De Novo Generation of Murine and Human MADR Recipient Cell Lines for Locus-Specific, Stable Integration of Transgenic Elements

Mosaic analysis by dual recombinase-mediated cassette exchange (MADR) is a technology that allows stable and locus-specific integration of transgenic elements into recipient cells carrying loxP and FRT sites. Nevertheless, most cell lines lack these recombination-specific sites. This protocol describes a method to introduce the minimum requirements into cells, leading to the generation of de novo primary MADR recipient cells or MADR "Proxy" cells. These cell lines allow the combinatorial use of a wide range of transgenic elements through MADR. For complete details on the use and execution of this protocol, please refer to Kim et al. (2019)

Oral TNFα Modulation Alters Neutrophil Infiltration, Improves Cognition and Diminishes Tau and Amyloid Pathology in the 3xTgAD Mouse Model.

Cytokines such as TNFα can polarize microglia/macrophages into different neuroinflammatory types. Skewing of the phenotype towards a cytotoxic state is thought to impair phagocytosis and has been described in Alzheimer's Disease (AD). Neuroinflammation can be perpetuated by a cycle of increasing cytokine production and maintenance of a polarized activation state that contributes to AD progression. In this study, 3xTgAD mice, age 6 months, were treated orally with 3 doses of the TNFα modulating compound isoindolin-1,3 dithione (IDT) for 10 months. We demonstrate that IDT is a TNFα modulating compound both in vitro and in vivo. Following long-term IDT administration, mice were assessed for learning & memory and tissue and serum were collected for analysis. Results demonstrate that IDT is safe for long-term treatment and significantly improves learning and memory in the 3xTgAD mouse model. IDT significantly reduced paired helical filament tau and fibrillar amyloid accumulation. Flow cytometry of brain cell populations revealed that IDT increased the infiltrating neutrophil population while reducing TNFα expression in this population. IDT is a safe and effective TNFα and innate immune system modulator. Thus small molecule, orally bioavailable modulators are promising therapeutics for Alzheimer's disease

Early intervention with a small molecule inhibitor for tumor necrosis factor-alpha prevents cognitive deficits in a triple transgenic mouse model of Alzheimer's disease

AbstractBackgroundChronic neuroinflammation is an important component of Alzheimer’s disease and could contribute to neuronal dysfunction, injury and loss that lead to disease progression. Multiple clinical studies implicate tumor necrosis factor-α as an inflammatory mediator of neurodegeneration in patients with Alzheimer’s because of elevated levels of this cytokine in the cerebrospinal fluid, hippocampus and cortex. Current Alzheimer’s disease interventions are symptomatic treatments with limited efficacy that do not address etiology. Thus, a critical need exists for novel treatments directed towards modifying the pathophysiology and progression.MethodsTo investigate the effect of early immune modulation on neuroinflammation and cognitive outcome, we treated triple transgenic Alzheimer’s disease mice (harboring PS1M146V, APPSwe, and tauP301L transgenes) with the small molecule tumor necrosis factor-α inhibitors, 3,6′-dithiothalidomide and thalidomide, beginning at four months of age. At this young age, mice do not exhibit plaque or tau pathology but do show mild intraneuronal amyloid beta protein staining and a robust increase in tumor necrosis factor-α. After 10 weeks of treatment, cognitive performance was assessed using radial arm maze and neuroinflammation was assessed using biochemical, stereological and flow cytometric endpoints.Results3,6′-dithiothalidomide reduced tumor necrosis factor-α mRNA and protein levels in the brain and improved working memory performance and the ratio of resting to reactive microglia in the hippocampus of triple transgenic mice.In comparison to non-transgenic controls, triple transgenic Alzheimer’s disease mice had increased total numbers of infiltrating peripheral monomyelocytic/granulocytic leukocytes with enhanced intracytoplasmic tumor necrosis factor-α, which was reduced after treatment with 3,6′-dithiothalidomide.ConclusionsThese results suggest that modulation of tumor necrosis factor-α with small molecule inhibitors is safe and effective with potential for the long-term prevention and treatment of Alzheimer’s disease

Fold regulation (compared to 3xTg Con mice) values for real time PCR.

<p>Data shown as mean ± SEM. Bold font indicates P < 0.05 compared to CON diet 3xTgAD mice. ARG1, arginase 1; FcγR, Fc gamma receptor; IL, interleukin; SPHK1, sphingosine kinase 1; TGF, transforming growth factor. N = 3–6/group.</p><p>Fold regulation (compared to 3xTg Con mice) values for real time PCR.</p

Effect of IDT effect on Aβ.

