Figure 1.
Establishment of a neurodegenerating model of primary cerebrocortical cultures: Association of different APP isoforms with glial and neuronal cell types.
A: Cell lysates and conditioned media samples of primary cortical cultures were collected at day 12 and day 20 for Western blot analysis. At day 12 compared to day 20, levels of the neuronal marker NSE are high, and levels of the glial marker GFAP are low. This indicates that at day 12 the neuronal population is intact, whereas the glial population becomes more pronounced at day 20, and neurons have degenerated by day 20. In the sAPP blot, reduced levels of low molecular weight sAPP coincide with the loss of neurons, while the increased high molecular weight sAPP parallels the increase in glia abundance. Approximate molecular weights are indicated. Day 12 and day 20 samples were examined on the same blots with irrelevant samples cropped from the images. B: A detailed view of sAPP bands in single representative lanes of Western blots from neuron-rich (day 12) and glia-rich (day 20) cell cultures reveals that the lower band (∼110 kDa) is predominant over the upper band (∼130 kDa) in neuron-rich cultures This pattern is reversed in glia-rich cultures, with a predominant upper band, and very little sAPP present at the lower molecular weight. C: Densitometric quantification of the top vs. bottom bands from day 12 and day 20 samples is also shown. Levels of the high molecular weight top band are relatively stable with time, while levels of the low molecular weight bottom band decrease dramatically between day 12 and day 20, concurrent with neurodegeneration.
Figure 2.
Rivastigmine preserves neuronal morphology and alters sAPP secretion.
A: At day 12 or day 20, cells were fixed for immunocytochemistry analysis and probed with anti-MAP2 (green) and anti-GFAP (red) antibodies. Neuronal MAP2 immunoreactivity declines to almost undetectable levels between day 12 and day 20 in untreated cells, whereas neuronal morphology is preserved in rivastigmine treated cultures. Glial GFAP was observed to increase in both treated and untreated cells between day 12 and day 20. These results confirm the degeneration of neurons by day 20. All samples were examined on the same blot with irrelevant samples cropped from the images. B: In a separate experiment, media samples were taken before the onset of neurodegeneration at day 12 and during the degenerating phase of the cell culture at day 16. The lower molecular weight neuronal form of sAPP declined significantly in untreated cells, but was rescued by 10 µM rivastigmine treatment.
Figure 3.
Rivastigmine enhances cell viability in neurodegenerating primary cultures.
A: Rivastigmine is a parasympathomimetic cholinergic agent used for the treatment of mild to moderate AD. Its systematic (IUPAC) name is (S)-N-Ethyl-N-methyl-3-[1-(dimethylamino)ethyl]-phenyl carbamate. B: Primary cultures were treated with rivastigmine at the indicated concentrations for 4 days starting at day 12, and were then subjected to the Cell Titer-Glo (CTG) assay to assess cell viability and metabolic activity. Significant increases in ATP concentrations were observed in the lysates of cells treated with 5 µM, 10 µM and 20 µM rivastigmine (both p<0.05), while no effect was observed with 2 µM rivastigmine. The potential of drug-induced toxicity was assessed by measuring LDH release, but no significant difference from vehicle was observed, suggesting that all rivastigmine doses were well tolerated.
Figure 4.
Rivastigmine increases the proportion of neuron-associated sAPP isoform to total secreted amyloid precursor protein (sAPP).
A: Relative levels of total sAPP were measured by Western blot using the monoclonal 22C11 antibody. Densitometric quantification of the blots indicates that total sAPP is increased significantly at both rivastigmine concentrations tested (both p<0.05). The intensity of each band relative to the total sAPP was calculated and the top and bottom bands show diverging patterns, with the top band decreasing with rivastigmine treatment, and the bottom band increasing with rivastigmine treatment relative to total sAPP. B: Levels of the longer KPI-containing sAPP isoform were determined by Western blot using a KPI-specific antibody. A single band was detected in the media samples of these cells, and the molecular weight of this band coincides with the higher molecular weight band detected with the 22C11 antibody. Thus, the distinguishing characteristic between the top and bottom bands observed with 22C11 are differences in splicing. Levels of KPI containing APP are not altered by rivastigmine treatment.
Figure 5.
Rivastigmine shifts APP processing toward the α-secretase pathwayI.
Levels of sAPPα and Aβx-40 were measured in media samples of cultures treated with rivastigmine using ELISA techniques. A: sAPPα increased significantly in response to both 5 µM and 10 µM rivastigmine (both p<0.01), but the treatments were not significantly different rom each other suggesting a maximum effect of rivastigmine at or below 5 µM. B: Aβx-40 levels were decreased by both concentrations of rivastigmine (both p<0.01). This effect was also not dose-dependent, suggesting a maximum effect at 5 µM or lower concentrations. Together, these data suggest a shift in the relative activities of the α- and β-secretase pathways, favoring production of the neurotrophic α-secretase product sAPPα while simultaneously reducing the neurotoxic β-secretase product Aβ.
Figure 6.
Rivastigmine does not alter APP holoprotein.
APP levels in lysates of cerebrocortical cultures treated with vehicle or rivastigmine were measured by Western blot using the 22C11 antibody. No difference in APP holoprotein was found in either the 5 µM or 10 µM rivastigmine treatments, indicating that changes in sAPP levels are not the result of changes in total APP production in these cells.
Figure 7.
Rivastigmine-mediated increase in neuron-associated sAPP and sAPPα parallels increases in neuronal protein markers.
Levels of low molecular weight neuronal sAPP (LMW-sAPP), high molecular weight glial sAPP (HMW-sAPP), and sAPPα were compared to levels of the presynaptic protein markers SNAP-25 and syntaxin-4, and the postsynaptic protein marker PSD-95. All values are expressed as a % of vehicle-treated cells for comparison. PSD-95 and SNAP-25 levels increased dose-dependently with rivastigmine treatment (both p<0.05), and syntaxin-4 levels increased but this change did not reach significance (7A and 7B). LMW-sAPP was increased with 5 µM rivastigmine but plateaued at the higher concentration. HMW-sAPP increased to a lesser extent at 5 µM, but was similar to vehicle at the higher concentration (7C). These data suggest that increased neuronal and decreased glial sAPP may be involved in the enhanced neuronal and synaptic marker stability that results from rivastigmine treatment, and that modulation of α-secretase may be involved in these effects.
Figure 8.
Rivastigmine elevates CSF sAPP levels in rodents.
Separate groups of Fischer-344 male rats (n = 8–12) were administered saline (control) or rivastigmine (0.75 mg/kg) i.p. once daily for 21 days, and CSF was collected within 120 min of the final dose for quantification of sAPP by Western blot analysis probing with anti-APP antibody, 22C11. The APP synthesis inhibitor, phenserine (2.5 mg/kg, i.p. for 21 days) was administered to a concurrent group of animals as a positive control (*<0.05 vs control, Dunnett's t-test).