Loss of LRP1 in Adult Neural Stem Cells Impairs Migration to Ischemic Lesions.

After ischemia, cells in the brain parenchyma upregulate stromal derived factor 1 (SDF1), driving chemokine receptor CXCR4-mediated migration of adult neural stem cells to the ischemic injury. We discovered a novel regulator of CXCR4 in neural stem cells, low-density lipoprotein receptor related protein 1 (LRP1). We used Nestin-driven knockout of LRP1 and induction of td-tomato in neural stem cells of adult mice. We observed reduced localization of td-tomato positive cells to the lesion, and find disrupted CXCR4-mediated neural stem cell migration in vitro, which is likely driven by LRP1-mediated loss of CXCR4 expression in vivo. Our results suggest that LRP1 is a novel regulator of CXCR4 in neural stem cells. This heretofore unknown interaction between LRP1 and CXCR4 could have significant consequences for multiple aspects of neural stem cell physiology.

Antiviral function and viral antagonism of the rapidly evolving dynein activating adaptor NINL.

Viruses interact with the intracellular transport machinery to promote viral replication. Such host-virus interactions can drive host gene adaptation, leaving signatures of pathogen-driven evolution in host genomes. Here, we leverage these genetic signatures to identify the dynein activating adaptor, ninein-like (NINL), as a critical component in the antiviral innate immune response and as a target of viral antagonism. Unique among genes encoding components of active dynein complexes, NINL has evolved under recurrent positive (diversifying) selection, particularly in its carboxy-terminal cargo-binding region. Consistent with a role for NINL in host immunity, we demonstrate that NINL knockout cells exhibit an impaired response to interferon, resulting in increased permissiveness to viral replication. Moreover, we show that proteases encoded by diverse picornaviruses and coronaviruses cleave and disrupt NINL function in a host- and virus-specific manner. Our work reveals the importance of NINL in the antiviral response and the utility of using signatures of host-virus genetic conflicts to uncover new components of antiviral immunity and targets of viral antagonism.

Humans and viruses are locked in an evolutionary arms race. Viruses hijack cells, using their resources and proteins to build more viral particles; the cells fight back, calling in the immune system to fend off the attack. Both actors must constantly and quickly evolve to keep up with each other. This genetic conflict has been happening for millions of years, and the indelible marks it has left on genes can serve to uncover exactly how viruses interact with the organisms they invade. One hotspot in this host-virus conflict is the complex network of molecules that help to move cargo inside a cell. This system transports elements of the immune system, but viruses can also harness it to make more of themselves. Scientists still know very little about how viruses and the intracellular transport machinery interact, and how this impacts viral replication and the immune response. Stevens et al. therefore set out to identify new interactions between viruses and the transport system by using clues left in host genomes by evolution. They focused on dynein, a core component of this machinery which helps to haul molecular actors across a cell. To do so, dynein relies on adaptor molecules such as 'Ninein-like', or NINL for short. Closely examining the gene sequence for NINL across primates highlighted an evolutionary signature characteristic of host-virus genetic conflicts; this suggests that the protein may be used by viruses to reproduce, or by cells to fend off infection. And indeed, human cells lacking the NINL gene were less able to defend themselves, allowing viruses to grow much faster than normal. Further work showed that NINL was important for a major type of antiviral immune response. As a potential means to sabotage this defence mechanism, some viruses cleave NINL at specific sites and disrupt its role in intracellular transport. Better antiviral treatments are needed to help humanity resist old foes and new threats alike. The work by Stevens et al. demonstrates how the information contained in host genomes can be leveraged to understand what drives susceptibility to an infection, and to pinpoint molecular actors which could become therapeutic targets.

The requirement for pyruvate dehydrogenase in leukemogenesis depends on cell lineage.

Cancer cells are metabolically similar to their corresponding normal tissues. Differences between cancers and normal tissues may reflect reprogramming during transformation or maintenance of the metabolism of the specific normal cell type that originated the cancer. Here, we compare glucose metabolism in hematopoiesis and leukemia. Thymus T cell progenitors were glucose avid and oxidized more glucose in the tricarboxylic acid cycle through pyruvate dehydrogenase (PDH) as compared with other hematopoietic cells. PDH deletion decreased double-positive T cell progenitor cells but had no effect on hematopoietic stem cells, myeloid progenitors, or other hematopoietic cells. PDH deletion blocked the development of Pten-deficient T cell leukemia, but not the development of a Pten-deficient myeloid neoplasm. Therefore, the requirement for PDH in leukemia reflected the metabolism of the normal cell of origin independently of the driver genetic lesion. PDH was required to prevent pyruvate accumulation and maintain glutathione levels and redox homeostasis.

