Nitrosylation of Vesicular Transporters in Brain of Amyloid Precursor Protein/Presenilin 1 Double Transgenic Mice

Abstract

Nitric oxide can attack thiol groups of cysteine residues in proteins and induce protein cysteine S-nitrosylation. Cholinergic and glutamatergic systems are dysregulated in Alzheimer’s disease. Vesicular acetylcholine transporter (VAChT) and vesicular glutamate transporter 1 (VGLUT1) are important in packaging acetylcholine and glutamate into vesicles, which is an important step for neurotransmission. Previously we found that VAChT and VGLUT1 can be nitrosylated and that S-nitrosylation of these transporters inhibits vesicular uptake of acetylcholine and glutamate. To understand the role of VAChT and VGLUT1 nitrosylation in the pathophysiological development of Alzheimer’s disease, we analyzed nitrosylation of VAChT and VGLUT1 in brain of amyloid precursor protein (APP) and presenilin 1 (PS1) double transgenic mice, an animal model for Alzheimer’s disease. Using a Morris water maze test, we found that 9- and 12-month-old APP/PS1 mice showed memory deficit, compared to wild type mice. We further found that total protein nitrosylation was increased in frontal cortex and hippocampus of 9- and 12-month-old APP/PS1 mice. Although nitrosylation of VAChT and VGLUT1 was not changed in hippocampus of 9- and 12-month-old APP/PS1 mice, nitrosylation of VAChT and VGLUT1 was significantly increased in frontal cortex of APP/PS1 mice at these ages. We also found that nitrosylation of VAChT and VGLUT1 was increased in hippocampus (but not frontal cortex) of 3-month-old APP/PS1 mice. These findings suggest that nitrosylation of VAChT and VGLUT1 may be associated with dysfunctional acetylcholinergic and glutamatergic neurotransmission in Alzheimer’s disease.

Keywords

Alzheimer’s disease oxidative stress protein nitrosylation vesicular acetylcholine transporter vesicular glutamate transporter

INTRODUCTION

In the past one hundred years, Alzheimer’s disease (AD) has become the most common and the leading cause of dementia in the aged population. AD patients experience memory loss, cognitive impairments, and behavioral changes such as aphasia, apraxia, agnosia, and executive dysfunction. Although the disease mechanisms of AD are not fully understood, amyloid-β (Aβ) plaques, neurofibrillary tangles, and deficits of neurotransmitter systems have been identified as major pathological factors of AD [1 –3].

The dysfunction of cholinergic and glutamatergic systems contributes to the cognitive changes and neuropathology seen in AD [4]. Acetylcholine is an essential neurotransmitter that modulates multiple cognitive processes including selective attention, sensory input, and associative thinking [5, 6]. These functions are largely supported by projections from cholinergic nuclei in the basal forebrain into virtually all cortical areas. Studies have shown that cholinergic neurotransmission is severely damaged in early stages of AD [7, 8]. Glutamate is the main excitatory neurotransmitter involved in long-term memory and many other cognitive processes. In AD, chronic hyperactivation of NMDA receptors caused by glutamate, leading to neurodegeneration, is one of the main factors in excitotoxicity [9, 10].

Nitric oxide (NO) is produced from L-arginine catalyzed by nitric oxide synthases (NOS) including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) [11 –13]. NO can attack thiol groups of cysteine residues in proteins and induce protein cysteine nitrosylation [14 –17]. Studies have shown that Aβ treatment increases NO production and NOS protein levels in cultured rat cortical neurons and rat brain [18, 19], suggesting that NO may contribute to Aβ-induced toxicity. Vesicular acetylcholine transporter (VAChT) is responsible for packaging acetylcholine, while vesicular glutamate transporters (VGLUTs) are responsible for packaging glutamate. VGLUT1 is predominant and expressed exclusively in the central nervous system [20]. These transporters play important roles in the process of acetylcholinergic and glutamatergic neurotransmission. Previously we found that VAChT and VGLUT1 can be nitrosylated by NO donor nitrosoglutathione and that nitrosylation of VAChT and VGLUT1 inhibits vesicular uptake acetylcholine and glutamate respectively in mouse brain [21]. To understand the role of nitrosylation in the pathophysiological development of AD, in the present study we analyzed total protein nitrosylation and nitrosylation of VAChT and VGLUT1 in frontal cortex and hippocampus of 3-, 6-, 9-, and 12-month-old amyloid precursor protein (APP) and presenilin 1 (PS1) double transgenic mice, an animal model for AD, compared to wild type mice.

