Maria H. Jäntti

OX1 Orexin/Hypocretin Receptor Signaling through Arachidonic Acid and Endocannabinoid Release

We showed previously that OX1 orexin receptor stimulation produced a strong 3H overflow response from [3H]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 OX1 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 A2 (cPLA2) 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 CB1 receptors in an artificial cell-cell communication assay that was developed. The cPLA2 cascade, in contrast, was involved in the activation of orexin receptor-operated Ca2+ influx. 2-AG was also released upon OX1 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).

Autocrine Endocannabinoid Signaling through CB1 Receptors Potentiates OX1 Orexin Receptor Signaling

It has been proposed that OX1 orexin receptors and CB1 cannabinoid receptors can form heteromeric complexes, which affect the trafficking of OX1 receptors and potentiate OX1 receptor signaling to extracellular signal–regulated kinase (ERK). We have recently shown that OX1 receptor activity releases high levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), suggesting an alternative route for OX1-CB1 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 OX1 receptor–expressing cells acted as a potent paracrine messenger stimulating ERK activity in neighboring CB1 receptor–expressing cells. When OX1 and CB1 receptors were expressed in the same cells, OX1 stimulation–induced ERK phosphorylation and activity were strongly potentiated. The potentiation but not the OX1 response as such was fully abolished by specific inhibition of CB1 receptors or the enzyme responsible for 2-AG generation, diacylglycerol lipase (DAGL). Although the results do not exclude the previously proposed OX1-CB1 heteromerization, they nevertheless unequivocally identify DAGL-dependent 2-AG generation as the pivotal determinant of the OX1-CB1 synergism and thus suggest a functional rather than a molecular interaction of OX1 and CB1 receptors.

OX1 orexin/hypocretin receptor activation of phospholipase D.

Orexin receptors potently signal to lipid messenger systems, and our previous studies have suggested that PLD would be one of these. We thus wanted to verify this by direct measurements and clarify the molecular mechanism of the coupling.

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.

Protein Kinase C Activation as a Potential Therapeutic Strategy in Alzheimer's Disease: Is there a Role for Embryonic Lethal Abnormal Vision-like Proteins?

Alzheimers disease (AD), the most common cause of dementia, is an irreversible and progressive neurodegenerative disorder. It affects predominantly brain areas that are critical for memory and learning and is characterized by two main pathological hallmarks: extracellular amyloid plaques and intracellular neurofibrillary tangles. Protein kinase C (PKC) has been classified as one of the cognitive kinases controlling memory and learning. By regulating several signalling pathways involved in amyloid and tau pathologies, it also plays an inhibitory role in AD pathophysiology. Among downstream targets of PKC are the embryonic lethal abnormal vision (ELAV)-like RNA-binding proteins that modulate the stability and the translation of specific target mRNAs involved in synaptic remodelling linked to cognitive processes. This MiniReview summarizes the current evidence on the role of PKC and ELAV-like proteins in learning and memory, highlighting how their derangement can contribute to AD pathophysiology. This last aspect emphasizes the potential of pharmacological activation of PKC as a promising therapeutic strategy for the treatment of AD.

Transient Receptor Potential Channels and Their Role in Modulating Radial Glial–Neuronal Interaction: A Signaling Pathway Involving mGluR5

The guidance of developing neurons to the right position in the central nervous system is of central importance in brain development. Canonical transient receptor potential (TRPC) channels are thought to mediate turning responses of growth cones to guidance cues through fine control of calcium transients. Proliferating and 1- to 5-day-differentiated neural progenitor cells (NPCs) showed expression of Trpc1 and Trpc3 mRNA, while Trpc4-7 was not clearly detected. Time-lapse imaging showed that the motility pattern of neuronal cells was phasic with bursts of rapid movement (>60 μm/h), changes in direction, and intermittent slow phases or stallings (<40 μm/h), which frequently occurred in close contact with radial glial processes. Genetic interference with the TRPC3 and TRPC1 channel enhanced the motility of NPCs (burst frequency/stalling frequency). TRPC3-deficient cells or cells treated with the TRPC3 blocker pyr3 infrequently changed direction and seldom contacted radial glial processes. TRPC channels are also activated by group I metabotropic glutamate receptors (mGluR1 and mGluR5). As shown here, pyr3 blocked the calcium response mediated through mGluR5 in radial glial processes. Furthermore, 2-methyl-6-(phenylethynyl)pyridine, a blocker of mGluR5, affected the motility pattern in a similar way as TRPC3/6 double knockout or pyr3. The results suggest that radial glial cells exert attractant signals to migrating neuronal cells, which alter their motility pattern. Our results suggest that mGluR5 acting through TRPC3 is of central importance in radial glial-mediated neuronal guidance.

