Neuronal Sigma-1 Receptors: Signaling Functions and Protective Roles in Neurodegenerative Diseases

1Sigma-1 receptor (S1R) is a ligand-operated protein that modulates activity of several client proteins from its position within the membrane of the endoplasmic reticulum (ER). It is widely expressed in multiple organs including the nervous system and it has important roles in modulation of neuronal physiology and synaptic plasticity. Autosomal recessive loss-of-function mutations in S1R are primarily associated with amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD), but polymorphisms in S1R also affect risk of developing Alzheimer’s disease (AD). Many S1R agonists are neuroprotective and loss of S1R accelerates neurodegenerative phenotypes. Neuroprotection from S1R activation is achieved by a diversity of signaling functions that promote cellular homeostasis and synaptic stability. In this review we summarize therapeutically relevant mechanisms by which the ligand-operated chaperone S1R modulates neurophysiology, counteracting its dysregulation from pathogenic stressors.

2Sigma-1 receptor is a 223 amino acid-long transmembrane protein residing in the ER membrane. S1R preferentially localizes to the specific microdomains of the ER called mitochondrial-associated membranes (MAM), where it can regulate InsP3R-dependent calcium flux from the ER to mitochondria, lipid dynamics, MAM stability, and the ER stress response. The MAM domain is also important for synthesis and transport of lipids and protein folding. Under resting conditions, S1R forms an inert complex with GRP78/BiP protein. When activated by agonists or ER calcium depletion, S1R dissociates from BiP and redistributes to the entire ER network, freeing it to interact with and modulate several client proteins including InsP3Rs inside and outside of the MAM domain as well as plasmalemmal ion channels, GPCRs, and kinases (summarized in Table 1).

3Sigma-1 receptor agonists do not noticeably alter ER calcium signaling under resting conditions, but they can influence ER calcium release triggered by Gq-coupled receptors. S1R chaperones InsP3R3 to the MAM domain and prevents its degradation, enhancing Ca2+ flux into mitochondria. This augments ATP production, but, in excess, it could also trigger the mitochondrial permeability transition. By contrast, InsP3R1, which is the predominant InsP3R isoform in neurons and has important signaling functions outside of the MAM, is negatively regulated by agonist-stimulated S1R in certain cell types like striatal medium spiny neurons (MSNs). Regulation ER calcium homeostasis and signaling by S1R has important implications for neurodegenerative diseases and this will be discussed later.

4Engagement of S1R with plasmalemmal channels and receptors is responsible for S1R-dependent fine-tuning of neuronal excitability. As many of S1R’s interaction partners function in the plasma membrane, it was proposed that activated S1R translocates from the ER to the plasma membrane where it binds to client proteins and acts as a chaperone or an auxiliary subunit. However, this assumption is often based on experiments in which S1R and/or its interaction partner are overexpressed, raising several caveats that call this interpretation into question. Alternatively, S1R may interact with plasma membrane proteins from its position in the ER like STIM proteins and/or regulate the maturation and/or trafficking of certain proteins to the plasma membrane.

5Sigma-1 receptor activation alters neuronal excitability through its interactions with voltage-gated ion channels. Voltage-gated sodium (Nav) channels augment neuronal depolarization and mediate action potentials. S1R ligands dissociate S1R from Nav1.5, leading to suppressed Nav1.5 activity. This action can be evoked by the endogenous S1R agonist N,N-dimethyltryptamine (DMT) and is partially opposed by the endogenous S1R antagonist progesterone. S1R agonists also limit excitability by inhibiting other Nav channels including Nav1.2 and Nav1.4. Voltage-gated potassium (Kv) channels respond to membrane depolarization during action potentials by releasing positively charged potassium ions from the cytosol to restore a hyperpolarized resting membrane potential and limit hyperexcitability. When S1R is activated by cocaine, S1R binds to the voltage-gated potassium channel Kv1.2 and enhances trafficking of Kv1.2 to the plasma membrane, decreasing excitability of dopamine D1 receptor (D1R)-expressing MSNs in the shell of the nucleus accumbens. S1R expression and activity also regulates the cardiac Kv channel hERG through control of maturation and trafficking and this function appears to be dependent on cholesterol and not S1R ligands, possibly suggesting a role for lipid rafts in S1R client protein assembly and trafficking. S1R appears to basally regulate Kv1.3 and Kv1.4 independent of agonist-stimulation. By means of such interactions, S1R regulates neuronal excitability.

6Sigma-1 receptor also influences synaptic functions through modulation of the NMDA receptor (NMDAR) activity. Physiological NMDAR activation can induce hippocampal long-term potentiation (LTP), spine maturation and learning, but pathophysiological levels of NMDAR activity triggers excitotoxicity. S1R facilitates NMDA receptor signaling and neurotransmission in hippocampal neurons, possibly through altering responses to calcium signals (e.g., inhibiting Ca2+-activated SK channels) and promoting expression of NMDA receptor subunits and their trafficking to the plasma membrane. S1R can also obviate negative-regulation of NDMARs by cannabinoid 1 receptor (CB1R). These interactions enhance neuronal firing and maturation of mushroom spines from NMDA receptor activation. Modulation of calcium signaling by S1R may regulate synaptic plasticity through stimulation of CaMKII, PKC, and ERK.

7Sigma-1 receptor agonists may promote synaptic plasticity and neuronal resilience in large part through their common ability to upregulate BDNF secretion and TrkB receptor signaling both in vitro and in vivo. For example, pridopidine, a potent S1R receptor agonist, promotes neurotrophic signaling via BDNF, ERK, and AKT pathways. S1R agonists appear to activate TrkB both through BDNF-dependent and independent mechanisms. This may involve regulation of BDNF expression and processing as well as direct interactions of S1R with the TrkB receptor. S1R also stimulates signaling by other receptor tyrosine kinases including the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR). Stimulation of neurotrophic receptors confers neuroprotection through control of gene expression.

