High- and low-sensitivity subforms of α4β2 and α3β2 nAChRs

Previous studies in other laboratories have shown that alpha4beta2 nicotinic acetylcholine receptor (nAChR) exhibits a biphasic concentration-response relationship for ACh with low and high EC50 components, and that the low EC50 component can be augmented by decreasing the alpha4:beta2 message ratio or incubating overnight in nicotine or at low temperature (Zwart and Vijverberg, 1998; Covernton and Connolly, 2000; Buisson and Bertrand, 2001; Nelson et al., 2003; Zhou et al., 2003). In the process of cloning ferret nAChR subunits, we found alpha4 and beta2 messages with long untranslated regions (UTRs), as well as those with no UTRs. Combinations of these messages revealed that the presence of UTRs influenced the ability to exclusively express high-sensitivity subforms of alpha4beta2 and alpha3beta2 nAChRs. Injection of oocytes with alpha4 and beta2 RNAs lacking UTRs (1:1 ratio) led to expression of a biphasic concentration-response relationship for ACh with EC50 values of 0.5 (high sensitivity) and 114 microM(low sensitivity). Decreasing the alpha4:beta2 message ratio to as much as 1:120 increased the high-sensitivity component slightly, but the ACh concentration response remained biphasic. In contrast, injection of messages with UTRs (1:1 ratio) led to expression of a monophasic concentration response to ACh and a high-sensitivity EC50 value of 2.3 microM, as shown in Fig. 1.     

Heparin modulates the conformation and signaling of platelet integrin αIIbβ3

Introduction

The glycosaminoglycan heparin has been shown to bind to platelet integrin αIIbβ3 and induce platelet activation and aggregation, although the relationship between binding and activation is unclear. We analyzed the interaction of heparin and αIIbβ3 in detail, to obtain a better understanding of the mechanism by which heparin acts on platelets.

Methods

We assessed conformational changes in αIIbβ3 by flow cytometry of platelets exposed to unfractionated heparin. In human platelets and K562 cells engineered to express αIIbβ3, we assayed the effect of heparin on key steps in integrin signaling: phosphorylation of the β3 chain cytoplasmic tail, and activation of src kinase. We measured the heparin binding affinity of purified αIIbβ3, and of recombinant fragments of αIIb and β3, by surface plasmon resonance.

Results and conclusions

Heparin binding results in conformational changes in αIIbβ3, similar to those observed upon ligand binding. Heparin binding alone is not sufficient to induce tyrosine phosphorylation of the integrin β3 cytoplasmic domain, but the presence of heparin increased both β3 phosphorylation and src kinase activation in response to ligand binding. Specific recombinant fragments derived from αIIb bound heparin, while recombinant β3 did not bind. This pattern of heparin binding, compared to the crystal structure of αIIbβ3, suggests that heparin-binding sites are located in clusters of basic amino acids in the headpiece and/or leg domains of αIIb. Binding of heparin to these clusters may stabilize the transition of αIIbβ3 to an open conformation with enhanced affinity for ligand, facilitating outside-in signaling and platelet activation.     

Phosphorylation and function of α4β2 receptor

The neuronal nicotinic acetylcholine receptor (nAChR) alpha4 and beta2 subunits expressed in heterologous expression systems assemble into high- and low-affinity receptors (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001; Nelson et al., 2003), which reflects the assembly of two distinct subunit stoichiometries of alpha4beta2 receptor (Nelson et al., 2003). The high-affinity receptor ([alpha4]2[beta2]3) is about 100-fold more sensitive to ACh than the low-affinity receptor ([alpha4]3[beta2]2) (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001; Nelson et al., 2003). Recent evidence implicated 14-3-3 proteins as modulators of the relative abundance of nAChR subunits in the endoplasmic reticulum (ER), where ligand-gated ion channels assemble. The 14-3-3 proteins influence ER-to-plasma membrane trafficking of multimeric cell-surface proteins (O'Kelly et al., 2002). 14-3-3 proteins bind components of these multimeric proteins, and this interaction overrides dibasic COP1 retention signal to permit forward transport of the protein (O'Kelly et al., 2002). In the case of alpha4beta2 nAChRs, 14-3-3 binds the alpha4 subunit, and this association is dependent on phosphorylation of a serine residue within a protein kinase A(PKA) consensus sequence in the large cytoplasmic domain of the alpha4 subunit, which is also a binding motif recognized by 14-3-3 (Jeancloss et al., 2001; O'Kelly et al., 2002). The interplay among PKA, alpha4 subunits, and 14-3-3 proteins increases cell-surface expression of alpha4beta2 nAChRs by increasing steady-state levels of the alpha4 subunit available for assembly with beta2 subunits (Jeancloss et al., 2001). Because it is not known how 14-3-3-dependent changes in the steady-state levels of the alpha4 subunit might affect the functional type of alpha4beta2 receptors, we have investigated the effects of mutations of the 14-3-3 binding motif in the alpha4 subunit on alpha4beta2 nAChR function.     

