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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 βIV 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.
Factors contributing to nervous system dysfunction after injury or disease include glial scarring, axon degeneration, demyelination, axon outgrowth inhibitors, and neuronal cell death (Yiu and He, 2003; Benn and Woolf, 2004; Franklin and Ffrench-Constant, 2008). Proteolysis of the axonal cytoskeletal proteins αII spectrin, βII spectrin, and ankyrinB by calpain, a calcium-dependent cysteine protease, has been proposed to be a major contributor to axon degeneration (Harada et al., 1997; Yoshida and Harada, 1997; White et al., 2000; Czogalla and Sikorski, 2005; Bevers and Neumar, 2008). Indeed, αII spectrin breakdown products are biomarkers for nervous system injury (Pike et al., 2004; Pineda et al., 2007). Although these cytoskeletal components are localized throughout the axon, there is a unique cytoskeleton associated with the most proximal segment of the axon (i.e. the axon initial segment or AIS) comprised of βIV spectrin and ankyrinG (ankG) (Kordeli et al., 1995; Berghs et al., 2000). However, the consequence of nervous system injury for this specialized cytoskeleton has not been explored.
The AIS functions as both a physiological and physical bridge between axonal and somatodendritic domains. It is responsible for the final integration of synaptic inputs and initiation and modulation of the action potential (AP) due to the high density of voltage-gated Na+ (Nav) and K+ (Kv) channels (Khaliq and Raman, 2006; Palmer and Stuart, 2006; Meeks and Mennerick, 2007; Goldberg et al., 2008; Kole et al., 2008; Shah et al., 2008). These ion channels are restricted to the AIS through interactions with ankG, which links AIS membrane proteins to the underlying βIV spectrin scaffold. βIV spectrin tethers ankG to the plasma membrane and actin cytoskeleton (Garrido et al., 2003; Pan et al., 2006; Dzhashiashvili et al., 2007; Hedstrom et al., 2007; Yang et al., 2007). Mice lacking ankG in Purkinje neurons fail to cluster AIS ion channels, develop ataxia, and fail to initiate APs (Zhou et al., 1998; Jenkins and Bennett, 2001; Pan et al., 2006). Similarly, βIV spectrin mutant mice develop ataxia, tremors, and deafness (Komada and Soriano, 2002; Lacas-Gervais et al., 2004; Yang et al., 2004). These behavioral deficits are coincident with progressive loss of AIS Nav channel immunoreactivity (Uemoto et al., 2007; Yang et al., 2007).
Morphologically the AIS is the transition zone from the soma to the axon compartments, and the AIS cytoskeleton functions to maintain neuronal polarity and proper axonal trafficking (Winckler et al., 1999; Song et al., 2009). For example, loss of ankG causes axons to acquire molecular and structural characteristics of dendrites including spines (Hedstrom et al., 2008). Together, these observations emphasize the importance of the AIS cytoskeleton in normal nervous system function.
Since genetic loss of the AIS cytoskeleton disrupts neuronal function and organization, we considered whether nervous system injury also disrupts this essential axonal domain. Using in vitro and in vivo injury models, we elucidate a new mechanism for neuronal injury: rapid, preferential, and irreversible proteolysis of the AIS cytoskeleton by calpain, resulting in disrupted neuronal polarity and loss of the ion channel clusters necessary for AP initiation.
Rats and mice were housed at the University of Connecticut Health Center and Baylor College of Medicine. All experiments were approved by institutional animal care and use committees and were performed in accordance with all National Institutes of Health guidelines for the humane treatment of animals.
The polyclonal βIV spectrin antibody was previously described (Ogawa et al., 2006). The polyclonal ankG antibody was kindly provided by Dr. V. Bennett (Duke University), and mouse monoclonal anti-ankG was purchased from Zymed Laboratories. Mouse monoclonal pan-neurofascin and Nav channel antibodies were previously described (Schafer et al., 2004; Hedstrom et al., 2008). The chicken polyclonal MAP2 antibody was purchased from EnCor Biotechnology. The mouse monoclonal αII spectrin antibody was purchased from Chemicon International. The mouse monoclonal β-actin and GFAP antibodies were purchased from Sigma. The mouse monoclonal NeuN and calpastatin antibodies were purchased from Millipore. All fluorescent secondary antibodies were purchased from Invitrogen except for AMCA-conjugated anti-chicken antibody (Accurate Chemical).
Primary cortical neurons were prepared from E18 rat embryos. Embryonic cortices were dissected and collected in HBSS (Invitrogen) followed by a 30 min trypsinization (0.25%) at 37°C. Cells were collected by centrifugation (600 × g for 3 min), resuspended in plating media (Neurobasal (Invitrogen) supplemented with 10% FBS (Mediatech)), and triturated with a fire-polished Pasteur pipette. The cell suspension was left to settle for 3 min, and the subsequent supernatnant was filtered through a 70 µm cell strainer (BD Biosciences). Cells were then plated on cover glass coated with 1 mg/ml poly-L-lysine (Sigma) and 10 µg/ml laminin (Invitrogen) at 1 × 106 cells/35 mm dish. Neurons were incubated in a humidified 5% CO2 incubator at 37°C. After 2–3 hrs, the media was exchanged to maintenance media (Neurobasal, 2% B27 (Invitrogen), 0.5 mM L-glutamine; 1× Pen-Strep) with addition of 25 µM L-glutamate. On day in vitro 4 (DIV4), the media was replaced with maintenance media without L-glutamate. Cells were fed every 3 days by replacing half the media with fresh maintenance media. In some cases, primary cortical neurons were transfected with membrane-bound GFP by lipofectamine 2000 (Invitrogen) as previously described for primary hippocampal neurons (Hedstrom et al., 2007).
