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By Emőke Borbély, Viktória Varga, Titanilla Szögi, Ildikó Schuster, Zsolt Bozsó, Botond Penke and Lívia Fülöp

Excerpt from the article published in Int. J. Mol. Sci. 2022, 23, 2514. https://doi.org/10.3390/ijms23052514

Editor’s Highlights

• Adult neurogenesis is essential for CNS plasticity. In early AD, neurogenic impairment can be observed, accompanied by hyperreactive astrogliosis.
• The highly selective S1R agonist, PRE084 (K_i = 2.2 nM), improved the proliferation and differentiation of hippocampal stem cells, manifesting in a quantitative increase in progenitor cells and immature neurons.

Abstract

Alzheimer’s disease (AD) is the most common form of dementia characterized by cognitive dysfunctions. Pharmacological interventions to slow the progression of AD are intensively studied. A potential direction targets neuronal sigma‐1 receptors (S1Rs). S1R ligands are recognized as promising therapeutic agents that may alleviate symptom severity of AD, possibly via preventing amyloid‐β‐(Aβ‐) induced neurotoxicity on the endoplasmic reticulum stress‐associated pathways.
Furthermore, S1Rs may also modulate adult neurogenesis, and the impairment of this process is reported to be associated with AD. We aimed to investigate the effects of two S1R agonists, dimethyltryptamine (DMT) and PRE084, in an Aβ‐induced in vivo mouse model characterizing neurogenic and anti‐neuroinflammatory symptoms of AD, and the modulatory effects of S1R agonists were analyzed by immunohistochemical methods and western blotting. DMT, binding moderately to S1R but with high affinity to 5‐HT receptors, negatively influenced neurogenesis, possibly as a result of activating both receptors differently. In contrast, the highly selective S1R agonist PRE084 stimulated hippocampal cell proliferation and differentiation. Regarding neuroinflammation, DMT and PRE084 significantly reduced Aβ1‐42‐induced astrogliosis, but neither had remarkable effects on microglial activation. In summary, the highly selective S1R agonist PRE084 may be a promising therapeutic agent for AD. Further studies are required to clarify the multifaceted neurogenic and anti‐neuroinflammatory roles of these agonists.