8Indirect regulation of transcriptional activity by S1R contributes to its neuroprotective properties. For example, S1R may prevent neuronal death by upregulating expression of the antiapoptotic mitochondrial protein Bcl-2. S1R regulates transcription through interactions with inositol-requiring enzyme 1 (IRE1) and emerin. S1R facilitates dimerization of the ER stress sensor and endonuclease IRE1 at the MAM domain, leading to splicing-dependent activation of the transcription factor XBP1, which goes on to upregulate several ER chaperones. S1R also decreases IRE1-driven inflammation, which may be important for microglial reactivity and migration to and from injury sites. As the ER membrane is contiguous with the nuclear envelope, activated S1R can move to the nuclear envelope where it regulates transcription through its recruitment of emerin and then chromatin-remodeling factors.

9A microarray study involving knockdown of S1R in cultured hippocampal neurons revealed altered transcription in pathways controlling protein ubiquitination, sterol biosynthesis, oxidative stress, and actin dynamics. Knockdown of S1R reduces the size of dendritic spine size in hippocampal neurons, indicating that it actively supports stability of mature spines. This was initially proposed to involve its role in regulating oxidative stress and Rac-GTP signaling, but may also involve modulation of calcium homeostasis in conditions of disease. Knockout of S1R is associated with increased formation of reactive oxygen species (ROS) and decreased expression and activity of NRF2, which promotes expression and activation of antioxidant molecules under conditions of stress. This may explain how S1R suppresses generation of ROS. Interestingly, spine shrinkage from knocking down S1R was prevented by reducing oxidative stress.

10Finally, in addition to protein–protein modulation, S1R was shown to interact with lipids. S1R localizes to lipid rafts – detergent-resistant microdomains of the ER – where it binds cholesterol at sterol-binding sites and S1R agonists such as SKF-10047 displace S1R and its binding partners from lipid rafts possibly through out-competing cholesterol binding. S1R targets to galactosyl-rich microdomains of the ER and is potentially involved in regulation of the differentiation of oligodendocytes and myelination as well as lipid transport to the myelin membrane. S1R also supports axonal growth through promoting myristoylation of p35, which increases its degradation and thereby decreases p25/CDK5-dependent hyperphosphorylation of Tau.

11This review only scratches the surface with regard to S1R’s multiplicitous roles in neurophysiology/neuroprotection and provides a glimpse into the specificity of its actions in differing cell types. It is tempting to speculate that the nature of modulation by S1R depends on the levels of S1R and its interaction partners in a given cell type (e.g., preferential interactions of S1R with cell-type abundant effectors) as well as many circumstantial factors such as the current physiological/pathophysiological state of the cell and signaling events (e.g., presence/absence of S1R ligands). To better understand possible actions of S1R, we summarized S1R interaction partners and the effects of S1R ligands and knockdown/overexpression on these protein–protein interactions (Figure 1 and Table 1).

12Sigma-1 receptor is a promising therapeutic target in the treatment of neurodegenerative diseases as it stabilizes the function of several intracellular systems through its role as a chaperone when activated by a variety of ligands with neuroprotective properties. Despite S1R pluripotency, relatively little is known regarding the mechanisms of receptor functioning and regulation at the molecular level. There is evidence that the structural organization of S1R and its conformational state are important determinants of S1R activity. However, the structural basis for S1R functionality remains poorly understood.

13Originally, S1R was characterized as a type 1 transmembrane protein with only one transmembrane domain. Hydrophobicity analysis confirmed a single-pass transmembrane topology of S1R, however, subsequent studies predicted a two transmembrane domain model of S1R topology. For example, Aydar and colleagues proposed two-transmembrane domains (TM1 a.a. 11–29 and TM2 a.a. 80–100) based on antibody staining experiments with expression of S1R fused to GFP at the N- or C-terminus in Xenopus oocytes. Immunolabeling of the GFP-tags was absent without membrane permeabilization, but was detected after permeabilization with 0.5% acetone, leading them to conclude that both the N- and the C-termini are located near the plasma membrane but within the cytoplasm. By contrast, the topological model proposed by Hayashi and Su situates S1R in ER membranes with both N- and C-terminal regions oriented to the ER lumen. This was based on immunocytochemical staining of endogenous S1R in CHO cells with antibodies targeting N- and C-termini. Similar to results of, no labeling was detected without permeabilization, suggesting that S1R is not in the plasma membrane. Permeabilization of plasma and ER membranes with CHAPS or Triton X-100 enabled staining for all antibodies with a distribution similar to the shape of the ER. When the plasma membrane was permeabilized with streptolysin-O, staining was present only for the antibody targeting the loop domain. The discrepancy between the topology models of and may have arisen from altered membrane insertion of GFP-fused S1R and/or cell type specific differences in S1R localization. For example, Hayashi and Su note that fusion of YFP to the C-terminus of S1R, but not the N-terminus, mirrors the distribution of endogenous S1R.

14The two-pass transmembrane model was widely accepted for a long time and has been used as a structural basis for molecular modeling and ligand docking studies. However, the crystal structure for human S1R was recently solved revealing a single transmembrane domain structure. According to this study, a short N-terminus faces the ER lumen while the C-terminal domain of protein is oriented to the cytosolic side.

15Adding another possible model of S1R topology to the mix, fused ascorbate peroxidase 2 (APEX2) to the N- or C-terminus of S1R and used electron microscopy to visualize deposition of 3,3′-diaminobenzidine outside or inside of the ER of ND7/23 cells and dorsal root ganglion (DRG) neurons. This experiment indicated that the N-terminus of S1R faces the cytosol, whereas the C-terminus is located in the ER lumen. This is consistent with the one transmembrane model suggested by the crystal structure, but suggests the orientation of S1R positions the bulk of its structure in the lumen of the ER with only a short N-terminus facing the cytosol. All of the experiments probing the topology and orientation of S1R have caveats that are important to keep in mind including specificity of antibodies and membrane permeabilization as well as alterations to S1R from protein fusions and crystallization conditions.