Deprivation experiments were performed as previously described with minor modifications (Uliasz and Hewett, 2000). Primary cortical neurons (DIV10) were transferred to an anaerobic chamber (Forma Scientific, Marietta, OH) containing a gas mixture of 5% CO2, 10% H2, 85% N2 (≤ 0.2 % O2). Once in the chamber, half the media was taken out of each well and stored for future use (conditioned media). The remaining media was aspirated and replaced by washing cultures three times with deoxygenated, glucose-free balanced salt solution [BSS0: 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 1 mM NaH2PO4, 26.2 mM NaHCO3, 1.8 mM CaCl2, 0.01 mM glycine, 2 mM glutamax, and 1× MEM amino acids (Gibco)]. Cultures were then placed in a 37°C incubator within the chamber for up to 3hr. Control cultures were washed 3 times in BSS containing 20 mM glucose (BSS20) and maintained at 37 °C in normoxic conditions for up to 3 hrs. Cultures were subsequently removed from the chamber and media was replaced with half conditioned media and half fresh maintenance media for up to 72 hrs.
For pharmacological experiments, the following compounds were used: MDL 28170 (MDL; Calbiochem), CA-074 (CA; Calbiochem), MK-801 (Sigma). Stock solutions were made in either DMSO (MDL and CA) or water (MK-801). These stock solutions were diluted into BSS and then added to cultures after the third wash (MDL: 0–100 µM; CA: 0–100 µM; MK-801 10 µM). Following deprivation, fresh drug was added to the maintenance media at the appropriate concentration. Vehicle controls (0 µM drug; DMSO or water) were included for each experiment.
Recombinant adenovirus containing calpastatin and/or EGFP were gifts from D.S. Park (Sedarous et al., 2003). Primary cortical neurons (DIV7) were infected for 6 hrs in maintenance media (multiplicity of infection=20). Cultures were washed once and left in maintenance media until deprivation experiments (DIV10). Infection efficiency was quantified in sister cultures by counting the number of gfp-positive cells versus total Hoechst-positive nuclei. Cell death and immunofluorescence were quantified as described below.
Cell death was analyzed in vitro using propidium iodide (PI; Invitrogen). PI was added to cultures 24 hrs after deprivation at 10 µg/ml. Cells were incubated at 37°C for 10 min followed by fixation in ice cold 1% or 4% PFA for 30 min followed by subsequent Hoechst labeling (Invitrogen) and immunostaining (see below). For the majority of the quantification, three 10× fields of view were collected per coverslip (2–3 coverslips per experiment). In some cases (see Immunostaining and Quantification), PI was quantified using ten 40× fields of view. For each field, the total numbers of PI and Hoechst-positive cells were counted using ImageJ software (National Institutes of Health). For each coverslip, the number of PI-positive cells in each view was pooled and expressed as a percentage of Hoechst-positive cells.
Cultured neurons were fixed in 1% (ankG or Nav channel staining) or 4% PFA (all other staining) and immunolabelled as previously described (Hedstrom et al., 2007). For quantification of in vitro immunofluorescence, the same fields of view used for PI quantification (see above) were used for quantifying AIS immunofluorescence (βIV spectrin, ankG, pan-Nav, or pan-neurofascin). An AIS was defined as a fluorescent segment of at least 10 µm in length and is expressed as a percentage of total Hoechst-positive cells. In some cases in which immunofluorescence was faint (pan-Nav and pan-neurofascin), AIS and Hoechst-positive cells were quantified using ten 40× fields of view per coverslip.
For in vivo experiments, animals were sacrificed and brains were rapidly dissected and flash frozen in 2-methylbutane pre-cooled over dry ice. Brains were stored at −80°C until sectioning. Brains were sectioned using a cryostat (15 µm) and mounted on glass slides. Subsequently, sections were fixed with 1% (ankG or Nav channel staining) or 4% PFA. Immunolabelling was performed as previously described (Schafer et al., 2004). In a few cases, immunostaining was performed using brains from perfused animals. Briefly, anesthetized animals were perfused with 4% PFA. Brains were dissected and post-fixed in 4% PFA for 4hr followed by equilibration in 20% sucrose, sectioning, and immunolabelling. In vivo immunofluorescence was quantified using five 20× fields collected for each ipsilateral and contralateral cortex of each animal. In some cases tissue sections were labeled using fluorescent NeuroTrace (invitrogen). The number of βIV spectrin-positive AIS, NeuN-positive cells, and Hoechst-positive cells were counted for each field. A βIV spectrin-positive AIS was defined as a fluorescent segment of at least 10 µm in length. Both βIV spectrin and NeuN staining were expressed as a percentage of total Hoechst-positive cells. Data are representative of 4 animals per condition. For staining of retinas, tissue from rats experiencing optic nerve crush was kindly provided by Drs. Yang Yang and Larry Benowitz.
Rat or mouse brain homogenates were prepared from freshly dissected tissue. Brains were homogenized in ice-cold buffer containing 0.32 M sucrose and 10 mM Tris-HCl (pH 7.5) (10 ml/gm wet brain weight). Crude brain homogenates were subsequently centrifuged to remove debris and nuclei (600 × g for 10 min). The resulting supernatant was used for protein estimation using the BCA method (Pierce Biotechnology). For the cleavage assay, equal volumes of supernatant (8–10 mg/ml) were aliquoted into separate tubes for each condition. To each supernatant an equal volume of cleavage buffer [Final concentration: 0–10 mM CaCl2, 20 mM Tris-HCl pH 7.5, 25 mM NaCl, 1 mM DTT] was added with or without protease inhibitors [Final Concentration: 0–100 µM MDL ; 0–10 µM calpastatin (Calbiochem); 0–100 µM calpeptin (Calbiochem); 0–100 µM ALLN (Calbiochem);0–100 µM CA; 0–100 µM GM-6001 (Calbiochem); 0–100 µM Q-VD-OPH (MP Biomedicals)]. The solution was incubated at 37°C for 30 min. The reaction was quenched with an equal volume of 2× reducing sample buffer and western blot analysis was performed as previously described (Schafer et al., 2004).
