Introduction
Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disease, is characterized by the progressive loss of motor neurons. Death typically occurs within 2–5 years of onset, usually due to respiratory failure [
1] and currently there is no efficacious therapy available [
2]. ALS is associated with mutations in a wide number of genes, with
C9ORF72 and
SOD1 mutations being the most common [
3]. However, the mechanisms by which these mutations cause ALS remain elusive.
ALS is a heterogeneous condition, and defects in several molecular pathways have been identified, including oxidative stress [
4], impaired axonal transport [
5], glutamate excitotoxicity [
6] and the secretion of toxic factors by non-neuronal cells [
7]. In addition to neuronal degeneration, accumulation of misfolded ubiquitinated protein aggregates is a hallmark of most forms of ALS [
8]. The compositions of these aggregates varies between ALS cases, but often comprise proteins known to be causative of ALS, including TDP-43, SOD1, p62, FUS and OPTN1, as well as dipeptide repeats generated due to a repeat expansion in the
C9ORF72 gene [
3]. This suggests that impaired proteostasis may be central to the pathogenesis of ALS.
Autophagy is a cellular mechanism required for the degradation of long-lived and misfolded proteins. Autophagy is negatively regulated by mTOR, which phosphorylates p70S6K, in turn dephosphorylating ULK1, a component of the autophagy initiation complex [
9]. Under certain conditions, including nutrient starvation or pharmacological treatment, mTOR may be inhibited, resulting in dephosphorylation of p70S6K, activation of ULK1 and initiation of autophagy. The autophagy initiation complex further comprises ATG13, ATG101 and FIP200 which together act to phosphorylate BECLIN-1 and activate VPS34 to induce autophagosome formation [
10]. A range of other autophagy related proteins (ATG) including ATG5, ATG12, ATG7, ATG10 and AGT16-L work together to promote elongation of the developing autophagosome [
11]. LC3B is a key protein involved in selective autophagy. Following conjugation of phosphatidylethanolamine to generate LC3-II, it is targeted to developing autophagosomes by interaction with ATG5-ATG12 where it subsequently recruits cargo destined for autophagic degradation into the autophagosome. An autophagy adapter protein, p62, contains a ubiquitin-binding domain in addition to an LC3B interacting domain, allowing the selective degradation of ubiquitinated proteins [
12]. Completed autophagosomes are transported along microtubules by Dynein proteins to the lysosomal rich perinuclear region where SNARE proteins regulate the fusion of autophagosome and lysosome [
13,
14]. Contents within the completed autophagosome, including p62, are degraded via lysosomal hydrolases and subsequently recycled back into the cytoplasm.
Several studies have implicated altered autophagy in various ALS models. Decreased ULK1 mRNA in ALS patient samples suggests an impaired initiation of autophagy [
15], whereas increased LC3B and p62 accumulation indicates impaired autophagic flux [
16‐
18]. However, the strongest evidence for a role of autophagy in the pathogenesis of ALS may arise from the function of proteins encoded by many of the genes associated with the development of ALS – the majority of them may in some way function as part of the autophagy pathway. SOD1 may activate autophagy through activation of BECLIN-1 [
19]. C9ORF72 has been shown to regulate endosomal trafficking, as well as possibly interacting with LC3B [
20]. Additionally, mutations in several autophagy receptor proteins, including p62, Optineurin and Ubiquilin 2, have been identified in ALS patients [
3]. Furthermore, activation of the autophagy pathway by pharmacological means has been demonstrated to increase survival in ALS animal models [
21‐
24].
In recent years, an increasing focus has been placed on non-neuronal cells in the pathogenesis of ALS. Several studies have indicated that patient astrocytes may secrete factors which are directly toxic to motor neurons [
7,
25,
26]. However, the identities of these factors remain largely unknown.
