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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC Aug 9, 2011.
Published in final edited form as:
PMCID: PMC3075005

AKT suppresses retrograde degeneration of dopaminergic axons by inhibition of macroautophagy


Axon degeneration is a hallmark of neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. Such degeneration is not a passive event, but rather is an active process, mediated by mechanisms that are distinct from the canonical pathways of programmed cell death that mediate destruction of the cell soma. Little is known of the diverse mechanisms involved, particularly those of retrograde axon degeneration. We have previously observed in living animal models of degeneration in the nigro-striatal projection that a constitutively active form of the kinase Akt (Myr-Akt) demonstrates an ability to suppress programmed cell death and preserve the soma of dopamine neurons. Here we show in both neurotoxin and physical injury (axotomy) models that Myr-Akt is also able to preserve their axons due to suppression of acute retrograde axon degeneration. This cellular phenotype is associated with increased mTor activity, and can be recapitulated by a constitutively active form of the small GTPase Rheb, an upstream activator of mTor. Axon degeneration in these models is accompanied by the occurrence of macroautophagy, which is suppressed by Myr-Akt. Conditional deletion of the essential autophagy mediator Atg7 in adult mice also achieves striking axon protection in these acute models of retrograde degeneration. The protection afforded by both Myr-Akt and Atg7 deletion is robust and lasting, because it is still observed as protection of both axons and dopaminergic striatal innervation weeks after injury. We conclude that acute retrograde axon degeneration is regulated by Akt/Rheb/mTor signaling pathways.

Keywords: degeneration, Parkinson’s disease, apoptosis, axon, autophagy, substantia nigra


The brain pathology observed in the adult-onset neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease (PD), is characterized not only by loss of neurons in select, vulnerable brain regions, but also by loss of axon projections. Indeed, for both of these disorders, some have proposed that axons and their synaptic terminals are the initial locus of the disease process, and that it is this pathology, not the loss of neurons, that results in the first manifestations and clinical progression of disease (Selkoe, 2002; Hornykiewicz, 1998; Cheng et al., 2010).

In spite of the importance of axon degeneration in these disorders, little is known of the underlying mechanisms. There is an emerging consensus that while the molecular mechanisms of axon degeneration may share some of the components of the canonical pathways of programmed cell death in some contexts (El-Khodor and Burke, 2002; Nikolaev et al., 2009), for the most part these processes are separate and distinct (Finn et al., 2000; Raff et al., 2002). Indeed, in the case of models of degeneration induced in the dopaminergic nigro-striatal system, many experimental approaches targeting the pathways of programmed cell death have succeeded in preserving neuron cell bodies, but not their axon projections (Eberhardt et al., 2000; Chen et al., 2008; Ries et al., 2008). The concept that the molecular mechanisms of axon and cell soma degeneration are distinct has been especially supported by observations in the Wlds mouse, which displays a remarkable resistance to Wallerian anterograde axon degeneration (Lunn et al., 1989). Much of what we do know about the mechanisms of axon degeneration derive from studies of the Wlds mutant protein (reviewed in Coleman and Freeman, 2010). However, how this mutant chimeric protein interacts with endogenous signaling pathways remains largely unknown. Additionally, in the nigro-striatal dopaminergic pathway, while Wlds abrogates classic anterograde Wallerian degeneration, it does not prevent retrograde degeneration, the form postulated to occur in PD (Sajadi et al., 2004; Cheng et al., 2010).

In view of these considerations, we noted with interest the ability of a constitutively active form of the survival signaling kinase Akt, myristoylated-Akt (Myr-Akt), which has diverse anti-apoptotic effects, to remarkably preserve the dopaminergic nigro-striatal axon projection in a highly destructive neurotoxin model of PD (Ries et al., 2006). Given not only the extent of axon preservation, but also the precise preservation of normal patterns of striatal innervation, we considered that Myr-Akt may provide protection not only of neuron cell bodies (Ries et al., 2006) but also their axons. To examine this possibility directly, we have employed a novel confocal imaging approach to visualize dopaminergic axons in the medial forebrain bundle (MFB) in mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase (TH) promoter. We have used this approach to monitor pathologic changes and to quantify axons during the acute injury period in both a neurotoxin and an axotomy model of induced retrograde axon degeneration in the dopaminergic nigro-striatal system.


