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Cell Cycle
ISSN: 1538-4101 (Print) 1551-4005 (Online) Journal homepage: https://www.tandfonline.com/loi/kccy20
Involvement of peroxisome proliferator-activated
receptor
β
/
δ
(PPAR
β
/
δ
) in BDNF signaling during
aging and in Alzheimer disease: Possible role of
4-hydroxynonenal (4-HNE)
Elisabetta Benedetti, Barbara D'Angelo, Loredana Cristiano, Erica Di
Giacomo, Francesca Fanelli, Sandra Moreno, Francesco Cecconi, Alessia
Fidoamore, Andrea Antonosante, Roberta Falcone, Rodolfo Ippoliti, Antonio
Giordano & Annamaria Cimini
To cite this article: Elisabetta Benedetti, Barbara D'Angelo, Loredana Cristiano, Erica Di Giacomo,
Francesca Fanelli, Sandra Moreno, Francesco Cecconi, Alessia Fidoamore, Andrea Antonosante, Roberta Falcone, Rodolfo Ippoliti, Antonio Giordano & Annamaria Cimini (2014) Involvement of peroxisome proliferator-activated receptor β/δ (PPAR β/δ) in BDNF signaling during aging and in Alzheimer disease: Possible role of 4-hydroxynonenal (4-HNE), Cell Cycle, 13:8, 1335-1344, DOI: 10.4161/cc.28295
To link to this article: https://doi.org/10.4161/cc.28295
Published online: 04 Mar 2014. Submit your article to this journal
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RepoRtCell Cycle 13:8, 1335–1344; April 15, 2014; © 2014 Landes Bioscience
RepoRt
Introduction
Aging is a multifactorial process characterized by a broad func-tional decline, including cognitive impairment, associated with alterations to neocortical and hippocampal areas, both involved in learning and memory processes. In general, cognitive pro-cesses require neuronal plasticity, which markedly decreases dur-ing agdur-ing and in neurodegenerative disorders, such as Alzheimer disease (AD). AD is the most common form of dementia, char-acterized by a progressive loss of synapses and neurons, result-ing in gradual impairment of memory, language, and reasonresult-ing skills.1 The main histopathological features of AD are extracellu-lar plaques, formed by amyloid-β (Aβ) deposits and intracelluextracellu-lar neurofibrillary tangles of hyperphosphorylated tau protein. Both types of deposits are caused by the conversion of soluble forms of the proteins into insoluble, fibrillary polymers.2,3
Aging and many neurological disorders are linked to oxidative stress, which is considered the common effector of the cascade of degenerative events.4,5 In this phenomenon, reactive oxygen spe-cies (ROS) play a fundamental role in the oxidative decomposition
of polyunsaturated fatty acids (i.e., lipid peroxidation), resulting in the formation of a complex mixture of aldehydic end products, such as malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and other alkenals.6,7 Among such aldehydes, 4-HNE has become the most studied cytotoxic product of lipid peroxidation8 and is considered one of the main signaling molecules in the pathogen-esis of neurodegenerative diseases. Moderate 4-HNE concentra-tions can induce differentiation, damage to proteasome,9,10 and apoptosis11,12 by modifying signal transduction, including activa-tion of adenylate cyclase, JNK, PKC, and caspase 3, while lower concentrations of the aldehyde appear to even promote cell pro-liferation and survival.8,13
Interestingly, 4-HNE has been also indicated as an intracel-lular agonist of peroxisome proliferator-activated receptor β/δ (PPAR β/δ).14
PPARs, which comprise isotypes α, β/δ, and γ, are ligand-activated transcription factors playing important physiological and pathological roles in different tissues, including the nervous tissue, both during development and in the pathogenesis of vari-ous disorders. Indeed, their involvement in neurodegenerative
*Correspondence to: Annamaria Cimini; Email: annamaria.cimini@univaq.it; Antonio Giordano; Email: antonio.giordano@unisi.it Submitted: 01/20/2014; Revised: 02/18/2014; Accepted: 02/19/2014; Published Online: 03/04/2014
http://dx.doi.org/10.4161/cc.28295
Involvement of peroxisome proliferator-activated
receptor β/δ (PPAR β/δ) in BDNF signaling
during aging and in Alzheimer disease
Possible role of 4-hydroxynonenal (4-HNE)
elisabetta Benedetti1, Barbara D’Angelo1, Loredana Cristiano1, erica Di Giacomo1, FrancescaFanelli2, Sandra Moreno2, Francesco Cecconi3, Alessia Fidoamore1, Andrea Antonosante1,Roberta Falcone1, Rodolfo Ippoliti1, Antonio Giordano4,5,*,
and Annamaria Cimini1,5,*
1Department of Life, Health, and environmental Sciences; University of L’Aquila; Coppito L’Aquila, Italy; 2Department of Science-LIMe; Roma tre University; Rome, Italy; 3Department of Biology; University of Rome “tor Vergata”; Rome, Italy; 4Department of Medical and Surgical Sciences and Neurosciences; University of Siena; Siena, Italy;
5Sbarro Institute for Cancer Research and Molecular Medicine and Center for Biotechnology; temple University; philadelphia, pA USA
Keywords: aging, neurodegenerative disease, transcription factors, oxidative stress, BDNF, TrkB, p75, JNK
Aging and many neurological disorders, such as AD, are linked to oxidative stress, which is considered the common effector of the cascade of degenerative events. In this phenomenon, reactive oxygen species play a fundamental role in the oxidative decomposition of polyunsaturated fatty acids, resulting in the formation of a complex mixture of aldehydic end products, such as malondialdehyde, 4-hydroxynonenal, and other alkenals. Interestingly, 4-HNe has been indicated as an intracellular agonist of peroxisome proliferator-activated receptor β/δ. In this study, we examined, at early and advanced AD stages (3, 9, and 18 months), the pattern of 4-HNe and its catabolic enzyme glutathione S-transferase p1 in relation to the expression of ppARβ/δ, BDNF signaling, as mRNA and protein, as well as on their pathological forms (i.e., precursors or truncated forms). the data obtained indicate a novel detrimental age-dependent role of ppAR β/δ in AD by increasing pro-BDNF and decreasing BDNF/trkB survival pathways, thus pointing toward the possibility that a specific ppARβ/δ antagonist may be used to counteract the disease progression.
