Sylwia Łukasiewicz

Hetero-dimerization of serotonin 5-HT2A and dopamine D2 receptors

In the present study, detailed information is presented on the hetero-dimerization of the serotonin 5-HT(2A) receptor and the dopamine D(2) receptor. Biophysical approaches (fluorescence spectroscopy as well as fluorescence lifetime microscopy) were used to determine the degree of fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent protein labeled receptor variants co-expressed in human embryonic kidney 293 cells (HEK293). Recorded data demonstrate the existence of energy transfer between the wild-type forms of 5-HT(2A)R and D(2)R, pointing toward the formation of hetero-5-HT(2A)R/D(2)R dimers and homo-5-HT(2A)R/5-HT(2A)R dimers. Moreover, the present study investigates the role of specific motifs (one containing adjacent arginine residues (217RRRRKR222) in the third intracellular loop (ic3) of D(2)R, and the other consisting of acidic glutamate residues (454EE455) in the C-tail of (5-HT(2A)R) in the formation of noncovalent complexes between these receptors. Our results suggest that these regions of 5-HT(2A)R and D(2)R may be involved in the interaction between these two proteins. On the other hand, the above-mentioned motifs do not play an important role in the homo-dimerization of these receptors. Furthermore, we estimated the influence of specific receptor ligands on the dimerization processes. Agonists (DOI and quinpirole) and antagonists (ketanserin and butaclamol) cause different effects on FRET efficiency depending on whether homo- or hetero-complexes are present. These data may have therapeutic implications, since (using the immunofluorescence double labeling protocols) the co-localization of these two receptors was demonstrated in the medial prefrontal cortex and pars reticulate of the substantia nigra of the rat brain.

Studies on the role of the receptor protein motifs possibly involved in electrostatic interactions on the dopamine D1 and D2 receptor oligomerization

We investigated the influence of an epitope from the third intracellular loop (ic3) of the dopamine D2 receptor, which contains adjacent arginine residues (217RRRRKR222), and an acidic epitope from the C‐terminus of the dopamine D1 receptor (404EE405) on the receptors’ localization and their interaction. We studied receptor dimer formation using fluorescence resonance energy transfer. Receptor proteins were tagged with fluorescence proteins and expressed in HEK293 cells. The degree of D1–D2 receptor heterodimerization strongly depended on the number of Arg residues replaced by Ala in the ic3 of D2R, which may suggest that the indicated region of ic3 in D2R might be involved in interactions between two dopamine receptors. In addition, the subcellular localization of these receptors in cells expressing both receptors D1–cyan fluorescent protein, D2–yellow fluorescent protein, and various mutants was examined by confocal microscopy. Genetic manipulations of the Arg‐rich epitope induced alterations in the localization of the resulting receptor proteins, leading to the conclusion that this epitope is responsible for the cellular localization of the receptor. The lack of energy transfer between the genetic variants of yellow fluorescent protein‐tagged D2R and cyan fluorescent protein‐tagged D1R may result from differing localization of these proteins in the cell rather than from the possible role of the D2R basic domain in the mechanism of D1–D2 receptor heterodimerization. However, we find that the acidic epitope from the C‐terminus of the dopamine D1 receptor is engaged in the heterodimerization process.

Effect of clozapine on the dimerization of serotonin 5-HT2A receptor and its genetic variant 5-HT2AH425Y with dopamine D2 receptor

Oligomerization of G protein-coupled receptors has become a very important issue in a present molecular pharmacology. In the present study the level of the serotonin 5-HT(2A) and the dopamine D(2) receptor interactions have been studied since it may have a key significance in understanding the mechanism of action of drugs used to treat schizophrenia. With the use of fluorescence resonance energy transfer we demonstrated that the serotonin 5-HT(2A) receptors form homo- and hetero-dimers with the dopamine D(2) receptors and polymorphism H452Y within the 5-HT(2A) receptor, implicated as a cause of altered response to antipsychotic treatment, disturbs both processes. Clozapine affected the hetero-dimers (5-HT(2A)H452Y/D(2)) complexes and increased the otherwise weakened dimerization to the value observed for combination of both wild type receptors, and had no effect on the serotonin receptor homo-dimers (5-HT(2A)H452Y/5-HT(2A)), while haloperidol has restored the weakened interaction within homo-complexes and did not effect the hetero-complexes. The obtained data suggest that H452Y polymorphism has an influence not only on the level of constitutive oligomerization of investigated receptors but also it changes their pharmacological properties within both homo- and hetero-complexes.

