Protein Sumoylation in Brain Development, NeuronalMorphology and Spinogenesis

Carole Gwizdek • Frederic Casse • Stephane Martin

Received: 29 May 2013 / Accepted: 22 July 2013

� Springer Science+Business Media New York 2013

Abstract Small ubiquitin-like modifiers (SUMOs) are

polypeptides resembling ubiquitin that are covalently

attached to specific lysine residue of target proteins through

a specific enzymatic pathway. Sumoylation is now seen as

a key posttranslational modification involved in many

biological processes, but little is known about how this

highly dynamic protein modification is regulated in the

brain. Disruption of the sumoylation enzymatic pathway

during the embryonic development leads to lethality

revealing a pivotal role for this protein modification during

development. The main aim of this review is to briefly

describe the SUMO pathway and give an overview of the

sumoylation regulations occurring in brain development,

neuronal morphology and synapse formation.

Keywords Posttranslational modification �Sumoylation � Brain development � Synapse

formation

Introduction

An accurate brain functioning depends on the establish-

ment of a highly organized network of interconnected

neuronal cells. The assembly and maintenance of these

networks rely on neuronal differentiation and on the

formation of synapses between axons and dendrites during

brain development. This neuronal connectivity enables the

rapid transfer of information in the brain. Synapse forma-

tion and elimination as well as synaptic transmission and

plasticity largely depend on the correct organization of

complex protein networks on both sides of the synapse.

These are structured in an array of scaffolding and adaptor

molecules, presenting many protein–protein interaction

domains to anchor and position effectors such as neuro-

transmitter receptors and their associated regulators or

components of multiple signalling pathways. These neu-

ronal networks are developmentally regulated and at a later

stage dynamically modified at the synaptic level to mod-

ulate synaptic plasticity. All these events involve multiple

protein–protein interactions that are often regulated by

posttranslational modifications (PTMs) such as phosphor-

ylation, acetylation or ubiquitination. Sumoylation is an

additional PTM that is clearly emerging as a key mecha-

nism essential for the regulation of protein function during

brain development, synaptic formation, transmission and

plasticity.

Small Ubiquitin-like Modifiers

In mammalian systems, there are at least four SUMO

paralogs, SUMO1–4. These are small proteins of *100

amino acids (*11 kDa) sharing only 18 % sequence

homology with the polypeptide ubiquitin. SUMO1–3 are

ubiquitously expressed (Geiss-Friedlander and Melchior

2007; Hay 2005), whereas SUMO4 is poorly characterized

and mainly expressed in lymphatic node, kidney and spleen

(Guo et al. 2004; Bohren et al. 2004). SUMO2 and SUMO3

are almost similar with *97 % identity and are referred as

SUMO2/3. SUMO1 shares only *47 % identity with

C. Gwizdek � F. Casse � S. Martin (&)

Institut de Pharmacologie Moleculaire et Cellulaire, Laboratory

of Excellence ‘Network for Innovation on Signal Transduction

Pathways in Life Sciences’, UMR7275, Centre National de la

Recherche Scientifique, University of Nice—Sophia-Antipolis,

660 route des lucioles, 06560 Valbonne, France

e-mail: martin@ipmc.cnrs.fr

123

Neuromol Med

DOI 10.1007/s12017-013-8252-z

SUMO2/3 and unlike SUMO2/3 cannot form poly-SUMO

chains (Johnson 2004).

The Sumoylation Machinery

SUMO moieties are conjugated to substrate proteins

through a dedicated enzymatic pathway (Fig. 1). Sumoy-

lation occurs at lysine residues and predominantly at a

WKx[D/E] consensus motif in substrate proteins where Wis a large hydrophobic residue, K is the target lysine, x can

be any residue, and D/E are aspartate and glutamate

(Rodriguez et al. 2001; Sampson et al. 2001). Importantly,

not all SUMO substrates are modified within this motif,

and not all WKx[D/E] motifs are sumoylated. Several

extended sumoylation consensus sites were identified by

sequencing analysis and revealed that the sequences

flanking the consensus motif are important to determine

whether a site can be sumoylated or not (for a recent

review, see Gareau and Lima 2010).

SUMO paralogs are synthesized as inactive precursors

that are matured by the hydrolase activity of SUMO-specific

proteases called SENPs (sentrin protease; Mukhopadhyay

and Dasso 2007; Hickey et al. 2012), resulting in the

exposure of a di-glycine C-terminal sequence. This step is a

prerequisite to allow the subsequent conjugating step that

leads to the SUMO conjugation to target proteins (Fig. 1).

Following this essential step, SUMO is activated via an

ATP-dependent process linking the activated SUMO to the

E1 ‘activating’ complex, formed by a heterodimer of SAE1

and SAE2 (SUMO-activating enzyme subunits 1/2, also

named AoS1 and Uba2, respectively; Gong et al. 1999). The

E1 enzyme will then pass the SUMO moiety onto the cat-

alytic cysteine residue (in position 93) of the sole E2 ‘con-

jugating’ enzyme of the SUMO system, Ubc9 (Johnson

2004; Hay 2005). Although an E3-ligating enzyme is gen-

erally essential for the ubiquitination process (although there

may be exceptions, Hoeller et al. 2007), sumoylation can be

achieved with just the E2 Ubc9 (Sampson et al. 2001).

Indeed, Ubc9 can bind directly to the core consensus

sumoylation sequence in a way that aligns the active site of

Ubc9 directly next to the e amino group of the lysine residue

to which the SUMO is to be conjugated (Bernier-Villamor

et al. 2002). However, an efficient sumoylation reaction

often requires the presence of an E3-ligating protein (Fig. 1).

A few of them have been identified and characterized so far,

and despite a restricted number of proteins possessing an E3

activity, they are involved in the sumoylation of a significant

number of target proteins (for a review, see Gareau and

Lima 2010). These E3 proteins facilitate the sumoylation

process either by orchestrating the substrate and the SUMO-

loaded Ubc9 in close proximity or by enhancing the SUMO

transfer rate onto a specific substrate when the E2 Ubc9 can

directly bind its target protein.

SUMO E3 proteins can be separated into distinct groups.