We hypothesize that impaired autophagy may be central to the pathogenesis of ALS. Herein we established induced pluripotent stem cells (iPSCs) from ALS patients and age-matched healthy controls. We demonstrate that patient astrocyte conditioned medium (ACM) decreases the viability of motor neurons derived from both control and patient iPSCs. To investigate the mechanisms by which astrocytes may mediate cell death we cultured HEK293T cells with ACM from both control and patient iPSC-derived astrocytes. We demonstrate that cells treated with patient ACM show lower expression of LC3-II, a protein required for selective degradation of p62. We further show a concomitant accumulation of p62 puncta, suggesting impaired autophagic flux. Additionally, we demonstrate increased accumulation of SOD1, the most widely studied protein in relation to ALS pathogenesis. These results indicate that patient astrocytes mediate an imbalance in the autophagy pathway, which may result in the accumulation of ALS-related proteins, causing pathological effects.
Discussion
The majority of ALS research using animal models during the previous two decades has focused on
SOD1 transgenic mouse models. However, SOD1 mutations account for only ~2% of ALS cases [
40]. Notably, there is accumulating evidence suggesting that
SOD1 mutations may give rise to ALS cases with divergent pathology relative to other mutations. Whereas the vast majority of ALS cases present with TDP-43 inclusions, patients harboring
SOD1 mutations are characteristically devoid of these inclusions [
41]. iPSC-derived astrocytes and motor neurons offer the opportunity to investigate glial-neuronal interactions, and impairments therein, which may be attributed to a wide variety of genetic mutations and will be highly advantageous in elucidating the precise molecular mechanisms leading to the development of ALS.
This research demonstrates ALS patient ACM decreases the viability of motor neurons, consistent with previous reports [
25]. This has been recapitulated using both animal model derived cells and patient derived cells harboring
SOD1 and
C9ORF72 mutations, as well as cells from patients with unknown genetic defects [
7,
27]. This intriguing pathogenic mechanism offers the potential to identify novel therapeutic targets. However, the precise factors which induce motor neuron toxicity remain to be elucidated. Increased expression of prostaglandin D2 was identified as a possible culprit, but inhibition of this protein offered only mild improvements in motor neuron survival [
42]. More recent evidence has suggested a complex comprising α2-Na/K ATPase/α-adducin as responsible for motor neuron cell death in a mtSOD1 mouse model [
29]. Knockdown of this complex in astrocytes, or its pharmacological inhibition, is sufficient to rescue motor neurons from astrocyte mediated cell death. However, this has only been demonstrated in
SOD1 transgenic models. It remains to be seen whether this mechanism is applicable to all ALS cases.
For cellular homeostasis, a fine balance between autophagosome formation and degradation needs to be maintained. Either over production of autophagosomes or decreased degradation of autophagosomes in neuronal cells can lead to impaired proteostasis and subsequent neurodegeneration [
43]. To our knowledge, only one study has demonstrated autophagic regulation by ACM. Perucho et al. demonstrated that transfusion of ACM into an animal model of Huntington’s disease is sufficient to induce autophagy and regulate the expression of mutant huntingtin inclusions in vivo [
35].
Our research demonstrates that cells treated with ALS patient ACM demonstrate significantly decreased levels of LC3-II. LC3-II is required for the selective autophagic degradation of p62 and targeted cargo. Decreased availability of LC3-II in response to increased autophagosome formation may result in accumulation of developing autophagosomes, disrupting the balance between autophagosome formation and degradation, leading to impaired proteostasis. Indeed, we observe increased numbers of p62 puncta awaiting degradation. p62 is regularly observed to be increased in ALS models [
16], and we suggest that this phenomenon may be mediated by astrocytes.
Autophagic degradation of several ALS-related proteins, including SOD1 and TDP-43, is mediated via p62 and LC3B [
37,
38]. Although no difference in TDP-43 expression was observed, a significant increase in SOD1 protein expression was demonstrated in response to ACM. Increased SOD1 cytoplasmic aggregations are often observed in patients, both with and without mutations within the
SOD1 gene [
44]. Whether the increase in SOD1 protein expression in response to patient ACM is a precursor to the development of pathological insoluble aggregations will require further study. Additionally, it will be interesting to observe whether more prolonged treatment with patient ACM would alter the expression of other ALS-related proteins.