Surgeries were performed as previously described (Ardelt et al., 2005; McCullough et al., 2005). Briefly, the middle cerebral artery was occluded in male C57BL/6 mice or Sprague-Dawley rats (Charles River Laboratory). The animals were awakened from anesthesia to confirm neurological deficits associated with occlusion. After 90 min, the animals were briefly re-anesthetized for removal of the suture. Animals were sacrificed for analyses at various times post-occlusion (3–72 hrs from the onset of 90 min occlusion). Separate non-survival cohorts were performed to confirm equivalency of blood flow reduction (laser Doppler) and to ensure physiological variables (pO2/pCO2/blood pressure) were equivalent between groups. During all surgical procedures, animals were maintained at 37°C. Immunofluorescence was performed as described above (see Immunofluorescence and Quantification). Infarction was confirmed by staining brain sections with Fluoro-Jade B (Chemicon), cresyl violet (Sigma), and/or anti-NeuN (Davoli et al., 2002; Tureyen et al., 2004; Duckworth et al., 2005).
For western blot analysis, brains were rapidly dissected and flash frozen in 2-methylbutane pre-cooled over dry-ice. Brains were stored at −80°C until homogenization. Immediately prior to homogenization, brains were divided into contralateral and ipsilateral hemispheres. Each hemisphere was homogenized separately in ice-cold 0.32 M sucrose and 5 mM sodium phosphate (pH 7.4) containing 10 mM EDTA, 0.5 mM sodium phenylmeythylsufonyl fluoride, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 2 µg/ml antipain, and 10 µg/ml benzamidine (10 ml/mg wet brain weight). Crude hemisphere homogenates were centrifuged to remove debris and nuclei (600 × g for 10 min). To ensure equal protein loading, protein concentrations were estimated from the resulting supernatant and resolving sample buffer was added to a final concentration of 2 mg/ml. Samples were boiled (5 min) or heated at 37°C (15 min) and subjected to western blot analysis as previously described (McCullough et al., 2005).
In some cases, rats subjected to MCAO received MDL, MK-801, or vehicle. MDL or vehicle [PEG 300/ EtOH (9:1)] was administered IV as previously described (Markgraf et al., 1998). Briefly, administration began at the time of reperfusion with a 20 mg/kg bolus (1 mL final volume) via an indwelling catheter. After which time, the catheter was connected to an infusion pump (Kent Scientific) and animals were infused for an additional 6 hrs (3.33 mg/kg/hr, total volume 5 mL). Animals received MK-801 (1 mg/kg) or vehicle (phosphate buffered saline; PBS) IP 30 min before the onset of occlusion (Gerriets et al., 2003). After the first dose, the animals received a dose of MK-801 (1mg/kg) or vehicle at 3 hrs and 6 hrs after reperfusion. All animals were sacrificed 24 hrs after occlusion for immunofluorescence, western blot analysis, and/or infarct assessment.
Primary hippocampal neurons were obtained from E18 timed pregnant Sprague-Dawley rat pups. For this, dissected hippocampi were trypsinized (0.25%) in Hank’s buffered salt solution (HBSS) (Gibco) for 15 min at 37°C, triturated and centrifuged at 800 × g for 4 min. Cells were then resuspended in maintenance media (Neurobasal with 2% B-27 supplement and 2 mM Glutamax) and plated on photo-etched glass coverslips (23 mm, square; Electron Microscopy Sciences, Hatfield, PA) at 1–5 × 105 cells/ well.
For live labeling of the AIS, a mouse monoclonal antibody (A12/18; (Hedstrom et al., 2008)) recognizing an extracellular epitope neurofascin was used in all the experiments. Conditioned media was removed from DIV 9–10 hippocampal neurons and stored for later use. After this, cells were washed twice in HBSS and incubated with primary antibody (1:200 dilution, 30 min, 37°C) in fresh maintenance media. This was followed by 3 washes in HBSS and incubation with secondary antibody (1:500, 30 min, 37°C). After final washes in HBSS, fresh and conditioned media was added to the cells in a 1:1 ratio. Images of immunostained neurons (red fluorescence) were acquired on a Zeiss Imager Z1 microscope. Etched grid numbers on the coverslips allowed for tracking of the same neurons after OGD induced injury. OGD was carried out on DIV 10 neurons as described previously. Modifications included use of modular incubation chamber (Billups-Rothenberg, Del Mar, CA) and use of Neurobasal-A (Gibco) instead of glucose free-BSS. The neurons were fixed and immunostained for AIS (βIV spectrin) and dendritic (MAP2) markers 8–10 days after OGD.
