Kanvara Kitiyadisai (1313168) | 6BBB0321 Biochemistry and

Molecular Genetics Library Project B | March 24, 2016

Will identification of UBE3A substrates allow therapeutic

intervention in Angelman Syndrome

SUBMITTED AS PART OF THE DEGREE OF B.SC. MOLECULAR GENETICS,

KING’S COLLEGE LONDON

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Contents

Section 1: Introduction to Angelman Syndrome

1.1) Causes and Symptoms

1.2) Imprinting and Prader-Willi syndrome

1.3) UBE3A protein and its putative role

Section 2: Neural substrates of UBE3A

2.1) Pbl/Ect2

2.1.1) Identification of Pbl as a UBE3A substrate

2.1.2) Function of Pbl and Ect2

2.1.3) Ect2 role in neuronal morphological differentiation

2.1.4) Ect2 and its association with autism in AS

2.2) GAT1

2.2.1) Identification of GAT1 and UBE3A interaction

2.2.2) Function of GAT1 and UBE3A interaction

2.2.3) GAT1 and its association with motor dysfunction in AS

2.3) SK2 Channel

2.3.1) Identification of SK2 channel as a UBE3A substrate

2.3.2) Function of SK2 channel in long-term potentiation

2.3.3) SK2 channel and its association with learning and memory in AS

2.4) Arc

2.4.1) Identification of Arc as a UBE3A substrate

2.4.2) The role of Arc in synaptic AMPA receptor trafficking

2.4.3) Arc and its association with cognitive dysfunction in AS

Section 3: Therapeutic potential in Angelman Syndrome

3.1) Current treatment

3.2) Potential therapy

3.2.1) GAT1 inhibitor improves learning and motor dysfunction in mice

3.2.2) Blocking SK channels enhances contextual learning and memory in AS

mice model

3.2.3) Reducing Arc expression improves seizures in AS mice model

3.3) Future work

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AS Research Timeline (Foundation for

Angelman Syndrome Therapeutics)

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Abstract

Angelman Syndrome (AS) is a genetic disorder resulting from a mutation in the

maternally imprinted UBE3A gene on chromosome 15. Symptoms of AS include speech

impairments, motor dysfunction, seizures and excessive laughter etc. Most AS patients

have microdeletions in the UBE3A gene, which encodes an E3 ubiquitin ligase. This E3

ubiquitin ligase functions to add ubiquitin molecules onto its substrates, which are

thereby targeted for degradation by the proteasome. Therefore, in AS patients, this non-

functional E3 ubiquitin ligase will lead to an increased expression of certain substrates

which are supposed to be ubiquitinated. In this project we explore the identification and

function of these identified neural UBE3A substrates (Pbl/Ect2, Arc, GAT1, SK2), and

how an increase in these substrate levels could possibly contribute to the neurological

dysfunction seen in AS patients. Even though there are currently no effective therapy for

AS, current research for AS therapies have focused on development of drugs targeting

these UBE3A substrates and the results displayed in this paper shows promising potential

for AS therapy in the imminent future.

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Abbreviations

AS – Angelman Syndrome

ASD – Autism Spectrum Disorder

BP – Breakpoints

Ect2 – Epithelial cell transforming 2

EPSP – Excitatory post-synaptic potential

GABA – Gamma-Aminobutyric acid

GABA-AR – GABA type-A receptors

GAT1 – GABA transporter 1

GEF – Guanine exchange factor

HS – Heatshock

IC – Imprinting Center

IEG – Immediate-early genes

LTD – Long-term depression

LTP – Long-term potentiation

Pbl – Pebble (Drosophila)

PWS – Prader-Willi Syndrome

shRNA – short hairpin RNA

SK channel – Small conductance calcium-activated potassium channel

SVZ – Subventricular zone

UAS – Upstream activating sequence

UBD – Ubiquitin binding domain

UPD – Uniparental disomy

VZ – Ventricular zone

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Section 1: Introduction to Angelman Syndrome

1.1) Causes and Symptoms

Angelman Syndrome (AS) is a rare neurodevelopmental disorder first identified in 1965

(Angelman, 2008) that affects approximately 1 in 15000 individuals. The most common

characteristics of AS patients include speech impairments, severe intellectual disability,

excessive laughter, happy demeanour, seizures and motor dysfunction (Margolis et al.,

2015). Some symptoms such as ataxia and microcephaly are only present in more severe

cases of AS. The severity of AS can vary as far as in terms of how many words a child

can speak (1-3 words in severe cases and 10-20 in less severe cases) to patients who may

lose their ability to walk. AS is often diagnosed in childhood between the ages of 3 to 7

years when neurodevelopmental abnormalities start to develop. There are no dominant

physical or craniofacial characteristics that define the disorder, and it is therefore often

confused with patients with autism spectrum disorder (ASD) (Williams et al., 1995).

AS arises due to a disruption of an imprinted gene UBE3A on chromosome 15. UBE3A is

expressed only from the maternal copy in the brain, whereas expression in other tissues

are biallelic. The AS gene disruptions are classified into 5 classes according to the

molecular mechanism resulting in the disorder. The majority of AS patients have class I

mutations (65-75%) where they have interstitial microdeletions in the maternally

inherited UBE3A 15q11-q13 region (Clayton-Smith, 2003). These deletions occur in the

three common chromosomal breakpoints (BP1, BP2 and BP3) where Type 1 patients

with larger deletions (BP1-BP3) have more severe phenotypes than those in Type 2 with

smaller break points (BP2-BP3). Patients with these microdeletions have mild to

moderately severe learning deficits and have behaviours similar to autistic individuals.