Activation of autophagy has been demonstrated to be beneficial to the survival of ALS animal models [
21‐
24,
45]. This research aimed to determine whether activation of autophagy, in addition to ACM treatment, would modify the accumulation of p62 puncta. Rapamycin induces autophagy via inhibition of mTOR and activation of ULK1 [
9], whereas the mechanism by which Trehalose modulates autophagy remains unclear [
46]. We show that Rapamycin treatment decreases p62 accumulation in cells treated with patient ACM. No additive effect is observed in cells treated with control conditioned medium and Rapamycin. Conversely, treatment with Trehalose has no effect on p62 accumulation in cells treated with patient ACM. Surprisingly, in cells treated with both control ACM and Trehalose, we observe a stark increase in p62 accumulation. It is unclear why this occurs, but may be due to dual activation of autophagy by ACM and Trehalose. Regulation of autophagy in the presence of patient ACM may aid in the identification of pharmacological agents with therapeutic potential.
Although Rapamycin was able to ameliorate the increased accumulation of p62 in cells treated with patient ACM, no changes in SOD1 expression were observed following the same treatment. It remains to be determined whether this increase in SOD1 expression is mediated via impaired autophagy, and whether prolonged activation of autophagic mechanisms may counter the accumulation in cells treated with patient ACM.
Several reports have suggested that astrocytes mediate cell death by the secretion of toxic factors. Our results suggest that ACM may disrupt the balance between autophagosome formation and degradation, notably by decreased expression of LC3-II. The mechanism by which these cells fail to induce, or possibly inhibit, expression of this key autophagy protein is unclear, but may represent a significant factor contributing to impaired autophagy in ALS models. Future studies will focus on further elucidating the mechanisms by which patient ACM mediates impaired autophagy, and fully establishing cellular methodologies for investigating therapeutic agents using patient and control derived cells.
Methods
Generation of iPSCs
The iPS04c3, iPS21c1, iPS21cx and iPS31c8 lines were generated using STEMCCA lentivirus reprogramming kit (SCR544, Merck Millipore). . Briefly, 2.0 × 104 fibroblasts on a γ-irradiated mouse embryonic feeder layer were transduced with the polycistronic lentivirus containing OCT4, SOX2, KLF4 and c-MYC in the presence of 5 μg/ml Polybrene in complete fibroblast medium. Transduction was repeated after 24 h. On day 3 the medium was replaced with knockout replacement medium comprised of KnockOut™ DMEM, supplemented with 10% KnockOut™ Serum Replacement (Gibco), 0.1 mM MEM Non-Essential Amino Acids Solution (Gibco), 0.1 mM β-mercaptoethanol, 1 mM L-glutamine and 10 ng/ml basic fibroblast growth factor (Peprotech, UK). Media were changed daily. Colonies with an ES-like morphology were picked after day 21 of reprogramming and expanded for further characterization.
The iPSC1cx1, iPSC3c2 and iPS24c1 generated from fibroblasts using the oriP/EBNA1-based episomal vector from the Epi5™ Episomal iPSC Reprogramming Kit, containing the OCT, SOX2, LIN28, L-MYC and KLF4 plus mp53DD with additional EBNA (Thermo Fisher, UK). Cells were transfected via nucleofection using program U-023. 5.0 × 104 fibroblasts were transfected per line and transferred to Geltrex™ coated wells. Cells were cultured in knockout replacement medium as listed above, supplemented with 100 ng/ml basic fibroblast growth factor for the following 14 days. On day 15 the medium was replaced with Essential 8™ Medium (Gibco) with daily change. Colonies appeared by day 12 and the first clones were isolated for expansion on day 21 of reprogramming.
iPSCs were maintained in Pluristem culture medium (Pluristem therapeutics) on plates coated with Geltrex (Thermo Fisher).
Cell differentiation
To differentiate cells towards motor neurons, the protocol by Du et al. was followed [
30]. For the generation of astrocytes, motor neuron progenitor cells generated using the protocol by Du et al. were cultured in DMEM/Glutamax (Life Technologies) supplemented with 10% FBS (Fisher Scientific). These cells were passaged 1:3 using trypsin/EDTA when 80% confluency was reached.
HEK 293T cell culture
HEK293T cells were maintained in DMEM/Glutamax supplemented with 10% FBS. These were routinely passaged using trypsin/EDTA at a ratio of 1:10 when ~90% confluency was reached.
