α6β2*‐subtype nicotinic acetylcholine receptors are more sensitive than α4β2*‐subtype receptors to regulation by chronic nicotine administration

Nicotinic acetylcholine receptors (nAChR) of the α6β2* subtype (where *indicates the possible presence of additional subunits) are prominently expressed on dopaminergic neurons. Because of this, their role in tobacco use and nicotine dependence has received much attention. Previous studies have demonstrated that α6β2*‐nAChR are down‐regulated following chronic nicotine exposure (unlike other subtypes that have been investigated – most prominently α4β2* nAChR). This study examines, for the first time, effects across a comprehensive chronic nicotine dose range. Chronic nicotine dose–responses and quantitative ligand‐binding autoradiography were used to define nicotine sensitivity of changes in α4β2*‐nAChR and α6β2*‐nAChR expression. α6β2*‐nAChR down‐regulation by chronic nicotine exposure in dopaminergic and optic‐tract nuclei was ≈three‐fold more sensitive than up‐regulation of α4β2*‐nAChR. In contrast, nAChR‐mediated [³H]‐dopamine release from dopamine‐terminal region synaptosomal preparations changed only in response to chronic treatment with high nicotine doses, whereas dopaminergic parameters (transporter expression and activity, dopamine receptor expression) were largely unchanged. Functional measures in olfactory tubercle preparations were made for the first time; both nAChR expression levels and nAChR‐mediated functional measures changed differently between striatum and olfactory tubercles. These results show that functional changes measured using synaptosomal [³H]‐DA release are primarily owing to changes in nAChR, rather than in dopaminergic, function.

     

Insulin induces phosphorylation of the AMPA receptor subunit GluR1, reversed by ZIP,and over‐expression of Protein Kinase M zeta,reversed by amyloid beta

Insulin receptor (IR) in the brain plays a role in synaptic plasticity and cognitive functions. Phosphorylation of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) receptors GluR1 subunit at Serine 831 is regulated by calcium–calmodulin‐dependent protein kinase II and protein kinase C that underlie long‐term potentiation and learning/memory. Recent studies have shown that the novel Protein Kinase M zeta (PKMζ) underlies synaptic plasticity and may regulate AMPAr. In this study, we show that insulin induces phosphorylation of Serine 831 GluR1 subunit of AMPAr and induces over‐expression of PKMζ; pre‐treatment with either the IR inhibitor 3‐Bromo‐5‐t‐butyl‐4‐hydroxy‐benzylidenemalonitrile (AG1024) or PKMζ inhibitor protein kinase C zeta pseudo‐substrate inhibitor returned the phosphorylation value of GluR1 to control level. Amyloid beta (Aβ) peptide in the form of oligomers interferes with IR signaling. Pre‐treating neuronal cultures with Aβ following incubation with insulin, we found a reduction of insulin‐dependent PKMζ over‐expression and MAPK/Erk (1/2) phosphorylation, i.e., signaling pathways involved in synaptic plasticity and learning/memory. These results indicate a new intracellular insulin signaling pathway, and, additionally, that insulin resistance in Alzheimer's disease is a response to the production and accumulation of Aβ.

     

Phenolic compounds prevent the oligomerization of α‐synuclein and reduce synaptic toxicity

