Identifying Mechanisms of Normal Cognitive Aging Using a Novel Mouse Genetic Reference Panel.

Developing strategies to maintain cognitive health is critical to quality of life during aging. The basis of healthy cognitive aging is poorly understood; thus, it is difficult to predict who will have normal cognition later in life. Individuals may have higher baseline functioning (cognitive reserve) and others may maintain or even improve with age (cognitive resilience). Understanding the mechanisms underlying cognitive reserve and resilience may hold the key to new therapeutic strategies for maintaining cognitive health. However, reserve and resilience have been inconsistently defined in human studies. Additionally, our understanding of the molecular and cellular bases of these phenomena is poor, compounded by a lack of longitudinal molecular and cognitive data that fully capture the dynamic trajectories of cognitive aging. Here, we used a genetically diverse mouse population (B6-BXDs) to characterize individual differences in cognitive abilities in adulthood and investigate evidence of cognitive reserve and/or resilience in middle-aged mice. We tested cognitive function at two ages (6 months and 14 months) using y-maze and contextual fear conditioning. We observed heritable variation in performance on these traits

The MHV68 M2 Protein Drives IL-10 Dependent B Cell Proliferation and Differentiation

Murine gammaherpesvirus 68 (MHV68) establishes long-term latency in memory B cells similar to the human gammaherpesvirus Epstein Barr Virus (EBV). EBV encodes an interleukin-10 (IL-10) homolog and modulates cellular IL-10 expression; however, the role of IL-10 in the establishment and/or maintenance of chronic EBV infection remains unclear. Notably, MHV68 does not encode an IL-10 homolog, but virus infection has been shown to result in elevated serum IL-10 levels in wild-type mice, and IL-10 deficiency results in decreased establishment of virus latency. Here we show that a unique MHV68 latency-associated gene product, the M2 protein, is required for the elevated serum IL-10 levels observed at 2 weeks post-infection. Furthermore, M2 protein expression in primary murine B cells drives high level IL-10 expression along with increased secretion of IL-2, IL-6, and MIP-1α. M2 expression was also shown to significantly augment LPS driven survival and proliferation of primary murine B cells. The latter was dependent on IL-10 expression as demonstrated by the failure of IL10−/− B cells to proliferate in response to M2 protein expression and rescue of M2-associated proliferation by addition of recombinant murine IL-10. M2 protein expression in primary B cells also led to upregulated surface expression of the high affinity IL-2 receptor (CD25) and the activation marker GL7, along with down-regulated surface expression of B220, MHC II, and sIgD. The cells retained CD19 and sIgG expression, suggesting differentiation to a pre-plasma memory B cell phenotype. These observations are consistent with previous analyses of M2-null MHV68 mutants that have suggested a role for the M2 protein in expansion and differentiation of MHV68 latently infected B cells—perhaps facilitating the establishment of virus latency in memory B cells. Thus, while the M2 protein is unique to MHV68, analysis of M2 function has revealed an important role for IL-10 in MHV68 pathogenesis—identifying a strategy that appears to be conserved between at least EBV and MHV68

Cross-Species Analyses Identify Dlgap2 as a Regulator of Age-Related Cognitive Decline and Alzheimer\u27s Dementia.

Genetic mechanisms underlying age-related cognitive decline and dementia remain poorly understood. Here, we take advantage of the Diversity Outbred mouse population to utilize quantitative trait loci mapping and identify Dlgap2 as a positional candidate responsible for modifying working memory decline. To evaluate the translational relevance of this finding, we utilize longitudinal cognitive measures from human patients, RNA expression from post-mortem brain tissue, data from a genome-wide association study (GWAS) of Alzheimer\u27s dementia (AD), and GWAS results in African Americans. We find an association between Dlgap2 and AD phenotypes at the variant, gene and protein expression, and methylation levels. Lower cortical DLGAP2 expression is observed in AD and is associated with more plaques and tangles at autopsy and faster cognitive decline. Results will inform future studies aimed at investigating the cross-species role of Dlgap2 in regulating cognitive decline and highlight the benefit of using genetically diverse mice to prioritize novel candidates

The Murine Gammaherpesvirus 68 M2 Gene Is Required for Efficient Reactivation from Latently Infected B Cells