(Williams, Driscoll and Dagli, 2010)

Figure 1. Organization of the AS-associated 15q11.2-q13 genomic region on Chromosome 15. BP1, BP2

and BP3 are the breakpoints associated with AS. The UBE3A gene and the IC is located between BP2 and

BP3 and disruptions in this could also lead to AS. (Williams, Driscoll and Dagli, 2010)

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3-5% of AS occurs due to paternal uniparental disomy (UPD) where the two copies of

chromosome 15 come from paternal origin, which arises due to postzygotic errors. These

Class II patients tend to present a less severe phenotype. (Jiang et al., 1999)

Class III AS patients (3-5%) have genetic and epigenetic defects in the Imprinting Center

(IC) on Chromosome 15 (Figure 1). Half of the Class III patients have mutations within

the IC which is involved in imprinting during gametogenesis, whereas the other half have

no identified mutation. Therefore, in these groups it is thought that imprinting defects

arise by sporadic pre or post-zygotic events. Epigenetic defects within the IC can change

DNA methylation patterns and affect the transcription of the maternally-inherited gene to

render it inactive.

Class IV are patients who have a mutation in the UBE3A gene located on Chromosome

15 resulting in protein truncation (5-11%). Patients with mutations such as missense or in

frame deletions are shown to have a milder phenotype than patients with intragenic

deletions within the UBE3A gene. The remaining patients who do not show any Class I-

Class IV gene disruption on UBE3A are grouped into Class V, which are individuals who

do not show any abnormalities on Chromosome 15. (Clayton-Smith, 2003).

1.2) Imprinting in Angelman and Prader-Willi syndromes

Prader-Willi syndrome (PWS) is also a neurological disorder associated with the gene on

chromosome 15 as in AS, with a prevalence of 1 in 25000. Symptoms of PWS for

example include severe hyptonia, developmental delay and high-pitched nasal voices.

Similarly to AS, PWS also occurs due to defects in imprinted genes (Fryer, 1997).

Imprinting is when gene expression is dependent on the chromosomal parent origin. AS

and Prader-Willi syndrome are two phenotypically different disorders but are similar in

origin, as both disorders arise from an imprinting defect on the same locus on

chromosome 15 (often called the PWS/AS locus). The IC on this highly conserved

15q11.2-q13 genomic region is bipartite, meaning it is composed of both the AS-IC and

the PWS-IC. The only mechanism that distinguishes these two disorders apart is which

parental origin in which it is imprinted. In AS, the genes affected are expressed only from

the maternal copy, but in PWS the genes affected are only from the paternal copy.

Therefore, AS mutations in the maternal chromosome 15 shows phenotype. On the

contrary, in PWS, mutations in the paternal chromosome 15 shows phenotype. (Smith et

al., 2011). An individual with paternal UPD will have AS, as two copies of the silenced

UBE3A will be inherited. Likewise, an individual with maternal UPD will have PWS, as

two copies of the silenced PWS gene (Fryer, 1997).

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1.3) UBE3A protein and its putative role

UBE3A gene located on the short arm of chromosome 15 is the principle gene responsible

for AS patients and is implicated in other autism-related disorders. Even though the

mechanism by which aberrant UBE3A expression leads to these neurological disorders is

unknown, it is known that UBE3A gene encodes an E3 ubiquitin ligase, E6-associated

protein (E6AP) also known as the UBE3A protein (Huibregtse, Scheffner and Howley,

1993). An E3 ubiquitin ligase functions by adding ubiquitin molecules to its substrates,

which is then targeted for degradation by the proteasome. The ubiquitin transfer of

UBE3A is catalysed by the HECT domain of the protein (Cooper et al., 2004). Therefore,

in theory, a faulty UBE3A gene must affect its role in targeting its substrates with

ubiquitin, leaving these substrates which were supposed to be degraded present, and this

change in protein turnover by an unknown mechanism must lead to the neurological

phenotypes seen in AS patients. Consequently, much of the AS research to date aims to

identify these UBE3A substrates and how an increase in these substrates could potentially

affect the brain (Lee et al., 2013).

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Section 2: Neural Substrates of UBE3A

2.1) Pbl/Ect2

2.1.1) Identification of Pbl as a UBE3A substrate

Pebble (Pbl) was first identified as a UBE3A substrate in an experiment by Reiter et al

(2006). They aimed to identify UBE3A substrates by utilizing the Drosophila Gal4-UAS

(upstream activating sequence) expression vector, into which the human UBE3A is

cloned, and integrates into the fly genome. To specifically analyse UBE3A gene

expression in Drosophila heads, they used heatshock-GAL4-specific (HS) lines which

were specifically expressed in the heads. These HS-GAL4 flies were crossed with flies

carrying the UAS-UBE3A lines, which induce UBE3A specific expression in Drosophila

head in response to heat induction. The protein extracts from Drosophila heads of these

UBE3A-expressing flies and control flies were then analysed by two-dimensional gel

electrophoresis. Differential UBE3A expression was compared between controls and

GAL4/UAS-UBE3A flies. This identified a highly downregulated protein spot which was

then determined by mass spectrometry to be the Drosophila Rho-GEF Pbl. Further

studies showed that Pbl is a direct target of UBE3A by confirming the physical

interaction of Pbl and Dube3a (Drosophila ortholog of UBE3A) by immunoprecipitation

(Reiter, 2006).

2.1.2) Function of Pbl and Ect2

Pbl is a Rho-GEF (guanine exchange factor), which are known to be involved in cell

signalling by which it acts to facilitate the exchange of Rho-GDP for Rho-GTP producing

the activated form of the Rho-GTPase protein, which can thereby recognize and signal

downstream effector targets (Schmidt, 2002). Ect2, a mammalian orthlog of the

Drosophila Pbl, like Pbl also functions to activate Rho-GTPases during cytokinesis. RNA

interference studies on Ect2 knockdown in mouse neuroblastoma shows a rapid

outgrowth of neurites and Ect2 involvement in neuronal differentiation through cell cycle

regulation by an unknown mechanism. (Tsuji et al., 2011).