16determined the first crystal structure of full-length human S1R using X-ray analysis. They expressed FLAG-tagged S1R in Sf9 insect cells, purified it using the detergent lauryl maltose neopentyl glycol (LMNG) and crystallized it using the lipidic cubic phase (LCP) method. According to the crystal structure, S1R homomers consist of three protein subunits, with each protomer having one transmembrane domain (Figure 2A). The transmembrane alpha-helices of trimers (encompassing amino acid residues 8 to 32) are separated from each other and located in the corners of the complex, while the C-terminal cytosolic domains of each protomer organize the trimeric structure and form highly conserved ligand-binding sites. The membrane proximal surface of each C-terminal domain is tightly associated with the cytosolic surface of the ER membrane. Each C-terminal domain contains a cupin-like β-barrel that can envelop a ligand (Figure 2A).

17An accurate model of the S1R ligand binding pocket is necessary for rational drug design aimed at the targeted treatment of neurodegenerative diseases. Mapping of the S1R ligand binding site was carried out in a large number of studies using mutational analysis and photoreactive probe labeling. The first identified amino acids that are important for ligand binding include Ser99, Tyr103, Leu105, and Leu106. The results of these studies are highly consistent with the structural model of S1R obtained via X-ray crystal analysis.

18Sigma-1 receptor crystal structures harboring chemically distinct ligands (the high-affinity, selective S1R ligands PD144418 and 4-IBP) show that both ligands bind in similar positions, forming electrostatic interactions with the highly conserved amino acid residue Glu172 (Figure 2B). The amino acid Asp126 which is also essential for ligand binding forms a hydrogen bond with Glu172. With the exception of only two amino acid residues, the S1R active site is hydrophobic and is occluded from aqueous solution. Other amino residues involved in ligand coordination are as follows: Val84, Trp89, Met93, Leu95, Tyr103, Leu105, Phe107, Ile124, Trp164, and Leu182 (Figure 2B). Additionally, Tyr103 creates a hydrogen bond with Glu172, which is apparently necessary for the formation of the binding pocket (Figure 2B). Indeed, in earlier works a significant decrease in the binding activity of the mutant Tyr103Phe was shown. The highly occluded structure of the binding pocket raises the questions about the pathway of ligand entry and explains the very slow ligand binding kinetics.

19conducted additional structural studies and molecular dynamics (MD) simulation experiments to reconstruct the ligand binding mechanism in detail. They solved crystal structures of S1R bound to the classical antagonists haloperidol and NE-100 as well as the agonist (+)-pentazocine. The obtained structures were highly similar to each other and did not differ significantly from the previously determined trimeric structures of S1R. They share a similar organization of the ligand-binding pocket. The overall conformations of S1R in complex with the antagonists or agonist remain almost identical with the exception of a difference in the position of (+)-pentazocine in the ligand binding site. On the basis of structural data and MD simulations the authors suggest that agonist binding leads to conformational changes of S1R compared to the unliganded form of receptor or antagonist bound S1R. MD simulations were used to characterize conformational rearrangements occurring during ligand association. Ligand association was characterized as a two-step process: (1) the hydrogen bonds between Trp136 and Ala161 break, leading to receptor “lid” opening, and (2) the interior of the receptor separates, exposing the binding pocket and allowing ligand entry.

20provided valuable insights on the structural basis for a ligand binding mechanism and describe the potential conformational differences between agonist and antagonist bound S1R. However, they do not explain the functional role of agonist/antagonist actions and the physiological relevance of agonist-induced structural rearrangements of S1R. The relationship between the ligand-receptor association and the subsequent biological response remains unclear.

21Extensive evidence indicates that S1R exists in multiple oligomeric states and recent studies suggest that ligand binding induces a shift in the oligomeric state of S1R, which could in turn lead to the various functional responses. For example, high-molecular weight forms of S1R have been detected in rat liver microsomal membranes using radioactive photosensitive labels. Oligomeric forms of S1R corresponding to hexamers, tetramers, octamers were also identified by size-exclusion chromatography (SEC). Analysis by SDS-PAGE after chemical crosslinking of individual oligomeric forms of MBP fused S1R has also confirmed the presence of monomers, tetramers and high molecular weight S1R oligomers. Cell-based fluorescence resonance energy transfer (FRET) studies have also confirmed the existence of several oligomeric states and revealed that agonists stabilize low-molecular-weight species, whereas antagonists favor oligomers. This model suggests that monomeric form of S1R is an “active” conformation involved in interactions with client proteins (Figure 3). On the other hand, found similar stabilizing effects of agonists and antagonists on S1R oligomeric states. However, the detergents used in the purification procedure of their experiments do not reflect the native lipid environment of membrane proteins, potentially limiting the validity of this finding. While the crystal structure of S1R has a trimeric fold, size-exclusion, cross-linking and multi-angle light scattering (SEC-MALS) experiments have revealed a wide range of oligomeric states from 140 up to 400 kDa. Thus, it remains unclear which oligomeric form(s) of S1R exists in vivo and which state(s) is(are) functionally active.

22Sigma-1 receptor oligomerization is disrupted by mutations in the GXXXG motif corresponding to amino acid residues 87–91. The GXXXG motif was previously through to reside in the second transmembrane domain and mediate subunit association via transmembrane alpha helices. However, the crystal structure suggests that it forms a beta-hairpin structure inside the oligomerization interface. The distance between Cα atoms of Gly88 in each protomer is about 6 Å. Thus, mutations of this residue can sterically interfere with subunit association. demonstrated that mutations within the GXXXG motif cause a shift toward the monomeric state of S1R. Interestingly, this is associated with a significant decrease in ligand binding, suggesting that ligand binding affinity may depend on S1R oligomerization processes. While oligomeric forms of S1R have a demonstrated ability to bind ligands, S1R monomers may lose this property. Mutations within the GXXXG motif also decrease S1R expression, which may indicate reduced stability of GXXXG mutants.

