Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

Loss of Protein Kinase C-αβ in Brain of Heroin Addicts and Morphine-Dependent Rats

Abstract: The biochemical status of human brain protein kinase C (PKC)-αβ during opiate dependence was studied by means of immunoblotting techniques in postmortem brain of heroin addicts who had died by opiate overdose. In the frontal cortex, a marked decrease (53%, p < 0.05) in the immunoreactivity of PKC-αβ was found in heroin addicts compared with matched controls. The loss of PKC-αβ in the brain of human addicts paralleled that observed in the frontal cortex of rats after chronic treatment with morphine (10–100 mg/kg i.p. for 5 days) (PKC-αβ decreased by 34%, p < 0.05). Chronic treatment with naloxone (1 mg/kg i.p. every 12 h for 5 days) did not alter PKC-αβ immunoreactivity in the rat brain. However, in morphine-dependent rats, naloxone-precipitated withdrawal induced a rapid and strong behavioral reaction with a concomitant up-regulation of PKC-αβ immunoreactivity to control values. These results indicated that the decrease of brain PKC-αβ induced by heroin/morphine is a μ-opioid receptor-mediated effect. The chronic administration of opiates has been associated with a marked sensitization of the adenylyl cyclase/cyclic AMP system, although this phenomenon is not exclusive of the opioid system but the general cellular adaptation to chronic inhibition of adenylyl cyclase. In this context, chronic treatment of rats with other inhibitory agonists (e.g., clonidine, 1 mg/kg i.p. every 12 h for 14 days) acting through receptors (e.g., α₂-adrenoceptors) also coupled to adenylyl cyclase did not alter brain PKC-αβ immunoreactivity. Together these findings suggest that the brain PKC system might play a major role in opiate addiction.     

β-Amyloid-Induced Neurotoxicity of a Hybrid Septal Cell Line Associated with Increased Tau Phosphorylation and Expression of β-Amyloid Precursor Protein

Abstract: Recent evidence suggests that β-amyloid peptide (β-AP) may induce tau protein phosphorylation, resulting in loss of microtubule binding capacity and formation of paired helical filaments. The mechanism by which β-AP increases tau phosphorylation, however, is unclear. Using a hybrid septal cell line, SN56, we demonstrate that aggregated β-AP_1–40 treatment caused cell injury. Accompanying the cell injury, the levels of phosphorylated tau as well as total tau were enhanced as detected immunochemically by AT8, PHF-1, Tau-1, and Tau-5 antibodies. Alkaline phosphatase treatment abolished AT8 and PHF-1 immunoreactivity, confirming that the tau phosphorylation sites were at least at Ser^199/202 and Ser³⁹⁶. In association with the increase in tau phosphorylation, the immunoreactivity of cell-associated and secreted β-amyloid precursor protein (β-APP) was markedly elevated. Application of antisense oligonucleotide to β-APP reduced expression of β-APP and immunoreactivity of phosphorylated tau. Control peptide β-AP_1–28 did not produce significant effects on tau phosphorylation, although it slightly increased cell-associated β-APP. These results suggest that βAP_1–40-induced tau phosphorylation may be associated with increased β-APP expression in degenerated neurons.     

Structure-Activity Analyses of β-Amyloid Peptides: Contributions of the β25–35 Region to Aggregation and Neurotoxicity

Abstract: The neurodegeneration of Alzheimer's disease has been theorized to be mediated, at least in part, by insoluble aggregates of β-amyloid protein that are widely distributed in the form of plaques throughout brain regions affected by the disease. Previous studies by our laboratory and others have demonstrated that the neurotoxicity of β-amyloid in vitro is dependent upon its spontaneous adoption of an aggregated structure. In this study, we report extensive structure-activity analyses of a series of peptides derived from both the proposed active fragment of β-amyloid, β25–35, and the full-length protein, β1–42. We examine the effects of amino acid residue deletions and substitutions on the ability of β-amyloid peptides to both form sedimentable aggregates and induce toxicity in cultured hippocampal neurons. We observe that significant levels of peptide aggregation are always associated with significant β-amyloid-induced neurotoxicity. Further, both N- and C-terminal regions of β25–35 appear to contribute to these processes. In particular, significant disruption of peptide aggregation and toxicity result from alterations in the β33–35 region. In β1–42 peptides, aggregation disruption is evidenced by changes in both electrophoresis profiles and fibril morphology visualized at the light and electron microscope levels. Using circular dichroism analysis in a subset of peptides, we observed classic features of β-sheet secondary structure in aggregating, toxic β-amyloid peptides but not in nonaggregating, nontoxic β-amyloid peptides. Together, these data further define the primary and secondary structures of β-amyloid that are involved in its in vitro assembly into neurotoxic peptide aggregates and may underlie both its pathological deposition and subsequent degenerative effects in Alzheimer's disease.     

