International Union of Basic and Clinical Pharmacology CXIV: Orexin Receptor Function, Nomenclature and Pharmacology.

The orexin system consists of the peptide transmitters orexin-A and -B and the G protein-coupled orexin receptors OX1 and OX2 Orexin receptors are capable of coupling to all four families of heterotrimeric G proteins, and there are also other complex features of the orexin receptor signaling. The system was discovered 25 years ago and was immediately identified as a central regulator of sleep and wakefulness; this is exemplified by the symptomatology of the disorder narcolepsy with cataplexy, in which orexinergic neurons degenerate. Subsequent translation of these findings into drug discovery and development has resulted to date in three clinically used orexin receptor antagonists to treat insomnia. In addition to sleep and wakefulness, the orexin system appears to be a central player at least in addiction and reward, and has a role in depression, anxiety and pain gating. Additional antagonists and agonists are in development to treat, for instance, insomnia, narcolepsy with or without cataplexy and other disorders with excessive daytime sleepiness, depression with insomnia, anxiety, schizophrenia, as well as eating and substance use disorders. The orexin system has thus proved an important regulator of numerous neural functions and a valuable drug target. Orexin prepro-peptide and orexin receptors are also expressed outside the central nervous system, but their potential physiological role there remains unknown. Significance Statement The orexin system was discovered 25 years ago and immediately emerged as an essential sleep-wakefulness regulator. This discovery has tremendously increased the understanding of these processes and has thus far resulted in the market approval of three orexin receptor antagonists, which promote more physiological sleep than previous hypnotics. Further, orexin receptor agonists and antagonists with different pharmacodynamic properties are in development since research has revealed additional potential therapeutic indications. Orexin receptor signaling is complex and may represent novel features.

Azulene as a biphenyl mimetic in orexin/hypocretin receptor agonists.

Azulene is a rare ring structure in drugs, and we investigated whether it could be used as a biphenyl mimetic in known orexin receptor agonist Nag 26, which is binding to both orexin receptors OX1 and OX2 with preference towards OX2. The most potent azulene-based compound was identified as an OX1 orexin receptor agonist (pEC50 = 5.79 ± 0.07, maximum response = 81 ± 8% (s.e.m. of five independent experiments) of the maximum response to orexin-A in Ca2+ elevation assay). However, the azulene ring and the biphenyl scaffold are not identical in their spatial shape and electron distribution, and their derivatives may adopt different binding modes in the binding site.

Allosteric interactions via the orthosteric ligand binding sites in a constitutive G-protein-coupled receptor homodimer.

I interpret some recent data to indicate that co-operative effects take place between the (identical) orthosteric binding sites in a G-protein-coupled receptor dimer. In the current study, the reasonability of this concept was tested by creating a mathematical model. The model is composed of a symmetrical constitutive receptor dimer in which the protomers are able to affect each other allosterically, and it includes binding, receptor activation and signal amplification steps. The model was utilized for analyses of previous data as well as simulations of predicted behaviour. The model demonstrates the behaviour stated in the hypotheses, i.e. even an apparently neutral receptor ligand can allosterically affect agonist binding or receptor activation by binding to the normal orthosteric ligand binding site. Therewith the speculated allosteric action originating from the orthosteric binding site of the dimeric receptor is a realistic possibility. The results of the simulations and curve fitting constitute a reasonable starting point for further studies, and the model can be utilized to design meaningful experiments to investigate these questions.

CRK2 Enhances Salt Tolerance by Regulating Callose Deposition in Connection with PLDα1.

High salinity is an increasingly prevalent source of stress to which plants must adapt. The receptor-like protein kinases, including members of the Cys-rich receptor-like kinase (CRK) subfamily, are a highly expanded family of transmembrane proteins in plants that are largely responsible for communication between cells and the extracellular environment. Various CRKs have been implicated in biotic and abiotic stress responses; however, their functions on a cellular level remain largely uncharacterized. Here we have shown that CRK2 enhances salt tolerance at the germination stage in Arabidopsis (Arabidopsis thaliana) and also modulates root length. We established that functional CRK2 is required for salt-induced callose deposition. In doing so, we revealed a role for callose deposition in response to increased salinity and demonstrated its importance for salt tolerance during germination. Using fluorescently tagged proteins, we observed specific changes in the subcellular localization of CRK2 in response to various stress treatments. Many of CRK2's cellular functions were dependent on phospholipase D activity, as were the subcellular localization changes. Thus, we propose that CRK2 acts downstream of phospholipase D during salt stress, promoting callose deposition and regulating plasmodesmal permeability, and that CRK2 adopts specific stress-dependent subcellular localization patterns that allow it to carry out its functions.

p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3.

Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF.

G-protein inhibition profile of the reported Gq/11 inhibitor UBO-QIC.

UBO-QIC (FR900359) is the only currently available Gq/11 protein inhibitor. However, its characterization has not been published, and we thus set out to do this. Gi, Gs and Gq protein-mediated responses were assessed utilizing endogenous or heterologously expressed receptors in Chinese hamster ovary cells. UBO-QIC, at 1 µM, was an effective inhibitor of the Gq-mediated responses, but was inactive at Gi- and Gs-mediated responses. Gq/11 and G16 responses were additionally compared in HEL92.1.7 cells, showing inhibition of Gq/11 responses. However, UBO-QIC also appeared to inhibit G16. Further studies are required to establish its profile with respect to the different Gq-family proteins.

G-protein-dependency of orexin/hypocretin receptor signalling in recombinant Chinese hamster ovary cells.

Multiple signalling pathways for orexin receptors have been discovered, and most thoroughly mapped in Chinese hamster ovary K1 (CHO-K1) cells. It is also known that orexin receptors can couple to the G-protein families Gi, Gs and Gq. However, the connection between the G-proteins and the downstream signals is only vaguely established, and we now set out to resolve this for human orexin receptors expressed in CHO-K1 cells. Adenylyl cyclase (AC), phospholipase A2, C and D, and diacylglycerol lipase activities were assessed by precursor radiolabelling and chromatographic separation, and calcium by fluorescent methods. Pertussis toxin, cholera toxin and the cyclic depsipeptide, UBO-QIC a.k.a. FR900359, were used to assess the involvement of Gi-, Gs- and Gq-family G-proteins, respectively. Calcium elevations as well as activation of the phospholipases and diacylglycerol lipase were dependent on Gq, as they were fully blocked by UBO-QIC. The low-potency AC activation fully depended on Gs. Surprisingly, the assumed Gi-dependent inhibition of AC was (fully or partially) inhibited by UBO-QIC, in opposition to the previous findings of no sensitivity of Gi proteins to UBO-QIC. Orexin receptor signalling is indeed mostly Gq-driven in CHO-K1 cells, even with respect to the less clearly mapped cascades such as phospholipase A2 and C and calcium influx, underlining the importance of Gq even under physiological conditions. AC regulation warrants more studies.

Human orexin/hypocretin receptors form constitutive homo- and heteromeric complexes with each other and with human CB1 cannabinoid receptors.

Human OX1 orexin receptors have been shown to homodimerize and they have also been suggested to heterodimerize with CB1 cannabinoid receptors. The latter has been suggested to be important for orexin receptor responses and trafficking. In this study, we wanted to assess the ability of the other combinations of receptors to also form similar complexes. Vectors for expression of human OX1, OX2 and CB1 receptors, C-terminally fused with either Renilla luciferase or GFP(2) green fluorescent protein variant, were generated. The constructs were transiently expressed in Chinese hamster ovary cells, and constitutive dimerization between the receptors was assessed by bioluminescence energy transfer (BRET). Orexin receptor subtypes readily formed homo- and hetero(di)mers, as suggested by significant BRET signals. CB1 receptors formed homodimers, and they also heterodimerized with both orexin receptors. Interestingly, BRET efficiency was higher for homodimers than for almost all heterodimers. This is likely to be due to the geometry of the interaction; the putatively symmetric dimers may place the C-termini in a more suitable orientation in homomers. Fusion of luciferase to an orexin receptor and GFP(2) to CB1 produced more effective BRET than the opposite fusions, also suggesting differences in geometry. Similar was seen for the OX1-OX2 interaction. In conclusion, orexin receptors have a significant propensity to make homo- and heterodi-/oligomeric complexes. However, it is unclear whether this affects their signaling. As orexin receptors efficiently signal via endocannabinoid production to CB1 receptors, dimerization could be an effective way of forming signal complexes with optimal cannabinoid concentrations available for cannabinoid receptors.

