3,4-Methylenedioxymethamphetamine induces differential regulation of tryptophan hydroxylase 2 protein and mRNA levels in the rat dorsal raphe nucleus.

Previous investigations with 3,4-methylenedioxymethamphetamine (MDMA) have suggested that administration of this drug results in a degeneration of 5-HT nerve terminals and subsequent alterations in 5-HT neurotransmission. However, only limited investigations have examined the effects of MDMA on the dorsal raphe nucleus. The present study was designed to assess the effect of MDMA on the rate-limiting enzyme in 5-HT biosynthesis, tryptophan hydroxylase (TPH), by measuring TPH2 protein and mRNA levels in rat dorsal raphe (DR) nucleus. Rats were administered MDMA (20 mg/kg, s.c.) or saline twice daily for 4 days and killed 14 days later. Tissue sections of the DR were processed for quantitative immunoautoradiography and in situ hybridization histochemistry for measurements of the levels of TPH2-immunoreactivity (IR) and TPH2 mRNA. To assess 5-HT axon terminal integrity after MDMA treatment, the density of 5-HT transporter (SERT) binding sites was measured by quantitative autoradiography using [125I]RTI-55 ((-)-2beta-carbomethoxy-3 beta-(4-iodophenyl) tropane) ((125)I-RTI-55) as a ligand. TPH2-IR levels were significantly decreased by 45% in the mid DR and by 40% in the caudal DR in the MDMA-treated rats compared with saline-injected rats. In contrast, TPH2 mRNA levels were significantly increased by 24% in the mid DR and by 12% in the caudal DR. MDMA treatment significantly decreased (125)I-RTI-55 labeled SERT binding sites in the striatum, nucleus accumbens and cingulate cortex demonstrating a loss of 5-HT terminals. The increase in TPH2 mRNA levels in both the mid DR and caudal DR of MDMA-treated rats may reflect a compensatory mechanism in the injured 5-HT neurons to increase TPH2 protein synthesis. Taken together, our results suggest that a serious defect occurs in the biosynthesis of TPH2 in the DR following MDMA administration.

Receptor-G-protein signalling in Alzheimer's disease.

Based on radioligand binding studies, it has long been assumed that the neurochemical pathology of Alzheimer's disease (AD) does not involve widespread changes in post-synaptic neurotransmitter function. However, more recent studies suggest that receptor function in AD may be compromised due to disrupted post-receptor signal transduction, in particular that mediated by the G-protein regulated phosphoinositide hydrolysis and adenylate cyclase (AC) pathways. The phosphoinositide hydrolysis pathway has been shown to be altered at a number of levels in AD post-mortem brains, including impaired agonist and G-protein regulation of phospholipase C, decreased protein kinase C (PKC) levels and activity, and a reduced number of receptor sites for the second messenger, Ins(1,4,5)P3. Of these, loss of Ins(1,4,5)P3 receptors and PKC in the entorhinal cortex and hippocampus correlates with AD-related neurofibrillary changes, as staged according to Braak's protocol. Disregulation of the phosphoinositide hydrolysis pathway may therefore have consequences for the progression of AD pathology. In contrast to the extensive pattern of disruption seen with the phosphoinositide hydrolysis pathway, changes to AC signalling in AD appear more circumscribed. Disruptions include a lesion at the level of Gs-protein stimulation of AC and, at least in the hippocampus, reduced enzyme activities in response to forskolin stimulation. Of these, the latter change has been shown to precede neurofibrillary changes. Apart from a loss of calcium/calmodulin sensitive AC isoforms, other components of this signalling pathway, including G-protein levels, Gi-protein mediated inhibition and protein kinase A levels and activity, remain relatively preserved in the disorder.

Dysfunctional intracellular calcium homoeostasis: a central cause of neurodegeneration in Alzheimer's disease.

The clinical symptoms of all forms of Alzheimer's disease (AD) result from a slowly progressive neurodegeneration that is associated with the excessive deposition of beta-amyloid (A beta) in plaques and in the cerebrovasculature, and the formation of intraneuronal neurofibrillary tangles, which are composed primarily of abnormally hyperphosphorylated tau protein. The sequence of cellular events that cause this pathology and neurodegeneration is unknown. It is, however, most probably linked to neuronal signal transduction systems that become misregulated in the brains of certain individuals, causing excessive A beta to be formed and/or deposited, tau to become aggregated and hyperphosphorylated and neurons to degenerate. We hypothesize that a progressive alteration in the ability of neurons to regulate intracellular calcium, particularly at the level of the endoplasmic reticulum, is a crucial signal transduction event that is linked strongly to the initiation and development of AD pathology. In this chapter we will discuss the key findings that lend support to this hypothesis.