MATERIALS AND METHODS

Cell culture

Mouse hippocampal HT22 cell lines were generously provided by the Salk Institute (La Jolla, CA, USA). HT22 cells were maintained in Dulbecco’s Modified Eagle Media (Life Technologies Inc, Burlington, Canada) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum, and cultured at 37°C under 5% CO₂.

APP/PS1 double transgenic mice and genotyping

The APP/PS1 double transgenic mice were obtained from cross breeding single transgenic mice expressing human APP_K670N/M671L with single transgenic mice expressing human PS1_M146L [22]. The mouse colonies were kept and maintained in a pathogen free environment in the Central Animal Care Service at the University of Manitoba. All procedures of animal study were in accordance with the guidelines from the Canadian Council on Animal Care.

APP/PS1 transgenic mice were selected by the genotyping using PCR. At 3 weeks after birth, DNA sample was isolated from the tail tip of each mouse. The human APP primers and human PS1 oligo primers were used for genotyping. For APP, forward primer is 5’-GCC GTT GAC AAG TAT CTC GAG ACA CCT GG-3’, while reverse primer is 5’-GTG TCT CCA CCA GCT GCT GTC TCT CGT TGG C-3’. For PS1, forward primer is 5’-GAC AAC CAC CTG AGC AAT AC-3’ while reverse primer is 5’-CAT CTT GCT CCA CCA CCT GCC-3’. APP/PS1 double transgenic mice displayed two target bands, while wild type mice displayed no bands. APP/PS1 mice and wild type mice were grown up to 3, 6, 9, or 12 months in age.

Morris water maze test

The Morris water maze test was conducted in a circular tank, which is 120 cm in diameter and 60 cm high. EthoVision XT system was used to track the movement of mice throughout the experiment. The water level was approximately 40 cm high and the temperature was kept at 20–23°C. The water tank was divided into 4 quadrants with one visual cue for each quadrant. A square platform (10×10 cm) was located in one quadrant area and submerged to 1 cm below the water surface. The water was mixed with milk to block the vision of the platform. First, mice were trained to locate and escape onto the platform using distal cues for 4 trials per day for 4 consecutive days. On each trial, a different starting position was used. Mice were allowed to swim for maximum 60 s. If mice could not find the platform within 60 s, they were guided to the platform and kept on the platform for 30 s in order to memorize the platform location. At day 5, the platform was removed and mice were placed in a position that was 180° from the original platform and allowed to swim for 60 s. Latency to reach platform site, numbers of platform-site crossings, swim speed, and swim distance were recorded and calculated.

Protein extraction

After placed in CO₂ chamber for 20 s, mice were immediately decapitated and their brains were removed. Frontal cortex and hippocampus were dissected on ice, immediately frozen in dry ice and stored in –80°C until further use. Decapitation and dissection processes took approximately 2 min for each mouse. Frontal cortex and hippocampus of mice were homogenized in 10:1 (ml/g) ice-cold lysis buffer containing 20 mM HEPES (pH 7.5), 250 mM NaCl, 20% glycerol, 30 mM MgCl₂, 0.5 mM EDTA, 0.1 mM EGTA, 1% nonidet P40, and 1×protease inhibitor cocktail (Thermo Scientific, Marietta, OH, USA). The homogenized tissues were kept on ice for 1 h and then centrifuged at 10,000× g for 15 min at 4°C. The supernatant was collected as protein extract. Protein concentrations were determined by Bradford protein assay [23].

