- Email: [email protected]
INT131 increases dendritic arborization and protects against Aβ toxicity by inducing mitochondrial changes in hippocampal neurons
Biochemical and Biophysical Research Communications xxx (2017) 1e8
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons Juan A. Godoy a, c, Juan M. Zolezzi a, Nibaldo C. Inestrosa a, b, c, * a Center lica Cato b Centre c Centro
gicas, Pontificia Universidad for Aging and Regeneration (CARE UC), Departamento de Biología Celular y Molecular, Facultad de Ciencias Biolo de Chile, Santiago, Chile for Healthy Brain Ageing, School of Psychiatry, Faculty of Medicine, University of New South Wales, Sydney, Australia de Excelencia en Biomedicina de Magallanes (CEBIMA), Universidad de Magallanes, Punta Arenas, Chile
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 May 2017 Accepted 23 June 2017 Available online xxx
In previous studies, we have demonstrated the beneficial effects of classic PPARg agonists on neuroprotection against Ab oligomer neurotoxicity in a double transgenic mouse model of Alzheimer' disease (AD). INT-131, a novel, non-thiazolidinedione compound that belongs to a new family of drugs, selective PPARg modulators (SPPARMs), has provided an emerging opportunity for the treatment of type 2 diabetes mellitus and metabolic syndrome. However, its role in the central nervous system has not been studied. The aim of this study was to evaluate the putative neuroprotective role of INT131 in hippocampal neurons. We found that INT131 increased dendritic branching, promoted neuronal survival against Ab amyloid, increased expression of PGC1-1a and modulated neuronal mitochondrial dynamics. Our results suggest that INT131, a drug that has been shown to be safe and effective in metabolic disorders, may constitute a new therapeutic alternative for AD. © 2017 Published by Elsevier Inc.
Keywords: SPPARM PPARg Amyloid-b Hippocampal neurons Mitochondria
1. Introduction Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptors involved in energy homeostasis and include the PPARa, b/d and g isoforms. This type of receptor forms obligate heterodimers with the retinoid X receptor to modulate gene transcription [1,2]. These receptors are activated by fatty acids, eicosanoids, fibrates and thiazolidinediones (TZD) [3]. PPARg is a clinically validated target for the treatment of type 2 diabetes mellitus (T2DM) due to its critical roles in the functions of the liver, muscle, adipocytes and macrophages and has a direct impact on dyslipidemia, atherosclerosis, cardiovascular disease and insulin resistance [4]. In this regard, TZDs are small molecules that act as full PPARg agonists, increasing insulin sensitivity and restoring insulin and glucose levels in hyperglycemic patients, and they have been clinically validated. However, the modulation of a wide spectrum of target genes also elicits undesirable effects, such as increased weight gain, cardiomegaly, hemodilution, bone fracture and plasma volume expansion [5,6].
* Corresponding author. (CARE UC) Biomedical Center, Pontificia Universidad lica de Chile, Av. Libertador Bernardo OHiggins Cato 340, Santiago, Chile. E-mail address: [email protected] (N.C. Inestrosa).
Recently, next-generation ligands called selective PPARg modulators (SPPARMs), which exert partial agonist activity when compared with TZDs, have been described. INT131 is a potent, nonTZD SPPARM developed for the treatment of T2DM. A variety of biochemical and cell-based assays of INT131 have shown that over a 4 week treatment period, compared with placebo, INT131 was well tolerated and significantly improved insulin sensitivity and glucose tolerance without any undesirable effects in T2DM patients [5,7]. For more than 25 years, the “amyloid cascade hypothesis” has dominated our understanding of the etiology and progression of Alzheimer's disease (AD), which is characterized by the extracellular deposition of amyloid-b peptide (Ab) and the formation of intracellular neurofibrillary tangles (NFTs), triggering several molecular changes within the brain and leading to the development of the pathological and clinical hallmarks observed in AD [8]. Previously, we have shown that PPARg at both the mRNA and protein levels is present in rat primary hippocampal neurons, and PPARg full agonists, such as troglitazone and rosiglitazone (Rosi), but not the PPARg antagonist GW-9662, prevent Ab-dependent neurotoxicity [9,10]. Concomitant activation of the PPARg co-activator 1-alpha (PGC1a), a cold-inducible co-activator of nuclear receptors that has been associated with mitochondrial biogenesis, might contribute to the
http://dx.doi.org/10.1016/j.bbrc.2017.06.146 0006-291X/© 2017 Published by Elsevier Inc.
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
2
J.A. Godoy et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
modulation of neuronal mitochondrial density and functionality, enhancing the PPARg-neuroprotective activity in AD [11]. The aim of our work was to evaluate the effects of INT131 and Rosi as PPARg agonists in primary hippocampal neurons, including neuronal survival under Ab challenge, PGC-1a protein expression level and the putative role in mitochondrial localization within synapses in the hippocampal CA1 region. Here, we report that INT131, a drug that has been shown to be safe, had better effects on neurons than other agonists such as Rosi, including increased dendritic branching, promotion of neuronal survival against Ab amyloid toxicity, increased expression of PGC1-1a and modulation of neuronal mitochondrial dynamics. 2. Materials and methods 2.1. Chemicals and reagents Culture media, serum and supplements were from Sigma (St. Louis, MO) and Gibco BRL (Paisley, UK). Rosi, INT131 and GW-9662 (GW) were obtained from Cayman Chemical (Ann Arbor, MI). An anti-COX IV antibody was purchased from Santa Cruz Biotechnology (San Diego, CA). MitoTracker Orange was purchased from Molecular Probes (Carlsbad, CA). An anti-PGC-1a antibody was obtained from Abcam Inc. (Cambridge, MA). A BCA Protein Assay Kit was purchased from Pierce Chemical Co. (Rockford, IL). Western Lightning Plus ECL was from PerkinElmer (Waltham, MA). 2.2. Rat primary hippocampal neurons Hippocampal neurons were isolated from 18-day-old SpragueDawley rat embryos. The neurons were cultured with Neurobasal medium supplemented with B27 supplement until 15DIV, when the experiments were performed [12]. 2.3. Amyloid-b oligomer (Abo) preparation A synthetic Ab1e42 peptide corresponding to wild-type human Ab was obtained from Genemed Synthesis, Inc. (San Francisco, CA). For Abo preparation, the Ab peptide was dissolved in DMSO at 5 mM and then diluted into distilled water to a final concentration of 100 mM; this preparation was incubated overnight for Abo formation. Ab oligomers were visualized by electron microscopy and analyzed by Tris-Tricine SDS gel electrophoresis, as previously described [13]. 2.4. Neuronal viability in hippocampal neurons Neuronal viability was evaluated using a MTT reduction assay. Briefly, 15DIV rat primary hippocampal neurons (10 104 cells/ 100 ml in a 96-well plate) were incubated under the following conditions: Rosi (10 mM), INT131 (1-10-100 nM) or Abo (5 mM) in supplement- and phenol red-free medium at 37 C for 24 h [14]. 2.5. Scholl analysis Hippocampal neurons, which were seeded onto coverslips in 24-well culture plates at a density of 6 104 cells per well, were transfected at 3DIV with EGFP using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and cultured until 15DIV. Statistical analysis was performed with ANOVA followed by the appropriate post hoc test [15]. 2.6. Mitochondrial length measurement Primary hippocampal neurons were labeled with MitoTracker
