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. 2012:3:1250.
doi: 10.1038/ncomms2238.

Involvement of PGC-1α in the formation and maintenance of neuronal dendritic spines

Aiwu Cheng  1 , Ruiqian Wan, Jenq-Lin Yang, Naomi Kamimura, Tae Gen Son, Xin Ouyang, Yongquan Luo, Eitan Okun, Mark P Mattson
Affiliations

Involvement of PGC-1α in the formation and maintenance of neuronal dendritic spines

Aiwu Cheng et al. Nat Commun. 2012.

Abstract

The formation, maintenance and reorganization of synapses are critical for brain development and the responses of neuronal circuits to environmental challenges. Here we describe a novel role for peroxisome proliferator-activated receptor γ co-activator 1α, a master regulator of mitochondrial biogenesis, in the formation and maintenance of dendritic spines in hippocampal neurons. In cultured hippocampal neurons, proliferator-activated receptor γ co-activator 1α overexpression increases dendritic spines and enhances the molecular differentiation of synapses, whereas knockdown of proliferator-activated receptor γ co-activator 1α inhibits spinogenesis and synaptogenesis. Proliferator-activated receptor γ co-activator 1α knockdown also reduces the density of dendritic spines in hippocampal dentate granule neurons in vivo. We further show that brain-derived neurotrophic factor stimulates proliferator-activated receptor γ co-activator-1α-dependent mitochondrial biogenesis by activating extracellular signal-regulated kinases and cyclic AMP response element-binding protein. Proliferator-activated receptor γ co-activator-1α knockdown inhibits brain-derived neurotrophic factor-induced dendritic spine formation without affecting expression and activation of the brain-derived neurotrophic factor receptor tyrosine receptor kinase B. Our findings suggest that proliferator-activated receptor γ co-activator-1α and mitochondrial biogenesis have important roles in the formation and maintenance of hippocampal dendritic spines and synapses.

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Conflict of interest statement

Competing interests: The authors have no competing interests to declare.

