Dopamine D<Subscript>2</Subscript> agonist priming in intact and dopamine-lesioned rats

Receptor priming is a recently discovered phenomenon by which receptor agonists produce abrupt and long-lived supersensitization of receptors. Induction of dopamine (DA) D2 receptor supersensitivity by the agonist quinpirole was discovered approximately 15 years ago, and was found to occur consistently if rats were treated repeatedly at daily or weekly or monthly intervals with low or high doses of quinpirole. In this review we summarize and discuss some of the major studies that underlie DA D2 receptor supersen-sitivity, describe behavioral processes that are known to be altered by DA D2 receptor supersensitivity, and discuss the importance of DA innervation on expression of enhanced behaviors. DA D2 receptor supersen-sitivity represents one of the neural mechanisms implicated in psychiatric disorders. Also, DA D₂ receptor supersensitivity and increased DA D₃ receptor expression are associated with motor dyskinesias, as in L-DOPA-treated Parkinson’s disease patients. An understanding of receptor priming, a knowledge of the types of behavioral expression associated with DA D₂ receptor supersensitivity, and an understanding of mechanisms associated with receptor supersen-sitization, can lead to improvements in the treatments of psychiatric and neurological disorders.     

Modulation of Postnatal Neurogenesis by Perinatal Asphyxia: Effect of D<Subscript>1</Subscript> and D<Subscript>2</Subscript> Dopamine Receptor Agonists

Perinatal asphyxia (PA) is associated to delayed cell death, affecting neurocircuitries of basal ganglia and hippocampus, and long-term neuropsychiatric disabilities. Several compensatory mechanisms have been suggested to take place, including cell proliferation and neurogenesis. There is evidence that PA can increase postnatal neurogenesis in hippocampus and subventricular zone (SVZ), modulated by dopamine, by still unclear mechanisms. We have studied here the effect of selective dopamine receptor agonists on cell death, cell proliferation and neurogenesis in organotypic cultures from control and asphyxia-exposed rats. Hippocampus and SVZ sampled at 1–3 postnatal days were cultured for 20–21 days. At day in vitro (DIV) 19, cultures were treated either with SKF38393 (10 and 100 µM, a D₁ agonist), quinpirole (10 µM, a D₂ agonist) or sulpiride (10 μM, a D₂ antagonist) + quinpirole (10 μM) and BrdU (10 μM, a mitosis marker) for 24 h. At DIV 20–21, cultures were processed for immunocytochemistry for microtubule-associated protein-2 (MAP-2, a neuronal marker), and BrdU, evaluated by confocal microscopy. Some cultures were analysed for cell viability at DIV 20–21 (LIVE/DEAD kit). PA increased cell death, cell proliferation and neurogenesis in hippocampus and SVZ cultures. The increase in cell death, but not in cell proliferation, was inhibited by both SKF38393 and quinpirole treatment. Neurogenesis was increased by quinpirole, but only in hippocampus, in cultures from both asphyxia-exposed and control-animals, effect that was antagonised by sulpiride, leading to the conclusion that dopamine modulates neurogenesis in hippocampus, mainly via D₂ receptors.     

Presence of D<Subscript>1</Subscript>- and D<Subscript>2</Subscript>-like dopamine receptors in the rat,mouse and bovine multiciliated ependyma

Summary The multiciliated ependyma forms an epithelial-like layer that could act as a selective barrier between the brain parenchyma and cerebrospinal fluid. In the present study, tyrosine hydroxylase-containing fibres have been detected in the basal pole of the ependymal cells of the lateral ventricles of rat, mouse and calf. The use of antibodies against at least two different peptide sequences of each D₂, D₃, D₄ and D₅ dopamine receptor subtype has allowed their detection in: (i) sections of mouse, rat and bovine lateral ventricles, by means of immunocytochemistry; and (ii) membrane protein extracts obtained from the ependymal layer of the bovine lateral ventricles, using immunoblotting. The immunocytochemical study has shown the presence of all these subtypes of dopamine receptors in the ependymal cells. Immunoblotting demonstrated similar immunoreactive bands for all receptor subtypes in both ependymal and corpus striatum membrane extracts.     

