or knockdown of the tumor suppressor gene PTEN enables regenerative growth of adult CNS axons after injury, which is accompanied by enhanced recovery of function. Consequently, PTEN represents a potential target for therapeutic interventions to enhance recovery after CNS injury. Here we show that activity-dependent IEG induction is attenuated in PTEN-depleted neurons. These findings raise the intriguing possibility that functional recovery due to regenerative growth may be limited by the disruption of plasticity-related signaling pathways, and that recovery might be enhanced by restoring PTEN expression after regenerative growth has been achieved.

The transmission soil transmitted helminths (STH) occurs via ingestion of or contact with infective stages present in soil contaminated with human faeces. It follows therefore that efforts to reduce faecal contamination of the environment should help to reduce risk of parasite exposure and improvements in water, sanitation and hygiene (WASH) are seen as essential for the long-term, sustainable control of STH. However, the link between WASH and STH is not always supported by the available evidence from randomised controlled trials, which report mixed effects of WASH intervention on infection risk. This review critically summarises the available trial evidence and offers an interpretation of the observed heterogeneity in findings. The review also discusses the implications of findings for control programmes and highlights three main issues which merit further consideration: intervention design, exposure assessment, and intervention fidelity assessment.

Advances in DNA sequencing technologies have reshaped our understanding of the molecular basis of cancer, providing a precise genomic view of tumors. Complementary biochemical and biophysical perspectives of cancer point toward profound shifts in nutrient uptake and utilization that propel tumor growth and major changes in the structure of the plasma membrane of tumor cells. The molecular mechanisms that bridge these fundamental aspects of tumor biology remain poorly understood. Here, we show that the lysophosphatidylcholine acyltransferase LPCAT1 functionally links specific genetic alterations in cancer with aberrant metabolism and plasma membrane remodeling to drive tumor growth. Growth factor receptor-driven cancers are found to depend on LPCAT1 to shape plasma membrane composition through enhanced saturated phosphatidylcholine content that is, in turn, required for the transduction of oncogenic signals. These results point to a genotype-informed strategy that prioritizes lipid remodeling pathways as therapeutic targets for diverse cancers.

High-fat diet (HFD) causes renal lipotoxicity that is ameliorated with AMP-activated protein kinase (AMPK) activation. Although bioactive eicosanoids increase with HFD and are essential in regulation of renal disease, their role in the inflammatory response to HFD-induced kidney disease and their modulation by AMPK activation remain unexplored. In a mouse model, we explored the effects of HFD on eicosanoid synthesis and the role of AMPK activation in ameliorating these changes. We used targeted lipidomic profiling with quantitative MS to determine PUFA and eicosanoid content in kidneys, urine, and renal arterial and venous circulation. HFD increased phospholipase expression as well as the total and free pro-inflammatory arachidonic acid (AA) and anti-inflammatory DHA in kidneys. Consistent with the parent PUFA levels, the AA- and DHA-derived lipoxygenase (LOX), cytochrome P450, and nonenzymatic degradation (NE) metabolites increased in kidneys with HFD, while EPA-derived LOX and NE metabolites decreased. Conversely, treatment with 5-aminoimidazole-4-carboxamide-1-β-D-furanosyl 5'-monophosphate (AICAR), an AMPK activator, reduced the free AA and DHA content and the DHA-derived metabolites in kidney. Interestingly, kidney and circulating AA, AA metabolites, EPA-derived LOX, and NE metabolites are increased with HFD; whereas, DHA metabolites are increased in kidney in contrast to their decreased circulating levels with HFD. Together, these changes showcase HFD-induced pro- and anti-inflammatory eicosanoid dysregulation and highlight the role of AMPK in correcting HFD-induced dysregulated eicosanoid pathways.

Eicosanoids and related metabolites (oxylipins) possess potent signaling properties, elicit numerous important physiologic responses, and serve as biomarkers of disease. In addition to their presence in free form, a considerable portion of these bioactive lipids is esterified to complex lipids in cell membranes and plasma lipoproteins. We developed a rapid and sensitive method for the analysis of esterified oxylipins using alkaline hydrolysis to release them followed by ultra-performance LC coupled with mass spectrometric analysis. Detailed evaluation of the data revealed that several oxylipins are susceptible to alkaline-induced degradation. Nevertheless, of the 136 metabolites we examined, 56 were reproducibly recovered after alkaline hydrolysis. We classified those metabolites that were resistant to alkaline-induced degradation and applied this methodology to quantify metabolite levels in a macrophage cell model and in plasma of healthy subjects. After alkaline hydrolysis of lipids, 34 metabolites could be detected and quantified in resting and activated macrophages, and 38 metabolites were recovered from human plasma at levels that were substantially greater than in free form. By carefully selecting internal standards and taking the observed experimental limitations into account, we established a robust method that can be reliably employed for the measurement of esterified oxylipins in biological samples.

Cancer testis antigens (CTA) are expressed in testis and placenta and anomalously activated in a variety of tumors. The mechanistic contribution of CTAs to neoplastic phenotypes remains largely unknown. Using a chemigenomics approach, we find that the CTA HORMAD1 correlates with resistance to the mitochondrial complex I inhibitor piericidin A in non-small cell lung cancer (NSCLC). Resistance was due to a reductive intracellular environment that attenuated the accumulation of free radicals. In human lung adenocarcinoma (LUAD) tumors, patients expressing high HORMAD1 exhibited elevated mutational burden and reduced survival. HORMAD1 tumors were enriched for genes essential for homologous recombination (HR), and HORMAD1 promoted RAD51-filament formation, but not DNA resection, during HR. Accordingly, HORMAD1 loss enhanced sensitivity to γ-irradiation and PARP inhibition, and HORMAD1 depletion significantly reduced tumor growth in vivo These results suggest that HORMAD1 expression specifies a novel subtype of LUAD, which has adapted to mitigate DNA damage. In this setting, HORMAD1 could represent a direct target for intervention to enhance sensitivity to DNA-damaging agents or as an immunotherapeutic target in patients.Significance: This study uses a chemigenomics approach to demonstrate that anomalous expression of the CTA HORMAD1 specifies resistance to oxidative stress and promotes HR to support tumor cell survival in NSCLC. Cancer Res; 78(21); 6196-208. ©2018 AACR.

