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https://www.mdpi.com/2073-4409/8/3/211/htm


Sigma-1 Receptor Activation Induces Autophagy and Increases Proteostasis Capacity In Vitro and In Vivo

Maximilian G. Christ

, Heike Huesmann

, Heike Nagel

, Andreas Kern

 and Christian Behl * 

Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Duesbergweg 6, 55128 Mainz, Germany

*

Author to whom correspondence should be addressed.

Received: 29 January 2019 / Accepted: 27 February 2019 / Published: 2 March 2019

Abstract

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Dysfunction of autophagy and disturbed protein homeostasis are linked to the pathogenesis of human neurodegenerative diseases and the modulation of autophagy as the protein clearance process has become one key pharmacological target. Due to the role of sigma-1 receptors (Sig-1R) in learning and memory, and the described pleiotropic neuroprotective effects in various experimental paradigms, Sig-1R activation is recognized as one potential approach for prevention and therapy of neurodegeneration and, interestingly, in amyotrophic lateral sclerosis associated with mutated Sig-1R, autophagy is disturbed. Here we analyzed the effects of tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine hydrochloride (ANAVEX2-73), a muscarinic receptor ligand and Sig-1R agonist, on autophagy and proteostasis. We describe, at the molecular level, for the first time, that pharmacological Sig-1R activation a) enhances the autophagic flux in human cells and in Caenorhabditis elegans and b) increases proteostasis capacity, ultimately ameliorating paralysis caused by protein aggregation in C. elegans. ANAVEX2-73 is already in clinical investigation for the treatment of Alzheimer’s disease, and the novel activities of this compound on autophagy and proteostasis described here may have consequences for the use and further development of the Sig-1R as a drug target in the future. Moreover, our study defines the Sig-1R as an upstream modulator of canonical autophagy, which may have further implications for various conditions with dysfunctional autophagy, besides neurodegeneration.

Keywords:

 sigma-1 receptor; autophagy; proteostasis; neurodegeneration; C. elegans

1. Introduction

The pathogenesis of neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease (AD, PD) as well as amyotrophic lateral sclerosis (ALS), has been linked to a disturbed protein homeostasis [1,2,3]. Therefore, the control and maintenance of proteome integrity and proteostasis is of utmost importance. Cellular proteostasis includes protein folding, protein assembly, refolding of damaged proteins, as well as protein degradation, and is under the control of a fine-tuned network of factors, including chaperones, such as heat shock protein 70 (HSP70), and distinct co-chaperones [4]. For intact function and long-term survival of the cell it is crucial to remove misfolded proteins via specialized processes; the two major cellular degradation pathways are the ubiquitin proteasome system (UPS) and autophagy [5,6,7]. The UPS is of particular importance for the physiological protein turnover, but is limited in the degradation substrates, and the autophagic-lysosomal pathway is responsible for the clearance of aggregated and disease-associated proteins, especially under pathogenic and aging conditions.

Autophagy is a highly dynamic vesicle-mediated cellular degradation pathway involving double-membraned vesicles, called autophagosomes, which sequester large protein complexes (protein aggregates), and even whole organelles, and deliver them to lysosomes for degradation [8]. Under low nutrition and energy conditions, autophagy guarantees energy supply by generating amino acid building blocks via recycling. In addition, autophagy plays an important role as a stress and adaptive response and rescue mechanism to maintain cell survival and function [8]. Canonical autophagy responds to environmental cues via a variety of factors that mainly belong to homologs of autophagy-related (ATG) genes, originally identified in yeast [9]. The mammalian target of rapamycin mTOR complex 1 (mTORC1) negatively regulates autophagic activity via inhibitory phosphorylation of ULK1, and is the key initial regulator of canonical autophagy. More downstream membrane expansion is modulated by two ubiquitin-like conjugation systems (ATG12-ATG5 and ATG8/LC3) and the ATG18 protein family members of WD repeat domain phosphoinositide interacting 1-4 (WIPI1-4), as recently excellently reviewed [10].

There is a great amount of data linking dysfunction and malfunction of autophagy to neurodegenerative disease and, consistent with its role in proteostasis, to the accumulation of protein aggregates. Thus, the modulation of autophagy has become one key pharmacological target in neurodegeneration [11,12,13]. In fact, there are multiple overlaps of autophagy and pathogenesis pathways in AD, PD, and ALS [12]. Recently different alternative views and new pharmacological targets towards AD prevention and treatment are evolving, and include a strong focus on the autophagy process.

