PET imaging of acetylcholinesterase inhibitor‐induced effects on α4β2 nicotinic acetylcholine receptor binding

Acetylcholinesterase inhibitors (AChEIs) are drugs that increase synaptic acetylcholine (ACh) concentrations and are under investigation as treatments for symptoms accompanying Alzheimer's disease. The goal of this work was to use PET imaging to evaluate alterations of in vivo α4β2 nicotinic acetylcholine receptor (nAChR) binding induced by the AChEIs physostigmine (PHY) and galanthamine (GAL). The α4β2 nAChR‐specific radioligand [¹⁸F]nifene was used to examine the effects of 0.1–0.2 mg/kg PHY, 5 mg/kg GAL, and saline in three separate experiments all performed on each of two rat subjects. A 60‐min bolus‐infusion protocol was used with drug administered after 30 min. Data from the thalamus and cortex were analyzed with a graphical model accounting for neurotransmitter activation using the cerebellum as a reference region to test for transient competition with bound [¹⁸F]nifene. Significant [¹⁸F]nifene displacement was detected in both regions during one PHY and both GAL studies, while no significant competition was observed in both saline studies. This preliminary work indicates the viability of [¹⁸F]nifene in detecting increases in synaptic ACh induced by AChEIs. Synapse 67:882–886, 2013. © 2013 Wiley Periodicals, Inc.     

Comparing α7 nicotinic acetylcholine receptor binding,amyloid‐β deposition,and mitochondria complex‐I function in living brain: A PET study in aged monkeys

This study was aimed to assess the correlations among α7 nicotinic acetylcholine receptor (α7‐nAChR) binding, amyloid‐β (Aβ) deposition, and mitochondrial complex I (MC‐I) activity in the brain of aged monkeys (Macaca mulatta). Positron emission tomography (PET) measurements with [¹¹C](R)‐MeQAA, [¹¹C]PIB, and [¹⁸F]BCPP‐EF were conducted in monkeys in a conscious condition. [¹¹C](R)‐MeQAA binding was analyzed by a simplified reference tissue model to calculate nondisplaceable binding potential (BP_ND), [¹¹C]PIB uptake was calculated by standard uptake value ratio (SUVR), and [¹⁸F]BCPP‐EF binding was determined by Logan graphical analysis to calculate total distribution volume (V_T) with arterial blood sampling. Higher brain uptake was determined in the thalamus, hippocampus, striatum, and cortical regions for [¹¹C](R)‐MeQAA, while being lower in the cerebellum. Significant age‐related reduction of [¹¹C](R)‐MeQAA binding to α7‐nAChR was determined only in the occipital cortex. The plot of Vt of [¹⁸F]BCPP‐EF against BP_ND of [¹¹C](R)‐MeQAA indicated a significant negative correlation in the hippocampus and cortical regions in aged animals. Plotting of SUVR of [¹¹C]PIB against BP_ND of [¹¹C](R)‐MeQAA showed a positive correlation. The in vivo binding of [¹¹C](R)‐MeQAA could reflect the upregulation of α7‐nAChR induced by neurodegenerative damage determined by Aβ deposition as well as impaired MC‐I activity in living brain. Synapse 69:475–483, 2015. © 2015 Wiley Periodicals, Inc.     

Engineering neuronal nicotinic acetylcholine receptors with functional sensitivity to α‐bungarotoxin: a novel α3‐knock‐in mouse

