Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease characterized by motor neuron cell death and subsequent paralysis of voluntary muscles. Although ALS specifically affects motor neurons, some cells are resistant to disease progression. Most ALS studies have focused on the cellular mechanisms that cause loss of motor neuron viability. Less is known about the surviving neurons, and most of that information has come from gene expression profiling. In this study, we functionally characterize the surviving spinal motor neurons by culturing them from SOD1 ALS mouse models at various stages of disease progression. Surprisingly, we found that in comparison to non-transgenic controls, ALS resistant motor neurons from adult SOD1G93A mice have enhanced axonal outgrowth and dendritic branching. Further, the enhanced outgrowth occurs before the mice become symptomatic, but increases with disease progression. Motor neurons from SOD1G93A mice also display an increase in the number and size of actin-based structures such as growth cones and filopodia. The increased outgrowth and branching phenotype is predominantly cell-intrinsic and can be induced in motor neurons from non-transgenic mice by exogenous expression of SOD1G93A. These results indicate that expression of mutant SOD1 in ALS-resistant adult motor neurons can enhance their regenerative capability via a mechanism that is not directly correlated with the onset of ALS symptoms. Understanding the positive effects that mutant SOD1 has on motor neuron regeneration could lead to new therapeutic strategies that capitalize on this mechanism.
- ALS
- Amyotrophic lateral sclerosis
- MN
- motor neuron
- SOD1
- superoxide dismutase 1
- GFP
- green fluorescent protein
- YFP
- yellow fluorescent protein
- NTg
- non-transgenic
- G93A
- transgenic mouse line expressing SOD1 with a glycine-to-alanine mutation at position 93
- G93A-DL
- transgenic mouse line expressing SOD1 with a glycine-to-alanine mutation at position 93 with a lower copy number than the G93A mouse line
- G85R
- transgenic mouse line expressing SOD1 with a glycine-to-arginine mutation at position 85 fused to YFP
Introduction
Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurodegenerative disorder in which there is selective loss of motor neurons (MNs) in the cerebral cortex, brainstem and spinal cord [35]. Not all MNs are equally susceptible to cell death during ALS disease progression. ALS mostly targets MNs required for voluntary movement, whereas MNs of the autonomic system are less affected [57]. Certain groups of somatic motor neurons, including those in the oculomotor nucleus and Onuf’s nucleus, are also generally spared [25, 32, 51, 69]. Furthermore, there is a gradient of vulnerability among spinal MNs, where faster motor units become affected before slower muscle types [58]. MNs that are less ALS-susceptible can compensate for the cells that initially die by establishing new connections with the motor endplate, although many of these will eventually succumb to the disease [64].This selective neuronal vulnerability is present in both sporadic ALS and familial ALS and is also recapitulated in rodent models, such as the SOD1G93A mouse [51]. The mechanism that renders certain subgroups of MNs more susceptible to ALS is largely unknown, although studies have shown that ALS resistant MNs differentially express GABA and glutamate receptor subunits [45], [44], [13] and show a higher expression of EGR1, IGF2 [3] and OPN [48]. On the other hand, ALS vulnerable MNs have high expression of matrix metalloproteinase-9 (MMP9) [11] which was identified as a modulator of ER stress [37, 39], supporting previous studies showing that such ALS vulnerable MNs are selectively prone to ER stress [63].
Approximately 90% of ALS cases are sporadic with unknown etiology; the remaining 10% are inherited and known as familial ALS (fALS), of which over 20% have mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD1) [14]. To date, over 155 different mutations have been identified in SOD1 either in isolated cases of ALS or more commonly in patients from families showing autosomal dominant patterns of inheritance [4, 55]. ALS-linked SOD1 mutations are thought to induce a toxic gain-of-property of the protein, which becomes prone to misfolding and subsequent aggregation [38, 61]. While the exact cause of SOD1-induced MN degeneration is unknown, a number of pathogenic processes, including excitotoxicity, oxidative stress, mitochondrial dysfunction, dysregulation of the cytoskeleton, axonal transport defects, and inflammation are considered to play important roles in eventually inducing cell death [22, 34]. Some of the most significant advances in our understanding of how mutations in the SOD1 gene cause ALS have been achieved through rodent models. Since the generation of the first SOD1G93A transgenic mouse line [31], several other mutant SOD1 models now exist with slight variation in their pathologies, including time to onset of symptoms and death [15, 36, 50]. The SOD1G93A mouse (referred to here as G93A) carries 22 copies of a causative mutation (a glycine-to-alanine replacement at residue 93) in the human SOD1 gene, inserted randomly into chromosome 12 of the mouse genome [2]. This mouse model has an onset of paralysis at ~90 days, accompanied by degenerative motor neuron loss similar to that of human ALS pathology [66], and death by 130-180 days depending on the genetic background and gender of the mice. The G93A mouse remains the most widely used model of human ALS [31] to date.