There are two subtypes of sigma receptors, sigma receptor-1 and sigma receptor-2, both highly expressed in the central nervous system; sigma-1 receptor is present also in various tumor cell lines, including HEK293 and HeLa cells employed in this study [14,15,16]. Sigma-1 receptor (Sig-1R) was cloned in 1996 [17,18] and represents an integral membrane protein of 223 amino acids localized to the endoplasmic reticulum (ER) (and the ER–mitochondria interface) suggesting a role as ER chaperone. Sig-1R was shown to promote cellular survival by (1) ensuring Ca2+signaling from the ER into mitochondria; (2) enhancing the signaling of ER to the nucleus; and (3) attenuating free radical damage by modulation of the activity of Nrf2, a redox-responsive transcription factor [19,20]. Structurally, Sig-1R ligand binding is characterized [21] and the crystal structure of the human receptor is solved [22].

In general, deficits in Sig-1R expression or activity are linked to neurodegeneration and the activation of Sig-1R is associated with neuroprotection in different in vitro and in vivo models, employing different types of pharmacological Sig-1R activators with different pharmacological profiles [15,23]. The pharmacological activation of Sig-1R leads to pluripotent modulatory downstream effects, and incorrect function of Sig-1R is strongly suggested to be also involved in the pathogenesis of neurodegeneration [24]. This is the basis of an effort to design novel and highly specific pharmacological Sig-1R activators for the therapy of neurodegenerative disease, including AD [25].

In this context a novel Sig-1R agonist, tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine hydrochloride (ANAVEX2-73), was developed. Pharmacologically, ANAVEX2-73 shows a dual activity on Sig-1 as well as on muscarinic receptors, acting with described affinities in the low micromolar range [26,27]. Previously, pre-clinical studies in animal models demonstrated robust disease-modifying activities of ANAVEX2-73. Regarding AD, ANAVEX2-73 has undergone testing in Phase 2a trial of patients demonstrating a favorable safety profile and a concentration-dependent improvement against exploratory endpoints [28,29,30]. A variety of neuromodulatory and neuroprotective effects are also already known for ANAVEX2-73, including mitochondrial protection in mouse models of AD, regulation of ERK activation and promotion of survival of astrocytes, as well as protection against oxidative stress [31,32,33].

First evidence for a possible link of Sig-1R, autophagy, and neurodegeneration has been recently shown in the context of ALS. It was discovered that ALS-linked mutant Sig1-R causes an accumulation of autophagic material and actually reduced autophagy [34], and that mice with genetically-altered Sig-1R show defective autophagy [35]. Moreover, it was demonstrated that cocaine-mediated autophagy in astrocytes involves Sig-1R [36]. In addition, it was found that a small-molecule Sig-1R modulator induces autophagic degradation of programmed-death ligand 1 (PD-L1) in cancer cells [37]. These findings prompted us to study the potential of ANAVEX2-73 to have an effect on autophagy in human HeLa and HEK293 cells (in vitro) as well as in C. elegans (in vivo), employing standard measures to analyze autophagic activity [38,39,40,41]. Moreover, the effects of ANAVEX2-73 on protein aggregation and, subsequently, the impact of protein aggregates on movement behavior in C. elegans were studied. Excitingly, ANAVEX2-73 is a potent inducer of autophagic flux in vitro and in vivo and ameliorates protein aggregate formation and paralysis in C. elegans.

2. Materials and Methods

2.1. Cell Culture and Microscopy

HeLa and HEK293A cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA, 41965062) supplemented with active FBS (Life Technologies GmbH, Carlsbad, CA, USA, 10270106), 1× ABAM (Invitrogen, 15240-062) and 1 mM sodium pyruvate (Invitrogen, 1136-088). After medium change, they were treated for 2 h with 10, 1, and 0.1 µM ANAVEX2-73 and PRE-084 (Tocris, Bristol, UK, 0589), respectively; ANAVEX2-73 was provided by ANAVEX Life Sciences Corp, New York, NY, USA. Afterwards Bafilomycin A1 (BafiA1) (Toronto Research Chemicals, North York, ON, Canada, B110000) was added for a further 2 h and the cells were eventually harvested. Western blot analyses were performed as described previously [40,41]. Briefly, cells were subjected to SDS–PAGE using precast NuPAGE 4%–12% Bis-Tris gels (Invitrogen, NPO322). Proteins were detected by chemiluminescence using the Amersham Imager 600 (GE).

Confocal fluorescence microscopical analyses of HEK293A cells stably expressing GFP-LC3B (kind gift of Dr. Sharon Tooze) were performed with the laser scanning microscope LSM 710 (Zeiss, Oberkochen, Germany).

