Cognitive Deficits in Neuropsychiatric Disorders: A Schizophrenia Model Associated with Neuronal Alpha7

Nicotinic Acetylcholine Receptor Localization

Savina Dine Kim

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AbstractCognitive deficits are prevalent symptoms in central nervous system disorders such as

Alzheimer’s disease, Parkinson’s disease, and schizophrenia. In schizophrenia, where cigarette

smoking occurs at a particularly high rate (~90%), it is thought that nicotine "self administration"

may ameliorate symptoms of attention deficits and thought disorder. Despite the well known

links between α7 nicotinic acetylcholine receptor (α7 nAChR) activation and cognitive

improvements, the mechanism(s) underlying this association are not known. This study aimed to

link the molecular level of α7 nAChR localization to its higher cognitive network level.

The alteration of α7nAChR localization by a valine to leucine polymorphism (V321L) of

Neuregulin-1 (Nrg1), a schizophrenia susceptibility gene, was observed. Using a Neuroblastoma-

2a cell line and immunofluorescence labeling, I found that V321L-Nrg1 decreases α7 nAChR

surface expression by ~50% but not its internal α7 pool, suggesting that Nrg1 affects receptor

insertion but not assembly. Next, signaling pathways that direct receptor trafficking were

investigated. V321L-Nrg1 triggers a ~30% decrease in PI3K signaling, suggesting the decrease

in surface α7nAChRs results from attenuated PI3K-Akt signaling.

Understanding how α7 nAChRs traffic to the axonal surface to modulate

neurotransmitters and maintain synaptic transmission is essential. It is a prerequisite for

synthesizing new treatments targeting cognition. This proposed model of α7 nAChR localization

raises the possibility of developing novel pharmacotherapeutic strategies aimed at modifying α7

nAChR expression in different brain regions. It will be of considerable interest to manipulate its

localization pathways to alter receptor properties and normalize cognitive deficiencies caused by

neuropsychiatric and neurodegenerative diseases.

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1. IntroductionCognitive deficits are prevalent in a variety of neuropsychiatric and neurodegenerative

disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), dementia and

schizophrenia. Unfortunately there are limited to no viable treatment options currently available,

thus research for novel therapies to improve cognitive function warrants investigation. Recent

studies have provided evidence targeting α7 nicotinic acetylcholine receptors (α7 nAChRs) to be

associated with this growing list of diseases [1,2].

To this day, surprisingly little is known about the specific roles of nAChRs in cognition,

partly due to the extremely limited understanding of its localization and function [2]. I aim to

investigate the molecular mechanisms by which α7 nAChRs become functional receptors to

modulate the release of neurotransmitters. It will target receptor localization beginning in the

nucleus for transcription to the axon for membrane insertion. This study hopes to enhance our

awareness of α7 nAChRs in order to advance research targeted to enhance cognition.

Cognition is a highly complex central nervous system (CNS) function which includes

domains such as learning, associative and working memory, attention and executive processes

[3,4]. Acetylcholine is an important participant in the maintenance of these functions for the

cholinergic system modulates GABAergic and glutamatergic synapses in neural circuits [5].

Evidence derived from psychopharmacological studies has revealed dysfunction of sensory

pathways and the “gating” of sensory information. This includes deficient P50 gating and pre-

pulse inhibition (PPI) in psychiatric patients, which were only slightly attenuated with drug use

[1,6]. The failure to inhibit these auditory event-evoked responses implicates cholinergic

dysfunction. This provides evidence to support the modulation of α7 nAChRs as a possible

treatment for these deficits [1-2,4].

Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the CNS.

These receptors regulate processes such as transmitter release, cell excitability and neuronal

integration, which are crucial for network operations and influence a number of physiological

functions, including neuronal development and synaptic plasticity [7]. Neuronal nAChRs belong

to a large superfamily of homologous receptors, known as the Cys-loop ion channel receptors,

which also include muscle-type nAChRs, anionic channels (e.g., gamma-amino-butyric [GABA]

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and glycine) and cationic channels (e.g., serotonin and acetylcholine). These membrane-bound

receptors comprise of a pentameric assembly formed from heteromeric or homomeric

combinations of various alpha (α2-α10) and beta (β2-β4) subunits (Fig. 1) [8].

One of the most abundant nAChR subunits in the mammalian brain is the α7 subunit [7].

Found differentially targeted to presynaptic sites, activated α7 nAChRs increases intraterminal

Ca2+ levels and facilitates the release of neurotransmitters such as dopamine, glutamate, g-

aminobutyric acid (GABA) and acetylcholine [10]. These α7 nAChRs are also the targets of

natural ligands and toxins including nicotine, the most widespread drug of abuse [11-12].

