Conditional transgenic mice expressing C-terminally truncated human α-synuclein (αSyn119) exhibit reduced striatal dopamine without loss of nigrostriatal pathway dopaminergic neurons
Conditional transgenic mice expressing C-terminally truncated human -synuclein (Syn119) exhibit reduced striatal dopamine without loss of nigrostriatal pathway dopaminergic neurons
Joo Paulo L Daher 0 1 2
Mingyao Ying 1 2
Rebecca Banerjee 6
Rebecca S McDonald 6
Myriam Dumas Hahn 0
Lichuan Yang 6
M Flint Beal 6
Bobby Thomas 6
Valina L Dawson 1 2 4 5
Ted M Dawson 1 2 5
Darren J Moore 1 2 3
0 Department of Pathology, School of Medicine, Fluminense Federal University , Niteroi , Brazil
1 Department of Neurology, Johns Hopkins University School of Medicine , Baltimore , USA
2 NeuroRegeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine , Baltimore , USA
3 Brain Mind Institute, School of Life Sciences, Ecole Polytechnique Federale de Lausanne , Lausanne , Switzerland
4 Department of Physiology, Johns Hopkins University School of Medicine , Baltimore , USA
5 Department of Neuroscience, Johns Hopkins University School of Medicine , Baltimore , USA
6 Department of Neurology and Neuroscience, Weill Medical College of Cornell University , New York , USA
Background: Missense mutations and multiplications of the -synuclein gene cause autosomal dominant familial Parkinson's disease (PD). -Synuclein protein is also a major component of Lewy bodies, the hallmark pathological inclusions of PD. Therefore, -synuclein plays an important role in the pathogenesis of familial and sporadic PD. To model -synuclein-linked disease in vivo, transgenic mouse models have been developed that express wild-type or mutant human synuclein from a variety of neuronal-selective heterologous promoter elements. These models exhibit a variety of behavioral and neuropathological features resembling some aspects of PD. However, an important deficiency of these models is the observed lack of robust or progressive nigrostriatal dopaminergic neuronal degeneration that is characteristic of PD. Results: We have developed conditional -synuclein transgenic mice that can express A53T, E46K or C-terminally truncated (1-119) human -synuclein pathological variants from the endogenous murine ROSA26 promoter in a Cre recombinase-dependent manner. Using these mice, we have evaluated the expression of these -synuclein variants on the integrity and viability of nigral dopaminergic neurons with age. Expression of A53T -synuclein or truncated Syn119 selectively in nigrostriatal pathway dopaminergic neurons for up to 12 months fails to precipitate dopaminergic neuronal loss in these mice. However, Syn119 expression in nigral dopaminergic neurons for up to 12 months causes a marked reduction in the levels of striatal dopamine and its metabolites together with other subtle neurochemical alterations.
Conclusion: We have developed and evaluated novel conditional -synuclein transgenic mice with
transgene expression directed selectively to nigrostriatal dopaminergic neurons as a potential new
mouse model of PD. Our data support the pathophysiological relevance of C-terminally truncated
-synuclein species in vivo. The expression of Syn119 in the mouse nigrostriatal dopaminergic
pathway may provide a useful model of striatal dopamine depletion and could potentially provide a
presymptomatic model of PD perhaps representative of the earliest derangements in dopaminergic
neuronal function observed prior to neuronal loss. These conditional -synuclein transgenic mice
provide novel tools for evaluating and dissecting the age-related effects of -synuclein pathological
variants on the function of the nigrostriatal dopaminergic pathway or other specific neuronal
Parkinson's disease (PD) is the most common
neurodegenerative movement disorder characterized by the
cardinal symptoms of muscular rigidity, resting tremor and
bradykinesia [1,2]. Underlying these motor deficits is the
progressive loss of dopaminergic neurons of the
substantia nigra pars compacta in addition to several other
neuronal populations, the corresponding reduction of striatal
dopamine levels, and the appearance of Lewy bodies and
Lewy neurites in surviving neurons of the brainstem [1-3].
Lewy bodies are round eosinophilic intracytoplasmic
proteinaceous inclusions composed of filamentous material,
a major component of which is the protein -synuclein
. Missense mutations (A30P, E46K and A53T) and
multiplications of the -synuclein gene cause rare autosomal
dominant familial forms of PD that often manifest disease
in some families with an extended clinical and
pathological spectrum resembling dementia with Lewy bodies
(DLB) [5-9]. Thus, -synuclein most likely plays a key role
in the pathogenesis of familial and sporadic PD in
addition to DLB.
It is not clear how mutations in -synuclein, or increased
levels of the wild-type -synuclein protein due to gene
multiplications, precipitate the demise of nigral
dopaminergic neurons in familial PD. How -synuclein
contributes to the pathogenesis of the more common sporadic
form of PD is also not known. It has been postulated that
post-translational modifications of normal -synuclein,
including oxidation, nitration, phosphorylation or
C-terminal truncation [10-13], could contribute to -synuclein
dysfunction and neuropathology in sporadic PD. The
appearance of some of these modifications correlates well
with neuronal loss and/or pathology in sporadic PD
brains [11-13]. Furthermore, the exact role of Lewy bodies
in dopaminergic neuronal degeneration in PD remains
enigmatic. Therefore, clarifying the mechanisms
underlying -synuclein-induced dopaminergic
neurodegeneration in vivo is critical to understanding the pathogenesis of
familial and sporadic PD.
It is now clear from studies of transgenic mouse models
that -synuclein pathogenic mutations induce neuronal
dysfunction and degeneration through a toxic
gain-offunction mechanism [14-16]. A number of transgenic
mice have been created that express wild-type or mutant
human -synuclein in neurons from an array of different
heterologous promoter elements. These include the
broadly expressing mouse prion protein, mouse Thy-1
and human platelet-derived growth factor- promoters
[17-23], or the catecholaminergic-specific rat tyrosine
hydroxylase (TH) promoter [24-27]. A wide range of
synuclein-related neurological phenotypes have been
described in these mouse models that resemble some
features of PD, such as behavioral motor deficits, neuronal
loss, reduced striatal dopamine levels, and the formation
of filamentous and non-filamentous -synuclein-positive
inclusions or aggregates. Many of these phenotypes,
however, are not usually observed in the same mouse model.