Biotin-switch and immunoblotting analysis

Biotin-switch followed by immunoblotting analysis was used to detect S-nitrosylated proteins. Biotin-switch assay was performed as previously described [21]. Briefly, free thiols in unmodified cysteine residues were first blocked by the thiol-specific methylthiolating agent methyl methanethiosulfonate (MMTS). 50 μg of protein was incubated with a blocking solution containing 2.5% sodium dodecyl sulphate (SDS) and 40 mM MMTS for 40 min at 50°C, and further incubated with 1 ml of acetone for 15 min at –20°C. Then samples were centrifuged at 15,000× g and the pellets were resuspended with 5 μl of HEN buffer (250 mM HEPES pH 7.7, 1 mM EDTA and 0.1 mM neocuproine) with 1% SDS. Second, the nitrosylated thiols in cysteine were reduced back to free thiols by ascorbate and labeled with N-[6-(biotinamido) hexyl]-3’- (2’-pyridyldithio) propionamide (biotin-HPDP). The samples were incubated with 50 mM ascorbate and 4 mM biotin-HPDP at room temperature for 1 h. Then the proteins were run on 10% SDS polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were first blocked with 5% milk in TBST buffer containing 10 mM Tris-HCl (pH 7.5) and 0.1% Tween-20 for 1 h at room temperature, then incubated with a polyclonal anti-biotin antibody (Sigma, St. Louis, MO, USA) overnight at 4°C, and further incubated for 1 h at room temperature with a secondary antibody conjugated to horseradish peroxidase (Abcam, Eugene, Oregon, USA). Immunoreactive bands were detected with enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA, USA) and imaged with the ChemiDoc MP System (Bio-Rad, Dreieich, Germany). Band signal intensity was quantified by using Image Lab (Bio-Rad).

To detect nitrosylation of VAChT and VGLUT1, the biotinylated proteins were purified by streptavidin-agarose beads and eluted with 100 μl of elution buffer containing 20 mM HEPES (pH 7.7), 100 mM NaCl, 1 mM EDTA, and 100 mM 2-mercaptoethanol. The eluted proteins were separated by SDS-PAGE and transferred to PVDF. Nitrosylated VAChT and VGLUT1 were detected with a polyclonal anti-VAChT antibody (Abcam) and a polyclonal anti-VGLUT1 antibody (Abcam) respectively using immunoblotting analysis. Before purification with streptavidin beads, an aliquot of protein mixture was used to determine protein input for normalizing nitrosylated VAChT and VGLUT1. Molecular size approximately is 72 kD for VAChT and 62 kD for VGLUT1.

Statistical analysis

Statistical analysis was performed using the SPSS13.0 software (IBM, Armonk, New York, USA). All the results were expressed as means± standard error of the mean (SEM). Statistical significance of differences among means was determined by one-way analysis of variance (ANOVA) with Tukey post hoc comparisons. Student’s t-tests were also performed for statistical analysis of two groups. A p value of less than 0.05 was regarded as statistically significant.

RESULTS

First, we investigated spatial memory in 3-, 6-, 9-, and 12-month-old APP/PS1 double transgenic mouse using Morris water maze test. As shown in Fig. 1A and B, the swimming speed and swimming distance were not different between APP/PS1 mice and their wild type littermates at ages of 3, 6, 9, and 12 months, suggesting that there was no hyperactive or hypoactive physiological conditions during the test between groups. Although the escape latency to reach platform and crossing number for the platform were not significantly different between 3- and 6-month-old APP/PS1 mice and their wild type littermates, the escape latency was significant increased while crossing number was significantly decreased in 9- and 12-month-old APP/PS1 mice when compared to their wild type littermates (Fig. 1C, D). This result suggests that the spatial memory in the 9- and 12-month-old APP/PS1 mice was impaired.

Second, to determine whether Aβ affects protein nitrosylation, we measured total nitrosylated protein levels in Aβ-treated HT22 mouse hippocampal cells. As shown in Fig. 2A, when only blocking agent MMTS but not reducing agent ascorbate was added (lane 1) or biotin-HPDP was not added (lane 2),nitrosylated proteins were not detected. When blocking agent, reducing agent and biotin-HPDP were used, basal nitrosylated protein could be detected (lane 3). NO donor nitrosoglutathione at 20 μM increased total nitrosylated protein levels (lane 4). The result verified that MMTS blocked free thiols, ascorbate reduced nitrosylated thiols, and nitrosoglutathione increased protein nitrosylation. Further, we found that although treatment with Aβ_1 - 42 at 0.25 μM for 24 h had no effect on total nitrosylated protein levels, treatment with Aβ_1 - 42 at 0.5 and 1 μM significantly increased total nitrosylated protein levels (Fig. 2B), suggesting that Aβ can induce protein nitrosylation.

