IQGAP2 regulates blood-brain barrier immune dynamics.

Brain endothelial cells (BECs) play an important role in maintaining central nervous system (CNS) homeostasis through blood-brain barrier (BBB) functions. BECs express low baseline levels of adhesion receptors, which limits entry of leukocytes. However, the molecular mediators governing this phenotype remain mostly unclear. Here, we explored how infiltration of immune cells across the BBB is influenced by the scaffold protein IQ motif containing GTPase activating protein 2 (IQGAP2). In mice and zebrafish, we demonstrate that loss of Iqgap2 increases infiltration of peripheral leukocytes into the CNS under homeostatic and inflammatory conditions. Using single-cell RNA sequencing and immunohistology, we further show that BECs from mice lacking Iqgap2 exhibit a profound inflammatory signature, including extensive upregulation of adhesion receptors and antigen-processing machinery. Human tissue analyses also reveal that Alzheimer's disease is associated with reduced hippocampal IQGAP2. Overall, our results implicate IQGAP2 as an essential regulator of BBB immune privilege and immune cell entry into the CNS.

Arteriolar degeneration and stiffness in cerebral amyloid angiopathy are linked to ß-amyloid deposition and lysyl oxidase.

Cerebral amyloid angiopathy (CAA) is a vasculopathy characterized by vascular ß-amyloid (Aß) deposition on cerebral blood vessels. CAA is closely linked to Alzheimer's disease (AD) and intracerebral hemorrhage. CAA is associated with the loss of autoregulation in the brain, vascular rupture, and cognitive decline. To assess morphological and molecular changes associated with the degeneration of penetrating arterioles in CAA, we analyzed post-mortem human brain tissue from 26 patients with mild, moderate, and severe CAA end neurological controls. The tissue was optically cleared for three-dimensional light sheet microscopy, and morphological features were quantified using surface volume rendering. We stained Aß, vascular smooth muscle (VSM), lysyl oxidase (LOX), and vascular markers to visualize the relationship between degenerative morphological features, including vascular dilation, dolichoectasia (variability in lumenal diameter) and tortuosity, and the volumes of VSM, Aß, and LOX in arterioles. Atomic force microscopy (AFM) was used to assess arteriolar wall stiffness, and we identified a pattern of morphological features associated with degenerating arterioles in the cortex. The volume of VSM associated with the arteriole was reduced by around 80% in arterioles with severe CAA and around 60% in cases with mild/moderate CAA. This loss of VSM correlated with increased arteriolar diameter and variability of diameter, suggesting VSM loss contributes to arteriolar laxity. These vascular morphological features correlated strongly with Aß deposits. At sites of microhemorrhage, Aß was consistently present, although the morphology of the deposits changed from the typical organized ring shape to sharply contoured shards with marked dilation of the vessel. AFM showed that arteriolar walls with CAA were more than 400% stiffer than those without CAA. Finally, we characterized the association of vascular degeneration with LOX, finding strong associations with VSM loss and vascular degeneration. These results show an association between vascular Aß deposition, microvascular degeneration, and increased vascular stiffness, likely due to the combined effects of replacement of VSM by ß-amyloid, cross-linking of extracellular matrices (ECM) by LOX, and possibly fibrosis. This advanced microscopic imaging study clarifies the association between Aß deposition and vascular fragility. Restoration of physiologic ECM properties in penetrating arteries may yield a novel therapeutic strategy for CAA.

Fatal iatrogenic cerebral ß-amyloid-related arteritis in a woman treated with lecanemab for Alzheimer's disease.

We report the case of a 79-year-old woman with Alzheimer's disease who participated in a Phase III randomized controlled trial called CLARITY-AD testing the experimental drug lecanemab. She was randomized to the placebo group and subsequently enrolled in an open-label extension which guaranteed she received the active drug. After the third biweekly infusion, she suffered a seizure characterized by speech arrest and a generalized convulsion. Magnetic resonance imaging revealed she had multifocal swelling and a marked increase in the number of cerebral microhemorrhages. She was treated with an antiepileptic regimen and high-dose intravenous corticosteroids but continued to worsen and died after 5 days. Post-mortem MRI confirmed extensive microhemorrhages in the temporal, parietal and occipital lobes. The autopsy confirmed the presence of two copies of APOE4, a gene associated with a higher risk of Alzheimer's disease, and neuropathological features of moderate severity Alzheimer's disease and severe cerebral amyloid angiopathy with perivascular lymphocytic infiltrates, reactive macrophages and fibrinoid degeneration of vessel walls. There were deposits of ß-amyloid in meningeal vessels and penetrating arterioles with numerous microaneurysms. We conclude that the patient likely died as a result of severe cerebral amyloid-related inflammation.

