Cystatin C protects neuronal cells from amyloid-beta-induced toxicity

Abstract

Multiple studies suggest that cystatin C (CysC) has a role in Alzheimer's disease (AD) and a decrease in CysC secretion is linked to the disease in patients with a polymorphism in the CysC gene. CysC binds amyloid-beta (Abeta) and inhibits formation of Abeta fibrils and oligomers both in vitro and in mouse models of amyloid deposition. Here we studied the effect of CysC on cultured primary hippocampal neurons and a neuronal cell line exposed to either oligomeric or fibrillar cytotoxic forms of Abeta. The extracellular addition of the secreted human CysC together with preformed either oligomeric or fibrillar Abeta increased cell survival. While CysC inhibits Abeta aggregation, it does not dissolve preformed Abeta fibrils or oligomers. Thus, CysC has multiple protective effects in AD, by preventing the formation of the toxic forms of Abeta and by direct protection of neuronal cells from Abeta toxicity. Therapeutic manipulation of CysC levels, resulting in slightly higher concentrations than physiological could protect neuronal cells from cell death in AD.

Figure 1. CysC protects N2a neuroblastoma cells from Aβ-induced cell-death

N2a cells were incubated for 40–44 hours in serum-free medium, in the presence or absence of either fibrillar (A) or oligomeric (B) Aβ, in the presence or absence of human CysC. MTS analysis of live cells, presented as mean ± standard deviation of percent of live cells incubated in serum-containing medium without either Aβ or CysC (mean of 4 experiments; *p≤.02 and **p≤.003 for the difference from cells with serum; ***p≤.03 and ****p≤.002 for the difference from cells with Aβ without CysC).

Figure 2. CysC protects rat primary hippocampal neurons from Aβ-induced cell-death

Cells were incubated for 24 hours, in the presence or absence of either fibrillar (A) or oligomeric (B) Aβ, in the presence or absence of human CysC. Additionally, Aβ was preincubated with CysC at conditions in which Aβ forms oligomers and the mix was added to the cells (B). Quantification of live cells, presented as mean ± standard deviation of percent of live cells incubated in serum-containing medium without either Aβ or CysC (mean of 4 experiments; *p≤.05 and **p≤.0003 for the difference from cells without Aβ and CysC; ***p≤.003 for the difference from cells with Aβ without CysC).

Figure 3. CysC inhibits Aβ oligomerization in vitro

Dot blot analysis with the antibody specific for oligomeric conformation (A11) of Aβ42 (100 μM) incubated with CysC (0, 7, 14 or 34 μM). Mean ± standard deviation of quantification of the intensity of the blots from 3 experiments.

Figure 4. CysC prevents Aβ-induced caspase-2 activation

Rat primary hippocampal neurons were pre-treated for 2 hours with 50 μM bVADfmk. After 2 hours cells were incubated for an additional 3 hours in the presence or absence of 3 μM oligomeric Aβ and in the presence or absence of 0.8 μM CysC. Active caspase-2 was pulled down using streptavidin beads and identified by Western blot analysis with a monoclonal caspase-2 specific antibody. Representative blots are shown; the experiments were replicated 3 times.