1. Introduction

Alzheimer’s disease (AD) is the most common form of dementia, characterized by progressive memory loss, impaired learning, and cognitive dysfunction. The main pathological hallmarks of AD are extracellular amyloid plaques and intracellular neurofibrillary tangles accumulated in the cerebral tissue [1], which first appear in the hippocampal and entorhinal regions of the brain, explaining the impairment of cognitive functions [2].
These changes are accompanied by the damage of synaptic connections, and neuronal death. The abnormal cleavage of amyloid precursor protein (APP) by β‐ and γ‐secretases predominantly yields 40 to 43 amino acid long amyloid‐β (Aβ) peptides, which aggregate, and manifest as cerebral deposits. Besides forming plaques, these oligomeric forms of Aβ are also thought to be neurotoxic [3–6]. These short oligomers might interfere with crucial intracellular mechanisms and signaling pathways. Thus, they may affect cell homeostasis, proliferation, differentiation, and survival [7–10]. Another significant symptom of AD is neuroinflammation, which involves various inflammatory components, such as immune cells, cytokines, and chemokines. Neuroinflammation might significantly alter neurogenesis, as well as enhancing Aβ production and plaque formation [11–13]. Currently, there is no cure for AD, and its progression cannot be prevented; at present, only symptomatic treatments of mild to moderate efficiency are available. Therefore, effective disease‐modifying therapeutics that may halt the progression of AD and contribute to the protection of neuronal integrity are eagerly awaited. A potentially new direction of the research aiming to find novel disease‐modulating agents targets the sigma receptors (SRs). SRs have received considerable attention for their potential role in the prevention of Aβ‐induced neurotoxicity, as well as in the regulation of the pathophysiology of AD. Furthermore, SRs may be essential for modulating neurogenesis in adulthood, and the stimulation of this process has been linked to AD. Thus, SR ligands are being recognized as promising therapeutic agents for treating or alleviating AD [6,14–16].
Two subtypes of SRs are distinguished, sigma‐1 receptor (S1R) and sigma‐2 receptor [17–19]. S1R is broadly expressed in the central nervous system (CNS), especially in the dentate gyrus (DG) region of the hippocampus (HC), both in neurons and glial cells. S1Rs are mainly located in a specific part of the cell where the endoplasmic reticulum (ER) and the mitochondria establish a tight interplay; this area is called the mitochondria‐associated ER membrane (MAM) [16,20–23]. S1R is known to influence neuronal survival, proliferation, neurite growth, plasticity, as well as learning and memory functions [24–27]. It has been reported that the expression level of S1R decreases in patients with neurodegenerative diseases like AD [16,22,23,28–33].
S1R binds a diverse set of molecules, for example, antipsychotics, antidepressants, and neurosteroids [34–37]. A non‐specific endogenous ligand of S1R is N,N‐dimethyltryptamine (DMT), a hallucinogenic agent assumed to be produced in small quantities and accumulated in the CNS [16,38–40]. Previous studies have shown that the administration of DMT modulates many ion channels [39], protects against hypoxia‐induced damage [41], alleviates neuroinflammation [42,43], increases the density of dendritic spines [44], as well as promotes neurogenesis and neuritogenesis [45–49]. However, DMT might also exert anxiogenic, neuro‐ and cytotoxic effects [47,50–52]. DMT is known to bind to several receptors with different affinities: 5‐hydroxytryptamine (5‐HT)1A‐B, 5‐HT1D, 5‐HT2A‐C, 5‐HT5A, 5‐HT6, 5‐HT7 receptors, S1R, SERT, dopamine (D)1‐5 receptors, α1AR, I1‐3, TAAR, NMDA [53–55]. Several adverse effects of DMT are primarily associated with the stimulation of 5‐HT2A receptors [47,50,51,53,56], while its positive impacts are rather related to the activation of S1Rs [40–44,46,49,50,52,57]. Moreover, the inflammation regulatory and plasticity promoting activities of DMT are also considered to result from its binding to both the S1Rs and 5‐HT receptors. Identifying the valid contributor molecules and signaling pathways behind this assumption requires more convincing evidence.
Many exogenous ligands of S1R have been identified, including (+)‐pentazocine, fluvoxamine, ANAVEX2‐73, and 2‐(4‐morpholinethyl)‐1‐phenylcyclohexanecarboxylate (PRE084) [16,22,24,58]. The antidepressant and nootropic properties of PRE084 are also recognized [59]. Based on our current knowledge, PRE084 may promote neuroprotection and neurite growth by stimulating the expression of different neurotrophic factors, as well as by activating signaling pathways involved in cell survival [60–65]. Previous studies suggest that this S1R‐agonist might positively impact learning and memory, as demonstrated in animal models of neurodegenerative diseases or traumatic brain injuries [63,64,66]. It is also reported that after the administration of Aβ25‐35 infusion into the right lateral ventricle of mice, PRE084 administration has moderated the adverse behavioral effects of Aβ25‐35 [27] via reducing neurotoxicity‐induced cell death [32,64]. Moreover, PRE084 may also promote neurogenesis [9] and cell survival by attenuating excitotoxicity and reducing microglial activity, as well as diminishing the expression of proinflammatory factors [67,68].
As mentioned above, in addition to its ability to support cell survival under stress conditions, activated S1Rs may also stimulate the formation of new neurons, even in the adult brain. In adulthood, mammalian neurogenesis is derived from neuronal stem cells (NSCs) located in the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus (HC), as well as from NSCs in the subventricular region of the lateral ventricles [69,70]. After differentiation and migration, these newly formed neurons can integrate into local neuronal circuits of the HC; thus, they might have a significant role in plasticity, cognitive functions, learning, and memory processes [71]. An optimal microenvironment is essential for the division, differentiation, migration, and maturation of NSCs. Physiologically, the activity of adult hippocampal neurogenesis decreases with aging, leading to a usually mild, age‐associated cognitive decline. However, a growing body of evidence indicates that the extent of adult neurogenesis is sharply diminished in the early stages of AD, even before the appearance of senile plaques [72–78]. This finding raises the question of whether impaired neurogenesis may initiate and/or contribute to more severe cognitive deficits, thus mediating AD’s pathogenesis. Furthermore, these findings suggest that the stimulation of neurogenesis might serve as a therapeutic target in AD, with a potential to improve cognitive functions and promote neural adaptability, thereby it might prevent or even treat AD.
In this study, two main objectives were addressed. First, to induce early acute AD like impairments in neurogenesis and generate neuroinflammation in adult wild‐type C57BL/6 mice by the intracerebroventricular (ICV) administration of Aβ1‐42 oligomers. In this experimental paradigm, we followed the administration protocol described by Li et al., who examined the effects of Aβ25‐35 on the same processes [9]. They reported that Aβ25‐35 stimulated the proliferation of neuronal progenitor cells, while enhancing the death of newly formed neurons and impaired neurite growth. Secondly, we attempted to restore the normal functioning of adult neurogenesis and reduce neuroinflammation by activating S1Rs with two different ligands, PRE084 and DMT. The intraperitoneally‐(IP‐)‐injected compounds were tested in wild‐type mice, either treated with Aβ1‐42‐oligomers or injected with vehicle (phosphate buffered saline (PBS)) as a control. Based on previously published articles on the beneficial effects of these S1R modulators, we expected to detect an obvious positive impact of the tested agents on the Aβ1‐42‐induced impairments in adult neurogenesis and neuroinflammation [41–43,49,52,57,60–64,79].