Detection, visualization and quantification of protein complexes in human Alzheimer's disease brains using proximity ligation assay.

Examination of healthy and diseased human brain is essential to translational neuroscience. Protein-protein interactions play a pivotal role in physiological and pathological processes, but their detection is difficult, especially in aged and fixed human brain tissue. We used the in-situ proximity ligation assay (PLA) to broaden the range of molecular interactions assessable in-situ in the human neuropathology. We adapted fluorescent in-situ PLA to detect ubiquitin-modified proteins in human brains with Alzheimer's disease (AD), including approaches for the management of autofluorescence and quantification using a high-content image analysis system. We confirmed that phosphorylated microtubule-associated protein tau (Serine202, Threonine205) aggregates were modified by ubiquitin and that phospho-tau-ubiquitin complexes were increased in hippocampal and frontal cortex regions in AD compared to non-AD brains. Overall, we refined PLA for use in human neuropathology, which has revealed a profound change in the distribution of ubiquitin in AD brain and its association with characteristic tau pathologies.

Combined treatment with Sigma1R and A2AR agonists fails to inhibit cocaine self-administration despite causing strong antagonistic accumbal A2AR-D2R complex interactions: the potential role of astrocytes.

Previous studies have indicated that acute treatment with the monoamine stabilizer OSU-6162 (5 mg/kg), which has a high affinity for Sigma1R, significantly increased the density of accumbal shell D2R-Sigma1R and A2AR-D2R heteroreceptor complexes following cocaine self-administration. Ex vivo studies using the A2AR agonist CGS21680 also suggested the existence of enhanced antagonistic accumbal A2AR-D2R allosteric interactions after treatment with OSU-6162 during cocaine self-administration. However, a 3-day treatment with OSU-6162 (5 mg/kg) failed to alter the behavioral effects of cocaine self-administration. To test these results and the relevance of OSU-6162 (2.5 mg/kg) and/or A2AR (0.05 mg/kg) agonist interactions, we administered low doses of receptor agonists during cocaine self-administration and assessed their neurochemical and behavioral effects. No effects were observed on cocaine self-administration; however, marked and highly significant increases using the proximity ligation assay (PLA) were induced by the co-treatment on the density of the A2AR-D2R heterocomplexes in the nucleus accumbens shell. Significant decreases in the affinity of the D2R high- and low-affinity agonist binding sites were also observed. Thus, in low doses, the highly significant neurochemical effects observed upon cotreatment with an A2AR agonist and a Sigma1R ligand on the A2AR-D2R heterocomplexes and their enhancement of allosteric inhibition of D2R high-affinity binding are not linked to the modulation of cocaine self-administration. The explanation may be related to an increased release of ATP and adenosine from astrocytes in the nucleus accumbens shell in cocaine self-administration. This can lead to increased activation of the A1R protomer in a putative A1R-A2AR-D2R complex that modulates glutamate release in the presynaptic glutamate synapse. We hypothesized that the integration of changes in presynaptic glutamate release and postjunctional heteroreceptor complex signaling, where D2R plays a key role, result in no changes in the firing of the GABA anti-reward neurons, resulting in no reduction in cocaine self-administration in the present experiments.

Detection, Visualization and Quantification of Protein Complexes in Human Alzheimer's Disease Brains using Proximity Ligation Assay.