Carnivore predation on livestock often leads people to retaliate. Persecution by humans has contributed strongly to global endangerment of carnivores. Preventing livestock losses would help to achieve three goals common to many human societies: preserve nature, protect animal welfare, and safeguard human livelihoods. Between 2016 and 2018, four independent reviews evaluated >40 years of research on lethal and nonlethal interventions for reducing predation on livestock. From 114 studies, we find a striking conclusion: scarce quantitative comparisons of interventions and scarce comparisons against experimental controls preclude strong inference about the effectiveness of methods. For wise investment of public resources in protecting livestock and carnivores, evidence of effectiveness should be a prerequisite to policy making or large-scale funding of any method or, at a minimum, should be measured during implementation. An appropriate evidence base is needed, and we recommend a coalition of scientists and managers be formed to establish and encourage use of consistent standards in future experimental evaluations.

Axons within the peripheral nervous system are capable of regeneration, but full functional recovery is rare. Recent work has shown that conditional deletion of two key signaling inhibitors of the PI3K and Jak/Stat pathways-phosphatase and tensin homolog (PTEN) and suppressor of cytokine signaling-3 (SOCS3), respectively-promotes regeneration of normally non-regenerative central nervous system axons. Moreover, in studies of optic nerve regeneration, co-deletion of both PTEN and SOCS3 has an even greater effect. Here, we test the hypotheses (1) that PTEN deletion enhances axon regeneration following sciatic nerve crush and (2) that PTEN/SOCS3 co-deletion further promotes regeneration. PTENfl/fl and PTEN/SOCS3fl/fl mice received direct injections of AAV-Cre into the fourth and fifth lumbar dorsal root ganglia (DRG) two weeks prior to sciatic nerve crush. Western blot analysis of whole cell lysates from DRG using phospho-specific antibodies revealed that PTEN deletion did not enhance or prolong PI3K signaling following sciatic nerve crush. However, PTEN/SOCS3 co-deletion activated PI3K for at least 7 days post-injury in contrast to controls, where activation peaked at 3 days. Quantification of SCG10-expressing regenerating sensory axons in the sciatic nerve after crush injury revealed longer distance regeneration at 3 days post-injury with both PTEN and PTEN/SOCS3 co-deletion. Additionally, analysis of noxious thermosensation and mechanosensation with PTEN/SOCS3 co-deletion revealed enhanced sensation at 14 and 21 days after crush, respectively, after which all treatment groups reached the same functional plateau. These findings indicate that co-deletion of PTEN and SOCS3 results in modest but measureable enhancement of early regeneration of DRG axons following crush injury.

Deletion of the phosphatase and tensin (PTEN) gene in neonatal mice leads to enlargement of the cell bodies of cortical motoneurons (CMNs) in adulthood (Gutilla et al., 2016). Here, we assessed whether PTEN deletion in adult mice would trigger growth of mature neurons. PTEN was deleted by injecting AAV-Cre into the sensorimotor cortex of adult transgenic mice with a lox-P flanked exon 5 of the PTEN gene and Cre-dependent reporter gene tdTomato. PTEN-deleted CMN's identified by tdT expression and retrograde labeling with fluorogold (FG) were significantly enlarged four months following PTEN deletion, and continued to increase in size through the latest time intervals examined (12-15 months post-deletion). Sholl analyses of tdT-positive pyramidal neurons revealed increases in dendritic branches at 6 months following adult PTEN deletion, and greater increases at 12 months. 12 months after adult PTEN deletion, axons in the medullary pyramids were significantly larger and G-ratios were higher. Mice with PTEN deletion exhibited no overt neurological symptoms and no seizures. Assessment of motor function on the rotarod and cylinder test revealed slight impairment of coordination with unilateral deletion; however, mice with bilateral PTEN deletion in the motor cortex performed better than controls on the rotarod at 8 and 10 months post-deletion. Our findings demonstrate that robust neuronal growth can be induced in fully mature cortical neurons long after the developmental period has ended and that this continuous growth occurs without obvious functional impairments.

We achieve automated and reliable annotation of lipid species and their molecular structures in high-throughput data from chromatography-coupled tandem mass spectrometry using decision rule sets embedded in Lipid Data Analyzer (LDA; http://genome.tugraz.at/lda2). Using various low- and high-resolution mass spectrometry instruments with several collision energies, we proved the method's platform independence. We propose that the software's reliability, flexibility, and ability to identify novel lipid molecular species may now render current state-of-the-art lipid libraries obsolete.

