Direct detection of alpha synuclein oligomers in vivo
- Hemi Dimant1,
- Suneil K Kalia†1, 2,
- Lorraine V Kalia†1, 3,
- Liya N Zhu1,
- Laura Kibuuka1,
- Darius Ebrahimi-Fakhari1, 4,
- Nikolaus R McFarland1, 5,
- Zhanyun Fan1,
- Bradley T Hyman1 and
- Pamela J McLean1, 6Email author
© Dimant et al.; licensee BioMed Central Ltd. 2013
Received: 07 February 2013
Accepted: 14 March 2013
Published: 09 May 2013
Rat models of Parkinson’s disease are widely used to elucidate the mechanisms underlying disease etiology or to investigate therapeutic approaches. Models were developed using toxins such as MPTP or 6-OHDA to specifically target dopaminergic neurons resulting in acute neuronal loss in the substantia nigra or by using viral vectors to induce the specific and gradual expression of alpha synuclein in the substantia nigra. The detection of alpha- synuclein oligomers, the presumed toxic species, in these models and others has been possible using only indirect biochemical approaches to date. Here we coinjected AAVs encoding alpha-synuclein fused to the N- or C-terminal half of VenusYFP in rat substantia nigra pars compacta and describe for the first time a novel viral vector rodent model with the unique ability to directly detect and track alpha synuclein oligomers ex vivo and in vivo.
Viral coinjection resulted in widespread VenusYFP signal within the nigrostriatal pathway, including cell bodies in the substantia nigra and synaptic accumulation in striatal terminals, suggestive of in vivo alpha-synuclein oligomers formation. Transduced rats showed alpha-synuclein induced dopaminergic neuron loss in the substantia nigra, the appearance of dystrophic neurites, and gliosis in the striatum. Moreover, we have applied in vivo imaging techniques in the living mouse to directly image alpha-synuclein oligomers in the cortex.
We have developed a unique animal model that provides a tool for the Parkinson’s disease research community with which to directly detect alpha- synuclein oligomers in vivo and screen therapeutic approaches targeting alpha-synuclein oligomers.
KeywordsParkinson’s disease Alpha synuclein Oligomers Animal model Live imaging
Alpha synuclein (α-syn) aggregation has been closely linked to Parkinson’s disease (PD) etiology. Lewy Bodies, the pathological hallmark of PD are enriched with alpha synuclein fibrils [1, 2] and point mutations and gene multiplications of the SNCA gene, which are associated with increased α-syn accumulation, result in familial PD [3–5]. Genome wide association studies have also demonstrated that single nucleotide polymorphisms in the SNCA loci may be a risk factor for idiopathic PD (Edwards 2010). Although the exact mechanism of α-syn induced toxicity remains unknown, recent observations allude to soluble α-syn oligomers being neurotoxic [6–9].
Rat models of PD have been developed based on the putative link between alpha synuclein and PD. First generation PD rat models employed neurotoxins such as 6-OHDA (6-hyroxydopamine) or MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) which acutely degenerate dopaminergic (DA) neurons in the substanatia nigra (SN), but do not result in significant α-syn pathology . Second generation models use viral vectors to target α-syn expression in the SN, resulting in a more gradual expression of α-syn with accompanying dopaminergic cell loss, which more closely resembles the chronic pathology of PD [11–14]. In these models and others, the detection of α-syn oligomers is based upon indirect approaches and biochemical techniques, but none offers direct detection of α-syn oligomers in vivo. In this study we present a third generation rat model of PD using bimolecular protein complementation assay (PCA) to enable the direct detection and visualization of α-syn oligomers along the nigrostriatal pathway. PCAs have been successfully applied to image protein-protein interactions in vivo based on a chemiluminescence signal from protein-luciferase conjugates, typically by transplanting immortalized cells expressing the protein-protein complementation pairs into organs of living mice . While supporting the feasibility of complementation in vivo, none of the PCAs described to date have directly introduced each protein fragment separately and none have done so using fluorescence as an output rather than chemiluminescence.
The PCA approach, successfully applied in our laboratory to detect and image α-syn oligomers in vitro [7, 8, 16–18], is demonstrated here in vivo by viral mediated expression of human α-syn fused to either the N- or C- terminus half of venusYFP. Formation of fluorescently labeled α-syn oligomers is directly visualized along the nigrostriatal pathway ex-vivo in rat brain and in cortical neurons in vivo in a living mouse brain. Our novel approach for the direct detection of α-syn oligomers in vivo provides a powerful tool to study the role of α-syn oligomers in PD and to explore therapeutic approaches targeting α-syn oligomerization.