Characterization of a putative orexin receptor in Ciona intestinalis sheds light on the evolution of the orexin/hypocretin system in chordates.

Tunicates are evolutionary model organisms bridging the gap between vertebrates and invertebrates. A genomic sequence in Ciona intestinalis (CiOX) shows high similarity to vertebrate orexin receptors and protostome allatotropin receptors (ATR). Here, molecular phylogeny suggested that CiOX is divergent from ATRs and human orexin receptors (hOX1/2). However, CiOX appears closer to hOX1/2 than to ATR both in terms of sequence percent identity and in its modelled binding cavity, as suggested by molecular modelling. CiOX was heterologously expressed in a recombinant HEK293 cell system. Human orexins weakly but concentration-dependently activated its Gq signalling (Ca2+ elevation), and the responses were inhibited by the non-selective orexin receptor antagonists TCS 1102 and almorexant, but only weakly by the OX1-selective antagonist SB-334867. Furthermore, the 5-/6-carboxytetramethylrhodamine (TAMRA)-labelled human orexin-A was able to bind to CiOX. Database mining was used to predict a potential endogenous C. intestinalis orexin peptide (Ci-orexin-A). Ci-orexin-A was able to displace TAMRA-orexin-A, but not to induce any calcium response at the CiOX. Consequently, we suggested that the orexin signalling system is conserved in Ciona intestinalis, although the relevant peptide-receptor interaction was not fully elucidated.

Physiology of the orexinergic/hypocretinergic system: a revisit in 2012.

The neuropeptides orexins and their G protein-coupled receptors, OX(1) and OX(2), were discovered in 1998, and since then, their role has been investigated in many functions mediated by the central nervous system, including sleep and wakefulness, appetite/metabolism, stress response, reward/addiction, and analgesia. Orexins also have peripheral actions of less clear physiological significance still. Cellular responses to the orexin receptor activity are highly diverse. The receptors couple to at least three families of heterotrimeric G proteins and other proteins that ultimately regulate entities such as phospholipases and kinases, which impact on neuronal excitation, synaptic plasticity, and cell death. This article is a 10-year update of my previous review on the physiology of the orexinergic/hypocretinergic system. I seek to provide a comprehensive update of orexin physiology that spans from the molecular players in orexin receptor signaling to the systemic responses yet emphasizing the cellular physiological aspects of this system.

Autocrine endocannabinoid signaling through CB1 receptors potentiates OX1 orexin receptor signaling.

It has been proposed that OX(1) orexin receptors and CB(1) cannabinoid receptors can form heteromeric complexes, which affect the trafficking of OX(1) receptors and potentiate OX(1) receptor signaling to extracellular signal-regulated kinase (ERK). We have recently shown that OX(1) receptor activity releases high levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), suggesting an alternative route for OX(1)-CB(1) receptor interaction in signaling, for instance, in retrograde synaptic transmission. In the current study, we set out to investigate this possibility utilizing recombinant Chinese hamster ovary K1 cells. 2-AG released from OX(1) receptor-expressing cells acted as a potent paracrine messenger stimulating ERK activity in neighboring CB(1) receptor-expressing cells. When OX(1) and CB(1) receptors were expressed in the same cells, OX(1) stimulation-induced ERK phosphorylation and activity were strongly potentiated. The potentiation but not the OX(1) response as such was fully abolished by specific inhibition of CB(1) receptors or the enzyme responsible for 2-AG generation, diacylglycerol lipase (DAGL). Although the results do not exclude the previously proposed OX(1)-CB(1) heteromerization, they nevertheless unequivocally identify DAGL-dependent 2-AG generation as the pivotal determinant of the OX(1)-CB(1) synergism and thus suggest a functional rather than a molecular interaction of OX(1) and CB(1) receptors.

OX1 orexin/hypocretin receptor signaling through arachidonic acid and endocannabinoid release.