Animal models of nigro-striatal axon injury

For these studies four models of nigro-striatal axon injury were used (supplemental Fig. S1A, available at www.jneurosci.org as supplemental material). Adult (8 week) male C57Bl/6 mice were obtained from Charles River Laboratories. The neurotoxin 6OHDA was used to induce retrograde axonal degeneration by injection into the striatum, or anterograde degeneration by injection into the MFB just anterior to the SN. Axotomy of the MFB was performed either distal to the SN, near striatal target, to induce retrograde degeneration, or proximal to the SN to induce anterograde degeneration. The intrastriatal 6OHDA model was induced as previously described (Silva et al., 2005). Briefly, a solution of 6OHDA was injected by microliter syringe at a rate of 0.5 μL/min by pump for a total dose of 15.0 μg/3 μl. Injection was performed into the left striatum at coordinates AP: +0.09 cm; ML: +0.22 cm; DV: −0.25 cm relative to bregma. After a wait of 2 minutes, the needle was slowly withdrawn. For lesion of the MFB, 6OHDA was infused at a rate of 0.2 μl/min for 5 min (total dose: 5 μg/1μL). Axotomy proximal to the SN was performed as previously described (El-Khodor and Burke, 2002) by use of a retractable wire knife (Kopf Instruments, Tujunga, CA) at coordinates: AP: −0.10 cm, ML: +0.20 cm. Axotomy distal to the SN was performed in a similar fashion at AP: −0.030 cm, ML: +0.20 cm. All surgical procedures were approved by the Columbia University Animal Care and Use Committee.

Intra-nigral injection of AAV vectors

Mice were anesthetized with ketamine/xylazine solution and placed in a stereotaxic frame (Kopf Instruments) with a mouse adapter. The tip of 5.0 μL syringe needle (26S) was inserted to stereotaxic coordinates AP: − 0.35cm; ML: +0.11cm; DV: −0.37cm, relative to bregma. These coordinates place the needle tip dorsal to the posterior SN. Viral vector suspension in a volume of 2.0 μL was injected at 0.1 μL/min over 20 minutes. After a wait of 5 minutes, the needle was slowly withdrawn. Successful transduction of DA neurons of the SN was confirmed histologically by double immunolabeling for FLAG and tyrosine hydroxylase (TH) or by fluorescent single-labeling of TH in combination with fluorescent detection of GFP or dsRed.

Production of AAV viral vectors

All vectors used for these studies were AAV1 serotype. Myr-Akt was produced as previously described (Ries et al., 2006). A plasmid encoding a 5′ src myristoylation signal in frame with mouse Akt1 was kindly provided by Dr. Thomas Franke (Franke et al., 1995; Ahmed et al., 1997). The myristoylated-Akt1 (Myr-Akt) sequence was modified to incorporate a FLAG-encoding sequence at the 3′ end and inserted into an AAV packaging construct that utilizes the CBA promoter, and contains a WPRE (Olson et al., 2006). AAV1 control injections, as specified for each experiment, were subcloned into the same viral backbone. A plasmid containing GFP-LC3 was kindly provided by Drs. N Mizushima and T. Yoshimori (Kabeya et al., 2000) and subcloned into the same viral backbone. AAV1 dsRed-LC3 was created by exchange of dsRed for GFP in the GFP-LC3 plasmid, and it was subcloned into a pFBGR viral backbone provided by the Gene Transfer Vector Core of the University of Iowa. This backbone utilizes the CMV promoter and lacks a WPRE. AAV1 Cre was also produced in the pFBGR viral backbone. AAV1 hRheb(S16H) was produced by the University of North Carolina Vector Core by use of an AAV packaging construct that utilizes the CBA promoter, and contains a 3′ WPRE (pBL).

Conditional deletion of Atg7 in SN neurons

Atg7fl/fl mice were crossed with TH-GFP mice to obtain Atg7fl/wt:TH-GFP mice. These mice were then crossed with Atg7fl/fl to obtain the Atg7fl/fl:TH-GFP genotype. Local deletion of Atg7 was achieved by intra-nigral injection of AAV Cre. Preliminary experiments revealed that AAV Cre achieved expression of Cre in SN neurons and recombination in ROSA26-LacZ mice (supplemental Fig. S2, available at www.jneurosci.org as supplemental material). Selective deletion of Atg7 mRNA in the SN was confirmed by non-radioactive in situ hybridization with a riboprobe complementary to exon 14 sequence (Komatsu et al., 2005) (Figure S2D), performed as described (Oo et al., 2009).


Mice were perfused intracardially with 0.9% NaCl followed by 4.0% paraformaldehyde in 0.1 M phosphate buffer (pH 7.1). The brain was removed carefully and blocked into midbrain and forebrain regions. The region containing the midbrain was postfixed for 1 week, cryoprotected in 20% sucrose overnight, and then rapidly frozen by immersion in isopentane on dry ice. A complete set of serial sections then was cut through the SN at 30 μm. Sections were processed free-floating. For TH immunofluorescent staining, the primary antibody was rabbit anti-TH (Calbiochem, La Jolla, CA) at 1:750. Sections then were treated with goat-anti-rabbit Texas Red conjugated secondary antibody.