To date, the cellular distribution and molecular mechanisms in which these receptors

traffic to the surface to bind to neurotransmitters have received little attention. Given that the α7

subunit is widely distributed throughout the brain, understanding its changes in cellular

distribution through production, assembly, trafficking and recycling is essential [13].

Furthermore, the brain regions (hippocampus and prefrontal cortex) associated with cognitive

deficits seen in disorders such as schizophrenia and AD overlap with regions of α7 nAChR

expression, exhibiting the direct link between the receptors and cognition [14]. In order to

comprehend the onset of cognitive deficits which are similarly found in symptoms of several

neurological diseases, schizophrenia will be of particular interest to this study. This investigation

will serve as a model for investigating α7 nAChR expression and localization, as results can be

associated with other diseases.

Schizophrenia is a complex, heritable psychiatric disorder, affecting approximately 1% of

the world’s population, with patients occupying 40% of hospital beds set aside for long term care

and 25% of all hospital beds [15]. It is characterized by chronic positive symptoms

(hallucinations, delusions and thought disorders), negative symptoms (social withdrawal, apathy

Figure 1. Organization and structure of α7 nAChRs. (A) Topology of nAChR transmembrane subunit. (B) Pentameric assembly of nAChR subunits in an assembled receptor. (C) Subunit arrangement of homomeric α7 subtypes and location of Ach binding sites [9].

Figure 2. Type III Nrg1 back signaling. (A) B-site App Cleaving Enzyme/β-secretase (BACE) cleavage event which separates the epidermal growth factor domain (EGF) and the transmembrane domain (TM). (B) γ-secretase cleavage event between the TM and intracellular domain (ICD). (C) ICD nuclear translocation.

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and emotional blunting) and cognitive deficits [16]. Schizophrenia patients suffer from cognitive

symptoms such as the inability to focus due to the “flooding” of extraneous sensory stimuli

which overwhelm the patient’s ability to think coherently [4,10]. Poor cognitive functioning

contributes to poor role-functioning as well as high costs of care and rates of inpatient

hospitalization along with drastic losses in employability and productivity for family members

[10].

Another common characteristic among schizophrenics is their exceptionally high

smoking rate (60-90%) [3]. Post mortem studies have reported a decrease in the normally high

levels of α7 nAChRs in the brain of schizophrenic patients, suggesting the use of nicotine as a

form of self medication to compensate for their lack of α7 nAChRs and therefore, proper

synaptic transmission [1,14]. Current antipsychotics attempt to alleviate symptoms associated

with schizophrenia; however, many have not reached their final goal due to their poor efficacy

and/ or toxic effects which cause undesirable side effects such as seizures and depression

[10,12]. Given increasing evidence of the cholinergic system’s role in the cognitive symptoms

characterized in schizophrenics, α7 nAChRs have been proposed as a candidate for the

development of new medications targeting these impairments.

Although schizophrenia has been studied as a major neuropsychiatric disorder over the

past century, its causes and pathogenesis still remain unclear [15]. However, extensive fine-

mapping of the 8p locus has identified Neuregulin-1 (Nrg1), a gene with pleotropic roles in

neurodevelopment and plasticity, as a candidate gene for schizophrenia onset [17-18]. The Nrg1

gene encodes ligands for the ErbB receptor tyrosine kinases [19]. Through alternative splicing

and alternate promoter, it gives rise

to six structurally and functionally

distinct isoforms (I-VI). Types I, II,

and III are expressed in all

vertebrates; however, the type III

isoform (cysteine-rich domain

[CRD] containing) is unique in that

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its expression is mainly restricted to neurons and is involved in juxtacrine signaling and can also

act as receptors (Fig. 2) [20].

In particular, a single nucleotide polymorphism (SNP), a valine to leucine substitution at

residue 321 (V321L), has been implicated in schizophrenia susceptibility (Fig. 3). V321L results

in ineffective Type III Nrg1 processing, as a consequence of Nrg1 proteins failing to undergo γ-

secretase cleavage and ICD nuclear translocation [21-22]. The downstream effects of this

cleavage are thought to target the axon and regulate expression and trafficking of neuronal

presynaptic α7 nAChRs [11,23]. These results inspired this investigation to focus specifically on

the molecular mechanisms underlying the V321L effect on functional α7 nAChRs along axonal

projections.

The aim of this study was to link together the molecular level of α7 nAChR trafficking to

its higher cognitive network level. This was accomplished by investigating the trafficking events

and mechanisms in the biosynthetic pathway as well as those in endocytic pathway of the

receptors. This paper elucidates the role of α7 nAChRs in cognitive function by focusing on a

schizophrenia-associated Type III Nrg1 gene and its effect on α7 nAChR expression as a model

for other disorders. Comprehending α7 nAChR localization in a particular neuronal pathway is a

prerequisite for understanding the role of the receptors in cognitive deficits, and for the rational

design of new drugs to target these symptoms. The attenuation or reversal of these disease

symptoms gives hope towards improved cognition and increased quality of life for

neuropsychiatric patients.