The largest family is the SP-RING domain-containing E3s

Fig. 1 The SUMO enzymatic pathway. SUMOs are synthesized as

precursors that are matured by the hydrolase activity of specific

SUMO proteases called SENPs. SUMO activation is achieved in an

ATP-dependent manner leading to the formation of a thioester bond

between SUMO and the SUMO-activating enzyme subunit SAE2

(Uba2). SUMO is then transferred to the active cysteine site of the

sole conjugating enzyme of the system, Ubc9. Ubc9 can directly

recognize substrate proteins and catalyze an isopeptide bond between

SUMO and the target lysine residue. However, in most cases, SUMO

conjugation is achieved in conjunction with an E3 protein. Both of

these reactions are reversible, and SUMO can be dynamically

removed from their substrates by the isopeptidase activity of the

SUMO proteases SENPs and DeSI1/2

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that binds SUMO non-covalently via their SUMO-inter-

acting motifs (SIMs). A SIM domain consists of a short

sequence of less than 10 residues that contains a core

hydrophobic amino acid stretch that interacts with the b2

strand of SUMO (Kerscher 2007; Namanja et al. 2012). SP-

RING E3s therefore act as adaptor protein to bring Ubc9

and the target protein in close proximity, allowing the

transfer of SUMO from Ubc9 to the target protein. Known

SP-RING E3s include the protein inhibitor of STAT

(PIAS) proteins. To date, there are five known members in

the mammalian PIAS family, PIAS1, PIAS3, PIASxa,

PIASxb and PIASy (Kahyo et al. 2001; Schmidt and

Muller 2002; Nishida and Yasuda 2002; Kotaja et al. 2002;

Sachdev et al. 2001). All PIAS members have been shown

to facilitate SUMO-linked protein assemblies (for a recent

review on PIAS, see (Rytinki et al. 2009).

Other SP-RING domain-containing E3s have been

identified in mammals. These include the nuclear MMS21/

NS2 (Potts and Yu 2005, 2007), the mitochondrial MUL1/

MAPL (Braschi et al. 2009) and the TOPORS (Hammer

et al. 2007; Weger et al. 2005), the latter being the first-

reported SUMO E3 bearing a dual SUMO–ubiquitin ligase

activity (Rajendra et al. 2004).

The second group of E3 is formed by the sole nucleoporin

Ran-binding protein 2 RanBP2 (Pichler et al. 2002). This

protein is unique in a sense that it has no known homology

with other E3s (Pichler et al. 2004). It is known to enhance

the in vitro sumoylation of various protein substrates

including HDAC4, PML and Sp100. This protein is part of

the nuclear pore complex where it interacts directly with the

SUMO-modified form of Ran GTPase-activating protein 1

(RANGAP1) in a stable complex with Ubc9 (Reverter and

Lima 2005). Although the subcellular localization of Ran-

BP2 is clearly defined, it does not require a direct interaction

with any of its substrates that pass through the nuclear pore.

The last group of E3s is the polycomb Pc proteins that

are involved in gene silencing. Among this family, it was

reported that Pc2 functions as an E3 protein. Pc2 does not

contain a RING domain but is believed to act as a scaf-

folding protein since it interacts directly with Ubc9, SUMO

and its substrate CtBP (Kagey et al. 2003). Other proteins

that do not contain any RING domain but bear an E3

activity have been reported in recent years. These include

the histone deacetylase 4 and 7 (HDAC4, Gregoire and

Yang 2005; HDAC7, Gao et al. 2008), the small GTPase

Ras homologue enriched in striatum protein (Rhes),

(Subramaniam et al. 2009; Subramaniam et al. 2010) and

the TNF-receptor-associated factor 7 (Morita et al. 2005).

The precise mechanisms by which all these SUMO E3

proteins enhance sumoylation are still largely unknown,

but they may be particularly important for the specificity of

sumoylation toward SUMO paralogs, formation of SUMO

chains and/or protein substrate recognition.

The SUMO Deconjugation System

Sumoylation is a dynamic process, and both SUMO con-

jugation to substrates and its reversible deconjugation must

be finely regulated to tightly control protein function (for a

recent comprehensive review on SUMO proteases, see

Hickey et al. 2012). Despite a covalent SUMO binding, this

is a reversible process due to the isopeptidase activity of

specific desumoylation enzymes. These enzymes cleave

sumoylated substrates between the last glycine residue of

SUMO and the lysine residue modified on the target pro-

tein leaving a matured SUMO that is free to reenter the

sumoylation pathway (Fig. 1).

The first identified class of SUMO proteases is called

sentrin (the other name of SUMO) protease (SENPs). In

humans, six SENPs (SENP1–3 and SENP5–7) have been

identified exhibiting specific subnuclear and subcellular

localization patterns and distinct specificity toward SUMO

paralogs (Mukhopadhyay and Dasso 2007; Hickey et al.

2012). These enzymes are responsible for the maturation of

SUMO precursors (with the exception of SENP6 and 7)

and for the deconjugation of SUMO from their sumoylated

substrates. Therefore, these enzymes are key to regulate the

available pool of conjugatable SUMO with the amount of

free available SUMO2/3 being much larger than that of

SUMO1.

SENP desumoylases also show distinct specificity

toward SUMO paralogs (Gong et al. 2000), and their

specificities are often driven by their subcellular localiza-

tion (Kolli et al. 2010). Interestingly, Kolli and collabora-

tors compared the relative selectivity for SUMO1 and

SUMO2 between all SENP catalytic domains and the

human recombinant full-length SENP1 and SENP2. They

reported differences in SUMO selectivity, revealing that

the SENP specificity is dictated by the variable N-terminal

domain of SENPs and not through their catalytic domains.

Their analyses also demonstrated that all isolated SENP

catalytic domains were reacting with all SUMO paralogs,

whereas all native SENPs except the endogenous SENP1 in

mammalian cells displayed higher selectivity for SUMO2.