For dopamine transporter (DAT), FLAG, phophorylated 4EBP1 and cathepsin D immunostaining, 30 μm sections were used. For DAT immunohistochemistry, sections were blocked with 0.1M Tris-buffered saline and 3% normal rabbit serum for 48hr at 4°C. Then they were washed and incubated with rat anti-DAT (Chemicon) at 1:1000. Sections then were incubated with biotinylated anti-mouse IgG (Vector Labs, Burlingame, CA), followed by ABC (Vector Labs). For FLAG, sections were initially treated with Mouse-on-Mouse Blocking Reagent (Vector Labs) and then processed free-floating with a mouse monoclonal anti-FLAG antibody (Sigma, St. Louis, MO) at 1:1,000. Sections then were incubated with biotinylated anti-mouse IgG (Vector Labs), followed by ABC (Vector Labs). For immunofluorescent staining, fluorescein conjugated avidin was used after secondary antibody.

For phosphorylated 4EBP1, immunostaining was performed on 30 μm sections with a rabbit anti-phospho-4EBP1 (Thr37/46) antibody (Cell Signaling, Beverly, MA) at 1:200. Sections then were treated with biotinylated protein A and avidin-biotinylated horseradish peroxidase complexes (ABC; Vector Labs). After immunoperoxidase staining, sections were thionin counterstained. For immunofluorescent staining, Texas Red conjugated goat-anti-rabbit secondary antibody or Texas Red conjugated donkey-anti-rabbit secondary antibody was used in double immunoflurescent staining. For cathepsin D immunofluorescent staining, goat-anti-cathepsin D (Santa Cruz Biotechnology, Santa Cruz, CA) was used at 1:100. Sections were then treated with biotinylated horse-anti-goat antibody, followed by fluorescein conjugated avidin.

Immunohistochemistry for Atg7 was performed with three different rabbit anti-Atg7 antibodies (Sigma, St. Louis, MO; Novus Biologicals, Littleton, CO; US Biological, Swampscott, MA) at 1:1000 (Sigma) and 1:200 (Novus Biologicals and US Biological). Sections were then treated with biotinylated protein A and avidin-biotinylated horseradish peroxidase complexes (ABC; Vector Labs). For immunofluorescent staining, Alexa 568 conjugated goat-anti rabbit secondary antibody (Invitrogen, Carlsbad, CA) was used. Atg7 recombinant protein (Abnova, Taiwan) and blocking peptide (US Biologicals) were used for primary antibody absorption at the molar ration of 5:1. Those sections were then processed with pre-absorbed primary antibody reagent, followed by biotinylated protein A and avidin-biotinylated horseradish peroxidase complexes.

Quantification of dopaminergic axons in the MFB

Quantification of axons was performed on TH-GFP transgenic mice, which express green fluorescent protein driven by tyrosine hydroxylase promoter (Sawamoto et al., 2001). Mice were perfused intracardially with 0.9% NaCl followed by 4.0% paraformaldehyde in 0.1 M phosphate buffer (pH 7.1). Following postfixation and cryoprotection, the brains were sectioned horizontally at 30 μm. A section containing the posterior third ventricular recess and the A13 dopamine cell group was selected for analysis (supplemental Fig. S1B, available at www.jneurosci.org as supplemental material). Confocal microscopy (Leica TCS SP5 AOBS MP System) was used to acquire images through the entire medial-to-lateral extent of the MFB. Proceeding from a point midway between the anterior A13 cells and the posterior third ventricle recess, images were acquired with a 20X objective with a zoom factor of 8 applied. Depending on the site of the MFB under analysis, five to six contiguous fields (97 μm × 97 μm) were scanned. Each field was scanned in the Z-axis with twenty 0.1 μm thickness optical planes from dorsal to ventral, for a total vertical distance of 2.0 μm in the center of the section. These twenty optical planes were then merged to obtain a single maximal projection of the sampled volume. In order to count the number of axons passing in the rostro-caudal dimension through each sample volume, two horizontal sampling lines were drawn on the image at a separation distance of 10 μm in the center of the maximal projection. Every intact axon crossing both lines was counted as positive.

Quantification of autophagic vacuoles

AAV GFP-LC3 was injected with AAV MyrAKT or dsRed into the SN of wildtype mice. AAV dsRed-LC3 was injected with AAV MyrAKT or vehicle into SN of TH-GFP mice. Mice were then lesioned with 6OHDA or axotomy. Lesioned mice were then sacrificed at different time points as indicated. A complete set of serial sections then was cut through the SN at 30 μm. Beginning with a random section between 1 and 4, every fourth section was selected, in keeping with the fractionator method of sampling. Every section was then examined under epifluorescence with 60X objective lens and each GFP-LC3 or dsRed-LC3-labeled autophagic vacuole present in SN was counted. AAV dsRed-LC3 was injected in TH-GFP mouse, and dsRed labeled autophagic vacuoles were counted in GFP positive dopaminergic neurons. Control injections of AAV GFP-LC3, AAV dsRed-LC3 and AAV dsRed into adult, non-lesioned, wildtype C57Bl/6 mice revealed that in the absence of injury, transduced neurons did not reveal distinct puncta or intra-cellular inclusions (supplemental Fig. S3B,C available at www.jneurosci.org as supplemental material).

