Loss of Sigma-2 Receptor/TMEM97 Is Associated with Neuropathic Injury-Induced Depression-Like Behaviors in Female Mice.

Previous studies have shown that ligands that bind to sigma-2 receptor/TMEM97 (s2R/TMEM97), a transmembrane protein, have anxiolytic/antidepressant-like properties and relieve neuropathic pain-like effects in rodents. Despite medical interest in s2R/TMEM97, little affective and pain behavioral characterization has been done using transgenic mice, which limits the development of s2R/TMEM97 as a viable therapeutic target. Using wild-type (WT) and global Tmem97 knock-out (KO) mice, we sought to identify the contribution of Tmem97 in modulating affective and pain-like behaviors using a battery of affective and pain assays, including open field, light/dark preference, elevated plus maze, forced swim test, tail suspension test, and the mechanical sensitivity tests. Our results demonstrate that female Tmem97 KO mice show less anxiety-like and depressive-like behaviors in light/dark preference and tail suspension tests but not in an open field, elevated plus maze, and forced swim tests at baseline. We next performed spared nerve injury in WT and Tmem97 KO mice to assess the role of Tmem97 in neuropathic pain-induced anxiety and depression. WT mice, but not Tmem97 KO mice, developed a prolonged neuropathic pain-induced depressive-like phenotype when tested 10 weeks after nerve injury in females. Our results show that Tmem97 plays a role in modulating anxiety-like and depressive-like behaviors in naive animals with a significant change in the presence of nerve injury in female mice. Overall, these data demonstrate that Tmem97 could be a target to alleviate affective comorbidities of pain disorders.

Recent Developments in Sigma-2 Receptor Compounds for Pain.

After years of enigmatic pharmacology, non-selective ligands, and uncertain clinical application, sigma receptors have emerged as interesting therapeutic drug discovery-development targets. Two subtypes of sigma receptors have now been cloned, sigma-1 receptor (S1R) and sigma-2 receptor (S2R), and there has been much complementary and converging information from advances in molecular biology, computer modeling, virtual screening, and in vitro and in vivo testing. One of several evolving areas of therapeutic potential is for the treatment of pain. In particular, there is accumulating recent evidence from preclinical models that the demonstrated positive effects of S2R compounds in these models suggest possible positive implications for clinical effectiveness against pains that have a neuropathic component. Such pain conditions have imperfect therapeutic options currently. The addition of new drugs to the now available armamentarium would represent a very significant advance for the large number of patients who suffer from these types of intractable pain. Further research is needed to identify and characterize compounds that have not only good in vitro activity but also the characteristics needed to enter clinical trials. Here, we summarize some of the recent reports of the analgesic activity of S2R compounds.

TMEM97 knockdown inhibits 5-fluorouracil resistance by regulating epithelial-mesenchymal transition and ABC transporter expression via inactivating the Akt/mTOR pathway in 5-fluorouracil-resistant colorectal cancer cells.

Resistance to 5-fluorouracil (5-FU) is still a primary setback to the success of colorectal cancer (CRC) chemotherapy. Transmembrane protein 97 (TMEM97) functions as an oncogene in CRC. However, the role and mechanism of TMEM97 in regulating 5-FU resistance in CRC cells remains unclear. TMEM97 expression in CRC samples was analyzed by GEPIA and human protein atlas (HPA) databases. TMEM97, E-cadherin, Vimentin, N-cadherin, P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1)/ABCC1, ABCC2, and the changes of protein kinase B/mammalian target of rapamycin (mTOR) pathway were explored by western blot analysis. IC50 value for 5-FU and cell viability was examined by MTT assay. Apoptosis was evaluated by flow cytometry. TMEM97 was highly expressed in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) based on GEPIA and HPA databases. TMEM97 knockdown attenuated 5-FU resistance in HCT116/R and SW480/R cells, as evidenced by the reduced IC50 value for 5-FU and the increased apoptosis. TMEM97 knockdown suppressed epithelial-mesenchymal transition (EMT), expression of ATP-binding cassette (ABC) transporters, and the Akt/mTOR pathway. Mechanistically, activation of Akt/mTOR pathway abolished the inhibitory effects of TMEM97 knockdown on 5-FU resistance, EMT, and ABC transporter expression. In conclusion, TMEM97 knockdown inhibited 5-FU resistance in CRC by regulating EMT and ABC transporter expression via inactivating the Akt/mTOR pathway.