For local calcium uncaging, a cell permeant acetoxymethyl (AM) ester derivative of the caged compound o-nitrophenyl EGTA (NP-EGTA AM, Molecular probes, Eugene, OR) was used at a final concentration of 25 µM. The change in intracellular calcium concentration was documented using the cell permeant AM ester of calcium green-1 (CG-1 AM, 10 µM, Molecular probes). First, neurons were live labeled with the AIS marker A12/18 as described above. This was followed by a simultaneous incubation with NP-EGTA AM and CG-1 AM (45 min at room temperature) in the dark. For this, HBSS containing 1.2 mM CaCl2 was used as the loading solution. Also, Pluronic F-127 (20% w/v, Molecular probes) was added to the loading solution for better cellular uptake of these compounds (final concentration < 0.02%). Stock solutions (5 mM) of NP-EGTA AM and CG-1 AM were prepared in DMSO. After loading, the neurons were washed and replenished with fresh maintenance media.
For photolysis and subsequent imaging of neurofascin, the AIS was first identified under low magnification (20×) using Alexa-594 secondary antibodies. A pinhole diaphragm was then closed so that the light cone of the UV lamp was focused only onto the AIS at higher magnification (40×). Calcium was then photolytically uncaged by 2 consecutive 500 ms UV flashes. Fluorescence images of the labeled AIS and intracellular calcium were acquired immediately before and after photolysis and subsequently at 5 min intervals. To measure changes in fluorescence, we calculated the ratio of the average fluorescence intensity for 6 control and 6 uncaged AIS at 30 minutes after uncaging to the fluorescence intensity immediately before uncaging at t=0.
Staining was visualized and images were collected using Axiovert 200M, Axio-observer Z1, and Axio-imager Z1 microscopes (Carl Zeiss MicroImaging, Inc.) fitted with AxioCam digital cameras (Carl Zeiss MicroImaging, Inc.). Images were taken using 10× (0.45 NA), 20× (0.8 NA), 40× (1.0 NA), 40× (0.75 NA), or 63× (1.4 NA) objectives. AxioVision (Carl Zeiss MicroImaging, Inc.) acquisition software was used for collection of images. Measurement of fluorescence intensity was performed using imageJ (NIH). In some images, contrast and brightness were subsequently adjusted using Photoshop (Adobe). No other processing of the images was performed. All figures were assembled using CorelDraw.
Prior to statistical analyses, all percentage data was transformed as previously described (Uliasz and Hewett, 2000). A one-way analysis of variance (ANOVA) was performed. If p<0.05, subsequent post-hoc analyses were performed (Newman-Keuls). All statistical analyses and graphs were compiled using GraphPad Prism software.
To determine if the AIS is disrupted following injury, we subjected mice to ischemic injury by middle cerebral artery occlusion (MCAO). The MCA was occluded using a nylon suture as previously described (McCullough et al., 2005). After 90 min of occlusion, the block was removed to allow blood to reperfuse the affected hemisphere. Animals were sacrificed at various times after the onset of MCAO (time post-occlusion) for immunohistochemistry and immunoblot analyses. In the uninjured (contralateral) side of the cerebral cortex neurons were identified as brightly stained Nissl-positive cells (Figure 1A, red), most of which were associated with robust βIV spectrin immunoreactivity at the AIS (Figure 1A, green; inset and arrow). In contrast, in the injured (ipsilateral) cortex there was remarkable loss of βIV spectrin staining at the AIS within 6 hrs after onset of occlusion. Further, there was an abrupt transition from neurons with disrupted initial segments to those with preserved βIV spectrin staining (Figure 1B, dashed line). Similar to βIV spectrin, ankG and Nav channel immunoreactivity at the AIS were also undetectable in the injured cortex (Figure 2). Thus, one consequence of ischemic neuronal injury is the rapid disruption of the molecular organization of the AIS.
Since nodes of Ranvier and axon initial segments share a common cytoskeletal organization (Kordeli et al., 1995; Berghs et al., 2000), we assumed that nodes would also be disrupted by ischemic injury. To test this possibility, we examined nodal and AIS βIV spectrin immunoreactivity in contralateral and ipsilateral striatum since this region is strongly affected by MCAO and has many neuronal cell bodies and bundles of myelinated axons. Strikingly, whereas βIV spectrin labeled the AIS (Figures 1C and 1E) and nodes (Figures 1C and 1F) in the contralateral striatum, only the AIS was disrupted in the ipsilateral striatum (Figures 1D and 1G). In the ipsilateral striatum large bundles of myelinated axons were robustly labeled for βIV spectrin indicating preserved nodes of Ranvier (Figures 1D and 1H). Together, these results show that despite a common molecular organization, the AIS cytoskeleton is preferentially susceptible to disruption after injury.
We next considered whether early disruption of the AIS cytoskeleton is a common phenomenon in different kinds of nervous system injury. To test this possibility, we examined the AIS of retinal ganglion cells (RGCs) 5 days after optic nerve crush injury. Whereas control NeuN-stained RGCs (red) had robust AIS βIV spectrin immunoreactivity (Figure 3A and 3C, green; we used βIV spectrin as an indicator of the AIS cytoskeleton since its localization depends on ankG (Yang et al., 2007)), RGCs with injured axons lost virtually all AIS βIV spectrin immunostaining (Figure 3B and 3D). Together, these results suggest a common consequence of neuronal injury is the preferential disruption of the AIS cytoskeleton.
