Peter P. Li

Cerebral cortex Gsα protein levels and forskolin-stimulated cyclic AMP formation are increased in bipolar affective disorder

Abstract: Experimental animal and peripheral blood cell studies point to guanine nucleotide regulatory (G) protein disturbances in bipolar affective disorder. We have previously reported elevated prefrontal cortex Gsα protein in bipolar affective disorder and have now extended these preliminary observations in a larger number of subjects, assessing the brain regional specificity of these changes in greater detail, determining the functional biochemical correlates of such changes, and evaluating their diagnostic specificity. Membrane G protein (Gsα, Giα, Goα, and Gβ) immunoreactivities were estimated by western blotting in postmortem brain regions obtained from 10 patients with a DSMIII‐R diagnosis of bipolar affective disorder and 10 nonpsychiatric controls matched on the basis of age, postmortem delay, and brain pH. To examine whether there were functional correlates to the observed elevated Gsα levels, basal and GTPγS‐and forskolin‐stimulated cyclic AMP production was determined in the same brain regions. Compared with controls, Gsα (52‐kDa species) immunoreactivity was significantly (p < 0.05) elevated in prefrontal (+36%), temporal (+65%), and occipital (+96%) cortex but not in hippocampus (+28%), thalamus (‐23%), or cerebellum (+21%). In contrast, no significant differences were found in the other G protein subunits (Giα, Goα, Gβ) measured in these regions. Forskolin‐stimulated cyclic AMP production was significantly increased in temporal (+31%) and occipital (+96%) cortex but not in other regions. No significant differences were apparent in basal or GTPγS‐stimulated cyclic AMP production. A significant correlation (r= 0.60, p < 0.001) was observed between forskolin‐stimulated cyclic AMP formation and Gsα (52 kDa) immunoreactivity when examined across these cortical regions. The observed increase in Gsα may be specific to bipolar disorders as no significant differences were detected in Gsα levels in temporal cortex from patients with either schizophrenia (n = 7) or Alzheimers disease (n = 7). In summary, the present study confirms and extends our earlier findings and supports the notion that increased Gsα levels and possibly Gsα‐adenylyl cyclase‐mediated signal transduction are relevant to the pathophysiology of bipolar affective disorder.

Postmortem cerebral cortex Gs α-subunit levels are elevated in bipolar affective disorder

We examined the relative abundance of G-protein subunits in postmortem brain obtained from 7 patiebts with bipolar affective disorder (BAD) compared with 7 age- and sex-matched controls. G-protein subunit immunoreactivities were determined in membranes prepared from postmortem prefrontal cortex using SDS-PAGE and immunoblotting with specific polyclonal antisera against selected G-protein subunits: Gsα, Gi(1&2)α, Goα and Gβ(1&2). Of these G-protein subunits, only Gsα immunoreactivity was found to be significantly elevated in frontal (+ 34%), and occipital (+ 80%) cortex (P < 0.05) in BAD compared with control subjects. Smaller increments (+ 22%) in cerebellar Gsα immunoreactivity were also found but were not statistically significant. On the basis that increased Gsα immunoreactivity may reflect enhanced functional responsiveness of the receptor-effector units to which this coupling protein is integral, the present findings suggest that disturbances in Gs-mediated signal transduction may be involved in the pathophysiology of BAD.

The phosphoinositide signal transduction system is impaired in bipolar affective disorder brain.

Abstract: The function of the phosphoinositide second messenger system was assessed in occipital, temporal, and frontal cortex obtained postmortem from subjects with bipolar affective disorder and matched controls by measuring the hydrolysis of [3H]phosphatidylinositol ([3H]PI) incubated with membrane preparations and several different stimulatory agents. Phospholipase C activity, measured in the presence of 0.1 mM Ca2+ to stimulate the enzyme, was not different in bipolar and control samples. G proteins coupled to phospholipase C were concentration‐dependently activated by guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) and by NaF. GTPγS‐stimulated [3H]PI hydrolysis was markedly lower (50%) at all tested concentrations (0.3–10 µM GTPγS) in occipital cortical membranes from bipolar compared with control subjects. Responses to GTPγS in temporal and frontal cortical membranes were similar in bipolars and controls, as were responses to NaF in all three regions. Brain lithium concentrations correlated directly with GTPγS‐stimulated [3H]PI hydrolysis in bipolar occipital, but not temporal or frontal, cortex. Carbachol, histamine, trans‐1‐aminocyclopentyl‐1,3‐dicarboxylic acid, serotonin, and ATP each activated [3H]PI hydrolysis above that obtained with GTPγS alone, and these responses were similar in bipolars and controls except for deficits in the responses to carbachol and serotonin in the occipital cortex, which were equivalent to the deficit detected with GTPγS alone. Thus, among the three cortical regions examined there was a selective impairment in G protein‐stimulated [3H]PI hydrolysis in occipital cortical membranes from bipolar compared with control subjects. These results directly demonstrate decreased activity of the phosphoinositide signal transduction system in specific brain regions in bipolar affective disorder.