Transmembrane protein 97 is a potential synaptic amyloid beta receptor in human Alzheimer's disease.

Synapse loss correlates with cognitive decline in Alzheimer's disease, and soluble oligomeric amyloid beta (Aß) is implicated in synaptic dysfunction and loss. An important knowledge gap is the lack of understanding of how Aß leads to synapse degeneration. In particular, there has been difficulty in determining whether there is a synaptic receptor that binds Aß and mediates toxicity. While many candidates have been observed in model systems, their relevance to human AD brain remains unknown. This is in part due to methodological limitations preventing visualization of Aß binding at individual synapses. To overcome this limitation, we combined two high resolution microscopy techniques: array tomography and Förster resonance energy transfer (FRET) to image over 1 million individual synaptic terminals in temporal cortex from AD (n = 11) and control cases (n = 9). Within presynapses and post-synaptic densities, oligomeric Aß generates a FRET signal with transmembrane protein 97. Further, Aß generates a FRET signal with cellular prion protein, and post-synaptic density 95 within post synapses. Transmembrane protein 97 is also present in a higher proportion of post synapses in Alzheimer's brain compared to controls. We inhibited Aß/transmembrane protein 97 interaction in a mouse model of amyloidopathy by treating with the allosteric modulator CT1812. CT1812 drug concentration correlated negatively with synaptic FRET signal between transmembrane protein 97 and Aß. In human-induced pluripotent stem cell derived neurons, transmembrane protein 97 is present in synapses and colocalizes with Aß when neurons are challenged with human Alzheimer's brain homogenate. Transcriptional changes are induced by Aß including changes in genes involved in neurodegeneration and neuroinflammation. CT1812 treatment of these neurons caused changes in gene sets involved in synaptic function. These data support a role for transmembrane protein 97 in the synaptic binding of Aß in human Alzheimer's disease brain where it may mediate synaptotoxicity.

Transmembrane protein TMEM97 and epigenetic reader BAHCC1 constitute an axis that supports pro-inflammatory cytokine expression.

Pro-inflammatory cytokine production by the retinal pigment epithelium (RPE) is a key etiology in retinal degenerative diseases, yet the underlying mechanisms are not well understood. TMEM97 is a scarcely studied transmembrane protein recently implicated in retinal degeneration. BAH domain coiled coil 1 (BAHCC1) is a newly discovered histone code reader involved in oncogenesis. A role for TMEM97 and BAHCC1 in RPE inflammation was not known. Here we found that they constitute a novel axis regulating pro-inflammatory cytokine expression in RPE cells. Transcriptomic analysis using a TMEM97-/- ARPE19 human cell line and the validation via TMEM97 loss- and gain-of-function revealed a profound role of TMEM97 in promoting the expression of pro-inflammatory cytokines, notably IL1ß and CCL2, and unexpectedly BAHCC1 as well. Moreover, co-immunoprecipitation indicated an association between the TMEM97 and BAHCC1 proteins. While TMEM97 ablation decreased and its overexpression increased NFκB (p50, p52, p65), the master transcription factor for pro-inflammatory cytokines, silencing BAHCC1 down-regulated NFκB and downstream pro-inflammatory cytokines. Furthermore, in an RPE-damage retinal degeneration mouse model, immunofluorescence illustrated down-regulation of IL1ß and CCL2 total proteins and suppression of glial activation in the retina of Tmem97-/- mice compared to Tmem97+/+ mice. Thus, TMEM97 is a novel determinant of pro-inflammatory cytokine expression acting via a previously unknown TMEM97- > BAHCC1- > NFκB cascade. SYNOPSIS: Retinal pigment epithelium (RPE) inflammation can lead to blindness. We identify here a previously uncharacterized cascade that underlies RPE cell production of pro-inflammatory cytokines. Specifically, transmembrane protein TMEM97 positively regulates the recently discovered histone code reader BAHCC1, which in turn enhances pro-inflammatory cytokine expression via the transcription factor NFκB.

TMEM97/Sigma 2 Receptor Increases Estrogen Receptor α Activity in Promoting Breast Cancer Cell Growth.