While most studies have focused on the cellular mechanisms and genes that induce MN death in ALS, less is known about the neurons that do survive, including their ability to resist stress-induced cell death and to compensate for dying MNs. Most of our current knowledge about surviving spinal MNs in ALS mouse models has largely been generated by gene expression profiling of both whole spine and micro-dissected tissue and cells [6, 13, 20, 21, 41, 63]. However, these studies provide just a single snapshot of the MN’s biology and only allow for inferences to be made about how changes in gene expression alter MN physiology, allow them to resist degeneration, or compensate for dying neurons by forming new motor endplate attachments. In the current study, we sought to functionally characterize ALS-resistant MNs by culturing them in vitro, where we would be able to directly assess dynamic cellular properties such as outgrowth, branching, and regulation of the cytoskeleton. We found that in comparison to age-matched, non-transgenic controls, ALS-resistant motor neurons cultured from G93A mouse models have enhanced axonal outgrowth and dendritic outgrowth/branching. They also have an increase in both number and size of actin-based cell structures such as growth cones and filopodia. Interestingly, enhanced outgrowth and branching occurs in pre-symptomatic mice but increases with the onset of ALS symptoms. This phenotype occurs independently of SOD1 enzymatic activity and can only slightly be enhanced by non-cell autonomous factors. Exogenous expression of mutant SOD1 in non-transgenic adult motor neurons is sufficient to induce the enhanced outgrowth phenotype. These results indicate that expression of mutant SOD1 in adult motor neurons can enhance their regenerative capability via a cell intrinsic mechanism that is not directly correlated with ALS onset. Identifying the signaling pathways and functional mechanisms involved in this regenerative response could open up novel avenues for experimental treatments for ALS patients.
Materials and Methods
Mouse colony housing and breeding
All studies involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida in accordance with NIH guidelines. Adult mice were housed one to five per cage and maintained on ad libitum food and water, with a 12 h light/dark cycle. Transgenic mouse strains SOD1-G93A and SOD1-G93A-DL (JAX stock #004435 and #002299 respectively [31] were purchased from Jackson laboratory (Bar Harbor, Maine), and bred by the Rodent Breeding Services offered by the Animal Care Services at the University of Florida. SOD1-G93A and SOD1-G93-DL colonies were maintained by breeding hemizygous mice either to wild type siblings, or to C57BL/6J inbred mice (Jax Stock # 000664). Additional transgenic mice strains used in this study were G85R-SOD1 :YFP [74] and WT-SOD1 (JAX stock #002297), both strains were generously supplied by Dr. Borchelt. The G85R-SOD1:YFP mice were maintained as heterozygotes on the FVB/NJ background and whereas the WT-SOD1 were maintained on a C57Bl/6J and C3H/HeJ hybrid background. For the studies involving G85R-SOD1 :YFP mice which were induced to develop ALS, please refer to a full description of the procedure by Ayers et al [5].In addition to the adult mice, we also used timed-pregnant C57BL/6J and SOD1-G93A-DL mice at gestational day 14, which were also generated by the Rodent Breeding Services offered by the Animal Care Services at the University of Florida.
Colony maintenance genotyping for all strains was performed as previously described [31, 73]. Furthermore, to control for possible transgene copy loss due to meiotic rearrangement, breeders were regularly screened by RT-PCR as previously described [33] and replaced with fresh founder stocks from Jackson laboratory (Bar Harbor, Maine) every 5 generations. In our colony SOD1-G93A and SOD1-G93-DL mice reached late disease stage at 150-180 days and 240-330 days of age respectively.
Assessment of ALS disease progression
Mice were considered symptomatic if they displayed a 15% loss of bodyweight or showed signs of leg paralysis, whichever was reached first. In our hands, the majority of mice (~70%) were euthanized because of leg paralysis, and the rest due to decreased body weight. All mice were euthanized by CO2 inhalation following the guidelines provided by the University of Florida Animal Care Services (ACS) and approved by the Institutional Animal Care and Use Committee (IACUC).
Study design
To control for gender differences in disease progression and phenotype of SOD1-G93A mice, symptomatic adult G93A and G93A-DL mice were always paired with non-transgenic (NTg) mice of the same gender and of similar age for each experiment, in most cases using littermates. Age and gender matching also allowed us to control for batch differences in the conditioned medium used to culture adult MN as described below under “cell culture conditions”.
