BACKGROUND
synucleinopathic diseases, like parkinson’s disease , dementia with lewy bodies  and multiple system atrophy , are all characterized by a pathologic aggregation of α-synuclein  protein in distinct brain regions .
increased expression of α-syn can be caused by a dominant heritable form of pd due to duplication or triplication of the α-syn gene
<cit> .
in order to model such synucleinopathies in vivo different mouse models were developed that overexpress human wild type α-syn
<cit> .
when expression is driven by the murine thy <dig> promoter, transgenic mice accumulate wild type hα-syn in cortical and subcortical regions including the nigrostriatal system
<cit>  whereas under the human pdgf-β promoter hα-syn accumulates preferentially in the neocortex and limbic system
<cit> .
abnormal accumulation of hα-syn in d-line transgenic mice is accompanied by alterations in mglur <dig> and autophagy similar to what has been observed in patients with dementia with lewy bodies
<cit> .
moreover the behavioral and neurodegenerative pathology in d-line mice can be reversed with mglur <dig> antagonists
<cit>  or by promoting the clearance of α-syn with rapamycin
<cit> , beclin 1
<cit>  and neurosin
<cit> .
given the behavioral phenotype and the predominant accumulation of α-syn in the neocortex and limbic system, these studies suggest that the pdgf-β hα-syn transgenic mouse model reproduces some aspects of synucleinopathies such as dlb.
recent studies have tested compounds developed to ameliorate pd-like pathology in the d-line mouse model and were able to show a reduction in the accumulation of α-syn, total and oxidized α-syn levels and behavioral deficits
<cit> .
taken together, these results indicate that this transgenic model might be useful for studies of α-syn target validation.
however, the progression of the behavioral deficits and pathology in d-line mice has not been fully characterized.
for this purpose we investigated the time course of behavioral deficits and neuropathology in d-line mice.
the occurrence of motor deficits was investigated using a challenging beam walk paradigm.
we also quantified hα-syn expression in the hippocampus and striatum using a biochemical approach.
moreover, by quantitative immunofluorescence at different ages we compared expression levels of both murine α-syn and hα-syn in the cortex, hippocampus, striatum and substantia nigra relative to non-transgenic littermates.
furthermore, we identified several cell populations that express transgenic hα-syn in different areas of the adult brain.
RESULTS
progressive motor deficits of d-line mice
motor coordination, in particular of the hind limbs, and balance of all mice was evaluated with the challenging beam walk test at  <dig>   <dig> and  <dig> months of age to evaluate progression of motor deficits in adult, mature and aging mice.
the time to cross the beam, the number of steps and slips, and the ratio of slips per step was recorded.
no significant changes in the number of slips were observed in  <dig> months old d-line and control animals .
<dig> months old transgenic and non-transgenic male mice made significantly more slips compared to the corresponding female mice .
transgenic female mice of the same age performed significantly worse compared to the corresponding female non-transgenic controls as analyzed by t-test.
the same trend was observed in male mice, but the results were not statistically significant .
the observed deficits faded at the age of  <dig> months and only the significant difference between female d-line and non-transgenic mice remained .
the significant differences shown in figure
1a-c were detected by analyzing data with t-test.
analyses of the presented data for the factor gender by two way anova followed by bonferroni’s post hoc test revealed a significant influence of the gender for transgenic as well as non transgenic mice .
analyses of data by two way anova followed by bonferroni’s post hoc test for the factor transgene and by one way anova did not reveal any significances.
analysis of the number of slips for progressive changes over age separated by sex showed only a significant improvement of non-transgenic female mice over age .
the time and number of steps required to traverse the beam and the ratio of slips per step did not differ significantly between d-line and non-transgenic littermates .
in order to verify that the observed motor differences between the d-line mice and non-transgenic littermates were not caused by modifications of spontaneous activity or deficits in spatial learning, the cylinder test and the two-choice swim test were performed, and no significant effects were detected .
general health of each mouse was determined using the irwin test—including the wire hanging test and vertical pole test, assessing physical characteristics and abnormalities, sensorimotor reflexes and motor abilities—but no significant differences were observed.
overall, all animals appeared healthy over the course of the study.
the nest building test was used to analyze d-line mice compared to non-transgenic littermates at  <dig> months of age.