Third, we investigated total nitrosylated protein levels in frontal cortex and hippocampus of APP/PS1 transgenic mice. As shown in Fig. 3A, although total nitrosylated protein levels were not increased in frontal cortex of 3- and 6-month-old APP/PS1 mice, they were significantly increased in frontal cortex of 9- and 12-month-old APP/PS1 mice when compared to their wild type littermates. In the hippocampus region, total nitrosylated protein levels were also significantly increased in 9- and 12-month-old APP/PS1 mice, but not in 3- and 6-month-old APP/PS1 mice (Fig. 3B).

Finally, we investigated nitrosylation of VAChT and VGLUT1 in frontal cortex and hippocampus of APP/PS1 mice. Although nitrosylation of VAChT was not increased in frontal cortex of 3-month-old APP/PS1 mice, it was significantly increased in frontal cortex of 6-, 9-, and 12-month-old APP/PS1 mice when compared to their wild type littermates (Fig. 4A). In contrast to frontal cortex region, nitrosylation of VAChT was only increased in hippocampus of 3-month-old APP/PS1 mice, but not in hippocampus of 6-, 9-, and 12-month-old APP/PS1 mice when compared to their wild type littermates (Fig. 4B). We also found that nitrosylation of VGLUT1 was also significantly increased in frontal cortex of 6-, 9-, and 12-month-old, but not in 3-month-old APP/PS1 mice when compared to their wild type littermates (Fig. 5A). Nitrosylation of VGLUT1 was also found in hippocampus of 3-, but not 6-, 9-, and 12-month-old APP/PS1 mice when compared to their wild type littermates (Fig. 5B).

DISCUSSION

In the current study, Morris water maze test revealed that the latency to reach platform area was significantly increased while the swimming crossings passing platform area were significantly decreased in 9- and 12-month-old APP/PS1 transgenic mice. This result is consistent with previous studies [24 –26], indicating that memory is impaired in 9- and 12-month-old APP/PS1 transgenic mice. We also found that nitrosylated protein levels were significantly increased in the frontal cortex and hippocampus of the 9- and 12-month-old APP/PS1 mice. Because previous studies have shown that Aβ was increased in APP/PS1 mice [26 –29], the current finding suggests that increased Aβ in these mice may further trigger protein nitrosylation. Further supporting this is our finding that nitrosylated protein levels were also increased in Aβ-treated cultured neuronal cells.

S-nitrosylation is induced by NO radicals. Many studies have shown that Aβ can increase NO production. For example, it was reported that exposure to oligomeric Aβ_1 - 42 led to a significant increase in NO production while nNOS inhibitor L-NAME completely eliminated Aβ-triggered NO production in cultured rat cerebrocortical neurons [29]. Aβ was also found to activate iNOS in glial cells, generating high levels of NO [30]. When Aβ_1 - 42 was injected into temporal cortex of rats, an increased nNOS expression in the same brain was detected [31]. Injection of Aβ_25 - 35 into rat temporal cortex not only caused a significant increase of iNOS expression but also reduced spatial memory in these rats [32]. In addition, expression of iNOS was found to be increased in hippocampus of APP/PS1 mice [33, 34]. These studies, together with our current finding, suggest that Aβ may activate NOS, subsequently increasing NO production, resulting in protein nitrosylation, which may contribute to memory deficiency in AD. Cholinergic deficiency and glutamate-mediated excitotoxicity have been recognized in the development of AD [35, 36]. VAChT and VGLUT1 are important in mediating acetylcholinergic and glutamatergic neurotransmission. Previously we have found that both VAChT and VGLUT1can be nitrosylated [21]. In the present study, we found that nitrosylation of VAChT and VGLUT1 was also increased in frontal cortex of 6-, 9-, and 12-month-old APP/PS1 mice, suggesting that nitrosylation may contribute to impairment of cholinergic system and glutamatergic system in AD.

