Repeated administration of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) modulates neuroinflammation and amyloid plaque load in mice bearing amyloid precursor protein and presenilin-1 mutant transgenes

BACKGROUND: Data indicates anti-oxidant, anti-inflammatory and pro-cognitive properties of noradrenaline and analyses of post-mortem brain of Alzheimer's disease (AD) patients reveal major neuronal loss in the noradrenergic locus coeruleus (LC), the main source of CNS noradrenaline (NA). The LC has projections to brain regions vulnerable to amyloid deposition and lack of LC derived NA could play a role in the progression of neuroinflammation in AD. Previous studies reveal that intraperitoneal (IP) injection of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) can modulate neuroinflammation in amyloid over-expressing mice and in one study, DSP-4 exacerbated existing neurodegeneration. METHODS: TASTPM mice over-express human APP and beta amyloid protein and show age related cognitive decline and neuroinflammation. In the present studies, 5 month old C57/BL6 and TASTPM mice were injected once monthly for 6 months with a low dose of DSP-4 (5 mg kg(-1)) or vehicle. At 8 and 11 months of age, mice were tested for cognitive ability and brains were examined for amyloid load and neuroinflammation. RESULTS: At 8 months of age there was no difference in LC tyrosine hydroxylase (TH) across all groups and cortical NA levels of TASTPM/DSP-4, WT/Vehicle and WT/DSP-4 were similar. NA levels were lowest in TASTPM/Vehicle. Messenger ribonucleic acid (mRNA) for various inflammatory markers were significantly increased in TASTPM/Vehicle compared with WT/Vehicle and by 8 months of age DSP-4 treatment modified this by reducing the levels of some of these markers in TASTPM. TASTPM/Vehicle showed increased astrocytosis and a significantly larger area of cortical amyloid plaque compared with TASTPM/DSP-4. However, by 11 months, NA levels were lowest in TASTPM/DSP-4 and there was a significant reduction in LC TH of TASTPM/DSP-4 only. Both TASTPM groups had comparable levels of amyloid, microglial activation and astrocytosis and mRNA for inflammatory markers was similar except for interleukin-1 beta which was increased by DSP-4. TASTPM mice were cognitively impaired at 8 and 11 months but DSP-4 did not modify this. CONCLUSION: These data reveal that a low dose of DSP-4 can have varied effects on the modulation of amyloid plaque deposition and neuroinflammation in TASTPM mice dependent on the duration of dosing

Perturbation of microRNAs in Rat Heart during Chronic Doxorubicin Treatment

<div><p>Anti-cancer therapy based on anthracyclines (DNA intercalating Topoisomerase II inhibitors) is limited by adverse effects of these compounds on the cardiovascular system, ultimately causing heart failure. Despite extensive investigations into the effects of doxorubicin on the cardiovascular system, the molecular mechanisms of toxicity remain largely unknown. MicroRNAs are endogenously transcribed non-coding 22 nucleotide long RNAs that regulate gene expression by decreasing mRNA stability and translation and play key roles in cardiac physiology and pathologies. Increasing doses of doxorubicin, but not etoposide (a Topoisomerase II inhibitor devoid of cardiovascular toxicity), specifically induced the up-regulation of miR-208b, miR-216b, miR-215, miR-34c and miR-367 in rat hearts. Furthermore, the lowest dosing regime (1 mg/kg/week for 2 weeks) led to a detectable increase of miR-216b in the absence of histopathological findings or alteration of classical cardiac stress biomarkers. <em>In silico</em> microRNA target predictions suggested that a number of doxorubicin-responsive microRNAs may regulate mRNAs involved in cardiac tissue remodeling. In particular miR-34c was able to mediate the DOX-induced changes of Sipa1 mRNA (a mitogen-induced Rap/Ran GTPase activating protein) at the post-transcriptional level and in a seed sequence dependent manner. Our results show that integrated heart tissue microRNA and mRNA profiling can provide valuable early genomic biomarkers of drug-induced cardiac injury as well as novel mechanistic insight into the underlying molecular pathways.</p> </div

Ambra1 expression was induced by DOX treatment <i>in vivo</i> and miR-34c could control its endogenous expression levels in H9c2 cardiac myoblasts.

