Mlc1-Expressing Perivascular Astrocytes Promote Blood-Brain Barrier Integrity.

In the mammalian brain, perivascular astrocytes (PAs) closely juxtapose blood vessels and are postulated to have important roles in the control of vascular physiology, including regulation of the blood-brain barrier (BBB). Deciphering specific functions for PAs in BBB biology, however, has been limited by the ability to distinguish these cells from other astrocyte populations. In order to characterize selective roles for PAs in vivo, a new mouse model has been generated in which the endogenous megalencephalic leukoencephalopathy with subcortical cysts 1 (Mlc1) gene drives expression of Cre fused to a mutated estrogen ligand-binding domain (Mlc1-T2A-CreERT2). This knock-in mouse model, which we term MLCT, allows for selective identification and tracking of PAs in the postnatal brain. We also demonstrate that MLCT-mediated ablation of PAs causes severe defects in BBB integrity, resulting in premature death. PA loss results in aberrant localization of Claudin 5 and -VE-Cadherin in endothelial cell junctions as well as robust microgliosis. Collectively, these data reveal essential functions for Mlc1-expressing PAs in regulating endothelial barrier integrity in mice and indicate that primary defects in astrocytes that cause BBB breakdown may contribute to human neurologic disorders.SIGNIFICANCE STATEMENT Interlaced among the billions of neurons and glia in the mammalian brain is an elaborate network of blood vessels. Signals from the brain parenchyma control the unique permeability properties of cerebral blood vessels known as the blood-brain barrier (BBB). However, we understand very little about the relative contributions of different neural cell types in the regulation of BBB functions. Here, we show that a specific subpopulation of astrocyte is essential for control of BBB integrity, with ablation of these cells leading to defects in endothelial cell junctions, BBB breakdown, and resulting neurologic deficits.

Memory Reactivation during Learning Simultaneously Promotes Dentate Gyrus/CA2,3 Pattern Differentiation and CA1 Memory Integration.

Events that overlap with previous experience may trigger reactivation of existing memories. However, such reactivation may have different representational consequences within the hippocampal circuit. Computational theories of hippocampal function suggest that dentate gyrus and CA2,3 (DG/CA2,3) are biased to differentiate highly similar memories, whereas CA1 may integrate related events by representing them with overlapping neural codes. Here, we tested whether the formation of differentiated or integrated representations in hippocampal subfields depends on the strength of memory reactivation during learning. Human participants of both sexes learned associations (AB pairs, either face-shape or scene-shape), and then underwent fMRI scanning while they encoded overlapping associations (BC shape-object pairs). Both before and after learning, participants were also scanned while viewing indirectly related elements of the overlapping memories (A and C images) in isolation. We used multivariate pattern analyses to measure reactivation of initial pair memories (A items) during overlapping pair (BC) learning, as well as learning-related representational change for indirectly related memory elements in hippocampal subfields. When prior memories were strongly reactivated during overlapping pair encoding, DG/CA2,3 and subiculum representations for indirectly related images (A and C) became less similar, consistent with pattern differentiation. Simultaneously, memory reactivation during new learning promoted integration in CA1, where representations for indirectly related memory elements became more similar after learning. Furthermore, memory reactivation and subiculum representation predicted faster and more accurate inference (AC) decisions. These data show that reactivation of related memories during new learning leads to dissociable coding strategies in hippocampal subfields, in line with computational theories.SIGNIFICANCE STATEMENT The flexibility of episodic memory allows us to remember both the details that differentiate similar events and the commonalities among them. Here, we tested how reactivation of past experience during new learning promotes formation of neural representations that might serve these two memory functions. We found that memory reactivation during learning promoted formation of differentiated representations for overlapping memories in the dentate gyrus/CA2,3 and subiculum subfields of the hippocampus, while simultaneously leading to the formation of integrated representations of related events in subfield CA1 Furthermore, memory reactivation and subiculum representation predicted success when inferring indirect relationships among events. These findings indicate that memory reactivation is an important learning signal that influences how overlapping events are represented within the hippocampal circuit.

Systematic Review of Aptamer Sequence Reporting in the Literature Reveals Widespread Unexplained Sequence Alterations.

