Laurence A. Brown

Nicotinic Acetylcholine Receptor Signalling: Roles in Alzheimer's Disease and Amyloid Neuroprotection

Alzheimers disease (AD), the major contributor to dementia in the elderly, involves accumulation in the brain of extracellular plaques containing the β-amyloid protein (Aβ) and intracellular neurofibrillary tangles of hyperphosphorylated tau protein. AD is also characterized by a loss of neurons, particularly those expressing nicotinic acetylcholine receptors (nAChRs), thereby leading to a reduction in nAChR numbers. The Aβ1–42 protein, which is toxic to neurons, is critical to the onset and progression of AD. The discovery of new drug therapies for AD is likely to be accelerated by an improved understanding of the mechanisms whereby Aβ causes neuronal death. We examine the evidence for a role in Aβ1–42 toxicity of nAChRs; paradoxically, nAChRs can also protect neurons when activated by nicotinic ligands. Aβ peptides and nicotine differentially activate several intracellular signaling pathways, including the phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene homolog pathway, the extracellular signal-regulated kinase/mitogen-activated protein kinase, and JAK-2/STAT-3 pathways. These pathways control cell death or survival and the secretion of Aβ peptides. We propose that understanding the differential activation of these pathways by nicotine and/or Aβ1–42 may offer the prospect of new routes to therapy for AD.

Neonicotinoid insecticides display partial and super agonist actions on native insect nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) are present in high density in insect nervous tissue and are targeted by neonicotinoid insecticides. Improved understanding of the actions of these insecticides will assist in the development of new compounds. Here, we have used whole‐cell patch‐clamp recording of cholinergic neurons cultured from the central nervous system of 3rd instar Drosophila larvae to examine the actions of acetylcholine (ACh) and nicotine, as well as the neonicotinoids imidacloprid, clothianidin and P‐CH‐clothianidin on native nAChRs of these neurons. Dose–response data yield an EC50 value for ACh of 19 μm. Both nicotine and imidacloprid act as low efficacy agonists at native nAChRs, evoking maximal current amplitudes 10–14% of those observed for ACh. Conversely, clothianidin and P‐CH‐clothianidin evoke maximal current amplitudes up to 56% greater than those evoked by 100 μm ACh in the same neurons. This is the first demonstration of ‘super’ agonist actions of an insecticide on native insect nAChRs. Cell‐attached recordings indicate that super agonism results from more frequent openings at the largest (63.5 pS) conductance state observed.

The CRTC1-SIK1 Pathway Regulates Entrainment of the Circadian Clock

Summary Retinal photoreceptors entrain the circadian system to the solar day. This photic resetting involves cAMP response element binding protein (CREB)-mediated upregulation of Per genes within individual cells of the suprachiasmatic nuclei (SCN). Our detailed understanding of this pathway is poor, and it remains unclear why entrainment to a new time zone takes several days. By analyzing the light-regulated transcriptome of the SCN, we have identified a key role for salt inducible kinase 1 (SIK1) and CREB-regulated transcription coactivator 1 (CRTC1) in clock re-setting. An entrainment stimulus causes CRTC1 to coactivate CREB, inducing the expression of Per1 and Sik1. SIK1 then inhibits further shifts of the clock by phosphorylation and deactivation of CRTC1. Knockdown of Sik1 within the SCN results in increased behavioral phase shifts and rapid re-entrainment following experimental jet lag. Thus SIK1 provides negative feedback, acting to suppress the effects of light on the clock. This pathway provides a potential target for the regulation of circadian rhythms.

Neurovascular relationships in hippocampal slices: physiological and anatomical studies of mechanisms underlying flow-metabolism coupling in intraparenchymal microvessels.