How does neuronal injury cause disruption of the AIS? One possible explanation is that like ankB and αII spectrin, ankG and βIV spectrin are susceptible to proteolysis after injury. To test this possibility, we performed immunoblot analyses of brain homogenates from sham, or MCAO-treated mice with antibodies against various AIS components (βIV spectrin, ankG, Nav channels, and neurofascin) or αII spectrin, a spectrin previously shown to be proteolyzed following ischemic injury (White et al., 2000; Czogalla and Sikorski, 2005). We identified distinct breakdown products associated with the AIS cytoskeleton. In each ipsilateral hemisphere, we found low molecular weight fragments from βIV spectrin (~45 kD) and ankG (~95 kD and 72 kD; Figure 4A, asterisks indicate breakdown products, arrows indicate full-length proteins). These bands could be detected at every time point analyzed (3 hrs-72 hrs post-occlusion). Coincident with their proteolysis, we also observed reductions in the amounts of full-length βIV spectrin and ankG (Figure 4A, arrows). In contrast to these AIS cytoskeletal proteins, neurofascin, a CAM highly enriched at the AIS, showed no detectable loss of the full-length protein or appearance of proteolytic fragments (Figure 4A). On the other hand, when homogenates were probed with an antibody against Nav channels, low molecular weight fragments could be detected in the ipsilateral hemisphere (Figure 4A, bracket and asterisk). However, these were not prominent until 12 h post-occlusion suggesting these proteins are proteolyzed after the AIS cytoskeleton. Importantly, immunoblots for αII spectrin demonstrated a clear ~150 kD breakdown product associated with each ipsilateral hemisphere (Figure 4A, asterisk). This product could not be detected in the contralateral hemisphere, sham control, or naïve mouse brain homogenates and is the product of proteolysis by the calcium-dependent protease calpain (Siman et al., 1984). Taken together, these results indicate that components of the AIS cytoskeleton are rapidly proteolyzed following injury.
What is the mechanism underlying proteolysis of the AIS cytoskeleton? Exposing naïve brain homogenate in vitro to increasing amounts of Ca2+ caused a concentration dependent increase in the proteolysis of βIV spectrin and ankG (Figure 5A), and their proteolytic fragments had molecular weights identical to those observed after in vivo ischemic injury (Figure 4A). Thus, proteolysis of the AIS cytoskeleton occurs under conditions of elevated Ca2+.
Since ankG and βIV spectrin proteolysis occurs under conditions of elevated Ca2+, we next sought to determine if a local, AIS restricted increase in Ca2+ concentration could also disrupt the molecular organization of the AIS. To do this we live-labeled the AIS using antibodies against an extracellular epitope of neurofascin and fluorescent conjugated secondary antibodies to visualize the AIS by fluorescence microscopy. We then loaded the labeled neurons with NP-EGTA, to permit photolytic uncaging of Ca2+. Finally, after identifying the AIS by neurofascin immunofluorescence, we uncaged Ca2+ only at the AIS and monitored the fluorescence intensity of neurofascin for 30 minutes. We found that in contrast to control initial segments in the same cultures uncaging of Ca2+ at the AIS resulted in a rapid decrease in NF immunofluorescence (Figures 5B and 5C). These observations are consistent with the idea that Ca2+-dependent proteolysis of ankG and βIV spectrin leads to loss of AIS membrane proteins.
Alpha II spectrin proteolysis after injury depends on the calcium-dependent cysteine protease calpain (Siman et al., 1984). To test whether calpain also proteolyzes βIV spectrin and ankG, we incubated naïve brain homogenates in 1 mM Ca2+ and increasing concentrations of different calpain inhibitors including MDL 28170, calpastatin, calpeptin, and ALLN (Figures 4B and S1; calpeptin and ALLN not shown). The addition of calpain inhibitors resulted in a concentration dependent reduction in both βIV spectrin and ankG proteolysis. However, some reports suggest that calpain inhibitors lack specificity and cross-inhibit cathepsins, another class of calcium-dependent cysteine proteases (Wang and Yuen, 1997; Donkor, 2000; Neffe and Abell, 2005; Cuerrier et al., 2007). To exclude cathepsins and other proteases as mediators of βIV spectrin and ankG proteolysis, we tested CA-074, a cathepsin B inhibitor (Figure S1), Q-VD-OPH, an inhibitor of caspases (data not shown) and GM-6001, an inhibitor of matrix metalloproteases (data not shown). Unlike calpain inhibitors, these inhibitors all failed to attenuate proteolysis of βIV spectrin or ankG at all concentrations tested (0–100 µM). As an important control, when these same samples were tested for αII spectrin proteolysis, production of the calpain-mediated ~150 kD breakdown product was inhibited by addition of calpain inhibitors but not other protease inhibitors (Figures 4B and S1). Taken together, these data strongly suggest that proteolysis of βIV spectrin and ankG is mediated by calpain.
The considerable neuronal damage induced in the MCAO model made it difficult to determine whether AIS disruption is a consequence of cell death, or a specific early response to neuronal injury. Therefore, to elucidate the chronology and molecular mechanism underlying AIS disassembly after neuronal injury, we analyzed AIS disruption in a culture model of ischemic injury: oxygen-glucose deprivation (OGD). At DIV10, we deprived cortical neuron cultures of oxygen (0–0.02%) and glucose (0 mM) for up to 3 hrs. After deprivation, cultures were returned to normoxia and glucose-containing media was added back to the cultures. Cell death and AIS integrity were then analyzed 20–24 hrs later. Cells deprived of oxygen and glucose for 1 hr were indistinguishable from control cultures. However, similar to our in vivo results, 2h of OGD resulted in a >80% decrease in neurons with βIV spectrin immunoreactivity at the AIS (Figure 6A). Comparable reductions in ankG, Nav channel, and neurofascin immunoreactivity were also observed with an identical timecourse (Figure 6B; quantification for βIV spectrin not shown). We also performed OGD experiments using hippocampal neurons and obtained similar results (data not shown).