Cardiac arrest (CA) affects >550,000 people annually in the United States whereas 80-90% of survivors suffer from a comatose state. Arousal from coma is critical for recovery, but mechanisms of arousal are undefined. Orexin-A, a hypothalamic excitatory neuropeptide, has been linked to arousal deficits in various brain injuries. We investigated the orexinergic system's role in recovery from CA-related neurological impairments, including arousal deficits. Using an asphyxial CA and resuscitation model in rats, we examine neurological recovery post-resuscitation in conjunction with changes in orexin-A levels in cerebrospinal fluid (CSF) and orexin-expressing neurons. We also conduct pharmacological inhibition of orexin post-resuscitation. We show that recovery from neurological deficits begins between 4 and 24 h post-resuscitation, with additional recovery by 72 h post-resuscitation. Orexin-A levels in the CSF are lowest during periods of poorest arousal post-resuscitation (4 h) and recover to control levels by 24 h. Immunostaining revealed that the number of orexin-A immunoreactive neurons declined at 4 h post-resuscitation, but increased to near normal levels by 24 h. There were no significant changes in the number of neurons expressing melanin-concentrating hormone, another neuropeptide localized in similar hypothalamus regions. Last, administration of the dual orexin receptor antagonist, suvorexant, during the initial 24 h post-resuscitation, led to sustained neurological deficits. The orexin pathway is critical during early phases of neurological recovery post-CA. Blocking this early action leads to persistent neurological deficits. This is of considerable clinical interest given that suvorexant recently received U.S. Food and Drug Administration approval for insomnia treatment.

High-frequency stimulation of the medial perforant path triggers robust phosphorylation of ribosomal protein S6 (rpS6) in activated dendritic domains and granule cell bodies. Here we dissect the signaling pathways responsible for synaptically driven rpS6 phosphorylation in the dentate gyrus using pharmacological agents to inhibit PI3-kinase/mTOR and MAPK/ERK-dependent kinases. Using phospho-specific antibodies for rpS6 at different sites (ser235/236 versus ser240/244), we show that delivery of the PI3-kinase inhibitor, wortmannin, decreased rpS6 phosphorylation throughout the somatodendritic compartment (granule cell layer, inner molecular layer, outer molecular layer), especially in granule cell bodies while sparing phosphorylation at activated synapses (middle molecular layer). In contrast, delivery of U0126, an MEK inhibitor, attenuated rpS6 phosphorylation specifically in the dendritic laminae leaving phosphorylation in the granule cell bodies intact. Delivery of the mTOR inhibitor, rapamycin, abolished activation of rpS6 phosphorylation in granule cell bodies and dendrites, whereas delivery of a selective S6K1 inhibitor, PF4708671, or RSK inhibitor, SL0101-1, attenuated rpS6 phosphorylation throughout the postsynaptic cell. These results reveal that MAPK/ERK-dependent signaling is predominately responsible for the selective induction of rpS6 phosphorylation at active synapses. In contrast, PI3-kinase/mTOR-dependent signaling induces rpS6 phosphorylation throughout the somatodendritic compartment but plays a minimal role at active synapses. Collectively, these results suggest a potential mechanism by which PI3-kinase/mTOR and MAPK/ERK pathways regulate translation at specific subcellular compartments in response to synaptic activity.

Although the BCL6 transcriptional repressor is frequently expressed in human follicular lymphomas (FL), its biological role in this disease remains unknown. Herein, we comprehensively identify the set of gene promoters directly targeted by BCL6 in primary human FLs. We noted that BCL6 binds and represses NOTCH2 and NOTCH pathway genes. Moreover, BCL6 and NOTCH2 pathway gene expression is inversely correlated in FL. Notably, BCL6 upregulation is associated with repression of NOTCH2 and its target genes in primary human and murine germinal center (GC) cells. Repression of NOTCH2 is an essential function of BCL6 in FL and GC B cells because inducible expression of Notch2 abrogated GC formation in mice and killed FL cells. Indeed, BCL6-targeting compounds or gene silencing leads to the induction of NOTCH2 activity and compromises survival of FL cells, whereas NOTCH2 depletion or pathway antagonists rescue FL cells from such effects. Moreover, BCL6 inhibitors induced NOTCH2 expression and suppressed growth of human FL xenografts in vivo and primary human FL specimens ex vivo These studies suggest that established FLs are thus dependent on BCL6 through its suppression of NOTCH2Significance: We show that human FLs are dependent on BCL6, and primary human FLs can be killed using specific BCL6 inhibitors. Integrative genomics and functional studies of BCL6 in primary FL cells point toward a novel mechanism whereby BCL6 repression of NOTCH2 drives the survival and growth of FL cells as well as GC B cells, which are the FL cell of origin. Cancer Discov; 7(5); 506-21. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 443.

We recently reported that stressed adipocytes release extracellular vesicles (EVs) that act as "find-me" signals to promote macrophage migration and activation. In this study, we performed a comprehensive characterization of stressed adipocyte-derived EVs, assessing their antigenic composition, lipidomics, and RNA profiles. Perilipin A was identified as one of the adipose-specific proteins and studied as a potential novel biomarker to detect adipocyte-derived EVs in circulation. Circulating EVs were significantly increased in mice with diet-induced obesity (DIO) and in obese humans with metabolic syndrome compared to lean controls. This increase was associated with decreased glucose tolerance in the DIO mice and metabolic dysfunction, elevated insulin, and homeostatic model assessment of insulin resistance (HOMA-IR) in the obese humans. EVs from both DIO mice and obese humans were enriched in perilipin A, a central gatekeeper of the adipocyte lipid storehouse and a marker of adipocyte differentiation. In obese humans, circulating levels of EVs enriched in perilipin A were dynamic, decreasing 35 % (p

Small-molecule inhibitors targeting growth factor receptors have failed to show efficacy for brain cancers, potentially due to their inability to achieve sufficient drug levels in the CNS. Targeting non-oncogene tumor co-dependencies provides an alternative approach, particularly if drugs with high brain penetration can be identified. Here we demonstrate that the highly lethal brain cancer glioblastoma (GBM) is remarkably dependent on cholesterol for survival, rendering these tumors sensitive to Liver X receptor (LXR) agonist-dependent cell death. We show that LXR-623, a clinically viable, highly brain-penetrant LXRα-partial/LXRβ-full agonist selectively kills GBM cells in an LXRβ- and cholesterol-dependent fashion, causing tumor regression and prolonged survival in mouse models. Thus, a metabolic co-dependency provides a pharmacological means to kill growth factor-activated cancers in the CNS.