Figure 3. Forms of Type III Nrg1 (A) WT-Type III Nrg1 form (B) Valine to leucine substitution at amino acid residue 321 in the TM (V321L-Type III Nrg1).

Figure 4. Methodology outline. N2a cells will be transduced with AAV, treated with ErbB2/ErbB4 then tested to investigate Nrg1 signaling, α7 nAChR surface and total expression, and the signaling pathways which regulate receptor localization.

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2. Methodology

2.1 Neuronal cultures

The mouse neuroblastoma-2a (N2a) cell line was obtained from the American Tissue

Culture Collection (ATCC). N2a cells were plated on 18 mm glass coverslips (precoated with 1

mg/ ml poly-d-lysine and 100 ug/ ml laminin) at 30% confluence to allow for visualization of

individual neurons. Cells were maintained in DMEM medium (GIBCO) supplemented with 10%

fetal bovine serum (FBS), 1 mM L-glutamine (GIBCO), 50 U/ ml each penicillin and

streptomycin (Sigma-Aldrich). For differentiation, N2a culture media was replaced with serum-

starved media (1% serum) for 1 week before transduction. Cultures were maintained at 37°C in

an atmosphere containing 5% CO2.

2.2 Generation of virus and transduction

Viruses were generated using the AAV Helper System. The provided AAV-293 cell line

(derived from HEK293 cells) was cultured until 40-50% confluence was reached. Cells were co-

transfected with AAV plasmid expression vectors using standard calcium phosphate methods.

The three vectors used were the following: AAV-GFP (pHelper, pAAV-RC2, pAAV-IRES-

hrGFP), AAV-WT (pHelper, pAAV-RC2, pAAV-HA-tagged WT Nrg1 [tag located in ICD])

and AAV-V321L (pHelper, pAAV-RC2, pAAV-V321L Nrg1) Vectors pHelper, pRC and pAAV

expression vector were co-transfected at a 1:1:1 ratio (Fig. 5). The pAAV-RC plasmid contains the rep

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and cap genes, encoding viral capsid structural proteins. The pHelper plasmid contains the subset of

adenovirus genes, VA, E2A and E4, which are necessary for the production of high-titer AAVs. These

cells also provide the adenovirus proteins E1A and E1B, which are also necessary for AAV production.

Cells and media were harvested 72 h post transfection. Four freeze-thaw cycles in a dry

ice/ ethanol bath and a 37°C bath were performed. The supernatant was collected as crude AAV

lysate. The N2a were transduced with equal quantities of HA-tagged [located in ICD] Type III

Nrg1. A 100 μl of virus medium (virus and DMEM growth medium [FBS, glutamine and penn:

strep]) was added to each well. After 4 h, 400 μl of fresh media were added and after 2-3 days,

another 500 μl of fresh media were added. Cells were incubated in virus medium at 37°C/ 5%

CO2 for 7-10 days prior to experimentation.

2.3 Protein Expression

Soluble ErbBs were prepared by transfecting HEK293 cells using standard calcium

phosphate methods [24] with plasmids encoding chimeras between human ErbB2 or ErbB4 and

the Fc domain of human IgG [20]. Conditioned media were harvested 5-7 days post transfection

and purified using protein A columns and eluted with 0.5 M citric acid into tubes containing 1 M

tris pH 8.0. The eluted proteins were subsequently dialyzed against phosphate-buffered saline

(PBS) medium and concentrated using Centri-prep-30 filters (Amicon). Protein concentrations

were determined by a bicinchoninic acid (BCA) protein assay. Soluble ErbB2-ECD and ErbB4-

ECD were used to treat N2a neurons at 2 nM final concentrations for 16 h prior to

experimentation.

2.4 Reagents

Where indicated, the following reagents were applied to the cells: 2 nM soluble ErbB2-

ECD or ErbB4-ECD [11], 50 nM Wortmannin (Invitrogen), 500 nM Akt Inhibitor IV

Figure 5. Adeno-Associated Virus (AAV) Creation. HEK293 cells were co-transfected with three vectors: pAAV expression, pHelper and pRC at a 1:1:1 ratio for AAV production.

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(Calbiochem), 500 nM phenylarsine oxide (Sigma-Aldrich). Inhibitors were added to the media

for 45 min prior to soluble ErbBs addition for an additional hour.

2.5 Immunostaining and fluorescent visualization

For standard immunodetection, neurons were fixed with 4% Paraformaldehyde (PFA) for

15 min at RT, permeabilized with 0.25% Triton X-100/ PBS for 5 min at RT, blocked with 5%

normal donkey serum (NDS)/ PBS and then incubated with primary antibodies for 2 h at 37°C.

