The goal of the present study was to determine whether activation of α7 nAChR had a neuroprotective effect against SOD1G85R aggregate-induced toxicity in a cellular model of ALS. We demonstrated that PNU282987, an α7 nAChR selective agonist, induces significant neuroprotective effects via reduction of SOD1G85R 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 findings identify α7 nAChR as a novel neuroprotective target against the SOD1G85R 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 identification of numerous genes associated with familial ALS also involved in regulation of autophagy, including SQSTM1 , OPTN , TBK1 , and VCP . The presence of intracellular, insoluble inclusions composed of misfolded proteins is a hallmark of ALS pathology . 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 . 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 SOD1G93A mice . In our previous studies, autophagy induction has demonstrated beneficial 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 SOD1G85R mice enhanced the clearance of mutant SOD1 aggregates and increased survival via activation of autophagy . Moreover, bosutinib, which boosts autophagy, can improve the survival of iPSC-derived motor neurons from patients with familial ALS caused by mutations in SOD1 .
Conversely, abnormalities (activations) in autophagy have been observed in numerous neurodegenerative diseases, including ALS . 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 specific effects of each individual mutation, pathology, and possibly other context-dependent influences. These results suggest the need for developing autophagy inducers with higher specificity and lower cytotoxicity based on ALS pathology . In the present study, PNU282987 exerted neuroprotective effects against SOD1G85R-induced toxicity via autophagy activation. Although it is necessary to explore this finding 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 Ca2+ signaling and plays an important role in the regulation of autophagy in response to different stresses . In support of this, we showed that AMPK and mTOR phosphorylation were significantly affected by pre-treatment with BAPTA-AM and STO609, indicating activation of the AMPK pathway and inhibition of the mTOR pathway via Ca2+ influx 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 . 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 significantly 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 significantly 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 SOD1G93A mice . 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 . 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 . 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.