Orange (20 nM) for 30 min at 37 C. Then, the neurons were exposed to INT131 and GW-9662 for 12 h and photographed using a Zeiss LSM 5 Pa confocal microscope. Mitochondrial length was measured using Feret's diameter tool in ImageJ software (NIH) and compared among the experimental conditions [16,17]. 2.7. Immunofluorescence and image analysis Hippocampal neurons were plated on poly-L-lysine-coated coverslips in 24-well plates (2.5 105 cells/coverslip) and cultured at 15DIV. Then, hippocampal neurons were incubated with specific antibodies. Images were captured with a Zeiss LSM 5 Pa confocal microscope, using a 63X/1.4 numerical aperture oil-immersion objective. Somatic and neuritic PGC-1a signal intensity was analyzed and expressed as corrected total cell fluorescence (CTCF), the micrographs was setting by Imaje J [18]. 2.8. Electron microscopy from CA1 hippocampal slices Hippocampal slices were prepared from the brains of 2-monthold C57BL/6J mice as previously described. Briefly, the animals were anesthetized with isoflurane and then sacrificed. Brains were surgically extracted, and transverse slices of the hippocampus (350 mm) were cut with a Leica VT 1000s vibratome. Slices were incubated in cold, oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid until treatment. Ultrathin sections (CA1 region of the hippocampus) were examined using a Phillips Tecnai 12 transmission electron microscope (Philips Electron Optics, Holland) at 80 kV. The obtained digital images were analyzed in ImageJ software (NIH) [19]. 2.9. Western blotting Treated hippocampal neurons were washed twice with ice-cold PBS and homogenized in 100 ml of RIPA buffer. Sample were resolved on 8% SDS-PAGE gels and transferred to PVDF membranes. Membranes were incubated overnight with rabbit anti-PGC-1a, rabbit anti-COX IV, and mouse anti-actin antibodies. The membranes were then incubated with horseradish peroxidaseconjugated secondary antibodies and developed using an enhanced chemiluminescence method and quantified [20]. 2.10. Ethics statement The Sprague-Dawley rats used in our experiments were housed and handled according to the guidelines outlined and approved by the Institutional Animal Care and Use Committee at the Faculty of lica de Chile. Biological Sciences of the Pontificia Universidad Cato 2.11. Quantification and statistical analysis The data are presented as the mean ± SEM from 4 to 6 independent experiments. Data were analyzed in GraphPad Prism software v5.01. Significance was established at p < 0.05. Error bars indicate SEM. *p < 0.05; **p < 0.01. 3. Results 3.1. INT131 treatment induces an increase in dendritic arborization To study the complexity of the dendritic tree, a main feature of mature neurons, we used neuronal cultures transfected with EGFP and subjected them to Scholl analysis, enabling us to determine the number, length and complexity of neuronal processes. Primary hippocampal neurons were treated with increasing doses of the full
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
J.A. Godoy et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
PPARg agonist Rosi (1e10 mM) or the specific agonist INT131 (1-10100 nM) (Fig. 1A shows representative micrographs). Our results showed an increase in the complexity of dendritic trees in neurons treated with INT131 compared with both the control condition and Rosi treatment, especially with 10 and 100 nM concentrations of INT131. In fact, 10 nM INT131 significantly increased the number of intersections evaluated at 30e40 mm from the soma (Fig. 1B, **p < 0.01 at 30e40 mm). Additionally, Scholl analysis revealed a significant effect of INT131 on the arborization of the neuritic tree at a lower dose than that of the full PPARg agonist. 3.2. INT131 prevents the neurotoxic effects of Ab species in cultured hippocampal neurons Accordingly, primary hippocampal neurons were challenged with 5 mM Ab, 10 mM Rosi (as full PPARg agonist) and INT131 (0.010.1-1 mM) to evaluate the effect of the treatment on neuronal
3
viability. Importantly, INT131 did not affect cell survival at different concentrations (Fig. 2A, light gray bar); moreover, increasing concentrations of INT131 seemed to protect hippocampal neurons from the harmful effects of Ab species (compare black bar vs gray bars). When compared with Rosi, the full PPARg agonist, INT131, exhibited the same or better viability performance at lower concentrations. We also studied the effect of Ab and INT131 on apoptotic nuclei quantification using Hoechst dye staining. Different concentrations of INT131 induced no changes in the number of apoptotic nuclei in hippocampal neuron cultures (Fig. 2B, see micrographs, light gray bars). By contrast, compared with control, the hippocampal neurons treated with 5 mM Abo exhibited several pyknotic nuclei, and quantification showed up to a 30% increase in apoptotic nuclei (Fig. 2B, graph, black bar). Moreover, the number of apoptotic nuclei diminished by 15e20% when the culture was pretreated with INT131 (Fig. 2B, gray bars). Under the same conditions, Rosi exerts the same effects as INT131
Fig. 1. INT131 treatment induces an increase in the dendritic arborization of hippocampal neurons. Hippocampal neurons at 15DIV were treated with different concentrations of INT131 or Rosi (Rosiglitazone) for 14e16 h. A. Representative images for neurons transfected with EGFP under treatment with different concentrations of INT131 or Rosi. B. Graphs show Sholl analysis for dendritic tree complexity under different concentrations of INT131 or Rosi. The data are expressed as the mean ± S.E.M. of three independent experiments. *p < 0.1, bar represents 10 mm.
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
4
J.A. Godoy et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
Fig. 2. INT131 protects hippocampal neurons from Ab oligomer-induced damage. A. The upper graph shows neuronal survival measurement with MTT under the same early conditions. B. The micrographs show a representative control neuronal culture, Abo treated and culture treated with Ab oligomers plus INT131 and stained with Hoechst dye to visualize apoptotic nuclei. The side graph shows the number of apoptotic nuclei under the specified conditions. The results are the mean ± SEM. n ¼ 4e6 experiments, Student's ttest, *p < 0.01. Bar represents 10 mm.
but at a higher dose. These results show that the maintenance and/ or improvement of neuronal viability and the decrease in apoptotic nuclei are due to the activity of INT131, suggesting that hippocampal neurons are protected from the apoptotic effects of Ab oligomers.
3.3. INT131 modulates PGC-1a levels in hippocampal neurons To determine whether INT131 modulates PGC-1a, a cofactor involved in mitochondrial biogenesis, we analyzed the expression of this factor along with the mitochondrial protein COX IV.
