The neuroprotective effects of α7 nicotinic acetylcholine receptor against mutant copper-zinc superoxide dismutase 1-mediated toxicity

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective and progressive loss of motor neurons. Although many drugs have entered clinical trials, few have shown effectiveness in the treatment of ALS. Previous studies searching for causal genes associated with familial ALS identied the copper-zinc superoxide dismutase 1 (SOD1). Other studies have shown that the stimulation of α7 nicotinic acetylcholine receptor (nAChR) can have neuroprotective effects in some models of neurodegenerative disease, as well as prevent glutamate-induced motor neuronal death. However, the effect of α7 nAChR agonists on mutant SOD1 aggregates in motor neurons remains unclear. We examined whether α7 nAChR activation had a neuroprotective effect against SOD1 G85R -induced toxicity in a cellular model for ALS. Furthermore, the mechanism was also examined by Western blot analysis and qRT-PCR.


Abstract Background
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective and progressive loss of motor neurons. Although many drugs have entered clinical trials, few have shown effectiveness in the treatment of ALS. Previous studies searching for causal genes associated with familial ALS identi ed the copper-zinc superoxide dismutase 1 (SOD1). Other studies have shown that the stimulation of α7 nicotinic acetylcholine receptor (nAChR) can have neuroprotective effects in some models of neurodegenerative disease, as well as prevent glutamate-induced motor neuronal death.
However, the effect of α7 nAChR agonists on mutant SOD1 aggregates in motor neurons remains unclear.

Methods
We examined whether α7 nAChR activation had a neuroprotective effect against SOD1 G85R -induced toxicity in a cellular model for ALS. Furthermore, the mechanism was also examined by Western blot analysis and qRT-PCR.

Results
We found that α7 nAChR activation showed signi cant neuroprotective effects against SOD1 G85R -induced toxicity via the reduction of intracellular protein aggregates. This reduction also correlated with the activation of autophagy through the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling pathway. Furthermore, the activation of α7 nAChRs was found to increase the biogenesis of lysosomes by inducing translocation of the transcription factor EB (TFEB) into the nucleus.

Conclusions
These results support the therapeutic potential of α7 nAChR activation in diseases that are characterized by SOD1 G85R aggregates, such as ALS.

Background
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurological disorder that is characterized by muscle weakness and atrophy, paralysis, and eventual death by respiratory failure. Symptoms result from the selective degeneration of upper and lower motor neurons. While 90-95% of ALS cases arise sporadically, 5-10% are familial in Japan. In previous studies searching for causal genes associated with familial ALS, copper-zinc superoxide dismutase 1 (SOD1), TAR DNA binding protein of 43 kDa (TDP43), fused in sarcoma (FUS), and optineurin (OPTN) have been identi ed as playing roles in pathological cascades and phase transitions in sporadic ALS [1][2][3][4][5][6][7]. Among familial ALS patients, mutation in SOD1 is a major autosomal dominant inherited allele associated with disease [8].
Several clinical studies suggest a role for glutamate-induced excitotoxicity in familial ALS pathology, although the potential pathogenesis in sporadic ALS is still unclear [9][10][11]. Previous experiments have shown that rat spinal cord cultures exposed to long-term low-dose glutamate show selective motor neuronal death [12][13][14]. For this reason, riluzole, a glutamate release inhibitor, has been developed as a therapeutic agent for ALS patients, and has been shown to prolong patient life by several months [15]. Although many drugs have entered into clinical trials, few have demonstrated any effectiveness in the treatment of ALS. Therefore, novel drugs from a new prospective are strongly desired in the clinical eld.
Nine α (α2 − α10) and three β (β2 − β4) nicotinic acetylcholine receptor (nAChR) subunits are expressed in the vertebrate brain [16]. These subunits coassemble to form a family of functionally diverse nAChRs. Among nAChR subunits, the most abundant subtypes in the mammalian nervous system are homomeric α7 nAChRs and heteromeric β2 nAChRs, including α4β2 nAChRs [16][17][18][19]. These receptors are also widely expressed in the dorsal and ventral horns in the spinal cord [14,[20][21][22]. Loss of nAChRs is associated with a number of disease states, including Alzheimer's disease, Parkinson's disease (PD), Lewy body disease, schizophrenia, autism, and attention de cit/hyperactivity disorder (ADHD) [23]. In addition, accumulating evidence suggests that α7 nAChRs are important targets in the development of therapeutics for PD. We have previously demonstrated that α7 nAChR activation protects against dopaminergic neuronal death in both acute and chronic animal models of PD induced by 6hydroxydopamine (6-OHDA) and rotenone, respectively [24,25].
Nagano et al. have also reported an early decrease in cholinergic input in motor neurons in the spinal cords of patients with ALS [26]. nAChRs have been implicated in neuroprotection mechanisms against acute cell stress induced by excitotoxicity, namely overactivation of glutamate receptors [27]. The stimulation of α7 nAChRs by nicotine actually prevented glutamate-induced motor neuronal death [14]. Therefore, α7 nAChRs represent an important therapeutic target for ALS as well as for PD. Recently, a candidate ALS drug ropinirole (a dopamine D 2 and D 3 receptor agonist used as an antiparkinsonian drug) has been discovered in drug screenings using ALS patient-derived iPS cells, and is now being evaluated in clinical trials for effectiveness [28]. Although the mechanistic details are still unknown, there may be commonalities in the pathological mechanisms of PD and ALS. However, the effect of α7 nAChR agonists against mutant SOD1 aggregates in neurons remains unclear. A pathological hallmark of ALS is the presence of cytoplasmic inclusions or protein aggregates in affected motor neurons, suggesting that impairment of protein degradation may play a role in the disease pathology [29]. SOD1 aggregates are present in both sporadic and familial ALS [30,31]. In addition, we found that most wild-type SOD1 proteins assume misfolded conformations in cerebrospinal uid of ALS patients regardless of SOD1 mutation status [32]. Thus, removal of SOD1 aggregates may be a potential therapeutic approach for ALS treatment. Currently, more than 180 types of SOD1 pathogenic mutations have been identi ed in ALS patients [33]. Among those, the pathogenic SOD1 G85R mutation has been most frequently studied [34,35,36,37].
In the present study, we examined whether α7 nAChR activation exhibited neuroprotective effects against SOD1 G85R -induced neurotoxicity in a cellular model of ALS.