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diseases, such as multiple sclerosis, amyotrophic lateral scle-rosis, Alzheimer, Parkinson, and Huntington diseases, is well recognized.15-22
Even though PPAR β/δ is the most abundant isotype in the developing and adult central nervous system (CNS),23-25 its role in neurodegenerative diseases remains unclear. We have previously demonstrated that PPAR β/δ is crucial for neuronal maturation, and that its expression affects the BDNF signaling pathway.26,27
Neurotrophins and their receptors are expressed in brain areas involved in plasticity (i.e., the hippocampus, cerebral cortex) and are considered the mediators of synaptic plasticity. The expres-sion of BDNF and its receptors, the full-length catalytic receptor (TrkB-fl), the truncated isoform (TrkB-t), lacking intracellular tyrosine kinase activity, and the unselective low-affinity p75NGFR receptor has been described during normal brain aging and in Alzheimer disease.28
The aim of the present work was to investigate the role of 4-HNE and PPAR β/δ in relation to BDNF signaling during AD progression and in physiological aging. To this purpose, we used the Tg2576 (Tg) mouse model, as compared with its wild-type (Wt) counterpart. Differently from other AD mouse models, this strain is characterized by a slowly progressive pathol-ogy, offering the opportunity to study even subtle age-dependent alterations.17,29,30,31 In the present study, we focused on the neo-cortex, which is more exposed to ROS, compared with other brain regions, owing to its high aerobic metabolism and content in PUFAs and redox-active transition metals.5,32 To this pur-pose, we examined at early and advanced AD stages (3, 9, and 18 mo) the pattern of 4-HNE and its catabolic enzyme glutathi-one S-transferase P1 (GSTP1)33 in relation to the expression of PPARβ/δ, BDNF, and its receptors, as mRNA and protein, as well as on their pathological forms (i.e., precursors or truncated forms).
The data obtained indicate a detrimental age-dependent role of PPAR β/δ in AD by increasing pro-BDNF and decreasing BDNF/TrkB survival pathway, thus suggesting that a specific PPARβ/δ antagonist may be used to counteract the disease progression.
Results
Oxidative damage has been reported in AD mice17,22,34,35 and lipid peroxidation products, such as 4-HNE,36 have been found to play an important role in AD-related neurodegenera-tion. Since 4-HNE has been recognized as an endogenous PPAR β/δ-ligand,14 the levels of its protein adducts were investigated. Immunofluorescence and WB results (Fig. 1A and B) show that HNE adduct levels in 9- and 18-mo-old neocortex are higher in Tg mice than in their Wt counterparts. At these ages, an addi-tional 48/52-kDa band, denser in the Tg than in Wt samples, is observed. By contrast, at the onset of disease (3 mo), 4-HNE lev-els are much lower in Tg than in Wt neocortex. These data may be related to the expression pattern of GSTP-1 (Fig. 1C), known to remove 4-HNE.33 In fact, in 3-mo-old Tg animals, the enzymatic protein is upregulated, while at 9 and 18 mo, it appears progres-sively downregulated. This event does not happen in Wt animals,
where the GSTP1 levels appear upregulated during aging, and not significant increases of HNE adducts are observed.
In Figure 2A and B the RT-PCR and WB analyses of PPAR β/δ in the neocortex of Wt and Tg mice at the age of 3, 9, and 18 mo are shown. It is possible to observe an increase of the tran-scription factor during normal and pathological aging. When comparing the 2 genotypes, upregulation of PPAR β/δ mRNA (at 3 mo) and of respective protein levels (at all ages) in Tg neo-cortex is detected. PPAR β/δ immunofluorescence in 18-mo-old samples shows a prevalent, perinuclear localization in Wt cortical neurons, while appearing more concentrated in discrete peri- and intra-nuclear regions in the Tg animals (Fig. 2C).
