의학 박사학위 논문

Studies on the Cytoplasmic Inclusions

Detected in Parkinson's disease

아 주 대 학 교 대 학 원

의 학 과

류 명 이

Studies on the Cytoplasmic Inclusions Detected in Parkinson's disease

by

Myung-Yi Ryu

A Dissertation Submitted to The Graduate School of Ajou University

in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY IN NEUROSCIENCE

Supervised by

Soo-Han Yoon, M.D., Ph.D.

Department of Medical Sciences

The Graduate School, Ajou University

February, 2007

류명이의 의학 박사학위 논문을 인준함.

심사위원장 이 광 인

심 사 위 원 윤 수 한 인

심 사 위 원 신 용 삼 인

심 사 위 원 최 동 국 인

심 사 위 원 이 필 휴 인

아 주 대 학 교 대 학 원

2006 년 12 월 22 일

ACKNOWLEDGEMENTS

A special thanks to my family and husband, Jong Pil for their loving support to

complete my Ph.D. This dissertation would not have been possible without them. I am

grateful to Dr. Yoon Soo Han and Lee Gwang, my advisor, for giving me guidance and

counsel and my committee members for their comments and suggestions. It has been a

pleasure working with my colleagues, in particular, Wen Yu and Kyoung-a. I am deeply

grateful to them for investing time and energy discussing idea with me.

ABSTRACT

Studies on the Cytoplasmic Inclusions Detected

in Parkinson’s disease

α-Synuclein-positive cytoplasmic inclusions are a pathological hallmark of several

neurodegenerative disorders, including Parkinson’s disease (PD), dementia with Lewy

bodies (DLB), and multiple system atrophy (MSA). Synphilin-1, interaction partner of α-

Synuclein is a major component of inclusion bodies, but it is unknown how synphiln-1

contributes to the cellular and biochemical mechanisms of PD, and its normal functions and

biochemical properties are poorly understood. To determine the protein interaction partners

of synphilin-1, we performed a yeast two-hybrid screen. We identified a new interacting

protein LIM domain only 7 protein, LMO7. This protein localized in the nucleus, cytoplasm

and cell surface, particularly adhesion junctions and contains a PDZ and LIM domain, both

of which mediate protein–protein interactions. In this study, LMO7 interacts with α-

synuclein interacting protein, synphilin-1 and revealed that the co-expression with synphilin-

1 results in the formation of cytoplasmic inclusions in cultured HEK293 and SY5Y cells.

Synphilin-1 interacts preferentially with the C-terminal LIM domain of LMO7 and LMO7

interacts with the ankyrin domain of synphilin-1. These findings have important implications

for understanding the molecular mechanism by which Lewy-body–associated proteins

interact through synphilin-1. We immunostained sections of brains from patients with

Parkinson’s disease and demonstrated that LMO7, as well as synphilin-1, accumulates in the

i

inclusion bodies. To define the role of LMO7 in the formation of these inclusion bodies, we

performed a co-transfection with synphilin-1 and LMO7 using cultured HEK293 cells. This

assay showed that LMO7 in the formation of these inclusion bodies promotes the formation

of cytoplasmic inclusions.

α-Synuclein, synphilin-1 and its interacting partner LMO7 are among constituent

proteins in these aggregates. The presence of ubiquitin and proteasome subunits in these

inclusions supports a role for this protein degradation pathway in the processing of proteins

involved in this disease. Treatment with proteasome inhibitors resulted in attenuation of

degradation and the accumulation of high molecular weight ubiquitinated LMO7 in

immunoprecipitation /immunoblot experiments. Additionally, proteasome inhibitors

stimulated the formation of peri-nuclear inclusions which were immunoreactive for LMO7,

ubiquitin and synphilin-1. These observations indicate that LMO7 is ubiquitinated and

degraded by the proteasome. Accumulation of ubiquitinated LMO7 due to impaired

clearance results in its aggregation as peri-nuclear inclusions.

These results suggest that LMO7 could serve as a neuropathological marker in patients

with α-synucleinopathies because it is strongly accumulated with synphilin-1 in the

inclusions of their brain cells. They also suggest that LMO7 could be a potential therapeutic

target for α-synucleinopathies.

Keywords: Parkinson’s disease, Lewy body, inclusion, α-synuclein, synphilin-1, PDZ and

LIM domain, LIM domain, LIM domain only 7 (LMO7), proteasome, ubiquitin,

neurodegenerative disorder, α-synucleinopathies

ii

TABLE OF CONTENTS

● PART I

ABSTRACT ···················································································································· i TABLE OF CONTENTS ···························································································· iii LIST OF FIGURES ······································································································ v I. INTRODUCTION ·································································································· 1 II. MATERIALS AND METHODS········································································· 14 A. MATERIALS ····································································································· 14 B. METHODS ········································································································· 14 1. Yeast two-hybrid screening ············································································· 14 2. X-gal assay and ONPG assay ········································································· 15 3. Generation of LMO7 complementary DNA ·················································· 16 5. In vitro binding assay ····················································································· 17

8. Western immunoblot analysis ········································································· 19 staining ···································································· 19

10. Primary culture of rat brain cortex ···························································· 20

13. Immunohistochemistry on human brain tissues ······································· 21 23

III. RESULTS ·············································································································· 24 DISCUSSION ········································································································ 42

CONCLUSION ······································································································· 45 ES ············································································································ 46

4. Cell culture, transfection and generation of stable cell lines ····················· 16

6. Immunocytochemistry ···················································································· 18 7. Preparation of LMO7 polyclonal antibodies ··············································· 18

9. Hematoxylin and eosin

11. LMO7-siRNA treatment ·············································································· 20 12. Quantitation of cells containing inclusions ·················································· 21

14. Statistical analysis ·························································································

IV.V.REFERENC

iii

● PART II

I. INTRODUCTION ·································································································· 55

II. MATERIALS AND METHODS········································································· 60 A. MATERIALS ····································································································· 60 B. METHODS ········································································································· 60 1. Cell culture and generation of stable cell line ·············································· 60 2. Inhibition of LMO7 protein synyhesis ························································· 60 3. Immunoprecipitation and immunoblot analysis ········································· 61 4. Immunostaining of LMO7-293 cell treated protease inhibitor ················· 61 III. RESULTS ·············································································································· 63 IV. DISCUSSION ········································································································ 68 V. CONCLUSION ······································································································· 70 REF

● P

DISCUSSION ········································································································ 82 CONCLUSION ······································································································· 83

ERENCES ············································································································ 84 ·················································································································· 87

ERENCES ············································································································ 71

ART III

I. INTRODUCTION ·································································································· 77 II. MATERIALS AND METHODS········································································· 79 A. METHODS ········································································································ 79 1. Case material ····································································································· 79 2. Immunohistochemical analysis of human brain tissues ······························· 79

ULTS ·············································································································· 80 III. RESIV.V.REF국문요약 ·····

iv

LIST OF FIGURES

ion of synphilin-1 with LMO7 in yeast. ··············································· 25

ig. 7. RNA interference of LMO7 by siRNA······························································ 36

Fig. 8. LMO7-mediated regulation in the formation of synphilin-1-positive inclusions 38

ig. 9. Location of overexpressed LMO7 in synphilin-1-positive inclusions ··············· 39 Fig. 10. Localization of LMO7 ains ····························· 41

● PART I Fig. 1. Schematic view of the synphilin-1 ····································································· 6

Fig. 2. Associat

Fig. 3. Determination of the interacting domains of LMO7 and synphilin-1 in a yeast ytwo-hybrid s stem ····························································································· 27

Fig. 4. Interaction of synphilin-1 and LMO7 ································································ 30 Fig. 5. Synphilin-1 interacts preferentially with the C-terminal LIM domain of LMO7

and LMO7 interacts with the ankyrin domain of synphilin-1. ···························· 32 Fig. 6. Formation of LMO7-positive inclusion in primary cortical neuron cultures····· 34

F

F

in inclusions of PD patient br

● PART II

Fig. 1. The ubiquitin proteasome-mediated pathway ·················································· 56

Fig. 2. Stability of LMO7 protein ················································································ 63

v

Fig. 3. Attenuation of LMO7 degradation by proteasome inhibitor ···························· 64

ig. 4. Ptoreasomal degradation and ubiquitination of LMO7······································ 66

ig. 5. Co-localization of ubiquitin in LMO7 positive inclusions ································ 67

PART III

ig. 1. CKII β subunits are present in Lewy bodies of aged human brains··················· 81

ig. 2. CKII β subunit co-localized with α-synuclein in aged human brain ················ 81

F F

●

F F

vi

RART I

LMO7 associates with synphilin-1 and promotes

the formation of cytosolic inclusion

PART II

LMO7 degradation

by the ubiquitin-proteasome pathway

PART III

Localization of CKII-β subunits

in Lewy bodies of aged human brains

I. INTRODUCTION 1. The cellular pathology of Parkinson’s disease

Parkinson’s disease (PD) is the second most common progressive neurodegenerative

brain disorder of humans, after Alzheimer’s disease. PD affects approximately 1% of the

population by the age of 65 years (Tanner, 1992), with a higher prevalence in men (Dluzen

and McDermott, 2000), it usually manifests itself in the fifth or sixth decade of life. PD is

characterized clinically by severe motor symptoms including uncontrollable resting tremor,

muscular rigidity, impaired postural reflexes, and bradykinesia, which vary between patients

(Lotharius and Brundin, 2002; Siderowf and Stern, 2003). These abnormalities can be

accompanied by other symptoms, such as autonomic dysfunction, depression, and a general

slowing of intellectual processes (Berrios et al., 1995). Pathologically, PD is characterized

by the marked degeneration of dopaminergic neurons in the substantia nigra pars compacta,

which leads to the depletion of dopamine (DA) in its striatal projections, and of other

brainstem neurons, with consequent disruption of the cerebral neuronal systems responsible

for motor functions (Lotharius and Brundin, 2002; Siderowf and Stern, 2003). This

neurodegeneration is accompanied by the presence of cytoplasmic (Lewy bodies, LBs) and

neuritic (Lewy neurites, LNs) inclusions (Gomez-Tortosa et al., 1999) in the surviving

dopaminergic neurons and other affected regions of the central nervous system (CNS), but

the mechanism underlying their formation is unclear, as is their pathogenic relevance.

PD is an essentially sporadic neurodegenerative disease whose pathogenesis remains

largely unknown, despite years of intense research in an attempt to explain the complexity

1

and the relative selectivity of dopaminergic neurodegeneration. Genetic and environmental

risk factors (Warner and Schapira, 2003) have gained more attention of late as possible

causes of PD, but their relative contributions in initiating the neurodegenerative process

continue to be debated. Based on current knowledge, late-onset idiopathic PD is thought to

result from a complex interaction among multiple predisposing genes and environmental

factors. In addition to sporadic forms of PD, several rare monogenic familial forms of the

disease, characterized by early-onset and an autosomal dominant or recessive pattern of

inheritance have been identified. Mutations in four genes have been clearly linked to PD

encoding α-synuclein (α-syn) (Polymeropoulos et al., 1997), parkin (Shimura et al., 2001),

ubiquitin carboxy-terminal hydrolase L-1 (Leroy et al., 1998), and DJ-1 (Bonifati et al.,

2003). Other genes or loci that may cause PD have been mapped in families (Siderowf and

stern, 2003; Polymeropoulos, 2000; Nussbaum and Ellis, 2003; Dawson TM and Dawson

VL, 2003).

Although familial forms of PD with specific genetic defects represent only a minor

part (~ 10%) of all cases, they may help to identify key abnormalities in protein pathways

that are likely to be involved in the more common, multifactorial sporadic form of the

disease. Mutations in the gene encoding for α-syn have received a great deal of attention

with the discovery that fibrillar α-syn aggregates are the major components of both LBs and

LNs (Spillantini et al., 1997; Spillantini et al., 1998), characterizing most familial and

sporadic PD. This observation suggests that although α-syn is infrequently mutated in PD,

other cellular processes that could lead to abnormal metabolism and accumulation of this

protein might play an important role in the pathogenesis of sporadic as well as familial

2

disease.

2. Mechanisms of α-synuclein aggregation

Abnormal protein aggregation appears to be a common feature in aging brain and in

several neurodegenerative diseases, although a clear role in the disease process remains to be

defined. In in vitro models, α-synuclein (or some of its truncated forms) readily assembles

into filaments resembling those isolated from brain of patients with LB dementia and

familiar PD (Crowther et al., 1998). The peptide derived from the central hydrophobic

region of α-syn (NAC) represents a second major intrinsic constituent of Alzheimer’s

plaques (Uversky and Fink, 2002).

Normal α-synuclein and its mutated forms (A53T and A30P) have a random coil

conformation and do not form significant secondary structure in aqueous solution at low

concentrations; however, at higher concentrations they are prone to self-aggregate,

producing amyloid fibrils (Wood et al., 1999). Several differences in the aggregation

behavior of the PD-linked mutants and the wildtype protein have been documented.

Monomeric α-synuclein aggregates in vitro to form stable fibrils via a metastable oligomeric

(i.e., protofibril) state (Volles et al., 2002). The protofibrillization rate of both mutants is

higher than that of wild-type protein; the fibrillation rate is lower in A30P and higher in

A53T (Conway et al., 2000; Conway et al., 2000).

Several mechanisms for α-syn aggregation have been proposed; those involving the

ubiquitin proteasome system (UPS) and oxidative stress have gained the most prominence

until now. UPS is the primary biochemical pathway responsible for the degradation of

3

normal and abnormal (mutated, misfolded, or unassembled) intracellular proteins

(Ciechanover, 2001). Failure of this system leads to protein accumulation and cell death

(McNaught et al., 2003). Degradation via UPS involves two successive steps: initially, the

protein substrate is tagged by covalent attachment of multiple ubiquitin molecules through

the action of ubiquitin-conjugating enzymes. Subsequently, the tagged protein is degraded by

the 26S proteasome, with release of reusable ubiquitin (Ciechanover, 2001). The 26S

proteasome belongs to the proteasome family of multicatalytic proteases and is located in the

cytoplasm, endoplasmic reticulum, perinuclear region, and nucleus of eukaryotic cells

(Voges et al., 1999). A growing body of evidence suggests that ubiquitin-dependent protein

degradation may be impaired in many neurodegenerative diseases, including PD and diffuse

LB disease (DLBD).

A key pathological feature in PD and DLBD is the formation of ubiquitinated

cytoplasmic inclusions (Gibb and Lees, 1988). In PD, LBs are formed within the

dopaminergic neurons of the substantia nigra pars compacta. On the other hand, the LB-

ubiquitin is in the form of polyubiquitin chains rather than ubiquitin monomers in DLBD, as

shown by biochemical analyses of isolated cortical LBs from postmortem tissue (Iwatsubo et

al., 1996). This observation suggests that poly-ubiquitinated proteins may accumulate in

inclusions as a result of a dysfunction in the proteasome degradation process. Besides

ubiquitin, LBs contain α-syn, subunits of the 26S proteasome, and other proteins including

parkin, 14-3-3 protein (Xu et al., 2002), and synphilin-1 (Spillantini et al., 1997). The last

forms a complex with α-synuclein, which is then ubiquitinated by the E3 ubiquitin ligase

activity (Chung et al., 2001). A mutation in parkin leads to autosomal recessive juvenile

4

parkinsonism, which commonly lacks microscopic α-syn Lewy-type aggregates (West et al.,

2002). Despite the absence of LBs, selective accumulation of the putatively toxic αSp22 has

been demonstrated in parkin-linked PD brains (Shimra et al., 2001), suggesting that parkin

mutations may predispose to accumulation of α-synuclein in a soluble nonfibrillar form.

Taken together, these findings propose that UPS inactivity may contribute to the

development of neurodegeneration in PD forms either or not characterized by LB formation.

Another mechanism underlying α-synuclein aggregation may involve the action of

oxidants, which cause α-synuclein to aggregate and thereby perhaps initiate formation of

toxic intermediate oligomers (Hashimoto et al., 1999; Goldberg and Lansbury, 2000),

probably due to a kinetic stabilization of the α-syn protofibril by a dopamine-α-synuclein

adduct (Conway et al., 2001). Other in vitro studies have revealed that overexpression of α-

synuclein can induce iron-dependent aggregation (Ostrerova-Golts et al., 2000).

3. Synphilin-1, binding partner of alpha-synuclein

3.1 Synphilin-1, protein family and structural implications

Synphilin-1 is a 919-amino-acid protein with a molecular mass of 115–140 kDa. The

physiological function of the protein is currently unknown, although several protein domains

have been defined, including six ankyrin-like repeats, a coiled-coil domain, and an

ATP/GTP-binding motif (Fig. 1). These domains are known to be present in a variety of

proteins mediating protein–protein interactions and underscore the relevance of defining

synphilin-1-interacting proteins. The highly conserved peptide sequence argues in favor of

critical domains for the physiological functioning of synphilin-1. Indeed, studies defining the

5

critical domains of synphilin-1 for interaction with alpha-synuclein, parkin, and dorfin have

identified fragments containing the central region of synphilin-1 (Fig. 1). A fragment

harboring amino acid residues 349–555 has been shown to be necessary and sufficient to

mediate interaction with alpha-synuclein (Neystat et al., 2002). The strongest interaction of

synphilin-1 with the E3 ligase parkin has been observed by using fragments encompassing

amino acid residues 214–556 (Chung et al., 2001). As expected from previous binding

studies, no differential interaction has been found with the known interacting proteins

alphasynuclein and parkin by using R621C mutant synphilin-1 protein. In contrast to

previously reported synphilin-1-interacting proteins, the ankyrin-like repeats and coiledcoil

domain located at amino acids 350–549 are not essential for the interaction of synphilin-1

with the E3 ligases SIAH-1 and SIAH-2 (Nagano et al. 2003; Liani et al. 2004; Fig. 1). For

these proteins, the minimal binding region of synphilin-1 has been narrowed to the first 202

(SIAH-1) or the first 227 (SIAH-2) amino acid residues, respectively (Nagano et al., 2003;

Liani et al., 2004).