<p>(A) Fold change of Ambra1 probe set in rat heart tissue treated with DOX. Fold change and statistical significance were assessed <i>vs.</i> each vehicle group. n = 4 to 6 (except #, n = 2) (B) Endogenous levels of Ambra1 were measured after miR-34c over-expression (miR-34c mimic) or inhibition (miR-34c HI) in absence of presence of DOX 0.1. Fold change value were normalized <i>vs.</i> the respective negative transfection controls in the untreated condition (n = 3). *P<0.05, **P<0.01, ***P<0.005. FC  =  fold change.</p

Chronic DOX treatment (3 mg/kg/week) alters levels of 25 microRNAs from week 2 onwards.

<p>Variation of cardiac microRNA levels versus vehicle are reported for animals treated with DOX 3 mg/kg/week for 2 and 4 weeks. Values were calculated via the relative quantification (ΔΔCt) method by using the mammalian U6 snRNA as a normalizer. MicroRNAs showing same trend at 2 and 4 weeks are italicized. # indicates microRNAs selected for further analysis. Significant P values (<0.05) are in bold. FC  =  fold change.</p

Study design and representative micrograph showing DOX-related vacuolation in the myocardium.

<p>(A) Six adult male rats were injected with the indicated doses of vehicle, doxorubicin (DOX), dexrazoxane (DZR), etoposide (EPS) or a combination of DOX and DZR for 2, 4 or 6 weeks. Cardiac tissue was excised and deep frozen for gene expression and microRNA profiling experiments. A representative micrograph of a toluidine blue stained myocardial section of a control (B) and of a DOX treated animal (C). Black arrows indicate sarcoplasmic micro- and macro- vacuolation of cardiomyocytes.</p

DOX 3 mg/kg/week altered levels of genomic cardiomyopathy indicators (Ankrd/Carp, Nppb, Myh7 and Myh6).

<p>Expression fold change relative to vehicle were represented for DOX 3 mg/kg/week at 2, 4 and 6 weeks time point (n = 6) for (A) Ankrd/Carp, (B) Nppb, (C) Myh7 and (D) Myh6. For each time point and each probe set, vehicle values were averaged and normalized to 1. The same correction was applied to the DOX treated values. Affymetrix probe-set number is indicated in brackets. Error bars represent standard deviation. T-test was performed for vehicle- vs. DOX-treated at each time point. *P<0.05, **P<0.01, ***P<0.005, NS = Non-Significant. (No t-test for #, as n = 2).</p

Relative quantification of DOX-responder microRNAs in rat heart across all groups.

<p>Relative quantification of (A) miR-208b, (B) miR-215, (C) miR-216b, (D) miR-367 and (E) miR-34c in DOX, DOX + DZR, EPS groups, normalized versus vehicle treated animals. Expression levels were measured by single assay qPCR (n = 3, except #, n = 2). DOX: Doxorubicin, DZR: dexrazoxane, EPS: etoposide; numbers indicate the weekly dose of each compound in mg/kg/week. Empty spaces represent non-sampled animals. The vehicle treated is the first column of each time-point. The animals used in this experiment were distinct from the ones represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040395#pone-0040395-t001" target="_blank">Table 1</a>. Error bars represent SD. T-test results are indicated by asterisks for significant DOX-treated groups vs. their own vehicle-treated, unless otherwise specified by horizontal range bars; *P<0.05, **P<0.01, ***P<0.005, NS = Non-Significant).</p

DOX-induced upregulation of miR-216 and miR-367 expression precedes the detection of overt histopathological lesions in cardiac tissue.

<p>Blue bars show cumulative vacuolation grade. X axis shows the DOX regimen in mg/kg/week received by the animals at 2 weeks. Y axes report cumulative histopathological scores and microRNA fold change vs. untreated cardiac tissues (normalized at value 1). Path grading  =  cumulative vacuolation score. FC  =  fold change. SEM  =  standard error on the mean.</p