Effect of β-FNA on Opiate δ Receptor Binding

Abstract: (β-FNA, the β -fumaramate methyl ester of naltrexone, has been shown to antagonize irreversibly the actions of morphine on the guinea pig ileum and mouse vas deferens bioassays but does not affect the actions of δ-receptor ligands on the mouse vas deferens bioassay, suggesting that the compound does not irreversibly bind to the S receptor. In this paper we examine the effect of (β -FNA on the binding of the prototypic δ agonists, Leuenkephalin and d -Ala²- d -Leu⁵-enkephalin, its metabolically stable analogue, and show that treatment of membranes with β -FNA does lead to alterations in the in vitro properties of δ receptors.     

Generation and Regulation of β-Amyloid Peptide Variants by Neurons

Abstract: Studies of processing of the Alzheimer β-amyloid precursor protein (βAPP) have been performed to date mostly in continuous cell lines and indicate the existence of two principal metabolic pathways: the "β-secretase" pathway, which generates β-amyloid (Aβ_1–40/42; ∼4 kDa), and the "α-secretase" pathway, which generates a smaller fragment, the "p3" peptide (Aβ_17–40/42; ∼3 kDa). To determine whether similar processing events underlie βAPP metabolism in neurons, media were examined following conditioning by primary neuronal cultures derived from embryonic day 17 rats. Immunoprecipitates of conditioned media derived from [³⁵S]methionine pulse-labeled primary neuronal cultures contained 4- and 3-kDa Aβ-related species. Radiosequencing analysis revealed that the 4-kDa band corresponded to conventional Aβ beginning at position Aβ(Asp¹), whereas both radio-sequencing and immunoprecipitation-mass spectrometry analyses indicated that the 3-kDa species in these conditioned media began with Aβ(Glu¹¹) at the N terminus, rather than Aβ(Leu¹⁷) as does the conventional p3 peptide. Either activation of protein kinase C or inhibition of protein phosphatase 1/2A increased soluble βAPP_α release and decreased generation of both the 4-kDa Aβ and the 3-kDa N-truncated Aβ. Unlike results obtained with continuously cultured cells, protein phosphatase 1/2A inhibitors were more potent at reducing Aβ secretion by neurons than were protein kinase C activators. These data indicate that rodent neurons generate abundant Aβ variant peptides and emphasize the role of protein phosphatases in modulating neuronal Aβ generation.     

β-Amyloid Protein Precursor and τ mRNA Levels Versus β-Amyloid Plaque and Neurofibrillary Tangles in the Aged Human Brain

Abstract: To learn whether or not the levels of β-amyloid protein precursor (APP) and τ mRNAs are related to the formation of β-amyloid and neurofibrillary tangles, we quantified these mRNA levels in three cortical regions of 38 aged human brains, which were examined immunocyto-chemically for β-amyloid and tangles. Marked individual variabilities were noted in APP and τ mRNA levels among elderly individuals. The mean APP mRNA level was slightly reduced in the β-amyloid plaque (++) group, but not in the plaque (+) group, compared to the plaque (−) group. Some brains in the plaque (−) group showed increased APP expression, the extent of which was not seen in the plaque (+)or(++) group. The differences in the mean τ mRNA levels were not statistically significant among the tangle (−), (+), and (++) groups. These results show that β-protein and τ deposition do not accompany increased expression of the APP and τ genes, respectively, and thus suggest that factors other than gene expression may be at work in the progression of β-amyloid and/or tangle formation in the aged human brain.     

In Vivo Aggregation of β-Amyloid Peptide Variants

Abstract: Transgenic Caenorhabditis elegans animals have been engineered to express wild-type and single-amino acid variants of a long form of human β-amyloid peptide (Aβ 1–42). These animals express high levels (∼300 ng of Aβ/mg of total protein) of apparently full-length peptide, as determined by quantitative immunoblot. Expression of wild-type Aβ in these animals leads to rapid production of amyloid deposits reactive with Congo red and thioflavin S. This model system has been used to examine the effect of Leu¹⁷Pro, Leu¹⁷Val, Ala³⁰-Pro, Met³⁵Cys, and Met³⁵Leu substitutions on the in vivo production of amyloid deposits. We find that the Leu¹⁷Pro and Met³⁵Cys substitutions completely block the formation of thioflavin S-reactive deposits, implicating these as key residues for in vivo amyloid formation. We have also constructed transgenic strains expressing a novel Aβ variant, the single-chain dimer. Animals expressing high levels of this variant also fail to produce thioflavin S-reactive deposits.     