Murine gammaherpesvirus 68 (γHV68) infection of mice provides a tractable small-animal model system for assessing the requirements for the establishment and maintenance of gammaherpesvirus latency within the lymphoid compartment. The M2 gene product of γHV68 is a latency-associated antigen with no discernible homology to any known proteins. Here we focus on the requirement for the M2 gene in splenic B-cell latency. Our analyses showed the following. (i) Low-dose (100 PFU) inoculation administered via the intranasal route resulted in a failure to establish splenic B-cell latency at day 16 postinfection. (ii) Increasing the inoculation dose to 4 × 10(5) PFU administered via the intranasal route partially restored the establishment of B-cell latency at day 16, but no virus reactivation was detected upon explant into tissue cultures. (iii) Although previous data failed to detect a phenotype of the M2 mutant upon high-dose intraperitoneal inoculation, decreasing the inoculation dose to 100 PFU administered intraperitoneally revealed a splenic B-cell latency phenotype at day 16 that was very similar to the phenotype observed upon high-dose intranasal inoculation. (iv) After low-dose intraperitoneal inoculation, fractionated B-cell populations showed that the M2 mutant virus was able to establish latency in surface immunoglobulin D-negative (sIgD(−)) B cells; by 6 months postinfection, equivalent frequencies of M2 mutant and marker rescue viral genome-positive sIgD(−) B cells were detected. (v) Like the marker rescue virus, the M2 mutant virus also established latency in splenic naive B cells upon low-dose intraperitoneal inoculation, but there was a significant lag in the decay of this latently infected reservoir compared to that seen with the marker rescue virus. (vi) After low-dose intranasal inoculation, by day 42 postinfection, latency was observed in the spleen, although at a frequency significantly lower than that in the marker rescue virus-infected mice; by 3 months postinfection, nearly equivalent levels of viral genome-positive cells were observed in the spleens of marker rescue virus- and M2 mutant virus-infected mice, and these cells were exclusively sIgD(−) B cells. Taken together, these data convincingly demonstrate a role for the M2 gene product in reactivation from splenic B cells and also suggest that disruption of the M2 gene leads to dose- and route-specific defects in the efficient establishment of splenic B-cell latency

M2 expressing cells have an activated, pre-plasma memory phenotype.

<p>(A) M2-transduced cells were CD19⁺, CD25^high, GL7^high, B220^low, I-A^{b low}, sIgD⁻, sIgG⁺, and CD138^low when compared to untransduced cells within the culture. Representative flow cytometry histograms at day 4 post-transduction. The black line open histograms reflect staining of the transduced (Thy1.1⁺) B cell population, while the filled gray histograms reflect staining of the untransduced (Thy1.1⁻) population. The top panels depict data from M2-transduced cultures; bottom panels depict data from M2.Stop-transduced cells. Data is representative of three samples per time-point with at least three independent experiments per stain. (B and C) ELISA quantitation of the levels of IgM and IgG in supernatants of M2 and M2.Stop transduced B cell cultures. Three samples were analyzed per time point, and the data shown is representative of three independent experiments. Significance of differences in IgG secretion was determined by two-tailed, unpaired Student's T test with a confidence level of 95%. * p = 0.0182, ** p = 0.0352, *** p = 0.0118.</p

Loss of M2 expression <i>in vivo</i> correlates with reduced serum IL-10 at the onset of latency.

<p>The reduction in serum IL-10 is independent of the route of infection and correlates with a significant reduction in reactivation from latency in the absence of M2. Groups of four to five mice were infected with 1000 pfu of M2 null mutant (MHV68/M2.Stop) or the M2 marker rescue virus (MHV68/M2.MR) via intranasal inoculation or 100 pfu via intraperitoneal inoculation (or naïve mice). On days 14 or 15 post-infection, mice were injected with biotin labeled anti-IL-10 antibody i.p. Twenty-four hours later, serum was collected for analysis of IL-10 levels (A and B), and splenocytes were recovered for determinations of the frequency of viral latency and reactivation (C and D). Data shown for intranasal inoculations is representative of two independent experiments, 4–5 mice per group, with IL-10 levels measured in individual mice and splenocytes pooled for viral latency assays (as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000039#s4" target="_blank">Materials and Methods</a>). Data shown for intraperitoneal inoculations is representative of three independent experiments, 4–5 mice per group, with IL-10 levels measured in individual mice and splenocytes pooled for viral latency assays. (A and B) Total serum IL-10 levels were significantly lower in mice infected with MHV68/M2.Stop compared to MHV68/M2.MR virus, independent of the route of infection. Intranasal infection data is from 10 individual mice from two independent infections; intraperitoneal data is from 15 individual mice from three independent infections. Two naïve animals were analyzed per infection * p = 0.0117, ** p = 0.0005. Significance of IL-10 data was determined by two-tailed, unpaired Student's T test with a confidence level of 95%. (C and D) Frequencies of splenocytes from MHV68/M2.MR and MHV68/M2.Stop infected mice harboring latent viral genomes and reactivating from latency upon explant. The frequency of latency was determined by nested, limiting-dilution PCR (LD-PCR) as previously described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000039#ppat.1000039-Weck1" target="_blank">[3]</a>. The frequency of cells reactivating from latency upon in vitro culture was determined by plating serial dilutions of live, intact splenocytes on mouse embryonic fibroblast monolayers and scoring cytopathic effect 14–21 days post-explant, as previously described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000039#ppat.1000039-Weck1" target="_blank">[3]</a>. Data from both assays was subjected to nonlinear regression analysis with a sigmoidal dose-response algorithm for best fit. Data points represent individual experiments with splenocytes pooled from 4–5 mice per condition.</p

M2 expressing B cells fail to expand in the absence of IL-10.

