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. 2009 Oct 21;29(42):13242-54.
doi: 10.1523/JNEUROSCI.3376-09.2009.

Disruption of the axon initial segment cytoskeleton is a new mechanism for neuronal injury

Dorothy P Schafer  1 , Smita Jha, Fudong Liu, Trupti Akella, Louise D McCullough, Matthew N Rasband
Affiliations

Disruption of the axon initial segment cytoskeleton is a new mechanism for neuronal injury

Dorothy P Schafer et al. J Neurosci. .

Abstract

Many factors contribute to nervous system dysfunction and failure to regenerate after injury or disease. Here, we describe a previously unrecognized mechanism for nervous system injury. We show that neuronal injury causes rapid, irreversible, and preferential proteolysis of the axon initial segment (AIS) cytoskeleton independently of cell death or axon degeneration, leading to loss of both ion channel clusters and neuronal polarity. Furthermore, we show this is caused by proteolysis of the AIS cytoskeletal proteins ankyrinG and betaIV spectrin by the calcium-dependent cysteine protease calpain. Importantly, calpain inhibition is sufficient to preserve the molecular organization of the AIS both in vitro and in vivo. We conclude that loss of AIS ion channel clusters and neuronal polarity are important contributors to neuronal dysfunction after injury, and that strategies to facilitate recovery must preserve or repair the AIS cytoskeleton.