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
J.A. Godoy et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
Hippocampal neurons at 14DIV were treated with 50 nM INT131 and 100 mM GW-9662 for 12 h, and the PGC-1a content in both the soma and neuritic regions was measured by CTCF. Our results showed an increase in PGC-1a labeling in the soma and neurites, while GW-9662 induced a significant decrease in both regions (Fig. 3A, see micrographs and graphs) [18]. When we quantified PGC-1a with western blotting in a dose-response experiment, the protein levels showed a 30% increase in total expression in neurons treated with 10e50 nM of INT131 compared with untreated neurons (Fig. 3B). Moreover, an increase in the protein levels of COX IV, a marker of the total population of mitochondria, was observed in neurons treated with 0.01 and 0.1 nM INT131 (Fig. 3B; graph). These results indicate that INT131 modulates the levels of PGC-1a, controlling mitochondrial biogenesis and density in hippocampal neurons. 3.4. INT131 induces mitochondrial morphological changes in hippocampal neurons and increases the number of mitochondria in the CA1 region of the hippocampus To study potential modulation of mitochondrial morphology, we stimulated primary hippocampal neuronal cultures with different concentrations of INT131 and the PPARg antagonist GW-9662. We then quantified the average length of mitochondria stained with MitoTracker Orange. The quantification revealed significant changes in mitochondrial length with 50 and 100 nM INT131 compared with control (Fig. 4, compare control vs black bar). Interestingly, the mitochondrial length quantification after GW9662 treatment showed a decrease in the average length (Fig. 4, compare control vs gray bar). To determine mitochondrial ability to localize at the synapse, the presence of mitochondria was evaluated in the hippocampal CA1 region of the rat brains by electron microscopy. The number of synapses and the presence and number of mitochondria in the pre- and post-synaptic regions were determined. Representative micrographs of the CA1 region are shown (Fig. 4B). Dose-response stimulation of hippocampal slices of the CA1 region with INT131 showed a significant increase in the total number of mitochondria (area: 16 mm2) at the end of treatment with 1 nM (2-fold over control) and 10 nM (4-fold over control). In addition, the increase in the total number of mitochondria observed with Rosi was 2-fold and 4-fold higher than control at 1 mM and 10 mM, respectively (Fig. 4B,a). Interestingly, the length of the synaptic region (defined as the synaptic bouton) increased up to 2-fold with 100 nM INT131 and close to 5-fold with 1 nM compared with controls. Rosi induced no changes in this parameter (Fig. 4B,b). We also evaluated the number of mitochondria in response to INT131 in the presynaptic region, which exhibited no changes until the dose was increased to 0.1 mM. The same result was observed for Rosi, which only reduced the mitochondrial number in the presynaptic region at a higher dose (Fig. 4B and c, see graphs). Additionally, the number of mitochondria in the post-synaptic region increased close to 2-fold with 0.01 and 0.1 mM INT131 compared with control. Rosi induced only a small increase in the number of mitochondria in this region and only at the higher dose (Fig. 4B,d). 4. Discussion AD is a neurodegenerative disorder with progressive dementia in the elderly and is accompanied by two main structural changes in the brain: senile plaque formation and intracellular protein deposits as neurofibrillary tangles. Consistent with the “amyloid cascade hypothesis”, we have previously shown that PPARg agonists can activate PPARg and reverse the morphological neurodegenerative changes in neurons exposed to Ab. Moreover, we showed that the full agonists, such as troglitazone and Rosi, protect
5
the neuritic network from Ab-induced loss, as detected by immunochemical and electron microscopic studies [9]. Previous research has shown that PPARs are a promising therapeutic target for several neurodegenerative disorders, including Parkinson's disease, AD, Huntington's disease and amyotrophic lateral sclerosis. PPARs have an important role in the improvement of mitochondrial dysfunction, dendritic spine density, proteasomal dysfunction, oxidative stress, and neuroinflammation, which are the major causes of the pathogenesis of neurodegenerative disorders [21,22]. However, exploratory analyses suggested that APOE e4 non-carriers exhibited cognitive and functional improvement in response to Rosi, whereas APOE e4 allele carriers showed no improvement, and some decline was noted [23]. In the current work, we demonstrate that compared with the full agonist Rosi, INT131, a synthetic SPPARM, has a marked effect on the morphology of the neuritic tree and significantly increases the number of neurites. In a similar manner, compared with the full PPARg agonists, INT131 increases viability and decreases the number of apoptotic nuclei at a lower concentration, even when hippocampal neurons are challenged with Ab species. Furthermore, regarding the “mitochondrial cascade hypothesis” of AD, many studies have demonstrated that Ab can induce mitochondrial abnormalities [24]. In AD, studies have shown that decreased glucose metabolism precedes clinical diagnosis and reduces energy metabolism [25]. The latter could be interpreted as an early sign of mitochondrial failure. Indeed, brains from AD patients showed a 50e65% decrease in the mitochondrial-encoded cytochrome oxidase COX I, III mRNA levels that contributed to mitochondrial failure, but nuclear-encoded lactate dehydrogenase B mRNA, a marker of glycolytic metabolism, exhibited normal levels, suggesting that the decrease in the COX I and III subunits contributes to the brain oxidative metabolism in AD [26]. In fact, multiple approaches include strategies aimed at increasing mitochondrial mass, promoting the mitochondrial fusion-fission dynamic, avoiding mitochondrial swelling, preventing mitochondrial Caþ2 overload and improving the overall redox status [27]. In this regard, we observed that INT131 induces an increase in mitochondrial length, a parameter that allows us to suggest that mitochondrial dynamics are active, indicating a type of PPARg-mediated enhanced mitochondrial function that can help serve to prevent mitochondrial failure in the presence of stressor stimuli, such as increased Ab levels. Importantly, in order to maintain and generate functional neuronal circuits within the CNS, neurons demand high levels of energy, which is provided by mitochondria. Moreover, mitochondria not only provide ATP but also participate in the formation of the neuronal circuitry architecture, as it changes location within the pre- or post-synaptic domains during developmental processes, suggesting a role for mitochondrial dynamics in dendritic spine formation and plasticity [28]. Consequently, in recent decades, PGC-1a, a key regulator of mitochondrial biogenesis and respiration, has demonstrated important roles in the formation and maintenance of hippocampal dendritic spines and synapses in hippocampal neurons [29,30]. In our study, we measured the presence of this co-activator under the presence of INT131. Our results indicate that INT131 modulates the levels of PGC-1a, suggesting that it controls mitochondrial biogenesis and modulates hippocampal neuron density. Moreover, the mitochondrial position at the synapse and its trafficking to both pre- and post-synaptic regions are essential for the optimal function of the synapses [28,31]. The most interesting finding of our work was the relative movement of the mitochondria towards the post-synaptic region under INT131 treatment and the increase in the synaptic region, which was measured as the length of the synaptic bouton. Relative
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
6
J.A. Godoy et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
Fig. 3. INT131 modulates PGC-1a in cultured hippocampal neurons. A. Hippocampal neurons at 14DIV were stimulated with different concentration of INT131 and GW-9662 for 12 h at 37 C. Then, the neurons were stained with phalloidin (green) and PGC-1a (blue). The intensity in the soma and neuritic process was quantified in response to each concentration by corrected total cell fluorescence (CTCF). ***p 0.001, ANOVA. (See representative micrographs and graphs). B. PGC-1a and COX IV protein expression levels in hippocampal neurons were analyzed by WB. Different concentrations of INT131 and GW-9662 were used. Representative blots of four independent experiments are shown (n ¼ 4). Data are expressed as the mean ± SEM of normalized data. *p < 0.1, **p 0.01 between untreated control and treated neurons. Bar represents 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
J.A. Godoy et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
7
Fig. 4. INT131 induces mitochondrial dynamics in hippocampal neurons. A. Hippocampal neuron cultures from 15 DIV were labeled with MitoTracker. Micrographs show the mitochondria population analyzed in terms of length of the mitochondria assessed by Feret's diameter. The data are expressed as the mean ± S.E.M. of three independent experiments. *p < 0.1, **p 0.01 between untreated control and treated neurons. Bar represents 10 mm. B. Micrographs from electron microscopy show representative slices of the CA1 region of hippocampus treated with INT131 and Rosi in a dose-response experiment. White arrow shows the pre- and post-synaptic zone in the CA1 region. In these experiments, the total mitochondria number was quantified in response to INT131 or Rosi treatment (a); the length of the synaptic region (pre- and post-synaptic junction) (b) and the number of mitochondria from the pre- (c) or post-synaptic (d) region were measured. The total synapses and synaptic area length were evaluated after INT131 or Rosi stimulus. Quantitative analysis was performed with n ¼ 3, one-way ANOVA, post hoc Bonferroni; *, p < 0.05; Bar, 500 nm.
to mitochondrial localization and morphology, the change in mitochondrial number was correlated with the relative movement of this organelle to the post-synaptic region, suggesting that the change in mitochondrial morphology, an increased number of small, round-shaped mitochondria, could correspond to mitochondrial dynamic events. Together, these results seem to suggest that mitochondrial changes are aimed to sustain the energy demand of strengthened synapses, considering the increase in the length of the synaptic bouton [32]. Based on these findings, studying whether an increase or activation of mitochondria will increase the amount of ATP will be of considerable interest as part of new therapeutic interventions against neurodegenerative disorders, particularly AD. According to the current literature, INT131 is a drug with a safe
therapeutic range that is effective against metabolic diseases. In this regard, our work describes, for the first time, that INT131 can exert similar effects to those reported for the full PPARg agonists in hippocampal neurons. Most importantly, compared with the full PPARg agonists, INT131 has no reported adverse effects; thus, our results suggest that INT131 should be further tested as a potential therapeutic alternative against AD and other neurodegenerative disorders. Acknowledgments *This work was supported by grants CONICYT-PFB 12/2007 from the Basal Centre for Excellence in Science and Technology and FONDECYT 1160724, both to NCI. A pre-doctoral fellowship was
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
8
J.A. Godoy et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
awarded to JAR from CONICYT. JAG is a PhD student from Universitat Pompeu Fabra, Barcelona, Spain.
