APOE4 impairs the microglial response in Alzheimer's disease by inducing TGFß-mediated checkpoints.

The APOE4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). The contribution of microglial APOE4 to AD pathogenesis is unknown, although APOE has the most enriched gene expression in neurodegenerative microglia (MGnD). Here, we show in mice and humans a negative role of microglial APOE4 in the induction of the MGnD response to neurodegeneration. Deletion of microglial APOE4 restores the MGnD phenotype associated with neuroprotection in P301S tau transgenic mice and decreases pathology in APP/PS1 mice. MGnD-astrocyte cross-talk associated with ß-amyloid (Aß) plaque encapsulation and clearance are mediated via LGALS3 signaling following microglial APOE4 deletion. In the brains of AD donors carrying the APOE4 allele, we found a sex-dependent reciprocal induction of AD risk factors associated with suppression of MGnD genes in females, including LGALS3, compared to individuals homozygous for the APOE3 allele. Mechanistically, APOE4-mediated induction of ITGB8-transforming growth factor-ß (TGFß) signaling impairs the MGnD response via upregulation of microglial homeostatic checkpoints, including Inpp5d, in mice. Deletion of Inpp5d in microglia restores MGnD-astrocyte cross-talk and facilitates plaque clearance in APP/PS1 mice. We identify the microglial APOE4-ITGB8-TGFß pathway as a negative regulator of microglial response to AD pathology, and restoring the MGnD phenotype via blocking ITGB8-TGFß signaling provides a promising therapeutic intervention for AD.

Apolipoprotein E4 impairs the response of neurodegenerative retinal microglia and prevents neuronal loss in glaucoma.

The apolipoprotein E4 (APOE4) allele is associated with an increased risk of Alzheimer disease and a decreased risk of glaucoma, but the underlying mechanisms remain poorly understood. Here, we found that in two mouse glaucoma models, microglia transitioned to a neurodegenerative phenotype characterized by upregulation of Apoe and Lgals3 (Galectin-3), which were also upregulated in human glaucomatous retinas. Mice with targeted deletion of Apoe in microglia or carrying the human APOE4 allele were protected from retinal ganglion cell (RGC) loss, despite elevated intraocular pressure (IOP). Similarly to Apoe-/- retinal microglia, APOE4-expressing microglia did not upregulate neurodegeneration-associated genes, including Lgals3, following IOP elevation. Genetic and pharmacologic targeting of Galectin-3 ameliorated RGC degeneration, and Galectin-3 expression was attenuated in human APOE4 glaucoma samples. These results demonstrate that impaired activation of APOE4 microglia is protective in glaucoma and that the APOE-Galectin-3 signaling can be targeted to treat this blinding disease.

Nasal administration of anti-CD3 mAb (Foralumab) downregulates NKG7 and increases TGFB1 and GIMAP7 expression in T cells in subjects with COVID-19.

T cells are present in early stages of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and play a major role in disease outcome and long-lasting immunity. Nasal administration of a fully human anti-CD3 monoclonal antibody (Foralumab) reduced lung inflammation as well as serum IL-6 and C-reactive protein in moderate cases of COVID-19. Using serum proteomics and RNA-sequencing, we investigated the immune changes in patients treated with nasal Foralumab. In a randomized trial, mild to moderate COVID-19 outpatients received nasal Foralumab (100 µg/d) given for 10 consecutive days and were compared to patients that did not receive Foralumab. We found that naïve-like T cells were increased in Foralumab-treated subjects and NGK7+ effector T cells were reduced. CCL5, IL32, CST7, GZMH, GZMB, GZMA, PRF1, and CCL4 gene expression were downregulated in T cells and CASP1 was downregulated in T cells, monocytes, and B cells in subjects treated with Foralumab. In addition to the downregulation of effector features, an increase in TGFB1 gene expression in cell types with known effector function was observed in Foralumab-treated subjects. We also found increased expression of GTP-binding gene GIMAP7 in subjects treated with Foralumab. Rho/ROCK1, a downstream pathway of GTPases signaling was downregulated in Foralumab-treated individuals. TGFB1, GIMAP7, and NKG7 transcriptomic changes observed in Foralumab-treated COVID-19 subjects were also observed in healthy volunteers, MS subjects, and mice treated with nasal anti-CD3. Our findings demonstrate that nasal Foralumab modulates the inflammatory response in COVID-19 and provides a novel avenue to treat the disease.

Somatic loss of ATM is a late event in pancreatic tumorigenesis.