Examination of healthy and diseased human brain is essential to translational neuroscience. Protein-protein interactions play a pivotal role in physiological and pathological processes, but their detection is difficult, especially in aged and fixed human brain tissue. We used the proximity ligation assay (PLA) to broaden the range of molecular interactions assessable in-situ in human neuropathology. We adapted fluorescent in-situ PLA to detect ubiquitin-modified proteins in human brains with Alzheimer's disease (AD), including approaches for the management of autofluorescence and quantification using a high-content image analysis system. We confirmed that hyperphosphorylated microtubule-associated protein tau (Serine202, Threonine205) aggregates were modified by ubiquitin and that phospho-tau-ubiquitin complexes were increased in hippocampal and frontal cortex regions in AD compared to non-AD brains. Overall, we refined PLA for use in human neuropathology, which has revealed a profound change in the distribution of ubiquitin in AD brain and its association with characteristic tau pathologies.

CRISPRi screens in human iPSC-derived astrocytes elucidate regulators of distinct inflammatory reactive states.

Astrocytes become reactive in response to insults to the central nervous system by adopting context-specific cellular signatures and outputs, but a systematic understanding of the underlying molecular mechanisms is lacking. In this study, we developed CRISPR interference screening in human induced pluripotent stem cell-derived astrocytes coupled to single-cell transcriptomics to systematically interrogate cytokine-induced inflammatory astrocyte reactivity. We found that autocrine-paracrine IL-6 and interferon signaling downstream of canonical NF-κB activation drove two distinct inflammatory reactive signatures, one promoted by STAT3 and the other inhibited by STAT3. These signatures overlapped with those observed in other experimental contexts, including mouse models, and their markers were upregulated in human brains in Alzheimer's disease and hypoxic-ischemic encephalopathy. Furthermore, we validated that markers of these signatures were regulated by STAT3 in vivo using a mouse model of neuroinflammation. These results and the platform that we established have the potential to guide the development of therapeutics to selectively modulate different aspects of inflammatory astrocyte reactivity.

The mGlu5 Receptor Protomer-Mediated Dopamine D2 Receptor Trans-Inhibition Is Dependent on the Adenosine A2A Receptor Protomer: Implications for Parkinson's Disease.

The adenosine A2A receptor (A2AR), dopamine D2 receptor (D2R) and metabotropic glutamate receptor type 5 (mGluR5) form A2AR-D2R-mGluR5 heteroreceptor complexes in living cells and in rat striatal neurons. In the current study, we present experimental data supporting the view that the A2AR protomer plays a major role in the inhibitory modulation of the density and the allosteric receptor-receptor interaction within the D2R-mGluR5 heteromeric component of the A2AR-D2R-mGluR5 complex in vitro and in vivo. The A2AR and mGluR5 protomers interact and modulate D2R protomer recognition and signalling upon forming a trimeric complex from these receptors. Expression of A2AR in HEK293T cells co-expressing D2R and mGluR5 resulted in a significant and marked increase in the formation of the D2R-mGluR5 heteromeric component in both bioluminescence resonance energy transfer and proximity ligation assays. A highly significant increase of the the high-affinity component of D2R (D2RKi High) values was found upon cotreatment with the mGluR5 and A2AR agonists in the cells expressing A2AR, D2R and mGluR5 with a significant effect observed also with the mGluR5 agonist alone compared to cells expressing only D2R and mGluR5. In cells co-expressing A2AR, D2R and mGluR5, stimulation of the cells with an mGluR5 agonist like or D2R antagonist fully counteracted the D2R agonist-induced inhibition of the cAMP levels which was not true in cells only expressing mGluR5 and D2R. In agreement, the mGluR5-negative allosteric modulator raseglurant significantly reduced the haloperidol-induced catalepsy in mice, and in A2AR knockout mice, the haloperidol action had almost disappeared, supporting a functional role for mGluR5 and A2AR in enhancing D2R blockade resulting in catalepsy. The results represent a relevant example of integrative activity within higher-order heteroreceptor complexes.

Increased density and antagonistic allosteric interactions in A2AR-D2R heterocomplexes in extinction from cocaine use, lost in cue induced reinstatement of cocaine seeking.