Aberrant estrogen receptor (ER) signaling is a major driver of breast tumor growth and progression. Sigma 2 receptor has long been implicated in breast carcinogenesis based on pharmacological studies, but its molecular identity had been elusive until TMEM97 was identified as the receptor. Herein, we report that the TMEM97/sigma 2 receptor is highly expressed in ER-positive breast tumors and its expression is strongly correlated with ERs and progesterone receptors (PRs) but not with HER2 status. High expression levels of TMEM97 are associated with reduced overall survival of patients. Breast cancer cells with increased expression of TMEM97 had a growth advantage over the control cells under both nutrition-limiting and sufficient conditions, while the knockdown of TMEM97 expression reduced tumor cell proliferations. When compared to their vector control cells, MCF7 and T47D cells with increased TMEM97 expression presented increased resistance to tamoxifen treatment and also grew better under estrogen-depleted conditions. The TMEM97/sigma 2 receptor enhanced the ERα transcriptional activities and increased the expression of genes responsive to estrogen treatment. Increased TMEM97 also stimulated the mTOR/S6K1 signaling pathways in the MCF7 and T47D cells. The increased level of active, phosphorylated ERα, and the enhanced resistance to tamoxifen treatment with increased TMEM97, could be blocked by an mTOR inhibitor. The knockdown of TMEM97 expression reduced the ERα and mTOR/S6K1 signaling activities, rendering the cells with an increased sensitivity to tamoxifen. The observations suggest that the TMEM97/sigma 2 receptor is a novel regulator of ERα activities in breast tumor cell growth.

Highly specific σ2R/TMEM97 ligand FEM-1689 alleviates neuropathic pain and inhibits the integrated stress response.

The sigma 2 receptor (σ2R) was described pharmacologically more than three decades ago, but its molecular identity remained obscure until recently when it was identified as transmembrane protein 97 (TMEM97). We and others have shown that σ2R/TMEM97 ligands alleviate mechanical hypersensitivity in mouse neuropathic pain models with a time course wherein maximal antinociceptive effect is approximately 24 h following dosing. We sought to understand this unique antineuropathic pain effect by addressing two key questions: do these σ2R/TMEM97 compounds act selectively via the receptor, and what is their downstream mechanism on nociceptive neurons? Using male and female conventional knockout mice for Tmem97, we find that a σ2R/TMEM97 binding compound, FEM-1689, requires the presence of the gene to produce antinociception in the spared nerve injury model in mice. Using primary mouse dorsal root ganglion neurons, we demonstrate that FEM-1689 inhibits the integrated stress response (ISR) and promotes neurite outgrowth via a σ2R/TMEM97-specific action. We extend the clinical translational value of these findings by showing that FEM-1689 reduces ISR and p-eIF2α levels in human sensory neurons and that it alleviates the pathogenic engagement of ISR by methylglyoxal. We also demonstrate that σ2R/TMEM97 is expressed in human nociceptors and satellite glial cells. These results validate σ2R/TMEM97 as a promising target for further development for the treatment of neuropathic pain.

Regulation of cholesterol metabolism: New players for an old physiological process.

Identified more than two centuries ago, cholesterol plays a pivotal role in human physiology. Since cholesterol metabolism is a physiologically significant process, it is not surprising that its alterations are associated with several pathologies. The discovery of new molecular targets or compounds able to modulate this sophisticated metabolism has been capturing the attention of research groups worldwide since many years. Endogenous and exogenous compounds are known to regulate cellular cholesterol synthesis and uptake, or reduce cholesterol absorption at the intestinal level, thereby regulating cholesterol homeostasis. However, there is a great need of new modulators and diverse new pathways have been uncovered. Here, after illustrating cholesterol metabolism and its well-known regulators, some new players of this important physiological process are also described.

BS148 Reduces the Aggressiveness of Metastatic Melanoma via Sigma-2 Receptor Targeting.