d-line mice showed a significantly reduced nest building behavior .
while non-transgenic mice built an almost perfect nest with the provided material, the d-line mice shredded only about 50% of the nesting material and an easily identifiable nest site was missing.
since no significant differences between male and female d-line mice were observed , data of both sexes were pooled for figure
1d.
taken together, our behavioral analysis argues for an impairment of motor coordination in transgenic mice during execution of challenging tasks, whereas overall motor activity is largely unaffected.
quantification of α-synuclein protein levels over age
analyses of hα-syn protein levels by elisa showed a significant increase in the hippocampus at  <dig> months of age compared to  <dig> and  <dig> months old d-line mice .
the increase was less pronounced in the striatum but was significant at  <dig> months compared to  <dig> and  <dig> months .
in order to investigate whether the progressive accumulation of hα-syn in brain homogenates using elisa could also be detected in situ we next analyzed expression levels of human and murine protein isoforms in the cortex, hippocampus, striatum and substantia nigra by quantification of immunofluorescent labeling using isoform-specific antibodies.
in all analyzed brain areas the pan  α-syn levels of d-line mice did not differ from non-transgenic littermates, and in both genotypes the total protein levels did not change significantly over age in cerebral cortical  and subcortical  areas.
however, analyses restricted to transgenic hα-syn, the same isoform that was analyzed biochemically by elisa, showed significantly higher immunofluorescence signal intensity  in d-line mice compared to ntg mice already at  <dig> months of age in cortical areas .
expression levels of hα-syn in the striatum and the substantia nigra were also above baseline in d-line mice  but were generally lower than in cortical areas.
despite these regional differences in total levels, an age-dependent significant increase of hα-syn immunofluorescence could be observed in all four brain areas of d-line mice , consistent with the data obtained by elisa.
this result, together with the absence of any age-related change in pan α-syn, raises the possibility that adaptive mechanisms may downregulate murine α-syn expression to ensure homeostatically appropriate protein levels throughout life.
an alternative explanation for the finding of constant pan α-syn levels is that the pan-specific monoclonal antibody may have lower affinity for the human isoform compared to murine α-syn.
this could potentially be assessed using an antibody that specifically detects only murine α-syn, but to our knowledge such a reagent is not available.
distribution pattern of human, murine and phosphorylated α-synuclein protein
next we investigated the distribution patterns of hα-syn and pan α-syn isoforms by double-immunofluorescent labeling throughout the brain .
high levels of hα-syn were detected in the pontine nucleus and in the cortex, including the cortical part of the amygdala and the hippocampus.
high levels of hα-syn accumulation were also detected in the striatum, ventral hypothalamus and in the olfactory bulb.
double-immunofluorescence for hα-syn and a n-terminal epitope of both murine and human α/β-synuclein  at higher magnification revealed that transgenic hα-syn obviously accumulates in somata and proximal dendrites.
this is also evident in layers with few neuronal somata but high density of neuropil , suggesting subcellular targeting of overexpressed hα-syn to both dendrites and axons.
in contrast, n-α/β-syn is preferentially expressed in synaptic terminals, notably in the stratum lucidum of hippocampal ca <dig>  and less obvious in the neocortex , consistent with previous reports on largely presynaptic expression in wild type mice
<cit> .
next, we investigated the expression pattern of phosphorylated protein  using an antibody specifically directed against an epitope including the phosphorylated-serine  <dig> residue of the human α-synuclein isoform .
phosphorylation at residues s <dig> and s <dig> is of particular interest for synucleinopathy research because it has been argued that phosphorylation at these residues might modulate the formation of protein aggregations, e.g.
inclusion bodies and fibrillary structures
<cit> .
we found that in the hippocampus and neocortex, the subcellular expression of phα-syn is largely restricted to neuronal nuclei , as evident from the overlay with the nuclear dye dapi .
qualitative analysis of ps129-hα-syn suggests that the vast majority of neuronal somata expressing the hα-syn isoform show easily detectable levels of ps129-hα-syn.
however, we found no evidence for expression in synaptic terminals or pathological intracellular accumulations in d-line mice of up to  <dig> months.
in contrast to the ps129s residue, phosphorylated s <dig> hα-syn is undetectable in d-line mice
<cit> .
