Zelan Wei

The ability of atypical antipsychotic drugs vs. haloperidol to protect PC12 cells against MPP+-induced apoptosis.

The present study examined the effects of the atypical antipsychotic drugs clozapine, olanzapine, quetiapine and risperidone, on N‐methyl‐4‐phenylpyridinium ion‐induced apoptosis and DNA damage in PC12 cells, and explored the molecular mechanisms underlying these effects. Haloperidol, a typical antipsychotic drug, was used for comparison. Exposure of PC12 cells to 50 µm N‐methyl‐4‐phenylpyridinium ion for 24 h resulted in a 35–45% loss of cells in culture. Pretreatment with the aforementioned atypical antipsychotic drugs significantly reduced the N‐methyl‐4‐phenylpyridinium ion‐induced cell loss, whereas haloperidol (10–100 µm) did not have this protective effect. Hoechst 33258 staining revealed the apoptotic nuclear features of the N‐methyl‐4‐phenylpyridinium ion‐induced cell death, and showed that the atypical antipsychotic drugs, but not haloperidol, effectively prevented PC12 cells from this N‐methyl‐4‐phenylpyridinium ion‐induced apoptosis. DNA fragmentation assays further confirmed the N‐methyl‐4‐phenylpyridinium ion‐induced nuclear fragmentation. Pretreatment with the atypical antipsychotic drugs completely prevented this nuclear fragmentation, whereas haloperidol only partially prevented it. In vitro oligonucleotide assays indicated an activation of a specific glycosylase that recognizes and cleaves bases (at the 8‐hydroxyl‐2‐deoxyguanine site) that were damaged by N‐methyl‐4‐phenylpyridinium ion. Pretreatment with the atypical antipsychotic drugs more effectively attenuated this N‐methyl‐4‐phenylpyridinium ion‐induced activation than did haloperidol. Northern blot analyses showed that the atypical antipsychotic drugs, but not haloperidol, blocked the N‐methyl‐4‐phenylpyridinium ion‐induced substantial increase of copper/zinc superoxide dismutase mRNA in PC12 cells. Atypical antipsychotic drugs slightly up‐regulated the expression of copper/zinc superoxide dismutase mRNA, whereas haloperidol strongly increased the expression of copper/zinc superoxide dismutase mRNA. These data may account for the different therapeutic effects and side‐effect profiles of typical and atypical antipsychotic drugs in schizophrenia.

Atypical antipsychotics attenuate neurotoxicity of β-amyloid(25–35) by modulating Bax and Bcl-Xl/s expression and localization

We have demonstrated recently that atypical antipsychotics possess neuroprotective actions in H2O2‐mediated and serum‐withdrawal models of cell death. In the present study, we compared the ability of atypical and typical antipsychotics to protect against an insult mediated by Aβ(25–35), an apoptogenic fragment of the Alzheimers disease‐related β‐amyloid (Aβ) peptide. Treatment of PC12 cell cultures with Aβ(25–35) did not significantly alter total cellular expression levels of Bax, a proapoptotic Bcl‐2 family member, or levels of Bcl‐XL, an antiapoptotic analogue. Treatment with Aβ(25–35), however, did result in mitochondrial translocation of Bax, which effectively increased the mitochondrial ratio of Bax to Bcl‐XL. This relative increase in proapoptotic molecules was reduced by pretreatment with atypical (quetiapine and olanzapine) and typical (haloperidol) antipsychotics. We also observed a selective increase in proapoptotic Bcl‐XS immunodetection in haloperidol‐treated cells, which was evident particularly in the mitochondrial compartment. This increase in proapoptotic molecules may account for the lower neuroprotective potential of haloperidol, as determined by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium (MTT) reduction assay. The disparate neuroprotective effects of atypical and typical antipsychotics/neuroleptics may be due to their respective abilities to regulate pro‐ and anti‐apoptotic protein translocation and expression.

Demonstration of an anti-oxidative stress mechanism of quetiapine: implications for the treatment of Alzheimer's disease.

We have shown that quetiapine, a new antipsychotic drug, protects cultured cells against oxidative stress‐related cytotoxicities induced by amyloid β (Aβ)25‐35, and that quetiapine prevents memory impairment and decreases Aβ plaques in the brains of amyloid precursor protein (APP)/presenilin‐1 (PS‐1) double‐mutant mice. The aim of this study was to understand why quetiapine has these protective effects. Because the cytotoxicity of both Aβ(25‐35) and Aβ(1‐40) requires fibril formation, our first experiments determined the effect of quetiapine on Aβ(25‐35) aggregation. Quetiapine inhibited Aβ(25‐35) aggregation in cell‐free aqueous solutions and blocked the fibrillar aggregation of Aβ(25‐35), as observed under an electron microscope. We then investigated why quetiapine inhibits Aβ(25‐35) aggregation. During the aggregation of Aβ(25‐35), a hydroxyl radical (OH•) was released, which in turn amplified Aβ(25‐35) aggregation. Quetiapine blocked OH•‐induced Aβ(25‐35) aggregation and scavenged the OH• produced in the Fenton system and in the Aβ(25‐35) solution, as analyzed using electron paramagnetic resonance spectroscopy. Furthermore, new compounds formed by quetiapine and OH• were observed in MS analysis. Finally, we applied Aβ(25‐35) to PC12 cells to observe the effect of quetiapine on living cells. Aβ(25‐35) increased levels of intracellular reactive oxygen species and calcium in PC12 cells and caused cell death, but these toxic effects were prevented by quetiapine. These results demonstrate an anti‐oxidative stress mechanism of quetiapine, which contributes to its protective effects observed in our previous studies and explains the effectiveness of this drug for Alzheimer’s disease patients with psychiatric and behavioral complications.

