Effect of oxidative stress on TRPM2 and TRPC3 channels in B lymphoblast cells in bipolar disorder.

OBJECTIVES: Recent findings implicate the calcium-permeable nonselective ion channels transient receptor potential (TRP) melastatin subtype 2 (TRPM2) and canonical subtype 3 (TRPC3) in the pathogenesis of bipolar disorder (BD). These channels are involved in calcium and oxidative stress signaling, both of which are disrupted in BD. Thus, we sought to determine if these channels are differentially affected by oxidative stress in cell lines of BD patient origin. METHODS: B lymphoblast cell lines (BLCLs) from bipolar I disorder (BD-I) patients (n = 6) and healthy controls (n = 5) were challenged with the oxidative stressor rotenone (2.5 µM and 10 µM) or vehicle for acute (24 hours) and chronic (four days) intervals. Cell viability was measured using propidium iodide, while TRPM2- and TRPC3-mediated calcium fluxes were measured in the presence of their respective activators (H(2) O(2) and 1-oleoyl-2-acetyl-sn-glycerol) using Fluo-4. Changes in TRPM2 and TRPC3 expression levels were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blotting. RESULTS: Cell viability decreased with increasing dose and duration of rotenone treatment, with BD-I patient BLCLs more susceptible than controls acutely (p < 0.001). A dose-dependent decrease in TRPC3 protein expression occurred after chronic (24%, p = 0.008) but not acute rotenone treatment. Interestingly, H(2) O(2) -provoked TRPM2-dependent calcium fluxes revealed an interaction between the effects of stressor addition and diagnostic subject group (p = 0.003). CONCLUSIONS: These data support an important role for TRPM2 and TRPC3 in sensing and responding to oxidative stress and in transducing oxidative stress signaling to intracellular calcium homeostasis and cellular stress responses, all of which have been implicated in the pathophysiology of BD.

Bcl-2 SNP rs956572 associates with disrupted intracellular calcium homeostasis in bipolar I disorder.

OBJECTIVES: Disrupted intracellular calcium (Ca(2+) ) homeostasis (ICH) related to mitochondrial and/or endoplasmic reticulum (ER) dysfunction has been implicated in bipolar disorder (BD). The anti-apoptotic protein B-cell CLL/lymphoma 2 (Bcl-2), encoded in a putative BD susceptibility locus, modulates ER-Ca(2+) dynamics. Recently, an intronic single-nucleotide polymorphism (SNP) in the Bcl-2 gene, rs956572, was suggested as a functionally active SNP that influences its messenger RNA (mRNA) and protein level as well as human gray matter volume. We sought to evaluate the impact of this variant on ICH in BD. METHODS: Basal intracellular Ca(2+) concentrations ([Ca(2+) ](B) ) and rs956572 genotypes were determined in B lymphoblast cell lines (BLCLs) from bipolar I disorder (BD-I) (n=150), bipolar II disorder (BD-II) (n=65), and major depressive disorder (n=30) patients, and from healthy subjects (n=70). Bcl-2 mRNA and protein levels were determined by quantitative reverse transcriptase polymerase chain reaction and immunoblotting, respectively. Functional interactions of rs956572 with ICH were assessed by thapsigargin- and lysophosphatidic acid (LPA)-stimulated Ca(2+) responses. RESULTS: Although rs956572 variation was not significantly associated with BD, BD-I, or BD-II, BLCL [Ca(2+) ](B) was significantly higher in BD-I G/G patients compared with other genotypes and with healthy subjects. Bcl-2 mRNA and protein levels were lowest in BD-I G/G patients. Compared with A carriers, BD-I patients with G/G variants showed a modest enhancing effect on thapsigargin- and LPA-stimulated Ca(2+) responses. CONCLUSIONS: These findings support the notion that genetic variation in Bcl-2 affecting its expression impacts ICH in BD. Moreover, we show here for the first time that this interactive effect is diagnostically specific to BD-I.

Differential modulation of intracellular Ca2+ responses in B lymphoblasts by mood stabilizers.