Electron microscopy

At 24 hours following injection of 6OHDA into either the striatum or the MFB, adult mice were perfused with 0.9% saline followed by 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 10 min at 4° C. Brains were post-fixed in the same fixative for 1–2 weeks. Sections through the SN were cut on a Vibratome and collected into 0.1 M Sorensen’s buffer. Sections were stained in 1% OsO4/0.1 M Sorensen’s for 60 min, and then washed. After dehydration in ascending concentrations of alcohols, sections were flat embedded in Durcupan between two pieces of Aclar. The sections were then trimmed to include the medial portion of the SNpc, mounted on a Durcupan peg, and then cut at 8 μm for examination under phase contrast. Sections of interest were then re-embedded, thin sectioned, and examined on a JEOL 1200EX electron microscope as previously described (Oo et al., 1996).

Western analysis

For analysis of proteins associated with autophagy, mice were unilaterally lesioned with 6OHDA and then sacrificed at indicated time points. Blots were probed with anti-LC3 Ab1 (courtesy of Dr Y. Uchiyama), anti-LC3 Ab2 (courtesy of Drs W. Haung Yu and R Nixon), or anti-Rab24 (BD Transduction Laboratories) or anti-Beclin 1 antibodies (Novus Biologicals). Phospho-mTor was detected with anti-p-mTor(Ser2448) (Cell Signaling, Beverly, MA). Proteins with detected with appropriate secondary antibodies, conjugated with horseradish peroxidase, and chemiluminescent substrate (Pierce). Densitometric analysis of band intensity was performed by using a FluorChem 8800 Imaging System (Alpha Innotech, San Leandro, CA).

Statistical Analysis

All data in the Figures is presented as mean ± standard error of the mean (SEM). Multiple group comparisons were performed by one way analysis of variance (ANOVA) unless otherwise indicated in the Figure legend, followed by a Tukey all pairwise multiple comparison procedure post hoc analysis. For two group comparisons, a Students t statistic was performed.


Myr-Akt suppresses retrograde axonal degeneration in the dopaminergic nigro- striatal projection

Following injection of the neurotoxin 6-hydroxydopamine (6OHDA) into the striatum, the principal target of the nigro-striatal dopaminergic projection, intense axon terminal degeneration can be demonstrated by the suppressed silver stain technique within 24–48 hours (Ries et al., 2008). The degeneration proceeds retrograde such that by 3 to 4 days postlesion, degenerating axons can be identified within the medial forebrain bundle (MFB), the principal tract conveying dopaminergic axons to the striatum. At this postlesion time, many dopaminergic axons within the MFB have lost expression of the tyrosine hydroxylase (TH) phenotype such that their number and morphology cannot be examined by immunostaining (supplemental Fig. S1C, available at www.jneurosci.org as supplemental material). However, in mice that express green fluorescent protein under the TH promoter (TH-GFP mice) (Sawamoto et al., 2001), it remains possible to continue to monitor these axons by the visualization of GFP by confocal microscopy in horizontal sections through the MFB (supplemental Fig. S1C, available at www.jneurosci.org as supplemental material). This analysis reveals that mice given a control (dsRed) AAV injection into the SN, there is a 42% loss of GFP-positive axons by 3 days postlesion (Figure 1A,B). In addition, the injured axons are thickened and fragmented, and show the formation of spheroids, a hallmark of axon injury (Figure 1A). In contrast, in mice given AAV Myr-Akt, there is only a 15% loss of axons (Figure 1A,B), not a significant change in comparison to the non-injected, contralateral control side. In addition to preservation of axon number, treatment with AAV Myr-Akt also preserves axon morphology, as fragmentation and spheroid formation are rarely observed (Figure 1A). We considered the possibility that treatment with AAV Myr-Akt may increase the number of axons in the MFB, due to sprouting prior to 6OHDA injection, and thereby confound an analysis of axon preservation. However, we found that this had not occurred (Figure 1B). Myr-Akt induces an increase in the caliber of dopaminergic axons (Ries et al., 2006), but this effect does not influence the number of axons counted by this methodology, which is based on continuity over a distance of 10 microns.

Figure 1
Myr-Akt suppresses retrograde degeneration in dopaminergic axons

Given the wide spectrum of cellular effects of Akt, we considered the possibility that it may act by preventing the primary neurotoxicity of 6OHDA, rather than acting specifically on mechanisms of axon degeneration. To address this possibility, we examined effects on retrograde degeneration induced by direct axotomy injury. In this model, made by knife cut in the anterior projection of the MFB, a postlesion interval of 6 days is required to observe a substantial loss of axons due to retrograde degeneration. At this time, mice injected with control AAV demonstrate a 33% loss of axons (Figure 1A,B). In contrast, mice injected with AAV Myr-Akt demonstrate only 5% loss (Figure 1A,B), not a significant difference compared to the contralateral, non-lesioned side. We conclude that the ability of constitutively active Akt to suppress retrograde axon degeneration is general to diverse forms of axon injury, and, in the case of 6OHDA, is unlikely to be due to prevention of neurotoxicity.