Lewy bodies, mainly composed of α‐synuclein (αS), are pathological hallmarks of Parkinson's disease and dementia with Lewy bodies. Epidemiological studies showed that green tea consumption or habitual intake of phenolic compounds reduced Parkinson's disease risk. We previously reported that phenolic compounds inhibited αS fibrillation and destabilized preformed αS fibrils. Cumulative evidence suggests that low‐order αS oligomers are neurotoxic and critical species in the pathogenesis of α‐synucleinopathies. To develop disease modifying therapies for α‐synucleinopathies, we examined effects of phenolic compounds (myricetin (Myr), curcumin, rosmarinic acid (RA), nordihydroguaiaretic acid, and ferulic acid) on αS oligomerization. Using methods such as photo‐induced cross‐linking of unmodified proteins, circular dichroism spectroscopy, the electron microscope, and the atomic force microscope, we showed that Myr and RA inhibited αS oligomerization and secondary structure conversion. The nuclear magnetic resonance analysis revealed that Myr directly bound to the N‐terminal region of αS, whereas direct binding of RA to monomeric αS was not detected. Electrophysiological assays for long‐term potentiation in mouse hippocampal slices revealed that Myr and RA ameliorated αS synaptic toxicity by inhibition of αS oligomerization. These results suggest that Myr and RA prevent the αS aggregation process, reducing the neurotoxicity of αS oligomers.

     

Chronic sazetidine‐A maintains anxiolytic effects and slower weight gain following chronic nicotine without maintaining increased density of nicotinic receptors in rodent brain

Chronic nicotine administration increases the density of brain α4β2* nicotinic acetylcholine receptors (nAChRs), which may contribute to nicotine addiction by exacerbating withdrawal symptoms associated with smoking cessation. Varenicline, a smoking cessation drug, also increases these receptors in rodent brain. The maintenance of this increase by varenicline as well as nicotine replacement may contribute to the high rate of relapse during the first year after smoking cessation. Recently, we found that sazetidine‐A (saz‐A), a potent partial agonist that desensitizes α4β2* nAChRs, does not increase the density of these receptors in brain at doses that decrease nicotine self‐administration, increase attention in rats, and produce anxiolytic effects in mice. Here, we investigated whether chronic saz‐A and varenicline maintain the density of nAChRs after their up‐regulation by nicotine. In addition, we examined the effects of these drugs on a measure of anxiety in mice and weight gain in rats. After increasing nAChRs in the rodent brain with chronic nicotine, replacing nicotine with chronic varenicline maintained the increased nAChR binding, as well as the α4β2 subunit proteins measured by western blots. In contrast, replacing nicotine treatments with chronic saz‐A resulted in the return of the density of nAChRs to the levels seen in saline controls. Nicotine, saz‐A and varenicline each demonstrated anxiolytic effects in mice, but only saz‐A and nicotine attenuated the gain of weight over a 6‐week period in rats. These findings suggest that apart from its modest anxiolytic and weight control effects, saz‐A, or drugs like it, may be useful in achieving long‐term abstinence from smoking.

     

Long‐term nicotine treatment down‐regulates α6β2* nicotinic receptor expression and function in nucleus accumbens

Long‐term nicotine exposure induces alterations in dopamine transmission in nucleus accumbens that sustain the reinforcing effects of smoking. One approach to understand the adaptive changes that arise involves measurement of endogenous dopamine release using voltammetry. We therefore treated rats for 2–3 months with nicotine and examined alterations in nAChR subtype expression and electrically evoked dopamine release in rat nucleus accumbens shell, a region key in addiction. Long‐term nicotine treatment selectively decreased stimulated α6β2* nAChR‐mediated dopamine release compared with vehicle‐treated rats. It also reduced α6β2* nAChRs, suggesting the receptor decline may contribute to the functional loss. This decreased response in release after chronic nicotine treatment was still partially sensitive to the agonist nicotine. Studies with an acetylcholinesterase inhibitor demonstrated that the response was also sensitive to increased endogenous acetylcholine. However, unlike the agonists, nAChR antagonists decreased dopamine release only in vehicle‐ but not nicotine‐treated rats. As antagonists function by blocking the action of acetylcholine, their ineffectiveness suggests that reduced acetylcholine levels partly underlie the dampened α6β2* nAChR‐mediated function in nicotine‐treated rats. As long‐term nicotine modifies dopamine release by decreasing α6β2* nAChRs and their function, these data suggest that interventions that target this subtype may be useful for treating nicotine dependence.