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Figures

Figure 1.
Figure 1.
Ischemic injury causes rapid disruption of the AIS cytoskeleton, but not nodes of Ranvier. A, Coronal, contralateral sections of cortex from 6 h MCAO treated mouse, labeled with a Nissl stain (NeuroTrace, red) to detect neuronal cell bodies, Hoechst (blue) to label nuclei, and anti-βIV spectrin (green) to label the AIS. The white arrow indicates the location of the pial surface. The inset shows a higher magnification of a neuron with a βIV spectrin-positive AIS (arrow). B, Ipsilateral, injured cortex from 6 h MCAO treated mouse. Labeled as in A. The dashed line delineates the border of AIS staining. The inset shows higher magnification of neurons without βIV spectrin immunoreactivity. Note that a few neurons in the ischemic region retain their AIS βIV spectrin immunoreactivity. C, Coronal, contralateral sections of Striatum from 6 h MCAO treated mouse, labeled as in A. Boxes indicate regions of higher magnification shown in E, F. D, Ipsilateral, injured striatum from 6 h MCAO treated mouse. Labeled as in A. Boxes indicate regions of higher magnification shown in G, H. Note the complete absence of AIS βIV spectrin immunoreactivity, but preserved nodal βIV spectrin staining. Scale bar, 50 μm. E, F, Higher magnification of contralateral striatum shown in C to illustrate βIV spectrin immunostaining at the AIS (E) and nodes of Ranvier (F). G, H, Higher magnification of ipsilateral striatum shown in D to illustrate loss of βIV spectrin immunostaining from the AIS (G) and nodes of Ranvier (H). Scale bars: A–D, 50 μm.
Figure 2.
Figure 2.
Nav channels are lost from the AIS following ischemic injury. A, B, After MCAO, contralateral (left) and ipsilateral (right) regions of rat cortex were immunolabeled for Nav channels (Pan-Nav, red), βIV spectrin (A, green) or ankG (B), and Hoechst to label nuclei (blue). The sections shown are from a brain collected 24 h after MCAO. Arrows indicate labeled AIS in layer 2/3 cortex. Scale bar, 50 μm.
Figure 3.
Figure 3.
Optic nerve crush results in loss of βIV spectrin staining at the AIS. A, Immunostaining of control retina for βIV spectrin (green), NeuN (red), and Hoechst (blue). B, Retina corresponding to the crushed optic nerve immunostained for βIV spectrin (green), NeuN (red), and Hoechst (blue). C, Higher magnification of the boxed region shown in A reveals intact AIS (green). D, Higher magnification of the boxed region shown in B demonstrates the loss of βIV spectrin immunostaining. Scale bars: A, B, 50 μm.
Figure 4.
Figure 4.
βIV spectrin and ankyrinG are proteolyzed after neuronal injury. A, Immunoblots of brain homogenates made from naive mouse brain (MBr) and ipsilateral (I) and contralateral (C) hemispheres of sham and occluded animals (time post-occlusion = 3, 6, 12, 24, or 72 h). Immunoblots were prepared using antibodies against αII spectrin, AIS cytoskeleton (ankG and βIV spectrin), and AIS membrane proteins (Nav channels and Neurofascin). β-actin was used as a loading control. Arrows indicate full-length forms of each protein and asterisks indicate major breakdown products (BDP). Breakdown products can be observed as early as 3 h after the onset of occlusion. The lower immunoblots (breakdown products) shown for βIV spectrin and ankG correspond to exposure times that were 3 and 10 times longer, respectively, than the immunoblots showing full-length proteins. Data are representative of 3 animals/occlusion time point. B, Mouse brain homogenate was incubated at 25°C for 30 min in the presence (1 mm CaCl2) or absence (−CaCl2) of calcium together with 0–100 μm of the calpain inhibitor MDL 28170. Homogenates were then analyzed by immunoblot to detect βIV spectrin, ankyrinG, or as a control for calpain mediated proteolysis αII spectrin. The control condition (0 μm drug) was incubated with vehicle alone (DMSO). Full-length proteins are indicated by arrows while breakdown products are indicated by asterisks.
Figure 5.
Figure 5.
Ca2+ dependent disruption of the AIS cytoskeleton. A, The AIS cytoskeleton is proteolyzed under conditions of elevated Ca2+. Naive mouse brain homogenate (MBr) was incubated at 25°C for 30 min in the presence of increasing concentrations of calcium (0–5 mm CaCl2). There is a concentration-dependent increase in proteolysis of full-length AIS and axonal cytoskeletal components. Full-length proteins are designated by an arrow and breakdown products are designated by asterisks. These breakdown products are the same molecular weights as observed following MCAO (n = 3). B, Live imaging of 10 DIV hippocampal neuron AIS using anti-neurofascin (A12/18) and after photolysis of NP-EGTA to uncage Ca2+ only at the AIS. AIS were imaged at 10 min intervals. Scale bar, 10 μm. C, The ratio of fluorescence intensity 30 min after Ca2+ uncaging (F (t) = 30) to the fluorescence intensity immediately before Ca2+ uncaging (F (t) = 0). 6 control and 6 uncaged cells were used for this analysis. Error bars, SD.
Figure 6.
Figure 6.
AIS disassembly is rapid and occurs before cell death. A, Cultured cortical neurons (DIV10) were deprived of oxygen and glucose for increasing times, followed by return to normal oxygen and glucose levels for 24 h. 24 h after OGD the molecular integrity of the AIS was analyzed by immunostaining neurons with anti-βIV spectrin (green). Nuclei are indicated by Hoechst staining (blue). Cell death was assessed using propidium iodide (PI, red). B, Quantification of AIS integrity (lines) and cell death (bars) as a percentage of total Hoechst-positive cells (±SEM). The AIS was labeled for ankG, Nav channels, neurofascin, or βIV spectrin (data not shown). Asterisks (red, AIS; black, cell death) denote mean values significantly different from control (CTL; p < 0.05). N = 9 in 3 independent experiments. C, Loss of the AIS does not cause axon degeneration. Neuronal process integrity was analyzed by transfecting neurons with GFP (GFP fluorescence is inverted) and staining for MAP2 (blue) and βIV spectrin (green and middle panel). The top panels show a control neuron. After 2 h of OGD (lower 3 panels), live neurons (PI-negative, red) lose their AIS, but retain neuronal processes (MAP2, blue and GFP). The axon is indicated by an arrow. Note: βIV spectrin staining (green) was labeled using a far-red fluorophore and is pseudocolored green in these images for visualization. Scale bar, 20 μm.