Diffuse large B cell lymphomas (DLBCLs) arise from proliferating B cells transiting different stages of the germinal center reaction. In activated B cell DLBCLs (ABC-DLBCLs), a class of DLBCLs that respond poorly to current therapies, chromosomal translocations and amplification lead to constitutive expression of the B cell lymphoma 6 (BCL6) oncogene. The role of BCL6 in maintaining these lymphomas has not been investigated. Here, we designed small-molecule inhibitors that display higher affinity for BCL6 than its endogenous corepressor ligands to evaluate their therapeutic efficacy for targeting ABC-DLBCL. We used an in silico drug design functional-group mapping approach called SILCS to create a specific BCL6 inhibitor called FX1 that has 10-fold greater potency than endogenous corepressors and binds an essential region of the BCL6 lateral groove. FX1 disrupted formation of the BCL6 repression complex, reactivated BCL6 target genes, and mimicked the phenotype of mice engineered to express BCL6 with corepressor binding site mutations. Low doses of FX1 induced regression of established tumors in mice bearing DLBCL xenografts. Furthermore, FX1 suppressed ABC-DLBCL cells in vitro and in vivo, as well as primary human ABC-DLBCL specimens ex vivo. These findings indicate that ABC-DLBCL is a BCL6-dependent disease that can be targeted by rationally designed inhibitors that exceed the binding affinity of natural BCL6 ligands.

Corticospinal tract (CST) axons from one hemisphere normally extend and terminate predominantly in the contralateral spinal cord. We previously showed that deleting the gene phosphatase and tensin homolog (PTEN) in the sensorimotor cortex enables CST axons to regenerate after spinal cord injury and that some regenerating axons extend along the "wrong" side. Here, we characterize the degree of specificity of regrowth in terms of laterality. PTEN was selectively deleted via cortical adeno-associated virus (AAV)-Cre injections in neonatal PTEN-floxed mice. As adults, mice received dorsal hemisection injuries at T12 or complete crush injuries at T9. CST axons from one hemisphere were traced by unilateral biotinylated dextran amine (BDA) injections in PTEN-deleted mice with spinal cord injury and in noninjured PTEN-floxed mice that had not received AAV-Cre. In noninjured mice, 97.9 ± 0.7% of BDA-labeled axons in white matter and 88.5 ± 1.0% of BDA-labeled axons in gray matter were contralateral to the cortex of origin. In contrast, laterality of CST axons that extended past a lesion due to PTEN deletion varied across animals. In some cases, regenerated axons extended predominantly on the ipsilateral side; in other cases, axons extended predominantly contralaterally, and in others, axons were similar in numbers on both sides. Similar results were seen in analyses of cases from previous studies using short hairpin (sh)RNA-mediated PTEN knock-down. These results indicate that CST axons that extend past a lesion due to PTEN deletion or knock-down do not maintain the contralateral rule of the noninjured CST, highlighting one aspect of how the resultant circuitry from regenerating axons may differ from that of the uninjured CST. J. Comp. Neurol. 524:2654-2676, 2016. © 2016 Wiley Periodicals, Inc.

Limb girdle muscular dystrophy 2A is due to loss-of-function mutations in the Calpain 3 (CAPN3) gene. Our previous data suggest that CAPN3 helps to maintain the integrity of the triad complex in skeletal muscle. In Capn3 knock-out mice (C3KO), Ca2+ release and Ca2+/calmodulin kinase II (CaMKII) signaling are attenuated. We hypothesized that calpainopathy may result from a failure to transmit loading-induced Ca2+-mediated signals, necessary to up-regulate expression of muscle adaptation genes. To test this hypothesis, we compared transcriptomes of muscles from wild type (WT) and C3KO mice subjected to endurance exercise. In WT mice, exercise induces a gene signature that includes myofibrillar, mitochondrial and oxidative lipid metabolism genes, necessary for muscle adaptation. C3KO muscles fail to activate the same gene signature. Furthermore, in agreement with the aberrant transcriptional profile, we observe a commensurate functional defect in lipid metabolism whereby C3KO muscles fail to release fatty acids from stored triacylglycerol. In conjunction with the defects in oxidative metabolism, C3KO mice demonstrate reduced exercise endurance. Failure to up-regulate genes in C3KO muscles is due, in part, to decreased levels of PGC1α, a transcriptional co-regulator that orchestrates the muscle adaptation response. Destabilization of PGC1α is attributable to decreased p38 MAPK activation via diminished CaMKII signaling. Thus, we elucidate a pathway downstream of Ca2+-mediated CaMKII activation that is dysfunctional in C3KO mice, leading to reduced transcription of genes involved in muscle adaptation. These studies identify a novel mechanism of muscular dystrophy: a blunted transcriptional response to muscle loading resulting in chronic failure to adapt and remodel.

Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6) locally near active synapses. Using antibodies specific for phosphorylation at different sites (ser235/236 versus ser240/244), we show that strong synaptic activation led to dramatic increases in immunostaining throughout postsynaptic neurons with selectively higher staining for p-ser235/236 in the activated dendritic lamina. Following LTP induction, phosphorylation at ser235/236 was detectable by 5 min, peaked at 30 min, and was maintained for hours. Phosphorylation at both sites was completely blocked by local infusion of the NMDA receptor antagonist, APV. Despite robust induction of p-rpS6 following high frequency stimulation, assessment of protein synthesis by autoradiography revealed no detectable increases. Exploration of a novel environment led to increases in the number of p-rpS6-positive neurons throughout the forebrain in a pattern reminiscent of immediate early gene induction and many individual neurons that were p-rpS6-positive coexpressed Arc protein. Our results constrain hypotheses about the possible role of rpS6 phosphorylation in regulating postsynaptic protein synthesis during induction of synaptic plasticity.