2.2. C. elegans Strains, Maintenance, and Methods

C. elegans were maintained according to standard procedures on nematode growth medium (NGM) plates seeded with HB101 Escherichia coli. The following strains were employed in this study: GFP-LGG-1 (ex[Plgg1-lgg1-GFP]/pRF4; kind gift of Beth Levine), maintained at 20 °C; and the strain CL2006 (dvIs2 [pCL12(unc-54/human Aß peptide 1-42) + pRF4]), maintained at 15 °C, as previously described [40,42]. The latter strain was obtained from the Caenorhabditis Genetic Center (USA).

For analysis of paralysis rate, synchronous CL2006 nematodes were cultivated at 15 °C on plates seeded with HB101 E. coli, resuspended in M9 buffer (control) or 100 µM or 50 µM ANAVEX2-73, respectively. Starting at the first day of adulthood, worms were transferred to fresh plates daily and were tested for paralysis by tapping their nose with a platinum wire. Worms that moved their nose but failed to move their bodies were scored as paralyzed. Dead worms or worms showing other phenotypes were not included into the statistics. Staining of amyloid ß42 aggregates using thioflavine S (Sigma T1892) were carried out as previously described [40]. Worms were mounted on 2% agar pads on a glass slide and confocal fluorescence microscopical analyses were performed with the laser scanning microscope LSM 710 (Zeiss, Oberkochen).

For analysis of autophagic activity, synchronous nematodes expressing GFP-LGG-1 were cultivated at 20 °C. At first day of adulthood, worms were transferred to 80 µM ANAVEX2-73 or control M9 liquid culture medium for 2 h, and were subsequently treated with BafiA1 or dimethyl sulfoxide (DMSO) (control) for 2 h. Thereafter, worms were lysed for Western blotting or analyzed by confocal fluorescence microscopy.

Western blot analyses were performed as described previously [40]. Generally, 12 worms were subjected to SDS–PAGE using precast NuPAGE 4%–12% Bis-Tris gels (Invitrogen, NPO322). Proteins were detected by chemiluminescence using the Amersham Imager 600 (GE).

2.3. Quantitative Real-Time PCR

RNA extraction, reverse transcription, and real-time PCR were performed as described previously [40].

2.4. Statistical Methods

Statistical significance was determined by Student t-test using SIGMA STAT (SPSS Science) as well as the log-rank test using SPSS Statistics (IBM). Statistical significance was accepted at a level of p ≤ 0.05. The results are expressed as mean ± standard deviation (SD).

3. Results and Discussion

3.1. Sig1-R Agonist ANAVEX2-73 Enhances Autophagic Activity

To study the effect of ANAVEX2-73 on autophagy, we treated human HeLa cells with the compound and analyzed autophagic activity by investigating the flux of LC3-II. LC3-II is the lipidated form of LC3, which (partially) stays attached to autophagosomes and thus gets degraded by lysosomes. Therefore, the quantification of the LC3-II flux, using BafiA1 for inhibition of lysosomal degradation, directly corresponds to cellular autophagic activity following the appropriate guidelines [38]. As displayed in Figure 1, ANAVEX2-73 significantly induces autophagic flux when compared to control conditions. There is a concentration-dependent and significant increase in the autophagic flux following application of ANAVEX2-73: An increase of over 2-fold at 10 µM and over 1.5-fold at 1 µM ANAVEX2-73 (Figure 1A). As standard positive control to provoke the induction of autophagy, HeLa cells were incubated with EBSS medium, which resembles nutrient deprivation as autophagy stimulus.

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Figure 1. Sig-1R activation enhances autophagic activity. Western blot analyses of the autophagic flux upon addition of ANAVEX2-73 (A) or PRE-084 (B). HeLa cells were treated with indicated concentrations of ANAVEX2-73, PRE-084, and BafiA1 (2 µM) or DMSO. Statistics are depicted as mean +/- SD. *** p ≤ 0.001, ** p ≤ 0.01, t-test, n = 4. (C) Representative confocal fluorescence microscopic images of HEK293 cells stably transfected with a GFP-LC3B reporter construct. Cells were treated with 1 µM ANAVEX2-73, BafiA1, and/or DMSO. Scale bar = 20 µm or 10 µm, respectively. GFP-positive autophagosomal structures (indicated by arrowheads) were counted in approx. thirty cells per treatment in three independent experiments. *** p ≤ 0.001, t-test.

Here we focused on ANAVEX2-73 as Sig-1R agonist but, of course, other common and highly selective Sig-1R agonists are available and were already studied in different cellular and animal models. Such compounds include (+)-pentazocine, (+)-SKF10,047, SA4503 (1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine), and PRE-084 (2-morpholin-4-ylethyl 1-phenylcyclohexane-1-carboxylate) [14]. Since the Sig-1R