These data therefore suggest that the SUMO2 turnover is

far more dynamic than for SUMO1 since most SENPs

displayed a marked preference for SUMO2 rather than for

SUMO1 (Kolli et al. 2010). Very recently, Sharma and

collaborators used biochemical and genetic approaches to

demonstrate that SENP1 has an essential function to

desumoylate SUMO1-modified proteins during mouse

embryonic development (Sharma et al. 2013). SENP1

specificity for SUMO1-modified proteins represents an

intrinsic enzymatic function and is therefore not simply due

to a subcellular localization mechanism.

The second class of SUMO deconjugation enzymes was

discovered recently and is called DeSI for desumoylating

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isopeptidase (Shin et al. 2012). This group is composed of

two isoforms (DeSI-1 and DeSI-2) initially cloned from

murine T cell lymphoma. DeSI-1 and DeSI-2 orthologues

are also expressed in humans and rats. Murine DeSI-1 and

DeSI-2 comprise, respectively, 168 and 194 residues

(between 18 and 21 kDa), share only 23 % protein

sequence identity and possess a peptidase domain. DeSI-1

is localized both in the nucleus and in the cytoplasm, while

DeSI-2 distribution is mainly restricted to the cytoplasm

(Shin et al. 2012). The only known substrate of DeSI-1 is

the transcriptional repressor BZEL (BTB-ZF protein

expressed in effector lymphocytes). Importantly, the active

DeSI-1 overexpression decreases BZEL transcriptional

repressor activity (Shin et al. 2012). PML and DNp63, two

well-characterized substrates of the SENPs, are not desu-

moylated by DeSI-1, suggesting that SENP and DeSI

enzymes likely recognize different subsets of sumoylated

proteins (Shin et al. 2012). However, the regulatory

mechanisms as well as the substrate recognition properties

of these particular enzymes are still not known.

While the SUMO deconjugation process starts to be better

understood, many questions remain regarding the regulatory

processes governing their substrate specificity, interacting

partners and subcellular localization. Future work will now

be required to assess the physiological and pathophysiolog-

ical implications of these deconjugating enzymes.

What Does Sumoylation Do?

Sumoylation is essential in all eukaryotes, and the genetic

removal of Ubc9, the sole conjugating enzyme of the system,

causes cell death (Hayashi et al. 2002; Saracco et al. 2007)

and early embryonic lethality in mice (Nacerddine et al.

2005; Table 1). The main molecular function of the su-

moylation process is undoubtedly the regulation of protein–

protein interactions either by promoting novel subsets of

SIM-containing protein interactors (Kerscher 2007; Namanja

et al. 2012), by preventing specific interactions or by mod-

ulating the kinetics of complex formation and composition at

the molecular level (for a recent comprehensive review, see

Gareau et al. 2012). The balance between a sumoylated and

desumoylated state of a particular protein will tightly regu-

late the function of the targeted protein and will greatly

depend on the substrate, the subcellular localization and the

cell type in which the target protein is expressed.

The presumed function of sumoylation is the dynamic

regulation of protein solubility. SUMO has been charac-

terized as one of the most soluble proteins (Marblestone

et al. 2006), and this biochemical property established su-

moylation as a potential regulator of protein aggregation

and therefore as a putative causing agent in neurological

disorders characterized by reduced protein solubility and

pathological aggregation (Krumova and Weishaupt 2013).

Consistent with this view, many aggregation-prone pro-

teins involved in neurological disorders were found to be

highly sumoylated (Riley et al. 2005; Shinbo et al. 2006;

Steffan et al. 2004; Li et al. 2003; Fei et al. 2006; Janer

et al. 2010; Krumova et al. 2011). However, considerable

work remains to be done to determine whether the role of

sumoylation is detrimental or beneficial to pathologies

involving protein aggregation, and this will likely represent

an exciting avenue for future research.

Another thrilling aspect of the SUMO field comes from

the existing cross talk between sumoylation and other

posttranslational modifications (Gareau et al. 2012) and

particularly between sumoylation and ubiquitination path-

ways (Huang and Figueiredo-Pereira 2010). Indeed,

sumoylation has been shown to compete with ubiquitina-

tion for the modification of the same target lysine residues

to protect proteins from degradation. However, it is now

clear that this view of the interplay between these post-

translational modifications is highly reductive (Denuc and

Marfany 2010; Hunter and Sun 2009; Ulrich 2005). Several

reports on the cross talk between these two PTMs for the

functional regulation of the same target protein are now

available. As an example, several target proteins are

modified with poly-SUMO chains, leading to the detection

by SUMO-targeted ubiquitin ligases (STUbLs) ultimately

causing their proteasomal degradation (Geoffroy et al.

2010; Tatham et al. 2008; Abed et al. 2011).

Functional Consequences of Specific Protein

Sumoylation in Brain Development, Neuronal

Maturation and Synapse Formation

The massive capacity of the mammalian CNS to process and

store information is believed to rely on the formation, adapt-

ability or plasticity of functional synapses. Since sumoylation

participates in a multitude of cellular processes such as DNA

transcription, intracellular transport and cell signalling, it is

clearly emerging as a key determinant in the regulation of

neuronal maturation, synapse formation and activity.

Sumoylation in Neuronal Specification

The Family of Pax Transcription Factors

Pax genes encode a family of transcription factors that are

key to embryonic development of the CNS. Pax family

contains nine members that are evolutionarily conserved

and contribute to the pre- and postnatal CNS development

by orchestrating cell specification from very early stages of

the brain development (see Thompson and Ziman 2011 for

a comprehensive review on Pax proteins).