2.1.3) Ect2 role in neuronal morphological differentiation

Previous studies using RNAi screening in Drosophila have identified Pbl as a gene

involved in Drosophila neuronal development (Kraut, Menon and Zinn, 2001).

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In an experiment by Tsuji et al., (2012) using PC12 cells (from rat adrenal medulla) and

cultures of primary cortical neurons from E14 mice, knockdown of Ect2 was done by

RNAi and analysis of cell morphology showed that there was an increased in number of

cells with neurite outgrowth in Ect2 knockdown cell, compared to control. This raises the

suggestion that Ect2 is a negative regulator of neurite outgrowth in PC12 cells.

Immunohistochemistry of cells stained with DAPI also shows high Ect2 expression in the

ventricular (VZ) and subventricular zones (SVZ) in the brain cortex (Figure 2B). These

two layers (VZ and SVZ) are known to be high in progenitor cells responsible for

neurogenesis of neurons (Doetsch et al., 1999). Furthermore, Ect2 knockdown

experiments were used to investigate the role of Ect2 in neuronal differentiation in

cortical neurons. Morphological studies at different stages of neuronal differentiation

allows the distinguishing and the physical characterization of cells into their

corresponding stage (De Lima, Merten and Voigt, 1997).

Comparison between the control RNAi culture and Ect2 RNAi knockdown culture

identifies no differences in the numbers of neuron at each differentiation stage and no

effect on the axon length. However, the number of growth cones/neurons were

significantly increased in Ect2-knockdown neurons compared to controls (Figure 2A).

The increased in number of growth cones signify a possible role of Ect2 in neuronal

morphological differentiation (Tsuji et al., 2012). A possible explanation for the

Figure 2. (A) The effects of Ect2-depletion in cortical mouse neurons. Ect2-deleted neurons showed an increase in number

of growth cones and growth cones-like structures (right) compared to control mouse neurons (left). The white arrows

represent growth cones or growth cones-like structures. The yellow arrow represents the tip of neurite with outgrowth less

than 10µm therefore is not scored as a growth cone. (B) The increase in number of growth cones shown in stage 2 and

stage 3 of neuronal differentiation between control and Ect2-RNAi mouse neurons. (C) Staining for Ect2 expression in

mouse cerebral cortex. Sections stained with anti-Ect2 (left) and DAPI (middle) and the combination of the two staining

showed Ect2 concentration in ventricular (VZ) and subventricular (SVZ) zones. (Adapted from Tsuji et al., 2012)

A

B

C

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increased number of growth cones following Ect2 depletion is due to the inhibition of the

Rho-ROCK pathway (Hall and Lalli, 2010). The Rho-ROCK pathway is a major

signalling pathway involved in inhibiting neuronal regeneration, repair and development

in the central nervous system. The mechanism by which this happens involves the

signalling of inhibitory factors which activates the Rho/ROCK pathway through

activation of Rho-GTPases, consequently leading to an impact on actin cytoskeletal

dynamics and resulting in the collapse and retraction of growth cone (Liu, Gao and

Wang, 2015). Therefore, not only is Ect2 involved in the catalysing the activation of

Rho-GTPase in the Rho/ROCK pathway, but it is thought that Ect2 also has a role in

actin reorganization of the growth cones.

This correlates with previous findings in whole brain sections of UBE3A knockout mice,

which shows not only a significant increase in Ect2 expression in the hippocampus but

also mislocalization of Ect2, unrestricted from its usual perinuclear localization pattern.

Whereas, in wildtype mice UBE3A and Ect2 are expressed within the same cellular

regions of the hippocampus. These findings suggest that UBE3A plays a crucial role in

Ect2 expression levels as well as the cellular distribution of Ect2 in the cerebellum and

hippocampus which could possibly lead to an expansion in Ect2 signal in knockout mice.

(Reiter, 2006).

2.1.4) Ect2 and its association with autism in AS

Mental retardation (autism) is the result of abnormal brain development in children, by

which the process involves modification of synapses, as well as the remodelling of

neuronal networks through loss of function. This is also a primary phenotype seen in AS

children. Ect2 is thought to be involved in regulating the remodelling of the neuronal

circuit (Ramocki and Zoghbi, 2008). Because Ect2 functions as a Rho-GEF to activate

Rho-GTPases during cytokinesis, this should only happen at a certain time when cells

need to regenerate or repair their population, hence it is targeted by UBE3A for

ubiquitination once cells no longer need to repopulate. Therefore, microdeletions of the

UBE3A in AS patients would result in production of non-functional UBE3A protein

which thereby does not target Ect2 for degradation at a certain stage leading to more

cellular Ect2. To conclude, the hypothesis proposed is that UBE3A might be involved in

growth of neuronal processes or synapse formation. This corresponds with findings of

increased Ect2 expression in hippocampus and the mislocalization of Ect2 to an ectopic

region in the brain, where this expansion of Ect2 signal could be detrimental to the

neuronal circuit (Reiter, 2006).

With increased Ect2 levels, there will be fewer cells with neurite outgrowth due to the

activation of the Rho-ROCK pathway which mediate cytoskeleton remodelling resulting

in collapse of growth cones (Liu, Gao and Wang, 2015). These results propose that

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dysregulation of Ect2 might possibly contribute to the cause of autism in AS.

Additionally, association studies on Ect2 in autism families might provide more evidence

for this hypothesis. As well as further knockout studies of Ect2 and UBE3A in vivo will

provide further validation for the causes of autism in AS and ASDs (Ramocki and

Zoghbi, 2008).