Since we previously demonstrated26,27 that PPAR β/δ activa-tion results in a decrease of the BDNF-specific receptor, TrkB full-length (TrkBfl), RT-PCR, and WB analyses for TrkBfl and its truncated form (TrkB-t) were performed (Fig. 3A–C). Levels of mRNA (including both full-length and truncated forms) dis-play no genotype-based differences, while significantly decreas-ing at the latest stage considered. On the other hand, WB results of TrkBfl and TrkB-t show different patterns depending on the age and genotype. TrkBfl protein levels progressively decrease during pathological aging and are consistently lower in Tg ani-mals than in Wt. By contrast, the truncated form significantly increases with age, being always more abundant in Tg than in Wt cortices. Immunofluorescence (Fig. 3D) shows a decrease in Trkfl positivity, particularly at neuritic level in 9-mo-old Tg animals.
Regarding BDNF, particular attention was devoted to its immature form (pro-BDNF), owing to its negative effects on cell survival.37 This protein strongly increases in Tg animals at 18 mo, reaching significantly higher levels with respect to Wt. A partially different pattern is displayed by the mature form (mBDNF), which decreases during aging in Wt mice (Fig. 4A and B); in the Tg neocortex, mBDNF increases starting from 3 mo (Fig. 4A and B) and constantly displays higher levels that in its Wt counter-part. These data are probably related to an amyloid-related astro-glia-derived neurotrophin secretion, previously described.38-40 In agreement, the WB analysis for the astrocyte marker GFAP on Wt and Tg cortices (Fig. 4C) shows a significant increase of the protein in Tg samples, starting from 9 mo, thus suggesting the ongoing of astrogliosis. Figure 4D of the same figure shows WB analysis for the activated form of ERK5, known to be involved in the neuronal survival pathway mediated by TrkBfl-BDNF. At 3 mo p-ERK5 is significantly more abundant in Tg animals than in Wt. At 9 mo, a strong decrease of the activated protein is observed in both genotypes, particularly dramatic in Tg neocor-tex, where its levels are significantly lower than in the respective Wt. At the latest age considered, the concentration of p-ERK5 is still decreased in the Tg animals, while it appears at the same level of the Wt 3-mo animals in 18-mo Wt genotype.
It is known that pro-BDNF detrimental effects on cell sur-vival are mediated by the aspecific neurotrophin receptor p75NTR, which interacts with sortilin, resulting in cell death.37 Moreover, it is also known that pro-BDNF stimulates the processing of p75NTR by α- and γ-secretases, yielding a 20-kDa intracellular domain (ICD) that, upon translocation to the nucleus, triggers
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a delayed apoptosis. Therefore, as ICD and pro-BDNF are both mediators of apoptosis,41 we investigated the levels of p75NTR and ICD, in the neocortex of Wt and Tg mice (Fig. 5A and B).
Interestingly, the ratio ICD/ p75 NTR is much higher in Tg vs. Wt animals at all stages and increases with aging (Fig. 5C). TUNEL analysis supports this notion, since apoptotic nuclei and
Figure 1. HNe adducts in Wt and tg cortices at different ages. (A) HNe immunolocalization in 3-, 9- and 18-mo Wt and tg cortex sections. three tg mice
and 3 Wt littermates for each age considered were deeply anesthetized before rapid killing by transcardial perfusion. Mice were perfusion-fixed, and brains were paraffin-embedded. Sagittal brain sections from tg and Wt animals were deparaffinized and incubated with pBS containing 0.01% trypsin, for 10 min at 37° and processed for the antigen-retrieval procedure, using a microwave oven operated at 720 W. After cooling, slides were transferred to pBS containing 4% BSA for 2 h at Rt, then incubated overnight at 4 °C with mouse monoclonal anti-HNe. Sections were thoroughly rinsed with pBS, then incubated for 2 h at Rt with goat anti-mouse IgG Alexa Fluor 488/546 conjugated diluted 1:2000 in blocking solution. Controls were performed in parallel by omitting the primary antibody. Slides were observed at florescence microscope AXIopHot Zeiss microscope equipped with Leica DFC 350 FX camera. Image acquisition was performed with Leica IM500 program. Bar = 50 μm. (B) Western blotting analysis for HNe protein adducts in Wt and tg animals at the indicated ages. SDS-pAGe was performed running samples (20 μg protein) on 7.5–15% polyacrylamide denaturing gels. protein bands were blotted onto polyvinylidene fluoride sheets. Non-specific binding sites were blocked for 1 h at Rt with 5% (w/v) non-fat dry milk in tBS-t. Membranes were incubated overnight at 4 °C with mouse monoclonal anti-HNe (1:400), followed by incubation with 1:2000 HRp-conjugated anti-mouse IgG secondary antibody in blocking solution, for 1 h at 4 °C. After rinsing, immunoreactive bands were visualized by eCL. the relative densities of the immunoreactive bands were determined and normalized with respect to β-actin, using a semiquantitative densitometric analysis. Data are mean ± SD of 4 different experiments. **, P < 0.001, AD vs. the respective Wt. (C) Western blotting analysis for gluthatione S-transferase (GStp1) in Wt and tg animals at the indicated ages. SDS-pAGe was performed running samples (20 μg protein) on 7.5–15% polyacrylamide denaturing gels. protein bands were blot-ted onto polyvinylidene fluoride sheets. Non-specific binding sites were blocked for 1 h at Rt with 5% (w/v) non-fat dry milk in tBS-t. Membranes were incubated overnight at 4 °C with rabbit polyclonal anti-GStp1 (1:1000), followed by incubation with 1:2000 HRp-conjugated anti-rabbit IgG secondary antibody in blocking solution, for 1 h at 4 °C. After rinsing, immunoreactive bands were visualized by eCL. the relative densities of the immunoreactive bands were determined and normalized with respect to β-actin, using a semiquantitative densitometric analysis. Data are mean ± SD of 4 different experiments. **P < 0.001, AD vs. the respective Wt .