Author Correction: Maternal vitamin C regulates reprogramming of DNA methylation and germline development.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Maternal vitamin C regulates reprogramming of DNA methylation and germline development.

Development is often assumed to be hardwired in the genome, but several lines of evidence indicate that it is susceptible to environmental modulation with potential long-term consequences, including in mammals1,2. The embryonic germline is of particular interest because of the potential for intergenerational epigenetic effects. The mammalian germline undergoes extensive DNA demethylation3-7 that occurs in large part by passive dilution of methylation over successive cell divisions, accompanied by active DNA demethylation by TET enzymes3,8-10. TET activity has been shown to be modulated by nutrients and metabolites, such as vitamin C11-15. Here we show that maternal vitamin C is required for proper DNA demethylation and the development of female fetal germ cells in a mouse model. Maternal vitamin C deficiency does not affect overall embryonic development but leads to reduced numbers of germ cells, delayed meiosis and reduced fecundity in adult offspring. The transcriptome of germ cells from vitamin-C-deficient embryos is remarkably similar to that of embryos carrying a null mutation in Tet1. Vitamin C deficiency leads to an aberrant DNA methylation profile that includes incomplete demethylation of key regulators of meiosis and transposable elements. These findings reveal that deficiency in vitamin C during gestation partially recapitulates loss of TET1, and provide a potential intergenerational mechanism for adjusting fecundity to environmental conditions.

Calorie restriction protects neural stem cells from age-related deficits in the subventricular zone.

The brain can generate new neurons from neural stem cells throughout life. However, the capacity for neurogenesis declines with age, reducing the potential for learning and repair. We explored the effects of calorie restriction, an established anti-aging intervention, on neural stem cells in the subventricular zone of young and aged mice. Calorie restriction transiently enhanced proliferation of neural progenitor cells in young, but not aged mice. However, calorie restriction prevented the age-related loss of neurogenesis in the aged brain. Calorie-restricted mice showed enhanced olfactory memory compared with ad libitum-fed controls, suggesting that calorie restriction can produce functional improvements in the aged brain. Calorie restriction also mitigated the age-related activation of microglia and subsequent increase in pro-inflammatory cytokines. Likewise, calorie restriction prevented increases in senescent cells normally observed in the subventricular zone in aged mice, further protecting this neurogenic niche from pro-inflammatory signals. Together, these data suggest that calorie restriction protects the subventricular zone microenvironment from age-related inflammation, thereby preserving neurogenesis into old age.

Heterogeneous Expression of SDF1 Retains Actively Proliferating Neural Progenitors in the Capillary Compartment of the Niche.

The vascular compartment of the adult brain ventricular-subventricular zone (V-SVZ) is a critical regulator of neural stem cell and progenitor function. Blood enters the V-SVZ via arteries and arterioles to capillaries that then connect with venules and veins to return blood to the heart. We found that stromal cell-derived factor 1 (SDF1) is expressed by a subpopulation of V-SVZ vessels, the capillaries, and that actively proliferating neural stem cells (NSCs) and progenitors are preferentially associated with these SDF1-positive vessels. In contrast, slowly dividing or quiescent NSCs are most prevalent near SDF1-negative vessels. By conditional knockout, we found that loss of SDF1 signaling in NSCs stimulates lineage progression and NSC displacement from the vessel niche. With aging, SDF1/CXCR4 signaling is dysregulated, coincident with reduced proliferation and increased displacement of dividing cells from the vasculature. Our findings demonstrate SDF1-based vascular heterogeneity in the niche and suggest that reduced SDF1 signaling contributes to age-related declines in adult neurogenesis.

Neurogenic Niche Microglia Undergo Positional Remodeling and Progressive Activation Contributing to Age-Associated Reductions in Neurogenesis.

Neural stem cells (NSCs) exist throughout life in the ventricular-subventricular zone (V-SVZ) of the mammalian forebrain. During aging NSC function is diminished through an unclear mechanism. In this study, we establish microglia, the immune cells of the brain, as integral niche cells within the V-SVZ that undergo age-associated repositioning in the V-SVZ. Microglia become activated early before NSC deficits during aging resulting in an antineurogenic microenvironment due to increased inflammatory cytokine secretion. These age-associated changes were not observed in non-neurogenic brain regions, suggesting V-SVZ microglia are specialized. Using a sustained inflammatory model in young adult mice, we induced microglia activation and inflammation that was accompanied by reduced NSC proliferation in the V-SVZ. Furthermore, in vitro studies revealed secreted factors from activated microglia reduced proliferation and neuron production compared to secreted factors from resting microglia. Our results suggest that age-associated chronic inflammation contributes to declines in NSC function within the aging neurogenic niche.

miR-124, -128, and -137 Orchestrate Neural Differentiation by Acting on Overlapping Gene Sets Containing a Highly Connected Transcription Factor Network.

