The Temporal Expression Pattern of Alpha-Synuclein Modulates Olfactory Neurogenesis in Transgenic Mice

Background Adult neurogenesis mirrors the brain´s endogenous capacity to generate new neurons throughout life. In the subventricular zone/ olfactory bulb system adult neurogenesis is linked to physiological olfactory function and has been shown to be impaired in murine models of neuronal alpha-Synuclein overexpression. We analyzed the degree and temporo-spatial dynamics of adult olfactory bulb neurogenesis in transgenic mice expressing human wild-type alpha-Synuclein (WTS) under the murine Thy1 (mThy1) promoter, a model known to have a particularly high tg expression associated with impaired olfaction. Results Survival of newly generated neurons (NeuN-positive) in the olfactory bulb was unchanged in mThy1 transgenic animals. Due to decreased dopaminergic differentiation a reduction in new dopaminergic neurons within the olfactory bulb glomerular layer was present. This is in contrast to our previously published data on transgenic animals that express WTS under the control of the human platelet-derived growth factor β (PDGF) promoter, that display a widespread decrease in survival of newly generated neurons in regions of adult neurogenesis, resulting in a much more pronounced neurogenesis deficit. Temporal and quantitative expression analysis using immunofluorescence co-localization analysis and Western blots revealed that in comparison to PDGF transgenic animals, in mThy1 transgenic animals WTS is expressed from later stages of neuronal maturation only but at significantly higher levels both in the olfactory bulb and cortex. Conclusions The dissociation between higher absolute expression levels of alpha-Synuclein but less severe impact on adult olfactory neurogenesis in mThy1 transgenic mice highlights the importance of temporal expression characteristics of alpha-Synuclein on the maturation of newborn neurons.


Results
Survival of newly generated neurons (NeuN-positive) in the olfactory bulb was unchanged in mThy1 transgenic animals. Due to decreased dopaminergic differentiation a reduction in new dopaminergic neurons within the olfactory bulb glomerular layer was present. This is in contrast to our previously published data on transgenic animals that express WTS under the control of the human platelet-derived growth factor β (PDGF) promoter, that display a widespread decrease in survival of newly generated neurons in regions of adult neurogenesis, resulting in a much more pronounced neurogenesis deficit. Temporal and quantitative expression analysis using immunofluorescence co-localization analysis and Western blots revealed that in comparison to PDGF transgenic animals, in mThy1 transgenic animals WTS is expressed from later stages of neuronal maturation only but at significantly higher levels both in the olfactory bulb and cortex.

Introduction
Dementia with Lewy bodies (DLB), Parkinson`s disease (PD), and multisystem atrophy (MSA) share the neuropathological hallmark of aggregation of alpha-Synuclein (a-syn) in various cell populations throughout the brain. The mechanisms leading to the accumulation and aggregation of this protein are still a matter of ongoing studies [1].
In the adult brain, neurogenesis is present throughout life in two distinct regions: the hippocampal dentate gyrus and the subventricular zone (SVZ) [2]. All mammals share this phenomenon to variable degrees [3,4]. In different neurodegenerative diseases reduced numbers of proliferating cells were observed in regions of adult neurogenesis (reviewed in [5]), indicating that besides the loss of existing neurons, the adult brain's endogenous capacity for cell renewal might be impaired as well. While neurodegeneration is commonly associated with dysfunction of cells, impaired adult neurogenesis is therefore another phenomenon observed in patients with synucleinopathies and models of a-syn related neurodegeneration [5]. This is in particular important since a-syn has a regulatory role in dendritic arborization and spine development in the hippocampal dentate gyrus [6].
In rodents, a number of studies suggest that neurogenesis in the olfactory bulb (OB) contributes to olfactory learning and memory [7,8]. A-syn transgenic (tg) animal models mimic certain neuropathological aspects of synucleinopathies such as PD and have been used to examine the physiological as well as pathological role of a-syn, with respect to adult neurogenesis [9]. Mice expressing WTS under the regulatory control of the human platelet-derived growth factor β (PDGF) promoter accumulate WTS in neurogenic niches resulting in compromised adult neurogenesis in the hippocampal as well as the OB system [10,11].
Tg animals expressing human wild-type a-syn (WTS) under the regulatory control of the murine Thy1 (mThy1) promoter overexpress WTS in a neuron-specific manner and are known to have a particularly high tg expression. Moreover, accumulation of WTS causes progressive, age-dependent neurodegenerative changes with an L-Dopa-reversible sensorimotor phenotype and olfactory dysfunction [10,[12][13][14]. The aim of this study was to investigate the degree and temporal dynamics of adult neurogenesis in these animals.
Compared to PDGF WTS tg mice we found a high expression of a-syn in the olfactory bulb of mThy1 tg mice, but only adult dopaminergic olfactory neurogenesis in mThy1 animals was impaired. Analysis of the temporal expression pattern during stages of adult neurogenesis revealed a later tg expression under mThy1 than PDGF promoter control, while a-syn protein expression is higher in mTHy1 mice. We conclude, that specifically the temporal expression of asyn during the generation of new neurons, and not only the absolute level of a-syn expression, crucially influences the survival and differentiation of newly generated neurons.