2. Results

2.1. Effects of PRE084 and DMT on Adult Neurogenesis in Aβ_1–42 and Vehicle-Treated Mice

Aβ_1–42 and DMT impair, while PRE084 promotes the survival of progenitor cells in DG.Proliferating cells were labeled by three IP injections of 5-Bromo-2′-Deoxyuridine (BrdU) with a 6 h interval, which was administered 24 h after the stereotaxic surgery. BrdU is a synthetic thymidine analog, which incorporates into the DNA strand, and can be detected by specific antibodies. We counted BrdU+ cells 14 days after the surgery. According to our results, the quantity of BrdU+ stem cells in the SGZ of the DG significantly differed among the six groups (ANOVA: p ≤ 0.0001). Aβ_1–42 infusion significantly reduced the number of progenitor cells compared to the respective control group (PBS-PBS vs. Aβ_1–42-PBS p = 0.001). Interestingly, significantly more severe negative changes were detected in animals treated with DMT. In those co-treated with both Aβ_1–42 and DMT, hardly any BrdU+ stem cells were detected in the SGZ (Aβ_1–42-DMT vs. PBS-PBS p ≤ 0.0001, vs. Aβ_1–42-PBS p = 0.005, vs. Aβ_1–42-PRE084 p ≤ 0.0001; PBS-DMT vs. PBS-PBS p = 0.001, vs. PBS-PRE084 p ≤ 0.0001). PRE084 treatment increased the amount of BrdU+ cells; the difference between the Aβ_1–42-infused groups was significant (Aβ_1–42-PBS vs. Aβ_1–42-PRE084 p ≤ 0.0001) (Figure 1).

Aβ_1–42 and PRE084 increase the number of premature cells, while DMT does not affect their quantity.To understand the effects of PRE084 and DMT on the maturation of granule cells, we quantified immature neurons in the SGZ of DG. To label premature cells, we stained a microtubule-associated protein called doublecortin (DCX), which is expressed specifically in migrating neuronal precursors. The measured DCX densities were significantly different among the six groups (ANOVA: p ≤ 0.0001). In those treated with Aβ_1–42-PBS and PBS-PRE084, the number of immature neurons was significantly higher compared to the control group (PBS-PBS vs. Aβ_1–42-PBS p = 0.037, vs. PBS-PRE084 p ≤ 0.0001, vs. Aβ_1–42-PRE084 p ≤ 0.0001). We also detected a significant difference between the Aβ_1–42-PBS and Aβ_1–42-PRE084 mice groups (p = 0.007). DMT administration did not affect the number of premature neurons compared to PBS-PBS mice (Figure 2).