The most severe phenotypes tend to be observed with
expression of the A53T variant whereas the wild-type
protein is only ever rarely toxic to neurons [15,17,19].
Despite the plethora of -synuclein mouse models that
have been created, only very few of these exhibit a loss of
nigral dopaminergic neurons, and in general this
neuronal loss is not progressive or robust [26,28]. Therefore,
synuclein mouse models do not currently exist that
reliably recapitulate essential features of PD especially the loss
of nigral dopaminergic neurons, the neuropathological
hallmark of the disease. Thus, there is a requirement for
the development of new -synuclein transgenic mouse
models that can faithfully recapitulate the dysfunction of
the nigrostriatal dopaminergic pathway in PD. Such
models may arise through improvements in transgenic
technology to increase transgene expression levels and
dopaminergic neuronal specificity, as well as through the
identification of more toxic -synuclein pathogenic
variants. A combination of these approaches could prove to
be advantageous in developing faithful -synuclein
mouse models of PD.
Recent studies have highlighted the pathophysiological
significance of C-terminally truncated -synuclein
species. Most recently, C-terminal truncation variants,
including Syn119, Syn122 and Syn123, have been detected
in brain tissue from human PD cases and -synuclein
transgenic mice, and were found to be enriched in Lewy
bodies [13,29]. Moreover, in vitro studies demonstrate
that human -synuclein lacking the C-terminal 20 or 30
amino acids (Syn120 or Syn130) assemble more
readily into filaments resembling the -synuclein filaments
observed in Lewy bodies [13,29-31]. Disease-associated
mutations in -synuclein promote the accumulation of
these truncated filaments compared to the wild-type
protein, and C-terminally truncated species can enhance the
aggregation of wild-type -synuclein at low
substoichiometric ratios suggestive of a seeding capacity [13,29,31].
Therefore, C-terminal truncations of -synuclein could
potentially advance disease progression or propagation
through promoting the pathological aggregation and
accumulation of -synuclein. At this juncture, however,
the role of C-terminally truncated -synuclein species in
the pathogenesis of PD is poorly understood. To further
understand the pathophysiological significance of
C-terminally truncated -synuclein species, transgenic mice
have recently been developed that express wild-type
Syn120 or A53T mutant Syn130 truncation variants
from the rat TH promoter [25,26]. Collectively, these mice
exhibit filamentous and non-filamentous
-synucleinpositive aggregates, deficits in locomotion, reduced
striatal dopamine levels, and in the A53T-Syn130 model a
developmental loss of TH-positive nigral neurons and
striatal nerve terminals.
Prior to the publication of these -synuclein truncation
models, we set out to develop a collection of transgenic
mice capable of producing robust expression of human
synuclein variants selectively in neurons of the
nigrostriatal dopaminergic pathway. Encouraged by the promising
neurodegenerative phenotype exhibited by conditional
transgenic mice expressing a polyglutamine-expanded
huntingtin protein , we chose to create similar
conditional transgenic mice through gene targeting of a
CreloxP-based transgene cassette at the ROSA26 genomic
locus to express E46K or A53T mutant human
-synuclein, or the putative pathogenic C-terminal truncation,
Syn119, in a Cre recombinase-dependent manner. It is
worth noting that transgenic mouse models expressing
E46K -synuclein do not currently exist, and that previous
-synuclein truncation models rely upon artificial
truncations (Syn120, Syn130) [25,26] rather than
truncations associated with disease pathology (Syn119) .
Here, we combine conditional transgenic technology with
new pathological variants of -synuclein to evaluate the
effects of expressing these -synuclein variants directly in
nigrostriatal pathway dopaminergic neurons.
Development of Cre-loxP Conditional Transgenic Mice for
Human -Synuclein Variants
To develop new -synuclein mouse models of PD, our
strategy was to create conditional transgenic mice that
selectively express pathological human -synuclein
variants in dopaminergic neurons of the nigrostriatal
pathway. Therefore, conditional transgenic mice were created
that express human A53T or E46K -synuclein or
C-terminally truncated Syn119 variants from the endogenous
murine ROSA26 promoter in a Cre
recombinase-dependent manner; hereafter referred to as ROSA26-Syn mice
(Fig. 1A). In these models, a conditional cassette
containing an -synuclein transgene immediately preceded by a
loxP-flanked transcriptional termination sequence
(neotpA), has been targeted to the murine ROSA26 locus
through homologous recombination [33,34]. Hence,
expression of the -synuclein transgene is driven by the
ubiquitous, neuronal-selective murine ROSA26 promoter
in discrete neuronal populations following Cre-mediated
excision of the neo-tpA cassette (Fig. 1A). Following
successful germ line transmission from chimeric mice for
each line, the resulting heterozygous progeny
(ROSA26Syn+/-, where + denotes the transgene) were intercrossed
and analyzed by Southern blot to confirm correct
targeting of the transgene at the ROSA26 locus (Fig. 1B). In
general, heterozygous and homozygous ROSA26-Syn mice
are viable and fertile, exhibit normal survival, and
manifest no gross behavioral or phenotypic abnormalities.
To induce conditional -synuclein transgene expression, a
two step breeding strategy was developed with either
THCre or nestin-Cre transgenic mice to derive homozygous
ROSA26-Syn+/+ mice with or without Cre expression
(Fig. 1C). TH-Cre mice display Cre expression and activity
restricted to central catecholaminergic neuronal
populations within the substantia nigra pars compacta, ventral
tegmental area, locus ceruleus, olfactory bulb,
hypothalamic nuclei, and superior cervical ganglia .
NestinCre mice show widespread expression of Cre activity in
neuronal and glial cells throughout the brain .
