Claude Walzer

Up-Regulation of Immunolabeled α2A-Adrenoceptors, Gi Coupling Proteins, and Regulatory Receptor Kinases in the Prefrontal Cortex of Depressed Suicides

Abstract : Suicide and depression are associated with an increased density of α2‐adrenoceptors (radioligand receptor binding) in specific regions of the human brain. The function of these inhibitory receptors involves various regulatory proteins (Gi coupling proteins and G protein‐coupled receptor kinases, GRKs), which work in concert with the receptors. In this study we quantitated in parallel the levels of immunolabeled α2A‐adrenoceptors and associated regulatory proteins in brains of suicide and depressed suicide victims. Specimens of the prefrontal cortex (Brodmann area 9) were collected from 51 suicide victims and 31 control subjects. Levels of α2A‐adrenoceptors, Gα1/2 proteins, and GRK 2/3 were assessed by immunoblotting techniques by using specific polyclonal antisera and the immunoreactive proteins were quantitated by densitometry. Increased levels of α2A‐adrenoceptors (31‐40%), Gα1/2 proteins (42‐63%), and membrane‐associated GRK 2/3 (24‐32%) were found in the prefrontal cortex of suicide victims and antidepressantfree depressed suicide victims. There were significant correlations between the levels of GRK 2/3 (dependent variable) and those of α2A‐adrenoceptors and Gα1/2 proteins (independent variables) in the same brain samples of suicide victims (r = 0.56, p = 0.008) and depressed suicide victims (r = 0.54, p = 0.041). Antemortem antidepressant treatment was associated with a significant reduction in the levels of Gα1/2 proteins (32%), but with modest decreases in the levels of α2A‐adrenoceptors (6%) and GRK 2/3 (18%) in brains of depressed suicide victims. The increased levels in concert of α2A‐adrenoceptors, Gα1/2 proteins, and GRK 2/3 in brains of depressed suicide victims support the existence of supersensitive α2A‐adrenoceptors in subjects with major depression.

Excess of serotonin affects embryonic interneuron migration through activation of the serotonin receptor 6

The discovery that a common polymorphism (5-HTTLPR, short variant) in the human serotonin transporter gene (SLC6A4) can influence personality traits and increase the risk for depression in adulthood has led to the hypothesis that a relative increase in the extracellular levels of serotonin (5-HT) during development could be critical for the establishment of brain circuits. Consistent with this idea, a large body of data demonstrate that 5-HT is a strong neurodevelopmental signal that can modulate a wide variety of cellular processes. In humans, serotonergic fibers appear in the developing cortex as early as the 10th gestational week, a period of intense neuronal migration. In this study we hypothesized that an excess of 5-HT could affect embryonic cortical interneuron migration. Using time-lapse videometry to monitor the migration of interneurons in embryonic mouse cortical slices, we discovered that the application of 5-HT decreased interneuron migration in a reversible and dose-dependent manner. We next found that 5-HT6 receptors were expressed in cortical interneurons and that 5-HT6 receptor activation decreased interneuron migration, whereas 5-HT6 receptor blockade prevented the migratory effects induced by 5-HT. Finally, we observed that interneurons were abnormally distributed in the cerebral cortex of serotonin transporter gene (Slc6a4) knockout mice that have high levels of extracellular 5-HT. These results shed new light on the neurodevelopmental alterations caused by an excess of 5-HT during the embryonic period and contribute to a better understanding of the cellular processes that could be modulated by genetically controlled differences in human 5-HT homeostasis.

Postsynaptic density protein PSD-95 expression in Alzheimer's disease and okadaic acid induced neuritic retraction

In order to understand how plasticity is related to neurodegeneration, we studied synaptic proteins with quantitative immunohistochemistry in the entorhinal cortex from Alzheimer patients and age-matched controls. We observed a significant decrease in presynaptic synaptophysin and an increase in postsynaptic density protein PSD-95, positively correlated with beta amyloid and phosphorylated Tau proteins in Alzheimer cases. Furthermore, Alzheimer-like neuritic retraction was generated in okadaic acid (OA) treated SH-SY5Y neuroblastoma cells with no decrease in PSD-95 expression. However, in a SH-SY5Y clone with decreased expression of transcription regulator LMO4 (as observed in Alzheimers disease) and increased neuritic length, PSD-95 expression was enhanced but did not change with OA treatment. Therefore, increased PSD-95 immunoreactivity in the entorhinal cortex might result from compensatory mechanisms, as in the SH-SY5Y clone, whereas increased Alzheimer-like Tau phosphorylation is not related to PSD-95 expression, as suggested by the OA-treated cell models.

Regulation of nonphosphorylated and phosphorylated forms of neurofilament proteins in the prefrontal cortex of human opioid addicts.