[16]
Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.06.146.
[17]
[18]
References [19] [1] J. Berger, D.E. Moller, The mechanisms of action of PPARs, Annu. Rev. Med. 53 (2002) 409e435. [2] J.M. Zolezzi, M.J. Santos, S. Bastías-Candia, C. Pinto, J.A. Godoy, N.C. Inestrosa, PPARs in the central nervous system: roles in neurodegeneration and neuroinflammation, Biol. Rev. Camb Philos. Soc. (2017), http://dx.doi.org/10.1111/ brv.12320. rniak, Peroxisome proliferator-activated receptors and their li[3] B. Grygiel-Go gands: nutritional and clinical implications, Nutr. J. 14 (2014) 13e17. [4] B. Gross, M. Pawlak, P. Lefebvre, B. Staels, PPARs in obesity-induced T2DM, dyslipidaemia and NAFLD, Nat. Rev. Endocrinol. 13 (2017) 36e49. [5] F.L. Dunn, L.S. Higgins, J. Fredrickson, A.M. DePaoli, INT131-004 study group. Selective modulation of PPARg activity can lower plasma glucose without typical thiazolidinedione side-effects in patients with Type 2 diabetes, J. Diabetes Complicat. 25 (2011) 151e158. [6] A. Abbas, J. Blandon, J. Rude, A. Elfar, D. Mukherjee, PPAR-g agonist in treatment of diabetes: cardiovascular safety considerations, Cardiovasc Hematol. Agents Med. Chem. 10 (2012) 124e134. [7] J.P. Taygerly, L.R. McGee, S.M. Rubenstein, et al., Discovery of INT131: a selective PPARg modulator that enhances insulin sensitivity, Bioorg Med. Chem. 15 (2013), 979e9. [8] J.A. Hardy, G.A. Higgins, Alzheimer's disease: the amyloid cascade hypothesis, Science 256 (1992) 184e185. [9] N.C. Inestrosa, J.A. Godoy, R.A. Quintanilla, et al., Peroxisome proliferatoractivated receptor gamma is expressed in hippocampal neurons and its activation prevents beta-amyloid neurodegeneration: role of Wnt signaling, Exp. Cell Res. 304 (2005) 91e104. [10] N.C. Inestrosa, F.J. Carvajal, J.M. Zolezzi, C. Tapia-Rojas, F. Serrano, D. Karmelic, E.M. Toledo, A. Toro, J. Toro, M.J. Santos, Peroxisome proliferators reduce spatial memory impairment, synaptic failure, and neurodegeneration in brains of a double transgenic mice model of Alzheimer's disease, J. Alzheimers Dis. 33 (4) (2013) 941e959. [11] L. Katsouri, C. Parr, N. Bogdanovic, et al., PPARgamma co-activator-1alpha (PGC-1alpha) reduces amyloid-beta generation through a PPARgammadependent mechanism, J. Alzheimers Dis. 25 (2011) 151e162. [12] G.A. Banker, W.M. Cowan, Rat hippocampal neurons in dispersed cell culture, Brain Res. 126 (1977) 397e442. [13] M.C. Dinamarca, J.P. Sagal, R.A. Quintanilla, J.A. Godoy, et al., Amyloid-bAcetylcholinesterase complexes potentiate neurodegenerative changes induced by the Ab peptide. Implications for the pathogenesis of Alzheimer's disease, Mol. Neurodegener. 5 (2010) 4. [14] M.J. Santos, R.A. Quintanilla, A. Toro, et al., Peroxisomal proliferation protects from b-amyloid neurodegeneration, J. Biol. Chem. 280 (49) (2005) 41057e41068. [15] N.C. Inestrosa, J.A. Godoy, J.Y. Vargas, M.S. Arr azola, Nicotine prevents synaptic
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27] [28]
[29] [30]
[31] [32]
impairment induced by amyloid-b oligomers through a7-nicotinic acetylcholine receptor activation, Neuromolecular Med. 15 (2013) 549e569. J.M. Zolezzi, C. Silva-Alvarez, D. Ordenes, J.A. Godoy, et al., Peroxisome proliferator-activated receptor (PPAR) g and PPARa agonists modulate mitochondrial fusion-fission dynamics: relevance to reactive oxygen species (ROS)-related neurodegenerative disorders? PLoS One 8 (2013) e64019. J.A. Godoy, M.S. Arr azola, D. Ordenes, C. Silva-Alvarez, N. Braidy, N.C. Inestrosa, Wnt-5a ligand modulates mitochondrial fission-fusion in rat hippocampal neurons, J. Biol. Chem. 289 (52) (2014) 36179e36193. R.A. McCloy, S. Rogers, C.E. Caldon, et al., Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events, Cell Cycle 13 (9) (2014) 1400e1412. M.S. Arr azola, N.C. Inestrosa, Monitoring mitochondrial membranes permeability in live neurons and mitochondrial swelling through electron microscopy analysis, Methods Mol. Biol. 1254 (2015) 87e97. A. Degasperi, M.R. Birtwistle, N. Volinsky, J. Rauch, W. Kolch, B.N. Kholodenko, Evaluating strategies to normalise biological replicates of Western blot data, PLoS One 9 (1) (2014) e87293, 27. J. Brodbeck, M.E. Balestra, A.M. Saunders, A.D. Roses, R.W. Mahley, Y. Huang, Rosiglitazone increases dendritic spine density and rescues spine loss caused by apolipoprotein E4 in primary cortical neurons, Proc. Natl. Acad. Sci. U. S. A. 29 105 (4) (2008) 1343e1346. S. Agarwal, A. Yadav, R.K. Chaturvedi, Peroxisome proliferator-activated receptors (PPARs) as therapeutic target in neurodegenerative disorders, Biochem. Biophys. Res. Commun. (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.08.043. M.E. Risner, A.M. Saunders, J.F. Altman, G.C. Ormandy, S. Craft, I.M. Foley, M.E. Zvartau-Hind, D.A. Hosford, A.D. Roses, Efficacy of rosiglitazone in a genetically defined population with mild-to-moderate Alzheimer'sdisease, Pharmacogenomics J. 6 (4) (2006) 246e254. R.H. Swerdlow, J.M. Burns, S.M. Khan, The Alzheimer's disease mitochondrial cascade hypothesis: progress and perspectives, Biochim. Biophys. Acta 1842 (2014) 1219e1231. M.A. Daulatzai, Cerebral hypoperfusion and glucose hypometabolism: key pathophysiological modulators promote neurodegeneration, cognitive impairment, and Alzheimer's disease, J. Neurosci. Res. (2016), http:// dx.doi.org/10.1002/jnr.23777. K. Chandrasekaran, T. Giordano, D.S.R. Brady, et al., Impairment in mitochondrial cytochrome oxidase gene expression in Alzheimer disease, Brain Res. Mol. Brain Res. 24 (1994) 336e340. R.K. Chaturvedi, M. Flint Beal, Mitochondrial diseases of the brain, Free Radic. Biol. Med. 63 (2013) 1e29. Z. Li, K. Okamoto, Y. Hayashi, M. Sheng, The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses, Cell 119 (2004) 873e887. R. Anderson, T. Prolla, PGC-1alpha in aging and anti-aging interventions, Biochim. Biophys. Acta 1790 (10) (2009) 1059e1066. A. Cheng, R. Wan, J.L. Yang, N. Kamimura, et al., Involvement of PGC-1a in the formation and maintenance of neuronal dendritic spines, Nat. Commun. 3 (2012) 250. D.T. Chang, A.S. Honick, I.J. Reynolds, Mitochondrial trafficking to synapses in cultured primary cortical neurons, J. Neurosci. 26 (2006) 7035e7045. T. Branco, V. Marra, K. Staras, Examining size-strength relationships at hippocampal synapses using an ultrastructural measurement of synaptic release probability, J. Struct. Biol. 172 (2) (2010) 203e210.