Adult and embryonic mouse spinal cord isolation
Embryo spinal cords were obtained from timed pregnant G93A-DL and C57BL/6J mice at embryonic day 14 as previously described in detail [7]. Once embryos were removed from the uterus, spinal cords were extracted under sterile conditions in a laminar flow hood with the aid of a dissecting microscope (SMZ800, NIKON INSTRUMENTS INC.) and small forceps and placed into cold Leibovitz’s L-15 medium (Life Technologies, Grand Island, NY) supplemented with 25 μg ml−1 penicillin-streptomycin (Life Technologies). The meninges and dorsal root ganglia (DRG) were peeled off and individual spinal cords were transferred into a 12 wells plate, identified and kept in cold L-15 medium on ice. Tails from each embryo were also harvested at this point for genotyping (G93A-DL mice).
Adult spinal cords were isolated by cutting the vertebrate column with scissors in front of the back legs and just below the medulla oblongata and flushed out of the spinal column using a syringe filled with cold supplemented DMEM/F12-medium with 18G needle (BD Biosciences). The DMEM/F12-medium used for this purpose consisted of DMEM/F12 in a 3:1 ratio supplemented with 36.54 mM NaHCO3 (Fisher Scientific), 0.18 mM L-adenine (Sigma), 312.5 μl L−1 2N HCL (Fisher Scientific), 10% of fetal calf serum (Hyclone, GE Healthcare Life Sciences, South Logan, Utah) and 25 μg ml−1 of penicillin-streptomycin (Life Technologies). The adult spinal cords were transferred into cold DMEM/F12-medium.
MN cell extraction and separation
Both embryonic and adult motor neurons were extracted using the method and reagents described in detail by Beaudet et al. [7] with a few modifications. Briefly, individual spinal cords were cut into small pieces and incubated for 30 min at 37°C in digestion buffer consisting of Dulbecco’s PBS (DPBS, Life Technologies, Grand Island, NY) containing 10 U/ml−1 papain (Worthington, Lakewood, NJ, USA), 200 μg/ml−1 L-cysteine (Sigma St. Louis, MO) and 250 U/ml−1 DNase (Sigma, St. Louis, MO). The digestion buffer was then removed and replaced with DPBS containing 8 mg/ml Ovomucoid trypsin inhibitor (Sigma), 8 mg/ml bovine serum albumin (BSA, Sigma), and 250 U/ml DNase. The tissue was then triturated using glass pipettes to obtain a single-cell suspension This step was repeated trice before all cells were collected and filtered through a 40 μm cell strainer (BD Falcon) and centrifuged at 280 g for 10 min at 4 °C for MN. Adult mixed motor neuron cultures were ready to plate after this step. Embryonic MN pellets were enriched by resuspending in 6 ml of cold Leibovitz’s L-15 medium (Life Technologies) and laid over a 1.06 g ml-1 Nycoprep density solution (Axis-Shield, Dundee, Scotland) and spun at 900 g for 20 min at 4 °C without brake in a swinging bucket centrifuge (Eppendorf, Hauppauge, NY). MN were collected at the interface of the Nycoprep solution and poured in a new 50 ml collection tube which was then filled with cold L-15. MN cells were counted at this step. MN collecting tubes were centrifuged at 425 g for 10 min in a swinging bucket centrifuge at 4°C.
Cell culture
Embryonic MN pellets were gently resuspended at 200,000 cells/cm2 in freshly prepared Motor Neuron Growth Medium (MNGM), which is described in detail by Graber DJ et al [27]. Briefly, the MNGM consists of Neurobasal A medium (NB-medium, Life Technologies) supplemented with 1X B-27 Serum-Free Supplement (Gibco/Life Technologies), 1X SATO supplement, 5 μg mL−1 Insulin (Gibco/Life Technologies), 1 mM Sodium pyruvate (Gibco/Life Technologies), 2 mM L-Glutamine (Gibco/Life Technologies), 40 ng mL−1 of 3,3,5-triiodo-L-thyronine sodium salt (T3; Sigma-Aldrich), 1 μg mL−1 Mouse laminin (Gibco/Life Technologies), 417 ng mL−1 Forskolin (Sigma-Aldrich), 5 μg mL−1 N-acetyl-L-cysteine (NAC, Sigma-Aldrich) and 1x Penicillin-streptomycin (Gibco/Life Technologies). After filter-sterilization using a 22 μm syringe filter, 10 ng mL−1 of each of the following growth factors was added to the medium: brain-derived neurotrophic factor (BDNF; Sigma-Aldrich), ciliary neurotrophic factor (CNTF; Peprotech, Rocky Hill, NJ) and glial-derived neurotrophic factor (GDNF; Peprotech). Embryonic MNs were either seeded on to Poly-D-lysine (PDL) coated 6cm tissue culture plates (10 μg mL−1 PDL, Sigma-Aldrich) to generate conditioned medium used for adult MN cultures, or on to 1.5 cm glass coverslips pre-coated first with PDL (10 μg mL−1 for 1h at RT) then with Human Placental Laminin for 3 h at 37°C (1.67 μg mL−1 laminin in NB-Medium, Sigma-Aldrich). Embryonic motor neurons grown on coverslips were cultured for 3 days prior to fixation in 4% PFA and immunostaining for imaging and growth analysis.