Peripheral nerve injury induces increased expression of thrombospondin-4 (TSP4) in spinal cord and dorsal root ganglia that contributes to neuropathic pain states through unknown mechanisms. Here, we test the hypothesis that TSP4 activates its receptor, the voltage-gated calcium channel Cavα2δ1 subunit (Cavα2δ1), on sensory afferent terminals in dorsal spinal cord to promote excitatory synaptogenesis and central sensitization that contribute to neuropathic pain states. We show that there is a direct molecular interaction between TSP4 and Cavα2δ1 in the spinal cord in vivo and that TSP4/Cavα2δ1-dependent processes lead to increased behavioral sensitivities to stimuli. In dorsal spinal cord, TSP4/Cavα2δ1-dependent processes lead to increased frequency of miniature and amplitude of evoked excitatory post-synaptic currents in second-order neurons as well as increased VGlut2- and PSD95-positive puncta, indicative of increased excitatory synapses. Blockade of TSP4/Cavα2δ1-dependent processes with Cavα2δ1 ligand gabapentin or genetic Cavα2δ1 knockdown blocks TSP4 induced nociception and its pathological correlates. Conversely, TSP4 antibodies or genetic ablation blocks nociception and changes in synaptic transmission in mice overexpressing Cavα2δ1 Importantly, TSP4/Cavα2δ1-dependent processes also lead to similar behavioral and pathological changes in a neuropathic pain model of peripheral nerve injury. Thus, a TSP4/Cavα2δ1-dependent pathway activated by TSP4 or peripheral nerve injury promotes exaggerated presynaptic excitatory input and evoked sensory neuron hyperexcitability and excitatory synaptogenesis, which together lead to central sensitization and pain state development.

Targeted deletion of the phosphatase and tensin homolog on chromosome ten (PTEN) gene in the sensorimotor cortex of neonatal mice enables robust regeneration of corticospinal tract (CST) axons following spinal cord injury as adults. Here, we assess the consequences of long-term conditional genetic PTEN deletion on cortical structure and neuronal morphology and screen for neuropathology. Mice with a LoxP-flanked exon 5 of the PTEN gene (PTENf/f mice) received AAV-Cre injections into the sensorimotor cortex at postnatal day 1 (P1) and were allowed to survive for up to 18months. As adults, mice were assessed for exploratory activity (open field), and motor coordination using the Rotarod®. Some mice received injections of Fluorogold into the spinal cord to retrogradely label the cells of origin of the CST. Brains were prepared for neurohistology and immunostained for PTEN and phospho-S6, which is a downstream marker of mammalian target of rapamycin (mTOR) activation. Immunostaining revealed a focal area of PTEN deletion affecting neurons in all cortical layers, although in some cases PTEN expression was maintained in many small-medium sized neurons in layers III-IV. Neurons lacking PTEN were robustly stained for pS6. Cortical thickness was significantly increased and cortical lamination was disrupted in the area of PTEN deletion. PTEN-negative layer V neurons that give rise to the CST, identified by retrograde labeling, were larger than neurons with maintained PTEN expression, and the relative area occupied by neuropil vs. cell bodies was increased. There was no evidence of tumor formation or other neuropathology. Mice with PTEN deletion exhibited open field activity comparable to controls and there was a trend for impaired Rotarod performance (not statistically significant). Our findings indicate that early postnatal genetic deletion of PTEN that is sufficient to enable axon regeneration by adult neurons causes neuronal hypertrophy but no other detectable neuropathology.

Rodent models of spinal cord injury are critical for the development of treatments for upper limb motor impairment in humans, but there are few methods for measuring forelimb strength of rodents, an important outcome measure. We developed a novel robotic device--the Robotic Rehabilitator of the Rodent Upper Extremity (RUE)--that requires rats to voluntarily reach for and pull a bar to retrieve a food reward; the resistance of the bar can be programmed. We used RUE to train forelimb strength of 16 rats three times per week for 23 weeks before and 38 weeks after a mild (100 kdyne) unilateral contusion at the cervical level 5 (C5). We measured maximum force produced when RUE movement was unexpectedly blocked. We compared this blocked pulling force (BPF) to weekly measures of forelimb strength obtained with a previous, well-established method: the grip strength meter (GSM). Before injury, BPF was 2.6 times higher (BPF, 444.6 ± 19.1 g; GSM, 168.4 ± 3.1 g) and 4.9 times more variable (p < 0.001) than pulling force measured with the GSM; the two measurement methods were uncorrelated (R(2) = 0.03; p = 0.84). After injury, there was a significant decrease in BPF of 134.35 g ± 14.71 g (p < 0.001). Together, our findings document BPF as a repeatable measure of forelimb force production, sensitive to a mild spinal cord injury, which comes closer to measuring maximum force than the GSM and thus may provide a useful measure for quantifying the effects of treatment in rodent models of SCI.