We report here the construction of a novel knock‐in mouse expressing chimeric α3 nicotinic acetylcholine receptor (nAChR) subunits with pharmacological sensitivity to α‐bungarotoxin (αBTX). Sensitivity was generated by substituting five amino acids in the loop C (β9–β10) region of the mouse α3 subunit with the corresponding residues from the α1 subunit of the muscle type receptor from Torpedo californica. To demonstrate the utility of the underlying concept, expressed α3[5] subunits were characterized in the superior cervical ganglia (SCG) of homozygous knock‐in mice, where the synaptic architecture of postsynaptic α3‐containing nAChR clusters could now, for the first time, be directly visualized and interrogated by live‐staining with rhodamine‐conjugated αBTX. Consistent with the postsynaptic localization of ganglionic nAChRs, the αBTX‐labeled puncta colocalized with a marker for synaptic varicosities. Following in vivo deafferentation, these puncta persisted but with significant changes in intensity and distribution that varied with the length of the recovery period. Compound action potentials and excitatory postsynaptic potentials recorded from SCG of mice homozygous for α3[5] were abolished by 100 nm αBTX, even in an α7 null background, demonstrating that synaptic throughput in the SCG is completely dependent on the α3‐subunit. In addition, we observed that the genetic background of various inbred and outbred mouse lines greatly affects the functional expression of α3[5]‐nAChRs, suggesting a powerful new approach for exploring the molecular mechanisms underlying receptor assembly and trafficking. As αBTX‐sensitive sequences can be readily introduced into other nicotinic receptor subunits normally insensitive to αBTX, the findings described here should be applicable to many other receptors.     

3‐[(2,4‐dimethoxy)benzylidene]‐anabaseine dihydrochloride protects against 6‐hydroxydopamine‐induced parkinsonian neurodegeneration through α7 nicotinic acetylcholine receptor stimulation in rats

To explore a novel therapy against Parkinson's disease through enhancement of α7 nicotinic acetylcholine receptor (nAChR), we evaluated the neuroprotective effects of 3‐[(2,4‐dimethoxy)benzylidene]‐anabaseine dihydrochloride (DMXBA; GTS‐21), a functionally selective α7 nAChR agonist, in a rat 6‐hydroxydopamine (6‐OHDA)‐induced hemiparkinsonian model. Microinjection of 6‐OHDA into the nigrostriatal pathway of rats destroys dopaminergic neurons selectively. DMXBA dose dependently inhibited methamphetamine‐stimulated rotational behavior and dopaminergic neuronal loss induced by 6‐OHDA. The protective effects were abolished by methyllycaconitine citrate salt hydrate, an α7 nAChR antagonist. Immunohistochemical study confirmed abundant α7 nAChR expression in the cytoplasm of dopaminergic neurons. These results indicate that DMXBA prevented 6‐OHDA‐induced dopaminergic neuronal loss through stimulating α7 nAChR in dopaminergic neurons. Injection of 6‐OHDA elevated immunoreactivities to glial markers such as ionized calcium binding adaptor molecule 1, CD68, and glial fibrillary acidic protein in the substantia nigra pars compacta of rats. In contrast, these immunoreactivities were markedly inhibited by comicroinjection of DMXBA. Microglia also expressed α7 nAChR in both resting and activated states. Hence, we hypothesize that DMXBA simultaneously affects microglia and dopaminergic neurons and that both actions lead to dopaminergic neuroprotection. The findings that DMXBA attenuates 6‐OHDA‐induced dopaminergic neurodegeneration and glial activation in a rat model of Parkinson's disease raisethe possibility that DMXBA could be a novel therapeutic compound to prevent Parkinson's disease development. © 2012 Wiley Periodicals, Inc.     

Enhancement of 18F‐fluorodeoxyglucose metabolism in rat brain frontal cortex using a β3 adrenoceptor agonist

We report the use of β3 adrenergic receptor mediated activation of rat brain frontal cortex using mirabegron (a selective β3 adrenoceptor agonist), measured by ¹⁸F‐FDG PET/CT. Another β3 agonist, CL 316,243, did not have this effect due to impermeability through the blood brain barrier (BBB), while atomoxetine, a norepinephrine transporter blocker, did increase ¹⁸F‐FDG uptake in the frontal cortex. Mirabegron exhibited a dose‐dependent increase in frontal cortex ¹⁸F‐FDG uptake. These findings suggest a possible use of selective β3 adrenoceptor agonists in reversing regional glucose hypometabolism in the brain. Synapse 69:96–98, 2015. © 2014 Wiley Periodicals, Inc.     

