Botulinum neurotoxin C initiates two different programs for neurite degeneration and neuronal apoptosis

Abstract

Clostridial neurotoxins are bacterial endopeptidases that cleave the major SNARE proteins in peripheral motorneurons. Here, we show that disruption of synaptic architecture by botulinum neurotoxin C1 (BoNT/C) in central nervous system neurons activates distinct neurodegenerative programs in the axo-dendritic network and in the cell bodies. Neurites degenerate at an early stage by an active caspase-independent fragmentation characterized by segregation of energy competent mitochondria. Later, the cell body mitochondria release cytochrome c, which is followed by caspase activation, apoptotic nuclear condensation, loss of membrane potential, and, finally, cell swelling and lysis. Recognition and scavenging of dying processes by glia also precede the removal of apoptotic cell bodies, in line with a temporal and spatial segregation of different degenerative processes. Our results suggest that, in response to widespread synaptic damage, neurons first dismantle their connections and finally undergo apoptosis, when their spatial relationships are lost.

Figure 1.

BoNT/C induces apoptosis in CGCs. (A) Time course of syntaxin and SNAP-25 cleavage analyzed by Western blot in CGC lysates. Cells were exposed to 20ng/ml BoNT/C for the time indicated. Concentration–response (B) and time course (C) of BoNT/C-induced cell death. In B, cell death was evaluated 30 h after treatment with the toxin, whereas in C, cells were exposed to 20 ng/ml BoNT/C in the absence or the presence of cycloheximide (CHX; 2 μM). Apoptosis is expressed as a percentage of condensed nuclei in cultures stained by H-33342 (D). Shown are HMM DNA fragmentation (E) and DNA laddering (F) in cultures exposed to 20 ng/ml BoNT/C for the indicated times. Data are means ± SD for triplicate determinations. Bar, 30 μm.

Figure 2.

Apoptotic demise of the cell bodies is a caspase-dependent process. (A) Cytochrome c release analyzed by confocal microscopy at the level of the cell bodies in BoNT/C-treated CGCs. The release, denoted by the diffuse staining, was not prevented by caspase inhibition (e.g., by 100 μM zVAD-fmk). Cells were exposed to 20 ng/ml BoNT/C for 30 h. Shown are the time course of caspase activation measured by enzymatic assay (B) and fodrin cleavage (C) in CGC lysates exposed to 20 ng/ml BoNT/C for the indicated times. Caspase inhibition by 100 μM zVAD-fmk prevented the increase in DEVD-afc cleaving activity (B), fodrin cleavage and apoptosis (C), nuclear condensation (A), HMM DNA fragmentation (D), and DNA laddering (E). The caspase-2 inhibitor zVDVAD-fmk (1 μM) partially protected from apoptosis. Bar, 10 μm.

Figure 3.

BoNT/C-induced apoptosis is independent of the block of neurotransmitter release and is not prevented by glutamatergic stimulation. (A) Dose–response of BoNT/C-induced block of neurotransmitter release and correlation with apoptosis. Neurotransmitter release was triggered by potassium depolarization in l-[G-³H]glutamine–loaded cultures. (B) Comparison of the block of neurotransmitter release with apoptosis induced by 20 ng/ml BoNT/C and 2 μg/ml BoNT/A. (C) CGCs were incubated with 20 ng/ml BoNT/C. After 12 h, 1 μM AMPA or 0.5 μM glutamate was added, and, after 36 h, apoptosis was evaluated by H-33342 staining. (D) CGCs were incubated with 20 ng/ml BoNT/C for 15 h. Then, they were exposed to 300 μM glutamate for 1 h, in the presence or in the absence of 2 μM MK801. Apoptosis was evaluated as a percentage of condensed nuclei. Data are means ± SD for triplicate determinations.

Figure 4.

A caspase-independent axonal breakdown preceded the apoptotic demise of the neuronal bodies. Appearance of the axo-dendritic network in CGC cultures as shown by phase-contrast (A) and scanning electron microscopy (B). The disassembly of the neuronal projections in the presence of 20 ng/ml BoNT/C is noteworthy. The magnification used in B allows the visualization of the only neuronal network. The p17 fragment of caspase-3 was detected along the neuronal projections (C), as well as within the cell bodies (D), by confocal microscopy. Note the absence of staining when the antibody was preabsorbed with p20/p17 caspase-3. Fluorescence images in C represent single focal planes acquired at the level of the neurites. The arrow indicates the cell shown in D to illustrate the presence of processed caspase-3 at the level of the cell body at 24 h. The Western blot shows small amounts of caspase-3 processing in untreated neurons and 18 h after BoNT/C. Substantial activation is seen after 32 h, when most neurons have undergone apoptosis (E). Note that caspase inhibition by zVAD-fmk prevented nuclear condensation, but did not prevent the dismantling of the neurites (A). Neurotrophic factors (100 ng/ml BDNF and 200 ng/ml IGF-1) and cAMP (1 mM) did not prevent axonal degeneration (not depicted) and only BDNF partially protected from apoptosis (E). Neurotrophic factors efficacy was monitored in parallel cultures in which apoptosis was induced by K⁺ withdrawal. The percentage of apoptosis was evaluated by scoring condensed, H33342-positive nuclei. (F) Data are means ± SD for triplicate determinations; *, P < 0.005. Bars: (A) 10 μm; (B) 5 μm; (C) 30 μm; (D) 10 μm.

