Ataxin-1 oligomers induce local spread of pathology and decreasing them by passive immunization slows Spinocerebellar ataxia type 1 phenotypes

Previously, we reported that ATXN1 oligomers are the primary drivers of toxicity in Spinocerebellar ataxia type 1 (SCA1; Lasagna-Reeves et al., 2015). Here we report that polyQ ATXN1 oligomers can propagate locally in vivo in mice predisposed to SCA1 following intracerebral oligomeric tissue inoculation. Our data also show that targeting these oligomers with passive immunotherapy leads to some improvement in motor coordination in SCA1 mice and to a modest increase in their life span. These findings provide evidence that oligomer propagation is regionally limited in SCA1 and that immunotherapy targeting extracellular oligomers can mildly modify disease phenotypes. DOI: http://dx.doi.org/10.7554/eLife.10891.001


Introduction
We recently demonstrated that soluble oligomers play an important role in Spinocerebellar ataxia type 1 (SCA1), a neurodegenerative disease caused by expansion of a CAG repeat that encodes for glutamine (Q) in ataxin-1 (ATXN1) (Lasagna-Reeves et al., 2015). In the same study, we showed that ATXN1 oligomers are internalized into the cell and seed the formation of new ATXN1 oligomers (Lasagna-Reeves et al., 2015). This 'seeding' concept has been explored in several other proteinopathies and is believed to be one of the drivers of neurodegeneration (Frost et al., 2009;Munch et al., 2011;Holmes and Diamond, 2012). What's more, evidence has mounted that soluble aggregates of amyloidogenic proteins including Tau, Ab and a-synuclein, can propagate pathology from cell-to-cell in a prion-like fashion, thereby causing pathology progression from one brain region to the another in a disease-specific pattern (Hardy and Revesz, 2012;Holmes and Diamond, 2012;Guo and Lee, 2014). Passive immunotherapy has been recently proposed as a feasible strategy to inhibit pathology propagation in mouse models for proteinopathy (Banks et al., 2007;Chai et al., 2011;Masliah et al., 2011;Yanamandra et al., 2013;Castillo-Carranza et al., 2014;Games et al., 2014;Tran et al., 2014). Despite these advances, it is not clear whether a mostly nuclear protein like ATXN1 will propagate from cell-to-cell in vivo and whether passive immunotherapy targeting oligomers will modify disease course.
In the present study, we report that polyQ ATXN1 oligomers act as a seed by inducing disease propagation to neighboring but not to distal cells and demonstrate that this cell-to-cell spread can be blocked using passive immunotherapy.