Are cells dead after 2 hr of OGD, and does this explain the loss of AIS proteins? Staining cultures with propidium iodide (PI), an indicator of cell death, showed that significant neuron death did not occur until 3 hrs of OGD (Figures 6A and 6B, bars). Cell viability as assessed by the MTT assay yielded similar results (data not shown). Alternatively, the loss of AIS βIV spectrin immunoreactivity could be a consequence of rapid and general axon degeneration. Therefore, we examined neuronal morphology by transfecting neurons with GFP and by staining for MAP2, a dendritic marker that has previously been shown to be proteolyzed and redistributed in neurons following ischemic injury (Pettigrew et al., 1996; Buddle et al., 2003; Bevers and Neumar, 2008). After 2 hrs of OGD, loss of AIS immunofluorescence occurred without axonal or dendritic retraction or degeneration (Figure 6C). Taken together, these results demonstrate that proteolysis of the AIS cytoskeleton and loss of clustered AIS proteins (e.g. Nav channels and neurofascin) is an early consequence of neuronal injury, occurs independently of cell death, and is not a consequence of axon degeneration.
Since neuronal injury results in loss of the AIS proteins required for action potential initiation and the maintenance of neuronal polarity, any functional recovery must include the re-establishment of the AIS cytoskeleton. To determine if the AIS recovers after injury, or if 2 hr OGD eventually results in widespread cell death, we examined neurons for βIV spectrin immunofluorescence and PI staining at 24 and 72 hrs after 2 hr of OGD (Figures 7A and 7B). By 72 hrs after OGD, we failed to observe any recovery in the number of cells with clustered AIS proteins despite no significant increase in cell death compared to the 24 hr timepoint.
Our previous studies have shown that it takes nearly a full week in culture for all hippocampal neurons to develop an AIS (Yang et al., 2007). Therefore, we considered the possibility that more than 72 hr might be needed for the reestablishment of the AIS. Alternatively, it is also possible that disruption of the AIS may result in conversion of dendrites into axons (Gomis-Ruth et al., 2008), and this may require longer recovery times. To test these possibilities, we live-immunolabled 10 DIV hippocampal neurons using antibodies against neurofascin. We visualized these neurons and their AIS using immunofluorescence microscopy and carefully noted their location using gridded microscope coverglass (Figure 7C, arrows). We then exposed these cells to OGD for 2 hr and returned them to the incubator for 8–10 days. These cells were then fixed and labeled for both βIV spectrin immunoreactivity and PI staining. Similar to the 72 hr timepoint (Figure 7A) we found the loss of initial segments persisted even 8–10 days after OGD (Figures 7D and 7E). Eight days after OGD βIV spectrin immunostaining was very weak and randomly distributed throughout neurons; when we examined the location of the former AIS, we did not detect any recovery of βIV spectrin immunoreactivity (Figure 7D, arrows).
We previously showed that dismantling of the AIS by silencing expression of ankG disrupted neuronal polarity and led to axons acquiring the molecular characteristics of dendrites (Hedstrom et al., 2008). To determine if injury-induced disruption of the AIS cytoskeleton also leads to loss of neuronal polarity, we live-labeled 10 DIV hippocampal neuron AIS using anti-neurofascin antibodies to identify the AIS and axon (Figures 7F and 7H, arrows), and MAP2 to define somatodendritic domains. At this time, MAP2 is excluded from axons (Figure 7G). However, after 8 d post OGD treatment, neuronal polarity was disrupted and MAP2 was detected in the axon. Taken together, these results suggest that proteolysis of the AIS cytoskeleton leads to an irreversible loss of clustered ion channels and neuronal polarity.
Most neurons lose their AIS after 2 hrs of OGD and die after 3 hrs of OGD (Figures 6A and 6B). If calpain proteolyses the AIS cytoskeleton, then calpain inhibitors should preserve the AIS cytoskeleton even after 3 hr of OGD. To test this, we treated neurons with the calpain inhibitor MDL 28170, and compared this to treatment using the NMDA receptor antagonist MK-801, a well-known inhibitor of neuronal excitotoxicity. In the presence of the calpain inhibitor, AIS integrity in vitro was significantly protected in a concentration-dependent manner: at 100 µM MDL 28170, 70.2% of cells had a βIV spectrin-positive AIS. However, cell death was not significantly inhibited since 53.2% of cells were PI+ (Figures 8A and 8B). In contrast to these results, treatment using the cathepsin B inhibitor CA-074 resulted in significant protection from cell death but not preservation of the AIS cytoskeleton (Figure S2A). Treatment with 10 µm MK-801 resulted in significant protection from cell death (34.4% cells were PI+; Figures 8A and 8B), but AIS integrity was only modestly protected (46.3% of cells had an identifiable, βIV spectrin labeled AIS). When both MDL 28170 and MK-801 were added to cultures, we observed both protection from cell death and preservation of the AIS (Figures 8A and 8B). Since the improved viability with the combined drug treatment was similar to MK-801 treatment alone, and the amount of AIS preservation was similar to the MDL 28170 treatment alone, we conclude that cell death and disruption of the AIS cytoskeleton are independent events.
To further confirm the role of calpain in disruption of the AIS cytoskeleton after OGD, we used adenovirus to overexpress enhanced green fluorescent protein (egfp) or the highly specific endogenous inhibitor of calpain, calpastatin (egfp-clpst; Figures 8C and 8D). While infection efficiencies were nearly identical (data not shown) and infection itself did not affect the AIS cytoskeleton or cell death (Figures 8C and 8D), overexpression of egfp-clpst significantly protected the AIS cytoskeleton after OGD compared to overexpression of egfp alone (39% and 5% of neurons retain βIV spectrin immunoreactivity, respectively; Figure 8C). Furthermore, neither egfp nor egfp-clpst protected against cell death after OGD (Figure 8D). Taken together, these results suggest that after neuronal injury, increased calpain activity causes cytoskeletal breakdown, which in turn disrupts the molecular organization of the AIS.