Calcium-sensitive regulation of monoamine oxidase-A contributes to the production of peroxyradicals in hippocampal cultures: implications for Alzheimer disease-related pathology

BackgroundCalcium (Ca2+) has recently been shown to selectively increase the activity of monoamine oxidase-A (MAO-A), a mitochondria-bound enzyme that generates peroxyradicals as a natural by-product of the deamination of neurotransmitters such as serotonin. It has also been suggested that increased intracellular free Ca2+ levels as well as MAO-A may be contributing to the oxidative stress associated with Alzheimer disease (AD).ResultsIncubation with Ca2+ selectively increases MAO-A enzymatic activity in protein extracts from mouse hippocampal HT-22 cell cultures. Treatment of HT-22 cultures with the Ca2+ ionophore A23187 also increases MAO-A activity, whereas overexpression of calbindin-D28K (CB-28K), a Ca2+-binding protein in brain that is greatly reduced in AD, decreases MAO-A activity. The effects of A23187 and CB-28K are both independent of any change in MAO-A protein or gene expression. The toxicity (via production of peroxyradicals and/or chromatin condensation) associated with either A23187 or the AD-related β-amyloid peptide, which also increases free intracellular Ca2+, is attenuated by MAO-A inhibition in HT-22 cells as well as in primary hippocampal cultures.ConclusionThese data suggest that increases in intracellular Ca2+ availability could contribute to a MAO-A-mediated mechanism with a role in AD-related oxidative stress.

Serine 209 resides within a putative p38(MAPK) consensus motif and regulates monoamine oxidase-A activity

The p38 mitogen‐activated protein kinase (MAPK) cascade as well as the enzyme monoamine oxidase‐A (MAO‐A) have both been associated with oxidative stress. We observed that the specific inhibition of the p38(MAPK) protein [using either a chemical inhibitor or a dominant‐negative p38(MAPK) clone] selectively induces MAO‐A activity and MAO‐A‐sensitive toxicity in several neuronal cell lines, including primary cortical neurons. Over‐expression of a constitutively active p38(MAPK) results in the phosphorylation of the MAO‐A protein and inhibition of MAO‐A activity. The MAO‐A(Ser209Glu) phosphomimic – bearing a targeted substitution within a putative p38(MAPK) consensus motif – is neither active nor neurotoxic. In contrast, the MAO‐A(Ser209Ala) variant (mimics dephosphorylation) does not associate with p38(MAPK), and is both very active and very toxic. Substitution of the homologous serine in the MAO‐B isoform, i.e. Ser200, with either Glu or Ala does not affect the catalytic activity of the corresponding over‐expressed proteins. These combined in vitro data strongly suggest a direct p38(MAPK)‐dependent inhibition of MAO‐A function. Based on published observations, this endogenous means of selectively regulating MAO‐A function could provide for an adaptive response to oxidative stress associated with disorders as diverse as depression, reperfusion/ischemia, and the early stages of Alzheimer’s disease.

Beneficial effects of quetiapine in a transgenic mouse model of Alzheimer's disease.

Previous studies have suggested that quetiapine, an atypical antipsychotic drug, may have beneficial effects on cognitive impairment, and be a neuroprotectant in treating neurodegenerative diseases. In the present study, we investigated the effects of quetiapine on memory impairment and pathological changes in an amyloid precursor protein (APP)/presenilin-1 (PS-1) double transgenic mouse model of Alzheimers disease (AD). Non-transgenic and transgenic mice were treated with quetiapine (0, 2.5, or 5mg/(kg day)) for 1, 4, and 7 months in drinking water from the age of 2 months. After 4 and 7 months of continuous quetiapine administration, memory impairment was prevented, and the number of beta-amyloid (Abeta) plaques decreased in the cortex and hippocampus of the transgenic mice. Quetiapine also decreased brain Abeta peptides, beta-secretase activity and expression, and the level of C99 (an APP C-terminal fragment following cleavage by beta-secretase) in the transgenic mice. Furthermore, quetiapine attenuated anxiety-like behavior, up-regulated cerebral Bcl-2 protein, and decreased cerebral nitrotyrosine in the transgenic mice. These findings suggest that quetiapine can alleviate cognitive impairment and pathological changes in an APP/PS1 double transgenic mouse model of AD, and further indicate that quetiapine may have preventive effects in the treatment of AD.

