Wnt Signaling Alteration in the Spinal Cord of Amyotrophic Lateral Sclerosis Transgenic Mice: Special Focus on Frizzled-5 Cellular Expression Pattern

Abstract

Background: Amyotrophic lateral sclerosis is a chronic neurodegenerative disease characterized by progressive paralysis due to degeneration of motor neurons by unknown causes. Recent evidence shows that Wnt signaling is involved in neurodegenerative processes, including Amyotrophic Lateral Sclerosis. However, to date, little is known regarding the expression of Wnt signaling components in this fatal condition. In the present study we used transgenic SOD1G93A mice to evaluate the expression of several Wnt signaling components, with special focus on Frizzled-5 cellular expression alteration along disease progression.

Findings: Based on previous studies demonstrating the expression of Wnts and their transcriptional regulation during Amyotrophic lateral sclerosis development, we have analyzed the mRNA expression of several Wnt signaling components in the spinal cord of SOD1G93A transgenic mice at different stages of the disease by using real time quantitative PCR analysis. Strikingly, one of the molecules that seemed not to be altered at mRNA level, Frizzled-5, showed a clear up-regulation at late stages in neurons, as evidenced by immunofluorescence assays. Moreover, increased Frizzled-5 appears to correlate with a decrease in NeuN signal in these cells, suggesting a correlation between neuronal affectation and the increased expression of this receptor.

Conclusions: Our data suggest the involvement of Wnt signaling pathways in the pathophysiology of Amyotrophic Lateral Sclerosis and, more specifically, the implication of Frizzled-5 receptor in the response of neuronal cells against neurodegeneration. Nevertheless, further experimental studies are needed to shed light on the specific role of Frizzled-5 and the emerging but increasing Wnt family of proteins research field as a potential target for this neuropathology.

Fig 1. Temporal mRNA expression pattern of different Wnt family members in the spinal cord of both WT and ALS transgenic mice.

The temporal mRNA expression pattern of Fz1, Fz2, Fz3, Fz4, Fz5, Fz8, Ror2, Ryk, Wif1, Wnt2, Wnt4, Wnt5a and Wnt7b was quantified using real time quantitative PCR using specific primers, both in the WT and ALS transgenic mice spinal cord and at different stages of the disease. Differences were calculated by setting the expression values of the WT samples at 1 and normalising against ribosomal 18S rRNA. In all cases, the data are presented as the mean ± SEM; * p < 0.05 versus WT.

Fig 2. Protein expression of Fz1, Fz4 and Fz5 receptors in the spinal cord of both WT and ALS transgenic mice.

This figure shows representative images obtained from the microscopic evaluation of sections processed by simple immunofluorescence to visualize Fz2, Fz4 and Fz5. Fz1 and Fz4 receptors do not undergo changes in their cellular expression pattern during disease progression. Instead, Fz5 immunoreactivity begins to increase at 12w in some cells, and clearly observed at 16w. The analysis was performed in WT and ALS mice spinal cords at 8w, 12w and 16w. Scale bars = 200 μm.

Fig 3. Neuronal location of Fz5 receptor in the spinal cord of both WT and ALS transgenic mice.

This figure shows representative images obtained from the microscopic evaluation of sections processed by double immunohistochemistry to visualize Fz5 in neurons (NeuN). Fz5 was expressed in most of neurons at differents spinal cord laminae in WT mice and also at different neuropathological stages of ALS transgenic mice. The analysis was performed in WT and ALS mice spinal cords at 8w, 12w and 16w. The squares in the images showing the entire spinal cord sections represent the areas where the corresponding higher magnification micrographs were obtained. Scale bars = 50 μm.

Fig 4. Increased expression of Fz5 receptor matched with a decrease in NeuN immunoreactivity at 16w.

This figure shows representative images obtained from the microscopic evaluation of sections processed by double immunohistochemistry to visualize Fz5 in neurons (NeuN). Cells with increased immunoreactivity for Fz5 showed low levels of NeuN staining (arrowheads). However, neurons with a baseline of Fz5 immunoreactivity exhibited normal NeuN expression (arrows). The analysis was performed in 16 ALS mice spinal cords. Scale bars = 50 μm.

Fig 5. Fz5 receptor was localized in motoneurons.

This figure shows representative images obtained from the microscopic evaluation of sections processed by double immunohistochemistry to visualize Fz5 in MNs (Chat). Fz5 receptor was expressed by MNs immunopositive for Chat staining both WT and ALS transgenic mice. No changes were found regarding to any increase in Fz5 immunoreactivity associated with MNs. The analysis was performed in WT and ALS mice spinal cords at 8w, 12w and 16w. The squares in the images showing the entire spinal cord sections represent the areas where the corresponding higher magnification micrographs were obtained. Scale bars = 50 μm.

Fig 6. Fz5 receptor was localized in astrocytes.

This figure shows representative images obtained from the microscopic evaluation of sections processed by double immunohistochemistry to visualize Fz5 in astrocytes (GFAP). Fz5 receptor was located in astrocytes both WT and ALS transgenic mice, mainly in contacting pial surface regions, but no changes were observed during disease progression. The analysis was performed in WT and ALS mice spinal cords at 8w, 12w and 16w. The squares in the images showing the entire spinal cord sections represent the areas where the corresponding higher magnification micrographs were obtained. Scale bars = 50 μm.

Fig 7. Location of Fz5 receptor and the mitochondrial enzyme SOD2.

This figure shows representative images obtained from the microscopic evaluation of sections processed by double immunohistochemistry to visualize Fz5 and mitochondrial enzyme SOD2. We did not observe a correlation between increased immunoreactivity of Fz5 and SOD2 in these neuronal cells, but an increase of SOD2 signal with the progression of the disease was observed. The analysis was performed in WT and ALS mice spinal cords at 8w, 12w and 16w. Scale bars = 50 μm.