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons Juan A. Godoy a, c, Juan M. Zolezzi a, Nibaldo C. Inestrosa a, b, c, * a Center lica Cato b Centre c Centro
gicas, Pontificia Universidad for Aging and Regeneration (CARE UC), Departamento de Biología Celular y Molecular, Facultad de Ciencias Biolo de Chile, Santiago, Chile for Healthy Brain Ageing, School of Psychiatry, Faculty of Medicine, University of New South Wales, Sydney, Australia de Excelencia en Biomedicina de Magallanes (CEBIMA), Universidad de Magallanes, Punta Arenas, Chile
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 May 2017 Accepted 23 June 2017 Available online xxx
In previous studies, we have demonstrated the beneficial effects of classic PPARg agonists on neuroprotection against Ab oligomer neurotoxicity in a double transgenic mouse model of Alzheimer' disease (AD). INT-131, a novel, non-thiazolidinedione compound that belongs to a new family of drugs, selective PPARg modulators (SPPARMs), has provided an emerging opportunity for the treatment of type 2 diabetes mellitus and metabolic syndrome. However, its role in the central nervous system has not been studied. The aim of this study was to evaluate the putative neuroprotective role of INT131 in hippocampal neurons. We found that INT131 increased dendritic branching, promoted neuronal survival against Ab amyloid, increased expression of PGC1-1a and modulated neuronal mitochondrial dynamics. Our results suggest that INT131, a drug that has been shown to be safe and effective in metabolic disorders, may constitute a new therapeutic alternative for AD. © 2017 Published by Elsevier Inc.
Keywords: SPPARM PPARg Amyloid-b Hippocampal neurons Mitochondria
1. Introduction Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptors involved in energy homeostasis and include the PPARa, b/d and g isoforms. This type of receptor forms obligate heterodimers with the retinoid X receptor to modulate gene transcription [1,2]. These receptors are activated by fatty acids, eicosanoids, fibrates and thiazolidinediones (TZD) [3]. PPARg is a clinically validated target for the treatment of type 2 diabetes mellitus (T2DM) due to its critical roles in the functions of the liver, muscle, adipocytes and macrophages and has a direct impact on dyslipidemia, atherosclerosis, cardiovascular disease and insulin resistance [4]. In this regard, TZDs are small molecules that act as full PPARg agonists, increasing insulin sensitivity and restoring insulin and glucose levels in hyperglycemic patients, and they have been clinically validated. However, the modulation of a wide spectrum of target genes also elicits undesirable effects, such as increased weight gain, cardiomegaly, hemodilution, bone fracture and plasma volume expansion [5,6].
* Corresponding author. (CARE UC) Biomedical Center, Pontificia Universidad lica de Chile, Av. Libertador Bernardo OHiggins Cato 340, Santiago, Chile. E-mail address: [email protected] (N.C. Inestrosa).
Recently, next-generation ligands called selective PPARg modulators (SPPARMs), which exert partial agonist activity when compared with TZDs, have been described. INT131 is a potent, nonTZD SPPARM developed for the treatment of T2DM. A variety of biochemical and cell-based assays of INT131 have shown that over a 4 week treatment period, compared with placebo, INT131 was well tolerated and significantly improved insulin sensitivity and glucose tolerance without any undesirable effects in T2DM patients [5,7]. For more than 25 years, the “amyloid cascade hypothesis” has dominated our understanding of the etiology and progression of Alzheimer's disease (AD), which is characterized by the extracellular deposition of amyloid-b peptide (Ab) and the formation of intracellular neurofibrillary tangles (NFTs), triggering several molecular changes within the brain and leading to the development of the pathological and clinical hallmarks observed in AD [8]. Previously, we have shown that PPARg at both the mRNA and protein levels is present in rat primary hippocampal neurons, and PPARg full agonists, such as troglitazone and rosiglitazone (Rosi), but not the PPARg antagonist GW-9662, prevent Ab-dependent neurotoxicity [9,10]. Concomitant activation of the PPARg co-activator 1-alpha (PGC1a), a cold-inducible co-activator of nuclear receptors that has been associated with mitochondrial biogenesis, might contribute to the
http://dx.doi.org/10.1016/j.bbrc.2017.06.146 0006-291X/© 2017 Published by Elsevier Inc.
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
2
J.A. Godoy et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
modulation of neuronal mitochondrial density and functionality, enhancing the PPARg-neuroprotective activity in AD [11]. The aim of our work was to evaluate the effects of INT131 and Rosi as PPARg agonists in primary hippocampal neurons, including neuronal survival under Ab challenge, PGC-1a protein expression level and the putative role in mitochondrial localization within synapses in the hippocampal CA1 region. Here, we report that INT131, a drug that has been shown to be safe, had better effects on neurons than other agonists such as Rosi, including increased dendritic branching, promotion of neuronal survival against Ab amyloid toxicity, increased expression of PGC1-1a and modulation of neuronal mitochondrial dynamics. 2. Materials and methods 2.1. Chemicals and reagents Culture media, serum and supplements were from Sigma (St. Louis, MO) and Gibco BRL (Paisley, UK). Rosi, INT131 and GW-9662 (GW) were obtained from Cayman Chemical (Ann Arbor, MI). An anti-COX IV antibody was purchased from Santa Cruz Biotechnology (San Diego, CA). MitoTracker Orange was purchased from Molecular Probes (Carlsbad, CA). An anti-PGC-1a antibody was obtained from Abcam Inc. (Cambridge, MA). A BCA Protein Assay Kit was purchased from Pierce Chemical Co. (Rockford, IL). Western Lightning Plus ECL was from PerkinElmer (Waltham, MA). 2.2. Rat primary hippocampal neurons Hippocampal neurons were isolated from 18-day-old SpragueDawley rat embryos. The neurons were cultured with Neurobasal medium supplemented with B27 supplement until 15DIV, when the experiments were performed [12]. 2.3. Amyloid-b oligomer (Abo) preparation A synthetic Ab1e42 peptide corresponding to wild-type human Ab was obtained from Genemed Synthesis, Inc. (San Francisco, CA). For Abo preparation, the Ab peptide was dissolved in DMSO at 5 mM and then diluted into distilled water to a final concentration of 100 mM; this preparation was incubated overnight for Abo formation. Ab oligomers were visualized by electron microscopy and analyzed by Tris-Tricine SDS gel electrophoresis, as previously described [13]. 2.4. Neuronal viability in hippocampal neurons Neuronal viability was evaluated using a MTT reduction assay. Briefly, 15DIV rat primary hippocampal neurons (10 104 cells/ 100 ml in a 96-well plate) were incubated under the following conditions: Rosi (10 mM), INT131 (1-10-100 nM) or Abo (5 mM) in supplement- and phenol red-free medium at 37 C for 24 h [14]. 2.5. Scholl analysis Hippocampal neurons, which were seeded onto coverslips in 24-well culture plates at a density of 6 104 cells per well, were transfected at 3DIV with EGFP using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and cultured until 15DIV. Statistical analysis was performed with ANOVA followed by the appropriate post hoc test [15]. 2.6. Mitochondrial length measurement Primary hippocampal neurons were labeled with MitoTracker
