The Neuro-Glial Properties of Adipose-Derived Adult Stromal (ADAS) Cells Are Not Regulated by Notch 1 and Are Not Derived from Neural Crest Lineage

We investigated whether adipose-derived adult stromal (ADAS) are of neural crest origin and the extent to which Notch 1 regulates their growth and differentiation. Mouse ADAS cells cultured in media formulated for neural stem cells (NSC) displayed limited capacity for self-renewal, clonogenicity, and neurosphere formation compared to NSC from the subventricular zone in the hippocampus. Although ADAS cells expressed Nestin, GFAP, NSE and Tuj1 in vitro, exposure to NSC differentiation supplements did not induce mature neuronal marker expression. In contrast, in mesenchymal stem cell (MSC) media, ADAS cells retained their ability to proliferate and differentiate beyond 20 passages and expressed high levels of Nestin. In neuritizing cocktails, ADAS cells extended processes, downregulated Nestin expression, and displayed depolarization-induced Ca2+ transients but no spontaneous or evoked neural network activity on Multi-Electrode Arrays. Deletion of Notch 1 in ADAS cell cultures grown in NSC proliferation medium did not significantly alter their proliferative potential in vitro or the differentiation-induced downregulation of Nestin. Co-culture of ADAS cells with fibroblasts that stably expressed the Notch ligand Jagged 1 or overexpression of the Notch intracellular domain (NICD) did not alter ADAS cell growth, morphology, or cellular marker expression. ADAS cells did not display robust expression of neural crest transcription factors or genes (Sox, CRABP2, and TH); and lineage tracing analyses using Wnt1–Cre;Rosa26R-lacZ or -EYFP reporter mice confirmed that fewer than 2% of the ADAS cell population derived from a Wnt1-positive population during development. In summary, although media formulations optimized for MSCs or NSCs enable expansion of mouse ADAS cells in vitro, we find no evidence that these cells are of neural crest origin, that they can undergo robust terminal differentiation into functionally mature neurons, and that Notch 1 is likely to be a key regulator of their cellular and molecular characteristics

Molecular basis for retinol binding by serum amyloid A during infection

Serum amyloid A (SAA) proteins are strongly induced in the liver by systemic infection and in the intestine by bacterial colonization. In infected mice, SAA proteins circulate in association with the vitamin A derivative retinol, suggesting that SAAs transport retinol during infection. Here we illuminate a structural basis for the retinol-SAA interaction. In the bloodstream of infected mice, most SAA is complexed with high-density lipoprotein (HDL). However, we found that the majority of the circulating retinol was associated with the small fraction of SAA proteins that circulate without binding to HDL, thus identifying free SAA as the predominant retinolbinding form in vivo. We then determined the crystal structure of retinol-bound mouse SAA3 at a resolution of 2.2 angstrom. Retinol-bound SAA3 formed a novel asymmetric trimeric assembly that was generated by the hydrophobic packing of the conserved amphipathic helices alpha 1 and alpha 3. This hydrophobic packing created a retinol-binding pocket in the center of the trimer, which was confirmed by mutagenesis studies. Together, these findings illuminate the molecular basis for retinol transport by SAA proteins during infection.Y

The basic leucine zipper transcription factor NFIL3 directs the development of a common innate lymphoid cell precursor

Abstract Innate lymphoid cells (ILCs) are recently identified lymphocytes that limit infection and promote tissue repair at mucosal surfaces. However, the pathways underlying ILC development remain unclear. Here we show that the transcription factor NFIL3 directs the development of a committed bone marrow precursor that differentiates into all known ILC lineages. NFIL3 was required in the common lymphoid progenitor (CLP), and was essential for the differentiation of αLP, a bone marrow cell population that gives rise to all known ILC lineages. Clonal differentiation studies revealed that CXCR6 + cells within the αLP population differentiate into all ILC lineages but not T-and B-cells. We further show that NFIL3 governs ILC development by directly regulating expression of the transcription factor TOX. These findings establish that NFIL3 directs the differentiation of a committed ILC precursor that gives rise to all ILC lineages and provide insight into the defining role of NFIL3 in ILC development

AAV-Dominant Negative Tumor Necrosis Factor (DN-TNF) Gene Transfer to the Striatum Does Not Rescue Medium Spiny Neurons in the YAC128 Mouse Model of Huntington's Disease

