Neurodegeneration and contralateral α-synuclein induction after intracerebral α-synuclein injections in the anterior olfactory nucleus of a Parkinson’s disease A53T mouse model

Forty-four adult male wild-type (WT, B6C3F1/J) and homozygous transgenic (TG, B6; C3-Tg-Prnp-SNCA*A53T- 83Vle/J; 004479, The Jackson Laboratory, USA) mice were initially used for this study. One group of WT animals was used for tract-tracing experiments. Behavioral analyses were carried out on the remaining animals pre- and post-injection of α-synuclein or saline prior to sacrifice after either 2 or 4 months (Fig. 1a). The initial design of the study thus included nine experimental groups: n = 4 for tract-tracing experiments and n = 5 each for α-synuclein or saline injections, in TG or WT animals, surviving either 2 or 4 months. At the time of the injection, the average age of animals was 46.63 ± 0.18 (SEM) weeks. Given the high degree of mortality among animals after the injection (especially among TG), TG groups to be sacrificed after 4 months were not included in the final analysis; instead, they were incorporated into the group to be sacrificed after 2 months. Mice were thus divided into seven groups at the end of the investigation. Group 1: WT tract-tracing dextran amine experiments (n = 4); groups 2 and 3: TG α-synuclein (n = 3) or saline (n = 4) surviving 2 months; groups 4 and 5: WT α-synuclein (n = 4) or saline (n = 4) surviving 2 months; and groups 6 and 7: WT α-synuclein (n = 4) or saline (n = 5) surviving 4 months; totaling 28 animals at the end of the study. The animals were housed in experimental groups on a standard 12 h/12 h light/dark cycle, at 21 °C, with food and water ad libitum. All experiments carried out were in agreement with European (Directive 2010/63/EU) and Spanish (RD 53/2013) regulations on the protection of animals used for scientific purposes, and they were also approved by the Ethical Committee for Animal Research of the University of Castilla-La Mancha (SAF2016–75768-R).

Four animals were anesthetized with a combined dose of ketamine hydrochloride (Ketolar, Parke-Davis, Madrid, Spain; 1.5 mL/kg, 75 mg/kg) and xylazine (Xilagesic, Calier, Barcelona, Spain; 0.5 mL/kg, 10 mg/kg). Eye drops (Lacryvisc, Alcon, Barcelona) were applied to prevent eye ulceration during surgery. Under stereotaxic control (anterior 2.8 mm; lateral 1 mm; depth − 2.7 mm, from dura mater) [22], rhodamine-labeled dextran amine (RDA) and fluorescein-labeled dextran amine (FDA) (10,000 mW, lysine fixable, Molecular Probes, Eugene, OR; 10% diluted in PBS) were iontophoretically injected (30–80 μm tip diameter; positive current pulses 7/7 s; 2–7 μA; 8–20 min) at the right and left dorsal AON, respectively.

Animals injected with tracer were perfused one week post-injection. Animals injected with α-synuclein or saline were perfused either 2 or 4 months after injection. Mice were anesthetized (as described above) and perfused with saline solution followed by 4% w/v para-formaldehyde fixative (phosphate buffered; 0.1 M sodium phosphate, pH 7.2). Brains were post-fixed in 4% w/v para-formaldehyde and cryoprotected in 30% w/v sucrose. Finally, brains were frontally sectioned using a freezing microtome, and 50-μm sections were sequentially collected in eight-section series.

Sections were counterstained with 4′6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Co. Ltd.) for 5 min in the dark, mounted and coverslipped with antifading polyvinyl alcohol mounting medium (Sigma-Aldrich Co. Ltd.). Images were taken using a confocal microscope LSM 800 (Zeiss, Jena, Germany). Contrast and brightness were adjusted using Image J software.