Direct detection of alpha synuclein oligomers in the rat nigrostriatal pathway
Alpha synuclein associated toxicity
Stereological analyses revealed 43% TH cell loss in animals co-injected with V1S + SV2 (Figure 2b). As expected a significant 51% TH cell loss was observed in the group of animals expressing only SV2, where oligomers are presumed to form but are not detectable (Figure 2b). A reduction in TH staining is also noticeable in the striatum, further conforming lesion formation (Figure 2c). In control animals expressing full length venus protein we observed an insignificant 15% TH cell loss consistent with the absence of a lesion (Figure 2b). These data demonstrates an α-syn induced toxicity resulting in neuronal cell loss.
Direct visualization of alpha synuclein oligomers in the striatum
Neuroinflammation in the striatum
In vivo imaging of alpha synuclein oligomers in the living mouse
We herein present a novel in vivo protein complementation approach to directly detect α-syn oligomers in rodent brain. We demonstrate the successful targeted delivery of two AAVs into the substantia nigra (SN) and the direct ex-vivo imaging of α-syn oligomers in the nigrostriatal pathway including the SN and the striatum. We demonstrate α-syn induced toxicity as measured by a reduction in TH-positive neurons in the SN as well as gliosis (astrocytosis) in the striatum. We also demonstrate that multi-photon imaging can be applied to specifically image α-syn oligomers in the brains of living mice.
In recent years studies have shown that α-syn oligomers play a key role in PD etiology, shifting the blame from mature α-syn fibrils, such as in Lewy bodies, to soluble oligomers [6, 23, 24]. Previous studies conducted in our laboratory have shown the applicability of PCA to detect α-syn oligomers in vitro and have provided a means to further dissect the role of α-syn oligomers in PD [7, 8, 16–18]. Here we show for the first time the advantage of this powerful technique to detect and visualize α-syn oligomerization in vivo.
AAVs (V1S + SV2) were injected into the SN of Sprague Dawley rats and incubated for a period of 8 weeks, at the end of which histological analysis was conducted to validate the detection of α-syn oligomers. VenusYFP fluorescence is detected in the SN including the pars compacta (SNpc) and pars reticulata (SNpr) and to some extent in the ventral tegmental area (VTA). This demonstrates the direct delivery and expression of 2 viruses which results in in vivo α-syn oligomer formation. Control animals injected with only one of the viruses, either V1S or SV2, did not exhibit any fluorescence in the injection site, even though they presumably form oligomers as well . Looking at neurons in the SN, we detect fluorescence in neurites, neuronal cell bodies, and the nucleus, as suggested by our inability to delineate nuclei . Quantitation of tyrosine hydroxylase immunopositive neurons in the SN revealed an average lesion of 45% in animals expressing α-syn regardless of whether we could directly detect oligomers via fluorescence or not, in accordance with previously reported α-syn-induced lesions in viral vector rat models [11–14, 27]. These results indicate that bimolecular fluorescent PCA with α-syn-Venus constructs does not artificially enhance α-syn toxicity, but rather is strictly an assay for α-syn oligomer formation. Furthermore, in animals expressing only full length venusYFP, no significant TH cell death was detected, demonstrating that neither the expression of full length venusYFP nor the injection procedure itself promotes a lesion. The ability to directly detect α-syn oligomers in vivo offers a powerful tool to elucidate the molecular mechanisms of α-syn mediated neurotoxicity and to examine α-syn based therapies.
Dopaminergic neurons project to the striatum along the nigrostraital pathway. Accordingly, we detect α-syn oligomers in the striatum of rats injected in the SN, with venusYFP fluorescence. This result demonstrates our ability to detect and visualize α-syn oligomers along the nigrostraital pathway but also confirms the successful targeting of the AAVs to neurons in the SN. Interestingly, in the striatum of rats expressing α-syn we observed distinct punctate fluorescence and dystrophic axons, compared to the diffuse venusYFP fluorescence and the healthy appearing axons observed in control animals expressing only venusYFP. The punctate staining, observed only in the striatum of α-syn expressing rats, was suggestive of α-syn aggregation, an intriguing observation in light of recent evidence demonstrating the synaptic localization of small α-syn aggregates in DLB patients, linking α-syn aggregation to synaptic pathology . Proteinase K (PK) treatment reduced venusYFP fluorescence in the striatum including puncta and axons. α-Syn staining was likewise markedly decreased following PK treatment suggesting that α-syn accumulation in the striatum represent soluble α-syn oligomers.