Activation and modulation of human α4β2 nicotinic acetylcholine receptors by the neonicotinoids clothianidin and imidacloprid

Neonicotinoids are synthetic, nicotine-derived insecticides used for agricultural and household pest control. Though highly effective at activating insect nicotinic receptors, many neonicotinoids are also capable of directly activating and/or modulating the activation of vertebrate nicotinic receptors. In this study, we have investigated the actions of the neonicotinoids clothianidin (CTD) and imidacloprid (IMI) on human neuronal α4β2 nicotinic acetylcholine receptors. The data demonstrate that the compounds are weak agonists of the human receptors with relative peak currents of 1-4% of the response to 1 mM acetylcholine (ACh). Coapplication of IMI strongly inhibited currents elicited by ACh. From Schild plot analysis, we estimate that the affinity of IMI for the human α4β2 receptor is 18 μM. The application of low concentrations of CTD potentiated responses to low concentrations of ACh, suggesting that receptors occupied by one ACh and one CTD molecule have a higher gating efficacy than receptors with one ACh bound. Interestingly, subunit stoichiometry affected inhibition by CTD, with (α4)(2) (β2)(3) receptors significantly more strongly inhibited than the (α4)(3) (β2)(2) receptors.     

A novel family of RGD-containing disintegrins (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets αIIbβ3 or αVβ3 and inhibits platelet aggregation and angiogenesis

A novel family of RGD-containing molecules (Tablysin-15) has been molecularly characterised from the salivary gland of the haematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity binding for platelet αIIbβ3 and endothelial cell αVβ3 integrins, but not for α5β1 or α2β1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~1 nM) and marginally to fibronectin (IC50 ~1 μM), but not to collagen. It also inhibits fibroblast growth factor (FGF)-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilised fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that αIIbβ3 binds to Tablysin-15. Moreover, immobilised Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin αIIbβ3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block αIIbβ3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.     

Ethanol consumption produces changes in behavior and on hippocampal α7 and α4β2 nicotinic receptors

Ethanol consumption produces a wide range of effects on the central nervous system, most of them related to changes in neural receptors. In vitro studies have demonstrated that ethanol increases neural nicotinic acetylcholine receptor (nnAChR) affinity for ACh (Narahashi et al., 1991) and have also reported differences in sensitivity of nnAChRs for ethanol, depending on the subunit composition of the receptor (Cardoso et al., 1999). There is evidence that ethanol induces changes in density of nnAChRs in cellular cultures (Gorbounova et al., 1998). However, there are no clear results concerning the effects of chronic ethanol on nicotinic receptors and on behavior in rats nonselected by their preference to ethanol. A number of studies demonstrate that nnAChRs participate in a variety of functions, including memory and learning processes, neurodegeneration, and neuroprotection (Picciotto et al., 2000). In the present work, we found significant alterations in the Fixed-Interval Behavioral Test, as well as in density and affinity parameters of hippocampal main subtypes of nnAChRs: alpha7 homopentamers and alpha4beta2 heteropentamers of ethanol-drinking rats nonselected by their preference to ethanol.     

Blocking α4β2 and α7 nicotinic acetylcholine receptors inhibits the reinstatement of morphine-induced CPP by drug priming in mice

Investigating the interaction between nicotinic and opioid receptors is of great interest for both basic mechanistic and clinical reasons. Morphine and nicotine, two common drugs of abuse, share several behavioral and rewarding properties. However, little is known about the subtypes of nicotinic acetylcholine receptors (nAChR) in the reinstatement of morphine-induced conditioned place preference (CPP). In this study, we found that a non-specific nAChR agonist, nicotine (0.5mg/kg), had no effects on the reinstatement of morphine-induced CPP. However, we found that pretreatment with specific α(4)β(2) and α(7) nAChR subtype antagonists, dihydroxy-β-erithroidine (DHβE, 5mg/kg) and methyllycaconitine (MLA, 4 mg/kg), 20 min prior to administration of morphine, inhibited the reinstatement of morphine-induced CPP by drug priming in mice. Furthermore, depression of the reinstatement of morphine-induced CPP by a single DHβE or MLA treatment lasted at least three days later when the reinstatement was induced by morphine priming. The data suggest that specific nAChR subtypes, i.e., α(4)β(2) and α(7), may contribute to the reinstatement of morphine-induced CPP by drug priming in mice.     