The neurofilament (NF) proteins (NF‐H, NF‐M, and NF‐L for high, medium, and low molecular weights) play a crucial role in the organization of neuronal shape and function. In a preliminary study, the abundance of total NF‐L was shown to be decreased in brains of opioid addicts. Because of the potential relevance of NF abnormalities in opioid addiction, we quantitated nonphosphorylated and phosphorylated NF in postmortem brains from 12 well‐defined opioid abusers who had died of an opiate overdose (heroin or methadone). Levels of NF were assessed by immunoblotting techniques using phospho‐independent and phospho‐dependent antibodies, and the relative (% changes in immunoreactivity) and absolute (changes in ng NF/μg total protein) amounts of NF were calculated. Decreased levels of nonphosphorylated NF‐H (42–32%), NF‐M (14–9%) and NF‐L (30–29%) were found in the prefrontal cortex of opioid addicts compared with sex, age, and postmortem delay‐matched controls. In contrast, increased levels of phosphorylated NF‐H (58–41%) and NF‐M (56–28%) were found in the same brains of opioid addicts. The ratio of phosphorylated to nonphosphorylated NF‐H in opioid addicts (3.4) was greater than that in control subjects (1.6). In the same brains of opioid addicts, the levels of protein phosphatase of the type 2A were found unchanged, which indicated that the hyperphosphorylation of NF‐H is not the result of a reduced dephosphorylation process. The immunodensities of GFAP (the specific glial cytoskeletol protein), α‐internexin (a neuronal filament related to NF‐L) and synaptophysin (a synapse‐specific protein) were found unchanged, suggesting a lack of gross changes in glial reaction, other intermediate filaments of the neuronal cytoskeletol, and synaptic density in the prefrontal cortex of opioid addicts. These marked reductions in total NF proteins and the aberrant hyperphosphorylation of NF‐H in brains of opioid addicts may play a significant role in the cellular mechanisms of opioid addiction. J. Neurosci. Res. 61:338–349, 2000.

Contribution of neural networks to Alzheimer disease's progression

The neuropathology of Alzheimer disease is characterized by senile plaques, neurofibrillary tangles and cell death. These hallmarks develop according to the differential vulnerability of brain networks, senile plaques accumulating preferentially in the associative cortical areas and neurofibrillary tangles in the entorhinal cortex and the hippocampus. We suggest that the main aetiological hypotheses such as the beta-amyloid cascade hypothesis or its variant, the synaptic beta-amyloid hypothesis, will have to consider neural networks not just as targets of degenerative processes but also as contributors of the diseases progression and of its phenotype. Three domains of research are highlighted in this review. First, the cerebral reserve and the redundancy of the networks elements are related to brain vulnerability. Indeed, an enriched environment appears to increase the cerebral reserve as well as the threshold of diseases onset. Second, diseases progression and memory performance cannot be explained by synaptic or neuronal loss only, but also by the presence of compensatory mechanisms, such as synaptic scaling, at the microcircuit level. Third, some phenotypes of Alzheimer disease, such as hallucinations, appear to be related to progressive dysfunction of neural networks as a result, for instance, of a decreased signal to noise ratio, involving a diminished activity of the cholinergic system. Overall, converging results from studies of biological as well as artificial neural networks lead to the conclusion that changes in neural networks contribute strongly to Alzheimer diseases progression.

Regulation of immunolabelled μ-opioid receptors and protein kinase C-α and ζ isoforms in the frontal cortex of human opiate addicts

Abstract To assess the status of opioid receptors in the human brain during the process of opiate addiction, the abundance of immunoreactive μ -opioid receptors was quantitated in postmortem brains of chronic opiate addicts who had died of a heroin or methadone overdose. The immunoreactive levels of the associated enzyme protein kinase C (PKC- α and ζ isoforms) and G proteins (G α i1/2 subunits) were also assessed in the same brains. In the frontal cortex of opiate addicts, the abundance of μ -opioid receptors was not different from that obtained in matched controls. The level of Ca 2+ -dependent PKC- α was decreased (25%), whereas that of the atypical PKC- ζ remained unchanged. The density of G α i1/2 proteins also was found to be increased (40%). The results indicate that opiate addiction in humans does not appear to be associated with a reduced density of brain μ -opioid receptors. The sustained down-regulation of PKC- α in the brain of opiate addicts would allow the up-regulation of G α i1/2 proteins aimed at compensating the postulated desensitization of the μ -opioid receptor system.

Density of Guanine Nucleotide-Binding Proteins in Platelets of Patients with Major Depression: Increased Abundance of the Gαi2 Subunit and Down-Regulation by Antidepressant Drug Treatment

The aim of this study was to quantitate the density of guanine nucleotide-binding (G) protein subunits (inhibitory G alpha i, stimulatory G alpha s, G alpha q/11, and G beta) in platelets of unipolar depressed patients to assess the status of these signal transduction proteins in depression and the effects of antidepressant drug treatment. Blood platelets were collected from 22 drug-free depressed patients and 22 age- and sex-matched healthy controls. The levels of the various G protein subunits were assessed by immunoblotting techniques. The immunoreactivity of G alpha 12 was increased (41%) and that of G alpha i3 decreased (25%) in platelets of depressed patients. The levels of other G protein subunits (G alpha s, G alpha q/11, G beta) did not change significantly with respect to those of control subjects. Chronic administration of cyclic antidepressant drugs (citalopram, clomipramine, imipramine) decreased the immunoreactivity of the up-regulated G alpha i2 protein (31%). Since platelet G alpha i2 is in line with the existence of supersensitivity of these receptors in major depression.