Understanding the timing and spectrum of genetic alterations that contribute to the development of pancreatic cancer is essential for effective interventions and treatments. The aim of this study was to characterize somatic ATM alterations in noninvasive pancreatic precursor lesions and invasive pancreatic adenocarcinomas from patients with and without pathogenic germline ATM variants. DNA was isolated and sequenced from the invasive pancreatic ductal adenocarcinomas and precursor lesions of patients with a pathogenic germline ATM variant. Tumor and precursor lesions from these patients as well as colloid carcinoma from patients without a germline ATM variant were immunolabeled to assess ATM expression. Among patients with a pathogenic germline ATM variant, somatic ATM alterations, either mutations and/or loss of protein expression, were identified in 75.0% of invasive pancreatic adenocarcinomas but only 7.1% of pancreatic precursor lesions. Loss of ATM expression was also detected in 31.0% of colloid carcinomas from patients unselected for germline ATM status, significantly higher than in pancreatic precursor lesions [pancreatic intraepithelial neoplasms (p = 0.0013); intraductal papillary mucinous neoplasms, p = 0.0040] and pancreatic ductal adenocarcinoma (p = 0.0076) unselected for germline ATM status. These data are consistent with the second hit to ATM being a late event in pancreatic tumorigenesis. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

DEPhT: a novel approach for efficient prophage discovery and precise extraction.

Advances in genome sequencing have produced hundreds of thousands of bacterial genome sequences, many of which have integrated prophages derived from temperate bacteriophages. These prophages play key roles by influencing bacterial metabolism, pathogenicity, antibiotic resistance, and defense against viral attack. However, they vary considerably even among related bacterial strains, and they are challenging to identify computationally and to extract precisely for comparative genomic analyses. Here, we describe DEPhT, a multimodal tool for prophage discovery and extraction. It has three run modes that facilitate rapid screening of large numbers of bacterial genomes, precise extraction of prophage sequences, and prophage annotation. DEPhT uses genomic architectural features that discriminate between phage and bacterial sequences for efficient prophage discovery, and targeted homology searches for precise prophage extraction. DEPhT is designed for prophage discovery in Mycobacterium genomes but can be adapted broadly to other bacteria. We deploy DEPhT to demonstrate that prophages are prevalent in Mycobacterium strains but are absent not only from the few well-characterized Mycobacterium tuberculosis strains, but also are absent from all ∼30 000 sequenced M. tuberculosis strains.

Sex-specific microglia state in the Neuroligin-4 knock-out mouse model of autism spectrum disorder.

Neuroligin-4 (NLGN4) loss-of-function mutations are associated with monogenic heritable autism spectrum disorder (ASD) and cause alterations in both synaptic and behavioral phenotypes. Microglia, the resident CNS macrophages, are implicated in ASD development and progression. Here we studied the impact of NLGN4 loss in a mouse model, focusing on microglia phenotype and function in both male and female mice. NLGN4 depletion caused lower microglia density, less ramified morphology, reduced response to injury and purinergic signaling specifically in the hippocampal CA3 region predominantly in male mice. Proteomic analysis revealed disrupted energy metabolism in male microglia and provided further evidence for sexual dimorphism in the ASD associated microglial phenotype. In addition, we observed impaired gamma oscillations in a sex-dependent manner. Lastly, estradiol application in male NLGN4-/- mice restored the altered microglial phenotype and function. Together, these results indicate that loss of NLGN4 affects not only neuronal network activity, but also changes the microglia state in a sex-dependent manner that could be targeted by estradiol treatment.

Sex-specific transcriptome of spinal microglia in neuropathic pain due to peripheral nerve injury.

Neuropathic pain is a prevalent and debilitating chronic disease that is characterized by activation in glial cells in various pain-related regions within the central nervous system. Recent studies have suggested a sexually dimorphic role of microglia in the maintenance of neuropathic pain in rodents. Here, we utilized RNA sequencing analysis and in vitro primary cultures of microglia to identify whether there is a common neuropathic microglial signature and characterize the sex differences in microglia in pain-related regions in nerve injury and chemotherapy-induced peripheral neuropathy mouse models. While mechanical allodynia and behavioral changes were observed in all models, transcriptomic analysis of microglia revealed no common transcriptional changes in spinal and supraspinal regions and in the different neuropathic models. However, there was a substantial change in microglial gene expression within the ipsilateral lumbar spinal cord 7 days after chronic constriction injury (CCI) of the sciatic nerve. Both sexes upregulated genes associated with inflammation, phagosome, and lysosome activation, though males revealed a prominent global transcriptional shift not observed in female mice. Transcriptomic comparison between male spinal microglia after CCI and data from other nerve injury models and neurodegenerative microglia demonstrated a unique CCI-induced signature reflecting acute activation of microglia. Further, in vitro studies revealed that only male microglia from nerve-injured mice developed a reactive phenotype with increased phagocytotic activity. This study demonstrates a lack of a common neuropathic microglial signature and indicates distinct sex differences in spinal microglia, suggesting they contribute to the sex-specific pain processing following nerve injury.

pdm_utils: a SEA-PHAGES MySQL phage database management toolkit.