<p>Following 38 weeks of IDT treatment, analysis of 6E10 (APP/Aβ) or Aβ42 immunolabeling in the hippocampus of 16 month-old 3xTgAD mice was performed on 50 μm sections, every 12th section through the hippocampus, by densitometry and % area covered by immunostaining. (A) Representative pictures of 6E10 staining in each treatment group (10x magnification; Bar equals 50 μm) with corresponding % Area and densitometry. Similarly, Aβ42 immunolabeling and quantification is shown in (B). Elimination of the primary antibody from the staining protocol resulted in loss of specific staining (data not shown). Data represent mean ± SEM of n = 8–10/group. (C) Results from soluble and insoluble hippocampal fractions analyzed by Aβ40 or 42-specific ELISA. One-way ANOVA did not reveal an overall significant effect of IDT treatment on either soluble Aβ40 (p = 0.0604) or Aβ42 (p = 0.1181). High dose IDT did significantly reduce soluble Aβ40 versus Con (*p < 0.05). There was an overall significant effect of IDT dose on insoluble Aβ40 levels (p = 0.0135) but insoluble Aβ42 did not quite reach significance (p = 0.0649). Both Low and High dose IDT reduced insoluble Aβ40 versus Con (*p< 0.05 and **p< 0.01). High dose IDT reduced insoluble Aβ42 (*p<0.05).</p

BACE1 Protein.

<p>(A) Representative western blot of hippocampal homogenates for BACE1. (B) Densitometry results for BACE1 and Actin were obtained and the changes in BACE1/Actin expressed as % Control. No significant effect of IDT was observed.</p

Schematic of IDT’s proposed mechanism of action.

<p>IDT destabilizes TNFα mRNA and reduces TNFα protein levels. Reduction of TNFα alters the expression of M1/M2 (N1/N2) genes and produces a novel mixed M1/M2 (N1/N2) phenotype. This may allow for improved microglia/macrophage (blue stars) function, recruiting of healthy neutrophils (beige circles) and improvement (+) of CNS reparative processes.</p

IDT increases p40 gene expression.

<p>Relative gene expression for a diverse array of phenotypic markers. Data are shown as fold regulation relative to Con 3xTgAD mice. ARG1, arginase 1; FcγR, Fc gamma receptor; IL, interleukin; SPHK1, sphingosine kinase 1; TGF, transforming growth factor. All gene fold regulation values are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137305#pone.0137305.t001" target="_blank">Table 1</a> as mean ± SEM of N = 3–6/group.</p

Effects of isoindolin-1,3 dithione (IDT) on TNFα protein and mRNA.

<p>(A) Structure of IDT. (B) Initial studies in BV2 cells demonstrate that IDT is effective at attenuating LPS-induced TNFα release into culture media (One-way ANOVA, p < 0.0001. p < 0.001 LPS vs. all IDT doses) with an IC₅₀ value of ~5 μM. Green bars at the top of the y-axis and right side of the x-axis indicate the 100% LPS and no LPS (NS) responses, respectively. n = 4/group. (C) Lactate dehydrogenase (LDH) release into the culture media was measured after 24 hr exposure to LPS ± IDT. No significant increase in LDH was detected compared to DMSO control. (D) Effect of IDT on TNFα mRNA stability in BV-2 cells. Cells were pre-treated for 4 hr with 100 ng/mL LPS followed by treatment with 25 μM IDT and 5 mg/mL actinomycin D (ActD) and collected at intervals of 15 min. Total RNAs were isolated and TNFα mRNA was quantified using qPCR. Values represent mean ± s.e.m. for two separate experiments performed in triplicate and relative quantification was performed using the Rq method 2 ^-ΔΔCt. Asterisks indicate significant difference from LPS/ActD-treated BV-2 cells (**P < 0.01). n = 3/group. (E) IDT inhibited LPS-stimulated Cortical TNFα protein expression in vivo. Mice were treated by oral gavage with a single dose (50 mg/kg) of IDT 30 minutes prior to a peripheral 5 mg/kg dose of LPS (i.p.). Cortical tissue was harvested 4 hours after LPS injection. n = 6/group. One-way ANOVA, p < 0.001; ***p < 0.001 vs. Con and **p < 0.01 vs. LPS. Data represent mean ± SEM of n = 3/group.</p

Flow cytometric analysis of leukocytes recovered from the brains of Con and IDT-treated 3xTgAD mice.

<p>(A) Representative flow cytometry profiles of the cell surface markers and gating strategy are shown for Con and Low IDT brains. (B) The percentage of leukocytes (CD45Hi+), microglia (CD45Dim+), granulocytes (CD45Hi+/CD11b+/GR1+) and neutrophils (CD45Hi+/CD11b+/GR1+/1A8+) among all cells analyzed across the four treatment groups. One-way ANOVA of the neutrophil populations revealed a significant IDT effect (F_{2, 10} = 6.468, P = 0.0023). Both Low/Med and High IDT treatment groups significantly increased neutrophil infiltration into the brain relative to Con 3xTgAD brains (*p < 0.05; **p < 0.01).</p