Fig. 1. Schematic view of the synphilin-1. Red bars critical domains for interaction with alpha-synuclein, parkin, dorfin, and SIAH-1, ANK ankyrin-repeat, Coil coiled-coil domain

6

3.2 Synphilin-1 and cellural function

Although synphilin-1 identified 7 years ago, only little is known about the

physiological function of synphilin-1. Like its interacting protein, alpha-synuclein,

synphilin-1 shows predominant neuronal expression and is enriched in presynaptic nerve

terminals during development (Ribeiro et al., 2002). This presynaptic localization is a result

of the developmental redistribution from the cell body to the nerve terminals and therefore

might reflect the maturation of synapses. Interestingly, contrasting observations concerning

this intracellular compartmentalization have been made in rat substantia nigra neurons, in

which synphilin-1 persists in the soma (Ribeiro et al., 2002). This indicates that synphilin-1

is available for the formation of pathological protein inclusion in susceptible areas. The

redistribution to axons in rat neurons is associated with a shift of molecular mass for

synphilin-1 from 115–140 kDa to 80–90 kDa, indicating the processing of the synphilin-1

protein (Ribeiro et al. 2002). In normal human brain tissue, synphilin-1 is primarily observed

in large neurons including Purkinje, nigral, and pyramidal neurons (Engelender et al., 2000;

Ribeiro et al., 2002; Murray et al. 2003). Analyses of human brain extracts have revealed

synphilin-1 predominantly as a 90-kDa band, but it also occurs as a 120-kDa fragment, and

lower molecular bands of 50 kDa and 65 kDa have been observed, supporting alternative

splicing or post-translational processing as mechanisms of synphilin-1 diversity (Murray et

al., 2003). The identification of the respective alternative fragments of synphilin-1 is of

interest for the determination of the mechanisms of intracellular redistribution and synaptic

function. Biochemical data indicating the presence of synphilin-1 in lipid fractions of brain

extracts are in agreement with the role of synphilin-1 as a synaptic vesicle-binding protein

7

(Ribeiro et al., 2002; Murray et al., 2003). The apparent intracellular co-localization with the

interacting alpha-synuclein protein also implies functional consequences. Synphilin-1

binding to synaptic vesicles has been shown to be negatively modulated by alphasynuclein

(Ribeiro et al., 2002). Moreover, synphilin-1 has been suggested to mediate the synaptic

functions of alphasynuclein, possibly by anchoring alpha-synuclein to the vesicle membrane.

Evidence for the relevance of the tightly regulated interaction with alpha-synuclein and of

binding to synaptic vesicles has come from observations of cultured cells. These studies

indicate that synphilin-1 is phosphorylated by casein kinase II and that inhibition of this

modification reduces its ability to interact with alphasynuclein and to form cytoplasmic

inclusions (Lee et al., 2004). The phosphorylation of synphilin-1 by glycogen synthase

kinase-3beta has been described based on a candidate approach (Tanji et al., 2003).

Glycogen synthase kinase-3beta is known to phosphorylate tau-protein, which is involved in

Alzheimer’s disease pathogenesis. This supports the role of post-translational modifications

of synphilin-1, although the functional implications of glycogen synthase kinase-3beta-

mediated phosphorylation remain to be determined. In this context, Tanji et al. (2003) have

speculated that synphilin-1 phosphorylation might trigger the ubiquitination and degradation

of synphilin-1. The identification of synphilin-1 as a substrate of the ubiquitin E3 ligase

parkin, the second known protein involved in PD pathogenesis, established the first link to

the ubiquitin-mediated protein degradation pathway (Chung et al., 2001). Several studies

have found that synphilin-1 is poly-ubiquitinated and subsequently degraded by the

proteasome (Chung et al., 2001; Lee et al., 2002). Recently, three other E3 ligases have been

found to ubiquitinate synphilin-1 and to mediate synphilin-1 degradation: dorfin, SIAH-1

8

and SIAH-2 (Ito et al., 2003; Nagano et al., 2003; Liani et al., 2004). Interestingly, all these

ubiquitin ligases, parkin, dorfin, and SIAH-1, are components of Lewy bodies in the brains

of PD patients and therefore are related to neurodegeneration (Schlossmacher et al., 2002;

Ito et al., 2003; Liani et al., 2004). This group has established a direct functional link of

synphilin-1 with the proteasome by the identification of the proteasomal protein S6 as a

novel synphilin-1-interacting protein (Krüger et al., 2003). S6 ATPase is a regulatory subunit

of the 19S proteasome responsible for the degradation of ubiquitinated proteins in the cell.

This underscores the potential physiological role of synphilin-1 in modulating the ubiquitin-

proteasome system.

3.3 Synphilin-1 and protein aggregation

The co-transfection of synphilin-1 and alpha-synuclein in cell culture results in the

formation of cytoplasmic protein inclusions resembling Lewy bodies in PD. Subsequent cell

culture experiments have revealed that the overexpression of synphilin-1 alone is sufficient

for the formation of proteinaceous inclusions in transfected cells in vitro and that inclusion

formation increases after proteasomal inhibition (O’Farrell et al., 2001; Lee et al., 2002;

Marx et al., 2003; Ito et al., 2003). Initial electronmicroscopical studies of the nature of these

inclusions have demonstrated membrane-bound lamellar-like phospholipid accumulations

(O’Farrell et al., 2001). This is an interesting finding, since lipids are components of Lewy

bodies, and since synphilin-1 has been identified as a vesicle-binding protein (Gai et al.,

2004; Ribeiro et al., 2002). However, whether the respective multilayered phospholipid

accumulations develop into Lewy bodies remains uncertain. Subsequent studies of synphilin-

9

1 inclusions formed in cell culture have revealed that they resemble so-called aggresomes.

Aggresomes are juxtanuclear inclusion bodies frequently observed after the expression of

misfolded proteins (Kopito, 2000; Johnston et al., 1998). These structures develop at the

microtubuleorganizing center and contain ubiquitinated proteins and components of the 26S

proteasome. The overexpression of several proteins involved in neurodegeneration, i.e.,

parkin in PD, presenilin-1 in Alzheimer’s disease, or androgen receptor in spinobulbar

muscular atrophy, results in the formation of aggresomes (Junn et al., 2002; Johnston et al.,

1998; Taylor et al., 2003). Synphilin-1-positive inclusions have been shown to display key

features of aggresomes, including positive staining for vimentin, gamma-tubulin, ubiquitin,

and proteasomal subunits (Tanaka et al., 2003; Ito et al., 2003; Krüger et al., 2003). The

function of aggresomes is currently the subject of debate. It is unclear whether aggresome

formation is causative or reactive to the proteasomal defect or whether proteasomal function

is improved or made worse in the absence of aggresomes. However, experimental data on

synphilin-1 overexpression argue in favor of a cytoprotective function of aggresomes formed

by synphilin-1 (Marx et al., 2003; Tanaka et al., 2003). These studies suggest that synphilin-

1-containing aggresomes represent an active protective response to the accumulation of

unwanted proteins. Indeed, for other proteins known to be sequestered into aggresomes, a

toxic effect on cells after the inhibition of aggresome formation has been demonstrated

(Taylor et al., 2003). The dissociation of aggresome formation and cell death has also been

observed on investigation of the R621C mutation in the synphilin-1 protein identified in PD.

In vitro assays have revealed a reduced propensity to form aggresomes of mutant synphilin-1

compared with wild-type protein. The reduced number of aggresome bearing cells is

10

associated with increased susceptibility to cellular stress (Marx et al., 2003). Aggresomes

associate with lysosomal structures implicating autophagy as a possible way of removal of

accumulating proteins; this may link aggresomes with the ultrastructural observations of

lamellar phospholipid-containing synphilin-1 inclusions reported previously (Fortun et al.,

2003; O’Farrell et al., 2002). These observations in cell culture have important implications

for the understanding of molecular mechanisms in PD. Characteristic Lewy bodies in brains

of PD patients exhibit several features of aggresomes resulting from proteasomal inhibition.

These features include a core and halo organization and the presence of alpha-synuclein and

of other members of the protein degradation and refolding machinery (Junn et al., 2002).

Indeed, proteasomal dysfunction involving proteolytic stress has been described in sporadic

PD patients (McNaught and Jenner, 2001). Despite quantitatively variable results depending

on the different antibodies used in immunohistochemistry, it is generally accepted that

synphilin-1 is a component of Lewy bodies in brains of sporadic PD patients (Wakabayashi

et al., 2002; Iseki et al., 2002; Murray et al., 2003). Synphilin-1 has been observed in up to

90% of Lewy bodies of PD patients. Its predominant localization in the central core suggests

a key role in Lewy body formation (Wakabayashi et al., 2000). The occurrence of synphilin-

1 in Lewy bodies in dementia with Lewy bodies (Wakabayashi et al., 2002; Iseki et al.,

2002; Katsuse et al., 2003; Murray et al., 2003) and in glial cytoplasmic inclusions in

multiple system atrophy (Wakabayashi et al., 2002; Murray et al., 2003) parallels the

occurrence of alpha-synuclein positive lesions. This might reflect its more general

involvement in the formation of cytoplasmic inclusions and neurodegeneration

(Wakabayashi et al., 2002).

11

4. LMO7, interaction partner of synphilin-1

LMO7 is a ~195 kDa protein (James et al., 2006) that is localized to human

chromosome 13q22.2 (Rozenblum et al., 2002). However, the precise role of LMO7 in

normal conditions is unclear. Previous studies have shown that LMO7 localizes in the

nucleus, cytoplasm and cell surface, particularly adhesion junctions (Ooshio et al., 2004).

Also, LMO7 is expressed throughout embryogenesis and in multiple adult tissues. LMO7

contains a predicted calponin homology (CH) domain, a putative F-box, a PDZ domain and

the LIM domain (Ooshio et al., 2004). The CH domain is predicted to bind actin and the

PDZ and LIM domains are each protein-protein interaction domains (Ooshio et al., 2004).

LMO7 contains a PDZ and LIM domain, both of which mediate protein–protein interactions

(Bach, 2000; Fanning and Anderson, 1999). There are at least six proteins in addition to

LMO7 that contain an N-terminal PDZ and a C-terminal LIM domain. The LIM domains of

several of these proteins were shown to interact with various kinases (Dueick et al., 1996;

Kuroda, 1996), whereas PDZ domains often associate with the cytoskeleton (Guy et al.,

1999; Vallenius et al., 2000). LIM/PDZ-containing proteins are likely to have important

roles in signal transduction, cell shape changes, motility, and cell adhesion, all of which are

essential for normal embryogenesis. As an example, mice homozygous for a mutation in the

PDZ domain containing protein Afidin showed developmental defects during and after

gastrulation, including impaired migration of mesoderm similar to Acrg-deletion embryos

(Ikeda, 1999). It is interesting to note that some splice forms of LMO7 also contain an N-

terminal F-box, which is yet another protein interaction domain that was shown to recruit

phosphorylated substrates to the SCF ubiquitin–ligase complexes (Skowyra et al., 1997).

12

Through the F-box, LMO7 may recruit LIM and PDZ domain-binding proteins for

degradation.

LIM domain only (LMO) is a special cystein-rich metal-binding structure that

consisted of two distinct zinc-binding subdomains (Dawid, Breen, & Toyama, 1998). This

motif mediates protein–protein interaction through the formation of dimers with identical or

different LMO, or by its binding to other protein motif. LIM proteins have been implicated

in a variety of functions such as transcription, differentiation, cytoskeletal interactions, signal

transduction, and cell adhesion through protein–protein interactions (Dawid, Breen, &

Toyama., 1998; Putilina et al., 1998). Therefore, inappropriate expression of LMO genes

may lead to disturbances in intracellular signaling, cell differentiation, cell adhesion,

cytoskeletal integration by interfering protein–protein integration.

PURPOSE

In the study described here, I demonstrated the interaction between LMO7 and

synphilin-1 and investigated the possible role of LMO7 in the formation of inclusion bodies

in the neurodegenerative disorders relate to synphilin-1, such as PD.

13

II. MATERIALS AND MRTHODS

A. MATERIALS

Normal and Parkinson’s disease human brain tissues were kindly provided by the

national center of neurology and psychiatry musashi hospital, Japan. ProQuestTM two-hybrid

system, transformed human brain cDNA library, X-Gal and all other yeast two-hybrid

components were purchased from Gibco-BRL (Rockville, MD, USA). Restriction enzymes,

DNA ligase and klenow were purchased from NEB (Beverly, MA, USA) and PCR Product

Purification Kit was obtained from Qiagen. Secondary antibody conjugated rhodamine and

FITC were purchased from Calbiochem (San Diego, CA, USA); Secondary antibody

conjugated horse radish peroxidase (HRP) from Zymed (San Francisco, CA, USA); antibody

conjugated biotin, avidin-biotin ABC kit, and Vectashield mounting solution from Vector

Laboratories (Burilingame, CA, USA); aan enhanced chemiluminescence kit from Pierce

Chemical Co. (Rockford, Illinois, USA); complete inhibitor cocktail from Roche (Mannheim,

Germany); PVDF membrane from Schleicher & Schuell Bioscience (Keene, NH, USA). All

other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

B. METHODS

1. Yeast two-hybrid screening

Yeast two-hybrid screening was performed using ProQuestTM two-hybrid system

according to the manufacturer’s instruction (Gibco BRL). Primary transformants (5 to 7 ×

105) were selected for growth on histidine dropout plates containing 25mM 3-aminotriazole

14

(3-AT). His+ colonies were subsequently analyzed for β-galactosidase activity by filter-lift

experiments. The interaction was then quantified by o-nitrophenyl-β-D-galactopyranoside

(ONPG) assays. After incubation for 2-3 days at 30℃, the yeast colonies showing blue color

were selected as positive clones. Plasmids from positive clones were extracted from yeast in

lysis buffer containing 2% Triton X-100, 1% SDS, 100mM NaCl, 10mM Tris, pH 8.0, and

1.0mM EDTA and then transformed into Eschericia coli DH5α using electroporation.

Sequences of the inserts in positive library plasmids were analyzed by automatic DNA

sequencer (ALF express, Amersham Pharmacia Biotech).

2. X-Gal assay and ONPG assay

In order to confirm the positive reactions both of the assays were performed in the

Mav203 yeast strains to detect the initiation of LacZ reporter gene transcription qualitatively

and quantitatively as described before (Klein et al., 1997; Hirasawa et al., 2001). In X-Gal

assay, Colony-lift Filter assay was used to check the activity of β-galactosidase. Briefly,

fresh colonies grown to about 1-3 mm in diameter were transferred completely to a sterile

filter and submerged in a pool of liquid nitrogen for 10 s and thawed at room temperature,

then it was put on a pre-soaked filter in the Z buffer/X-Gal solution (100 mL Z buffer, 0.27

mL β-mercaptoethanol, 1.67 mL X-gal stock solution; Z buffer: Na2HPO4·7H2O 16.1 g/L,

NaH2PO4·H2O 5.50 g/L, KCl 0.75 g/L, MgSO4·H2O 0.246 g/L, pH=7.0), then the filters were

incubated at 30 ºC and the colors of colonies were checked periodically.

In ONPG assay, ONPG was used as the substrate of β-galactosidase for the liquid

culture assay. In brief, at least three independent clones were selected, grown, harvested,

15

centrifuged, and resuspended in Z buffer, frozen in liquid nitrogen, and thawed at 37 ºC in a

water bath. Then the reaction systems (ONPG+Z buffer+β-mercaptoethanol+yeast cells

resuspension) were placed in a 30 ºC incubator. After the yellow color developed Na2CO3

1 mol/L was added to the reaction and blank tubes. Relapsed time was recorded in minutes.

Reaction tubes were centrifuged at 12,000 rpm/min for 10 min and supernatants were

carefully transferred to clean cuvettes and OD420 of the samples relative to the blank was

recorded. At last, the β-galactosidase units were calculated as:

β-galactosidase units=1000×OD420/(t×V×OD600)

where t =elapsed time (in min) of incubation, V= 0.1 mL×concentration factor (the

concentration factor is 5), OD600 =Opital density at 600 nm of 1 mL of culture.

3. Generation of LMO7 complementary DNA

Full-length LMO7 cDNA was constructed by a combination of PCR from a human

brain and kidney cDNA library (Invitrogen Life Technologies, Carlsbad, CA, USA). Full-

length LMO7 cDNA was cloned into pCMV-flag or -myc vector between srfI and kpntI sites.

The cDNA sequences of fragment encoding amino acids 1-274, 275-492, 493-798 of LMO7

were cloned into pCMV-FLAG vector between srfI and kpntI sites to generate pCMV-

FLAG-LMO7-F1, pCMV-FLAG-LMO7-F2, and pCMV-FLAG-LMO7-F3, respectively. All

plasmid constructs were sequenced in entirety to confirm sequence integrity.

4. Cell culture and transfection

HEK293 and SH-SY5Y cells were obtained from the American Type Culture

16

Collection (ATCC). All cell lines including synph-293 were grown in Dulbecco’s modified

Eagle’s medium containing with 10% fetal bovine serum (Gibco-BRL) and grown at 37 °C

in a humidified atmosphere containing 10% CO2. We transiently transfected cells by the

calcium phosphate precipitation method using 10 µg of plasmid DNA per 10-cm plate or 0.5

µg of each plasmid DNA per well 14 (4-well chamber slides). Full-length LMO7 and

synphilin-1 were inserted into a vector containing a FLAG- and myc-tag each. We processed

cells 48 h after transfection. The HEK293 cells were transfected with pFLAG-LMO7,

selected, cloned, and maintained in medium containing 1 mg/ml G418 (Invitrogen) to

generate LMO7-293 cells. All transfections used the calcium phosphate transfection kit

(Invitrogen) according to the supplier’s instructions.