Adult mixed motor neuron cultures were seeded onto PDL coated cover slips (10 μg mL−1) and cultured in MNGM which had been pre-conditioned for 4 days by embryonic motor neurons isolated from NTg C57BL/6J mice. Given that adult MN pallets contain considerable amount of debris when first plated, cells were not counted prior to seeding. After the cells were allowed to attach to the coverslips in a humidified 37°C incubator for 1 h, they were washed twice with warm NB-medium to remove debris and cultured in 1 ml of conditioned MNGM mixed 1:1 with freshly prepared MNGM. Adult MNs were cultured for 2 days prior to fixation and immunostaining for imaging and growth analysis. MN’s where selected for analysis based on their expression of Tuj1, their large size, multipolarity, and stellate cell shape.
Immunofluorescence
Cells were fixed with 4% electron microscopy grade paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA) for 10 min at RT, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 3 min, and washed twice with 1X DPBS. Cells were stained overnight at 4°C with primary antibodies diluted in immunofluorescence staining buffer. They were then washed twice with DPBS for 5 min, incubated with secondary antibodies (diluted 1:1000) for 1 hr at room temperature in immunofluorescence staining buffer. F-actin was stained with Phalloidin-568 (diluted 1:100, Life Technologies) for 30 min at room temperature in immunofluorescence staining buffer. Finally, cells were washed three times with DPBS before mounting with Prolong Diamond W/ DAPI (Life Technologies). We used the following antibodies/stains: Mouse anti-β3 Tubulin (TUJ1 1:500 dilution, Covance, Princeton, NJ), Goat anti-ChAT ( 1:1000, AB144, ED Millipore), Mouse anti-Tau (1:25000, gifted from the Giasson lab [65]) Alexa Fluor™ Phalloidin 568, anti-mouse IgG 488 and anti-rabbit IgG 488 (Life Technologies) used at 1:1000.
Imaging
Imaging of IF stained motor neurons was performed on a Nikon A1R+ laser scanning confocal microscope with 40X 1.3 NA and 60X 1.49 NA objectives and the GaAsP multi-detector unit. Imaging of cells for outgrowth and branching pattern analysis was done using the EVOS XL digital inverted microscope (Life Technologies).
Image analysis
Neurite growth cone analysis
Confocal z-stacks were converted into a single maximum intensity projection image. Terminal neurite growth cone size, filopodia length, and filopodia number were analyzed using Fiji (ImageJ) software. Filopodia length was defined as the distance between lamellipodium edge to the furthest end of the extending filopodia. Values were exported into Microsoft Excel and Graphpad Prism for statistical analysis.
Neurite tracing and branching analysis
Images were taken on the EVOS XL digital inverted microscope and imported into Fiji (ImageJ) software. All visible projections in these images were traced using the Simple Neurite Tracer plugin [43]. An image stack was created from the tracing which was then analyzed using the Sholl analysis plugin [23]. To compare the relative change in neuron radius between NTg and SOD1 mutants across different experiments each set was normalized to the average radius of the age matched NTg control group. The Sholl profile containing the number of branches per given distance from soma and overall neuron radius was then exported to Microsoft Excel or Graphpad Prism for analysis.
Axon tracing and analysis
Staining of motor neuron cultures were performed as previously described with a mouse-anti Tau antibody. Imaging was performed on the EVOS XL digital inverted microscope and the images were imported into Fiji (ImageJ) software. The 16-bit color lookup table was applied to each image to visualize the intensity of Tau-staining in the neurites. The neurite that was most intensely stained for Tau was then measured from the center of the soma to determine if it was the longest projection from the cell.