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2.2) GAT1

2.2.1) Identification of GAT1 and UBE3A interaction

GABA transporter 1 (GAT1) interaction with UBE3A was first noted in an experiment

by Egawa et al (2012). Endogenous UBE3A protein taken from wildtype mouse

cerebellum was immunoprecipitated with either anti-UBE3A antibody or the control

immunoglobulin (IgG). Immunoprecipitates from wildtype and UBE3A knockout mice

were separated by SDS-PAGE, followed by immunoblotting with either anti-GAT1 or

anti-UBE3A antibody. SDS-PAGE shows that endogenous GAT1 was detected in

immunoprecipitates with anti-UBE3A antibody but not with control IgG in wildtype

mouse. UBE3A knockout mice shows increased GAT1 expression. Therefore, this

indicates that endogenous UBE3A must bind to endogenous GAT1 in mouse cerebellum.

Additionally, further in vitro degradation assays found that GAT1 protein is rapidly

degraded in cerebellar lysates derived from wild-type mice, but was stable in those

derived from UBE3A knockout mice suggesting that UBE3A may be involved in the

degradation of GAT1 in mouse cerebellum (Egawa et al., 2012).

2.2.2) Function of GAT1 in tonic inhibition

Gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the

central nervous system (CNS) which controls neuronal excitability in the hippocampus

(Kersanté et al., 2013). GABA operates through GABA type-A receptors (GABA-AR) by

the process of tonic inhibition. Tonic inhibition is a type of inhibitory transmission which

relies on GABA concentrations in the extracellular space binding to extrasynaptic

GABA-AR. This in turns regulates neuronal excitability through a chloride channel and

its effects on membrane potential depends on the chloride gradient (Farrant and Nusser,

2005). Tonic GABA inhibition could also be a result of increased extracellular GABA

concentration by the action of GAT-1 (Li, Yu and Jiang, 2013). GAT-1 (GABA

transporter-1) is a membrane glycoprotein by which functions to regulate extracellular

GABA levels to maintain this level near thermodynamic equilibrium. Even though the

exact mechanism of how GAT1 does this is still unknown, it is shown that when spillover

of GABA at the synaptic cleft occurs, GAT-1 is able to remove this ambient GABA and

in reverse, once vesicular release of GABA is low, GAT1 replaces this spillover

providing ambient GABA for tonic inhibition. GABA conductance is particularly studied

as previous findings have implicated a role for extrasynaptic GABA-AR receptor

signalling in many neurological and psychiatric disorders (Kersanté et al., 2013).

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2.2.3) GAT1 and its association with motor dysfunction in AS

Cerebellar ataxia is commonly found in AS patients, where it is mainly involved with

characteristic features of abnormal movement and balance. It is hypothesized that GABA

signaling is involved in the neurological symptoms caused by UBE3A deficiency. This is

supported by evidence showing that AS patients with deletion on chromosome 15

containing the gene encoding for GABA-AR subunits in addition to the UBE3A gene

showed a more severe phenotype than other AS patients without these GABA-AR

deletions (Egawa et al., 2008).

Because GAT1 is associated with tonic inhibition, an experiment by Egawa et al (2012)

aims to evaluate the effects of GAT1 on tonic inhibition in UBE3A knockout mice.

Whole-cell voltage clamp recordings were used to detect GABA-AR mediated tonic

currents in cerebellar brain slices from wildtype mice and UBE3A knockout mice.

Results show that tonic inhibition currents in UBE3A knockout mice decreased by

around 34% compared to wildtype mice. Further studies showed that this decreased tonic

inhibition was due to lower ambient GABA concentration and not GABA-AR

downregulation. The significant increase in GAT1 protein concentration in knockout

mice confirmed that GAT1 was responsible for this decreased tonic inhibition (Egawa et

al., 2012). Tonic GABA-AR mediated conductance is known to control the excitability of

cellular granule cells by decreasing the total membrane input resistance (Hamann, Rossi

and Attwell, 2002). Cerebellar granule cells of UBE3A knockout mice required lower

current injections to reach action potential threshold suggesting that the decreased tonic

inhibition in knockout mice contributes to the increased membrane excitability to near

action potential threshold.

Firing patterns of Purkinje cells are also investigated, as firing patterns in Purkinje cells

are thought to be involved in coordination and movement. The three representative firing

patterns are shown in Figure 3A. Tonic firing consists of a stable and fixed rate without

any bursting behaviour or any long silent periods. Trimodal firing consist of a period of

tonic firing followed by a period of bursting and then a silent period. Tonic silent pattern

consists of a short firing pattern with repetitive pauses followed by a silent period

(Womack and Khodakhah, 2002). Interestingly, the action potential firing patterns of

Purkinje cells taken from UBE3A knockout mice predominantly shows tonic firing

patterns, whereas Purkinje cells from wildtype mice shows the same affinity for tonic and

trimodal firing patterns (Figure 3B). This suggests there is a change in firing pattern in

UBE3A knockout mice which could be the result of decreased tonic inhibition (Egawa et

al., 2012).

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Similar cases of firing pattern changes from tonic to trimodal have been previously linked

to the blockage of excitatory or inhibitory synaptic transmission (Womack and

Khodakhah, 2002). This correlates with findings of decreased tonic inhibition by GAT1,

which leads to increased membrane excitability. Therefore, this decrease in tonic

inhibition could be the cause of the change in firing patterns in Purkinje cells which in

turns could result in motor dysfunction in AS. Therefore, this underpins decreased tonic

inhibition as a crucial mechanism underlying movement disorder in AS.