Myeloid-derived cells such as monocytes, dendritic cells (DCs), and macrophages are at the heart of the immune effector function in an inflammatory response. But because of the lack of an efficient imaging system to trace these cells live during their migration and maturation in their native environment at sub-cellular resolution, our knowledge is limited to data available from specific time-points analyzed by flow cytometry, histology, genomics and other immunological methods. Here, we have developed a ratiometric imaging method for measuring monocyte maturation in inflamed mouse lungs in situ using real-time using 2-photon imaging and complementary methods. We visualized that while undifferentiated monocytes were predominantly found only in the vasculature, a semi-differentiated monocyte/macrophage population could enter the tissue and resembled more mature and differentiated populations by morphology and surface phenotype. As these cells entered and differentiated, they were already selectively localized near inflamed airways and their entry was associated with changes in motility and morphology. We were able to visualize these during the act of differentiation, a process that can be demonstrated in this way to be faster on a per-cell basis under inflammatory conditions. Finally, our in situ analyses demonstrated increases, in the differentiating cells, for both antigen uptake and the ability to mediate interactions with T cells. This work, while largely confirming proposed models for in situ differentiation, provides important in situ data on the coordinated site-specific recruitment and differentiation of these cells and helps elaborate the predominance of immune pathology at the airways. Our novel imaging technology to trace immunogenic cell maturation in situ will complement existing information available on in situ differentiation deduced from other immunological methods, and assist better understanding of the spatio-temporal cellular behavior during an inflammatory response.

Studies of axon regeneration in the spinal cord often assess regeneration of the corticospinal tract (CST). Emx1-Cre x Thy1-STOP-YFP mice have been reported to have yellow fluorescent protein (YFP) selectively expressed in forebrain neurons leading to genetic labeling of CST axons in the spinal cord, and it was suggested that these CST-YFP mice would be useful for studies of CST regeneration. Because regeneration past a lesion may involve only a few axons, the presence of labeled non-CST axons compromises interpretation. We show here that in CST-YFP mice, some YFP-labeled axons are not from the CST. Specifically, YFP-labeled axons are present in regions beyond those with anterogradely labeled CST axons, most YFP-labeled axons beyond established CST locations do not undergo Wallerian degeneration following a large lesion of the sensorimotor cortex, some rubrospinal and reticulospinal neurons are labeled with YFP, and some YFP-labeled cells in the spinal gray matter have YFP-labeled projections into the spinal cord white matter. We further demonstrate that the density of YFP-labeled axon arbors hinders tracing of single axons to their point of origin in the main descending tracts. In light of recent advances in 3D imaging for visualizing axons in unsectioned blocks of spinal cord, we also assessed CST-YFP mice for 3D imaging and found that YFP fluorescence in CST-YFP mice is faint for clearing-based 3D imaging in comparison with fluorescence in Thy1-YFP-H mice and fluorescence of mini-ruby biotinylated dextran amine (BDA). Overall, the nonspecific and faint YFP labeling in CST-YFP mice limits their utility for assessments of CST axon regeneration.

Because age is the greatest risk factor for sporadic Alzheimer's disease (AD), phenotypic screens based upon old age-associated brain toxicities were used to develop the potent neurotrophic drug J147. Since certain aspects of aging may be primary cause of AD, we hypothesized that J147 would be effective against AD-associated pathology in rapidly aging SAMP8 mice and could be used to identify some of the molecular contributions of aging to AD. An inclusive and integrative multiomics approach was used to investigate protein and gene expression, metabolite levels, and cognition in old and young SAMP8 mice. J147 reduced cognitive deficits in old SAMP8 mice, while restoring multiple molecular markers associated with human AD, vascular pathology, impaired synaptic function, and inflammation to those approaching the young phenotype. The extensive assays used in this study identified a subset of molecular changes associated with aging that may be necessary for the development of AD.

The oral drug FTY720 affects sphingosine-1-phosphate (S1P) signaling on targeted cells that bear the S1P receptors S1P1, S1P3, S1P4, and S1P5. We examined the effect of FTY720 treatment on the biology of mouse neural progenitor cells (NPCs) after transplantation in a viral model of demyelination. Intracerebral infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in an acute encephalomyelitis, followed by demyelination similar in pathology to the human demyelinating disease, multiple sclerosis. We have previously reported that intraspinal transplantation of mouse NPCs into JHMV-infected animals resulted in selective colonization of demyelinated lesions, preferential differentiation into oligodendroglia accompanied by axonal preservation, and increased remyelination. Cultured NPCs expressed transcripts for S1P receptors S1P1, S1P2, S1P3, S1P4, and S1P5. FTY720 treatment of cultured NPCs resulted in increased mitogen-activated protein kinase phosphorylation and migration after exposure to the chemokine CXCL12. Administration of FTY720 to JHMV-infected mice resulted in enhanced migration and increased proliferation of transplanted NPCs after spinal cord engraftment. FTY720 treatment did not improve clinical disease, diminish neuroinflammation or the severity of demyelination, nor increase remyelination. These findings argue that FTY720 treatment selectively increases NPC proliferation and migration but does not either improve clinical outcome or enhance remyelination after transplantation into animals in which immune-mediated demyelination is initiated by the viral infection of the central nervous system.

Spinal cord injury (SCI) disrupts the long axonal tracts of the spinal cord leading to devastating loss of function. Cell transplantation in the injured spinal cord has the potential to lead to recovery after SCI via a variety of mechanisms. One such strategy is the formation of neuronal relays between injured long tract axons and denervated neurons. The idea of creating a neuronal relay was first proposed over 25 years ago when fetal tissue was first successfully transplanted into the injured rodent spinal cord. Advances in labeling of grafted cells and the development of neural stem cell culturing techniques have improved the ability to create and refine such relays. Several recent studies have examined the ability to create a novel neuronal circuit between injured axons and denervated targets. This approach is an alternative to long-distance regeneration of damaged axons that may provide a meaningful degree of recovery without direct recreation of lost pathways. This brief review will examine the contribution of fetal grafting to current advances in neuronal grafting. Of particular interest will be the ability of transplanted neurons derived from fetal grafts, neural precursor cells and neural stem cells to reconnect long distance motor and sensory pathways of the injured spinal cord. This article is part of a Special Issue entitled SI: Spinal cord injury.