Primary antibodies used included the following: anti-HA Type III Nrg1 Antibody (1:1000;

Covance), anti-Pan-Axonal Neurofilament Marker Monoclonal Antibody (1:500; Covance) and

anti-PI3,4,5P3 (1:50; Echelon). Neurons were washed and incubated in secondary antibodies

conjugated to Alexa Fluor 350 (1:1000; Invitrogen), Alexa Fluor 594 (1:1000; Invitrogen) or

Biotin-SP-conjugated IgM antibodies (1:500; Jackson ImmunoResearch Laboratories)/

Strepavidin (1:1000; Molecular Probles) for 1 h at 37°C. Slips were mounted using Anti-Fade

Fluoromount-G (SouthernBiotech) and images were captured using a confocal microscope (Axio

Imager; Carl Zeiss, Inc. and Olympus FluoView 1000 Confocal) at 60x with oil. ImageJ 1.44

was used for image quantification and analysis. Brightness and contrast were adjusted using

Photoshop software (Version 7.0; Adobe)

To label surface α7 nAChRs, N2a neurons were stained with Alexa Fluor 594-conjugated

α-bungarotoxin (αBgTx-594) (1:1000; Invitrogen) for 30 min at 37°C prior to fixation and

permeabilization. To label total α7 nAChR protein, cells were stained with αBgTx-488 after

permeabilization and brief fixation. Staining was followed by primary and secondary antibody

incubation. Surface fluorescence intensity and area were measured along the NF-positive axons

using ImageJ. The lengths of axonal processes were measured by manually tracing the NF-

positive axons. Surface quantification was determined by dividing the intensity arbitrary unit

(AU) value by the area (3.040 pixels/ 1 μm).

2.6 Analysis of gene expression

Prior to RNA isolation, N2a were incubated with either Wortmannin (50 nM) or Akt

Inhibitor (500 nM) for 45 min. ErbB4 (2 nM) were added to the wells for an additional hour.

Reverse transcription and qRT-PCR were performed using primers detecting the CHRNA7 gene.

Housekeeping gene GAPDH was used as a control for normalization.

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3. Results3.1 Valine at residue 321 (V321) is required for Type III Nrg1 back signaling to induce

intracellular domain nuclear translocation

Type III Nrg 1 acts as a bidirectional signaling molecule [17]. Upon stimulation, Nrg1

undergoes γ-secretase-dependent intramembranous cleavage, inducing intracellular domain

(ICD) nuclear translocation [20]. To determine if V321L-Nrg1 contributes to defective Nrg1

receptor signaling via the ICD, WT-Nrg1 ICD and V321L-Nrg1 ICD levels in the nucleus were

compared (Fig. 6 A). In order to initiate back signaling, cells were treated with either the

extracellular domain (ECD) of ErbB2 (B2-ECD; control) or ErbB4 (B4-ECD) prior to

experimentation. B4-ECD, unlike B2-ECD, binds with high affinity to the EGF-domain of Nrg1,

triggering back signaling to occur [24].

Figure 6. Valine at residue 321 is required for Type III Nrg1 back signaling to induce ICD nuclear translocation. (A) Confocal images were collected using N2a transduced with AAVs expressing HA-tagged [ICD tag] WT-Nrg1 or V321L-Nrg1. Cells were incubated with Anti-HA Type III Nrg1 and stained with DAPI (nuclei). Images were obtained with a 60x oil objective. Bar, 10µm. (B) Quantification of Type III Nrg1 ICD fluorescence intensity in the nuclei, within the DAPI stained area. Samples without B4-ECD treatment (-) were treated with B2-ECD as a control. The graph shows means ± SEM. Statistical significance was determined using a student’s t-test. *, P < 0.0001.

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Results indicated a ~1.8 fold increase of ICD fluorescence in the nuclei of B4-ECD

treated N2a compared to B2-ECD controls (WT B2-ECD [control], 12.95 ± 0.58 AU; WT B4-

ECD, 24.13 ± 1.29 AU; V321L B2-ECD [control], 5.98 ± 0.47 AU; V321L B4-ECD, 12.48 ±

0.58 AU; Fig. 6 B). Initial activation of Nrg1 back signaling is required for the valine-regulated

ICD translocation. B4-ECD treatment also led to a ~50% decrease of Nrg1 ICD in the V321L

N2a compared to WT N2a (Fig. 6 B). This suggests that the V321 residue is important for Nrg1-

ICD translocation.