We showed previously that OX(1) orexin receptor stimulation produced a strong (3)H overflow response from [(3)H]arachidonic acid (AA)-labeled cells. Here we addressed this issue with a novel set of tools and methods, to distinguish the enzyme pathways responsible for this response. CHO-K1 cells heterologously expressing human OX(1) receptors were used as a model system. By using selective pharmacological inhibitors, we showed that, in orexin-A-stimulated cells, the AA-derived radioactivity was released as two distinct components, i.e., free AA and the endocannabinoid 2-arachidonoyl glycerol (2-AG). Two orexin-activated enzymatic cascades are responsible for this response: cytosolic phospholipase A(2) (cPLA(2)) and diacylglycerol lipase; the former cascade is responsible for part of the AA release, whereas the latter is responsible for all of the 2-AG release and part of the AA release. Essentially only diacylglycerol released by phospholipase C but not by phospholipase D was implicated as a substrate for 2-AG production, although both phospholipases were strongly activated. The 2-AG released acted as a potent paracrine messenger through cannabinoid CB(1) receptors in an artificial cell-cell communication assay that was developed. The cPLA(2) cascade, in contrast, was involved in the activation of orexin receptor-operated Ca(2+) influx. 2-AG was also released upon OX(1) receptor stimulation in recombinant HEK-293 and neuro-2a cells. The results directly show, for the first time, that orexin receptors are able to generate potent endocannabinoid signals in addition to arachidonic acid signals, which may explain the proposed orexin-cannabinoid interactions (e.g., in neurons).

Involvement of cyclin-dependent kinase-5 in the kainic acid-mediated degeneration of glutamatergic synapses in the rat hippocampus.

Increased levels of glutamate causing excitotoxic damage accompany neurological disorders such as ischemia/stroke, epilepsy and some neurodegenerative diseases. Cyclin-dependent kinase-5 (Cdk5) is important for synaptic plasticity and is deregulated in neurodegenerative diseases. However, the mechanisms by which kainic acid (KA)-induced excitotoxic damage involves Cdk5 in neuronal injury are not fully understood. In this work, we have thus studied involvement of Cdk5 in the KA-mediated degeneration of glutamatergic synapses in the rat hippocampus. KA induced degeneration of mossy fiber synapses and decreased glutamate receptor (GluR)6/7 and post-synaptic density protein 95 (PSD95) levels in rat hippocampus in vivo after intraventricular injection of KA. KA also increased the cleavage of Cdk5 regulatory protein p35, and Cdk5 phosphorylation in the hippocampus at 12 h after treatment. Studies with hippocampal neurons in vitro showed a rapid decline in GluR6/7 and PSD95 levels after KA treatment with the breakdown of p35 protein and phosphorylation of Cdk5. These changes depended on an increase in calcium as shown by the chelators 1,2-bis(o-aminophenoxy)ethane-N,N,N ',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and glycol-bis (2-aminoethylether)-N,N,N ',N '-tetra-acetic acid. Inhibition of Cdk5 using roscovitine or employing dominant-negative Cdk5 and Cdk5 silencing RNA constructs counteracted the decreases in GluR6/7 and PSD95 levels induced by KA in hippocampal neurons. The dominant-negative Cdk5 was also able to decrease neuronal degeneration induced by KA in cultured neurons. The results show that Cdk5 is essentially involved in the KA-mediated alterations in synaptic proteins and in cell degeneration in hippocampal neurons after an excitotoxic injury. Inhibition of pathways activated by Cdk5 may be beneficial for treatment of synaptic degeneration and excitotoxicity observed in various brain diseases.

Downregulation of NF-kappaB signaling by mutant huntingtin proteins induces oxidative stress and cell death.