23The oligomerization interface was further characterized with crystallographic data, leading to identification of key amino acids involved in subunit interactions. The sequence of the oligomerization interface is highly conserved among species, confirming its physiological importance. The trimerization surface is formed largely by hydrophobic residues within the C-terminal cupin domain. For example, a Phe191 residue from each protomer forms inter-subunit contacts. There are the polar interactions between sidechains of Thr141, His54, and Glu55. The amino acids Trp81, Phe83, Met90, Ala92 and Leu111, His116, Arg119, Trp136, Ala161, Trp169, Asp188, Phe191, Ser192, Gln194, Asp195, and Thr198 are also engaged in formation of the trimerization interface.

24Despite the detailed characterization of oligomerization interface and comprehensive studies of ligand-induced structural rearrangements, the significance of S1R oligomer-monomer transitions in the regulation of S1R functions remains unknown. To correlate structural rearrangements observed in vitro with physiological responses, it has been proposed that the physiological significance of S1R oligomerization may be linked to the protein–protein interactions of S1R with its partners. For example, the monomeric form of S1R interacts with Nav1.5, acid-sensing channels and D1R. Two groups identified a direct and agonist-dependent interaction between S1R and the dopamine transporter (DAT), resulting in attenuated DA efflux and calcium signals evoked by methamphetamine. suggested that agonists induce dissociation of S1R multimers into monomers which in turn interact with DAT. Mutational analyses have shown that the interaction site is located in the transmembrane domain of S1R.

25As mentioned above, initiation of S1R activity was also proposed to involve ligand-, calcium-, or ER stress-dependent dissociation of S1R from binding immunoglobulin protein (BiP), which is a chaperone located in the lumen of the ER. Recently, used a novel bioluminescence resonance energy transfer (BRET) assay to study the ligand-mediated oligomerization of S1R. They revealed the distinct effects of agonists and antagonists on S1R homomerization, consistent with previous results. Interestingly, while the agonist pentazocine facilitated interaction of BiP and S1R, haloperidol induced the dissociation of S1R from BiP. Thus, S1R ligands may regulate the association between S1R and BiP through controlling S1R oligomerization and monomerization. This is likely also relevant to S1R interactions with its client proteins (Figure 3).

26It would be interesting to know more clearly how S1R associates with various proteins located in the ER lumen, ER membrane, cytoplasm and plasma membrane and to resolve the conflicting models of S1R topology and orientation. Given the topology model proposed by, the bulk of S1R may face the ER lumen. This topology is consistent with the well-described interaction of S1R with BiP, but raises it questions about how S1R interacts with proteins in the cytosol with only a small cytosolic N-terminal tail. Perhaps S1R has two or more structural elements or configurations responsible for the binding of S1R to different proteins. The structural and biological mechanisms of such interactions remain to be fully elucidated.

27Many S1R agonists are anti-amnestic, synaptogenetic, and neuroprotective in conditions of neuronal stress. They also mitigate disease and symptoms in experimental models of ALS, Alzheimer’s disease (AD), Parkinson’s disease (PD) Huntington’s disease (HD) stroke and traumatic brain injury. By contrast, S1R deficiency exacerbates progression of neurological disorders as well as symptoms commonly associated with neurodegenerative diseases. For example, pharmacological inhibition of S1R leads to mushroom spine loss in hippocampal cultures and this could be related to memory impairments from the anti-psychotic drug and S1R antagonist haloperidol (KD for S1R ∼3 nM). S1R knockout (KO) mice have several phenotypes resulting from neuronal dysfunction and late-onset neurodegeneration. These data collectively highlight the innate, neuroprotective properties of S1R activity. The following sections summarize genetic associations of S1R mutations/polymorphisms with neurodegenerative diseases, examples of neuroprotection in respective disorders by S1R agonists, and possible mechanisms of action.

28Amyotrophic lateral sclerosis is a fatal neurodegenerative disease featuring progressive weakness of skeletal muscles due to upper and/or lower motor neuron dysfunction and loss. Several recessive, loss-of-function mutations in S1R have been associated with ALS, distal hereditary motor neuropathy and/or FTD. S1R is highly expressed in motor neurons, suggesting a possible cell autonomous mechanism of motor neuron degeneration in these patients. Although S1R KO mice do not develop an overt ALS phenotype, they have deficits in locomotion and motor performance related to muscle weakness, axonal degeneration, and motor neuron loss. KO of S1R accelerates the onset and progression of ALS in the SOD1G93A mouse model of ALS, whereas the S1R agonists PRE-084 and SA4503 are neuroprotective and extend survival of SOD1G93A mice. PRE-084 is also protective in wobbler mice, which develop spontaneous motor neuron degeneration. PRE-084 is a derivative of phencyclidine (PCP) with nanomolar affinity for S1R and negligible affinity for PCP receptors and GPCRs. SA4503 has low nanomolar affinity for S1R, low micromolar affinity for S2R, and little affinity for 36 other receptors, ion channels and second messenger systems. Treatment of SOD1G93A mice with the S1R agonist pridopidine improves axonal transport (e.g., of BDNF, GDNF, and mitochondria) and BDNF secretion while attenuating atrophy of neuromuscular junctions, muscle fibers and motor neurons (pharmacological properties summarized in HD section). Pridopidine treatment reduces the prevalence of SOD1 aggregates in spinal cords of SOD1G93A mice. S1R activity may additionally protect motor neurons by reducing their excitability through facilitation of potassium channel activity. Motor neuron degeneration from the absence of S1R is associated with reduced contacts between mitochondria and ER, ER stress, calcium dysregulation and this may help to explain pathology in ALS patients with mutations in S1R.