identification of α-synuclein-expressing neurons
in order to identify neuronal populations that accumulate hα-syn, several multichannel immunofluorescence experiments were performed.
using antibodies against gad <dig> and tyrosine hydroxylase we found that hα-syn is expressed in subpopulations of gabaergic and dopaminergic neurons, respectively, of the olfactory bulb , and in gabaergic interneurons of cortical areas .
since some gabaergic somata in the hippocampal formation show little or even no gad <dig> immunoreactivity
<cit>  we alternatively identified interneurons by expression of the neuregulin receptor erbb <dig> that is a selective marker for a subset of cortical gabaergic neurons
<cit> .
we detected hα-syn in a small number of erbb4-positive interneurons in the cornu ammonis and dentate gyrus but not on microglia identified by iba <dig> immunoreactivity .
with regard to glutamatergic principal neurons, hα-syn was not detectable in mitral cells of the olfactory bulb , but strong expression was evident in subsets of neocortical and hippocampal pyramidal cells .
we noted the occurrence of strong hα-syn immunoreactivity in some regions of the adult brain featuring immature and migrating neurons , such as the ventricular zone and the rostral migratory stream.
using multichannel immunofluorescence, we confirmed hα-syn expression in immature migrating neurons that were identified by several established markers such as doublecortin, psa-ncam and erbb <dig> , suggesting that hα-syn expression is not a unifying feature of migrating or maturing neurons in adult d-line mice.
taken together, our data show that strong hα-syn immunofluorescence occurs region-specifically in heterogeneous populations of neurons of the adult brain.
discussion
this study was designed to characterize the time course and progression of the pathology in d-line mice, a model of α-syn accumulation similar to dlb.
we found progressive motor coordination deficits at  <dig> and  <dig> months of age complemented by alterations in the nest building behavior.
these deficits were accompanied by a parallel age-dependent increase in the levels of hα-syn in somata and neuritic processes of a subset of neocortical, limbic and nigrostriatal system cells.
the mechanisms by which accumulation of hα-syn in these circuitries might result in functional deficits are not completely clear.
recent findings on changes in expression levels of neurotrophic factors in hα-syn transgenic mice
<cit>  suggest that altered bdnf levels could be linked to onset and progression of parkinson’s disease
<cit> .
the progressive motor alterations in the challenging beam walk test detected in d-line mice are consistent with previous studies using the rotarod and pole test
<cit>  and support a dysfunction of the nigrostriatal system.
also supporting this possibility, previous studies have shown that d-line mice of either sex generally perform worse compared to non-transgenic littermates; however, at the age of  <dig> months d-line and non-transgenic male mice consistently slip more often during the challenging beam walk test than females.
in transgenic mice the sex difference seems to increase up to the age of  <dig> months, suggesting that hα-syn has an earlier impact on motor performance in male than in female mice.
interestingly, these results are reminiscent of sex differences in humans where female parkinson patients show a delayed onset of the disease compared to males
<cit> .
nest building behavior is an intrinsic behavior in both female and male mice that requires proper fine motor skills.
the nest is important for sustaining body temperature and provides shelter during birth and rearing of offspring.
different lesioning studies provide evidence that nest building behavior strongly depends on the proper function of the hippocampus, caudate putamen and ventral mesencephalic tegmentum
<cit> .
moreover, nest building was shown to be dopamine- and enkephalin-dependent
<cit> .
all these brain systems are known to be highly relevant for pd
<cit> , suggesting that the nest building test is an appropriate tool for assessing parkinson-associated impairments.
therefore, our here presented data of nest building and motor deficits in the beam walk test suggest, that d-line transgenic mice represent with the most common behavioral symptoms of pd.
analyses of hα-syn levels in the brain by elisa showed an age dependent increase of transgenic protein levels in the hippocampus and striatum between  <dig> and  <dig> months.
using quantitative analysis of immunofluorescence against hα-syn we could verify this result in situ.
interestingly, however, we detected neither genotype-dependent nor age-dependent increases in total  α-syn levels.
given that under the pdgf-β promoter the mrna expression of α-syn is stable throughout life, the progressive accumulation of hα-syn protein in the brains of the transgenic mice indicates deficits in clearance.