Irregularities of intracellular calcium (Ca2+) homeostasis have been implicated in the pathophysiology of bipolar disorder (BD). Findings that chronic ex-vivo treatment with lithium modifies lysophosphatidic acid (LPA)-stimulated Ca2+ responses in B lymphoblast cell lines (BLCLs) from BD-I patients and healthy controls, and differentially decreases levels of the type-3 canonical transient receptor potential Ca2+-permeable channel in BLCLs from BD-I patients, support the view that the amelioration of these abnormalities is important in the therapeutic action of lithium. To determine whether other clinically efficacious mood stabilizers share these effects, LPA (100 mum)- and thapsigargin (TG, 200 nm)-stimulated Ca2+ responses were determined in BLCLs from BD-I patients and healthy controls treated acutely (24 h) and chronically (7 d) ex vivo with therapeutically relevant concentrations of lithium (0.75 mm), valproate (0.5 mm), lamotrigine (15 mum) or respective vehicles. Chronic treatment with valproate significantly attenuated LPA-stimulated Ca2+ responses ([downward arrow]8%: F's=9.1-9.4, d.f.=1, 9, p's<0.05) compared to vehicle in BLCLs from BD-I patients and healthy controls, similar to chronic lithium treatment ([downward arrow]8%: F=6.2, d.f.=1, 21, p<0.05), but also attenuated TG-evoked Ca2+ responses ([downward arrow]10% to [downward arrow]19%: F's=5.5-15.5, d.f.=1, 12, p's<0.05). However, chronic lamotrigine treatment did not affect LPA- or TG-stimulated Ca2+ responses. These results suggest that chronic lithium and valproate treatments act differently from lamotrigine in respect of modulation of receptor- and/or capacitance-mediated Ca2+ flux. These differential effects on Ca2+ responses may be relevant to the distinctive clinical profiles of these mood stabilizers.

TRPM2 variants and bipolar disorder risk: confirmation in a family-based association study.

OBJECTIVE: Recent case-control studies implicate the transient receptor potential melastatin 2 (TRPM2) channel in conferring risk for bipolar disorder (BD), though the risk variants differed. As confounding effects of population structure could not be unequivocally ruled out as the basis for the discordance, we tested the association of TRPM2 with BD in a family design, which is immune to population stratification, for those TRPM2 single nucleotide polymorphisms (SNPs) previously reported as associated with BD. METHODS: The exon 11 SNP (rs1556314) and four informative intronic SNPs (rs1785437, rs1618355, rs933151, and rs749909) were genotyped in 300 BD families by TaqMan allelic discrimination and results were analyzed using chi(2) test, transmission disequilibrium test, and pedigree-based association. SNP rs1556314 was also genotyped in our case-control sample set comprised of 184 BD and 195 healthy Caucasian subjects. RESULTS: The SNP rs1556314 in exon 11 was significantly associated with bipolar disorder type I (BD-I) (p = 0.011, p(permutation) = 0.015) in the case-control dataset and in the family design (p = 0.018, p(permutation) = 0.052, TDTPHASE). Interestingly, the C-T-A haplotype of SNPs rs1618355, rs933151, and rs749909 was significantly associated with early age at onset in BD-I families. CONCLUSION: Significant association of TRPM2 genetic variants with BD in case-control and family datasets further supports a role for TRPM2 in the pathogenesis of this disorder. Overtransmission of the G allele of rs1556314 at exon 11 of TRPM2 in BD-I but not bipolar disorder type II (BD-II) further supports different genetic contributions to the pathogenesis of these bipolar phenotypes.

Analysis of BDNF Val66Met allele-specific mRNA levels in bipolar disorder.