Disulfide bond exchanges in integrins αIIbβ3 and αvβ3 are required for activation and post-ligation signaling during clot retraction

Background

Integrin αIIbβ3 mediates platelet adhesion, aggregation and fibrin clot retraction. These processes require activation of αIIbβ3 and post-ligation signaling. Disulfide bond exchanges are involved in αIIbβ3 and αvβ3 activation.

Methods

In order to investigate the role of integrin activation and disulfide bond exchange during αIIbβ3- and αvβ3-mediated clot retraction, we co-expressed in baby hamster kidney cells wild-type (WT) human αIIb and WT or mutated human β3 that contain single or double cysteine substitutions disrupting C523-C544 or C560-C583 bonds. Flow cytometry was used to measure surface expression and activation state of the integrins. Time-course of fibrin clot retraction was examined.

Results

Cells expressed WT or mutated human αIIbβ3 as well as chimeric hamster/human αvβ3. The αIIbβ3 mutants were constitutively active and the thiol blocker dithiobisnitrobenzoic acid (DTNB) did not affect their activation state. WT cells retracted the clot and addition of αvβ3 inhibitors decreased the retraction rate. The active mutants and WT cells activated by anti-LIBS6 antibody retracted the clot faster than untreated WT cells, particularly in the presence of αvβ3 inhibitor. DTNB substantially inhibited clot retraction by WT or double C523S/C544S mutant expressing cells, but minimally affected single C523S, C544S or C560S mutants. Anti-LIBS6-enhanced clot retraction was significantly inhibited by DTNB when added prior to anti-LIBS6.

Conclusions

Both αIIbβ3 and αvβ3 contribute to clot retraction without prior activation of the integrins. Activation of αIIbβ3, but not of αvβ3 enhances clot retraction. Both αIIbβ3 activation and post-ligation signaling during clot retraction require disulfide bond exchange.     

Immunotherapeutic Approaches Targeting Amyloid-β, α-Synuclein,and Tau for the Treatment of Neurodegenerative Disorders

Disease-modifying alternatives are sorely needed for the treatment of neurodegenerative disorders, a group of diseases that afflict approximately 50 million Americans annually. Immunotherapy is one of the most developed approaches in this direction. Vaccination against amyloid-β, α-synuclein, or tau has been extensively explored, specially as the discovery that these proteins may propagate cell-to-cell and be accessible to antibodies when embedded into the plasma membrane or in the extracellular space. Likewise, the use of passive immunization approaches with specific antibodies against abnormal conformations of these proteins has also yielded promising results. The clinical development of immunotherapies for Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, dementia with Lewy bodies, and other neurodegenerative disorders is a field in constant evolution. Results to date suggest that immunotherapy is a promising therapeutic approach for neurodegenerative diseases that progress with the accumulation and prion-like propagation of toxic protein aggregates. Here we provide an overview of the most novel and relevant immunotherapeutic advances targeting amyloid-β in Alzheimer’s disease, α-synuclein in Alzheimer’s disease and Parkinson’s disease, and tau in Alzheimer’s disease and frontotemporal dementia.     

Involvement of postsynaptic α1- and α2-adrenoceptors in the flexor reflex activity in the spinal Rats

Summary Clonidine (0.2 mg/kg i.v.) and St 587 (1 mg/kg i.v.), selective agonsists of ₂- and ₁-adrenoceptors, respectively, increased the flexor reflex amplitude in the spinal rat. Yohimbine and rauwolscine, ₂-adrenoceptor antagonists, inhibited dose-dependently the effect of clonidine but not of St 587. However, prazosin and clozapine, ₁-adrenoceptor antagonists, diminished the action of both agonists in a dose-dependent manner. After central chemosympathectomy caused by 6-OH-DA or DSP-4 the response to clonidine did not differ from that in the sham-operated animals. In 6-OH-DA treated rats yohimbine (1 mg/kg i.v.) antagonized the effect of clonidine to the same extent as it did in unlesioned animals. It is concluded that the results are functional evidence that ₁- and ₂-adrenoceptors are present in the rat spinal cord, and stimulation of each receptor increases flexor reflex activity.     