29Dopamine receptors play important roles in learning and memory, motivation and movement and S1R agonists modulate dopaminergic signaling through multiple mechanisms. This has primarily been studied in the context of psychostimulant research, but these results may be important for understanding regulation of dopamine neurotransmission and its dysregulation in HD and PD. S1R appears to differentially regulate dopamine D1 and D2 receptors, as S1R activation by cocaine inhibits D2R and prevents histamine H3 receptor-dependent inhibition of the dopamine D1 receptor, stimulating Gs, recruitment of β-arrestin and phosphorylation of ERK1/2. Although S1R activation does not affect basal dopamine neurotransmission, it attenuates methamphetamine-induced and DAT-dependent increases in firing of dopamine neurons. It also interacts directly with the DAT and attenuates calcium signals evoked by methamphetamine. As a result, S1R limits hyperactivity, motivated behavior and reinforcement from methamphetamine.

30Abnormalities in movement and cognition in PD result from degeneration of dopaminergic neurons projecting from the substantia nigra to the striatum. S1R is expressed in these neurons and it can bidirectionally modulate NMDAR-dependent release of dopamine in striatal brain slice experiments. S1R may be decreased in striatal regions that are preferentially affected in PD, which could contribute to neuropathology as indicated by studies with S1R KO mice. Similar to PD patients, S1R KO mice have age-related deficits in motor behavior and degeneration of dopaminergic neurons. This appears to be related to aggregation and phosphorylation of α-synuclein which may be driven by phosphorylation of eIF2α from ER stress and proteasomal dysfunction. Pharmacological inhibition of ER stress prevented oligomerization of α-synuclein, dopaminergic neuron loss and motor impairments in S1R KO mice.

31Recent studies found that S1R agonists are protective in PD models. For example, chronic treatment with PRE-084 gradually improves PD-like motor deficits from unilateral intrastriatal 6-hydroxydopamine (6-OHDA) lesions (hemiparkinsonian model) when treatment onset was prompt. This treatment suppressed neuroinflammation while increasing levels of neurotrophic factors, monoamines (e.g., dopamine and serotonin), dopaminergic innervation of the striatum, and nigral neuron survival. Low dose pridopidine treatment (0.3 mg/kg) of unilaterally 6-OHDA-lesioned mice partially protected nigral dopaminergic cell bodies and increased dopaminergic fiber density in the motor striatum. This was associated with a gradual restoration of forelimb use (cylinder test, stepping test) and prevention of rotational bias toward the ipsilateral side. The delayed recovery of motor function corresponds roughly with the expected timeline of pridopidine-dependent dopaminergic axon sprouting. Treatment efficacy was absent in S1R KO mice, which had reduced loss of dopaminergic neurons in the substantia nigra pars compacta, but a greater loss of dopaminergic fibers in the striatum compared to wild-type mice. The increased vulnerability of S1R knockout mice to axonal degeneration in the nigrostriatal pathway could relate S1R’s ability to promote growth and repair of neurites. The neuroresorative effects of pridopidine were associated with upregulation of neurotrophic factors (BDNF, GDNF, pERK1/2) and associated signaling in the striatum and substantia nigra as well as reduced microglial activation.

32Huntington’s disease (HD) patients suffer from psychiatric, motor and cognitive disturbances that gradually worsen, leading to dementia, cachexia and eventually death. HD is a dominantly inherited neurodegenerative disease resulting from a CAG trinucleotide repeat expansion in exon 1 of the Huntingtin gene (>35 CAG repeats), leading to expression of mutant Huntingtin (mHtt) protein with an elongated polyglutamine tract. mHtt is broadly expressed throughout the body, but striatal MSNs are preferentially vulnerable in HD. The most significant contributions of mHtt to HD pathology remains a matter of debate and intense investigation. The CAG expansion compromises normal functions of Htt and disrupts cellular functioning through gain of mHtt function mechanisms, with possible toxic contributions from repeat-associated non-AUG translation. This results in oxidative damage, glial reactivity, altered intracellular signaling, metabolism and energy levels, impaired axonal transport, transcriptional dysregulation, aberrant calcium regulation associated with ER stress, synapse loss and excitotoxicity.

33Initial studies on the potential role of S1R in HD pathology were carried out with cellular models of HD., found that expression of mHtt (N-terminal huntingtin fragment proteins with 120 polyQ repeats or full-length Htt protein with 75 repeats) downregulates S1R expression in neuronal PC6.3 cells. There were no differences in S1R expression in control cells expressing the N-terminal fragment of Htt with 18 polyQ repeats or wild-type Htt. Administration of the selective S1R agonist PRE-084 prevented mHtt-dependent downregulation of S1R, SOD1, SOD2, thioredoxin 2, and Bcl-XL in neuronal PC6.3 cells. However, S1R expression appears to be upregulated in the striatum of YAC128 HD mice at 12 months of age and in the striatum of patients with advanced HD, possibly as an effort to compensate for ER calcium dysregulation and stress. PRE-084 also decreased caspase-3 cleavage and oxidative stress and upregulated calpastatin, NF-κB-p65 levels and NF-κB signaling in mHtt expressing PC6.3 cells, enhancing their viability. proposed that the neuroprotective properties of S1R activity involved modulation of the calpastatin/calpain system, increasing NF-κB signaling and thereby upregulating antioxidants and decreasing ROS levels.

34Another study demonstrated that large neuronal nuclear inclusions were strongly positive for S1R in human brains affected by polyglutamine diseases and intranuclear inclusion body disease. Also, S1R immunostaining colocalized with most intranuclear mHtt aggregates in HeLa cells expressing the N-terminal fragment of mHtt. Downregulation of S1R with antisense RNA increased the amount of mHtt aggregates in both the cytoplasm and nucleus. This was reproduced by treatment with the proteasome inhibitor epoxomicin. Moreover, proteasome activity was significantly lower following knockdown of S1R.

