Inhibition of neuroinflammatory nitric oxide signaling suppresses glycation and prevents neuronal dysfunction in mouse prion disease.

Several neurodegenerative diseases associated with protein misfolding (Alzheimer's and Parkinson's disease) exhibit oxidative and nitrergic stress following initiation of neuroinflammatory pathways. Associated nitric oxide (NO)-mediated posttranslational modifications impact upon protein functions that can exacerbate pathology. Nonenzymatic and irreversible glycation signaling has been implicated as an underlying pathway that promotes protein misfolding, but the direct interactions between both pathways are poorly understood. Here we investigated the therapeutic potential of pharmacologically suppressing neuroinflammatory NO signaling during early disease progression of prion-infected mice. Mice were injected daily with an NO synthase (NOS) inhibitor at early disease stages, hippocampal gene and protein expression levels of oxidative and nitrergic stress markers were analyzed, and electrophysiological characterization of pyramidal CA1 neurons was performed. Increased neuroinflammatory signaling was observed in mice between 6 and 10 wk postinoculation (w.p.i.) with scrapie prion protein. Their hippocampi were characterized by enhanced nitrergic stress associated with a decline in neuronal function by 9 w.p.i. Daily in vivo administration of the NOS inhibitor L-NAME between 6 and 9 w.p.i. at 20 mg/kg prevented the functional degeneration of hippocampal neurons in prion-diseased mice. We further found that this intervention in diseased mice reduced 3-nitrotyrosination of triose-phosphate isomerase, an enzyme involved in the formation of disease-associated glycation. Furthermore, L-NAME application led to a reduced expression of the receptor for advanced glycation end-products and the diminished accumulation of hippocampal prion misfolding. Our data suggest that suppressing neuroinflammatory NO signaling slows functional neurodegeneration and reduces nitrergic and glycation-associated cellular stress.

Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability.

Nitric oxide (NO) regulates neuronal function and thus is critical for tuning neuronal communication. Mechanisms by which NO modulates protein function and interaction include posttranslational modifications (PTMs) such as S-nitrosylation. Importantly, cross signaling between S-nitrosylation and prenylation can have major regulatory potential. However, the exact protein targets and resulting changes in function remain elusive. Here, we interrogated the role of NO-dependent PTMs and farnesylation in synaptic transmission. We found that NO compromises synaptic function at the Drosophila neuromuscular junction (NMJ) in a cGMP-independent manner. NO suppressed release and reduced the size of available vesicle pools, which was reversed by glutathione (GSH) and occluded by genetic up-regulation of GSH-generating and de-nitrosylating glutamate-cysteine-ligase and S-nitroso-glutathione reductase activities. Enhanced nitrergic activity led to S-nitrosylation of the fusion-clamp protein complexin (cpx) and altered its membrane association and interactions with active zone (AZ) and soluble N-ethyl-maleimide-sensitive fusion protein Attachment Protein Receptor (SNARE) proteins. Furthermore, genetic and pharmacological suppression of farnesylation and a nitrosylation mimetic mutant of cpx induced identical physiological and localization phenotypes as caused by NO. Together, our data provide evidence for a novel physiological nitrergic molecular switch involving S-nitrosylation, which reversibly suppresses farnesylation and thereby enhances the net-clamping function of cpx. These data illustrate a new mechanistic signaling pathway by which regulation of farnesylation can fine-tune synaptic release.

Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction.

G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR). By using a genetically engineered mouse expressing a G protein-biased M3-mAChR mutant, we reveal the first evidence, to our knowledge, of a role for M3-mAChR phosphorylation in bronchial smooth muscle contraction in health and in a disease state with relevance to human asthma. Furthermore, this mouse model can be used to distinguish the physiological responses that are regulated by M3-mAChR phosphorylation (which include control of lung function) from those responses that are downstream of G protein signaling. In this way, we present an approach by which to predict the physiological/therapeutic outcome of M3-mAChR-biased ligands with important implications for drug discovery.

An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory.

Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser(228)) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser(228) These data supported the hypothesis that phosphorylation at Ser(228) was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser(228) on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser(228) phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser(228) not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning.