The management of advanced-stage melanoma is clinically challenging, mainly because of its resistance to the currently available therapies. Therefore, it is important to develop alternative therapeutic strategies. The sigma-2 receptor (S2R) is overexpressed in proliferating tumor cells and represents a promising vulnerability to target. Indeed, we have recently identified a potent S2R modulator (BS148) that is effective in melanoma. To elucidate its mechanism of action, we designed and synthesized a BS148 fluorescent probe that enters SK-MEL-2 melanoma cells as assessed using confocal microscopy analysis. We show that S2R knockdown significantly reduces the anti-proliferative effect induced by BS148 administration, indicating the engagement of S2R in BS148-mediated cytotoxicity. Interestingly, BS148 treatment showed similar molecular effects to S2R RNA interference-mediated knockdown. We demonstrate that BS148 administration activates the endoplasmic reticulum stress response through the upregulation of protein kinase R-like ER kinase (PERK), activating transcription factor 4 (ATF4) genes, and C/EBP homologous protein (CHOP). Furthermore, we show that BS148 treatment downregulates genes related to the cholesterol pathway and activates the MAPK signaling pathway. Finally, we translate our results into patient-derived xenograft (PDX) cells, proving that BS148 treatment reduces melanoma cell viability and migration. These results demonstrate that BS148 is able to inhibit metastatic melanoma cell proliferation and migration through its interaction with the S2R and confirm its role as a promising target to treat cancer.

Highly specific σ2R/TMEM97 ligand alleviates neuropathic pain and inhibits the integrated stress response.

The Sigma 2 receptor (σ2R) was described pharmacologically more than three decades ago, but its molecular identity remained obscure until recently when it was identified as transmembrane protein 97 (TMEM97). We and others have shown that σ2R/TMEM97 ligands alleviate mechanical hypersensitivity in mouse neuropathic pain models with a time course wherein maximal anti-nociceptive effect is approximately 24 hours following dosing. We sought to understand this unique anti-neuropathic pain effect by addressing two key questions: do these σ2R/TMEM97 compounds act selectively via the receptor, and what is their downstream mechanism on nociceptive neurons? Using male and female conventional knockout (KO) mice for Tmem97, we find that a new σ2R/TMEM97 binding compound, FEM-1689, requires the presence of the gene to produce anti-nociception in the spared nerve injury model in mice. Using primary mouse dorsal root ganglion (DRG) neurons, we demonstrate that FEM-1689 inhibits the integrated stress response (ISR) and promotes neurite outgrowth via a σ2R/TMEM97-specific action. We extend the clinical translational value of these findings by showing that FEM-1689 reduces ISR and p-eIF2α levels in human sensory neurons and that it alleviates the pathogenic engagement of ISR by methylglyoxal. We also demonstrate that σ2R/TMEM97 is expressed in human nociceptors and satellite glial cells. These results validate σ2R/TMEM97 as a promising target for further development for the treatment of neuropathic pain.

Sigma-2 Receptors-From Basic Biology to Therapeutic Target: A Focus on Age-Related Degenerative Diseases.

There is a large unmet medical need to develop disease-modifying treatment options for individuals with age-related degenerative diseases of the central nervous system. The sigma-2 receptor (S2R), encoded by TMEM97, is expressed in brain and retinal cells, and regulates cell functions via its co-receptor progesterone receptor membrane component 1 (PGRMC1), and through other protein-protein interactions. Studies describing functions of S2R involve the manipulation of expression or pharmacological modulation using exogenous small-molecule ligands. These studies demonstrate that S2R modulates key pathways involved in age-related diseases including autophagy, trafficking, oxidative stress, and amyloid-ß and α-synuclein toxicity. Furthermore, S2R modulation can ameliorate functional deficits in cell-based and animal models of disease. This review summarizes the current evidence-based understanding of S2R biology and function, and its potential as a therapeutic target for age-related degenerative diseases of the central nervous system, including Alzheimer's disease, α-synucleinopathies, and dry age-related macular degeneration.

Sigma-2 Receptor Ligand Binding Modulates Association between TSPO and TMEM97.