The in vitro results described above indicate that calpain disrupts the clustering of AIS ion channels and neuronal polarity by proteolysis of the AIS cytoskeleton. To confirm that calpain mediates the disruption of the AIS in vivo, we subjected rats to 90 min of MCAO followed by reperfusion. Then, at the time of reperfusion, we infused either vehicle or the calpain inhibitor MDL 28170 for 6 hrs. For comparison, in a separate cohort of animals, we administered MK-801 30 min prior to MCAO, and then every 3 hrs beginning at the time of reperfusion for 6 hrs. We collected brains for immunohistochemical analysis 24 hrs after MCAO. For both MDL 28170 and MK-801 treated animals, we observed a 40–50% reduction in total infarct size as measured by Fluoro-Jade staining (data not shown), cresyl violet staining (Figure S2B), and reduction in NeuN immunoreactivity.
Because cell death and AIS cytoskeleton disruption after OGD are distinct events, we examined the AIS in brain regions that were still clearly infarcted following drug administration in vivo (all quantification and analysis of AIS immunoreactivity was made from infarcted brain regions with at least 50% reduction in NeuN staining; Figure 9A, red, and Figure 9C;(Igarashi et al., 2001; Davoli et al., 2002)). Similar to the in vitro OGD results, calpain inhibition increased the number of immunolabeled AIS in the ipsilateral cortex (Figure 9A, green, arrows) by 36.1% (p<0.01) over vehicle and 24.6% (p<0.01) over MK-801 treated animals (Figure 9B). Within the infarcted region analyzed, NeuN staining did not differ between groups (Figure 9C), suggesting that increased AIS immunoreactivity was not due to increased cell survival within the infarcted region. Similar to βIV spectrin, we also found increased levels of AIS Nav channel immunoreactivity in MDL 28170 treated animals (Figure 9D; arrows), but not vehicle or MK-801 treated animals. Thus, calpain inhibition protects the molecular organization of the AIS following ischemic injury in vivo.
An AP is initiated when sufficient excitatory synaptic input depolarizes the AIS membrane to threshold. AP initiation, duration, and modulation also depend on the variety of Nav and Kv channels found at the AIS (Figure 10A). The AIS also maintains neuronal polarity by preserving the distinction between axonal and somatodendritic domains (Hedstrom et al., 2008). Given its central role in nervous system function, diseases and injuries that specifically affect the molecular organization and/or integrity of the AIS are expected to have profound neurological effects. Even mild injuries without overt cell death may lead to disruption of the AIS cytoskeleton and modest reductions in ankG at the AIS. This could have significant effects on brain function due to decreased amounts of Nav channels and the corresponding changes in neuronal excitability. Consistent with this idea several AIS proteins, including ankG, have been implicated as susceptibility loci in diverse psychiatric disorders such as autism and bipolar disorder (Alarcon et al., 2008; Ferreira et al., 2008). In the present study we provide evidence that i) neuronal injury causes rapid and preferential disruption of the AIS, ii) the ankG and βIV spectrin-based AIS cytoskeleton is proteolyzed after injury, iii) proteolysis of the AIS cytoskeleton causes loss of ion channel clusters and neuronal polarity, iv) injury-induced proteolysis of ankG and βIV spectrin depends on the calcium-dependent cysteine protease calpain, v) calpain inhibitors preserve the molecular organization of the AIS and the integrity of its cytoskeleton after neuronal injury, vi) disruption of the AIS occurs independently of neuron death, and vii) disruption of the AIS is an irreversible event. Together our results suggest that increased Ca2+ influx resulting from neuronal injury (Figure 10B) activates the Ca2+ dependent protease calpain, which in turn preferentially proteolyses ankG and βIV spectrin (Figure 10C). Loss of the intact ankG and βIV spectrin based cytoskeleton results in failure to retain the ion channels and CAMs at the AIS leading to their dispersion and/or degradation (Figure 10D; Hedstrom et al., 2008). Although we focused our analyses on the AIS cytoskeleton, both αII and βII spectrin are also proteolyzed after injury. Whether disruption of these cytoskeletal proteins can also contribute to loss of neuronal polarity is an active area of investigation.
The AIS also functions to maintain neuronal polarity since its disruption through pharmacological means or through silencing of ankG expression causes disruption in proper protein trafficking and axons acquire the molecular characteristics of dendrites. For example, Winckler et al. (1999) showed that axonal membrane proteins are restricted in their mobility within the AIS membrane when compared to distal axons. Upon pharmacological disruption of the axonal cytoskeleton, this diffusion barrier was lost and membrane proteins were no longer restricted to distinct domains. To further elucidate the molecular mechanisms underlying maintenance of neuronal polarity, Hedstrom et al. (2008) silenced the expression of ankG in fully polarized hippocampal neurons. This dismantled the AIS and caused axons to acquire the molecular characteristics of dendrites including the presence of the somatodendritic protein MAP2. Finally, the ankG-based AIS functions as a filter to exclude dendritic carrier vesicles, and permit entry of axonal carrier vesicles (Song et al., 2009). Together, these experiments demonstrated that ankG is necessary to maintain neuronal polarity and the correct molecular composition of the AIS. Futhermore, these previous studies support our observation that an important consequence of the injury-dependent disruption of the ankG/βIV spectrin-based cytoskeleton is loss of neuronal polarity. We propose that disrupted neuronal polarity represents a previously unappreciated and major consequence of neuronal injury.