Figures

Figure 1
Figure 1. Numbers of mitochondria increases during the differentiation and maturation of hippocampal neurons
(a) Confocal images of primary cultured hippocampal neurons at 3 and 10 days (3 DIV and 10 DIV) in culture showing mt-Dsred fluorescence in individual neurons. Scale bars = 10 μm. (b) Results of counts of mitochondrial number in individual neurons at different culture days, as indicated. (c) Results of measurements of mitochondrial mass determined by mitotracker-green fluorescence intensity normalized to cellular protein concentration (see Methods). (d) Results of measurements of ATP concentration (normalized to cellular protein levels) at different culture days as indicated. Values are mean ± SD (n = 4–5 separate cultures). *p<0.05 and **p<0.01 (ANOVA with Student Newman–Keuls post-hoc tests). (e) The length of mitochondria in neurites was measured and the frequencies of the mitochondrial size were plotted (10–20 neurons were analyzed for each condition).
Figure 2
Figure 2. siRNA-mediated knockdown of PGC-1α reduces mitochondrial content and dendritic spine formation in cultured hippocampal neurons
(a) Quantitative PCR using specific primers to PGC-1α demonstrates a large reduction of PGC-1α mRNA in Ad-GFP-Si-PGC-1α infected cells and increased PGC-1α mRNA levels in Ad-GFP-PGC1α infected cells (48 h infections) compared to uninfected neurons and neurons expressing a scrambled shRNA (Ad-GFP-Si-Con). (b) Immunoblot analysis showing that levels of PGC-1α (~95 kd) are elevated in neurons infected with Ad-GFP-PGC1α and are reduced in neurons infected with Ad-GFP-Si-PGC-1α compared to uninfected neurons and neurons infected with Ad-GFP-Si-Con. (c, d) Hippocampal neurons at 5 days in culture were transfected with constructs of GFP-Si- PGC-1α, GFP-Si-Con or GFP-PGC-1α, for 48 h and then fixed for immunostaining using an antibody againt PGC-1α (red). (c) GFP+ cells (arrows) in GFP-Si-PGC-1α transfected neurons exhibit little or no PGC-1α fluorescence compared to the surrounding GFP− cells or to GFP-Si-Con transfected neurons. (d) GFP+ cells (arrows) in GFP- PGC-1α transfected neurons exhibit a stronger PGC-1α fluorescence intensity, with the PGC-1α concentrated in the nucleus, compared to surrounding GFP− cells. (e) Representative confocal images of mito-DsRed and GFP in the dendritic arbors of 12 day-old hippocampal neurons that were co-transfected with mito-DsRed and either GFP-Si-con, GFP-Si-PGC-1α or GFP-PGC-1α constructs on culture day 5. The lower panels show higher magnification of dendrites. Bars = 20 μm. (f, g) Results of counts of mitochondrial numbers in the neurites of individual neurons (e) and dendritic mitochondrial density index (mitochondria/neurite length) (g) (in hippocampal neurons that were co-transfected with mitoDsRed and either GFP-Si-con or GFP-Si-PGC-1α (n = 10–15 neurons analyzed for each condition). Values are the mean ± SD. **p<0.01 compared to the corresponding GFP-Si-Con value (Student’s t-test).. (h) Results of measurements of ATP levels in 12 day-old hippocampal neurons that were infected with Ad-GFP-Si-con, Ad-GFP-Si-PGC-1α or Ad-GFP-PGC-1α on culture day 5. The results are normalized to protein concentration. Values are mean ± SD (n = 4–5 separate cultures). *p<0.05 and **p<0.01 (Student’s t-test). (i) Spine density (number of spines per 100 μm) were quantified in hippocampal neurons that were transfected with either GFP-Si-con or GFP-Si-PGC-1α. n = 10–15 neurons analyzed for each condition. Values are the mean ± SD. **p<0.01 compared to the corresponding GFP-Si-Con value (Student’s t-test).
Figure 3
Figure 3. Evidence that PGC-1α plays a pivotal role in the molecular differentiation of synapses in developing hippocampal neurons
(a, b) Representative confocal images of dendritic segments at high magnification in 12 day-old neurons fixed and immunostained with PSD95 (a) or synapsin I (b) antibodies after transfecting GFP-Si-con or GFP-Si-PGC1α constructs on culture day 5. Bars = 20 μm. (c) numbers of PSD95 and Synapsin I immunoreactive puncta (number of puncta per 100 μm) (n = 10–15 neurons analyzed for each condition). Values are the mean ± SD. **p<0.001 compared to the corresponding GFP-Si-Con value(Student’s t-test). (d) Images of hippocampal neurons at 12 days in culture that had been transfected with GFP-Si-con or GFP-Si-PGC1α on culture day 5. Neurons were then processed for evaluation of activity-dependent uptake of the fluorescent probe FM4-64FX into presynaptic terminals (see Methods). (e) FM4-64FX-labeled puncta (red) were quantified (number of FM4-64 puncta per 100 μm) in 10–15 neurons/culture/condition. Values are mean ± SD (n = 4–5 separate cultures). **p<0.01 (Student’s t-test). (f) A representative immunoblot of PGC-1α, Cyt-c, Synapsin 1, PSD95 and actin in 12 day-old hippocampal neurons that were infected with Ad-GFP or Ad-GFP-PGC-1α on culture day 5. (g) Results of densitometric analysis of blots showing Cyt-c, Synapsin 1 and PSD95 protein levels (fold change compared to the value for neurons infected with Ad-GFP-Con) as in (f). Values are mean ± SD (n = 4–5 separate cultures). **p<0.01 (Student’s t-test).
Figure 4
Figure 4. PGC-1α is required for the maintenance of dendritic spines in adult mouse hippocampal dentate gyrus granule neurons