The density of mature granule cells is unaffected by Aβ_1–42 or PRE084 administration, while DMT induces a decrease in neuronal density.To detect and evaluate mature granule cells in the HC, we performed neuronal nuclei (NeuN) immunostaining (Figure 3). Again, significant differences were observed among the groups (ANOVA: p = 0.001). In DMT-treated animals, significantly lower NeuN+ cell densities were evident in the HC compared to the PBS-PBS and Aβ_1–42-PBS group (PBS-PBS vs. PBS-DMT p = 0.001, vs. Aβ_1–42-DMT p = 0.022; Aβ_1–42-PBS vs. PBS-DMT p ≤ 0.0001, vs. Aβ_1–42-DMT p = 0.003).

2.2. Effects of PRE084 and DMT on Neuroinflammation Induced by Aβ_1–42

Aβ_1–42 stimulates microglia activation, and neither PRE084, nor DMT alleviate this effect, while DMT alone significantly decreases microglial density.Neuroinflammation results from the activation of an immune response in the CNS, mediated by microglia and astrocytes. This process is induced by infective agents, neurodegenerative diseases, or injuries. To identify activated microglia in the HC, we stained ionized calcium-binding adapter molecule 1 (Iba1), expressed explicitly by monocyte-derived and resident macrophages, including microglia. Our results showed a significant difference in the density of Iba1+ microglia among the groups (ANOVA: p = 0.002). Aβ_1–42 administration significantly increased the density of activated microglia compared to the vehicle-treated control groups (PBS-PBS vs. Aβ_1–42-PBS p = 0.015; PBS-PRE084 vs. Aβ_1–42-PRE084 p = 0.035; PBS-DMT vs. Aβ_1–42-DMT p = 0.039). In the PBS-DMT group, the density of Iba1+ microglia was significantly reduced compared to PBS-PBS-treated animals (PBS-PBS vs. PBS-DMT p = 0.031). Still, none of the treatments were found to be able to alleviate the proinflammatory effect of Aβ_1–42 (Figure 4).

Aβ_1–42 stimulates astrocyte reactivation, while the administration of DMT or PRE084 reduces this effect.Reactive astrocytes were immunostained for glial fibrillary acidic protein (GFAP), an intermediate filament protein expressed by different cell types, mainly reactive astrocytes, in the CNS. Significantly different GFAP+ cell densities were detected in the HC of the different groups (ANOVA: p = 0.002). A significant increase in the rate of reactivated astrocytes was detected in the Aβ_1–42-PBS group compared to PBS-PBS-treated mice (p ≤ 0.0001). Furthermore, GFAP+ cell densities were significantly lower in all other groups compared to Aβ_1–42-PBS-treated mice (Aβ_1–42-PBS vs. PBS-PRE084 p = 0.013, vs. Aβ_1–42-PRE084 p = 0.013, vs. PBS-DMT p ≤ 0.0001, vs. Aβ_1–42-DMT, p = 0.001). The stimulatory effect of Aβ_1–42 on astrocyte reactivation was alleviated by PRE084 and DMT administration (Figure 5).

The activation of inflammatory processes was assessed by the determination of certain proinflammatory cytokines (IL1β and TNFα). The levels of both pro- IL1β and soluble IL1β, as well as membrane-bound TNFα and soluble TNFα, were determined by western blot analyses (see Supplement Figure S1). These results corroborate our findings regarding the activation of the glial immunodefense system in response to the Aβ_1–42 stimulus. The production of the active cytokine forms could be modulated by DMT-treatment; however, only the change in TNFα-level was significant.

