Short-term odor habituation was impaired in Tg2576 mice compared to WT controls.

<p>For example, as shown here, 12 MO Tg2576 mice (n = 12) showed less habituation over the course of four repeated odor stimuli than age-matched WT controls (n = 12).</p

Behavioral discrimination of overlapping mixtures in WT mice.

<p>The pseudo-color panels (color corresponds to proportion correct) show performance for individual mice in each task. Mean performance for each task (top right). Different mice were used for each task. As previously shown in rats, the 10c vs. 10cR1 discrimination was significantly easier to acquire than 10c vs 10c-1. However, so few individual animals acquired the 10c vs. 10c-1 task it was not feasible to use it to test the effects of APP over-expression. There was no significant difference between WT and Tg2576 mice in performance on an odor mixture discrimination task (10c vs. 10cR1) across age (bottom right). Of the initial 7 WT and 6 Tg2576 mice, 1 WT and 2 Tg2576 animals died prior to 12 months and are not included here. Animals were trained in the two-alternative forced choice task prior to 5 months of age and then tested bimonthly until 12 months. Initial acquisition of the discrimination was not affected by genotype. Furthermore, there was no significant effect of genotype on performance of this well learned odor discrimination task through 12 months of age.</p

Single unit activity in Tg2576 versus age-matched WT mice at 3, 6 and 12 MO.

<p>Tg2576 showed a trend towards higher baseline activity (A) versus age-matched WT (<i>p</i> = .06) but no difference in highest odor-evoked response (B). Unit entrainment to respiration (C) was diminished in Tg2576, though this did not emerge until 12 MO (* = <i>p</i><.05). Data presented as mean ± SEM.</p

Summary of data obtained for 20+ month old Tg2576.

<p>(A) Mice over 20 months of age showed abundant thioflavin S positive staining in (from left to right) OB, anterior piriform cortex (aPCX), hippocampus (HPX) and lateral entorhinal cortex (LEC). Scale bar is 500 µm. (B) Thioflavin S area fractions for 20MO animals in OB, aPCX, HPX and LEC. (C) 20 MO Tg2576 showed no difference in baseline or maximal odor-evoked unit activity and (D) no difference in unit entrainment respiration. (E) Finally, no difference was observed in single-unit receptive field specificity compared to age-matched WT.</p

Single unit receptive fields in aPCX.

<p>(A) Representative single-unit response to stimulus set. Rasters represent unit activity from a single cell tested with multiple odors and histogram indicates tally of rasters for each odor. Shaded area indicates 2 seconds starting at the onset of stimulus delivery. (B) Odor receptive fields in Tg2576 versus age-matched WT at 3, 6 and 12 MO. X-axis is odor stimuli organized by response strength. Y-axis is odor-evoked spikes per second normalized to the highest response of the six odors. Tg2576 single-units showed no difference in receptive field specificity compared to age-matched WT mice.</p

MiR-21 in Extracellular Vesicles Leads to Neurotoxicity via TLR7 Signaling in SIV Neurological Disease

<div><p>Recent studies have found that extracellular vesicles (EVs) play an important role in normal and disease processes. In the present study, we isolated and characterized EVs from the brains of rhesus macaques, both with and without simian immunodeficiency virus (SIV) induced central nervous system (CNS) disease. Small RNA sequencing revealed increased miR-21 levels in EVs from SIV encephalitic (SIVE) brains. In situ hybridization revealed increased miR-21 expression in neurons and macrophage/microglial cells/nodules during SIV induced CNS disease. In vitro culture of macrophages revealed that miR-21 is released into EVs and is neurotoxic when compared to EVs derived from miR-21^-/- knockout animals. A mutation of the sequence within miR-21, predicted to bind TLR7, eliminates this neurotoxicity. Indeed miR-21 in EV activates TLR7 in a reporter cell line, and the neurotoxicity is dependent upon TLR7, as neurons isolated from TLR7^-/- knockout mice are protected from neurotoxicity. Further, we show that EVs isolated from the brains of monkeys with SIV induced CNS disease activates TLR7 and were neurotoxic when compared to EVs from control animals. Finally, we show that EV-miR-21 induced neurotoxicity was unaffected by apoptosis inhibition but could be prevented by a necroptosis inhibitor, necrostatin-1, highlighting the actions of this pathway in a growing number of CNS disorders.</p></div

In vitro neurotoxicity assays with exosomes from bone marrow derived macrophage (BMDM) cultures.