Homozygous ROSA26-Syn+/+ mice were first crossed
with heterozygous ROSA26-Syn+/-/TH-Cre or
ROSA26Syn+/-/nestin-Cre mice in order to eventually produce
homozygous ROSA26-Syn+/+/TH-Cre or ROSA26-Syn+/
+/nestin-Cre mice and their non-floxed ROSA26-Syn+/+
littermate controls at an expected frequency of 25% per
genotype per litter (Fig. 1C). We notice however that
passing the Cre transgene through the germline in this final
breeding step results in germline excision of the neo-tpA
cassette for one of the transgenic alleles in 100% of the
progeny from crosses with nestin-Cre and ~50% of the
progeny from crosses with TH-Cre i.e. ROSA26-Syn+/flox/
Cre (Fig. 1C, D). This produces homozygous mice with
conditional transgene expression from one allele but
widespread, unrestricted expression from the opposite
allele. This was unexpected since expression of Cre
transgenes in these mice is considered to be largely restricted to
CNS tissues but they also appear to exhibit some activity
in the germline in our mice. Thus, we were restricted to
producing cohorts of heterozygous
+/fl +/- +/- +/fl+/++/fl +/++/-
Tg: -/- +/- +/+
FGiegnuerreat1ion of Cre-loxP Conditional -Synuclein Transgenic Mice
Generation of Cre-loxP Conditional -Synuclein Transgenic Mice. (A) Gene targeting strategy for generating
conditional ROSA26-Syn transgenic mice. Human -synuclein cDNAs were placed downstream of a loxP-flanked cassette
containing a neomycin resistance gene (neo) and a triple transcriptional termination sequence (tpA). The entire neo-tpA-Syn
conditional transgene cassette is situated adjacent to the endogenous ROSA26 promoter. Following Cre-mediated excision of
the neo-tpA cassette, the -synuclein transgene is expressed from the adjacent ROSA26 promoter. ROSA26-Syn mice were
genotyped using the indicated PCR primers P1, P2 and P3. Primers P1/P3 produce a wild-type band of ~500 bp whereas
primers P1/P2 produce a transgenic band of ~250 bp. Following Cre-mediated excision, primers P1/P3 can also amplify a ~1.5 kb
band from the recombined transgenic allele. SA, splice acceptor; pA, polyadenylation sequence. (B) Southern blot analysis of
EcoRV-digested tail genomic DNA derived from either wild-type (-/-), heterozygous (+/-) or homozygous (+/+)
ROSA26Syn119 mice. Blots were hybridized with a [32P]-labeled DNA probe as indicated in (A). The probe detects the wild-type
allele at ~11 kb and the transgenic allele at ~3.8 kb owing to an additional EcoRV site in the neo gene, as indicated. (C) Breeding
strategy to produce floxed ROSA26-Syn119+/+/TH-Cre mice and their non-floxed ROSA26-Syn+/+ littermates at a frequency
of 25% per genotype (italics). Notice that homozygous ROSA26-Syn119+/+/TH-Cre mice (bold) were often produced with a
germ line deletion (flox) of one allele (ROSA26-Syn119+/flox/TH-Cre) at a ratio of 1:1 (+/+ to +/flox) following crossing to
TH-Cre mice. (D) PCR genotyping strategy for ROSA26-Syn119/TH-Cre mice using primers P1, P2 and P3 to distinguish the
transgenic allele (Tg, ~250 bp, P1+P2), wild-type allele (WT, ~500 bp, P1+P3), or the transgenic allele following germline
deletion of the neo-tpA cassette (Tgflox, ~1.5 kb, P1+P3).
tin-Cre mice but could successfully produce homozygous
ROSA26-Syn+/+/TH-Cre mice following screening and
elimination of mice with germline excision of the neo-tpA
cassette (Fig. 1C, D). Collectively, we have created an
important resource of conditional transgenic mice
capable of expressing A53T, E46K or C-terminally truncated
human -synuclein from the endogenous murine
ROSA26 promoter in a Cre recombinase-dependent
Cre-Dependent Expression of Human A53T -Synuclein
We next sought to verify that -synuclein transgene
expression in these mice can be induced in a
Cre-dependent manner. Protein extracts were prepared from brain
tissue of 10 month-old floxed ROSA26-Syn/Cre mice and
their non-floxed ROSA26-Syn littermates followed by
Western blot analysis with human-specific -synuclein
antibodies. Human Syn119 expression is confirmed in
brain regions with TH-positive neurons or nerve terminals
including the olfactory bulb, striatum, cerebral cortex and
ventral midbrain of floxed homozygous
ROSA26Syn119+/+/TH-Cre mice that is completely absent from
non-floxed littermate mice (Fig. 2A). Similarly, the
expression of A53T human -synuclein is confirmed in brain
regions with nestin-positive neurons from floxed
heterozygous ROSA26-Syn-A53T+/-/nestin-Cre mice that is
absent from the brains of their non-floxed littermate mice
(Fig. 2A). Thus, both A53T -synuclein and Syn119
transgenes are expressed in a Cre-dependent manner in
these mice. The E46K -synuclein transgenic mice were
not characterized further in this study.
To compare the expression level of human Syn119 in the
ROSA26-Syn mice with endogenous -synuclein,
proteins extracted from various brain regions of 10
monthold ROSA26-Syn119/Cre mice and their non-floxed
littermates were analyzed by Western blotting with the
synuclein antibody, Syn-1. Syn119 is expressed at less
than 10% of the levels of endogenous -synuclein in these
brain regions (Fig. 2B). This is perhaps not surprising
since Syn119 is expressed selectively in most TH-positive
catecholaminergic neurons in these brain regions whereas
endogenous -synuclein is known to be expressed
broadly within many neuronal populations throughout
the brain . It is possible to observe a doubling of
Syn119 expression between heterozygous and
homozygous floxed ROSA26-Syn119/TH-Cre mice as
expected (Fig. 2B). We could also confirm a broader
distribution and higher levels of Syn119 in
ROSA26Syn119+/-/nestin-Cre mice compared to
ROSA26Syn119+/+/TH-Cre mice reflecting the widespread
distribution and greater number of nestin-positive neurons
compared to the more restricted TH-positive
catecholaminergic neuronal population in each brain region (Fig.