3.2 Decreased axonal α7 nAChR surface expression results from V321L-Type III Nrg

To further explore the possibility that defective Nrg1 processing may alter α7 nAChR

numbers, axonal surface expression was examined. N2a were treated with ErbB4 and labeled

with αBgTx-594 before fixation and permeabilization. αBgTx is a selective antagonist of α7

nAChRs [25]. Anti-HA Type III Nrg1 and anti-neurofilament (NF) proteins were applied to

allow visualization of α7 nAChRs relative to the neuron location (Fig. 7 A).

Figure 7. Valine to leucine substitution results in decreased axonal α7 nAChR surface expression. (A) Confocal images were collected from WT and V321L transduced N2a treated with either B2-ECD (control) or B4-ECD for 16 hours.

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Quantification of αBgTx-594 fluorescence along NF-positive axons revealed a ~50%

decrease in α7 nAChR surface expression in V321L N2a compared to WT N2a (WT B4-ECD,

4.16 ± 0.44 AU; V321l B4-ECD, 1.98 ± 0.36 AU; Fig. 7 B). In addition to the leucine

substitution, ErbB tyrosine kinase signaling has been shown to also alter α7 nAChR levels [11].

To determine whether the Nrg1-ICD translocation pathway alone is sufficient to regulate

receptor trafficking, membrane targeting of α7 nAChRs in response to either B2-ECD (control)

or B4-ECD were also assessed.

Treating WT N2a with B4-ECD led to a ~1.6 fold increase in α7 nAChR surface

expression compared to B2-ECD treated controls (from 2.43 ± 0.84 AU in control cultures to

4.16 ± 0.44 AU; Fig. 7 C). When I repeated this experiment in V321L N2a, there was no

significant change in α7 nAChR surface expression between the control and B4-ECD treated

cultures (B2-ECD [control], 1.79 ± 0.24 AU; B4-ECD, 1.98 ± 0.36 AU; Fig. 7 C). Collectively

these results indicate that V321 is necessary for α7 nAChR membrane targeting.

3.3 Decrease in surface α7 nAChR expression is due to decreased membrane insertion

Prior studies have shown that an increase in endocytosis, recycling of receptors, can be

another cause of the decreased receptor expression [11]. To assess whether the decrease in α7

nAChR surface expression in V321L N2a is attributable to decreased membrane insertion, a

pharmacological inhibitor of endocytosis, phenylarsine oxide (PAO) was used.

Figure 8. Valine to leucine substitution decreases α7 nAChR surface expression in the absence of endocytosis. (A) Quantification of aBgTx-594 for surface α7 nAChR fluorescence intensity with and without PAO treatment. The graph shows means ± SEM. Statistical significance was determined using a student’s t-test. *, P < 0.0001. (B) WT to V321L surface α7 nAChR ratio with and without PAO treatment. The graph shows means ± SEM. No statistical significance was determined between the non-treated and treated N2a.

Images were obtained with a 60x oil objective. Bar, 10µm. (B) Intensity of WT + B4-ECD and V321L + B4-ECD images are shown in pseudocolour in the lower panels (blue, low values (0=lowest); white, high values (255=highest). (C) Quantification of aBgTx -594 surface fluorescence intensity on NF-positive axons. Samples without B4-ECD (-) treatment were treated with B2-ECD as control cultures. The graph shows means ± SEM. Statistical significance was determined using a student’s t-test. *, P < 0.05; **, P < 0.0001.

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After PAO treatment, the surface α7 nAChR expression was quantified. PAO treatment

induced a ~2-fold increase in the number of receptors on the axon surface (from 4.16 ± 0.44 AU

under WT control conditions to 10.49 ± 0.81 AU after PAO treatment and from 1.98 ± 0.36 AU

under V321L control conditions to 3.84 ± 0.41 AU after PAO treatment; Fig. 8 A) However, the

ratio of WT to V321L surface expression remained constant between N2a treated with and

without PAO (2.10 ± 0.40 under control conditions to 2.73 ± 0.61 after PAO treatment; Fig. 8

B). These results confirm that the decreased surface α7 nAChR expression in V321L-Nrg1 N2a

is due to decreased membrane insertion and not increased endocytosis.

3.4 Total internal pool of α7 nAChRs is not altered by V321L-Type III Nrg1

The next question I asked was whether the V321L-Nrg1 only alters surface expression of

α7 nAChRs or also total α7 subunit protein levels inside the neuron. To label the internal pool of

α7 nAChRs, B4-ECD treated N2a were labeled for surface α7 nAChRs with αBgTx-594 and

total pool of α7 nAChRs with αBgTx-488 (Fig. 9 A).