5. In vitro binding assays

HEK293 cells constitutively expressing FLAG-tagged LMO7 and Myc-tagged

synphilin-1 were harvested by scraping and were centrifuged at 3000X g for 1 min at 4 °C.

The cell pellet was washed with Dulbecco’s phosphate-buffered saline (PBS) twice and

resuspended in lysis buffer containing Hepes (50 mM, pH 7.4), NaCl (150 mM), EDTA (3

mM), 1% Triton X-100, 0.1% SDS and a protease inhibitors cocktail (Calbiochem). FLAG-

tagged LMO7 was immunoprecipitated by anti-FLAG M2 agarose-affinity gel (Sigma) at

4 °C overnight. Next day beads were washed 5 times with lysis buffer containing NaCl (500

mM). And then the reaction was stopped by adding SDS gel-loading buffer (Invitrogen) and

boiling at 95 °C for 5 min. Proteins were separated by SDS-PAGE and detected using anti-

FLAG (1:3000, sigma) and anti-Myc (1:1000, santa cruz) antibody.

17

6. Immunocytochemistry

To visualize immunostaining, transfected cells were grown in glass-bottom culture

chamber dishes (MatTek Corp., Ashland, MA, USA), fixed with 4% paraformaldehyde in

0.1M PBS. For double-staining, both primary antibodies were diluted in PBS containing

blocking buffer (3% bovine serum albumin and 0.3% Triton X-100) and incubated overnight

at 4°C. After three washes in PBS, the cultures were incubated in appropriate fluorescein or

rhodamine red-labeled secondary antibodies (1 : 200, Vector Laboratories, Burlingame, CA,

USA) for 30 min at room temperature and mounted with Vectashield (Vector Laboratories,

Burlingame, CA, USA). Anti-flag (M2 monoclonal 1:500) antibody and anti-α-synuclein

(1:200, monoclonal) were obtained from Sigma (Sigma-Aldrich St Louis, MO, USA). Anti-

synphilin-1 (1:400, polyclonal) and anti-neuronal nuclei (NeuN) (1:100, monoclonal) were

obtained from Chemicon International, Inc. (Temecula, CA, USA). Calbiochem supplied

antibodies to anti-myc (1:100, polyclonal).

7. Preparation of LMO7 polyclonal antibodies

LMO7 antibody was produced by immunizing rabbits (Lab Frontier, Korea). This

antibody was against the full-length recombinant LMO7 with an N-terminal 6× His tag . The

resulting immune sera were screened against recombinant 6× His LMO7 and FLAG-LMO7

transfected HEK 293 cell lysates. The antibodies were further affinity purified against the

recombinant His-tagged LMO7 coupled to Affigel 10 (Pierce, Ill.).

18

8. Western immunoblot analysis

Cells at ~95% confluence were harvested with trypsin. Medium and cell suspensions

were centrifuged; pellets were washed, resuspended in lysis buffer containing Hepes (50 mM,

pH 7.4), NaCl (150 mM), EDTA (3 mM), 1% Triton X-100, 0.1% SDS and a protease

inhibitors cocktail (Calbiochem), held on ice for 40 min and centrifuged at 12,000 rpm for 10

min. Supernatants were saved at -20°C as detergent-soluble samples; pellets were

resuspended in urea lysis buffer, held at RT for 3 h, and spun at 12,000 rpm for 10 min

30ug/lane of each protein sample was electrophoresed on 16% Tris–Tricine or 15% Tris–

HCl Criterion gels (Bio-Rad, Hercules, CA, USA), transferred to polyvinylidene fluoride

membranes, washed with 0.05% Tween-20 in Tris-buffered saline (T-TBS), blocked in 5%

non-fat milk/T-TBS and incubated overnight at 4°C in primary antibody in 5% milk T-TBS.

Following three TBS-T washes, secondary antibodies linked to horseradish peroxidase

(HRP-conjugated, all used at a dilution of 1 : 2000) were incubated at room temperature for

1 h. Immunoblots were washed in TBS-T three times and processed with a

chemiluminescent detection system (NEN Life Science Products, Boston, MA, USA)

according to the manufacturer's instructions. Chemiluminescence was detected for 5 min in a

Bio-Rad Fluor-S MultiImager and the band density determined using Bio-Rad Quantity One

software.

9. Hematoxylin and eosin staining

For hematoxylin and eosin staining of cells and tissues, samples were washed with

PBS twice and incubated with hematoxylin (Vector Laboratories, Burlingame, CA) at room

19

temperature for 3 min. Cells were then rinsed with deionized water three times and destained

with acidic alcohol for a few seconds. After rinsing the cells again with deionized water,

bicarbonate solution (1 g/liter) was added, and cells were incubated for 3 min. After this

bluing step, cells were washed again with deionized water and placed in 70% ethanol for 3

min, followed by staining with eosin (0.5 g of Eosin Y, 2.5 ml of acetic acid, 500 ml of 70%

ethanol) for 1 min. Cells were then washed with three changes of 95% ethanol and

dehydrated with absolute ethanol. Slides were dried, mounted, and analyzed under a light

microscope.

10. Primary culture of rat brain cortex

Rat brain embryos were recovered at day 18 from gestating Sprague-Dawley rats and

primary cultures were performed as described (Dawson et al., 1993). The various brain

regions were dissected under a microscope, incubated for 20 min in 0.027% trypsin/saline

solution (5% phosphate buffered saline, 40 mM sucrose, 30 mM glucose, 10mM HEPES, pH

7.4) and transferred to modified Eagle’s medium (MEM), 10% horse serum, 10% fetal

bovine serum, 2 mM glutamine. Cells were dissociated by trituration, counted, and plated in

15 mm multiwell (Nunc) plates coated with polyomithine at a density of 2 x 104 cells per

well. The medium was changed twice a week. 12 days after plating, the cells were treated

with 10µM MG132 for 24h and then analysed by immunocytochemistry.

11. LMO7-siRNA treatment

To inhibit the expression of endogenous LMO7 in HEK293 cells, RNA interference

20

(RNAi) was used. Four short interfering RNAs (siRNAs) were designed, synthesized and

mixed by Dharmacon (Lafayette, CO). The effect of siRNAs was tested in our lab. The

LMO7 siRNA or control siRNA was transfected for RNAi. Briefly, the 50nM siRNA was

transfected into LMO7-293 cells by Lipofectamine 2000 (Invitrogen).

12. Quantitation of cells containing inclusions

The number of cells containing LMO7 immunopositive inclusions were assessed

following immunocytochemistry using a Nikon Eclipse TE300 microscope with a 20X

objective as follows: Cells were assessed by an observer blind to the transfection conditions

(i.e. the cotransfected plasmid). Thirty-nine fields at 20 X were assessed for each well and

two wells were assessed for each experiment. Each field contained between 1 and 10

transfected cells and between 300 and 400 cells were assessed for each experiment. A total

of four experiments were performed with each condition. A positively transfected cell was

scored on the presence of significant a-synuclein immunostaining compared to background

(which in all cases was negligible). A transfected cell containing inclusions was scored on

the presence of a detectable aggregate of a-synuclein immunostaining. A cell was considered

positive for inclusions regardless of the size or number of inclusions, however, the inclusions

had to be detectable at 20 X. The numbers of cells containing inclusions were expressed as a

percentage of the total number of transfected cells.

13. Immunohistochemistry on human brain tissues

Parkinson cases (2 females, 1 males; age of 76~83 years) and control cases (2 male,

21

age of 73 years) were obtained from the National center of neurology and psychiatry

musashi hospital (Japan). Sections (6 µm) were cut from the midbrain of patients with PD

and controls. After rinsing in PBS, the sections were exposed to 0.3% H2O for 30 min to

quench endogenous peroxidase activities. Before incubation of primary antibodies, non-

specific binding was blocked with normal serum (0.1M PBS with 1% BSA and 0.2% Triton

X-100) from species in which the secondary antibody was raised. The duration of the

blocking was 30 min. The sections were then incubated with the primary antibodies diluted

by 0.1% fixation using 0.1M PBS with 0.5% BSA overnight at 4 °C. The primary antibodies

used for this study Rabbit anti-LMO7 (1:200) and goat anti-synphilin-1 (1:500), a polyclonal

antibody, was employed as well. After rinsing in PBS, the sections were incubated with

biotinylated secondary antibodies diluted 0.1M PBS with 0.5% BSA for 1 h at room

temperature and the reaction products were visualized by the avidin–biotin–peroxidase

complex method (ABC kit) using 3,3-diaminobenzidine-tetra-hydrochloride as the

chromogen. The adjacent sections served as negative controls. All the procedures for

negative controls markers were processed in the same manner except the primary antibodies

were omitted.

For immunofluorescent double staining, nonspecific binding was blocked by normal

goat serum. The sections were incubated overnight at 4 °C with the first primary antibodies:

synphilin-1 (1:500) and LMO7 (1:200). Rabbit anti-myc (1:100) and goat anti-synphilin-1

(1:400), a polyclonal antibody, was employed as well. After rinsing in PBS, the sections

were exposed to rhodamine conjugated goat anti-mouse IgG (1:200), the secondary antibody,

for 2 h at room temperature in the dark. After blocking nonspecific binding, the sections

22

were then incubated with other proper primary antibodies at 4°C overnight. Finally, the

sections were incubated with FITC conjugated goat anti-rabbit IgG (1:200) at room

temperature for 2 h in the dark, rinsed with TBS and mounted with glycerol containing n-

propyl gallate. The adjacent sections were used as negative controls. All the procedures for

negative controls were processed in the same manner except the primary antibodies were

omitted. Immunoperoxidase reactivity was assessed with light microscopy, and fluorescent

staining was evaluated using a fluorescence microscope (Nikon).

14. Statistical Analysis

All values were presented as mean ± SE. Statistical significance of the data were

evaluated using analysis of variance, followed by post hoc tests using the Fisher’s

adjustment or the Student’s t-test when comparing two conditions. Probability values less

than 0.05 (P < 0.05) were considered significant and probability values less than 0.01 (P

<0.01) were considered highly significant.

23

III. RESULTS

1. Screening of binding partner of synphilin-1 in the yeast two-hybrid system

To identify novel proteins that interact with synphilin-1, yeast two hybrid assay was

performed with full-length synphilin-1 as a bait (Fig. 2A). Synphilin-1 and human fetal brain

cDNA library was fused to the Gal-4 DNA binding domain in the vector pDBleu (pDB-

synph1) and the Gal-4 activation domain in the vector pPC86 respectively. All constructed

vectors were introduced into the host strain, MaV203, and the transformed strains were grow

on SC-Leu-+Trp-+His- for 4~5 days at 30 °C. To detect background activation of the HIS3

reporter gene, all transformants plated on various concentrations of 3-AT. And then 3-AT of

25 mM was used for selection of interactions of synphilin-1 protein with other proteins that

were expressed by the cDNA library. After screening of human fetal brain cDNA library,

synpilin-1-interacting protein, LMO7 were identified. Two independent LOM7 clones were

identified (clones 1 and 2; fig. 2B) In a liquid culture assay, the interaction of LMO7 with

synphilin-1 was very stronger than with negative control (P <0.01) (Fig. 2C).

24

Y2H bait ( 1 ~ 919 a.a)

919 a.a

A

Y2H bait ( 1 ~ 919 a.a)

919 a.a

A

B

Leu /trp /his +25mM 3AT

positivecontrol

negativecontrol

Clone 1

Clone 2

C

0

0.3

0.6

0.9

1.2

1.5

1.8Mav203 Clone1

Synph1+ LMO7 Fragments

Clone2ga

lacto

sidas

eact

ivity

/mille

r uni

ts

**

B

Leu /trp /his +25mM 3AT

positivecontrol

negativecontrol

Clone 1

Clone 2

C

0

0.3

0.6

0.9

1.2

1.5

1.8Mav203 Clone1

Synph1+ LMO7 Fragments

Clone2ga

lacto

sidas

eact

ivity

/mille

r uni

ts

**

Fig. 2. Association of synphilin-1 with LMO7 in yeast. (A) The box represents full-length synphilin-1 cDNA with its domains. Lines represent the fragments of α-synuclein used as baits for the yeast two-hybrid screenings that yielded positive signals. (B) Yeast growth of defined media plate (Lue-/trp-/his-+25mM 3AT) showing that LMO7 interacts specifically with synphilin-1 in yeast (clone 1 and 2). There was no interaction of either synphilin-1 or LMO7 with vector alone (negative control). (C) β-galactosidase liquid assays (ONPG assay) showing an increase in interaction between synphilin-1 and LMO7 (clone1 and 2) when compared with negative control. Error bars represent standard errors; n = 4. *P <0.01 vs control.

25

2. Determination of the interacting domains of synphilin-1 and LMO7 in a yeast two

hybrid system

We precisely identified the synphilin-1-binding of synphilin-1 and LMO7 in a yeast

two-hybrid interaction assay. We first overexpressed full-length synphilin-1 and LMO7 in

yeast under selective growth media and noted that they interact (Fig. 3A). To further map the

critical regions involved in the interaction, we constructed several plasmids with different

synphilin-1 and LMO7 domains and expressed them in yeast. As shown in Figure 3B and 3C,

we generated five fragments of LMO7, LMO7-F1 to F5, to examine the interaction with

synphilin-1 (fig. 3B), and four fragments of synphilin-1, synph1-F1 to F4, to examine the

interaction with LMO7 (fig. 3C). Each fragment has a C-terminal deletion and/or an N-

terminal deletion. Using these LMO7 fragments, we then examined the interaction with

synphilin-1 in yeast cells. In the yeast two-hybrid assay, synphilin-1 fused to the Gal4

binding domain was used for the interaction with a panel of LMO7 fragments fused to the

Gal4 DNA-activating domain. As shown in Figure 3A, synphilin-1 interacted with LMO7-F4

(600-797 a.a) and LMO7-F5 (730-797a.a), but not with LMO7-F1 (1-220 a.a), LMO7-F2 (1-

366 a.a) and LMO7-F3 (1-600 a.a). These results indicated that a synphilin-1-binding site

was located at the C-terminus of LMO7 between amino acid residues 730 and 797 (Fig. 3B).

And a deletion series of synphilin-1 in pDBLeu, constructs of synphilin-1 corresponding to

synph1-F1 (1-115 a.a) and synph-F2 (1-249 a.a) did not interact with LMO7. Strong

interactions with constructs corresponding to synph1-F3 (1-641 a.a) and synph1-F4 (301-

919) indicate that ankyrin-like repeats and coiled-coil domain were required for interaction

with LMO7 (Fig. 3C).

26

ACoiled-coil

ATP, GTP-binding domainAnkyrin-like repeatspDBLeu-synph1

PDZ LIMpPC86-LMO7

+

Interaction with pDBLeu-synph1 and pPC86-LMO7β-gal filter assay

Negative control

Positive control

Interaction of pDBLeu-synph1 and pPC86-LMO7+++

Coiled-coilATP, GTP-binding domainAnkyrin-like repeats

Coiled-coilATP, GTP-binding domainAnkyrin-like repeats

pDBLeu-synph1

PDZ LIMPDZ LIMpPC86-LMO7

+

Interaction with pDBLeu-synph1 and pPC86-LMO7β-gal filter assay

Negative control

Positive control

Interaction of pDBLeu-synph1 and pPC86-LMO7+++

ACoiled-coil

ATP, GTP-binding domainAnkyrin-like repeatspDBLeu-synph1

PDZ LIMpPC86-LMO7

+

Interaction with pDBLeu-synph1 and pPC86-LMO7β-gal filter assay

Negative control

Positive control

Interaction of pDBLeu-synph1 and pPC86-LMO7+++

Coiled-coilATP, GTP-binding domainAnkyrin-like repeats

Coiled-coilATP, GTP-binding domainAnkyrin-like repeats

pDBLeu-synph1

PDZ LIMPDZ LIMpPC86-LMO7

+

Interaction with pDBLeu-synph1 and pPC86-LMO7β-gal filter assay

Negative control

Positive control

Interaction of pDBLeu-synph1 and pPC86-LMO7+++

Coiled-coilATP, GTP-binding domainAnkyrin-like repeats

Coiled-coilATP, GTP-binding domainAnkyrin-like repeats

pDBLeu-synph1

PDZ LIMPDZ LIMpPC86-LMO7

+

Interaction with pDBLeu-synph1 and pPC86-LMO7β-gal filter assay

Negative control

Positive control

Interaction of pDBLeu-synph1 and pPC86-LMO7+++

Coiled-coilATP, GTP-binding domainAnkyrin-like repeats

Coiled-coilATP, GTP-binding domainAnkyrin-like repeats

pDBLeu-synph1

PDZ LIMPDZ LIMpPC86-LMO7

+

Interaction with pDBLeu-synph1 and pPC86-LMO7β-gal filter assay

Negative control

Positive control

Interaction of pDBLeu-synph1 and pPC86-LMO7+++

Deletions of synphilin-1 in pDBLeu

Interaction with pPC86-LMO7 β-gal activityc

+

-

+++

+++

Deletions of LMO7 in pPC86

Interaction with pDBLeu-synph1 β-gal activityB

+

+

+

+++

+++

Deletions of synphilin-1 in pDBLeu

Interaction with pPC86-LMO7 β-gal activityc

+

-

+++

+++

Deletions of synphilin-1 in pDBLeu

Interaction with pPC86-LMO7 β-gal activityc

+

-

+++

+++

Deletions of LMO7 in pPC86

Interaction with pDBLeu-synph1 β-gal activityB

+

+

+

+++

+++

Deletions of LMO7 in pPC86

Interaction with pDBLeu-synph1 β-gal activityB

+

+

+

+++

+++

27

Fig. 3. Determination of the interacting domains of LMO7 and synphilin-1 in a yeast two-hybrid system. (A) LMO7 was fused to pPC86 and synphilin-1was fused to pDBLeu. The criteria for positive interaction were based on their growth on X-gal plates free of leucine and tryptopan . β-galactosidase was measured by liquid assay using O-nitrophenyl-b-D-galactose as the substrate (left of figures). (B) and (C): The LMO7 deletion (or synphilin-1) mutants and β-galactosidase activity for each construct in the presence of pDBLeu-synphilin1 (or pPC86-LMO7) in the yeast two-hybrid system are shown. b-Galactosidase activity was determined by colony-lift filter assay. The level of interaction is defined as: +++, very strong; +, weak; -, undetectable. Measurement of β-galactosidase levels was done in triplicate from three independent colonies in three separate experiments.