Material And Methods
Plasmid, cell culture, and transfection Expression plasmids (pmCherry-N1, Clontech Laboratories Inc., CA, USA) harboring human SOD1 (Wildtype (SOD1 WT ) or mutant (SOD1 G85R )) were prepared as previously reported [36,37] for 24 h. The number of live cells was estimated by Cell Counting Kit-8, following the manufacturer's instructions (Wako Pure Chemical Industries Ltd.). Brie y, the reagent was added into the wells and the plate was incubated at 37 °C for 3 h. Cell viability was calculated through the detection of the optical density of formazan at 450 nm using GloMax® (Promega). A 600 nm wavelength was used as reference.

Aggregation rate analysis
We performed aggregation assay based on previous studies [36,37]. Brie y, after 24 h of mCherry-fused SOD1 WT or SOD1 G85R vector transfection, the cells were treated with PNU282987 in the presence or absence of 20 µM MC or 20 µM CQ for 24 h. Subsequently, the cells were washed twice with PBS for 5 min and xed with 4% paraformaldehyde for 15 min. Fluorescent microscopy images were acquired with a confocal uorescence microscope (LSM700, Carl Zeiss). For counting the number of aggregates, we used IN Cell Investigator 2200 (GE Healthcare). In each experiment, at least 3000 cells were counted.

RNA preparation and qRT-PCR
Reverse transcription was performed using the ReverTra Ace qPCR RT Master Mix, in accordance with the manufacturer's instructions (TOYOBO). qRT-PCR was performed using SYBR Green on a StepOne Real-Time PCR System, in accordance with the manufacturer's instructions (Life Technologies). The sequences of gene-speci c primer sets are shown in Table 1. The expression levels of mRNA were normalized to the expression levels of β-actin mRNA.

Immunostaining
At 24 h after transfection of each vector into N2a cells, the cells were treated with 1 µM PNU298987 in the presence or absence 1 µM STO609 or 1 µM 1,2-bis (2-aminophenoxy) ethane-N,N,N0,N0-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM). At 24 h after the treatment, cells were xed by 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 diluted in PBS. To block the reaction, the cells were treated with 2% goat serum for 60 min. The cells were incubated with rabbit polyclonal antibody against TFEB (1:200, Proteintech) at 4 °C for overnight. Subsequently, the cells were incubated with secondary antibody (goat anti-rabbit antibody Alexa 488) at room temperature for 1 h. In addition, nuclear staining was performed with Hoechst 33342 (Molecular Probes). Fluorescent microscopy images were acquired with a confocal uorescence microscope (LSM700, Carl Zeiss).