Adeno-associated virus (AAV) mediated overexpression of mutant SOD1 in adult MNs
MNs from adult (9-12 months) NTg mice were isolated and plated as described above. On the day they were plated, 20 μl of AAV2/8 (titer 1×109) expressing WT SOD1-YFP, G93A SOD1-YFP and GFP only was added to the wells and the cells were cultured for 10 days (growth medium was refreshed every 5 days). For these experiments we used a self-complementary virus (scAAV), driven by the chick beta actin promotor (CBA) as described in [60]. After 10 days in culture, cells were fixed, stained for Tuj1 and imaged/analyzed as described below.
Results and Discussion
ALS-resistant G93A motor neurons have enhanced neurite outgrowth
Using a well characterized protocol for adult motor neuron extraction [7], we isolated MNs from whole spinal cords from SOD1G93A high copy number mice (referred to as G93A) at late disease stage (defined by hind leg paralysis/weight loss), occurring between postnatal days 150 and 180. Cells were cultured for two days in vitro and immunostained for β3 tubulin (Tuj1) and Choline Acetyltransferase (ChAT) to confirm our success in isolating MNs (Supplemental Fig. 1) as described by others [7]. For imaging and analysis purposes, we stained the cells with Tuj1 and phalloidin (for actin visualization) and selected MNs based on their large soma size, multi-polarity and Tuj1 positive staining.
Importantly, MN cultures from ALS mouse models were always made in parallel with MN cultures from sex- and age-matched, non-transgenic littermates (NTg) to control for experimental variability. During cell isolation, all established neuronal projections are severed, thus this assay is a direct measurement of the cells’ ability to generate new processes. We performed a large-scale quantitative analysis of MN size and branching complexity by tracing the neurons and performing Sholl analysis. Interestingly, MNs from G93A mice displayed substantially increased outgrowth in comparison to NTg MNs, both in neurite length (~40% longer than NTg cells) and number of branches per cell (Fig. 1a-d). While it is known that ALS-resistant MNs have altered RNA expression profiles, including some genes involved in cytoskeletal pathways, the genetic changes observed are usually associated with the negative regulation of outgrowth [56]. Hence, we were surprised to discover that ALS-resistant MNs actually have an enhanced capacity for outgrowth following neurite severing.
While the G93A mouse is the most extensively studied rodent model of ALS, there is some concern about its use given that such a high copy number of the transgene is required to generate the rapid onset of ALS symptoms. This is in contrast to human SOD1-based ALS, where individuals expressing 1 copy of mutant SOD1 at endogenous levels can develop the disease. To determine if the enhanced outgrowth of G93A MNs could occur with a more physiologically relevant level of expression, we cultured cells from another mouse model, SOD1G93A-DL (referred to as G93A-DL), which only has 6-8 transgene copies per cell, hence referred to as dilute (DL) [1]. These mice still get ALS, but disease progression is slower, with a delayed end-stage occurring when the mice are between 8-11 months of age. Surprisingly, MNs isolated from end-stage G93A-DL mice exhibited an even greater outgrowth capacity over age-matched NTg MNs than that observed for the G93A MNs. Compared to controls, G93A-DL exhibit a ~55% median outgrowth length increase (Fig. 1f, g) and had more than twice the number of branches >40 μm away from the cell body (Fig. 1f, h), with a continuous increase to more than 3 times as many branches at 70μm away from the cell body (Fig. 1f). Thus, the enhanced outgrowth phenotype was still present in motor neurons from a G93A mouse with more moderate overexpression of the transgene.
To determine if the increased outgrowth of neurites also meant an increase in axonal regeneration, we stained the cells with anti-Tau to distinguish axons from dendrites. Based on having the highest Tau fluorescence intensity, we determined that 63% of the cells analyzed had a process that could be identified as the axon. Of these cells, the neurite with the highest Tau fluorescence was also the longest process 79% of the time (Supplemental Fig. 2). This implies that, in addition to the observed increase in dendritic arborization as defined by the number of processes, the enhanced increase in overall cell radius (reported as length) corresponds to enhanced axonal regeneration.
The G93A mutation preserves the enzymatic activity of SOD1, which is to remove superoxide radicals. Reactive oxygen species (ROS) such as superoxide (O2·-, hydrogen peroxide (H2O2) and the hydroxyl radical (OH−) are used by neurons in intracellular signaling events during apoptosis, differentiation, and cell migration [46, 68]. Hence, a potential explanation for the enhanced outgrowth seen in ALS-resistant MNs from G93A and G93A-DL mice might be that the neurons have altered redox signaling that promotes neurite extension. To test for this, we cultured MNs from heterozygote mice overexpressing wild-type human SOD1 (referred to as WT-SOD1). Additionally, WT-SOD1 mice do not get ALS [24, 29], which reduces the possibility of an activated MN stress response. Relative to age-matched NTg control MNs, WT-SOD1 MNs actually exhibited a slight decrease in outgrowth and branching (Fig. 1i-l). This is consistent with previous findings where ROS depletion in neurons has been shown to have negative effects on neurite outgrowth [49]. Therefore, it is unlikely that the enhanced outgrowth of G93A MNs is attributed to increasing the level of enzymatically active SOD1.