Figure 9. Valine to leucine substitution does not significantly alter total α7 nAChR internal pool levels. (A) Confocal images were collected from WT and V321L N2a treated with B4-ECD for 16 hours. N2a were labeled for surface and total α7 nAChRs using aBgTx-594 and αBgTx-488 respectively. Images were obtained with a 60x oil objective. Bar, 10µm. (B) Quantification of aBgTx-488 for total α7 nAChR fluorescence intensity. The graph shows means ± SEM. No statistical significance was determined between the WT and V321L N2a using a student’s t-test. (C) Surface to total α7 nAChR quantification ratio. The graph shows means ± SEM. No statistical significance was determined between the WT and V321L N2a for surface to total α7 nAChR ratios.

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While V321L-Nrg1decreases surface α7 nAChRs, an overall change in total α7 nAChRs

was not detected. The internal pool level of α7 nAChRs was not significantly different between

the WT and V321L N2a (WT, 6.26 ± 1.05 AU; V321L, 5.46 ± 1.38 AU; Fig. 9 B). In addition,

the ratio of surface α7 nAChRs to total internal pool levels of α7 nAChRs was also not

significantly different (WT, 0.41 ± 0.04; V321L, 0.31 ± 0.04; Fig. 9 C).

These results, combined with surface α7 nAChR expression in WT and V321L neurons

(Fig. 7 C) and PAO results (Fig. 8), indicates that V321L-Nrg1 targets only the insertion of α7

nAChRs to the axon surface, not the total production of α7 protein.

3.5 Disruption of PI3K signaling results from V321L-Type III Nrg1

Stimulated by the findings that α7 nAChR expression is affected by V321L-Nrg1, my

next objective was to test for signaling pathways downstream of Nrg1 which direct the receptor

membrane insertion. The phosphoinositide 3-kinases (PI3K) pathway, a major regulator of

membrane protein trafficking, has been shown to be regulated by Nrg1 [11]. However, the

mechanism by which this process occurs is unknown. To delineate the signaling pathways

involved in α7 nAChR membrane targeting, I began by focusing on PI3K.

Figure 10. Valine to leucine mutation results in decreased axonal PIP3 expression; however, somatic expression remains constant. (A) Confocal images were collected from WT and V321L transduced N2a treated with B4-ECD for 16 hours. N2a were incubated with anti-PIP3 antibody and stained with DAPI. Images were obtained with a 60x oil objective. Bar, 10µm. (B) Quantification of PIP3 fluorescence intensity in the axon. A significant decrease of PIP3 expression was found in V321L N2a. The graph shows means ± SEM. *, P < 0.05. (C) Quantification of PIP3 fluorescence intensity in the N2a soma. There was no significant difference between the WT and V321L N2a.

Figure 11. Gel using α7 CHRNA7 primers. Messenger RNA was collected in N2a without transduction, WT-Nrg1 expressing and V321L-Nrg1 expressing N2a cells. Associated DNA was analyzed using α7 primers. N2a with and without transduction endogenously express α7 nAChR mRNA.

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In order to observe changes in PI3K signaling, the PtdIns 3K product,

phosphatidylinositol 3,4,5 trisphosphate (PIP3) was quantified (Fig. 10 A). In V321L N2a, a

significant decrease of ~30% in PIP3 expression was detected along the axon (WT, 8.47 ± 1.02

AU; V321L, 5.68 ± 0.88 AU; Fig. 10 B). However, this decrease was not present in the soma

(WT, 12.98 ± 1.33 AU; V321L, 12.09 ± 1.48 AU; Fig. 10 C). This finding brings arise new

questions; whether Nrg1 only affects signaling pathways found on the axon requires further

investigation. Thus the evidence above shows that the decrease in surface α7 nAChRs results

from attenuated PI3K signaling along the axon.

3.6 Does V321L-Type III Nrg1 affect α7 nAChR gene expression?

The observations that Nrg1 gene disruption results in the loss of surface α7 nAChRs, led

me to ask whether the effects of V321L-Nrg1 begins at the gene level. An additional test of

whether V321L-Nrg1 also affects α7 mRNA transcript expression was undertaken. N2a

endogenously express the α7 gene CHRNA7; however, at extremely low levels (Fig. 11).

This experiment was designed to test the gene expression in

N2a with and without B4-ECD treatment. Next, in order to

further investigate the signaling pathways involved in α7

nAChR membrane targeting, I individually tested the effects

of the PI3K and Akt pathway, which is activated

downstream of PI3K and regulates gene expression [26].

N2a were pretreated with 50 nM of PI3K inhibitor

wortmannin and 500 nM of Akt Inhibitor IV. When qRT-

PCR was used to quantify gene expression levels using N2a,

B4-ECD treated N2a, WM treated N2a and Akt-Inhibitor

treated N2a, complete data could not be retrieved. Numerous trials have revealed that N2a do not

express significant amount of α7 mRNA for quantification. Fig. 11 shows a gel of N2a

expressing WT-Nrg1 or V321L-Nrg1 as well as N2a without transduction. This image was only

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able to be assembled after three 35 PCR cycles of amplification, the first two cycles yielding a

gel with little to no visible bands. Currently, this experiment is being repeated using ventral

hippocampal primary neurons to yield better results. Collectively, these results will indicate if

V321L-Nrg1 alters α7 nAChR localization beginning in the nucleus and determine if signaling

pathways such as PI3K or Akt are associated with changes in gene expression.