Experiments were carried out to investigate the functional and anatomical relationships between neuronal elements and cerebral microvessels in 300-350-microm thick coronal hippocampal slices maintained at 33-35 degrees C, obtained from 150-200 g male Wistar rats. Cerebral arterioles (9-22 microm in diameter) were visualized in situ and pre-constricted by 22.0+/-6.6% by the addition of the thromboxane A2 agonist U46619 (75 nM), to the bathing medium. The glutamate agonist N-methyl-D-aspartate (0.01-1 mM) produced a dose-related increase in luminal diameter of pre-constricted vessels. In the presence of 4 microM haemoglobin to scavenge nitric oxide from the extravascular environment of the slice, the increase in diameter evoked by 0.1 mM N-methyl-D-aspartate was significantly reduced from 17.5+/-4.6% to 4.8+/-1.7% indicating that N-methyl-D-aspartate-induced vasodilatation of cerebral microvessels is mediated via a mechanism which involves neuronally-derived nitric oxide. In a parallel anatomical study, beta-nicotinamide adenine dinucleotide phosphate-dependent diaphorase staining was used to reveal the enzyme nitric oxide synthase in vascular endothelium and neurons in slices. A small subpopulation (< 11 cells per slice) of darkly-stained multipolar neurons, 21-32 microm in diameter was observed to give rise to a dense network of fine diaphorase-reactive nerve fibres that ramified throughout the whole of the hippocampus and appeared to come into close apposition with arterioles. Morphometric analysis of the relationship between cerebral microvessels, beta-nicotinamide adenine dinucleotide phosphate, reduced form-dependent diaphorase-reactive neuronal elements and individual pyramidal layer neurons, identified by filling with biocytin, revealed that for a given point on a pyramidal layer neuron, the proximity of the nearest diaphorase-reactive nerve fibre was less than 10 microm, whilst the distance to the nearest arteriole (the smallest functional unit for controlling blood flow) was in excess of 70 microm. Such a distance would probably preclude diffusion of vasoactive metabolites in effective concentrations from the area of increased neuronal activity. We therefore propose that the diaphorase-reactive nerve network constitutes the functional link. It is possible that during periods of increased neuronal activity, spillover of glutamate from synapses may activate the diaphorase-reactive network. Release of nitric oxide from the network in the vicinity of local cerebral arterioles may then produce relaxation of the vascular smooth muscle, enabling increased blood flow into the capillary network supplying the region of increased metabolic activity. This study has shown that the process whereby increases in neuronal activity elicit a local change in cerebral blood flow remains functionally intact in hippocampal slice preparations. Nitric oxide of neuronal origin appears to be involved in mediating the coupling between neurons and cerebral arterioles. Stereological analysis of the relationship between neuronal and vascular elements within hippocampal slices suggested that a small subpopulation of nitric oxide synthase-containing neurons which give rise to a diffuse network of fine nitric oxide synthase-containing nerve fibres that lie in close apposition to cerebral arterioles may provide the anatomical substrate for coupling of blood flow to metabolism.

The nicotinic acetylcholine receptors of the parasitic nematode Ascaris suum: formation of two distinct drug targets by varying the relative expression levels of two subunits.

Parasitic nematodes are of medical and veterinary importance, adversely affecting human health and animal welfare. Ascaris suum is a gastrointestinal parasite of pigs; in addition to its veterinary significance it is a good model of the human parasite Ascaris lumbricoides, estimated to infect ∼1.4 billion people globally. Anthelmintic drugs are essential to control nematode parasites, and nicotinic acetylcholine receptors (nAChRs) on nerve and muscle are the targets of cholinergic anthelmintics such as levamisole and pyrantel. Previous genetic analyses of nematode nAChRs have been confined to Caenorhabditis elegans, which is phylogenetically distinct from Ascaris spp. and many other important parasites. Here we report the cloning and expression of two nAChR subunit cDNAs from A. suum. The subunits are very similar in sequence to C. elegans UNC-29 and UNC-38, are expressed on muscle cells and can be expressed robustly in Xenopus oocytes to form acetylcholine-, nicotine-, levamisole- and pyrantel-sensitive channels. We also demonstrate that changing the stoichiometry of the receptor by injecting different ratios of the subunit cRNAs can reproduce two of the three pharmacological subtypes of nAChR present in A. suum muscle cells. When the ratio was 5∶1 (Asu-unc-38∶Asu-unc-29), nicotine was a full agonist and levamisole was a partial agonist, and oocytes responded to oxantel, but not pyrantel. At the reverse ratio (1∶5 Asu-unc-38∶Asu-unc-29), levamisole was a full agonist and nicotine was a partial agonist, and the oocytes responded to pyrantel, but not oxantel. These results represent the first in vitro expression of any parasitic nicotinic receptor and show that their properties are substantially different from those of C. elegans. The results also show that changing the expression level of a single receptor subunit dramatically altered the efficacy of some anthelmintic drugs. In vitro expression of these subunits may permit the development of parasite-specific screens for future anthelmintics.

Bio-imaging of nitric oxide-producing neurones in slices of rat brain using 4,5-diaminofluorescein

4,5-Diaminofluorescein (DAF-2) was used to identify individual nitric oxide (NO)-producing neurones in brain slices in vitro. Coronal slices of midbrain or hippocampus, 300 microm thick from young adult rats, were incubated for 30 min in 1 microM DAF-2 diacetate (DAF-2 DA) and maintained in ACSF at 33 degrees C. Illumination at 450-490 nm revealed punctate fluorescence in neurones in the lateral tegmental nucleus, dorsal raphe nucleus, dorsolateral periaqueductal grey matter, deep collicular layers and cortical areas. Neurones in the hippocampal pyramidal cell layer, molecular layer of the dentate gyrus and the hilus fluoresced also. The fluorescence was abolished by pre-incubation of slices with L-NAME (100 microM-1 mM), the inhibitor of constitutive nitric oxide synthase (NOS), but not by D-NAME (100 microM) or L-NIL (5-50 microM), an inhibitor of inducible NOS. In some superficially located arterioles, there were small regions of bright fluorescence close to the outer smooth muscle wall and diffuse fluorescence within the adjacent smooth muscle cells. A diffuse fluorescence was also seen in some superficially located capillaries. Basal production of NO was not seen within deeper blood vessels. DAF-2 DA offers a sensitive indicator for visualising basal production of NO with high spatial resolution and could provide a means of identifying NOS-containing neurones in brain slices in vitro prior to neurophysiological study.