Neuropsin cleaves EphB2 in the amygdala to control anxiety.

A minority of individuals experiencing traumatic events develop anxiety disorders. The reason for the lack of correspondence between the prevalence of exposure to psychological trauma and the development of anxiety is unknown. Extracellular proteolysis contributes to fear-associated responses by facilitating neuronal plasticity at the neuron-matrix interface. Here we show in mice that the serine protease neuropsin is critical for stress-related plasticity in the amygdala by regulating the dynamics of the EphB2-NMDA-receptor interaction, the expression of Fkbp5 and anxiety-like behaviour. Stress results in neuropsin-dependent cleavage of EphB2 in the amygdala causing dissociation of EphB2 from the NR1 subunit of the NMDA receptor and promoting membrane turnover of EphB2 receptors. Dynamic EphB2-NR1 interaction enhances NMDA receptor current, induces Fkbp5 gene expression and enhances behavioural signatures of anxiety. On stress, neuropsin-deficient mice do not show EphB2 cleavage and its dissociation from NR1 resulting in a static EphB2-NR1 interaction, attenuated induction of the Fkbp5 gene and low anxiety. The behavioural response to stress can be restored by intra-amygdala injection of neuropsin into neuropsin-deficient mice and disrupted by the injection of either anti-EphB2 antibodies or silencing the Fkbp5 gene in the amygdala of wild-type mice. Our findings establish a novel neuronal pathway linking stress-induced proteolysis of EphB2 in the amygdala to anxiety.

The M3-muscarinic receptor regulates learning and memory in a receptor phosphorylation/arrestin-dependent manner.

Degeneration of the cholinergic system is considered to be the underlying pathology that results in the cognitive deficit in Alzheimer's disease. This pathology is thought to be linked to a loss of signaling through the cholinergic M(1)-muscarinic receptor subtype. However, recent studies have cast doubt on whether this is the primary receptor mediating cholinergic-hippocampal learning and memory. The current study offers an alternative mechanism involving the M(3)-muscarinic receptor that is expressed in numerous brain regions including the hippocampus. We demonstrate here that M(3)-muscarinic receptor knockout mice show a deficit in fear conditioning learning and memory. The mechanism used by the M(3)-muscarinic receptor in this process involves receptor phosphorylation because a knockin mouse strain expressing a phosphorylation-deficient receptor mutant also shows a deficit in fear conditioning. Consistent with a role for receptor phosphorylation, we demonstrate that the M(3)-muscarinic receptor is phosphorylated in the hippocampus following agonist treatment and following fear conditioning training. Importantly, the phosphorylation-deficient M(3)-muscarinic receptor was coupled normally to G(q/11)-signaling but was uncoupled from phosphorylation-dependent processes such as receptor internalization and arrestin recruitment. It can, therefore, be concluded that M(3)-muscarinic receptor-dependent learning and memory depends, at least in part, on receptor phosphorylation/arrestin signaling. This study opens the potential for biased M(3)-muscarinic receptor ligands that direct phosphorylation/arrestin-dependent (non-G protein) signaling as being beneficial in cognitive disorders.

Redox stress and metal dys-homeostasis appear as hallmarks of early prion disease pathogenesis in mice.

Neurodegenerative diseases are associated with a multitude of dysfunctional cellular pathways. One major contributory factor is a redox stress challenge during the development of several protein misfolding conditions including Alzheimer's (AD), Parkinson's disease (PD), and less common conditions such as Creutzfeldt Jakob disease (CJD). CJD is caused by misfolding of the neuronal prion protein and is characterised by a neurotoxic unfolded protein response involving chronic endoplasmic reticulum stress, reduced protein translation and spongiosis leading subsequently to synaptic and neuronal loss. Here we have characterised prion disease in mice to assess redox stress components including nitrergic and oxidative markers associated with neuroinflammatory activation. Aberrant regulation of the homeostasis of several redox metals contributes to the overall cellular redox stress and we have identified altered levels of iron, copper, zinc, and manganese in the hippocampus of prion diseased mice. Our data show that early in disease, there is evidence for oxidative stress in conjunction with reduced antioxidant superoxide dismutase mRNA and protein expression. Moreover, expression of divalent metal transporter proteins was reduced for Atp7b, Atox1, Slc11a2, Slc39a14 at 6-7 weeks but increased for Slc39a14 and Mt1 at 10 weeks of disease. Our data present evidence for a strong pro-oxidant environment and altered redox metal homeostasis in early disease pathology which both may be contributory factors to aggravating this protein misfolding disease.