2.3. S1R Protein Level Is Elevated by Aβ_1–42 Treatment, as Well as by the Co-Administration of Aβ_1–42 and PRE084 or DMT

To determine the effects of Aβ_1–42 and PRE084 or DMT on the expression of S1R, a western blot (WB) analysis using GAPDH loading control was performed on HC and cerebral cortex samples of three animals per group. Our findings revealed a significant difference in the S1R levels among the groups (ANOVA: p ≤ 0.0001). S1R protein levels were significantly elevated in all groups, except in PBS-DMT-treated animals, as compared to control subjects (PBS-PBS vs. Aβ_1–42-PBS p ≤ 0.0001, vs. PBS-PRE084 p = 0.018, vs. Aβ_1–42-PRE084 p ≤ 0.0001, vs. PBS-DMT p = 0.540; vs. Aβ_1–42-DMT p ≤ 0.0001, respectively). In comparison with Aβ_1–42-PBS-treated mice, the Aβ_1–42-PRE084 (p = 0.004) and Aβ_1–42-DMT (p = 0.673) groups showed higher protein levels, while significantly lower levels of S1R were detected in PBS-PRE084 (p = 0.032) and PBS-DMT (p = 0.001) treated mice. As expected, the co-administration of Aβ_1–42 and either of the S1R agonists increased the S1R protein level compared to the respective control group (Aβ_1–42-PRE084 vs. PBS-PRE084 p ≤ 0.0001; Aβ_1–42-DMT vs. PBS-DMT p = 0.015). Notably, the expression of S1R was significantly increased in Aβ_1–42-PRE084-treated animals compared to the Aβ_1–42-DMT group (p ≤ 0.0001). (Figure 6).

3. Discussion

During neurogenesis in adulthood, new neurons continuously develop and differentiate from hippocampal stem cells, and are integrated into existing neuronal networks to maintain plasticity of the CNS, and thereby preserve learning and memory functions. It has been recognized that the formation of new neurons reduces with age, manifesting in impaired cognitive functions [80]. In certain neurodegenerative diseases this cluster of mental symptoms is much more pronounced due to a decreased rate of neurogenesis, increased destruction of mature neurons, and enhanced neuroinflammatory responses. The most prevalent disease of this kind is AD, characterized by progressive dementia. Early alternations in adult neurogenesis and neuroinflammation may appear several years or even a decade before the diagnosis of AD, and probably contributes to the onset of neurological symptoms. It is hypothesized that an intensive stimulation of hippocampal neurogenesis and the reduction in neuroinflammation in adulthood could slow down the rate of decline of cognitive skills. Moreover, the uniquely structured S1R protein, functioning as a ligand‐operated chaperone, is known to play a major role in both neurogenesis and neuroinflammation. Thus, it is assumed that the activation of S1Rs may be a promising therapeutic strategy to stimulate adult neurogenesis and alleviate neuroinflammatory processes.
The first objective of our study was to model these early alternations appearing in AD. Our experimental paradigm was based on the work of Li et al., in a modified way: instead of Aβ25‐35, we injected Aβ1‐42 ICV to induce early AD‐like changes [9]. The reason for this modification is that Aβ25‐35 is a non‐natural, truncated sequence, and although it is prone to aggregation, its kinetics for aggregation differ from that of the native Aβ1‐42 peptide.
Therefore, using this latter peptide should yield biologically more relevant findings [81]. In the work of Li et al., neurogenesis was assessed 14 and 28 days after the peptide injections, and significant differences were detected on day 28 in neurogenic markers compared to baseline (reduced proliferation and neurite growth, increased death of newly formed cells) [9]. In our experimental model, AD‐like cerebral neurogenic and neuroinflammatory changes could be detected as early as two weeks after the administration of Aβ1‐42. We demonstrated that a single administration of Aβ1‐42, directly into the lateral ventricles, significantly impaired the proliferation and increased the number of immature cells in mice. The effects of Aβ on neurogenesis are highly controversial in the literature.
Numerous reports indicate that Aβ significantly decreases the formation of new neurons, possibly by impairing their ability to divide, as well as by diminishing the survival of neuronal stem cells in DG [7–9,75–77,82]. However, some research groups have published that Aβ can induce the initial proliferation step of neuron formation in different transgenic mouse strains [9,78,83–85] or in cellular models of AD [86–91]. In our experiments, an increase in the number of differentiating immature neurons was observed in Aβ1‐42‐treated animals, which may be explained by a compensatory cerebral mechanism [77,92]. Specifically, this enhancement of neuronal cell differentiation may be a response to the disturbed homeostasis resulting from the decrease in the stem cell population, aiming to restore the balance within the CNS. As we expected, in our experimental model, no significant reduction was detected in the density of mature, functional neurons in HC two weeks after the administration of Aβ1‐42, indicating that the existing neuronal system may remain unaffected.
Regarding neuroinflammation, we found that a single administration of Aβ1‐42 stimulated neuroinflammatory processes, causing a significant increase in the densities of activated microglia and hyperreactive astrocytes. In line with our observations, several in vivo experiments have demonstrated the neuroinflammation‐inducing effects of Aβ fibrils and oligomers injected into the brain tissue in different experimental models [93–95].
This neuroinflammatory environment may affect adult neurogenesis either positively or negatively [11,12,96–101]. It is known that cytokines and chemokines produced by activated microglia and astrocytes play an important role in neuroinflammatory processes.
Certain anti‐(IL‐4, IL‐10) and proinflammatory (IL‐6, TNF‐α) factors substantially influence neurogenesis, e.g., they can diminish proliferation and cell survival, while they may also stimulate cell differentiation [13]. Thus, beyond its direct effects on immature neurons, Aβ1‐42 may also affect neurogenesis by generating a relatively mild, but chronic neuroinflammatory environment. Further research is needed to clarify the relative contribution of these two processes (direct and indirect) to the final decline of adult neurogenesis in AD.
Since the S1R protein plays a major role in neurogenesis and neuroinflammation, and changes in S1R expression levels have not been studied in exogenous Aβ‐induced AD models, we examined the expression levels of this protein. In our case, the expression of S1R increased after a single administration of Aβ1‐42. This finding may contradict some literature data, which report on the down‐regulation of S1R in the early stage of human AD [24]. In the reported cases, both the amount and the binding potential of S1R were found to be decreased, presumably as a consequence of hippocampal neuronal death [24,102–105]. In contrast, other studies indicate that AD‐related ER‐stress can lead to an up‐regulation of S1R [16,29,106,107], which, serving as a chaperon, modulates the canonical unfolded protein response (UPR) pathways (PERK, IRE1a, ATF6) [16,108]. In our study, the observed elevation of the level of S1R may be a consequence of the cytotoxic effect of Aβ1–42, which induces ER stress, and thus activates the UPR pathways and upregulates S1R expression.
To date, the biological effects of DMT and PRE084 have not been studied in an Aβ‐induced model of early AD with demonstrated changes in neurogenesis and S1R expression levels, as well as neuroinflammation. Therefore, we aimed to assess whether the modulation of S1Rs with selected ligands can restore Aβ1‐42‐induced alternations in adult neurogenesis and reduce neuroinflammation.