The ventricular-subventricular zone harbors neural stem cells (NSCs) that can differentiate into neurons, astrocytes, and oligodendrocytes. This process requires loss of stem cell properties and gain of characteristics associated with differentiated cells. miRNAs function as important drivers of this transition; miR-124, -128, and -137 are among the most relevant ones and have been shown to share commonalities and act as proneurogenic regulators. We conducted biological and genomic analyses to dissect their target repertoire during neurogenesis and tested the hypothesis that they act cooperatively to promote differentiation. To map their target genes, we transfected NSCs with antagomiRs and analyzed differences in their mRNA profile throughout differentiation with respect to controls. This strategy led to the identification of 910 targets for miR-124, 216 for miR-128, and 652 for miR-137. The target sets show extensive overlap. Inspection by gene ontology and network analysis indicated that transcription factors are a major component of these miRNAs target sets. Moreover, several of these transcription factors form a highly interconnected network. Sp1 was determined to be the main node of this network and was further investigated. Our data suggest that miR-124, -128, and -137 act synergistically to regulate Sp1 expression. Sp1 levels are dramatically reduced as cells differentiate and silencing of its expression reduced neuronal production and affected NSC viability and proliferation. In summary, our results show that miRNAs can act cooperatively and synergistically to regulate complex biological processes like neurogenesis and that transcription factors are heavily targeted to branch out their regulatory effect.

Microwave & magnetic (M2) proteomics reveals CNS-specific protein expression waves that precede clinical symptoms of experimental autoimmune encephalomyelitis.

Central nervous system-specific proteins (CSPs), transported across the damaged blood-brain-barrier (BBB) to cerebrospinal fluid (CSF) and blood (serum), might be promising diagnostic, prognostic and predictive protein biomarkers of disease in individual multiple sclerosis (MS) patients because they are not expected to be present at appreciable levels in the circulation of healthy subjects. We hypothesized that microwave &magnetic (M(2)) proteomics of CSPs in brain tissue might be an effective means to prioritize putative CSP biomarkers for future immunoassays in serum. To test this hypothesis, we used M(2) proteomics to longitudinally assess CSP expression in brain tissue from mice during experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Confirmation of central nervous system (CNS)-infiltrating inflammatory cell response and CSP expression in serum was achieved with cytokine ELISPOT and ELISA immunoassays, respectively, for selected CSPs. M(2) proteomics (and ELISA) revealed characteristic CSP expression waves, including synapsin-1 and α-II-spectrin, which peaked at day 7 in brain tissue (and serum) and preceded clinical EAE symptoms that began at day 10 and peaked at day 20. Moreover, M(2) proteomics supports the concept that relatively few CNS-infiltrating inflammatory cells can have a disproportionally large impact on CSP expression prior to clinical manifestation of EAE.

Microwave & Magnetic (M2) Proteomics of a Mouse Model of Mild Traumatic Brain Injury.

Short-term increases in oxidative stress and decreases in motor function, including debilitating effects on balance and motor control, can occur following primary mild traumatic brain injuries (mTBI). However, the long-term effects on motor unit impairment and integrity as well as the molecular mechanisms underlying secondary injuries are poorly understood. We hypothesized that changes in central nervous system-specific protein (CSP) expression might correlate to these long-term effects. To test our hypothesis, we longitudinally assessed a closed-skull mTBI mouse model, vs. sham control, at 1, 7, 30, and 120 days post-injury. Motor impairment was determined by rotarod and grip strength performance measures, while motor unit integrity was determined using electromyography. Relative protein expression was determined by microwave & magnetic (M2) proteomics of ipsilateral brain tissue, as previously described. Isoprostane measurements were performed to confirm a primary oxidative stress response. Decoding the relative expression of 476 ± 56 top-ranked proteins for each specimen revealed statistically significant changes in the expression of two well-known CSPs at 1, 7 and 30 days post-injury: P < 0.001 for myelin basic protein (MBP) and P < 0.05 for myelin associated glycoprotein (MAG). This was confirmed by Western blot. Moreover, MAG, αII-spectrin (SPNA2) and neurofilament light (NEFL) expression at 30 days post-injury were directly related to grip strength (P < 0.05). While higher-powered studies of larger cohorts merit further investigation, this study supports the proof-of-concept that M2 proteomics is a rapid method to quantify putative protein biomarkers and therapeutic targets of mTBI and suggests the feasibility of CSP expression correlations to long-term effects on motor impairment.