Cholinergic deficits in AD mainly result from decreased acetylcholine and death of cholinergic neurons. Aβ has consistently been found to impair cholinergic neurotransmission. For example, intracerebroventricular injection with Aβ_1 - 40 has been found to decrease acetylcholine levels and synthesis enzyme choline acetyltransferase activity, but increase metabolism enzyme acetylcholine esterase activity in mouse hippocampus [37]. Chronic treatment with Aβ_25 - 35 has also been found to decrease acetylcholine release from rat frontal cortex and hippocampus [38]. Tg2576 mouse is an animal model for AD. In 9-11-month-old Tg2576 mice, not only memory is impaired, but also potassium-induced acetylcholine release in hippocampus is also decreased [39]. APP/PS1mice also show decreased expression of choline acetyltransferase in hippocampus [37]. These studies suggest deficiency of cholinergic system in AD. Previously we found that NO donor nitrosoglutathione inhibited vesicular uptake of ³[H] acetylcholine and this uptake could be reversed by either VAChT inhibitor vesamicol or reducing agent dithiothreitol in mouse brain [21], suggesting that nitrosylation of VAChT may inhibit vesicular acetylcholine uptake, impairing cholinergic neurotransmission. Increased nitrosylation of VAChT in brain of APP/PS1 mice found in the current study suggests that the nitrosylation process may also contribute to deficiency of cholinergic neurotransmission in AD.

Glutamate-induced excitotoxicity contributes significantly to the process of neurodegeneration in AD pathophysiology. Increased nitrosylation of VGLUT1 in APP/PS1 mice may also impair glutamatergic neurotransmission. We have found that NO radical inhibits vesicular glutamate uptake and this uptake can be reversed by reducing agent and VGLUT inhibitor [21]. Our results suggest that nitrosylation of VGLUT1 in APP/PS1 may inhibit vesicular glutamate uptake, subsequently causing accumulation of glutamate in cytosol. It is interesting that excitatory amino acid transporter 3 (EAAT3) has been found to reversely transport glutamate out of pre-synapse [40, 41]. EAAT3 is located in axon terminals. Under physiological conditions, extracellular glutamate can be uptaken by presynapse through EAAT3 and then re-uptaken by synaptic vesicles. However, under pathological conditions, EAAT3 can reversely transport glutamate and cause exitotoxicity [40]. It is possible that increased Aβ in APP/PS1 mice may induce nitrosylation of VGLUT1, resulting in inhibition of VGLUT1. Inhibited VGLUT1 can decrease vesicular uptake of glutamate and increase cytosolic glutamate levels. Accumulated cytosolic glutamate may be further transported reversely out of pre-synapse, resulting in the elevated extracellular glutamate levels. However, further study is needed to verify this suggestion.

In the present study, we also found that nitrosylation of VAChT and VGLUT1 was increased in hippocampus of 3-month-old but not in 6-, 9-, and 12-month-old APP/PS1 mice when compared to their littermates. It has been reported that neuronal nitric oxide synthase was significantly increased in hippocampus of 14-month-old mice when compared to 2-month-old mice [42], suggesting that nitric oxide production may be increased during the aging process. It is possible that aging-induced increase of nitrosylation in control mice may dilute differences of nitrosylation in hippocampus between control and APP/PS1 mice at late ages. Protein cysteines can be oxidatively modified not only by nitrosylation, but also by sulfenylation, sulfinylation, and sulfonylation [43]. It is also possible that these oxidative modifications rather than nitrosylation may occur at later ages. It will be interesting to study if sulfenylation, sulfinylation, and sulfonylation of VAChT and VGLUT1 are increased in hippocampus of older APP/PS1 mice. Our study also showed that nitrosylation of VAChT and VGLUT1 was increased in hippocampus but not in frontal cortex of 3-month-old APP/PS1 mice. Since free radical production is lower in early ages than later ages, this suggests that VAChT and VGLUT1 in hippocampus are more sensitive to modification by nitrosylation than in frontal cortex. Biological consequences of nitrosylation discrepancy at different ages in the same region or in a different region at the same age require further investigation. Nitrosylation can be reversed by small redox protein thioredoxin. It will be interesting to determine if delivery of thioredoxin into brain improves memory deficiency of APP/PS1 mice in the future.

