Figure Dissection 2

Overview:

A primary symptom of Alzheimer's Disease is damage to neuronal connections. This experiment demonstrates the relationship between exercise and Bdnf levels in the hippocampus and explores the potential mechanistic pathway for BDNF to elicit neuroprotection and neurogenesis. This pathway is important to understanding how exercise may have a preventative effect on AD via neuroprotection and neurogenesis. Furthermore, this experiment connects the activation of PGC-1ɑ via the Fndc5 gene to increased levels of Brain-derived neurotrophic factors (BDNF) levels in the neurons in response to exercise. This mechanistic exploration provided a possible pathway to answer the question: What is the effect of exercise on neurodegeneration?

Background Information:

BDNF Role in Neuroprotection and Neurogenesis

BDNF is a growth factor of the nervous tissues that is upregulated in connection to aerobic exercise. BDNF has a central role in protecting existing neurons and stimulating the formation of new neurons from neural stem cells during the process of neurogenesis. Research shows that BDNF appears to coordinate its action with at least two other growth factors: vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1). The relationship between IGF-1 and exercise is discussed in Figure Dissection 1. Both BDNF and IGF-1 are involved in the induction of neurogenesis. These processes protect existing neurons, produce new neurons, and enhance brain plasticity creating the exercise-induced protective effect against AD. Furthermore, BDNF levels are most concentrated in the hippocampus; thus, they have been linked to improved cognition. For additional information on BDNF in video format watch the video on the left (0:50-2:52) created by ©Alila Medical Media.

Proposed PGC-1ɑ/FNDC5/BDNF Pathway

Below is the proposed mechanistic pathway of how exercise induces increased BNDF levels via the PGC-1ɑ/Errɑ transcriptional complex as proposed by Wrann et al.

Figure 1. Model of PGC-1ɑ/FNDC5/BDNF pathway in exercise.

Endurance exercise stimulates increased hippocampal Fndc5 gene expression via a PGC-1ɑ/Errɑ transcriptional complex. This elevated Fndc5 gene expression stimulates Bdnf gene expression. BDNF is the master regulator of nerve cell survival, differentiation, and plasticity in the brain; therefore, leading to improved cognitive function, learning, and memory. (Wrann et al., 2013)

Figure 1 Analysis:

This figure proposes the pathway revealed in the experiments below. Endurance exercise induces PGC-1α. PGC-1α is a transcriptional coactivator that has been linked to reduced excitotoxicity and the formation and maintenance of neuronal dendritic spines in animal models. But, PGC-1α does not bind to the DNA itself but interacts with the estrogen-related receptor alpha (ERRα), a central metabolic regulator. It is suggested that ERRα is involved in the induction of FNDC5 by PGC-1α. The exact role of individual ERRα binding sites in the Fndc5 gene is unknown. The Fndc5 gene is transcribed to produce FNDC5 which is a PGC-1α-dependent protein that is cleaved and secreted from muscles during exercise. The secreted form of FNDC5 is called irisin (not shown in the diagram). FNDC5 is a positive regulator of BDNF gene expression: therefore, an increase of FNDC5 is correlated with an increase in BDNF expression. There is a negative feedback loop in where a high concentration of BDNF inhibits FNDC5 expression. This is assumed to be a part of a homeostatic loop. The increased BDNF levels have a positive effect on neuron cell survival, differentiation, and plasticity. These effects lead to enhanced cognition, learning, and memory. For more details on the effect of BDNF on the brain see the above background information.

Experiment 1: Evaluation of the relationship between hippocampal Fndc5 gene expression and exercise.

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Experimental Design

In order to develop the proposed pathway above, the researchers tested the effect of exercise on Fndc5 gene expression in a mouse model. Six-week-old male C57/B16 wild-type mice were individually housed with access to a (A) running wheel or (B) without for 30 days. The experimental group was sacrificed approximately 10 hours after their last exercise. The mRNA levels of Fndc5 and the transcriptional regulators Pgc1a and Erra were measured in the hippocampus, and brain in both groups of mice. Additionally, the mRNA levels of Bdnf, lgf1, Arc, cFos, Npas4, and Zif268 were measured in the hippocampus and brain of both groups of mice. (Wrann et al., 2013)

Figure 2. Endurance Exercise Induces Hippocampal Fndc5 Gene Expression

(B–E) Male six-week-old C57/Bl6 wild-type mice were individually housed in cages with access to a running wheel (free-running wheel) or without (sedentary). Mice were exercised for 30 days and sacrificed approximately 10 hr after their last bout of exercise. Data are shown as mRNA levels relative to Rsp18 expression, expressed as mean ± SEM. *p < 0.05 compared to sedentary control group. (Wrann et al., 2013)