We have previously reported an association between the BDNF Val66Met polymorphism and bipolar disorder (BD). However, the possibility that genomic imprinting in BDNF gene affects risk for BD has not been investigated. To examine the possibility of genomic imprinting in the BDNF gene in BD, we analyzed the parent-of-origin effect (POE) and differential expression of the BDNF Val66Met alleles in BD. We performed a family-based association study and ETDT analyses of the Val66Met polymorphism in 312 BD nuclear families, and compared allele-specific mRNA levels in both post-mortem brain samples and B lymphoblasts from BD patients and controls. The BDNF Val66 allele was transmitted significantly more often to patients with BD (maternal transmissions: 46/22, p=0.003; paternal transmissions: 55/30, p=0.006). There was no significant difference between maternal and paternal transmission ratios. There was no significant difference in the ratio of Val/Met-specific mRNA expression between BD and controls, in either brain or B lymphoblasts. The Val/Met ratio was much lower in the brain vs. B lymphoblasts. These data do not support a role for genomic imprinting as a modifier of the contribution of BDNF gene to risk of susceptibility to BD.

Impaired endoplasmic reticulum stress response in B-lymphoblasts from patients with bipolar-I disorder.

BACKGROUND: Aberrant intracellular calcium (Ca2+) signaling in patients with bipolar-I disorder (BD-I) suggests disturbed endoplasmic reticulum (ER) function in BD. We examined whether the ER stress response is altered in BD-I patients and the relationship to basal intracellular Ca2+ levels ([Ca2+]B), in B lymphoblasts (BLCLs) from BD-I patients. METHODS: Endoplasmic reticulum stress-induced X-box binding protein 1 (XBP1), C/EBP homologous protein (CHOP), and GRP78 expression in BLCLs from BD-I subjects stratified on elevated or normal [Ca2+]B and control subjects were determined by real-time quantitative reverse transcription polymerase chain reaction. The XBP1 -116C/G polymorphism, which impairs the XBP1 loop in the ER stress response, were genotyped with a TaqMan-based assay. RESULTS: Compared with control subjects, thapsigargin- and tunicamycin-induced increases in XBP1 and CHOP but not GRP78 messenger RNA levels were significantly lower in BD-I patients. However, induction of these genes did not differ significantly in the two BD-I subgroups stratified on [Ca2+]B. Furthermore, the attenuated XBP1 induction cannot be explained solely by differences of XBP1 -116C/G genotype frequency. CONCLUSIONS: Our findings suggest that the ER stress response is impaired in BD-I patients but irrespective of altered intracellular Ca2+ homeostasis as reflected in elevated [Ca2+]B. Moreover, an effect of XBP1 -116C/G polymorphism could not account for the attenuated XBP1 induction in bipolar-I disorder observed in this study.

Cytoprotection by lithium and valproate varies between cell types and cellular stresses.

Despite much evidence that lithium and valproate, two commonly used mood stabilizers, exhibit neuroprotective properties against an array of insults, the pharmacological relevance of such effects is not clear because most of these studies examined the acute effect of these drugs in supratherapeutic doses against insults which were of limited disease relevance to bipolar disorder. In the present study, we investigated whether lithium and valproate, at clinically relevant doses, protects human neuroblastoma (SH-SY5Y) and glioma (SVG and U87) cells against oxidative stress and endoplasmic reticulum stress in a time-dependent manner. Pretreatment of SH-SY5Y cells for 7 days, but not 1 day, with 1 mM of lithium or 0.6 mM of valproate significantly reduced rotenone and H2O2-induced cytotoxicity, cytochrome c release and caspase-3 activation, and increased Bcl-2 levels. Conversely, neither acute nor chronic treatment of SH-SY5Y cells with lithium or valproate elicited cytoprotective responses against thapsigargin-evoked cell death and caspase-3 activation. Moreover, inhibitors of glycogen synthase kinase-3 (GSK-3), kenpaullone and SB216763, abrogated rotenone-induced, but not H2O2-induced, cytotoxicity. Thus the cytoprotective effects of lithium and valproate against H2O2-induced cell death is likely independent of GSK-3 inhibition. On the other hand, chronic lithium or valproate treatment did not ameliorate cytotoxicity induced by rotenone, H2O2, and thapsigargin in SVG astroglial and U87 MG glioma cell lines. Our results suggest that lithium and valproate may decrease vulnerability of human neural, but not glial, cells to cellular injury evoked by oxidative stress possibly arising from putative mitochondrial disturbances implicated in bipolar disorder.

Characterization of the transient receptor potential channels mediating lysophosphatidic acid-stimulated calcium mobilization in B lymphoblasts.