Previous studies have shown that postmortem delay due to mild ischemic conditions after decapitation may affect the levels of protein modification, free thiols, and other biochemical factors. For example, postmortem delay of 30 minutes after decapitation was found to decrease tau protein phosphorylation in rat brain [44]. Although postmortem delay of 30 minutes had no effect on oxidized glutathione levels, this delay decreased total glutathione levels by 7% [45]. It has also been reported that total thiol, catalase, and glutathione reductase were significantly decreased in rat heart at postmortem delay of 1 hour [46]. A recent study using Fourier transform infrared spectroscopy and X-ray absorption spectroscopy has shown that even postmortem delay of 2 minutes can decrease thiol to disulfide ratio in rat cerebellum [47]. In the present study, the process of anesthesia, decapitation, and dissection took approximately 2.5 minutes. The postmortem delay of 2.5 minutes may increase protein nitrosylation, subsequently reduce the differences of nitrosylation between control and APP/PS1 mice that were present in vivo. In the future, a quicker isolation process which can reduce postmortem delay is required to verify our current findings.

In summary, we found in the present study that memory was impaired in 9- and 12-month-old APP/PS1 transgenic mice. We also found in these mice that total protein nitrosylation was increased in frontal cortex and hippocampus. Further nitrosylation of VAChT and VGLUT1 was increased in frontal cortex of these mice. Because previously we found that nitrosylation of VAChT and VGLUT1 can inhibit their transporter activities, these findings suggest that nitrosylation of VAChT and VGLUT1 may inhibit vesicular uptake of acetylcholine and glutamate, contributing to dysfunctional cholinergic and glutamatergic neurotransmission in AD. Inhibition of vesicular uptake of acetylcholine may further reduce acetylcholine synaptic release. Because axon terminal EAAT3 can reversely transport glutamate at pathological conditions, inhibition of vesicular uptake of glutamate may increase cytosolic glutamate levels that activate EAAT3 reverse transport process, subsequently causing exitotoxicity (Fig. 6).

Footnotes

ACKNOWLEDGMENTS

This work was supported by a discovery grant from Natural Sciences and Engineering Research Council of Canada (JFW) and an Establishment grant from Manitoba Health Research Council (JFW).

Authors’ disclosures available online ().

References

Kumar

, Walter

(2011) Phosphorylation of amyloid beta (Aβ) peptides-a trigger for formation of toxic aggregates in Alzheimer’s disease. Aging 3, 803–812.

Gozes

(2010) Tau pathology and future therapeutics. Curr Alzheimer Res 7, 685–686.

Chun

, Johnson

(2007) The role of tau phosphorylation and cleavage in neuronal cell death. Front Biosci 12, 733–756.

Sultana

, Butterfield

(2008) Alterations of some membrane transport proteins in Alzheimer’s disease: Role of amyloid beta-peptide. Mol Biosyst 4, 36–41.

Mesulam

(2004) The cholinergic lesion of Alzheimer’s disease: Pivotal factor or side show? Learn Mem 11, 43–49.

Sarter

, Hasselmo

, Bruno

, Givens

(2005) Unraveling the attentional functions of cortical cholinergic inputs: Interactions between signal-driven and cognitive modulation of signal detection. Brain Res Brain Rev 48, 98–111.

Bierer

, Haroutunian

, Gabriel

, Knott

, Carlin

, Purohit

, Perl

, Schmeidler

, Kanof

, Davis

(1995) Neurochemical correlates of dementia severity in Alzheimer’s disease: Relative importance of the cholinergic deficits. J Neurochem 64, 749–760.

Reinikainen

, Soininen

, Riekkinen

(1990) Neurotransmitter changes in Alzheimer’s disease: Implications to diagnostics and therapy. J Neurosci Res 27, 576–586.

Dodd

, Scott

, Westphalen

(1994) Excitotoxic mechanisms in the pathogenesis of dementia. Neurochem Int 25, 203–219.

10.

Butterfield

, Pocernich

(2003) The glutamatergic system and Alzheimer’s disease: Therapeutic implications. CNS Drugs 17, 641–652.

11.

Popp

, Fleming

, Busse

(1998) Pulsatile stretch in coronary arteries elicits release of endothelium-derived hyperpolarizing factor: A modulator of arterial compliance. Circ Res 82, 696–703.

12.

Ignarro

, Cirino

, Casini

, Napoli

(1999) Nitric oxide as a signaling molecule in the vascular system: An overview. J Cardiovasc Pharmacol 34, 879–886.

13.