this is consistent with previous studies showing alterations in the autophagy pathway in the d-line mice
<cit> , these alterations were reversed pharmacologically with rapamycin
<cit>  or genetically with beclin-1
<cit>  or atg7
<cit>  and worsen with shatg7
<cit>  or bafilomycin-a1
<cit> .
in addition, the finding of constant total α-syn levels despite the parallel accumulation of transgenic hα-syn protein raises the possibility that the expression of endogenous murine α-syn might be a tightly regulated mechanism.
overexpression of hα-syn could therefore be a trigger for homeostatic downregulation of the murine isoform.
moreover, total α-syn immunoreactivity even decreased slightly in the substantia nigra of non-transgenic animals suggesting that reduction of α-syn levels in the nigrostriatal dopaminergic pathway of older mice is a normal event during aging in healthy rodents, consistent with reports by other groups
<cit> .
we identified subsets of transgene expressing neurons as inhibitory gabaergic interneurons or excitatory glutamatergic pyramidal neurons in cortical areas.
with respect to dopaminergic cells, we found co-expression of hα-syn and tyrosine hydroxylase in many neurons of the olfactory bulb, in contrast to the substantia nigra where only low levels of hα-syn were detectable.
we also detected hα-syn immunoreactivity in some gabaergic interneurons of the olfactory bulb but, in contrast to cortical areas, not in glutamatergic principal neurons .
overall, this pattern is consistent with the reported expression of pdgf-β in different types of neurons
<cit> .
our investigation of hα-syn phosphorylation at residue s <dig> shows that immunoreactivity is largely restricted to the nuclei of neurons that express high levels of the transgenic protein.
of note, we found no indication of granular or fibrillary-like accumulations of ps129-hα-syn, opening the question whether this nuclear immunoreactivity reflects regular physiological events or rather indicates progression towards neuropathology consistent with previous reports
<cit> .
finally, consistent with previous studies
<cit>  we found that hα-syn accumulates in areas featuring immature neurons, such as the subventricular zone and the rostral migratory stream.
accumulation of hα-syn in these areas results in reduced proliferation and survival of neuroblasts
<cit> , however, the functional relevance of these alterations is yet unknown.
interestingly, we found no indication of hα-syn immunoreactivity in maturing neurons of the dentate gyrus, indicating that hα-syn expression is not a common feature of all populations of immature or migrating neurons.
additional studies are required to investigate whether these regional differences have functional relevance that may affect survival rates or maturation of these cells.
CONCLUSIONS
our data suggest that hα-syn accumulates in subsets of glutamatergic, dopaminergic, and gabaergic, neuronal populations in the neocortex, limbic system and nigrostriatal system similar to the distribution of hα-syn in dlb.
immunoreactivity for human protein phosphorylated at residue s <dig> is restricted to neuronal somata, whereas much of the additional immunofluorescent signal from hα-syn protein not phosphorylated at the s <dig> residue comes from areas with few neuronal somata but dense neuropil, thus mimicking the distribution of the endogenous murine α-syn isoform.
we also detected progressively increasing levels of hα-syn protein during adulthood using different experimental approaches, and concomitant impairment of motor coordination.
in contrast, the level of total α-syn is constant between 3– <dig> months, possibly due to a compensatory downregulation of the endogenous α-syn isoform.
we conclude that different readouts, i.e.
behavioral, biochemical and histological, can detect structural and functional pathology in the d-line mouse model, and that this animal model is highly valuable for pd-related research and drug development.