For α-synuclein and ionized calcium-binding adaptor molecule 1 (Iba-1) immunohistochemistry, endogenous peroxidase activity was inhibited by bathing twice for 15 min in 1% H₂O₂ in phosphate-buffered saline (PBS) (0.01 M pH 7.4). After washes, sections were incubated for 2 h in blocking buffer (0.3% TX-100, 5% normal horse serum with PBS 0.01, pH 7.4). Subsequently, sections were incubated at 4 °C overnight in blocking buffer with primary antibodies (Additional file 1: Table S1). For neuronal marker (NeuN) and glial fibrillary acidic protein (GFAP) immunohistochemistry, endogenous peroxidase activity was inhibited by a 20-min bath in 1% H₂O₂ in PBS. After washes, sections were incubated for 2 h in PBS with 0.1% TX-100 (Iba-1) or 0.3% TX-100 (NeuN). Then sections were incubated in PBS with 0.1% TX-100 (Iba-1) or 0.3% TX-100 (NeuN) with primary antibodies at 4 °C overnight (Additional file 1: Table S1).

After washes, sections were incubated for 2 h in secondary biotinylated antibodies in blocking buffer (Additional file 1: Table S1); for NeuN and GFAP, sections were incubated without normal horse serum. Sections for immunohistochemistry were incubated in avidin-biotin complex (ABC Standard, Vector Laboratories), reacted using 0.025% 3,3′-diaminobenzidine and 0.1% H₂O₂. Then sections were cresyl violet counterstained (Sigma Aldrich, C5042). The protocol was as follows: dip in distilled H₂O for 2 min; incubate slides in cresyl violet 0.1% for 1 min; take out the slides, removing the excess by tapping on the container; quickly dip the slides 8 times in 96% ethanol, and then 8 times in 100% ethanol; leave slides in xylene I for 1 min and xylene II for 2 min; coverslip with DPX (Sigma Aldrich, 06522). Controls included omitting primary antibodies, secondary antibodies, and using tissue from WT animals.

Quantification was carried out using Stereo Investigator software (MBF Bioscience; microscope Zeiss Axio Imager M2), which is composed of an optical fractionator stereology probe. Stereological techniques are unbiased three-dimensional counting methods using random frames over regions of interest. This probe estimates the number of human α-synuclein aggregates found in cell bodies (aggregates in neurites were not considered), NeuN or Iba-1-positive cells and sub-volumes in the optical dissector (virtual space) in a thick tissue section. Afterwards, these sub-volumes were extrapolated to estimate the entire cell population (cells/mm³). The methodology is based on detection of the top-edge of the cell in the dissector and having enough focal planes to determine whether it is in the dissector, using a 63× oil objective and z-axis. This rule is useful to prevent overestimation. Prior to counting aggregates or cells, data such as the number of sections, thickness (50 μm) and interval between sections (eight) were added to the software [26].

OB, AON and piriform cortex (Pir) boundaries were traced using cresyl violet staining at 5× magnification. In addition, OB layers (glomerular, GL; external plexiform, EPL; mitral, MiL; internal plexiform, IPL and granule, GrL layers) were also drawn for Iba-1 staining. Tissue was then examined at high magnification (Plan Apochromat, 63×/ 1.4, Oil lens, Ref: 420782–9900). The parameters of grid size and counting frame were adapted to α-synuclein, NeuN and Iba-1 markers and the size of different layers (Additional file 1: Table S2). Section thickness was measured at each counting site. Both α-synuclein aggregates, NeuN and Iba-1-positive cells were counted by placing a marker on each within the counting frame whilst not touching the red exclusion line.

Given the number of fibers in GFAP-positive cells, optical dissector analysis was ruled out. The images, taken with a Nikon Eclipse 80i, were captured from each section and hemisphere (OB n = 2, 6–9 sections per hemisphere; AON n = 2, 3–4 sections per hemisphere; Pir n = 2, 4–5 sections per hemisphere; 24 animals; N = 1550 pictures). Images were processed with ImageJ and converted to 8-bit gray scale. Afterwards, we analyzed the generated histogram. The histogram mode was multiplied by 0.7 to obtain the threshold and measure the area fraction.