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chromatin margination are more frequently detected in Tg corti-ces than in their Wt counterparts, starting from 9 mo (Fig. 5D).
Discussion
Oxidative stress is present early in the pathogenesis of Alzheimer disease,17 and overproduction of reactive oxygen
species (ROS) induces excitotoxicity, neuro-inflammation, neuronal death, and defects in glial cells.42 However, the biological role of ROS has recently been better elucidated, and ROS have been recognized as ubiquitous endogenous signaling molecules.43 Moderate increase of ROS was shown to act as a molecu-lar signal exerting downstream effects that ultimately induce endogenous defense mecha-nisms, culminating in enhanced stress resis-tance and increased life span.44 Specific targets for a balanced ROS regulation are peroxisome-proliferator-activated receptors (PPARs). The 3 PPAR isoforms (α, β/δ and γ) are members of the nuclear receptors superfamily, which are expressed throughout the brain at differ-ent levels and localizations.25 Since we have previously demonstrated that PPAR β/δ is a crucial factor in neuronal maturation and in BDNF pathway regulation27 and the oxida-tive stress mediator 4-HNE is an intracellular agonist of PPAR β/δ),14 we decided to study the expression of this nuclear receptor and of 4-HNE adducts during aging and in AD in relation to BDNF pathway. Particularly, since a moderate increase of ROS was shown in neo-cortex compared with hippocampus,17,22 our study were focused on this former brain area in Wt and Tg mice. We observed during nor-mal aging and in AD conditions a parallelism between the increase of PPAR β/δ and of HNE adducts, suggesting a possible activation of the nuclear receptor that is known to modulate the BDNF pathway. In agreement, we observed a decrease of TrKBfl starting from 3 mo of age in Tg animals with the concomitant increase of the ratio of ICD/p75 NTR; pro-BDNF appears significantly increase at 18 mo in Tg animals. The level of expression of these proteins with normal aging is interesting; in fact, TrkBfl and p75 NTR appear decreased in favor of their truncated counterpart, thus indicating that the same mechanisms may act during aging and AD, but with different timing.
PPARβ/δ seems to works as sensor of oxida-tive stress, this phenomenon being apparent at 3 mo age in Tg mice, typically characterized by the appearance of oxidative stress,17-22 even though in the present study this event is not correlated with cell death, as apoptotic nuclei are not detected by TUNEL analysis at this age. Therefore, it appears that in the neocortex at 3 mo age, the increased levels of PPAR β/δ and the absence of an increase of 4-HNE adducts protect this brain area from the early perturbation of the redox status. These data are further supported, in neocortex, by the BDNF pathway analysis: in fact, although at 3 mo a decrease of TrkBfl and an increase Figure 2. ppARβ/δ expression in Wt and tg animals at different ages. (A) Real-time pCR in 3-,
9-, and 18-mo cortices for peroxisome proliferator-activated receptor (ppARβ/δ). the gene expressions were quantified in a 2-step Rt-pCR. Complimentary DNA was reverse transcribed from total RNA samples using High-Capacity cDNA Reverse trancription Kit. pCR prod-ucts were synthesized from cDNA using the taqMan universal pCR master mix and Assays on Demand gene expression reagents for mouse ppARβ/δ (Assay ID: Mm01305432-m1). Measurements were made using the ABI prism 7300Ht sequence detection system. As ref-erence, tBp gene expression assay was used. Results represent normalized ppARβ/δ mRNA amounts relative to 3 mo healthy tissue using the 2-ΔΔCt method. Data are mean ± SD of 4
different experiments. **P < 0.001, AD vs. the respective Wt. (B) Western blotting analysis for ppARβ/δ. SDS-pAGe was performed running samples (20 μg protein) on 7.5–15% poly-acrylamide denaturing gels. protein bands were blotted onto polyvinylidene fluoride sheets. Non-specific binding sites were blocked for 1 h at Rt with 5% (w/v) non-fat dry milk in tBS-t. Membranes were incubated overnight at 4 °C with rabbit polyclonal anti-ppARβ/δ (1:1000), followed by incubation with 1:2000 HRp-conjugated anti-rabbit IgG secondary antibody in blocking solution, for 1 h at 4 °C. After rinsing, immunoreactive bands were visualized by eCL. the relative densities of the immunoreactive bands were determined and normalized with respect to β-actin, using a semiquantitative densitometric analysis. Data are mean ± SD of 4 different experiments. **P < 0.001, AD vs. the respective Wt. (C) ppARβ/δ immunolocalization in 18-mo Wt and tg brain sections. three tg mice and 3 Wt littermates for each age con-sidered were deeply anesthetized before rapid killing by transcardial perfusion. Mice were perfusion-fixed, and brains were paraffin-embedded. Sagittal brain sections from tg and Wt animals were deparaffinized and incubated with pBS containing 0.01% trypsin, for 10 min at 37° and processed for the antigen-retrieval procedure, using a microwave oven operated at 720 W. After cooling, slides were transferred to pBS containing 4% BSA for 2 h at Rt, then incu-bated overnight at 4 °C with rabbit polyclonal anti-ppARβ/δ (1:100). Sections were thoroughly rinsed with pBS, then incubated for 2 h at Rt with goat anti-rabbit IgG Alexa Fluor 488/546 conjugated diluted 1:2000 in blocking solution. Controls were performed in parallel by omit-ting the primary antibody. Slides were observed at florescence microscope AXIopHot Zeiss microscope equipped with Leica DFC 350 FX camera. Image acquisition was performed with Leica IM500 program. Bar = 15 μm.