28

3. LMO7 interacts with synphilin-1 in HEK293 cells

Interaction of LMO7 with synphilin-1 was examined in mammalian cells using an

immunoprecipitation assay. We conducted co-transfection experiments with myc-tagged

synphilin-1 and flag-tagged LMO7 followed by co-immunoprecipitation. From lysates of

HEK 293 cells co-expressing myc-tagged synphilin-1 (myc-synphilin-1) and FLAG-tagged

LMO7 (flag-LMO7), an anti-flag M2 agarose immunoprecipitated flag-LMO7 (Fig. 4A). In

addition, an anti-Myc agarose immunoprecipitated Myc-synphilin-1 in a similar manner

(data not shown). To confirm if endogenous LMO7 interact with synphilin-1 in mammalian

cell in vitro, synph-293 cell line were lysed and then flag-tagged synphilin-1 was

immunoprecipitated by anti-FLAG M2 agarose-affinity gel at 4 °C overnight. And

endogenous LMO7 binding with flag-tagged synphilin-1 were detected by western blotting

using anti-LMO7 polyclonal antibody (fig. 4B). Synphilin-1 and endogenous LMO7

coimmunoprecipitated, suggesting physiologic relevance of the interaction. It was concluded

that LMO7 and synphilin-1 proteins interact with each other in mammalian cells and

confirming that the interaction between LMO7 and synphilin-1 is specific.

29

flag-LMO7myc-synphilin-1

Anti-mycINPUT

Flag-IP

Anti-myc

Anti-flag

- +-+

++

130kD

130kD

180kD

Aflag-LMO7myc-synphilin-1

Anti-mycINPUT

Flag-IP

Anti-myc

Anti-flag

- +-+

++

130kD

130kD

180kD

flag-LMO7myc-synphilin-1

Anti-mycINPUT

Flag-IP

Anti-myc

Anti-flag

- +-+

++flag-LMO7myc-synphilin-1

Anti-mycINPUT Anti-mycINPUT

Flag-IP

Anti-myc

Anti-flag

Flag-IP

Anti-myc

Anti-flag

- +-+

++

130kD130kD

130kD130kD

180kD180kD

A

anti-flag

anti-flag

anti-LMO7

Input

IP flag

130

130

200150

293Synph-293

B

anti-flag

anti-flag

anti-LMO7

Input

IP flag

130

130

200150

293Synph-293

anti-flag

anti-flag

anti-LMO7

Input

IP flag

anti-flag

anti-flag

anti-LMO7

Input

IP flag

130130

130130

200200150150

293Synph-293

B

Fig. 4. Interaction of synphilin-1 and LMO7. (A) Lysates prepared from HEK293 cells transfected with Myc-tagged synphilin-1 and FLAG-tagged LMO7, respectively, were subjected to IP with anti-FLAG and subsequently immunoblotted with anti-myc antibodies. The blot was also stripped and reprobed with anti-FLAG (lower panel) to illustrate that relatively equivalent amounts of synphilin-1 were expressed. (B) Endogenous LMO7 interact with synphiln-1 in synph-293 stable cell line.

30

4. Identification of domains involved in LMO7-synphilin-1 association

To identify which portion of synphilin-1 binds to LMO7, we expressed a series of

deletion mutants of Myc-tagged synphilin-1 and FLAG-tagged LMO7 in HEK293 cells (Fig.

5). HEK293 cells co-transfected with expression vectors for Myc-synphilin-1 and FLAG-

LMO7-fragments were lysed and precipitated with various FLAG-tagged proteins, such as

Flag-LMO7 (full), Flag-LMO7-Fragment 1(F1), Flag-LMO7-F2 and Flag-LMO7-F3. Native

HEK293 cells were used as a control (Fig. 5A). Anti-FLAG immunoblotting revealed that

FLAG-tagged LMO7 fragment proteins and anti-myc immunoblotting revealed that

synphilin-1 bind with LMO7 fragments. We found that LMO-F3 (containing LIM domain),

but not LMO-F1 (containing PDZ domain), specifically bound synphilin-1, indicating that

LMO7 binds to synphilin-1 via its C-terminal region containing LIM domain.

To identify the binding site of synphilin-1 to LMO7, HEK293 cells co-transfected with

expression vectors for Myc-LMO7 and FLAG-synphilin-1-fragments were lysed and

precipitated with various FLAG-tagged synphilin-1 proteins (FLAG-synph1, FLAG-synph1-

F1~F5) (Fig.5B). After FLAG-immunoprecipitation, Anti-Myc immunoblotting revealed

that only the FLAG-synph1-F2 and F5 could precipitate LMO7 (Fig. 5B). Thus, LMO7

interacts with synphilin-1 mainly through its central portion, which contains the ankyrin-like

repeat, the coiled-coil domain, and the ATP/GTP-binding domain.

31

+ + + + +- fla

g-LMO7

flag-L

MO7-F1

flag-L

MO7-F2

flag-L

MO7-F3

kD

anti-myc

anti-myc

anti-flagIP flag

Input

37

50

100

130

130

myc-synph1

A

+ + + + ++ + + + +- fla

g-LMO7

flag-L

MO7-F1

flag-L

MO7-F2

flag-L

MO7-F3

- flag-L

MO7fla

g-LMO7-F

1fla

g-LMO7-F

2fla

g-LMO7-F

3

kD

anti-myc

anti-myc

anti-flagIP flag

Input

37

50

100

130

130

myc-synph1

A

PDZ domain LIM domain

F1

F2

F3

PDZ domain LIM domain

F1

F2

F3

Fig. 5. Synphilin-1 interacts preferentially with the C-terminal LIM domain of LMO7 and LMO7 interacts with the ankyrin domain of synphilin-1. (A) Lysates prepared from HEK293 cells transfected with myc-tagged synphilin-1 and various flag-tagged LMO7 domain constructs were subjected to IP with anti-flag M2 agarose followed by anti-myc immunoblotting. The blot was also stripped and reprobed with the anti-flag (lower panel) to illustrate the relative amounts of the LMO7 constructs that were expressed. Putative functional domains of LMO7 used in the mapping experiments are shown.

32

130

+ + + + +- fla

g-syn

ph1

flag-s

ynph

1-F1

flag-s

ynph

1-F2

flag-s

ynph

1-F3

kD+ +

flag-s

ynph

1-F4

flag-s

ynph

1-F5

110

110

100

75

37

25

anti-myc

anti-myc

anti-flagIP flag

Input

myc-LMO7

B

130

+ + + + +- fla

g-syn

ph1

flag-s

ynph

1-F1

flag-s

ynph

1-F2

flag-s

ynph

1-F3

kD+ +

flag-s

ynph

1-F4

flag-s

ynph

1-F5

110

110

100

75

37

25

anti-myc

anti-myc

anti-flagIP flag

Input

myc-LMO7

130130

+ + + + +- fla

g-syn

ph1

flag-s

ynph

1-F1

flag-s

ynph

1-F2

flag-s

ynph

1-F3

kD+ +

flag-s

ynph

1-F4

flag-s

ynph

1-F5

110110

110110

100100

7575

3737

2525

anti-myc

anti-myc

anti-flagIP flag

Input

myc-LMO7

B

(B) Lysates prepared from HEK293 cells transfected with myc-tagged LMO7 and various flag-tagged fragments of synphilin-1 were subjected to IP with anti-flag M2 agarose followed by anti-myc immunoblotting. The blot was also stripped and reprobed with the anti-flag antibody (lower panel) to illustrate the relative amounts of the synphilin-1 constructs that were expressed. A schematic representation of the different fragments of synphilin-1 used in the mapping experiments is shown at the bottom of the figure. Both experiments were replicated 3 times with similar results.

33

5. LMO7 positive inclusion in primary cortical neuron

Hematoxylin and eosin staining of primary cortical neuron cultures (13 DIV) was not

shown cytoplasmic eosinophilic inclusions (Fig.6A, a), but after treatment of 10µM MG132,

primary culture cells were shown inclusions (fig. 6A, b). Figure 6B demonstrates double

staining with anti-LMO7 and neuron-specific marker NeuN staining of rat cortical neurons

grown in primary culture cells (13 DIV). Many NeuN-positive cells were detected (Fig. 6B,

a), whereas a few LMO7-stained cells could be identified and revealed positive cytoplasimc

inclusion (Fig. 6B, b and c).

a b

a b c

A

B

a b

a b c

A

B

Fig. 6. Formation of LMO7-positive inclusion in primary cortical neuron cultures. (A) After treatment of 10µM MG132, primary culture cells develop cytoplasmic eosinophilic inclusions when stained with H & E. (B) Double staining with anti-NeuN and anti-LMO7 antibody staining on primary culture cells (13 DIV). a, The anti-NeuN antibody has been labeled with FITC. b, LMO7 antibody was labeled with rhodamine. c, Double exposure with a and b. a, Scale bar, 50 µm; b, 25 µm.

34

6. Effect of endogenous LMO7 siRNA on the Formation of Synphilin-1-Positive

Inclusions in HEK293 Cells

We knocked down the endogenous LMO7 by transfecting LMO7-siRNA in HEK293

cells. As shown in Figure 00, the siRNA of LMO7 completely inhibited the expression of

endogenous LMO7 in HEK293 cells (Figure 7, lane 2 versus lane 1). Using this system, we

further investigated the role of LMO7 in the formation of synphilin-1-positive inclusions in

HEK293 cells. As shown in Figure 7, the transfection with NUB1 siRNA did not cause any

effects on the formation of synphilin-1-positive inclusions in HEK293 cells (Figure 7, lane 2

versus lane 1). This is probably because other proteins compensate for the function of LMO7

in HEK293 cells.

35

Con

trol

LMO

7

siRNA

LMO7

*

kDa

200

150

a

Con

trol

LMO

7

siRNA

LMO7

*

kDa

200

150

Con

trol

LMO

7

siRNA

LMO7

*

kDa

200

150

a

wt-α-synuclein : + +flag-synphilin-1 : + +

control siRNA :LMO7 siRNA :

b

+ -- +1 2

wt-α-synuclein : + +flag-synphilin-1 : + +

control siRNA :LMO7 siRNA :

b

+ -- +1 2

Fig. 7. RNA interference of LMO7 by siRNA. (a) Effect of LMO7 siRNA on the expression of endogenous LMO7. HEK293 cells were transfected with a siRNA of control or LMO7 and a plasmid encoding FLAG-synphilin-1 and wt-α-synuclein. Twenty-four hours after transfection, cells were lysed. The expression level of endogenous LMO7 was then determined by Western blotting using anti-NUB1 antibody. A nonspecific band is indicated by an asterisk. (b) Effect of LMO7 siRNA on the formation of synphilin-1-positive inclusions. HEK293 cells were transfected with a siRNA of control or LMO7 and a plasmid encoding both FLAG-synphilin-1 and wt-α-synuclein. Twenty-four hours after transfection, cells were fixed, and then the transfected cells containing cytoplasmic inclusions were counted under a fluorescence microscope. Each bar represents the mean ± SE (*P ± 0.5, not significant).

36

7. LMO7 promotes the Formation of Synphilin-1-Positive Inclusions in HEK293 Cells

To determine whether LMO7 is involved in the formation or breakdown of synphilin-

1-positive inclusions, we overexpressed FLAG-tagged LMO7 in HEK293 cells. Because

inclusions were formed in cells co-expressing synphilin-1 and NAC of α-synuclein, we

estimated the effect of the overexpression of FLAG-LMO7 on inclusion formation using this

assay system. Inclusions were not generated when Myc or FLAG alone (data not shown)

were expressed. In contrast, inclusions were generated when Myc-synphilin-1 was expressed

(fig.8). Specifically, when Myc-synphilin-1 was expressed alone, 5.2% of cells generated

inclusions. When Myc-synphilin-1 and wt-α-synuclein were expressed, 12.8% of cells

generated inclusions. Importantly, when Myc-synphilin-1 and wt-α-synuclein were co-

expressed with FLAG-LMO7, the number of inclusion-positive cells was increased to 5.2%

(Fig. 8a). This result suggests that LMO7 promotes the formation of synphilin-1-positive

inclusions in HEK293 cells. When we transfected HEK 293 cells with vectors encoding

Myc-synphilin-1 and full-length FLAG-tagged LMO7 each, we did not observe any

morphological change. Cytosolic inclusions were eosinophilic when stained with

haematoxylin and eosin, but control HEK293 cells non-transfected had no inclusions (Fig.

8b).

37

0

5

10

15

20

% ce

lls w

ith in

clusio

ns

-

++-

+

-

-

- -

flag-sph1:

wt-syn:

LMO7:

a

+

+

+

* *

0

5

10

15

20

% ce

lls w

ith in

clusio

ns

-

++-

+

-

-

- -

flag-sph1:

wt-syn:

LMO7:

a

+

+

+

% ce

lls w

ith in

clusio

ns

-

++-

+

-

-

- -

flag-sph1:

wt-syn:

LMO7:

a

+

+

+

% ce

lls w

ith in

clusio

ns

-

++-

+

-

-

- -

flag-sph1:

wt-syn:

LMO7:

a

+

+

+

* *

bb

Fig. 8. LMO7-mediated regulation in the formation of synphilin-1-positive inclusions. a. Effect of LMO7 overexpression on the formation of synphilin-1-positive inclusions. In HEK293 cells transfected with various constructs, cytoplasmic inclusions with green fluorescence and H&E stain eosin-positive inclusion were quantified. The transfected cells containing cytoplasmic inclusions were counted. The value of percent cells with inclusions was calculated as the ratio of the number of transfected cells containing inclusions to the total number of transfected cells. Each bar represents the mean ± SE (*P±0.001). b. HEK 293 cells co-transfected with constructs encoding Myc-synph1 and full-length FLAG-LMO7 develop cytosolic eosinophilic inclusion. Scale bar, 50 µm.

38

8. Colocalization of LMO7 and synphilin-1

We determined the location of Myc-synphilin-1 and FLAG-LMO7 in HEK293 cells

shown in Figure 9. As shown in Figure 9, Myc-tagged synphilin-1 was mainly located in the

inclusions (red), and was robust present in the cytoplasm. The over-expressed FLAG-tagged

LMO7 was located in the nucleus, cytoplasm, and inclusions (green). This results show that

LMO7 and synphilin-1 were colocalized in inclusion.

Synphilin-1 LMO7 MergeSynphilin-1 LMO7 Merge

Fig. 9. Location of overexpressed LMO7 in synphilin-1-positive inclusions. FLAG-LMO7 was co-expressed with Myc-synphilin-1 in HEK293 cells. After 24 hours, the cells were fixed and immunostained with anti-flag (1:500) and anti-myc (1:100) antibody. The primary antibody was then labeled with Texas Red-conjugated and FITC secondary antibody (1:200). The immunostained cells were treated with DAPI for the nuclear staining and then analyzed under a fluorescence microscope (data mot shown). The location of myc-synphilin-1 was shown by the green fluorescence, and the location of FLAG-LMO7 was shown by the red fluorescence of Texas Red . Their co-localization was shown by the merging of both fluorescences. Top, HEK293 cell; bottom, SH-SY5Y cell lines.

39

9. LMO7, as well as synphilin-1, is accumulated in inclusions of PD patients

The normal brain and the brain from patients with PD sections were stained with

hematoxylin and eosin (H & E stain) (Fig. 10A). In the normal brain tissues as control,

eosinophilic inclusions were not observed (Fig. 10A, a). An H & E stain demonstrates a

rounded pink cytoplasmic Lewy body in substantia nigra with Parkinson's disease (Fig. 10A,

b and c, arrow).

α-Synuclein and synphilin-1 are major components of Lewy bodys found in the brains

of patients with PD (Arima et al., 1998; Baba et al., 1998; Wakabayashi et al., 2002).

Because LMO7 interacts with synphilin-1, we hypothesized that LMO7 is also present in the

inclusion bodies in the brains of patients with PD. To determine this, immunohistochemical

investigations were performed on normal brains (Figure 10B, a) and the brains from patients

with PD (Figure 10B, b and c) using anti-synphilin-1 and anti-LMO7 antibody. As shown in

Figure 10B-a, the anti-LMO7 antibody weakly immunostained the neuronal cytoplasm in the

normal brains. In the brains of patients with PD, brainstem type LBs (Fig. 10B, b) were

positive for LMO7. These findings together showed that LMO7, as well as synphilin-1 (Fig.

10B, c) is present in LBs of PD. Because synphilin-1 is a major component in inclusions of

α-synucleinopathies, we examined the relationship between synphilin-1 and LMO7. PD

patient tissue sections were double labeld with anti-synphilin-1 and anti-LMO7. As shown in

Figure 10B, inclusions were immunopositive to both antibodies of human synphilin-1 (red)

and LMO7 (green) and double-labeling immunofluorescence revealed colocalization of

synphilin-1 and LMO7 in inclusions of PD patients tissues. Thus, the vast majority of

inclusions in the brains of patients with PD contained LMO7.