Biocompatible Polymeric Nanoparticles as Promising Candidates for Drug Delivery

The use of polymeric nanoparticles (NPs) in pharmacology provides many benefits because this approach can increase the efficacy and selectivity of active compounds. However, development of new nanocarriers requires better understanding of the interactions between NPs and the immune system, allowing for the optimization of NP properties for effective drug delivery. Therefore, in the present study, we focused on the investigation of the interactions between biocompatible polymeric NPs and a murine macrophage cell line (RAW 264.7) and a human monocytic leukemia cell line (THP-1). NPs based on a liquid core with polyelectrolyte shells were prepared by sequential adsorption of polyelectrolytes (LbL) using AOT (docusate sodium salt) as the emulsifier and the biocompatible polyelectrolytes polyanion PGA (poly-l-glutamic acid sodium salt) and polycation PLL (poly l-lysine). The average size of the obtained NPs was 80 nm. Pegylated external layers were prepared using PGA-g-PEG (PGA grafted by PEG poly(ethylene glycol)). The influence of the physicochemical properties of the NPs (charge, size, surface modification) on viability, phagocytosis potential, and endocytosis was studied. Internalization of NPs was determined by flow cytometry and confocal microscopy. Moreover, we evaluated whether addition of PEG chains downregulates particle uptake by phagocytic cells. The presented results confirm that the obtained PEG-grafted NPs are promising candidates for drug delivery.

In Vitro Interaction of Polyelectrolyte Nanocapsules with Model Cells

The nanocapsules based on a liquid core with polyelectrolyte shells prepared by the technique of sequential adsorption of polyelectrolytes (LbL) were investigated to verify capsules bioacceptance. Using AOT (docusate sodium salt) as emulsifier, we obtained liquid cores, stabilized by the interfacial complex AOT/PLL (poly-l-lysine hydrobromide). These liquid cores were encapsulated by sequential adsorption of polyelectrolytes using biocompatible polyanion PGA (poly-l-glutamic acid sodium salt) and biocompatible polycation PLL. The average size of the formed capsules was 60-80 nm. The influence of a number of polyelectrolytes layer in the shell (thickness of polyelectrolytes shell), surface charge, and capsule doses on cell viability was studied in a cellular coculture assay. In order to improve nanocapsules biocompatibility, the PEG-ylated external layers were prepared using PGA-g-PEG (PGA grafted by PEG poly(ethylene glycol)). For the most toxic nanocapsules (with only one polycation layer) about 90% of cells could survive when the concentration of nanocapsules was below 0.2 × 10(6) per one cell. That suggests that they use as a delivery vehicles is quite safe for living cells. Analysis of internalization of AOT(PLL/PGA)4-g-PEG in HEK 293 cells indicates that tested nanocapsules can easily penetrate cells membrane.

Encapsulation of clozapine in polymeric nanocapsules and its biological effects.

Clozapine is an effective atypical antipsychotic drug that unfortunately exhibits poor oral bioavailability. Moreover, the clinical use of the compound is limited because of its numerous unfavorable and unsafe side effects. Therefore, the aim of the present study was the development of a new nanocarrier for a more effective clozapine delivery. Here, clozapine was encapsulated into polymeric nanocapsules (NCs). Polyelectrolyte multilayer shells were constructed by the technique of sequential adsorption of polyelectrolytes (LbL) using biocompatible polyanion PGA (Poly-L-glutamic acid, sodium salt) and polycation PLL (poly-L-lysine) on clozapine-loaded nanoemulsion cores. Pegylated external layers were prepared using PGA-g-PEG (PGA grafted by PEG (polyethylene glycol)). Clozapine was successfully loaded into the PLL-PGA nanocarriers (CLO-NCs) with an average size of 100 nm. In vitro analysis of the interactions of the CLO-NCs with the cells of the mononuclear phagocytic system (MPS) was conducted. Cell biocompatibility, phagocytosis potential, and cellular uptake were studied. Additionally, the biodistribution and behavioral effects of the encapsulated clozapine were also studied. The results indicate that surface modified (by PEG grafting) polymeric PLL-PGA CLO-NCs are very promising nanovehicles for improving clozapine delivery.

Dopamine D2 and serotonin 5-HT1A receptor interaction in the context of the effects of antipsychotics - in vitro studies.

The serotonin 5‐HT1A receptor (5‐HT1AR) and dopamine D2 receptor (D2R) have been implicated as important sites of action in antipsychotics. Several lines of evidence indicate the key role of G protein‐coupled receptors (GPCRs) heteromers in pathophysiology of schizophrenia and highlight these complexes as novel drug targets. Because heterodimers can form only on those cells co‐expressing constituent receptors, they present a target of high pharmacological specificity in the context of biochemical effects induced by antipsychotic drugs. In studies conducted in the HEK 293 cell line, we demonstrated that 5‐HT1AR and D2R are able to form constitutive heterodimers, and antipsychotic drugs (clozapine, olanzapine, aripiprazole, and lurasidone) enhanced this process, with clozapine being most effective. Various functional tests (cAMP and IP1 as well as ERK activation) indicated that the drugs had different effects on signal transduction by the heteromer. Interestingly, co‐incubation of heterodimer‐expressing HEK 293 cells with clozapine and the 5‐HT1AR agonist 8‐OH DPAT potentiated post‐synaptic effects, especially with respect to ERK activation. Our results indicate that the D2‐5‐HT1A complex possesses biochemical, pharmacological, and functional properties distinct from those of mono‐ and homomers. This result has implications for the development of improved pharmacotherapy for schizophrenia or other disorders (activating the heteromer might be cognitive enhancing, since it is expressed in frontal cortex) through the specific targeting of heterodimers.