Neurochemical studies were previously performed on the effects of a 10 day extinction learning from cocaine self-administration on D2R and A2AR recognition and D2R Gi/o coupling in the ventral striatum. In the present study biochemical receptor binding and proximity ligation assay were used to study possible changes in the allosteric receptor-receptor interactions and the density of the A2AR-D2R heterocomplexes in the ventral striatum (nucleus accumbens shell) in extinction from cocaine self-administration including cue induced reinstatement of cocaine seeking. A significant and clear-cut reduction of active lever pressing was observed in extinction on day 10 from cocaine use. In cue induced reinstatement of cocaine self-administration a significant return in active lever presses developed. In extinction, significant increases in the density of A2AR-D2R and D2R-Sigma1R heterocomplexes were observed in nucleus accumbens shell. In contrast, cue-induced reinstatement of cocaine seeking produced no significant changes in these heteroreceptor complexes of the nucleus accumbens shell. In the 3H raclopride/quinpirole competition binding experiments, the extinction led to a significant increase in the D2R Ki, High dissociation constant in the ventral striatum upon ex vivo exposure to CGS 21680 (100 nM), compared to the same exposure performed in membrane preparations from yoked saline rats. No significant changes in D2R Ki, High values were observed in membrane preparations from rats after cue-induced reinstatement of cocaine-seeking undergoing the same exposure ex vivo to CGS 21680 when compared with membrane preparations from yoked saline rats undergoing the same procedures. It seems likely that increased formation of A2AR-D2R and putative A2AR-D2R-Sigma1R heterocomplexes in the nucleus accumbens shell is part of the mechanism for the enhanced antagonistic allosteric A2AR-D2R interactions developed in extinction learning from cocaine. It reduces cocaine reward through reduced D2R function, and these inhibitory mechanisms are no longer in operation in cue induced reinstatement of cocaine seeking.

The Balance of MU-Opioid, Dopamine D2 and Adenosine A2A Heteroreceptor Complexes in the Ventral Striatal-Pallidal GABA Antireward Neurons May Have a Significant Role in Morphine and Cocaine Use Disorders.

The widespread distribution of heteroreceptor complexes with allosteric receptor-receptor interactions in the CNS represents a novel integrative molecular mechanism in the plasma membrane of neurons and glial cells. It was proposed that they form the molecular basis for learning and short-and long-term memories. This is also true for drug memories formed during the development of substance use disorders like morphine and cocaine use disorders. In cocaine use disorder it was found that irreversible A2AR-D2R complexes with an allosteric brake on D2R recognition and signaling are formed in increased densities in the ventral enkephalin positive striatal-pallidal GABA antireward neurons. In this perspective article we discuss and propose how an increase in opioid heteroreceptor complexes, containing MOR-DOR, MOR-MOR and MOR-D2R, and their balance with each other and A2AR-D2R complexes in the striatal-pallidal enkephalin positive GABA antireward neurons, may represent markers for development of morphine use disorders. We suggest that increased formation of MOR-DOR complexes takes place in the striatal-pallidal enkephalin positive GABA antireward neurons after chronic morphine treatment in part through recruitment of MOR from the MOR-D2R complexes due to the possibility that MOR upon morphine treatment can develop a higher affinity for DOR. As a result, increased numbers of D2R monomers/homomers in these neurons become free to interact with the A2A receptors found in high densities within such neurons. Increased numbers of A2AR-D2R heteroreceptor complexes are formed and contribute to enhanced firing of these antireward neurons due to loss of inhibitory D2R protomer signaling which finally leads to the development of morphine use disorder. Development of cocaine use disorder may instead be reduced through enkephalin induced activation of the MOR-DOR complex inhibiting the activity of the enkephalin positive GABA antireward neurons. Altogether, we propose that these altered complexes could be pharmacological targets to modulate the reward and the development of substance use disorders.

Acute cocaine treatment enhances the antagonistic allosteric adenosine A2A-dopamine D2 receptor-receptor interactions in rat dorsal striatum without increasing significantly extracellular dopamine levels.