35Clinical trials with pridopidine indicate that it has efficacy in treating motor symptoms of HD and recent evidence suggests that the therapeutic effect of pridopidine involves S1R. Pridopidine was originally dubbed a “dopamine stabilizer” based on behavioral experiments showing that it can both decrease locomotion in hyperactive rodents (e.g., from D-amphetamine or MK-801) and increase locomotion in hypoactive rodents (e.g., animals that have habituated to their environment or co-treated with the VMAT inhibitor tetrabenazine). Increased locomotion in hypoactive rodents may relate to pridopidine’s ability to increase dopamine and norepinephrine in the cortex and subcortical areas, which may also explain increased firing in prefrontal pyramidal neurons. Pridopidine’s ability to bidirectionally normalize activity levels may have particular utility in the treatment of HD in which patients develop hyperkinetic motor disturbances followed by hypoactivity later in disease. The mechanism of action was initially proposed to involve low-affinity/fast-off negative modulation of dopamine D2 receptors with a slight binding preference for the agonist binding site when the receptor is in the active, catalytic, high-affinity state, but the affinity of pridopidine for D2 receptors of dopamine is relatively low being in the micromolar range. Unlike classical D2 receptor antagonists, pridopidine does not induce hypoactivity or catalepsy. Pridopidine also has micomolar affinity for several additional GPCRs including adrenergic alpha 2A/C receptors, serotonergic 5HT1A and 5HT2A receptors, and histamine H3 receptors and these interactions may influence levels of extracellular monoamines and glutamatergic neurotransmission. The effects of pridopidine in behavioral assays are not fully blocked in D2R receptor knockout mice, prompting further investigation into potential molecular targets of pridopidine. More recently, pridopidine was found to have a high affinity (kD = ∼80 nM) for S1R and it primarily binds S1R rather than D2 receptors in vivo at behaviorally relevant doses, suggesting that S1R might mediate the therapeutic effects of pridopidine.

36Recent studies show that activation of S1R by pridopidine might be disease-modifying in HD. found that pridopidine reduces motor symptoms of R6/2 mice, improving performance on the horizontal ladder task and open-field locomotor measurements when treatment was started presymptomatically (5–6 mg/kg via daily intraperitoneal injections). Pridopidine also extends their lifespan. In vivo treatment also normalized striatal BDNF and DARPP32 levels, while decreasing the size and amount of mHtt aggregates. 150 μM pridopidine reduced apoptosis and restored pERK1/2 levels in a mouse striatal knock-in cellular HD model (STHdh111/111) and these effects were blocked by the S1R antagonist NE-100. found that low nanomolar concentrations of pridopidine and the structurally similar S1R agonist (+)3-PPP are neuroprotective in another cellular model of HD. Both compounds stabilized synaptic connections between cortical and striatal MSNs in primary corticostriatal co-cultures prepared from from YAC128 HD mouse pups. Deletion of S1R with Cas9 prevented pridopidine and 3-PPP from rescuing dendritic spine loss in HD MSNs. Interestingly, S1R deletion also resulted in significant spine loss in WT MSNs. This observation indicated an important role for S1R in maintaining MSN spine stability. A synaptoprotective action of pridopidine was further supported by a series of Ca2+ imaging experiments. Previous studies demonstrated that abnormal Ca2+ signaling in post-synaptic spines is responsible for their destabilization in HD MSNs. Decreased ER Ca2+ levels due to mHtt-induced InsP3R1 hyperactivity increases neuronal store-operated calcium entry (nSOC) in HD MSNs to synaptotoxic levels. Pridopidine treatment of corticostriatal co-cultures from YAC128 mice prevented InsP3R1 hyperactivity, restored ER Ca2+ levels, and decreased nSOC in HD MSNs. Deletion of S1R WT MSNs resulted in depletion of ER Ca2+ content, suggesting that it might stabilize MSN spines through homeostatic control of ER Ca2+ levels. Deletion of S1R in HD MSNs prevented the normalization of ER Ca2+ by pridopidine. The selective S1R agonist PRE-084 also prevents dendritic spine loss in HD MSNs and this rescue is blunted by the S1R antagonist NE-100. These findings suggest that in addition to the ability of pridopidine to mitigate motor symptoms of HD, it may also foster synaptic and neuronal viability via activation of S1R.

37Consistent with this, found that pridopidine and 3-PPP protects mouse primary striatal and cortical neurons from expression of mHtt (22 vs. 58 CAG repeats with the first 586 amino acids of Htt), as measured by imaging nuclear condensation in apoptotic cells and neuronal morphology. Pridopidine also rescued HD patient-derived induced pluripotent stem cells. The S1R antagonist NE-100 or genetic ablation of S1R blocked the neuroprotective effects. Although BDNF was also protective and is upregulated by S1R stimulation, blockade of BDNF signaling with the TrkB receptor antagonist ANA-12 did not impede the neuroprotective effects of pridopidine. However, ANA-12, like NE-100, suppressed pridopidine’s ability to prevent mitochondrial depolarization from mHtt, as measured using tetramethyl rhodamine methyl ester (TMRM). These data indicate that S1R activation by pridopidine or 3-PPP is neuroprotective, but neuroprotection is not entirely mediated by BDNF signaling.

38Pridopidine activates neuroprotective pathways that are compromised in HD (e.g., BDNF and AKT pathways), improving behavioral and transcriptional deficits in mouse models of HD. Consistent with its ability to promote neuronal plasticity and survival, pridopidine upregulates expression BDNF, dopamine D1 receptor, AKT/PI3K and glucocorticoid pathway components and stimulates BDNF secretion in an S1R-dependent fashion. Microarray and qPCR studies showed that pridopidine upregulates several genes downstream of BDNF including EGR1, EGR2, KLF5, CDKN1A, Homer1a, and Arc. BDNF overexpression is sufficient to rescue many phenotypic characteristics of YAC128 HD mice (e.g., motor performance, cognitive deficits, synaptic density), further suggesting that BDNF signaling could be an important contributor to neuroprotection following S1R activation. The idea that some of the beneficial effects of pridopidine in HD models can be mediated through BDNF signaling was supported by recent experimental evidence from. Synaptic scaling was suppressed in YAC128 cultures, as determined by recording the amplitude and frequency of mEPSCs after blockade of activity-dependent neurotransmission with TTX. Synaptic scaling was restored in YAC128 neurons by pharmacological activation of S1R with pridopidine or 3-PPP through BDNF-TrkB signaling. Given that AKT is a potent pro-survival kinase, its upregulation may help promote neuronal resilience by phosphorylating apoptotic proteins (e.g., BAD and GSK3) and forkhead family transcription factors (e.g., FOXOs). Also, the calcium regulating genes calbindin and Homer1a are downregulated in the striatum of Q175 and YAC128 HD mice and they are both upregulated by pridopidine treatment. These results indicate that when activated by pridopidine S1R acts on several transcriptional networks to foster neuronal function and survival in HD models.