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Several members of the Pax family are targeted by the

sumoylation system. Among them, Pax6, an evolutionarily

conserved transcription factor playing major roles in brain

and eye development, was shown to be sumoylated. Su-

moylation of Pax6 at lysine residue 91 allows the binding

of the sumoylated protein to the target gene promoter and

was required to positively regulate expression of specific

target genes (Yan et al. 2010). They further demonstrated

that sumoylation of Pax6 occurs in vivo at embryonic days

E9.5 and E11.5 and showed that SUMO-Pax6 is localized

Table 1 Developmental

involvement of sumoylation in

genetically-modified mouse

models

SUMO

machinery

Mutation Phenotype Reference

Ubc9 KO Early embryonic lethality Nacerddine et al. 2005

Inducible KO Small intestinal epithelium defects Demarque et al. 2011

Constitutive

overexpression

No developmental defects

Protection from focal cerebral

ischemic damage

Lee et al. 2011

SUMO1 KO Palatogenesis defects

Embryonic lethality

Alkuraya et al. 2006

KO No phenotype under physiological

conditions

Evdokimov et al. 2008

KO No phenotype under physiological

conditions

Impaired adipogenesis under high-fat diet

Increased hepatic acute-phase responses

after lipopolysaccharide exposure

Zhang et al. 2008

Mikkonen et al. 2013

Venteclef et al. 2010

KO Embryonic lethality associated with

congenital heart defects

Wang et al. 2011

Pias1 KO Perinatal lethality

Interferon-inducible gene expression and

innate immune response misregulations

Liu et al. 2004

Pias2/Piasx KO No phenotype except reduced testis weight Santti et al. 2005

Pias4/Piasy KO No phenotype Wong et al. 2004

RanBP2 KO Embryonic lethality Aslanukov et al. 2006

Dawlaty et al. 2008

Haploinsufficiency Chromosome missegregation

Metabolic disorders

Age-dependent neuroprotection to

photosensory neurons

Dawlaty et al. 2008

Aslanukov et al. 2006

Cho et al. 2009

TOPORS KO Perinatal lethality Marshall et al. 2010

SENP1 KO Early embryonic lethality associated with

placental abnormalities

Yamaguchi et al. 2005

Sharma et al. 2013

KO Embryonic lethality

Erythropoiesis defects in fetal liver

Endothelial cell angiogenesis defects

Lymphoid development defects

Cheng et al. 2007

Xu et al. 2010

Van Nguyen et al. 2012

KO Embryonic lethality

Erythropoiesis defects in fetal liver

Yu et al. 2010

SENP2 KO Early embryonic lethality associated with

placental abnormalities

Chiu et al. 2008

KO Embryonic lethality associated with

congenital heart defects

Kang et al. 2010

Cardiac

overexpression

Embryonic/postnatal lethality associated

with congenital heart defects

Cardiac dysfunction in adult mice

Kim et al. 2012

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in the mouse embryonic eye, suggesting an important role

for Pax sumoylation in controlling mammalian eye and

brain development.

The transcription factor Pax7, an important player in

neural crest and muscle development, was very recently

reported to interact with the E2-conjugating SUMO

enzyme Ubc9 leading to its sumoylation at the lysine res-

idue 85 (Luan et al. 2013). Interestingly, Luan and col-

laborators further showed that the sumoylation machinery

is highly enriched within neural crest precursors. Contrary

to the results obtained with WT Pax7, the unilateral elec-

troporation of Pax7-invalidated chick embryos with a non-

sumoylatable form of Pax7 (K85R) was not able to rescue

the normal neural crest development, demonstrating that

sumoylation is essential to the function of Pax7 in neural

crest development (Luan et al. 2013). These data therefore

indicate that the posttranslational modification of Pax7 by

SUMO is a critical mechanism for the regulation of Pax7

activity at early stages of the brain development.

The transcription factor Pax8 was also shown to be su-

moylated (de Cristofaro et al. 2009). Pax8 is involved in

the morphogenesis of the thyroid gland and in the main-

tenance of the differentiated thyroid phenotype. Pax8 is

sumoylated on its lysine residue 309, and the E3 ligase

PIASy was able to increase the fraction of sumoylated

Pax8. Pax8 is targeted in the SUMO nuclear bodies, which

are structures that regulate the nucleoplasmic concentration

of transcription factors by SUMO trapping. Pax8 sumoy-

lation regulates the steady-state protein level of Pax8, and

although not directly linked to the brain development, Pax8

sumoylation may play important roles during thyroid

development and differentiation.

The Photoreceptor-specific Nr2e3 Transcription Factor

The vertebrate retina is a tractable and powerful model sys-

tem to assess neuronal cell specification. Development of rod

and cone photoreceptors from a common progenitor in the

retina is critically dependent on transcription factors includ-

ing the nuclear hormone receptor Nr2e3 and the basic motif-

leucine zipper transcription factor Nrl (neural retina leucine

zipper; Haider et al. 2000; Mears et al. 2001). Specification of

retinal rod photoreceptors is determined by these transcrip-

tion factors, which activate expression of rod-specific genes

and repress expression of cone-photoreceptor-specific genes

to promote rod differentiation. The group of Seth Blackshaw

has reported another important role for the sumoylation

process in the regulation of gene transcription during CNS

development. Based on their initial observations that the E3

SUMO ligase Pias3 mRNA was selectively expressed in

developing photoreceptors (Blackshaw et al. 2004), they

showed that PIAS3 is selectively expressed in developing rod

and cone photoreceptors and interacts with the transcription

factor NR2e3 (Onishi et al. 2009). They further demonstrated

that PIAS3 is required for the sumoylation of NR2e3 at three

lysine residues (K178, K315 and K322). Sumoylation of

Nr2e3 promotes rod photoreceptor differentiation by con-

verting Nr2e3 into a potent repressor of cone-specific gene

expression (Onishi et al. 2009). Blocking sumoylation in

photoreceptors results in cells with morphological and

molecular features of cones and an absence of rod-specific

markers consistent with a key role for the sumoylation pro-

cess in photoreceptor differentiation.

The Neural Retina Leucine Zipper NRL Transcription

Factor

The transcription factor Nrl is also central to the develop-

ment of rod photoreceptor. Indeed, in the absence of Nrl,

photoreceptor precursors in mouse retina produce only

cones that primarily express S-opsin. Conversely, ectopic

expression of Nrl in postmitotic precursors leads to a rod-

only retina. Nrl is sumoylated at lysine-20 and lysine-24

residues (Roger et al. 2010). Non-sumoylatable Nrl mutant

showed reduced transcriptional activation of two direct

targets of Nrl, the Nr2e3 and rhodopsin promoters. In vivo

electroporation of non-sumoylatable Nrl mutant protein

into mouse retina invalidated for Nrl leads to reduced

Nr2e3 activation and altered conversion of cones to rod

photoreceptors (Roger et al. 2010), suggesting a complex

role for the sumoylation process in the regulation of the

gene networks involved in photoreceptor development and

specification.