Altered 1-oleoyl-lysophosphatidic acid (LPA, 100 microM)-stimulated calcium responses occur in B-lymphoblast cell lines from bipolar disorder patients, but the mechanism(s) involved is uncertain. Lysophosphatidic acid shares a structurally similar fatty acid side chain with the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of subtypes 3, 6 and 7 of the canonical transient receptor potential (TRPC) cation channel subfamily. Accordingly, the objective of this study was to determine whether the LPA-stimulated calcium response in B-lymphoblasts is mediated, in part, through this TRPC channel subfamily. Divalent cation selectivity in response to thapsigargin, LPA and OAG were used to distinguish TRPC-like character of the responses to these agents in BLCLs. The sensitivity to gadolinium, an inhibitor of capacitative calcium channels, was used to determine the store-operated nature of the responses. The TRPC isoforms that are present in BLCLs as identified by immunoblotting and/or PCR include TRPC1, 3 and 5. Minimal barium influx in calcium-free buffer was observed following thapsigargin stimulation. However, LPA stimulated barium influx of a magnitude similar to that induced by OAG. Thapsigargin-provoked calcium influx was completely inhibited by gadolinium (10 microM), whereas LPA and OAG-stimulated responses were partially inhibited and potentiated, respectively. The results suggest that 100 microM LPA stimulates calcium entry through channels with characteristics similar to TRPC3, as TRPC6 and 7 are absent in B-lymphoblasts.

Chronic lithium treatment attenuates intracellular calcium mobilization.

Elevated basal intracellular calcium (Ca(2+)) levels ([Ca(2+)](B)) in B lymphoblast cell lines (BLCLs) from bipolar I disorder (BD-I) patients implicate altered Ca(2+) homeostasis in this illness. Chronic lithium treatment affects key proteins modulating intracellular Ca(2+) signaling. Thus, we sought to determine if chronic exposure to therapeutic lithium concentrations also modifies intracellular Ca(2+) homeostasis in this surrogate cellular model of signal transduction disturbances in BD. BLCLs from BD-I (N=26) and healthy subjects (N=17) were regrown from frozen stock and incubated with 0.75 mM lithium or vehicle for 24 h (acute) or 7 days (chronic). [Ca(2+)](B), lysophosphatidic acid (LPA)-stimulated Ca(2+) mobilization ([Ca(2+)](S)), and thapsigargin-induced store-operated Ca(2+) entry (SOCE) were determined using ratiometric fluorometry with Fura-2. Compared with vehicle, chronic lithium exposure resulted in significantly higher [Ca(2+)](B) (F=8.47; p=0.006) in BLCLs from BD-I and healthy subjects. However, peak LPA-stimulated [Ca(2+)](S) and SOCE were significantly reduced (F=11.1, p=0.002 and F=8.36, p=0.007, respectively). Acute lithium exposure did not significantly affect measured parameters. In summary, the effect of chronic lithium to elevate [Ca(2+)](B) in BLCLs while attenuating both receptor-stimulated and SOCE components of intracellular Ca(2+) mobilization in BLCLs suggests that modulation of intracellular Ca(2+) homeostasis may be important to the therapeutic action of lithium.

cAMP-Dependent protein kinase (PKA) subunit mRNA levels in postmortem brain from patients with bipolar affective disorder (BD).

Earlier findings of elevated basal and stimulated PKA activities, and increased immunoreactive levels of PKA regulatory and catalytic subunits in discrete postmortem brain regions from bipolar disorder (BD) patients suggest that disturbances in PKA are involved in the pathophysiology of BD. PKA subunit mRNA levels were measured using SYBR Green real-time RT-PCR to determine if previously observed differences in immunoreactive levels of PKA RIIbeta and Calpha subunits were associated with corresponding changes in mRNA levels in temporal and frontal cortices from the same BD patients and matched controls. In distinct contrast to the higher immunolabeling levels of the PKA subunits previously reported in the BD brain, there were no significant differences in RIIbeta and Calpha subunit mRNA levels in the temporal and frontal cortices of BD patients compared with controls. These findings infer that the elevated PKA immunolabeling and activity found in the selected cerebral cortical regions of BD postmortem brain were due to a posttranscriptional mechanism, rather than changes in regulation of gene transcription and/or mRNA stability of the PKA subunits.