Sigma-2 receptor (S2R) is a S2R ligand-binding site historically associated with reportedly 21.5 kDa proteins that have been linked to several diseases, such as cancer, Alzheimer's disease, and schizophrenia. The S2R is highly expressed in various tumors, where it correlates with the proliferative status of the malignant cells. Recently, S2R was reported to be the transmembrane protein TMEM97. Prior to that, we had been investigating the translocator protein (TSPO) as a potential 21.5 kDa S2R candidate protein with reported heme and sterol associations. Here, we investigate the contributions of TMEM97 and TSPO to S2R activity in MCF7 breast adenocarcinoma and MIA PaCa-2 (MP) pancreatic carcinoma cells. Additionally, the role of the reported S2R-interacting partner PGRMC1 was also elucidated. Proximity ligation assays and co-immunoprecipitation show a functional association between S2R and TSPO. Moreover, a close physical colocalization of TMEM97 and TSPO was found in MP cells. In MCF7 cells, co-immunoprecipitation only occurred with TMEM97 but not with PGRMC1, which was further confirmed by confocal microscopy experiments. Treatment with the TMEM97 ligand 20-(S)-hydroxycholesterol reduced co-immunoprecipitation of both TMEM97 and PGRMC1 in immune pellets of immunoprecipitated TSPO in MP cells. To the best of our knowledge, this is the first suggestion of a (functional) interaction between TSPO and TMEM97 that can be affected by S2R ligands.

Barbamide Displays Affinity for Membrane-Bound Receptors and Impacts Store-Operated Calcium Entry in Mouse Sensory Neurons.

Marine cyanobacteria are a rich source of bio-active metabolites that have been utilized as leads for drug discovery and pharmacological tools for basic science research. Here, we describe the re-isolation of a well-known metabolite, barbamide, from Curaçao on three different occasions and the characterization of barbamide's biological interactions with targets of the mammalian nervous system. Barbamide was originally discovered as a molluscicidal agent from a filamentous marine cyanobacterium. In our hands, we found little evidence of toxicity against mammalian cell cultures. However, barbamide showed several affinities when screened for binding affinity for a panel of 45 receptors and transporters known to be involved in nociception and sensory neuron activity. We found high levels of binding affinity for the dopamine transporter, the kappa opioid receptor, and the sigma receptors (sigma-1 and sigma-2 also known as transmembrane protein 97; TMEM97). We tested barbamide in vitro in isolated sensory neurons from female mice to explore its functional impact on calcium flux in these cells. Barbamide by itself had no observable impact on calcium flux. However, barbamide enhanced the effect of the TRPV1 agonist capsaicin and enhanced store-operated calcium entry (SOCE) responses after depletion of intracellular calcium. Overall, these results demonstrate the biological potential of barbamide at sensory neurons with implications for future drug development projects surrounding this molecule.

Development of a CRISPRi Human Retinal Pigmented Epithelium Model for Functional Study of Age-Related Macular Degeneration Genes.

Age-related macular degeneration (AMD) is a blinding disease characterised by dysfunction of the retinal pigmented epithelium (RPE) which culminates in disruption or loss of the neurosensory retina. Genome-wide association studies have identified >60 genetic risk factors for AMD; however, the expression profile and functional role of many of these genes remain elusive in human RPE. To facilitate functional studies of AMD-associated genes, we developed a human RPE model with integrated CRISPR interference (CRISPRi) for gene repression by generating a stable ARPE19 cell line expressing dCas9-KRAB. We performed transcriptomic analysis of the human retina to prioritise AMD-associated genes and selected TMEM97 as a candidate gene for knockdown study. Using specific sgRNAs, we showed that knockdown of TMEM97 in ARPE19 reduced reactive oxygen species (ROS) levels and exerted a protective effect against oxidative stress-induced cell death. This work provides the first functional study of TMEM97 in RPE and supports a potential role of TMEM97 in AMD pathobiology. Our study highlights the potential for using CRISPRi to study AMD genetics, and the CRISPRi RPE platform generated here provided a useful in vitro tool for functional studies of AMD-associated genes.

Unde venisti PGRMC? Grand-Scale Biology from Early Eukaryotes and Eumetazoan Animal Origins.