Figure 2 Analysis:

Figure 2B shows the mice with access to the running wheel had a significantly higher expression of Fndc5 in the hippocampus. There was also a significant increase in the expression of PGC-1α and ERRα which form the transcriptional complex that induces the expression of Fndc5. These changes in Fndc5, PGC-1α, and ERRα were not observed in the whole brain as shown by figure 2C. Therefore, exercise significantly increases Fndc5, PGC-1α, and ERRα specifically in the hippocampus.

Additionally, the researchers evaluated the expression of genes typically induced by neuronal activity. Figure 2 D & E demonstrates that Arc, cFos, and Zif268 were upregulated in both the remainder of the brain and the hippocampus. Arc, cFos, and Zif268 are genes that control cell growth and neural plasticity. Npas4 is a transcriptional component in hippocampal function and regulator of activity-induced Bdnf expression (Wann et al., 2013). In both the brain and hippocampus there was no increase in Npas4 expression suggesting that the increased levels of Bdnf expression are due to a transcriptional response to exercise in the hippocampus.

Experiment 2: Building the PGC-1ɑ/FNDC5/BDNF pathway in exercise

Now that the experiments confirmed that Fndc5 gene expression is elevated in response to endurance exercise in the mouse model they aim to build the proposed PGC-1ɑ/FNDC5/BDNF pathway. Note this pathway is built via experiments with primary cortical neurons. Experiment 1 shows that Fndc5 expression is highly induced in the hippocampus. Therefore, this pathway can be assumed to be true in hippocampal neurons as well.

Experimental Design:

To build the PGC-1ɑ/FNDC5/BDNF pathway in exercise the experimenters tested if FNDC5 is required for BDNF gene expression and assessed the role of PGC-1ɑ in controlling BDNF gene expression. First, the experimenters used the brains of PGC-1ɑ knockout mice to assess the role of PGC-1ɑ in controlling BDNF genes. Then the experimenters transduced primary cortical neurons with either PGC-1ɑ adenovirus(experimental) or a GFP adenovirus (control) and assessed the relative gene expression in Erra, Errb, Errg, and FNDC5 to understand the role Erra plays in the transcription of FNDC5. Then the exerimentors transduced primary cortical neurons with either FNDC5 adenovirus(experimental) or a GFP adenovirus (control) and assessed the relative gene expression in activity-induced genes involved in hippocampal function (BDNF, ARC, cFos, Npas4, Zif268) by qPCR.To test if FNDC5 is required for BDNF gene expression, FNDC5 was knocked out in primary cortical neurons using lentivirally delivered shRNA. Experimenters used a luminescence/ATP-based assay to assess the effects of the gain- and loss-of-function of FNDC5 on cell viability of cultured neurons. The experimenters treated primary cortical neurons with varying concentrations of BDNF to understand the type of pathway. Experimenters used the brains of PGC-1ɑ knockout mice to assess the role of PGC-1ɑ in controlling BDNF genes. (Wrann et al., 2013)

Figure 3. Neuronal Fndc5 Gene Expression Is Regulated by a PGC-1ɑ

(E) Cortices were harvested from either male five-month-old PGC-1ɑ KO (PGC-1ɑ-/-) or wild-type mice (PGC-1ɑ +/+). mRNA was prepared and gene expression was assessed by qPCR. All data are shown as mRNA level relative to Rsp 18 expression, expressed as mean +/- SEM. *p<0.05 compared to the corresponding control group. (Wrann et al., 2013)

Figure 3 Analysis:

Figure 3 determines if PGC-1ɑ regulated Fndc5 gene expression. The experimenters conduct a knockout experiment using PGC-1ɑ-/- mice and analyze the difference in relative gene expression of both Fndc5 and the downstream expression of BDNF. Figure 3 shows that neuronal Fndc5 gene expression is regulated on by PGC-1ɑ because the knockout mice (PGC-1ɑ-/-) had lower expression of Fndc5.