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of TrkB-t and of ICD/p75NTR ratio are observed with respect to Wt animals, a concomitant upregulation of p-ERK5 and BDNF levels were apparent, indicating the activation of a survival pathway and suggesting that a com-pensatory response to an early pertur-bation of the physiological status was acting in the neocortex, as previously described for peroxisomal proteins by us in the same animal model.17 On the other hand, an increase of mBDNF is observed at any age considered in the Tg mice in agreement with previ-ous observations in APP23 transgenic mice, where higher BDNF contents were observed in the cortex.38-40 These findings are probably related to an amyloid-related glia-derived neuro-trophin secretion,38-40 supported in our experimental conditions by the WB analysis for the astrocyte marker GFAP (Fig. 4C), where a significant increase of the protein in Tg samples, starting from 9 mo, is observed, thus suggesting the ongoing of astrogliosis.
It is known that Tg2576 mice develop amyloid plaques and declin-ing cognitive functions startdeclin-ing from 6 mo of age,45 so in our 9- and 18-mo-old mice the AD was full blown, and higher levels of oxidative stress mark-ers and apoptotic nuclei are observed. At these times an increase of 4-HNE adducts and PPARβ/δ and pro-BDNF levels were observed, particularly at 18 mo, suggesting a detrimental effects of the concomitant increase of 4-HNE and PPAR β/δ, pro-BDNF on cell sur-vival. In addition the analysis of BDNF pathway shows a decrease of p-ERK5 starting at 9 mo in Tg mice, suggesting an impairment of pro-survival signal-ing mediated by BDNF, further sup-ported by the TUNEL analysis.
Overall, it appears that the possible detrimental role of PPAR β/δ in AD depends on the oxidative stress level and particularly on the physiological/patho-logical levels of HNE adducts. These data may be useful for further studies on the use of PPARβ/δ antagonist as possible adjuvant for AD therapy.
Conclusion
It has become clear that neuropro-tective strategies should accurately
Figure 3. trkB expression in Wt and tg cortices at different ages. (A) Real-time pCR in 3-, 9-, and 18-mo
cortices for trkBfl. the gene expressions were quantified in a 2-step Rt-pCR. Complimentary DNA was reverse transcribed from total RNA samples using High-Capacity cDNA Reverse trancription Kit. pCR products were synthesized from cDNA using the taqMan universal pCR master mix and Assays on Demand gene expression reagents for mouse trkB (Assay ID: Mm00435422-m1). Measurements were made using the ABI prism 7300Ht sequence detection system. As reference tBp gene expression assay was used. Results represent normalized trkB mRNA amounts relative to 3-mo healthy tissue using the 2-ΔΔCt method. Data are mean ± SD of 4 different experiments. +P < 0.01, 9 mo vs. 18 mo, both Wt and
tg. (B) Western blotting analysis for trkBfl SDS-pAGe was performed running samples (20 μg protein) on 7.5–15% polyacrylamide denaturing gels. protein bands were blotted onto polyvinylidene fluoride sheets. Non-specific binding sites were blocked for 1 h at Rt with 5% (w/v) non-fat dry milk in tBS-t. Membranes were incubated overnight at 4 °C with rabbit polyclonal anti-trkBfl (1:200), followed by incubation with 1:2000 HRp-conjugated anti-rabbit IgG secondary antibody in blocking solution, for 1 h at 4 °C. After rinsing, immunoreactive bands were visualized by eCL. the relative densities of the immunoreactive bands were determined and normalized with respect to β-actin, using a semiquan-titative densitometric analysis. Data are mean ± SD of 4 different experiments. **P < 0.001, AD vs. the respective Wt. (C) Western blotting analysis for trkBt. Membranes were incubated overnight at 4 °C with rabbit polyclonal trkB-t (1:200), followed by incubation with 1:2000 HRp-conjugated anti-rabbit IgG secondary antibody in blocking solution for 1 h at 4 °C. After rinsing, immunoreactive bands were visualized by eCL. the relative densities of the immunoreactive bands were determined and nor-malized with respect to β-actin, using a semiquantitative densitometric analysis. Data are mean ± SD of 4 different experiments. **P < 0.001, AD vs. the respective Wt. (D) Immunolocalization in 9 mo Wt and tg brain sections. three 9 tg mice and 3 Wt littermates, 9 mo of age were deeply anesthetized before rapid killing by transcardial perfusion. Mice were perfusion-fixed, and brains were paraffin-embedded. Sagittal brain sections from tg and Wt animals were deparaffinized and incubated with pBS containing 0.01% trypsin, for 10 min at 37° and processed for the antigen-retrieval procedure, using a microwave oven operated at 720 W. After cooling, slides were transferred to pBS containing 4% BSA for 2 h at Rt, then incubated overnight at 4 °C with rabbit polyclonal anti-trkB (1:50). Sections were thoroughly rinsed with pBS, then incubated for 2 h at Rt with goat anti-rabbit IgG Alexa Fluor 488/546 conjugated diluted 1:2000 in blocking solution. Controls were performed in parallel by omitting the primary anti-body. Slides were observed at florescence microscope AXIopHot Zeiss microscope equipped with Leica DFC 350 FX camera. Image acquisition was performed with Leica IM500 program. Bar = 50 μm.