 synucleinopathic diseases, like parkinson’s disease, dementia with lewy bodies and multiple system atrophy, are all characterized by a pathologic aggregation of α-synuclein protein in distinct brain regions. increased expression of α-syn can be caused by a dominant heritable form of pd due to duplication or triplication of the α-syn gene. in order to model such synucleinopathies in vivo different mouse models were developed that overexpress human wild type α-syn. when expression is driven by the murine thy promoter, transgenic mice accumulate wild type hα-syn in cortical and subcortical regions including the nigrostriatal system whereas under the human pdgf-β promoter hα-syn accumulates preferentially in the neocortex and limbic system. abnormal accumulation of hα-syn in d-line transgenic mice is accompanied by alterations in mglur and autophagy similar to what has been observed in patients with dementia with lewy bodies. moreover the behavioral and neurodegenerative pathology in d-line mice can be reversed with mglur antagonists or by promoting the clearance of α-syn with rapamycin, beclin 1 and neurosin. given the behavioral phenotype and the predominant accumulation of α-syn in the neocortex and limbic system, these studies suggest that the pdgf-β hα-syn transgenic mouse model reproduces some aspects of synucleinopathies such as dlb. recent studies have tested compounds developed to ameliorate pd-like pathology in the d-line mouse model and were able to show a reduction in the accumulation of α-syn, total and oxidized α-syn levels and behavioral deficits. taken together, these results indicate that this transgenic model might be useful for studies of α-syn target validation. however, the progression of the behavioral deficits and pathology in d-line mice has not been fully characterized. for this purpose we investigated the time course of behavioral deficits and neuropathology in d-line mice. the occurrence of motor deficits was investigated using a challenging beam walk paradigm. we also quantified hα-syn expression in the hippocampus and striatum using a biochemical approach. moreover, by quantitative immunofluorescence at different ages we compared expression levels of both murine α-syn and hα-syn in the cortex, hippocampus, striatum and substantia nigra relative to non-transgenic littermates. furthermore, we identified several cell populations that express transgenic hα-syn in different areas of the adult brain.  progressive motor deficits of d-line mice motor coordination, in particular of the hind limbs, and balance of all mice was evaluated with the challenging beam walk test at and months of age to evaluate progression of motor deficits in adult, mature and aging mice. the time to cross the beam, the number of steps and slips, and the ratio of slips per step was recorded. no significant changes in the number of slips were observed in months old d-line and control animals. months old transgenic and non-transgenic male mice made significantly more slips compared to the corresponding female mice. transgenic female mice of the same age performed significantly worse compared to the corresponding female non-transgenic controls as analyzed by t-test. the same trend was observed in male mice, but the results were not statistically significant. the observed deficits faded at the age of months and only the significant difference between female d-line and non-transgenic mice remained. the significant differences shown in figure 1a-c were detected by analyzing data with t-test. analyses of the presented data for the factor gender by two way anova followed by bonferroni’s post hoc test revealed a significant influence of the gender for transgenic as well as non transgenic mice. analyses of data by two way anova followed by bonferroni’s post hoc test for the factor transgene and by one way anova did not reveal any significances. analysis of the number of slips for progressive changes over age separated by sex showed only a significant improvement of non-transgenic female mice over age. the time and number of steps required to traverse the beam and the ratio of slips per step did not differ significantly between d-line and non-transgenic littermates. in order to verify that the observed motor differences between the d-line mice and non-transgenic littermates were not caused by modifications of spontaneous activity or deficits in spatial learning, the cylinder test and the two-choice swim test were performed, and no significant effects were detected. general health of each mouse was determined using the irwin test—including the wire hanging test and vertical pole test, assessing physical characteristics and abnormalities, sensorimotor reflexes and motor abilities—but no significant differences were observed. overall, all animals appeared healthy over the course of the study. the nest building test was used to analyze d-line mice compared to non-transgenic littermates at months of age. d-line mice showed a significantly reduced nest building behavior. while non-transgenic mice built an almost perfect nest with the provided material, the d-line mice shredded only about 50% of the nesting material and an easily identifiable nest site was missing. since no significant differences between male and female d-line mice were observed, data of both sexes were pooled for figure 1d. taken together, our behavioral analysis argues for