Conditioned medium
To generate astrocyte conditioned medium (ACM), astrocytes were cultured to confluency at which point cells were washed three times with PBS and the medium replenished. ACM was collected after 5 days of culture, filtered through 0.45um syringe filter, and stored at −80 °C until use. For experiments of motor neuron viability, motor neuron medium was used for the preparation of ACM. For experiments on HEK293T cells, DMEM supplemented with 10% FBS was used for the preparation of conditioned medium.
Culture with ACM
To assess the effects of ACM on target cells, conditioned medium obtained from individual iPSC-astrocyte lines was cultured on cells for 5 days, then cells were fixed using 4% paraformaldehyde for immunocytochemistry analysis, or protein was harvested for western blot analysis. To assess the effects of drug treatments, cells were treated with 200nM Rapamycin (Sigma, #R8781) or 100nM Trehalose (Sigma, #T9531) for the final 48 h of co-culture.
Motor neuron viability assessment
To generate motor neurons, 106 motor neuron progenitor cells were cultured in suspension using Aggrewell 400 plates (Stem Cell Technologies) to form uniform sized motor neuron progenitor spheres. These were cultured in suspension for 6 days. After 6 days, 30 spheres were placed into each well of a 24-well plate coated with Geltrex. Neurons were allowed to grow from spheres for 4 days prior to culture in ACM. Cells were cultured with ACM for a total of 5 days, after which cells were fixed for immunocytochemistry analyses and quantification.
Immunocytochemistry
For immunocytochemistry analysis, cells were washed with PBS and fixed in 4% paraformaldehyde (Santa Cruz, sc-253236) for 15 min at room temperature. Cells were then washed twice with 0.1% Tween 20 (Sigma, P9416) followed by permeablising in 1% Triton X-100 (Sigma, T8787) for 30 min. Cells were then blocked in 10% goat serum (Sigma) with 1% BSA (Sigma, A2153) and 1% Triton X-100. After, cells were washed three times with PBS and incubated overnight with primary antibodies as follows: SSEA4, SOX2, TRA-180 and OCT4 (CST, #9656S, each 1:200 dilution), MNX1 (DSHB, 81.5C10, 1:50 dilution), TUJ1 (Sigma, T2200, 1:200), GFAP (Dako, Z033401-2, 1:200 dilution), p62 (Abcam - ab56416, 1:100). Hoechst was used to stain nuclei (BD Pharmingen, #561908). Secondary antibodies (Thermo Fisher cat: A-11001 and A-11037) were used at a dilution of 1:2000.
Western blot analysis
For western blot analysis Protein was harvested using RIPA buffer (Sigma, R0278) supplemented with protease inhibitors (Santa Cruz, sc-29131). Protein concentration was calculated using BCA kit (Thermo Fisher Cat #23225). 10ug of protein was loaded on 12% SDS polyacrylamide gels. The following primary antibodies were used: SOD1 (Sigma, HPA001401, 1:1000 dilution), TDP-43 (CST - #3448, 1:1000 dilution), ATG12 (CST, #4180, 1:1000 dilution), BECLIN-1 (CST, #3495, 1:1000 dilution), pULK1 (CST, #5869, 1:1000 dilution), LC3B (CST, #12741, 1:1000 dilution), mTOR (CST - #2972, 1:1000 dilution), ATG3 (CST - #3415, 1:1000 dilution), β-Actin (Sigma, A3854, 1:50000 dilution). Secondary antibodies were used at a dilution of 1:2000 (CST, #7074 and #7076). Blots were detected using chemiluminescence substrate (Millipore Cat# WBKLS0500).
Imaging and analyses
The Operetta imaging hardware and Harmony software (Perkin Elmer) were used to image and quantify cells/puncta. Nuclei were identified by staining with Hoechst and MNX1 (for motor neurons). After identifying cytoplasmic regions based on p62 expression, p62 puncta were identified using the spot finder tool. For the quantification of motor neurons the entirety of each well was visualized and MNX1 positive cells were quantified. For the quantification of p62 puncta a minimum of 30 fields of view were used for each condition.
Statistical analyses
All statistical analyses were performed using Graph Pad Prism. *p < 0.05 and **p < 0.01 were deemed statistically significant.
Acknowledgements
The authors acknowledge Dr. Enda O’Connell for scientific and technical assistance and the facilities of the Genomics Core at the NUI Galway.