β-Amyloid1–40 Increases Expression of β-Amyloid Precursor Protein in Neuronal Hybrid Cells

Abstract: Studies of cell injury and death in Alzheimer's disease have suggested a prominent role for β-amyloid peptide (β-AP), a 40–43-amino-acid peptide derived from a larger membrane glycoprotein, β-amyloid precursor protein (β-APP). Previous experiments have demonstrated that β-AP induces cytotoxicity in a neuronal hybrid cell line (MES 23.5) in vitro. Here, we demonstrate that β-APP mRNA content is increased 3.5-fold in 24 h after treatment with β-AP_1–40. Accompanying β-AP_1–40-induced cell injury, levels of cell-associated β-APP and a C-terminal intermediate fragment are increased up to 15-fold, and levels of secreted forms of β-APP and 12- and 4-kDa fragments are also increased. Application of β-APP antisense oligodeoxynucleotide reduces both cytotoxicity and β-APP expression. 6-Hydroxydopamine application or glucose deprivation causes extensive cell damage, but they do not increase β-APP expression. These results suggest a selective positive feedback mechanism whereby β-AP may induce cytotoxicity and increase levels of potentially neurotrophic as well as amyloidogenic fragments of β-APP with the net consequence of further neuronal damage.     

β-Endorphin Is a Potent Inhibitor of Thymidine Incorporation into DNA via μ and κ-Opioid Receptors in Fetal Rat Brain Cell Aggregates in Culture

Abstract: Thymidine incorporation into DNA was inhibited dose-dependently by β-endorphin in rat fetal brain cell aggregate cultures. The inhibition was reversed partially by μ (cyclic D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide) or k (norbinaltorphimine) antagonists. Complete blockade of the β-endorphin inhibitory effect was achieved only on concomitant exposure to both antagonists. Eadie–Hofstee analysis revealed that β-endorphin inhibited thymidine incorporation noncompetitively. In the presence of protease inhibitors, β-endorphin decreased thymidine incorporation with an IC₅₀ of 0.7 n M . Truncated and N -acetylated β-endorphin derivatives, which bind with low affinity to opioid receptors, did not affect thymidine incorporation. These findings indicate that β-endorphin at physiological concentrations can regulate thymidine incorporation in cultured brain cells.     

Apolipoprotein E Attenuates β-Amyloid-Induced Astrocyte Activation

Abstract: A common feature of Alzheimer's disease pathology is an abundance of activated glia, indicative of an inflammatory reaction in the brain. The relationship between glial activation and neurodegeneration is not known, although several cytokines and inflammatory mediators produced by activated glia have the potential to initiate or exacerbate the progression of neuropathology. As β-amyloid (Aβ) is one of several stimuli that can activate glia, it is important to determine how Aβ-induced glial activation is influenced by other proteins present in the plaque, such as apolipoprotein E (apoE). We examined the effect of native preparations of apoE on activation of rat cortical astrocyte cultures by Aβ1–42. The apoE source was conditioned medium from human embryonic kidney 293 cells stably transfected with human apoE3 or apoE4 cDNA. By morphological criteria, apoE inhibited Aβ-induced astrocyte activation in three experimental paradigms: apoE pretreatment blocked subsequent Aβ-induced activation, Aβ aged in the presence of apoE did not activate astrocytes, and apoE addition to activated astrocytes transiently reversed the activated phenotype. No apoE isoform selectivity was observed. The effect of apoE appears to be specific to Aβ, as apoE did not attenuate cyclic AMP-induced astrocyte activation. These data suggest that apoE may modulate the ability of Aβ to induce inflammatory responses in the brain.     