2B). A small proportion of truncated endogenous
-synuclein could also be detected in forebrain regions from
control ROSA26-Syn119+/+ mice that express the highest
levels of endogenous -synuclein (Fig. 2B), as noted
previously . It was not possible to directly compare the
expression level of human A53T -synuclein with
endogenous -synuclein in the ROSA26-Syn-A53T/Cre mice
since these proteins co-migrate and human -synuclein
expression does not appreciably influence the total level
of -synuclein detectable using the Syn-1 antibody (data
Finally, we compared the expression level of A53T
-synuclein in our ROSA26-Syn mice to a well-characterized
mouse prion promoter (mo.PrP)-based A53T human
synuclein neurodegenerative mouse model [19,38].
Despite the lack of obvious substantia nigra pathology in
this mo.PrP-A53T mouse model, it is possible to detect
more prominent -synuclein expression in the ventral
midbrain by Western blotting with the human-specific
LB509 antibody compared to the ROSA26-Syn-A53T
mice with transgene expression directed to either
TH-positive or nestin-positive neurons (Fig. 2C). This finding
most likely relates to the relatively small contribution of
TH-positive neurons to the total number of neurons
within the ventral midbrain region which is highlighted
by the observation that heterozygous ROSA26-Syn-A53T+/
-/nestin-Cre mice produced far greater transgene
expression than homozygous ROSA26-Syn-A53T+/+/TH-Cre
mice (Fig. 2C). Greater -synuclein expression observed
in the mo.PrP-A53T transgenic mice compared to the
ROSA26-Syn-A53T+/-/nestin-Cre mice most likely relates
to differences in the strength of the ROSA26 and mo.PrP
promoters and/or transgene copy number, both of which
are considered to be higher in the mo.PrP-A53T mice .
Due to the low level of transgene expression in the
ROSA26-Syn/Cre mice it was not possible to reliably
demonstrate the selective neuronal distribution pattern of
human -synuclein above background by
immunohistochemical methods with currently available
human-specific -synuclein antibodies or the Syn-1 antibody (data
not shown). It was also not possible to observe the
presence of -synuclein pathological inclusions or aggregates
in the substantia nigra of these mice up to 10 months of
age (data not shown). Collectively, this data demonstrates
that the ROSA26-Syn mice express human Syn119 or
human A53T -synuclein in a Cre-dependent manner but
at lower levels than those of endogenous, murine
-synuclein or A53T -synuclein expressed in mo.PrP transgenic
Lack of Nigral Dopaminergic Neuronal Degeneration in
Synuclein Transgenic Mice
To determine whether the expression of human Syn119
or A53T -synuclein in the ROSA26-Syn/Cre mice could
contribute to the degeneration of nigrostriatal
dopaminergic neurons with age, we generated suitably-sized
Syn-A53T+/- IB: LB509
FCirgeu-Dreep2endent Human -Synuclein Expression in ROSA26-Syn Mice
Cre-Dependent Human -Synuclein Expression in ROSA26-Syn Mice. (A) Cre-dependent expression of
-synuclein variants. Protein extracts (50 g) derived from distinct brain regions of 10 month-old ROSA26-Syn119+/+/TH-Cre mice
and their ROSA26-Syn119+/+ littermate controls were probed with the human-specific -synuclein antibody, Syn204, with
actin and TH antibodies as controls. HEK-293T cells expressing human Syn119 were used as a positive control. Equivalent
proteins derived from ROSA26-Syn-A53T+/-/nestin-Cre mice and their ROSA26-Syn-A53T+/- littermates were probed with
human-specific -synuclein antibody, LB509, and actin antibody. HEK-293T cells expressing human Syn-A53T were used as a
positive control. (B) Comparison of human Syn119 expression with endogenous -synuclein. Proteins (50 g) derived from
distinct brain regions of 10 month-old ROSA26-Syn119+/-/TH-Cre, ROSA26-Syn119+/+/TH-Cre,
ROSA26-Syn119+/-/nestin-Cre and their ROSA26-Syn119+/+ littermate controls were probed with -synuclein antibody, Syn-1, to detect
endogenous mouse (ms) -synuclein and human Syn119 expression. (C) Comparison of human A53T -synuclein expression in
ROSA26-Syn-A53T mice and mouse prion promoter (mo.PrP) A53T-Syn transgenic mice. Proteins extracted from ventral
midbrain tissue of adult ROSA26-Syn-A53T mice (50 g) and mo.PrP-A53T mice (5 g) were probed with LB509 antibody or
actin antibody as a loading control. HEK-293T cells expressing human Syn-A53T were used as a positive control.
cohorts of aged floxed and non-floxed mice. In particular,
we generated Syn119 or A53T -synuclein mice crossed
with TH-Cre (ROSA26-Syn+/+/TH-Cre) or nestin-Cre
(ROSA26-Syn+/-/nestin-Cre) transgenic mice and their
non-floxed age-matched littermates (ROSA26-Syn+/- or
ROSA26-Syn+/+ mice). It is anticipated that TH-Cre and
nestin-Cre mice would direct transgene expression in the
vast majority of nigral dopaminergic neurons in the
ROSA26-Syn/Cre mice [35,36]. Mice were aged to 1012
months and the numbers of TH-positive and
Nissl-positive neurons in the substantia nigra pars compacta were
counted using unbiased stereological methods (Fig. 3A).
At this age, we fail to observe a significant loss of nigral
dopaminergic neurons or Nissl-positive neurons due to
human Syn119 or A53T -synuclein expression (Fig.
3AE). In general, we do not observe alterations in the
survival of the ROSA26-Syn119/Cre mice up to 18
months of age. It is not possible to conclude whether or
not the Syn119 protein has pathophysiological
properties since comparable mice expressing pathogenic A53T
LFaigckuroef N3igral Dopaminergic Neuronal Degeneration in ROSA26-Syn Mice
Lack of Nigral Dopaminergic Neuronal Degeneration in ROSA26-Syn Mice. Stereological analysis of TH-positive
and Nissl-positive neurons in the substantia nigra pars compacta of male ROSA26-Syn mice with or without Cre expression.