Neuropathy target esterase: Immunolocalization to neuronal cell bodies and axons

Determination of the molecular mechanisms involved in organophosphate-induced axonopathy may help to elucidate those involved in normal axonal maintenance and in other neurodegenerative conditions. In this study we aimed to define the cellular distribution of neuropathy target esterase, the primary target protein for neuropathic organophosphates. A synthetic peptide corresponding to the sequence of a proteolytic fragment of neuropathy target esterase purified from chicken brain was used to raise a rabbit antiserum designated R28. The antiserum was shown by immunoprecipitation and western blotting of brain extracts to react with a polypeptide of the expected molecular size (155,000 mol. wt); this reaction was blocked by preincubating the antiserum with the immunizing peptide. Prominent intracellular immunostaining by R28 was seen in neuronal cell bodies and, in some cases, proximal axon segments in frozen sections of chicken brain cortex, optic tectum, cerebellum, spinal cord, and dorsal root ganglia. Cells with glial morphology were not immunostained, neither were normal sciatic nerve or motor end plates. However, 8-12 h following sciatic nerve ligation, immunoreactive material was seen to accumulate both proximal and, to a lesser extent, distal to the ligature, indicating that neuropathy target esterase undergoes fast axonal transport. No gross qualitative or quantitative changes in the above pattern of neuropathy target esterase immunoreactivity were detected in tissue obtained from chickens one or three days following treatment with a neuropathic organophosphate. The presence of neuropathy target esterase in essentially all neurons indicates that the selective vulnerability of long axons to neuropathic organophosphates is dependent on factors additional to the presence of the target protein.

Chronic Activation of γ2 AMPK Induces Obesity and Reduces β Cell Function

Summary Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.

Neuronal activity‐related coupling in cortical arterioles: involvement of astrocyte‐derived factors

Neuronal activity‐evoked dilatation was investigated in cortical arterioles in brain slices from mature rats maintained in vitro at 31–33°C. In the presence of the thromboxane A2 agonist U46619 (75 nm) to preconstrict vessels, internal diameter decreased by 14.2% and rhythmic contractile activity (vasomotion) developed. Addition of the epoxygenase inhibitor miconazole (20 μm) produced a further decrease in diameter and increase in the frequency of  vasomotion, suggesting that tonic release of epoxygenase products maintains a level of cerebrovascular dilator tone. Addition of 1 μm AMPA for 5 min evoked a 15.4 ± 3.7% increase in diameter and the frequency of vasomotion decreased by −6.7 ± 1.4 contractions min−1. The response persisted in the presence of 1 μm TTX, indicating that it was independent of neuronal activity and thus likely to have been evoked by activation of AMPA receptors on astrocytes rather than neurones. The response to the brief (5 min) application of AMPA remained unchanged in the presence of miconazole (20 μm). Prolonged (30 min) application of AMPA produced a +12.1 ± 1.5% increase in internal diameter and reduction in vasomotion (−8.4 ± 1.7 contractions min−1) that were sustained throughout the stimulation period. However, when AMPA was applied in the presence of miconazole (20 μm) it evoked only a transient increase in diameter (+9.8 ± 3.1%) and decrease in vasomotion (−6.6 ± 1.5 contractions min−1) that lasted for less than 10 min despite continued application of AMPA. The results suggest that products of epoxygenase activity, probably epoxyeicosatrienoic acids (EETs) are involved in activity‐related dilatation in cortical arterioles. Whilst epoxygenase activity is not required to initiate dilatation, it appears to be involved in sustaining the response. Thus EETs released from membrane stores could contribute to the initial stages, but once these have been depleted de novo synthesis of EETs is required to maintain the effect.

Fluorescent imaging of nitric oxide production in neuronal varicosities associated with intraparenchymal arterioles in rat hippocampal slices.

The fluorescent indicator 4,5-diaminofluorescein (DAF-2) has been used to investigate the production of nitric oxide in the vicinity of intraparenchymal cerebral blood vessels. Slices of rat hippocampus 300-350 microm thick, were loaded with 5 microM DAF-2 diacetate. On exposure to light of 450-490 nm wavelength, point sources of fluorescence, 1.8+/-0.2 microm in diameter (mean+/-SEM), were observed in close apposition to the outer surface of the vascular smooth muscle wall of 10/15 arterioles. In fixed slices, resectioned and processed for nicotinamide adenine dinucleotide phosphate-dependent diaphorase, stained varicose fibres were also seen in close association with the smooth muscle wall of small arterioles. These findings suggest that tonic activity in perivascular nitrergic nerve fibres lying in close proximity to intraparenchymal microvessels may be a source of dilator tone within the parenchyma.