ATXN1 oligomers seed the formation of new endogenous ATXN1 oligomers in vivo
We recently demonstrated that in Atxn1 154Q/+ mice ATXN1 oligomers are restricted to focal subpopulations of Purkinje cells (PCs) and are not evenly present throughout the cerebellum. Notably, this focal distribution coincided with cellular toxicity (Lasagna-Reeves et al., 2015). This observation, together with the finding that ATXN1 oligomers are able to penetrate cells in culture and seed the formation of new ATXN1 oligomers led us to hypothesize that if ATXN1 oligomers propagate in vivo, intracerebral injection of brain extract from a symptomatic SCA1 mouse into a disease-free mouse would predispose the latter to develop neuropathology. To test this hypothesis, we injected cerebellar extract (10 mg, 2.5 mL) either from Atxn1 154Q/+ or wild-type mice into the deep cerebellar nuclei of wild type, Atxn1 -/and Atxn1 78Q/+ mice ( Figure 1A). Atxn1 78Q/+ mice express one allele of murine Atxn1 with a 78Q expansion. These mice do not display behavioral abnormalities nor any neuropathology, including ATXN1 inclusions, indicating that a single copy of 78Q-Atxn1 is insufficient to generate disease within the short lifespan of a mouse (Lorenzetti et al., 2000). These characteristics make this mouse an ideal model to determine the seeding abilities of ATXN1 soluble oligomers from Atxn1 154Q/+ mice lysates.
Before performing injections, we confirmed by western blot analysis that the injected material from Atxn1 154Q/+ mouse cerebella was indeed enriched in oligomers ( Figure 1A). Nine different groups of mice (each constituting 8 animals) were injected at 3 months of age ( Figure 1B). Three months after injection, ELISA revealed that cerebellar extracts from the SCA1 Atxn1 154Q/+ mice caused a two-to-three fold increase in oligomer formation in the cerebellum and brain stem of Atxn1 78Q/+ mice compared to other groups ( Figure 1C, left panels). Oligomer levels in the cortex, which were lower to begin with than in the cerebellum and brainstem, did not significantly change in any of the groups following injection of cerebellar lysate. Neuropathology confirmed that oligomers were located in cerebellum and brain stem, but not in cortex, hippocampus, striatum or olfactory bulb (data not shown). ATXN1 oligomers thus appear to propagate only to neighboring areas, and, at least under these conditions, trans-synaptic propagation was not detected. Nevertheless, we can't exclude the possibility that a higher concentration of inoculum and/or a longer incubation time might lead to oligomer propagation to farther areas. In all of the groups of mice tested, we observed similar amounts of total ATXN1 ( Figure 1C, right panels), which indicates that the increase of oligomers in the cerebellum and brain stem of Atxn1 78Q/+ mice was produced by a seeding effect of ATXN1-154Q soluble oligomers rather than by inducing more expression of ATXN1-78Q. To evaluate whether or not pathology propagation was triggered by oligomers presented in the injected material from Atxn1 154Q/+, we performed a cell-based seeding assay using the injected materials as previously described (Lasagna-Reeves et al., 2015). Specifically, we added the injected material from Atxn1 154Q/+ alone or pre-incubated with the anti-oligomer antibody F11G3 to cells that express ATXN1(82Q) fused to mRFP. After a 10 hr incubation, we then quantified the amount of oligomeric inclusions. In comparison with cells incubated with the injected material from wild type or nontreated group, the cells exposed only to the injected material from Atxn1 154Q/+ developed the largest proportion of oligomeric inclusions (37.25%). Moreover, when cells were exposed to the injected material from Atxn1 154Q/+ pre-incubated with F11G3, the proportion of oligomeric inclusions (15.25%) did not increase in comparison with the control groups thus suggesting that the active material in the injected lysate are the oligomers (Figure 1-figure supplement 1). In addition, this oligomeric pathology did not result from the surgical procedure itself: only PCs of Atxn1 78Q/+ knockin mice injected with cerebellar extract from Atxn1 154Q/+ mice, not those injected with WT cerebellar extract, showed an increase in oligomers ( Figure 1D). The observation that no oligomers were detected in PCs from either wild-type or Atxn1 null mice injected with Atxn1 154Q/+ lysates suggests  that host expression of polyQ-expanded ATXN1 is necessary for the de novo formation of ATXN1 oligomers in PCs. Immunofluorescence confirmed that the oligomers detected in Atxn1 78Q/+ PCs were indeed ATXN1 oligomers ( Figure 1E). Overall, these data suggest that the ATXN1 oligomers present in the injected material from Atxn1 154Q/+ are responsible for inducing propagation of ATXN1 oligomers in Atxn1 78Q/+ mice. Nevertheless, it is possible that other factors present in the inoculum play an important role in promoting propagation. Further, downstream exploration of the precise biochemical composition of these lysates will surely shed insight into this matter.
To determine if oligomer propagation was accompanied by motor deficit, we performed the rotarod assay in mice harboring brain lysate for 3 months. Even though the performance varied during training, there was no evidence of sustained deficit in the Atxn1 78Q/+ knockin mice injected with cerebellar extract from Atxn1 154Q/+ mice (Figure 1-figure supplement 2). Despite the fact that oligomers propagate through the cerebellum in Atxn1 78Q/+ knockin mice, the newly formed 78Q ATXN1 oligomers appear insufficient to induce motor deficit or degeneration (data not shown). This lack of toxicity could be because in Atxn1 78Q/+ knockin adult mice, the neurons are relatively healthy and thus can counteract the toxic effect of newly formed oligomers. In contrast, in Atxn1 154Q/+ mice, the neurons become dysfunctional by four weeks (Lasagna-Reeves et al., 2015) rendering them more susceptible to the toxicity of oligomers. Another possibility is that the newly formed 78Q ATXN1 oligomers adopt a different structure or conformation that is less toxic than the 154Q ATXN1 oligomers. Further studies will be necessary to determine if these newly formed ATXN1 oligomers are solely derived from 78Q ATXN1 molecules or whether they contain some persistent 154Q ATXN1 seeds.