40

A

B

a b c

a b c

fed

A

B

a b c

a b c

fed

Fig. 10. Localization of LMO7 in inclusions of PD patient brains. (A) H&E-stained section of the brains of patients with PD showing an eosinophilic inclusion in a substantia nigra neurons (b and c, arrow) (B) Localization of synphilin-1 and LMO7 in inclusion bodies of the brains of patients with PD. Immunohistochemical studies were performed on the substantia nigra (a) from control subjects and the substantia nigra from PD (b and c) using anti-LMO7 antibody (a and b) and anti-synphilin-1 antibody (c). Anti-LMO7 (a) and anti-synphilin-1 antibodies (data not shown) weakly immunolabeled the neuronal cytoplasm in the normal brains. LBs in the brains of patients with PD were immunostained with anti-LMO7 (b), as well as anti-synphilin-1 (c). Double immunofluorescence staining showing co-localization of synphilin-1 and LMO7 in patients with PD. Synphilin-1 appears red (d) and LMO7 appears green (e). The overlap of synphilin-1 with LMO7 appears yellow (f). Scale bars = 10 µm.

41

IV. DISCUSSION

I have isolated here human LMO7 as an synphilin-1-binding protein. Although

previous papers had reported the partial sequence of LMO7 and tissue distribution of the

LMO7 mRNA (Putilina et al., 1998; Rozenblum et al., 2002; Kurihara et al., 2002;

Semenova et al., 200318–21), no functional analysis of LMO7 protein has been performed.

In this paper, we have provided several lines of evidence suggesting that LMO7 binds to

synphilin-1: (i) LMO7 binds to synphilin-1 as estimated by the yeast two-hybrid and

coimmunoprecipitation from the extracts of cells exogenously expressing the fragments of

LMO7; (ii) endogenous LMO7 and synphilin-1 are coimmunoprecipitated from the extracts

of synph-293 cells; and (iii) LMO7 co-localizes with synphilin-1 in the human PD brain.

Here I showed that LMO7 interacts with α-synuclein interacting protein, synphilin-1 and

revealed that the co-expression with synphilin-1 results in the formation of cytoplasmic

inclusions in cultured HEK293 and SY5Y cells. Synphilin-1 interacts preferentially with the

C-terminal LIM domain of LMO7 and LMO7 interacts with the ankyrin domain of

synphilin-1. These findings have important implications for understanding the molecular

mechanism by which Lewy-body–associated proteins interact through synphilin-1.

The Lewy body is morphologically composed of two major components, the dense

core and the surrounding halo (Pollanen et al., 1993; Galvin et al., 1999). α-Synuclein and

NUB1 are mainly concentrated in the surrounding halo whereas synphilin-1 is mainly

concentrated within the dense core (Irizarry et al., 1998; Wakabayashi et al., 1998;

Wakabayashi et al., 2000; Tanji et al., 2006). In the present study, I showed that LMO7 co-

42

localized with synphilin-1 in the human PD brain, concentrated in the dense core of Lewy

bodies where synphilin-1 expression predominates in the brains of patients with Parkinson’s

disease.

I demonstrated that RNA interference (RNAi) of endogenous LMO7 does not cause

any effects on the formation of synphilin-1-positive inclusions in HEK293 Cells. I knocked

down the endogenous LMO7 by transfecting siRNA and confirmed the siRNA of LMO7

completely inhibited the expression of endogenous LMO7 in HEK293 cells. Using this

system, we further investigated the role of LMO7 in the formation of synphilin-1-positive

inclusions in HEK293 cells. In my results, the transfection with LMO7 siRNA did not cause

any effects on the formation of synphilin-1-positive inclusions in HEK293 cells. This is

probably because other proteins compensate for the function of LMO7 in HEK293 cells as

previous report (Tanji et al., 2006).

Although I defined that LMO7 promotes the formation of cytosolic inclusion in

cultured cells, the function of LMO7 in human brain has not been elucidated. However, I

believe that LMO7 plays the same role in cells of human brain. Because both LMO7 and

synphilin-1 are expressed in the normal brain, LMO7 should also promote the formation of

cytosolic inclusion in the cells of normal brain through its interaction with synphilin-1. Thus,

LMO7 seems to play an important role in brain cells under both physiological and

pathological conditions.

The PDZ and LIM domains of LMO7 are predicted to correlate protein-protein

interaction domains (Ooshio et al., 2004). LMO7 may act as an adapter molecule that

anchors synphilin-1 to intracellular proteins involved in vesicle transport and cytoskeletal

43

functions (Engelender et al., 1999). These related LMO-proteins suggested a gene regulatory

role for LMO7. LMO7 is alternatively spliced and expressed in most tissues tested (Putilina

et al., 1998; Rozenblum et al., 2002). Analyses of rat brain extracts have revealed LMO7

predominantly as a 70-kDa band, but it also occurs as a 190 kDa fragment, and lower

molecular bands of 35 kDa has been observed, supporting alternative splicing or post-

translational processing as mechanisms of LMO7 diversity.

In addition to the basic science aspects, my findings on LMO7 have two important

bearings clinically. First, I suggested that LMO7 could serve as a neuropathological marker

in patients with α-synucleinopathies because it is strongly accumulated with synphilin-1 in

the inclusions of their brain cells. Second, LMO7 could be a potential therapeutic target for

α-synucleinopathies. Future studies on the function LMO7 may be helpful to understand the

normal function of α-synuclein and synphilin-1, as well as the potential therapeutic

implications in the Parkinson’s disease and other α-synucleinopathies.

44

V. CONCLUSION

Synphilin-1 interacts preferentially with the C-terminal LIM domain of LMO7 and

LMO7 interacts with the ankyrin domain of synphilin-1. These findings have important

implications for understanding the molecular mechanism by which Lewy-body–associated

proteins interact through synphilin-1. With the basic science aspects, LMO7 could serve as a

neuropathological marker in patients with α-synucleinopathies because it is strongly

accumulated with synphilin-1 in the inclusions of their brain cells. LMO7 also may be

helpful to understand the normal function of α-synuclein and synphilin-1, as well as the

potential therapeutic implications in the Parkinson’s disease and other α-synucleinopathies

45

REFERENCES

1. Berrios GE, Campbell C, Politynska BE: Autonomic failure, depression and anxiety in Parkinson's disease. Br J Psychiatry 166:789-792, 1995

2. Bonifati V, Rizzu P, van Baren MJ, Schaap O, Breedveld GJ, Krieger E, Dekker MC,

Squitieri F, Ibanez P, Joosse M, van Dongen JW, Vanacore N, van Swieten JC, Brice A, Meco G, van Duijn CM, Oostra BA, Heutink P: Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science 299:256-259, 2003

3. Chung KK, Zhang Y, Lim KL, Tanaka Y, Huang H, Gao J, Ross CA, Dawson VL,

Dawson TM: Parkin ubiquitinates the alpha-synuclein-interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease. Nat. Med. 7, 1144–1150, 2001

4. Ciechanover A: Linking ubiquitin, parkin and synphilin-1. Nat. Med. 7, 1108–1109,

2001

5. Conway KA, Harper JD, Lansbury PT, Jr: Fibrils formed in vitro from alpha-synuclein

and two mutant forms linked to Parkinson's disease are typical amyloid. Biochemistry 39, 2552–2563, 2000

6. Conway KA, Lee SJ, Rochet JC, Ding TT, Williamson RE, Lansbury PT, Jr:

Acceleration of oligomerization, not fibrillization, is a shared property of both alphasynuclein mutations linked to early-onset Parkinson's disease: implications for

pathogenesis and therapy. Proc. Natl. Acad. Sci. USA 97, 571–576, 2000

7. Crowther RA, Jakes R, Spillantini MG, Goedert M: Synthetic filaments assembled

from C-terminally truncated alpha-synuclein. FEBS Lett. 436, 309–312, 1998

8. Dawson TM and Dawson VL: Rare genetic mutations shed light on the pathogenesis

of Parkinson disease. J. Clin. Invest. 111, 145–151, 2003

46

9. Dawson VL, Dawson TM, Bartley DA, Uhl GR, Snyder SH: Mechanisms of nitric oxide-mediated neurotoxicity in primary brain cultures. J Neurosci 13:2651-2661, 1993

10. Dluzen DE, McDermott JL: Gender differences in neurotoxicity of the nigrostriatal

dopaminergic system: implications for Parkinson's disease. J Gend Specif Med 3:36-42, 2000

11. Engelender S, Wanner T, Kleiderlein JJ, Wakabayashi K, Tsuji S, Takahashi H,

Ashworth R, Margolis RL, Ross CA: Organization of the human synphilin-1 gene, a candidate for Parkinson’s disease. Mamm Genome 11:763–766, 2000

12. Fortun J, Dunn WA, Joy S, Li J, Notterpek L: Emerging role for autophagy in the

removal of aggresomes in Schwann cells. J Neurosci 23:10672–10680, 2003 13. Gai WP, Yuan HX, Li XQ, Power JTH, Blumbergs PC, Jensen PH: In situ and in vitro

study of colocalization and segregation of alpha-synuclein, ubiquitin and lipids in Lewy bodies. Exp Neurol 166:324–333, 2004

14. Galvin JE, Lee VM, Schmidt ML, Tu PH, Iwatsubo T, Trojanowski JQ: Pathobiology

of the Lewy body. Adv Neurol 80:313-324, 1999 15. Gibb WR and Lees AJ: The relevance of the Lewy body to the pathogenesis of

idiopathic Parkinson's disease. J. Neurol. Neurosurg. Psychiatry 51, 745–752, 1988 16. Goldberg MS and Lansbury PT, Jr: Is there a cause-and-effect relationship between

alpha-synuclein fibrillization and Parkinson's disease? Nat. Cell Biol. 2, E115–E119, 2000

17. Gomez-Tortosa E, Newell K, Irizarry MC, Albert M, Growdon JH, Hyman BT:

Clinical and quantitative pathologic correlates of dementia with Lewy bodies. Neurology 53:1284-1291, 1999

47

18. Hashimoto M, Hsu LJ, Xia Y, Takeda A, Sisk A, Sundsmo M, Masliah E: Oxidative stress induces amyloid-like aggregate formation of NACP/alpha-synuclein in vitro.

NeuroReport 10, 717–721, 1999 19. Hirasawa A, Awaji T, Xu Z, Shinoura H, Tsujimoto G: Regulation of subcellular

localization of alpha1-adrenoceptor subtypes. Life Sci 68:2259-2267, 2001 20. Irizarry MC, Growdon W, Gomez-Isla T, Newell K, George JM, Clayton DF, Hyman

BT: Nigral and cortical Lewy bodies and dystrophic nigral neurites in Parkinson's disease and cortical Lewy body disease contain alpha-synuclein immunoreactivity. J Neuropathol Exp Neurol 57:334-337, 1998

21. Iseki E, Takayama N, Furakawa Y, Marui W, Nakai T, Miura S, Ueda K, Kosaka K:

Immunohistochemical study of synphilin-1 in brains of patients with dementia with Lewy bodies—synphilin-1 is non-specifically implicated in the formation of different neuroanl cytoskeletal inclusions. Neurosci Lett 326:211–215, 2002

22. Ito T, Niwa J, Hishikawa N, Ishigaki S, Doyu M, Sobue G: Dorfin localizes to Lewy

bodies and ubiquitylates synphilin-1. J Biol Chem 278:29106-29114, 2003 23. Iwatsubo T, Yamaguchi H, Fujimuro M, Yokosawa H, Ihara Y, Trojanowski JQ, Lee

VM: Purification and characterization of Lewy bodies from the brains of patients with

diffuse Lewy body disease. Am. J. Pathol. 148, 1517–1529, 1996 24. James MH, Soroush Rais-Bahrami, Katherine LW: Lmo7 is an emerin-binding protein

that regulates the transcription of emerin and many other muscle-relevant genes: Human Molecular Genetics, 15:3459–3472, 2006

25. Johnston JA, Ward CL, Kopito RR: Aggresomes: a cellular response to misfolded

proteins. J Cell Biol 143:1883–1898, 1998 26. Junn E, Lee SS, Suhr UT, Mouradian MM: Parkin accumulation in aggresomes due to

proteasome impairment. J Biol Chem 277:47870–47877, 2002

48

27. Katsuse O, Iseki E, Marui W, Kosaka K: Developmental stages of cortical Lewy bodies and their relation to axonal transport blockage in brains of patients with dementia with Lewy bodies. J Neurol Sci 211:29–35, 2003

28. Klein U, Ramirez MT, Kobilka BK, von Zastrow M: A novel interaction between

adrenergic receptors and the alpha-subunit of eukaryotic initiation factor 2B. J Biol Chem 272:19099-19102, 1997

29. Kopito RR: Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol

10:524–530, 2000 30. Krüger R, Marx FP, Strauss KM, Dawson S, Riess O, Schulz JB: Identification of the

proteasomal subunit S6-ATPase as a novel synphilin-1 interacting protein. Progr No. 410.3. Abstract Viewer/Itinerary Planner. Society for Neuroscience, Washington, 2003

31. Kurihara LJ, Semenova E, Miller W, Ingram RS, Guan XJ, Tilghman SM: Candidate

genes required for embryonic development: a comparative analysis of distal mouse chromosome 14 and human chromosome 13q22. Genomics 79:154-161, 2002

32. Lee G, Junn E, Tanaka M, Kim YM, Mouradian MM: Synphilin-1 degradation by the

ubiquitin-proteasome pathway and effects on cell survival. J Neurochem 83:346–352, 2002

33. Lee G, Tanaka M, Park K, Lee SS, Kim YM, Junn E, Lee SH, Mouradian MM: Casein

kinase II-mediated phosphorylation regulates alpha-synuclein/synphilin-1 interaction and inclusion body formation. J Biol Chem 279:6834–6839, 2004

34. Leroy E, Boyer R, Auburger G, Leube B, Ulm G, Mezey E, Harta G, Brownstein MJ,

Jonnalagada S, Chernova T, Dehejia A, Lavedan C, Gasser T, Steinbach PJ, Wilkinson KD, Polymeropoulos MH: The ubiquitin pathway in Parkinson's disease. Nature 395:451-452, 1998

49

35. Liani E, Eyal A, Shemer R, Berg D, Franck T, Riess O, Ross CA, Rott R, Engelender S: Ubiquitylation of synphilin-1 and alpha-synuclein by SIAH proteins and their presence in Lewy bodies imply a role in Parkinson’s disease. Proc Nat Acad Sci USA 101:5500–5505, 2004

36. Lotharius J, Brundin P: Pathogenesis of Parkinson's disease: dopamine, vesicles and

alpha-synuclein. Nat Rev Neurosci 3:932-942, 2002 37. Marx FP, Holzmann C, Strauss KM, Li L, Eberhardt O, Cookson MR, Hernandez D,

Farrer MJ, Kachergus J, Engelender S, Ross CA, Berger K, Schöls L, Schulz JB, Riess O, Krüger R: Identification and functional characterization of a novel R621C mutation in the synphilin-1 gene in Parkinson’s disease. Hum Mol Genet 12:1223–1231, 2003

38. McNaught K, Belizaire R, Isacson O, Jenner P, Olanow CW: Altered proteasomal

function in sporadic Parkinson's disease. Exp. Neurol. 179, 38–46, 2003 39. McNaught KS, Jenner P: Proteasomal function is impaired in substantia nigra in

Parkinson’s disease. Neurosci Lett 297:191–194, 2001 40. Murray IVJ, Medford MA, Guan HP, Rueter SM, Trojanowski JQ, Lee VMY:

Synphilin in normal human brains and in synucleinopathies: studies with new antibodies. Acta Neuropathol 105:177–184, 2003

41. Nagano Y, Yamashita H, Takahashi T, Kishida S, Nakamura T, Iseki E, Hattori N,

Mizuno Y, Kikuchi A, Matsumoto M: Siah-1 facilitates ubiquitination and degradation of synphilin-1. J Biol Chem 278:51504–51514, 2003

42. Neystat M, Rzhetskaya M, Kholodilov N, Burke RE: Analysis of synphilin-1 and

synuclein interactions by yeast two-hybrid beta-galactosidase liquid assay. Neurosci Lett 325:119–123, 2002

43. Nussbaum RL, and Ellis CE: Alzheimer's disease and Parkinson's disease. N. Engl. J.

Med. 348, 1356–1364; erratum in. N. Engl. J. Med. 348, 2588, 2003

50

44. O’Farrell C, Murphy DD, Petrucelli L, Singleton AB, Hussey J, Farrer M, Hardy J, Dickson DW, Cookson MR: Transfected synphilin-1 forms cytoplasmic inclusions in HEK293 cells. Brain Res Mol Brain Res 97:94–102, 2001

45. O’Farrell C, Pickford F, Vink L, McGowan E, Cookson MR: Sequence conservation

between mouse and human synphilin-1. Neurosci Lett 322:9–12, 2002 46. Ostrerova-Golts N, Petrucelli L, Hardy J, Lee JM, Farer M, Wolozin B: The A53T

alpha-synuclein mutation increases iron-dependent aggregation and toxicity. J.