Statistical analysis was performed using Graphpad Prism software (v6.01; La Jolla, CA). A Shapiro-Wilk test (N < 50) was carried out to analyze the normality. A statistical comparison was analyzed using two-way ANOVA (Tukey post-hoc test) or Kruskal-Wallis non-parametric test (Dunn’s multiple comparisons test), with the following variables: treatment (saline or α-synuclein injection), genotype (WT or TG), time (only for WT, 2 or 4 months survival) or hemisphere (left, LH, or right, RH). Two-tailed and Mann-Whitney tests were only applied when the P-value of the source of variation (treatment, genotype, time or hemisphere effect) was significant. Differences were regarded as statistically significant at * or #P < 0.05; **or ##P < 0.01; *** or ###P < 0.001; **** or ####P < 0.0001. Significant results are described in the text and/or figures. Statistical data are included in the Additional file 2: Figure S1-S4, Additional file 3: Tables S3–S7 and Additional file 4: Tables S8-S17.

Animals were weighed during the experimental procedure. Weight loss was observed in TG as compared to WT mice, even before injections (Interaction: F (3, 64) = 1.120, P = 0.3478; Treatment effect: F (3, 64) = 0.5025, P = 0.6819; Genotype effect: F (1, 64) = 39.07, P < 0.0001). (Fig. 1b). However, no changes were noticed in WT mice as a function of post-injection time (Interaction: F (3, 86) = 0.4156, P = 0.7420; Treatment effect: F (3, 86) = 1.975, P = 0.1237; Time effect: F (1, 86) = 0.4801, P = 0.4902) (Fig. 1b).

In order to have our own material available to analyze the AON connections, RDA and FDA were injected in the right (Fig. 2a) and left (Fig. 2b) dorsal parts of the AON, respectively. This way, ipsi- and contralateral projections could be easily compared. Axons decussate through the anterior commissure (Fig. 2c). The AON is a secondary olfactory structure and connects to the contralateral AON (Fig. 2d, e). It also connects bilaterally with primary olfactory structures, such as the OB (Fig. 2f) and, more frequently, ipsilaterally with other secondary olfactory structures such as the Pir (Fig. 2g), the anterior cortical amygdala (Fig. 2h), the olfactory tubercle (Fig. 2i) and the entorhinal cortex and hippocampus (Fig. 2j).

The AON is preferentially involved in proteinopathies at an early stage, not only in PD [2, 15, 72, 76, 80], but also in Alzheimer’s disease [68], and it has been hypothesized that this is due to its multiple connections [67, 79]. The human AON is a structure composed of several subdivisions [77], whose connections are only inferred in the human brain from comparative data [79], which is scarce in primates [54] but more abundant in rodents [9, 53]. The results presented here (Fig. 2) confirm that the AON is a secondary bulbar structure, reciprocally and bilaterally connected to other secondary olfactory structures, and also to at least 27 non-olfactory structures [9]. Ipsilaterally, the AON is reciprocally connected to the OB [53] (Fig. 2). Contralaterally, afferent projections to the AONd are mainly originated in the AON and secondarily in the OB (Fig. 2). Accordingly, contralateral and trans-synaptic involvement or related connections should be considered in our α-synucleinopathy results. Spreading from AON to basal ganglia, including substantia nigra and caudate-putamen, cannot discarded via indirect pathways [79].

In order to evaluate α-synuclein spreading along the olfactory anatomical pathways [79], α-synuclein or saline were injected into the right AON. In fact, this nucleus was chosen because it could be the starting point for spreading the pathology in the brain. Actually, although analyzing nigro-striatal projection involvement was not the main objective of the study, exciting findings were reached (Additional file 4: Figure S7 and S8). Our observations confirmed that α-synucleinopathy was present in caudate-putamen and, in contrast to the original description of A53T mouse model, also in the substantia nigra [24]. Interestingly, this α-synucleinopathy in A53T model appears to be enhanced by α-synuclein injection. These aggregates were not found in WT animals (Additional file 4: Figure S7 and S8).