Figure 3. (A) The three representative firing patterns from a Purkinje cell. Top panel shows tonic firing pattern with

periods of stable firing with no bursts or silent firing. The middle panel shows a trimodal firing pattern where there is a

period of tonic firing followed by bursts and a short pause (silent) pattern. The bottom panel shows a tonic silent pattern

which consists of a short firing with repetitive pauses followed by a silent period. (B) The percentage of Purkinje cells

with each of the three firing patterns taken from wildtype and UBE3A knockout mice. (Egawa et al., 2012)

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2.3) SK2 Channel

2.3.1) Identification of SK2 channel as a UBE3A substrate

The SK2 channel was first identified as a UBE3A substrate in an experiment by Sun et al

(2015). Western blot analysis of proteins from various hippocampal subcellular fractions

were taken from wildtype and UBE3A-knockout mice (AS mice). A synaptic terminal

isolated from a neuron (synaptosome) showed higher levels of SK2 in these fractions in

AS mice compared to that from wildtype mice. This increase in synaptic SK2 levels from

AS mice was then tested for its relation to UBE3A. Immunofluorescent staining of

UBE3A and SK2 showed that they were co-localized along the dendrites of CA1

hippocampal neurons in wildtype mice. Further immunoprecipitation experiments were

done on anti-UBE3A and anti-SK2 blots using either anti-UBE3A antibodies or control

immunoglobulin (IgG) antibody. Results showed that SK2 co-immunoprecipitated with

UBE3A in wildtype mice but not in AS mice. Also, immunoprecipitation performed with

anti-SK2 antibodies and western blots labelled with anti-SK2 and anti-ubiquitin

antibodies showed that the amount of ubiquitinated protein in SK2 antibody pull-down

samples from AS mice was decreased compared to WT mice (Figure 4). Therefore, this

decreased SK2 ubiquitination in AS mice was hypothesized to be the result of the lack of

UBE3A ubiquitin ligase.

Evidence from UBE3A knockdown studies

confirms this hypothesis, as SK2 ubiquitination

was significantly reduced following UBE3A

siRNA treatment on COS-1 cells (derived from

monkey kidney tissue). This ubiquitination of

SK2 by UBE3A was then found to be on the C-

terminus of SK2, in which multiple lysine

residues are present. Mutation experiments on

lysine residues were able to identify three

critical lysine residues on the SK2 C-terminal

domain which are responsible for UBE3A-

mediated ubiquitination (Sun et al., 2015).

Figure 4. Immunoprecipitation performed with anti-SK2

antibodies and western blots labelled with anti-SK2 and

anti-ubiquitin (Ub) antibodies. Arrows indicate

ubiquitinated SK2 and/or SK2-associated protein (left).

Quantification of abundance of ubiquitinated SK2 in

hippocampus of wildtype and AS mice (Sun et al., 2015).

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2.3.2) Function of SK2 channel in long-term potentiation

Small conductance calcium-activated potassium channels (SK channels) are known to

modulate membrane excitability in hippocampal Cornu ammonis (CA1) neurons by

regulation of potassium cations across the cell membrane. This activation of the SK

channel is stimulated by intracellular concentrations of Ca2+ entering via NMDA

receptors (Hammond, 2006). The function of NMDA receptor (NMDAR) activation is

linked to long-term potentiation (LTP) and long-term depression (LTD), which are

thought to be the mechanism underlying associative learning and memory (Gruart et al.,

2015). LTP is a persistent and long-lasting increase in signal transmission between

neurons following synaptic stimulation, whereas LTD plays the opposite role. In

hippocampal CA1 neurons, SK2 channels are activated by NMDAR activation which

results in repolarization of the membrane, which in turns terminates NMDAR function.

This negative feedback loop is involved in determining the membrane excitability, as

well as the LTP induction threshold. LTP induction is also known to regulate synaptic

SK2 expression, as it triggers endocytosis of synaptic SK2s (Lin et al., 2011). From the

three subtypes of SK channels, SK2 was shown to be predominantly expressed in CA1

and CA3 layers and is hypothesized to be the channel that modulates hippocampal

synaptic plasticity and learning (Hammond, 2006).

2.3.3) SK2 channel and its association with learning and memory in AS

AS patients have phenotypes such as cognitive delay and speech impairments which are

associated with learning deficiencies and impaired memory (Jiang et al., 2010). Previous

research have shown that LTP is impaired in UBE3A-deficient mice, also known as AS

mice model (Van Woerden et al., 2007). Because synaptic SK2 is ubiquitinated by

UBE3A, levels of SK2 must increase in AS mice. It is hypothesized that this increase in

SK2 expression levels affect LTP which results in learning and memory impairments in

AS mice.

Theta-burst stimulation (TBS), a protocol used to stimulate neural pathways in the brain

was utilized to study the differential effects of LTP in wildtype and AS mice (Huang et

al., 2005). TBS induced a long-term potentiation in hippocampal slices of wildtype mice,

whereas in AS mice there was only a short-term facilitation. Further tests to confirm that

this change in LTP was a result of SK2 channels was done using Apamin (a selective

SK2 channel blocker). Apamin was incubated with AS hippocampal slices and TBS now

induced a long-term potentiation as seen previously in wildtype mice. Therefore, Apamin

was able to reverse the effects of LTP in AS mice suggesting that SK2 must be

responsible for this change in LTP (Sun et al., 2015).

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Additionally, LTD is also thought to be involved in learning and memory, therefore the

differential effects of LTD is also studied in wildtype and AS mice in a similar study to

the one mentioned previously (Sun et al., 2015). Low-frequency stimulations were

applied to wildtype and AS mice hippocampal slices. Results show that wildtype slices

showed a short-term synaptic depression (opposite to seen in LTP), which is what was

expected as described in previous literature (Dudek et al., 1993). AS mice on the other

hand, displayed a sustained long-term depression. Again, incubation with Apamin was

able to reverse the effects of LTD in AS mice, suggesting that SK2 is also responsible for

the change in LTD as seen from LTP (Figure 5).

Because SK2 channel activation resulting in membrane repolarization inhibits NMDAR

channel opening, this negative feedback loop is investigated in wildtype and AS

hippocampal slices. NMDAR-mediated synaptic response was measured and results show

that excitatory post-synaptic potential (EPSP) is significantly lower in AS mice compared

to wildtype mice. Also, pre-treatment with AP5 (an NMDAR antagonist) also reduced

LTD in AS mice suggesting that enhanced LTD in AS mice is also NMDAR dependent

(Sun et al., 2015).