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regulate the ROS levels to avoid either too-high or too-low ROS concentrations. Therefore, a protective drug should not just scav-enge ROS as much as possible, but also normalize the disease-evoked ROS deregulation.
This work highlights the level of HNE during aging and, par-ticularly in neurodegeneration, plays a crucial role in PPARβ/δ/ BDNF signaling, suggesting a further mechanism of oxidative action on cell survival, as proposed in Figure 6, which shows a
schematic representation depict-ing the interactions between the different molecular inter-mediates studied. The scheme proposes that the increase of oxi-dative stress activates, by 4-HNE, PPARβ/δ, which, in turn, triggers the decrease of TrkBfl. On the other hand, the oxidative stress, through a not well-identified pathway (PPARβ/δ-dependent?), increases pro-BDNF and the ratio ICD/p75NTR with concomitant increase of neuronal death.
Furthermore it appears inter-esting to study, in future work, the effects PPARβ/δ antagonist in counteracting this pathway.
Materials and Methods
Materials
DEPC water and Trizol reagent were purchased from Life Technologies. Triton X-100, sodium dodecylsulfate (SDS), Tween20, bovine serum albu-mine (BSA), nonidet P40, sodium deoxycolate, ethylen diamine tet-raacetate (EDTA), phenylmeth-anesulphonylfluoride (PMSF), sodium fluoride, sodium pyro-phosphate, ortovanadate, leu-peptin, aprotinin, pepstatin, NaCl, polyvinylidene difluoride (PVDF) sheets were all purchased from Sigma Chemical Co.; ECL kit was from Biorad Laboratories; Vectashield was purchased from Vector Laboratories Inc. All other chemicals were of the highest analytical grade.
Animals
Heterozygous female Tg2576 mice (Tg)29 and wild-type (Wt) littermates were used for all experiments. Male mice (C57B6 × SJL), hemizygous for human AβPP695 and expressing the human AβPP 695 with the double mutation K670N and M671L (FADSwedish mutation), were purchased from Taconic Farms, Inc. The Tg2576 colony is main-tained by breeding hemizygous transgenic male with female B6SJLF1 mice, and the genotyping is performed by established methods (Taconic PCR protocol).
The experiments were performed in accordance with the European Communities Council Directive of 24 November 1986 Figure 4. BDNF levels in Wt and tg cortices at different ages. (A and B) pro-BDNF and mBDNF expression,
respectively, in Wt and tg cortices at different ages. SDS-pAGe was performed running samples (20 μg pro-tein) on 7.5–15% polyacrylamide denaturing gels. protein bands were blotted onto polyvinylidene fluo-ride sheets. Non-specific binding sites were blocked for 1 h at Rt with 5% (w/v) non-fat dry milk in tBS-t. Membranes were incubated overnight at 4 °C with rabbit polyclonal anti-BDNF (1:100) (able to recognize both pro and mature BDNF forms), followed by incubation with 1:2000 HRp-conjugated anti-rabbit IgG sec-ondary antibody in blocking solution for 1 h at 4 °C. After rinsing, immunoreactive bands were visualized by eCL. the relative densities of the immunoreactive bands were determined and normalized with respect to β-actin, using a semiquantitative densitometric analysis. Data are mean ± SD of 4 different experiments. **P < 0.001, AD vs. the respective Wt. (C) Western blotting analysis for the astrocyte marker GFAp in Wt and tg cortices at different ages. Membranes were incubated overnight at 4 °C with rabbit polyclonal anti-GFAp (1:200) followed by incubation with 1:2000 HRp-conjugated anti-rabbit IgG secondary antibody in blocking solution, for 1 h at 4 °C. After rinsing, immunoreactive bands were visualized by eCL. the relative densities of the immunoreactive bands were determined and normalized with respect to β-actin, using a semiquantita-tive densitometric analysis. Data are mean ± SD of 4 different experiments. **P < 0.001, AD vs. the respec-tive Wt. (D) Western blotting analysis for p-eRK5 in Wt and tg cortices at different ages. Membranes were incubated overnight at 4 °C with rabbit polyclonal anti-p-eRK5 (1:200),followed by incubation with 1:2000 HRp-conjugated anti-rabbit IgG secondary antibody in blocking solution, for 1 h at 4 °C. After rinsing, immu-noreactive bands were visualized by eCL. the relative densities of the immuimmu-noreactive bands were deter-mined and normalized with respect to β-actin, using a semiquantitative densitometric analysis. Data are mean ± SD of 4 different experiments. **P < 0.001, AD vs. the respective Wt.