Neurosci.20, 6048–6054, 2000 47. Pollanen MS, Dickson DW, Bergeron C: Pathology and biology of the Lewy body. J

Neuropathol Exp Neurol 52:183-191, 1993

48. Polymeropoulos MH: Genetics of Parkinson's disease. Ann. N.Y. Acad. Sci. 920, 28–32, 2000

49. Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B, Root

H, Rubenstein J, Boyer R, Stenroos ES, Chandrasekharappa S, Athanassiadou A, Papapetropoulos T, Johnson WG, Lazzarini AM, Duvoisin RC, Di Iorio G, Golbe LI, Nussbaum RL: Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science 276:2045-2047, 1997

50. Putilina T, Jaworski C, Gentleman S, McDonald B, Kadiri M, Wong P: Analysis of a

human cDNA containing a tissue-specific alternatively spliced LIM domain. Biochem Biophys Res Commun 252:433-439, 1998

51. Ribeiro CS, Carneiro K, Ross CA, Menezes JR, Engelender S: Synphilin-1 is

developmentally localized to synaptic terminals, and its association with synaptic vesicles is modulated by alpha-synuclein. J Biol Chem 277:23927–23933, 2002

51

52. Rozenblum E, Vahteristo P, Sandberg T, Bergthorsson JT, Syrjakoski K, Weaver D, Haraldsson K, Johannsdottir HK, Vehmanen P, Nigam S, Golberger N, Robbins C, Pak E, Dutra A, Gillander E, Stephan DA, Bailey-Wilson J, Juo SH, Kainu T, Arason A, Barkardottir RB, Nevanlinna H, Borg A, Kallioniemi OP: A genomic map of a 6-Mb region at 13q21-q22 implicated in cancer development: identification and characterization of candidate genes. Hum Genet 110:111-121, 2002

53. Schlossmacher MG, Frosch MP, Gai WP, Medina M, Sharma N, Forno L, Ochiishi T,

Shimura H, Sharon R, Hattori N, Langston JW, Mizuno Y, Hyman BT, Selkoe DJ, Kosik KS: Parkin localizes to the Lewy bodies of Parkinson disease and dementia with Lewy bodies. Am J Pathol 160:1655–1667, 2002

54. Semenova E, Wang X, Jablonski MM, Levorse J, Tilghman SM: An engineered 800

kilobase deletion of Uchl3 and Lmo7 on mouse chromosome 14 causes defects in viability, postnatal growth and degeneration of muscle and retina. Hum Mol Genet 12:1301-1312, 2003

55. Shimura H, Schlossmacher MG, Hattori N, Frosch MP, Trockenbacher A, Schneider R,

Mizuno Y, Kosik KS, Selkoe DJ: Ubiquitination of a new form of alpha-synuclein by

parkin from human brain: implications for Parkinson's disease. Science 293, 263–269, 2001

56. Siderowf A, Stern M: Update on Parkinson disease. Ann Intern Med 138:651-658,

2003 57. Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M: Alpha-synuclein in

filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with

Lewy bodies. Proc. Natl. Acad. Sci. USA 95, 6469–6473, 1998 58. Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M: Alpha-

synuclein in Lewy bodies. Nature 388, 839–840, 1997

52

59. Tanaka M, Kim YM, Lee G, Junn E, Iwatsubo T, Mouradian MM: Aggresomes formed by alpha-synuclein and synphilin-1 are cytoprotective. J Biol Chem 279:4625–4631, 2003

60. Tanji K, Tanaka T, Mori F, Kito K, Takahashi H, Wakabayashi K, Kamitani T: NUB1

suppresses the formation of Lewy body-like inclusions by proteasomal degradation of synphilin-1. Am J Pathol 169:553-565, 2006

61. Tanji K, Toki T, Tamo W, Imaizumi T, Matsumiya T, Mori F, Takahashi H, Satoh K,

Wakabayashi K: Glycogen synthase kinase-3beta phosphorylates synphilin-1 in vitro. Neuropathology 23:199–202, 2003

62. Tanner CM: Occupational and environmental causes of parkinsonism. Occup Med

7:503-513, 1992 63. Taylor JP, Tanaka F, Robitschek J, Sandoval M, Taye A, Markovic-Plese S, Fischbeck

K: Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing protein. Hum Mol Genet 12:749–757, 2003

64. Uversky VN and Fink AL: Amino acid determinants of alpha-synuclein aggregation:

putting together pieces of the puzzle. FEBS Lett. 522, 9–13, 2002 65. Voges D, Zwickl P, Baumeister W: The 26S proteasome: a molecular machine

designed for controlled proteolysis. Annu. Rev. Biochem. 68, 1015–1068, 1999 66. Volles MJ and Lansbury PT, Jr: Vesicle permeabilization by protofibrillar alpha-

synuclein is sensitive to Parkinson's disease-linked mutations and occurs by a pore-like mechanism. Biochemistry 41, 4595–4602, 2002

67. Wakabayashi K, Engelender S, Tanaka Y, Yoshimoto M, Mori F, Tsuji S, Ross CA,

Takahashi H: Immunocytochemical localization of synphilin-1, an alpha-synuclein-associated protein, in neurodegenerative disorders. Acta Neuropathol 103:209–214, 2002

53

68. Wakabayashi K, Engelender S, Yoshimoto M, Tsuji S, Ross CA, Takahashi H: Synphilin-1 is present in Lewy bodies in Parkinson’s disease. Ann Neurol 47:521–523, 2000

69. Wakabayashi K, Hayashi S, Kakita A, Yamada M, Toyoshima Y, Yoshimoto M,

Takahashi H: Accumulation of alpha-synuclein/NACP is a cytopathological feature common to Lewy body disease and multiple system atrophy. Acta Neuropathol (Berl) 96:445-452, 1998

70. Warner TT, Schapira AH: Genetic and environmental factors in the cause of

Parkinson's disease. Ann Neurol 53 Suppl 3:S16-23; discussion S23-15, 2003 71. West A, Periquet M, Lincoln S, Lucking CB, Nicholl D, Bonifati V, Rawal N, Gasser T,

Lohmann E, Deleuze JF, Maraganore D, Levey A, Wood N, Durr A, Hardy J, Brice A, Farrer M: Complex relationship between Parkin mutations and Parkinson disease. Am J Med Genet 114:584-591, 2002

72. Wood SJ, Wypych J, Steavenson S, Louis JC, Citron M, Biere AL: α-Synuclein

fibrillogenesis is nucleation-dependent. Implications for the pathogenesis of

Parkinson's disease. J. Biol. Chem. 274, 19509–19512, 1999 73. Xu J, Kao SY, Lee FJ, Song W, Jin LW, Yankner BA: Dopamine-dependent

neurotoxicity of alphasynuclein: a mechanism for selective neurodegeneration in

Parkinson disease. Nat. Med. 8, 600–606, 2002

54

I. INTRODUCTION

1. Ubiquitin–proteosome system (UPS)

1.1. Ubiquitination of proteins

The Ubiquitin–proteosome system (UPS) is believed to play an important role in the

fine tuning and rapid degradation control of 30% or more of newly made proteins within the

cell (Schubert et al., 2000). This process plays a crucial role in a number of cellular events

such as cell cycling, signal transduction, metabolism and the immune response (Pagano,

1997; Ben-Neriah, 2002). Polyubiquitination of substrates is a priming event for

proteasomal-mediated degradation (Hershko and Ciechanover, 1998). Protein ubiquitination

occurs by three-enzyme system (Fig. 1). In this process the ATP dependent ubiquitin (Ub)-

activating enzyme E1 is believed to form a high-energy Ub intermediate. The activated Ub is

then accepted via a thioester bond by an Ub-conjugating E2 enzyme (UBC). Chains of Ub

molecules are linked by ε-amide bonds leading to the polyubiquitination of an E3-bound

substrate. For mammalian cells only a single E1 is known to exist at present (Handley et al.,

1991). At present, the E2 family of enzymes consists over 20 members (Pickart, 2001).

Hundreds of E3 enzymes are believed to be present which infer selectivity within the

ubiquitination pathway (Pickart, 2001). Once polyubiquitinated the targets are accepted as

substrate for 26S protease-memediated degradation. The 26S proteasome is a large

multiprotein complex (2.5 MDa) that requires ATP for protein degradation (Voges et al.,

1999).

55

Fig. 1. The ubiquitin proteasome-mediated pathway. 1) Activation of ubiquitin by E1. 2) Transfer of the activated ubiquitin moiety from E1 to E2. 3) Ubiquitin is further transferred in one of two ways. 3a) In the case of HECT-domain ligases (E3s), ubiquitin generates a third, high-energy intermediate with the ligase. 4a) Following specific recognition of the substrate and generation of an E3–substrate complex, multiple ubiquitin moieties are successively transferred to generate a substrate-anchored polyubiquitin chain that serves as a recognition marker for the 26S proteasome. 3b) In the case of RING-finger domain E3s, a ternary complex is generated between the substrate, E3, and E2, and the activated ubiquitin moieties are transferred directly from E2 to the E3-bound substrate. 5 and 6) Recycling of the E2s and E3s, respectively. 7) Recognition of the polyubiquitin chain by the 19S subcomplex of the 26S proteasome. 8) Degradation of the substrate to generate peptides with release of free and reusable ubiquitin

56

1.2. Protein aggregation and neurodegeneration

Many sporadic and inherited neurodegenerative diseases are characterised by the

presence of insoluble protein aggregates. These aggregates are essentially composed of

elevated levels of multiubiquitinated proteins (Alves-Rodrigues et al., 1998; Kopito, 2000).

Initial protein aggregation could lead to an accumulation of these aggregates by

progressively impairing function of the UPS, a pathway that is involved in protein

degradation (Bence et al., 2001). Of interest, proteosome subunits colocalize in inclusion

bodies associated with neurodegenerative diseases (Cummings et al., 1998). Thus, several

lines of evidence exist that suggest a linkage between dysfunction of the UPS and

neurodegeneration (Alves-Rodrigues et al., 1998; (Kopito, 2000). For example, in addition

to the forms of PD caused by UCHL1 and parkin mutations (Leroy et al., 1998; Kitada et al.,

1998; Shimura et al., 2000), a mutant form of Ub called Ub+1 has been detected in the

brains of Alzheimer’s patients, including those with nonfamilial Alzheimer’s Disease (AD)

(van Leuven et al., 1998). In this condition, polyubiquitin chains made by Ub+1 mutated Ub

are refractory to disassembly by deubiquitinating enzymes. This causes an accumulation and

aggregation of ubiquitinated proteins, leading to neurodegeneration (Lam et al., 2000).

2. Ubiquitination of proteins in Parkinson’s disease

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by

loss of dopaminergic neurons in the substantia nigra pars compacta and by eosinophilic

cytoplasmic inclusions known as Lewy bodies (Forno, 1996). Genetic and biochemical

analyses point to a central role for the ubiquitin-proteasome pathway in the pathogenesis of

57

this disease (McNaught et al., 2001). Poly-ubiquitination of proteins is a marker for their

degradation by proteasome, the proteolytic complex that degrades many cytoplasmic

proteins (Ciechanover et al., 2000). The three genes linked to date to inherited PD, namely

α-synuclein, parkin and ubiquitin C-terminal hydrolase L1 (UCH-L1), are either closely

involved in the proper functioning of the ubiquitin-proteasome pathway or are degraded by

this protein clearing machinery of cells (Mouradian, 2002). Both α-synuclein and Parkin are

ubiquitinated proteins (Bennett et al. 1999; Choi et al., 2000; Imai et al., 2000; Zhang et al.,

2000). At the same time, Parkin functions as an E3 ubiquitin ligase (Shimura et al., 2000;

Zhang et al., 2000) while UCH-L1 hydrolyzes small C-terminal adducts of ubiquitin to

generate ubiquitin monomers, which can then be recycled and used to clear other proteins

(Leroy et al. 1998). Additionally, the presence of Parkin (Shimura et al., 1999), poly-

ubiquitin chains (Iwatsubo et al., 1996), proteasome subunits (Ii et al., 1997) and UCH-L1

within Lewy bodies (Lowe et al., 1990) further support a pathogenetic role for this protein

degradation pathway in the pathogenesis of PD.

Screening for proteins that interact with disease gene products provides clues about the

function of pathogenic proteins and could elucidate cell-death pathways. For example, a-

synuclein, mutations in which result in autosomal dominant PD (Polymeropoulos et al.

1997), interacts with a number of molecules including synphilin-1 (Engelender et al., 1999).

Co-expression of α-synuclein and synphilin-1 in transfected cells results in the formation of

eosinophilic cytoplasmic inclusions that resemble Lewy bodies (Engelender et al., 1999).

Furthermore, both these proteins are present in Lewy bodies in the brains of patients with PD

or dementia with Lewy bodies (Spillantini et al. 1998; Wakabayashi et al. 2000). Recently,

58

Parkin was reported to be the E3 ubiquitin ligase for synphilin-1 and the co-expression of

Parkin was found to be required for synphilin-1 ubiquitination and aggregation into cytosolic

inclusions (Chung et al., 2001).

PURPOSE

In this part, I studied that degradation of LMO7 in cells and a tendency for this protein

to be poly-ubiquitinated and aggregated into inclusions.

59

II. MATERIALS AND MRTHODS A. MATERIALS

Chemicals were purchased from the following companies: cycloheximide (Sigma, St.

Louis, MO, USA) and MG132 (Calbiochem, La Jolla, CA, USA. Cycloheximide and

MG132 were dissolved in dimethyl sulfoxide (DMSO).

B. METHODS

1. Cell culture and generation of stable cell line

Human embryonic kidney 293 (HEK293) cells (ATCC) were cultured in Dulbecco’s

modified eagle’s medium (DMEM, Gibco, Rockville, MD, USA),) supplemented with 10%

fetal bovine serum and grown at 37℃ in a humidified atmosphere containing 10% CO2.

pFLAG-LMO7 expressing full-length LMO7 with an N-terminal FLAG tag was generated as

described previous part I. Transfections were carried out with the Calcium Phosphate

Transfection Kit (Invitrogen, Carslbad, CA, USA) according to the supplier’s instructions.

Stably transfected HEK293 cells over-expressing FLAG-tagged LMO7 (LMO7-293) were

selected, cloned by dilution and maintained in the presence of 1 mg/mL G418 (Sigma, St.

Louis, MO, USA). Finally selected LMO7-293 cell lines were detected and confirmed by

western blotting with anti-FLAG antibody.

2. Inhibition of LMO7 protein synyhesis

Stably transfected LMO7-293 cells were treated with 100 uM cycloheximide

60

(Grunberg et al., 1998) for the indicated time points (0, 2, 4, 8, 16 h) and cell lysates were

analyzed by immunoblotting, using anti-FLAG antibody. The resultant cell lysates were

centrifuged for 10 min at 12,000 rpm to remove debris, and FLAG-tagged-LMO7 was

immunoprecipitated with anti-FLAG M2-Agarose Affinity gel (Sigma, St Louis, MO, USA)

at 4℃ for 16 h. Precipitates washed five times with lysis buffer were subjected to sodium

dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Data from triplicate

samples were normalized relative to time zero set at 100%.

3. Immunoprecipitation and immunoblot analysis

LMO7-293 cells were harvested, washed in PBS, and lysed in Hepes (50 mM, pH 7.4),

NaCl (150 mM), EDTA (3 mM), 1% Triton X-100, 0.1% SDS and a protease inhibitors

cocktail (Complete, Boehringer). Lysates were centrifuged at 12,000 rpm for 10 min, and the

supernatant was precleared before immunoprecipitation. Samples (300µg) were incubated

with 40 µl of anti-FLAG M2 affinity gel (Sigma) at 4 °C for 16 h with constant mixing.

Immunoprecipitates or total-cell lysates were subjected to western blot analysis using anti-

ubiquitin antibody (FL76) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or HRP

conjugated anti-FLAG (M2) antibody with ECL detection reagent (NEN, Boston, MA, USA).

4. Immunostaining of LMO7-293 cell treated protease inhibitor

LMO7-293, HEK293 and SH-SY5Y cells transiently transfected with FLAG-LMO7

were treated with proteasome inhibitors or DMSO for 13 h. After washing with PBS, cells

were fixed with 4% formaldehyde at room temperature (25℃) for 10 min, permeablized with

61

0.3% Triton X-100 for 10 min, and blocked with 3% BSA for 30 min. Cells were then

incubated overnight at 4℃ with the appropriate primary antibody diluted in PBS. For

staining control, primary antibody was omitted. After washing with PBS three times,

secondary antibody was added for 2 h at room temperature. Samples were visualized under a

Zeiss (LSM510) confocal microscope or epi-fluorescence microscope (Zeiss, Axiophot,

Thornwood, NY, USA). For quantification of inclusions, 10 microscopic fields were

randomly selected and the percentage of inclusion-positive cells was counted.

62

III. RESULTS

1. LMO7 is stable protein in vitro

The degradation of LMO7 was studied in both HEK293 and LMO7-293 cells. FLAG-

tagged LMO7 was expressed stably in HEK293 cells and the specificity of transgene

expression was verified by western blot analysis with anti-FLAG antibody. We inhibited de

novo protein synthesis with cycloheximide. After the indicated time points (0, 2, 4, 8, 16)

cells were lysed and FLAG-tagged- and endogenous LMO7 protein expression was analyzed

by immunoblotting. As shown in Fig. 2, LMO7 was stable over 16 h in both HEK293 and

LMO7-293 stable cell lines. Therefore LMO7 is remarkably stable and under the conditions

used in these experiments.

80 162 4

Cycloheximide (hour)

β-actin

80 162 4

Cycloheximide (hour)

FLAG-LMO7

β-actin

Endo. LMO7

80 162 4

Cycloheximide (hour)

β-actin

80 162 4

Cycloheximide (hour)

FLAG-LMO7

β-actin

Endo. LMO7

Fig. 2. Stability of LMO7 protein. De novo protein synthesis was inhibited with 10 µM cycloheximide for the indicated time points. Endogenous and overexpressed LMO7 was analyzed by immunoblotting using anti-LMO7 and FLAG antibody. LMO7 was very stable over long time periods, 16h.

63

2. Attenuation of LMO7 degradation by proteasome inhibitor

To study the proteasomal degradation of LMO7, LMO7-293 cells treated with two

different proteasome inhibitors, MG-132 and lactacystin. Both agents markedly attenuated

the degradation of FLAG-LMO7 in LMO7-293 cells compared with DMSO treated cells

(Fig. 3A). Over 40 h of incubation in the presence of MG-132 resulted in significant

cytotoxicity. The abrupt decline in LMO7 recovery at 40 h is likely due to cell death. In

addition, the expression level of FLAG-LMO7 was quantified in LMO7-293 cells treated

with MG132 for 24 h by western blotting with anti-FLAG antibody and compared with the

control protein β-actin (Fig. 3B). This experiment revealed a 1.8-fold accumulation of

FLAG-LMO7 in the presence of the proteasome inhibitor.