Therefore, conclusions can be made that increased SK2 levels contributes to the decrease

in LTP and LTD seen in AS mice. As previous studies on manipulating SK2 activity have

been shown to influence learning performances in mouse (Stackman et al., 2002), Sun et

al., proposed that in AS mice this increased levels of synaptic SK2 is responsible for the

Figure 5. (A) The changes in excitatory post-synaptic potential (EPSP) of long-term potentiation

(LTP) in hippocampal slices from wildtype and AS mice, as well as changes from Apamin pre-treated

AS and wildtype slices. (B) The changes in EPSP of long-term depression (LTD) in hippocampal

slices from wildtype and AS mice, as well as the changes from Apamin pre-treated AS and wildtype

slices. (Sun et al., 2015).

A B

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decrease in LTP and LTD which thereby contributes to hippocampal-dependent learning

deficit seen in AS patients.

2.4) Arc

2.4.1) Identification of Arc a UBE3A substrate

Arc was first noted as a UBE3A substrate in an experiment by Greer et al (2010) through

the identification of a UBE3A binding domain (UBD). The initial experiment aimed to

identify an E3 ubiquitin ligase substrate in mouse by using HA-tagged ubiquitin. Through

this experiment, the protein Sacsin was identified as a UBE3A candidate substrate. From

Sacsin, Greer et al recognized a notable amino acid sequence which was known to be

present in previously identified UBE3A substrates. This allowed them to formulate a

hypothesis that this amino acid sequence motif (HHR23A) could be a UBE3A-binding

domain, and this will therefore be present in other UBE3A substrates. This theory was

confirmed by mutation studies of HHR23A to assess its ability to be ubiquitinated by

UBE3A, as mutations in this putative motif were able to block UBE3A interaction.

Greer et al were able to align this UBD motif to

various mammalian genomes to identify

proteins containing this UBD (Figure 6). This

method identified the synaptic protein Arc

(activity-related cytoskeleton-associated

protein) as a possible UBE3A substrate. Co-

immunoprecipitation experiments were done to

confirm the identity of Arc as a UBE3A

substrate. In vitro ubiquitination assays were

performed using recombinant Arc, Arc without

UBD and GST-tagged UBE3A and these were

done on Western blots probed with anti-Arc

antibody. Results show that Arc was able to

bind to UBE3A dependent on the UBD and

UBE3A was shown to ubiquitinate Arc in

vitro. Therefore, interaction studies were able

to show that Arc protein is a UBE3A substrate

and is targeted by UBE3A for ubiquitination

(Greer et al., 2010).

Figure 6. Sequence alignment of the human protein Sacsin and

human UBD motif (HHR23A) and of the Arc protein with UBD.

Red are identical amino acid residue and blue are similar amino acid

residues (same amino acid property). (Greer et al., 2010)

19

2.4.2) The role of Arc in synaptic AMPA receptor trafficking

Arc is a member of the immediate-early genes (IEG) family which directly affects

neuronal function. Arc is shown to localize to activated synapse, dependent on NMDA

receptor activation. The exact function of Arc protein is unknown but due to its neural

localization, it is thought to be involved in neuronal plasticity, a process critical in

learning and memory (Mokin, 2005). AMPA receptors (AMPAR) are glutamate receptors

that mediates fast synaptic transmission in CNS. Previous studies have shown that Arc

plays a regulatory role in AMPAR trafficking. This occurs as Arc mRNA is upregulated

upon neuronal activity and is localized to these activated synapses. Arc protein then

recruits endophilin and dynamin and this complex accelerates endocytosis to selectively

recruit AMPAR receptors, thereby downregulating AMPAR surface expression levels.

Arc knockout studies have also shown an increase in AMPAR levels. Therefore, it is

formulated that Arc acts to regulate basal AMPAR level (Chowdhury et al., 2006).

2.4.3) Arc and its association with cognitive dysfunction in AS

Cognitive dysfunction is typically seen in AS patients (Dagli, Buiting and Williams,

2011). One mechanism contributing to this is thought to be due to increased Arc levels in

AS patients. Protein expression studies have identified higher Arc expression in brains of

UBE3A knockout mice compared to wildtype, however Arc mRNA levels remain similar

between AS and wildtype mice. This suggests that the increased Arc protein seen in the

brain of AS mice is due to a defect in the ability of UBE3A to ubiquitinate Arc. Arc is

involved in AMPAR trafficking, where a decrease in Arc levels leads to an increase in

the surface expression of AMPARs. This hypothesis was tested by using shRNAs

targeting UBE3A expression in hippocampal neurons to look at differential AMPAR

surface expression. Results were shown to correlate with hypothesis, where in cells with

shRNA targeted against UBE3A, reduced levels of GluR1 (subunit of AMPAR) at the

membrane were seen (Greer et al., 2010).

Previous literature has stated that increased Arc levels are shown to increase the rate of

endocytosis which eventually decreases AMPAR surface expression (Chowdhury et al.,

2006). Therefore, in this experiment, AMPAR endocytosis was tested in the absence of

UBE3A. Results correspond with the literature, showing an increase in levels of

endocytosed GluR1 in neurons with shRNA against UBE3A compared to control

neurons. This suggests that the decrease in AMPAR surface expression in UBE3A-

deficient neurons is due to increased endocytosis. Further studies looking at the function

of AMPAR at synapses showed a significant decrease in AMPAR mEPSC frequency

(measure of AMPAR-mediated synaptic transmission) recorded from neurons expressing

shRNA against UBE3A (Liu et al., 2013). This further implies an abnormal AMPAR

function observed at the synapses of AS neurons.