Recent preclinical advances highlight the therapeutic potential of treatments aimed at boosting regeneration and plasticity of spinal circuitry damaged by spinal cord injury (SCI). With several promising candidates being considered for translation into clinical trials, the SCI community has called for a non-human primate model as a crucial validation step to test efficacy and validity of these therapies prior to human testing. The present paper reviews the previous and ongoing efforts of the California Spinal Cord Consortium (CSCC), a multidisciplinary team of experts from 5 University of California medical and research centers, to develop this crucial translational SCI model. We focus on the growing volumes of high resolution data collected by the CSCC, and our efforts to develop a biomedical informatics framework aimed at leveraging multidimensional data to monitor plasticity and repair targeting recovery of hand and arm function. Although the main focus of many researchers is the restoration of voluntary motor control, we also describe our ongoing efforts to add assessments of sensory function, including pain, vital signs during surgery, and recovery of bladder and bowel function. By pooling our multidimensional data resources and building a unified database infrastructure for this clinically relevant translational model of SCI, we are now in a unique position to test promising therapeutic strategies' efficacy on the entire syndrome of SCI. We review analyses highlighting the intersection between motor, sensory, autonomic and pathological contributions to the overall restoration of function. This article is part of a Special Issue entitled SI: Spinal cord injury.

Previous studies indicate that conditional genetic deletion of phosphatase and tensin homolog (PTEN) in neonatal mice enhances the ability of axons to regenerate following spinal cord injury (SCI) in adults. Here, we assessed whether deleting PTEN in adult neurons post-SCI is also effective, and whether enhanced regenerative growth is accompanied by enhanced recovery of voluntary motor function. PTEN(loxP/loxP) mice received moderate contusion injuries at cervical level 5 (C5). One group received unilateral injections of adeno-associated virus expressing CRE (AAV-CRE) into the sensorimotor cortex; controls received a vector expressing green fluorescent protein (AAV-GFP) or injuries only (no vector injections). Forelimb function was tested for 14weeks post-SCI using a grip strength meter (GSM) and a hanging task. The corticospinal tract (CST) was traced by injecting mini-ruby BDA into the sensorimotor cortex. Forelimb gripping ability was severely impaired immediately post-SCI but recovered slowly over time. The extent of recovery was significantly greater in PTEN-deleted mice in comparison to either the AAV-GFP group or the injury only group. BDA tract tracing revealed significantly higher numbers of BDA-labeled axons in caudal segments in the PTEN-deleted group compared to control groups. In addition, in the PTEN-deleted group, there were exuberant collaterals extending from the main tract rostral to the lesion and into and around the scar tissue at the injury site. These results indicate that PTEN deletion in adult mice shortly post-SCI can enhance regenerative growth of CST axons and forelimb motor function recovery.

The development of intestinal permeability and the penetration of microbial products are key factors associated with the onset of metabolic disease. However, the mechanisms underlying this remain unclear. Here we show that, unlike liver or adipose tissue, high fat diet (HFD)/obesity in mice does not cause monocyte/macrophage infiltration into the intestine or pro-inflammatory changes in gene expression. Rather HFD causes depletion of intestinal eosinophils associated with the onset of intestinal permeability. Intestinal eosinophil numbers were restored by returning HFD fed mice to normal chow and were unchanged in leptin-deficient (Ob/Ob) mice, indicating that eosinophil depletion is caused specifically by a high fat diet and not obesity per se. Analysis of different aspects of intestinal permeability in HFD fed and Ob/Ob mice shows an association between eosinophil depletion and ileal paracelullar permeability, as well as leakage of albumin into the feces, but not overall permeability to FITC dextran. These findings provide the first evidence that a high fat diet causes intestinal eosinophil depletion, rather than inflammation, which may contribute to defective barrier integrity and the onset of metabolic disease.

Efforts to understand spinal cord injury (SCI) and other complex neurotrauma disorders at the pre-clinical level have shown progress in recent years. However, successful translation of basic research into clinical practice has been slow, partly because of the large, heterogeneous data sets involved. In this sense, translational neurological research represents a "big data" problem. In an effort to expedite translation of pre-clinical knowledge into standards of patient care for SCI, we describe the development of a novel database for translational neurotrauma research known as Visualized Syndromic Information and Outcomes for Neurotrauma-SCI (VISION-SCI). We present demographics, descriptive statistics, and translational syndromic outcomes derived from our ongoing efforts to build a multi-center, multi-species pre-clinical database for SCI models. We leveraged archived surgical records, postoperative care logs, behavioral outcome measures, and histopathology from approximately 3000 mice, rats, and monkeys from pre-clinical SCI studies published between 1993 and 2013. The majority of animals in the database have measures collected for health monitoring, such as weight loss/gain, heart rate, blood pressure, postoperative monitoring of bladder function and drug/fluid administration, behavioral outcome measures of locomotion, and tissue sparing postmortem. Attempts to align these variables with currently accepted common data elements highlighted the need for more translational outcomes to be identified as clinical endpoints for therapeutic testing. Last, we use syndromic analysis to identify conserved biological mechanisms of recovery after cervical SCI between rats and monkeys that will allow for more-efficient testing of therapeutics that will need to be translated toward future clinical trials.