Aptamers have been the subject of more than 144 000 papers to date. However, there has been a growing concern that discrepancies in the reporting of aptamer research limit the reliability of these reagents for research and other applications. These observations noting inconsistencies in the use of our RNA antilysozyme aptamer served as an impetus for our systematic review of the reporting of aptamer sequences in the literature. Our detailed examination of the literature citing the RNA antilysozyme aptamer revealed that 93% of the 61 publications reviewed reported unexplained altered sequences with 96% of those using DNA variants. The 10 most cited aptamers were examined using a standardized methodology in order to categorize the extent to which the sequences themselves and altered sequences were adequately described in the literature. Our review of 780 aptamer publications spanned decades, multiple journals, and research groups and revealed that 41% of the papers reported unexplained sequence alterations or omitted sequences. We identified 10 common categories of sequence alterations including deletions, substitutions, and additions, among others. Overall, our findings can be used as a starting point for building better practices in author submissions and publication standards, elevating the rigor and reproducibility of aptamer research.

A pragmatic clinical trial of cascade testing for familial hypercholesterolemia.

PURPOSE: We compared new cases detected per index case in familial hypercholesterolemia (FH) families with or without an identifiable monogenic etiology. METHODS: We enrolled 52 FH probands with a pathogenic variant (FHg+) in LDLR, APOB, or PCSK9 and 73 probands without such a variant (FHg-). After direct contact by the study team, family members (FMs) of FHg+ probands could opt-in for genetic testing and FMs of FHg- probands were asked to provide a lipid profile. New cases were defined as presence of a pathogenic variant in FHg+ families and as low-density lipoprotein cholesterol ≥155 mg/dL in FHg- families. RESULTS: Of 71 FHg+ probands seen by a genetic counselor, 52 consented and identified 253 FMs (111 consented and were tested, yielding 48 new cases). Of 101 FHg- probands who received counseling, 73 consented and identified 295 FMs (63 consented and were tested, yielding 17 new cases). New case detection per index case was significantly greater in FHg+ than in FHg- families (0.92 vs 0.23), a result of higher cascade testing uptake (43.9 vs 21.4%) and yield (43.2 vs 27.0%) in the former. CONCLUSION: New case detection rate was significantly higher in FH families with a monogenic etiology than in those without such an etiology owing to greater uptake and yield of cascade testing.

Targeting IGF2 to reprogram the tumor microenvironment for enhanced viro-immunotherapy.

BACKGROUND: The FDA approval of oncolytic herpes simplex-1 virus (oHSV) therapy underscores its therapeutic promise and safety as a cancer immunotherapy. Despite this promise, the current efficacy of oHSV is significantly limited to a small subset of patients largely due to the resistance in tumor and tumor microenvironment (TME). METHODS: RNA sequencing (RNA-Seq) was used to identify molecular targets of oHSV resistance. Intracranial human and murine glioma or breast cancer brain metastasis (BCBM) tumor-bearing mouse models were employed to elucidate the mechanism underlying oHSV therapy-induced resistance. RESULTS: Transcriptome analysis identified IGF2 as one of the top-secreted proteins following oHSV treatment. Moreover, IGF2 expression was significantly upregulated in 10 out of 14 recurrent GBM patients after treatment with oHSV, rQNestin34.5v.2 (71.4%; P = .0020) (ClinicalTrials.gov, NCT03152318). Depletion of IGF2 substantially enhanced oHSV-mediated tumor cell killing in vitro and improved survival of mice bearing BCBM tumors in vivo. To mitigate the oHSV-induced IGF2 in the TME, we constructed a novel oHSV, oHSV-D11mt, secreting a modified IGF2R domain 11 (IGF2RD11mt) that acts as IGF2 decoy receptor. Selective blocking of IGF2 by IGF2RD11mt significantly increased cytotoxicity, reduced oHSV-induced neutrophils/PMN-MDSCs infiltration, and reduced secretion of immune suppressive/proangiogenic cytokines, while increased CD8 + cytotoxic T lymphocytes (CTLs) infiltration, leading to enhanced survival in GBM or BCBM tumor-bearing mice. CONCLUSIONS: This is the first study reporting that oHSV-induced secreted IGF2 exerts a critical role in resistance to oHSV therapy, which can be overcome by oHSV-D11mt as a promising therapeutic advance for enhanced viro-immunotherapy.