Another potential explanation for the enhanced outgrowth phenomenon we observed is that ALS-resistant G93A cells grow bigger because they are actually larger cells to begin with. This notion is contradictory to published in vivo studies of spinal cord tissue from patients and mouse models with ALS, where surviving MNs have significantly reduced soma size due to the small γ fusimotor neurons (γ-MNs) being selectively disease resistant [40]. However, since no previous studies have characterized the size of cultured ALS-resistant MNs from adult mice, we sought to determine if the cells that generate the longest neurites were also the largest cells. We measured the soma of NTg, G93A, and G93A-DL MNs to determine if neurite growth correlated with the size of the cell body.
While the mean soma area did increase significantly in G93A and G93A-DL MNs compared to NTg cells, we found that within each group, soma area was not correlated with neurite length and could therefore not be used to predict outgrowth (Supplemental Fig. 3). Thus, cell size is not a potential explanation for the increased outgrowth and branching phenotype seen in G93A and G93A-DL MNs.
Mutant SOD1 MNs acquire enhanced ability to regenerate before onset of ALS symptoms
Having established that end-stage ALS-resistant MNs from both G93A and G93A-DL mouse models have an enhanced capacity for neurite outgrowth and branching, we next wanted to determine if this was a survival response to ALS-induced cell death or if the neurons acquired this phenotype before the onset of symptoms. To do so, we first cultured MNs from presymptomatic G93A-DL mice at one, two and six months of age, and measured the outgrowth and branching. We found that G93A-DL MNs from one month old mice had identical outgrowth compared to NTg cells, and then began to grow significantly larger beginning at two months of age. This trend continues as the mice age and grow closer to the onset of ALS symptoms at 9 months old (Fig. 2a, b). In comparison, MNs from 2-month-old G93A mice did not display any enhanced outgrowth capacity (Fig. 2c-e). It is interesting that a presymptomatic phenotype was only seen in cells expressing low levels of G93A, though perhaps this may reflect a difference in how the disease develops between the two mouse models: sudden onset with rapid progression for the G93A mouse and slower, more gradual progression of symptoms for the G93A-DL mouse. Alternatively, this might reflect that there is an age-dependent outgrowth response of this neuronal sub-population in the presence of SOD1G93A, and that the G93A mice die before their suriving motor neurons are old enough to have the strongest regenerative potential.
To further confirm that the enhanced outgrowth of ALS-resistant MNs is not triggered by disease onset, we cultured cells from heterozygous mice overexpressing SOD1G85R fused with yellow fluorescent protein (YFP) (referred to as G85R mice). G85R is another well-documented mutant of SOD1 where mice homozygote for the transgene develop ALS, whereas heterozygotes never develop symptoms [72]. Hence, this is a useful model for studying the effects of the mutation at low dosage in the absence of disease onset. Surprisingly we found that G85R MNs cultured from 6-month-old mice displayed a similar increase in branching and outgrowth as MNs from G93A mouse models, although the branching pattern was somewhat different, with the most significant differences (double the number of branches) occurring between 50-80 μm from the soma (Fig. 2f-h). Given that the heterozygote G85R mice do not develop ALS, the motor neurons extracted from these mice have not been subjected to selection through disease resistance and thus represent all adult MNs with SOD1 mutations, not just those resistant to ALS. Thus, the enhanced outgrowth phenotype is not directly caused by the onset of ALS. To determine if this inherent outgrowth phenotype would be further enhanced with ALS disease onset, we extracted MNs from adult G85R mice that had been injected with mutant SOD1 fibrils, which has been previously reported to induce ALS in these mice [5]. Interestingly, we found that these ALS-resistant MNs were twice as big and had twice as many branches at 40 μm from the soma compared to the un-induced G85R MNs (Fig. 2f-h). Furthermore, ALS-resistant MNs from induced G85R mice were 30-40% longer and more branched compared to NTg controls than end-stage G93A-DL MNs (Fig. 1e-h). This demonstrates that mutant SOD1 expression by itself induces enhanced MN outgrowth and that upon induction and progression of ALS, this phenotype is enhanced in disease resistant MNs. It is also of note than unlike G93A, the G85R mutant loses most of its enzymatic activity [12, 16], further confirming that the enhanced outgrowth seen in mutant SOD1 MNs is not due to altered redox signaling resulting from SOD1 enzymatic activity.