Iron and α-synuclein in the substantia nigra of MPTP-treated mice

One of the prominent pathological features of Parkinson's disease (PD) is the abnormal accumulation of iron in the substantia nigra pars compacta (SNpc), in the reactive microglia, and in association with neuromelanin, within the melanin-containing dopamine (DA) neurons. Lewy body, the morphological hallmark of PD, is composed of lipids, redox-active iron, and aggregated alpha-synuclein, concentrating in its peripheral halo and ubiquitinated, hyperphosphorylated, neurofilament proteins. The capacity of free iron to enhance and promote the generation of toxic reactive oxygen radicals has been discussed numerous times. Recent observations, that iron induces aggregation of inert alpha-synuclein to toxic aggregates, have reinforced the critical role of iron in oxidative stress-induced pathogenesis of DA neuron degeneration and protein degradation via ubiquitination. N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and 6-hydroxydopamine-induced neurodegeneration in rodents and nonhuman primates is associated with increased presence of iron and alpha-synuclein in the SNpc. The accumulation of iron in MPTP-induced neurodegeneration has been linked to nitric oxide-dependent mechanism, resulting in degradation of prominent iron regulatory proteins by ubiquitination. Radical scavengers such as R-apomorphine and green tea catechin polyphenol (-)-epigallocatechin-3-gallate, as well as the recently developed brain-permeable VK-28 series derivative iron chelators, which are neuroprotective against these neurotoxins in mice and rats, prevent the accumulation of iron and alpha-synuclein in SNpc. This study supports the notion that a combination of iron chelation and antioxidant therapy, as emphasized on several occasions, might be a significant approach to neuroprotection in PD and other neurodegenerative diseases.     

Aging and α-synuclein affect synaptic plasticity in the dentate gyrus

Although intracellular accumulation of α-synuclein (α-syn) is a characteristic pathological change in Parkinson’s disease, Lewy body dementia and Alzheimer’s disease, the normal function of this presynaptic protein is still unknown. To assess the contribution of α-syn to synaptic plasticity as well as to age-related synaptic degeneration in mice, we compared adult and aged mice overexpressing mutated (A30P) human α-syn with their nontransgenic littermates using behavioral tests and electrophysiological measures in the dentate gyrus. We found decreased basal synaptic transmission and paired-pulse facilitation in the perforant path-dentate granule cell synapses of aged mice. In addition, α-syn accumulation in aged A30P mice but not in aged wild-type mice led to long-term depression of synaptic transmission after a stimulation protocol that normally induces long-term potentiation. These findings suggest that overexpression of mutated α-syn exacerbates the aging process and leads to impaired synaptic plasticity.     

Selective loss of Purkinje cells from the rat cerebellum caused by acrylamide and the responses ofβ-glucuronidase andβ-galactosidase

Summary Acrylamide (30mg/kg) given daily to rats five times each week for 3 weeks leads to progressive loss of Purkinje cells. The necrotic cells begin to be visible from the third day and their numbers reach a peak at the time when the dosing ceases at 18 days. They are less frequent thereafter, but are still visible almost 3 weeks later in small numbers. The density of Purkinje cells per millimeter falls to about 70% of normal at the 7th day, and a similar degree of reduction of the neuronal marker enzyme, -galactosidase, is found over the same time scale. By contrast, while there is a brisk macrophage/microglial response in the molecular layer to the loss of the Purkinje cell dendrites, the increase in -glucuronidase activity is relatively minor and is not significantly different from normal until after the 21st day. These responses are discussed in the context of the use of lysosomal enzyme activities in the assay of certain neurotoxic lesions.     