     

Extracellular α‐synuclein alters synaptic transmission in brain neurons by perforating the neuronal plasma membrane

It has been postulated that the accumulation of extracellular α‐synuclein (α‐syn) might alter the neuronal membrane by formation of ‘pore‐like structures’ that will lead to alterations in ionic homeostasis. However, this has never been demonstrated to occur in brain neuronal plasma membranes. In this study, we show that α‐syn oligomers rapidly associate with hippocampal membranes in a punctate fashion, resulting in increased membrane conductance (5 fold over control) and the influx of both calcium and a fluorescent glucose analogue. The enhancement in intracellular calcium (1.7 fold over control) caused a large increase in the frequency of synaptic transmission (2.5 fold over control), calcium transients (3 fold over control), and synaptic vesicle release. Both primary hippocampal and dissociated nigral neurons showed rapid increases in membrane conductance by α‐syn oligomers. In addition, we show here that α‐syn caused synaptotoxic failure associated with a decrease in SV2, a membrane protein of synaptic vesicles associated with neurotransmitter release. In conclusion, extracellular α‐syn oligomers facilitate the perforation of the neuronal plasma membrane, thus explaining, in part, the synaptotoxicity observed in neurodegenerative diseases characterized by its extracellular accumulation.

     

Is the mTOR‐signalling cascade disrupted in Schizophrenia?

The mammalian target of rapamycin (mTOR) signalling cascade is involved in the intracellular regulation of protein synthesis, specifically for proteins involved in controlling neuronal morphology and facilitating synaptic plasticity. Research has revealed that the activity of the mTOR cascade is influenced by several extracellular and environmental factors that have been implicated in schizophrenia. Therefore, there is reason to believe that one of the downstream consequences of dysfunction or hypofunction of these factors in schizophrenia is disrupted mTOR signalling and hence impaired protein synthesis. This results in abnormal neurodevelopment and deficient synaptic plasticity, outcomes which could underlie some of the positive, negative and cognitive symptoms of schizophrenia. This review will discuss the functional roles of the mTOR cascade and present evidence in support of a novel mTOR‐based hypothesis of the neuropathology of schizophrenia.

     

Positive allosteric modulation of alpha‐7 nicotinic receptors promotes cell death by inducing Ca2+ release from the endoplasmic reticulum

Positive allosteric modulation of α7 isoform of nicotinic acetylcholine receptors (α7‐nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca²⁺ signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca²⁺ signaling and cell viability. We used human SH‐SY5Y neuroblastoma cells overexpressing α7‐nAChRs (α7‐SH) and their control (C‐SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca²⁺ with Fura‐2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch‐clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of α7‐SH but not of C‐SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca²⁺ concentration; (iii) released Ca²⁺ from the ER by a Ca²⁺‐induced Ca²⁺ release mechanism only in α7‐SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of α7‐nAChRs, ryanodine receptors, or IP₃ receptors. In conclusion, positive allosteric modulation of α7‐nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca²⁺, overloading of intracellular Ca²⁺, and neuronal cell death.

     

Expression of the synaptic exocytosis‐regulating molecule complexin 2 in taste buds and its participation in peripheral taste transduction

Taste information from type III taste cells to gustatory neurons is thought to be transmitted via synapses. However, the molecular mechanisms underlying taste transduction through this pathway have not been fully elucidated. In this study, to identify molecules that participate in synaptic taste transduction, we investigated whether complexins (Cplxs), which play roles in regulating membrane fusion in synaptic vesicle exocytosis, were expressed in taste bud cells. Among four Cplx isoforms, strong expression of Cplx2 mRNA was detected in type III taste cells. To investigate the function of CPLX2 in taste transduction, we observed taste responses in CPLX2‐knockout mice. When assessed with electrophysiological and behavioral assays, taste responses to some sour stimuli in CPLX2‐knockout mice were significantly lower than those in wild‐type mice. These results suggested that CPLX2 participated in synaptic taste transduction from type III taste cells to gustatory neurons.