Materials and Methods Animals
All animal procedures were performed in accordance with the protocols approved by the animal care use committee of the University of California, San Diego following National Institutes of Health guidelines for the humane treatment of animals. Animal euthanasia was performed using Xylazin/Ketamin i.p. injections for anaesthesia before transcardial paraformaldehyde perfusion for tissue fixation. All mice were kept in a 12 h light/dark cycle and had access to food and water ad libitum. Tg mice expressing WTS under the regulatory control of the mThy1 promoter belonging to the high expression line 61 described earlier [10] were compared to wildtype littermate controls (Ctrl) of the same C57BL6/DBA background (n = 6 per group). To assess the impact of tg expression on olfactory neurogenesis, a standard neurogenesis paradigm for survival of new neurons was applied as described previously [11,15]. Animals received daily intraperitoneal injections with 5-Bromo-2-deoxyuridine (BrdU; 50mg/kg) for 5 days at the age of 4 months and were sacrificed 1 month later (Fig 1A).

Primary antibodies and final dilutions
The following primary antibodies were used: rat-α-BrdU (OBT0030, 1:100, Accurate, Westbury, USA), mouse-α-PCNA (P8825, 1:1000, Sigma-Aldrich, St. Louis, USA), goat-α-DCX Control and mThy1 a-Synuclein transgenic animals were subjected to a paradigm with 5 consecutive days of i.p. BrdU injections at the age of 4 months and histological analysis at the age of 5 months (panel A). In the SVZ no difference in the number of neuronal progenitor (PCNA-positive) cells was detectable. In the granular cell layer of the olfactory bulb the numbers of surviving BrdU-positive cells, the percentage of BrdU/NeuN double-positive cells as well as the numbers of newly generated neurons were not significantly different in both groups, indicating that mThy1 human wild-type a-Synuclein had no effect on adult granular cell layer neurogenesis (panel B). Numbers are given as means ± standard error means.

Immunohistochemistry
Free-floating sections were stained as published previously [11]. The incubation in 0.6% H 2 O 2 in Tris-buffered saline (TBS: 0.15M NaCl, 0.1M Tris-HCl, pH 7.5, 30 min) was followed by blocking steps in TBS/0.25% Triton-X100/3% normal donkey serum for 30 min. Thereafter, the sections were incubated in primary antibody in TBS/donkey serum overnight at 4°C. Sections were then incubated for 1h with biotinylated secondary antibodies according to the primary antibody host. Amplification of signal intensity was achieved using avidin-biotin peroxidase complex for 1h (Vectastain Elite ABC kit; Vector Laboratories, Inc., Burlingame, USA) and signal visualization using diaminobenzidin (DAB; 25 mg/ml 0.01% H2O2, 0.04% NiCl in TBS) peroxidase reaction for 10 min.
For the detection of BrdU-labeled nuclei, the following DNA denaturation steps preceded the incubation with primary anti-BrdU antibody: 2 h incubation in 2M HCl at 37°C followed by a pH adjustment step using 10min rinse in 0.1 M boric acid, pH 8.5. All sections were mounted in anatomical order on glass slides and coverslipped with NeoMount (Merck, KGaA, Darmstadt, Germany).