A quantitative autoradiographic study of [3H]cAMP binding to cytosolic and particulate protein kinase A in post-mortem brain staged for Alzheimer's disease neurofibrillary changes and amyloid deposits.

The cAMP-dependent protein kinase (PKA) has been implicated in the Alzheimer's disease pathology of abnormal tau phosphorylation leading to neurofibrillary tangle (NFT) formation, as well as in amyloid precursor protein alpha-secretase processing. In the present study, we determined whether [3H]cAMP binding to cytosolic and particulate PKA showed any relationship to the extent of Alzheimer's disease pathology at post-mortem. Autoradiographic [3H]cAMP binding to cytosolic and particulate PKA was measured in sections of entorhinal cortex/hippocampal formation from 23 cases that had been staged for Alzheimer's disease-related neurofibrillary changes and amyloid deposits according to Braak and Braak [H. Braak, E. Braak, Neuropathological staging of Alzheimer's-related changes, Acta Neuropathol. 82 (1991) 239-259]. [3H]cAMP binding to cytosolic PKA showed statistically significant reductions in the entorhinal cortex (P<0.01, ANOVA) with respect to neurofibrillary changes. Post-hoc analysis with Fisher's PLSD test showed significant reductions of [3H]cAMP binding to cytosolic PKA at the isocortical stages (V and VI), compared to the non-pathological (O) (by 55%, P<0.01), transentorhinal (I and II) (by 58%, P<0.001) and limbic (III and IV) (by 45%, P<0.05) stages. A significant reduction (by 25%, P<0.05) was also seen in the transentorhinal compared to the limbic stages. [3H]cAMP binding to cytosolic PKA showed no significant alterations with respect to neurofibrillary changes in either the subiculum, CA1-CA4 subfields of the hippocampus or the dentate gyrus. [3H]cAMP binding to cytosolic PKA also showed significant declines in the entorhinal cortex (P<0.01) and subiculum (P<0.05) with respect to staging for amyloid deposits. Post-hoc analysis with Fisher's PLSD test showed significant reductions of [3H]cAMP binding to cytosolic PKA in the entorhinal cortex at amyloid stage C compared to stages O (by 41%, P<0.01) and A (by 38%, P<0.01). In the subiculum, there were significant reductions of [3H]cAMP binding at stages C (by 41%, P<0.01) and B (by 40%, P<0.05), respectively, compared to stage O. [3H]cAMP binding to particulate PKA did not show significant relationships to staging for either neurofibrillary changes or amyloid deposits in either the entorhinal cortex or any of the hippocampal subregions. These findings suggest that whereas [3H]cAMP binding to cytosolic PKA in the entorhinal cortex is reduced with progression of neurofibrillary and amyloid pathology, other hippocampal regions show a preservation of cytosolic and particulate PKA even in late stage pathologies.

Loss of inositol 1,4,5-trisphosphate receptor sites and decreased PKC levels correlate with staging of Alzheimer's disease neurofibrillary pathology.

Inositol 1,4,5-trisphosphate (IP3), inositol 1,3,4,5-tetrakisphosphate (IP4) and protein kinase C (PKC) play important roles in the phosphoinositide hydrolysis signal transducing pathway. Several studies have shown severe deficits in both IP3 receptor levels and PKC levels and activity in Alzheimer's disease brain, although the relationship of these changes to disease pathology is poorly understood. In the present study, we determined the autoradiographic localization of [3H]IP3 and [3H]IP4 binding to their calcium mobilizing receptor sites and [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding to PKC in sections of entorhinal cortex/hippocampal formation and cerebellum from 24 cases that had been staged for Alzheimer's disease-related neurofibrillary changes and amyloid deposition according to Braak and Braak [Acta Neuropathol. Berl., 82 (1991) 239-259]. Results indicated that [3H]IP3 binding showed a trend towards a decline with staging for neurofibrillary changes in the entorhinal region (0.05 < P < 0.10, ANOVA) and subiculum (0.05 < P < 0.10). In the former region, [3H]IP3 binding showed a significant decline with staging for amyloid deposition (P < 0.05). [3H]IP3 binding in the CA1 region showed statistically significant declines with respect to both neurofibrillary changes and amyloid staging (P < 0.05). [3H]IP3 binding levels in the other hippocampal subregions were too low to quantify accurately. The binding of [3H]IP4 showed no significant changes with either neurofibrillary changes or amyloid staging in any of the regions investigated. In contrast, [3H]PDBu binding showed significant declines with neurofibrillary staging in the entorhinal region (P < 0.01), subiculum (P < 0.001), CA1 (P < 0.001), CA2 (P < 0.001), CA3 (P < 0.001) and CA4 (P < 0.0001) regions and the dentate gyrus (P < 0.0001). Of these regions, only the subiculum showed a significant decline of [3H]PDBu binding with amyloid staging. There were no significant neurofibrillary or amyloid stage-related changes in either [3H]IP3, [3H]IP4 or [3H]PDBu binding in the molecular layer of the cerebellum. These findings suggest that reduced IP3 receptor and PKC levels in the entorhinal cortex/hippocampal formation reflect and may be important for the progression of Alzheimer's disease neurofibrillary pathology. The data also suggests that hippocampal IP3 receptor loss is related to the extent of amyloid deposition.