     

Ligand‐directed delivery of fluorophores to track native calcium‐permeable AMPA receptors in neuronal cultures

Subcellular trafficking of neuronal receptors is known to play a key role in synaptic development, homeostasis, and plasticity. We have developed a ligand‐targeted and photo‐cleavable probe for delivering a synthetic fluorophore to AMPA receptors natively expressed in neurons. After a receptor is bound to the ligand portion of the probe molecule, a proteinaceous nucleophile reacts with an electrophile on the probe, covalently bonding the two species. The ligand may then be removed by photolysis, returning the receptor to its non‐liganded state while leaving intact the new covalent bond between the receptor and the fluorophore. This strategy was used to label polyamine‐sensitive receptors, including calcium‐permeable AMPA receptors, in live hippocampal neurons from rats. Here, we describe experiments where we examined specificity, competition, and concentration on labeling efficacy as well as quantified receptor trafficking. Pharmacological competition during the labeling step with either a competitive or non‐competitive glutamate receptor antagonist prevented the majority of labeling observed without a blocker. In other experiments, labeled receptors were observed to alter their locations and we were able to track and quantify their movements.

     

Degradation of gamma secretase activating protein by the ubiquitin–proteasome pathway

A major hallmark feature of Alzheimer's disease is the accumulation of amyloid β (Aβ), whose formation is regulated by the γ‐secretase complex and its activating protein (also known as γ‐secretase activating protein, or GSAP). Because GSAP interacts with the γ‐secretase without affecting the cleavage of Notch, it is an ideal target for a viable anti‐Aβ therapy. GSAP derives from a C‐terminal fragment of a larger precursor protein of 98 kDa via a caspase 3‐mediated cleavage. However, the mechanism(s) involved in its degradation remain unknown. In this study, we show that GSAP has a short half‐life of approximately 5 h. Neuronal cells treated with proteasome inhibitors markedly prevented GSAP protein degradation, which was associated with a significant increment in Aβ levels and γ‐secretase cleavage products. In contrast, treatment with calpain blocker and lysosome inhibitors had no effect. In addition, we provide experimental evidence that GSAP is ubiquitinated. Taken together, our findings reveal that GSAP is degraded through the ubiquitin–proteasome system. Modulation of the GSAP degradation pathway may be implemented as a viable target for a safer anti‐Aβ therapeutic approach in Alzheimer's disease.

     

Ionizing radiation causes increased tau phosphorylation in primary neurons

Radiotherapy is the major treatment modality for primary and metastatic brain tumors which involves the exposure of brain to ionizing radiation. Ionizing radiation can induce various detrimental pathophysiological effects in the adult brain, and Alzheimer's disease and related neurodegenerative disorders are considered to be late effects of radiation. In this study, we investigated whether ionizing radiation causes changes in tau phosphorylation in cultured primary neurons similar to that in Alzheimer's disease. We demonstrated that exposure to 0.5 or 2 Gy γ rays causes increased phosphorylation of tau protein at several phosphorylation sites in a time‐ and dose‐dependent manner. Consistently, we also found ionizing radiation causes increased activation of GSK3β, c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase before radiation‐induced increase in tau phosphorylation. Specific inhibitors of these kinases almost fully blocked radiation‐induced tau phosphorylation. Our studies further revealed that oxidative stress plays an important role in ionizing radiation‐induced tau phosphorylation, likely through the activation of c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase, but not GSK3β. Overall, our studies suggest that ionizing radiation may cause increased risk for development of Alzheimer's disease by promoting abnormal tau phosphorylation.

