Insulin Receptor Dysfunction Impairs Cellular Clearance of Neurotoxic Oligomeric A��

Accumulation of amyloid β (Aβ) oligomers in the brain is toxic to synapses and may play an important role in memory loss in Alzheimer disease. However, how these toxins are built up in the brain is not understood. In this study we investigate whether impairments of insulin and insulin-like growth factor-1 (IGF-1) receptors play a role in aggregation of Aβ. Using primary neuronal culture and immortal cell line models, we show that expression of normal insulin or IGF-1 receptors confers cells with abilities to reduce exogenously applied Aβ oligomers (also known as ADDLs) to monomers. In contrast, transfection of malfunctioning human insulin receptor mutants, identified originally from patient with insulin resistance syndrome, or inhibition of insulin and IGF-1 receptors via pharmacological reagents increases ADDL levels by exacerbating their aggregation. In healthy cells, activation of insulin and IGF-1 receptor reduces the extracellular ADDLs applied to cells via seemingly the insulin-degrading enzyme activity. Although insulin triggers ADDL internalization, IGF-1 appears to keep ADDLs on the cell surface. Nevertheless, both insulin and IGF-1 reduce ADDL binding, protect synapses from ADDL synaptotoxic effects, and prevent the ADDL-induced surface insulin receptor loss. Our results suggest that dysfunctions of brain insulin and IGF-1 receptors contribute to Aβ aggregation and subsequent synaptic loss.Abnormal protein misfolding and aggregation are common features in neurodegenerative diseases such as Alzheimer (AD),² Parkinson, Huntington, and prion diseases (1–3). In the AD brain, intracellular accumulation of hyperphosphorylated Tau aggregates and extracellular amyloid deposits comprise the two major pathological hallmarks of the disease (1, 4). Aβ aggregation has been shown to initiate from Aβ1–42, a peptide normally cleaved from the amyloid precursor protein (APP) via activities of α- and γ-secretases (5, 6). A large body of evidence in the past decade has indicated that accumulated soluble oligomers of Aβ1–42, likely the earliest or intermediate forms of Aβ deposition, are potently toxic to neurons. The toxic effects of Aβ oligomers include synaptic structural deterioration (7, 8) and functional deficits such as inhibition of synaptic transmission (9) and synaptic plasticity (10–13), as well as memory loss (11, 14, 15). Accumulation of high levels of these oligomers may also trigger inflammatory processes and oxidative stress in the brain probably due to activation of astrocytes and microglia (16, 17). Thus, to understand how a physiologically produced peptide becomes a misfolded toxin has been one of the key issues in uncovering the molecular pathogenesis of the disease.Aβ accumulation and aggregation could derive from overproduction or impaired clearance. Mutations of APP or presenilins 1 and 2, for example, are shown to cause overproduction of Aβ1–42 and amyloid deposits in the brain of early onset AD (18, 19). Because early onset AD accounts for less than 5% of entire AD population, APP and presenilin mutations cannot represent a universal mechanism for accumulation/aggregation of Aβ in the majority of AD cases. With respect to clearance, Aβ is normally removed by both global and local mechanisms, with the former requiring vascular transport across the blood-brain barrier (20, 21) and the latter via local enzymatic digestions by several metalloproteases, including neprilysin, insulin-degrading enzyme (IDE), and endothelin converting enzymes 1 and 2 (22–24).The fact that insulin is a common substrate for most of the identified Aβ-degrading enzymes has drawn attention of investigators to roles of insulin signaling in Aβ clearance. Increases in insulin levels frequently seen in insulin resistance may compete for these enzymes and thus contribute to Aβ accumulation. Indeed, insulin signaling has been shown to regulate expression of metalloproteases such as IDE (25, 26), and influence aspects of Aβ metabolism and catabolism (27). In the endothelium of the brain-blood barrier and glial cells, insulin signaling is reported to regulate protein-protein interactions in an uptake cascade involving low density lipoprotein receptor-related protein and its ligands ApoE and α2-macroglobulin, a system known to bind and clear Aβ via endocytosis and/or vascular transport (28, 29). Similarly, circulating IGF-1 has been reported to play a role in Aβ clearance probably via facilitating brain-blood barrier transportation (30, 31).In the brain, insulin signaling plays a role in learning and memory (32–34), potentially linking insulin resistance to AD dementia. Recently we and others have shown that Aβ oligomers interact with neuronal insulin receptors to cause impairments of the receptor expression and function (35–37). These impairments mimic the Aβ oligomer-induced synaptic long term potentiation inhibition and can be overcome by insulin treatment (35, 38). Consistently, impairments of both IR and IGF-1R have been reported in the AD brain (39–41).Based on these results, we ask whether impairment of insulin and IGF-1 signaling contribute to Aβ oligomer build-up in brain cells. To address this question, we set out to test roles of IR and IGF-1R in cellular clearance and transport of Aβ oligomers (ADDLs) applied to primary neuronal cultures and cell lines overexpressing IR and IGF-1R. Our results show that insulin and IGF-1 receptors function to reduce Aβ oligomers to monomers, and prevent Aβ oligomer-induced synaptic toxicity both at the level of synapse composition and structure. By contrast, receptor impairments resulting from “kinase-dead” insulin receptor mutations, a tyrosine kinase inhibitor of the insulin and IGF-1 receptor, or an inhibitory IGF-1 receptor antibody increase ADDL aggregation in the extracellular medium. Our results provide cellular evidence linking insulin and IGF-1 signaling to amyloidogenesis.     