SUMMARY: Bacteriophages (phages) are incredibly abundant and genetically diverse. The volume of phage genomics data is rapidly increasing, driven in part by the SEA-PHAGES program, which isolates, sequences and manually annotates hundreds of phage genomes each year. With an ever-expanding genomics dataset, there are many opportunities for generating new biological insights through comparative genomic and bioinformatic analyses. As a result, there is a growing need to be able to store, update, explore and analyze phage genomics data. The package pdm_utils provides a collection of tools for MySQL phage database management designed to meet specific needs in the SEA-PHAGES program and phage genomics generally. AVAILABILITY AND IMPLEMENTATION: https://pypi.org/project/pdm-utils/.

Examination of ATM, BRCA1, and BRCA2 promoter methylation in patients with pancreatic cancer.

BACKGROUND: Pancreatic cancer is a lethal disease with a poor 5-year survival rate. Pathogenic germline variants in the coding regions of ATM, BRCA1, and BRCA2 are found in up to 4.8% of pancreatic cancer patients. Germline promoter methylation and gene silencing arising from a germline variant or through other mechanisms have been described as a cause of tumor suppressor gene inactivation. METHODS: We measured the level of promoter methylation of the ATM, BRCA1, and BRCA2 genes in peripheral blood lymphocytes from 655 patients with pancreatic cancer using real-time PCR. RESULTS: No evidence of germline promoter methylation of any of these genes was found. Promoter methylation levels were minimal with no patient having promoter methylation greater than 3.4%, 3.3%, and 7.6% for ATM, BRCA1 and BRCA2, respectively, well below levels found in patients who have inherited promoter methylation (∼50%). CONCLUSIONS: We found no evidence of germline promoter methylation for the pancreatic susceptibility genes ATM, BRCA1 and BRCA2 in patients with pancreatic cancer. This study reveals that constitutive germline methylation of promoter CpG islands is rare in pancreatic cancer.

Molecular characterization of organoids derived from pancreatic intraductal papillary mucinous neoplasms.

Intraductal papillary mucinous neoplasms (IPMNs) are commonly identified non-invasive cyst-forming pancreatic neoplasms with the potential to progress into invasive pancreatic adenocarcinoma. There are few in vitro models with which to study the biology of IPMNs and their progression to invasive carcinoma. Therefore, we generated a living biobank of organoids from seven normal pancreatic ducts and ten IPMNs. We characterized eight IPMN organoid samples using whole genome sequencing and characterized five IPMN organoids and seven normal pancreatic duct organoids using transcriptome sequencing. We identified an average of 11,344 somatic mutations in the genomes of organoids derived from IPMNs, with one sample harboring 61,537 somatic mutations enriched for T→C transitions and T→A transversions. Recurrent coding somatic mutations were identified in 15 genes, including KRAS, GNAS, RNF43, PHF3, and RBM10. The most frequently mutated genes were KRAS, GNAS, and RNF43, with somatic mutations identified in six (75%), four (50%), and three (37.5%) IPMN organoid samples, respectively. On average, we identified 36 structural variants in IPMN derived organoids, and none had an unstable phenotype (> 200 structural variants). Transcriptome sequencing identified 28 genes differentially expressed between normal pancreatic duct organoid and IPMN organoid samples. The most significantly upregulated and downregulated genes were CLDN18 and FOXA1. Immunohistochemical analysis of FOXA1 expression in 112 IPMNs, 113 mucinous cystic neoplasms, and 145 pancreatic ductal adenocarcinomas demonstrated statistically significant loss of expression in low-grade IPMNs (p < 0.0016), mucinous cystic neoplasms (p < 0.0001), and pancreatic ductal adenocarcinoma of any histologic grade (p < 0.0001) compared to normal pancreatic ducts. These data indicate that FOXA1 loss of expression occurs early in pancreatic tumorigenesis. Our study highlights the utility of organoid culture to study the genetics and biology of normal pancreatic duct and IPMNs. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.