Sumoylation in Dendritic and Synaptic Morphogenesis

The family of Myocyte Enhancer Factor 2 Transcription

Factors

Myocyte-specific enhancer factor 2 (MEF2) is a family of

transcription factors that plays critical roles in cellular dif-

ferentiation and embryonic development. MEF2 target genes

have diverse functions at synapses, revealing a broad role for

MEF2 in synapse development. Several of these MEF2

targets are mutated in human neurological disorders,

including epilepsy and autism spectrum disorders, suggest-

ing that these disorders may be caused by disruption of an

activity-dependent gene program that controls synapse

development (Flavell et al. 2008). There are four MEF2

proteins denoted as MEF2A, MEF2B, MEF2C and MEF2D.

MEF2A is highly expressed in the brain where it plays key

roles in cell differentiation, dendritic morphogenesis and

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spinogenesis (Whitford et al. 2002). Many studies have

reported that the activity of these transcription factors is

tightly modulated by posttranslational modifications, such as

activation by phosphorylation as well as repression by class

II histone deacetylases (HDACs).

In an elegant study, Shalizi and colleagues demonstrated

the sumoylation-dependent repression of the transcription

factor MEF2A in the developing cerebellar cortex. Following

neuronal activation and calcium signalling, there is a molec-

ular switch from MEF2A sumoylation at lysine-403 to its

acetylation, leading to MEF2A activation and inhibition of

dendritic claw differentiation and to synapse disassembly

(Fig. 2; Shalizi et al. 2006). The nature of the MEF2 de-

sumoylase involved in the molecular switch is still unknown

but appears to be stimulated upon calcineurin dephosphoryl-

ation of MEF2A at serine-408. This work also represents

the first demonstration for a role of the sumoylation pathway

in an activity-dependent process occurring during the brain

development. MEF2A was also reported to be sumoylated

at the lysine-395 residue both in vitro and in vivo. The E3

ligase PIAS1 was shown to enhance the sumoylation of

MEF2A. The non-sumoylatable K395R MEF2A mutant has

enhanced transcriptional activity compared to the wild-type

sumoylatable protein (Riquelme et al. 2006). However, the

physiological consequences of this particular lysine-395

sumoylation were not investigated. Interestingly, it was

reported that the PIASx protein is also a MEF2A SUMO E3

ligase. Overexpression and knockdown experiments showed

that PIASx drives the differentiation of granule neuron den-

dritic claws in rat cerebellar slices, revealing novel functions

for PIASx in the establishment of neuronal connectivity dur-

ing brain development (Shalizi et al. 2007). More recently, Lu

and collaborators added an additional level of activity-

dependent regulation for MEF2 activity (Lu et al. 2012). They

used specific shRNA for MEF2A, SENP2 knockout embryos

and in vivo sumoylation assays and identified SENP2 as the

desumoylation enzyme of MEF2A. SENP2 protein stability

was markedly increased in depolarized SHSY5Y neuroblas-

toma cells leading to an enhanced transcriptional activity of

MEF2A, consistent with the repressing role of MEF2A

sumoylation (Lu et al. 2012).

Other members of the MEF2 family are targeted by the

SUMO system. This is the case for the transcription factor

MEF2C that is sumoylated at lysine-391 leading to the

repression of its transcriptional activity without altering its

DNA-binding activity (Kang et al. 2006). Interestingly,

phosphorylation of residue S396 in MEF2C, five residues

downstream of the sumoylation site, led to an enhanced

MEF2C sumoylation. S396A mutation reduced sumoyla-

tion of MEF2C in vivo and enhanced the transcriptional

activity of MEF2C, indicating that phosphorylation of

MEF2C at S396 facilitates its sumoylation at K391 leading

to inhibition of transcription (Kang et al. 2006).

Finally, the last family member of the MEF2 family,

MEF2D, was also reported to be sumoylated in humans.

Sumoylation of MEF2D by SUMO2 and SUMO3 occurs

within the C-terminal transcriptional activation domain at

lysine-439, and its sumoylation inhibits transcription. It

was also shown that the SUMO protease SENP3 reverses

MEF2D sumoylation, leading to its increased transcrip-

tional activity (Gregoire and Yang 2005). The same group

reported that phosphorylation of MEF2D at serine-444 by

the cyclin-dependent kinase 5 (cdk5) stimulates the

sumoylation of the protein and inhibits its transcriptional

activity (Gregoire et al. 2006). Together, all these data

reveal a coordinated cross talk between three posttransla-

tional modifications phosphorylation, acetylation and su-

moylation, to tightly control gene transcription, protein

functions in vivo and subsequently cell differentiation and

dendritic morphogenesis during the brain development.

The Calcium/Calmodulin-dependent Serine Protein

Kinase (CASK)

The calcium/calmodulin-dependent serine protein kinase

(CASK) is a scaffolding protein required for the formation

of dendritic spines and interacting with various synaptic

proteins. CASK is an essential gene, and its deletion leads

to lethality in mice just after birth. Several mutations of the

CASK gene have recently been reported and are associated

with X-linked mental retardation in humans, suggesting

important roles for CASK in brain development (Hackett

et al. 2010). Chao and colleagues reported that CASK is

sumoylated at the lysine-679 residue (Chao et al. 2008).

They showed that the sumoylation of CASK reduces the

interaction between CASK and the protein 4.1. Overex-

pression of a CASK–SUMO1 fusion construct in neurons,

which mimics CASK sumoylation, impaired spine forma-

tion, indicating that CASK sumoylation contributes to

spinogenesis (Chao et al. 2008).

The Activity-regulated Cytoskeleton-associated

Protein (Arc)

Arc is now seen as a central regulator of protein-synthesis-

dependent forms of synaptic plasticity and neurogenesis.