Lysosomal staining
N2a cells were seeded at 1.0 × 10 5 cells/ml in CELLview™ cell culture dishes (Greiner Bio-one). At 24 h after transfection to N2a cells each vector, the cells were treated with 1 µM PNU298987 in the presence or absence 1 µM STO609 or 1 µM BAPTA-AM. At 24 h after the treatment, lysosomes were stained with LysoTracker™ Green DND-26 (Thermo Fisher Scienti c) and nucelei were stained with Hoechst 33342 (Thermo Fisher Scienti c), in accordance with the manufacturer's instructions (Thermo Fisher Scienti c).
Fluorescent microscopy images were acquired with a confocal uorescence microscope (LSM700, Carl Zeiss). Image analysis was performed using ImageJ.

Cell fractions preparation
At 24 h after transfection of each vector into N2a cells, the cells were treated with 1 µM PNU298987 in the presence or absence 1 µM STO609 or 1 µM BAPTA-AM. At 24 h after the treatment, 1.0 × 10 6 cells were harvested with trypsin-EDTA and nuclear extraction was performed using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents, in accordance with the manufacturer's instructions (Thermo Fisher Scienti c). To assess the purity of the fractionation, the cytoplasmic and nuclear fractions were con rmed by immunoblotting using anti-GAPDH (1:1000, MBL Ltd.) as a cytoplasmic marker and anti-Histone H3 (1:1000, Cell Signaling Technology Ltd.) as a nuclear marker.

Statistical analysis
Data are given as the mean ± standard error of the mean (SEM). Signi cance was determined using the Student's t test or analysis of variance. Further statistical analysis for post hoc comparisons was performed using the Bonferroni/Dunn test (StatView, Abacus). p-values of less than 0.05 were considered to be statistically signi cant.