     

A mutation protective against Alzheimer's disease renders amyloid β precursor protein incapable of mediating neurotoxicity

Expression of a familial Alzheimer's disease (AD)‐linked mutant of amyloid β precursor protein (APP) or the binding of transforming growth factor β2 to wild‐type (wt)‐APP causes neuronal death by activating an intracellular death signal (a APP‐mediated intracellular death signal) in the absence of the involvement of amyloid β (Aβ) toxicity in vitro. These neuronal death models may therefore be regarded as Aβ‐independent neuronal death models related to AD. A recent study has shown that the A673T mutation in the APP isoform APP₇₇₀, corresponding to the A598T mutation in the most prevalent neuronal APP isoform APP₆₉₅ (an AD‐protective mutant of APP), is linked to a reduction in the incidence rate of AD. Consistent with this, cells expressing the AD‐protective mutant of APP produce less Aβ than cells expressing wt‐APP. In this study, transforming growth factor β2 caused death in cultured neuronal cells expressing wt‐APP, but not in those expressing the AD‐protective mutant of APP. This result suggests that the AD‐protective mutation of APP reduces the incidence rate of AD by attenuating the APP‐mediated intracellular death signal. In addition, a mutation that causes hereditary cerebral hemorrhage with amyloidosis‐Dutch type also attenuated the APP‐mediated intracellular death signal.

     

Postnatal ethanol exposure alters levels of 2‐arachidonylglycerol‐metabolizing enzymes and pharmacological inhibition of monoacylglycerol lipase does not cause neurodegeneration in neonatal mice

The consumption of ethanol by pregnant women may cause neurological abnormalities, affecting learning and memory processes in children, and are collectively described as fetal alcohol spectrum disorders. However, the molecular mechanisms underlying these changes are still poorly understood. In our previous studies, we found that ethanol treatment of postnatal day 7 (P7) mice significantly enhances the anandamide levels but not the 2‐arachidonylglycerol (2‐AG) levels and induces widespread neurodegeneration, but the reason for the lack of significant effects of ethanol on the 2‐AG level is unknown. In this study, we examined developmental changes in diacylglycerol lipase‐α, β (DAGL‐α and β) and monoacylglycerol lipase (MAGL). We found that the levels of these proteins were significantly higher in adult brains compared to those detected early in brain development. Next, we examined the influence of P7 ethanol treatment on these enzymes, finding that it differentially altered the DAGL‐α protein and mRNA levels but consistently enhanced those of the DAGL‐β. Interestingly, the ethanol treatment enhanced MAGL protein and mRNA levels. Inhibition of MAGL with KML29 failed to induce neurodegeneration in P7 mice. Collectively, these findings suggest that ethanol significantly activates DAGL‐β and MAGL in the neonatal brain, resulting in no net change in 2‐AG levels.

     

Pre‐synaptic localization of the γ‐secretase‐inhibiting protein p24α2 in the mammalian brain

Dysregulated metabolism and consequent extracellular accumulation of amyloid‐β (Aβ) peptides in the brain underlie the pathogenesis of Alzheimer's disease. Extracellular Aβ in the brain parenchyma is mainly secreted from the pre‐synaptic terminals of neuronal cells in a synaptic activity‐dependent manner. The p24 family member p24α₂ reportedly attenuates Aβ generation by inhibiting γ‐secretase processing of amyloid precursor protein; however, the pattern of expression and localization of p24α₂ in the brain remains unknown. We performed immunohistochemical staining and subcellular fractionation for p24α₂ in the mouse brain. Immunostaining showed that p24α₂ is broadly distributed in the gray matter of the central nervous system and is predominantly localized to synapses. Subcellular fractionation revealed prominent localization of p24α₂ in the pre‐synaptic terminals. Immunoisolation of synaptic vesicles (SV) indicated that p24α₂ is condensed at active zone‐docked SV. During development, p24α₂ expression is highest in the post‐natal period and gradually decreases with age. We also confirmed that amyloid precursor protein and γ‐secretase components are localized at active zone‐docked SV. Our results suggest a novel functional role for p24α₂ in the regulation of synaptic transmission and synaptogenesis, and provide evidence for the participation of p24α₂ in the regulation of Aβ generation and secretion in the brain.