Cell-type specific regulation of calreticulin and Bcl-2 expression by mood stabilizer drugs.

Recent studies in rat brain and cell cultures have demonstrated that expression of the peptide-folding chaperone protein calreticulin is increased by valproate treatment, while the anti-apoptotic Bcl-2 is increased by both lithium and valproate. We asked whether a similar pattern of regulation by these drugs is evident in human neuronal and glial cells. One-week treatment with 1 mM valproate induced a significant (90%) increase in calreticulin mRNA and protein levels in SVG, a glial cell line, but reduced its mRNA levels by 38% in hNT neuronal cells. Valproate also markedly increased Bcl-2 mRNA levels by 260%, but only in hNT neurons. In contrast, lithium had no significant effect in either cell type. Valproate-induced increases in calreticulin may therefore show glial specificity in humans, while changes in Bcl-2 levels may be neuron specific. These results highlight the cell model dependence of outcomes in molecular studies of mood stabilizer effects and the need for caution in interpreting findings in model systems.

TRPC3 protein is expressed across the lifespan in human prefrontal cortex and cerebellum.

The canonical transient receptor potential type 3 (TRPC3) channel is a non-selective, voltage-independent cation channel that is expressed in both excitable and non-excitable cells. As little is known regarding its presence in human brain and the influence of age on its expression, we examined TRPC3 protein expression by immunoblotting in postmortem prefrontal cortex and cerebellum obtained from subjects (8 days to 83 years) with no history of psychiatric or neurological disorder. The expression of TRPC3 protein in the prefrontal cortex (Brodmann area A9/A10) of the neonates/infants (<2 y) was significantly higher (25%) than that in the adolescent to adult (11y-83y) age group, whereas cerebellar TRPC3 levels showed no age-related changes. The results indicate that TRPC3 may be developmentally regulated in prefrontal cortex, and its expression in discrete human brain regions throughout the lifespan suggests a physiological role for TRPC3 during postnatal and adult life.

Hyperactive intracellular calcium dynamics in B lymphoblasts from patients with bipolar I disorder.

Substantial evidence implicates abnormalities of intracellular calcium (Ca2+) dynamics in the pathophysiology of bipolar disorder (BD). However, the precise mechanisms underlying such disturbances are poorly understood. To further elaborate the nature of altered intracellular Ca2+ signalling dynamics that occur in BD, we examined receptor- and store-operated Ca2+ responses in B lymphoblast cell lines (BLCLs), which have been found in earlier studies to 'report' BD-associated disturbances. Basal Ca2+ concentrations ([Ca2+]B), and lysophosphatidic acid (LPA)- and thapsigargin-stimulated Ca2+ responses were determined in BLCLs from 52 BD-I patients and 30 healthy comparison subjects using fura-2, and ratiometric fluorometry. ANOVA revealed a significant effect of diagnosis, but not gender, on [Ca2+]B (F1,63=4.4, p=0.04) and the rate of rise (F1,63=5.2, p=0.03) of LPA-stimulated Ca2+ responses in BLCLs from patients compared with those from healthy subjects. A significant genderxdiagnosis interaction on the LPA-induced rate of rise (F1,63=4.6, p=0.03) was accounted for by a faster rate of rise (97%) in BLCLs from BD-I males compared with healthy males but not in those from female patients compared with healthy females. A genderxdiagnosis interaction in thapsigargin-evoked Ca2+ influx (F1,61=3.8, p=0.05) resulted from a significantly higher peak [Ca2+]influx (24%) in BLCLs from female compared with male patients. The results suggest more rapid LPA-stimulated Ca2+ responses occur in BLCLs from BD-I patients compared with controls, which are probably mediated, in part, by canonical transient receptor potential type 3 (TRPC3)-like channels. Additionally, this study highlights sex-dependent differences that can occur in the pathophysiological disturbances involved in BD.

Further support for association of the mitochondrial complex I subunit gene NDUFV2 with bipolar disorder.