Results
The α7 nAChR agonist reduces intracellular SOD1 G85R aggregates and prevents neurotoxicity of SOD1 G85R Previously, aggregate formation was con rmed in mCherry-fused SOD1 G85R (hereafter SOD1 G85R )transfected N2a cells, which contributed to cell death via the formation of triton X-100-insoluble aggregates [36][37][38][39]. To investigate the effect of α7 nAChR activation on SOD1 G85R aggregate formation, we evaluated the number of intracellular aggregates with an Imaging Cytometer after treatment with PNU282987, an α7 nAChR selective agonist (Fig. 1). SOD1 G85R formed intracellular aggregates in approximately 25% of transfected N2a cells. PNU282987 treatment signi cantly reduced the percentage of cells with intracellular SOD1 G85R aggregates (Fig. 1A,B). This reduction in aggregate formation was markedly blocked by methyllycaconitine (MC), an α7 nAChR antagonist (Fig. 1A, B). In order to examine the effect of PNU282987 on SOD1 G85R -mediated neurotoxicity, we performed a MTT assay in differentiated N2a cells (Fig. 1C). Although transfection of SOD1 WT did not affect cell survival, transfection of SOD1 G85R severely induced cell death. PNU282987 prevented SOD1 G85R -induced cell death; in addition, methyllycaconitine signi cantly inhibited the PNU282987-induced neuroprotection (Fig. 1C). These results suggest that α7 nAChR activation exerts signi cant neuroprotective effects against SOD1 G85R -induced toxicity via the reduction of intracellular protein aggregates.
The α7 nAChR agonist exerts neuroprotective effects against SOD1 G85R aggregation via activation of autophagy To analyze the mechanism of the reduction of intracellular aggregates upon α7 nAChR activation, we focused on autophagy, a prominent protein degradation pathway. Western blot analysis showed that PNU282987 treatment increased the formation of LC3-II, a marker of autophagy, in N2a cells ( Fig. 2A).
PNU282987 treatment also signi cantly decreased p62 protein levels, a marker of selective autophagy (Fig. 2B). In addition, chloroquine (CQ), an inhibitor of autophagy, prevented the reduction of cytoplasmic aggregation of SOD1 G85R induced by PNU282987 (Fig. 2C, D). To further investigate whether the neuroprotective effects of PNU282987 were associated with autophagy, we performed a MTT assay in the presence of chloroquine. The protective effect of PNU282987 was signi cantly inhibited by chloroquine treatment (Fig. 2E). These data suggest that PNU282987 treatment reduces the quantity of subcellular aggregates via the upregulation of autophagy, which prevented SOD1 G85R -associated neurotoxicity.
α7 nAChR activation promotes autophagy via Ca 2+ in ux and the AMP-activated protein kinase (AMPK)mammalian target of rapamycin (mTOR) signaling pathway α7 nAChR activation increases intracellular Ca 2+ in ux through the α7 receptor channel. It is wellestablished that Ca 2+ in ux is a regulator of autophagy [40]. To elucidate the pathways involved in elevated Ca 2+ in ux after α7 nAChR activation, we used the cell-permeable cytosolic Ca 2+ chelator BAPTA-AM. Pre-treatment with BAPTA-AM signi cantly inhibited the formation of LC3-II and the reduction in p62 protein induced by PNU282987 (Fig. 3A-C). CaMKKβ has been proposed as a potential target of cytosolic Ca 2+ signaling [41], although direct evidence remains elusive. Similar to the effects of BAPTA-AM, the formation of LC3-II and the reduction in p62 induced by PNU282987 were signi cantly inhbited by STO609, a CaMKKβ-speci c inhibitor (Figs. 3A-C).
The AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling pathway is downstream of Ca 2+ signaling and plays an important role in the regulation of autophagy in response to different stress conditions [42]. To further identify the signal transduction pathway mediated by PNU282987, we investigated the AMPK-mTOR pathway. PNU282987 signi cantly increased AMPK phosphorylation and signi cantly inhibited mTOR phosphorylation (Figs. 3A, D-E). In addition, BAPTA-AM and STO609 pre-treatment signi cantly decreased PNU282987-dependent AMPK phosphorylation and restored mTOR phosphorylation (Fig. 3A, D-E), suggesting activation of the AMPK pathway and inhibition of the mTOR pathway occurs via Ca 2+ in ux following α7 nAChR activation. These results suggest that α7 nAChR activation potentially induces autophagy via Ca 2+ in ux and signaling through the AMPK and mTOR pathways. α7 nAChR agonist promotes lysosomal biogenesis via nuclear translocation of transcription factor EB (TFEB) AMPK signaling activates transcription factor EB (TFEB), which is a potential key transcription in the induction of autophagy upon its dephosphorylation and nuclear translocation [43]. TFEB can enter the nucleus and bind to the E-box of the CLEAR element, which regulates the transcription of genes associated with the biogenesis of lysosomes and autophagosomes [44]. We rst analyzed mRNA levels of lysosomes and autophagy-related genes whose transcription could be regulated by TFEB using qRT-PCR. The mRNA levels of lysosomal-related genes (such as Lamp1, Lamp2, Npc1, and Tpp1) and autophagy-related genes (such as Becn1, Map1lc3b, Sqstm1, and Uvrag) were signi cantly up-regulated in response to PNU282987 treatment (Fig. 4). Upregulation of these mRNAs was markedly blocked by pretreatment with BAPTA-AM and STO609 (Fig. 4).
Next, we examined the distribution of TFEB in the SOD1 G85R -transfected N2a cells after PNU282987 treatment using immuno uorescence (Fig. 5A). TFEB levels in the nucleus were increased following PNU282987 treatment. Meanwhile, TFEB levels in the cytoplasm were decreased by PNU282987 (Fig. 5A). Western blot also demonstrated that PNU282987 treatment decreased and increased TFEB levels in cytoplasmic and nuclear fractions, respectively (Fig. 5B, C). In addition, nuclear translocation of TFEB induced by PNU282987 was signi cantly inhibited following pre-treatment with BAPTA-AM and STO609 (Fig. 5B, C). We next determined whether PNU282987 enhances lysosomal biogenesis using immuno uorescence (Fig. 5D, E). PNU282987 promoted lysosomal biogenesis as quanti ed by uorescence intensity in LysoTracker-stained cells. The effect of PNU282987 on lysosomal biogenesis was inhibited by pre-treatment with BAPTA-AM and STO609 (Fig. 5D, E). Together, these data suggest that α7 nAChRs activation promotes lysosomal biogenesis via the nuclear transposition of TFEB.