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4. Conclusions

Adult neurogenesis is essential for CNS plasticity. In early AD, neurogenic impairment can be observed, accompanied by hyperreactive astrogliosis. During the treatment of AD, neurogenesis should be promoted, while neuroinflammation should be suppressed.
S1R plays a role in both processes. In our experiments, we established a model of early AD induced by Aβ1‐42, in which acute neuroinflammation, impaired neurogenesis and elevated S1R levels were detected. In this model, two S1R agonists were tested. DMT, binding moderately to S1R but with a high affinity to 5‐HT receptors, negatively influenced neurogenesis in the Aβ1‐42‐induced rodent model, probably explained by its acting on the latter receptor class. In contrast, the highly selective S1R agonist, PRE084 improved the proliferation and differentiation of hippocampal stem cells, manifesting in a quantitative increase in progenitor cells and immature neurons. Further experiments are required to investigate the main molecular pathways targeted by DMT, through which it affects neurogenesis and the survival of mature neurons. Moreover, DMT and PRE084 were found to significantly reduce Aβ1‐42‐induced hyperreactive astrogliosis. However, none of these ligands had a remarkable effect on microglial activation. Therefore, further studies are needed to clarify the role of DMT and PRE084 in neuroinflammatory processes induced by Aβ1‐42, resembling the changes characteristic of AD.