Microglia, the brain’s immune sentinels, have garnered much attention in recent years. Researchers have begun to identify the manifold roles that these cells play in the central nervous system (CNS), ...and this work has been greatly facilitated by microglial depletion paradigms. The varying degrees of spatiotemporal manipulation afforded by such techniques allow microglial ablation before, during, and/or following insult, injury, or disease. We review the major methods of microglial depletion, including toxin-based, genetic, and pharmacological approaches, which differ in key factors including depletion onset, duration, and off-target effects. We conclude that pharmacological CSF1R inhibitors afford the most extensive versatility in manipulating microglia, making them ideal candidates for future studies investigating microglial function in health and disease.
Microglial depletion animal models provide insight into microglial cell function in the healthy and diseased brain.Microglia are dependent on colony-stimulating factor 1 receptor (CSF1R) signaling for survival in the vertebrate brain.CSF1R inhibition allows rapid and titratable myeloid cell population manipulation in the rodent CNS.We posit that optimal microglial depletion models (e.g., CSF1R inhibitors) should ideally enable the elimination of these cells for any duration of time in different cell subsets. These models might also stimulate microglial repopulation in any model or species, and act in a clinically relevant fashion.
Combinatorial strategies are needed to overcome the resistance of pancreatic cancer to immune checkpoint blockade (ICB). DNA damage activates the innate immune response and improves ICB efficacy. ...Because ATM is an apical kinase in the radiation-induced DNA damage response, we investigated the effects of ATM inhibition and radiation on pancreatic tumor immunogenicity. ATM was inhibited through pharmacologic and genetic strategies in human and murine pancreatic cancer models both
and
. Tumor immunogenicity was evaluated after ATM inhibition alone and in combination with radiation by assessing TBK1 and Type I interferon (T1IFN) signaling as well as tumor growth following PD-L1/PD-1 checkpoint inhibition. Inhibition of ATM increased tumoral T1IFN expression in a cGAS/STING-independent, but TBK1- and SRC-dependent, manner. The combination of ATM inhibition with radiation further enhanced TBK1 activity, T1IFN production, and antigen presentation. Furthermore, ATM silencing increased PD-L1 expression and increased the sensitivity of pancreatic tumors to PD-L1-blocking antibody in association with increased tumoral CD8
T cells and established immune memory. In patient pancreatic tumors, low ATM expression inversely correlated with PD-L1 expression. Taken together, these results demonstrate that the efficacy of ICB in pancreatic cancer is enhanced by ATM inhibition and further potentiated by radiation as a function of increased tumoral immunogenicity, underscoring the potential of ATM inhibition in combination with ICB and radiation as an efficacious treatment strategy for pancreatic cancer. SIGNIFICANCE: This study demonstrates that ATM inhibition induces a T1IFN-mediated innate immune response in pancreatic cancer that is further enhanced by radiation and leads to increased sensitivity to anti-PD-L1 therapy.
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Abstract
We present a protocol to prepare extracted DNA for sequencing on the Illumina sequencing platform that has been optimized for ancient and degraded DNA. Our approach, the Santa Cruz Reaction ...or SCR, uses directional splinted ligation of Illumina’s P5 and P7 adapters to convert natively single-stranded DNA and heat denatured double-stranded DNA into sequencing libraries in a single enzymatic reaction. To demonstrate its efficacy in converting degraded DNA molecules, we prepare 5 ancient DNA extracts into sequencing libraries using the SCR and 2 of the most commonly used approaches for preparing degraded DNA for sequencing: BEST, which targets and converts double-stranded DNA, and ssDNA2.0, which targets and converts single-stranded DNA. We then compare the efficiency with which each approach recovers unique molecules, or library complexity, given a standard amount of DNA input. We find that the SCR consistently outperforms the BEST protocol in recovering unique molecules and, despite its relative simplicity to perform and low cost per library, has similar performance to ssDNA2.0 across a wide range of DNA inputs. The SCR is a cost- and time-efficient approach that minimizes the loss of unique molecules and makes accessible a taxonomically, geographically, and a temporally broader sample of preserved remains for genomic analysis.
Microglia shape the synaptic environment in health and disease, but synapses do not exist in a vacuum. Instead, pre- and postsynaptic terminals are surrounded by extracellular matrix (ECM), which ...together with glia comprise the four elements of the contemporary tetrapartite synapse model. While research in this area is still just beginning, accumulating evidence points toward a novel role for microglia in regulating the ECM during normal brain homeostasis, and such processes may, in turn, become dysfunctional in disease. As it relates to synapses, microglia are reported to modify the perisynaptic matrix, which is the diffuse matrix that surrounds dendritic and axonal terminals, as well as perineuronal nets (PNNs), specialized reticular formations of compact ECM that enwrap neuronal subsets and stabilize proximal synapses. The interconnected relationship between synapses and the ECM in which they are embedded suggests that alterations in one structure necessarily affect the dynamics of the other, and microglia may need to sculpt the matrix to modify the synapses within. Here, we provide an overview of the microglial regulation of synapses, perisynaptic matrix, and PNNs, propose candidate mechanisms by which these structures may be modified, and present the implications of such modifications in normal brain homeostasis and in disease.