Inflammatory cytokine tumor necrosis factor α suppresses neuroprotective endogenous erythropoietin from astrocytes mediated by hypoxia‐inducible factor‐2α

Interest in erythropoietin (EPO) as a neuroprotective mediator has grown since it was found that systemically administered EPO is protective in several animal models of disease. However, given that the blood–brain barrier limits EPO entry into the brain, alternative approaches that induce endogenous EPO production in the brain may be more effective clinically and associated with fewer untoward side‐effects. Astrocytes are the main source of EPO in the central nervous system. In the present study we investigated the effect of the inflammatory cytokine tumor necrosis factor α (TNFα) on hypoxia‐induced upregulation of EPO in rat brain. Hypoxia significantly increased EPO mRNA expression in the brain and kidney, and this increase was suppressed by TNFα in vivo. In cultured astrocytes exposed to hypoxic conditions for 6 and 12 h, TNFα suppressed the hypoxia‐induced increase in EPO mRNA expression in a concentration‐dependent manner. TNFα inhibition of hypoxia‐induced EPO expression was mediated primarily by hypoxia‐inducible factor (HIF)‐2α rather than HIF‐1α. The effects of TNFα in reducing hypoxia‐induced upregulation of EPO mRNA expression probably involve destabilization of HIF‐2α, which is regulated by the nuclear factor (NF)‐κB signaling pathway. TNFα treatment attenuated the protective effects of astrocytes on neurons under hypoxic conditions via EPO signaling. The effective blockade of TNFα signaling may contribute to the maintenance of the neuroprotective effects of EPO even under hypoxic conditions with an inflammatory response.     

β‐Amyloid activates presynaptic α7 nicotinic acetylcholine receptors reconstituted into a model nerve cell system: involvement of lipid rafts

Beta amyloid (Aβ) plays a central role in the pathogenesis of Alzheimer’s disease. Aβ is the major constituent of senile plaques, but there is a significant presence of Aβ in the brain in soluble forms. The results of functional studies indicate that soluble Aβ interacts with the α7 nicotinic acetylcholine receptor (nAChR) complex with apparent high affinity. However, conflicting data exist as to the nature of the Aβ–α7 nAChR interaction, and whether it is the result of specific binding. Moreover, both agonist‐like and antagonist‐like effects have been reported. In particular, agonist‐like effects have been observed for presynaptic nAChRs. Here, we demonstrate Aβ_1‐42‐evoked stimulatory changes in presynaptic Ca²⁺ level via exogenous α7 nAChRs expressed in the axonal varicosities of differentiated hybrid neuroblastoma NG108‐15 cells as a model, presynaptic system. The Aβ_1‐42‐evoked responses were concentration‐dependent and were sensitive to the highly selective α7 nAChR antagonist α‐bungarotoxin. Voltage‐gated Ca²⁺ channels and internal Ca²⁺ stores were both involved in Aβ_1‐42‐evoked increases in presynaptic Ca²⁺ following activation of α7 nAChRs. In addition, disruption of lipid rafts by cholesterol depletion led to substantially attenuated responses to Aβ_1‐42, whereas responses to nicotine were largely intact. These results directly implicate the nicotinic receptor complex as a target for the agonist‐like action of pico‐ to nanomolar concentrations of soluble Aβ_1‐42 on the presynaptic nerve terminal, including the possible involvement of receptor‐associated lipid rafts. This interaction probably plays an important neuromodulatory role in synaptic dynamics.     

δ‐Catenin/NPRAP: A new member of the glycogen synthase kinase‐3β signaling complex that promotes β‐catenin turnover in neurons

Through a multiprotein complex, glycogen synthase kinase‐3β (GSK‐3β) phosphorylates and destabilizes β‐catenin, an important signaling event for neuronal growth and proper synaptic function. δ‐Catenin, or NPRAP (CTNND2), is a neural enriched member of the β‐catenin superfamily and is also known to modulate neurite outgrowth and synaptic activity. In this study, we investigated the possibility that δ‐catenin expression is also affected by GSK‐3β signaling and participates in the molecular complex regulating β‐catenin turnover in neurons. Immunofluorescent light microscopy revealed colocalization of δ‐catenin with members of the molecular destruction complex: GSK‐3β, β‐catenin, and adenomatous polyposis coli proteins in rat primary neurons. GSK‐3β formed a complex with δ‐catenin, and its inhibition resulted in increased δ‐catenin and β‐catenin expression levels. LY294002 and amyloid peptide, known activators of GSK‐3β signaling, reduced δ‐catenin expression levels. Furthermore, δ‐catenin immunoreactivity increased and protein turnover decreased when neurons were treated with proteasome inhibitors, suggesting that the stability of δ‐catenin, like that of β‐catenin, is regulated by proteasome‐mediated degradation. Coimmunoprecipitation experiments showed that δ‐catenin overexpression promoted GSK‐3β and β‐catenin interactions. Primary cortical neurons and PC12 cells expressing δ‐catenin treated with proteasome inhibitors showed increased ubiquitinated β‐catenin forms. Consistent with the hypothesis that δ‐catenin promotes the interaction of the destruction complex molecules, cycloheximide treatment of cells overexpressing δ‐catenin showed enhanced β‐catenin turnover. These studies identify δ‐catenin as a new member of the GSK‐3β signaling pathway and further suggest that δ‐catenin is potentially involved in facilitating the interaction, ubiquitination, and subsequent turnover of β‐catenin in neuronal cells. © 2010 Wiley‐Liss, Inc.     