Passive immunotherapy decreases ATXN1 oligomer pathology and improves motor coordination
Given the ability of the anti-oligomer antibody F11G3 to inhibit the internalization of ATXN1 oligomer complexes from Atxn1 154Q/+ cerebellum in vitro (Lasagna-Reeves et al., 2015), we postulated that anti-oligomer (F11G3) immunotherapy could reduce the load of ATXN1 oligomers and mitigate the motor impairment observed in the Atxn1 154Q/+ model. Because Atxn1 154Q/+ mice develop motor incoordination as early as 5 weeks of age (Watase et al., 2002), we began treatment at 4 weeks of age, where Atxn1 154Q/+ mice already have a modest accumulation of oligomers in the cerebellum (Figure 2-figure supplement 1). Wild-type and Atxn1 154Q/+ mice were injected intraperitoneally with F11G3 or control IgM antibodies (5 mg/Kg) once a week for 6 weeks. One week after the last injection, we performed the rotarod assay, sacrificed the mice and performed pathological and biochemical analyses. Injected mice were separated into two cohorts, one for biochemical and pathological analysis and one for behavioral and survival analysis. We focused our pathological examination on the cerebellum. Brain sections were immunostained with the anti-oligomer antibody, A-11 (Kayed et al., 2003), to ensure depletion of oligomers at the site of interest. Atxn1 154Q/+ mice treated with the anti-oligomer antibody showed fewer PCs with oligomers than the control group (Figure 2A and B). We further analyzed the cerebella by ELISA and noted a decrease in the amount of oligomers and ATXN1 ( Figure 2C and D). Notably, the immunotherapy did not affect the formation and total number of nuclear inclusions in the cortex ( Figure 2E): either these cortical inclusions are not preceded by oligomers or the antibody did not get internalized into these cells in vivo. Considering our previous study where we demonstrated that the anti-oligomer antibody blocks the seeding effect of ATXN1 oligomers in cell culture without getting internalized into the cell (Lasagna-Reeves et al., 2015), we suggest that the anti-oligomer antibody arrests the propagation of ATXN1 oligomer complexes to neighboring areas by targeting extracellular oligomeric entities rather than directly targeting intracellular oligomers. To confirm that the immunotherapy was indeed targeting ATXN1 oligomers, we analyzed the treated and control samples by western blot using F11G3 and anti-ATXN1 antibody ( Figure 3A and B). The western blot analysis revealed a decrease in ATXN1 oligomers in the treated group, but no observable change at the level of monomeric ATXN1 ( Figure 3A and B).
To determine the functional consequences of this immunotherapy, we tested motor performance in treated and control Atxn1 154Q/+ mice. Rotarod analysis revealed a mildly significant improvement in coordination following six weeks of anti-oligomer immunotherapy ( Figure 3C). Although treated mice still possessed a motor deficit in comparison with wild type mice, continued weekly immunotherapy treatments in Atxn1 154Q/+ mice throughout their lifespan extended survival by approximately 3.5 weeks in comparison with the control-treated Atxn1 154Q/+ mice ( Figure 3D). Rotarod assay in all treatment groups over a four-day period (four trials per day, averaged) 6 weeks following onset of immunotherapy (mice 10 weeks of age). n = 12 per genotype; ** denotes p<0.01, ANOVA followed by Tukey's post hoc test. (D) Kaplan-Meier survival curve shows that animals treated with anti-oligomer immunotherapy (blue line) lived, on average, 3.5 weeks longer than control animals (red line). No death was observed in WT mice receiving immunotherapy (black and grey lines). *** denotes p<0.001, Log-rank (Mantel-Cox) test. n = 12 per genotype. DOI: 10.7554/eLife.10891.008

