Activation of Wnt Signaling Pathway Reverses Cognitive Deficits in an Alzheimer’s Disease Model

A descriptive image depicting the primary differences in the appearance and characteristics of a healthy brain and a brain of a patient suffering from Alzheimer’s Disease in mild and advanced stages. Photo Credit: http://sierram.web.unc.edu/2011/04/22/caffeine-and-alzheimers-disease/

Introduction:

Alzheimer’s disease is a neurodegenerative disorder that results in  impairment of memory and  cognition. As the disease progresses, symptoms such as confusion, mood swings, and trouble with language become prevalent and can complicate simple daily tasks. In most people with Alzheimer’s, symptoms first appear after age 60. Alzheimer’s disease is the most common cause of dementia among the elderly. One feature common to all patients suffering from dementia is the accumulation of amyloid-β (Aβ ) peptides which are the main component of amyloid plaques. These plaques are known to cause synaptic failure and eventually, neuronal deterioration.

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Magnetic resonance image (MRI) of a healthy human brain. Magnetic resonance imaging is a primary method for visualizing the progression of effects resulting from Alheimer’s Disease.
Photo credit: http://en.wikipedia.org/wiki/Magnetic_resonance_imaging

In past studies, Wnt ligands, a class of lipid-modified signaling glycoproteins involved in various cellular processes, were found to be a potential remedy to the disease. These ligands are involved in the development of the nervous system and there has been evidence suggesting that Wnt signaling significantly influences the synaptic transmission. More specifically, activation of the Wnt/β-catenin signaling pathway using the Wnt3a ligand protects against the toxic effects of Aβ peptides. The difficulty that arises in conducting studies regarding the condition lies in the fact that human subjects may only be examined to an extent. As with studies regarding many other diseases, the use of human subjects becomes an issue of morality. One remedy to this issue is the use of an organism with a brain similar to that of humans. Mice are an ideal choice because of their short generation time and an accelerated lifespan. In the following study, the in-vivo activation of this pathway will be evaluated in the hippocampus of adult wild type and APP/PS1 (amyloid precursor protein) mice. This protein produces Aβ peptides to simulate the plaques of an Alzheimer’s patient.

Experimental Methods:

To study the role of Wnt signaling in the cognitive function of adult mice, WASP-1, a potentiator of Wnt/β-catenin signaling  and FOXY-5, an activator of both Wnt/JNK and Wnt/Ca+2 signaling, were used. Both healthy mice and those with the APP/PS1 protein were used in the assessments. The first measure of assessment was a behavioral test. For this test, mice with the protein were implanted with an infusion system designed to stimulate Wnt-pathway activation. All of the mice were trained in a circular water maze (1.2 m diameter, opaque water, 50 cm deep, 19–21°C). The training was conducted everyday until the subjects were able to complete three successive trials within <20 s. A video-tracking system was used to collect the resulting data. Upon completion of the trials, hippocampi of treated and control mice were dissected and homogenized to conduct a western blot. Hippocampal slices were also used to obtain electrophysiological measurements. To do so, the brain samples were subject to electrical stimulation to simulate excitatory synaptic potentials and long term potentiation.

Results:

Figure 1. A–B, Immunoblotting for Wnt pathway proteins of hippocampal homogenates from WASP-1 (n = 3), FOXY-5 (n = 3), and control (n = 3) mice. A, Top, Western blot of control and WASP-1-treated mice for the canonical Wnt signaling proteins β-catenin, GSK-3β, and its inhibitory isoform phospho-Ser-9 (p-GSK-3β), and the target gene c-myc. Bottom: Quantification of protein band intensities after β-actin normalization. Graphic shows fold increase versus control. B, Top, Western blot of control and FOXY-5-treated mice for β-catenin and the noncanonical Wnt signaling proteins JNK and CaMKII and their corresponding phosphorylated isoforms. Bottom, Quantitative analysis as described in A. C–G, Behavioral performance of WASP-1 (n = 7), FOXY-5 (n = 6), and control (n = 7) mice in the memory flexibility test. C, Daily progression on each platform location. D, Comparison of the mean number of trials necessary to reach criterion. Graphic shows the mean value of the 4 d of testing for each group. E, Representative tracks of the last day of testing showing different swimming strategies among groups. F, Comparison of the mean swim speed showing no significant differences among groups. G, Comparison of the escape latency in the visible platform test showing no significant differences among groups for any trial. H–I, Behavioral performance of WASP-1 (n = 7), FOXY-5 (n = 6), and control (n = 7) mice in the novel object recognition test. H, Total object exploration time during acquisition memory phase of the test showing no significant differences among groups. I, Comparison of the preference index for a novel object during retention memory phase of the test.Figure 1. A–B, Immunoblotting for Wnt pathway proteins of hippocampal homogenates from WASP-1 (n = 3), FOXY-5 (n = 3), and control (n = 3) mice. A, Top, Western blot of control and WASP-1-treated mice for the canonical Wnt signaling proteins β-catenin, GSK-3β, and its inhibitory isoform phospho-Ser-9 (p-GSK-3β), and the target gene c-myc. Bottom: Quantification of protein band intensities after β-actin normalization. Graphic shows fold increase versus control. B, Top, Western blot of control and FOXY-5-treated mice for β-catenin and the noncanonical Wnt signaling proteins JNK and CaMKII and their corresponding phosphorylated isoforms. Bottom, Quantitative analysis as described in A. C–G, Behavioral performance of WASP-1 (n = 7), FOXY-5 (n = 6), and control (n = 7) mice in the memory flexibility test. C, Daily progression on each platform location. D, Comparison of the mean number of trials necessary to reach criterion. Graphic shows the mean value of the 4 d of testing for each group. E, Representative tracks of the last day of testing showing different swimming strategies among groups. F, Comparison of the mean swim speed showing no significant differences among groups. G, Comparison of the escape latency in the visible platform test showing no significant differences among groups for any trial. H–I, Behavioral performance of WASP-1 (n = 7), FOXY-5 (n = 6), and control (n = 7) mice in the novel object recognition test. H, Total object exploration time during acquisition memory phase of the test showing no significant differences among groups. I, Comparison of the preference index for a novel object during retention memory phase of the test.
 Figure 2. A–E, Field recordings of hippocampal slices from WASP-1 (n = 10), FOXY-5 (n = 10), and control (n = 10) mice. A, Representative synaptic responses for equal fiber volley (FV) amplitude (black arrow). B, Correlation of fEPSP and FV amplitudes for each group. Black lines are the regression line for fEPSP (output) and FV (input). The correlation coefficients (R2) in the control, WASP-1, and FOXY-5 mice were 0.9855, 0.9714, and 0.9869, respectively. C, Average traces of fEPSP at three different intensities of stimulation (gray arrows). Dotted lines indicate FV amplitude of 1 mV. D, Plot of fEPSP amplitude versus stimulus intensity (from 200 to 900 μA). E, Plot of FV amplitude versus stimulus intensity (from 200 to 900 μA).Figure 2. A–E, Field recordings of hippocampal slices from WASP-1 (n = 10), FOXY-5 (n = 10), and control (n = 10) mice. A, Representative synaptic responses for equal fiber volley (FV) amplitude (black arrow). B, Correlation of fEPSP and FV amplitudes for each group. Black lines are the regression line for fEPSP (output) and FV (input). The correlation coefficients (R2) in the control, WASP-1, and FOXY-5 mice were 0.9855, 0.9714, and 0.9869, respectively. C, Average traces of fEPSP at three different intensities of stimulation (gray arrows). Dotted lines indicate FV amplitude of 1 mV. D, Plot of fEPSP amplitude versus stimulus intensity (from 200 to 900 μA). E, Plot of FV amplitude versus stimulus intensity (from 200 to 900 μA).
Figure 3. A–C, Field recordings of hippocampal slices from WASP-1 (n = 9), FOXY-5 (n = 9), and control (n = 10) mice. A, Average traces of fEPSP before (dotted lines) and after (filled lines) HFS. B, Plot of fEPSP amplitude versus time (from 10 min before HFS (arrow) up to 90 min after). Graphic shows percent increase of fEPSP amplitude compared with baseline (dotted line). C, Comparison of mean fEPSP amplitude among groups at 30 or 90 min after HFS showing percent increase versus baseline.
Figure 3. A–C, Field recordings of hippocampal slices from WASP-1 (n = 9), FOXY-5 (n = 9), and control (n = 10) mice. A, Average traces of fEPSP before (dotted lines) and after (filled lines) HFS. B, Plot of fEPSP amplitude versus time (from 10 min before HFS (arrow) up to 90 min after). Graphic shows percent increase of fEPSP amplitude compared with baseline (dotted line). C, Comparison of mean fEPSP amplitude among groups at 30 or 90 min after HFS showing percent increase versus baseline.
Figure 4. A–C, Field recordings of hippocampal slices from WASP-1 (n = 9), TCS-183 (n = 6), and TCS-183 + WASP-1 (n = 6)-treated mice. A, Representative traces of fEPSP before (dotted lines) and after (filled lines) HFS. B, Time course of normalized fEPSP amplitude from 15 min before HFS (arrow) up to 60 min after. Graphic shows percent increase of fEPSP amplitude compared with baseline (dotted line). C, Comparison of mean fEPSP amplitude among treated mice groups at 30 or 60 min after HFS showing percent increase versus baseline. (D–F) Field recordings of hippocampal slices from FOXY-5 (n = 9), TAT-TI-JIP (n = 6), and TAT-TI-JIP + FOXY-5 (n = 5)-treated mice. D, Representative traces of fEPSP before (dotted lines) and after (filled lines) HFS. E, Time course of normalized fEPSP amplitude as described in B. F, Comparison of mean fEPSP amplitude as described in C.
Figure 4. A–C, Field recordings of hippocampal slices from WASP-1 (n = 9), TCS-183 (n = 6), and TCS-183 + WASP-1 (n = 6)-treated mice. A, Representative traces of fEPSP before (dotted lines) and after (filled lines) HFS. B, Time course of normalized fEPSP amplitude from 15 min before HFS (arrow) up to 60 min after. Graphic shows percent increase of fEPSP amplitude compared with baseline (dotted line). C, Comparison of mean fEPSP amplitude among treated mice groups at 30 or 60 min after HFS showing percent increase versus baseline. (D–F) Field recordings of hippocampal slices from FOXY-5 (n = 9), TAT-TI-JIP (n = 6), and TAT-TI-JIP + FOXY-5 (n = 5)-treated mice. D, Representative traces of fEPSP before (dotted lines) and after (filled lines) HFS. E, Time course of normalized fEPSP amplitude as described in B. F, Comparison of mean fEPSP amplitude as described in C.
Figure 5. A, B, Immunoblotting of presynaptic (Syn-1 and Syp) and postsynaptic (PSD-95 and NR2B) proteins. A, Top, Western blot of hippocampal homogenates from WASP-1 (n = 3) and control (n = 3) mice. Bottom, Quantification of protein band intensities after β-actin normalization. Graphic shows fold increase versus control. B, Top, Western blot of hippocampal homogenates from FOXY-5 (n = 3) and control (n = 3) mice. Bottom, Quantitative analysis as described in A. n.s., No significant differences.
Figure 5. A, B, Immunoblotting of presynaptic (Syn-1 and Syp) and postsynaptic (PSD-95 and NR2B) proteins. A, Top, Western blot of hippocampal homogenates from WASP-1 (n = 3) and control (n = 3) mice. Bottom, Quantification of protein band intensities after β-actin normalization. Graphic shows fold increase versus control. B, Top, Western blot of hippocampal homogenates from FOXY-5 (n = 3) and control (n = 3) mice. Bottom, Quantitative analysis as described in A. n.s., No significant differences.
Discussion:

Through the completion of this study, the following key points were supported:

  1. WASP-1 and FOXY-5 activate Wnt signaling in vivo (Figure 1A and 1B)
  2. WASP-1 and FOXY-5 improve episodic memory (Figure 1C – 1I)
  3. WASP-1 and FOXY-5 enhance synaptic function and plasticity (Figure 2, 3 and 4)
  4. WASP-1 and FOXY-5 increase synaptic protein levels (Figures 1A, 2, 3, and 5A)

The results of this study support the conclusion that Wnt signaling could be an important pathway regulating memory of adults. The mechanism for this memory improvement occurs involves synaptic repair and as a result, becomes relevant to the topic of Alzheimer’s disease. Remarkable improvements in both long and short term memory were noted in the study which reveals a promising break-through in identifying a method of treatment for this detrimental disease. The results of this study are the first of its kind  in the sense that this was an in vivo measure and as such, more studies similar to this one are a necessity. The paper this page is based on was very well written and easy to follow but would be improved greatly if the rationale for the selected methods were fully explained.

References:

  1. Vargas, J.,  Fuenzalida, M., & Inestrosa, N. (2014) In vivo Activation of Wnt Signaling Pathway Enhances Cognitive Function of Adult Mice and Reverses Cognitive Deficits in an Alzheimer’s Disease Model. Journal of Neuroscience. 34(6): 2191-2202
  2. Alzheimer’s Association (2007) What is Alzheimer’s? Retrieved from: http://www.alz.org/alzheimers_disease_alzheimers_disease.asp
  3. National Institute on Aging (2012) Alheimer’s Disease Fact Sheet. Retrieved from: http://www.nia.nih.gov/alzheimers/publication/alzheimers-disease-fact-sheet

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