Figure 5.

Cytoskeletal disassembly and abnormal tau phosphorylation accompanied the early degeneration of the neurites. (A) CGCs were exposed to 20 ng/ml BoNT/C for the times indicated and immunostained for actin and β_III-tubulin. Confocal images were acquired from the same field. The bulblike structures present at 8 h along the neurites are shown at higher magnification in B (arrows). (C) The total amount of tau protein was detected by the K9JA antibody, which recognizes tau independently of its phosphorylation. In the same cell extracts, abnormal tau phosphorylation was detected by using the S202/205 phosphospecific antibody 11b. Neurons were exposed to 20 ng/ml BoNT/C for the indicated times. (D) The pattern of tau distribution was determined by a different antibody (Innogenetics) and changed in cells exposed to 20 ng/ml BoNT/C. Images in A, B, and D are extended focus images derived by merging 50–60 focal planes acquired in the z dimension with a 63× oil 1.3 NA apochromat objective. Bars, 20 μm.

Figure 6.

Mitochondrial integrity is retained during the early axonal degeneration, but it is lost in the late demise of the cell bodies. BoNT/C-treated CGCs were loaded with 20 nM TMRE and imaged by confocal microscopy (A and B). Time series imaging of mitochondria at the level of the cell bodies (A, a–e; time [h] a: 30; b: 37, c: 42, d: 42.5, and e: 43.5; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200406126/DC1). (B) CGC treated with 20 ng/ml BoNT/C for 18 h were loaded with calcein-AM (green) and TMRE (red) and imaged by confocal microscopy. The bottom panels show the detail of the related insets. Note the presence of TMRE-positive mitochondria, which is indicative of a retained membrane potential in the degenerating neurites and the different mitochondrial morphology/distribution in BoNT/C-treated neurons versus control. (C and D) Analysis of cytochrome c release along the neurites in BoNT/C-treated cultures. CGCs were immunostained for cytochrome c and mtHSP-70. Confocal images were acquired from the same field (C) and colocalization quantified by ImarisColoc software (D). Scattergrams show the percentage of the green channel voxels (cytochrome c [cyt c]) that colocalize with the red channel ones (mtHSP-70). Bars: (A) 5 μm; (B) 10 μm; (C) 15 μm.

Figure 7.

Effect of kinases inhibitors on BoNT/C-induced degeneration of the neurites and death of the cell bodies. Time course of BoNT/C-induced cell death in the presence of the MLK inhibitor CEP1347 (100 nM; A), GSK-3 inhibitor (2 μM), alsterpaullone, ALS (1 μM), and roscovitine, (10 μM; B). Apoptosis was evaluated as a percentage of condensed nuclei stained by H-33342. (C) CGCs were exposed to 20 ng/ml BoNT/C and different kinase inhibitors for 18 h and were loaded with calcein-AM before being imaged by confocal microscopy. Bar, 30 μm.

Figure 8.

Selective phosphatidylserine exposure on degenerating neurites preluded their clearance by glial cells. BoNT/C-treated CGCs were followed for 48 h by conventional time-lapse microscopy (A; and Videos 2 and 3, available at http://www.jcb.org/cgi/content/full/jcb.200406126/DC1). As neurites degenerated and fragmented, a glial cell inhabiting the layer below the neuronal network became activated and begun clearing damaged projections. At a later stage (36–48 h), when cell bodies succumbed to apoptosis, the glial cell scavenged cell corpses (Videos 2 and 3, respectively). Images in A are extracted from Video 2 at the following times (h): a, 1; b, 23; c, 34; and d, 40. (B) BoNT/C-treated CGCs were loaded with Alexa 633–conjugated Annexin V (red) and calcein-AM (green) and imaged by time-lapse confocal microscopy (B and Fig. S1). The image in B and the rotation in Fig. S1 are three-dimensional renderings from multiple z-stacked images collected at 12 (top) and 18 h (bottom). The calcein stain is masked by the appearance of annexin positivity along the surface of neurites. Image stacks were collected by laser scanning microscopy and rendered using the Imaris software. Bars: (A) 25 μm; (B) 10 μm.