Having established that adult MNs from multiple backgrounds exhibited an enhanced outgrowth independent of ALS onset, we then sought to determine if this phenotype was innate to MNs expressing mutant SOD1 by culturing embryonic MNs. We isolated MNs from individual G93A-DL and NTg pups at E14 and measured outgrowth after 3 days in culture. No significant difference was observed in outgrowth or branching compared to NTg controls (Fig. 2i-k). This data suggests that, the enhanced outgrowth phenotype may be acquired in early in adulthood, rather than exist as an inherent trait of MNs expressing low levels of mutant SOD1. However, given that embryonic MN pools contain both ALS-resistant and ALS-vulnerable motor neurons it could reflect the opposing effects of mutant SOD1 on these different populations of MNs. Alternatively the lack of enhanced outgrowth in embryonic motor neurons might suggest a selective effect on postnatal/adult versus embryonic MNs.
Expression of SOD1G93A in non-transgenic MNs is sufficient to induce increased outgrowth
To determine if SOD1G93A expression in itself could induce enhanced outgrowth in MNs, independently from any potential priming for regeneration that may occur from the surviving MNs responding to ALS onset, we used adeno-associated virus (AAV) to overexpress G93A-YFP, WT-SOD1-YFP and GFP-only in adult MNs isolated from 9-12 month old NTg mice. Viral infections were performed the day the cells were isolated from the spinal cord and expression of the viral vectors was first detectable after 3 days. Because of the delayed expression, we let the cells grow for 10 days in culture. Expressoin of G93A-YFP induced significant outgrowth of MNs compared to WT-SOD1-YFP or GFP-alone. Furthermore, based on fluorescence intensity, we found expression levels of G93A-YFP positively correlated with outgrowth (Fig. 3d). Interestingly, although the population comparisons were not statistically significant (Fig. 3c) , expression of WT-SOD1 was correlated with decreased outgrowth (Fig. 3d), mimicking the trend seen from analyzing outgrowth of MNs isolated from the WT-SOD1 overexpressing transgenic mouse model (Figure 1i-l). In addition to demonstrating that the regenerative capacity of adult motor neurons could be increased by the expression SOD1G93A of independently of ALS or even long-term priming by cell stress, these experiments validated the findings made from comparisons between SOD1 transgenic and non-transgenic mice. The virus experiments also alleviate the concern that we were potentially selecting for different cellular subpopulations when culturing cells from different mouse models or disease states, since in these experiments, the expressed transgene was the only variable. Given that exogenous expression of SOD1G93A enhances regeneration compared to control infected cells strongly argues that this is a cell-intrinsic phenomenon of ALS-resistant motor neurons.
Enhanced outgrowth of ALS-resistant MNs is predominantly cell-intrinsic
There is considerable evidence showing that the SOD1 mutant-mediated ALS disease process is both cell and non-cell autonomous [10, 18, 74, 76]. While synthesis of mutant SOD1 within motor neurons is a primary driver of the disease, expression of mutant SOD1 by other cells such as interneurons, microglia, astrocytes, Schwan cells and T-lymphocytes substantially contribute to the progression of ALS [8–10, 17, 26, 42, 76]. Although our data showing induced outgrowth on adult MNs induced by exogenous overexpression of mutant SOD1 strongly suggests that contaminating non-neural cells do not contribute to this phenotype, our data comparing adult MN cultures from mutant SOD1 and NTg mice might be influenced by non-cell autonomous effects since the cultures contain other cells like microglia and astrocytes. Hence, it is possible that non-neuronal cells were present in different ratios in NTg vs. mutant SOD1 cultures and/or are differentially secreting factors that contribute to the increased outgrowth seen in mutant SOD1 MNs. To test this possibility, we harvested conditioned medium from 3-day-old G93A, G93A-DL, and NTg adult MN cultures and used this medium to culture NTg MNs to see if it could influence neurite out growth. While conditioned media from G93A and G93A-DL MNs did slightly increase outgrowth of NTg MNs, the median difference was only ~5% (Fig. 4 a-c) increase, or roughly 10-20 times less than the median outgrowth differences of G93A, G93A-DL, (Fig. 1a-h), or G85R MNs (Fig. 2g-i) relative to their NTg controls. These data suggest that the observed enhanced outgrowth and branching phenotype of ALS-resistant MNs is not a result of secreted factors from non-neuronal cells, but rather a predominantly intrinsic quality of these MNs. Further, since mutant SOD1 can undergo intercellular transmission [28], it is possible that these experiments reflect a response to internalized SOD1G93A secreted by the transgenic MNs and not outgrowth factors secreted by non-neuronal cells.