To ascertain whether the axon protection provided by Myr-Akt in the acute period of degeneration following 6OHDA affords a robust and lasting preservation of the dopaminergic nigro-striatal projection, we performed immunostaining for the high-affinity dopamine transporter (DAT), a specific marker for dopaminergic terminals, at 4 weeks postlesion in the striatum. This analysis demonstrated that acute protection did indeed result in a lasting preservation of striatal dopaminergic innervation (Figure 1C).

Macroautophagy occurs in the axons and cell bodies of dopamine neurons of the SN following axon injury and is suppressed by Myr-Akt

We have previously shown that activation of caspase-3 occurs in axons in the immature nervous system following either axotomy (El-Khodor and Burke, 2002) or intra-striatal injection of 6OHDA, but there is neither activation of caspase-3 nor calpain following intrastriatal 6OHDA in the mature nervous system (Ries et al., 2008). Given the evidence of a role for macroautophagy (hereafter referred to as ‘autophagy’) in the degeneration of axons and their terminals in the context of injury in tissue culture (Larsen et al., 2002; Yang et al., 2007) and both axon pruning (Song et al., 2008) and degeneration (Wang et al., 2006) in vivo, we performed ultrastructural analysis of the striatum following intra-striatal injection of 6OHDA. This analysis revealed the presence of numerous autophagic vacuoles (AVs) in striatal neuropil (Figure 2). AVs were identified as vesicular structures with a delimiting double membrane, often containing subcellular organelles such as mitochondria (Mizushima, 2004). A number of these vesicles also contained multilamellar membranous structures, another characteristic ultrastructural feature of AVs (Hornung et al., 1989; Jia et al., 1997; Hariri et al., 2000; Nixon et al., 2005; Borsello et al., 2003) (Figure 2B,C). Occasional AVs were observed within nerve terminals, identified by adjacent synaptic vesicles (Figure 2B). We considered the possibility that the formation of AVs within the striatum may be a response unique to the proximate injection of 6OHDA, rather than a general feature of the degeneration of nigro-striatal axons. To assess this possibility, we also performed an analysis following a distant injection of 6OHDA into the MFB, which induces anterograde degeneration. In these mice as well, AVs were identified within striatal neuropil (Figure 2B,C). Examination of numerous striatal sections from control, non-lesioned adult C57Bl/6 mice failed to reveal AVs (supplemental Fig. S3A, available at www.jneurosci.org as supplemental material).

Figure 2
Ultrastructural features of autophagy in striatal neuropil following 6OHDA injection

To assess whether autophagy occurs specifically within defined dopaminergic neurons in these neurotoxin models, and to assess the general occurrence of autophagy following axon injury in these neurons, we made use of a viral vector approach to transduce them with dsRed-LC3 in TH-GFP mice. Microtubule-associated protein 1 light chain 3 (LC3) is a mammalian homologue of yeast Apg8p, a critical component of a ubiquitin-like conjugation system required for autophagosome formation (Kabeya et al., 2000; Mizushima, 2004), and it associates with isolation membranes in an Atg5-Atg12-dependent manner and through a process catalyzed by Atg7 (Mizushima, 2004). The use of dsRed-LC3 therefore permits demonstration of AVs as intracellular red puncta in dopaminergic neurons. Induction of retrograde axon degeneration by intra-striatal 6OHDA caused the appearance of multiple discrete puncta in the axons and cell bodies of dopaminergic neurons (Figure 3A and Movie S1, available at www.jneurosci.org as supplemental material)). Such puncta were also observed following induction of retrograde axon degeneration by anterior MFB axotomy, and anterograde degeneration by posterior axotomy (Figure 3A). Thus autophagy occurs in both axons and cell bodies of dopaminergic neurons following either neurotoxin or physical injury. DsRed-LC3 red puncta were not observed in these neurons in the absence of 6OHDA or axotomy lesion (supplemental Fig. S3C, available at www.jneurosci.org as supplemental material)

Figure 3
Autophagy occurs in dopamine neuron cell bodies and axons in diverse models of axon injury