Bredt

, Snyder

(1990) Isolation of nitric oxide synthetase a calmodulin- requiring enzyme. Proc Natl Acad Sci U S A 87, 682–685.

14.

Drew

, Leeuwenburgh

(2002) Aging and the role of reactive nitrogen species. Ann N Y Acad Sci 959, 66–81.

15.

Wink

, Miranda

, Espey

(2000) Effects of oxidative and nitrosative stress in cytotoxicity. Semin Perinatol 24, 20–23.

16.

Lei

, Pan

, Aggarwal

, Chen

, Hartman

, Sucher

, Lipton

(1992) Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex. Neuron 8, 1087–1099.

17.

Nakamura

, Tu

, Akhtar

, Sunic

, Okamoto

, Lipton

(2013) Aberrant protein s-nitrosylation in neurodegenerative diseases. Neuron 78, 596–614.

18.

Lipton

, Singel

, Stamler

(1994) Nitric oxide in the central nervous system. Prog Brain Res 103, 359–364.

19.

Stamler

, Toone

, Lipton

, Sucher

(1997) (S)NO Signals: Translocation, regulation and a concensus motif. Neuron 18, 691–696.

20.

Moriyama

, Yamamoto

(2004) Glutamatergic chemical transmission: Look! Here there, and anywhere. J Biochem 135, 155–163.

21.

Wang

, Zhou

, Leylek

, Tan

, Sun

, Parkinson

, Wang

(2015) Protein cysteine S-nitrosylation inhibits vesicular uptake of neurotransmitters. Neuroscience 311, 374–381.

22.

Holcomb

, Gordon

, McGowan

, Yu

, Benkovic

, Jantzen

, Wright

, Saad

, Mueller

, Morgan

, Sanders

, Zehr

, O’Campo

, Hardy

, Prada

, Eckman

, Younkin

, Hsiao

, Duff

(1998) Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes. Nat Med 4, 97–100.

23.

Bradford

(1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–252.

24.

, Du

, Pick

, Sui

, Brownlee

, Klann

(2012) Glucagon-like peptide-1 cleavage product GLP-1(9-36) amide rescues synaptic plasticity and memory deficits in Alzheimer’s disease model mice. J Neurosci 32, 13701–13708.

25.

Lalonde

, Kim

, Maxwell

, Fukuchi

(2005) Exploratory activity and spatial learning in 12-month-old APP(695)SWE/co+PS1/DeltaE9 mice with amyloid plaques. Neurosci Lett 390, 87–92.

26.

Zhang

, Hao

, Liu

, Zhang

, Lei

, Su

, Miao

, Li

(2011) Soluble Aβ levels correlate with cognitive deficits in the 12-month-old APPswe/PS1dE9 mouse model of Alzheimer’s disease. Behav Brain Res 222, 342–350.

27.

Zhu

, Shi

, Li

, Wang

, Zhang

, Tempier

, He

, Kong

, Wang

, Li

(2014) Quetiapine attenuates glial activation and proinflammatory cytokines in APP/PS1 transgenic mice via inhibition of nuclear factor-κB pathway. Int J Neuropsychopharmacol 18, pii: pyu022.

28.

Schmöle

, Lundt

, Ternes

, Albayram

, Ulas

, Schultze

, Bano

, Nicotera

, Alferink

, Zimmer

(2015) Cannabinoid receptor 2 deficiency results in reduced neuroinflammation in an Alzheimer’s disease mouse model. Neurobiol Aging 36, 710–719.

29.

Cetin

, Yazihan

, Dincer

, Akbulut

(2013) The effect of intracerebroventricular injection of beta amyloid peptide (1-42) on caspase-3 activity, lipid peroxidation, nitric oxide and NOS expression in young adult and aged rat brain. Turk Neurosurg 23, 144–150.

30.

Ayasolla

, Khan

, Singh

(2004) Inflammatory mediator and beta-amyloid (25-35)-induced ceramide generation and iNOS expression are inhibited by vitamin E. Free Radic Biol Med 37, 325–338.

31.

Molokanova

, Akhtar

, Sanz-Blasco

, Tu

, Piña-Crespo

, McKercher

, Lipton

(2014) Differential effects of synaptic and extrasynaptic NMDA receptors on Aβ-induced nitric oxide production in cerebrocortical neurons. J Neurosci 34, 5023–5028.