However, the mechanisms controlling Arc functions are

only beginning to be elucidated (Bramham et al. 2010). Arc

is essential to regulate actin cytoskeletal dynamics, which

underlie synaptic plasticity with the consolidation of long-

term potentiation (LTP) and the regulation of AMPA-type

glutamate receptor (AMPAR) endocytosis underlying long-

term depression (LTD). Arc has been suggested to be su-

moylated on two lysine residues, and the mutation of these

SUMO sites disrupts the subcellular localization of Arc in

dendrites (Bramham et al. 2010). More recently, it was

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123

reported by the Henley group that suppression of network

activity with the sodium channel blocker tetrodotoxin

(TTX) reduces the protein level of desumoylation enzyme

SENP1 leading to the increase in protein sumoylation. Arc

was also confirmed to be a SUMO substrate involved in the

TTX-induced increase in surface expression of AMPAR

that underlies homeostatic scaling (Craig et al. 2012). The

functional consequences of Arc sumoylation in other forms

of synaptic plasticity, in neurogenesis and in brain devel-

opment are still missing and will likely represent out-

standing issues to the scientific community.

The Metabotropic Glutamate Receptor mGluR8

Dysfunction of the seven transmembrane G-protein-cou-

pled receptor mGluR may have an important impact on

signal transduction, resulting in various neurological dis-

orders including, but not restricted to, night blindness,

addiction, epilepsy, schizophrenia and autism spectrum

disorders. mGluR8 belongs to the Group III metabotropic

glutamate receptors and plays key roles in regulating pre-

synaptic activity through G-protein-dependent signalling

pathways and intracellular protein–protein interactions

(Enz 2012). mGluR8 presents an extended intracellular

C-terminal domain, and using this C-terminal part in an

initial yeast two-hybrid screen, they identified the SUMO-

conjugating E2 enzyme Ubc9 and three SUMO E3s, Pias1,

PIASc and Piasxb, as interactors of mGluR8 (Tang et al.

2005). All Group III mGluR C-termini can be modified by

SUMO1 in vitro (Wilkinson et al. 2008). More recently, it

was shown that the splice variant mGluR8b is co-expressed

with Ubc9, SUMO1, PIAS1 and PIAS3L in cell bodies of

the ganglion cell layer, a particular subregion of the retina

(Dutting et al. 2011). mGluR8 seems to be sumoylated in

heterologous HEK293 cells; however, the demonstration

that endogenously expressed mGluR8 is sumoylated in

neurons as well as the functional consequences of this

modification is still missing.

Developmental Impact of Sumoylation in Transgenic

Mouse Models

Several animal models have been engineered in the recent

years to assess the significance, the developmental regu-

lation and the physiological roles of protein sumoylation

(Table 1). Consistent with its unique function in sumoy-

lation, the sole SUMO E2-conjugating enzyme Ubc9 is

essential to the embryonic development in mammals since

Ubc9-null mouse embryos die at early postimplantation

stage (Nacerddine et al. 2005). Ubc9 mutant blastocyst

presents abnormalities in nuclear and subnuclear architec-

ture and mislocalization of RanGAP1 and Ran, two

essential factors involved in nucleocytoplasmic exchanges

and mitotic defects in the structure and segregation of

chromosomes. To overcome these deleterious defects and

Fig. 2 A calcium-regulated MEF2A sumoylation switch dictates

postsynaptic differentiation in the mammalian brain. The transcrip-

tional activity of MEF2A is regulated by posttranslational modifica-

tions. In the absence of calcium influx, MEF2A is phosphorylated at

serine-408 and sumoylated at lysine-403, leading to the transcrip-

tional repression of the transcription factor Nur77. Activity-dependent

calcium signalling events promote the dephosphorylation of MEF2A

and its subsequent desumoylation. MEF2A is then acetylated at the

available lysine-403, leading to inhibition of dendritic claw differen-

tiation through the activation of specific genes that promote synapse

disassembly

Neuromol Med

123

study the developmental impact of the SUMO E2 enzyme,

Dejean’s group generated mice with inducible Ubc9

knockout (Demarque et al. 2011). Loss of Ubc9 expression

in adult mice induces a rapid and dramatic intestinal fail-

ure. However, the sudden death of Ubc9-deficient mice did

not allow the study of later defects in other tissues or

organs. Dissecting the consequences of Ubc9 inactivation

on other physiological processes such as brain development

and neuronal activity will therefore require the engineering

of tissue-specific knockout animals. In an alternative

strategy, Hallenbeck and collaborators generated trans-

genic mice in which Ubc9 expression is under the control

of the chicken b-actin promoter and thus highly synthe-

sized in all tissues. The expression levels varied greatly

among the analyzed mouse lines but led to an overall

enhanced cerebral sumoylation (Lee et al. 2011). Some of

the lines did not breed well and were eventually lost, but

the inability to breed was not correlated to Ubc9 expression

levels. Highly expressing Ubc9 mice subjected to focal

cerebral ischemia showed reduced cerebral infarct volume

compared to their wild-type littermates, indicating that an

elevated global sumoylation protects the brain from

ischemic damages.

The importance of SUMO1 in mammalian development is

still not fully elucidated. Several SUMO1 mouse lines have

been investigated with highly controversial results (Table 1).

The role of SUMO1 in embryonic development was initially

reported by Alkuraya and collaborators (Alkuraya et al.

2006). They showed palatogenesis defects and significant

modifications of Mendelian ratios from heterozygous

breeding with embryonic or immediate postnatal death for

both SUMO1 hetero- and homozygote mice. SUMO1 null

mouse lines were then generated by two groups using distinct

strategies (Zhang et al. 2008; Evdokimov et al. 2008).

Homozygous null mice were found to thrive normally under

classical living conditions, leading to the conclusion that

SUMO1 function is dispensable for embryonic development

or viability of adult animals. In those mice, RanGAP1, a

major SUMO1-modified protein, was found to be sumoy-

lated by SUMO2/3 at a much higher level than in wild-type

mice, suggesting that loss of SUMO1 expression could be

compensated for by SUMO2/3. Finally, the various SUMO1

mouse lines described earlier (Alkuraya et al. 2006; Evd-

okimov et al. 2008) were reexamined on a different genetic

background by an independent group (Wang et al. 2011). In

this work, SUMO1 null mice exhibited high mortality rates

with congenital heart defects that were rescued by cardiac

reexpression of the SUMO1 gene. Altogether, these reports

suggest that SUMO1 modifications may be important for

mammalian development, in particular for acquiring normal

cardiac functions. Deleterious effects in SUMO1-deficient

animals may be compensated by SUMO2/3 modifications

but greatly depend on the genetic background. Importantly,

SUMO1 null mice that are viable under physiological con-

ditions may present significant defects under challenging

conditions. Accordingly, SUMO1-deficient mice from the

Janne’s group showed impaired adipogenesis in high-fat diet

(Mikkonen et al. 2013) and increased hepatic acute-phase

responses after lipopolysaccharide exposure (Venteclef et al.