(a) Low magnification images showing GFP fluorescence in neurons in the dentate gyrus. Adenoviruses that contain GFP-Si-Con or GFP-Si-PGC1α were injected into the dentate gyrus of the hippocampus of 2 month-old male mice. Two weeks later the mice were euthanized, and brains were sectioned and confocal images of GFP fluorescence were acquired. (b) Representative confocal images showing PGC-1α immunostaining (red) and GFP (green) in the dentate gyrus of the hippocampus of mice in which granule neurons were infected with either Ad-GFP-Si-Con or Ad-GFP-Si-PGC1α. (c, d) Representative confocal images showing dendritic trees (c) and dendritic spines (d) of neurons infected with either GFP-Si-Con or GFP-Si-PGC1α. The dendritic spines were quantified in the secondary and tertiary segment of dendrites which are illustrated in b (bracelets). Scale bars in (a)=100 μm, (b) = 50 μm and in (c, d) =10 μm. (e–g) Results of quantitative analysis of total dendritic length, number of bifurcations, and dendritic spine density in dentate granule neurons infected with either Ad-GFP-Si-Con or Ad-GFP-Si-PGC1α. Values are the mean ± SD (n = 5 mice; 15–20 neurons analyzed per condition. **p<0.01 (Student’s t-test).
Figure 5
Figure 5. BDNF stimulates PGC-1α promoter activity via activation of MAP kinases and CREB
(a) Illustration of the structure of the 2 kb PGC-1α promoter linked to a firefly luciferase reporter gene harboring no mutations (PGC-1α-2kb) or a CRE site deletion (PGC1α-ΔCRE), and the Renilla luciferase-expressing plasmid (pRL-TK) used as an internal control. (b) Relative luciferase activity of PGC-1α-2kb and PGC-1α-ΔCRE in neurons treated with or without BDNF for 32 h. Dual luciferase activity was measured at 48 h of transfection and the firefly luciferase activity was normalized to the level of pRL-TK Renilla luciferase activity and plotted as the fold of control (PGC1α-2kb, no BDNF treatment). Values are mean ± SD. *p<0.05 and **p<0.001 (n=5) (ANOVA with Student Newman–Keuls post-hoc tests). (c) Immunoblot analysis of phosphorylation of Erk1/2 and CREB in control and BDNF-treated neurons. Neurons were pretreated with vehicle or kinase inhibitors 30 min prior to exposure to BDNF (40 ng/ml) for 30 min: MAPK inhibitor, PD980059 (PD, 20 μM); PI3K inhibitor LY 290043 (LY, 20 μM). p-Erk1/2 and p-CREB are bands in blots probed with antibodies that selectively recognize phospho-Erk1/2 and phospho-CREB; the same blots were re-probed with an antibody against total CREB (phosphorylation-independent antibody). (d) Relative luciferase activity of PGC1α-2kb in cells incubated under the indicated conditions. The analysis and plot are same as that described in panel b. Values are mean ± SD, *p<0.05 and **p<0.001 (n=5) (ANOVA with Student Newman–Keuls post-hoc tests).
Figure 6
Figure 6. BDNF enhances PGC-1α-mediated mitochondrial biogenesis
(a, b) Effects of BDNF on PGC-1α, NRF1 and TFAM mRNA and protein levels in cultured hippocampal neurons. Cultured hippocampal neurons (7 days in culture) were treated with 40 ng/ml BDNF for the indicated time periods. mRNA (by quantitative PCR) and protein levels (densitometry analysis) are expressed as fold change compared to the value for vehicle-treated control neurons. Values are mean ± SD. *p<0.05, **p<0.001 (n=4) (Student’s t-test). (c) A representative gel showing mtDNA (left) and results of measurement of the amount of mtDNA normalized to protein concentration in cultured hippocampal neurons after treating with BDNF at indicated concentrations, or vehicle control, for 7 days (from culture days 5 – 12). *p<0.05 compared to the 0 ng/ml BDNF value (ANOVA with Student Newman–Keuls post-hoc tests). (d) Mitochondrial mass determined by mt-green fluorescence intensity normalized to cellular protein concentration (see Methods). (e) Results of measurements of ATP concentrations in hippocampal neurons after 7 days of treatment with BDNF or vehicle; values were normalized to protein concentration. Values are mean ± SD (n = 3 separate cultures). *p<0.05 (Student’s t-test). (f) A representative immunoblot of the mitochondrial proteins Cox1 and Cyt-c, and actin, in neurons that had been treated for 7 days with the indicated concentrations of BDNF. The graph shows the results of densitometric analysis of the blots (n = 3 separate cultures). For graphs in panels d, e and f: *p<0.05, **p<0.01 (Student’s t-test).
Figure 7
Figure 7. PGC-1α knockdown inhibits BDNF-induced synaptogenesis without affecting TrkB expression or activation
(a) Confocal images showing PSD95 immunostaining in cultured hippocampal neurons (12 days in culture) that had been treated with vehicle (Con) or BDNF (40 ng/ml) (BDNF) or transfected with GFP-Si-PGC1α on culture day 5 and then treated with 40 ng/ml BDNF (GFP-Si-PGC-1α + BDNF). Scale bars = 20 μm. (b) Results of quantitative analysis of PSD95 immunoreactive puncta in dendrites of neurons from the indicated treatment groups. Bars represent the mean ± SD (n =5 cultures with 15–30 neurons analyzed per condition). **p<0.01 (Student’s t-test). (c) Immunoblot analysis of PSD95 protein levels in neurons from the indicated treatment groups. (d) Immunoblot analysis of TrkB in hippocampal neurons 7 days after infection with Ad-GFP-Si-con or Ad-GFP-Si-PGC1α, compared to uninfected neurons. (e) Immunoprecipitation and quantitative analysis of TrkB physophorylation. TrkB in protein extracts (0.5 mg) from cultured hippocampal neurons in the indicated treatment groups was immunoprecipitated using a rabbit anti-TrkB antibody, and then subjected to immunoblot analysis using the anti phopho-tyrosine antibody PY20 (Cell Signaling). **p<0.01 (n = 3) (Student’s t-test).
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