Immunofluorescence
After the blocking steps in TBS/0.25% Triton-X100/3% normal donkey serum for 30 min the primary antibodies were applied in TBS-donkey serum overnight at 4°C, followed by incubation with secondary fluorochrome-coupled antibodies for 60 min at room temperature and DAPI (D9542, Sigma-Aldrich, St. Louis, USA) counterstaining (1:1000, 5 min) in order to identify cell nuclei. Sections were then dried, mounted and coverslipped with NeoMount. All sections were processed simultaneously under the same conditions.
Every 12th 40μm section (480μm interval) of the left hemisphere was selected from each animal and processed for immunohistochemistry or-fluorescence. The reference volume was determined by tracing the areas using the respective StereoInvestigator software tool. The number of PCNA-positive cells in the SVZ and BrdU-positive cells in the GCL and GLOM of the OB respectively, were counted exhaustively. In the OB GCL, the optical fractionator, a systematic, random counting procedure combining the advantages of an optical dissector and systematic uniform sampling, was used [16]. BrdU-positive nuclei were counted within a 30μm × 30μm counting frame in a 300μm × 300μm counting grid to achieve a Gundersen coefficient of error <0.1. A guard distance of 2.0μm was used during all cell-counting procedures to avoid introduction of errors due to sectioning artefacts. The total counts of positive profiles were multiplied by the ratio of reference volume to sampling volume in order to obtain the estimated number of positive cells for each structure.
To determine the frequency of neuronal and dopaminergic differentiation of newly generated cells, as well as coexpression of a-syn with cell markers of stages of adult neurogenesis, sections were examined using a confocal laser microscope (Nikon Eclipse TE 300 microscope, Plan Fluor 40x objective (Nikon Instruments Inc. Melville, USA), BIORAD Radiance 2700 laser system and Lasersharp Confocal Assistant Software (BioRad, Hercules, USA) at a pinhole setting that corresponded to a focal plane of 2μm or less.
Within the anatomical structure of interest, microscopy frames were chosen randomly by the investigator from among every 12 th 40μm section as to allow for equal representation of all parts of the structure. To avoid bias, all BrdU positive cells within a confocal frame were analyzed for colocalization. To exclude false positive results, these cells were analyzed using confocal imaging along the full z-axis and sequentially at each fluorophore wavelength to ensure that coexpression of fluorophores was present in identical focal planes.

Western Blot analysis
For Western blot analysis, cortex and olfactory bulb of mouse brains were homogenized in RIPA Buffer (50mM Tris/HCl pH7.4, 150 mM NaCl, 2 mM EDTA, 1% (v/v) NP-40, 0.1% (w/v) SDS) in the presence of a proteases inhibitor cocktail (Roche) with a douncer homogenizer Potter at 4°C. Protein levels in the tissue homogenates were quantified via BCA protein assay (Thermo Scientific). 80 μg of total proteins were mixed with the same volume of SDS sample buffer (0.125 M Tris/HCl pH 6.8, 4% SDS, 20% glycerol), separated on 15% SDS-PAGE, and blotted onto nitrocellulose membranes (Millipore). The blots were probed with a mouse antialpha-synuclein-1 antibody (1:2000, BD Transduction Laboratories) and a rabbit anti GAPDH antibody (1:2000, FL335, Santa Cruz Technology), followed by the incubation with secondary goat anti mouse and goat anti rabbit antibodies coupled to horseradish peroxidase (1:10000 Dianova, Hamburg, Germany). For the detection of proteins, membranes were incubated with the SuperSignal West Pico Sensitivity Substrate (Thermo Scientific) and visualized by VersaDoc gel imaging system (BioRad).

Statistical analysis
All data are expressed as mean values ± standard error means (SEM). Comparison of the level of neurogenesis with animals of the PDGF strain sacrificed at the age of 4 months was performed based on previously published data generated under identical experimental conditions [11]. Quantified data was normalized to respective wildtype littermate controls. Unpaired, two-sided Student t-test or one-way ANOVA followed by a Tukey's multiple comparison posthoc test was applied with Prism 4 statistics software (GraphPad, San Diego, USA). The significance level was set at p < 0.05 ( Ã ), p<0.005 ( ÃÃ ) and p<0.0005 ( ÃÃÃ ) as indicated.

Comparison to a previously published murine WTS tg model
In PDGF WTS tg mice we previously found a 34% reduction in the number of newly generated GCL neurons, as well as a 59% reduction in newly generated GLOM dopaminergic neurons [11,15]. To compare OB neurogenesis in mThy1 and PDGF WTS animals, we normalized the previously published data [11] and current data with the respective control group of each tg strain and calculated changes in percentage (Fig 3).

WTS expression in mThy1 and PDGF tg mice
The expression levels of WTS mRNA and protein have been reported to be 4-8x higher in mThy1 than in PDGF animals [10,20]. To specifically compare protein levels of a-syn in the OB, we performed Western blot analysis of cortical and OB areas. We found, that expression of a-syn is significantly higher in mThy1 WTS tg mice than in PDGF WTS tg mice (Fig 4), both in the cortex (Fig 4A and 4B) and in the OB (Fig 4C and 4D). We therefore conclude, that the quantity of tg expression has only a minor impact on modulation of adult OB neurogenesis in this model.