The title usage of Unde venisti 'from where have you come' is from a now dead language (Latin) that foundationally influenced modern English (not the major influence, but an essential formative one). This is an apt analogy for how both the ancient eukaryotic and eumetazoan functions of PGRMC proteins (PGRMC1 and PGRMC2 in mammals) probably influence modern human biology: via a formative trajectory from an evolutionarily foundational fulcrum. There is an arguable probability, although not a certainty, that PGRMC-like proteins were involved in eukaryogenesis. If so, then the proto-eukaryotic ancestral protein is modelled as having initiated the oxygen-induced and CYP450 (Cytochrome P450)-mediated synthesis of sterols in the endoplasmic reticulum to regulate proto-mitochondrial activity and heme homeostasis, as well as having enabled sterol transport between endoplasmic reticulum (ER) and mitochondria membranes involving the actin cytoskeleton, transport of heme from mitochondria, and possibly the regulation/origins of mitosis/meiosis. Later, during animal evolution, the last eumetazoan common ancestor (LEUMCA) acquired PGRMC phosphorylated tyrosines coincidentally with the gastrulation organizer, Netrin/deleted in colorectal carcinoma (DCC) signaling, muscle fibers, synapsed neurons, and neural recovery via a sleep-like process. Modern PGRMC proteins regulate multiple functions, including CYP450-mediated steroidogenesis, membrane trafficking, heme homeostasis, glycolysis/Warburg effect, fatty acid metabolism, mitochondrial regulation, and genomic CpG epigenetic regulation of gene expression. The latter imposes the system of differentiation status-sensitive cell-type specific proteomic complements in multi-tissued descendants of the LEUMCA. This paper attempts to trace PGRMC functions through time, proposing that key functions were involved in early eukaryotes, and were later added upon in the LEUMCA. An accompanying paper considers the implications of this awareness for human health and disease.

Quo vadis PGRMC? Grand-Scale Biology in Human Health and Disease.

The title usage of Latin Quo vadis 'where are you going' extends the question Unde venisti from where 'did you come?' posed in the accompanying paper and extends consideration of how ancient eukaryotic and eumetazoan functions of progesterone receptor membrane component (PGRMC) proteins (PGRMC1 and PGRMC2 in mammals) could influence modern human health and disease. This paper attempts to extrapolate to modern biology in terms of extensions of hypothetical ancestral functional states from early eukaryotes and the last eumetazoan common ancestor (LEUMCA), to relativize human metabolic physiology and disease. As novel cell types and functional specializations appeared in bilaterian animals, PGRMC functions are hypothesized to have continued to be part of the toolkit used to develop new cell types and manage increasingly complex tasks such as nerve-gut-microbiome neuronal and hormonal communication. A critical role of PGRMC (as one component of a new eumetazoan genetic machinery) is proposed in LEUMCA endocrinology, neurogenesis, and nerve-gut communication with possible involvement in circadian nicotinamide adenine dinucleotide synthesis. This model would explain the contribution of PGRMC to metabolic and differentiation/behavioral changes observed in age-related diseases like diabetes, cancer and perhaps aging itself. Consistent with proposed key regulation of neurogenesis in the LEUMCA, it is argued that Alzheimer's disease is the modern pathology that most closely reflects the suite of functions related to PGRMC biology, with the 'usual suspect' pathologies possibly being downstream of PGRMC1. Hopefully, these thoughts help to signpost directions for future research.

Neuroprotective Effects of σ2R/TMEM97 Receptor Modulators in the Neuronal Model of Huntington's Disease.

Huntington's disease (HD) is a genetic neurodegenerative disease caused by an expanded CAG repeat in the Huntingtin (HTT) gene that encodes for an expanded polyglutamine (polyQ) repeat in exon-1 of the human mutant huntingtin (mHTT) protein. The presence of this polyQ repeat results in neuronal degeneration, for which there is no cure or treatment that modifies disease progression. In previous studies, we have shown that small molecules that bind selectively to σ2R/TMEM97 can have significant neuroprotective effects in models of Alzheimer's disease, traumatic brain injury, and several other neurodegenerative diseases. In the present work, we extend these investigations and show that certain σ2R/TMEM97-selective ligands decrease mHTT-induced neuronal toxicity. We first synthesized a set of compounds designed to bind to σ2R/TMEM97 and determined their binding profiles (Ki values) for σ2R/TMEM97 and other proteins in the central nervous system. Modulators with high affinity and selectivity for σ2R/TMEM97 were then tested in our HD cell model. Primary cortical neurons were cultured in vitro for 7 days and then co-transfected with either a normal HTT construct (Htt N-586-22Q/GFP) or the mHTT construct Htt-N586-82Q/GFP. Transfected neurons were treated with either σ2R/TMEM97 or σ1R modulators for 48 h. After treatment, neurons were fixed and stained with Hoechst, and condensed nuclei were quantified to assess cell death in the transfected neurons. Significantly, σ2R/TMEM97 modulators reduce the neuronal toxicity induced by mHTT, and their neuroprotective effects are not blocked by NE-100, a selective σ1R antagonist known to block neuroprotection by σ1R ligands. These results indicate for the first time that σ2R/TMEM97 modulators can protect neurons from mHTT-induced neuronal toxicity, suggesting that targeting σ2R/TMEM97 may lead to a novel therapeutic approach to treat patients with HD.