Effects of dopamine D<Subscript>4</Subscript> receptor antagonist on spontaneous alternation in rats

Background  

The present study was a component of a series of studies scrutinising the neuroreceptor substrate of behavioural flexibility in a rat model. Spontaneous alternation paradigms model the natural tendency of rodents to spontaneously and flexibly shift between alternative spatial responses. In the study it was tested for the first time if the neurochemical substrate mediating spontaneous alternation behaviour includes the dopamine D₄ receptor.     

Extrastriatal dopamine D<Subscript>2</Subscript> receptors in Parkinson's disease: a longitudinal study

Most antiparkinsonian drugs are known to act through central dopamine D(2) receptor agonism. A previous longitudinal positron emission tomography (PET) study has indicated that, in the striatum of Parkinson's disease (PD) patients, dopamine D(2) receptor binding declines at a relatively fast annual rate of 2-4% (compared to the rate of <1%/year in healthy individuals). In the present study, the examination of longitudinal changes in D(2) receptors was extended to extrastriatal brain regions in PD. Eight early PD patients were examined twice with PET, approximately 3 years apart, using a high-affinity extrastriatal D(2)/D(3) receptor tracer, [(11)C]FLB 457. Both the MRI-referenced region-of-interest method and the voxel-based statistical analysis method were used independently in the analysis. Regional D(2)-like availabilities (binding potentials) in the left dorsolateral prefrontal cortex, the left temporal cortex and the left and right medial thalami were significantly decreased at the second examination by 20-37% (corresponding to an annual decline of 6-11%). Thus, the annual loss of extrastriatal D(2) availability in PD is up to three times faster than the rate previously reported in the putamen. Our longitudinal study shows first evidence concerning cortical D(2) receptor loss in the progression of PD, although it is not possible to distinguish between the effects of the therapy and the disease.     

Requirement of hippocampal phospholipase A<Subscript>2</Subscript> activity for long-term memory retrieval in rats

Summary. In rats, the inhibition of phospholipase A₂ (PLA₂) in hippocampus was reported to impair memory acquisition. In the present study we investigated in rats whether PLA₂ inhibition in hippocampus is also related to impairment of memory retrieval. Rats were bilaterally implanted with cannulae in hippocampal CA1 region. After recovery, animals were submitted to one-trial step-down inhibitory avoidance task and tested for long-term memory (LTM) 24 h later. Before test session, animals received infusions of vehicle or the PLA₂ inhibitor PACOCF₃. Inhibition of PLA₂ activity impaired LTM retrieval. Memory impairment was fully reversed once PLA₂ activity was recovered. Moreover, LTM retrieval per se increased PLA₂ activity. To our knowledge, we demonstrated for the first time that PLA₂ activity is required for memory retrieval. Because reduced PLA₂ activity has been found in Alzheimer’s disease brains, the present results may be relevant to clarify at least part of the biology of this disorder.     

Conditioning training and retrieval increase phospholipase A<Subscript>2</Subscript> activity in the cerebral cortex of rats

In rats, phospholipase A₂ (PLA₂) activity was found to be increased in the hippocampus immediately after training and retrieval of a contextual fear conditioning paradigm (step-down inhibitory avoidance [IA] task). In the present study we investigated whether PLA₂ is also activated in the cerebral cortex of rats in association with contextual fear learning and retrieval. We observed that IA training induces a rapid (immediately after training) and long-lasting (3 h after training) activation of PLA₂ in both frontal and parietal cortices. However, immediately after retrieval (measured 24 h after training), PLA₂ activity was increased just in the parietal cortex. These findings suggest that PLA₂ activity is differentially required in the frontal and parietal cortices for the mechanisms of contextual learning and retrieval. Because reduced brain PLA₂ activity has been reported in Alzheimer disease, our results suggest that stimulation of PLA₂ activity may offer new treatment strategies for this disease.     