Scaffold hopping from (5-hydroxymethyl) isophthalates to multisubstituted pyrimidines diminishes binding affinity to the C1 domain of protein kinase C

Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. Utilizing the crystal structure of the PKCδ C1B domain, we have developed hydrophobic isophthalic acid derivatives that modify PKC functions by binding to the C1 domain of the enzyme. In the present study, we aimed to improve the drug-like properties of the isophthalic acid derivatives by increasing their solubility and enhancing the binding affinity. Here we describe the design and synthesis of a series of multisubstituted pyrimidines as analogs of C1 domain–targeted isophthalates and characterize their binding affinities to the PKCα isoform. In contrast to our computational predictions, the scaffold hopping from phenyl to pyrimidine core diminished the binding affinity. Although the novel pyrimidines did not establish improved binding affinity for PKCα compared to our previous isophthalic acid derivatives, the present results provide useful structure-activity relationship data for further development of ligands targeted to the C1 domain of PKC.

Protein kinase C -activating isophthalate derivatives mitigate Alzheimer's disease-related cellular alterations

ABSTRACT Abnormal protein kinase C (PKC) function contributes to many pathophysiological processes relevant for Alzheimers disease (AD), such as amyloid precursor protein (APP) processing. Phorbol esters and other PKC activators have been demonstrated to enhance the secretion of soluble APP&agr; (sAPP&agr;), reduce the levels of &bgr;‐amyloid (A&bgr;), induce synaptogenesis, and promote neuroprotection. We have previously described isophthalate derivatives as a structurally simple family of PKC activators. Here, we characterised the effects of isophthalate derivatives HMI‐1a3 and HMI‐1b11 on neuronal viability, neuroinflammatory response, processing of APP and dendritic spine density and morphology in in vitro. HMI‐1a3 increased the viability of embryonic primary cortical neurons and decreased the production of the pro‐inflammatory mediator TNF&agr;, but not that of nitric oxide, in mouse neuron‐BV2 microglia co‐cultures upon LPS‐ and IFN‐&ggr;‐induced neuroinflammation. Furthermore, both HMI‐1a3 and HMI‐1b11 increased the levels of sAPP&agr; relative to total sAPP and the ratio of A&bgr;42/A&bgr;40 in human SH‐SY5Y neuroblastoma cells. Finally, bryostatin‐1, but not HMI‐1a3, increased the number of mushroom spines in proportion to total spine density in mature mouse hippocampal neuron cultures. These results suggest that the PKC activator HMI‐1a3 exerts neuroprotective functions in the in vitro models relevant for AD by reducing the production of TNF&agr; and increasing the secretion of neuroprotective sAPP&agr;. HighlightsHMI‐1a3 promotes neuronal viability upon the induction of neuroinflammation.HMI‐1a3 decreases the production of the TNF&agr;, but not nitric oxide.HMI‐1a3 and HMI‐1b11 affect processing and maturation of APP in neuroblastoma cells.Bryostatin‐1, but not HMI‐1a3, increases the number of mushroom spines in primary neurons.

Anticancer activity of the protein kinase C modulator HMI‐1a3 in 2D and 3D cell culture models of androgen‐responsive and androgen‐unresponsive prostate cancer

Prostate cancer is one of the most common cancers in men. Although it has a relatively high 5‐year survival rate, development of resistance to standard androgen‐deprivation therapy is a significant clinical problem. Therefore, novel therapeutic strategies are urgently needed. The protein kinase C (PKC) family is a putative prostate cancer drug target, but so far no PKC‐targeting drugs are available for clinical use. By contrast to the standard approach of developing PKC inhibitors, we have developed isophthalate derivatives as PKC agonists. In this study, we have characterized the effects of the most potent isophthalate, 5‐(hydroxymethyl)isophthalate 1a3 (HMI‐1a3), on three prostate cancer cell lines (LNCaP, DU145, and PC3) using both 2D and 3D cell culture models. In 2D cell culture, HMI‐1a3 reduced cell viability or proliferation in all cell lines as determined by the metabolic activity of the cells (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide assay) and thymidine incorporation. However, the mechanism of action in LNCaP cells was different to that in DU145 or PC3 cells. In LNCaP cells, HMI‐1a3 induced a PKC‐dependent activation of caspase 3/7, indicating an apoptotic response, whereas in DU145 and PC3 cells, it induced senescence, which was independent of PKC. This was observed as typical senescent morphology, increased β‐galactosidase activity, and upregulation of the senescence marker p21 and downregulation of E2F transcription factor 1. Using a multicellular spheroid model, we further showed that HMI‐1a3 affects the growth of LNCaP and DU145 cells in a 3D culture, emphasizing its potential as a lead compound for cancer drug development.