Orange (20 nM) for 30 min at 37 C. Then, the neurons were exposed to INT131 and GW-9662 for 12 h and photographed using a Zeiss LSM 5 Pa confocal microscope. Mitochondrial length was measured using Feret's diameter tool in ImageJ software (NIH) and compared among the experimental conditions [16,17]. 2.7. Immunofluorescence and image analysis Hippocampal neurons were plated on poly-L-lysine-coated coverslips in 24-well plates (2.5 105 cells/coverslip) and cultured at 15DIV. Then, hippocampal neurons were incubated with specific antibodies. Images were captured with a Zeiss LSM 5 Pa confocal microscope, using a 63X/1.4 numerical aperture oil-immersion objective. Somatic and neuritic PGC-1a signal intensity was analyzed and expressed as corrected total cell fluorescence (CTCF), the micrographs was setting by Imaje J [18]. 2.8. Electron microscopy from CA1 hippocampal slices Hippocampal slices were prepared from the brains of 2-monthold C57BL/6J mice as previously described. Briefly, the animals were anesthetized with isoflurane and then sacrificed. Brains were surgically extracted, and transverse slices of the hippocampus (350 mm) were cut with a Leica VT 1000s vibratome. Slices were incubated in cold, oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid until treatment. Ultrathin sections (CA1 region of the hippocampus) were examined using a Phillips Tecnai 12 transmission electron microscope (Philips Electron Optics, Holland) at 80 kV. The obtained digital images were analyzed in ImageJ software (NIH) [19]. 2.9. Western blotting Treated hippocampal neurons were washed twice with ice-cold PBS and homogenized in 100 ml of RIPA buffer. Sample were resolved on 8% SDS-PAGE gels and transferred to PVDF membranes. Membranes were incubated overnight with rabbit anti-PGC-1a, rabbit anti-COX IV, and mouse anti-actin antibodies. The membranes were then incubated with horseradish peroxidaseconjugated secondary antibodies and developed using an enhanced chemiluminescence method and quantified [20]. 2.10. Ethics statement The Sprague-Dawley rats used in our experiments were housed and handled according to the guidelines outlined and approved by the Institutional Animal Care and Use Committee at the Faculty of lica de Chile. Biological Sciences of the Pontificia Universidad Cato 2.11. Quantification and statistical analysis The data are presented as the mean ± SEM from 4 to 6 independent experiments. Data were analyzed in GraphPad Prism software v5.01. Significance was established at p < 0.05. Error bars indicate SEM. *p < 0.05; **p < 0.01. 3. Results 3.1. INT131 treatment induces an increase in dendritic arborization To study the complexity of the dendritic tree, a main feature of mature neurons, we used neuronal cultures transfected with EGFP and subjected them to Scholl analysis, enabling us to determine the number, length and complexity of neuronal processes. Primary hippocampal neurons were treated with increasing doses of the full
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
J.A. Godoy et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
PPARg agonist Rosi (1e10 mM) or the specific agonist INT131 (1-10100 nM) (Fig. 1A shows representative micrographs). Our results showed an increase in the complexity of dendritic trees in neurons treated with INT131 compared with both the control condition and Rosi treatment, especially with 10 and 100 nM concentrations of INT131. In fact, 10 nM INT131 significantly increased the number of intersections evaluated at 30e40 mm from the soma (Fig. 1B, **p < 0.01 at 30e40 mm). Additionally, Scholl analysis revealed a significant effect of INT131 on the arborization of the neuritic tree at a lower dose than that of the full PPARg agonist. 3.2. INT131 prevents the neurotoxic effects of Ab species in cultured hippocampal neurons Accordingly, primary hippocampal neurons were challenged with 5 mM Ab, 10 mM Rosi (as full PPARg agonist) and INT131 (0.010.1-1 mM) to evaluate the effect of the treatment on neuronal
3
viability. Importantly, INT131 did not affect cell survival at different concentrations (Fig. 2A, light gray bar); moreover, increasing concentrations of INT131 seemed to protect hippocampal neurons from the harmful effects of Ab species (compare black bar vs gray bars). When compared with Rosi, the full PPARg agonist, INT131, exhibited the same or better viability performance at lower concentrations. We also studied the effect of Ab and INT131 on apoptotic nuclei quantification using Hoechst dye staining. Different concentrations of INT131 induced no changes in the number of apoptotic nuclei in hippocampal neuron cultures (Fig. 2B, see micrographs, light gray bars). By contrast, compared with control, the hippocampal neurons treated with 5 mM Abo exhibited several pyknotic nuclei, and quantification showed up to a 30% increase in apoptotic nuclei (Fig. 2B, graph, black bar). Moreover, the number of apoptotic nuclei diminished by 15e20% when the culture was pretreated with INT131 (Fig. 2B, gray bars). Under the same conditions, Rosi exerts the same effects as INT131
Fig. 1. INT131 treatment induces an increase in the dendritic arborization of hippocampal neurons. Hippocampal neurons at 15DIV were treated with different concentrations of INT131 or Rosi (Rosiglitazone) for 14e16 h. A. Representative images for neurons transfected with EGFP under treatment with different concentrations of INT131 or Rosi. B. Graphs show Sholl analysis for dendritic tree complexity under different concentrations of INT131 or Rosi. The data are expressed as the mean ± S.E.M. of three independent experiments. *p < 0.1, bar represents 10 mm.
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
4
J.A. Godoy et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
Fig. 2. INT131 protects hippocampal neurons from Ab oligomer-induced damage. A. The upper graph shows neuronal survival measurement with MTT under the same early conditions. B. The micrographs show a representative control neuronal culture, Abo treated and culture treated with Ab oligomers plus INT131 and stained with Hoechst dye to visualize apoptotic nuclei. The side graph shows the number of apoptotic nuclei under the specified conditions. The results are the mean ± SEM. n ¼ 4e6 experiments, Student's ttest, *p < 0.01. Bar represents 10 mm.
but at a higher dose. These results show that the maintenance and/ or improvement of neuronal viability and the decrease in apoptotic nuclei are due to the activity of INT131, suggesting that hippocampal neurons are protected from the apoptotic effects of Ab oligomers.
3.3. INT131 modulates PGC-1a levels in hippocampal neurons To determine whether INT131 modulates PGC-1a, a cofactor involved in mitochondrial biogenesis, we analyzed the expression of this factor along with the mitochondrial protein COX IV.