<p>(A) B cells from C57Bl6 and IL-10^−/− mice were isolated from splenocytes and transduced with either the M2 or M2.Stop retroviruses. Triplicate wells were analyzed per time-point. IL-10^−/− B cells transduced with M2 failed to expand to the same extent as wild-type B cells. Data shown are representative of 3 independent experiments. (B) Quantitative ELISA analyses confirm that IL-10^−/− B cells fail to secrete IL-10. (C) IL-10^−/− B cells transduced with M2 secrete significantly higher levels of IL-6 than C57Bl/6 B cells transduced with M2. However, IL-6 fails to compensate for the IL-10 deficiency. Data shown is representative of two independent experiments, each containing triplicate cultures. (D) Supernatants from C57Bl6 B cells transduced with M2 compliment for the IL-10^−/− B cell proliferation defect. B cells from C57Bl6 and IL-10^−/− mice were isolated and triplicate cultures transduced with M2 or M2.Stop retroviruses. On day 2 post-transduction, 500 µL of supernatant from the C57Bl6 B cells transduced with the M2.Stop retrovirus and IL-10^−/− B cells transduced with the M2 retrovirus were replaced with 500 µL of tissue culture supernatant recovered from the C57Bl6/M2 B cell cultures from that respective day. Triplicate wells were analyzed per time-point. High IL-10 levels fail to complement the lack of M2 expression in the C57Bl/6 B cells, but did drive proliferation of the M2-transduced IL-10^−/− B cells. Data are represented as the fold-change in percent Thy1.1 positive cells in the cultures over the percent Thy1.1 positive cells present at two days post-transduction. (E) Recombinant murine IL-10 rescues M2-mediated expansion of IL-10^−/− B cells, but fails to expand either the C57Bl/6 or IL-10^−/− M2.Stop transduced B cell populations. B cells from C57Bl6 and IL-10^−/− mice were isolated from splenocytes and transduced with either the M2 or M2.Stop retroviruses. IL-10 was added (final concentration of 20 ng/ml) to the indicated samples starting on day 2 post-transduction (see text for description). Triplicate wells were analyzed per time-point.</p

Systematic Mutagenesis of the Murine Gammaherpesvirus 68 M2 Protein Identifies Domains Important for Chronic Infection▿

Murine gammaherpesvirus 68 (MHV68) infection of inbred mice represents a genetically tractable small-animal model for assessing the requirements for the establishment of latency, as well as reactivation from latency, within the lymphoid compartment. By day 16 postinfection, MHV68 latency in the spleen is found in B cells, dendritic cells, and macrophages. However, as with Epstein-Barr virus, by 3 months postinfection MHV68 latency is predominantly found in isotype-switched memory B cells. The MHV68 M2 gene product is a latency-associated antigen with no discernible homology to any known cellular or viral proteins. However, depending on experimental conditions, the M2 protein has been shown to play a critical role in both the efficient establishment of latency in splenic B cells and reactivation from latently infected splenic B cells. Inspection of the sequence of the M2 protein reveals several hallmarks of a signaling molecule, including multiple PXXP motifs and two potential tyrosine phosphorylation sites. Here, we report the generation of a panel of recombinant MHV68 viruses harboring mutations in the M2 gene that disrupt putative functional motifs. Subsequent analyses of the panel of M2 mutant viruses revealed a functionally important cluster of PXXP motifs in the C-terminal region of M2, which have previously been implicated in binding Vav proteins (P. A. Madureira, P. Matos, I. Soeiro, L. K. Dixon, J. P. Simas, and E. W. Lam, J. Biol. Chem. 280:37310-37318, 2005; L. Rodrigues, M. Pires de Miranda, M. J. Caloca, X. R. Bustelo, and J. P. Simas, J. Virol. 80:6123-6135, 2006). Further characterization of two adjacent PXXP motifs in the C terminus of the M2 protein revealed differences in the functions of these domains in M2-driven expansion of primary murine B cells in culture. Finally, we show that tyrosine residues 120 and 129 play a critical role in both the establishment of splenic latency and reactivation from latency upon explant of splenocytes into tissue culture. Taken together, these analyses will aide future studies for identifying M2 interacting partners and B-cell signaling pathways that are manipulated by the M2 protein