an impairment of motor coordination in transgenic mice during execution of challenging tasks, whereas overall motor activity is largely unaffected. quantification of α-synuclein protein levels over age analyses of hα-syn protein levels by elisa showed a significant increase in the hippocampus at months of age compared to and months old d-line mice. the increase was less pronounced in the striatum but was significant at months compared to and months. in order to investigate whether the progressive accumulation of hα-syn in brain homogenates using elisa could also be detected in situ we next analyzed expression levels of human and murine protein isoforms in the cortex, hippocampus, striatum and substantia nigra by quantification of immunofluorescent labeling using isoform-specific antibodies. in all analyzed brain areas the pan α-syn levels of d-line mice did not differ from non-transgenic littermates, and in both genotypes the total protein levels did not change significantly over age in cerebral cortical and subcortical areas. however, analyses restricted to transgenic hα-syn, the same isoform that was analyzed biochemically by elisa, showed significantly higher immunofluorescence signal intensity in d-line mice compared to ntg mice already at months of age in cortical areas. expression levels of hα-syn in the striatum and the substantia nigra were also above baseline in d-line mice but were generally lower than in cortical areas. despite these regional differences in total levels, an age-dependent significant increase of hα-syn immunofluorescence could be observed in all four brain areas of d-line mice, consistent with the data obtained by elisa. this result, together with the absence of any age-related change in pan α-syn, raises the possibility that adaptive mechanisms may downregulate murine α-syn expression to ensure homeostatically appropriate protein levels throughout life. an alternative explanation for the finding of constant pan α-syn levels is that the pan-specific monoclonal antibody may have lower affinity for the human isoform compared to murine α-syn. this could potentially be assessed using an antibody that specifically detects only murine α-syn, but to our knowledge such a reagent is not available. distribution pattern of human, murine and phosphorylated α-synuclein protein next we investigated the distribution patterns of hα-syn and pan α-syn isoforms by double-immunofluorescent labeling throughout the brain. high levels of hα-syn were detected in the pontine nucleus and in the cortex, including the cortical part of the amygdala and the hippocampus. high levels of hα-syn accumulation were also detected in the striatum, ventral hypothalamus and in the olfactory bulb. double-immunofluorescence for hα-syn and a n-terminal epitope of both murine and human α/β-synuclein at higher magnification revealed that transgenic hα-syn obviously accumulates in somata and proximal dendrites. this is also evident in layers with few neuronal somata but high density of neuropil, suggesting subcellular targeting of overexpressed hα-syn to both dendrites and axons. in contrast, n-α/β-syn is preferentially expressed in synaptic terminals, notably in the stratum lucidum of hippocampal ca and less obvious in the neocortex, consistent with previous reports on largely presynaptic expression in wild type mice. next, we investigated the expression pattern of phosphorylated protein using an antibody specifically directed against an epitope including the phosphorylated-serine residue of the human α-synuclein isoform. phosphorylation at residues s and s is of particular interest for synucleinopathy research because it has been argued that phosphorylation at these residues might modulate the formation of protein aggregations, e.g. inclusion bodies and fibrillary structures. we found that in the hippocampus and neocortex, the subcellular expression of phα-syn is largely restricted to neuronal nuclei, as evident from the overlay with the nuclear dye dapi. qualitative analysis of ps129-hα-syn suggests that the vast majority of neuronal somata expressing the hα-syn isoform show easily detectable levels of ps129-hα-syn. however, we found no evidence for expression in synaptic terminals or pathological intracellular accumulations in d-line mice of up to months. in contrast to the ps129s residue, phosphorylated s hα-syn is undetectable in d-line mice. identification of α-synuclein-expressing neurons in order to identify neuronal populations that accumulate hα-syn, several multichannel immunofluorescence experiments were performed. using antibodies against gad and tyrosine hydroxylase we found that hα-syn is expressed in subpopulations of gabaergic and dopaminergic neurons, respectively, of the olfactory bulb, and in gabaergic interneurons of cortical areas. since some gabaergic somata in the hippocampal formation show little or even no gad immunoreactivity we alternatively identified interneurons by expression of the neuregulin receptor erbb that is a selective marker for a subset of cortical gabaergic neurons. we detected hα-syn in a small number of erbb4-positive interneurons in the cornu ammonis and dentate gyrus but not on microglia identified by iba immunoreactivity. with regard to glutamatergic principal neurons, hα-syn was not detectable in mitral cells of the olfactory bulb, but strong expression was evident in subsets of neocortical