L-745,870 suppresses the nighttime serotonin N-acetyltransferase activity in chick retina: in vivo evidence for agonist activity at D<Subscript>4</Subscript>-dopamine receptors

Summary. This study examined the in vivo activity of L-745,870 at dopamine (DA) D₄ receptors, using the chick retina as a model system. In dark-adapted retinas of various vertebrates, including hen, DA acting via D₄ receptors suppresses melatonin content and activity of serotonin N-acetyltransferase (AA-NAT, a key regulatory enzyme in melatonin synthesis). Systemic administration to chicks of quinpirole (0.1 mg/kg), a high affinity agonist of D₃/D₄-DA receptors, potently decreased the nighttime AA-NAT activity of the retina. The quinpirole-evoked decline in the enzyme activity was attenuated by L-745,870 (0.1–10 nmol/eye). In addition to this action, L-745,870 given to chicks either directly into the eye (0.03–10 nmol/eye) or intraperitoneally (0.5–5 mg/kg) decreased the nighttime AA-NAT activity of the retina in a dose-dependent manner. The suppressive effect of L-745,870 on retinal AA-NAT activity was blocked by 2-chloro-11-(4-methylpiperazino)dibenz[b,f]oxepin, an antagonist of D₄-DA receptors, but was not affected by raclopride, an antagonist of D₂/D₃-DA receptors. Altogether these results indicate that in chicks L-745,870, the potent putative D₄-DA receptor antagonist, behaves in vivo as a partial D₄ agonist. Received May 5, 2002; accepted September 16, 2002 Published online December 9, 2002 Authors' address: Dr. J. B. Zawilska, Department of Biogenic Amines, Polish Academy of Sciences, POB-225 Lodz-1, 90-950 Poland, e-mail: jolantaz@amina1.zabpan.lodz.pl     

Dopamine and glutamate dysfunctions in schizophrenia: Role of the dopamine D<Subscript>3</Subscript> receptor

Symptoms of schizophrenia are improved by dopamine antagonists and exacerbated by dopamine-releasing agents, suggesting hyperactivity of dopamine. However, chronic blockade of glutamate neurotransmission by antagonists at theN-methyl-D-aspartate (NMDA) receptor subtype produces a pathophysiological state resembling schizophrenia. A link between cortical glutamate/NMDA deficiency and subcortical dopamine hyperactivity, particularly in the mesolimbic pathway, has been hypothesized in schizophrenia. Here we show that hyperactivity produced by NMDA receptor blockade is dependent upon stimulation of the dopamine D₃ receptor subtype. Since D₃ receptor antagonists and antipsychotics produced very similar effects, our results add to the growing evidence suggesting that D₃ receptor blockade might produce antipsychotic effects.     

The α<Subscript>1</Subscript>,α<Subscript>2</Subscript>, and α<Subscript>3</Subscript> Subunits of GABA<Subscript>A</Subscript> Receptors: Comparison in Seizure-Prone and -Resistant Mice and during Development

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in brain, opens chloride channels through actions on GABAA receptors. We now report base and amino acid sequences of the alpha 1, alpha 2, and alpha 3 subunits from GABAA receptors of audiogenic seizure-prone (DBA/2J) and -resistant (C57BL/6J) inbred strains of mice. Inbreeding had fixed different alleles of the alpha 1 subunit in the two strains, giving five base differences in the cDNAs. None of these affected amino acid sequence, but one did create a NsiI restriction site potentially useful in mapping genomic DNA. No base or amino acid sequence differences between the strains were detected for the other two subunits. Northern blots revealed no apparent strain differences in message levels for these three subunits in whole brains of the mice at 3 weeks of age, the peak of seizure susceptibility in DBA/2J, but did reveal distinct regional and developmental patterns of expression among the subunits in mouse brain.     