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(86/609/EEC). Formal approval to conduct the experiments described was obtained from the Italian Ministry of Health (D.L.vo 116/92; Prot. No.155-VI-1.1.). All efforts were made to minimize the number of animals used and their suffering.
Real-time PCR and western blotting
For real-time PCR (RT-PCR) and western blotting (WB) analy-ses, Tg and Wt female mice age 3-, 9-, and 18-mo-old were used. Six animals from each group were killed by cervical dislocation; brains were rapidly excised on an ice-cold plate and sagittally cut into 2 halves to be used for RT-PCR and WB, respec-tively. The neocortices and hippo-campi were dissected out from each half, and 3 pools of 2 hippocampi or neocortices were obtained.
RT-PCR
Total cellular RNA was extracted by Trizol Reagent, according the manufacturer’s instructions. The total RNA concentration was determined spectrophotometri-cally in RNase-free water. The gene expressions were quanti-fied in a 2-step reverse transcrip-tion- polymerase chain reaction (RT-PCR). Complimentary DNA was reverse transcribed from total RNA samples using High-Capacity cDNA Reverse Trancription Kit (Life Technologies). PCR prod-ucts were synthesized from cDNA using the TaqMan universal PCR master mix and Assays on Demand gene expression reagents for mouse PPARβ/δ and TrkB (Assay ID: Mm01305432-m1 and Mm00435422-m1), (Life Technologies). Measurements were made using the ABI Prism 7300HT sequence detection system, accord-ing to the manufacturer’s protocol. As reference, TBP gene expres-sion assay was used. Results rep-resent normalized PPARβ/δ and TrkB mRNA amounts relative to 3-mo healthy tissue using the 2-ΔΔCt method.46 Data are mean of 4 inde-pendent experiments.
Figure 5. p75NtR levels in Wt and tg cortices at different ages. (A) Western blotting analysis for p75.NtR
SDS-pAGe was performed running samples (20 μg protein) on 7.5–15% polyacrylamide denaturing gels. protein bands were blotted onto polyvinylidene fluoride sheets. Non-specific binding sites were blocked for 1 h at Rt with 5% (w/v) non-fat dry milk in tBS-t. Membranes were incubated overnight at 4 °C with rabbit polyclonal anti- p75NtR (1:500), followed by incubation with 1:2000 HRp-conjugated anti-rabbit
IgG secondary antibody in blocking solution, for 1 h at 4 °C. After rinsing, immunoreactive bands were visualized by eCL. the relative densities of the immunoreactive bands were determined and normalized with respect to β-actin using a semiquantitative densitometric analysis. Data are mean ± SD of 4 different experiments. **P < 0.001, AD vs. the respective Wt. (B) Western blotting analysis for ICD. Membranes were incubated overnight at 4 °C with rabbit polyclonal anti- p75.NtR (1:500), (able to recognize also the cleaved
p75NtR form), followed by incubation with 1:2000 HRp-conjugated anti-rabbit IgG secondary antibody in
blocking solution, for 1 h at 4 °C. After rinsing, immunoreactive bands were visualized by eCL. the relative densities of the immunoreactive bands were determined and normalized with respect to β-actin, using a semiquantitative densitometric analysis. Data are mean ± SD of 4 different experiments. **P < 0.001, AD vs. the respective Wt. In (C) the graphic representation of the ratio ICD/p75NtR. In (D) tunel assay on
9- and 18-mo Wt and tg cortices paraffin-embedded sections were de-waxed in xylene, rehydrated in descending concentration of ethanol, and rinsed in pBS; sections were then subjected to 350 W micro-wave irradiation in 0.1 M citrate buffer pH 6.0 for 5 min for permeabilization and washed in pBS. Sections were incubated with tdt and fluorescein-labeled dUtp in tdt buffer in a dark humid chamber at 37 °C for 60 min. Negative controls were performed omitting tdt. Slides were mounted with Vectashield Mounting Medium and were observed at florescence microscope AXIopHot Zeiss microscope equipped with Leica DFC 350 FX camera. Image acquisition was performed with Leica IM500 program. Bar = 40 μm.
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Western blottingProtein extraction was performed by homogenizing tissue in lysis buffer (320 mM sucrose, 50 mM NaCl, 50 mM Tris–HCl, pH 7.5, 1% Triton X-100, 0.5 mM sodium orthovanadate, 5 mM β-glycerophosphate, 1% protease inhibitor), incubating on ice for 30 min. Homogenates were then centrifuged at 13 000 g for 10 min. The total protein content of the resulting supernatant was determined using a spectrophotometric assay, according to Bradford. Samples were then diluted 3:4 in 200 mM Tris–HCl, pH 6.8, containing 40% glycerol, 20%β-mercaptoethanol, 4% sodium dodecyl phosphate (SDS), bromophenol blue.