Stoichiometry and pharmacology of two human α4β2 nicotinic receptor types

The alpha4beta2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR subtype in the brain, where it forms the high-affinity binding site for nicotine. The alpha4beta2 nAChR belongs to a gene family of ligand-gated ion channels that also includes muscle nAChRs, GABAA receptors, and glycine receptors and that assembles into pentameric structures. alpha4 and beta2 nAChR subunits expressed heterologously in Xenopus laevis oocytes assemble into a mixture of high- and low-affinity functional receptors, giving rise to biphasic ACh concentration-response curves (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001). High- and low-affinity alpha4beta2 nAChRs differ significantly in their functional and pharmacological properties (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001; Nelson et al., 2003) and result from the assembly of alpha4 and beta2 subunits into two distinct stoichiometric arrangements: (alpha4)2(beta2)3(high-affinity subtype) and (alpha4)3(beta2)2 (low-affinity subtype) (Nelson et al., 2003). In this study we have examined the functional and pharmacological properties of high- and low-affinity alpha4beta2 receptors using two-electrode voltage clamp procedures on Xenopus oocytes transfected with high (1:10) or low (10:1) ratios of alpha4/beta2 cDNAs, which yield high (1:10)- or low (10:1)- affinity receptors with monophasic ACh concentration- response curves. Furthermore, to determine the stoichiometry of high- and low-affinity receptors expressed heterologously by Xenopus oocytes, we have determined the stoichiometry of high- and low-affinity alpha4beta2 receptors by mutating a highly conserved hydrophobic residue in the middle (position 9') of the pore-lining domain, which increases agonist potency in a manner that allows predictions on subunit composition (Cooper et al., 1991; Revah et al., 1991; Labarca et al., 1995; Boorman et al., 2000).     

Palmitoylation supports the association of tetraspanin CD63 with CD9 and integrin αIIbβ3 in activated platelets

CD63 and CD9 are members of the tetraspanin superfamily of integral membrane proteins that function as organizers of multi-molecular signaling complexes involved in cell morphology, motility and proliferation. Tetraspanin complexes cluster dynamically in unique cholesterol-rich tetraspanin-enriched microdomains (TEMs). In resting platelets, CD63 is located in the membranes of lysosomes and dense granules. Following platelet activation and granule exocytosis, CD63 is expressed on the plasma membrane, co-localizes with the αIIbβ3-CD9 complex and is incorporated into the Triton-insoluble actin cytoskeleton, dependent on fibrinogen binding to αIIbβ3. In nucleated cell lines, the assembly and maintenance of TEMs depends on the palmitoylation of both tetraspanins and some partner proteins. This study investigated the role of palmitoylation in platelet TEM assembly and maintenance. [³H]-palmitate-labeled, washed human platelets were studied at rest, or following activation with thrombin (0.1 U/ml). CD63 and CD9 were separated by density gradient centrifugation, isolated by immunoprecipitation, and [³H]-palmitate was measured in each fraction. Palmitate levels increased in all fractions following thrombin activation. However, the relative inter-fraction distribution of the tetraspanins did not change. 2-bromopalmitate (2-BP), an inhibitor of protein palmitoylation as demonstrated by decreased [³H]-palmitate labeling of platelet proteins, blocked both thrombin-induced platelet aggregation and platelet spreading on immobilized fibrinogen in a dose-dependent manner. 2-BP also inhibited the activation-dependent association of CD63 with CD9, and the incorporation of CD63 into the Triton-insoluble actin cytoskeleton. In contrast, 2-BP had no effect on the incorporation of αIIbβ3 into the activated platelet cytoskeleton. These results demonstrate that palmitoylation is required for platelet tetraspanin-tetraspanin and tetraspanin-integrin interaction and for complete platelet spreading on a fibrinogen substrate.     

Comparison of the behavioural and histological characteristics of the 6-OHDA and α-synuclein rat models of Parkinson's disease

Development of relevant models of Parkinson's disease (PD) is essential for a better understanding of the pathological processes underlying the human disease and for the evaluation of promising targets for therapeutic intervention. To date, most pre-clinical studies have been performed in the well-established rodent and non-human primate models using injection of 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydroxypyridine (MPTP). Overexpression of the disease-causing protein α-synuclein (α-syn), using adeno-associated viral (AAV) vectors, has provided a novel model that recapitulates many features of the human disease. In the present study we compared the AAV-α-syn rat model with models where the nigro-striatal pathway is lesioned by injection of 6-OHDA in the striatum (partial lesion) or the medial forebrain bundle (full lesion). Examination of the behavioural changes over time revealed a different progression and magnitude of the motor impairment. Interestingly, dopamine (DA) neuron loss is prominent in both the toxin and the AAV-α-syn models. However, α-syn overexpressing animals were seen to exhibit less cell and terminal loss for an equivalent level of motor abnormalities. Prominent and persistent axonal pathology is only observed in the α-syn rat model. We suggest that, while neuronal and terminal loss mainly accounts for the behavioural impairment in the toxin-based model, similar motor deficits result from the combination of cell death and dysfunction of the remaining nigro-striatal neurons in the AAV-α-syn model. While the two models have been developed to mimic DA neuron deficiency, they differ in their temporal and neuropathological characteristics, and replicate different aspects of the pathophysiology of the human disease. This study suggests that the AAV-α-syn model replicates the human pathology more closely than either of the other two 6-OHDA lesion models.     