Indeed this finding, taken together with accumulating evidence implicating α-syn in disease progression via a non-cell-autonomous neuroinflammatory process [29–31], led us to investigate whether inflammation occurs concomitantly with the aggregation of α-syn in the striatum and the presence of dystrophic neurites. Iba1 immunostaining showed no difference in microglial activation in response to V1S + SV2 expression or venusYFP control. However, GFAP analysis showed a small but significant elevation in GFAP expression ipsilateral in the V1S + SV2 group compared to that in control animals expressing only venusYFP. In accordance with our observation, asctrocytic gliosis is commonly reported in rodents models of α-synucleinopathies .
The uniquely designed PCA presented here has the advantage to detect and visualize α-syn oligomerization in post mortem tissue with minimal tissue processing, but could potentially be utilized to monitor the formation of α-syn oligomers over time in the brain of a living animal using two-photon microscopy. Recent advances in two-photon microscopy have enabled in vivo visualization of protein aggregation and neurodegeneration in the brain of an Alzheimer’s disease mouse model [22, 33, 34] and protein degradation in the brain of a PD animal model [35–37]. Here, using two-photon microscopy, we demonstrate the ability to image and detect α-syn oligomers in vivo. High-resolution images showed venusYFP fluorescence in neuronal cell bodies and axons, in agreement with a previous report examining cellular localization of GFP-tagged α-syn using two-photon microscopy [35, 36]. We have also imaged α-syn oligomers in dendrites and dendritic spines, although we cannot rule out the possibility that the presence of a-syn in dendritic spines is an artifact of overexpression. Nonetheless, although α-syn is a pre-synaptic protein, our ability to image α-syn in the synapse may provide insight into the effects of α-syn on dendritic spines, which have been shown to degenerate in DLB patients  and thus may play a role in disease progression through trans-synaptic mechanisms. It is important to emphasize that multi-photon imaging of neurons in the brain of a living animal bestows the ability to perform longitudinal studies on individual neurons which, coupled with our ability to directly detect α-syn oligomers using fluorescence, offers a powerful tool with which to monitor α-syn aggregation and examine its direct effect on neurons over time.
Taken together, we present here a unique animal model to detect and monitor α-syn oligomer formation in the brain of rodents. Our animal model will give researchers the opportunity to better understand α-syn associated pathogenesis specifically in the context of α-syn oligomers in vivo but also offers great therapeutic screening potential for therapeutic agents that target α-syn oligomers.
The viral vectors pAAV-CBA-VENUS1-SYNUCLEIN-WPRE (V1S) and pAAV-CBA-SYNUCLEIN-VENUS2-WPRE (SV2) were constructed by inserting the human SNCA gene, fused to either the N-terminus half of venusYFP (V1S) or the C-terminus half of venusYFP (SV2), into the EcoRV and NheI sites of the pAAV-CBA-WPRE vector . pAAV-CBA-VenusYFP-WPRE was constructed by inserting the venusYFP gene into the XhoI and NheI sites of pAAV-CBA-WPRE vector. AAV serotype 8 was produced by the Harvard Gene Therapy Initiative as previously described .
Sprague Dawley rats (300–350 gram) were anesthetized with intraperitoneal injection of ketamine/xylazine and placed in a stereotaxic frame. The surgical site was shaved and sterilized with betadine prior to making a 2 cm incision along the midline. The scalp was exposed and a unilateral injection targeting the SN was preformed at coordinates AP -5.2, ML -2 and DV -7.4 with bregma as a point of reference. For each animal, a total volume of 2 μl of virus was injected at a rate of 0.2 μl/min using a microinjection pump and 10 μl Hamilton syringe with a 30-gauge needle. For V1S + SV2 group 1 μl of AAV8- V1S (8.3•1012 viral genome/ml) and 1 μl of AAV8-SV2 (8.7•1012 viral genome/ml) were injected; for the venusYFP group and the SV2 group, 2 μl of AAV8-venus (1•1012 viral genome/ml) and 2 μl of AAV8-SV2 were injected, respectively. At the end of injection the needle remained in place for 5 minutes before gradual removal.