Intracerebral NMDA Injection Stimulates Production of Interleukin-1β in Perinatal Rat Brain

Abstract: Susceptibility to NMDA neurotoxicity peaks in the early postnatal period in rats. Although indirect evidence suggests that interleukin-1β is a mediator of NMDA neurotoxicity in perinatal rats, direct confirmation of NMDA-induced interleukin-1β production in the brain has not been reported previously. The primary goal of this study was to determine if intracerebral injection of a neurotoxic dose of NMDA stimulates interleukin-1β production acutely. We used a rat-specific interleukin-1β ELISA to quantify brain tissue homogenate interleukin-1β content, and an immunocytochemical assay with a monoclonal anti-rat interleukin-1β antibody to visualize its distribution. NMDA (10 nmol) was injected stereotaxically into 7-day-old rats, using coordinates that targeted the striatum and overlying dorsal hippocampus. Interleukin-1β concentrations were measured in samples from the injected and contralateral cerebral hemispheres 0–12 h later; in addition, the impact of treatment with the noncompetitive NMDA antagonist MK-801 on interleukin-1β production was assessed. We found marked increases in tissue content of interleukin-1β in the lesioned hemisphere; values peaked at 6 h post injection. Treatment with MK-801 (1 mg/kg) blocked NMDA-induced increases in interleukin-1β. Preliminary immunocytochemical analysis demonstrated high concentrations of interleukin-1β-immunoreactive cells in the lesioned hippocampus, and concurrent increases in interleukin-1β immunoreactivity diffusely in the ependyma at 6 h after NMDA administration. Our data provide the first direct evidence that NMDA-induced excitotoxic injury stimulates interleukin-1β production in vivo.     

Rat Frontal Cortex β1-Adrenoceptors Are Activated by the β3-Adrenoceptor Agonists SR 58611A and SR 58878A but Not by BRL 37344 or ICI 215,001

Abstract: SR 58611A, a selective agonist of gut and brown adipose tissue β₃-adrenoceptors (β₃ARs), has been reported to have antidepressant-like activity in rodents by indicating brain β₃ARs as the sites of this property. SR 58611A and its acid metabolite SR 58878A, as opposed to BRL 37344, ICI 215,001, and CGP 12177, increased cyclic AMP levels in rat frontal cortex. ICI 215,001, differently from BRL 37344, at concentrations in the millimolar range antagonized norepinephrine- or (−)-isoproterenol-stimulated adenylyl cyclase partially. The increase of cyclic AMP levels induced by SR 58878A was blocked selectively by β₁AR antagonist CGP 20712A but not by β₂AR antagonist ICI 118,551. In addition, PCR analysis did not reveal β₃AR mRNA, and no specific β₃AR binding sites were detected by [³H]CGP 12177 in rat frontal cortex. When down-regulation of the β₁AR ligand binding and mRNA levels had been induced in frontal cortex by chronic administration of imipramine, SR 58878A as well as norepinephrine and (−)-isoproterenol increased the cyclic AMP production less markedly. Our findings indicate that β₃ARs are absent in the adult rat frontal cortex, and that various β₃AR agonists differently affect the frontal cortex β₁ARs, indicating that SR 58611A may exert its putative antidepressant effect acting on the frontal cortex β₁ARs.     

Acetylcholinesterase Is Increased in the Brains of Transgenic Mice Expressing the C-Terminal Fragment (CT100) of the β-Amyloid Protein Precursor of Alzheimer's Disease

Abstract: Acetylcholinesterase (AChE) expression is markedly affected in Alzheimer's disease (AD). AChE activity is lower in most regions of the AD brain, but it is increased within and around amyloid plaques. We have previously shown that AChE expression in P19 cells is increased by the amyloid β protein (Aβ). The aim of this study was to investigate AChE expression using a transgenic mouse model of Aβ overproduction. The β-actin promoter was used to drive expression of a transgene encoding the 100-amino acid C-terminal fragment of the human amyloid precursor protein (APP CT100). Analysis of extracts from transgenic mice revealed that the human sequences of full-length human APP CT100 and Aβ were overexpressed in the brain. Levels of salt-extractable AChE isoforms were increased in the brains of APP CT100 mice. There was also an increase in amphiphilic monomeric form (G^A₁) of AChE in the APP CT100 mice, whereas other isoforms were not changed. An increase in the proportion of G^A₁ AChE was also detected in samples of frontal cortex from AD patients. Analysis of AChE by lectin binding revealed differences in the glycosylation pattern in APP CT100 mice similar to those observed in frontal cortex samples from AD. The results are consistent with the possibility that changes in AChE isoform levels and glycosylation patterns in the AD brain may be a direct consequence of altered APP metabolism.     