To further establish and characterize the occurrence of autophagy in the nigro-striatal system following intra-striatal 6OHDA lesion, we examined the expression of several proteins that have been associated with autophagy. LC3 exists in two forms in cells: LC3-I is identified at an apparent molecular weight of 18 kDa, and is localized to the cytosol; LC3-II is identified at 16 kDa, it is enriched in the AV fraction, and is present on both sides of AV membranes (Kabeya et al., 2000). Increase in conversion of LC3-I to II is an indicator of activation of autophagy. Utilizing two different antibodies to LC3, we observed that the LC3-II isoform is identified in mouse SN homogenates following intra-striatal 6OHDA, but not vehicle, injection (supplemental Fig. S4A, available at www.jneurosci.org as supplemental material). Rab24 is a GTP-binding protein that has also been localized to AVs (Munafo and Colombo, 2002), and identified in the central nervous system following axon injury (Egami et al., 2005). There is an induction of Rab24 protein following intra-striatal 6OHDA (supplemental Fig. S4B, available at www.jneurosci.org as supplemental material). This induction is maximal at postlesion day 2 (Fig. S4B). Beclin 1, a novel Bcl-2 interacting protein, is homologous to the yeast autophagy gene Atg6, and is a mediator of autophagy (Liang et al., 1999). Beclin 1 protein expression is also induced in SN following 6OHDA, and, like Rab24, induction is maximal at postlesion day 2 (supplemental Fig. S4C, available at www.jneurosci.org as supplemental material).

To explore the relationship between the ability of Myr-Akt to preserve nigro-striatal dopaminergic axons following distal injury, and the occurrence of autophagy during the degenerative process, we quantified both the number of neurons in the SNpc with AVs and the total number of AVs in the SNpc neuron population following Myr-Akt in models of retrograde degeneration induced by intra-striatal 6OHDA and anterior axotomy. For this analysis, wildtype, rather than TH-GFP mice, were used in the 6OHDA model and AVs were identified following transduction with AAV GFP-LC3 (supplemental Fig. S3B, available at www.jneurosci.org as supplemental material). Following treatment with Myr-Akt, fewer SNpc neurons contain AVs at postlesion days 1–3 after 6OHDA (Figure 3B). Similar results were observed in an analysis of the total number of AVs in the SNpc neuron population (Figure 3B). Treatment with Myr-Akt also diminished the number of dopaminergic neurons with AVs at postlesion day 2 following anterior axotomy (Figure 3B) determined as the number of GFP-positive neurons with red puncta following transduction with AAV dsRed-LC3.

Inhibition of retrograde axonal degeneration by Myr-Akt is associated with mTor signaling

In diverse contexts, the ability of Akt to inhibit autophagy has been attributed to signaling through the mTor protein complex (Figure 4A) (Lum et al., 2005; Meijer and Codogno, 2004; Mizushima et al., 2008). We determined that transduction of SN with AAV Myr-Akt results in increased abundance of phosphorylated mTor protein (Figure 4B). At the cellular level, expression of p-4E-BP1, an mTor substrate, was observed exclusively in the injected SN following transduction with AAV Myr-Akt (Figure 4C). Following transduction, immunostaining for p-4E-BP1 was observed not only in the cell bodies of neurons in the SNpc, but also in their axons within the MFB (Figure 4D). Expression of p-4E-BP1 was observed exclusively in neurons that also expressed Myr-Akt (Figure 4C). In such p-4E-BP1-positive neurons, there was a significant increase in neuron size (Figure 4C), confirming a biologic effect of enhanced Akt/mTor signaling. We observed a punctate appearance of p-4E-BP1 immunostaining in neurons, due to a precise co-localization of p-4E-BP1 immunostaining with that for cathepsin-D, a lysosomal marker (Figure 4C).

Figure 4
Myr-Akt mediates axon protection through mTor signaling

To assess whether the ability of Myr-Akt to protect axons is mediated through mTor signaling, we examined the possibility of also achieving protection by activation of Rheb, a GTPase that is activated by Akt and mediates activation of mTor (Figure 4A). We created a mutant form of human Rheb, hRheb(S16H), that had been identified in a screen for constitutively active mutants in yeast, and confirmed to be active in mammalian cells (Yan et al., 2006). Like Myr-Akt, transduction of dopamine neurons of the SNpc with hRheb(S16H) resulted in an increased abundance of p-4E-BP1 protein (Figure 4E). Transduction of dopamine neurons with hRheb(S16H) also conferred resistance of their axons to retrograde degeneration induced by intra-striatal 6OHDA (Figure 4F), suggesting that Myr-Akt acts, at least in part, through Rheb-mTor signaling to achieve axon protection.

Abrogation of autophagy signaling within dopamine neurons by regionally selective deletion of Atg7 confers resistance to retrograde axonal degeneration