BACKGROUND: Antagonistic adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) receptor-receptor interactions have previously been demonstrated in A2AR-D2R heteroreceptor complexes in the rat dorsal striatum. They mainly involve a reduction of affinity in the high-affinity component of the D2R agonist binding site upon activation in vivo of the A2AR by an A2AR agonist. Upon cocaine self-administration, this antagonistic A2AR-D2R interaction disappeared in the dorsal striatum. METHODS: In the current experiments, it was tested whether such modifications in the antagonistic A2AR-D2R receptor-receptor interactions can develop also after an acute systemic injection of a low cocaine dose (1 mg/kg; sc). RESULTS: Microdialysis experiments indicated that acute cocaine did not significantly alter the extracellular dopamine levels in the dorsal striatum of the awake Wistar rats. Competition dopamine receptor binding experiments demonstrated that in the acute cocaine group, the A2AR agonist CGS-21680 produced significantly larger increases in the D2R Ki, High values (reduction of high-affinity) versus the saline-injected (i.e. control) group. Furthermore, in the dorsal striatum membrane preparation from acute cocaine-injected rats, CGS-21680 also produced significant increases in the D2R Ki, Low values (reduction of low-affinity) and in the proportion of D2Rs in the high-affinity state (RH). Such significant effects were not observed with CGS-21680 in the control group. CONCLUSIONS: The molecular mechanism involved in the acute cocaine-induced increase in the antagonistic allosteric A2AR-D2R receptor-receptor interactions may be an increased formation of higher-order complexes A2AR-D2R-sigma1R in which cocaine by binding to the sigma1R protomer also allosterically enhances the inhibitory A2AR-D2R interaction in this receptor complex.

Dual disruption of aldehyde dehydrogenases 1 and 3 promotes functional changes in the glutathione redox system and enhances chemosensitivity in nonsmall cell lung cancer.

Aldehyde dehydrogenases (ALDHs) are multifunctional enzymes that oxidize diverse endogenous and exogenous aldehydes. We conducted a meta-analysis based on The Cancer Genome Atlas and Gene Expression Omnibus data and detected genetic alterations in ALDH1A1, ALDH1A3, or ALDH3A1, 86% of which were gene amplification or mRNA upregulation, in 31% of nonsmall cell lung cancers (NSCLCs). The expression of these isoenzymes impacted chemoresistance and shortened survival times in patients. We hypothesized that these enzymes provide an oxidative advantage for the persistence of NSCLC. To test this hypothesis, we used genetic and pharmacological approaches with DIMATE, an irreversible inhibitor of ALDH1/3. DIMATE showed cytotoxicity in 73% of NSCLC cell lines tested and demonstrated antitumor activity in orthotopic xenografts via hydroxynonenal-protein adduct accumulation, GSTO1-mediated depletion of glutathione and increased H2O2. Consistent with this result, ALDH1/3 disruption synergized with ROS-inducing agents or glutathione synthesis inhibitors to trigger cell death. In lung cancer xenografts with high to moderate cisplatin resistance, combination treatment with DIMATE promoted strong synergistic responses with tumor regression. These results indicate that NSCLCs with increased expression of ALDH1A1, ALDH1A3, or ALDH3A1 may be targeted by strategies involving inhibitors of these isoenzymes as monotherapy or in combination with chemotherapy to overcome patient-specific drug resistance.

OSU-6162, a Sigma1R Ligand in Low Doses, Can Further Increase the Effects of Cocaine Self-Administration on Accumbal D2R Heteroreceptor Complexes.

Cocaine was previously shown to act at the Sigma1R which is a target for counteracting cocaine actions. It therefore becomes of interest to test if the monoamine stabilizer (-) OSU-6162 (OSU-6162) with a nanomolar affinity for the Sigma1R can acutely modulate in low doses the effects of cocaine self-administration. In behavioral studies, OSU-6162 (5 mg/kg, s.c.) did not significantly change the number of active lever pressing and cocaine infusions. However, a trend to reduce cocaine readouts was found after 3 days of treatment. In contrast, in maintenance of cocaine self-administration, the proximity ligation assay performed on brains from rats pretreated with OSU-6162 showed highly significant increases in the density of the D2R-Sigma1R heteroreceptor complexes in the shell of the nucleus accumbens versus OSU-6162 induced increases in this region of yoked saline rats. In cocaine self-administration, highly significant increases were also induced by OSU-6162 in the A2AR-D2R heteroreceptor complexes in the nucleus accumbens shell versus vehicle-treated rats. Furthermore, ex vivo, the A2AR agonist CGS21680 (100 nM) produced a marked and significant increase of the D2R Ki high values in the OSU-6162-treated versus vehicle-treated rats under maintenance of cocaine self-administration. These results indicate a substantial increase in the inhibitory allosteric A2AR-D2R interactions following cocaine self-administration upon activation by the A2AR agonist ex vivo. The current results indicate that OSU-6162 via its high affinity for the Sigma1R may increase the number of accumbal shell D2R-Sigma1R and A2AR-D2R heteroreceptor complexes associated with further increases in the antagonistic A2AR-D2R interactions in cocaine self-administration.