Discussion
Our previous study showed that ATXN1 oligomeric complexes were able to penetrate cells in culture and seed the formation of new ATXN1 oligomers (Lasagna-Reeves et al., 2015). It is widely accepted that amyloid formation is characterized by such seeding (Serio et al., 2000;Pedersen et al., 2004). This phenomenon has been explored in cell culture assays, several of which have demonstrated that extracellular aggregates that are internalized into the cell induce aggregation of intracellular proteins (Frost et al., 2009;Munch et al., 2011;Holmes and Diamond, 2012). Moreover, the concept of cell-to-cell spread of many amyloidogenic proteins in vivo has been demonstrated by the progression of protein aggregate pathology from one brain region to another in a disease-specific pattern (Hardy and Revesz, 2012;Holmes and Diamond, 2012). In our study, however, we found that injected oligomers induced formation of new ATXN1 oligomers only in areas proximal to the injection site. It thus seems unlikely that ATXN1 oligomers propagate transsynaptically in SCA1, as has been suggested with other neurodegenerative diseases (Harris et al., 2010;de Calignon et al., 2012;Liu et al., 2012;Pecho-Vrieseling et al., 2014). Rather, we suggest ATXN1 propagates through a secretion and reuptake mechanism between neighboring cells as has been proposed for other proteinopathies (Guo and Lee, 2014). Furthermore, no obvious signs of degeneration or motor incoordination were observed in the injected mice. This suggests that the cell must continuously express the toxic entity to trigger degeneration and behavioral deficits in mice. This notion is supported by the observation that immunotherapy targeting the ATXN1 oligomers slightly modified the phenotype in the Atxn1 154Q/+ mice: the antibody hindered the local propagation of ATXN1 oligomers. This treatment is not curative, however, because neurons expressing polyQ ATXN1 will still form their own toxic oligomeric entities, and some non-oligomeric forms of PolyQ ATXN1 might contribute to toxicity.
Passive immunotherapy has been proposed as a feasible strategy to inhibit pathogenesis in mouse models for AD and PD (Banks et al., 2007;Chai et al., 2011;Masliah et al., 2011;Yanamandra et al., 2013;Castillo-Carranza et al., 2014;Games et al., 2014). Our findings suggest that such therapy might slightly impact disease progression by blocking the propagation of these oligomeric entities, but to halt or reverse symptoms it would likely be necessary to incorporate a joint therapy that targets the root cause of disease, the abnormal accumulation of polyQ ATXN1 (Park et al., 2013). Altogether, our findings revealed that ATXN1 oligomer propagation contributes to SCA1 pathogenesis, and that such propagation especially in the vicinity of affected cells, could be targeted through passive immunotherapy. That the benefit from such therapy was small highlights the need to develop therapies that target ATXN1, the protein driving the pathogenesis. The need for such combination therapy is likely to extend to other disease-driving proteins.

Experimental procedures
Mouse models and preparation of brain extracts All mouse procedures were approved by the Institutional Animal Care and Use Committee for Baylor College of Medicine and Affiliates. Atxn1 154Q/+ ,Atxn1 78Q/+ and Atxn1 -/mice have been previously described (Lorenzetti et al., 2000;Watase et al., 2002) and were backcrossed to C57BL/6 for more than ten generations. Mouse cerebella were dissected and lysed in 0.5% Triton buffer (0.5% Triton X-100, 50 mM Tris pH 8, 75 mM NaCl) supplemented with protease and phosphatase inhibitors (Sigma, St-Louis, Mo). The protein lysate was then incubated on ice for 20 min and centrifuged at 13,200 r.p.m. for 10 min at 4˚C, and the supernatants were portioned into aliquots, snap-frozen, and stored at -80˚C until used.

Rotarod assay
Motor coordination was assessed on the Rotarod assay as previously described (Park et al., 2013), with four trials a day (separated by 1 hr each) for 4 days. The tester was blinded to animal genotype and treatment.

Immunotherapy
We used F11G3 and a control mouse IgM as antibodies for immunotherapy. Antibodies were administered at 5 mg/kg via intraperitoneal (i.p) injection once a week for six weeks. One week after completion of the treatment 12 mice per group were tested on the rotarod assay and sacrificed immediately afterward so that brains could be collected for biochemical and histopathological analysis. For survival studies, 12 mice per group were vaccinated once a week (5 mg/Kg) throughout their lifespan.

Brain sections immunofluorescence
Paraffin sections were deparaffinized, rehydrated, and washed in 0.01 M PBS 3 times for 5 min each time. After blocking in normal goat serum for 1 hr, sections were incubated overnight with rabbit anti-ATXN1 antibody 11750 (1:700). The next day, the sections were washed in PBS 3 times for 10 min each and then incubated with goat anti-rabbit IgG Alexa Fluor 568 (1:700; Invitrogen) for 1 hr. The sections were then washed 3 times for 10 min each time in PBS before incubation overnight with mouse anti-oligomers F11G3 (1:300). The next day, the sections were washed in PBS 3 times for 10 min each before incubation with goat anti-IgM Alexa Fluor 488 (1:700; Invitrogen) for 1 hr. Sections were washed and mounted in Vectashield mounting medium with DAPI (Vector Laboratories). The sections were examined using a Zeiss LSM 710 confocal microscope.