Application of feedback‐controlled bolus plus infusion (FC‐B/I) method for quantitative PET imaging of dopamine transporters with [18F]β‐CFT‐FE in conscious monkey brain

The competitive inhibition of dopamine transporters (DAT) with cocaine, a specific DAT inhibitor, was evaluated with a feedback‐controlled bolus plus infusion (FC‐B/I) method using animal positron emission tomography (PET) in the living brain of conscious monkey. 2β‐Carbomethoxy‐3β‐(4‐fluorophenyl)‐8‐(2‐[¹⁸F]fluoroethyl) nortropane ([¹⁸F]β‐CFT‐FE; Harada et al. [2004] Synapse 54:37–45) was used for this study because it provided specific, fast, and reversible kinetic properties to DAT in the striatum. In FC‐B/I method, the real‐time image reconstruction was started just after intravenous bolus injection of [¹⁸F]β‐CFT‐FE to generate a time‐activity curve in the striatum, and the infusion rate was adjusted to achieve an equilibrium state of the striatal radioactivity concentrations by means of a feedback‐control algorithm. The first equilibrium state in the brain was reached within 20 min after the infusion start. Intravenous administration of cocaine at the doses of 0.02, 0.1, and 0.5 mg/kg shifted the equilibrium radioactivity level to the second equilibrium state in a dose‐dependent manner, while no significant alterations was observed in the cerebellum. The present results demonstrated that the combined use of FC‐B/I method and PET probe with fast kinetics like [¹⁸F]β‐CFT‐FE could be useful to assess the occupancy of drugs in the living brain with PET. Synapse, 2013. © 2012 Wiley Periodicals, Inc.     

Kynurenic acid,by targeting α7 nicotinic acetylcholine receptors,modulates extracellular GABA levels in the rat striatum in vivo

Kynurenic acid (KYNA) is an astrocyte‐derived non‐competitive antagonist of the α7 nicotinic acetylcholine receptor (α7nAChR) and inhibits the NMDA receptor (NMDAR) competitively. The main aim of the present study was to examine the possible effects of KYNA (30 – 1000 nm ), applied locally by reverse dialysis for 2 h, on extracellular GABA levels in the rat striatum. KYNA concentration‐dependently reduced GABA levels, with 300 nm KYNA causing a maximal reduction to ~60% of baseline concentrations. The effect of KYNA (100 nm ) was prevented by co‐application of galantamine (5 μm ), an agonist at a site of the α7nAChR that is very similar to that targeted by KYNA. Infusion of 7‐chlorokynurenic acid (100 nm ), an NMDAR antagonist acting selectively at the glycine_B site of the receptor, affected neither basal GABA levels nor the KYNA‐induced reduction in GABA. Inhibition of endogenous KYNA formation by reverse dialysis of (S)‐4‐(ethylsulfonyl)benzoylalanine (ESBA; 1 mm ) increased extracellular GABA levels, reaching a peak of 156% of baseline levels after 1 h. Co‐infusion of 100 nm KYNA abolished the effect of ESBA. Qualitatively and quantitatively similar, bi‐directional effects of KYNA on extracellular glutamate were observed in the same microdialysis samples. Taken together, the present findings suggest that fluctuations in endogenous KYNA levels, by modulating α7nAChR function, control extracellular GABA levels in the rat striatum. This effect may be relevant for a number of physiological and pathological processes involving the basal ganglia.     