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
J.A. Godoy et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
Hippocampal neurons at 14DIV were treated with 50 nM INT131 and 100 mM GW-9662 for 12 h, and the PGC-1a content in both the soma and neuritic regions was measured by CTCF. Our results showed an increase in PGC-1a labeling in the soma and neurites, while GW-9662 induced a significant decrease in both regions (Fig. 3A, see micrographs and graphs) [18]. When we quantified PGC-1a with western blotting in a dose-response experiment, the protein levels showed a 30% increase in total expression in neurons treated with 10e50 nM of INT131 compared with untreated neurons (Fig. 3B). Moreover, an increase in the protein levels of COX IV, a marker of the total population of mitochondria, was observed in neurons treated with 0.01 and 0.1 nM INT131 (Fig. 3B; graph). These results indicate that INT131 modulates the levels of PGC-1a, controlling mitochondrial biogenesis and density in hippocampal neurons. 3.4. INT131 induces mitochondrial morphological changes in hippocampal neurons and increases the number of mitochondria in the CA1 region of the hippocampus To study potential modulation of mitochondrial morphology, we stimulated primary hippocampal neuronal cultures with different concentrations of INT131 and the PPARg antagonist GW-9662. We then quantified the average length of mitochondria stained with MitoTracker Orange. The quantification revealed significant changes in mitochondrial length with 50 and 100 nM INT131 compared with control (Fig. 4, compare control vs black bar). Interestingly, the mitochondrial length quantification after GW9662 treatment showed a decrease in the average length (Fig. 4, compare control vs gray bar). To determine mitochondrial ability to localize at the synapse, the presence of mitochondria was evaluated in the hippocampal CA1 region of the rat brains by electron microscopy. The number of synapses and the presence and number of mitochondria in the pre- and post-synaptic regions were determined. Representative micrographs of the CA1 region are shown (Fig. 4B). Dose-response stimulation of hippocampal slices of the CA1 region with INT131 showed a significant increase in the total number of mitochondria (area: 16 mm2) at the end of treatment with 1 nM (2-fold over control) and 10 nM (4-fold over control). In addition, the increase in the total number of mitochondria observed with Rosi was 2-fold and 4-fold higher than control at 1 mM and 10 mM, respectively (Fig. 4B,a). Interestingly, the length of the synaptic region (defined as the synaptic bouton) increased up to 2-fold with 100 nM INT131 and close to 5-fold with 1 nM compared with controls. Rosi induced no changes in this parameter (Fig. 4B,b). We also evaluated the number of mitochondria in response to INT131 in the presynaptic region, which exhibited no changes until the dose was increased to 0.1 mM. The same result was observed for Rosi, which only reduced the mitochondrial number in the presynaptic region at a higher dose (Fig. 4B and c, see graphs). Additionally, the number of mitochondria in the post-synaptic region increased close to 2-fold with 0.01 and 0.1 mM INT131 compared with control. Rosi induced only a small increase in the number of mitochondria in this region and only at the higher dose (Fig. 4B,d). 4. Discussion AD is a neurodegenerative disorder with progressive dementia in the elderly and is accompanied by two main structural changes in the brain: senile plaque formation and intracellular protein deposits as neurofibrillary tangles. Consistent with the “amyloid cascade hypothesis”, we have previously shown that PPARg agonists can activate PPARg and reverse the morphological neurodegenerative changes in neurons exposed to Ab. Moreover, we showed that the full agonists, such as troglitazone and Rosi, protect
5
the neuritic network from Ab-induced loss, as detected by immunochemical and electron microscopic studies [9]. Previous research has shown that PPARs are a promising therapeutic target for several neurodegenerative disorders, including Parkinson's disease, AD, Huntington's disease and amyotrophic lateral sclerosis. PPARs have an important role in the improvement of mitochondrial dysfunction, dendritic spine density, proteasomal dysfunction, oxidative stress, and neuroinflammation, which are the major causes of the pathogenesis of neurodegenerative disorders [21,22]. However, exploratory analyses suggested that APOE e4 non-carriers exhibited cognitive and functional improvement in response to Rosi, whereas APOE e4 allele carriers showed no improvement, and some decline was noted [23]. In the current work, we demonstrate that compared with the full agonist Rosi, INT131, a synthetic SPPARM, has a marked effect on the morphology of the neuritic tree and significantly increases the number of neurites. In a similar manner, compared with the full PPARg agonists, INT131 increases viability and decreases the number of apoptotic nuclei at a lower concentration, even when hippocampal neurons are challenged with Ab species. Furthermore, regarding the “mitochondrial cascade hypothesis” of AD, many studies have demonstrated that Ab can induce mitochondrial abnormalities [24]. In AD, studies have shown that decreased glucose metabolism precedes clinical diagnosis and reduces energy metabolism [25]. The latter could be interpreted as an early sign of mitochondrial failure. Indeed, brains from AD patients showed a 50e65% decrease in the mitochondrial-encoded cytochrome oxidase COX I, III mRNA levels that contributed to mitochondrial failure, but nuclear-encoded lactate dehydrogenase B mRNA, a marker of glycolytic metabolism, exhibited normal levels, suggesting that the decrease in the COX I and III subunits contributes to the brain oxidative metabolism in AD [26]. In fact, multiple approaches include strategies aimed at increasing mitochondrial mass, promoting the mitochondrial fusion-fission dynamic, avoiding mitochondrial swelling, preventing mitochondrial Caþ2 overload and improving the overall redox status [27]. In this regard, we observed that INT131 induces an increase in mitochondrial length, a parameter that allows us to suggest that mitochondrial dynamics are active, indicating a type of PPARg-mediated enhanced mitochondrial function that can help serve to prevent mitochondrial failure in the presence of stressor stimuli, such as increased Ab levels. Importantly, in order to maintain and generate functional neuronal circuits within the CNS, neurons demand high levels of energy, which is provided by mitochondria. Moreover, mitochondria not only provide ATP but also participate in the formation of the neuronal circuitry architecture, as it changes location within the pre- or post-synaptic domains during developmental processes, suggesting a role for mitochondrial dynamics in dendritic spine formation and plasticity [28]. Consequently, in recent decades, PGC-1a, a key regulator of mitochondrial biogenesis and respiration, has demonstrated important roles in the formation and maintenance of hippocampal dendritic spines and synapses in hippocampal neurons [29,30]. In our study, we measured the presence of this co-activator under the presence of INT131. Our results indicate that INT131 modulates the levels of PGC-1a, suggesting that it controls mitochondrial biogenesis and modulates hippocampal neuron density. Moreover, the mitochondrial position at the synapse and its trafficking to both pre- and post-synaptic regions are essential for the optimal function of the synapses [28,31]. The most interesting finding of our work was the relative movement of the mitochondria towards the post-synaptic region under INT131 treatment and the increase in the synaptic region, which was measured as the length of the synaptic bouton. Relative
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
6
J.A. Godoy et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
Fig. 3. INT131 modulates PGC-1a in cultured hippocampal neurons. A. Hippocampal neurons at 14DIV were stimulated with different concentration of INT131 and GW-9662 for 12 h at 37 C. Then, the neurons were stained with phalloidin (green) and PGC-1a (blue). The intensity in the soma and neuritic process was quantified in response to each concentration by corrected total cell fluorescence (CTCF). ***p 0.001, ANOVA. (See representative micrographs and graphs). B. PGC-1a and COX IV protein expression levels in hippocampal neurons were analyzed by WB. Different concentrations of INT131 and GW-9662 were used. Representative blots of four independent experiments are shown (n ¼ 4). Data are expressed as the mean ± SEM of normalized data. *p < 0.1, **p 0.01 between untreated control and treated neurons. Bar represents 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
J.A. Godoy et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
7
Fig. 4. INT131 induces mitochondrial dynamics in hippocampal neurons. A. Hippocampal neuron cultures from 15 DIV were labeled with MitoTracker. Micrographs show the mitochondria population analyzed in terms of length of the mitochondria assessed by Feret's diameter. The data are expressed as the mean ± S.E.M. of three independent experiments. *p < 0.1, **p 0.01 between untreated control and treated neurons. Bar represents 10 mm. B. Micrographs from electron microscopy show representative slices of the CA1 region of hippocampus treated with INT131 and Rosi in a dose-response experiment. White arrow shows the pre- and post-synaptic zone in the CA1 region. In these experiments, the total mitochondria number was quantified in response to INT131 or Rosi treatment (a); the length of the synaptic region (pre- and post-synaptic junction) (b) and the number of mitochondria from the pre- (c) or post-synaptic (d) region were measured. The total synapses and synaptic area length were evaluated after INT131 or Rosi stimulus. Quantitative analysis was performed with n ¼ 3, one-way ANOVA, post hoc Bonferroni; *, p < 0.05; Bar, 500 nm.
to mitochondrial localization and morphology, the change in mitochondrial number was correlated with the relative movement of this organelle to the post-synaptic region, suggesting that the change in mitochondrial morphology, an increased number of small, round-shaped mitochondria, could correspond to mitochondrial dynamic events. Together, these results seem to suggest that mitochondrial changes are aimed to sustain the energy demand of strengthened synapses, considering the increase in the length of the synaptic bouton [32]. Based on these findings, studying whether an increase or activation of mitochondria will increase the amount of ATP will be of considerable interest as part of new therapeutic interventions against neurodegenerative disorders, particularly AD. According to the current literature, INT131 is a drug with a safe
therapeutic range that is effective against metabolic diseases. In this regard, our work describes, for the first time, that INT131 can exert similar effects to those reported for the full PPARg agonists in hippocampal neurons. Most importantly, compared with the full PPARg agonists, INT131 has no reported adverse effects; thus, our results suggest that INT131 should be further tested as a potential therapeutic alternative against AD and other neurodegenerative disorders. Acknowledgments *This work was supported by grants CONICYT-PFB 12/2007 from the Basal Centre for Excellence in Science and Technology and FONDECYT 1160724, both to NCI. A pre-doctoral fellowship was
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146
8
J.A. Godoy et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
awarded to JAR from CONICYT. JAG is a PhD student from Universitat Pompeu Fabra, Barcelona, Spain.
[16]
Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.06.146.