Stereotaxic surgery
Atxn1 78Q/+ , wild type or null Atxn1 mice were anaesthetized with a mixture of ketamine (110 mg/kg body weight) and xylazine (20 mg/kg body weight) in saline. Bilateral stereotaxic injections of 2.5 ml brain extract (3.9 mg/mL) from Atxn1 154Q/+ or Wild Type mice were placed with a Hamilton syringe into the cerebellum (From Bregma; Post -6.0 mm, lat +/-2.0 mm, dv -2.2 mm). Injection speed was 1.25 ml/minute and the needle was kept in place for an additional 2 min before it was slowly withdrawn. The surgical area was cleaned with sterile saline, the incision was sutured, and the mice were monitored until recovery from anesthesia. If not otherwise stated, 8 mice/group were used. Injected material was obtained from mouse brain extracts prepared from the cerebellum of aged 30-weekold Atxn1 154Q/+ and age-matched, non-transgenic wild-type control mice. All animal experiments were in compliance with protocols approved by the local Animal Care and Use Committee.

Cell-based seeding assay
A stable Daoy mRFP-ATXN1(82Q) cell line was generated as previously described (Park et al., 2013). Cells were plated in 24-well plates (2*104 cells/ml). 24 hr later, cells were treated with 2.5 ml brain extract (3.9 mg/mL) from Atxn1 154Q/+ or Wild Type mice. After 10 hr of treatment, cells were fixed with methanol at À80˚C for 45 min. RFP-positive inclusions ranging from 350 to 900 nm were considered oligomeric; inclusions larger than 900 nm and not detected by F11G3 were considered fibrillar. One hundred cells were quantified per group in triplicates. Analyses were manually performed with Image J. For blocking assays, each sample was mixed with F11G3 antibody (2.5 mg/ml) in a ratio 1:1 (vol/vol) for 1 hr and then added to the cells.

ELISA
For ELISA, plates were coated with 10 ml of the soluble fraction of brains using 0.1 M sodium bicarbonate (pH 9.6) as a coating buffer, followed by incubation for 1 hr at 37˚C, washing three times with Tris-buffered saline with very low (0.01%) Tween 20 (TBS-T), and then blocking for 1 hr at 37˚C with 10% BSA. The plates were then washed three times with TBS-T; F11G3 (1:500), A-11 (1:1000), 11750 (1:2000) or Tubulin (1:2000) antibodies (diluted in 5% nonfat milk in TBS-T) were added and allowed to react for 1 hr at 37˚C. The plates were then washed three times with TBS-T, and 100 ml of horseradish peroxidase-conjugated anti-mouse IgM, anti-mouse IgG or anti-rabbit IgG (diluted 1:10,000 in 5% nonfat milk in TBS-T; Promega, Madison, WI) were added, followed by incubation for 1 hr at 37˚C. Finally, plates were washed 3 times with TBS-T and developed with 3,3,5,5-tetramethylbenzidine (TMB-1 component substrate) from KPL (Gaithersburg, MD). The reaction was stopped with 100 ml of 1 M HCl, and samples were read at 450 nm.

Analysis of ATXN1 pathology
To determined the percent of PCs with ATXN1 oligomers, 5 mm brain sections were stained with anti-Calbindin antibody as described above to quantify the total number of PCs in the cerebellum. The number of PCs positive for Calbindin was consider as the 100%. The adjacent sections were immunostained for oligomers using F11G3 or A-11. For the quantification of nuclear inclusions, we stained ATXN1 using 11750 antibody and performed nuclear staining with hematoxylin. We considered the total amount of nuclei in the cortex as the 100% of nucleus. Data were analyzed using posthoc test.

Statistical analysis
Experimental analysis and data collection were performed in a blinded fashion. p-values were determined using the appropriate statistical method via GraphPad Prism, as described throughout the manuscript. For simple comparisons, Student's t-test was used. For multiple comparisons, ANOVA followed by the appropriate post hoc analysis were utilized. All data is presented as mean ± SEM. *, ** and *** denote p<0.05, p<0.01 and p<0.001, respectively.