CHA<Subscript>2</Subscript>DS<Subscript>2</Subscript>-VASc score and prognosis in ischemic strokes with atrial fibrillation

The CHA₂DS₂-VASc score was developed to improve stroke risk stratification in atrial fibrillation (AF) patients. We sought to analyze the distribution and prognostic value of the CHA₂DS₂-VASc score in a cohort of ischemic stroke patients with AF. In total, 439 consecutive stroke patients with AF were studied. The CHA₂DS₂-VASc score was calculated according to clinical status before stroke onset. Poor outcome was defined as a modified Rankin score of 3 to 6 at 3 months. Association between CHA₂DS₂-VASc score and poor outcome was analyzed using logistic regression analysis. In 95.6% of patients, CHA₂DS₂-VASc was >1 and only 41.8% of those with previously diagnosed AF were using oral anticoagulation at the time of the stroke. Poor outcome was found in 53.1% of the patients. In univariate analysis age, female sex, current smoking, previous stroke, CHA₂DS₂-VASc score, and stroke severity were associated with outcome. In multivariate analysis, CHA₂DS₂-VASc score was independently associated with poor outcome [OR 1.36 (95% CI: 1.14–1.62), P = 0.001] as well as NIHSS [OR 1.22 (95% CI: 1.17–1.26), P < 0.001]. After removing stroke severity, therapeutic anticoagulation was also associated with stroke prognosis [OR 0.45 (95% CI: 0.23–0.86), P = 0.016]. Most patients with ischemic stroke and AF have a high CHA₂DS₂-VASc score. Independent of stroke severity, CHA₂DS₂-VASc score is associated with 3-month outcome. Despite all the available information and guidelines, our AF patients are clearly undertreated.     

Evaluation of the Profile and Mechanism of Neurotoxicity of Water-Soluble [Cu(P)<Subscript>4</Subscript>]PF<Subscript>6</Subscript> and [Au(P)<Subscript>4</Subscript>]PF<Subscript>6</Subscript> (P = thp or PTA) Anticancer Complexes

[Cu(thp)₄]PF₆, [Cu(PTA)₄]PF₆, [Au(thp)₄]PF₆ and [Au(PTA)₄]PF₆ are phosphane (thp = tris(hydroxymethyl)phosphane; PTA = 1,3,5-triaza-7-phosphaadamantane) copper(I) and gold(I) water-soluble complexes characterized by high anticancer activity in a wide range of solid tumors, often able to overcome drug resistance of platinum-based compounds. For these reasons, they have been proposed as a valid alternative to platinum-based chemotherapeutic drugs (e.g., cisplatin and oxaliplatin). In vitro experiments performed on organotypic cultures of dorsal root ganglia (DRG) from 15-day-old rat embryos revealed that copper-based compounds were not neurotoxic even at concentrations higher than the IC₅₀ obtained in human cancer cells while [Au(PTA)₄]PF₆ was neurotoxic at lower concentration than IC₅₀ in cancer cell lines. The ability of these compounds to hinder the proteasome machinery in DRG neurons was tested by fluorimetric assay showing that the non-neurotoxic copper-based complexes do not inhibit proteasome activity in DRG primary neuron cultures. On the contrary, the neurotoxic complex [Au(PTA)₄]PF₆, induced a significant inhibition of proteasome activity even at concentrations lower than the IC₅₀ in cancer cells. The proteasome inhibition induced by [Au(PTA)₄]PF₆ was associated with a significant increase in α-tubulin polymerization that was not observed following the treatment with copper-based compounds. Uptake experiments performed by atomic absorption spectrometry showed that both copper-based complexes and [Au(PTA)₄]PF₆ are internalized in neuron cultures. In vitro and in vivo preliminary data confirmed copper-based complexes as the most promising compounds, not only for their anticancer activity but also concerning the peripheral neurotoxicity profile.     