Autoradiographic characterization of [3H]cGMP binding sites in the rat brain.

Quantitative autoradiography was used to characterize and localize [3H]cGMP binding sites in the rat brain. [3H]cGMP binding was found to be pH-sensitive (with two optima at 7.4 and 5.0) and Mg2+-dependent. At pH 7.4, the binding was dependent on inclusion of the phosphodiesterase inhibitor IBMX. In contrast, at pH 5.0, IBMX had little effect on binding. The binding of [3H]cGMP was reversible and saturable with a Kd of 22 nM at pH 7.4 and 36 nM at pH 5.0. Bmax values were 172 fmol/mg at pH 7.4 and 462 fmol/mg at pH 5.0. [3H]cGMP binding was inhibited by cGMP and its analogues, with cGMP and cAMP being the most potent at pH 7.4 and cGMP and 8-Br-cGMP being the most potent at pH 5.0. Using an extracellular pH 7.4 buffer, the selective cGMP-dependent protein kinase (PKG) inhibitor Rp-8pCPT-cGMPS had very little effect on [3H]cGMP binding. In contrast, with a cytosolic pH 5.0 buffer, Rp-8pCPT-cGMPS displaced binding in the cerebellum. This indicates that PKG is localized in the cerebellum, and that the binding to PKG is favored under cytosolic conditions. Autoradiographic localization of [3H]cGMP binding sites revealed a heterogeneous distribution with the highest densities in the substantia nigra and interpeduncular nucleus. High densities were also observed in the basal ganglia, the medial habenular nucleus, the frontoparietal cortex, the lateral amygdaloid nucleus and the subiculum. It is concluded that the nature of [3H]cGMP binding is complex, with one site probably being related to cytosolic PKG mainly found in the cerebellum, and one site probably representing cGMP-stimulated phosphodiesterase mainly located in the forebrain.

Impaired G-protein-stimulated adenylyl cyclase activity in Alzheimer's disease brain is not accompanied by reduced cyclic-AMP-dependent protein kinase A activity.

Previous studies have shown that the regulation of adenylyl cyclase activity is disrupted in Alzheimer's disease postmortem brain. In the present study, we determined whether disrupted adenylyl cyclase is accompanied by altered cAMP-dependent protein kinase activity in Alzheimer's disease superior temporal cortex and cerebellum. GTP gamma S-stimulated adenylyl cyclase activity was significantly lower in Alzheimer's disease superior temporal cortex, but not cerebellum, compared to values from a series of matched control cases. Neither basal or forskolin-stimulated adenylyl cyclase activities were significantly different between the Alzheimer's disease and control brain regions. No significant differences were seen in either particulate or soluble fraction cAMP-dependent protein kinase activities between the Alzheimer's disease and control brain regions. It is concluded that disrupted adenylyl cyclase signalling in Alzheimer's disease brain occurs specifically at the level of Gs-protein-enzyme interactions and is not accompanied by an altered cAMP-dependent protein kinase activity.

Reduced nitric oxide responsive soluble guanylyl cyclase activity in the superior temporal cortex of patients with Alzheimer's disease.

Particulate and soluble guanylyl cyclase activities were studied in postmortem temporal cortex from a series of Alzheimer's disease patients and matched control subjects. Particulate guanylyl cyclase activity was not significantly different between groups. In contrast, the Vmax values for basal and sodium nitroprusside-stimulated soluble guanylyl cyclase activities were approximately 50% lower in the Alzheimer's disease cases, compared to controls. This difference between groups was statistically significant for sodium nitroprusside-stimulated, but not for the basal, enzyme activities. These results provide the first evidence for a loss of nitric oxide responsive guanylyl cyclase activity in Alzheimer's disease brain.