Huntington's disease is associated with a reactive microglial response and consequent inflammation. To address the role of these cells in disease pathogenesis, we depleted microglia from R6/2 mice, a ...rapidly progressing model of Huntington's disease marked by behavioural impairment, mutant huntingtin (mHTT) accumulation, and early death, through colony-stimulating factor 1 receptor inhibition (CSF1Ri) with pexidartinib (PLX3397) for the duration of disease. Although we observed an interferon gene signature in addition to downregulated neuritogenic and synaptic gene pathways with disease, overt inflammation was not evident by microglial morphology or cytokine transcript levels in R6/2 mice. Nonetheless, CSF1Ri-induced microglial elimination reduced or prevented disease-related grip strength and object recognition deficits, mHTT accumulation, astrogliosis, and striatal volume loss, the latter of which was not associated with reductions in cell number but with the extracellular accumulation of chondroitin sulphate proteoglycans (CSPGs)-a primary component of glial scars. A concurrent loss of proteoglycan-containing perineuronal nets was also evident in R6/2 mice, and microglial elimination not only prevented this but also strikingly increased perineuronal nets in the brains of naïve littermates, suggesting a new role for microglia as homeostatic regulators of perineuronal net formation and integrity.
Low-protein diets promote metabolic health in rodents and humans, and the benefits of low-protein diets are recapitulated by specifically reducing dietary levels of the three branched-chain amino ...acids (BCAAs), leucine, isoleucine, and valine. Here, we demonstrate that each BCAA has distinct metabolic effects. A low isoleucine diet reprograms liver and adipose metabolism, increasing hepatic insulin sensitivity and ketogenesis and increasing energy expenditure, activating the FGF21-UCP1 axis. Reducing valine induces similar but more modest metabolic effects, whereas these effects are absent with low leucine. Reducing isoleucine or valine rapidly restores metabolic health to diet-induced obese mice. Finally, we demonstrate that variation in dietary isoleucine levels helps explain body mass index differences in humans. Our results reveal isoleucine as a key regulator of metabolic health and the adverse metabolic response to dietary BCAAs and suggest reducing dietary isoleucine as a new approach to treating and preventing obesity and diabetes.
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•Reduced isoleucine or valine, but not leucine, promotes metabolic health in mice•Reduced isoleucine is required for the metabolic benefits of a low-protein diet•The benefits of isoleucine restriction are mediated in part by FGF21•Dietary levels of isoleucine are positively associated with BMI in humans
Yu and Richardson et al. find that restriction of dietary isoleucine or valine promotes metabolic health in mice and that restriction of dietary isoleucine is required for the metabolic benefits of a low-protein diet. Furthermore, higher dietary isoleucine levels are associated with increased BMI in humans.
Microglia, the brain's principal immune cell, are increasingly implicated in Alzheimer's disease (AD), but the molecular interfaces through which these cells contribute to amyloid beta (Aβ)-related ...neurodegeneration are unclear. We recently identified microglial contributions to the homeostatic and disease-associated modulation of perineuronal nets (PNNs), extracellular matrix structures that enwrap and stabilize neuronal synapses, but whether PNNs are altered in AD remains controversial.
Extensive histological analysis was performed on male and female 5xFAD mice at 4, 8, 12, and 18 months of age to assess plaque burden, microgliosis, and PNNs. Findings were validated in postmortem AD tissue. The role of neuroinflammation in PNN loss was investigated via LPS treatment, and the ability to prevent or rescue disease-related reductions in PNNs was assessed by treating 5xFAD and 3xTg-AD model mice with colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia.
Utilizing the 5xFAD mouse model and human cortical tissue, we report that PNNs are extensively lost in AD in proportion to plaque burden. Activated microglia closely associate with and engulf damaged nets in the 5xFAD brain, and inclusions of PNN material are evident in mouse and human microglia, while aggrecan, a critical PNN component, deposits within human dense-core plaques. Disease-associated reductions in parvalbumin (PV)+ interneurons, frequently coated by PNNs, are preceded by PNN coverage and integrity impairments, and similar phenotypes are elicited in wild-type mice following microglial activation with LPS. Chronic pharmacological depletion of microglia prevents 5xFAD PNN loss, with similar results observed following depletion in aged 3xTg-AD mice, and this occurs despite plaque persistence.
We conclude that phenotypically altered microglia facilitate plaque-dependent PNN loss in the AD brain.
The NIH (NIA, NINDS) and the Alzheimer's Association.
The chance discovery of a 1.5-3.5 years old mummified girl presents a unique opportunity to further our understanding of health and disease among children in 19th Century San Francisco. This study ...focuses on carbon and nitrogen stable isotope signatures in serial samples of hair that cover the last 14 months of her life. Results suggest an initial omnivorous diet with little input from marine resources or C4 plants. Around six months before death δ15N starts a steady increase, with a noticeable acceleration just two months before she died. The magnitude of δ15N change, +1.5‰ in total, is consistent with severe undernourishment or starvation. Cemetery records from this time period in San Francisco indicate high rates of infant and child mortality, mainly due to bacterial-borne infectious diseases, about two orders of magnitude higher than today. Taken together, we hypothesize that the girl died after a prolonged battle with such an illness. Results highlight the tremendous impacts that modern sanitation and medicine have had since the 1800s on human health and lifespan in the United States.
Abstract
Hybridization capture approaches allow targeted high-throughput sequencing analysis at reduced costs compared to shotgun sequencing. Hybridization capture is particularly useful in analyses ...of genomic data from ancient, environmental, and forensic samples, where target content is low, DNA is fragmented and multiplex PCR or other targeted approaches often fail. Here, we describe a DNA bait synthesis approach for hybridization capture that we call Circular Nucleic acid Enrichment Reagent, or CNER (pronounced ‘snare’). The CNER method uses rolling-circle amplification followed by restriction digestion to discretize microgram quantities of hybridization probes. We demonstrate the utility of the CNER method by generating probes for a panel of 23 771 known sites of single nucleotide polymorphism in the horse genome. Using these probes, we capture and sequence from a panel of ten ancient horse DNA libraries, comparing CNER capture efficiency to a commercially available approach. With about one million read pairs per sample, CNERs captured more targets (90.5% versus 66.5%) at greater mean depth than an alternative commercial approach.
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