Discussion
The goal of the present study was to determine whether activation of α7 nAChR had a neuroprotective effect against SOD1 G85R aggregate-induced toxicity in a cellular model of ALS. We demonstrated that PNU282987, an α7 nAChR selective agonist, induces signi cant neuroprotective effects via reduction of SOD1 G85R intracellular aggregates. This reduction strongly correlated with the activation of autophagy via the AMPK-mTOR signaling pathway. Furthermore, PNU282987 increased lysosome biogenesis by promoting TFEB translocation into the nucleus. These ndings identify α7 nAChR as a novel neuroprotective target against the SOD1 G85R aggregate-mediated neurotoxicity.
Autophagy dysfunction has been implicated in various neurodegenerative diseases, including ALS [45,46]. The notion that autophagy dysfunction contributes to ALS pathogenesis is strongly supported by the identi cation of numerous genes associated with familial ALS also involved in regulation of autophagy, including SQSTM1 [47], OPTN [4], TBK1 [48], and VCP [49]. The presence of intracellular, insoluble inclusions composed of misfolded proteins is a hallmark of ALS pathology [29]. Therefore, removal of SOD1 aggregates may represent one potential therapeutic approach for ALS treatment.
There are two major pathways for cellular protein degradation: the ubiquitin proteasome system (UPS), and autophagy. Autophagy has been shown to degrade both soluble and aggregated protein substrates that are too large to pass through the pore of the proteasome, such as the toxic SOD1 protein aggregates, while UPS primarily degrades soluble SOD1, suggesting that autophagy regulation is critical for improving ALS pathology [46,50].
Treatment of mutant SOD1 transgenic mice with trehalose resulted in increased life span, improved neuronal survival, reduced astrogliosis, and delayed disease onset via activation of autophagy [51]. Similarly, carbamazepine treatment activated autophagy via the AMPK-ULK1 signaling pathway and promoted the clearance of mutant SOD1 aggregates. Carbamazepine treatment also delayed disease onset and extended life span of SOD1 G93A mice [52]. In our previous studies, autophagy induction has demonstrated bene cial effects in cells harboring pathogenic SOD1 mutations [36,37,53]. In addition to pharmacological studies, genetic ablation of XBP-1 (X-box-binding protein) in motor neurons of SOD1 G85R mice enhanced the clearance of mutant SOD1 aggregates and increased survival via activation of autophagy [54]. Moreover, bosutinib, which boosts autophagy, can improve the survival of iPSC-derived motor neurons from patients with familial ALS caused by mutations in SOD1 [55].
Conversely, abnormalities (activations) in autophagy have been observed in numerous neurodegenerative diseases, including ALS [56]. Pharmacological and genetic modulation of autophagy may result in diverse and even detrimental outcomes to the survival of ALS models; interventions targeting genes including mSOD1, FUS and TDP-43 [46,50] have shown that it may be necessary to jointly consider the speci c effects of each individual mutation, pathology, and possibly other context-dependent in uences. These results suggest the need for developing autophagy inducers with higher speci city and lower cytotoxicity based on ALS pathology [50]. In the present study, PNU282987 exerted neuroprotective effects against SOD1 G85R -induced toxicity via autophagy activation. Although it is necessary to explore this nding further with iPS cells and animal models, among candidates of autophagy inducers, α7 nAChR may be a promising candidate.
Our study indicates that PNU282987 decreased mTOR phosphorylation and increased AMPK phosphorylation, and subsequently induced autophagy. The AMPK-mTOR signaling pathway is a downstream target of Ca 2+ signaling and plays an important role in the regulation of autophagy in response to different stresses [42]. In support of this, we showed that AMPK and mTOR phosphorylation were signi cantly affected by pre-treatment with BAPTA-AM and STO609, indicating activation of the AMPK pathway and inhibition of the mTOR pathway via Ca 2+ in ux following α7 nAChR activation. In addition, AMPK phosphorylation activates TFEB, which is a potential key transcription for autophagy induction upon its dephosphorylation and nuclear translocation [43]. It has been reported that under stress conditions or upon loss of function, TDP43 can regulate the nuclear translocation of TFEB in order to promote the transcription of autophagic genes. This indicates that TFEB may play a role in potential strategies for ALS treatment. In the present study, PNU282987 signi cantly increased the mRNA levels of Lamp1, Lamp2, Npc1, Tpp1, Becn1, Map1lc3b, Sqstm1, and Uvrag the transcription of which could be regulated by TFEB. In addition, PNU282987 signi cantly increased the TFEB translocation into the nucleus and promoted lysosomal biogenesis. Previously, trehalose, an enhancer of mTOR-independent autophagy, was shown to delay ALS onset and reduce motor neuron loss in SOD1 G93A mice [57]. In contrast, mTOR-dependent activation of autophagy resulted in loss of motor neurons and reduced survival in the same ALS mouse model, which may be due to other physiological functions of mTOR inhibition [58]. The neuroprotective effects of α7 nAChR activation may be due to pleiotropic effects, it will be necessary to conduct further studies.
ALS is a multifactorial disease encompassing a network of cellular pathways [59]. Drugs with pleiotropic effects may be practically more effective than drugs with a single effect for patients with ALS. As α7 nAChR activation has various neuroprotective effects including autophagy activation, α7 nAChR activation may possess novel therapeutic potential for ALS.

Conclusion
Stimulation of α7 nAChR showed a neuroprotective effect against SOD1 G85R aggregate-induced toxicity in a cellular model of ALS. It was suggested that this effect is due to activation of autophagy by AMPK-mTOR pathway and TFEB translocation into the nucleus. These results support the therapeutic potential of α7 nAChR activation in diseases that are characterized by SOD1 G85R aggregates, such as ALS.

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The authors declare that they have no competing interests.

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