SDS-PAGE was performed running samples (5–20 μg pro-tein) on 7.5–15% polyacrylamide denaturing gels. Protein bands were transferred onto polyvinylidene fluoride (PVDF) sheets by wet electrophoretic transfer. Non-specific binding sites were blocked for 1 h at room temperature (RT) with 5% (w/v) non-fat dry milk (Bio-Rad Laboratories) in Tris-buffered saline contain-ing 0.1% (v/v) Tween 20 (TBS-T). Membranes were incubated overnight at 4˚C with the following primary antibodies appropri-ately diluted in blocking solution: mouse monoclonal anti-HNE (1:400, generous gift of Prof Koji Uchida, Nagoya University), rabbit polyclonal antibodies to: GSTP-1 (1:1000, Oxford
Biomedical Research), PPARβ/δ (1:1000, Novus Biologicals), TrkBfl and Trkt (1:200, SantaCruz Biotechnology), BDNF (1:100, SantaCruz), GFAP (1:200 Sigma-Aldrich), ERK5 (1:200, Millipore [Upstate]), p75NTR (1:500 Sigma-Aldrich), β-actin (1:2000 Sigma-Aldrich).
This was followed by incubation with 1:2000 HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibody (SantaCruz Biotechnology) in blocking solution, for 1 h at 4˚C. After rinsing, immunoreactive bands were visualized by enhanced chemilumi-nescence (ECL) according to the manufacturer’s instructions. The relative densities of the immunoreactive bands were deter-mined and normalized with respect to β-actin, using a semiquan-titative densitometric analysis. Data are mean of 4 independent experiments.
Immunofluorescence
For morphological studies, 3 Tg mice and 3 Wt littermates for each age considered were deeply anesthetized with urethane (1 g/kg body weight, injected i.p.), before rapid killing by trans-cardial perfusion. Mice were perfusion-fixed, and brains were paraffin-embedded, as previously described.17
Serial, 5-μm-thick sagittal brain sections from Tg and Wt animals were deparaffinized and incubated with PBS con-taining 0.01% trypsin for 10 min at 37° then immersed in 10 mM sodium citrate buffer, pH 6.1, and processed for the anti-gen-retrieval procedure, using a microwave oven operated at 720 W for 10 min.17 After cooling, slides were transferred to phosphate buffered saline (PBS) containing 4% bovine serum albumine (BSA, blocking solution) for 2 h at RT, then incu-bated overnight at 4 °C with either of the following antibodies, all diluted in blocking solution: mouse monoclonal anti-HNE (1:400, generous gift of Prof Uchida, Nagoya university, Japan), rabbit polyclonal anti-PPARβ/δ (1:100, lot number 230-107, Affinity BioReagents, ABR), rabbit polyclonal anti-TrkBfl, 1:50, (SantaCruz Biotechnology), rabbit polyclonal anti-GFAP (1:200, Sigma-Aldrich).
Sections were thoroughly rinsed with PBS then incubated for 2 h at RT with goat anti-rabbit IgG Alexa Fluor 488/546 conjugated or anti-mouse IgG Alexa Fluor 488/546 conju-gated (Life Technologies), diluted 1:2000 in blocking solution. Controls were performed in parallel by omitting the primary antibody.
Slides were observed at florescence microscope AXIOPHOT Zeiss microscope equipped with Leica DFC 350 FX camera. Image acquisition was performed with Leica IM500 program.
Tunel
The TUNEL assay was performed using the In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics Gmgh) following the manufacturer’s instructions with modifications. Briefly, paraffin-embedded sections were de-waxed in xylene, rehydrated in descending concentration of ethanol (100%, 95%, 80%, 70%), and rinsed in PBS; sections were then subjected to 350 W microwave irradiation in 0.1 M citrate buffer pH 6.0 for 5 min for permeabilization and washed in PBS. Sections were incu-bated with TdT and fluorescein-labeled dUTP in TdT buffer in a dark humid chamber at 37 °C for 60 min. Negative controls were performed omitting TdT. Slides were mounted with Vectashield Figure 6. Schematic representation depicting the interactions between
the different molecular intermediates studied. the scheme proposes that the increase of oxidative stress activates, by 4-HNe, ppARβ/δ, which, in turn, triggers the decrease of trkBfl. on the other hand, the oxidative stress, through a not well-identified pathway (ppARβ/δ-dependent?), increases pro-BDNF and the ratio ICD/p75NtR with concomitant increase
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Mounting Medium and were observed at florescence microscope AXIOPHOT Zeiss microscope equipped with Leica DFC 350 FX camera. Image acquisition was performed with Leica IM500 program.
Statistical analysis
Statistical analysis for RT-PCR and western blotting experi-ments was performed by t test. These statistical calculations were performed using the Statistical Package for the Social Sciences (SPSS) software. For all statistical analyses, P < 0.01 was consid-ered as statistically significant.
Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.
Acknowledgments
This work has been supported by Relevant Interest University (RIA) fund (Prof Cimini). The Authors thank to Human Health Foundation for the support. Many of the experiments have been performed in the Research Center for Molecular Diagnostics and Advanced therapies granted by the Abruzzo Earthquacke Relief Fund (AERF).
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