Figure 4. ERRα Is a Key Interacting Transcription Factor with PGC-1α for Regulating Fndc5 Gene Expression in Neurons

(A) Primary cortical neurons at DIV 7 were transduced with either PGC-1α or GFP adenovirus and harvested 48 hr later. ∗p < 0.05 compared to control group. (Wrann et al., 2013)

Figure 4 Analysis

Figure 4 shows that ERRα is a key interacting transcription factor with PGC-1α. The relative mRNA expression of ERRα significantly increases in the primary cortical neurons that were transduced with PGC-1α compared to primary cortical neurons transduced with GFP adenovirus. This did change did not occur in Errb or Errg expression. There was also a significant increase in Fndc5 expression in the primary cortical neurons transduced with PGC-1α. Therefore, ERRα is involved in the transcription factors regulating Fndc5 gene expression.

Figure 5 (B-E, G) FNDC5 Regulates Bdnf Gene Expression in a Cell-Autonomous Manner, and Recombinant BDNF Decreases Fndc5 Gene Expression as Part of a Negative Feedback Loop

(B) Primary cortical neurons at DIV 7 were transduced with either FNDC5 or GFP adenovirus. Forty-eight hours later mRNA was prepared and gene expression was assessed by qPCR. (C) Primary cortical neurons at DIV 5 were transduced with lentivirus carrying the specified shRNA hairpins against Fndc5 or luciferase (Luc) as control. Four days later, mRNA was prepared and gene expression was assessed by qPCR. (D) Primary cortical neurons at DIV 7 were transduced with either FNDC5 or GFP adenovirus. Cell viability was assessed 3 days later using the CellTiter-Glo Luminescent Cell Viability Assay. AU, arbitrary unit. (E) Primary cortical neurons at DIV 5 were transduced with lentivirus carrying the specified shRND hairpins against Fndc5 or luciferase (Luc) as control. Cell viability was assessed 3 days later using the CellTiter-Glo Luminescent Cell Viability Assay. AU, arbitrary unit. (G) Primary cortical neurons at DIV 7 were stimulated overnight with human recombinant BDNF at the indicated concentrations or vehicle, mRNA was prepared and gene expression was assessed by qPCR. Data (B, C) are shown as mRNA levels relative to Rsp18 expression. All data are expressed as mean +/- SEM *p< 0.05 compared to the corresponding control group. (Wrann et al., 2013)

Figure 5 Analysis:

Figure 5B shows that BDNF and related genes are expressed at increased levels in the presence of FNDC5 in the primary cortical neurons. This confirms that BDNF expression is dependent on FNDC5 expression as shown in figure 5C. In figure 5C as the Fndc5 is inhibited by the shRNS hairpins the expression of BDNF decreases. These two tests confirm that BDNF expression is dependent on Fndc5 expression.

Figure 5D addresses the cell viability of primary cortical neurons transduced with either Fndc5 or GFP adenovirus. There was a significant increase in cell viability in the primary cortical neurons transduced with Fndc5. In figure 5E the shRNA hairpins that inhibit Fndc5 decrease the primary cortical cell viability. Therefore, it can be concluded that Fndc5 expression increases cell viability and has the predicted neuroprotective properties.

Figure 5G shows the existence of a homeostatic feedback loop. Between 0.1-1 ng/ml of BNDF concentration the relative gene expression of Fndc5 begins to decrease. This suggests the negative feedback loop that is indicated in the proposed PGC-1ɑ/FNDC5/BDNF pathway.

Overall Conclusion:

These experiments confirm the correlation between exercise and increased neural plasticity via the BDNF gene expression pathway providing confirmation of the beneficial effects of exercise on neuroprotection in the hippocampus. This mechanism provides some evidence that exercise is a preventative measure against AD by linking aerobic exercise directly to the growth factor BDNF. The mechanistic is approach is critical to start to form a causation between exercise and BDNF instead of an unexplained correlation. Since a hallmark characteristic of AD is neuronal connection damage the increased levels of neuroprotection and neurogenesis via exercise, as outlined by this pathway, would potentially help build up protection against AD neurodegeneration or slow the rate of AD. It is important to note that the mechanism of how endurance exercise induces PGC-1ɑ is not outlined in this literature. Regardless these experiments do confirm that endurance exercise do induce BDNF gene expression that leads to neuroprotection and neurogenesis.

References

Wrann, C. D., White, J. P., Salogiannnis, J., Laznik-Bogoslavski, D., Wu, J., Ma, D., Lin, J. D., Greenberg, M. E., & Spiegelman, B. M. (2013). Exercise induces hippocampal BDNF through a PGC-1α/FNDC5 pathway. Cell metabolism, 18(5), 649–659. https://doi.org/10.1016/j.cmet.2013.09.008

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