ALS-resistant MNs have increased growth cone size, number, and filopodia length
Previous studies have implicated that SOD1 mutations may be linked to cytoskeletal defects in MNs vulnerable to ALS [47, 75]. Furthermore, G93A has been shown to bind actin and negatively disrupt the actin cytoskeleton [67]. However, enhanced branching and neurite extension are usually associated with actin-based structures, such as the growth cone’s lamellipodia and filopodia, which are positive regulators of neurite outgrowth. To investigate whether ALS resistant G93A MNs displayed abnormalities in their actin cytoskeleton, we co-stained end-stage cells for filamentous actin (F-actin) and Tuj-1. Surprisingly, ALS-resistant MNs from G93A and G93A-DL mice had significantly larger growth cones than NTg MNs (Fig. 5). We also found that there was twice as many filopodia per growth cone and the filopodia were 50% longer in ALS-resistant MNs (Fig. 5d, e). Interestingly, this upregulation of actin structures was not present MNs from presymptomatic 2- or 6-month-old mice (Fig. 5f, g), suggesting that as a late-stage adaptation response, MNs upregulate actin-based processes that favor the formation of structures that enhance outgrowth and regeneration.
As mentioned previously, there are several published studies characterizing genetic changes in MNs from G93A mice at various stages of disease progression [19–21, 30, 52, 56, 62, 77]. However, these studies have not reached consensus regarding the underlying genetics promoting ALS resistance, probably due to the variation in experimental design and tissue sampling. For example, using G93A mice, one study found a massive up-regulation of genes involved in cell growth and/or maintenance from micro-dissected motor neurons from the lumbar spinal cord [56], whereas another study found Wnt signaling to be significantly activated when homogenized whole spinal cord was used for RNA extraction [77]. Apart from a single study showing SOD1 binding of actin [67], little is known about actin dynamics in SOD1 driven ALS, although it has been shown that upregulation of axonal guidance genes and actin cytoskeletal genes (including alpha-and beta-actin) occurs from the lumbar spinal cord of pre-symptomatic G93A mouse [20]. This could explain our results showing that well before symptoms become present, G93A MNs are actually primed for outgrowth and regeneration, at least in part due to a positive activation of the actin cytoskeleton. It is also worth noting that Lobsiger et al. found that regenerative/injury pathways genes were upregulated both in dismutase active and inactive (G37R and G85R) MNs at disease onset , including ATF3, a gene also known to be upregulated in end stage in G93A mice [11, 41, 59, 70, 71].
In addition, work by Saxena et al. demonstrated that gastrocnemius-innervating MNs (presumed to be ALS-vulnerable) have a differential upregulation of endoplamismic reticulum (ER) stress and unfolded protein response (UPR) pathways compared to soleus-innervating MNs (presumed to be ALS-resistant) [63]. Other recent studies have shown that under non-pathological conditions, activation of the UPR can promote axonal regeneration after peripheral nerve injury [54] and that axonal ER-signalling and the UPR upregulate regeneration and the formation of axonal growth cones [53]. Thus, one might speculate that the stronger ER-stress/UPR response of ALS-sensitive MNs to mutant SOD1 overwhelms the MNs and leads to cell death, whereas the more minimal response of ALS-resistant MNs allows for the pro-regenerative signaling downstream of the UPR to take precedence. While it has been speculated that upregulation of regenerative/injury pathways is merely a compensatory response to mutant SOD1G93A-dependent impairment of axonal regeneration [41, 58] we don’t believe this explains the enhanced outgrowth of ALS-resistant MNs since even heterozygote G85R-SOD1:YFP mice, which never develop ALS and therefore do not experience SOD1 mutant dependent MN degeneration, still exhibit the phenotype. Moreover, acute overexpression of mutant SOD1 in cultured adult MNs undergoing regeneration enhances outgrowth, suggesting that a mere compensation response can not soley be responsible for this phenotype. Further studies are needed to determine if the enhanced outgrowth seen in adult ALS resistant MNs is preceded by transcriptional regulation of actin cytoskeletal proteins and if so which stress signals induce this response in early pre-symptomatic stages.
Acknowledgments
This project was supported by a Pathway to Independence Award from the National Institutes of Health (R00 NS087104) to E.A.V.