Imidazoline receptor proteins in brains of patients with Alzheimer's disease

Imidazoline receptors (29/30- and 45-kDa proteins) were quantitated in postmortem brains of patients with Alzheimers disease (AD) by using immunoblotting techniques and a specific antiserum. Increased levels of the 29/30-kDa protein (30%), 45-kDa protein (36%) and glial fibrillary acidic protein (88%) were found in the frontal cortex of AD patients. These findings are in line with the reported higher density of imidazoline receptors labelled by [3H]idazoxan in AD brains, suggesting that these imidazoline receptor proteins are related to the I2-imidazoline receptor located in mitochondria of glial (astrocyte) cells.

Influence of genetic variation in alcohol and aldehyde dehydrogenase on serotonin metabolism.

The influence of genetic variation in alcohol dehydrogenase (ADH; EC 1.1.1.1) and aldehyde dehydrogenase (ALDH; EC 1.2.1.3) on the metabolic pattern of serotonin (5-hydroxytryptamine, 5-HT) in humans was examined from the relative urinary concentrations of the end products 5-hydroxyindole-3-acetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTOL). Healthy Caucasian (Swedish) and Oriental (Chinese) subjects were genotyped for ADH2, ADH3 and ALDH2 by a PCR/SSCP technique. The 5-HTOL/5-HIAA ratios ranged between 0.9-9.4 pmol/nmol (4.4 +/- 1.8, mean +/- SD, n = 143). No significant difference in the 5-HT metabolic pattern was observed between Caucasians and Orientals (4.3 +/- 1.8 and 4.4 +/- 1.8 pmol/nmol, respectively), nor between any of the ADH2, ADH3 and ALDH2 genotypes. Despite the modulatory effects of genetic variation of these enzymes on ethanol metabolism, the present results indicate that the individual isozyme composition of ADH2, ADH3 and ALDH2 is not important for the metabolic pattern of 5-HT.

G protein-coupled receptor kinases, β-arrestin-2 and associated regulatory proteins in the human brain: postmortem changes, effect of age and subcellular distribution

G protein-coupled receptor kinases (GRKs) and beta-arrestin-2 play a crucial role in the regulation of neurotransmitter receptors in brain. In this study, GRK2, GRK6, beta-arrestin-2 and associated regulatory proteins (Gbeta proteins and protein phosphatase (PP)-2A) were quantitated in human brains (immunodensity with specific antibodies) to assess for postmortem changes (pattern of protein degradation) and to investigate the effect of aging on these regulatory proteins as well as their subcellular distribution (cytosol and membrane fractions). In brain (prefrontal cortex, total homogenate) of healthy subjects (n=14) the immunodensities of GRK2 (r=-0.76), GRK6 (r=-0.64), beta-arrestin-2 (r=-0.57), Gbeta proteins (r=-0.59) and neurofilament (NF)-L (r=-0.64), but not PP-2A, declined markedly with the length of postmortem delay (PMD, 3-81 h). With these linear decay models, the average decreases per 12 h of PMD (from 12 to 72 h) were 7-11% for the various proteins. The immunodensities of GRK2 (r=-0.71), GRK6 (r=-0.61), and beta-arrestin-2 (r=-0.54) in human brain (n=12) also declined with aging (16 to 87 years) and the average decreases per decade (from 20 to 80 years) were 3-5%. In contrast, the immunodensities of PP-2A, Gbeta and NF-L in brain did not correlate significantly with the age of the subject at death (16-87 years). The immunodensities of GRK2/6 and beta-arrestin-2 showed marked individual variations and were strongly reduced after several freeze/thaw cycles. In the prefrontal cortex the subcellular distribution (cytosol/membrane) of the two GRKs differed markedly (GRK2: 60%/40%; GRK6: 5%/95%), and that of beta-arrestin-2 was as expected for a soluble protein (60%/40%). In brains of healthy subjects, the immunodensities of cytosolic GRK2 and beta-arrestin-2 correlated, respectively, with those of membrane-associated GRK2 (r=0.67, P=0.049, n=9) and membrane-associated beta-arrestin-2 (r=0.77, P=0.01, n=9). The results of this study emphasize the importance of examining relevant variables (PMD, age) and potential artifacts (individual variation, freeze-thawing effect) when designing signal transduction studies in neuropsychiatric disorders using the postmortem human brain.