Many previous investigations have identified the occurrence of autophagy in the presence of axon degeneration (Larsen et al., 2002; Wang et al., 2006; Yang et al., 2007; Komatsu et al., 2007; Chen et al., 2009), but its role has been unclear. Based on our observation that Myr-Akt may be acting through Rheb-mTor signaling to provide axon protection, we considered that there may be a relationship between the ability of Myr-Akt to suppress autophagy, through mTor signaling, and to protect axons. In order to assess this possible relationship, we sought to selectively disrupt autophagy signaling by genetic deletion of an essential autophagy mediator, Atg7, in mice with a floxed allele (Komatsu et al., 2005). Furthermore, to avoid disturbance of the development of the nigro-striatal system, we sought to ablate Atg7 in mature mice since there is extensive evidence that intact autophagy signaling is required during development for normal axon maintenance (Komatsu et al., 2006; Hara et al., 2006; Komatsu et al., 2007). In normal adult mice, we observed Atg7 protein expression in numerous axons in brain (supplemental Fig. S5A, available at www.jneurosci.org as supplemental material), but in dopaminergic axons of the MFB, protein expression was observed only following injury (Fig. S5B). Regionally selective deletion of Atg7 in mature mice was achieved by intra-nigral injection of AAV Cre (supplemental Fig. S2A-D, available at www.jneurosci.org as supplemental material). In mice homozygous for the Atg7fl allele, and injected with AAV Cre, but not control mice, there was protection from retrograde axon degeneration induced by either intra-striatal 6OHDA (Figure 5A) or axotomy (Figure 5B). This axon protection was observed not only as preservation of axon number, but also by the absence of axonal pathology such as fragmentation and spheroid formation (Figure 5A,B). Deletion of Atg7 provided a lasting protection of axons, as shown by an increase in their remaining number at 4 weeks after 6OHDA lesion (Figure 5C). A comparable degree of protection was observed for striatal dopaminergic innervation, demonstrated by immunostaining for DAT (not shown).

Figure 5
Following deletion of Atg7, axons of SNpc dopamine neurons are resistant to retrograde axon degeneration


Akt has a diverse array of cellular phenotypes, including effects on cell death, growth and metabolism (Manning and Cantley, 2007). Within post-mitotic neurons, it not only has these general cellular effects on survival (Brunet et al., 2001) and size (Kwon et al., 2006; Ries et al., 2006), but also effects that are unique to neurons, including regulation of axon sprouting (Namikawa et al., 2000; Ries et al., 2006; Park et al., 2008), caliber and branching (Markus et al., 2002), synaptic strength (Wang et al., 2003) and dendritic growth (Kwon et al., 2006). We herein describe a novel neuronal phenotype of Akt, the ability to regulate axon degeneration. Demonstration of this phenotype was achieved with an imaging approach that allows quantification of dopaminergic axons in spite of loss of endogenous TH protein expression due to injury and permits the conclusion that the apparent protection of axons is due to their actual structural preservation. This conclusion is supported by the observation that Akt diminishes the occurrence of axon swelling and fragmentation. In addition, because this method of axon quantification requires identification of individual fibers, and is not a population measure, it is not influenced by either amount of GFP protein expression or caliber of the axons, both of which may be affected by Akt. We therefore conclude that Akt acts to preserve the structural integrity of axons following distal injury. Our observations have been made with a mutant, constitutively active form of Akt that incorporates a myristoylation signal at its N-terminus (Ahmed et al., 1997). This mutation is believed to mediate constitutive activity by targeting Akt protein to cellular membranes, where it is then activated by phosphorylation (Ahmed et al., 1997). While Myr-Akt shares most cellular phenotypes with both overexpressed wildtype and other non-myristoylated constitutively active forms of Akt, nevertheless, some phenotypes are unique to the myristoylated form (Dufner et al., 1999). Whether the myristoylation signal is necessary for the axon protection phenotype that we have observed will require future study.

Little is yet known of the mechanisms of axon degeneration, but there is now abundant evidence that they are subject to cellular regulation and control (Coleman, 2005; Luo and O’Leary, 2005). There is also growing evidence that these pathways are diverse. For example, while the mutant protein Wlds suppresses axon degeneration due to injury, it does not affect normal developmental axon pruning (Hoopfer et al., 2006). The mechanisms of axon degeneration also change with maturation. While we observed activation of caspase-3 in nigro-striatal axons in the intra-striatal 6OHDA model in immature mice, it is not activated in mature animals (Ries et al., 2008). In mature mice, we observe a robust induction of autophagy following 6OHDA. AVs were identified by ultrastructural analysis (Mizushima, 2004) in striatal neuropil and specifically within nerve terminals. Autophagy was further confirmed by the identification of both GFP-LC3 and dsRed-LC3 positive puncta (Kabeya et al., 2000; Mizushima, 2004), the latter specifically within the cell soma and axons of dopaminergic neurons following four different types of axon injury. These observations are in keeping with those of other investigators who have reported the occurrence of autophagy in the course of neurite or axon degeneration (Larsen et al., 2002; Wang et al., 2006; Yang et al., 2007; Komatsu et al., 2007; Plowey et al., 2008; Chen et al., 2009).