and hippocampal pyramidal cells. we noted the occurrence of strong hα-syn immunoreactivity in some regions of the adult brain featuring immature and migrating neurons, such as the ventricular zone and the rostral migratory stream. using multichannel immunofluorescence, we confirmed hα-syn expression in immature migrating neurons that were identified by several established markers such as doublecortin, psa-ncam and erbb, suggesting that hα-syn expression is not a unifying feature of migrating or maturing neurons in adult d-line mice. taken together, our data show that strong hα-syn immunofluorescence occurs region-specifically in heterogeneous populations of neurons of the adult brain. discussion this study was designed to characterize the time course and progression of the pathology in d-line mice, a model of α-syn accumulation similar to dlb. we found progressive motor coordination deficits at and months of age complemented by alterations in the nest building behavior. these deficits were accompanied by a parallel age-dependent increase in the levels of hα-syn in somata and neuritic processes of a subset of neocortical, limbic and nigrostriatal system cells. the mechanisms by which accumulation of hα-syn in these circuitries might result in functional deficits are not completely clear. recent findings on changes in expression levels of neurotrophic factors in hα-syn transgenic mice suggest that altered bdnf levels could be linked to onset and progression of parkinson’s disease. the progressive motor alterations in the challenging beam walk test detected in d-line mice are consistent with previous studies using the rotarod and pole test and support a dysfunction of the nigrostriatal system. also supporting this possibility, previous studies have shown that d-line mice of either sex generally perform worse compared to non-transgenic littermates; however, at the age of months d-line and non-transgenic male mice consistently slip more often during the challenging beam walk test than females. in transgenic mice the sex difference seems to increase up to the age of months, suggesting that hα-syn has an earlier impact on motor performance in male than in female mice. interestingly, these results are reminiscent of sex differences in humans where female parkinson patients show a delayed onset of the disease compared to males. nest building behavior is an intrinsic behavior in both female and male mice that requires proper fine motor skills. the nest is important for sustaining body temperature and provides shelter during birth and rearing of offspring. different lesioning studies provide evidence that nest building behavior strongly depends on the proper function of the hippocampus, caudate putamen and ventral mesencephalic tegmentum. moreover, nest building was shown to be dopamine- and enkephalin-dependent. all these brain systems are known to be highly relevant for pd, suggesting that the nest building test is an appropriate tool for assessing parkinson-associated impairments. therefore, our here presented data of nest building and motor deficits in the beam walk test suggest, that d-line transgenic mice represent with the most common behavioral symptoms of pd. analyses of hα-syn levels in the brain by elisa showed an age dependent increase of transgenic protein levels in the hippocampus and striatum between and months. using quantitative analysis of immunofluorescence against hα-syn we could verify this result in situ. interestingly, however, we detected neither genotype-dependent nor age-dependent increases in total α-syn levels. given that under the pdgf-β promoter the mrna expression of α-syn is stable throughout life, the progressive accumulation of hα-syn protein in the brains of the transgenic mice indicates deficits in clearance. this is consistent with previous studies showing alterations in the autophagy pathway in the d-line mice, these alterations were reversed pharmacologically with rapamycin or genetically with beclin-1 or atg7 and worsen with shatg7 or bafilomycin-a1. in addition, the finding of constant total α-syn levels despite the parallel accumulation of transgenic hα-syn protein raises the possibility that the expression of endogenous murine α-syn might be a tightly regulated mechanism. overexpression of hα-syn could therefore be a trigger for homeostatic downregulation of the murine isoform. moreover, total α-syn immunoreactivity even decreased slightly in the substantia nigra of non-transgenic animals suggesting that reduction of α-syn levels in the nigrostriatal dopaminergic pathway of older mice is a normal event during aging in healthy rodents, consistent with reports by other groups. we identified subsets of transgene expressing neurons as inhibitory gabaergic interneurons or excitatory glutamatergic pyramidal neurons in cortical areas. with respect to dopaminergic cells, we found co-expression of hα-syn and tyrosine hydroxylase in many neurons of the olfactory bulb, in contrast to the substantia nigra where only low levels of hα-syn were detectable. we also detected hα-syn immunoreactivity in some gabaergic interneurons of the olfactory bulb but, in contrast to cortical areas, not in glutamatergic principal neurons. overall, this pattern is consistent with the reported expression of pdgf-β in different types of neurons. our investigation of hα-syn phosphorylation at residue s shows that