2010). Interestingly, those mice present mild alteration in

nociception that could be further explored. Furthermore,

given the protective effect of sumoylation against cerebral

ischemia damages, testing SUMO1 null mice for their sen-

sitivity to focal ischemia would be of great interest to better

understand the role of SUMO1 sumoylation in such

conditions.

SUMO conjugation is facilitated by different SUMO E3

ligases, including members of the PIAS family, TOPORS,

Pc2 and RanBP2 (see above for further details). PIAS1,

PIASx or PIASy null mice have been generated, but these

KO animals display no obvious cellular sumoylation

defects (Santti et al. 2005; Wong et al. 2004; Liu et al.

2004). Moreover, PIASx and PIASy are not essential for

embryonic development and viability in adult mice.

PIAS1-deficient mice, however, showed a slight increase in

perinatal lethality compared to their wild-type littermates.

These subtle phenotypes may reflect a functional redun-

dancy between the members of the PIAS family during

mouse embryonic development. Consistent with this view,

double-knockout embryos for PIAS1 and PIASy died early

during embryonic development. However, since PIAS

transcriptional regulation activity does not necessarily rely

on their SUMO E3 ligase activity, the effective impact of

an impaired sumoylation in these developmental defects

remains to be evaluated. Similarly, given the dual ubiquitin

and SUMO ligase activities of the E3 TOPORS, it is dif-

ficult to solely attribute perinatal lethality found in TO-

PORS-null mice to SUMO-mediated deregulation

(Marshall et al. 2010). Finally, RanBP2-null mutant mice

showed embryonic lethality, revealing a key role for this

multifunctional protein in mammalian development.

Interestingly, reduced expression levels of RanBP2 led to

different deficits with a missegregation of chromosomes

(Aslanukov et al. 2006; Dawlaty et al. 2008) or alterations

in glucose and lipid metabolisms (Aslanukov et al. 2006).

Importantly, RanBP2 haplo-insufficiency also conferred an

age-dependent neuroprotection to photosensory neurons

upon a prolonged light-elicited oxidative stress, a delete-

rious factor involved in the pathological degeneration of

the retina (Cho et al. 2009). However, it is important to

stress here that the mechanisms by which these RanBP2

effects occur and in particular with respect to the sumoy-

lation process are far from being elucidated.

As mentioned above, SENPs are key deconjugating

enzymes important for the tight control of the cellular su-

moylation status in mammalian cells. To elucidate the

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123

biological functions of SENP1 and SENP2, several groups

generated KO mice (Table 1). All reports revealed that the

disruption of either SENP1 or SENP2 causes strong

developmental defects leading to embryonic or early

postnatal lethality depending on the knockout strategy and/

or the genetic background. The most precocious death was

associated with severe abnormalities in placental organi-

zation (Yamaguchi et al. 2005; Sharma et al. 2013; Chiu

et al. 2008). In other studies, SENP1 disruption led to

multiple defects in fetus affecting erythropoiesis in fetal

liver (Cheng et al. 2007; Yu et al. 2010), endothelial cell

angiogenesis (Xu et al. 2010) or early lymphoid develop-

ment (Van Nguyen et al. 2012). SENP2 disruption or tis-

sue-specific overexpression induced embryonic heart

malformation and cardiac dysfunction in adult mice.

SENP1 or SENP2 knockout effects resulted at least par-

tially from an altered sumoylation level of key transcription

regulators, including the hypoxia-inducible factor 1a(HIF1a), p53 and polycomb group proteins leading to

modification of their stability, localization or promoter-

binding activities. Whether the brain development is

affected in SENP1 or SENP2-null mice was not specified

in these reports and therefore remains as an opened excit-

ing question.

Developmental and Activity-dependent Regulation

of Sumoylation in the Vertebrate CNS

The involvement of sumoylation in neuronal functions has

been extensively reviewed elsewhere (Krumova and We-

ishaupt 2013; Martin 2009; Wilkinson et al. 2010; Martin

et al. 2007b), but the developmental regulation of this

essential protein modification has received very little atten-

tion despite essential impacts on embryonic life (Table 1).

Neuronal activity events are tightly associated with the

regulation of the location, the number and the strength of

synapses that are formed during the CNS development

(Flavell and Greenberg 2008). These events rely on a myriad

of cellular processes occurring back and forth and ranging

from transcriptional regulations in the nucleus to the for-

mation and organization of complex protein networks locally

at synapses. Sumoylation is acting throughout the cell and is

now clearly emerging as a potent regulator of protein inter-

actions, degradation/stability and/or subcellular targeting of

modified target proteins. However, an accurate posttransla-

tional regulatory function implies that the SUMO regulator

must be regulated. In this respect, the levels of expression as

well as the spatiotemporal distribution of the sumoylation

machinery are emerging as efficient ways to dynamically

modulate protein function in the developing brain.

Indeed, mRNA levels of Ubc9 and SUMO1 are develop-

mentally regulated in the rat brain, with higher expression

levels before birth (Watanabe et al. 2008). More recently, we

reported that the protein expression levels of SUMO-modified

substrates as well as several components of the sumoylation

machinery are temporally and spatially regulated in the

developing rat brain (Loriol et al. 2012). High levels of nuclear

protein sumoylation were detected early in the development

with a transient sumoylation increase peaking at embryonic

day 12 for both SUMO1 and SUMO2/3 sumoylation and a

second increasing phase at birth for SUMO2/3 substrates

consistent with an important role for sumoylation over the

synaptogenesis period. The overall sumoylation is then slowly

decreased in aging brain except in synaptic compartments

where there are progressively more SUMO substrates (Loriol

et al. 2012). The relative increase in synaptic sumoylation in

adult brain is consistent with an enrichment of the sumoylation

machinery in dendritic spines of fully mature rat hippocampal

neurons with an increase in synaptic sumoylation enzymes

AoS1 and Ubc9 immunoreactivities and a concomitant

decrease in synaptic desumoylase SENP1 immunoreactivity

(Loriol et al. 2012). Our data from Fig. 3 show that AoS1 and

Ubc9 SUMO enzymes as well as SENP1 and SENP6 decon-

jugating proteins are expressed in dendrites and partially

colocalized at synapses in mature mouse cortical neurons

consistent with our initial demonstration that sumoylation

occurs at synapses (Martin et al. 2007a).