A2AR Transmembrane 2 Peptide Administration Disrupts the A2AR-A2AR Homoreceptor but Not the A2AR-D2R Heteroreceptor Complex: Lack of Actions on Rodent Cocaine Self-Administration.

It was previously demonstrated that rat adenosine A2AR transmembrane V peptide administration into the nucleus accumbens enhances cocaine self-administration through disruption of the A2AR-dopamine (D2R) heteroreceptor complex of this region. Unlike human A2AR transmembrane 4 (TM4) and 5 (TM5), A2AR TM2 did not interfere with the formation of the A2AR-D2R heteroreceptor complex in cellular models using BRET1 assay. A2AR TM2 was proposed to be part of the of the receptor interface of the A2AR homomer instead and was therefore tested in the current article for effects on rat cocaine self-administration using rat A2AR synthetic TM2 peptide bilaterally injected into the nucleus accumbens. The injected A2AR TM2 peptide failed to significantly counteract the inhibitory action of the A2AR agonist CGS 21680 (0.1 mg/Kg) on cocaine self-administration. In line with these results, the microinjected A2AR TM2 peptide did not reduce the number of proximity ligation assay blobs identifying A2AR-D2R heteroreceptor complexes in the nucleus accumbens. In contrast, the A2AR TM2 peptide significantly reduced the number of A2AR-A2AR homoreceptor complexes in the nucleus accumbens. As to effects on the receptor-receptor interactions in the A2AR-D2R heteroreceptor complexes, the A2AR TM2 peptide did not alter the significant increase in the D2R Ki, high values produced by the A2AR agonist CGS 21680 ex vivo in the ventral striatum. The results indicate that the accumbal A2AR-A2AR homomeric complexes are not involved in mediating the A2AR agonist-induced inhibition of cocaine self-administration.

Acute Cocaine Enhances Dopamine D2R Recognition and Signaling and Counteracts D2R Internalization in Sigma1R-D2R Heteroreceptor Complexes.

The current study was performed to establish the actions of nanomolar concentrations of cocaine, not blocking the dopamine transporter, on dopamine D2 receptor (D2R)-sigma 1 receptor (δ1R) heteroreceptor complexes and the D2R protomer recognition, signaling and internalization in cellular models. We report the existence of D2R-δ1R heteroreceptor complexes in subcortical limbic areas as well as the dorsal striatum, with different distribution patterns using the in situ proximity ligation assay. Also, through BRET, these heteromers were demonstrated in HEK293 cells. Furthermore, saturation binding assay demonstrated that in membrane preparations of HEK293 cells coexpressing D2R and δ1R, cocaine (1 nM) significantly increased the D2R Bmax values over cells singly expressing D2R. CREB reporter luc-gene assay indicated that coexpressed δ1R significantly reduced the potency of the D2R-like agonist quinpirole to inhibit via D2R activation the forskolin induced increase of the CREB signal. In contrast, the addition of 100 nM cocaine was found to markedly increase the quinpirole potency to inhibit the forskolin-induced increase of the CREB signal in the D2R-δ1R cells. These events were associated with a marked reduction of cocaine-induced internalization of D2R protomers in D2R-δ1R heteromer-containing cells vs D2R singly expressing cells as studied by means of confocal analysis of D2R-δ1R trafficking and internalization. Overall, the formation of D2R-δ1R heteromers enhanced the ability of cocaine to increase the D2R protomer function associated with a marked reduction of its internalization. The existence of D2R-δ1R heteromers opens up a new understanding of the acute actions of cocaine.

Mapping the Interface of a GPCR Dimer: A Structural Model of the A2A Adenosine and D2 Dopamine Receptor Heteromer.