(A) Example of immunohistochemical staining for TH with Nissl counterstain on coronal midbrain sections highlighting the
substantia nigra region from 12 month-old ROSA26-Syn119+/+/TH-Cre mice and their ROSA26-Syn119+/+ littermate controls.
(B-E) Stereological analysis revealing a normal number of TH-positive and Nissl-positive neurons in the substantia nigra pars
compacta (SNpc) of 1012 month-old ROSA26-Syn119+/-/nestin-Cre (B), ROSA26-Syn119+/+/TH-Cre (C),
ROSA26-SynA53T+/-/nestin-Cre (D) and ROSA26-Syn-A53T+/+/TH-Cre (E) mice compared to their age-matched non-floxed
ROSA26Syn littermates (Cre -). Data are expressed as the mean SEM (n = 46 mice per genotype). There are no significant
differences (p > 0.05) between genotypes following statistical analysis by unpaired, two-tailed Student's t test.
synuclein at identical levels in the same neurons also
failed to influence dopaminergic neuronal viability in
Reduced Striatal Dopamine Induced by Syn119
Expression in Catecholaminergic Neurons
To determine whether the expression of human Syn119
in nigral dopaminergic neurons leads to nigrostriatal
neuronal dysfunction, we monitored the levels of striatal
dopamine and its metabolites in aged ROSA26-Syn119/
Cre mice and their non-floxed littermate control mice by
HPLC. No differences in the levels of striatal dopamine or
its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC)
and homovanillic acid (HVA), are observed in
heterozygous ROSA26-Syn119+/-/nestin-Cre mice compared to
their non-floxed littermates at 1213 months of age (Fig.
4A, B). However, in homozygous ROSA26-Syn119+/+/
TH-Cre mice with an approximate doubling of Syn119
expression in TH-positive dopaminergic neurons, we
observe a significant marked reduction in the levels of
striatal dopamine (~32%), DOPAC (~46%) and HVA
(~33%) compared to their non-floxed
ROSA26Syn119+/+ littermates at 1011 months of age (Fig. 4C,
D). This effect on the nigrostriatal dopaminergic system is
specific since the levels of striatal 5-hydroxytryptamine
(5HT) in these ROSA26-Syn119+/+/TH-Cre mice are
normal (Fig. 4D). No further alterations in dopamine levels
in other regions of the brain are observed in these mice,
including the olfactory bulb, cerebral cortex and
prefrontal cortex (data not shown). Furthermore, striatal
dopamine turnover in the ROSA26-Syn119/Cre mice is
also normal (data not shown). Taken together, this data
demonstrates that the expression of human Syn119 in
TH-positive catecholaminergic neurons leads to a
reduction in striatal dopamine content in the absence of nigral
dopaminergic neuronal loss. This effect would appear to
be dependent on transgene dosage since the reduced
expression of Syn119 in heterozygous
ROSA26Syn119+/-/nestin-Cre mice fails to induce deficits in
striatal dopamine levels. Alternatively, this effect could
potentially relate to mosaic expression of Cre
recombinase in nigral dopaminergic neurons of TH-Cre and
nestin-Cre transgenic mice. Our data suggest that human
Syn119 may be of pathophysiological relevance in vivo.
Neurochemical Alterations in Syn119 Transgenic Mice
To further evaluate the potential pathophysiological
effects of Syn119 expression in the brain, the levels of
the neurotransmitters norepinephrine (NE) and 5-HT
were monitored in the olfactory bulb, cerebral cortex,
prefrontal cortex, striatum and brainstem. A significant
increase in the level of NE in the striatum (Fig. 5A) and
5HT in the prefrontal cortex (Fig. 5B) is observed in
homozygous ROSA26-Syn119+/+/TH-Cre mice
compared to their non-floxed littermates at 1011 months of
age. NE and 5-HT levels in these brain regions are normal
in heterozygous ROSA26-Syn119+/-/nestin-Cre mice at
1213 months of age suggesting that the alterations in
neurotransmitter levels are specific to the homozygous
ROSA26-Syn119+/+/TH-Cre mice (data not shown).
Thus, Syn119 expression in TH-positive
catecholaminergic neurons induces additional subtle neurochemical
alterations in vivo.
Here, we describe the development and evaluation of
conditional transgenic mice expressing human -synuclein
pathological variants in dopaminergic neurons of the
nigrostriatal pathway from the endogenous ROSA26
promoter. Our investigations have focused on the potential
contribution of two pathologic variants, A53T mutant
synuclein and C-terminally truncated Syn119, to the
normal integrity and function of nigral dopaminergic
neurons with age. Our studies demonstrate that A53T
synuclein and Syn119 are expressed in a Cre
recombinase-dependent manner in these ROSA26-Syn mice. We
show that neither A53T -synuclein nor Syn119
expression in mice aged for up to 12 months is sufficient to
induce the degeneration of nigral dopaminergic neurons.
However, we demonstrate that the expression of Syn119
in nigral dopaminergic neurons for up to 12 months in
this mouse model causes a marked reduction in the levels
of striatal dopamine and its metabolites in addition to
other subtle neurochemical alterations. Thus, at ages up to
12 months we observe nigrostriatal dopaminergic
pathway dysfunction in the absence of frank neuronal loss.
Further deficits in nigrostriatal dopaminergic function
may be revealed in these mice with advanced age. The
ROSA26-Syn mice therefore represent a useful model of
striatal dopamine depletion and could provide a potential
presymptomatic model of PD perhaps representative of
the earliest derangements in dopaminergic neuronal
function observed prior to neuronal loss.
Our data suggest that the C-terminally truncated
-synuclein variant, Syn119, is of pathophysiological relevance
in vivo. The expression of Syn119 in nigral TH-positive
dopaminergic neurons precipitates striatal dopamine loss
without compromising dopaminergic neuronal viability.
Syn119, together with Syn122 and Syn123, are
normally present in mammalian brain at low levels but are
enriched as soluble and aggregated species in Lewy
bodypositive PD and DLB brains and in transgenic mouse
brains expressing human A53T -synuclein [13,29].