The persistence of a pool of SUMO1- and SUMO2/3-

modified proteins at synapses and the synaptic distribution

of the sumoylation machinery in adult brains (Loriol et al.

2012) suggests a role for the sumoylation process in the

regulation of pre- and postsynaptic functions. Consistent

with this view, it was shown that sumoylation of presynaptic

proteins modulates neurotransmitter release. Increasing

protein sumoylation by entrapping recombinant SUMO1 in

synaptosomes decreased glutamate release evoked by KCl,

whereas decreasing sumoylation with the catalytic domain

of SENP1 enhanced KCl-evoked release (Feligioni et al.

2009). Dai and colleagues also reported that SUMO1 asso-

ciates with the exocytotic calcium sensor synaptotagmin VII

and that SENP1 overexpression enhances insulin exocytosis

in pancreatic b-cells (Dai et al. 2011). Consistent with a

functional role of sumoylation at presynaptic terminals, we

reported that a neuronal depolarization triggers the redistri-

bution of the SUMO machinery with a sustained accumu-

lation of AoS1 and Ubc9 sumoylation enzymes and a

transient decrease in the desumoylase SENP1 immunore-

activity at presynaptic terminals of rat hippocampal neurons

(Loriol et al. 2013). Postsynaptic SUMO enzyme levels are

also transiently decreased upon neuronal activation, result-

ing in a transient decrease in SUMO2/3-sumoylated protein

levels in synaptoneurosomes, whereas SUMO1-modified

protein levels remain unchanged. Interestingly, this transient

synaptic drop occurs without affecting global sumoylation

levels, indicating that this regulation happens locally at

Neuromol Med

123

synapses in an activity-dependent manner (Loriol et al.

2013).

Another potential way to locally regulate neuronal su-

moylation has been recently reported by the Henley group.

They showed an increased level of both SUMO1 and Ubc9

mRNAs in dendrites upon a chemical long-term potentiation

(ChemLTP) protocol suggesting an activity-dependent

translation of the sumoylation machinery in dendrites near

synaptic sites (Jaafari et al. 2013). Interestingly, ChemLTP

led to an increased colocalization of SUMO-1 with Ubc9 at

PSD95 synaptic sites consistent with the recruitment of su-

moylated materials to dendritic spines. Using this ChemLTP

protocol, the same group showed that sumoylation by both

SUMO1 and SUMO2/3 of the brain-specific G-protein-cou-

pled receptor interacting scaffold protein GISP occurs in

neurons, revealing GISP as a novel neuronal SUMO sub-

strate (Kantamneni et al. 2011). The functional consequences

of this activity-dependent GISP sumoylation are still not

known. Overall, these data are consistent with an important

role for the sumoylation/desumoylation process in synaptic

functions. However, the regulatory mechanisms underlying

the regulation of the sumoylation machinery transport in

axon, dendrites and spines are still largely unspecified.

The overall understanding of the molecular events occur-

ring in the activity-dependent and spatiotemporal regulation

of the sumoylation machinery is of particular interest to the

field of neurological disorders. For instance, a link between

the sumoylation process and the devastating brain disorder

schizophrenia was recently reported (Rubio et al. 2013). The

level of the SUMO E3 ligase PIAS3 protein is significantly

decreased in the superior temporal gyrus of schizophrenia

compared to control subjects. The authors propose that given

the role of sumoylation in spinogenesis (Chao et al. 2008) and

the synaptic redistribution of the sumoylation machinery

occurring in an activity-dependent manner (Loriol et al.

2013), a delayed redistribution of SUMO components during

early adulthood may lead to changes in the synaptic pruning

observed in schizophrenia (Rubio et al. 2013). Alterations in

the sumoylation pathway in the developing brain could

therefore underlie some of the critical features of the disease.

Conclusion

As highlighted in this review, sumoylation emerges as a major

reversible posttranslational process that contributes to a

Fig. 3 Subcellular localization of the sumoylation machinery in

mouse cortical neurons. Confocal images show the partial colocal-

ization (yellow) between the postsynaptic markers Homer1 in green

and in red, the sumoylation enzymes AoS1 (a) and Ubc9 (b), the

desumoylases SENP1 (c), SENP6 (d), SUMO1 (e) and SUMO2/3

(f) in 15 DIV cultured mouse cortical neurons. Higher magnification

of dendrite is also depicted. Scale bars 20 lm (Color figure online)

Neuromol Med

123

variety of regulatory actions at different stages of the brain

development. The future discovery and functional character-

ization of novel sumoylated substrates at all stages of the brain

development will undoubtedly help our understanding on the

role of the sumoylation process in brain development, neu-

ronal morphology and spinogenesis. Further studies are now

required to unravel how this important posttranslational reg-

ulatory mechanism is orchestrated in the developing brain.

Acknowledgments We thank F. Aguila for excellent artwork. We

gratefully acknowledge the ‘Fondation pour la Recherche Medicale’

(Equipe labellisee ‘Fondation pour la Recherche Medicale’

DEQ20111223747), the National Research Agency (ANR JCJC), the

‘Jerome Lejeune’ and ‘Bettencourt-Schueller’ foundations for finan-

cial support. S.M. is a fellow from Young Investigator CNRS ATIP/

ATIP? and ANR JCJC programs. This work was also supported by

the French Government through the ‘Investments for the Future,’

LabEx SIGNALIFE # ANR-11-LABX-0028-01.

Conflict of interest None.

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