The role of cytokines in modulating learning and memory and brain plasticity.

Cytokines are proteins secreted in the central nervous system by neurons, microglia, astrocytes and infiltrating peripheral immune cells under physiological and pathological conditions. Over the last 20 years, a growing number of reports have investigated the effects of these molecules on brain plasticity. In this review, we describe how the key cytokines interleukin 1ß, interleukin 6 and tumour necrosis factor α were found to support long-term plasticity and learning and memory processes in physiological conditions. In contrast, during inflammation where cytokines levels are elevated such as in models of brain injury or infection, depression or neurodegeneration, the effects of cytokines are mostly detrimental to memory mechanisms, associated behaviours and homeostatic plasticity.

The metabolome identity: basis for discovery of biomarkers in neurodegeneration.

Neurodegenerative disorders are often associated with cellular dysfunction caused by underlying protein-misfolding signalling. Numerous neuropathologies are diagnosed at late stage symptomatic changes which occur in response to these molecular malfunctions and treatment is often too late or restricted only to the slowing of further cell death. Important new strategies to identify early biomarkers with predictive value to intervene with disease progression at stages where cell dysfunction has not progressed irreversibly is of paramount importance. Thus, the identification of these markers presents an essential opportunity to identify and target disease pathways. This review highlights some important metabolic alterations detected in neurodegeneration caused by misfolded prion protein and discusses common toxicity pathways identified across different neurodegenerative diseases. Thus, having established some commonalities between various degenerative conditions, detectable metabolic changes may be of extreme value as an early diagnostic biomarker in disease.

Dysregulation of stress systems and nitric oxide signaling underlies neuronal dysfunction in Alzheimer's disease.

Stress is a multimodal response involving the coordination of numerous body systems in order to maximize the chance of survival. However, long term activation of the stress response results in neuronal oxidative stress via reactive oxygen and nitrogen species generation, contributing to the development of depression. Stress-induced depression shares a high comorbidity with other neurological conditions including Alzheimer's disease (AD) and dementia, often appearing as one of the earliest observable symptoms in these diseases. Furthermore, stress and/or depression appear to exacerbate cognitive impairment in the context of AD associated with dysfunctional catecholaminergic signaling. Given there are a number of homologous pathways involved in the pathophysiology of depression and AD, this article will highlight the mechanisms by which stress-induced perturbations in oxidative stress, and particularly NO signaling, contribute to neurodegeneration.

Alterations in neuronal metabolism contribute to the pathogenesis of prion disease.

Neurodegenerative conditions are characterised by a progressive loss of neurons, which is believed to be initiated by misfolded protein aggregations. During this time period, many physiological and metabolomic alterations and changes in gene expression contribute to the decline in neuronal function. However, these pathological effects have not been fully characterised. In this study, we utilised a metabolomic approach to investigate the metabolic changes occurring in the hippocampus and cortex of mice infected with misfolded prion protein. In order to identify these changes, the samples were analysed by ultrahigh-performance liquid chromatography-tandem mass spectroscopy. The present dataset comprises a total of 498 compounds of known identity, named biochemicals, which have undergone principal component analysis and supervised machine learning. The results generated are consistent with the prion-inoculated mice having significantly altered metabolic profiles. In particular, we highlight the alterations associated with the metabolism of glucose, neuropeptides, fatty acids, L-arginine/nitric oxide and prostaglandins, all of which undergo significant changes during the disease. These data provide possibilities for future studies targeting and investigating specific pathways to better understand the processes involved in neuronal dysfunction in neurodegenerative diseases.

M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss.

The current frontline symptomatic treatment for Alzheimer's disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR-selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.