[17]
[18]
References [19] [1] J. Berger, D.E. Moller, The mechanisms of action of PPARs, Annu. Rev. Med. 53 (2002) 409e435. [2] J.M. Zolezzi, M.J. Santos, S. Bastías-Candia, C. Pinto, J.A. Godoy, N.C. Inestrosa, PPARs in the central nervous system: roles in neurodegeneration and neuroinflammation, Biol. Rev. Camb Philos. Soc. (2017), http://dx.doi.org/10.1111/ brv.12320. rniak, Peroxisome proliferator-activated receptors and their li[3] B. Grygiel-Go gands: nutritional and clinical implications, Nutr. J. 14 (2014) 13e17. [4] B. Gross, M. Pawlak, P. Lefebvre, B. Staels, PPARs in obesity-induced T2DM, dyslipidaemia and NAFLD, Nat. Rev. Endocrinol. 13 (2017) 36e49. [5] F.L. Dunn, L.S. Higgins, J. Fredrickson, A.M. DePaoli, INT131-004 study group. Selective modulation of PPARg activity can lower plasma glucose without typical thiazolidinedione side-effects in patients with Type 2 diabetes, J. Diabetes Complicat. 25 (2011) 151e158. [6] A. Abbas, J. Blandon, J. Rude, A. Elfar, D. Mukherjee, PPAR-g agonist in treatment of diabetes: cardiovascular safety considerations, Cardiovasc Hematol. Agents Med. Chem. 10 (2012) 124e134. [7] J.P. Taygerly, L.R. McGee, S.M. Rubenstein, et al., Discovery of INT131: a selective PPARg modulator that enhances insulin sensitivity, Bioorg Med. Chem. 15 (2013), 979e9. [8] J.A. Hardy, G.A. Higgins, Alzheimer's disease: the amyloid cascade hypothesis, Science 256 (1992) 184e185. [9] N.C. Inestrosa, J.A. Godoy, R.A. Quintanilla, et al., Peroxisome proliferatoractivated receptor gamma is expressed in hippocampal neurons and its activation prevents beta-amyloid neurodegeneration: role of Wnt signaling, Exp. Cell Res. 304 (2005) 91e104. [10] N.C. Inestrosa, F.J. Carvajal, J.M. Zolezzi, C. Tapia-Rojas, F. Serrano, D. Karmelic, E.M. Toledo, A. Toro, J. Toro, M.J. Santos, Peroxisome proliferators reduce spatial memory impairment, synaptic failure, and neurodegeneration in brains of a double transgenic mice model of Alzheimer's disease, J. Alzheimers Dis. 33 (4) (2013) 941e959. [11] L. Katsouri, C. Parr, N. Bogdanovic, et al., PPARgamma co-activator-1alpha (PGC-1alpha) reduces amyloid-beta generation through a PPARgammadependent mechanism, J. Alzheimers Dis. 25 (2011) 151e162. [12] G.A. Banker, W.M. Cowan, Rat hippocampal neurons in dispersed cell culture, Brain Res. 126 (1977) 397e442. [13] M.C. Dinamarca, J.P. Sagal, R.A. Quintanilla, J.A. Godoy, et al., Amyloid-bAcetylcholinesterase complexes potentiate neurodegenerative changes induced by the Ab peptide. Implications for the pathogenesis of Alzheimer's disease, Mol. Neurodegener. 5 (2010) 4. [14] M.J. Santos, R.A. Quintanilla, A. Toro, et al., Peroxisomal proliferation protects from b-amyloid neurodegeneration, J. Biol. Chem. 280 (49) (2005) 41057e41068. [15] N.C. Inestrosa, J.A. Godoy, J.Y. Vargas, M.S. Arr azola, Nicotine prevents synaptic
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27] [28]
[29] [30]
[31] [32]
impairment induced by amyloid-b oligomers through a7-nicotinic acetylcholine receptor activation, Neuromolecular Med. 15 (2013) 549e569. J.M. Zolezzi, C. Silva-Alvarez, D. Ordenes, J.A. Godoy, et al., Peroxisome proliferator-activated receptor (PPAR) g and PPARa agonists modulate mitochondrial fusion-fission dynamics: relevance to reactive oxygen species (ROS)-related neurodegenerative disorders? PLoS One 8 (2013) e64019. J.A. Godoy, M.S. Arr azola, D. Ordenes, C. Silva-Alvarez, N. Braidy, N.C. Inestrosa, Wnt-5a ligand modulates mitochondrial fission-fusion in rat hippocampal neurons, J. Biol. Chem. 289 (52) (2014) 36179e36193. R.A. McCloy, S. Rogers, C.E. Caldon, et al., Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events, Cell Cycle 13 (9) (2014) 1400e1412. M.S. Arr azola, N.C. Inestrosa, Monitoring mitochondrial membranes permeability in live neurons and mitochondrial swelling through electron microscopy analysis, Methods Mol. Biol. 1254 (2015) 87e97. A. Degasperi, M.R. Birtwistle, N. Volinsky, J. Rauch, W. Kolch, B.N. Kholodenko, Evaluating strategies to normalise biological replicates of Western blot data, PLoS One 9 (1) (2014) e87293, 27. J. Brodbeck, M.E. Balestra, A.M. Saunders, A.D. Roses, R.W. Mahley, Y. Huang, Rosiglitazone increases dendritic spine density and rescues spine loss caused by apolipoprotein E4 in primary cortical neurons, Proc. Natl. Acad. Sci. U. S. A. 29 105 (4) (2008) 1343e1346. S. Agarwal, A. Yadav, R.K. Chaturvedi, Peroxisome proliferator-activated receptors (PPARs) as therapeutic target in neurodegenerative disorders, Biochem. Biophys. Res. Commun. (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.08.043. M.E. Risner, A.M. Saunders, J.F. Altman, G.C. Ormandy, S. Craft, I.M. Foley, M.E. Zvartau-Hind, D.A. Hosford, A.D. Roses, Efficacy of rosiglitazone in a genetically defined population with mild-to-moderate Alzheimer'sdisease, Pharmacogenomics J. 6 (4) (2006) 246e254. R.H. Swerdlow, J.M. Burns, S.M. Khan, The Alzheimer's disease mitochondrial cascade hypothesis: progress and perspectives, Biochim. Biophys. Acta 1842 (2014) 1219e1231. M.A. Daulatzai, Cerebral hypoperfusion and glucose hypometabolism: key pathophysiological modulators promote neurodegeneration, cognitive impairment, and Alzheimer's disease, J. Neurosci. Res. (2016), http:// dx.doi.org/10.1002/jnr.23777. K. Chandrasekaran, T. Giordano, D.S.R. Brady, et al., Impairment in mitochondrial cytochrome oxidase gene expression in Alzheimer disease, Brain Res. Mol. Brain Res. 24 (1994) 336e340. R.K. Chaturvedi, M. Flint Beal, Mitochondrial diseases of the brain, Free Radic. Biol. Med. 63 (2013) 1e29. Z. Li, K. Okamoto, Y. Hayashi, M. Sheng, The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses, Cell 119 (2004) 873e887. R. Anderson, T. Prolla, PGC-1alpha in aging and anti-aging interventions, Biochim. Biophys. Acta 1790 (10) (2009) 1059e1066. A. Cheng, R. Wan, J.L. Yang, N. Kamimura, et al., Involvement of PGC-1a in the formation and maintenance of neuronal dendritic spines, Nat. Commun. 3 (2012) 250. D.T. Chang, A.S. Honick, I.J. Reynolds, Mitochondrial trafficking to synapses in cultured primary cortical neurons, J. Neurosci. 26 (2006) 7035e7045. T. Branco, V. Marra, K. Staras, Examining size-strength relationships at hippocampal synapses using an ultrastructural measurement of synaptic release probability, J. Struct. Biol. 172 (2) (2010) 203e210.
Please cite this article in press as: J.A. Godoy, et al., INT131 increases dendritic arborization and protects against Ab toxicity by inducing mitochondrial changes in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.146