Anti-CRLF2 Antibody-Armored Biodegradable Nanoparticles for Childhood B-ALL.

B-precursor acute lymphoblastic leukemia (B-ALL) lymphoblast (blast) internalization of anti-cytokine receptor-like factor 2 (CRLF2) antibody-armored biodegradable nanoparticles (AbBNPs) are investigated. First, AbBNPsaere synthesized by adsorbing anti-CRLF2 antibodies to poly(D,L-lactide- co -glycolide) (PLGA) nanoparticles of various sizes and antibody surface density (Ab/BNP) ratios. Second, AbBNPs are incubated with CRLF2-overexpressing (CRLF2+) or control blasts. Third, internalization of AbBNPs by blasts is evaluated by multicolor flow cytometry as a function of receptor expression, AbBNP size, and Ab/BNP ratio. Results from these experiments are con-firmed by electron microscopy, fluorescence microscopy, and Western blotting. The optimal size and Ab/BNP for internalization of AbBNPs by CRLF2+ blasts is 50 nm with 10 Ab/BNP and 100 nm with 25 Ab/BNP. These studies show that internalization of AbBNPs in childhood B-ALL blasts is AbBNP size-and Ab/BNP ratio-dependent. All AbBNP combinations are non-cytotoxic. It is also shown that CD47 is very slightly up-regulated by blasts exposed to AbBNPs. CD47 is "the marker of self" overexpressed by blasts to escape phagocytosis, or "cellular devouring", by beneficial macrophages. The results indicate that precise engineering of AbBNPs by size and Ab/BNP ratio may improve the internalization and selectivity of future biodegradable nanoparticles for the treatment of leukemia patients, including drug-resistant minority children and Down's syndrome patients with CRLF2+B-ALL.

Immunoenrichment microwave and magnetic proteomics for quantifying CD47 in the experimental autoimmune encephalomyelitis model of multiple sclerosis.

We hypothesized that quantitative MS/MS-based proteomics at multiple time points, incorporating immunoenrichment prior to rapid microwave and magnetic (IM(2) ) sample preparation, might enable correlation of the relative expression of CD47 and other low abundance proteins to disease progression in the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis. To test our hypothesis, anti-CD47 antibodies were used to enrich for low abundance CD47 prior to microwave and magnetic proteomics in EAE. Decoding protein expression at each time point, with CD47-immunoenriched samples and targeted proteomic analysis, enabled peptides from the low abundance proteins to be precisely quantified throughout disease progression, including: CD47: 86-99, corresponding to the "marker of self" overexpressed by myelin that prevents phagocytosis, or "cellular devouring," by microglia and macrophages; myelin basic protein: 223-228, corresponding to myelin basic protein; and migration inhibitory factor: 79-87, corresponding to a proinflammatory cytokine that inhibits macrophage migration. While validation in a larger cohort is underway, we conclude that IM(2) proteomics is a rapid method to precisely quantify peptides from CD47 and other low abundance proteins throughout disease progression in EAE. This is likely due to improvements in selectivity and sensitivity, necessary to partially overcome masking of low abundance proteins by high abundance proteins and improve dynamic range.

Microwave and magnetic (M(2) ) proteomics of the experimental autoimmune encephalomyelitis animal model of multiple sclerosis.

We hypothesized that quantitative MS/MS-based proteomics at multiple time points, incorporating rapid microwave and magnetic (M(2) ) sample preparation, could enable relative protein expression to be correlated to disease progression in the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis. To test our hypothesis, microwave-assisted reduction/alkylation/digestion of proteins from brain tissue lysates bound to C8 magnetic beads and microwave-assisted isobaric chemical labeling were performed of released peptides, in 90 s prior to unbiased proteomic analysis. Disease progression in EAE was assessed by scoring clinical EAE disease severity and confirmed by histopathologic evaluation for central nervous system inflammation. Decoding the expression of 283 top-ranked proteins (p <0.05) at each time point relative to their expression at the peak of disease, from a total of 1191 proteins observed in four technical replicates, revealed a strong statistical correlation to EAE disease score, particularly for the following four proteins that closely mirror disease progression: 14-3-3ε (p = 3.4E-6); GPI (p = 2.1E-5); PLP1 (p = 8.0E-4); PRX1 (p = 1.7E-4). These results were confirmed by Western blotting, signaling pathway analysis, and hierarchical clustering of EAE risk groups. While validation in a larger cohort is underway, we conclude that M(2) proteomics is a rapid method to quantify putative prognostic/predictive protein biomarkers and therapeutic targets of disease progression in the EAE animal model of multiple sclerosis.