We reported previously the formation of ectopic colonies in widespread areas of the nervous system after transplantation of fetal neural stem cells (NSCs) into spinal cord transection sites. Here, we characterize the incidence, distribution, and cellular composition of the colonies. NSCs harvested from E14 spinal cords from rats that express GFP were treated with a growth factor cocktail and grafted into the site of a complete spinal cord transection. Two months after transplant, spinal cord and brain tissue were analyzed histologically. Ectopic colonies were found at long distances from the transplant in the central canal of the spinal cord, the surface of the brainstem and spinal cord, and in the fourth ventricle. Colonies were present in 50% of the rats, and most rats had multiple colonies. Axons extended from the colonies into the host CNS. Colonies were strongly positive for nestin, a marker for neural precursors, and contained NeuN-positive cells with processes resembling dendrites, GFAP-positive astrocytes, APC/CC1-positive oligodendrocytes, and Ki-67-positive cells, indicating ongoing proliferation. Stereological analyses revealed an estimated 21,818 cells in a colony in the fourth ventricle, of which 1005 (5%) were Ki-67 positive. Immunostaining for synaptic markers (synaptophysin and VGluT-1) revealed large numbers of synaptophysin-positive puncta within the colonies but fewer VGluT-1 puncta. Continuing expansion of NSC-derived cell masses in confined spaces in the spinal cord and brain could produce symptoms attributable to compression of nearby tissue. It remains to be determined whether other cell types with self-renewing potential can also form colonies.

It is well known that the ω-3 fatty acids (ω-3-FAs; also known as n-3 fatty acids) can exert potent anti-inflammatory effects. Commonly consumed as fish products, dietary supplements and pharmaceuticals, ω-3-FAs have a number of health benefits ascribed to them, including reduced plasma triglyceride levels, amelioration of atherosclerosis and increased insulin sensitivity. We reported that Gpr120 is the functional receptor for these fatty acids and that ω-3-FAs produce robust anti-inflammatory, insulin-sensitizing effects, both in vivo and in vitro, in a Gpr120-dependent manner. Indeed, genetic variants that predispose to obesity and diabetes have been described in the gene encoding GPR120 in humans (FFAR4). However, the amount of fish oils that would have to be consumed to sustain chronic agonism of Gpr120 is too high to be practical, and, thus, a high-affinity small-molecule Gpr120 agonist would be of potential clinical benefit. Accordingly, Gpr120 is a widely studied drug discovery target within the pharmaceutical industry. Gpr40 is another lipid-sensing G protein-coupled receptor, and it has been difficult to identify compounds with a high degree of selectivity for Gpr120 over Gpr40 (ref. 11). Here we report that a selective high-affinity, orally available, small-molecule Gpr120 agonist (cpdA) exerts potent anti-inflammatory effects on macrophages in vitro and in obese mice in vivo. Gpr120 agonist treatment of high-fat diet-fed obese mice causes improved glucose tolerance, decreased hyperinsulinemia, increased insulin sensitivity and decreased hepatic steatosis. This suggests that Gpr120 agonists could become new insulin-sensitizing drugs for the treatment of type 2 diabetes and other human insulin-resistant states in the future.

Conditional genetic deletion of phosphatase and tensin homolog (PTEN) in the sensorimotor cortex of neonatal mice enables regeneration of corticospinal tract (CST) axons after spinal cord injury (SCI). The present study addresses three questions: (1) whether PTEN knockdown in adult rats by nongenetic techniques enables CST regeneration, (2) whether interventions to enable CST regeneration enhance recovery of voluntary motor function, and (3) whether delivery of salmon fibrin into the injury site further enhances CST regeneration and motor recovery. Adult rats were trained in a staircase-reaching task and then received either intracortical injections of AAVshPTEN to delete PTEN or a control vector expressing shRNA for luciferase (AAVshLuc). Rats then received cervical dorsal hemisection injuries and salmon fibrin was injected into the injury site in half the rats, yielding four groups (AAVshPTEN, AAVshLuc, AAVshPTEN + fibrin, and AAVshLuc + fibrin). Forepaw function was assessed for 10 weeks after injury and CST axons were traced by injecting biotin-conjugated dextran amine into the sensorimotor cortex. Rats that received AAVshPTEN alone did not exhibit improved motor function, whereas rats that received AAVshPTEN and salmon fibrin had significantly higher forelimb-reaching scores. Tract tracing revealed that CST axons extended farther caudally in the group that received AAVshPTEN and salmon fibrin versus other groups. There were no significant differences in lesion size between the groups. Together, these data suggest that the combination of PTEN deletion and salmon fibrin injection into the lesion can significantly improve voluntary motor function after SCI by enabling regenerative growth of CST axons.

As part of the NIH "Facilities of Research Excellence-Spinal Cord Injury" project to support independent replication, we repeated key parts of a study reporting robust engraftment of neural stem cells (NSCs) treated with growth factors after complete spinal cord transection in rats. Rats (n=20) received complete transections at thoracic level 3 (T3) and 2weeks later received NSC transplants in a fibrin matrix with a growth factor cocktail using 2 different transplantation methods (with and without removal of scar tissue). Control rats (n=9) received transections only. Hindlimb locomotor function was assessed with the BBB scale. Nine weeks post injury, reticulospinal tract axons were traced in 6 rats by injecting BDA into the reticular formation. Transplants grew to fill the lesion cavity in most rats although grafts made with scar tissue removal had large central cavities. Grafts blended extensively with host tissue obliterating the astroglial boundary at the cut ends, but in most cases there was a well-defined partition within the graft that separated rostral and caudal parts of the graft. In some cases, the partition contained non-neuronal scar tissue. There was extensive outgrowth of GFP labeled axons from the graft, but there was minimal ingrowth of host axons into the graft revealed by tract tracing and immunocytochemistry for 5HT. There were no statistically significant differences between transplant and control groups in the degree of locomotor recovery. Our results confirm the previous report that NSC transplants can fill lesion cavities and robustly extend axons, but reveal that most grafts do not create a continuous bridge of neural tissue between rostral and caudal segments.