20

Even though results suggest that UBE3A mediates AMPAR surface expression levels, it

is not conclusive whether this is done through Arc-mediated degradation. To test

whether Arc is the regulating factor, they hypothesize that overexpression of Arc would

lead to reduced AMPAR expression. Results show that co-expression of UBE3A with

Arc in neurons was able to inhibit the ability of Arc to promote endocytosis of AMPAR

subunit, GluR1 (Shepherd et al., 2006). Where experiments with Arc lacking the UBD in

neurons were able to promote endocytosis, its co-expression with UBE3A did not reverse

this effect (Figure 7A). These results suggest that the ability of UBE3A to regulate

AMPAR surface expression is dependent on its ability to ubiquitinate Arc.

Further experiments were carried out to determine whether UBE3A promotes AMPAR

expression at synapse is through Arc-mediated degradation. Neurons were transfected

with either shRNAs targeting UBE3A or shRNAs targeting Arc or both. Again, AMPAR

cell surface expression is assessed and results are consistent with previous hypothesis.

ShRNA against Arc transfected into neurons cause a small increase in surface AMPAR

expression. Although this increase was not statistically significant, the authors suggested

that this is due to the low neuronal activity in neuron culture. Neurons with both shRNA

targeting UBE3A and shRNA targeting Arc were able to rescue the decrease in AMPAR

expression seen in neurons with shRNA against UBE3A alone, to near control levels

(Figure 7B) (Greer et al., 2010).

In conclusion, these results suggest that UBE3A is able to mediate AMPAR surface

expression in neurons where this ability is dependent upon ubiquitination of Arc protein

Figure 7. (A) The levels of surface AMPA receptor (GluR1 subunit) on hippocampal neurons

transfected with control, Arc, UBE3A+Arc, Arc without UBD and UBE3A+Arc without UBD. (B) The

levels of surface AMPA receptor (GluR1 subunit) on hippocampal neurons transfected with vector

control, UBE3A RNAi, UBE3A scrambled RNAi, Arc RNAi, Arc scrambled RNAi, UBE3A RNAi+Arc

RNAi and UBE3A RNAi+Arc scrambled RNAi. (Greer et al., 2010)

A B

21

levels. Therefore, AS patients with UBE3A deletions would lead to an increase in Arc

levels therefore promoting endocytosis and resulting in a decrease in the surface

expression of AMPARs. This induction of Arc is thought to be critical for regulating

neuronal excitation of AMPARs and must be regulated effectively for normal function of

synapses (Flavell, 2006). Aberrant Arc levels are postulated to lead to defects in synaptic

transmission which could possibly result in cognitive dysfunction observed in AS

patients.

22

Section 3: Therapeutic potential of Angelman Syndrome

3.1) Current Treatment

There is currently no effective therapy for Angelman Syndrome. Clinical treatments often

focus on minimizing the behavioural symptoms of AS (Bailus and Segal, 2014). Highly

occurring phenotypes of AS include seizures (about 80% of AS patients), which can be

problematic. Clinical prescription of AS patients include anti-epileptic drugs such as

sodium valproate or clonazepam etc. to reduce epileptic seizures. Also in certain patients

with severe movement disorder, physical therapy is often established to help with

movement and coordination, or if progressive and severe, surgeries might be required.

Further therapies to attenuate symptoms can also include behavioural therapy to

overcome hyperactivity, typically seen in children with AS. AS patients also have speech

impairments or absence of speech, therefore communication therapy is also established

for a more effective communication with AS patients. Simple sign or hand signals are

introduced as well as Picture Exchange Communication System (PECS) which could also

improve communication in AS patients (Clayton-Smith, 2003).

3.2) Potential Therapies

Even though there is currently no effective therapy for Angelman Syndrome, the

therapeutic potential for AS is highly promising, considering the progress made within

the last couple of years. The mass of current research have successfully identified

UBE3A neural substrates and the pathological mechanisms in which these substrates

intervene are intensively studied (Bailus and Segal, 2014). There can be various

approaches towards AS therapy, some research tackles AS in the aspect of directly

reactivating the UBE3A gene on the paternal chromosome and restoring UBE3A

expression (Bailus et al., 2016), whereas other research aim to introduce inhibitors of

these UBE3A substrates as potential pharmalogical drug targets. In this project we

focused on the research which have successfully introduced inhibitors of known UBE3A

substrates (mentioned in Section 2) and how they could potentially be introduced into

clinical trials as a treatment for AS.

23

3.2.1) GAT1 inhibitor improves learning and motor dysfunction in mice

GAT1 was identified as a UBE3A substrate and is linked with decreased tonic inhibition

in AS mice model, this is thought to play a role in motor dysfunction in AS patients (see

Section 2.1). In this fairly recent research by Sałat et al (2015) they hypothesize that if

ambient GABA transmission is reduced in an AS mice model due to decreased tonic

inhibition, then an increase in GABA may reverse these neurological effects. Therefore,

in this particular experiment they assess the effect of Tiagabine (a selective GAT1

inhibitor) on memory and learning in mice. Three behavioural assays commonly used to

assess memory and learning in mice are used. These include the Morris Water Maze

(MWM), the Radial arm water maze and the Passive avoidance test (PA).

In the PA task, wildtype mice were able to explore the two compartments separated by a

door, one in light and one in dark (Ader, Weijnen and Moleman, 1972). An hour before

the conditioning phase, mice were pretreated with either Tiagabine or a control injection,

and 30 minutes later they were treated with Scopolamine (a drug known to induce

memory impairments or cognitive defects in animals). In the conditioning phase, a mild

foot shock is exerted only in the dark compartment, and this stage is repeated for a certain

period of time to allow memory acquisition. Later during the testing trials, this same

compartment was introduced and the latency time between door opening and entry into

dark compartment of mice is recorded. Results show that mice treated with Scopolamine

and Tiagabine had a significantly longer latency time than mice treated with Scopolamine

and control injection. Similar results were obtained for the MWM and radial arm water

maze test. This suggests an ability for Tiagabine to reverse memory impairments in

cognitive dysfunction mice, further suggesting that inhibition of GAT1 function could

possibly improve memory and learning in AS mice model (Sałat et al., 2015).