The combination of vitamin D<Subscript>3</Subscript> and dehydroascorbic acid administration attenuates brain damage in focal ischemia

The aim of this study is to determine the protective effects of vitamin D₃ and dehydroascorbic acid (DHA), a blood-brain barrier transportable form of vitamin C, against ischemia/reperfusion (I/R) injury on a middle cerebral artery occlusion/reperfusion model of brain since reactive oxygen species play an important role in the pathophysiology of I/R injury in brain. In order to examine antioxidant status and lipid peroxidation, we assayed malondialdehyde (MDA) levels as a marker of lipid peroxidation, and reduced glutathione (GSH) and superoxide dismutase (SOD) enzyme activities as free radical scavenging enzymes in cortex and corpus striatum (CS). Wistar albino rats were divided into five equal groups of each consisting of seven rats: control, I/R, I/R + DHA, I/R + vitamin D₃, and I/R + vitamin D₃ + dehydroascorbic acid groups. MDA levels were found to be increased in the I/R group, I/R + DHA, and I/R + vitamin D₃ groups compared with the control group in both cortex and corpus striatum. However, MDA level were found to be significantly decreased in only I/R + vitamin D₃ + DHA group compared with the I/R group in cortex (P < 0.0001). MDA levels were not significantly different in I/R + DHA, and I/R + vitamin D₃ groups compared with the I/R group. GSH and SOD enzyme activities were significantly decreased in I/R, I/R + DHA, and I/R + vitamin D₃ groups compared with the control group in both cortex and corpus striatum (CS) (P < 0.0001). Whereas, both GSH and SOD activity were increased in I/R + vitamin D₃ + DHA group compared with the I/R group in both cortex and CS (P < 0.001 in cortex, P < 0.001 in CS for SOD P < 0.002 in cortex P < 0.03 in CS for GSH). Our results demonstrate that the combination of vitamin D₃ and DHA treatment prevent free radical production and dietary supplementation of vitamin D₃ and DHA which may be useful in the ischemic cerebral vascular diseases.     

Differential Sensitivity of A<Subscript>2A</Subscript> and Especially D<Subscript>2</Subscript> Receptor Trafficking to Cocaine Compared with Lipid Rafts in Cotransfected CHO Cell Lines. Novel Actions of Cocaine Independent of the DA Transporter

The effects of low and high concentrations of cocaine have been studied in vitro on the trafficking of plasma membrane A_2A and D₂ immunoreactivities in previously characterized A_2A-D₂ CHO cell lines. Receptor double immunofluorescence staining was performed with D₂ and A_2A antibodies, planar lipid rafts immunolabeling with biotinylated cholera toxin subunit B and membrane invaginations with an anti-caveolin-1 antibody. A computer-assisted image analysis demonstrated a substantial and highly significant rise of membrane-associated D₂ immunoreactivity (IR) after 8 h of exposure to a low concentration of cocaine (150 nM). At this low concentration of cocaine, there was also an increase of membrane associated A_2A immunoreactivity but smaller and less significant. However, this increase became considerably larger and highly significant at 150 μM at which concentration the rise of D₂ immunoreactivity had begun to disappear. It may be suggested that an allosteric action of cocaine at 150 nM on the D₂ receptors may primarily increase the insertion of D₂ monomers, homomers and also of a subpopulation of A_2A-D₂ heteromers from the cytoplasm into the plasma membrane due to the conformational change induced by cocaine in the D₂ receptor. The planar lipid rafts and the caveolae are only affected by the higher concentrations of cocaine. It is proposed that changes in D₂ and A_2A-D₂ trafficking induced by allosteric actions of cocaine at D₂ receptors may contribute to the alterations of D₂ signaling found in cocaine abusers.     