Glycosidases of apple fruit: A multi-functional β-galactosidase

Extraction of Spartan apple ( Malus domestica ) fruit acetone powder and fractionation of the extract on DEAE-agarose allowed detection and quantification of 10 glycosidases active toward 4-methylumbelliferyl glycosides. Hydrolysis was measured fluorimetrically. The predominant activity, a β- d -galactosidase (EC 3.2.1.23), labile upon purification, was stabilized by soluble PVP. Molecular weights, measured by gel permeation HPLC, pH optima and K_m values were obtained for most glycosidase activities. Multiple forms of several activities were found. The major α- d - and β- d -galactosidases were resolved on phosphocellulose. The β- d -galactosidase so obtained had associated α- l -arabinopyranosidase and β- d -fucosidase activities which were retained upon GP-HPLC. Mixed substrate kinetic analysis and inhibition analysis of this fraction indicated that the enzyme has 3 catalytic sites, 1 for each substrate, whose substrates mutually influence each other's activity positively.     

G Protein independent phosphorylation and internalization of the δ-opioid receptor

Agonist activation of the δ-opioid receptor leads to internalization via Gβγ recruitment of G protein coupled receptor kinase-2, which phosphorylates the receptor at several sites, including Ser363, allowing β-arrestin binding and localization to clathrin coated pits. Using human embryonic kidney cells expressing a δ-opioid receptor we tested the hypothesis that prevention of receptor coupling to G protein by treatment with pertussis toxin (PTX) will block these processes. PTX treatment did not reduce phosphorylation of δ-opioid receptor Ser363 in response to the agonist [ d -Pen2, d -Pen5]enkephalin, or recruitment of β-arrestin 2-green fluorescent protein to the membrane and only slowed, but did not prevent, [ d -Pen2, d -Pen5]enkephalin-induced internalization. Similarly, PTX treatment only partially prevented the ability of the δ-opioid peptide agonists deltorphin II and [Met5]enkephalin and the non-peptide agonist BW373U86 to induce receptor internalization. No internalization was seen with morphine, oxymorphindole or the putative δ₁-opioid agonist TAN-67 in the presence or absence of PTX, even though TAN-67 showed a strong activation of G protein, as measured by guanosine-5'-O-(3-[³⁵S]thio)triphosphate binding. The ability of an agonist to stimulate phosphorylation at Ser363 was predictive of its capacity to induce internalization. The results suggest a role for G protein in δ-opioid receptor internalization, but show that alternative G protein independent pathways exist.     

Role of Src in ligand-specific regulation of δ-opioid receptor desensitization and internalization

The opioid receptors are a member of G protein-coupled receptors that mediate physiological effects of endogenous opioid peptides and structurally distinct opioid alkaloids. Although it is well characterized that there is differential receptor desensitization and internalization properties following activation by distinct agonists, the underlying mechanisms remain elusive. We investigated the signaling events of δ-opioid receptor (δOR) initiated by two ligands, DPDPE and TIPP. We found that although both ligands inhibited adenylyl cyclase (AC) and activated ERK1/2, only DPDPE induced desensitization and internalization of the δOR. We further found that DPDPE, instead of TIPP, could activate GRK2 by phosphorylating the non-receptor tyrosine kinase Src and translocating it to membrane receptors. Activation of GRK2 led to the phosphorylation of serine residues in the C-terminal tail, which facilitates β-arrestin1/2 membrane translocation. Meanwhile, we also found that DPDPE promoted β-arrestin1 dephosphorylation in a Src-dependent manner. Thus, DPDPE appears to strengthen β-arrestin function by dual regulations: promoting β-arrestin recruitment and increasing β-arrestin dephosphorylation at the plasma membrane in a Src-dependent manner. All effects initiated by DPDPE could be abolished or suppressed by PP2, an inhibitor of Src. Morphine, which has been previously shown to be unable to desensitize or internalize δOR, also behaved as TIPP in failure to utilize Src to regulate δOR signaling. These findings point to the existence of agonist-specific utilization of Src to regulate δOR signaling and reveal the molecular events by which Src modulates δOR responsiveness.     

Secreted Forms of β-Amyloid Precursor Protein Protect Against Ischemic Brain Injury

Abstract: The β-amyloid precursor protein (βAPP) is the source of the amyloid β-peptide that accumulates in the brain in Alzheimer's disease. A major processing pathway for βAPP involves an enzymatic cleavage within the amyloid β-peptide sequence that liberates secreted forms of βAPP (APP^Ss) into the extracellular milieu. We now report that postischemic administration of these APP^Ss intracerebroventricularly protects neurons in the CA1 region of rat hippocampus against ischemic injury. Treatment with APP^S695 or APP^S751 resulted in increased neuronal survival, and the surviving cells were functional as demonstrated by their ability to synthesize protein. These data provide direct evidence for a neuroprotective action of APP^Ss in vivo.