In our study we used aged Tg2576 animals, herein referred to as APP animals, and compared their plaque pathology to that of APP animals lacking α-synuclein (APP/Snca-/-) at various ages. In general the mice are indistinguishable from their littermates in terms of appearance and viability. In order to determine whether we could detect differences in the production of two main Aβ species we performed Sandwich ELISA experiments at an early age. At 6 months of age neither the APP nor the APP/Snca-/-exhibit Aβ plaque pathology (data not shown). As expected, no difference in Aβ40 or Aβ42 levels could be detected by Sandwich ELISA (Figure ) when comparing 6-month-old APP and APP/Snca-/- animals. We then focused our study to determine the effects of α-synuclein on the onset of plaque deposition, and the progression of the pathology by comparing these parameters between APP and APP/Snca-/-.
Sandwich ELISA of 6-month-old APP and APP/Snca-/- animals. Absence of α-synuclein did not lead to significant alterations in the levels total Aβ40 and Aβ42.
In order to determine whether the age of onset of plaque deposition is affected, we studied 9-month-old APP and APP/Snca-/- animals as that is the earliest time when plaques can be detected in APP mice. Using immunohistochemistry with 6E10 antibody to human APP, we detected a slight increase in the number of plaques (Figure ), but the difference failed to reach statistical significance (Figure ). The number of plaques increased in both APP and APP/Snca-/- mice at 12 months (Figure ). Similar to 9-month-old animals, there was a slight, but not statistically significant, difference between the two genotypes in the number or size of plaques (Figure ). As a homologous protein to α-synuclein, we studied the protein levels of β-synuclein as well as another synaptic vesicle protein, synaptophysin, in the brain homogenates of APP and APP/Snca-/- mice. We were unable to detect significant differences in the expression of β-synuclein or synaptophysin in brain homogenates, limiting the possibility that these presynaptic proteins play a compensatory role for the loss of α-synuclein (Figure ). Overall, it appears that α-synuclein is not a large player in initiation/seeding of the plaque pathology in APP mice.
Figure 2 Immunostaining of 9- and 12-month-old APP and APP/Snca-/- brains. A. Sections were stained with 6E10 antibody to detect the total number of amyloid beta plaques. Scale bar denotes 100 μm. B. Representation of the number of plaques per section (more ...)
At 18 months of age, however, differences in the number of plaques became obvious. When performing 6E10 and thioflavine S staining of comparable coronal sections, we observed a vastly increased number of plaques in the APP/Snca-/- animals when compared to APP animals (Figure ). Because the number of plaques in APP/Snca-/- mice reached a level that was difficult to be counted manually, we chose to quantify the plaque density by assessing the signal intensity of matched areas. Statistical analysis revealed that both total (6E10-positive) and dense-core plaque (thioflavin S-positive) load in the APP/Snca-/- animals were increased 3–4 fold when compared to the number of plaques found in age-matched Tg2576 animals (p < 0.001).
Figure 3 Plaque analysis of 18-month-old APP and APP/Snca-/- animals. A. Sections were stained with 6E10 antibody or thioflavine S as indicated. 6E10 staining revealed the total plaque load while thioflavine S detected mature plaques only. The plaque density is (more ...)
As expected, Western blot analysis showed that α-synuclein protein was missing from the brain homogenates of the APP/Snca-/- animals (Figure ) and the levels of full-length APP were not affected by the absence of α-synuclein. Once again we were unable to detect an increase in the β-synuclein (Figure ). Unexpectedly, we found that synaptophysin, which was unchanged at 12 months, was up-regulated by two-fold in APP/Snca-/- animals when compared to APP animals (Figure , p < 0.01). This could be attributed to a compensatory mechanism activated due to α-synuclein deficiency. However, the reason for the selective increase of synaptophysin, but not β-synuclein, is not clear.
Figure 4 Western blotting of 18-month-old of APP and APP/Snca-/- animals. A. 10 μg of forebrain lysates were separated on SDS-PAGE, transferred to a nitrocellulose membrane and probed with antibodies against α-synuclein, APP, synaptophysin and (more ...)