Although multiple studies have injected different kinds of α-synuclein in mice (preformed fibrils, oligomers, human extracts and sick mice extracts), those focused on OB/AON injections are scarce [51, 63, 64, 74]. On the other hand, A53T is a useful α-synucleinopathy model that expresses α-synuclein in olfactory structures. Previous reports of our work describe maximum synucleinopathy levels over 40 weeks [20, 21, 78, 80]. As far as we know, the present report is the first to include injections restricted to the AON in both WT and TG mice. Our results show that α-synuclein aggregates were mostly found in the contralateral hemisphere (left OB and AON) after saline injections, which could be attributable to basal interhemispheric differences (Fig. 4). This observation is very relevant in the context of unresolved motor asymmetry reported in PD [16]. Another interesting observation is that there is a trend for a denser labeling in the contralateral hemisphere of α-synuclein-injected TG as compared to saline-injected TG animals, particularly in the OB (Fig. 4). This could be related to the previously described contralateral projections (Fig. 2), indicating α-synucleinopathy retrograde induction (seeding and/or spreading). Endogenous α-synuclein expressed in TG animals would be necessary to allow exogenous injected α-synuclein to increase synucleinopathy in the contralateral hemisphere. Thus, we suggest that exogenous α-synuclein is able to induce seeding and/or spreading to the contralateral hemisphere in TG animals. In WT animals, α-synuclein was only detected at the injection site. These results could be due to different factors: susceptibility to a prior seeding, time-dependent spreading or reactivity of different molecular α-synuclein species. First, it has been reported that α-synuclein preformed fibrils-inoculated A53T mice enhanced the conversion of endogenous α-synuclein into pathological forms, acting as a template. This has been reported not only in vivo, but also in vitro, since α-synuclein aggregation could occur as a nucleation-dependent mechanism [48]. Second, the post-injection periods vary in different studies, ranging from 72 h to 30, 90 or 180 days [48, 64, 66]. Third, despite α-synuclein oligomerizing and aggregating into fibrils under pathological conditions, it has been reported that monomers and oligomers were more rapidly transferred from OB of WT to interconnected regions as compared to fibrils [64]. Therefore, it would be interesting to have this kind of molecule available to improve the injection results; α-synuclein recombinants are larger, which possibly makes spreading more difficult, thus requiring more time. In agreement with our results, α-synuclein fibrils have been intrahippocampally injected in TG and WT mice, without α-synuclein spread from the injection site having been detected in WT after 2 months [66].

PD is characterized by neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta and the corresponding denervation in the striatum [39]. Interestingly, in patients, this has been correlated with neuronal loss in the OB [35].

Neurodegeneration has been checked using NeuN labeling in different mouse PD models, including 6-OHDA injections [46], adeno-associated-virus vector (AAV9) for α-synuclein [57] and transgenic mouse overexpressing human α-synuclein under the Thy-1 promoter (Thy-α-syn) [11]. Bilaterally, in OB, AON and Pir, our results showed that the number of NeuN-positive cells/mm³ (density) was higher in saline-injected WT group as compared to saline-injected TG mice at 2 months. Given that these differences are not observed in α-synuclein injected animals, we suggested that, in WT mice, α-synuclein injection might be also involved in neurodegeneration. In fact, in Pir, NeuN density decreased in α-synuclein injected WT as compared to saline-injected WT group.

No differences were noticed in TG animals, regardless of injection and hemispheres. It could be due to the fact that A53T mouse model constitutively expresses human α-synuclein gene, therefore α-synuclein injection could provoke minor induction on neurodegeneration. As far as we know, this is the first report describing unbiased NeuN counting in olfactory structures in A53T mouse model.

Regarding α-synucleinopathy and neurodegeneration, despite of our results show basal interhemispheric differences in α-synuclein aggregates in saline-injected TG in OB and AON, there is not correlation to neuronal loss. Likewise, although data show an increase of α-synuclein between saline-injected TG and α-synuclein-injected TG in left OB, no differences were observed in NeuN counting.

The density of NeuN differences between saline-injection TG and saline-injection WT could be due to transgene. These differences disappeared in α-synuclein injected animals. So, it could be as a consequence of exogenous α-synuclein inoculation. In WT mice, although our findings indicated that α-synuclein was restricted to the injection site (right AON), neuronal loss was significantly observed in Pir. It could be due to the fact that our α-synuclein antibody is able to label α-synuclein aggregates but no other conformations such as fibrils or soluble forms, which could act on neurodegeneration as well.

Both microglia and astroglia have been involved in PD progression [32] and associated α-synucleinopathy [7].