<p><b>(A)</b> Quantitative reat-time PCR (qRT-PCR) for miR-21 was performed on EVs isolated from WT and miR-21^-/- BMDMs. Raw CT values confirm the absence of miR-21 in the EVs isolated from miR-21^-/- BMDMs. <b>(B)</b> WT mouse hippocampal neurons were incubated with 1 μg of EVs isolated from WT (WT-Exo) and miR-21 ^-/- (miR-21KO-Exo) littermate BMDMs for 24 hr. LDH assay was performed to assess the neuronal viability Results indicate a significantly higher in cell death with WT EVs than with miR-21^-/- EVs. Statistical analyses were performed on data from six independent experiments. Error bars = SEM; ^**<i>P</i> < 0.01; unpaired t-test. <b>(C)</b> Cultured hippocampal neurons (DIV 7) from WT and TLR7-/- mice were treated with CL075 (6μM) and vehicle for 6h and harvested for real time PCR using GAPDH as an internal control to quantify the levels of IL6 and TNFα. Error bars = SEM; ^*<i>P</i> < 0.05; ****P < 0.0001; Two-way ANOVA with Bonferroni post-hoc test. <b>(D)</b> LDH assay was performed on WT and TLR7^-/- mice hippocampal cultures with WT and miR-21^-/- littermate BMDM derived EVs. A significant increase in neuronal cell death is seen with WT-EVs when compared to miR-21^-/- EVs. No miR-21-EV induced toxicity was found when hippocampal neurons from TLR7^-/- mice were used. Error bars = SEM; ^**<i>P</i> < 0.01; Two-way ANOVA with Bonferroni post-hoc test.</p

Isolation and characterization of brain derived EVs.

<p><b>(A)</b> Left, Electron microscopic (TEM) morphological analysis of EVs derived from uninfected (control) macaque brain. EVs show a size range from 100–150 nm. Scale bar = 100 nm. Right, Western blots for flotillin, CD9, CD63, CD81, HSP70, TSG101 markers for EVs. Non-EV fractions from sucrose gradients were used as negative controls for the EV proteins, brain lysates were used as positive controls for the synaptic proteins. <b>(B)</b> Small RNA sequencing performed on RNA isolated from uninfected, SIV and SIVE brains. Analysis revealed significantly increased expression of miR-21-5p, miR-100-5p and miR-146-5p, and decreased expression miR-126-5p, in SIVE. Error bars = SEM; * <i>P</i> <0.05, ** <i>P</i> <0.01*** <i>P</i> <0.001, **** <i>P</i> < 0.0001; ANOVA with Tukey’s post-hoc test. <b>(C)</b> qRT-PCR validation of miR-21 expression in EVs. Relative quantification was performed based on a standard curve. Statistical analyses were performed on log-transformed values. One-way ANOVA showed p = 0.0024 with Tukey’s <0.01 for uninfected vs. SIVE, and SIV vs. SIVE.</p

In vitro neurotoxicity assays with artificial EVs.

<p><b>(A)</b> Wildtype (WT) mouse hippocampal neurons were incubated with 1 μg of synthetic miRNAs; miR-21 (miR21-WT), miR-21 containing a mutation in TLR7 binding site (miR21-Mut) and a known TLR7 activator, Let-7b, and DOTAP artificial EVs. Neurons were incubated for 24 hr and then stained with NeuN, a cell body marker for neurons (Left). The number of NeuN positive neurons was counted and the relative neuronal viability to untreated cultures was calculated (Middle). The result indicates no significant cell death by naked synthetic miRNAs. LDH assay was performed to assess the neuronal viability (Right). Results indicate no difference in cell death. <b>(B)</b> Synthetic miRNAs were mixed with DOTAP liposomal formulations creating “artificial EVs” and WT hippocampal neurons were incubated with 1 μg of synthetic miRNAs within artificial EVs for 24 hr. NeuN staining was performed, and the results indicated increased neuronal loss as seen in fewer numbers of green NeuN positive neurons in miR21-WT and Let-7b treated cultures when compared to miR21-mut and DOTAP treated hippocampal cultures (Left). Quantification (middle bar graph) shows a significant cell death in cultures treated with miR-21-WT and Let-7b when compared to DOTAP control. LDH assay was performed to assess the neuronal viability. Results indicate a significantly higher cell death with miR-21-WT and Let-7b than with miR-21-Mut and DOTAP control. Statistical analyses were performed on data from six independent experiments for NeuN counting and three independent experiments for LDH assay. Error bar = SEM; ^***<i>P</i><0.001; One-way ANOVA with Dunnett’s post-hoc test. <b>(C)</b> Immunostaining was performed for the neuronal (neurite) marker, MAP2 and staining reveals loss of neurites in cultures treated with miR-21-WT and Let-7b artificial EVs compared to DOTAP only or in the miR-21-Mut treated cultures. Statistical analyses were performed on data from three independent experiments. Error bar = SEM; ^***<i>P</i><0.001; One-way ANOVA with Dunnett’s post-hoc test. Scale bar = 20 μm.</p

Combined FISH and IF for miR-21, CD163 and GFAP.

<p>In SIVE brain sections containing macrophage/microglial nodules, miR-21 (magenta) partially colocalized with the macrophage/microglia marker CD163 (green). No colocalization was observed with GFAP (red), an astrocyte marker. DAPI (blue) was used to label nuclei; a scrambled miRNA probe (Scrm) was used as negative control for hybridization; and U6 probe (a non-coding snRNA) was used as a positive control. Scale bars = 20 μm for all panels except 5 μm for SIVE-Mag panels.</p