<div><p>CNS inflammation is a hallmark of neurodegenerative disease, and recent studies suggest that the inflammatory response may contribute to neuronal demise. In particular, increased tumor necrosis factor (TNF) signaling is implicated in the pathology of both Parkinson's disease (PD) and Alzheimer's disease (AD). We have previously shown that localized gene delivery of dominant negative TNF to the degenerating brain region can limit pathology in animal models of PD and AD. TNF is upregulated in Huntington's disease (HD), like in PD and AD, but it is unknown whether TNF signaling contributes to neuronal degeneration in HD. We used <i>in vivo</i> gene delivery to test whether selective reduction of soluble TNF signaling could attenuate medium spiny neuron (MSN) degeneration in the YAC128 transgenic (TG) mouse model of Huntington's disease (HD). AAV vectors encoding cDNA for dominant-negative tumor necrosis factor (DN-TNF) or GFP (control) were injected into the striatum of young adult wild type WT and YAC128 TG mice and achieved 30–50% target coverage. Expression of dominant negative TNF protein was confirmed immunohistologically and biochemically and was maintained as mice aged to one year, but declined significantly over time. However, the extent of striatal DN-TNF gene transfer achieved in our studies was not sufficient to achieve robust effects on neuroinflammation, rescue degenerating MSNs or improve motor function in treated mice. Our findings suggest that alternative drug delivery strategies should be explored to determine whether greater target coverage by DN-TNF protein might afford some level of neuroprotection against HD-like pathology and/or that soluble TNF signaling may not be the primary driver of striatal neuroinflammation and MSN loss in YAC128 TG mice.</p></div

Serum amyloid A delivers retinol to intestinal myeloid cells to promote adaptive immunity

Vitamin A and its derivative retinol are essential for the development of intestinal adaptive immunity. Retinoic acid (RA)-producing myeloid cells are central to this process, but how myeloid cells acquire retinol for conversion to RA is unknown. Here, we show that serum amyloid A (SAA) proteins-retinol-binding proteins induced in intestinal epithelial cells by the microbiota-deliver retinol to myeloid cells. We identify low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) as an SAA receptor that endocytoses SAA-retinol complexes and promotes retinol acquisition by RA-producing intestinal myeloid cells. Consequently, SAA and LRP1 are essential for vitamin A-dependent immunity, including B and T cell homing to the intestine and immunoglobulin A production. Our findings identify a key mechanism by which vitamin A promotes intestinal immunity.Y

Experimental Design.

<p>YAC128 TG and WT male and female mice received injections of AAV-DN-TNF-GFP (GFP is expressed from an IRES) or AAV-GFP (control) in the striatum at 2 months of age (green arrow). A cohort of animals underwent motor function testing on balance beam and rotarod (yellow arrowheads) at 3, 5, 7, 9, 11 and 12 months. Then, medium spiny neurons in the striatum were quantified by unbiased stereology (black arrowhead). At 3, 9 and 12 months of age, additional cohorts of animals were used to measure DN-TNF protein in the striatum by ELISA (red arrowheads) and for analysis of inflammation-related gene expression by qPCR and protein by multiplexed ELISA (blue arrowhead).</p

AAV-DN-TNF did not significantly reduce markers of inflammation in YAC128 TG mice.

<p>12-month TG or WT male and female mice that received AAV-DN-TNF or AAV-GFP viral vector injections at 2 months were compared using (A) quantitative PCR to measure expression of inflammatory markers in dissected striata. Mice injected with AAV-DNTNF had reduced CCL5 compared to those injected with AAV-GFP, regardless of genotype (main effect of AAV-injection: F(1,14) = 5.6, p<0.05). (B) Levels of inflammation-related proteins in serum in the same animals were compared by multiplexed ELISA. Transgenic mice had increased KC, regardless of AAV-injection (main effect: F(1,14), 12.7, p<0.05) whereas DNTNF reduced IFNγ protein levels in both WT and transgenic mice (main effect of AAV-injection: F(1,14) = 10, p<0.05). 2-way ANOVA.</p

YAC128 TG mice display a moderately increased neuroinflammatory profile relative to WT mice.

<p>(A) Quantitative PCR analysis using mRNA isolated from dissected striatum in unmanipulated YAC128 transgenic (TG) and WT male and female mice age 3, 9 and 12 months, relative to 3-month old WT animals. There is a significant interaction between age and genotype for MCP1 and GFAP (p = 0.0019, and p = 0.03, respectively), a significant effect of age for TNF, IL-6, and CD45 (p = 0.01, p = 0.02 and p = 0.03, respectively) and a significant effect of genotype for RANTES (p = 0.01). 2-way ANOVA, Bonferroni's post test, *p<0.05, **p<0.01, ***p<0.001. (B) Multiplex ELISA for inflammatory mediators in serum samples taken from 12-month un-operated mice (t-test, *p<0.05). (C) Quantification of activated microglia in the striatum in un-operated mice age 9 and 12 months. 2-way ANOVA (genotype, age) revealed no differences between groups.</p

AAV-DN-TNF gene transfer in the striatum did not reduce MSN degeneration in YAC128 TG mice.

<p>NeuN-positive neuronal nuclei in striata of 12-month animals were quantified using unbiased stereology and compared across treatment groups. YAC128 TG mice had significantly less neurons (main effect of genotype F(1,34) = 4.7, p<0.05) that were not affected by AAV-DNTNF injection (no significant interaction: F(1,34) = 0.99, p>0.05).</p