Accumulation of abnormal proteins and endoplasmic reticulum stress accompany neurodegenerative diseases including Huntington's disease. We show that the expression of mutant huntingtin proteins with extended polyglutamine repeats differentially affected endoplasmic reticulum signaling cascades linked to the inositol-requiring enzyme-1 (IRE1) pathway. Thus, the p38 and c-Jun N-terminal kinase pathways were activated, while the levels of the nuclear factor-kappaB-p65 (NF-kappaB-p65) protein decreased. Downregulation of NF-kappaB signaling was linked to decreased antioxidant levels, increased oxidative stress, and enhanced cell death. Concomitantly, calpain was activated, and treatment with calpain inhibitors restored NF-kappaB-p65 levels and increased cell viability. The calpain regulator, calpastatin, was low in cells expressing mutant huntingtin, and overexpression of calpastatin counteracted the deleterious effects caused by N-terminal mutant huntingtin proteins. These results show that calpastatin and an altered NF-kappaB-p65 signaling are crucial factors involved in oxidative stress and cell death mediated by mutant huntingtin proteins.

Cellular Signaling Mechanisms of Hypocretin/Orexin.

Orexin receptors (OXRs) are promiscuous G-protein-coupled receptors that signal via several G-proteins and, putatively, via other proteins. On which basis the signal pathways are selected and orchestrated is largely unknown. We also have an insufficient understanding of the kind of signaling that is important for specific types of cellular responses. OXRs are able to form complexes with several other G-protein-coupled receptors in vitro, and one possibility is that the complexing partners regulate the use of certain signal transducers. In the central nervous system neurons, the main acute downstream responses of OXR activation are the inhibition of K+ channels and the activation of the Na+/Ca2+ exchanger and non-selective cation channels of unknown identity. The exact nature of the intracellular signal chain between the OXRs and these downstream targets is yet to be elucidated, but the Gq-phospholipase C (PLC) protein kinase C pathway - which is a significant signaling pathway for OXRs in recombinant cells - may be one of the players in neurons. The Gq-PLC pathway may also, under certain circumstances, take the route to diacylglycerol lipase, which leads to the production of the potent endocannabinoid (eCB), 2-arachidonoyl glycerol, and thereby connects orexins with eCB signaling. In addition, OXRs have been studied in the context of neurodegeneration and cancer cell death. Overall, OXR signaling is complex, and it can change depending on the cell type and environment.

Orexin-A measurement in narcolepsy: A stability study and a comparison of LC-MS/MS and immunoassays.

BACKGROUND: Orexin-A and -B are neuropeptides involved in sleep-wake regulation. In human narcolepsy type 1, this cycle is disrupted due to loss of orexin-producing neurons in the hypothalamus. Cerebrospinal fluid (CSF) orexin-A measurement is used in the diagnosis of narcolepsy type 1. Currently available immunoassays may lack specificity for accurate orexin quantification. We developed and validated a liquid chromatography mass spectrometry assay (LC-MS/MS) for CSF orexin-A and B. METHODS: We used CSF samples from narcolepsy type 1 (n = 22) and type 2 (n = 6) and non-narcoleptic controls (n = 44). Stable isotope-labeled orexin-A and -B internal standards were added to samples before solid-phase extraction and quantification by LC-MS/MS. The samples were also assayed by commercial radioimmunoassay (RIA, n = 42) and enzymatic immunoassay (EIA, n = 72) kits. Stability of orexins in CSF was studied for 12 months. RESULTS: Our assay has a good sensitivity (10 pmol/L = 35 pg/mL) and a wide linear range (35-3500 pg/mL). Added orexin-A and -B were stable in CSF for 12 and 3 months, respectively, when frozen. The median orexin-A concentration in CSF from narcolepsy type 1 patients was <35 pg/mL (range < 35-131 pg/mL), which was lower than that in CSF from control individuals (98 pg/mL, range < 35-424 pg/mL). Orexin-A concentrations determined using our LC-MS/MS assay were five times lower than those measured with a commercial RIA. Orexin-B concentrations were undetectable. CONCLUSIONS: Orexin-A concentrations measured by our LC-MS/MS assay were lower in narcolepsy type 1 patients as compared to controls. RIA yielded on average higher concentrations than LC-MS/MS.

Orexin receptors in GtoPdb v.2021.3.

Orexin receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Orexin receptors [42]) are activated by the endogenous polypeptides orexin-A and orexin-B (also known as hypocretin-1 and -2; 33 and 28 aa) derived from a common precursor, preproorexin or orexin precursor, by proteolytic cleavage and some typical peptide modifications [109]. Currently the only orexin receptor ligands in clinical use are suvorexant and lemborexant, which are used as hypnotics. Orexin receptor crystal structures have been solved [134, 133, 54, 117, 46].