Furthermore, disease-associated mutations in -synuclein
promote the accumulation of Syn119 and other
truncation species compared to the wild-type protein in neural
cell lines and -synuclein transgenic mice [13,29]. Further
supporting the pathogenicity of truncated Syn119 and
related species, in vitro studies demonstrate that
C-terminally truncated -synuclein variants fibrillize and
aggregate more readily than the full-length protein and can
FiSgyunr1e194Expression in Catecholaminergic Neurons Leads to a Reduction of Striatal Dopamine and its Metabolites
Syn119 Expression in Catecholaminergic Neurons Leads to a Reduction of Striatal Dopamine and its
Metabolites. Analysis of striatal biogenic amines in ROSA26-Syn119 mice by HPLC with electrochemical detection. The level of
dopamine (A, C), or the dopamine metabolites, DOPAC and HVA, and 5-HT (B, D) were measured by HPLC in striatal tissue
from (A, B) 1213 month-old female ROSA26-Syn119+/-/nestin-Cre mice (Cre +) and their ROSA26-Syn119+/- littermate
controls (Cre -) and from (C, D) 1011 month-old female ROSA26-Syn119+/+/TH-Cre mice (Cre +) and their
ROSA26Syn119+/+ littermate controls (Cre -). The concentration of each biogenic amine is expressed as ng per mg of protein, and
data represent the mean SEM (n = 79 mice for nestin-Cre and n = 56 mice for TH-Cre). Significant differences between
Cre- and Cre + genotypes were determined following statistical analysis by unpaired, two-tailed Student's t test (*p < 0.05, **p
seed the aggregation of full-length -synuclein
[13,2931]. The mechanism through which -synuclein
C-terminal truncations arise is not known. -Synuclein can be
proteolytically processed at its C-terminus via a number of
proteases in vitro, including cathepsin D and calpain I, but
as yet none of these proteases have been shown to
specifically generate the Syn119 variant [39-42]. However,
Syn119 expression has been demonstrated in vivo and
this variant accumulates in diseased brain tissue [13,29].
The normal and pathophysiological mechanism
responsible for the generation of Syn119 and similar truncation
variants in vivo remains to be clarified.
iFNniegCuuarrtoecch5heomlaimcailnAerltgeicraNtioeunrsoCnasused by Syn119 Expression
Neurochemical Alterations Caused by Syn119
Expression in Catecholaminergic Neurons. Analysis of
biogenic amines in discrete brain regions of
ROSA26Syn119 mice by HPLC with electrochemical detection. The
level of NE (A) and 5-HT (B) were measured by HPLC in
olfactory bulb, cerebral cortex, prefrontal cortex, striatum
and brainstem tissues of 1011 month-old female
ROSA26Syn119+/+/TH-Cre mice (Cre +) and their
ROSA26Syn119+/+ littermate controls (Cre -). The concentration of
NE and 5-HT is expressed as ng per mg of tissue, and data
represent the mean SEM (n = 57 mice per genotype).
Significant differences between Cre - and Cre + genotypes were
determined following statistical analysis by unpaired,
twotailed Student's t test (*p < 0.05).
Our data in the ROSA26-Syn mice provides the first in
vivo demonstration of a pathophysiological effect of the
disease-associated Syn119 species on the nigrostriatal
dopaminergic pathway. It is not yet clear how the
expression of Syn119 in dopaminergic neurons produces
deficits in striatal dopamine levels in the absence of frank
neuronal loss. Syn119 could potentially induce the
dysfunction or degeneration of striatal dopaminergic nerve
terminals without compromising the integrity of the
neuronal soma. At the cellular level, Syn119 could impair
pre-synaptic dopaminergic nerve terminals through the
accumulation of submicroscopic toxic oligomers or
aggregates [13,29], or could potentially sensitize nerve
terminals to secondary insults such as oxidative and nitrosative
stress that accumulate with age . Future analyses of
these mice will aim to clarify the mechanism through
which Syn119 could potentially act at nigrostriatal
dopaminergic nerve terminals. The effects of Syn119
expression in vivo are reminiscent to those of Syn120
expressed in transgenic mice from the rat TH promoter on
an -synuclein null background . Syn120 transgenic
mice exhibit a reduction in striatal dopamine and HVA
levels that are first evident between 13 months but do
not worsen up to 12 months of age . Accompanying
these alterations are late-onset motoric deficits and the
formation of pathological -synuclein inclusions. A
caveat of this mouse model is that the Syn120 truncated
species has not yet been observed in vivo in brain tissue
and may therefore represent a non-physiological
truncated -synuclein species . Furthermore, the Syn120
mice were generated on an -synuclein null background
which complicates the interpretation of their phenotype.
Nevertheless, the ROSA26-Syn119 mice and the
Syn120 transgenic mice suggest that C-terminal
truncation of -synuclein could represent a pathophysiological
process during the development and/or propagation of
PD that potentially contributes to the dysfunction of the
nigrostriatal dopaminergic pathway.