Decreased brain [3H]inositol 1,4,5-trisphosphate binding in Alzheimer's disease

Inositol 1,4,5-trisphosphate (IP3) receptor binding sites were studied in autopsied brains from 10 subjects with dementia of the Alzheimer type (DAT) and 10 age-matched controls. In the parietal cortex and hippocampus, there was a 50-70% loss of [3H]IP3 binding whereas no significant changes were observed in frontal, occipital and temporal cortices, caudate or amygdala. Scatchard analysis confirmed a reduction in receptor density rather than a change in affinity. Since muscarinic receptors are robustly coupled to IP3 formation, our data demonstrate abnormalities beyond the muscarinic receptor recognition site in DAT.

Rat Brain and Plasma Norepinephrine Glycol Metabolites Determined by Gas Chromatography‐Mass Fragmentography

Abstract: A gas chromatographic‐mass fragmentographic (GC‐MF) procedure is described for the simultaneous quantitation of 3,4‐dihydroxyphenyl‐ethyleneglycol (DHPG) and 3‐methoxy‐4‐hydroxyphenylethyleneglycol (MHPG) in brain tissue and plasma. DHPG and MHPG were assayed as their respective acetyl‐trifluoroacyl esters, using [2H2]DHPG and [2H3]MHPG as internal standards. Assay sensitivities of at least 1 ng per sample were attainable for the quantitation of free glycols, whereas for determination of total DHPG, assay sensitivity was 2.5 ng. Whole rat brain total (99.2 ±4.11 ng/g) and free (13.0 ± 1.14 ng/g) DHPG concentrations were similar to respective total (86.0 ± 3.70 ng/g) and free (12.3 ± 0.41 ng/g) MHPG levels. Total DHPG concentrations exceeded total MHPG levels in hypothalamus (3.0:1), midbrain (1.4:1), pons plus medulla (1.3:1), and hippocampus (1.5:1), whereas in other brain regions the levels of these metabolites were similar. In plasma, however, total DHPG levels were only 20% as high as MHPG concentrations. In mouse brain, DHPG and MHPG occurred almost entirely in free form (>90%), but total DHPG levels were only 50% as high as respective MHPG concentrations. These results emphasize the substantial formation of DHPG compared with MHPG in rat and mouse brain and suggest that DHPG formation and eMux may be of equal or greater importance than MHPG in the metabolic clearance of CNS norepinephrine in some species.

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

Disturbed intracellular calcium (Ca2+) homeostasis has been implicated in bipolar disorder (BD). Reduced mRNA levels of the transient receptor potential Ca2+ permeable channel melastatin type 2, TRPM2, in B lymphoblast cell lines (BLCL) from bipolar I disorder (BD‐I) patients showing elevated basal intracellular Ca2+ ([Ca2+]B), an index of altered intracellular Ca2+ homeostasis, along with its location within a putative BD susceptibility locus (21q22.3), implicates the involvement of this gene in the Ca2+ 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 [Ca2+]B, in a case control design. The 5′ TaqMan SNP assay was used to detect selected SNPs. BLCL [Ca2+]B was determined by ratiometric fluorometry. SNP rs1618355 in intron 18 was significantly associated with BD as a whole (P < 7.0 × 10−5; odds ratio (OR) = 2.60), and when stratified into BD‐I (P < 7.0 × 10−5, OR = 2.48) and BD‐II (P = 7.0 × 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 × 10−12). A weak relationship was also detected between BLCL [Ca2+]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.

Chronic Lithium Treatment Attenuates Intracellular Calcium Mobilization

Elevated basal intracellular calcium (Ca2+) levels ([Ca2+]B) in B lymphoblast cell lines (BLCLs) from bipolar I disorder (BD-I) patients implicate altered Ca2+ homeostasis in this illness. Chronic lithium treatment affects key proteins modulating intracellular Ca2+ signaling. Thus, we sought to determine if chronic exposure to therapeutic lithium concentrations also modifies intracellular Ca2+ 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). [Ca2+]B, lysophosphatidic acid (LPA)-stimulated Ca2+ mobilization ([Ca2+]S), and thapsigargin-induced store-operated Ca2+ entry (SOCE) were determined using ratiometric fluorometry with Fura-2. Compared with vehicle, chronic lithium exposure resulted in significantly higher [Ca2+]B (F=8.47; p=0.006) in BLCLs from BD-I and healthy subjects. However, peak LPA-stimulated [Ca2+]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 [Ca2+]B in BLCLs while attenuating both receptor-stimulated and SOCE components of intracellular Ca2+ mobilization in BLCLs suggests that modulation of intracellular Ca2+ homeostasis may be important to the therapeutic action of lithium.