Role of silent polymorphisms within the dopamine D1 receptor associated with schizophrenia on D1-D2 receptor hetero-dimerization.

Within the coding region of the dopamine D(1) receptor (D(1)R), two synonymous polymorphisms, D(1)R(G198A) and D(1)R(G1263), have been identified and postulated to correlate with the schizophrenia phenotype. Binding studies revealed that the density of these genetic variants was much lower than the density of wild type D(1)R in the human embryonic kidney (HEK) 293 cell line, used as a model system. From the data obtained using MFOLD software it is apparent that the G198A mutation has a greater impact on the secondary structure of the mRNA, which may affect its translation. However, the G1263A mutation is localized within the serine 421 codon of D(1)R, which is predicted to be a potential site of phosphorylation according to the PHOSIDA database. In order to determine whether the studied synonymous polymorphisms influence the process of dopamine D(1)-D(2) receptors heterodimerization, we employed fluorescence resonance energy transfer (FRET) technology. The dopamine D(2) receptor (D(2)R) was tagged with cyan fluorescence protein and the D(1)R and its genetic variants were tagged with yellow fluorescence protein. The degree of D(1)-D(2) receptor hetero-dimerization was significantly decreased when genetic variants of D(1)R were co-expressed with D(2)R. Since the D(1)R mutations affected the expression levels of the proteins in the cell membrane without affecting the cellular localization of the receptor proteins, we postulated that the D(1)R polymorphisms altered the translation rate and protein structure of the receptor. The altered hetero-dimerization that likely results from the lower expression of these genetic variants of D(1)R with D(2)R may be partially responsible for the association of both G198A and G1263A polymorphisms with the schizophrenia phenotype.

Polyelectrolyte-coated nanocapsules containing undecylenic acid: Synthesis, biocompatibility and neuroprotective properties

The main objectives of the present study were to investigate the biocompatibility of polyelectrolyte-coated nanocapsules and to evaluate the neuroprotective action of the nanoencapsulated water-insoluble neuroprotective drug-undecylenic acid (UDA), in vitro. Core-shell nanocapsules were synthesized using nanoemulsification and the layer-by-layer (LbL) technique (by saturation method). The average size of synthesized nanocapsules was around 80 nm and the concentration was 2.5 × 10(10) particles/ml. Their zeta potential values ranged from less than -30 mV for the ones with external polyanion layers through -4 mV for the PEG-ylated layers to more than 30 mV for the polycation layers. Biocompatibility of synthesized nanocarriers was evaluated in the SH-SY5Y human neuroblastoma cell line using cell viability/toxicity assays (MTT reduction, LDH release). The results obtained showed that synthesized nanocapsules coated with PLL and PGA (also PEG-ylated) were non-toxic to SH-SY5Y cells, therefore, they were used as nanocarriers for UDA. Moreover, studies with ROD/FITC-labeled polyelectrolytes demonstrated approximately 20% cellular uptake of synthetized nanocapsules. Further studies showed that nanoencapsulated form of UDA was biocompatible and protected SH-SY5Y cells against the staurosporine-induced damage in lower concentrations than those of the same drug added directly to the culture medium. These data suggest that designed nanocapsules might serve as novel, promising delivery systems for neuroprotective agents.

Antidepressant drugs promote the heterodimerization of the dopamine D2 and somatostatin Sst5 receptors – fluorescence in vitro studies

BACKGROUND The interaction between the dopaminergic and somatostatinergic systems and their role in mood regulation have been well-documented. Therefore, we decided to investigate the effect of antidepressant drugs on the heterodimerization of the dopamine D2 and somatostatin Sst5 receptors. METHODS The human receptor proteins were tagged with fluorescent proteins, expressed in the HEK 293 cells and incubated with antidepressant drugs: desipramine and citalopram. To determine the FRET efficiency, the fluorescence resonance energy transfer (FRET) and photobleaching confocal microscopy techniques were used. RESULTS We found that the efficiency of FRET is markedly increased in cells coexpressing the somatostatin Sst5 and dopamine D2 receptors after 48 h of incubation with desipramine and citalopram. CONCLUSIONS In the present study we provide physical evidence, based on FRET analysis, that antidepressants increase Sst5 and D2 receptors heterodimerization. The effect is specific because desipramine in the incubation medium uncouples other pairs of receptors, such as the dopamine D1-D2 receptors.