It is now well established that in response to axon injury polarity may be plastic such that dendrites can be converted into axons or supernumerary axons can grow from the cell body (Havton and Kellerth, 1987; Hoang et al., 2005). One particularly intriguing experiment showed that in cultured hippocampal neurons this depends on where the axonal transection takes place: transection at sites distal to the cell maintain axon identity, while transection close to the cell body (35 µm from the soma and about the same location as the AIS), causes a fate switch and the conversion of a dendrite into an axon (Gomis-Ruth et al., 2008). Although this change was attributed to altered microtubule stability, an alternative explanation may be the disruption of the AIS cytoskeleton. Our results demonstrating that AIS disruption leads to irreversible dismantling of the molecular organization of the AIS and loss of neuronal polarity supports the latter conclusion. Furthermore, we previously showed that loss of the AIS cytoskeleton converted axons into dendrites (Hedstrom et al., 2008). These differences in dendrite to axon and axon to dendrite conversion may reflect unique aspects of each injury model. In any case, both studies emphasize that preservation of the AIS cytoskeleton after injury may be one important way to reduce inappropriate synaptic connections by preserving the appropriate neuronal polarity and the identity of the axon.
Besides failure to properly initiate action potentials in the absence of Nav channel clusters (Kole et al., 2008) and maintain neuronal polarity (Hedstrom et al., 2008), what other consequences might result from disruption of the AIS cytoskeleton? In addition to ion channels, the AIS is highly enriched in cell adhesion molecules (CAMs; Figure S6A), accessory and signaling proteins, and a unique extracellular matrix (ECM; Ogawa and Rasband, 2008), all of which depend on an intact AIS cytoskeleton for their localization (Hedstrom et al., 2007). Therefore, loss of the AIS cytoskeleton would be expected to disrupt any molecular or cellular specializations that exist at this site. For example, some data suggest that the AIS CAM neurofascin (NF)-186 and/or ECM directs GABAergic innervation of the AIS (Ango et al., 2004; Szabadics et al., 2006). In addition, AIS recruitment of gephyrin, a postsynaptic scaffolding protein that regulates GABA receptor clustering, also depends on NF-186 (Burkarth et al., 2007). Thus, dispersion of NF-186 away from the AIS through loss of ankG and βIV spectrin might also disrupt GABAergic neurotransmission at this site.
Although we focused our experiments on the AIS, in myelinated axons the node of Ranvier functions to regenerate the AP and has a very similar molecular organization with the same ankG and βIV spectrin-based cytoskeleton (Susuki and Rasband, 2008). Nodes of Ranvier may be a particularly fragile point in the axon since they lack an overlying myelin sheath and could readily contribute to axonal Ca2+ overload. Consistent with this idea it is now appreciated that the permanent functional deficits associated with demyelination are a consequence of axon degeneration, and calpain is upregulated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (Bjartmar and Trapp, 2001; Guyton et al., 2005). Indeed, axonal degeneration is attenuated in EAE with the addition of calpain inhibitors (Hassen et al., 2006). However, in our analysis of nodal structure in the striatum after MCAO, although we observed profound loss of initial segments, we failed to observe disruption of nodes of Ranvier even 72 hr after injury. These results suggest that proximity of the axonal injury to the cell body may influence proteolysis of the cytoskeleton or that the nodal cytoskeleton is resistant to proteolysis. This may also reflect differences in the amount of calpain found in axonal or somatodendritic cellular compartments. Future experiments focused on white matter injury and responses at nodes should help to clarify these issues.
We have discussed proteolysis of the AIS cytoskeleton as mainly a maladaptive response to neuronal injury. However, it may be that dismantling of the AIS and loss of Nav channels before neuronal death is a neuroprotective response to injury. We demonstrated that 2 hrs of OGD caused disruption of the AIS, but not cell death. In stroke, waves of depolarization and excitotoxicity are major contributors to the development of the ischemic core and penumbra (the area of the brain bordering the ischemic core where the majority of therapeutic strategies are targeted). Inhibiting neuronal depolarization and a cell’s ability to fire APs may protect the cell from increased excitation and spread of an already excitotoxic cellular environment. Consistent with this idea, Nav channel blockers have been shown to be effective neuroprotective agents following ischemic and experimental spinal cord injury (Schwartz and Fehlings, 2001).
In conclusion, we have shown that neuronal injury disrupts the molecular organization of the AIS by calpain-dependent proteolysis of the AIS cytoskeleton. Although we have mainly used ischemia as a model injury, our results in the optic nerve crush model suggest that any injury or disease that causes increases in calpain activity by cytosolic Ca2+ overload will affect the AIS cytoskeleton and neuronal function. Finally, since normal nervous system function requires properly polarized neurons with high densities of ion channels at the AIS, any effort aimed at nervous system protection, repair or recovery must consider not only strategies to enhance cell survival, but also incorporate ways to preserve or repair the integrity of the AIS.
We thank Dr. Sandra Hewett for helpful discussions and the use of her anaerobic chamber. We thank Drs. Michael Stankewich, Stephen Crocker, Elior Peles, and Peter Saggau for helpful discussions. We thank Drs. Yang Yang and Larry Benowitz for providing retinas from animals with optic nerve crush. This research was supported by grants from the National Institutes of Health NS044916 (MNR), NS050505 (LDM), NS055215 (LDM), the Department of Defense W81XWH-08-2-0145 (MNR), the Dr. Miriam and Sheldon Adelson Medical Research Foundation, and Mission Connect. DPS was supported in part by National Institutes of Health training grant 5T32NS041224. MNR is a Harry Weaver Neuroscience Scholar of the National Multiple Sclerosis Society.