Exploration of Diazaspiro Cores as Piperazine Bioisosteres in the Development of σ2 Receptor Ligands.

A series of σ2R compounds containing benzimidazolone and diazacycloalkane cores was synthesized and evaluated in radioligand binding assays. Replacing the piperazine moiety in a lead compound with diazaspiroalkanes and the fused octahydropyrrolo[3,4-b] pyrrole ring system resulted in a loss in affinity for the σ2R. On the other hand, the bridged 2,5-diazabicyclo[2.2.1]heptane, 1,4-diazepine, and a 3-aminoazetidine analog possessed nanomolar affinities for the σ2R. Computational chemistry studies were also conducted with the recently published crystal structure of the σ2R/TMEM97 and revealed that hydrogen bond interactions with ASP29 and π-stacking interactions with TYR150 were largely responsible for the high binding affinity of small molecules to this protein.

(-)-Epicatechin Provides Neuroprotection in Sodium Iodate-Induced Retinal Degeneration.

Oxidative stress, mitochondrial impairment, and pathological amyloid beta (Aß) deposition are involved in the pathogenesis of dry age-related macular degeneration (AMD). The natural flavonoid (-)-epicatechin (EC) is known to be an antioxidant and neuroprotective compound. Whether EC plays a therapeutic role in AMD is unknown. In this work, we aimed to assess the efficacy and molecular mechanisms of EC against sodium iodate (NaIO3)-induced retinal degeneration in C57BL/6 mice via bioinformatic, morphological, and functional methods. We demonstrated that EC had no toxic effects on the retina and could ameliorate retinal deformation and thinning. EC treatment prevented outer retinal degeneration, reduced drusen-like deposits, increased b-wave amplitude in electroretinography, blocked retinal gliosis, and increased the number and quality of mitochondria. Importantly, EC increased the protein expression of OPA1 and decreased the expression of PINK1, indicating the role of EC in mitochondrial fusion that impaired by NaIO3. Moreover, EC downregulated APP and TMEM97 levels, upregulated PGRMC1 levels, and reduced subretinal Aß accumulation. This study illustrated that EC, which may become a promising therapeutic strategy for AMD, prevented NaIO3-induced retinal degeneration, and this improvement may be associated with the mitochondrial quality control and the TMEM97/PGRMC1/Aß signaling pathway.

The Sigma-2 Receptor/TMEM97 Agonist PB28 Suppresses Cell Proliferation and Invasion by Regulating the PI3K-AKT-mTOR Signalling Pathway in Renal Cancer.

Sigma-2 receptor/TMEM97 is overexpressed in many tumours, and sigma-2 receptor ligands are under investigation for cancer therapy. We intended to evaluate the effect of PB28 on renal cancer in proliferation, migration and invasion in vitro and in vivo. Invasive renal cancer cell lines treated with PB28 (or sigma-2 receptor antagonist 1) were subjected to cell proliferation, migration and invasion assays. The therapeutic effect of PB28 was performed on nude mice. Western blot for proteins in the PI3K-AKT-mTOR signalling pathway was conducted. A CCK-8 assay was used to examine the effect of the combination of PB28 and cisplatin on renal cancer cells. Significant inhibitory effects were observed on proliferation, migration and invasion of 786-O and ACHN cells after culturing with PB28. But, the outcomes of sigma-2 receptor antagonist 1 presented the opposite tendency. PB28 significantly inhibited the proliferative and invasive ability of OS-RC-2 cells in vivo. Treatment resulted in decreased phosphorylation of constituents of the PI3K-AKT-mTOR pathway. The combination of PB28 and cisplatin showed enhanced efficacy in the inhibition of renal cancer cell proliferation. Taken together, PB28 inhibited the tumorigenic behaviours of renal cancer cells by regulating the PI3K-AKT-mTOR signalling pathway and was expected to be a sensitizer of cisplatin.