Tau and caspase 3 as targets for neuroprotection

Abstract

The peptide drug candidate NAP (davunetide) has demonstrated protective effects in various in vivo and in vitro models of neurodegeneration. NAP was shown to reduce tau hyperphosphorylation as well as to prevent caspase-3 activation and cytochrome-3 release from mitochondria, both characteristic of apoptotic cell death. Recent studies suggest that caspases may play a role in tau pathology. The purpose of this study was to evaluate the effect of NAP on tau hyperphosphorylation and caspase activity in the same biological system. Our experimental setup used primary neuronal cultures subjected to oxygen-glucose deprivation (OGD), with and without NAP or caspase inhibitor. Cell viability was assessed by measuring mitochondrial activity (MTS assay), and immunoblots were used for analyzing protein level. It was shown that apoptosis was responsible for all cell death occurring following ischemia, and NAP treatment showed a concentration-dependent protection from cell death. Ischemia caused an increase in the levels of active caspase-3 and hyperphosphorylated tau, both of which were prevented by either NAP or caspase-inhibitor treatment. Our data suggest that, in this model system, caspase activation may be an upstream event to tau hyperphosphorylation, although additional studies will be required to fully elucidate the cascade of events.

Figure 1

Culture purity: at 5-6 DIV, cells were fixed and immunofluorescence staining was performed using the astrocyte marker GFAP (green), the neuronal marker NeuN (red), and DAPI stain (blue) for nuclei. Quantification of neuronal cells was done using 10 random fields from each of 3 experiments (×20 magnification). 95.9 ± 0.96% of the total cells (DAPI stain) were recognized as neurons (NeuN positive, GFAP negative).

Figure 2

NAP protects from cell death in a dose dependent manner following 2 hours of OGD insult, but not after additional 24 hours reperfusion. Primary neuronal cultures (5-6DIV) were subjected to 2 h of ischemic insult. Cell viability was evaluated using MTS viability assay. Data was normalized to % of control. Results are shown as mean ± SE. *Significantly different from OGD with no NAP, OGD + NAP10⁻¹⁵ M, OGD + NAP 10⁻¹³ M, OGD+NAP 10⁻¹¹ M (3 independent experiments are summarized, ANOVA, with post hoc Scheffe, significant difference considered to be P ≤ 0.05). Using LSD post hoc, NAP 10⁻⁵ M (+), 10⁻⁷ M are significantly different from the OGD group (P < 0.05).

Figure 3

Following 2 hours of OGD, cells died exclusively from apoptosis. Cultures were treated with either NAP (10⁻⁵ M) or QVD-OPH (2∗10⁻⁵ M) and subjected to 2 h of ischemic insult. Cell viability was evaluated immediately following the ischemic period using MTS viability assay. Data was normalized to % of control. Results are shown as mean ± SE; *significantly different from all other groups, P ≤ 0.05 (ANOVA, with post hoc Scheffe).

Figure 4

An increase in active caspase-3 levels induced by 2 hours of OGD was diminished by NAP treatment (10⁻⁵ M). Cultures were treated with 10⁻⁵  M NAP or with 20 μM QVD-OPH (broad spectrum caspase inhibitor) exposed to OGD insult for 2 hours. Proteins were extracted and analyzed using immunoblot with a specific antiactive caspase-3 antibody. A representative blot of active caspase-3 antibody is exhibited in (a). (ANOVA with post hoc LSD, P ≤ 0.05) (b).

Figure 5

2 hours of OGD caused an increase in p-tau²⁰² levels, prevented by either NAP or QVD-OPH treatment. Cultures were treated with 10⁻⁵ M NAP or with 20 μM QVD-OPH (broad spectrum caspase inhibitor, indicated as OGD + Q) and exposed to OGD insult for 2 hours. Proteins were extracted and analyzed using immunoblot with a specific anti-p-tau²⁰² and antitotal tau antibodies. A representative blot of p-tau²⁰² and total tau is exhibited in (a). P-tau levels were quantified and normalized to total tau levels (ANOVA with post hoc LSD, P ≤ 0.05) (b).