Choline Promotes Nicotinic Receptor ��4 + ��2 Up-regulation

Neuronal nicotinic acetylcholine receptors (nAChR) composed of α4 + β2 subunits, the high affinity nicotine-binding site in the mammalian brain, up-regulate in response to chronic nicotine exposure. The identities of endogenous mediators of this process are unknown. We find that choline also up-regulates α4 + β2 nAChRs stably expressed by HEK293 cells as measured by increased [³H]epibatidine density. Choline-mediated up-regulation is dose-dependent and corresponds with an increase in β2 subunit protein expression. The choline kinase inhibitor hemicholinium-3 inhibits ∼60% of choline-mediated up-regulation revealing both an HC3-dependent and -independent pathway. Furthermore, choline-mediated up-regulation is not additive with up-regulation agents such as nicotine, but it is additive with weaker promoters of the up-regulation process. When co-applied with the pro-inflammatory cytokine tumor necrosis factor α, choline-mediated up-regulation is increased further through a mechanism that includes an increase in both α4 and β2 protein expression, and this is inhibited by the p38 MAPK inhibitor SB202190. These findings extend the view that up-regulation of α4 + β2 nAChRs is a normal physiological response to altered metabolic and inflammatory conditions.     

Signaling Cascade of Diacylglycerol Kinase �� in the Pituitary Intermediate Lobe: Dopamine D2 Receptor/Phospholipase C��4/Diacylglycerol Kinase ��/Protein Kinase C��

The pituitary gland dynamically changes its hormone output under various pathophysiological conditions. One of the pathways implicated in the regulatory mechanism of this gland is a dopaminergic system that operates the phosphoinositide (PI) cycle to transmit downstream signal through second messengers. We have previously shown that diacylglycerol kinase β (DGKβ) is coexpressed with dopamine D1 and D2 receptors in medium spiny neurons of the striatum, suggesting a plausible implication of DGKβ in dopaminergic transmission. However, it remains elusive whether DGKβ is involved in the dopaminergic system in the pituitary gland. The aim of this study is to investigate the expression and localization of DGK in the pituitary gland, together with the molecular components involved in the PI signaling cascade, including dopamine receptors, phospholipase C (PLC), and a major downstream molecule, protein kinase C (PKC). Here we show that DGKβ and the dopamine D2 receptor are coexpressed in the intermediate lobe and localize to the plasma membrane side by side. In addition, we reveal that PLCβ4 and PKCα are the subtypes expressed in the intermediate lobe among those families. These findings will substantiate and further extend our understanding of the molecular-anatomical pathway of PI signaling and the functional roles of DGK in the pituitary intermediate lobe. (J Histochem Cytochem 58:119–129, 2010)     