[18F]Nifene test–retest reproducibility in first‐in‐human imaging of α4β2* nicotinic acetylcholine receptors

The aim of this study was to examine the suitability of [¹⁸ F]nifene, a novel α4β2* nicotinic acetylcholine receptor (nAChR) radiotracer, for in vivo brain imaging in a first‐in‐human study. Methods : Eight healthy subjects (4 M,4 F;21–69,44 ± 21 yrs) underwent a [¹⁸F]nifene positron emission tomography scan (200 ± 3.7 MBq), and seven underwent a second scan within 58 ± 31 days. Regional estimates of DVR were measured using the multilinear reference tissue model (MRTM2) with the corpus callosum as reference region. DVR reproducibility was evaluated with test–retest variability (TRV) and intraclass correlation coefficient (ICC). Results : The DVR ranged from 1.3 to 2.5 across brain regions with a TRV of 0–7%, and did not demonstrate a systematic difference between test and retest. The ICCs ranged from 0.2 to 0.9. DVR estimates were stable after 40 min. Conclusion : The binding profile and tracer kinetics of [¹⁸F]nifene make it a promising α4β2* nAChR radiotracer for scientific research in humans, with reliable DVR test–retest reproducibility.     

Regulation of GSK‐3β by calpain in the 3‐nitropropionic acid model

Glycogen synthase kinase‐3β (GSK‐3β) is a crucial component in the cascade of events that culminate in a range of neurodegenerative diseases. It is controlled by several pathways, including calpain‐mediated cleavage. Calpain mediates in cell death induced by 3‐nitropropionic acid (3‐NP), but GSK‐3β regulation has not been demonstrated. Here we studied changes in total GSK‐3β protein levels and GSK‐3β phosphorylation at Ser‐9 in this model. The 3‐NP treatment induced GSK‐3β truncation. This regulation was dependent on calpain activation, since addition of calpeptin to the medium prevented this cleavage. While calpain inhibition prevented 3‐NP‐induced neuronal loss, inhibition of GSK‐3β by SB‐415286 did not. Furthermore, inhibition of cdk5, a known target of calpain involved in 3‐NP‐induced cell death, also failed to rescue neurons in our model. Our results point to a new target of calpain and indicate possible cross‐talk between calpain and GSK‐3β in the 3‐NP toxicity pathway. On the basis of our findings, we propose that calpain may modulate 3‐NP‐induced neuronal loss. © 2009 Wiley‐Liss, Inc.     

Analysis of β‐amyloid (Aβ) deposition in the temporal lobe in Alzheimer's disease using Fourier (spectral) analysis

R. A. Armstrong and N. J. Cairns (2010) Neuropathology and Applied Neurobiology 36, 248–257
Analysis of β‐amyloid (Aβ) deposition in the temporal lobe in Alzheimer's disease using Fourier (spectral) analysis Aim: To determine the spatial pattern of β‐amyloid (Aβ) deposition throughout the temporal lobe in Alzheimer's disease (AD). Methods: Sections of the complete temporal lobe from six cases of sporadic AD were immunolabelled with antibody against Aβ. Fourier (spectral) analysis was used to identify sinusoidal patterns in the fluctuation of Aβ deposition in a direction parallel to the pia mater or alveus. Results: Significant sinusoidal fluctuations in density were evident in 81/99 (82%) analyses. In 64% of analyses, two frequency components were present with density peaks of Aβ deposits repeating every 500–1000 µm and at distances greater than 1000 µm. In 25% of analyses, three or more frequency components were present. The estimated period or wavelength (number of sample units to complete one full cycle) of the first and second frequency components did not vary significantly between gyri of the temporal lobe, but there was evidence that the fluctuations of the classic deposits had longer periods than the diffuse and primitive deposits. Conclusions: (i) Aβ deposits exhibit complex sinusoidal fluctuations in density in the temporal lobe in AD; (ii) fluctuations in Aβ deposition may reflect the formation of Aβ deposits in relation to the modular and vascular structure of the cortex; and (iii) Fourier analysis may be a useful statistical method for studying the patterns of Aβ deposition both in AD and in transgenic models of disease.