DMSO

MG132

FLAG-LMO7

β-actin

A

0

0.4

0.8

1.2

1.6

2

Rel

ativ

e in

tens

ity

DMSO MG132

*

B

DMSO MG132

*

DMSO

MG132

FLAG-LMO7

β-actin

A

DMSO

MG132

FLAG-LMO7

β-actin

A

0

0.4

0.8

1.2

1.6

2

Rel

ativ

e in

tens

ity

DMSO MG132

*

B

DMSO MG132

*

0

0.4

0.8

1.2

1.6

2

0

0.4

0.8

1.2

1.6

2

Rel

ativ

e in

tens

ity

DMSO MG132

*

B

DMSO MG132

*

Fig. 3. Attenuation of LMO7 degradation by proteasome inhibitor. (A) LMO7-293 cells were treated with 10 uM MG132 or DMSO for 24 h. LMO7 was detected by western blotting with anti-FLAG antibody (upper panel). An immunoblot for β-actin is shown as control (lower panel). (B) Band intensity was quantified from three separate experiments using the UN-SCAN-IT (Silk Scientific Corp., Orem, UT, USA) densitometric software and means ± SD are compared using student t-test. *p < 0.005.

64

3. LMO7 is ubiquitinated and interacts with ubiquitinated protein

Incubation with the proteasome inhibitor MG132 increased the amount pf LMO7 in

HEK293 cells (fig. 3), suggesting that this increased LMO7 contains ubiquitin-conjugated

proteins. We then examined if LMO7 could be covalently modified by ubiquitin as well. To

determine if synphilin-1 is modified by ubiquitin, LMO7-293 cells were treated with the

proteasome inhibitor MG-132 and FLAG-tagged LMO7 was immunoprecipitated with anti-

FLAG antibody and ubiquitinated proteins were detected by western blotting using anti-

ubiquitin antibody (Fig. 4). We found that LMO7 is ubiquitylated by MG132 treatment, as

shown by the significant anti-ubiquitin immunoreactivity in the form of smear, which is

characteristic of polyubiquitylated proteins. Much less ubiquitylation of LMO7 was observed

when cells were DMSO treated in LMO7-293 cells. These observations indicate that LMO7

is covalently modified by ubiquitin leading to its proteasomal degradation and that LMO7

interacts with a number of other ubiquitinated proteins.

4. Proteasomal inhibition leads to the formation of LMO7- and ubiquitin-positive

inclusion

In previous result, LMO7 is degraded by ubiquitin and interacts with a number of other

ubiquitinated proteins. To confirm these findings, we checked for intracellular co-

localization of LMO7 and ubiquitin by immunocytochemical staining with anti-FLAG

antibody followed by rhodamine conjugated secondary antibody, and anti-ubiquitin primary

antibody followed by fluorescein conjugated secondary antibody. In the absence of

proteasome inhibitor, LMO7 and ubiquitin colocalized diffusely in the cytoplasm of LMO7-

65

293 cells (Fig. 5A). Treatment of LMO7-293 cells with MG132 resulted in the formation of

relatively large peri-nuclear inclusions in a majority of cells (Fig. 5B). These were

immunoreactive to both LMO7 and ubiquitin. In view of my data in Fig. 5, the ubiquitin

immunoreactivity in these inclusions suggests that in addition to LMO7 being ubiquitinated

and aggregated other ubiquitinated proteins accumulate as well.

LMO7-(Ub)n

MG132 - -+ +

Total FLAG-IP

Ubi

quiti

nW

B

FLAG WB LMO7

LMO7-(Ub)n

MG132 - -+ +

LMO7-(Ub)n

MG132 - -+ +

Total FLAG-IP

Ubi

quiti

nW

B

FLAG WB LMO7

Fig. 4. Proteasomal degradation and ubiquitination of LMO7. LMO7-293 cells were treated with either 10 uM MG-132 (+) or DMSO (–) for 24 h and lysed in respective buffers as described in Materials and Methods. After immunoprecipitation with anti-FLAG antibody, western blotting was done with anti-ubiquitin antibody (upper panel) or with anti-FLAG antibody (lower panel) to verify proper immunoprecipitation of LMO7.

66

A

B

a

a b c

b cA

B

a

a b c

b c

Fig. 5. Co-localization of ubiquitin in LMO7 positive inclusions. LMO7-293 cells were treated with vehicle (DMSO) (A) or 10 uM MG132 (B) for 13 h and stained for LMO7 (rhodamine, red, a) and ubiquitin (fluorescein, green, b). There co-localization was shown by the merging of both fluorescences (C) Omission of primary antibody gave no signal in any of these experiments. Scale bar, 10 µm.

67

IV. DISCUSSION

Synphilin-1, which is present in Lewy bodies and interacts with α-synuclein

(Engelender et al., 1999; Wakabayashi et al., 2000), shares the same degradation pathway as

other PD associated gene products such as α-synuclein (Bennett et al., 1999; Imai et al.,

2000) and Parkin (Choi et al., 2000; Imai et al., 2000; Zhang et al., 2000), namely the

ubiquitin-proteasome pathway. LMO7, interaction partner of synphilin-1 is relatively stable

by 16 h. Additionally, similar to the aggregation of a-synuclein in PC12 cells (Rideout et al.,

2001), proteasomal inhibition leads to the formation of peri-nuclear inclusions which stained

for synphilin-1, a-synuclein and ubiquitin. LMO7 also promotes its polyubiquitylation and

proteasomal degradation by proteasome inhibitor and proteasomal inhibition leads to the

formation of peri-nuclear inclusions which stained for LMO7 and ubiquitin.

Immunostaining of Lewy bodies has revealed that a-synuclein is mainly present in the

peripheral halo while synphilin-1 is mainly concentrated within the central core (Irizarry et

al., 1998; Wakabayashi et al., 1998; Wakabayashi et al., 2000). In my present experiment,

the peri-nuclear inclusions formed in LMO7 over-expressing cells as a result of proteasomal

inhibition appeared quite large. It is conceivable that the aggregation of LMO7 is a primary

or initial event in Lewy body formation, which then recruits other proteins to accumulate in

these structures. Additionally, the proteasomal impairment found in the parkinsonian nigra

(McNaught and Jenner, 2001) could provide the necessary cellular and biochemical

environment for LMO7 to clump into large Lewy bodies. The previous report of increased

synphilin-1 positive inclusions in transiently transfected HEK293 cells by a proteasome

68

inhibitor supports my present observations (O’Farrell et al. 2001).

In previous reports, synphilin-1 was shown to be ubiquitinated by four RING-finger-

containing ubiquitin E3 ligases, parkin, siah-1 and -2, and dorfin (Engelender et al., 1999; Ito

et al., 2003; Nagano et al., 2003; Liani et al., 2004). The functional similarity of these E3

ligases indicates that multiple pathways are facilitating the ubiquitination of synphilin-1.

Interestingly, siah proteins ubiquitinate synphilin-1, promoting its degradation by the

ubiquitin-proteasome system (Nagano et al., 2003; Liani et al., 2004). Unlike siah proteins,

parkin assembles a lysine 63-linked polyubiquitin chain on synphilin-1 that is distinct from

the classical, degradation-associated, lysine 48-linked ubiquitination (Doss-Pepe et al., 2005;

Lim et al., 2006). So far, it has been unknown which type of polyubiquitin chain is

assembled on synphilin-1 by dorfin (Ito et al., 2003). In the past, some groups overexpressed

these E3 ligases in HEK293 cells to promote the formation of inclusions (Chung et al., 2001;

Liani et al., 2004). However, it is still unclear how the overexpression of these E3 ligases

plays a role in the formation of inclusions (Nagano et al., 2003; Liani et al., 2004; Doss-Pepe

et al., 2005; Lim et al., 2006). In my inclusion-formation assay, these E3 ligases were not

overexpressed, because we could efficiently generate LMO7-positive inclusions without the

overexpression of E3 ligases. In this way I was able to investigate the role of LMO7 in the

formation of inclusions under the physiological expression of E3 ligases. In my present

experiments, poly-ubiquitination of LMO7 by immunoprecipitation /immunoblotting assay

as well as the formation of ubiquitin-, and LMO7-positive inclusions were evident without

the need for parkin transfection. Inhibition of proteasome function revealed the ubiquitinated

nature of LMO7 in LMO7-293 cells. While endogenous parkin expression has been detected

69

by RT-PCR in HEK293 cells (Putilina et al., 1998), and my observations indicate that parkin

over-expression is not necessary for LMO7 ubiquitination or its aggregation into inclusions.

This finding suggests that endogenous levels of parkin may be sufficient to ligate ubiquitin

onto LMO7 or alternatively raises the possibility that LMO7 could be ubiquitinated by an E3

ligase other than parkin.

The above findings taken together reveal that LMO7 can aggregate in cells as

ubiquitinated inclusions containing synphilin-1. Whether these two protein partners promote

or seed each other’s aggregation is an interesting hypothesis that requires further testing. A

similar cross seeding has been demonstrated between Ab-amyloid and a-synuclein (Han et

al., 1995). While the cytotoxicity of such aggregates is not established, their presence

provides clues about the molecular properties of constituent proteins.

V. CONCLUSION

Synphilin-1, which is present in Lewy bodies and interacts with α-synuclein

(Engelender et al., 1999; Wakabayashi et al., 2000), shares the same degradation pathway as

other PD associated gene products such as α-synuclein (Bennett et al., 1999; Imai et al.,

2000) and Parkin (Choi et al., 2000; Imai et al., 2000; Zhang et al., 2000), namely the

ubiquitin-proteasome pathway. LMO7 also promotes its polyubiquitylation and proteasomal

degradation by proteasome inhibitor and proteasomal inhibition leads to the formation of

peri-nuclear inclusions which stained for LMO7 and ubiquitin.

70

REFERENCES 1. Alves-Rodrigues A, Gregori L, Figueiredo-Pereira ME: Ubiquitin, cellular inclusions

and their role in neurodegeneration. Trends Neurosci. 21, 516–520, 1998 2. Bence, N.F., Sampat, R.M., Kopito, R.R., 2001. Impairment of the ubiquitin-proteasome

system by protein aggregation. Science 292, 1552–1555. 3. Ben-Neriah Y (2002) Regulatory functions of ubiquitination in the immune system. Nat

Immunol 3:20-26. 4. Bennett MC, Bishop JF, Leng Y, Chock PB, Chase TN, Mouradian MM: Degradation of

alpha-synuclein by proteasome. J. Biol. Chem 274, 33855–33858, 1999 5. Choi P, Ostrerova-Golts N, Sparkman D, Cochran E, Lee JM, Wolozin B: Parkin is

metabolized by the ubiquitin/proteosome system. Neuroreport 11, 2635–2638 2000 6. Chung KK, Zhang Y, Lim KL, Tanaka Y, Huang H, Gao J, Ross CA, Dawson VL,

Dawson TM: Parkin ubiquitinates the alpha-synuclein-interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease. Nat. Med. 7, 1144–1150, 2001

7. Ciechanover A, Orian A, Schwartz AL: The ubiquitinmediated proteolytic pathway:

mode of action and clinical implications. J. Cell Biochem. 77, 40–51, 2000 8. Cummings CJ, Mancini MA, Antalffy B, DeFranco DB, Orr HT, Zoghbi HY (1998)

Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat Genet 19:148-154.

9. Doss-Pepe EW, Chen L, Madura K: Alpha-synuclein and parkin contribute to the

assembly of ubiquitin lysine 63-linked multiubiquitin chains. J Biol Chem 280:16619-16624, 2005

71

10. Engelender S, Kaminsky Z, Guo X, Sharp AH, Amaravi RK, Kleiderlein JJ, Margolis RL, Troncoso JC, Lanahan AA, Worley PF, Dawson VL, Dawson TM, Ross CA: Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions. Nat. Genet. 22, 110–114, 1999

11. Forno LS: Neuropathology of Parkinson’s disease. J. Neuropathol. Exp. Neurol. 55,

259–272, 1996 12. Grunberg J, Walter J, Loetscher H, Deuschle U, Jacobsen H, Haass C: Alzheimer's

disease associated presenilin-1 holoprotein and its 18-20 kDa C-terminal fragment are death substrates for proteases of the caspase family. Biochemistry 37:2263-2270, 1998

13. Han H, Weinreb PH, Lansbury PT. Jr: The core Alzheimer’s peptide NAC forms

amyloid fibrils which seed and are seeded by beta-amyloid: is NAC a common trigger or target in neurodegenerative disease? Chem. Biol. 2, 163–169, 1995

14. Handley, P.M., Mueckler, M., Siegel, N.R., Ciechanover, A., Schwartz, A.L., 1991.

Molecular cloning, sequence, and tissue distribution of the human ubiquitin-activating enzyme E1. Proceedings of the National Academy Science of the USA 88, 258–262.

15. Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425-479. 16. Ii K, Ito H, Tanaka K, Hirano A: Immunocytochemical co-localization of the proteasome

in ubiquitinated structures in neurodegenerative diseases and the elderly. J. Neuropathol. Exp. Neurol. 56, 125–131, 1997

17. Imai Y, Soda M, Inoue H, Hattori N, Mizuno Y, Takahashi R: An unfolded putative

transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of Parkin. Cell 105, 891–902, 2001

18. Imai Y, Soda M, Takahashi R: Parkin suppresses unfolded protein stress-induced cell

death through its E3 ubiquitin-protein ligase activity. J. Biol. Chem. 275, 35661–35664, 2000

72

19. Irizarry MC, Growdon W, Gomez-Isla T, Newell K, George JM, Clayton DF, Hyman B. T: Nigral and cortical Lewy bodies and dystrophic nigral neurites in Parkinson’s disease and cortical Lewy body disease contain alpha-synuclein immunoreactivity. J. Neuropathol. Exp. Neurol. 57, 334–337, 1998

20. Ito T, Niwa J, Hishikawa N, Ishigaki S, Doyu M, Sobue G: Dorfin localizes to Lewy

bodies and ubiquitylates synphilin-1. J Biol Chem 278:29106-29114, 2003 21. Iwatsubo T, Yamaguchi H, Fujimuro M, Yokosawa H, Ihara Y, Trojanowski JQ, Lee

VM: Purification and characterization of Lewy bodies from the brains of patients with diffuse Lewy body disease. Am. J. Pathol. 148, 1517–1529, 1996

22. Kawamata H, McLean P. J, Sharma N, Hyman BT: Interaction of alpha-synuclein and

synphilin-1: effect of Parkinson’s disease-associated mutations. J. Neurochem. 77, 929–934, 2001

23. Kopito RR (2000) Aggresomes, inclusion bodies and protein aggregation. Trends Cell

Biol 10:524-530. 24. Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, Minoshima S, Yokochi M,

Mizuno Y, Shimizu N: Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392, 605–608, 1998

25. Lam YA, Pickart CM, Alban A, Landon M, Jamieson C, Ramage R, Mayer RJ, Layfield

R: Inhibition of the ubiquitinproteasome system in Alzheimer’s disease. Proceedings of the National Academy Science of the USA 97, 9902–9906, 2000

26. Leroy E, Boyer R, Auburger G, Leube B, Ulm G, Mezey E, Harta G, Brownstein MJ,

Jonnalagada S, Chernova T, Dehejia A, Lavedan C, Gasser T, Steinbach PJ., Wilkinson KD, Polymeropoulos MH: The ubiquitin pathway in Parkinson’s disease. Nature 395, 451–452, 1998

73

27. Liani E, Eyal A, Shemer R, Berg D, Franck T, Riess O, Ross CA, Rott R, Engelender S: Ubiquitylation of synphilin-1 and alpha-synuclein by SIAH proteins and their presence in Lewy bodies imply a role in Parkinson’s disease. Proc Nat Acad Sci USA 101:5500–5505, 2004

28. Lim KL, Dawson VL, Dawson TM: Parkin-mediated lysine 63-linked

polyubiquitination: a link to protein inclusions formation in Parkinson's and other conformational diseases? Neurobiol Aging 27:524-529, 2006

29. Lowe J, McDermott H, Landon M, Mayer RJ, Wilkinson KD: Ubiquitin carboxyl-

terminal hydrolase (PGP 9.5) is selectively present in ubiquitinated inclusion bodies characteristic of human neurodegenerative diseases. J. Pathol. 161, 153–160, 1990

30. McNaught KS and Jenner P: Proteasomal function is impaired in substantia nigra in

Parkinson’s disease. Neurosci. Lett. 297, 191–194, 2001 31. McNaught KS, Olanow CW, Halliwell B, Isacson O, Jenner P: Failure of the ubiquitin-

proteasome system in Parkinson’s disease. Nat. Rev. Neurosci. 2, 589–594, 2001 32. Mouradian MM: Recent advances in the genetics and pathogenesis of Parkinson disease.

Neurology 58, 179–185, 2002

33. Nagano Y, Yamashita H, Takahashi T, Kishida S, Nakamura T, Iseki E, Hattori N, Mizuno Y, Kikuchi A, Matsumoto M: Siah-1 facilitates ubiquitination and degradation of synphilin-1. J Biol Chem 278:51504-51514, 2003

34. O’Farrell C, Murphy DD, Petrucelli L, Singleton AB, Hussey J, Farrer M, Hardy J, Dickson DW, Cookson MR: Transfected synphilin-1 forms cytoplasmic inclusions in HEK293 cells. Brain Res. Mol Brain Res. 97, 94–102, 2001

35. Pagano M (1997) Cell cycle regulation by the ubiquitin pathway. Faseb J 11:1067-1075.

74

36. Pickart, C.M., 2001. Mechanisms underlying ubiquitination. Annual Review of Biochemistry 70, 503–533.

37. Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B, Root H,

Rubenstein J, Boyer R, Stenroos ES, Chandrasekharappa S, Athanassiadou A, Papapetropoulos T, Johnson WG, Lazzarini AM, Duvoisin RC, Di Iorio G, Golbe LI, Nussbaum RL: Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 276, 2045–2047, 1997

38. Putilina T, Jaworski C, Gentleman S, McDonald B, Kadiri M, Wong P: Analysis of a

human cDNA containing a tissue-specific alternatively spliced LIM domain. Biochem Biophys Res Commun 252:433-439, 1998

39. Rideout HJ, Larsen KE, Sulzer D, Stefanis L: Proteasomal inhibition leads to formation

of ubiquitin/alpha-synuclein-immunoreactive inclusions in PC12 cells. J. Neurochem. 78, 899–908, 2001

40. Schubert U, Anton LC, Gibbs J, Norbury CC, Yewdell JW, Bennink JR (2000) Rapid

degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 404:770-774.