Functional Receptor Coupling to Gi Is A Mechanism of Agonist-Promoted Desensitization of the β2-Adrenergic Receptor

Abstract

The β₂-adrenergic receptor (β₂AR) couples to G_s, activating adenylyl cyclase (AC) and increasing cAMP. Such signaling undergoes desensitization with continued agonist exposure. β₂AR also couple to G_i after receptor phosphorylation by the cAMP dependent protein kinase A, but the efficiency of such coupling is not known. Given the PKA dependence of β₂AR-G_i coupling, we explored whether this may be a mechanism of agonist-promoted desensitization. HEK293 cells were transfected to express β₂AR or β₂AR and G_iα2, and then treated with vehicle or the agonist isoproterenol to evoke agonist-promoted β₂AR desensitization. Membrane AC activities showed that G_iα2 overexpression decreased basal levels, but the fold-stimulation of the AC over basal by agonist was not altered. However, with treatment of the cells with isoproterenol prior to membrane preparation, a marked decrease in agonist-stimulated AC was observed with the cells overexpressing G_iα2. in the absence of such overexpression, β₂AR desensitization was 23 ± 7%, while with 5-fold G_iα2 overexpression desensitization was 58 ± 5% (p<0.01, n=4). the effect of G_i on desensitization was receptor-specific, in that forskolin responses were not altered by G_iα2 overexpression. Thus, acquired β₂AR coupling to G_i is an important mechanism of agonist-promoted desensitization, and pathologic conditions that increase G_i levels contribute to β₂AR dysfunction.     

Scanning Mutagenesis of ��-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the ��9��10 Nicotinic Acetylcholine Receptor

Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the α9α10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp⁵–Arg⁷ and Asp¹¹–Ile¹⁵, because changes at these positions resulted in the loss of activity at the α9α10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K+N9A, all of which were more potent than Vc1.1 at both the rat α9α10 and the human α9/rat α10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the α3β2, α3β4, and α7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the α9α10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the α9α10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs.Marine snails belonging to the Conus genus produce a variety of neurotoxic peptides in their venom glands that they use for the capture of prey (1–3). Within this repertoire of conopeptides, those that are disulfide-rich are referred to as conotoxins. Conotoxins typically range in size from 12 to 30 amino acids, contain 4 or more Cys residues, and exhibit high potency and selectivity toward a variety of membrane receptors and ion channels (4, 5). The α-conotoxin subfamily members typically range in size from 12 to 19 amino acids, contain 2 disulfide bonds in a Cys^I–Cys^III and Cys^II–Cys^IV connectivity, and have an amidated C terminus, as depicted in Fig. 1. They interact with nicotinic acetylcholine receptors (nAChRs),⁴ of both the muscle and the neuronal type, which have been implicated in a range of neurological disorders varying from Alzheimer disease to addiction (6–8).Open in a separate windowFIGURE 1.α-Conotoxin sequences and structure of Vc1.1. a, the sequences of selected α-conotoxins relevant to this study are shown by one-letter amino acid codes. The asterisk indicates an amidated C terminus, which is a common post-translational modification found in α-conotoxins. The conserved cysteine residues are highlighted in yellow, and the Cys^I–Cys^III and Cys^II–Cys^IV disulfide connectivity is indicated by the connecting lines under the sequence. The number of residues between the cysteines define two backbone “loops,” which are used to classify α-conotoxins into subclasses. For example, RgIA has four residues in loop 1 and three residues in loop 2, making this a 4/3 loop subclass α-conotoxin. b, structural representation of Vc1.1 (PDB 2H8S), with disulfide bonds depicted in yellow. The cysteines, the loops, and the termini are labeled.The nAChRs are ligand-gated ion channels that respond to ACh, nicotine, and other competitive agonists/antagonists. They are composed of five subunits, with differing nAChR subunit composition according to the site of expression. The muscle-type nAChRs are composed of two α subunits, a β and δ subunit, and either an ϵ or a γ subunit (9–12). The neuronal forms exist either as homomeric channels composed of α subunits alone or αβ heteromeric channels. The wide variety of possible subunit combinations has led to unique subtypes with distinct pharmacological properties. This makes α-conotoxins valuable neuropharmacological tools and drug leads, because they have the ability to distinguish between different nAChR subtypes. Effectively, they are small rigid scaffolds that display amino acids on their surface to selectively target their receptors (13).Of particular interest in this study is the α-conotoxin Vc1.1, a synthetic derivative of a naturally occurring peptide from the venom of the marine cone snail, Conus victoriae. It was discovered using PCR screening of cDNA extracted from the snail venom duct (14). Fig. 1 depicts the sequences of selected α-conotoxins, including Vc1.1, which is 16 amino acids in length and displays the classic disulfide bond connectivity observed for α-conotoxins, together with a short helical segment as depicted in Fig. 1b. The conserved Cys framework of α-conotoxins defines two backbone loops, which vary in size and residue composition, and are classified by an n/m nomenclature to define subclasses of α-conotoxins. For example, Vc1.1 is a 4/7 subclass α-conotoxin, because it contains four residues in loop 1 and seven in loop 2. RgIA (15–17) is another conotoxin of interest in this study, because it is also selective for the α9α10 nAChR subtype, and has a 4/3 framework. Vc1.1 contains an amidated C terminus, a post-translational modification common to most α-conotoxins, but it is not present in RgIA. Vc1.1 lacks the post-translationally modified hydroxyproline and γ-carboxyglutamate residues present in the native peptide, vc1a, isolated from the venom duct of C. victoriae (18).Vc1.1 has been under development as a drug lead for neuropathic pain (19). When tested in rat models of neuropathic pain, Vc1.1 induced analgesia when injected intramuscularly near the site of injury (20). Initially, it was thought that α3-containing subtypes of nAChRs may be the target for Vc1.1 (21); however, it was then reported that Vc1.1 has a 100-fold higher affinity at the α9α10 nAChR subtype (22, 23). The α9α10 nAChR mediates synaptic transmission between efferent olivocochlear fibers and cochlear hair cells (24–26). The mRNA of these receptor subtypes is expressed in many different tissue types from the inner ear, dorsal root ganglion (27), skin keratinocytes (28), and lymphocytes (29) to the pituitary (26). The α10 subunit has to be expressed with the α9 subunit to form a functional receptor. In the auditory system, the α9α10 nAChR plays an important role in hair cell development, but its role in other tissues is yet to be characterized (22, 26, 30, 31).Owing to the promising antinociceptive effects of Vc1.1 in animals, its analogs are of interest as leads for the treatment of neuropathic pain (14, 20). To date, studies have predominantly focused on the α9α10 nAChR, but the very recent finding that Vc1.1 also targets the γ-aminobutyric acid, type B receptor (32) has raised interest in the molecular mode of action of Vc1.1 in analgesia. Hence there is a need to define structure-activity relationships of this peptide at several targets, including human and rat forms of the α9α10 nAChR. In particular, we were interested in analogs that maintain potency at the rat α9α10 nAChR but also show significant improvement in potency at human forms of the receptor, while maintaining selectivity over other nAChR subtypes.In this study we determined such structure-activity relationships for Vc1.1 at the α9α10 nAChR by successively mutating each non-Cys residue of Vc1.1 to either an “inert” residue (Ala), a negatively charged residue (Asp), or a positively charged residue (Lys) and observing the impact on the structure and functional activity of Vc1.1. Once the key residues had been identified, a second generation of analogs with new substitutions was synthesized and tested at the rat α9α10 nAChR. The analogs were also analyzed at the human α9/rat α10 (hα9rα10) hybrid clone, because a recent report⁵ suggested differences in the activity of Vc1.1 at the human and rat clones of the α9α10 nAChR. We also examined the effect of pH change on the structure of Vc1.1 using NMR αH chemical shift analysis. The results from this study provide valuable insight into the key residues involved in the interaction of Vc1.1 with the α9α10 nAChR subtype and have the potential to assist in the development of conotoxin analogs as drug leads for the treatment of neuropathic pain (4, 33).