4. DiscussionThe data from the present study confirm and extend earlier findings indicating that α7

nAChR localization to the axonal surface is regulated by Type III Nrg1, acting as a receptor for

ErbB4 [11,23]. The findings demonstrate that the valine to leucine polymorphism on reside 321

impairs γ-secretase-mediated intramembranous cleavage preventing ICD nuclear localization.

This mutation impairs proper membrane insertion of α7 nAChRs by impeding the activation of a

local PI3K signaling pathway and therefore the Akt pathway. It is striking that a schizophrenia-

susceptibility genetic mutation found in the TM of Nrg1 is essential for the entire trafficking

process beginning at mRNA transcription, α7 receptor assembly and eventually to α7 axonal

membrane targeting. These results expand the repertoire of identified mechanisms by which

Nrg1 signaling contributes to the establishment and maturation of functional presynaptic

terminals [21]. In summary, this study demonstrates that the V321L-Nrg1 mutation affects the

number of presynaptic receptors which modulate the release of a variety of neurotransmitters and

outlines the pathways which regulate its localization down the axon (Fig. 12).

The V321L mutation dramatically

decreases the number of surface receptors on

presynaptic terminals. Using PAO, I

confirmed that Nrg1 back signaling plays a

key role in the insertion of these receptors to

the membrane. The number of α7 nAChRs

present on the membrane surface to be

activated is imperative for they control the

Figure 12. Proposed model of a7 nAChR localization including signaling pathways involved (GBRC: GABAergic receptor/ GLUR: glutamatergic receptor).

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excitatory and inhibitory actions of GABA and glutamate, which also play key roles in learning

and memory [1,5].

In contrast to the insertion of receptors, the early stages of assembling α7 subunits is not

mediated by Nrg1 back signaling. V321L-Nrg1 was found to

not affect the total internal pool of α7 protein. Because αBgTx

binding requires at least partial assembly of α7 subunits into

pentamers [25], α7 nAChRs are being synthesized into functional receptors in the axon, shown

by fluorescing internal pools of α7 nAChRs. This experiment reveals the possibility that α7

subunits are not completely assembled and functional until they are inserted onto the membrane.

To gain more insight into the dynamics of regulated α7 nAChR localization, I targeted

the PI3K signaling pathway because PI3K and its product PIP3 are ubiquitous signaling

molecules that link cell surface receptors to intracellular downstream effects [26]. My results

show that this change in functional receptors on the surface may be accomplished by debilitating

the PI3K pathway on the axon, shown by the diminished levels of PIP3 on the V321L N2a axons.

However, this decrease was not present in the soma, revealing a possibility that Nrg1 back

signaling may only target the axon.

My results have also shown that V321L-Nrg1 affects back signaling by impeding the γ-

secretase cleavage for ICD translocation; demonstrating that the ICD, which lacks known

functional motifs, may mediate protein to protein interactions for PI3K activation. This raises the

intriguing possibility that altered Nrg1 intramembranous processing may support recent

speculations that receptor Nrg1 acts through Src, a non-receptor tyrosine kinase, which may

phosphorylate with ICD interaction and activate PI3K. Additionally, because the Akt pathway is

identified as a downstream target of PI3K, the inhibition of the PI3K pathway will then also lead

to an inhibition of Akt, which plays a key role in mediating signaling for cell growth and gene

regulation [17]. The lack of total α7 nAChR internal pool change between the WT and V321L

N2a further supports the notion that PI3K-Akt pathway only controls the insertion of receptors to

the surface and not assembly of functional receptors. The PI3K-Akt activity will increase our

understanding of the molecular mechanisms that regulate the localization of α7 nAChRs to

advance studies of presynaptic α7 nAChR targeting.

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Together, these findings support the proposal that normal levels of α7 nAChR expression

require presynaptic Nrg1 back signaling, which is impaired in the schizophrenia-susceptibility

polymorphism V321L. Clearly, these results are distinct from demonstrations of Nrg1

bidirectional signaling. My study specifically focused on the γ-secretase cleavage portion of

Nrg1 back signaling to understand its role in α7 nAChR localization, from the process of

synthesis down to the insertion of preformed receptors. It delineates the molecular details of how

Nrg1 communicates with PI3K then Akt to regulate α7 nAChR insertion, which were previously

unclear. Additionally, these findings support the proposal that genetic modifications of Nrg1

mediated-signaling in presynaptic inputs change the profile of nAChRs, which thereby alters the

temporal profile of responses to neurotransmitters, agonists and antagonists [23]. Because α7

nAChRs play a major role in regulating neurotransmitter release such as GABA and glutamate,

altering its temporal profile may lead to deficits in sensory gating by altering these transmissions.

