Axon degeneration is key component of neuronal death in amyloid-β toxicity

Abstract

Depending upon the stimulus, neuronal cell death can either be triggered from the cell body (soma) or the axon. We investigated the origin of the degeneration signal in amyloid β (Aβ) induced neuronal cell death in cultured in vitro hippocampal neurons. We discovered that Aβ1-42 toxicity-induced axon degeneration precedes cell death in hippocampal neurons. Overexpression of Bcl-xl inhibited both axonal and cell body degeneration in the Aβ-42 treated neurons. Nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) blocks axon degeneration in a variety of paradigms, but it cannot block neuronal cell body death. Therefore, if the neuronal death signals in Aβ1-42 toxicity originate from degenerating axons, we should be able to block neuronal death by inhibiting axon degeneration. To explore this possibility we over-expressed Nmnat1 in hippocampal neurons. We found that inhibition of axon degeneration in Aβ1-42 treated neurons prevented neuronal cell death. Thus, we conclude that axon degeneration is the key component of Aβ1-42 induced neuronal degeneration, and therapies targeting axonal protection can be important in finding a treatment for Alzheimer's disease.

Keywords: Alzheimer’s; Amyloid-β; Axon degeneration; Bcl-xl; Nmnat.

Figure 1. Axonal degeneration precedes somal death in Aβ_1–42 toxicity

Hippocampal neurons (7 DIV) were treated with 5 μM fibrillar Aβ_1-42 or oligomeric Aβ_1-42 or control reverse peptide Aβ_42-1, and phase-contrast microscopy was performed to analyze somal death and axonal degeneration. Quantitative analysis revealed that the Aβ1–42 oligomers were more neurotoxic then fibrillar Aβ_1-42 and axon degenerated at a significantly earlier time point than the soma death. The data were arcsine-transformed to meet the assumptions of parametric testing and Repeated Measures ANOVA was performed to see the effect of time (F=5850.9475, p<0.0001) and two-way ANOVA and Turkey methods for Post-hoc analysis were performed to compare the time dependent effects of fibrillar and oligomeric Aβ on cell body death and axonal degeneration. * indicates statistically different from cell body degeneration in each treatment group, # indicate significant different from fibrillar Aβ_1-42 treatment groups. (p < 0.0001).

Figure 2. Bcl-xl overexpression prevents Aβ-induced neuronal degeneration

Neurons (7 DIV) expressing Bcl-xl or empty vector were treated with 5 μM Aβ_1–42 for 48 h. Representative phase-contrast images revealed that Bcl-xl overexpression prevented axonal degeneration caused by Aβ_1–42 (B).

Figure 3. Bcl-xl over-expression prevents both axonal degeneration and cell death induced by fibrillar Aβ

Neurons (7 DIV) were treated with 5 μM fibrillar Aβ_1–42 (A, B and C), and fluorescence microscopy was performed to visualize Hoechst 33342 staining to score dead neurons [condensed chromatin (arrows) and surviving neurons (arrow heads)]. Axonal degeneration was measured via neuron-specific tubulin fragmentation. Bcl-xl overexpression blocked both axonal degeneration and somal death (arrowheads) in Aβ-induced neurodegeneration (D, E and F). Quantitative analysis showed significant axonal protection (G) and significant cell death protection (H) in Bcl-xl overexpressing neurons. * indicates statistically different from control (reverse peptide, Aβ_42-1), # indicates statistically different from Aβ_42-1-treated neurons.

Figure 4. Nmnat1 over-expression blocks fibrillar Aβ_1–42-induced axon degeneration

nuNmnat or cytNmnat expressing hippocampal neurons were treated with 5 μM Aβ_1–42 Phase-contrast microscopy was performed to visualize the protective effects of Nmnat1 on Aβ-treated neurons. Representative images of phase contrast microscopy revealed that fibrillar Aβ_1–42 caused extensive neuronal degeneration (A). nuNmnat overexpression (B) or cytNmnat overexpression (C) each prevented neuronal degeneration caused by Aβ_1–42.

Figure 5. Nmnat1 over-expression prevents both axon degeneration and cell death induced by fibrillar Aβ_1–42 toxicity

Hippocampal neurons were infected at 1 DIV with lentiviruses expressing nuNmnat (A, B and C) and cytNmnat (D, E and F). Six days after infection, neurons were treated with 5 μM fibrillar Aβ_1–42. After an additional 48 h, neurons were fixed, and immunofluorescence analysis of Tuj1 and Hoechst 33342 was performed. Representative images of Tuj1 (A), Hoechst 33342 (B) and C (merged image of A and C) of neurons over-expressing nuNmnat1 show that both the axon and cell body were protected in these neurons. Representative images of Tuj1 (D), Hoechst 33342 (E) and F (merged image of D and E) of neurons over-expressing cytNmnat1 show that both the axon and cell body were protected in these neurons as well. Quantitative analysis of axon integrity (G) and somal survival (H) reveals that Nmnat blocked both axonal degeneration and somal death in fibrillar Aβ-treated neurons. * indicates statistically different from control (reverse peptide, Aβ_42-1), # indicates statistically different from Aβ_42-1 treated neurons.

Figure 6. Bcl-xl and cytNmnat1 over-expression prevents both axon degeneration and cell death induced by oligomeric Aβ_1–42 toxicity

Hippocampal neurons were infected at 1 DIV with control lentiviruses (A, B and C) and lentiviruses expressing Bcl-xl (D, F and F) or cytNmnat (G, H and I). Six days after infection, neurons were treated with 5 μM oligomeric Aβ_1–42. After an additional 24 h, neurons were fixed, and immunofluorescence analysis of Tuj1 and Hoechst 33342 was performed. Representative images of Tuj1 (D), Hoechst 33342 (E) and F (merged image of D and E) of neurons over-expressing Bcl-xl show that both the axon and cell body were protected in these neurons. Representative images of Tuj1 (G), Hoechst 33342 (H) and I (merged image of G and H) of neurons over-expressing cytNmnat1 show that both the axon and cell body were protected in these neurons as well. Quantitative analysis of axon integrity (J) and somal survival (K) reveals that Bcl-xl and Nmnat blocked both axonal degeneration and somal death in oligomeric Aβ-treated neurons. * indicates statistically different from control (reverse peptide, Aβ_42-1), # indicates statistically different from Aβ_42-1 treated neurons.