Humanin attenuates Alzheimer-like cognitive deficits and pathological changes induced by amyloid β-peptide in rats

Amyloid β-peptide (Aβ) has been implicated as a key molecule in the neurodegenerative cascades of Alzheimer’s disease (AD). Humanin (HN) is a secretory peptide that inhibits the neurotoxicity of Aβ. However, the mechanism(s) by which HN exerts its neuroprotection against Aβ-induced ADlike pathological changes and memory deficits are yet to be completely defined. In the present study, we provided evidence that treatment of rats with HN increases the number of dendritic branches and the density of dendritic spines, and upregulates pre- and post-synaptic protein levels; these effects lead to enhanced long-term potentiation and amelioration of the memory deficits induced by Aβ1–42. HN also attenuated Aβ1–42-induced tau hyperphosphorylation, apparently by inhibiting the phosphorylation of Tyr307 on the inhibitory protein phosphatase-2A (PP2A) catalytic subunit and thereby activating PP2A. HN also inhibited apoptosis and reduced the oxidative stress induced by Aβ1–42. These findings provide novel mechanisms of action for the ability of HN to protect against Aβ1–42-induced AD-like pathological changes and memory deficits.

Alzheimer disease-related presenilin-1 variants exert distinct effects on monoamine oxidase-A activity in vitro

Monoamine oxidase-A (MAO-A) has been associated with both depression and Alzheimer disease (AD). Recently, carriers of AD-related presenilin-1 (PS-1) alleles have been found to be at higher risk for developing clinical depression. We chose to examine whether PS-1 could influence MAO-A function in vitro. Overexpression of selected AD-related PS-1 variants (wildtype, Y115H, ΔEx9 and M146V) in mouse hippocampal HT-22 cells affects MAO-A catalytic activity in a variant-specific manner. The ability of the PS-1 substrate-competitor DAPT to induce MAO-A activity in cells expressing either PS-1 wildtype or PS-1(M146V) suggests the potential for a direct influence of PS-1 on MAO-A function. In support of this, we were able to co-immunoprecipitate MAO-A with FLAG-tagged PS-1 wildtype and M146V proteins. This potential for a direct protein–protein interaction between PS-1 and MAO-A is not specific for HT-22 cells as we were also able to co-immunoprecipitate MAO-A with FLAG-PS-1 variants in N2a mouse neuroblastoma cells and in HEK293 human embryonic kidney cells. Finally, we demonstrate that the two PS-1 variants reported to be associated with an increased incidence of clinical depression [e.g., A431E and L235V] both induce MAO-A activity in HT-22 cells. A direct influence of PS-1 variants on MAO-A function could provide an explanation for the changes in monoaminergic tone observed in several neurodegenerative processes including AD. The ability to induce MAO-A catalytic activity with a PS-1/γ-secretase inhibitor should also be considered when designing secretase inhibitor-based therapeutics.

Aspartic acid substitutions in monoamine oxidase-A reveal both catalytic-dependent and -independent influences on cell viability and proliferation

Post-translational influences could underlie the ambiguous roles of monoamine oxidase-A (MAO-A) in pathologies such as depression, cancer and Alzheimer disease. In support of this, we recently demonstrated that the Ca2+-sensitive component of MAO-A catalytic activity is inhibited by a pro-survival p38 (MAPK)-dependent mechanism. We substituted three aspartic acid (D) residues in human MAO-A that reside in putative Ca2+-binding motifs and overexpressed the individual proteins in the human HEK293 cell line. We assayed the overexpressed proteins for catalytic activity and for their influence on cell viability (using MTT conversion and trypan blue exclusion) and proliferation/DNA synthesis [using bromodeoxyuridine (BrdU) incorporation]. Innate MAO-A catalytic activity (and the capacity for generating hydrogen peroxide) was unaffected by the D61A substitution, but inhibited moderately or completely by the D248A and D328G substitutions, respectively. The Ca2+-sensitive activities of wild-type and D248A MAO-A proteins were enhanced by treatment with the selective p38(MAPK) inhibitor, SB203580, but was completely abrogated by the D61A substitution. Monoamine oxidase-A(D61A) was toxic to cells and exerted no effect on cell proliferation, while MAO-A(D248A) was generally comparable to wild-type MAO-A. As expected, the catalytic-dead MAO-A(D328G) was not cytotoxic, but unexpectedly enhanced both MTT conversion and BrdU staining. Variant-dependent changes in Bax and Bcl-2/Bcl-XL protein expression were observed. A different pattern of effects in N2-a cells suggests cell line-dependent roles for MAO-A. A catalytic-dependent mechanism influences MAO-A-mediated cytotoxicity, whereas a catalytic-independent mechanism contributes to proliferation. Context-dependent inputs by either mechanism could underlie the ambiguous pathological contributions of MAO-A.