Recent work has revealed that glutamatergic transmission from the ventral hippocampus (vHipp)

to the nucleus accumbens shell (nAcc) is believed to be involved in the regulation of sensory

gating and PPI. This further explains the dramatic increase of smoking rates in schizophrenics,

supporting the “self-medication hypothesis” which states that the use of nicotine may be a form

of self-therapy for cognitive improvement [3,12].

There are also interesting parallels between my results and current pathophysiological

studies. Post mortem studies have revealed decreased expression of α7 nAChRs in several brain

regions, such as the hippocampus, thalamic reticular nucleus and prefrontal cortex in

schizophrenia patients [14]. These brain regions are also associated with risk alleles at the Nrg1

locus [11,27]. It is also important to note that these same regions are critically involved in

cognitive function and are tightly regulated by cholinergic projections from forebrain regions [1-

3]. Disruption of this function results in cognitive impairment in day-to-day tasks involving

working memory and attention. In addition, genetic linkage studies provide increasing evidence

identifying both the Nrg1 and α7 nAChR CHRNA7 gene as major susceptibility genes for

schizophrenia [10,22].

In summary, the working model of α7 nAChR localization shown through this study in

Fig. 12 outlines the proposed signaling pathways involved. The findings establish a link between

the valine 321, which is required for Nrg1 proteolytic cleavage and nuclear translocation, to α7

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nAChRs. By distinguishing the downstream effects of schizophrenia-associated mutation

V321L, this work sets the stage for further exploration of the finely tuned functionality of α7

nAChR receptors. Its associated signaling pathways can be used as treatment targets for

cognitive deficits found similarly among various neuropsychiatric disorders.

5. ConclusionTo the best of my knowledge, this is the first study to examine the effect of a Nrg1

polymorphism implicated in the etiopathogenesis of schizophrenia on α7 nAChR expression and

localization. Insight into the implication of nAChRs in schizophrenia can serve as the first step to

understanding their neurotransmitter receptor involvement in other neurological and psychiatric

diseases which consist of similar cognitive deficits found in schizophrenia. CNS disorders

involving a modification of α7 nAChRs include Tourette’s syndrome, ADHD, autism,

depression, and the neurodegenerative Alzheimer’s and Parkinson’s diseases [7,16]. Most

notably, the present study creates a working model of α7 nAChR localization, which indicates

that the same signaling pathways could operate in different types of neurons expressing Nrg1

throughout the brain. Because the main localization of nicotinic acetylcholine receptors is on

presynaptic structures that have a modulatory role in neurotransmission, a change in receptor

number can lead to drastic consequences on the modulation of synaptic transmission and

plasticity, which also play major roles for proper cognition.

Understanding the molecular mechanisms by which α7 nAChRs localize to the surface to

be activated and control the release of inhibitory and excitatory neurotransmitters are a

prerequisite for understanding the rational design of new drugs. Currently, most pharmacological

studies have focused on biophysical properties of the receptors [7,14]; however this study shows

the process of receptor synthesis and trafficking. Knowledge of receptor localization raises the

possibility of developing novel strategies aimed at modifying nAChR expression on different

neurotransmitter synapses and controlling transmitter release by targeting the specific pathways

which regulate its expression such as PI3K and Akt.

It will be of considerable interest to manipulate the Nrg1 ICD nuclear signaling pathway

to regulate α7 nAChRs on the axonal membrane surface, influencing receptor properties. For

instance, in patients with genetically inherited neuropsychiatric disorders which alter receptor

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properties, specific pathways such as PI3K or Akt that regulate its localization, as described in

my study, can be targeted. The main possibility is that enough receptors are being synthesized;

however, receptors are not being transferred to effective areas on the neuron. Instead of

manipulating the gene, researchers can specify certain signaling pathways through treatment

activation and deactivation to normalize the issue. This would be a more beneficial approach

than manipulating Nrg1, which regulates cell-cell interactions and is implicated in functions

ranging from maintenance of synapses to receptor expression [17]. It is not often that genetic

disorders can be cured through the use of pharmacology. However, my study holds great promise

in developing novel pharmaceutical strategies aimed at modifying α7 nAChR expression in

different brain and cell domains based on the molecular mechanisms which give rise to their

functionality. Insight into the implication of α7 nAChRs through a schizophrenia model can

serve as a viable approach to treating cognitive deficits that are hallmarks of neuropsychiatric

and neurodegenerative disorders.

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