Octopamine receptors from the barnacle balanus improvisus are activated by the alpha2-adrenoceptor agonist medetomidine.

G protein-coupled octopamine receptors of insects and other invertebrates represent counterparts of adrenoceptors in vertebrate animals. The alpha(2)-adrenoceptor agonist medetomidine, which is in clinical use as a veterinary sedative agent, was discovered to inhibit the settling process of barnacles, an important step in the ontogeny of this crustacean species. Settling of barnacles onto ship hulls leads to biofouling that has many harmful practical consequences, and medetomidine is currently under development as a novel type of antifouling agent. We now report that medetomidine induces hyperactivity in the barnacle larvae to disrupt the settling process. To identify the molecular targets of medetomidine, we cloned five octopamine receptors from the barnacle Balanus improvisus. We show by phylogenetic analyses that one receptor (BiOctalpha) belongs to the alpha-adrenoceptor-like subfamily, and the other four (BiOctbeta-R1, BiOctbeta-R2, BiOctbeta-R3, and BiOctbeta-R4) belong to the beta-adrenoceptor-like octopamine receptor subfamily. Phylogenetic analyses also indicated that B. improvisus has a different repertoire of beta-adrenoceptor-like octopamine receptors than insects. When expressed in CHO cells, the cloned receptors were activated by both octopamine and medetomidine, resulting in increased intracellular cAMP or calcium levels. Tyramine activated the receptors but with much lesser potency than octopamine. A hypothesis for receptor discrimination between tyramine and octopamine was generated from a homology three-dimensional model. The characterization of B. improvisus octopamine receptors is important for a better functional understanding of these receptors in crustaceans as well as for practical applications in development of environmentally sustainable antifouling agents.

Filtration assay for arachidonic acid release.

Arachidonic acid (AA) release is a central message in cell signaling. Fatty acid release is generally assessed by manual sampling of radioactivity release from cells prelabeled with a radiolabeled fatty acid. The assay is laborious, time-consuming, and susceptible to high noise. Here we present a fast and reproducible method for 96-well filter plates and cells in suspension, a method that is best suited for agonist concentration-response studies and, thus, for ligand screening. The method offers tremendous time and effort savings and enables execution of large experimental series previously unattainable for AA release studies.

Prolyl oligopeptidase inhibition by KYP-2407 increases alpha-synuclein fibril degradation in neuron-like cells.

Growing evidence emphasizes insufficient clearance of pathological alpha-synuclein (αSYN) aggregates in the progression of Parkinson's disease (PD). Consequently, cellular degradation pathways represent a potential therapeutic target. Prolyl oligopeptidase (PREP) is highly expressed in the brain and has been suggested to increase αSYN aggregation and negatively regulate the autophagy pathway. Inhibition of PREP with a small molecule inhibitor, KYP-2407, stimulates autophagy and reduces the oligomeric species of αSYN aggregates in PD mouse models. However, whether PREP inhibition has any effects on intracellular αSYN fibrils has not been studied before. In this study, the effect of KYP2407 on αSYN preformed fibrils (PFFs) was tested in SH-SY5Y cells and human astrocytes. Immunostaining analysis revealed that both cell types accumulated αSYN PFFs intracellularly but KYP-2047 decreased intracellular αSYN deposits only in SH-SY5Y cells, as astrocytes did not show any PREP activity. Western blot analysis confirmed the reduction of high molecular weight αSYN species in SH-SY5Y cell lysates, and secretion of αSYN from SH-SY5Y cells also decreased in the presence of KYP-2407. Accumulation of αSYN inside the SH-SY5Y cells resulted in an increase of the auto-lysosomal proteins p62 and LC3BII, as well as calpain 1 and 2, which have been shown to be associated with PD pathology. Notably, treatment with KYP-2407 significantly reduced p62 and LC3BII levels, indicating an increased autophagic flux, and calpain 1 and 2 levels returned to normal in the presence of KYP-2407. Our findings indicate that PREP inhibition can potentially be used as therapy to reduce the insoluble intracellular αSYN aggregates.