Previous research on GAT1 inhibitor in AS mice models has also showed potential. THIP

injections (GAT1 inhibitor) showed an increase in tonic inhibition currents in cerebellar

granule cells in UBE3A knockout mice. GAIT analysis in AS mice showed that there was

an abnormal hind paw angle, which could possibly result in motor dysfunction in AS

mice, also seen in AS patients (Egawa et al., 2012). However, once treated with in vivo

injections of THIP, there was a reduction in the abnormal hind paw angle seen in AS

mice to near normal levels (Figure 8).

Therefore, from these two experiments we can conclude that GAT1 inhibitors are shown

to reverse the characteristics of AS mice such as impaired learning and motor dysfunction

following a GAT1 increase in UBE3A-deficient mice. This information could aid in

developing a GAT1 inhibitor drug to improve these characteristics seen in AS patients.

24

3.2.2) Blocking SK channels enhances contextual learning and memory in

AS mice model

Apamin (a selective SK channel blocker) has previously been reported to reverse the

abnormal effects of LTP and LTD seen in AS mice (see Section 2.3.3). Therefore, the

same research group studied the effect of Apamin on learning in AS mice (Vick, Guidi

and Stackman, 2010). To assess learning in mice, the fear conditioning paradigm was

used. In this experiment mice were initially exposed to a loud tone, then instantly after

the tone a mild foot shock is exerted. AS and wildtype mice were pre-treated with

Apamin 30 minutes before this conditioning phase. After conditioning, the loud tone is

associated with foot shock, and before the foot shock is actually exerted, mice showed a

freezing behaviour. This experiment analyzes contextual memory, whether mice have the

ability to remember this association (tone with footshock) and freezes before shock is

applied. This freeze time was measure in wildtype and AS mice. Results showed that

Apamin application significantly improved learning and memory performance in AS

mice (Sun et al., 2015).

These results suggest that the increased levels of SK2 channels are responsible for

learning deficits and this could be improved by inhibiting SK2 channels using drugs such

as Apamin.

3.3.3) Reducing Arc expression improves seizures in AS mice model

Arc protein is associated with cognitive dysfunction in AS mice models (see section

2.4.3). Seizures associated with AS are often present in early ages from 6 months and this

varies according to severity in adult AS patients (Flavell, 2006). Therefore, in this

experiment Arc expression was studied in juvenile AS mice model. Seizure inductions

were performed using an audiogenic stimulus which elicited a seizure-like response in

AS mice. Arc knockout mice were achieved by breeding wildtype mice with

Figure 8. (A) The hind paw angle in wildtype and UBE3A knockout mice. (B) The decrease

in paw angle in UBE3A knockout mice treated with THIP injection compared to control.

(Egawa et al., 2012)

A B

25

heterozygous mice for the Arc allele. Arc levels were then confirmed by quantitative

PCR. Once mice was exposed to an audiogenic stimulus, the time it took for mice to

regain movement after the stimulus was ceased was assessed. Results showed that in AS

mice with reduced Arc expression, the seizure-like response previously seen in AS mice

was no longer present. Also, EEG recordings were analyzed to record the electrical

activity of the brain. Previous research has shown that AS mice have abnormal EEG

recordings with large spikes compared to the normal steady recordings seen in wildtype

mice. EEG recordings show that in Arc-knockout mice, the spiking events seen in AS

mice has completely disappeared to wildtype levels (Figure 9) (Mandel-Brehm et al.,

2015).

These results suggest that reducing Arc levels can reverse the abnormal seizure-like

responses in AS mice. This information could provide further research into potential

drugs that target Arc expression, which could potentially attenuate seizures in AS

patients.

3.3) Future work

Overall, the progression made in the search of a cure for Angelman Syndrome has been

highly successful. Research identifying UBE3A substrates and their associated function

to Angelman Syndrome has led to more in depth research on development of drugs to

inhibit these neurological functions and reverse the neurological defects seen in AS. The

successful experiments using GAT1 inhibitor THIP (mentioned in Section 3.2.1) to

reverse the effects of motor dysfunction in AS mice has led to its introduction into

clinical trials (Egawa et al., 2012). Currently, Ovid Therapeutics have developed a

GABA-AR agonist (Gaboxadol or THIP) which is able to restore tonic inhibition in AS

Figure 9. (A) Representative amplitudes shown for EEG recordings in wildtype and AS mice. (B)

The number of spiking events seen in wildtype, AS mice, Arc knockout in wildtype and Arc

knockout in AS mice. (Mandel-Brehm et al., 2015)

26

mice model and is expected to commence Phase 2 clinical trials in late 2016. This oral

drug is to be taken as a daily dose of once or twice, and is expected to be able to treat AS

symptoms in AS patients of all ages (OVID THERAPEUTICS INC., 2016).

Other drugs for the remaining UBE3A substrates have yet to be further introduced into

clinical trials. Even though Arc-knockdown drugs for reducing Arc expression and

Apamin for blocking SK channels were successfully shown to improve AS symptoms in

mice, the hesitation for further drug development might be due to the inappropriate doses

of these drugs for effective inhibition, where accumulation of these high doses could infer

toxicity issues in humans. Additionally, another developing cure for AS licensed by

Agilis Biotherapeutics is currently entering Phase I clinical trials. This drug utilizes gene

therapy for precise vector delivery of UBE3A DNA to cells in the brain. This is expected

to restore UBE3A gene expression in AS patients.

To conclude, potential AS drugs are starting to emerge in clinical trials after over 50

years since the first identification of AS symptoms, and only 10 years after the first

UBE3A substrate was identified. This suggests that the identification of UBE3A neural

substrates were able to provide further insight into the pathology and mechanisms

underlying AS symptoms, allowing subsequent development of AS drugs. Moreover,

intensive research of AS is still ongoing and the possibility of AS therapy in the future is

very favourable.

Word count: 6490

27

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