Figure 7.
Figure 7.
Loss of the AIS is irreversible. A, Cultured neurons were treated for 2 h of OGD, followed by recovery for 24 or 72 h. The AIS was labeled with anti-βIV spectrin (green), nuclei by Hoechst (blue), and cell death was assessed with PI (red). B, Quantification of AIS integrity (lines) and cell death (bars) as a percentage of total Hoechst-positive cells (±SEM) in control and 24 or 72 h post OGD treated cells. The AIS was labeled for βIV spectrin. Red asterisks indicate a significant difference in AIS immunoreactivity from control cultures (CTL). C, Live labeling of 10 DIV hippocampal neurons using anti-neurofascin antibodies (A12/18). Arrows indicate the location of axon initial segments. D, Same field of view as in C 8 d after treatment by OGD. Hippocampal neurons were fixed and stained for βIV spectrin immunoreactivity. Arrows indicate the location of the AIS determined 8 d previously. E, The percentage of control and 10 d OGD treated cells that also had βIV spectrin AIS immunoreactivity or were labeled with propidium iodide (PI) Error bars, SD. F, G, 10 DIV hippocampal neuron live-labeled using anti-neurofascin (A12/18; F) and anti-MAP2 (G). The axon is indicated by arrows. Same scale as in I. MAP2 is somatodendritic and excluded from the axon (arrows). H, 10 DIV hippocampal neuron live-labeled using anti-neurofascin (A12/18). The axon is indicated by arrows. Same scale as in I. I, Same neuron as in H 8 d after OGD, labeled for MAP2 immunoreactivity. After OGD the neuron loses polarity and MAP2 invades the axon (arrows). Scale bars: A, 20 μm; I, 5 μm.
Figure 8.
Figure 8.
AIS disassembly following OGD is blocked by the calpain inhibitor MDL 28170. A, Cultured cortical neurons (DIV10) were treated during (3 h) and after (24 h) OGD with a calpain inhibitor MDL 28170 (100 μm) and/or an NMDA receptor antagonist MK-801 (10 μm). AIS integrity was assessed with an antibody against βIV spectrin (green) and cell death was assessed with PI (red). Neurons treated with MDL 28170 retained AIS integrity (arrows) whereas protection from cell death was modest. White and red asterisks indicate live and dead neurons with an AIS, respectively. Nuclei are labeled by Hoechst staining. B, Quantification of immunofluorescence as a percentage of Hoechst labeled nuclei for OGD treated cultures in the presence of 0–100 μm MDL 28170, 10 μm MK-801, or 100 μm MDL 28170 + 10 μm MK-801. Asterisks (black corresponds to cell death measurements while red corresponds to AIS measurements) denote percentages that are statistically similar to control, i.e., no OGD treatment (CTL; black bar, red triangle). n = 9 in 3 independent experiments. Error bars, ±SEM. C, Neurons infected with egfp-calpastatin (white bars) or egfp alone (black bars) were deprived of oxygen and glucose for 3 h. AIS integrity was measured by immunostaining with anti-βIV spectrin and counting the number of Hoechst labeled cells with an AIS. After OGD, the AIS was significantly protected in egfp-calpastatin infected cultures (asterisk) compared to control (egfp infected) cultures. D, There was no difference in the percentage of Hoechst labeled nuclei that were also PI-positive between egfp-calpastatin-infected and egfp-infected cultures.
Figure 9.
Figure 9.
Calpain inhibition in vivo attenuates AIS disassembly following ischemic injury. A, Rats were subjected to MCAO in the presence or absence (vehicle) of a cell-permeable calpain inhibitor (MDL 28170) or an NMDA antagonist (MK-801). Coronal sections of rat cortex were stained (t = 24 h post-occlusion) with the neuronal marker NeuN (red); the AIS was labeled with anti-βIV spectrin (green) and nuclei were labeled with Hoechst (blue). White arrows indicate βIV spectrin labeled AIS, while black arrows indicate NeuN labeled neurons. B, Quantification of the preservation of the AIS (n = 4 animals for each group). Compared to MK-801 or vehicle treated animals, AIS integrity is significantly increased (asterisk) in the infarcted region of the ipsilateral cortex (white bars) of MDL 28170-treated animals. C, Within the infarcted region of the ipsilateral cortex (white bars), NeuN staining is significantly reduced as compared to the contralateral cortices (black bars) in all treatment groups. D, Within infarcted regions, there was a significant increase in Nav channel/βIV spectrin-labeled (yellow; arrow) AIS in animals treated with the calpain inhibitor (MDL 28170) versus MK-801 or vehicle-treated animals. Scale bars: A, D, 50 μm.
Figure 10.
Figure 10.
AIS disassembly following acute CNS injury. A, Nav channels, Kv channels, and cell adhesion molecules are clustered at high density at the AIS through interactions with the AIS cytoskeleton comprised of βIV spectrin and ankG. B, Upon injury, calcium enters the cell and activates calpain, a Ca2+ dependent cysteine protease. C, Calpain cleaves the spectrin/ankyrin-based cytoskeleton associated with the AIS. D, Destruction of the AIS cytoskeleton causes loss of high densities of ion channels at the AIS, impaired ability to initiate action potentials, and loss of neuronal polarity.
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