41. Shimura H, Hattori N, Kubo S, Yoshikawa M, Kitada T, Matsumine H, Asakawa S,

Minoshima S, Yamamura Y, Shimizu N, Mizuno Y: Immunohistochemical and subcellular localization of Parkin protein: absence of protein in autosomal recessive juvenile parkinsonism patients. Ann. Neurol. 45, 668–672, 1999

42. Shimura H, Hattori N, Kubo S, Mizuno Y, Asakawa S, Minoshima S, Shimizu N, Iwai K,

Chiba T, Tanaka K, Suzuki T: Familial Parkinson disease gene product, parkin, is a ubiquitinprotein ligase. Nat. Genet. 25, 302–305, 2000

43. Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M: Alpha-synuclein in

filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with lewy bodies. Proc. Natl Acad. Sci. USA 95, 6469–6473, 1998

75

44. van Leuven, F.W., Burbach, J.P., Hol, E.M., 1998. Mutations in RNA: a first example of molecular misreading in Alzheimer’s disease. Trends in Neuroscience 21, 331–335.

45. Voges, D., Zwickl, P., Baumeister, W., 1999. The 26S proteasome: a molecular machine

designed for controlled proteolysis. Annual Review of Biochemistry 68, 1015–1068. 46. Wakabayashi K, Hayashi S, Kakita A, Yamada M, Toyoshima Y, Yoshimoto M,

Takahashi H: Accumulation of alphasynuclein/NACP is a cytopathological feature common to Lewy body disease and multiple system atrophy. Acta Neuropathol. (Berl) 96, 445–452, 1998

47. Wakabayashi K, Engelender S, Yoshimoto M, Tsuji S, Ross CA, Takahashi H:

Synphilin-1 is present in Lewy bodies in Parkinson’s disease. Ann. Neurol. 47, 521–523, 2000

48. Zhang Y, Gao J, Chung KK, Huang H, Dawson VL, Dawson TM: Parkin functions as an

E2-dependent ubiquitin-protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1. Proc. Natl Acad. Sci. USA 97, 13354–13359, 2000

76

I. INTRODUCTION

The accumulation of pathogenic proteins in inclusions is characteristic of several

neurodegenerative disorders (Ince et al., 1998; Mckeith et al., 2004; Wilson et al., 2004;

Recchia et al., 2004). Although the molecular mechanisms that lead to the formation of these

inclusions are not completely understood, elucidating their constituents can provide clues

about the pathogenesis of the disease and about the genesis of the inclusions. For example,

α-synuclein, which is an abundant constituent of Lewy bodies (Baba et al., 1998; Spillantini

et al., 1998) appears to have an important role in the pathogenesis of Parkinson’s disease

(PD) and other α-synucleinopathies. In addition, synphilin-1, which interacts with α-

synuclein and induces the formation of cytoplasmic inclusion in cultured cells, is another

component of Lewy bodies in the brains of patients with PD (Wakabayashi et al., 2000;

Engelender et al., 1999).

Casein kinase II (CKII) is a ubiquitous seryl/threonyl protein kinase which has a vital

cellular role in eukaryotic cells (Pinna, 1990; Litchfield, 2003). The holoenzyme is generally

composed of two catalytic (α and/or α’) and two regulatory (β) subunits. α-Synuclein has

several consensus sites for this kinase and is strongly phosphorylated by CKII, particularly at

serine 129 (Okochi et al, 2000). CamKII, on the other hand, has only a weak

phosphorylating activity on α-synuclein in vitro (Okochi et al, 2000). We previously

reported that CKII mediated phosphorylation of synphilin-1 regulates α-synuclein/synphilin-

1 interaction and thereby inclusion body formation (Lee et al., 2004). We found that both

CKII α and β subunits are present in cytoplasmic inclusions of cells transfected with these

77

two protein partners. Therefore, CKII-induced phosphorylation may have an important role

in the formation of inclusions in the context of α-synuclein and synphilin-1 interaction.

However, the pathological relevance of this kinase to human α-synucleinopathies are

unknown.

In the present investigation, we demonstrate that CKII β-subunits are present in Lewy

bodies co-localizing with α-synuclein in aged human brains.

78

II. MATERIALS AND METHODS

1. Case material

Brain tissue samples were obtained from aged persons from the Department of

Neurosurgery, Ajou University Hospital. Four patients (1 man and 3 women) who were

performed emergency decompressive frontal lobectomy due to acute traumatic brain

swelling and injury. These patients had no known previous history of neurodegenerative

disorders. The mean age was 68.75 years (range, 67-72 years).

2. Immunohistochemical analysis of human brain tissues

Immediate after decompressive frontal craniectomy, part of the brain block that was

removed to control increased intracranial pressure was fixed with 4% paraformaldehyde

(PFA) solution. After fixation in 4% PFA, brain tissues were immersed in 0.1 M phosphate

buffer containing 30% sucrose at 4°C, and then frozen and sectioned in the coronal plane at

30-µm on a sliding cryostat (Leica CM 3000). Brain sections were permeabilized with 0.2%

Triton X-100 in PBS for 30 min, and washed with PBS. Endogenous peroxidase was blocked

by incubating sections in 3% hydrogen peroxide solution for 5 min, and then rinsed in PBS.

After blocking non-specific binding with 0.5% BSA in PBS, brain sections were incubated

with primary antibodies for 16 h at 4°C. Antibodies to α-synuclein (1:1,000, Sigma), CKII α

subunits (1:80, Calbiochem), and CKII β subunit (1: 100, Calbiochem) were used as primary

antibodies. Brain sections were stained by the immunoperoxidase technique using Vectastain

ABC kit (Vector) with diaminobenzidine tetrahydrochloride as chromogen or by double-

79

immunofluorescent staining procedures with fluorescein isothiocyanate (FITC) and

rhodamine-conjugated secondary antibodies. Samples were visualized with a fluorescence

confocal microscope (Olympus).

III. RESULTS

1. CKII β subunits immunoreactivity in Lewy bodies

Immunohistochemical staining of cerebral cortices from aged human brains with Lewy

bodies showed localization of CKII β subunits in these inclusions (Fig. 1), but did not show

localization of CKII α subunits (data not shown). Nearly all Lewy bodies were strongly

positive for CKII β. To further confirm the localization of CKII β subunits in Lewy bodies,

double-staining immunohistochemistry was carried out with antibodies to α-synuclein and

CKII β. Most Lewy bodies in the cortex were immunoreactive for both α-synuclein and

CKII β subunits (Fig. 2). The signal for α-synuclein was stronger than that for CKII β. These

observations suggest that CKII subunits are components of Lewy bodies co-localizing with

α-synuclein.

80

Fig. 1. CKII β subunits are present in Lewy bodies of aged human brains. Cerebral cortical tissues from aged human brains with Lewy bodies were immunostained with

antibodies to CKII β subunits. Positively stained Lewy bodies with antibody are indicated by arrow. Bars=10 µM

α-synuclein CKII-β Merseα-synuclein CKII-β Merseα-synuclein CKII-β Merse

Fig. 2. CKII β subunit co-localizes with α-synuclein in aged human brain. The cerebral cortex of an aged human brain was stained for α-synuclein (rhodamine, red) and CKII β (fluorescein isothiocyannte, green), and analyzed under a confocal microscope. Bars=10 µM

81

IV. DISCUSSION

The present study shows that Lewy bodies in aged human brains are immunoreactive

for CKII β subunits, this result are similar to our previous data that CKII β subunits localize

in cytoplasmic inclusions induced by the co-expression of α-synuclein and synphilin-1 in

293 cells (Lee et al., 2004). Our present in vivo finding may be an important clue for

understanding the molecular mechanisms that induce the formation of Lewy body-like

inclusions.

The α subunit of CKII is catalytically active, whereas the β subunit is inactive.

Although the function of CKII β is still not entirely understood, this subunit has the

specificity of interaction with substrate (Litchfield, 2003). Therefore, CKII β has a great

chance to interact with its substrates than CKII α. This may explain that CKII β was detected

in Lewy bodies, but CKII α subunit was not. To exclude the possibility of antibody

specificity, we used another CKII α antibody (Santa Cruz), and obtained the same result

(data not shown). These observations were made in older individuals with cortical Lewy

bodies, but we suspect that all Lewy bodies likely have CKII β subunits since this kinase is

present in most brain neurons (Girault et al., 1990; Martin et al., 1990).

Phosphorylation events have been implicated in certain neurodegenerative diseases.

For example, the hyperphosphorylation of tau is associated with the pathogenesis of

Alzheimer’s disease (Buee et al., 2000) and phosphorylated α–synuclein at Ser129 is

deposited in Lewy bodies of Dementia with Lewy bodies (DLB) and alpha-synuclein

transgenic Drosophila (Fujiwara et al., 2002; Takahashi et al., 2003). Our observations

82

suggest that CKII mediated phosphorylation as well as CKII kinase itself may be related to

the formation of protein aggregates in human α–synucleinopathies. These findings may be

helpful to understand the process of LB formation in neurodegenerative disorders.

V. CONCLUSION

The present in vivo study extends and substantiates our previous experiments in ellular

models demonstrating that CKII subunits are present in and regulate the formation of α–

synuclein inclusions in transfected 293 cells. Most Lewy bodies in aged human brains are

strongly stained by CKII β, but not by CKII α. Our results suggest CKII mediated

phosphorylation and CKII kinase itself contribute to the formation of α–synuclein inclusions.

The cellular and in vivo experiments collectively suggest an important pathogenetic role of

CKII in the aggregation of α–synuclein and synphilin-1 and in the formation of Lewy bodies.

83

REFERENCES

1. Baba M, Nakajo S, Tu PH, Tomita T, Nakaya K, Lee VM, Trojanowski JQ, Iwatsubo T:

Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies. Am J Pathol 152:879-884, 1998

2. Buee L, Bussiere T, Buee-Scherrer V, Delacourte A, Hof PR: Tau protein isoforms,

phosphorylation and role in neurodegenerative disorders. Brain Res 33:95-130, 2000 3. Engelender S, Kaminsky Z, Guo X, Sharp AH, Amaravi RK, Kleiderlein JJ, Margolis

RL, Troncoso JC, Lanahan AA, Worley PF, Dawson VL, Dawson TM, Ross CA: Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions. Nat Genet 22:110-114, 1999

4. Fujiwara H, Hasegawa M, Dohmae N, Kawashima A, Masliah E, Goldberg MS, Shen J,

Takio K, Iwatsubo T: alpha-Synuclein is phosphorylated in synucleinopathy lesions. Nat Cell Biol 4:160-164, 2002

5. Girault JA, Hemmings HC Jr, Zorn SH, Gustafson EL, Greengard P: Characterization in

mammalian brain of a DARPP-32 serine kinase identical to casein kinase II. J Neurochem 55:1772-1783, 1990

6. Ince PG, Perry EK, Morris CM: Dementia with Lewy bodies. A distinct non-Alzheimer

dementia syndrome? Brain Pathol 8:299-324, 1998 7. Lee G, Tanaka M, Park K, Lee SS, Kim YM, Junn E et al: Casein kinase II-mediated

phosphorylation regulates alpha-synuclein/synphilin-1 interaction and inclusion body formation. J Biol Chem 279:6834-6839, 2004

8. Litchfield DW: Protein kinase CK2: structure, regulation and role in cellular decisions of

life and death. Biochem J 369:1-15, 2003

84

9. Martin ME, Alcazar A, Salinas M: Subcellular and regional distribution of casein kinase II and initiation factor 2 activities during rat brain development. Int J Dev Neurosci 8:47-54, 1990

10. McKeith I, Mintzer J, Aarsland D, Burn D, Chiu H, Cohen-Mansfield J, Dickson D,

Dubois B, Duda JE, Feldman H, Gauthier S, Halliday G, Lawlor B, Lippa C, Lopez OL, Carlos Machado J, O’Brien J, Playfer J, Reid W: Dementia with Lewy bodies. Lancet Neurol 3:19-28, 2004

11. Okochi M, Walter J, Koyama A, Nakajo S, Baba M, Iwatsubo T, Meijer L, Kahle PJ,

Haass C: Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein. J Biol Chem 275:390-397, 2000

12. Pinna LA: Casein kinase 2: an 'eminence grise' in cellular regulation? Biochim Biophys

Acta. 1054:267-284, 1990 13. Recchia A, Debetto P, Negro A, Guidolin D, Skaper SD, Giusti P: Alpha-synuclein and

Parkinson's disease. FASEB J 18:617-626, 2004 14. Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M: alpha-Synuclein in

filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with lewy bodies. Proc Natl Acad Sci U S A 95:6469-73, 1998

15. Takahashi M, Kanuka H, Fujiwara H, Koyama A, Hasegawa M, Miura M, Iwatsubo T:

Phosphorylation of alpha-synuclein characteristic of synucleinopathy lesions is recapitulated in alpha-synuclein transgenic Drosophila. Neurosci Lett 336:155-158, 2003

16. Wakabayashi K, Engelender S, Yoshimoto M, Tsuji S, Ross CA, Takahashi H:

Synphilin-1 is present in Lewy bodies in Parkinson's disease. Ann Neurol 47:521-523, 2000

85

17. Wilson CA, Murphy DD, Giasson BI, Zhang B, Trojanowski JQ, Lee VM: Degradativeorganelles containing mislocalized alpha-and beta-synuclein proliferate in presenilin-1 null neurons. J Cell Biol 165:335-346, 2004

86

- 국문요약 -

파킨슨씨병에서 형성된 세포내 응집체에 관한 연구

아주대학교 대학원 의학과

류 명 이

(지도교수: 윤 수 한)

세포 내 응집체인 α-synuclein 은 다양한 퇴행성 신경질환의 병리학적

표식자로 보고되어져 있으며, 결합 단백질로 잘 알려져 있는 synphilin-1 은

파킨슨씨병을 포함한 신경퇴행성 질환에서 관찰되는 세포 내 응집체의 중요한

구성성분이다. 그러나 이러한 질환에서 synphilin-1 단백질의 세포학적, 생화학적

메커니즘과 세포 내에서의 역할은 여전히 규명되어 있지 않다. 따라서 본

연구에서는 synphilin-1 의 기능을 알기 위해 synphilin-1 과 결합하는 유전자를

yeast two-hybrid screen 을 통하여 탐색한 결과, LMO7 이라는 새로운 단백질을

얻을 수 있었다. LMO7 은 핵과 세포질, 그리고 세포 표면과 세포연접 부위에서도

발견되는 단백질이며, 서로 다른 단백질과 단백질 사이의 결합 도매인인 PDZ 와

LIM 도매인을 가진 단백질로서, 연접한 세포들끼리의 결합에 관여하여 많은

생화학적 반응에 관여하는 것으로 알려져 있다. Yeast two-hybrid screening 과

mammalian cell 에서의 binding assay 를 통하여, synphilin-1 의 ankyrin-like

repeats 와 coiled-coil domain 이 LMO7 의 LIM domain 을 포함한 C-terminal 에

결합한다는 것을 알았고, HEK293 세포에 synphilin-1 과 LMO7 을 같이 과발현한

87

후 이중세포염색을 통하여 두 단백질이 형성된 응집체 내에 같이 존재함을 알 수

있었다. LMO7 단백질의 발현은 α-synuclein 과 synhilin-1 으로 형성된 응집체의

수의 증가를 유도하였으나, LMO7 단백질의 과발현이 세포에게 toxic 하게

작용하지는 않았다. 파킨슨씨병을 가진 환자의 중뇌조직에서도 synphilin-1 과

LMO7 이 같은 응집체 내에서 관찰되는 것을 확인함으로써, LMO7 은 synphilin-

1 과 결합하여 퇴행성 신경질환에 관여하는 중요한 단백질일 것이라 시사되어진다.

퇴행성 신경질환에 형성되는 α-synuclein 과 synphilin-1 을 비롯한

응집체의 구성성분의 분해는 유비퀴틴 (ubiquitin)을 통한 단백질 분해경로

(protein degradation pathway) 를 통해 이루어진다고 알려져 있다. 따라서

synphilin-1 과 결합하여 루이소체의 응집체에서 발견되는 LMO7 단백질 또한

유비퀴틴에 의해 단백질의 분해과정과 연관이 있을 것이며, proteasome inhibitor 의

처리 후 LMO7 단백질을 유비퀴틴으로 확인을 한 결과, LMO7 단백질도

proteasom 에 의해 분해되고 유비퀴틴화 (ubiquitination) 되는 것을 확인할 수

있었다. 이로써 LMO7 단백질은 신경퇴행성 신경질환에 형성되는 응집체의 새로운

물질이며, 이 단백질의 연구는 퇴행성 신경질환의 새로운 접근 방법을 제시하였다.

핵심어 : 파킨슨씨병 (Parkinson’s disease), 루이소체 (Lewy body), 응집체

(inclusion), 알파-시누클레인 (α-synuclein), synphilin-1, LIM domain only 7

(LMO7), ubiquitin, 유비퀴틴 (ubiquitin), 프로테아솜 (proteasome), 신경퇴행성

질환 (neurodegenerative disorder), α-synucleinopathies

88