However, the precise role that autophagy plays in the setting of axon injury has been debated. Some investigators have proposed that it plays a direct role in axon destruction (Yang et al., 2007; Plowey et al., 2008), whereas other have proposed that AVs passively accumulate in injured axons due to impaired axon transport (Chen et al., 2009), or that the induction of autophagy represents an attempted protective response (Larsen et al., 2002; Komatsu et al., 2007). To explore a possible relationship between the ability of Myr-Akt to protect axons, and the robust occurrence of autophagy in these models of axon injury, we examined the effect of Myr-Akt expression on the prevalence of AVs, and observed that it resulted in a marked decrease in their number. This observation is in keeping with extensive evidence in other contexts that Akt signaling negatively regulates autophagy (Lum et al., 2005; Meijer and Codogno, 2004; Mizushima et al., 2008). To further evaluate a possible relationship between the ability of Myr-Akt to protect axons and its ability to suppress autophagy, we examined effects of a constitutively active form of Rheb, a GTPase that mediates downstream effects of Akt by direct activation of mTor. For this purpose, we used hRheb(S16H), a mutant that is resistant to negative regulation by TSC1/2 (Yan et al., 2006). Transduction of SN neurons with hRheb(S16H) resulted in a robust increase in the number of p-4E-BP1-positive neurons, and it successfully recapitulated the axon protection phenotype of Myr-Akt, thus supporting the possibility of a relationship between the ability of both Myr-Akt and hRheb(S16H) to protect axons and signaling through mTor.

mTor activation has a wide range of cellular effects (Swiech et al., 2008). To examine whether suppression of autophagy by mTor specifically mediates the axon protection phenotype of Myr-Akt and hRheb(S16H), we utilized a genetic approach to abrogate autophagy signaling selectively within SN neurons by transduction with AAV Cre in Atg7fl/fl mice (Komatsu et al., 2005). Deletion of Atg7 signaling in these neurons results in a striking resistance of their axons to retrograde degeneration. This resistance is robust and lasting, as it is observed as an increased number of preserved axons and striatal dopaminergic nerve terminals at four weeks after toxin injury. Thus, we conclude that the ability of Myr-Akt to protect axons from retrograde degeneration is likely to be due, at least in part, to its ability to suppress autophagy through mTor signaling.

Our observations in these in vivo models are consistent with those of Yang and colleagues who noted in several in vitro models of injury that both pharmacologic and genetic disruption of autophagy signaling protected from axon degeneration (Yang et al., 2007). They are also in keeping with the findings of Plowey and colleagues who observed that RNAi mediated knockdown of Atg7 expression prevented degeneration of neurites induced by a mutant form of LRRK2 that causes PD (Plowey et al., 2008). While our findings would appear to be at variance with several observations that genetic ablation of autophagy signaling leads to neurodegeneration (Komatsu et al., 2006; Hara et al., 2006) and specifically to axon degeneration in the Atg7fl/fl mice (Komatsu et al., 2007), all of these observations were made in a context in which autophagy signaling had been disrupted during development. Perhaps the most universal emerging theme for the cellular role of autophagy is that it is highly context dependent (Cherra and Chu, 2008; Levine and Yuan, 2005). We postulate that while autophagy may be essential for normal axon homeostasis during development, in the setting of the mature brain, in the context of injury or neurodegenerative disease, it can lead to destruction.

Our conclusion that Myr-Akt provides protection from retrograde axon degeneration in the mature brain has implications for the pathogenesis and treatment of neurodegenerative disease, the most important being that the cellular consequences of activation of autophagy can be deleterious or beneficial, depending on the precise context. Autophagy has been postulated to play an important role in the clearance of pathogenic protein aggregates in neurodegenerative diseases (Mizushima et al., 2008). Of particular relevance in relation to our studies is that it has been postulated to play a critical role in the clearance of α-synuclein (Cuervo et al., 2004) which has been postulated to form toxin aggregates in dopaminergic axons. However, our observations in acute models of retrograde axon degeneration suggest that, to the extent that similar mechanisms may underlie axon degeneration in chronic neurodegenerative diseases, then activation of autophagy signaling may be deleterious. Our findings also imply that activation of Akt signaling pathways not only provides important neuroprotection of the cell soma level by blockade of apoptosis, but also a novel ability to forestall degeneration of axons. Thus, if activated at the appropriate time in select cellular compartments, these pathways may provide attractive candidates for the development of therapeutics in the treatment of neurodegenerative diseases.

Supplementary Material




This work was supported by NIH NS26836 and NS38370, the Parkinson’s Disease Foundation and the RJG Foundation (REB). We thank D Sulzer, AM Cuervo, and A Yamamoto for critical review of the manuscript and useful discussions. We also thank D Sulzer, RA Nixon, WH Yu, and A Tagliaferro for helpful examination of the electron micrographs. We also wish to express our gratitude in memoriam to Anne M Cataldo who generously offered her assistance in the interpretation of these micrographs. We thank RA Nixon and WH Yu for making available to us their antibody to LC3, and Y Uchiyama and M Shibata for making available to us their antibody to LC3. We express our gratitude to N Mizushima and T Yoshimori for kindly providing us with their GFP-LC3 clone. We gratefully acknowledge the expert technical assistance of Ms. Mary Schoenebeck in the performance of electron microscopy.

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