     

Prion protein regulates glutathione metabolism and neural glutamate and cysteine uptake via excitatory amino acid transporter 3

Prion protein (PrP) plays crucial roles in regulating antioxidant systems to improve cell defenses against cellular stress. Here, we show that the interactions of PrP with the excitatory amino acid transporter 3 (EAAT3), γ‐glutamyl transpeptidase (γ‐GT), and multi‐drug resistance protein 1 (MRP1) in astrocytes and the interaction between PrP and EAAT3 in neurons regulate the astroglial and neuronal metabolism of the antioxidant glutathione. Ablation of PrP in astrocytes and cerebellar neurons leads to dysregulation of EAAT3‐mediated uptake of glutamate and cysteine, which are precursors for the synthesis of glutathione. In PrP‐deficient astrocytes, levels of intracellular glutathione are increased, and under oxidative stress, levels of extracellular glutathione are increased, due to (i) increased glutathione release via MRP1 and (ii) reduced activity of the glutathione‐degrading enzyme γ‐GT. In PrP‐deficient cerebellar neurons, cell death is enhanced under oxidative stress and glutamate excitotoxicity, when compared to wild‐type cerebellar neurons. These results indicate a functional interplay of PrP with EAAT3, MRP1 and γ‐GT in astrocytes and of PrP and EAAT3 in neurons, suggesting that these interactions play an important role in the metabolic cross‐talk between astrocytes and neurons and in protection of neurons by astrocytes from oxidative and glutamate‐induced cytotoxicity.

     

Sulforaphane enhances temozolomide‐induced apoptosis because of down‐regulation of miR‐21 via Wnt/β‐catenin signaling in glioblastoma

Temozolomide (TMZ) has been widely used in the treatment of glioblastoma (GBM), although inherent or acquired resistance restricts the application. This study was aimed to evaluate the efficacy of sulforaphane (SFN) to TMZ‐induced apoptosis in GBM cells and the potential mechanism. Biochemical assays and subcutaneous tumor establishment were used to characterize the function of SFN in TMZ‐induced apoptosis. Our results revealed that β‐catenin and miR‐21 were concordantly expressed in GBM cell lines, and SFN significantly reduced miR‐21 expression through inhibiting the Wnt/β‐catenin/TCF4 pathway. Furthermore, down‐regulation of miR‐21 enhanced the pro‐apoptotic efficacy of TMZ in GBM cells. Finally, we observed that SFN strengthened TMZ‐mediated apoptosis in a miR‐21‐dependent manner. In conclusion, SFN effectively enhances TMZ‐induced apoptosis by inhibiting miR‐21 via Wnt/β‐catenin signaling in GBM cells. These findings support the use of SFN for potential therapeutic approach to overcome TMZ resistance in GBM treatment.

     

Alpha‐ketoglutarate dehydrogenase complex‐dependent succinylation of proteins in neurons and neuronal cell lines

Reversible post‐translation modifications of proteins are common in all cells and appear to regulate many processes. Nevertheless, the enzyme(s) responsible for the alterations and the significance of the modification are largely unknown. Succinylation of proteins occurs and causes large changes in the structure of proteins; however, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins remain unknown. These studies focused on succinylation of mitochondrial proteins. The results demonstrate that the α‐ketoglutarate dehydrogenase complex (KGDHC) can serve as a trans‐succinylase that mediates succinylation in an α‐ketoglutarate‐dependent manner. Inhibition of KGDHC reduced succinylation of both cytosolic and mitochondrial proteins in cultured neurons and in a neuronal cell line. Purified KGDHC can succinylate multiple proteins including other enzymes of the tricarboxylic acid cycle leading to modification of their activity. Inhibition of KGDHC also modifies acetylation by modifying the pyruvate dehydrogenase complex. The much greater effectiveness of KGDHC than succinyl‐CoA suggests that the catalysis owing to the E2k succinyltransferase is important. Succinylation appears to be a major signaling system and it can be mediated by KGDHC.