GABA-B<Subscript>1</Subscript> Receptors are Coupled to the ERK1/2 MAP Kinase Pathway in the Absence of GABA-B<Subscript>2</Subscript> Subunits

In the current model of γ-aminobutyric acid (GABA) B receptor function, there is a requirement for GABA-B_1/2 heterodimerisation for targetting to the cell surface. However, different lines of evidence suggest that the GABA-B₁ subunit can form a functional receptor in the absence of GABA-B₂. We observed coupling of endogenous GABA-B₁ receptors in the DI-TNC1 glial cell line to the ERK pathway in response to baclofen even though these cells do not express GABA-B₂. GABA-B_1A receptors were also able to mediate a rapid, transient, and dose-dependent activation of the ERK1/2 MAP kinase pathway when transfected alone into HEK 293 cells. The response was abolished by G_i/o and MEK inhibition, potentiated by inhibitors of phospholipase C and protein kinase C and did not involve PI-3-kinase activity. Finally, using bioluminescence resonance energy transfer and co-immunoprecipitation, we show the existence of homodimeric GABA-B_1A receptors in transfected HEK293 cells. Altogether, our observations show that GABA-B_1A receptors are able to activate the ERK1/2 pathway despite the absence of surface targetting partner GABA-B₂ in both HEK 293 cells and the DI-TNC1 cell line. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Heart-rate response to alpha<Subscript>2</Subscript>-adrenergic receptor antagonism by antipsychotics

Purpose

To explore the relationship between antipsychotic-associated antagonism of alpha₂-adrenergic receptors and resting heart rate in individuals with schizophrenia.

Methods

Thirty-one inpatients treated with antipsychotics were included in this exploratory analysis. Antipsychotic doses were converted to haloperidol equivalents for alpha₂-adrenergic receptor antagonism. Resting heart rate was measured with the patient in the seated upright posture.

Results

After controlling for confounding variables, the relationship between alpha₂-adrenergic receptor antagonism and resting heart rate demonstrated a positive linear effect (P = 0.002) as well as a nonlinear effect that accounted for an additional 14% of the variability in resting heart rate (P = 0.005).

Conclusion

The observed inverted-U relationship between alpha₂-adrenergic receptor antagonism and resting heart rate can possibly be attributed to an altered response of beta₁-adrenergic receptors to increased norepinephrine release. Further investigations are required to confirm this exploratory finding, taking into account additional variables that include other receptors which either directly or indirectly influence heart rate.

ClinicalTrials.gov Identifier

NCT01392885.

     

Vitamin D<Subscript>3</Subscript> Reverses the Hippocampal Cytoskeleton Imbalance But Not Memory Deficits Caused by Ovariectomy in Adult Wistar Rats

The objective of study was to investigate changes caused by ovariectomy (OVX) on aversive and non-aversive memories, as well as on cytoskeleton phosphorylating system and on vitamin D receptor (VDR) immunocontent in hippocampus. The neuroprotective role of vitamin D was also investigated. Ninety-day-old female Wistar rats were divided into four groups: SHAM, OVX, VITAMIN D and OVX + VITAMIN D; 30 days after the OVX, vitamin D supplementation (500 IU/kg), by gavage, for 30 days was started. Results showed that OVX impaired short-term and long-term recognition, and long-term aversive memories. OVX altered hippocampal cytoskeleton phosphorylating system, evidenced by the hyperphosphorylation of glial fibrillary acidic protein (GFAP), low molecular weight neurofilament subunit (NFL), medium molecular weight neurofilament subunit (NFM) and high molecular weight neurofilament subunit (NFH), and increased the immunocontent of c-Jun N-terminal protein kinases (JNK), Ca²⁺/calmodulin-dependent protein kinase II (PKCaMII) and of the sites phosphorylated lysine–serine–proline (KSP) repeats, Ser55 and Ser57. Vitamin D reversed the effects caused by OVX on cytoskeleton in hippocampus, but it was not able to reverse the effects on memory.     