Altered TRPC7 gene expression in bipolar-I disorder.

BACKGROUND As altered storage-operated calcium (Ca(2+)) entry (SOCE) may affect Ca(2+) homeostasis in bipolar disorder (BD), we determined whether changes occur in the expression of TRPC7 and SERCA2s, proteins implicated or known to be involved in SOCE, in B lymphoblast cell lines (BLCLs) from BD-I patients and comparison subjects. METHODS mRNA levels were determined in BLCL lysates from BD-I, BD-II, and major depressive disorder patients, and healthy subjects by comparative reverse transcriptase-polymerase chain reaction, and BLCL basal intracellular Ca(2+) concentration ([Ca(2+)]B) was determined by ratiometric spectrophotometry using Fura-2, in aliquots of the same cell lines, at 13-16 passages in culture. RESULTS TRPC7 mRNA levels were significantly lower in BLCLs from BD-I patients with high BLCL [Ca(2+)]B compared with those showing normal [Ca(2+)]B (-33%, p =.017) and with BD-II patients (-48%, p =.003), major depressive disorder patients (-47%, p =.049) and healthy subjects (-33%, p =.038). [Ca(2+)]B also correlated inversely with TRPC7 mRNA levels in BLCLs from the BD-I group as a whole (r = -.35, p =.027). CONCLUSIONS Reduced TRPC7 gene expression may be a trait associated with pathophysiological disturbances of Ca(2+) homeostasis in a subgroup of BD-I patients.

Increased cyclic AMP-dependent protein kinase activity in postmortem brain from patients with bipolar affective disorder

Abstract : Previous observations of reduced [3H]cyclic AMP binding in postmortem brainregions frombipolar affective disorder subjects imply cyclic AMP‐dependent proteinkinase functionmay be altered in this illness. To test this hypothesis, basal and stimulated cyclic AMP‐dependent protein kinase activity was determined in cytosolic and particulate fractions of postmortem brain from bipolar disorder patients and matched controls. Maximal enzyme activity was significantly higher (104%) in temporal cortex cytosolic fractions from bipolar disorder brain compared with matched controls. In temporal cortex particulate fractions and in the cytosolic and particulate fractions of other brain regions, smaller but statistically nonsignificant increments in maximal enzyme activity were detected. Basal cyclic AMP‐dependent protein kinase activity was also significantly higher (40%) in temporal cortex cytosolic fractions of bipolar disorder brain compared with controls. Estimated EC50 values for cyclic AMP activation of this kinase were significantly lower (70 and 58%, respectively) in both cytosolic and particulate fractions of temporal cortex from bipolar disorder subjects compared with controls. These findings suggest that higher cyclic AMP‐dependent proteinkinase activity in bipolar disorder brain may be associated with a reduction of regulatory subunits of this enzyme, reflecting a possible adaptive response of this transducing enzyme to increased cyclic AMP signaling in this disorder.

Role of intracellular calcium signaling in the pathophysiology and pharmacotherapy of bipolar disorder: current status

Abstract Evidence implicating disturbances of intracellular Ca 2+ homeostasis has continued to accumulate, with a recent burst of new observations obtained using cultured cell lines from patients with bipolar disorder (BD) suggesting that disturbances occur in receptor-activated and store-operated calcium entry. The potential confounding effects of state of illness and medications on results obtained with various surrogate cellular models is reviewed, and the extent to which findings may reflect trait changes is considered. The role of ER and mitochondria in maintaining intracellular Ca 2+ homeostasis and in protecting against induction of apoptosis is now better understood. Disrupted Ca 2+ dynamics found in cell lines from BD patients point to disturbances in these homeostatic control modules in the pathophysiology of a subtype of BD. This notion is further supported by convergence of observations that, on the one hand, show therapeutic concentrations of lithium modifies intracellular Ca 2+ dynamics in non-human and human cell lines of different ontogeny, and on the other hand, demonstrate that this mood stabilizer modulates anti-apoptotic protein expression that counteracts mitochondrial/ER stress-induced impairment in Ca 2+ homeostasis.