Web-Based Tool (FH Family Share) to Increase Uptake of Cascade Testing for Familial Hypercholesterolemia: Development and Evaluation.

BACKGROUND: Familial hypercholesterolemia, a prevalent genetic disorder, remains significantly underdiagnosed in the United States. Cascade testing, wherein individuals diagnosed with familial hypercholesterolemia- probands-contact their family members to inform them of their risk for familial hypercholesterolemia, has low uptake in the United States. Digital tools are needed to facilitate communication between familial hypercholesterolemia probands and their family members and to promote sharing of familial hypercholesterolemia-related risk information. OBJECTIVE: We aimed to create and evaluate a web-based tool designed to enhance familial communication and promote cascade testing for familial hypercholesterolemia. METHODS: A hybrid type 1 implementation science framework and a user-centered design process were used to develop an interactive web-based tool-FH Family Share-that enables familial hypercholesterolemia probands to communicate information about their familial hypercholesterolemia diagnosis with at-risk relatives. Probands can also use the tool to draw a family pedigree and learn more about familial hypercholesterolemia through education modules and curated knowledge resources. Usability guidelines and standards were taken into account during the design and development of the tool. The initial prototype underwent a cognitive walkthrough, which was followed by usability testing with key stakeholders including genetic counselors and patients with familial hypercholesterolemia. Participants navigated the prototype using the think-aloud technique, and their feedback was used to refine features of the tool. RESULTS: Key themes that emerged from the cognitive walkthrough were design, format, navigation, terminology, instructions, and learnability. Expert feedback from the cognitive walkthrough resulted in a rebuild of the web-based tool to align it with institutional standards. Usability testing with genetic counselors and patients with familial hypercholesterolemia provided insights on user experience, satisfaction and interface design and highlighted specific modifications that were made to refine the features of FH Family Share. Genetic counselors and patients with familial hypercholesterolemia suggested inclusion of the following features in the web-based tool: (1) a letter-to-family-member email template, (2) education modules, and (3) knowledge resources. Surveys revealed that 6 of 9 (67%) genetic counselors found information within FH Family Share very easy to find, and 5 of 9 (56%) genetic counselors found information very easy to understand; 5 of 9 (56%) patients found information very easy to find within the website, and 7 of 9 (78%) patients found information very easy to understand. All genetic counselors and patients indicated that FH Family Share was a resource worth returning to. CONCLUSIONS: FH Family Share facilitates communication between probands and their relatives. Once informed, at-risk family members have the option to seek testing and treatment for familial hypercholesterolemia.

Genetic basis of hypercholesterolemia in adults.

We investigated monogenic and polygenic causes of hypercholesterolemia in a population-based cohort, excluding secondary hypercholesterolemia, and using an established framework to identify pathogenic variants. We studied 1682 individuals (50.2 ± 8.6 years, 41.3% males) from southeast Minnesota with primary hypercholesterolemia (low-density lipoprotein cholesterol (LDL-C) ≥155 mg/dl in the absence of identifiable secondary causes). Familial hypercholesterolemia (FH) phenotype was defined as a Dutch Lipid Clinic Network (DLCN) score ≥6. Participants underwent sequencing of LDLR, APOB, and PCSK9, and genotyping of 12 LDL-C-associated single-nucleotide variants to construct a polygenic score (PGS) for LDL-C. The presence of a pathogenic/likely pathogenic variant was considered monogenic etiology and a PGS ≥90th percentile was considered polygenic etiology. The mean LDL-C level was 187.3 ± 32.3 mg/dl and phenotypic FH was present in 8.4% of the cohort. An identifiable genetic etiology was present in 17.1% individuals (monogenic in 1.5% and polygenic in 15.6%). Phenotypic and genetic FH showed poor overlap. Only 26% of those who met the clinical criteria of FH had an identifiable genetic etiology and of those with an identifiable genetic etiology only 12.9% met clinical criteria for FH. Genetic factors explained 7.4% of the variance in LDL-C. In conclusion, in adults with primary hypercholesterolemia, 17.1% had an identifiable genetic etiology and the overlap between phenotypic and genetic FH was modest.