32.

Diaz

, Limon

, Chávez

, Zenteno

, Guevara

(2012) Aβ25-35 injection into the temporal cortex induces chronic inflammation that contributes to neurodegeneration and spatial memory impairment in rats. J Alzheimers Dis 30, 505–522.

33.

Kalifa

, Polston

, Allard

, Manaye

(2011) Distribution patterns of cannabinoid CB1 receptors in the hippocampus of APPswe/PS1ΔE9 double transgenic mice. Brain Res 1376, 94–100.

34.

Jimenez

, Baglietto-Vargas

, Caballero

, Moreno-Gonzalez

, Torres

, Sanchez-Varo

, Ruano

, Vizuete

, Gutierrez

, Vitorica

(2008) Inflammatory response in the hippocampus of PS1M146L/APP751SL mouse model of Alzheimer’s disease: Age-dependent switch in the microglial phenotype from alternative to classic. J Neurosci 28, 11650–11661.

35.

Auld

, Kornecook

, Bastianetto

, Quirion

(2002) Alzheimer’s disease and the basal forebrain cholinergic system: Relations to [beta]-amyloid peptides, cognition, and treatment strategies. Prog Neurobiol 68, 209–245.

36.

Butterfield

, Pocernich

(2003) The glutamatergic system and Alzheimer’s disease: Therapeutic implications. CNS Drugs 17, 641–652.

37.

Kim

, Shin

, Lee

, Choi

, Jung

, Cho

, Hwang

, Kim

, Han

, Chung

, Jang

, Rhim

, Kim

, Nah

(2015) Oral administration of gintonin attenuates cholinergic impairments by scopolamine, amyloid-β protein, and mouse model of Alzheimer’s disease. Mol Cells 38, 796–805.

38.

Olariu

, Tran

, Yamada

, Mizuno

, Hefco

, Nabeshima

(2001) Memory deficits and increased emotionality induced by beta-amyloid (25-35) are correlated with the reduced acetylcholine release and altered phorbol dibutyrate binding in the hippocampus. J Neural Transm (Vienna) 108, 1065–1079.

39.

Watanabe

, Yamagata

, Takasaki

, Sano

, Hayakawa

, Katsurabayashi

, Egashira

, Mishima

, Iwasaki

, Fujiwara

(2009) Decreased acetylcholine release is correlated to memory impairment in the Tg2576 transgenic mouse model of Alzheimer’s disease. Brain Res 1249, 222–228.

40.

Gebhardt

, Körner

, Heinemann

(2002) Delayed anoxic depolarizations in hippocampal neurons of mice lacking the excitatory amino acid carrier 1. Cereb Blood Flow Metab 22, 569–575.

41.

Vandenberg

, Ryan

(2013) Mechanisms of glutamate transport. Physiol Rev 93, 1621–1657.

42.

Adachi

, Abe

, Sasaki

, Kato

, Kasahara

, Araki

(2010) Role of inducible or neuronal nitric oxide synthase in neurogenesis of the dentate gyrus in aged mice. Metab Brain Dis 25, 419–424.

43.

Chung

, Wang

, Venkatraman

, Murray

, Van Eyk

(2013) Cysteine oxidative posttranslational modifications: Emerging regulation in the cardiovascular system. Circ Res 112, 382–392.

44.

Gärtner

, Janke

, Holzer

, Vanmechelen

, Arendt

(1998) Postmortem changes in the phosphorylation state of tau-protein in the rat brain. Neurobiol Aging 19, 535–543.

45.

Cooper

, Pulsinelli

, Duffy

(1980) Glutathione and ascorbate during ischemia and postischemic reperfusion in rat brain. J Neurochem 35, 1242–1245.

46.

Abo El-Noor

, Elhosary

, Khedr

, El-Desouky

(2016) Estimation of early postmortem interval through biochemical and pathological changes in rat heart and kidney. Am J Forensic Med Pathol 37, 40–46.

47.

Hackett

, Britz

, Paterson

, Nichol

, Pickering

, George

(2015) In situ biospectroscopic investigation of rapid ischemic and postmortem induced biochemical alterations in the rat brain. ACS Chem Neurosci 6, 226–238.