Allosteric Modulator Desformylflustrabromine Relieves the Inhibition of α2β2 and α4β2 Nicotinic Acetylcholine Receptors by β-Amyloid<Subscript>1–42</Subscript> Peptide

Nicotinic acetylcholine receptors (nAChRs) are pentameric transmembrane proteins that belong to the cys-loop ligand-gated ion channel family. These receptors are widely expressed in the brain and implicated in the pathophysiology of many neurological conditions, including Alzheimer’s disease (AD), where typical symptoms include the loss of cognitive function and dementia. The presence of extracellular neuritic plaques composed of β amyloid (Aβ_1–42) peptide is a characteristic feature of AD. Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) for α4β2 nAChRs since it increases peak ACh responses without inducing a response on its own. Previously, the effect of dFBr on the α2β2 nAChR subtype was not known. The action of dFBr was tested on α2β2 receptors expressed in Xenopus oocytes. It was found that dFBr is also a PAM for the α2β2 receptor. Next we tested whether dFBr had any effect on the previously known block of both the α4β2 and α2β2 receptors by Aβ_1–42. We found that the functional blockade of ACh-induced currents in oocytes expressing α4β2 and α2β2 receptors by Aβ_1–42 was prevented by dFBr. We conclude that dFBr is a positive allosteric modulator for both α4β2 and α2β2 subtypes of nAChRs and that it also relieves the blockade of these receptors by Aβ_1–42. This study demonstrates that PAMs for the non-α7 nAChRs have the potential to develop into clinically applicable drugs for AD and other disorders.     

Mouse Striatal Dopamine Nerve Terminals Express α4α5β2 and Two Stoichiometric Forms of α4β2*-Nicotinic Acetylcholine Receptors

Wild-type and α5 null mutant mice were used to identify nicotinic cholinergic receptors (nAChRs) that mediate α-conotoxin MII (α-CtxMII)-resistant dopamine (DA) release from striatal synaptosomes. Concentration–effect curves for ACh-stimulated release (20 s) were monophasic when wild-type synaptosomes were assayed but biphasic with synaptosomes from the α5 null mutant. Deleting the α5 gene also resulted in decreased maximal ACh-stimulated α-CtxMII-resistant DA release. When a shorter perfusion time (5 s) was used, biphasic curves were detected in both wild-type and α5 null mutants, indicative of high- and low-sensitivity (HS and LS) activity. In addition, DHβE-sensitive (HS) and DHβE-resistant (LS) components were found in both genotypes. These results indicate that α-CtxMII-resistant DA release is mediated by α4α5β2, (α4)₂(β2)₃ (HS), and (α4)₃(β2)₂ (LS) nAChRs.     

Distinct roles in vivo for the ubiquitin-proteasome system and the autophagy-lysosomal pathway in the degradation of α-synuclein

Increased intracellular levels of α-synuclein are implicated in Parkinson's disease and related disorders and may be caused by alterations in the ubiquitin-proteasome system (UPS) or the autophagy-lysosomal pathway (ALP). A critical question remains how α-synuclein is degraded by neurons in vivo. To address this, our study uses α-synuclein transgenic mice, expressing human α-synuclein or α-synuclein-eGFP under the (h)PDGF-β promoter, in combination with in vivo pharmacologic and multiphoton imaging strategies to systematically test degradation pathways in the living mouse brain. We demonstrate that the UPS is the main degradation pathway for α-synuclein under normal conditions in vivo while with increased α-synuclein burden the ALP is recruited. Moreover, we report alterations of the UPS in α-synuclein transgenic mice and age dependence to the role of the UPS in α-synuclein degradation. In addition, we provide evidence that the UPS and ALP might be functionally connected such that impairment of one can upregulate the other. These results provide a novel link between the UPS, the ALP, and α-synuclein pathology and may have important implications for future therapeutics targeting degradation pathways.