immunoreactivity is largely restricted to the nuclei of neurons that express high levels of the transgenic protein. of note, we found no indication of granular or fibrillary-like accumulations of ps129-hα-syn, opening the question whether this nuclear immunoreactivity reflects regular physiological events or rather indicates progression towards neuropathology consistent with previous reports. finally, consistent with previous studies we found that hα-syn accumulates in areas featuring immature neurons, such as the subventricular zone and the rostral migratory stream. accumulation of hα-syn in these areas results in reduced proliferation and survival of neuroblasts, however, the functional relevance of these alterations is yet unknown. interestingly, we found no indication of hα-syn immunoreactivity in maturing neurons of the dentate gyrus, indicating that hα-syn expression is not a common feature of all populations of immature or migrating neurons. additional studies are required to investigate whether these regional differences have functional relevance that may affect survival rates or maturation of these cells.  our data suggest that hα-syn accumulates in subsets of glutamatergic, dopaminergic, and gabaergic, neuronal populations in the neocortex, limbic system and nigrostriatal system similar to the distribution of hα-syn in dlb. immunoreactivity for human protein phosphorylated at residue s is restricted to neuronal somata, whereas much of the additional immunofluorescent signal from hα-syn protein not phosphorylated at the s residue comes from areas with few neuronal somata but dense neuropil, thus mimicking the distribution of the endogenous murine α-syn isoform. we also detected progressively increasing levels of hα-syn protein during adulthood using different experimental approaches, and concomitant impairment of motor coordination. in contrast, the level of total α-syn is constant between 3– months, possibly due to a compensatory downregulation of the endogenous α-syn isoform. we conclude that different readouts, i.e. behavioral, biochemical and histological, can detect structural and functional pathology in the d-line mouse model, and that this animal model is highly valuable for pd-related research and drug development. 
progressive accumulation of α-synuclein protein in different brain regions is a hallmark of synucleinopathic diseases, such as parkinson’s disease, dementia with lewy bodies and multiple system atrophy. α-syn transgenic mouse models have been developed to investigate the effects of α-syn accumulation on behavioral deficits and neuropathology. we also found human α-syn expression in immature neurons of both the ventricular zone and the rostral migratory stream, but not in the dentate gyrus. the present study demonstrates that the pdgf-β α-syn transgenic mouse model presents with early and progressive accumulation of human α-syn that is accompanied by motor deficits. these mice showed progressive impairment of motor coordination of the limbs that resulted in significant differences compared to non-transgenic littermates at and months of age. analyses of the phenotype of α-syn expressing cells revealed strong expression in dopaminergic olfactory bulb neurons, subsets of gabaergic interneurons and glutamatergic principal cells throughout the telencephalon. for this purpose we investigated the time course of behavioral deficits and neuropathology in pdgf-β human wild type α-syn transgenic mice between and months of age.
 progressive accumulation of α-synuclein protein in different brain regions is a hallmark of synucleinopathic diseases, such as parkinson’s disease, dementia with lewy bodies and multiple system atrophy. α-syn transgenic mouse models have been developed to investigate the effects of α-syn accumulation on behavioral deficits and neuropathology. however, the onset and progression of pathology in α-syn transgenic mice have not been fully characterized. for this purpose we investigated the time course of behavioral deficits and neuropathology in pdgf-β human wild type α-syn transgenic mice between and months of age.  these mice showed progressive impairment of motor coordination of the limbs that resulted in significant differences compared to non-transgenic littermates at and months of age. biochemical and immunohistological analyses revealed constantly increasing levels of human α-syn in different brain areas. human α-syn was expressed particularly in somata and neurites of a subset of neocortical and limbic system neurons. most of these neurons showed immunoreactivity for phosphorylated human α-syn confined to nuclei and perinuclear cytoplasm. analyses of the phenotype of α-syn expressing cells revealed strong expression in dopaminergic olfactory bulb neurons, subsets of gabaergic interneurons and glutamatergic principal cells throughout the telencephalon. we also found human α-syn expression in immature neurons of both the ventricular zone and the rostral migratory stream, but not in the dentate gyrus.  the present study demonstrates that the pdgf-β α-syn transgenic mouse model presents with early and progressive accumulation of human α-syn that is accompanied by motor deficits. this information is essential for the design of therapeutical studies of synucleinopathies. behaviorimmunofluorescencemotor deficitmouse modelparkinson’s diseasephosphorylationsynucleinopathyα-synucleintransgene 