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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
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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).
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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
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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,
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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.
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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
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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
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- 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
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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
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