For in vivo imaging, C57 mice were anesthetized with 2% isoflorane and placed in a stereotaxic frame above a heated blanket. The surgical site was sterilized with betadine and isopropanol prior to conducting a 1 cm incision along the midline. The scalp was incised and an injection targeting layers 2–3 of the cortex was preformed at coordinates AP -1, ML -1 and DV -0.7 with bregma as a point of reference. A total volume of 2 μl (1 μl V1S + 1 μl SV2) was injected at a rate of 0.1 μl/min using a microinjection pump and 10 μl Hamilton syringe with a 33-gauge needle. After 4 weeks a cranial window was installed as described previously . All studies were performed with the approval of the Massachusetts General Hospital Animal Care and Use Committee and in compliance with the National Institute of Health guidelines for the use of experimental animals.
At 8 weeks post-injection, rats were deeply anesthetized and transcardially perfused with 0.01 M phosphate buffered saline (PBS, pH7.4, Sigma) followed by 4% paraformaldehyde in PBS. Brains were post-fixed for 24–72 hours and transferred to a solution of 30% sucrose in PBS. Frozen brain were sectioned at 40 μm on a sliding microtome and kept in a cryoprotectant solution (30% sucrose, 30% ethylene glycol in PBS).
Free-floating coronal sections were washed overnight in PBS to remove cryoprotectant. Unless otherwise stated, all steps were performed at room temperature with three 10 min washes in PBS-TX (0.3% triton X-100) between each step. For diaminobenzidine (DAB) staining sections were treated with 10% methanol and 3% H2O2 to inhibit endogenous peroxidases, permeabilized in PBS-TX for 30 min and blocked in 5% NGS in PBS-TX for 30 min. Sections were incubated with primary antibody rabbit anti- TH (1:10 K, Millipore) or mouse anti α-syn (4B12 1:1000, Signet) over night at 4°C followed by secondary goat anti rabbit- biotin or goat anti mouse-biotin (1:200 Jackson ImmunoResearch) for 1 hour at room temperature and either avidin-biotin (Vectastain kit) or streptavidin-alexa555 for 1 hour at room temperature. Sections were incubated with DAB (Vector laboratories) to visualize TH positive cells, rinsed in PBS, mounted on superfrost slides (Fisher scientific) and coverslipped (Permount, Sigma). For immunofluorescence, sections were mounted onto superfrost, coverslipped with vectashield (Vector laboratories) and kept at 4°C.
Striatal coronal sections were mounted onto slides and dried prior to rehydration in 0.5 M Tris buffered saline containing 0.05% Tween-20 (TBST, pH7.4). Digestion of mounted sections was carried out with 50 μg/ml proteinase K at 55°C for 120 min. Sections were post fixed with 4% PFA for 30 min at room temperature and washed with TBST. Immunostaining of treated and non-treated sections with mouse anti α-syn clone 4B12 was conducted as described in the immunostaining section.
Microscopy and stereology
Fluorescence images were obtained on either a Zeiss LSM510 META confocal microscope with X20 magnification or a Zeiss Axio observer inverted microscope, X5 magnification. In vivo images of anesthetized mice were obtained using a Bio-Rad 1024ES multiphoton microscope mounted on an Olympus BX50WI upright microscope. Dopaminergic neuron analysis was performed with an Olympus BX51 microscope. At least 8 coronal sections per animal throughout the SN were analyzed to determine tyrosine hydroxylase cell loss by conducting unbiased stereology cell counting according to the optical fractionator principle  using the Olympus CAST stereology software. At least 4 coronal sections for GFAP and 3 for Iba1 per animal, were analyzed to determine the level of protein expression throughout the striatum using high definition images obtained by automated collection and stitching of X5 images using the MetaMorph software (MetaMorph). The striatum, ipsilateral and contralateral, was outlined and the mean intensity in the region of interest was determined using FIJI (NIH).
GFAP and Iba1 expression was analyzed using Mann Whitney test. Data is expressed as group median with interquartile range. TH cell count was analyzed using one tailed t-test. Data is expressed as group mean ± SD. Statistical analysis was performed using GraphPad Prism software.
The authors thank Michal Arbel and Eloise Hudry for their help with craniotomy surgeries and two-photon microscopy and Steve Rodriguez for his help with fluorescent microscopy. P.J.M is supported by NIH NS063963. H.D is supported by Ruth L. Kirschstein National Research Service Award (NRSA) Institutional Research Training Grants (T32). D.E.-F. received support from the German National Academic Foundation (Studienstiftung des deutschen Volkes e.V.) and the Parkinson’s Disease Foundation. N.R.M. is supported by K08 NS067024. L.V.K. is supported by a Canadian Institutes of Health Research Clinician-Scientist Award. This paper is dedicated to Laura Kibuuka.
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