The A2A adenosine (A2AR) and D2 dopamine (D2R) receptors form oligomers in the cell membrane and allosteric interactions across the A2AR-D2R heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of A2AR-D2R heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the A2AR-D2R heterodimer was generated using a combined experimental and computational approach. Regions involved in the heteromer interface were modeled based on the effects of peptides derived from the transmembrane (TM) helices on A2AR-D2R receptor-receptor interactions in bioluminescence resonance energy transfer (BRET) and proximity ligation assays. Peptides corresponding to TM-IV and TM-V of the A2AR blocked heterodimer interactions and disrupted the allosteric effect of A2AR activation on D2R agonist binding. Protein-protein docking was used to construct a model of the A2AR-D2R heterodimer with a TM-IV/V interface, which was refined using molecular dynamics simulations. Mutations in the predicted interface reduced A2AR-D2R interactions in BRET experiments and altered the allosteric modulation. The heterodimer model provided insights into the structural basis of allosteric modulation and the technique developed to characterize the A2AR-D2R interface can be extended to study the many other G protein-coupled receptors that engage in heteroreceptor complexes.

Receptor⁻Receptor Interactions in Multiple 5-HT1A Heteroreceptor Complexes in Raphe-Hippocampal 5-HT Transmission and Their Relevance for Depression and Its Treatment.

Due to the binding to a number of proteins to the receptor protomers in receptor heteromers in the brain, the term "heteroreceptor complexes" was introduced. A number of serotonin 5-HT1A heteroreceptor complexes were recently found to be linked to the ascending 5-HT pathways known to have a significant role in depression. The 5-HT1A⁻FGFR1 heteroreceptor complexes were involved in synergistically enhancing neuroplasticity in the hippocampus and in the dorsal raphe 5-HT nerve cells. The 5-HT1A protomer significantly increased FGFR1 protomer signaling in wild-type rats. Disturbances in the 5-HT1A⁻FGFR1 heteroreceptor complexes in the raphe-hippocampal 5-HT system were found in a genetic rat model of depression (Flinders sensitive line (FSL) rats). Deficits in FSL rats were observed in the ability of combined FGFR1 and 5-HT1A agonist cotreatment to produce antidepressant-like effects. It may in part reflect a failure of FGFR1 treatment to uncouple the 5-HT1A postjunctional receptors and autoreceptors from the hippocampal and dorsal raphe GIRK channels, respectively. This may result in maintained inhibition of hippocampal pyramidal nerve cell and dorsal raphe 5-HT nerve cell firing. Also, 5-HT1A⁻5-HT2A isoreceptor complexes were recently demonstrated to exist in the hippocampus and limbic cortex. They may play a role in depression through an ability of 5-HT2A protomer signaling to inhibit the 5-HT1A protomer recognition and signaling. Finally, galanin (1⁻15) was reported to enhance the antidepressant effects of fluoxetine through the putative formation of GalR1⁻GalR2⁻5-HT1A heteroreceptor complexes. Taken together, these novel 5-HT1A receptor complexes offer new targets for treatment of depression.

Medio de cultivo convencional enriquecido con extractos de quinoa, amaranto y chía promueve el crecimiento de Staphylococcus aureus / Conventional culture media enriched with extracts of quinoa, amaranth and chia promotes the Staphylococcus aureus growth

La identificación bacteriana durante las infecciones microbiológicas es un aspecto crítico a la hora de escoger un tratamiento específico para evitar complicaciones del paciente o en algunos casos su muerte. Por ello, incrementar el crecimiento celular durante el diagnóstico clínico por cultivo bacteriano puede reducir el tiempo para determinar el patógeno que causa la enfermedad. En este trabajo, se utilizó el cultivo bacteriano como metodología y se evaluó el crecimiento de Staphylococcus aureus en un medio de cultivo convencional enriquecido con extractos de Chenopodium quinoa, Amaranthus caudatus y Salvia hispanica. Los resultados obtenidos muestran que estos extractos, a bajas concentraciones, tienen un efecto protector contra la citotoxicidad que se podría generar por el estrés oxidativo producto del metabolismo celular de las bacterias cultivadas in vitro e incrementan significativamente el crecimiento bacteriano. La adicción de estos extractos a los medios convencionales podría mejorar el crecimiento bacteriano durante un diagnóstico bacteriológico y reducir el tiempo de identificación del patógeno.