Slow N-acetyltransferase 2 status leads to enhanced intrastriatal dopamine depletion in 6-hydroxydopamine-lesioned rats

We previously reported an association between the N-acetyltransferase 2 (NAT2) slow acetylator status and Parkinson's disease (PD). We have now investigated the possible functional relevance of this association by treating Fischer 344 (F344) rapid and Wistar-Kyoto (WKY) slow NAT2 acetylator rat strains with the neurotoxin 6-hydroxydopamine (6-OHDA). Intrastriatal treatment with either 10 or 20 microg of 6-OHDA lead to a significantly greater reduction of striatal dopamine concentrations in the WKY slow acetylator rat strain than in the F344 rapid acetylator rat strain (P < 0.004), reflecting a more marked degree of dopaminergic denervation. Nigral dopaminergic cell counts were also lower in the WKY rats, but this difference failed to reach statistical significance, suggesting that slow acetylation is especially deleterious at the level of striatal nerve endings.     

The effects of alpha-tocoferol and H<Subscript>2</Subscript>O<Subscript>2</Subscript> on the mitochondrial membrane potential and Bax/Bcl-xL ratio in PC12 cells

Using the flow-cytometry method, we demonstrated the ability of alpha-tocopherol (alpha-T) at nano- and micromolar concentrations to recover the mitochondrial membrane potential (ΔΨm), which decreased after H₂O₂ treatment in PC12 neuronal line cells. Alterations in the ΔΨm in PC12 cells were evaluated using the fluorescent probe tetramethylrhodamine (TMRM). The higher cell-fluorescence intensity corresponded to a higher ΔΨm value. Treatment with H₂O₂ resulted in a decrease in the fluorescence intensity from 100% in the control to 43.2 ± 5.1% (p < 0.001). Long-term 18-h preincubation with 100 nM or 100 μM alpha-T increased the fluorescence intensity in PC12 cells from 43.2 ± 5.2% up to 88.4 ± 4.8 and 89.6 ± 6.5% of the control value, respectively (p < 0.01 according to the one-way analysis of variances). Alpha-T at both 100 nM and 100 μM concentrations significantly and with similar efficacy decreased the ratio of the proapoptotic protein Bax to the antiapoptotic protein Bcl-xL (Bax/Bcl-xL) in the control PC12 cells. However, we did not reveal any significant effect of H₂O₂ or preincubation with alpha-T on this ratio in PC12 lysates. Furthermore, H₂O₂ increased both the Bax and Bcl-xL levels in PC12 cells.     

Evaluation of the Protective Effects of Sarains on H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Mitochondrial Dysfunction and Oxidative Stress in SH-SY5Y Neuroblastoma Cells

Sarains are diamide alkaloids isolated from the Mediterranean sponge Haliclona (Rhizoniera) sarai that have previously shown antibacterial, insecticidal and anti-fouling activities. In this study, we examined for the first time the neuroprotective effects of sarains 1, 2 and A against oxidative stress in a human neuronal model. SH-SY5Y cells were co-incubated with sarains at concentrations ranging from 0.01 to 10 μM, and the well-known oxidant hydrogen peroxide at 150 μM for 6 h and the protective effects of the compounds were evaluated. Among the sarains tested, sarain A was the most promising compound, improving mitochondrial function and decreasing reactive oxygen species levels in human neuroblastoma cells treated with the compound at 0.01, 0.1 and 1 μM. This compound was also able to increase the activity of the antioxidant enzymes superoxide dismutases by inducing the translocation of the nuclear factor E2-related factor 2 (Nrf2) to the nucleus at the lower concentrations tested (0.01 and 0.1 μM). Moreover, sarain A at 0.1 and 1 μM blocked the mitochondrial permeability transition pore (mPTP) opening through cyclophilin D inhibition. These results suggest that the protective effects produced by the treatment with sarain A are related with its ability to block the mPTP and to enhance the Nrf2 pathway, indicating that sarain A may be a candidate compound for further studies in neurodegenerative diseases.