The ROSA26-Syn mice offer an important resource for
modeling various aspects of PD. These mice allow the
expression of disease-associated -synuclein variants
selectively in nigrostriatal pathway dopaminergic neurons
in order to evaluate their potentially pathogenic affects
with age. Transgene expression in these mice is driven by
the well-characterized neuronal-specific, endogenous
murine ROSA26 promoter in a Cre-dependent manner
[33,34,44]. This system permits the expression of
-synuclein variants in virtually any neuronal population in the
brain depending upon the availability of suitable Cre
driver lines. Conceivably, these ROSA26-Syn mice could
also be used to model other -synucleinopathies, such as
DLB, by directing transgene expression to neurons of the
forebrain. A particularly attractive feature of these
ROSA26-Syn mice is that one can directly compare the
effects of different pathological variants of the -synuclein
protein since variants are expressed at equivalent levels
from the same stable promoter at identical copy number
in the same neurons. This provides a clear advance over
conventional transgenesis where individual founder lines
can often produce marked differences in transgene
expression levels and expression patterns due to the effects of
genomic integration site, epigenetic modifications and
transgene copy number. Of note, there are no known
phenotypes associated with disruption of the ROSA26
genomic locus [33,44]. However, an obvious caveat of the
ROSA26-Syn mice is reduced transgene expression since
only one to two transgene copies can be accommodated at
the ROSA26 locus. Using the ROSA26-Syn mice it is
possible to co-express two pathological -synuclein variants
from opposite alleles at equivalent levels in the same
neurons to investigate how they may interact to precipitate
pathology or compromise neuronal viability. This feature
allows one to examine, for example, whether Syn119
expression can seed and accelerate the aggregation of the
full-length -synuclein variants, A53T or E46K, in
dopaminergic neurons in vivo. Another useful feature of
the ROSA26-Syn mice is that they are ideally suited for
investigating potential gene-environment interactions
with dopamine neuron-specific toxins such as MPTP 
since -synuclein expression can be directed selectively to
nigrostriatal pathway dopaminergic neurons using the
TH-Cre driver line. In this scenario, one can directly
compare the potential differential interaction of dopaminergic
toxins with various pathogenic -synuclein variants.
Importantly, we have made these ROSA26-Syn mice
available as a unique resource to the PD research
community by distribution through The Jackson Laboratory. The
ROSA26-Syn mice provide a useful and unique tool for
evaluating the age-related effects of expressing
-synuclein pathogenic variants in neurons in vivo that will
hopefully provide important insight into the molecular
mechanisms underlying the pathogenesis of PD and DLB.
Materials and methods
Generation and Breeding of Conditional -Synuclein
Human -synuclein variants (E46K, A53T or 1119) were
targeted to the ROSA26 genomic locus in 129/SvJ
embryonic stem (ES) cells as described . Briefly, -synuclein
cDNAs were subcloned into XhoI and NotI sites of
acceptor plasmid pBigT thereby placing the transgene
downstream of a loxP-flanked neo-tpA transcriptional
termination cassette . The entire neo-tpA--synuclein
conditional transgene was excised and inserted into
targeting plasmid pROSA26PA via PacI and AscI sites .
The final pROSA26PA-Syn construct was linearized with
KpnI, gel purified with GELase reagent (Epicenter,
Madison, WI) and used for electroporation of mouse 129/SvJ
ES cells. Following G418 selection, at least six correctly
targeted ES cell clones were identified by PCR and
Southern blot analysis as described [33,34], and two clones
were microinjected into C57BL/6J mouse blastocysts to
derive chimeric mice. Four to six chimeras were bred with
C57BL/6J mice for each targeting construct with at least
one chimeric mouse producing germline transmission of
the correctly targeted allele to F1 progeny. ES cell
selection, blastocyst microinjection and chimera breeding was
conducted by the University of Cincinnati Gene-Targeted
Mouse Service. F1 heterozygous mice were routinely
identified by PCR of tail genomic DNA with primers: P1,
5'aaagtcgctctgagttgttat-3'; P2, 5'-gcgaagagtttgtcctcaacc-3'; P3,
5'-ggagcgggagaaatggatatg-3'; producing a ~500 bp product
from the WT allele and a ~250 bp product from the
transgenic allele [33,34]. Following intercrossing of F1
heterozygous mice, mice were further analyzed by Southern blot
to verify correct targeting of the transgene at the ROSA26
ROSA26-Syn mice were maintained as 129/SvJ and
C57BL/6J hybrids through backcrossing to C57BL/6J mice
for 12 generations and then intercrossing of
heterozygous progeny. TH-Cre  and nestin-Cre 
transgenic mice were maintained on a C57BL/6J background.
Nestin-Cre transgenic mice (stock number 003771) were
obtained from The Jackson Laboratory (Bar Harbor, ME).
To generate cohorts of mice for aging, heterozygous
ROSA26-Syn+/- mice were crossed with each hemizygous
Cre line, and the F1 ROSA26-Syn+/-/Cre progeny were
further crossed with homozygous ROSA26-Syn+/+ mice
to produce F2 mice. The F2 mice produced from these
crosses on a mixed 129/SvJ and C57BL/6J hybrid
background were used throughout this study. Age-matched
ROSA26-Syn+/- or, +/+ littermates without Cre were used as
controls. Cre transgenes were genotyped by an established
PCR protocol with the primers
5'-GGAAGGTGTCCAATTTACTGACCGTA-3' to produce a 300 bp transgenic fragment
. Adult hemizygous PrP-A53T-Syn transgenic mice
 were kindly provided by Dr. Michael K. Lee (Johns
Hopkins University). All mice were housed and treated in
strict accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals. Mice were
maintained in a pathogen-free facility and exposed to a 12
h light/dark cycle with food and water provided ad libitum.
The ROSA26-Syn mice are available from The Jackson
Laboratory (Bar Harbor, ME; http://www.jax.org); JAX
stock number, 008883 (ROAS26-Syn-A53T), 008886
(ROSA26-Syn-E46K) and 008889 (ROSA26-Syn119).