Increasing the bacterial growth rate reduces the time getting the bacteria identification. This is helpful to choose an accurate and quick therapeutic strategy during microbiological infections, avoiding illness complications or in some cases the death. Here, we used bacterial growth method and we evaluated the growth of Staphylococcus aureus including an extract of Chenopodium quinoa, Amaranthus caudatus and Salvia hispanica in the routine culture media. Results show that adding these extracts, at low concentrations, have a protective effect against the cytotoxicity that could be generated by the oxidative stress product of the cellular metabolism of the bacteria growing in vitro and significantly increase the bacterial growth. The addition of these extracts to conventional culture media could improve bacterial growth during a bacteriological diagnosis and to reduce the time of pathogen identification.

[The 1, 2, 3 of laboratory animal experimentation]. / El 1, 2, 3 de la experimentación con animales de laboratorio.

The slow scientific development in Latin America in recent decades has delayed the incorporation of laboratory animal experimentation; however, this situation has started to change. Today, extraordinary scientific progress is evident, which has promoted the introduction and increased use of laboratory animals as an important tool for the advancement of biomedical sciences. In the aftermath of this boom, the need to provide the scientific community with training and guidance in all aspects related to animal experimentation has arisen. It is the responsibility of each country to regulate this practice, for both bioethical and legal reasons, to ensure consideration of the animals' rights and welfare. The following manuscript is the result of papers presented at the International Workshop on Laboratory Animal Testing held at the Technical University of Ambato, Ecuador; it contains information regarding the current state of affairs in laboratory animal testing and emphasizes critical aspects such as main species used, ethical and legal principles, and experimental and alternative designs for animal use. These works aim to ensure good practices that should define scientific work. This document will be relevant to both researchers who aim to newly incorporate animal testing into their research and those who seek to update their knowledge.

El 1, 2, 3 de la experimentación con animales de laboratorio / The 1, 2, 3 of laboratory animal experimentation

RESUMEN El lento desarrollo científico experimentado en América Latina en las últimas décadas ha retrasado la incorporación de la experimentación con animales de laboratorio, sin embargo, esta realidad ha comenzado a cambiar. En la actualidad, se evidencia un extraordinario progreso científico que ha promovido la introducción e incremento del uso de animales de laboratorio como una importante herramienta para el avance de las ciencias biomédicas. A raíz de este auge, surge la necesidad de proporcionar a la comunidad científica la formación y directrices en todos los aspectos relacionados con la experimentación animal. Es responsabilidad de cada país legislar esta práctica para que no solo por razones bioéticas, sino también legales, se considere el derecho de los animales y con ello su bienestar. El siguiente manuscrito es el resultado de las comunicaciones presentadas en el Taller Internacional en Ciencias de Animales de Laboratorio celebrado en la Universidad Técnica de Ambato, Ecuador; contiene la actualidad en la ciencia de animales de laboratorio, haciendo énfasis en aspectos fundamentales, tales como: principales especies utilizadas y su biología, principios éticos y legales, diseño experimental y alternativas al uso de animales, todas ellas encaminadas a garantizar las buenas prácticas que deben caracterizar el quehacer científico. Sin duda, este documento será relevante tanto para aquellos investigadores que pretenden incorporar la experimentación animal a sus investigaciones, como para aquellos que, aun teniendo experiencia, pretendan actualizar sus conocimientos.

ABSTRACT The slow scientific development in Latin America in recent decades has delayed the incorporation of laboratory animal experimentation; however, this situation has started to change. Today, extraordinary scientific progress is evident, which has promoted the introduction and increased use of laboratory animals as an important tool for the advancement of biomedical sciences. In the aftermath of this boom, the need to provide the scientific community with training and guidance in all aspects related to animal experimentation has arisen. It is the responsibility of each country to regulate this practice, for both bioethical and legal reasons, to ensure consideration of the animals' rights and welfare. The following manuscript is the result of papers presented at the International Workshop on Laboratory Animal Testing held at the Technical University of Ambato, Ecuador; it contains information regarding the current state of affairs in laboratory animal testing and emphasizes critical aspects such as main species used, ethical and legal principles, and experimental and alternative designs for animal use. These works aim to ensure good practices that should define scientific work. This document will be relevant to both researchers who aim to newly incorporate animal testing into their research and those who seek to update their knowledge.