Temporal expression pattern of WTS in mThy1 tg animals
In PDGF animals it has been shown that during adult neurogenesis in the SVZ/OB system, tg a-syn is expressed from very early stages of cell maturation, as positive colocalization of a-syn with Doublecortin (DCX), a marker of migratory neuroblasts [21], has been shown in both SVZ and RMS [11,15].
To compare the temporal pattern of tg expression during adult olfactory neurogenesis we performed a detailed immunofluorescence coexpression analysis of the tg WTS with established markers of cell maturation in mThy1 animals (Fig 5). For this we used sex-determining region Y-box 2 (SOX2) as a marker of the stem cell stage [17,22], DCX as a marker for migratory neuroblasts, as well as Calretinin, neuronal nuclear antigen (NeuN) and tyrosine hydroxylase (TH) as markers for mature and dopaminergic OB neurons [17].
In mThy1 animals WTS did not colocalize with markers of early stages of adult OB neurogenesis such as SOX2 and DCX, while coexpression was positive in mature neurons. The temporal expression of WTS therefore profoundly differs between the two tg animal strains, such as that WTS is expressed in a much later maturation phase of a newly generated neuron in mThy1 than in PDGF animals.

Discussion
The neurobiological effect of a-syn on aspects of adult neurogenesis is still not well understood, although in recent years several studies addressed this in both the hippocampal dentate gyrus as well as the SVZ/OB system in tg animal models and murine embryonic stem cells [11,15,[23][24][25][26]. Specifically the use of conditional tg mouse models has proven that the impairment of Temporal Expression of a-Synuclein Modulates Olfactory Neurogenesis adult neurogenesis is reversibly modulated by a-syn [27][28][29]. In this study we investigated the effect of high levels of WTS expression under the control of the mThy1 promoter on different aspects of OB adult neurogenesis and delineated a dissociation of the expression level versus temporal expression pattern on survival and differentiation of new neurons by comparison to the PDGF WTS tg mouse.

Impairment of dopaminergic olfactory bulb neurogenesis may be linked to olfactory deficits in mThy1 animals
Adult OB neurogenesis is regulated by external cues such as odor exposure [18,[30][31][32]. It has been convincingly shown that a continuous turnover of newly generated neurons from the SVZ/OB system that integrate as interneurons to the network of the murine OB optimizes olfactory performance [8,18,33]. Our results indicate unchanged rates for SVZ proliferation and survival of newly generated cells in the GCL and GLOM of the OB in mThy1 animals, but a trend towards a reduction in dopaminergic differentiation in the GLOM (p = 0.07) and a significant reduction of newly generated dopaminergic neurons in the GLOM by 61% (p = 0.04).
Repeatedly studies have found a functional connection between dopaminergic OB neurogenesis and olfactory function in the past: In a murine model of impaired neural progenitor cell migration, reduced levels of dopaminergic OB neurogenesis were associated with deficits in fine olfactory discrimination tasks [34]. Furthermore the knock-out of midkine, a growth factor involved in cell migration and neurogenesis, has been shown to cause a partial loss of dopaminergic neurons and depletion of dopamine in the OB, resulting in pronounced olfactory discrimination deficits [35]. A loss of dopaminergic neurons in the OB GLOM by introducing a dopaminergic lesion (6-OHDA into the OB GLOM) caused olfactory deficits that were restored by endogenous adult neurogenesis within one month on a cellular and two months on a functional level [36].
Previous behavioral studies in mThy1 WTS tg animals used paradigms of odor detection, discrimination and identification and showed-among other sensorimotor deficits-a significant olfactory dysfunction, present at the age of 5-6 months [12,37]. At this time point protein-K resistant a-syn inclusions were reported throughout the OB [37]. Our results of decreased levels of OB neurogenesis in this murine model offer an additional path of explanation for this behavioral deficit and confirm previous speculations in this direction by Fleming and colleagues [38].