Western blot analysis
Brain regions, including the olfactory bulb, cerebral
cortex, striatum, ventral midbrain, brainstem and
cerebellum, from 10 month-old mice were dissected from fresh
brains, homogenized in ice-cold RIPA buffer (50 mM
TrisHCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton
X100, 0.1% SDS, 1 Complete Mini protease inhibitor
cocktail [Roche]), and then centrifuged at 20,000 g for
15 min at 4C. The RIPA-soluble supernatant fraction was
collected, quantified using BCA reagent (Pierce, Rockford,
IL) with BSA standards, and stored at -80C. Proteins
(100 g) were resolved by electrophoresis on 15%
SDSPAGE gels and transferred to nitrocellulose membranes
(0.2 m; Biorad). To detect -synuclein expression, blots
were probed with human-specific mouse monoclonal
antibodies, Syn204 (Cell Signaling Technology) and
LB509 (Zymed), or with the mouse monoclonal Syn-1
antibody (BD Bioscience). Antibodies to tyrosine
hydroxylase (Novus Biologicals) and actin (Sigma) were used to
control for regional dissection and protein loading,
respectively. Lysates derived from HEK-293T cells
transiently expressing untagged human -synuclein (A53T or
Syn119; pcDNA3.1 expression vector [Invitrogen] )
served as positive controls for -synuclein antibodies. To
compare A53T -synuclein expression in ventral midbrain
tissue from adult mo.PrP-A53T-Syn mice  and
ROSA26-Syn-A53T/Cre mice, proteins were prepared as
described above and analyzed by Western blotting with
Immunohistochemistry and Stereological Cell Counting
Perfusion-fixed coronal midbrain sections (40-m) were
cut on a sliding microtome (Microm, Kalamazoo, MI) and
collected free-floating in PBS. Sections were
permeabilized and blocked in PBS containing 0.4% Triton X-100
and 4% normal goat serum at room temperature for 30
min. Sections then were incubated in rabbit polyclonal
anti-TH antibody (1:1000; Novus Biologicals, Littleton,
CO) in PBS containing 0.2% Triton X-100 and 2% normal
goat serum overnight at 4C and washed with PBS
containing 0.2% Triton X-100 and 1% normal goat serum.
Next, sections were incubated for 1 hr in biotinylated goat
anti-rabbit antibody (1:1000; Jackson ImmunoResearch,
West Grove, PA) in PBS with 0.2% Triton X-100 and 1.5%
goat serum, washed, and visualized by incubation in
biotin-streptavidin-HRP complex (ABC; Vector
Laboratories, Burlingame, CA), followed by incubation with
3,3'diaminobenzidine per the manufacturer's instructions
(Sigma). Sections were mounted on glass slides and
allowed to air dry overnight before being counterstained
with Nissl, dehydrated with ethanol, and cover-slipped
for visualization. To evaluate dopaminergic neuronal loss,
unbiased stereological methodology was employed as
described previously [46,47] to count TH-positive and
Nissl-positive neurons in the left and right pars compacta
region of every fourth section throughout the entire
midbrain region. This method was carried out by using a
computer-assisted image analysis system, consisting of an
Axiophot 2 photomicroscope (Carl Zeiss Vision,
Hallbergmoos, Germany) equipped with a
computer-controlled motorized stage (Ludl Electronics, Hawthorne, NY), a
Hitachi HV C20 video camera, and interfaced with a
Stereo Investigator system (MicroBrightField, Williston,
VT, USA) with optical fractionator probe.
Measurement of Biogenic Amines by HPLC
HPLC with electrochemical detection was used to
measure the concentration of the biogenic amines, dopamine,
3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic
acid (HVA), 5-hydroxytryptamine (5-HT),
5-hydroxyindoleacetic acid (5-HIAA) and norepinephrine (NE) in
discrete brain regions, as previously described . Female
ROSA26-Syn/Cre mice and age-matched ROSA26-Syn
littermate controls were sacrificed by decapitation, brains
were quickly removed and frozen on dry ice, and stored at
-80C. Striata, prefrontal cortex, cerebral cortex, olfactory
bulb and brainstem were dissected from frozen tissue and
samples were weighed and sonicated in 0.2 ml of 0.1 M
perchloric acid (at 100 l/mg tissue) containing 0.01%
EDTA and 25 g/ml 3,4-dihydroxybenzylamine (DHBA,
Sigma) as an internal standard. Following centrifugation
at 15,000 g for 10 min at 4C, 20 l of supernatant was
injected onto a C-18 80 4.6 mm column (ESA, Inc
Chelmsford, MA). The mobile phase consisted of 0.1 M
LiH2PO4, 0.85 mM 1-octanesulfonic acid and 10% (v/v)
methanol. The flow rate was kept at 1 ml/min. Biogenic
amines and their metabolites were detected by a
2-channel Coulochem II electrochemical detector (ESA, Inc.
Chelmsford, MA) with the working electrode kept at 0.7
V. Data were collected and processed using external
standards for respective amines on a EZChrome Elite Client
Workstation (ESA, Inc. Chelmsford, MA). Concentrations
of biogenic amines are expressed as ng per mg protein.
The protein concentrations of tissue homogenates were
measured using BCA reagent (Pierce, Rockford, IL).
Throughout the experiments investigators were blinded to
genotype of the mice. Data represent mean S.E.M. from
groups of animals for each genotype. Statistical analysis
for stereology and HPLC assessments between genotypes
were assessed by unpaired, two-tailed Students t test.
Differences were considered significant when p < 0.05. All
statistical analyses were performed using GraphPad
InStatversion 3 and Prism software (San Diego, CA).
DLB: dementia with Lewy bodies; DOPAC:
3,4-dihydroxyphenylacetic acid; 5-HT: 5-hydroxytryptamine; HVA:
homovanillic acid; NE: norepinephrine; neo: neomycin;
PD: Parkinson's disease; TH: tyrosine hydroxylase; tpA:
transcriptional termination sequence.
The authors declare that they have no competing interests.
TMD and DJM designed the research, JPD, MY, RB, RSM,
LY and BT conducted the experiments, TMD and DJM
contributed new research tools, MDH, BT, MFB, VLD,
TMD and DJM supervised the experiments and provided
reagent and infrastructure support, JPD, MY, BT, TMD and
DJM analyzed the data, DJM prepared the manuscript, BT,
MFB, VLD and TMD critically reviewed the manuscript.
All authors read and approved the final manuscript.
We are grateful to Dr. Michael K. Lee (Johns Hopkins University) for
helpful discussions on this work and for providing mo.PrP-A53T-Syn
transgenic mice. This work was supported by grants from the Michael J. Fox
Foundation for Parkinson's Research (DJM, TMD, MFB), Department of
Defense (MFB), Parkinson's Disease Foundation (MFB), and NIH, NINDS,
R21 NS057795 (DJM, TMD), RO1 NS060885 (BT) and P50 NS038377
(TMD). TMD is the Leonard and Madlyn Abramson Professor in
Neurodegenerative Diseases at Johns Hopkins University.
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