39A recent study demonstrated that pridopidine improves motor performance in YAC128 HD mice as well as anxiety- and depressive-like phenotypes, but it was unable to prevent striatal and corpus callosum atrophy, indicating that S1R agonism is insufficient to completely mitigate HD neuropathology and complementary treatment strategies should be considered. While pridopidine might be insufficient to completely prevent HD progression, when taken together the data on its effects in HD models and in HD patients shows that it can mitigate symptoms and is likely has the capacity to modify disease.

40Alzheimer’s disease (AD) is the most pervasive cause of dementia in elderly people and it involves progressive impairment of memory and other cognitive faculties from damage to the hippocampus and other parts of the brain. Age is the main risk factor for the sporadic form of the disease. Early onset of AD is characterized by the development of the disease before the age of 65 and most of these cases result from autosomal dominant inherited mutations in amyloid precursor protein (APP), presenilin-1 (PSEN1) or presenilin-2 protein (PSEN2). Autosomal dominant inheritance accounts for about 1% of all cases of AD. When APP is cleaved by β- and γ-secretases, Aβ is formed with a length of 39 to 42 amino acid residues. Presenilins are part of the γ-secretase protease complex and are key catalytic subunits. In AD mutations in the APP and PSEN1, PSEN2 genes promote the formation of an extracellular fragment of Aβ with a length of 42 amino acid residues (Aβ42), the accumulation of which contributes to the formation of amyloid oligomers. Recently, it has been demonstrated that Aβ is generated intracellularly at the MAM domain and may influence functioning of the ER, mitochondria, and MAM. Given this finding and the importance of S1R at MAM domains, it is perhaps not surprising that common S1R polymorphisms influence risk of developing AD. In fact, certain genetic combinations of S1R and apolipoprotein E (APOE) genotypes synergistically increase the risk of AD.

41Several S1R agonists have anti-amnestic properties, overcoming learning and memory impairments from amyloid-β toxicity or scopolamine. S1R agonists promote neurogenesis in the hippocampus and they may mitigate memory impairment because they can stabilize mature, mushroom spines, which serve as sites of robust synaptic connections encoding lasting information. Mushroom spine loss may underlie memory defects in models of AD, as hippocampal neuron mushroom spines are lost in vitro and in vivo in both presenilin-1-M146V knock-in (PS1-KI) and APP knockin (APP-KI) models of AD. As postmortem and in vivo brain imaging studies have demonstrated a reduced density of S1R in the brains of patients with AD and S1R knockdown destabilizes mushroom spines, downregulation of S1R may contribute to AD pathology. Consistent with this, knockout of S1R in APPSwe AD mice increases oxidative stress within the hippocampus and exacerbates memory impairments. The novel positive S1R modulator (±)-2-(3-chlorophenyl)-3,3,5,5-tetramethyl-2-oxo-oxazaphosphinane (OZP002) was also neuroprotective in pharmacological and genetic models of AD. It potentiated the antidepressant effect of the S1R agonist igmesine and prevented scopolamine-induced learning deficits in the Y maze test and passive avoidance test. Its effect was blocked by NE-100 or in S1R knockout mice.

42Treatment of hippocampal cultures with Aβ42 oligomers induces loss of mushroom spines and Aβ42 accumulation in hippocampal cultures prepared from APP knock-in mice also causes mushroom spine loss. This is also observed in vivo. Pridopidine and 3-PPP prevent mushroom spine loss from both of these sources of Aβ toxicity in hippocampal neuronal cultures. Pridopidine treatment normalized synaptic functioning, preventing LTP deficits caused by Aβ42 oligomers. Pridopidine and 3-PPP also prevented mushroom spine loss in hippocampal cultures prepared from PS1-KI mice that model familial AD. Importantly, oral treatment with pridopidine rescued mushroom spines in vivo in PS1-KI mice, suggesting this as a viable treatment strategy for memory deficits in familial AD.

43AF710B also stabilized mushroom spines in vitro in hippocampal cultures prepared from AD mice (PS1-KI and APP-KI models). AF710B was found to potently and selectively stimulate the M1 muscarinic acetylcholine receptor (M1R) and S1R. AF710B binds to M1R outside of its orthosteric binding site, suggesting an allosteric mechanism of action. This is supported by data showing that 0.1 nM AF710B enhances the affinity and potency of the M1R agonist carbachol. The mechanism by which AF710B activates S1R is less clear, but the anti-amnestic properties of AF710B appear to require S1R, because the S1R antagonist NE-100 can suppress them. No significant binding was observed with other targets in a screen involving 83 GPCRs, ion channels and transporters. Treatment of 3xTg-AD mice with AF710B (10 μg/kg delivered by intraperitoneal injections daily for 2 months) reduced levels of BACE1, Aβ1–42, plaques, p25/CDK5, GSK-3β activity, Tau phosphorylation and memory deficits in the Morris water-maze. It was previously known that M1R activation improves cognition and reduces AD-like pathology in animal models, but the combined activity of AF710B at both M1R and S1R might make it particularly therapeutic. Indeed, in vivo treatment of McGill-R-Thy1-APP transgenic rats also reduced amyloid burden and inflammation while enhancing synaptogenesis and cognition. Another mixed muscarinic/σ1R agonist, ANAVEX2-73, was able to mitigate Aβ25–35-induced tau phosphorylation and Aβ1–42 seeding in mice and may help to preserve cognition in preliminary clinical trials clinical studies with AD patients.