Temporal expression of a-Synuclein separately influences cell survival and differentiation in newly generated OB neurons
We found higher levels of WTS expression in the OB of mThy1 than PDGF tg animals, confirming previous studies [10,14,37]. The significant reduction of newly generated dopaminergic neurons in the GLOM of mThy1 animals was due to a reduction in dopaminergic differentiation at unchanged levels of proliferation and survival. In contrast, the marked deficits in adult OB neurogenesis in PDGF animals were mainly due to decreased cell survival (-50%) at an unchanged level of neuronal (GCL) and dopaminergic (GLOM) cell differentiation resulting in decreased numbers of newly generated neurons by 50% in the GCL and 59% in the GLOM [11].
Reports from cell-free experimental systems, yeast, neurons derived from mouse embryonic stem cells, as well as human SCNA-gene duplications and triplications indicate a dose-dependent effect of a-syn toxicity [39][40][41][42][43]. WTS expression in mThy1 brains has previously been shown to be 4-8x higher than in the PDGF strain on an mRNA level [10,20], which we confirmed by demonstrating a significant difference in a-syn protein expression. In comparison to the severely impaired OB neurogenesis found in PDGF animals, the relatively mild impairment of adult olfactory neurogenesis in mThy1 animals shown in this work does not support a dosedependent a-syn toxicity for adult olfactory neurogenesis.
Our interpretation is that not only the expression level, but in particular the temporal expression of WTS in tg mThy1 and PDGF animals seems to influence aspects of adult neurogenesis. The coexpression analysis in this study showed that WTS tg expression during the maturation process of a newly generated neuron commences at a much later stage under mThy1, than under PDGF promoter control. In addition, data from the phylogenetic activity of both promoter elements point in the same direction: while mThy1 reaches a stable expression from postnatal day 7 onwards [44], PDGF is already prenatally active [45].
There are substantial differences in the temporal expression of WTS in PDFG and Thy-1 promoter mice within the neurogenic niche. We speculate that this difference in the temporal expression of WTS accounts for differences in neuroblast survival and differentiation between mThy1 and PDGF animals: earlier expression of WTS during a crucial phase of maturation has a more pronounced effect on cell survival, whereas later expression has a greater influence on neuronal differentiation (Fig 5). The sequence of changes during cell maturation in adult OB neurogenesis are well-known: during the DCX-positive neuroblast phase of neuronal maturation, migration of cells and dendritic growth are regulated while spine formation and growth follow later, when cells become NeuN-positive [46]. The time course of tg expression might therefore influence certain time-dependent sequences of neuronal maturation such as e.g. cell differentiation or spine formation. This is also corroborated by findings in human embryonic stem (ES) cell cultures that, when transfected with lenti-viral vectors coding for WTS during late stages of differentiation, have been found to exhibit a decreased rate of dopaminergic differentiation [47]. On the other hand several reports describe that murine ES cell cultures transfected with WTS at an undifferentiated, early stage did not show changes in their dopaminergic differentiation capacity [42,48].
One major difference between the PDGF and the mThy1 WTS mice is a different expression pattern of human alpha-synuclein. While in mThy1 mice (line 61) WTS expression can be found mainly in pyramidal neurons throughout the brain and in highest expression in neocortex, hippocampus, olfactory system, thalamus, substantia nigra and brain stem, in PDGF mice (line D), WTS is mainly expressed in the neuropil and non-pyramidal cells of the neocortex, hippocampus and olfactory system [10].
The PDGF promoter does not lead to a major expression of WTS in the nigral system and dopaminergic neurons, while the ubiquitous tg expression under mThy1 promoter control includes the areas containing TH-expressing cells and fibers like the olfactory bulb, striatum, and substantia nigra. It has been observed in dopaminergic lesion models that nigro-striatal denervation can modulate SVZ cell proliferation and increase dopaminergic neurogenesis in the GLOM of the olfactory bulb [49,50]. While one might speculate that widespread WTS expression in the brain and dopaminergic system might lead to major modulations in adult neurogenesis, our results in the Thy-1 promoter model show that this is not the case at the age of 5 months.
PDGF WTS tg mice have been reported to show glial expression of WTS [10]. Although this is found to a lesser extent in the line used in this study (line D), and the low WTS expression in neurogenic niches, it nevertheless cannot be excluded that glial WTS expression might add to the neurogenesis deficit in the PDGF WTS tg strain.

Conclusion
In conclusion, this study provides evidence for reduced dopaminergic adult OB neurogenesis in the murine tg mThy1 WTS model due to changed differentiation but not survival of newly generated neurons. This might explain the known olfactory deficits in these mice. Furthermore, the comparison of neurogenesis deficits and tg expression kinetics in different WTS tg mice points beyond a dose-dependent effect of WTS on adult OB neurogenesis. We for the first time show a dissociation of expression levels and their detrimental effect on adult OB neurogenesis in mouse models of synucleinopathies. In the two WTS models compared in this study, the early onset of WTS expression during the maturation of an individual neuron has a stronger effect on levels of adult neurogenesis than a later but much higher tg expression level, providing evidence for the importance of temporal expression characteristics in WTS related experimental neurodegeneration (Fig 6).
This finding may not only help to understand results of adult neurogenesis studies due to promoter differences, but also expands the dimensions of expression characteristics to be Temporal Expression of a-Synuclein Modulates Olfactory Neurogenesis studied in future tg models of synucleinopathies-along cell-specificity, anatomy and dose, also temporal tg expression characteristics should be addressed.