BACKGROUND: The nuclear-encoded mitochondrial complex I subunit gene, NDUFV2, has been implicated in the pathogenesis of bipolar disorder (BD) in Japanese by virtue of association of variants in its promoter with BD and decreased NDUFV2 messenger ribonucleic acid (mRNA) levels in B lymphoblasts (BLCL) in BD patients compared to controls. We sought to determine if these same changes occur in non-Japanese populations and, if so, their relationship to altered basal intracellular Ca(2+) ([Ca(2+)](B)) in BLCL from BD patients. METHODS: Bipolar disorder patients and healthy subjects included 298 subjects of European Caucasian descent. The 5'-nuclease allelic discrimination TaqMan assay was used to detect selected single nucleotide polymorphisms (SNPs) in promoter, introns and 3'UTR regions, spanning the NDUFV2 gene. NDUFV2 mRNA levels and [Ca(2+)](B) in BLCLs were determined. RESULTS: The A allele of the NDUFV2 SNP rs1156044 was significantly associated (Bonferroni-corrected) with BD (p = 0.013) but differed in allele (rs1156044 G allele) from that previously reported as associated with BD. There was a trend for elevated BLCL [Ca(2+)](B) associated with SNP rs977581 in BD patients, but NDUFV2 mRNA levels in BLCLs did not differ between patients and controls, nor represented genotypes. CONCLUSIONS: While genetic variants of NDUFV2 may increase risk for BD, the role of its altered expression and the link to intracellular Ca(2+) abnormalities in BD remains equivocal.

Galpha(s) sensitizes human SH-SY5Y cells to apoptosis independently of the protein kinase A pathway.

Disturbances in Galpha(s-L) levels and function have been implicated in the pathophysiology of bipolar disorder, but the role of these changes in the development of the illness is not clear. In view of the critical role of Galpha(s)-mediated cAMP signaling in regulating cell survival, we investigated the potential role of Galpha(s-L) in modulating susceptibility to cellular stressors in human SH-SY5Y neuroblastoma cells. Overexpression of Galpha(s-L) to a level twice that of the vector-transfected cells did not directly affect cell viability but significantly increased the sensitivity to induction of cell death by serum deprivation and other apoptotic stimuli, including staurosporine, H(2)O(2), and tunicamycin. This enhanced sensitivity was associated with increased caspase-3 activation and appearance of fragmented nuclei (Hoechst 33342 staining). The broad-spectrum caspase inhibitor z-VAD-fmk completely suppressed cell death evoked by these apoptotic insults in both vector-transfected and Galpha(s-L)-overexpressing cells. The increased vulnerability conferred by increased Galpha(s-L) expression was neither mimicked by cAMP analogs 8-Br-cAMP, 8-CPT-cAMP, and 8-CPT-2Me-cAMP nor attenuated by PKA inhibitors Rp-cAMPS and KT5720. These data indicate that Galpha(s-L) may modulate apoptotic processes in a caspase-dependent manner through a signaling cascade that is independent of the cAMP/PKA or cAMP/Epac pathway. These results suggest that enhanced Galpha(s-L) expression, as was observed in post-mortem brain of bipolar patients, may impair cellular resilience in response to intracellular stress signals resulting from mitochondrial and/or endoplasmic reticulum dysfunction implicated in this disorder.

Association of the putative susceptibility gene, transient receptor potential protein melastatin type 2, with bipolar disorder.