44In addition to S1R agonists, positive allosteric modulators that do not compete with the (+)-pentazocine binding site might have therapeutic value. For example, SKF-83959 shows promise in the 6-OHDA-induced rat model of Parkinson’s disease. Also, OZP002 attenuated learning deficits from scopolamine, ICV injection of amyloid Aβ25–35, or the APPSwe transgene and protected against neurotoxicity associated with ICV injection of amyloid Aβ25–35. Several selective allosteric modulators for S1R have been discovered (methylphenylpiracetam and SOMCL-668) (see for a review) and it will be interesting to see whether they are efficacious in models of neurodegenerative diseases. Hinting at potential utility, SOMCL-668 enhanced (+)-SKF-10047-stimulated neurite growth and BDNF production in an S1R-dependent manner. Although both direct agonists and positive allosteric modulators may have therapeutic promise, it is unclear which are better candidates for clinical trials.

45Recent data suggests that S1R functionally interacts with presenilin 1 (PS1) and presenilin 2 (PS2), which are implicated in AD. Although cleaved PS1 is the catalytic subunit in the γ-secretase complex, the holoprotein version of PS1 functions as a passive leak channel in the ER membrane. Similarly, PS2 forms a calcium leak channel in the ER. Many familial AD-causing mutations in either PS1 or PS2 disrupt tonic Ca2+ release from the ER via PS1 and PS2 leak channels, increasing the concentration of calcium in the ER. Pridopidine promotes ER calcium homeostasis by decreasing luminal calcium levels in cultured hippocampal neurons from WT, PS1-KI and conditional presenilin double-knockout mice (PS1flox/flox/PS2–/–) infected with lenti-NLS-GFP as well as in neurons expression Cas9 and gRNA targeting the PS1 gene. However, this effect is lost in PS1flox/flox/PS2–/– hippocampal neurons infected with lenti-NLS-GFP-Cre. Knockout of PS1, PS2, or PS1/2 causes mushroom spine loss in hippocampal neurons. Consistent with functional data, pridopidine can compensate for this and restore mushroom spine integrity in presenilin 1 or presenilin 2 knockout neurons, but not in PS1/2 double knockout neurons. Consistent with the spine loss phenotype in PS1 and/or PS2 KO neurons, inactivation of PS1 in the mouse forebrain causes mild cognitive deficits, whereas inactivation of both PS1 and PS2 severely impacts cognition and synaptic plasticity, leading to neurodegeneration. PS1 and PS2 both contribute to resting ER calcium homeostasis and form a redundant ER calcium leak pathway that is required for pridopidine to decrease the concentration of ER calcium and stabilize mushroom spines. The reason for this is unclear, but S1R agonists might decrease ER calcium in hippocampal neurons by enhancing ER calcium leakage either by modulating leak activity of PS channels or increasing the prevalence of PS holoprotein in the ER membrane. The later possibility is intriguing because S1R might impede catalytic cleavage of PS1, reducing its incorporation into the γ-secretase complex. This could underlie the ability of S1R agonists to reduce Aβ42 accumulation and aggregation. However, more work is needed to characterize how S1R activity normalizes ER calcium homeostasis and mushroom spine prevalence while mitigating other hallmarks of AD neuropathology.

46When ER calcium levels are chronically elevated from mutations in presenilin 1, suppression of neuronal store-operated calcium entry (nSOC) leads to destabilization of hippocampal mushroom spines. Upregulating nSOC either pharmacologically or via overexpression of STIM2 or EB3 prevents PS1-KI mushroom spine loss. Consistent with pridopidine’s ability to decrease ER calcium levels in hippocampal neurons, it increased nSOC in the spines of cultured PS1-KI neurons and activity of the nSOC pathway was required for the rescue of PS1-KI mushroom spines. This suggests that pridopidine rescues PS1-KI mushroom spines through decreasing ER calcium levels and thereby stimulating nSOC pathway activity.

47Highlighting the importance of the client protein milieu in determining the effects of S1R activity, S1R suppresses store-operated calcium entry (SOC) in other cell types outside of the hippocampus. In non-neuronal cells, treatment with S1R agonists or overexpression of S1R or suppresses SOC. This may involve S1R binding to STIM1 and disrupting the interaction of STIM1 and Orai1. Additionally, in MSNs from YAC128 mice that model Huntington’s disease pridopidine decreases supranormal nSOC, which is synaptotoxic to MSNs. This contrasts with data involving hippocampal neurons in which pridopidine decreased ER calcium levels and enhanced nSOC. This divergence indicates that the effect of S1R in a given cell type depends on the availability of S1R interaction partners. For instance, InsP3R1 constitutes the primary ER calcium release pathway in MSNs, whereas presenilins preferentially mediate leakage of ER calcium in hippocampal neurons. Additionally, although S1R suppresses STIM1-dependent SOC in HEK293 and CHO cells, STIM2 is the predominant regulator of nSOC in hippocampal neurons. S1R might not bind and sequester STIM2 the way it does with STIM1 or if S1R does bind STIM2, it might enhance or minimally effect STIM2-gated nSOC. Thus, regulation of synaptic plasticity by S1R is likely to be multifaceted and highly dependent on the cellular context.

48Sigma-1 receptor is incredibly versatile in its ability to foster neuronal homeostasis in the context of several neurodegenerative disorders. Several S1R agonists are FDA-approved, such as fluvoxamine and donepezil and they may be repurposed for the treatment of several neurodegenerative diseases. Additional S1R agonists such as pridopidine have shown promising results in preclinical studies and in clinical trials.

49All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

50The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.