Disturbed intracellular calcium (Ca(2+)) homeostasis has been implicated in bipolar disorder (BD). Reduced mRNA levels of the transient receptor potential Ca(2+) permeable channel melastatin type 2, TRPM2, in B lymphoblast cell lines (BLCL) from bipolar I disorder (BD-I) patients showing elevated basal intracellular Ca(2+) ([Ca(2+)](B)), an index of altered intracellular Ca(2+) homeostasis, along with its location within a putative BD susceptibility locus (21q22.3), implicates the involvement of this gene in the Ca(2+) abnormalities and the genetic diathesis to BD. We tested this hypothesis by examining the association of selected single nucleotide polymorphisms (SNPs) and their haplotypes, spanning the TRPM2 gene, with BD and BLCL [Ca(2+)](B), in a case control design. The 5' TaqMan SNP assay was used to detect selected SNPs. BLCL [Ca(2+)](B) was determined by ratiometric fluorometry. SNP rs1618355 in intron 18 was significantly associated with BD as a whole (P < 7.0 x 10(-5); odds ratio (OR) = 2.60), and when stratified into BD-I (P < 7.0 x 10(-5), OR = 2.48) and BD-II (P = 7.0 x 10(-5), OR = 2.88) subgroups. In addition, the alleles of the individual SNPs forming a seven marker at-risk haplotype were in excess in BD (12.0% in BD vs. 0.9% in controls; P = 2.3 x 10(-12)). A weak relationship was also detected between BLCL [Ca(2+)](B) and TRPM2 SNP rs1612472 in intron 19. These findings suggest genetic variants of the TRPM2 gene increase risk for BD and support the notion that TRPM2 may be involved in the pathophysiology of BD.

Altered cAMP-dependent protein kinase subunit immunolabeling in post-mortem brain from patients with bipolar affective disorder.

Previous findings of reduced [3H]cAMP binding and increased activities of cAMP-dependent protein kinase (PKA) in discrete post-mortem brain regions from patients with bipolar affective disorder (BD) suggest that PKA, the major downstream target of cAMP, is also affected in this illness. As prolonged elevation of intracellular cAMP levels can modify PKA regulatory (R) and catalytic (C) subunit levels, we sought to determine whether these PKA abnormalities are related to changes in the abundance of PKA subunits in BD brain. Using immunoblotting techniques along with PKA subunit isoform-specific polyclonal antisera, levels of PKA RIalpha, RIbeta, RIIalpha, RIIbeta and Calpha subunits were measured in cytosolic and particulate fractions of temporal, frontal and parietal cortices of post-mortem brain from BD patients and matched, non-neurological, non-psychiatric controls. Immunoreactive levels of cytosolic Calpha in temporal and frontal cortices, as well as that of cytosolic RIIbeta in temporal cortex, were significantly higher in the BD compared with the matched control brains. These changes were independent of age, post-mortem interval or pH and unrelated to ante-mortem lithium treatment or suicide. These findings strengthen further the notion that the cAMP/PKA signaling system is up-regulated in discrete cerebral cortical regions in BD.

Altered CTX-catalyzed and endogenous [32P]ADP-ribosylation of stimulatory G protein alphas isoforms in postmortem bipolar affective disorder temporal cortex.

Reports of elevated Gs alpha subunit (alpha(s)) immunolabeling and cAMP-mediated hyper-functionality in autopsied cerebral cortical brain regions from bipolar affective disorder (BD) patients suggest signal transduction abnormalities occur in this disorder. Because covalent modification of alpha(s) can affect its turnover and levels, we determined whether CTX-catalyzed and endogenous [(32)P] adenosine diphosphate (ADP)-ribosylation of alpha(s) isoforms are altered in temporal and occipital cortical regions, which show elevated alpha(s) levels in BD as compared to nonpsychiatric subjects. Reduced CTX-catalyzed [(32)P]ADP-ribosylated alpha(s-S) and endogenous [(32)P]ADP-ribosylation of a 39-kDa alpha(s)-like protein were found in BD temporal cortex compared to controls. These findings suggest that clearance of these alpha(s) isoforms through ADP-ribosylation may be decreased in BD temporal cortex. Although no differences were observed in mean levels of endogenous and CTX-catalyzed [(32)P]ADP-ribosylation of alpha(s-L) in BD temporal cortex, alpha(s-L) immunolabeling was elevated significantly and correlated inversely with the degree of endogenous [(32)P]ADP-ribosylation of this subunit. In addition, endogenous [(32)P]ADP-ribosylation of an exogenous substrate, myelin basic protein, was similar in BD and comparison subject temporal cortex. Taken together, these observations suggest that elevations of alpha(s) in BD brain are more likely related to factors affecting the disposition or availability of alpha(s) to this posttranslational enzymatic modification.